Discovery and development of a small-molecule inhibitor targeting the GAS41 YEATS domain in non-small cell lung cancer

Abstract

GAS41 frequently overexpressed in Non-Small Cell Lung Cancer (NSCLC). GAS41 contains YEATS domain, which recognizes acetylated lysine residues on histones to recruit protein complexes and facilitate transcription. Suppression of GAS41 in NSCLC models inhibits cellular proliferation and markedly reduces tumor growth in mouse xenografts, justifying development of small molecule inhibitors. We have employed structure-based design and medicinal chemistry optimization to discover DLG-41, a sub-micromolar inhibitor binding to GAS41 YEATS domain. DLG-41 potently disrupts the association of GAS41 YEATS with chromatin in mammalian cells and inhibits proliferation of NSCLC cell lines with sub-micromolar potency without significantly affecting normal lung fibroblasts. DLG-41 induces more effective growth inhibition in A549 versus GAS41-knockout cells, demonstrating on-target activity. DLG-41 treatment upregulates CDKN1A gene and downregulates pathways associated with lung cancer cell identity, tumor migration, and invasion. DLG-41 is a promising chemical probe for targeting GAS41 protein in NSCLC models and has potential for future development.

Graphical Abstract

Introduction

GAS41 (encoded by YEATS4 gene) is an epigenetic reader protein which binds to acylated lysine residues in chromatin and modulates gene transcription. Aberrant GAS41 expression and acyl-lysine recognition activity of its YEATS domain has been detected in various cancers, including glioma, colorectal, and lung cancers.^1-4 GAS41 protein contains two functional domains, the C-terminal coiled-coil domain and the N-terminal YEATS domain.⁵ The coiled-coil domain facilitates dimerization of the GAS41 protein and interacts with other subunits in large chromatin remodeling complexes, such as TIP60/p300 and SRCAP complexes.⁶ The YEATS domain contains an acylation recognition channel, formed by aromatic residues W94, Y74, and F96, that engages the acyl group of the modified lysine through a hydrogen bonding network.⁷ Characterization of the GAS41 YEATS binding with various acylated lysine residues has demonstrated a preference for acetylated or crotonylated lysine 27 on histone H3 (H3K27ac/cr mark).⁸ Outside of the acylation binding pocket, GAS41 YEATS binds to the H3 N-terminus through an acidic pocket unique to GAS41 over other YEATS domain containing proteins.⁹

In NSCLC, GAS41 protein is overexpressed in over 20% of samples in The Cancer Genome Atlas (TCGA).¹⁰ YEATS4 was identified as a component of the frequently amplified 12q15 amplicon and overexpressed GAS41 protein was identified in a cohort of NSCLC patient samples.⁴ YEATS4 overexpression in normal bronchial epithelial cells induced phenotypic changes indicative of NSCLC malignant transformation, suggesting a role of GAS41 in lung cancer tumorigenesis.⁴ In NSCLC cellular models, GAS41 colocalizes with H3K27ac marks at the promoters of actively transcribed oncogenes.¹¹ The recruitment of the SRCAP complex to exchange histone H2A for H2A.Z and increase gene expression at NSCLC-associated oncogenes is facilitated through GAS41 YEATS H3K27ac recognition activity.¹² Genetic silencing of GAS41 in NSCLC cell models inhibited cellular proliferation and cell cycle gene expression.¹² In mouse xenograft models of H1299 NSCLC cells, silencing of GAS41 dramatically reduced tumor growth. Both cellular and tumor proliferation could be re-established with ectopic expression of wild-type GAS41, but not the acetylation binding mutants W93A or Y74A.¹² Mechanistically, GAS41 has been shown to repress the p53 tumor suppressor pathway, and independently repress p21 expression, in NSCLC cellular models.⁴ Taken together, there is significant evidence that GAS41 YEATS activity is a critical driver of NSCLC onset and tumor growth.

Emerging data suggests that GAS41 is a promising target for the development of small molecule inhibitors as potential anti-cancer agents. The well-defined binding site on the YEATS domain, which interacts with H3K27ac, presents a suitable pocket for small molecule binding. To date, several small molecule inhibitors of GAS41 YEATS domain have been reported. We have discovered first-in-class GAS41 inhibitors by performing NMR-based fragment screening combined with medicinal chemistry optimization yielding low μM inhibitors.¹³ To improve the activity of these compounds, we developed dimeric analogs to bivalently engage two YEATS domains in dimeric GAS41 protein and achieved mid-nanomolar potency. However, these compounds have limited activity in cancer cell lines, likely due to their large size and limited permeability.¹³ More recently, a different series of potent, nanomolar small molecule GAS41 binders developed based on virtual screening have been disclosed, but no biological activity have been reported for these compounds.¹⁴ Additionally, benzamide based ligands were identified from a virtual screening campaign, and developed into modest binders (K_D = 32.6μM) with weak activity in U87 glioma cellular proliferation assays (GI₅₀ > 50μM).¹⁵ Despite these efforts, there remains a need for potent GAS41 YEATS domain inhibitors with improved drug-like properties and on-target activity in cancer cells.

Here, we have performed extensive optimization of the previously disclosed fragment-like aminothiophene scaffold to improve both binding affinity for the GAS41 YEATS domain and cell permeability. By performing structure-guided design combined with extensive medicinal chemistry, we developed DLG-41, a sub-micromolar GAS41 YEATS domain inhibitor. DLG-41 binds the GAS41 YEATS domain in acetylation binding pocket and engages GAS41 YEATS in mammalian cells. Importantly, DLG-41 is active in NSCLC cell models, exhibiting sub-micromolar antiproliferative activity, and inducing differential gene expression to inhibit the antioxidant response to oxidative stress and cellular migration.

Results and Discussion

Optimization of a fragment hit for GAS41 YEATS domain

We have previously described results of a fragment screening against GAS41 YEATS domain resulting in identification of 1 (6EE9) (5-(tert-butyl)thiophene-2-yl)(pyrrolidine-1-yl)methanone).¹³ Activity of the fragment hit in a fluorescence polarization assay yielded IC₅₀ = 210 μM (Table 1). This compound contains an aminothiophene amide moiety as a pharmacophore mimicking the acylated lysine from H3 derived peptides. We have attempted optimization of 1 by introducing substitutions on the thiophene and amide sides (Table 1 and Table 2, respectively).

Table 1.

Chemical structures and inhibitory activity of GAS41 YEATS inhibitors 1-8

Compound	R	IC50 FP (μM)
1	tert-butyl	210 ± 13.0
2		18.5 ± 3.06
3		146 ± 24.0
4		208 ± 39.6
5		22.8 ± 3.54
6		21.6 ± 3.92
7		4.57 ± 0.19
8		34.0 ± 3.50

IC₅₀ FP values are determined from FP assay with H3K23crK27cr-FAM probe; data shown as mean ± SD from a minimum of two independent experiments.

Table 2.

Chemical structures and inhibitory activity of GAS41 YEATS inhibitors 9-18, with optimization of azetidine substituents.

Compound	R	R1	IC50 FP (μM)
9		H	21.8 ± 1.1
10		H	10.4 ± 2.4
11		H	4.3 ± 1.4
12		H	13.0 ± 2.2
13		H	9.8 ± 0.9
14		H	8.5 ± 0.4
15		H	10.1 ± 1.5
16		H	2.0 ± 0.5
17		H	7.3 ± 1.0
18		H	8.9 ± 0.6

IC₅₀ FP values are determined from FP assay with H3K23crK27cr-FAM probe; data shown as mean ± SD from a minimum of two independent experiments.

In a first step, we replaced the tert-butyl group in 1 (6EE9) with L-proline yielding 2 with 10-fold improved activity (IC₅₀ = 18.5 μM, Table 1).¹³ Subsequently, we introduced methyl groups into various positions of the proline yielding several analogs, including 3 (IC₅₀ FP = 146 μM), 4 (IC₅₀ FP = 208 μM), 5 (IC₅₀ FP = 22.8 μM) and 6 (IC₅₀ FP = 21.6 μM), but no improvement was achieved. Interestingly, the simultaneous introduction of two methyl groups in the 4-position of the proline side chain led to compound 7 with improved potency (IC₅₀ FP = 4.57 μM). An attempt to introduce a bulky group in the 4-position of the (S)-proline ring in compound 8 did not improve the activity (IC₅₀ FP = 34.0 μM), likely because this part of the molecule is solvent-exposed.

In the next step, we fixed L-proline moiety on the thiophene, as in 2, and performed optimization of the amide fragment (Table 2). Since substitution of pyrrolidine ring creates stereoisomers and conformers, we introduced 3-phenylazetidine. It resulted in 9 (IC₅₀ FP = 21.8 μM) with activity comparable to 2, demonstrating that replacing the pyrrolidine with 3-substituted azetidine is tolerable and maintains potency. We have previously reported several analogs of 9, replacing phenyl with various heterocycles: thiazole 10 (IC₅₀ FP = 10.4 μM) and benzothiazole 11 (IC₅₀ FP = 4.3 μM) leading to few-fold improved potency.¹³ We also determined the crystal structure of GAS41 YEATS domain in complex with 10 and found that the thiazole ring is not involved in extensive interactions, suggesting that further optimization may create additional contacts with the YEATS domain.¹³

Here, we continued optimization of the aromatic substituents, and we introduced a benzylic moiety 12 (IC₅₀ FP = 13 μM), benzimidazole 13 (IC₅₀ FP = 9.8 μM), and benzothiophene 14 (IC₅₀ FP = 8.5 μM) but no improvement in the activity over 10 was achieved (Table 2). We further substituted the thiazole with the second phenyl giving rise to 15 (IC₅₀ FP = 10.1 μM), 16 (IC₅₀ FP = 2.0 μM) and 17 (IC₅₀ FP = 7.3 μM). In this series, we found that the 5-phenyl thiazole analog 16 is 5-fold more potent vs 10 (Table 2). To investigate the influence of the nitrogen atom in the thiazole ring of 16 we also synthesized thiophene analog 18 (IC₅₀ FP = 8.9 μM) and found that this change decreased the activity 4.5-fold (Table 2).

Optimization of thiazole substitutions

We selected the 16 scaffold and investigated substitutions of the phenyl ring. First, we synthesized several analogs with single Cl, OMe, and NH₂ substitutions introduced into ortho- and meta-positions and found that all these compounds have relatively similar activities varying ± 2-fold when compared to 16 (Table 2). The most potent compound in this series was 19 (IC₅₀ FP = 0.98 μM), suggesting that an amino group may form favorable interactions with the YEATS domain. Analysis of the crystal structure of 10 bound to the GAS41 YEATS domain revealed the presence of several negatively charged side chains (e.g., D41, E40, and E90) adjacent to the thiazole ring. Thus, to create favorable electrostatic interactions, we introduced several positively charged groups to the phenyl ring of 16 leading to a series of compounds (20-27). Surprisingly, all these compounds had nearly identical activity with IC₅₀ values ranging from 1.2 to 2.5 μM (Table 3). Importantly, these activities were close to the affinity of the fluorescein labeled peptide we used in the FP assay (K_D = 1.30 μM)¹³ , indicating that the current assay has reached its sensitivity threshold, suggesting a need for an improved assay to characterize more potent compounds.

Table 3.

Chemical structures and inhibitory activity of GAS41 YEATS inhibitors with optimization of thiazole substituents

Compound	R1	R2	IC50 FP (μM)	IC50 AL (μM)
16		H	2.00 ± 0.05	0.73 ± 0.04
19		H	0.98 ± 0.21	0.25 ± 0.05
20 (DLG-1)		H	2.51 ± 0.16	0.15 ± 0.04
21		H	1.32 ± 0.22	0.46 ± 0.12
22		H	1.21 ± 0.08	0.56 ± 0.06
23		H	2.33 ± 0.05	0.71 ± 0.15
24		H	1.23 ± 0.54	0.51 ± 0.17
25		H	1.65 ± 0.28	0.44 ± 0.05
26		H	1.65 ± 0.18	0.56 ± 0.001
27		H	1.68 ± 0.11	0.41 ± 0.08

IC₅₀ FP values are determined from FP assay with H3K23crK27cr-FAM probe. IC₅₀ AL values are determined from AlphaLISA assay with 21-Biot probe. Data shown as mean ± SD from a minimum of two independent experiments.

To develop a new biochemical assay, we have modified the amino group in 21, which we predicted to be partially solvent exposed, and introduced a biotin moiety yielding probe compound 21-biot (Figure S1A). Using this compound and his₆-GAS41 YEATS (13-158) we developed an AlphaLISA competition assay.¹³ This assay showed improved sensitivity, and we reduced the concentration of the GAS41 YEATS domain to 100 nM to quantify the activity of more potent compounds. First, we validated the AlphaLISA assay by performing competition with 21, which lacks the biotin group, and obtained an IC₅₀ = 460 ± 120 nM (Figure S1B). Subsequently, we tested the activity of compound 16 analogs, and we found that IC₅₀ values range between 2.4 to 0.15 μM, which are generally lower when compared to the FP assay (Table 3). Importantly, AlphaLISA activities span a broader range vs the FP assay, providing more accurate evaluation of compound activities. The most potent compounds in this series include 19-22, 24-25, 27 with IC₅₀ in the 150 - 500 nM range (Table 3).

Development of cell permeable GAS41 inhibitor

20 (DLG-1) is one of the most potent and soluble inhibitors we developed, with IC₅₀ = 150 nM in the AlphaLISA assay (Table 3). To establish the binding mode of DLG-1, we have determined the 2.3 Å crystal structure of the complex with GAS41 YEATS domain (Figure 1A, Table S1). Protein crystallized with 4 protein molecules in the asymmetric unit and we found that compound DLG-1 is bound to each protein molecule (Figure S2) in a channel-like site which constitutes the binding site of the acetylated histone peptides (Figure 1B).⁸ The central thiophene fits into an aromatic cage composed of three aromatic rings of H71, W93 and F96, and the amide carbonyl group forms two hydrogen bonds with backbone amide protons of G92 and Y93 (Figure 1C). The pyrrolidine moiety is partly solvent exposed with the positively charged amine forming a hydrogen bond with the backbone carbonyl of E95. On the other side of compound 18, the thiazole ring is engaged in aromatic pi-pi stacking interactions with the side chains of H43 and Y74 (Figure 1C). The phenyl ring in DLG-1, approaches the backbone of Y74 and the amino-methyl group interacts with the negatively charged carboxylic moiety of E90 (Figure 1C), which most likely contributes to the 4-fold improvement in the activity when compared to unsubstituted 16 (Table 3). The binding mode of DLG-1 (Figure 1C) is similar to 10 which we previously reported.¹³ We found that the DLG-1 phenyl group in two out of four molecules observed in the asymmetric unit of GAS41 YEATS-DLG-1 crystal structure form a slightly different orientation with the amino-methyl group involved in the intermolecular contacts, most likely representing crystal packing artifacts (Figure S2).

Figure 1. Crystal structure of GAS41 YEATS domain with bound DLG-1.

A. An electron density mFo-DFc map for DLG-1 contoured at 1.5 σ level. B. Overall binding mode of DLG-1 with GAS41 YEATS domain in surface representation and ligand shown in sticks. C. Details of DLG-1 binding mode. Selected GAS41 residues (white carbons) and DLG-1 (green carbons) are shown as sticks. Hydrogen bonds are indicated by dashed lines and pi-pi stacking interactions are shown as blue arrows. Structure of GAS41 YEATS domain with bound DLG-1 has been deposited in PDB under code 9O4Y.

While DLG-1 is a relatively potent GAS41 inhibitor, the strong positive charge and low cLogP = 1 renders this compound as a poor candidate for cellular studies. To explore more hydrophobic analogs, we have selected 21, with similar activity to DLG-1, as a scaffold for further modifications (Table 4). The terminal amino group in 21 represents a convenient moiety for subsequent functionalization and we synthesized corresponding acetyl amide 28 (IC₅₀ AL = 300 nM, Table 4), which shows comparable activity to 21. We have also developed additional amides based on iso-propyl (29, IC₅₀ AL = 200 nM), cyclopentyl (30, IC₅₀ AL = 600 nM) and phenyl (31, IC₅₀ AL = 200 nM) and found that all these compounds have comparable activity, suggesting that the hydrophobic amide moieties are solvent exposed. Potency was also maintained for analogs with secondary amides 32 (IC₅₀ AL = 900 nM), 33 (IC₅₀ AL = 900 nM) and 34 (IC₅₀ AL = 1.1 μM). Substitutions in the phenyl ring with Cl were also tolerated with only a slight reduction in potency for 35 (IC₅₀AL = 1.4 μM) and 36 (IC₅₀ AL = 900 nM). The crystal structure of the YEATS domain with bound DLG-1 revealed that the phenyl ring is partially solvent exposed, allowing us to introduce a more hydrophobic moiety and increase the cLogP even further, we replaced phenyl with the naphthyl group, such as 37 (IC₅₀ AL = 700 nM) and 38 (IC₅₀ AL = 3.5 μM).

Table 4.

Chemical structures and inhibitory activity of GAS41 YEATS inhibitors focused on optimization of activity and properties.

Compound	R1	R2	R3	R4	R5	IC50 FP (μM)	IC50 AL (μM)	cLogP
28	H	H	H	Me-	H	0.9 ± 0.06	0.3 ± 0.04	0.9
29	H	H	H	i-Pr-	H	2.8 ± 0.23	0.2 ± 0.06	1.7
30	H	H	H	Cyclopentyl-	H	1.3 ± 0.40	0.6 ± 0.08	2.3
31	H	H	H	Ph-	H	1.1 ± 0.01	0.2 ± 0.08	2.7
32	H	H	Me-	Me-	H	1.1 ± 0.01	0.9 ± 0.21	0.9
33					H	2.0 ± 0.03	0.9 ± 0.17	0.6
34					H	3.7 ± 0.5	1.1 ± 0.03	1.0
35	Cl	H	H	Me-	H	3.4 ± 0.13	1.4 ± 0.33	1.3
36	H	Cl	H	Me-	H	2.8 ± 0.03	0.9 ± 0.21	1.6
37				Me-	H	2.3 ± 0.56	0.7 ± 0.27	2.0
38				i-Pr-	H	3.7 ± 0.98	3.5 ± 0.43	2.9
39 (DLG-41)				Et-	Me	2.7 ± 0.13	0.6 ± 0.10	3.6
40 (DLG-41nc)						117.5 ± 7.7	> 50	3.0

IC₅₀ FP values are determined from FP assay with H3K23crK27cr-FAM probe. IC₅₀ AL values are determined from AlphaLISA assay with 21-biot probe. Data shown as mean ± SD from a minimum of two independent experiments. cLogP calculated using ChemDraw 19.0.

To develop compounds with a favorable cLogP maintaining strong activity, we modified 37, replacing the terminal methyl with an ethyl group and added di-methyl groups to the proline side chain as in 7 yielding 39 (DLG-41). This new compound shows potent activity in AlphaLISA (IC₅₀ AL = 600 nM) and favorable cLogP = 3.6 (Figure 2A, Table 4). We tested the binding of DLG-41 to GAS41 YEATS domain by ITC and found K_D = 1.0 ± 0.3 μM and N = 0.80 stoichiometry (Figure 2B). This affinity is slightly reduced, when compared to biochemical assays, most likely due to limited solubility of DLG-41 in ITC conditions. We also synthesized a negative control compound by replacing the central thiophene with a thiazole, resulting in compound 40 (DLG-41nc) showing no GAS41 YEATS domain inhibition up to 10 μM (Figure 2A). To confirm direct binding of DLG-41 to the GAS41 YEATS domain, we performed NMR experiments, which clearly showed extensive amide perturbations with intermediate to slow exchange binding kinetics for DLG-41 but no perturbations with the control DLG-41nc (Figure 2C,D). We also profiled DLG-41 against a diverse panel of protein kinases and found no significant off-target activity (Table S2). In summary, we selected DLG-41 as a lead compound for cellular studies, based on its potency and properties.

Figure 2. DLG-41 is a potent inhibitor that binds to the GAS41 YEATS domain.

A. AlphaLISA competition assay for DLG-41 and DLG-41nc with 21-Biot probe. Representative curves are shown, IC₅₀ (DLG-41) is shown as mean ± SD from two independent experiments. B. Characterization of the direct binding of DLG-41 to GAS41 YEATS by ITC. Representative titration is shown; K_D and N calculations represent the mean ± standard deviation from two independent experiments. C. ¹H-¹⁵N HSQC NMR spectra of 50 μM ¹⁵N GAS41 YEATS bound to 100 μM DLG-41 (red) or 100 μM DLG-41nc (green) superimposed over the spectra of 50μM ¹⁵N GAS41 YEATS with DMSO (black). D. ¹H-¹⁵N HSQC NMR spectra of 50 μM ¹⁵N GAS41 YEATS with DMSO (black) superimposed with spectra of 50 μM ¹⁵N GAS41 YEATS with indicated molar ratios of DLG-41. Arrow points to amides undergoing strong broadening upon DLG-41 binding, and the amide showing slow exchange is boxed.

DLG-41 blocks binding of GAS41 with chromatin in mammalian cells and inhibits growth of lung cancer cells (or NSCLC cells)

To assess whether DLG-41 blocks binding of GAS41 to chromatin in cells, we developed a NanoBRET (Nanoluciferase Bioluminescence Resonance Energy Transfer) assay. For this assay, we transfected constructs encoding NanoLUC-GAS41 and Halo-H3.3 into HEK293T cells to generate BRET signal upon binding of GAS41 to histone H3 incorporated into the chromatin and endogenously acetylated in cells. Treatment with DLG-41 inhibits the signal in this assay with IC₅₀ = 0.9 μM, demonstrating that our GAS41 inhibitor blocks binding of GAS41 with chromatin in cells (Figure 3A). The less potent compound DLG-41nc showed much weaker activity in this assay with IC₅₀ > 25 μM. To further validate this assay, we have tested JQ1, an inhibitor of BET bromodomains,¹⁶ and found no activity in GAS41 BRET assay (Figure S3). However, when tested in the NanoBRET assay utilizing NanoLuc-tagged BRD4-BD1 and cells transfected with Halo-tagged histone H3.3, JQ1 shows strong activity (IC₅₀ = 79 nM), while DLG-41 had no activity (Figure S3). Overall, DLG-41 is a potent inhibitor that blocks chromatin binding activity of GAS41 in cells.

Figure 3: DLG-41 inhibits GAS41 YEATS domain in cells and blocks proliferation of NSCLC cell lines in GAS41 dependent manner.

A. Activity of DLG-41 and DLG-41nc in the NanoBRET assay in HEK293T cells. Representative data of mean normalized % maximum change of NanoBRET ratio ± SD is shown. B. Representative growth inhibition of indicated cell lines treated with DLG-41 or DLG-41nc for 7 days C. Representative colony images from the SRB assay in H1299 and H1993 cells treated with DLG-41 (7 days). D. Quantification of the growth inhibition of indicated cell lines treated with DLG-41 or DLG-41nc for 7 days using the SRB staining cytotoxicity assay. GI₅₀ values calculated as mean ± SD from two independent experiments. E. Analysis of GAS41 expression in a panel of cell lines determined by Western blot. Representative blot of two independent experiments. F, G. Proliferation of A549 and A549 GAS41 KO cells treated with DLG-41 (F) or DLG-41nc (G) for 14 days. Data are calculated as mean ± SD from two independent experiments.

We selected DLG-41 for cellular studies and tested the activity in a panel of NSCLC cell lines (A549, H1299, H1993, H358, H23, H460) using an SRB cell staining assay. We found that treatment with DLG-41 blocks proliferation in a majority of these cells, with GI₅₀ ranging from 0.45 to 1.0 μM (Figure 3B, C, D). We also tested DLG-41 in normal lung fibroblasts (IMR-90) and observed much weaker activity with GI₅₀ > 10 μM (Figure 3B, D). Furthermore, comparing DLG-41 with DLG-41nc shows 3-8 fold weaker cell growth inhibition for the less potent compound. To test whether the antiproliferative activity of DLG-41 correlates with GAS41 protein levels, we analyzed the expression of GAS41 in these cell lines. The majority of tested cell lines have very strong GAS41 expression when compared to normal lung fibroblasts (IMR-90), which is consistent with GAS41 amplification in NSCLC (Figure 3E).⁴ Interestingly, H358 cells, which are not sensitive to the treatment with DLG-41, also show much lower GAS41 levels, supporting the correlation between GAS41 expression and activity of DLG-41.

To further confirm this correlation and the on-target activity of DLG-41, we have generated an A549 GAS41-knockout cell line (A549 GAS41 KO) and validated loss of GAS41 expression (Figure 3E).¹³ Treatment of A549 cells for 14 days showed a strong growth inhibition (GI₅₀ = 0.46 μM) while the A549 GAS41 KO cells were 8-fold less sensitive (GI₅₀ = 3.42 μM, Figure 3F). We also tested DLG-41nc and found that our weaker control compound shows similar activity in A549 and A549 GAS41 KO cell lines (GI₅₀ > 3 μM, Figure 3G). In summary, DLG-41 inhibits proliferation of NSCLC cell lines cells in GAS41 dependent manner.

DLG-41 disrupts NSCLC-associated gene expression and cellular phenotypes

GAS41 has been recently shown to repress CDKN1A encoding the tumor suppressor protein p21 in a p53-independent mechanism.¹⁷ We have tested whether treatment with our GAS41 inhibitor affects the expression of CDKN1A in H1299 cells. Indeed, DLG-41 but not DLG-41nc strongly enhanced levels of CDKN1A, supporting the on-target activity (Figure 4A). To further investigate the mechanism of action of DLG-41, we have performed RNA-seq studies in H1299 cells. We found that 7-day treatment with 1.5 μM DLG-41 resulted in 876 significantly downregulated and 236 significantly upregulated genes (Figure 4B, C). Gene-set Enrichment Analysis (GSEA, Broad Institute) revealed that differentially enriched genes were involved in pathways relevant to NSCLC disease signaling. Downregulated gene sets included those overexpressed in lung cancer relative to normal lung cells, as well as hallmark genes for the epithelial-mesenchymal transition pathway (Figure 4D). Notable genes that are upregulated in lung cancer cells, but downregulated with DLG-41 treatment, include SPP1 and TSPAN8, which are considered prognostic markers of lung cancer progression.^18,19 Upregulated gene sets included hallmark genes of DNA repair (Figure 4D). On the contrary, our control compound DLG-41nc showed minimal effects on gene expression (Figure 4B), in agreement with its weaker antiproliferative activity.

Figure 4: DLG-41 decreases global gene expression and ameliorates NSCLC phenotypes in H1299 cells.

A. Gene expression levels of CDKN1A in H1299 cells treated with indicated concentrations of DLG-41 or DLG-41nc for 7 days. Data shown as mean ± SD from two independent experiments. B. Volcano plots depicting differential gene expression analysis of RNA-sequencing data of H1299 cells treated with 1.5 μM DLG-41 (left) or 1.5 μM DLG-41nc (right) for 7 days relative to DMSO. C. Heatmap depicting RNA-seq Z-scores for DLG-41 and DMSO replicates. D. Selected enrichment plots of gene-set enrichment analysis of DLG-41 treated samples vs. DMSO. E. Growth of H1299 cells treated with DLG-41 for 96 hours in the presence or absence of 5 mM NAC (n=2) normalized to DMSO. Data shown as mean ± SD from two independent experiments. F. Representative images of the wound-healing assay in H1299 cells treated with indicated concentrations of DLG-41 or DMSO and two time points. G Quantification of wound-healing assay scratch closures. Average of n=3 measurements. Significance (panels A,E, F) calculated by two-tailed Student’s t-test: ns, not significant, *P < 0.05, **P < 0.005, ***P < 0.001, ****P < 0.0001.

To further validate the mechanism of action of DLG-41, we performed phenotypic experiments in H1299 cells. First, we tested whether the activity of DLG-41 is mediated by oxidative stress. GAS41 YEATS domain has been shown to enhance the activity of the transcription factor NRF2, which is considered a master regulator of the cellular antioxidant defense against oxidative stress.²⁰ Thus, inhibition of GAS41 YEATS is expected to diminish NRF2 activity by hindering NRF2 localization to antioxidant promoter regions.²¹ Concurrent treatment of H1299 cells with DLG-41 combined with n-acetylcysteine (NAC) rescued cell growth inhibition when compared to DLG-41 used alone (Figure 4E). NAC is a potent antioxidant, and rescue of cellular proliferation supports that the antiproliferative mechanism of action of DLG-41 is dependent on ROS and oxidative stress.²² These results are consistent with an upregulation of DNA repair pathways, suggesting DLG-41 induces oxidative stress and causes DNA damage through ROS. To assess the effect of DLG-41 on cell migration, we performed a wound healing assay in NSCLC cells. Treatment of H1299 cells with DLG-41 slowed scratch healing even at a relatively short 24 h time point (Figure 4F, G), suggesting that DLG-41 impairs cell migration, an important process in metastasis.²³

Chemistry

Synthesis of compounds 1-8 was performed using a two-step sequential general approach shown in Scheme 1 starting from (5-aminothiophen-2-yl)(pyrrolidin-1-yl)methanone. In short, this approach consisted of a HATU-assisted amide coupling and Boc-group removal leading to the target compounds in 43-84% yield in 2 steps. For 5, the intermediate was additionally methylated on amide nitrogen and then Boc-deprotected leading to the target compound in 38% yield.

Scheme 1. Synthesis of 1-8.

Reagents and conditions: i) acid (1,1 eq.), HATU (1.1 eq.), DIPEA (1.5 eq.), DCM, 40 °C, overnight, then TFA (30 eq.), DCM, 0 °C and 7N NH₃ in MeOH or for 5, MeI, LiHMDS, −20 °C, THF, then TFA (30 eq.), DCM, 0 °C and 7N NH₃.

For the synthesis of compounds bearing azetidine ring we adapted the two-step general approach with using BocProNHThCOOH (50) as a common intermediate and varying substitutions at the 3rd position of azetidine ring (Scheme 2) obtaining compounds 9-18 in 68-78% over 2 steps. Corresponding azetidines for 12 and 13 were synthesized as previously described.^24,25

Scheme 2. Synthesis of compounds 9-11, 14-18.

Reagents and conditions: i) a) Zn (1.8 eq.), TMSCl (0.3 eq.), 1,2-dibromoethane (0.3 eq.), DMA, b) het(aryl)bromide (1eq.), Pd(dppf)Cl₂ (0.04 eq.), CuI (0.08 eq.), DMA, 80 °C, 6; ii) 4N HCl in 1,4-dioxane, r.t., 4h; iii) amine hydrochloride (1.1 eq), HATU (1.1 eq.), DIPEA (3 eq.), DCM, 45 °C overnight, then TFA (30 eq.), DCM, 0 °C and 7N NH₃.

Synthesis of compounds 17-40 was performed through the common 2-(azetidine-3-yl)-5-bromothiazole-containing intermediate (51), which was readily prepared from the BocProNHThCOOH (50) and 3-(5-bromothiazol-2-yl)azetidin-1-ium trifluoroacetate by HATU-assisted amide coupling (Scheme 3).¹³

Scheme 3. Synthesis of 19-27.

Reagents and conditions: i) a) Zn (1.8 eq.), TMSCl (0.3 eq.), 1,2-dibromoethane (0.3 eq.), DMA, b) het(aryl)bromide (1eq.), Pd(dppf)Cl₂ (0.04 eq.), CuI (0.08 eq.), DMA, 80 °C, 6 h; ii) NBS, DMF, r.t., overnight, then TFA (30 eq.), DCM, 0 °C, 60 min; iii) TFA salt (1.1 eq), HATU (1.1 eq.), DIPEA (3 eq.), DCM, 40 °C overnight; iv) boronic acid or boronpinacolate (1.3 eq.), NaHCO₃ (3 eq.), Pd(dppf)Cl₂ (0.05 eq.), 1,4-dioxane:water=10:1, 80 °C, overnight, then TFA (30 eq.), DCM, 0 °C and 7N NH₃.

Having tert-butyl (S)-2-((5-(3-(5-bromothiazol-2-yl)azetidine-1-carbonyl)thiophen-2-yl)carbamoyl)pyrrolidine-1-carboxylate (51) in hands allowed us to use Suzuki-Miyaura coupling as a combinatorial step in the synthetic pathway. Corresponding boronic acids or boronopinacates were either commercially available or prepared according to adapted procedures described before, followed Boc-deprotection led to 19-27 in 36-86% yield over 2 steps starting from 2-(azetidine-3-yl)-5-bromothiazole-containing intermediate (51).

The amide installation in the DLG-41 scaffold was achieved by acylation with the corresponding acyl chloride, or NHS-ester for biotinylated probe 21-biot (see SI for the full structure), following Boc-group removal in the presence of TFA affording the acylated compounds in 58-86% yield over 2 steps.

The rest of the series from Table 4 was synthesized by combination of approaches from Schemes 3 and 4 (Scheme 5) from previously synthesized tert-butyl (S)-2-((5-(3-(5-bromothiazol-2-yl)azetidine-1-carbonyl)thiophen-2-yl)carbamoyl)pyrrolidine-1-carboxylate (51) and separately synthesized or purchased boronates.

Scheme 4. Synthesis of 28-31, 21-biot.

Reagents and conditions: i) RCOCl (1.2 eq.), DIPEA (2 eq.), DCM, overnight or RCONHS (1.1 eq.), DIPEA (2 eq.), DMF, overnight, then TFA (30 eq.), DCM, 0 °C and 7N NH₃.

Scheme 5. Synthesis of compounds (32-39) from Table 4.

Reagents and conditions: i) bromide (1 eq.), boronic acid or pinacolate (1.3 eq.), NaHCO₃ (3 eq.), PdCl₂(dppf) * DCM (5% mol.), 1,4-dioxane:water = 10:1, 80 °C, Ar, overnight; ii) TFA (30 eq.), DCM, 0 °C, then 7N NH₃.

Compound with decreased potency (DLG-41nc) was synthesized using approach analogous to DLG-40 starting from the corresponding 2-amino-5-thiazole methyl carboxylate (Scheme 6) providing the desired compound with 45% yield over 3 steps.

Scheme 6. Synthesis of 40.

Reagents and conditions: i) acid (1 eq), N-methylimidazole (2.5 eq.), DCM, 0 °C, then MsCl (1.2 eq.), then amine (1 eq.) overnight; ii) LiOH, MeOH/THF, 50 °C, overnight; iii) a) Zn (1.8 eq.), TMSCl (0.3 eq.), 1,2-dibromoethane (0.3 eq.), DMA, b) arylbromide (1eq.), Pd(dppf)Cl₂ (0.04 eq.), CuI (0.08 eq.), DMA, 80 °C, 6 h; iv) TFA (30 eq.), DCM, 0 °C, 60 min; v) TFA salt (1.1 eq), acid (1 eq.), HATU (1.1 eq.), DIPEA (3 eq.), DCM, 40 °C overnight; then TFA (30 eq.), DCM, 0 °C and 7N NH₃

All compounds used for activity evaluation had purity > 95% and HPLC traces for selected compounds are in Figure S4. Molecular strings for all the final compounds are in Table S3.

Conclusions

The family of human proteins containing YEATS domains represent druggable protein-protein interaction targets.²⁶ Among these proteins, GAS41 is amplified in several types of cancer, including brain and lung cancers, making GAS41 YEATS domain an attractive target for inhibitor development.^1,4 Indeed, small molecule inhibitors blocking GAS41 interactions have been reported, but there is a need to develop cell permeable GAS41 inhibitors with on-target activity in cancer models. Here, we have developed a series of GAS41 protein-protein interaction inhibitors derived from thiophene-amide scaffold we identified via fragment screening approach.¹³ By employing an extensive structure-based medicinal chemistry optimization, we developed compound DLG-41, which is a sub-μM and cell permeable GAS41 YEATS domain inhibitor and the negative control compound DLG-41nc. DLG-41 inhibits the binding of GAS41 to chromatin in cells and blocks growth of NSCLC with GI₅₀ ~ 0.5 μM. Importantly, we demonstrated that activity of DLG-41 is dependent on the expression of GAS41 protein, and growth inhibition is strongly reduced in cell lines with low levels of GAS41 or upon GAS41 knockout, indicating on-target activity. Treatment of NSCLC cell lines with DLG-41 shows phenotypic effects and modulation of pathways critical to cell proliferation and metastasis. Collectively, our studies suggest that small-molecule inhibition of the GAS41 YEATS domain is a viable strategy for targeting NSCLC cells, and DLG-41 exhibits potential for further optimization into anti-cancer agents.

Experimental Section

Materials

Protein Expression and Purification

The following GAS41 constructs were used for these studies: YEATS domain for the AlphaLISA experiments (residues 13 – 158 in pQE-80L vector with N-terminal his₆-tag); YEATS domain for Fluorescence Polarization and crystallization (residues 1 – 148 in pGST vector with N-terminal GST-tag and N-terminal TEV cleavage site); YEATS domain for NMR and ITC experiments (residues 18-190 in pNIC-CH vector with C-terminal his₆-tag). GAS41 (13-158) and GAS41 (1-148) were expressed and purified as previously described.¹³ Briefly, GAS41 YEATS constructs were expressed using E. Coli BL21(DE3) One Shot (ThermoFisher) or Rosetta DE3 (Millipore Sigma) cells in either LB or ¹⁵N M9 minimal media and induced with 0.25 mM or 0.4 mM IPTG for 16h at 18°C. Cells were re-suspended in lysis buffer (25 mM TRIS-HCl pH 7.5, 500 mM NaCl, 1 mM TCEP, 0.5 mM PMSF, 10% glycerol, with 30 mM imidazole for his₆-tagged constructs), lysed using a cell disrupter, and applied to HisTrap FF (Cytiva) affinity column or Glutathione Sepharose 4B column (Cytiva). Proteins were eluted using buffer containing 300 mM imidazole or 10mM reduced glutathione, and dialyzed against buffer containing 50 mM Tris (pH 7.5), 150 mM NaCl, and 1 mM TCEP; GAS41 (18-190) was eluted using a buffer containing 25 mM TRIS-HCl pH 7.5, 500 mM imidazole, 500 mM NaCl, 1 mM TCEP and dialyzed to a storage buffer containing 50 mM TRIS-HCl pH 7.5, 150 mM NaCl, 1 mM TCEP for NMR experiments or 50 mM sodium phosphate pH 7.5, 150 mM NaCl, 1 mM TCEP for ITC experiments. For crystallization experiments, GST-GAS41 YEATS (1-148) was proteolytically cleaved with TEV protease overnight and re-applied to Glutathione Sepharose 4B (Cytiva) columns to extract the GST- affinity tag. GAS41 YEATS (1-148) was then applied to S75 column for size exclusion chromatography purification and exchanged into buffer containing 20 mM Tris (pH 7.5) and 300 mM NaCl for storage.

NMR binding experiments

NMR ¹H-¹⁵N HSQC experiments were performed at 30°C with 50 μM GAS41 YEATS domain (residues 18-190) in buffer containing 50 mM Tris, pH 7.5, 150 mM NaCl, 1 mM TCEP, 7% D2O, and 5% DMSO. Compounds were added from DMSO stocks to final concentrations indicated in the figure labels. All NMR spectra were acquired on a 600 MHz Bruker Avance III spectrometer equipped with cryoprobe running Topspin version 3.7. Processing and spectral visualization were performed using NMRPipe and Sparky.^27,28

Fluorescence polarization assay

For fluorescence polarization competition assays, 1 μM GST-GAS41 YEATS domain (residues 1-148) was incubated with inhibitor dilutions at 1% DMSO in an assay buffer containing 50 mM Tris pH 7.5, 150 mM NaCl, 1 mM TCEP, 0.01% BSA, and 0.01% Tween-20 for 1 h in a 96-well black plate (Costar). 25 nM fluorescein labeled di-crotonylated histone H3 (FAM-H3K23crK27cr) was added and incubated for an additional hour. Fluorescence polarization data was measured at 525 nm on a Pherastar plate reader (BMG Labtech). Data was fit in Prism 7.0 (GraphPad) to determine IC₅₀ values.

Crystallization and structure determination

GAS41 YEATS domain protein (residues 1-148) at 7.8 mg/mL was incubated with 500 μM DLG-1 for two hours at room temperature in a 50 mM TRIS pH 7.5, 150 mM NaCl, 1 mM TCEP buffer. Crystals were harvested from a 96-well tray assembled using sitting drop vapor diffusion in final conditions of 1260 mM Ammonium sulfate, 100 mM HEPES/Sodium hydroxide pH 7.5, 25% Glycerol, covered in 10 μl Al’s oil. Crystals were harvested into a cryoprotectant solution containing 1.26 M Ammonium sulfate, 100 mM HEPES/ Sodium hydroxide pH 7.5, 25% glycerol, 500 μM DLG-1, and 1% DMSO, and stored in liquid nitrogen. Diffraction data for GAS41 YEATS-inhibitor complex were collected at the 21-ID-G beam line at the Life Sciences Collaborative Access Team at the Advanced Photon Source. Data were integrated and scaled using HKL-2000²⁹, and the structure was solved by molecular replacement with MOLREP³⁰ in CCP4 suite³¹, using native-GAS41 YEATS structure as a search model (PDB 5VNA). Further model building was performed in COOT³², and structure was refined using REFMAC and PHENIX program suite.³³ The coordinates of the GAS41 YEATS in complex with DLG-1 have been deposited in the Protein Data Bank under accession number 9O4Y.

AlphaLISA assay

Competition experiments were performed using his₆-GAS41(13-158). Briefly, 100 nM His₆-GAS41(13-158) was incubated with competitors diluted 100-fold from DMSO stocks into 50 mM HEPES pH 7.5, 100 mM NaCl, 1 mM TCEP, 0.05% BSA, 0.01% Tween-20 for 1 h at 1% DMSO in a 96-well ½-Area AlphaPlate (PerkinElmer). 21-biot was added to a final concentration of 25 nM and incubated for 1 h. Nickel Chelate Acceptor AlphaLISA beads (PerkinElmer) were added to a final concentration of 10 μg/mL and incubated for 1 h. Streptavidin Donor beads (PerkinElmer) were added to a final concentration of 10 μg/mL and incubated for 2 h. All AlphaLISA assay incubations were performed at room temperature. Luminescence signal was measured on a Pherastar plate reader (BMG Labtech).

Isothermal Calorimetry (ITC)

GAS41(18-190) was dialyzed at 4°C against a 50 mM sodium phosphate pH 7.5, 150 mM NaCl, 1 mM TCEP buffer and degassed prior to measurement. Titrations were performed using the VP-ITC system (MicroCal) at 25°C. The calorimetric cell, which contained DLG-41 (concentrations in the 15-20 μM range), was titrated with GAS41 (18-190) (concentrations in the 90-120 μM range) injected in 10 μL aliquots. Data was analyzed using Origin 7.0 (OriginLab) to obtain K_D, stoichiometry, entropy, and enthalpy. Experiments were run in duplicate.

NanoBRET assay

Full-length GAS41 was subcloned into the pNIC-NL vector via Flexi digest (Promega); Histone H3.3-HaloTag^® Fusion Vector was purchased from Promega (Madison, WI). HEK293T cells were transiently transfected for 16 h using FUGENE HD (Promega) reagent at a ratio of 1 ug of Halo-H3.3 to 10 ng Nanoluc-GAS41. After 24 h transfection, cells were trypsinized and treated with indicated compounds and concentrations; half of samples were also treated with 100 nM of Halo-618 ligand (Promega), then replated in a white 96-well plate (COSTAR) and incubated at 37°C for 24 hours. Furimazine substrate was added to each well to initiate the BRET reaction; the plate was immediately read and emission at both 480 nm and 618 nm on a Pherastar (BMG Labtech) plate reader using the NanoBRET module (BMG Labtech). Signal was calculated in milli-BRET units as a ratio of 618 nm/480 nm signal multiplied by 1000. IC₅₀ values were calculated in Prism 9.0 (Graphpad) using the variable slope, four parameters equation using background subtracted and normalized values.

Cell Viability Assays

H1299, A549, IMR-90, H522, H358, H23, and H460 cells were sourced from the American Type Culture collection (ATCC). For cell proliferation assay, 2000 cells/well of cell lines H1299, A549, IMR-90, H522, H358, H23 cells or 500 cells/well of H460, were seeded into 12 well plate in and treated with inhibitor or vehicle to final DMSO concentration at 0.25%. Cell culture media for H23, H1299, H1993, A549, H522, H358, H450, and A549-GAS41 KO was Roswell Park Memorial Institute (RPMI)-1640 supplemented with 10% heat inactivated FBS and 1% Penicillin-streptomycin or antibiotic-antimycotic. Media was changed after 3-4 days and on day 7, cell viability was assessed using the Sulforhodamine B (SRB) cell staining assay (Abcam). A549 GAS41-KO cell line was generated as previously described.¹³ For growth curves, 3000 cells/well of A549 or A549 GAS41-KO were seeded in 6-well tissue culture plates in RPMI-1640 + 10% FBS + 1% Pen-Strep and treated using indicated compound or vehicle with a final concentration of 0.25% DMSO. Cells were tryspinized, cell number was evaluated using a slide cell counter (Biorad) and replated to 3000 cells/well every 3 or 4 days through day 14.

For N-Acetylcysteine rescue experiments, H1299 cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum (FBS) and 1% antibiotic-antimycotic mix (Invitrogen). Cells were maintained in a humidified incubator at 37°C with 5% CO2. N-Acetylcysteine (NAC, Cayman Chemical, CAS Number 616-91-1) was used as a rescue agent at a final concentration of 5 mM. Cells were pretreated with NAC overnight before the addition of GAS41 inhibitors. Cell proliferation was monitored using the BioTek BioSpa 8 Automated Incubator, and data were analyzed using GraphPad Prism software.

RNA-seq library preparation

H1299 cells were treated with DMSO, 1.5 μM DLG-41, or 1.5 μM DLG-41nc for 7 days in triplicate. Total RNA was isolated using the RNEasy Mini kit according to the manufacturer protocol (Qiagen). mRNA was enriched using the NEBNext PolyA enrichment (NEB); sequencing libraries were prepared using the IDT xGen Library Preparation Kit using UDI adapters. Library quantity was assessed via Qubit (Thermo Fisher Scientific) and quality was assessed by TBE gel and using Agilent TapeStation bioanalyzer for <1% adapter content. Samples were pooled and sequenced on an Illumina NovaSeqX (150 bp x 2, paired-end) at the Advanced Genomics Core at the University of Michigan.

RNA-seq analysis

Sequencing data was trimmed using Trimgalore and evaluated for quality using FastQC.³⁴ Gene and transcript abundances were quantified from the trimmed reads using RSEM with Bowtie2, mapped against the hg38 (GRCh38) reference genome. ³⁵ Differential expression analysis was performed using the EdgeR package in R (4.3.3).³⁶ Significance cutoffs for genes were a false discovery rate < 0.05 and an absolute fold-change of 1.5. Gene-set enrichment analysis (http://www.broadinstitute.org/gsea/index.jsp) was performed on gene expression data for DLG-41 vs. DMSO-treated treated H1299 cells.³⁷

Wound Healing assay

H1299 cells were seeded at 10,000 c/mL in 3mL media (RPMI, 10% FBS, 1% Pen-strep) in 6-well tissue culture treated plates. Plates were incubated for 3 days at 37°C, 5% CO2 until cells reached 90% confluence. Media was aspirated and a scratch down the center of each well was created using a clean pipette tip. Wells were washed 3x with PBS and media was replaced with the addition of indicated treatment at a final concentration of 0.25% DMSO. Pictures of wounds were taken immediately (0 h) and locations were marked on cell plate. After 24 hours, media was aspirated, cells were washed 3x with PBS, and media and treatment were replaced. Pictures were immediately taken in consistent locations in wells and analyzed using Fiji (ImageJ); scratch areas were quantified using the MRI Wound Healing Tool (Montpellier Ressources Imagerie).^38,39

Immunoblotting

Whole-cell lysates were prepared using RIPA lysis buffer (Thermo Scientific) containing 1× protease inhibitor cocktail (Sigma) according to standard protocol and quantitated using a BCA protein assay kit (Thermo Scientific). A total of 10 μg lysate was loaded on 4-12% Bis-TRIS NuPAGE precast gels (Invitrogen) and transferred to PVDF membranes. All blots were probed with specific primary antibodies overnight at 4°C and with the appropriate HRP-conjugated secondary antibody for 1 h at room temperature. Membranes were visualized on with enhanced chemiluminescent (ECL) reagent (ECL Prime, GE Healthcare) using a GelDoc Imager (Biorad). Antibodies used for these blots include GAS41 (Santa Cruz, sc-393708, mouse monoclonal) and alpha-Tubulin (Cell Signaling #3873 mouse monoclonal).

Reverse Transcription-Quantitative PCR

H1299 cells were seeded at 3.0x10³ cells/well in a tissue-culture treated 6-well plate using RPMI + 10% FBS + 1% Pen-Strep media with the addition of the indicated treatment, in triplicate, at a final concentration of 0.25% DMSO. Media and treatment were changed after three days and treatment continued to 7 days, where cells were trypsinized, counted, and collected. RNA was isolated using the RNEasy Mini Kit (Qiagen) according to the manufacturer’s protocol. Total RNA was reverse transcribed to cDNA using a HICAP RT kit (Applied Biosystems). Quantitative real-time PCR was performed using Taqman Master Mix and probes (Applied Biosystems, Thermo Fisher Scientific) for indicated genes and the housekeeping genes GAPDH or HRPT1. Real-time PCR was processed using the CFX96 Touch Real-Time PCR Detection System (Bio-Rad).

Chemical synthesis

General details

All solvents and reagents were purchased from commercial sources and used without purification. Anhydrous solvents were purchased from Acros Organics and stored under a nitrogen atmosphere with activated molecular sieves. Standard vacuum line techniques were used, and glassware was flame dried prior to use. Deionized water obtained using MilliporeSigma^™ Milli-Q^™ Ultrapure Water Systems. Thin Layer Chromatography (TLC) was carried out using aluminum plates coated with 60 F254 silica gel. Plates were visualized using UV light (254 or 365 nm) or staining with 1% aq. KMnO₄. Normal-phase and reverse-phase silica gel chromatography was carried out using CombiFlashRf+ flash column chromatography system with pre-filled RediSep^® Rf and RediSep Rf Gold C18 columns. NMR spectra were recorded using a Bruker Avance III 600 MHz. Chemical shifts are reported in ppm relative to tetramethylsilane or residual solvent signal. Multiplicities are denoted as s- singlet, d- doublet, t- triplet, q- quartet, and quin- quintet and derivatives thereof (br denotes a broad resonance peak). Coupling constants recorded as Hz and round to the nearest 0.1 Hz. Some signals that overlapped with solvents or were too broad were identified using ¹H–¹³C HSQC spectrum (multiplicity not known). The exact mass measurements were determined on Agilent Q-TOF time-of-flight mass spectrometer using positive ion mode and electrospray ionization. The purity analysis of final compounds was determined on Shimadzu Prominence HPLC system (20 series: binary pump, UV/vis at 254 nm, heated column compartment 28°C), using Restek Ultra C18 (5 μm) 150 mm × 4.6 mm column. LC-MS was recorded on Shimadzu LC-2020 system (DUIS-ESI). The solvents were programmed to run at gradient starting 20% ACN in water to 80% during 8 min run. If not indicated, the purity of all final compounds was >95% as determined by HPLC via the integration of UV spectra at 254 nm. Compound names were generated using ChemBioDraw Ultra v16 systematic naming. All final compounds were additionally freeze-dried prior to use in biochemical and cellular experiments

Preparation of compounds

General procedure A

To a screw cap 10dr vial were added carboxylic acid (1.1 eq.), DIPEA (1.5 eq., 3 eq. in case of amine hydrochloride), HATU (1.1 eq.) and anhydrous DCM (5 mL). The mixture was stirred for 15min at RT, then corresponding amine (1 eq.) was added, the vial was sealed and the reaction mixture was heated at 40°C overnight. After cooling to RT, the mixture was diluted with DCM (20 mL), and sequentially washed with water, sat. aqueous sodium hydrocarbonate, and brine. Organic phase was dried over sodium sulfate, concentrated in vacuo and the residue was re-dissolved in the DCM (2 mL) at 0°C and TFA (30 eq.) was added dropwise, and the mixture was stirred at 0°C for 60 min. The mixture was concentrated in vacuo and the crude was triturated with 7N ammonia in methanol and concentrated again. Obtained residue was purified by silica gel column chromatography (0-100% Hexane/EtOAc to DCM/MeOH) then RPH (0-100% MeOH/Water) to afford the titled compound as an off white solid.

General procedure B

To a de-gassed suspension of zinc powder (217 mg, 3.338 mmol, 1.8 eq.) in DMA (2 mL) in the screw cap vial was added drop-wise a mixture of chlorotrimethylsilane (67.3 μL, 57.6 mg, 0.53 mmol, 0.3 eq.) and 1,2-dibromoethane (45.9 μL, 99.6 mg, 0.53 mmol, 0.3 eq.) and the resultant mixture was stirred at room temperature under Ar for 15 minutes. To this mixture was then added dropwise neat 3-iodoazetidine-1-carboxylic acid tert-butyl ester (753 mg, 2.661 mmol, 1.4 eq.) and the resultant mixture was stirred at room temperature for 15 minutes. In a separate vial, PdCl₂(dppf) * DCM (65.2 mg, 0.08 mmol, 0.04 eq.), and copper iodide (30 mg, 0.157 mmol, 0.08 mmol) were added to a degassed solution of corresponding het(aryl)bromide (1.862 mmol, 1 eq.) in DMA (1 mL). After stirring for 30 minutes, the zinc suspension above was added to the solution suspension of het(aryl)bromide, PdCl₂(dppf) * DCM and copper iodide and the reaction mixture was allowed to stir at 80°C for 2 hours under argon. The resultant mixture was cooled to r.t diluted with EtOAc and filtered through the pad with Celite, the pad was washed with EtOAc, and collected organics were washed with mixture of saturated ammonium chloride solution and ammonium hydroxide (15:1). The organic phase was dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The residue was purified by silica gel flash chromatography (0-100% ethyl acetate in hexanes), fractions with the corresponding pick in the LC-MS were combined, concentrated and used in the next step without further purification.

General procedure C

To a solution of Boc-protected substituted azetidine (1 mmol) in the 3 mL of 1,4-dioxane 4M HCl in 1,4-dioxane (3 mL) was added dropwise, and the mixture was stirred overnight at RT. After this all volatiles were removed under reduced pressure, the residue was triturated with dry ACN, and ACN was decanted, remaining solid was dried in vacuo providing the corresponding hydrochloride as a white solid with quantitative yield.

General procedure D

Step 1.

To a degassed suspension of boronic acid or boronpinacolate (1.3 eq., 0.65 mmol), bromide (1 eq., 0.5 mmol), NaHCO₃ (3 eq., 1.5 mmol) in mixture 1,4-dioxane:water = 10:1 PdCl₂(dppf) * DCM (0.025 mmol) was added in one portion. Obtained suspension was degassed one time more, refilled with Ar and allowed to stir at 80°C overnight under argon. The resultant mixture was cooled to RT diluted with EtOAc and filtered through the pad with Celite, the pad was washed with EtOAc, and collected organics were washed with brine, dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The residue was purified by silica gel flash chromatography (0-100% ethyl acetate in hexanes then 0-20% methanol in dichloromethane), fractions with the corresponding peak in the LC-MS were combined, concentrated and the residue was used without further purification, or

Step 2.

The residue was re-dissolved in the DCM (5 mL). To this mixture TFA (30 eq.) was added dropwise at 0°C, and the mixture was stirred at 0°C for 60 min. The mixture was concentrated in vacuo and the crude was triturated with 7N ammonia in methanol and concentrated again. Obtained residue was purified by silica gel column chromatography (0-100% Hexane/EtOAc to DCM/MeOH) then RPH (0-100% MeOH/Water) to afford the titled compound as an off white solid.

General procedure E

To a degassed suspension of B₂Pin₂ (1.3 eq., 1.3 mmol), bromide (1 eq., 1 mmol), KOAc (3 eq., 3 mmol) in 1,4-dioxane PdCl₂(dppf) * DCM (0.05 mmol) was added in one portion. Obtained suspension was degassed one time more, refilled with Ar and allowed to stir at 100°C overnight under argon. The resultant mixture was cooled to RT diluted with EtOAc and filtered through the pad with Celite, the pad was washed with EtOAc, and collected organics were washed with brine, dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The residue was used in the next step without further purification.

General procedure F

To a screw cap 10dr vial were added amine (1 eq.), DIPEA (2 eq.) and anhydrous DCM (DMF for NHS ester) (5 mL). The mixture was stirred for 15 min at 0°C, then corresponding acylchloride or NHS ester (1.5 eq.) was added, the vial was sealed and the reaction mixture was stirred at RT overnight. Then the mixture was diluted with DCM (20 mL), and sequentially washed with water, sat. aqueous NaHCO₃, and brine (for the NHS involving reaction solution was directly injected on RPH column and fractions with the corresponding pick in the LC-MS were combined and concentrated). Organic phase was dried over sodium sulfate, concentrated in vacuo and the residue was re-dissolved in the DCM (2 mL) at 0°C and TFA (30 eq.) was added dropwise, and the mixture was stirred at 0°C for 60 min. The mixture was concentrated in vacuo and the crude was triturated with 7N ammonia in methanol and concentrated again. Obtained residue was purified by silica gel column chromatography (0-100% Hexane/EtOAc to DCM/MeOH) then RPH (0-100% MeOH/Water) to afford the titled compound as an off white solid.

General procedure G⁴²

To a solution of corresponding bromophenylboronic acid (283 mg, 1.41 mmol) in IPA (2 mL) was added NiI₂ (13 mg, 0.04 mmol) and trans-2-aminocyclohexanol HCl (6.4 mg, 0.04 mmol) at RT A solution of NaHMDS (1 M in THF) (1.4 mL, 1.4 mmol) was added dropwise to the reaction mixture at RT The reaction mixture was degassed before addition of tert-butyl-3-iodoazetidine-carboxylate (200 mg, 0.7 mmol). The reaction mixture was heated at 90°C for 2 h. The resulting mixture was concentrated under pressure and diluted with water (10 mL). The resulting mixture was extracted with EtOAc (2-10 mL). The combined organic phases were dried over sodium sulfate and evaporated under reduced pressure. The residue was purified by silica gel column chromatography (0-30% Hexane/EtOAc), fractions with the corresponding pick in the LC-MS were combined, concentrated under reduced pressure and used in the next step without further purification.

General procedure H

Corresponding compound with phtalimide protection group in MeOH (5-10 mL) and treated with N₂H₄*H₂O (30 eq.), then the mixture was heated at 65°C for 2 h, cooled to RT and concentrated in vacuo. The residue was purified by silica gel flash chromatography (0-30% methanol in dichloromethane), fractions with the corresponding peak in the LC-MS were combined, concentrated, and the residue was used in the next step without further purification.

(S)-1-methyl-N-(5-(pyrrolidine-1-carbonyl)thiophen-2-yl)pyrrolidine-2-carboxamide (3)

Synthesized according to general procedure A with using (5-aminothiophen-2-yl)(pyrrolidin-1-yl)methanone and N-methyl-(S)-proline to afford 3 (10mg, 83%) as an off white solid.^{43 1}H NMR (600 MHz, CD₃OD): δ 7.44 (d, J = 4.2 Hz, 1H), 6.85 (d, J = 4.2 Hz, 1H), 3.82 (s, 2H), 3.60 (s, 2H), 3.23 – 3.16 (m, 1H), 3.11 – 3.03 (m, 1H), 2.48 – 2.37 (m, 4H), 2.32 – 2.23 (m, 1H), 2.04 (s, 2H), 1.96 (s, 2H), 1.93 – 1.82 (m, 3H). ¹³C NMR (125 MHz, CD₃OD): δ 173.8, 164.4, 145.4, 130.5, 129.9, 113.6, 70.0, 57.4, 48.5 (from HSQC), 47.5 (from HSQC), 41.5, 31.8, 27.6, 24.9, 24.8. HR-MS (ESI): [M+H⁺] calculated 308.1427, found 308.1439.

(S)-2-methyl-N-(5-(pyrrolidine-1-carbonyl)thiophen-2-yl)pyrrolidine-2-carboxamide (4)

Synthesized according to general procedure A with using (5-aminothiophen-2-yl)(pyrrolidin-1-yl)methanone and Boc-2-methyl-(S)-proline to afford 4 (11mg, 80%) as an off white solid.^{43 1}H NMR (600 MHz, CD₃OD): δ 7.44 (d, J = 4.2 Hz, 1H), 6.84 (d, J = 4.2 Hz, 1H), 3.82 (s, 2H), 3.60 (s, 2H), 3.13 (dt, 10.5, 6.4 Hz, 1H), 2.92 (dt, J = 10.5, 6.4 Hz, 1H), 2.28 – 2.20 (m, 1H), 2.04 (s, 2H), 1.96 (s, 2H), 1.87 – 1.79 (m, 1H), 1.77 – 1.67 (m, 2H), 1.44 (s, 3H). ¹³C NMR (125 MHz, CD₃OD): δ 177.4, 164.5, 145.6, 130.8, 129.9, 113.5, 67.6, 50.2, 47.8, 47.53 (from HSQC), 38.8, 27.6, 27.2, 25.6, 24.9. HR-MS (ESI): [M+H⁺] calculated 308.1427, found 308.1437.

(S)-N-methyl-N-(5-(pyrrolidine-1-carbonyl)thiophen-2-yl)pyrrolidine-2-carboxamide (5)

Synthesized starting from tert-butyl (S)-2-((5-(pyrrolidine-1-carbonyl)thiophen-2-yl)carbamoyl)pyrrolidine-1-carboxylate obtained according to general procedure A from (5-aminothiophen-2-yl)(pyrrolidin-1-yl)methanone and (S)-1-Boc-proline. To a stirred solution of tert-butyl (S)-2-((5-(pyrrolidine-1-carbonyl)thiophen-2-yl)carbamoyl)pyrrolidine-1-carboxylate (50 mg, 0.127 mmol, 1 eq.) in 10 mL of anhydrous THF 1M solution of LiHMDS in THF (140 μL, 0.14 mmol, 1.1 eq.) was added dropwise at −20°C. The mixture was stirred at this temperature for 30 min before MeI (21.6 mg, 9.5 μL, 0.15 mmol, 1.2 eq.) was added in one portion. The mixture was allowed to warm to room temperature and stirred overnight. The reaction was quenched with saturated ammonium chloride solution (20 mL) and extracted with EtOAc (3*10 mL). Gathered organic layers were dried over sodium sulfate, concentrated in vacuo and the residue was re-dissolved in the DCM (2 mL) at 0°C, TFA (10 eq.) was added dropwise, and the mixture was stirred at 0°C for 60 min. The mixture was concentrated in vacuo and the crude was triturated with 7N ammonia in methanol and concentrated again. Obtained residue was purified by silica gel column chromatography (0-100% Hexane/EtOAc to DCM/MeOH) then RPH (0-100% MeOH/Water) to afford 5 (13 mg, 38%) as an off white solid. ¹H NMR (600 MHz, CD₃OD), mixture of rotamers 1:1: δ 7.54 (d, J = 4.0 Hz, 0.5H), 7.51 (d, J = 4.3 Hz, 0.5H), 7.17 (d, J = 4.0 Hz, 0.5H), 6.94 (d, J = 4.2 Hz, 0.5H), 5.07 – 4.98 (m, 0.5H), 4.55 – 4.44 (m, 0.5H), 3.91 – 3.78 (m, 2H), 3.78 – 3.55 (m, 4H), 3.55 – 3.39 (m, 2H), 2.67 (q, J = 8.0 Hz, 0.5H), 2.25 – 1.77 (m, 8.5H). ¹³C NMR (125 MHz, CD₃OD) mixture of rotamers 1:1: δ 176.3, 176.0, 173.5, 164.4, 164.3, 164.3, 162.6, 150.0, 148.8, 139.4, 133.7, 132.0, 130.5, 128.9, 127.6, 120.8, 113.3, 102.9, 60.6, 59.8, 59.3, 54.8, 50.2, 49.9, 48.1, 39.5, 36.1, 33.13, 32.65, 31.26, 31.0, 27.6, 27.2, 26.3, 24.9. HR-MS (ESI): [M+H⁺] calculated 308.1427, found 308.1437.

(2S,4S)-4-methyl-N-(5-(pyrrolidine-1-carbonyl)thiophen-2-yl)pyrrolidine-2-carboxamide (6)

Synthesized according to general procedure A with using (5-aminothiophen-2-yl)(pyrrolidin-1-yl)methanone and (2S,4S)-1-(tert-butoxycarbonyl)-4-methylpyrrolidine-2-carboxylic acid to afford 6 (8mg, 77%) as an off white solid.^{43 1}H NMR (600 MHz, CD₃OD): δ 7.42 (d, J = 4.2 Hz, 1H), 6.77 (d, J = 4.2 Hz, 1H), 3.92 (t, J = 8.3 Hz, 1H), 3.79 (br.s, 2H), 3.59 (br.s, 2H), 3.15 (dd, J = 10.6, 7.0 Hz, 1H), 2.57 (dd, J = 10.3, 9.1 Hz, 1H), 2.42 (dt, J = 12.8, 7.7 Hz, 1H), 2.23 (dq, J = 8.6, 7.0 Hz, 1H), 2.02 (br.s, 2H), 1.94 (br.s, 2H), 1.42 (dt, J = 12.5, 9.0 Hz, 1H), 1.04 (d, J = 6.6 Hz, 3H). ¹³C NMR (125 MHz, CD₃OD): δ 173.7, 164.3, 145.4, 130.6, 129.8, 113.4, 62.0, 55.3, 50.1, 48.5 (from HSQC), 40.3, 36.7, 27.6, 24.9, 17.8. HR-MS (ESI): [M+H⁺] calculated 308.1427, found 308.1434.

(S)-4,4-dimethyl-N-(5-(pyrrolidine-1-carbonyl)thiophen-2-yl)pyrrolidine-2-carboxamide (7)

Synthesized according to general procedure A with using (5-aminothiophen-2-yl)(pyrrolidin-1-yl)methanone and (2S)-1-(tert-butoxycarbonyl)-4,4-dimethylpyrrolidine-2-carboxylic acid to afford 7 (25 mg, 93%) as an off white solid.^{43 1}H NMR (600 MHz, CD₃OD): δ 7.42 (d, J = 4.2 Hz, 1H), 6.79 (d, J = 4.2 Hz, 1H), 3.97 (dd, J = 8.7, 7.8 Hz, 1H), 3.80 (s, 2H), 3.59 (s, 2H), 2.79 – 2.67 (m, 2H), 2.07 – 1.98 (m, 3H), 1.94 (s, 2H), 1.64 (dd, J = 12.7, 7.8 Hz, 1H), 1.09 (s, 3H), 1.08 (s, 3H). ¹³C NMR (125 MHz, CD₃OD): δ 13C NMR (151 MHz, MeOD) δ 174.7, 164.3, 145.4, 130.5, 129.9, 113.4, 61.6, 61.2, 50.1, 46.2, 41.2, 27.6, 26.8, 26.7, 24.9. HR-MS (ESI): [M+H⁺] calculated 322.1584, found 322.1588.

(2S,4S)-4-phenoxy-N-(5-(pyrrolidine-1-carbonyl)thiophen-2-yl)pyrrolidine-2-carboxamide (8)

Synthesized according to general procedure A with using (5-aminothiophen-2-yl)(pyrrolidin-1-yl)methanone and (2S,4S)-1-(tert-butoxycarbonyl)-4-phenoxypyrrolidine-2-carboxylic acid to afford 8 (11mg, 81%) as an off white solid.^{43 1}H NMR (600 MHz, CD₃OD): δ 7.44 (d, J = 4.2 Hz, 1H), 7.18 (t, J = 8.0 Hz, 2H), 6.87 (t, J = 7.3 Hz, 1H), 6.80 (d, J = 4.2 Hz, 1H), 6.76 (d, J = 7.9 Hz, 2H), 4.96 (s, 1H), 3.96 (dd, J = 9.6, 3.2 Hz, 1H), 3.80 (br.s, 2H), 3.60 (br.s, 2H), 3.26 (m, 2H), 2.43 (ddd, J = 14.4, 9.7, 5.0 Hz, 1H), 2.31 (dd, J = 13.9, 1.4 Hz, 1H), 2.02 (br.s, 2H), 1.94 (br.s, 2H). ¹³C NMR (125 MHz, CD₃OD): δ 174.0, 164.4, 158.4, 145.6, 130.5, 130.4, 130.0, 122.1, 116.9, 113.3, 78.7, 60.6, 53.3, 50.2, 48.5 (from HSQC), 37.9, 27.6, 24.9. HR-MS (ESI): [M+H⁺] calculated 386.1533, found 386.1540.

(S)-5-(1-(tert-butoxycarbonyl)pyrrolidine-2-carboxamido)thiophene-2-carboxylic (51).

Step 1. tert-butyl (S)-2-((5-(methoxycarbonyl)thiophen-2-yl)carbamoyl)pyrrolidine-1-carboxylate.

The mixture of 5-bromothiophene-2-carboxylate (222 mg, 1 mmol), N-(tert-Butoxycarbonyl)proline amide (235 mg, 1.1 mmol), and K₂CO₃ (276 mg, 2 mmol) in anhydrous 1,4-dioxane (10 mL) was degassed and Pd₂dba₃ (46 mg, 0.05 mmol) and Xantphos (58 mg, 0.1 mmol) were added in one portion. The mixture was degassed again and heated at 100°C overnight. The resulting mixture was cooled to RT and filtered through a pad with Celite. The pad was washed with EtOAc (3*50 mL) and combined filtrates were concentrated under reduced pressure. The residue was purified by silica gel column chromatography (0-100% Hexane/(EtOAc-EtOH=3:1), fraction with the corresponding peak in the LC-MS were combined, concentrated under reduced pressure and the residue was additionally recrystallized from toluene. Yield 255 mg, 72%. ee > 99%. All physical characteristics are the same as described previously.¹³

Step 2. (S)-5-(1-(tert-butoxycarbonyl)pyrrolidine-2-carboxamido)thiophene-2-carboxylic.

Synthesized according to the procedure described previously.¹³

(S)-N-(5-(3-phenylazetidine-1-carbonyl)thiophen-2-yl)pyrrolidine-2-carboxamide (9)

Synthesized according to general procedure A with using (S)-5-(1-(tert-butoxycarbonyl)pyrrolidine-2-carboxamido)thiophene-2-carboxylic acid and 2-(azetidin-3-yl)phenyl hydrochloride (43, synthesized according to general procedures B and C and used as obtained) to afford 9 (8 mg, 72%) as an off white solid. ¹H NMR (600 MHz, CD₃OD): δ 7.43 – 7.35 (m, 5H), 7.29 – 7.24 (m, 1H), 6.80 (d, J = 4.2 Hz, 1H), 4.94 (s, 1H), 4.54 (s, 2H), 4.18 (s, 1H), 4.05 – 3.95 (m, 1H), 3.84 (dd, J = 8.8, 5.9 Hz, 1H), 3.05 (dt, J = 10.4, 6.5 Hz, 1H), 2.97 (dt, J = 10.4, 6.8 Hz, 1H), 2.20 (ddt, J = 12.4, 8.5, 7.1 Hz, 1H), 1.88 (dtd, J = 12.6, 7.4, 5.9 Hz, 1H), 1.80 (p, J = 6.7 Hz, 2H). ¹³C NMR (125 MHz, CD₃OD): δ 166.64, 165.17, 145.20, 143.0, 130.2, 129.9, 129.2, 128.3, 127.7, 114.5, 61.4, 57.5, 47.5, 35.5, 30.7, 25.0. HR-MS (ESI): [M+H⁺] calculated 356.1427, found 356.1439.

(S)-N-(5-(3-(pyridin-4-ylmethyl)azetidine-1-carbonyl)thiophen-2-yl)pyrrolidine-2-carboxamide (12)

Synthesized according to general procedure A with using (S)-5-(1-(tert-butoxycarbonyl)pyrrolidine-2-carboxamido)thiophene-2-carboxylic acid and 4-(azetidin-3-ylmethyl)pyridine dihydrochloride (corresponding Boc-protected amine was used in the General procedure C to obtain the deprotected hydrochloride) to afford 12 (8 mg, 66%) as an off white solid.^{25 1}H NMR (600 MHz, CD₃OD+CDCl₃): δ 8.43 (d, J = 5.4 Hz, 2H), 7.33 (d, J = 4.2 Hz, 1H), 7.26 (d, J = 5.4 Hz, 2H), 6.73 (d, J = 4.2 Hz, 1H), 4.60 (s, 1H), 4.26 (s, 1H), 4.20 (s, 1H), 3.88 (s, 1H), 3.81 (dd, J = 8.9, 5.8 Hz, 1H), 3.18-3.07 (m, 1H), 3.07 – 3.00 (m, 3H), 3.0-2.93 (m, 1H), 2.19 (td, J = 12.9, 6.6 Hz, 1H), 1.89 (td, J = 12.9, 6.6 Hz, 1H), 1.78 (p, J = 6.6 Hz, 2H). ¹³C NMR (125 MHz, CD₃OD+CDCl₃): δ 173.7, 165.1, 150.3, 149.9, 145.6, 130.2, 126.9, 125.2, 113.1, 61.1, 58.5 (CH₂ from azetidine ring), 54.6 (CH₂ from azetidine ring), 47.8, 39.8, 31.6, 30.5, 26.8. HR-MS (ESI): [M+H⁺] calculated 374.1333, found 374.1339.

(S)-N-(5-(3-(1H-benzo[d]imidazol-2-yl)azetidine-1-carbonyl)thiophen-2-yl)pyrrolidine-2-carboxamide (13)

Synthesized according to general procedure A with using (S)-5-(1-(tert-butoxycarbonyl)pyrrolidine-2-carboxamido)thiophene-2-carboxylic acid and 2-(azetidin-3-yl)-1H-benzo[d]imidazole dihydrochloride to afford 13 (8 mg, 55%) as an off white solid. ¹H NMR (600 MHz, CD₃OD): δ 7.55 (s, 2H), 7.41 (d, J = 4.2 Hz, 1H), 7.23 (dd, J = 6.1, 3.1 Hz, 2H), 6.81 (d, J = 4.2 Hz, 1H), 4.97 (s, 1H), 4.86 (s, 1H), 4.62 (s, 1H), 4.44 (s, 1H), 4.27 (tt, J = 8.9, 6.1 Hz, 1H), 3.88 (dd, J = 8.7, 6.0 Hz, 1H), 3.08 (dt, J = 10.2, 6.6 Hz, 1H), 3.00 (dt, J = 10.4, 6.8 Hz, 1H), 2.21 (ddd, J = 12.7, 8.3, 6.3 Hz, 1H), 1.90 (dq, J = 12.9, 6.6 Hz, 1H), 1.82 (p, J = 6.8 Hz, 2H). ¹³C NMR (125 MHz, CD₃OD): δ 173.7, 165.6, 155.6, 146.2, 130.3, 127.8, 123.7, 113.7, 61.6, 58.8, 55.3, 48.0, 31.9, 26.9. HR-MS (ESI): [M+H⁺] calculated 396.1489, found 396.1493.

(S)-N-(5-(3-(benzo[b]thiophen-3-yl)azetidine-1-carbonyl)thiophen-2-yl)pyrrolidine-2-carboxamide (14)

Step 1. 3-(benzo[b]thiophen-3-yl)azetidine hydrochloride (46).

Synthesized according to general procedures B and C to afford 3-(benzo[b]thiophen-3-yl)azetidine hydrochloride (94 mg, 38%) as an off white solid. ¹H NMR (600 MHz, CD₃OD) δ 7.92 (dd, J = 7.1, 1.8 Hz, 1H), 7.70 – 7.65 (m, 2H), 7.45 – 7.38 (m, 2H), 4.57 (td, J = 7.0, 3.5 Hz, 3H), 4.34 (q, J = 3.7, 3.1 Hz, 2H). ¹³C NMR (150 MHz, CD₃OD): δ 142.1, 138.7, 135.1, 126.1, 125.6, 124.1, 123.7, 122.3, 52.9, 32.2. MS (m/z) found for [M+H⁺]: 190 Da.

Step 2. (S)-N-(5-(3-(benzo[b]thiophen-3-yl)azetidine-1-carbonyl)thiophen-2-yl)pyrrolidine-2-carboxamide.

Synthesized according to general procedure A with using (S)-5-(1-(tert-butoxycarbonyl)pyrrolidine-2-carboxamido)thiophene-2-carboxylic acid and 3-(benzo[b]thiophen-3-yl)azetidine hydrochloride to afford 14 (13 mg, 23%) as an off white solid. ¹H NMR (600 MHz, CD₃OD) δ 7.90 (dt, J = 7.6, 1.0 Hz, 1H), 7.75 – 7.71 (m, 1H), 7.55 (s, 1H), 7.43 – 7.35 (m, 3H), 6.83 (dd, J = 4.2, 0.9 Hz, 1H), 5.04 (s, 1H), 4.66 (s, 2H), 4.49 (dd, J = 8.4, 6.3 Hz, 1H), 4.36 (dt, J = 9.4, 4.5 Hz, 2H), 3.51 – 3.45 (m, 1H), 3.45 – 3.37 (m, 1H), 2.59 – 2.49 (m, 1H), 2.19 – 2.07 (m, 3H). ¹³C NMR (150 MHz, CD₃OD): δ 166.7, 165.3, 145.2, 142.3, 139.2, 137.2, 130.2, 129.1, 125.8, 125.4, 124.1, 123.0, 122.5, 114.5, 61.4, 59.7, 55.6, 47.5, 30.8, 30.2, 25.0. HR-MS (ESI): [M+H⁺] calculated 412.1148, found 412.1148

(S)-N-(5-(3-(4-phenylthiazol-2-yl)azetidine-1-carbonyl)thiophen-2-yl)pyrrolidine-2-carboxamide (15)

Synthesized according to general procedure A with using (S)-5-(1-(tert-butoxycarbonyl)pyrrolidine-2-carboxamido)thiophene-2-carboxylic acid and 2-(azetidin-3-yl)-4-phenylthiazole hydrochloride (obtained according to general procedures B and C, and was used without further purification) to afford 15 (6 mg, 68%) as an off white solid. ¹H NMR (600 MHz, CCl₄+ CD₃OD) δ 7.93 – 7.87 (m, 2H), 7.59 (s, 1H), 7.42 – 7.36 (m, 3H), 7.33 – 7.28 (m, 1H), 6.76 (d, J = 4.2 Hz, 1H), 4H are in the pick of H₂O, 4.43 – 4.32 (m, 1H), 4.04 – 3.98 (m, 1H), 3.18 (dt, J = 10.7, 6.9 Hz, 1H), 3.11 (dt, J = 10.7, 6.9 Hz, 1H), 2.30 (dt, J = 15.5, 7.7 Hz, 1H), 1.96 (dt, J = 15.5, 7.7 Hz, 1H), 1.93 – 1.83 (m, 2H).¹³C NMR (150 MHz, CCl₄+ CD₃OD): δ 170.4, 164.8, 156.7, 145.5, 135.0, 130.1, 129.4, 129.0, 127.3, 127.2, 113.7, 113.3, 113.3, 60.9, 59.6, 56.2, 47.6, 33.4, 31.3, 26.1. HR-MS (ESI): [M+H⁺] calculated 439.1257, found 439.1260.

(S)-N-(5-(3-(5-phenylthiazol-2-yl)azetidine-1-carbonyl)thiophen-2-yl)pyrrolidine-2-carboxamide (16)

Step 1. 2-(azetidin-3-yl)-5-phenylthiazole hydrochloride (48).

Synthesized according to general procedures B and C to afford 2-(azetidin-3-yl)-5-phenylthiazole hydrochloride (70 mg, 15%) as an off white solid. ¹H NMR (600 MHz, DMSO-d6) δ 9.51 (s, 1H), 9.22 (s, 1H), 8.24 (s, 1H), 7.70 – 7.63 (m, 2H), 7.46 (t, J = 7.7 Hz, 2H), 7.41 – 7.34 (m, 1H), 4.50 (p, J = 8.3 Hz, 1H), 4.38 – 4.28 (m, 2H), 4.26 – 4.18 (m, 2H). ¹³C NMR (150 MHz, DMSO-d6): δ 166.6, 139.4, 138.5, 130.6, 129.3, 128.5, 126.4, 50.6, 33.5. MS (m/z) found for [M+H⁺]: 217 Da.

Step 2. (S)-N-(5-(3-(5-phenylthiazol-2-yl)azetidine-1-carbonyl)thiophen-2-yl)pyrrolidine-2-carboxamide.

Synthesized according to general procedure A with using (S)-5-(1-(tert-butoxycarbonyl)pyrrolidine-2-carboxamido)thiophene-2-carboxylic acid and 2-(azetidin-3-yl)-5-phenylthiazole hydrochloride to afford 16 (8 mg, 79%) as an off white solid. ¹H NMR (600 MHz, CD₃OD+CD₂Cl₂) δ 7.95 (s, 1H), 7.61 – 7.56 (m, 2H), 7.43 – 7.39 (m, 2H), 7.38 (d, J = 4.3 Hz, 1H), 7.36 – 7.31 (m, 1H), 6.78 (d, J = 4.2 Hz, 1H), 4.95 (br.s, 1H), 4.75 (br.s., 1H), 4.6 (br.s. overlapped with H₂O, 1H), 4.50-4.40 (br.s, 1H), 4.34 (tt, J = 8.8, 5.8 Hz, 1H), 3.85 (dd, J = 8.9, 5.8 Hz, 1H), 3.05 (dt, J = 10.5, 6.5 Hz, 1H), 2.98 (dt, J = 10.5, 6.8 Hz, 1H), 2.20 (ddt, J = 12.7, 8.9, 7.3 Hz, 1H), 1.90 (dtd, J = 12.7, 7.3, 5.9 Hz, 1H), 1.79 (p, J = 7.0 Hz, 2H). ¹³C NMR (150 MHz, CD₃OD+CD₂Cl₂): δ 173.6, 170.4, 165.2, 145.8, 141.0, 138.8, 131.9, 130.1, 130.0, 129.4, 127.5, 127.1, 113.3, 61.2, 59.9, 56.5, 54.1, 47.8, 33.4, 31.7, 26.8. HR-MS (ESI): [M+H⁺] calculated 439.1257, found 439.1259.

(S)-N-(5-(3-(2-phenylthiazol-5-yl)azetidine-1-carbonyl)thiophen-2-yl)pyrrolidine-2-carboxamide (17)

Synthesized according to general procedure A with using (S)-5-(1-(tert-butoxycarbonyl)pyrrolidine-2-carboxamido)thiophene-2-carboxylic acid and 5-(azetidin-3-yl)-2-phenylthiazole hydrochloride (obtained according to general procedures B and C, and was used without further purification) to afford 17 (6 mg, 71%) as an off white solid. ¹H NMR (600 MHz, CD₃OD) δ 7.97 – 7.90 (m, 2H), 7.78 (s, 1H), 7.47 (dd, J = 5.2, 2.0 Hz, 3H), 7.41 (d, J = 4.2 Hz, 1H), 6.81 (d, J = 4.2 Hz, 1H), 4.99 (s, 1H), 4.60 (br.s, 2H), 4.43 – 4.31 (m, 1H), 4.19 (br.s, 1H), 3.83 (dd, J = 8.8, 5.8 Hz, 1H), 3.05 (dt, J = 10.3, 6.5 Hz, 1H), 2.97 (dt, J = 10.4, 6.8 Hz, 1H), 2.24 – 2.16 (m, 1H), 1.94 – 1.84 (m, 1H), 1.80 (p, J = 6.9 Hz, 2H). ¹³C NMR (150 MHz, CD₃OD): δ 174.4, 165.7, 141.9, 141.8, 134.6, 131.5, 130.5, 130.2, 127.5, 113.6, 61.6, 48.1, 33.1, 32.0, 29.3, 27.1. HR-MS (ESI): [M+H⁺] calculated 439.1257, found 439.1259.

(S)-N-(5-(3-(5-phenylthiophen-2-yl)azetidine-1-carbonyl)thiophen-2-yl)pyrrolidine-2-carboxamide (18)

Synthesized according to general procedure A with using (S)-5-(1-(tert-butoxycarbonyl)pyrrolidine-2-carboxamido)thiophene-2-carboxylic acid and 5-(azetidin-3-yl)-2-phenylthiophene hydrochloride (obtained according to general procedures B and C, and was used without further purification) to afford 18 (8 mg, 70%) as an off white solid. ¹H NMR (600 MHz, CDCl3) δ 7.56 (d, J = 7.1 Hz, 2H), 7.39 (d, J = 4.1 Hz, 1H), 7.36 (t, J = 7.8 Hz, 2H), 7.27 (t, J = 7.4 Hz, 1H), 7.16 (d, J = 3.6 Hz, 1H), 6.92 (d, J = 3.7 Hz, 1H), 6.67 (d, J = 4.1 Hz, 1H), 4.73 (s, 2H), 4.39 (s, 2H), 4.14 (tt, J = 8.8, 6.1 Hz, 1H), 3.96 (dd, J = 9.3, 5.1 Hz, 1H), 3.10 (dt, J = 10.3, 6.8 Hz, 1H), 3.00 (dt, J = 10.3, 6.3 Hz, 1H), 2.27 – 2.17 (m, 1H), 2.09 – 1.99 (m, 1H), 1.77 (p, J = 6.8 Hz, 2H). ¹³C NMR (150 MHz, CD₃OD): δ 172.1, 163.7, 144.5, 143.9, 143.5, 134.3, 129.3, 129.0, 127.7, 126.8, 125.8, 125.5, 123.1, 111.8, 60.8, 60.4, 57.1, 47.5, 30.7, 30.6, 26.4. HR-MS (ESI): [M+H⁺] calculated 438.1304, found 438.1311.

tert-butyl 3-(5-bromothiazol-2-yl)azetidine-1-carboxylate.

The tert-butyl 3-(thiazol-2-yl)azetidine-1-carboxylate is synthesized according to general procedure B (Boc-intermediate was used directly) and was used in the next step without further purification. To a solution of tert-butyl 3-(thiazol-2-yl)azetidine-1-carboxylate (1g, 4.17 mmol, 1 eq.) in 20 mL of anhydrous DMF, NBS (890 mg, 5 mmol, 1.2 eq.) was added portion wise at RT. The mixture was stirred 12 h at RT, poured on ice and extracted with EtOAc (3*50 mL). Collected organics were washed with brine, dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The residue was purified by silica gel flash chromatography (0-60% ethyl acetate in hexanes) to afford tert-butyl 3-(5-bromothiazol-2-yl)azetidine-1-carboxylate (665 mg, 50%) as a clear oil. ¹H NMR (600 MHz, CDCl₃) δ 7.62 (s, 1H), 4.33 (t, J = 8.6 Hz, 2H), 4.14 (dd, J = 8.6, 5.9 Hz, 2H), 4.02 (tt, J = 8.7, 5.9 Hz, 1H), 1.45 (s, 9H). ¹³C NMR (150 MHz, CDCl₃): δ 172.3, 156.3, 144.1, 108.6, 80.1, 55.5, 32.4, 28.5. MS (m/z) found for [M+H⁺]: 262 and 264 Da.

tert-butyl (S)-2-((5-(3-(5-bromothiazol-2-yl)azetidine-1-carbonyl)thiophen-2-yl)carbamoyl)pyrrolidine-1-carboxylate (52)

Synthesized according to general procedure A with using (S)-5-(1-(tert-butoxycarbonyl)pyrrolidine-2-carboxamido)thiophene-2-carboxylic acid and 3-(5-bromothiazol-2-yl)azetidin-1-ium trifluoroacetate (obtained by treating of tert-butyl 3-(5-bromothiazol-2-yl)azetidine-1-carboxylate solution in DCM with TFA (30 eq.) at 0°C, after that the mixture was stirred at 0°C for 60 min, all volatiles were removed in vacuo and obtained residue was used in the HATU-assisted coupling reaction directly without further purification) to afford tert-butyl (S)-2-((5-(3-(5-bromothiazol-2-yl)azetidine-1-carbonyl)thiophen-2-yl)carbamoyl)pyrrolidine-1-carboxylate (58 mg, 57%) as an off white solid. ¹H NMR (600 MHz, CD₃OD) δ 7.73 (s, 1H), 7.39 (d, J = 4.3 Hz, 1H), 6.77 (d, J = 4.2 Hz, 1H), 4.90 (br.s., 1H), 4.60 (br.s., 2H), 4.39 – 4.31 (m, 2H), 4.31 – 4.25 (m, 1H), 3.61 – 3.53 (m, 1H), 3.51 – 3.43 (m, 1H), 2.38 – 2.22 (m, 1H), 2.06 – 1.95 (m, 2H), 1.96 – 1.84 (m, 1H), 1.46 (s, 3H), 1.33 (s, 6H). MS (m/z) found for [M+H-tert-Bu⁺]: 484 and 486 Da.

(S)-N-(5-(3-(5-(3-aminophenyl)thiazol-2-yl)azetidine-1-carbonyl)thiophen-2-yl)pyrrolidine-2-carboxamide trifluoroacetate (19)

Synthesized according to general procedure D with using tert-butyl (S)-2-((5-(3-(5-bromothiazol-2-yl)azetidine-1-carbonyl)thiophen-2-yl)carbamoyl)pyrrolidine-1-carboxylate and 3-aminophenylboronic acid to afford 19 (8 mg, 36%) as an off white solid. ¹H NMR (600 MHz, CD₃OD) δ 7.90 (s, 1H), 7.38 (d, J = 4.2 Hz, 1H), 7.12 (t, J = 7.8 Hz, 1H), 6.94 (t, J = 2.0 Hz, 1H), 6.91 – 6.86 (m, 1H), 6.80 (d, J = 4.2 Hz, 1H), 6.69 (ddd, J = 8.0, 2.3, 0.9 Hz, 1H), 4.94 (s, 1H), 4.76 – 4.53 (m, 2H), 4.48 – 4.24 (m, 2H), 4.16 (dd, J = 8.7, 6.1 Hz, 1H), 3.27 – 3.21 (m, 1H), 3.21 – 3.13 (m, 1H), 2.41 – 2.30 (m, 1H), 2.04 – 1.87 (m, 3H). ¹³C NMR (150 MHz, CD₃OD): δ 170.7, 170.3, 165.4, 163.5, 163.2, 163.0, 162.8, 149.8, 145.9, 142.0, 138.7, 132.8, 130.9, 130.3, 128.1, 121.1, 119.2, 117.1, 116.6, 115.3, 114.1, 114.0, 61.4, 60.2, 56.6, 49.8, 47.8, 33.5, 31.4, 26.1. HR-MS (ESI): [M+H⁺] calculated 454.1366, found 454.137.

(S)-N-(5-(3-(5-(3-(aminomethyl)phenyl)thiazol-2-yl)azetidine-1-carbonyl)thiophen-2-yl)pyrrolidine-2-carboxamide (20)

Synthesized according to general procedure D with using tert-butyl (S)-2-((5-(3-(5-bromothiazol-2-yl)azetidine-1-carbonyl)thiophen-2-yl)carbamoyl)pyrrolidine-1-carboxylate and (3-aminomethylphenyl)boronic acid hydrochloride to afford 20 (16 mg, 58%) as an off white solid. ¹H NMR (600 MHz, CD₃OD) δ 8.09 (s, 1H), 7.77 (t, J = 1.8 Hz, 1H), 7.69 (dt, J = 7.8, 1.5 Hz, 1H), 7.52 (t, J = 7.7 Hz, 1H), 7.45 (dt, J = 7.7, 1.4 Hz, 1H), 7.41 (d, J = 4.2 Hz, 1H), 6.83 (d, J = 4.2 Hz, 1H), 4.95 (s, 2H), 4.65 (s, 2H), 4.50 (m, 1H), 4.46 – 4.35 (m, 2H), 4.18 (s, 2H), 3.48 (dt, J = 10.7, 6.8 Hz, 1H), 3.41 (dt, J = 11.2, 6.8 Hz, 1H), 2.57 – 2.48 (m, 1H), 2.12 (qt, J = 4.7, 2.5 Hz, 2H). ¹³C NMR (150 MHz, CD₃OD): δ 174.0, 170.6, 165.3, 145.9, 144.3, 140.9, 139.0, 132.2, 130.3, 130.2, 128.5, 127.1, 126.5, 126.2, 113.3, 61.2, 59.9, 56.2, 47.9, 46.2, 33.4, 31.8, 26.9. HR-MS (ESI): [M+H⁺] calculated 468.1522, found 468.1532.

(S)-N-(5-(3-(5-(4-(aminomethyl)phenyl)thiazol-2-yl)azetidine-1-carbonyl)thiophen-2-yl)pyrrolidine-2-carboxamide (21)

Synthesized according to general procedure D with using tert-butyl (S)-2-((5-(3-(5-bromothiazol-2-yl)azetidine-1-carbonyl)thiophen-2-yl)carbamoyl)pyrrolidine-1-carboxylate and (4-aminomethylphenyl)boronic acid hydrochloride to afford 21 (8 mg, 53%) as an off white solid. ¹H NMR (600 MHz, CD₃OD) δ 8.09 (s, 1H), 7.71 (d, J = 7.8 Hz, 2H), 7.55 (d, J = 7.9 Hz, 2H), 7.41 (d, J = 4.1 Hz, 1H), 6.86 (d, J = 4.0 Hz, 1H), 4.99 (s, 1H), 2H are in the pick of H₂O, and are present in HSQC at 59.8, 4.65 (s, 1H), 4.57 – 4.49 (m, 1H), 4.49 – 4.25 (m, 1H), 4.16 (s, 2H), 3.48 (q, J = 6.5, 5.9 Hz, 1H), 3.44 (q, J = 5.6, 5.0 Hz, 1H), 2.65 – 2.48 (m, 1H), 2.20 – 2.08 (m, 3H). ¹³C NMR (150 MHz, CD₃OD): δ 171.5, 166.7, 165.3, 145.4, 140.3, 139.9, 134.7, 133.1, 131.0, 130.4, 128.6, 128.3, 114.6, 61.3, 60.2, 56.5, 47.5, 43.9, 33.6, 30.8, 25.0. HR-MS (ESI): [M+H⁺] calculated 468.1522, found 468.1499.

(S)-N-(5-(3-(5-(isoindolin-4-yl)thiazol-2-yl)azetidine-1-carbonyl)thiophen-2-yl)pyrrolidine-2-carboxamide trifluoroacetate (22)

Synthesized according to general procedure D with using tert-butyl (S)-2-((5-(3-(5-bromothiazol-2-yl)azetidine-1-carbonyl)thiophen-2-yl)carbamoyl)pyrrolidine-1-carboxylate and tert-butyl 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)isoindoline-2-carboxylate (obtained from tert-butyl 4-bromoisoindoline-2-carboxylate according to general procedure E) to afford 22 (25 mg, 29%) as an off white solid. ¹H NMR (600 MHz, CD₃OD) δ 7.93 (s, 1H), 7.61 (d, J = 7.6 Hz, 1H), 7.51 (t, J = 7.6 Hz, 1H), 7.46 (d, J = 7.7 Hz, 1H), 7.41 (d, J = 4.2 Hz, 1H), 6.83 (d, J = 4.2 Hz, 1H), 5.00 (s, 1H), 2H are in the pick of H₂O, and are present in HSQC at 51.7 ppm, 4.70 (s, 2H), 4.64 (s, 1H), 4.50 – 4.40 (m, 2H), 3.47 (dt, J = 11.0, 6.8 Hz, 1H), 3.39 (dt, J = 11.1, 6.7 Hz, 1H), 2.58 – 2.46 (m, 1H), 2.18 – 2.07 (m, 3H). ¹³C NMR (150 MHz, CD₃OD): δ 172.3, 167.0, 165.3, 163.1 (q, CO from TFA salt, q, J²C-F = 33.7 Hz), 145.5, 141.7, 137.6, 137.4, 133.2, 131.0, 130.2, 130.0, 128.8, 128.1, 124.2, 118.3 (CF3 from TFA salt, q, J¹C-F = 292.3 Hz), 114.5, 61.3, 59.9, 56.7, 52.2, 52.1, 47.5, 33.6, 30.8, 25.1. HR-MS (ESI): [M+H⁺] calculated 480.1522, found 480.1533.

tert-butyl 3-(2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)azetidine-1-carboxylate.

Synthesized according to general procedure to general procedures G and E. Yellowish oil (75 mg, 30% based on tert-butyl-3-iodoazetidine-carboxylate). ¹H NMR (600 MHz, CD₃OD+CD₂Cl₂) δ 7.77 (d, J = 7.5 Hz, 1H), 7.46 (d, J = 4.4 Hz, 2H), 7.22 (dt, J = 8.0, 4.2 Hz, 1H), 4.49 – 4.41 (m, 1H), 4.31 (t, J = 8.7 Hz, 2H), 3.93 (t, J = 7.3 Hz, 2H), 1.45 (s, 9H), 1.33 (s, 12H). ¹³C NMR (150 MHz, CD₃OD+CD₂Cl₂): δ 157.9, 148.7, 137.2, 132.4, 126.8, 125.8, 84.8, 80.7, 56.8, 54.1, 33.4, 28.7, 25.2. LR-MS (m/z) found for [M+H-tert-Bu⁺]: 304 Da

(S)-N-(5-(3-(5-(2-(azetidin-3-yl)phenyl)thiazol-2-yl)azetidine-1-carbonyl)thiophen-2-yl)pyrrolidine-2-carboxamide (23)

Synthesized according to general procedure D with using tert-butyl (S)-2-((5-(3-(5-bromothiazol-2-yl)azetidine-1-carbonyl)thiophen-2-yl)carbamoyl)pyrrolidine-1-carboxylate and tert-butyl 3-(2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)azetidine-1-carboxylate to afford 23 (7 mg, 48%) as an off white solid. ¹H NMR (600 MHz, CD₃OD) δ 7.59 (d, J = 7.9 Hz, 1H), 7.55 (s, 1H), 7.47 (ddd, J = 7.8, 6.8, 2.1 Hz, 1H), 7.40 (d, J = 4.2 Hz, 1H), 7.34 – 7.28 (m, 2H), 6.76 (d, J = 4.2 Hz, 1H), 4.97 (s, 3H), 4.52 (s, 1H), 4.45 – 4.24 (m, 3H), 3.85-3.75 (m, 4H), 3.06 (dt, J = 10.4, 6.5 Hz, 1H), 2.98 (dt, J = 10.4, 6.8 Hz, 1H), 2.20 (ddt, J = 12.6, 8.8, 7.3 Hz, 1H), 1.95 – 1.88 (m, 1H), 1.81 (p, J = 6.9 Hz, 2H). ¹³C NMR (150 MHz, CD₃OD), mixture of rotamers: δ 172.9, 171.5, 165.6, 165.52, 146.1, 142.5, 142.4, 140.6, 139.0, 138.2, 137.8, 132.8, 132.7, 131.0, 131.0, 130.8, 130.4, 130.3, 129.1, 128.6, 127.9, 127.53, 127.50, 114.0, 113.9, 61.5, 60.2, 56.8, 53.4, 47.9, 47.8, 35.8, 33.6, 33.1, 31.5, 31.2, 26.3, 26.1. HR-MS (ESI): [M+H⁺] calculated 494.1679, found 494.1681.

tert-butyl 3-(3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)azetidine-1-carboxylate.

Synthesized according to general procedure to general procedures G and E. White semi-solid (125 mg, 50% based on tert-butyl-3-iodoazetidine-carboxylate). ¹H NMR (600 MHz, CDCl₃) δ 7.74 – 7.68 (m, 2H), 7.42 (d, J = 7.9, 1.7 Hz, 1H), 7.36 (t, J = 7.5 Hz, 1H), 4.31 (t, J = 8.7 Hz, 2H), 4.05 – 3.97 (m, 2H), 3.81 – 3.72 (m, 1H), 1.47 (s, 9H), 1.35 (s, 12H). ¹³C NMR (150 MHz, CDCl₃): δ 156.5, 141.5, 133.6, 133.2, 129.7, 128.3, 84.0, 79.6, 56.6, 33.6, 28.6, 25.0. LR-MS (m/z) found for [M+H-tert-Bu⁺]: 304 Da

(S)-N-(5-(3-(5-(3-(azetidin-3-yl)phenyl)thiazol-2-yl)azetidine-1-carbonyl)thiophen-2-yl)pyrrolidine-2-carboxamide (24)

Synthesized according to general procedure D with using tert-butyl (S)-2-((5-(3-(5-bromothiazol-2-yl)azetidine-1-carbonyl)thiophen-2-yl)carbamoyl)pyrrolidine-1-carboxylate and tert-butyl 3-(3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)azetidine-1-carboxylate (obtained from tert-butyl-3-iodoazetidine-carboxylate according to general procedures G and E) to afford 24 (9 mg, 61%) as an off white solid. ¹H NMR (600 MHz, CD₃OD) δ 8.07 (s, 1H), 7.64 (s, 1H), 7.59 – 7.54 (m, 1H), 7.45 (t, J = 7.7 Hz, 1H), 7.42 – 7.36 (m, 2H), 6.81 (d, J = 4.2 Hz, 1H), 4.96 (s, 1H), 4.68 (s, 2H), 4.38 (m, 2H), 4.28 – 4.18 (m, 3H), 4.13 (m, 2H), 3.86 (dd, J = 8.7, 5.9 Hz, 1H), 3.06 (dt, J = 10.4, 6.5 Hz, 1H), 2.98 (dt, J = 10.5, 6.8 Hz, 1H), 2.27 – 2.15 (m, 1H), 1.88 (ddt, J = 12.5, 7.8, 6.1 Hz, 1H), 1.80 (p, J = 6.9 Hz, 2H). ¹³C NMR (150 MHz, CD₃OD): δ 174.0, 171.2, 164.9, 146.0, 142.8, 140.3, 140.2, 132.9, 131.0, 130.0, 128.3, 127.8, 126.9, 126.3, 113.8, 61.6, 60.5, 56.5, 53.8, 48.1, 37.7, 33.7, 31.8, 27.1. HR-MS (ESI): [M+H⁺] calculated 494.1679, found 494.1698.

tert-butyl 3-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)azetidine-1-carboxylate.

Synthesized according to general procedure to general procedures G and E. White semi-solid (135 mg, 54% based on tert-butyl-3-iodoazetidine-carboxylate). ¹H NMR (600 MHz, CD₃OD+CD₂Cl₂) δ 7.75 (d, J = 8.1 Hz, 2H), 7.29 (d, J = 8.0 Hz, 2H), 4.29 (t, J = 8.7 Hz, 2H), 3.92 (dd, J = 8.7, 6.1 Hz, 2H), 3.74 (tt, J = 8.6, 6.0 Hz, 1H), 1.45 (s, 9H), 1.30 (s, 12H). ¹³C NMR (150 MHz, CD₃OD+CD₂Cl₂): δ 157.7, 146.4, 136.0, 126.9, 84.7, 80.7, 57.1, 54.2, 34.4, 28.7, 25.2. LR-MS (m/z) found for [M+H-tert-Bu⁺]: 304 Da

(S)-N-(5-(3-(5-(4-(azetidin-3-yl)phenyl)thiazol-2-yl)azetidine-1-carbonyl)thiophen-2-yl)pyrrolidine-2-carboxamide (25).

Synthesized according to general procedure D with using tert-butyl (S)-2-((5-(3-(5-bromothiazol-2-yl)azetidine-1-carbonyl)thiophen-2-yl)carbamoyl)pyrrolidine-1-carboxylate and tert-butyl 3-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)azetidine-1-carboxylate (obtained from tert-butyl-3-iodoazetidine-carboxylate according to general procedures G and E) to afford 25 (9 mg, 36%) as an off white solid. ¹H NMR (600 MHz, CD₃OD+DMSO-d₆) δ 8.20 (s, 1H), 7.76 (d, J = 7.9 Hz, 2H), 7.55 (d, J = 7.9 Hz, 2H), 7.45 (d, J = 4.2 Hz, 1H), 6.94 (d, J = 4.1 Hz, 1H), 4.70 (s, 4H), 4.50 – 4.43 (m, 1H), 4.32 – 4.20 (m, 3H), 4.12 (t, J = 8.0 Hz, 2H), 3.92 (dd, J = 8.8, 5.8 Hz, 1H), 3.06 (t, J = 6.7 Hz, 2H), 2.26 – 2.19 (m, 1H), 1.96 – 1.90 (m, 1H), 1.83 (p, J = 7.0 Hz, 2H). ¹³C NMR (150 MHz, CD₃OD + DMSO-d₆): δ 174.1, 170.9, 165.0, 146.1, 142.2, 140.3, 139.8, 131.4, 130.0, 129.1, 128.9, 127.8, 113.8, 61.7, 53.9, 48.1, 38.0, 33.7, 31.9, 27.1. HR-MS (ESI): [M+H⁺] calculated 494.1679, found 494.1679.

(S)-N-(5-(3-(5-(4-(pyrrolidin-1-ylmethyl)phenyl)thiazol-2-yl)azetidine-1-carbonyl)thiophen-2-yl)pyrrolidine-2-carboxamide (26)

Synthesized according to general procedure D with using tert-butyl (S)-2-((5-(3-(5-bromothiazol-2-yl)azetidine-1-carbonyl)thiophen-2-yl)carbamoyl)pyrrolidine-1-carboxylate and (1-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzyl)pyrrolidine to afford 26 (15 mg, 70%) as an off white solid. ¹H NMR (600 MHz, CD₃OD) δ 8.21 (s, 1H), 7.80 (d, J = 8.2 Hz, 2H), 7.65 (d, J = 8.2 Hz, 2H), 7.44 (d, J = 4.2 Hz, 1H), 6.87 (d, J = 4.2 Hz, 1H), 5.00 (br.s, 1H), 4.69 (s, 1H), 4.59 – 4.22 (m, 6H), 3.60 – 3.47 (m, 3H), 3.47 – 3.40 (m, 1H), 3.28 – 3.19 (m, 2H), 2.63 – 2.50 (m, 1H), 2.27 – 2.18 (m, 2H), 2.19 – 2.12 (m, 3H), 2.11 – 2.02 (m, 2H). ¹³C NMR (150 MHz, CD₃OD): δ 172.2, 166.7, 165.3, 145.4, 140.3, 139.3, 133.7, 132.5, 130.3, 128.8, 128.6, 128.5, 114.5, 61.4, 60.1, 58.8, 57.0, 55.0, 47.5, 33.5, 30.8, 25.0, 23.8. HR-MS (ESI): [M+H⁺] calculated 522.1992, found 522.1994.

(S)-N-(5-(3-(5-(4-((4-methylpiperazin-1-yl)methyl)phenyl)thiazol-2-yl)azetidine-1-carbonyl)thiophen-2-yl)pyrrolidine-2-carboxamide (27).

Synthesized according to general procedure D with using tert-butyl (S)-2-((5-(3-(5-bromothiazol-2-yl)azetidine-1-carbonyl)thiophen-2-yl)carbamoyl)pyrrolidine-1-carboxylate and 1-methyl-4-[4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzyl]piperazine to afford 27 (13 mg, 68%) as an off white solid. ¹H NMR (600 MHz, CD₃OD) δ 8.23 (s, 1H), 7.81 (d, J = 8.2 Hz, 2H), 7.74 (d, J = 8.2 Hz, 2H), 7.42 (d, J = 4.2 Hz, 1H), 6.85 (d, J = 4.2 Hz, 1H), 4.99 (s, 1H), 4.70 (s, 1H), 4.55 (s, 2H), 4.53 – 4.28 (m, 4H), 3.69 (s, 8H), 3.53 – 3.46 (m, 1H), 3.44 – 3.39 (m, 1H), 3.02 (s, 3H), 2.59 – 2.52 (m, 1H), 2.18 – 2.10 (m, 3H). ¹³C NMR (150 MHz, CD₃OD): δ 172.6, 166.7, 165.3, 145.4, 140.3, 139.1, 134.1, 133.8, 133.7, 130.3, 129.8, 128.8, 128.5, 114.5, 61.4, 60.7, 56.9, 51.3, 49.1 (from HSQC), 47.5, 43.3, 33.5, 30.8, 25.0. HR-MS (ESI): [M+H⁺] calculated 551.2257, found 551.2263.

(S)-N-(5-(3-(5-(4-(acetamidomethyl)phenyl)thiazol-2-yl)azetidine-1-carbonyl)thiophen-2-yl)pyrrolidine-2-carboxamide (28).

Synthesized according to general procedure F with using (S)-N-(5-(3-(5-(4-(aminomethyl)phenyl)thiazol-2-yl)azetidine-1-carbonyl)thiophen-2-yl)pyrrolidine-2-carboxamide (obtained by general procedure D Boc-intermediate 53 was used directly, LR-MS (m/z) found for [M+H]⁺: 568 Da) and acetylchloride to afford 28 (8 mg, 86%) as an off white solid. ¹H NMR (600 MHz, CD₃OD+30%DMSO-d6) δ 8.07 (s, 1H), 7.64 – 7.58 (m, 2H), 7.39 – 7.33 (m, 3H), 6.84 (d, J = 4.2 Hz, 1H), 4.90 (br.s, 1H), 4.65 (br.s, 2H), 4.40 – 4.36 (m, 1H), 4.34 (s, 3H), 3.90 (dd, J = 8.8, 5.9 Hz, 1H), 3.02 (dtd, J = 17.1, 10.5, 6.7 Hz, 2H), 2.19 (dq, J = 12.6, 7.5 Hz, 1H), 1.96 (s, 3H), 1.93 – 1.84 (m, 1H), 1.78 (p, J = 7.0 Hz, 2H). ¹³C NMR (150 MHz, CD₃OD+30%DMSO-d6): δ 173.2, 172.2, 170.7, 165.0, 146.0, 141.1, 140.6, 139.5, 131.1, 130.0, 129.5, 128.0, 127.8, 113.8, 61.5, 60.0, 56.3, 48.0, 43.5, 33.6, 31.7, 26.8, 23.1. HR-MS (ESI): [M+H⁺] calculated 510.1628, found 510.1628.

(S)-N-(5-(3-(5-(4-(isobutyramidomethyl)phenyl)thiazol-2-yl)azetidine-1-carbonyl)thiophen-2-yl)pyrrolidine-2-carboxamide (29)

Synthesized according to general procedure F with using (S)-N-(5-(3-(5-(4-(aminomethyl)phenyl)thiazol-2-yl)azetidine-1-carbonyl)thiophen-2-yl)pyrrolidine-2-carboxamide (obtained by general procedure D Boc-intermediate 53 was used directly, LR-MS (m/z) found for [M+H]⁺: 568 Da) and 2-methylpropanoyl chloride to afford 29 (12 mg, 61%) as an off white solid. ¹H NMR (600 MHz, CD₃OD) δ 8.30 (s, 1H), 7.66 (d, J = 7.9 Hz, 2H), 7.42 (d, J = 4.1 Hz, 1H), 7.40 (d, J = 7.8 Hz, 2H), 6.86 (d, J = 4.0 Hz, 1H), 4.94 (s, 1H), 4.65 (s, 2H), 4.62 – 4.48 (m, 3H), 4.40 (s, 2H), 3.54 – 3.45 (m, 1H), 3.42 (dt, J = 11.3, 6.6 Hz, 1H), 2.61 – 2.48 (m, 2H), 2.13 (d, J = 5.2 Hz, 3H), 1.15 (d, J = 6.9 Hz, 6H). ¹³C NMR (150 MHz, CD₃OD): δ 180.2, 173.1, 166.7, 165.4, 145.5, 142.3, 142.2, 135,0, 133.2, 130.4, 129.5, 128.6, 128.0, 114.6, 61.4, 60.0, 57.1, 47.5, 43.5, 36.3, 32.9, 30.8, 25.0, 19.9. HR-MS (ESI): [M+H⁺] calculated 538.1941, found 538.1950.

(S)-N-(5-(3-(5-(4-(cyclopentanecarboxamidomethyl)phenyl)thiazol-2-yl)azetidine-1-carbonyl)thiophen-2-yl)pyrrolidine-2-carboxamide (30).

Synthesized according to general procedure F with using (S)-N-(5-(3-(5-(4-(aminomethyl)phenyl)thiazol-2-yl)azetidine-1-carbonyl)thiophen-2-yl)pyrrolidine-2-carboxamide (obtained by general procedure D Boc-intermediate 53 was used directly, LR-MS (m/z) found for [M+H]⁺: 568 Da) and cyclopentanoyl chloride (freshly prepared from cyclopentanecarboxylic acid and oxalylchloride to afford 30 (10 mg, 61%) as an off white solid.^{44 1}H NMR (600 MHz, CD₃OD) δ 8.14 (s, 1H), 7.62 (d, J = 8.2 Hz, 2H), 7.42 (d, J = 4.2 Hz, 1H), 7.37 (d, J = 8.0 Hz, 2H), 6.84 (d, J = 4.2 Hz, 1H), 4.96 (s, 1H), 4.75 – 4.43 (m, 4H), 4.39 (s, 3H), 3.48 (dt, J = 12.9, 6.9 Hz, 1H), 3.41 (dt, J = 10.9, 6.6 Hz, 1H), 2.76 – 2.64 (m, 1H), 2.59 – 2.50 (m, 1H), 2.17 – 2.08 (m, 3H), 1.94 – 1.83 (m, 2H), 1.79 – 1.72 (m, 4H), 1.65 – 1.57 (m, 2H). ¹³C NMR (150 MHz, CD₃OD): δ 179.3, 171.8, 166.7, 165.3, 145.4, 141.7, 141.6, 137.3, 130.4, 130.3, 129.4, 128.8, 127.9, 114.5, 61.4, 60.1, 56.8, 47.5, 46.6, 43.7, 33.3, 31.5, 30.8, 27.0, 25.0. HR-MS (ESI): [M+H⁺] calculated 564.2098, found 564.2105.

(S)-N-(5-(3-(5-(4-(benzamidomethyl)phenyl)thiazol-2-yl)azetidine-1-carbonyl)thiophen-2-yl)pyrrolidine-2-carboxamide (31)

Synthesized according to general procedure F with using (S)-N-(5-(3-(5-(4-(aminomethyl)phenyl)thiazol-2-yl)azetidine-1-carbonyl)thiophen-2-yl)pyrrolidine-2-carboxamide (obtained by general procedure D Boc-intermediate 53 was used directly, LR-MS (m/z) found for [M+H]⁺: 568 Da) and benzoylchloride 31 (15 mg, 58%) as an off white solid. ¹H NMR (600 MHz, CD₃OD) δ 8.14 (s, 1H), 7.88 (dt, J = 7.2, 1.4 Hz, 2H), 7.65 (d, J = 8.2 Hz, 2H), 7.59 – 7.54 (m, 1H), 7.52 – 7.45 (m, 4H), 7.43 (d, J = 4.2 Hz, 1H), 6.86 (d, J = 4.2 Hz, 1H), 5.02 (s, 1H), 4.74 (s, 2H), 4.63 (s, 2H), 4.60 – 4.34 (m, 3H), 3.50 (dt, J = 11.1, 6.9 Hz, 1H), 3.43 (dt, J = 11.2, 6.6 Hz, 1H), 2.63 – 2.50 (m, 1H), 2.21 – 2.08 (m, 3H). ¹³C NMR (150 MHz, CD₃OD): δ 171.7, 170.237, 166.7, 165.3, 145.4, 141.6, 141.5, 137.5, 135.5, 132.8, 130.5, 130.3, 129.6, 129.5, 128.8, 128.3, 127.9, 114.5, 61.4, 59.9, 56.7, 47.5, 44.2, 33.3, 30.8, 25.0. HR-MS (ESI): [M+H⁺] calculated 572.1785, found 572.1786.

(S)-N-(5-(3-(5-(4-(3,25-dioxo-29-((3aS,4S,6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)-6,9,12,15,18,21-hexaoxa-2,24-diazanonacosyl)phenyl)thiazol-2-yl)azetidine-1-carbonyl)thiophen-2-yl)pyrrolidine-2-carboxamide trifluoroacetate salt (21-biot)

Synthesized according to general procedure F with using (S)-N-(5-(3-(5-(4-(aminomethyl)phenyl)thiazol-2-yl)azetidine-1-carbonyl)thiophen-2-yl)pyrrolidine-2-carboxamide (obtained by general procedure D Boc-intermediate 53 was used directly, LR-MS (m/z) found for [M+H]⁺: 568 Da) and biotin-PEG6-NHS ester to afford 21-biot (22 mg, 73%) as an off white semi-solid. Obtained semi-solid was re-dissolved in anhydrous DCM and treated with TFA (10 eq.) at 0°C for 30 min, volatiles were removed in vacuo and the residue was lyophilized providing the TFA salt in quantitative yield as an off white semi-solid. ¹H NMR (600 MHz, CD₃OD) δ 8.02 (s, 1H), 7.59 (d, J = 8.2 Hz, 2H), 7.41 (d, J = 4.3 Hz, 1H), 7.37 (d, J = 8.0 Hz, 2H), 6.83 (d, J = 4.2 Hz, 1H), 4.97 (s, 1H), 4.62 (s, 2H), 4.52 – 4.45 (m, 2H), 4.45 – 4.32 (m, 4H), 4.29 (dd, J = 7.9, 4.5 Hz, 1H), 3.77 (t, J = 6.0 Hz, 2H), 3.65 – 3.55 (m, 20H), 3.52 (t, J = 5.5 Hz, 2H), 3.50 – 3.44 (m, 1H), 3.44 – 3.38 (m, 1H), 3.34 (t, J = 5.5 Hz, 2H), 3.21 – 3.14 (m, 1H), 2.91 (dd, J = 12.7, 5.0 Hz, 1H), 2.69 (d, J = 12.7 Hz, 1H), 2.52 (t, J = 5.9 Hz, 3H), 2.20 (t, J = 7.4 Hz, 2H), 2.18 – 2.09 (m, 3H), 1.77 – 1.52 (m, 4H), 1.43 (q, J = 7.6 Hz, 2H). ¹³C NMR (150 MHz, CD₃OD): δ 174.7, 172.7, 169.4, 165.4, 164.7, 163.9, 161.7 (q, CO from TFA salt, q, J²C-F = 34.4 Hz), 144.0, 139.6, 139.5, 137.8, 129.7, 128.9, 128.0, 127.4, 126.4, 117.0 (q, CO from TFA salt, q, J¹C-F = 293.2 Hz), 113.2, 70.1, 70.1, 70.1, 70.1, 70.1, 69.9, 69.9, 69.2, 66.9, 62.0, 60.2, 60.0, 58.7, 55.6, 55.2, 46.1, 42.3, 39.7, 39.0, 36.3, 35.4, 32.2, 29.4, 28.4, 28.1, 25.4, 23.7. HR-MS (ESI): [M+H⁺] calculated 1029.4242, found 1029.4245.

(S)-N-(5-(3-(5-(4-((N-methylacetamido)methyl)phenyl)thiazol-2-yl)azetidine-1-carbonyl)thiophen-2-yl)pyrrolidine-2-carboxamide (32)

Synthesized according to general procedure D with using tert-butyl (S)-2-((5-(3-(5-bromothiazol-2-yl)azetidine-1-carbonyl)thiophen-2-yl)carbamoyl)pyrrolidine-1-carboxylate and N-methyl-N-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzyl)acetamide 54 (obtained by general procedure E from N-[(4-bromophenyl)methyl]-N-methylacetamide to afford 32 (16 mg, 33%) as an off white solid.^{45 1}H NMR (600 MHz, CD₃OD+CDCl3) δ 8.01 (s, 0.4H), 7.98 (s, 0.6H), 7.64 (d, J = 8.2 Hz, 1H), 7.58 (d, J = 8.2 Hz, 1H), 7.40 (d, J = 4.2 Hz, 1H), 7.31 (d, J = 8.0 Hz, 1H), 7.28 (d, J = 8.0 Hz, 1H), 6.80 (d, J = 4.2 Hz, 1H), 4.97 (s, 1H), 4.69 (s, 1H), 4.64 (s, 1H), 4.61 (s, 2H), 4.48 (s, 1H), 4.37 (s, 1H), 3.86 (dd, J = 8.8, 5.9 Hz, 1H), 3.07 (dt, J = 10.4, 6.5 Hz, 1H), 3.02 (s, 2H), 2.98 (dd, J = 10.5, 6.8 Hz, 1H), 2.94 (s, 1H), 2.25 – 2.19 (m, 1H), 2.18 (s, 2H), 2.17 (s, 1H), 1.96 – 1.84 (m, 1H), 1.81 (p, J = 6.9 Hz, 2H). ¹³C NMR (150 MHz, CD₃OD+CDCl3): δ 173.8, 173.6, 173.4, 170.8, 170.7, 165.5, 146.1, 140.8, 140.6, 139.2, 139.1, 138.9, 138.4, 131.6, 131.3, 130.3, 129.6, 128.4, 128.2, 127.9, 127.3, 127.3, 113.5, 61.4, 60.1, 56.6, 54.8, 51.3, 47.9, 36.3, 34.3, 33.5, 33.5, 31.8, 26.9, 21.6, 21.4. HR-MS (ESI): [M+H⁺] calculated 524.1785, found 524.1794.

(S)-N-(5-(3-(5-(2-acetyl-1,2,3,4-tetrahydroisoquinolin-6-yl)thiazol-2-yl)azetidine-1-carbonyl)thiophen-2-yl)pyrrolidine-2-carboxamide (33)

Synthesized according to general procedure D with using tert-butyl (S)-2-((5-(3-(5-bromothiazol-2-yl)azetidine-1-carbonyl)thiophen-2-yl)carbamoyl)pyrrolidine-1-carboxylate and 1-(6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-3,4-dihydroisoquinolin-2(1H)-yl)ethan-1-one 55 (obtained by general procedure E from 1-(6-bromo-3,4-dihydro-2(1H)-isoquinolinyl)ethenone to afford 33 (8 mg, 30%) as an off white solid.^{46 1}H NMR (600 MHz, CD₃OD+CDCl₃) δ 7.94 (s, 0.4H), 7.93 (s, 0.6H), 7.41 (ddd, J = 14.9, 7.5, 1.9 Hz, 2H), 7.37 (d, J = 4.3 Hz, 1H), 7.20 (dd, J = 14.3, 8.0 Hz, 1H), 6.77 (d, J = 4.3 Hz, 1H), 4.93 (s, 2H), 4.68 (m, 3H), 4.54 (br.s, 1H), 4.33 (q, J = 5.7, 4.5 Hz, 1H), 3.82 (dd, J = 8.8, 5.8 Hz, 1H), 3.78 (t, J = 6.0 Hz, 1H), 3.74 (t, J = 5.9 Hz, 1H), 3.04 (dt, J = 10.5, 6.5 Hz, 1H), 3.01 – 2.93 (m, 2H), 2.87 (t, J = 6.0 Hz, 1H), 2.18 (m, 4H), 1.88 (dd, J = 13.1, 6.4 Hz, 1H), 1.79 (q, J = 6.9 Hz, 2H). ¹³C NMR (150 MHz, CD₃OD+CDCl₃): δ 173.9, 171.9, 171.8, 170.4, 170.4, 165.3, 145.9, 140.7, 140.6, 138.9, 138.8, 136.9, 136.4, 134.7, 134.3, 130.7, 130.4, 130.3, 128.3, 128.0, 127.7, 127.4, 127.1, 125.8, 125.6, 113.3, 61.3, 59.9, 56.3, 49.9, 47.9, 44.9, 44.9, 40.6, 33.4, 31.8, 30.0, 29.2, 26.9, 21.8, 21.5. HR-MS (ESI): [M+H⁺] calculated 536.1785, found 536.1793.

(S)-N-(5-(3-(5-(4-((2-oxopyrrolidin-1-yl)methyl)phenyl)thiazol-2-yl)azetidine-1-carbonyl)thiophen-2-yl)pyrrolidine-2-carboxamide (34)

Synthesized according to general procedure D with using tert-butyl (S)-2-((5-(3-(5-bromothiazol-2-yl)azetidine-1-carbonyl)thiophen-2-yl)carbamoyl)pyrrolidine-1-carboxylate and 1-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzyl)pyrrolidin-2-one 56 (obtained by general procedure E from 1-[(4-bromophenyl)methyl]-2-pyrrolidinone to afford 34 (11 mg, 38%) as an off white solid.^{46 1}H NMR (600 MHz, CD₃OD+CD₂Cl₂) δ 7.98 (s, 1H), 7.59 (d, J = 8.2 Hz, 2H), 7.39 (d, J = 4.2 Hz, 1H), 7.31 (d, J = 7.5 Hz, 2H), 6.79 (d, J = 4.1 Hz, 1H), 4.92 (s, 1H), 4.47 (s, 4H), 4.39 – 4.32 (m, 2H), 3.84 (dd, J = 8.9, 5.8 Hz, 1H), 3.36 (t, J = 7.1 Hz, 2H), 3.09 – 3.02 (m, 1H), 3.02 – 2.94 (m, 1H), 2.44 (t, J = 8.2 Hz, 2H), 2.23 – 2.17 (m, 1H), 2.07 – 2.00 (m, 3H), 1.92 – 1.86 (m, 1H), 1.82 – 1.76 (m, 2H). ¹³C NMR (150 MHz, CD₃OD+ CD₂Cl₂): δ 177.4, 173.9, 170.7, 165.4, 146.0, 144.5, 140.6, 139.1, 138.0, 131.5, 130.3, 129.7, 127.9, 127.2, 113.3, 61.3, 48.1, 47.9, 47.0, 33.5, 31.8, 31.7, 26.9, 18.5., 29.2, 26.9, 21.8, 21.5. HR-MS (ESI): [M+H⁺] calculated 536.1785, found 536.1789.

(S)-N-(5-(3-(5-(4-(acetamidomethyl)-2-chlorophenyl)thiazol-2-yl)azetidine-1-carbonyl)thiophen-2-yl)pyrrolidine-2-carboxamide (35)

Synthesized according to general procedure D with using tert-butyl (S)-2-((5-(3-(5-bromothiazol-2-yl)azetidine-1-carbonyl)thiophen-2-yl)carbamoyl)pyrrolidine-1-carboxylate and N-(3-chloro-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzyl)acetamide 57 (obtained by general procedure E from N-[(4-bromo-3-chlorophenyl)methyl]acetamide to afford 35 (8 mg, 30%) as an off white solid.^{47 1}H NMR (600 MHz, DMSO-D6) δ 8.41 (t, J = 6.1 Hz, 1H), 8.07 (s, 1H), 7.66 (d, J = 8.0 Hz, 1H), 7.48 (d, J = 1.8 Hz, 1H), 7.35 – 7.29 (m, 2H), 6.91 (d, J = 4.2 Hz, 1H), 4.72 (br.s., 4H), 4.42 (tt, J = 8.8, 5.7 Hz, 1H), 4.29 (m, 2H), 3.85 (dd, J = 8.8, 5.8 Hz, 1H), 2.98 – 2.89 (m, 2H), 2.14 – 2.04 (m, 1H), 1.90 (s, 3H), 1.80 (ddd, J = 12.9, 7.2, 5.8 Hz, 1H), 1.69 (p, J = 7.0 Hz, 2H). ¹³C NMR (150 MHz, DMSO-D6): δ 172.1, 170.8, 169.3, 162.9, 144.2, 142.3, 142.1, 134.0, 131.3, 130.9, 128.9, 128.2, 127.6, 126.7, 126.3, 112.6, 60.0, 46.6, 41.2, 31.8, 30.2, 25.5, 22.5. HR-MS (ESI): [M+H⁺] calculated 544.1238, found 544.1243.

((S)-N-(5-(3-(5-(4-(acetamidomethyl)-3-chlorophenyl)thiazol-2-yl)azetidine-1-carbonyl)thiophen-2-yl)pyrrolidine-2-carboxamide (36)

Synthesized according to general procedure D with using tert-butyl (S)-2-((5-(3-(5-bromothiazol-2-yl)azetidine-1-carbonyl)thiophen-2-yl)carbamoyl)pyrrolidine-1-carboxylate and N-(2-chloro-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzyl)acetamide 58 (obtained by general procedure E from N-[(4-bromo-2-chlorophenyl)methyl]acetamide to afford 36 (9 mg, 33%) as an off white solid.^{48 1}H NMR (600 MHz, DMSO-D6) δ 8.37 (t, J = 5.8 Hz, 1H), 8.26 (s, 1H), 7.78 (d, J = 1.9 Hz, 1H), 7.58 (dd, J = 8.0, 1.9 Hz, 1H), 7.38 (d, J = 8.1 Hz, 1H), 7.33 (d, J = 4.2 Hz, 1H), 6.86 (d, J = 4.2 Hz, 1H), 4.39 (ddd, J = 8.8, 7.1, 4.3 Hz, 1H), 4.32 (d, J = 5.8 Hz, 2H), 4.07 (dd, J = 8.7, 6.4 Hz, 1H), 3.07 (t, J = 6.9 Hz, 2H), 2.19 (ddd, J = 12.9, 8.5, 6.4 Hz, 1H), 1.95 – 1.83 (m, 4H), 1.80 (q, J = 7.0 Hz, 2H). ¹³C NMR (150 MHz, DMSO-D6): δ 169.8, 169.4, 162.8, 143.8, 139.5, 136.9, 136.4, 132.8, 131.2, 129.7, 128.2, 126.9, 126.3, 125.2, 112.8, 59.7, 46.3, 31.9, 29.8, 24.7, 22.4. HR-MS (ESI): [M+H⁺] calculated 544.1238, found 544.1246.

(S)-N-(5-(3-(5-(4-(acetamidomethyl)naphthalen-1-yl)thiazol-2-yl)azetidine-1-carbonyl)thiophen-2-yl)pyrrolidine-2-carboxamide (37)

Step 1. N-((4-bromonaphthalen-1-yl)methyl)acetamide.

The a suspension of (4-bromonaphthalen-1-yl)methanamine hydrochloride (271 mg, 1 mmol, 1 eq.) in 5 ml of anhydrous pyridine at 0°C was added AcCl (86 mg, 78 μL, 1.5 mmol, 1.5 eq.) dropwise. The mixture was stirred at RT for 2 h and concentrated in vacuo. The residue was diluted with EtOAc (50 mL) and washed with water and brine. The organic fraction was dried over sodium sulfate and concentrated in vacuo, obtained residue was used without further purification. Pale semi-solid (241 mg, 87%). LR-MS (m/z) found for [M+H⁺]: 278 and 280 Da.

Step 2. N-((4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)naphthalen-1-yl)methyl)acetamide (59).

Synthesized according to general procedure E with using N-((4-bromonaphthalen-1-yl)methyl)acetamide to afford N-((4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)naphthalen-1-yl)methyl)acetamide (192 mg, 68%). LR-MS (m/z) found for [M+H]: 326 Da.

Step 3. (S)-N-(5-(3-(5-(4-(acetamidomethyl)naphthalen-1-yl)thiazol-2-yl)azetidine-1-carbonyl)thiophen-2-yl)pyrrolidine-2-carboxamide.

Synthesized according to general procedure D with using tert-butyl (S)-2-((5-(3-(5-bromothiazol-2-yl)azetidine-1-carbonyl)thiophen-2-yl)carbamoyl)pyrrolidine-1-carboxylate and N-((4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)naphthalen-1-yl)methyl)acetamide to afford 37 (12 mg, 33%) as an off white solid. ¹H NMR (600 MHz, CD₃OD) δ 8.13 (d, J = 8.9 Hz, 1H), 8.08 (d, J = 8.8 Hz, 1H), 7.80 (s, 1H), 7.58 (t, J = 7.0 Hz, 1H), 7.55 – 7.47 (m, 3H), 7.38 (d, J = 4.2 Hz, 1H), 6.79 (d, J = 4.2 Hz, 1H), 4.94 (s, 1H), 4.85 (s, 2H), 4.69 (s, 2H), 4.48 – 4.28 (m, 2H), 3.84 (dd, J = 8.8, 5.9 Hz, 1H), 3.05 (dt, J = 10.4, 6.5 Hz, 1H), 2.97 (dt, J = 10.4, 6.8 Hz, 1H), 2.19 (ddt, J = 12.4, 8.5, 7.1 Hz, 1H), 2.01 (s, 3H), 1.91 – 1.84 (m, 1H), 1.79 (p, J = 6.9 Hz, 2H). ¹³C NMR (150 MHz, CD₃OD): δ 174.0, 173.0, 172.2, 165.6, 146.3, 142.7, 138.1, 136.8, 133.4, 132.9, 130.4, 129.5, 129.3, 127.9, 127.8, 127.5, 126.8, 126.4, 125.1, 113.6, 61.6, 60.2, 56.6, 48.0, 42.2, 33.6, 31.9, 26.9, 22.5. HR-MS (ESI): [M+H⁺] calculated 560.1785, found 560.1788.

(S)-N-(5-(3-(5-(4-((N-methylisobutyramido)methyl)naphthalen-1-yl)thiazol-2-yl)azetidine-1-carbonyl)thiophen-2-yl)pyrrolidine-2-carboxamide (38)

Step 1. 1-(4-bromonaphthalen-1-yl)-N-methylmethanamine.

The solution of 1-bromo-4-(bromomethyl)naphthalene (300 mg, 1 mmol, 1 eq.) in 1 ml of anhydrous THF was added to the 7N ammonia in methanol solution (20 mL, 140 eq.) dropwise over 30 min. The mixture was stirred at r.t. overnight and concentrated in vacuo. The residue was purified by silica gel flash chromatography (0-100% ethylacetate in hexane), fractions with the corresponding peak in the LC-MS were combined, concentrated and the residue was used without further purification. Yellowish oil (174 mg, 70%). LR-MS (m/z) found for [M+H⁺]: 250 and 252 Da.

Step 2. N-((4-bromonaphthalen-1-yl)methyl)-N-methylisobutyramide.

Synthesized according to the general procedure F with using 1-(4-bromonaphthalen-1-yl)-N-methylmethanamine and isobutyryl chloride to afford the titled compound as a white semisolid (156 mg, 70%) that was used without further purification. LR-MS (m/z) found for [M+H⁺]: 320 and 322 Da.

Step 3. N-methyl-N-((4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)naphthalen-1-yl)methyl)isobutyramide (60).

Synthesized according to general procedure E with using N-((4-bromonaphthalen-1-yl)methyl)-N-methylisobutyramide to afford N-methyl-N-((4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)naphthalen-1-yl)methyl)isobutyramide (145 mg, 81%). LR-MS (m/z) found for [M+H]: 368 Da.

Step 4. (S)-N-(5-(3-(5-(4-((N-methylisobutyramido)methyl)naphthalen-1-yl)thiazol-2-yl)azetidine-1-carbonyl)thiophen-2-yl)pyrrolidine-2-carboxamide.

Synthesized according to general procedure D with using tert-butyl (S)-2-((5-(3-(5-bromothiazol-2-yl)azetidine-1-carbonyl)thiophen-2-yl)carbamoyl)pyrrolidine-1-carboxylate and N-methyl-N-((4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)naphthalen-1-yl)methyl)isobutyramide to afford 38 (18 mg, 41%) as an off white solid. ¹H NMR (600 MHz, CD₃OD) δ 8.17 – 8.07 (m, 2H), 7.85 (s, 1H), 7.68 – 7.53 (m, 3H), 7.47 – 7.35 (m, 1.8H), 7.24 (d, J = 7.4 Hz, 0.2H), 6.81 (d, J = 4.2 Hz, 1H), 5.22 (s, 0.5H), 5.12 (s, 1.5H), 4.99 (s, 1H), 4.66 (s, 2H), 4.49 – 4.31 (m, 2H), 3.84 (dd, J = 8.8, 5.8 Hz, 1H), 3.12 – 2.80 (m, 6H), 2.19 (dt, J = 12.6, 7.8 Hz, 1H), 1.93 – 1.83 (m, 1H), 1.80 (q, J = 6.9 Hz, 2H), 1.17 (d, J = 6.7 Hz, 4H), 1.12 (d, J = 6.7 Hz, 2H). ¹³C NMR (150 MHz, CD₃OD): δ 180.8, 179.5, 174.2, 172.4, 172.3, 165.7, 146.4, 142.9, 142.8, 138.1, 138.0, 135.6, 135.3, 133.5, 133.5, 133.2, 132.5, 130.4, 129.5, 129.4, 129.3, 128.2, 128.0, 127.8, 127.5, 127.0, 126.9, 126.8, 125.2, 124.1, 123.0, 113.6, 66.6, 61.6, 56.7, 52.3, 49.8, 49.3, 35.4, 35.1, 33.6, 32.0, 31.8, 31.6, 27.0, 20.1, 19.6. HR-MS (ESI): [M+H⁺] calculated 602.2254, found 602.2258.

(S)-4,4-dimethyl-N-(5-(3-(5-(4-(propionamidomethyl)naphthalen-1-yl)thiazol-2-yl)azetidine-1-carbonyl)thiophen-2-yl)pyrrolidine-2-carboxamide (39)

Step 1. N-((4-bromonaphthalen-1-yl)methyl)propionamide.

The a suspension of (4-bromonaphthalen-1-yl)methanamine hydrochloride (271 mg, 1 mmol, 1 eq.) in 5 ml of anhydrous pyridine at 0°C was added propionyl chloride (139 mg, 131 μL, 1.5 mmol, 1.5 eq.) dropwise. The mixture was stirred at RT for 2h and concentrated in vacuo. The residue was diluted with EtOAc (50 mL) and washed with water and brine. The organic fraction was dried over sodium sulfate and concentrated in vacuo, obtained residue was used without further purification. Pale semi-solid (262 mg, 90%). LR-MS (m/z) found for [M+H⁺]: 292 and 294 Da.

Step 2. N-((4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)naphthalen-1-yl)methyl)propionamide (61).

Synthesized according to general procedure E with using N-((4-bromonaphthalen-1-yl)methyl)propionamide to afford N-((4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)naphthalen-1-yl)methyl)propionamide (173 mg, 57 %). LR-MS (m/z) found for [M+H]: 340 Da.

Step 3. tert-butyl 3-(5-(4-(propionamidomethyl)naphthalen-1-yl)thiazol-2-yl)azetidine-1-carboxylate.

Synthesized according to the Step 1 of general procedure D with using N-((4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)naphthalen-1-yl)methyl)propionamide and tert-butyl 3-(5-bromothiazol-2-yl)azetidine-1-carboxylate to afford tert-butyl 3-(5-(4-(propionamidomethyl)naphthalen-1-yl)thiazol-2-yl)azetidine-1-carboxylate (156 mg, 68%). LR-MS (m/z) found for [M+H-tBu]⁺: 396 Da.

Step 4. (S)-4,4-dimethyl-N-(5-(3-(5-(4-(propionamidomethyl)naphthalen-1-yl)thiazol-2-yl)azetidine-1-carbonyl)thiophen-2-yl)pyrrolidine-2-carboxamide.

Synthesized according to general procedure A with using (S)-5-(4,4-dimethylpyrrolidine-2-carboxamido)thiophene-2-carboxylic acid (prepared the same way as 49.¹³ and N-((4-(2-(azetidin-3-yl)thiazol-5-yl)naphthalen-1-yl)methyl)propionamide trifluoroacetate ( substituted azetidine was synthesized according procedures B and C from the corresponding bromide) to afford 39 (18 mg, 91%) as an off white solid. ¹H NMR (600 MHz, CD₃OD) δ 8.09 (d, J = 8.5 Hz, 1H), 8.03 (d, J = 8.4 Hz, 1H), 7.75 (s, 1H), 7.53 (t, J = 7.6 Hz, 1H), 7.48 (t, J = 7.6 Hz, 1H), 7.44 (s, 2H), 7.32 (d, J = 4.2 Hz, 1H), 6.77 (d, J = 4.3 Hz, 1H), 5.04 (s, 1H), 4.64 (s, 3H), 4.35 – 4.30 (m, 1H), 3.93 (t, J = 8.3 Hz, 1H), 2.71 (q, J = 10.6 Hz, 2H), 2.27 (q, J = 7.6 Hz, 2H), 1.99 (dd, J = 12.7, 8.7 Hz, 1H), 1.61 (dd, J = 12.7, 7.9 Hz, 1H), 1.14 (t, J = 7.6 Hz, 3H), 1.05 (s, 3H), 1.03 (s, 3H). Note: signal for NaphthylCH₂NH- overlaps with signal of H₂O, corresponding carbon is observable in HSQC. ¹³C NMR (150 MHz, CD₃OD): δ 176.7, 174.5, 172.0, 165.5, 146.2, 142.7, 138.1, 136.9, 133.3, 132.9, 130.4, 129.4, 129.2, 127.9, 127.7, 127.5, 126.8, 126.3, 125.1, 113.7, 61.6, 61.2, 60.1, 56.6, 46.2, 42.1, 41.2, 33.5, 30.2, 26.8, 26.7, 10.6. HR-MS (ESI): [M+H⁺] calculated 602.2254, found 602.2256.

(S)-4,4-dimethyl-N-(5-(3-(5-(4-(propionamidomethyl)naphthalen-1-yl)thiazol-2-yl)azetidine-1-carbonyl)thiazol-2-yl)pyrrolidine-2-carboxamide (40)

Step 1. Methyl (S)-2-(1-(tert-butoxycarbonyl)-4,4-dimethylpyrrolidine-2-carboxamido)thiazole-5-carboxylate 63.

Synthesized according to procedure previously described.¹³ To a stirred solution of (S)-1-(tert-butoxycarbonyl)-4,4-dimethylpyrrolidine-2-carboxylic acid 62 (243 mg, 1 mmol) in DCM (10 mL), was added 1-methylimidazole (205 mg, 199 μL, 2.5 mmol) and the mixture was cooled to 0°C under argon atmosphere. The reaction mixture was stirred for 10 min at 0°C and MsCl (137 mg, 92 μL, 1.2 mmol) was added dropwise at the same temperature and stirring continued for 1h at 0°C. Then the methyl 2-aminothiazole-5-carboxylate (158 mg, 1 mmol) was added, and the mixture was stirred at RT overnight. The reaction was quenched with water (10 mL), layers were separated, and the aqueous layer was extracted with DCM (3-10 mL). The gathered organic layers were washed with 1M HCl (aq), NaHCO₃ (sat. aq.) and brine, dried over sodium sulfate, concentrated in vacuo. The residue was purified by silica gel flash chromatography (0-20% methanol in DCM), fractions with the corresponding peak in the LC-MS were combined, concentrated and the residue was used without further purification. Clear semi-solid (260 mg, 68%). LR-MS (m/z) found for [M+H⁺]: 384 Da.

Step 2. (S)-2-(1-(tert-butoxycarbonyl)-4,4-dimethylpyrrolidine-2-carboxamido)thiazole-5-carboxylic acid.

Synthesized according to the Step 2 of the procedure prepared the same way as 49 with using Methyl (S)-2-(1-(tert-butoxycarbonyl)-4,4-dimethylpyrrolidine-2-carboxamido)thiazole-5-carboxylate to afford (S)-2-(1-(tert-butoxycarbonyl)-4,4-dimethylpyrrolidine-2-carboxamido)thiazole-5-carboxylic acid (251 mg, quant.).¹³ LR-MS (m/z) found for [M-H]⁻: 368 Da.

Step 3. (S)-4,4-dimethyl-N-(5-(3-(5-(4-(propionamidomethyl)naphthalen-1-yl)thiazol-2-yl)azetidine-1-carbonyl)thiazol-2-yl)pyrrolidine-2-carboxamide.

Synthesized according to general procedure A with using (S)-2-(1-(tert-butoxycarbonyl)-4,4-dimethylpyrrolidine-2-carboxamido)thiazole-5-carboxylic acid and N-((4-(2-(azetidin-3-yl)thiazol-5-yl)naphthalen-1-yl)methyl)propionamide trifluoroacetate substituted azetidine was synthesized according procedures B and C from the corresponding bromide) to afford 40 (24 mg, 67%) as an off white solid. ¹H NMR (600 MHz, CD₂Cl₂) δ 8.12 (d, J = 7.3 Hz, 1H), 8.09 (d, J = 8.0 Hz, 1H), 7.79 (s, 1H), 7.78 (s, 1H), 7.58 (ddd, J = 8.3, 6.8, 1.4 Hz, 1H), 7.54 (ddd, J = 8.2, 6.8, 1.4 Hz, 1H), 7.48 (d, J = 7.3 Hz, 1H), 7.45 (d, J = 7.2 Hz, 1H), 6.3 (br. t., 1H), 4.88 (d, J = 5.6 Hz, 2H), 4.77 (s, 2H), 4.59 (s, 1H), 4.44 (s, 1H), 4.31 (tt, J = 8.8, 5.9 Hz, 1H), 4.05 (dd, J = 9.1, 7.7 Hz, 1H), 2.81 (dd, J = 10.7, 1.3 Hz, 1H), 2.69 (d, J = 10.7 Hz, 1H), 2.24 (q, J = 7.6 Hz, 2H), 2.09 (ddd, J = 12.9, 9.1, 1.3 Hz, 1H), 1.69 (dd, J = 12.9, 7.8 Hz, 1H), 1.15 (t, J = 7.6 Hz, 3H), 1.06 (s, 3H), 1.05 (s, 3H). ¹³C NMR (150 MHz, CD₂Cl₂): δ 174.5, 173.8, 170.3, 162.6, 160.9, 142.3, 142.2, 141.7, 136.9, 136.1, 132.6, 132.1, 128.8, 128.7, 128.7, 127.2, 126.3, 125.9, 125.8, 124.4, 60.8, 60.6, 58.7, 55.6, 44.9, 41.7, 40.8, 32.9, 29.9, 26.2, 26.1, 10.1. HR-MS (ESI): [M+H⁺] calculated 603.2207, found 603.2208.

Supplementary Material

Supporting Information. This Supporting Information is available free of charge via the Internet at http://pubs.acs.org.

• Supplementary figures S1-S4 include probe titration for the AlphaLISA assay, supplemental crystal structure figures, NanoBRET assay data and HPLC traces, and Supplementary Tables S1-S3 include refinement statistics, kinase profiling and molecular strings (PDF).

Abbreviations

CDKN1A

cyclin dependent kinase inhibitor 1A

CSP

chemical shift perturbation

GAS41

glioma-amplified sequence 1

H3K27ac

histone H3 lysine 27 acetylation

H3K27cr

histone H3 lysine 27 crotonylation

HPLC

reverse phase high-performance liquid chromatography

HSQC

heteronuclear single quantum coherence

FP

fluorescence polarization

ITC

isothermal titration calorimetry

Kd

equilibrium dissociation constant

NanoBRET

nanoluciferase bioluminescence resonance energy transfer

NMR

nuclear magnetic resonance

NSCLC

non-small cell lung cancer

RT

room temperature

SAR

structure-activity relationship

YEATS4

YEATS-domain containing 4 (human)

Data Availability

Crystal structures of GAS41 YEATS cocrystalized in the presence of DLG-1 are available in the Protein Data Bank (PDB) under accession code 9O4Y and authors will release the atomic coordinates upon article publication. RNA-seq data (reads in fastq files and log2 counts-per-million matrices) for H1299 cells treated with DLG-41 and DLG-41nc have been deposited in the Gene Expression Omnibus (GEO) under the accession code GSE295002.