A Druglike Small Molecule that Targets r(CCUG) Repeats in Myotonic Dystrophy Type 2 Facilitates Degradation by RNA Quality Control Pathways

Abstract

Myotonic dystrophy type 2 (DM2) is one of >40 microsatellite disorders caused by RNA repeat expansions. The DM2 repeat expansion, r(CCUG)^exp (where “exp” denotes expanded repeating nucleotides), is harbored in intron 1 of the CCHC-type zinc finger nucleic acid binding protein (CNBP). The expanded RNA repeat causes disease by a gain-of-function mechanism, sequestering various RNA-binding proteins including the pre-mRNA splicing regulator MBNL1. Sequestration of MBNL1 results in its loss-of-function and concomitant deregulation of the alternative splicing of its native substrates. Notably, this r(CCUG)^exp causes retention of intron 1 in the mature CNBP mRNA. Herein, we report druglike small molecules that bind the structure adopted by r(CCUG)^exp and improve DM2-associated defects. These small molecules were optimized from screening hits from an RNA-focused small-molecule library to afford a compound that binds r(CCUG)^exp specifically and with nanomolar affinity, facilitates endogenous degradation of the aberrantly retained intron in which it is harbored, and rescues alternative splicing defects.

INTRODUCTION

RNA repeat expansions cause >40 microsatellite disorders, including Huntington’s disease [HD, r(CAG)^exp], C9orf72 amyotrophic lateral sclerosis/frontotemporal dementia [c9ALS/FTD, r(G₄C₂)^exp], and myotonic dystrophy types 1 and 2 [DM1, r(CUG)^exp and DM2, r(CCUG)^exp, respectively]. (1) Repeat expansions cause disease through various mechanisms dependent upon its location within a gene. For example, an RNA gain-of-function mechanism occurs when repeat sequesters functionally inactivate proteins that regulate alternative pre-mRNA splicing. (2) Repeat expansions can also contribute to disease via an aberrant translational mechanism that generates toxic proteins, named repeat-associated non-ATG translation. (3) Recently, it was discovered that GC-rich RNA repeat expansions located in introns, such as r(CCUG)^exp, r(CUG)^exp, and r(G₄C₂)^exp, cause retention of the intron in which they are harbored in mature mRNA species. (4)

The DM2 repeat expansion, r(CCUG)^exp, is harbored in intron 1 of CCHC-type zinc finger nucleic acid binding protein gene (CNBP). (5) This r(CCUG)^exp folds into a structure containing repeating 2 × 2 CU/UC internal loops (Figure 1A), (6) which bind and sequester muscleblind-like 1 (MBNL1), an important regulator of alternative pre-mRNA splicing, in nuclear foci. (7,8) Sequestration of MBNL1 by r(CCUG)^exp results in pre-mRNA splicing defects of MBNL1-regulated genes. For example, insulin receptor (IR) exon 11 is excluded too frequently in DM2 cells (Figure 1B). (9) r(CCUG)^exp can also cause dysfunction through intron retention, where CNBP itself is mis-spliced, and the r(CCUG)^exp-containing intron 1 is retained in mature mRNA species (Figure 1C). (4)

Figure 1.

r(CCUG)^exp-mediated defects in DM2. (A) DM2 is caused by r(CCUG)^exp in CNBP intron 1, which folds into a structure with repeating 2 × 2 nucleotide internal loops that sequester regulatory proteins involved in pre-mRNA splicing such as MBNL1. (B) Sequestration of MBNL1 by r(CCUG)^exp results in splicing defects in its native pre-mRNAs substrates. For example, IR exon 11 is included ∼70% of the time in healthy cells, but only ∼50% in DM2-affected cells. (C) r(CCUG)^exp also causes intron retention where intron 1 is aberrantly retained in CNBP mature RNA.

We have previously shown that intron retention is due to MBNL1 binding; knock-down of MBNL1 reduces intron retention, whereas forced expression of MBNL1 increases intron retenion. (10) Thus, small-molecule recognition of r(CCUG)^exp liberates MBNL1 and rescues deregulated splicing events and intron retention. In particular, we have shown that kanamycin A derivatives that bind r(CCUG)^exp can specifically improve DM2-associated defects and reduce the levels of intron 1 containing r(CCUG)^exp. (10) In another example of an intron-retained RNA repeat [r(CUG)^exp in intron 3 of transcription factor 4 mRNA (TCF4) in Fuchs endothelial corneal dystrophy (FECD)], (11) small-molecule binding results in decay of the r(CUG)^exp-containing intron 3 through the RNA exosome. (12) Thus, small-molecule recognition of RNA repeat expansions in retained introns can allow for targeted RNA degradation through stimulation of RNA quality control mechanisms. In addition to interfacing of disease-causing RNAs with endogenous decay pathways, other methods for targeted RNA degradation rely on chimeric compounds comprising an RNA-binding ligand attached to a cleaving module. However, this strategy increases compound molecular weight and can affect various pharmacological and physicochemical properties such as membrane permeability and solubility. (13–17)

Although our previous studies targeting r(CCUG)^exp showed that small molecules can indeed interface the toxic RNA with endogenous degradation pathways, the aminoglycosides from which they are derived are not particularly druglike and provide little opportunity for optimization. (10,18) Herein, we studied a collection of RNA-focused, druglike small molecules to target r(CCUG)^exp. Medicinal chemistry optimization of screening hits afforded a druglike small molecule that binds r(CCUG)^exp with nanomolar affinity, specifically degrades r(CCUG)^exp-containing intron 1, and improves DM2-associated defects.

RESULTS & DISCUSSION

Studying an RNA-Focused Small-Molecule Compound Collection for Targeting r(CCUG)

To identify novel small molecules that bind r(CCUG)^exp, a druglike RNA-focused small-molecule library containing 3271 compounds was employed. (19) This library was previously designed to contain structurally diverse small molecules that have chemotypes that confer avidity toward RNA. (19) These chemotypes include benzimidazoles, indoles, thiazoles, and quinazolines. Furthermore, the compounds in this library are druglike as defined by satisfying Lipinksi’s Rule of 5, (20) where Log P is < 5, molecular weight is < 500, the number of hydrogen bond donors is <5, and the number of hydrogen bond acceptors is <10 (Figure 2A). Historically, druglikeness has been defined by satisfying the Rule of 5; however, an increasing number of approved orally bioavailable drugs fall outside of these guidelines, and parameters such as molecular weight are increasing. (21,22) On average, the library also contains similar physicochemical properties to known drugs in DrugBank (Figure 2A). (19) Thus, this library can be used to identify druglike small molecules that bind to RNA structures.

Figure 2.

Small molecules that inhibit r(CCUG)^exp–MBNL1 complex formation in vitro. (A) Characteristics of an RNA-focused small molecule library compared to characteristics of known drugs in DrugBank. (B) IC₅₀ values of hit compounds from the RNA-focused library screen for disrupting the r(CCUG)₁₂–MBNL1 complex (n = 2). Molecules in red have IC₅₀s < 50 μM. (C) Chemical structures of compounds with IC₅₀s < 50 μM. (D) Rescue of the IR exon 11 splicing defect by 3, 4, 5, and 7 in DM2 patient-derived fibroblasts (n = 2). Error bars represent standard deviation (SD) for all panels.

To identify compounds that bind r(CCUG)^exp, a previously reported time-resolved fluorescence resonance energy transfer (TR-FRET) assay was used that measures disruption of a preformed r(CCUG)₁₂–MBNL1 complex by small molecules or other modalities. (23,24) The 3271 member library was screened at 200 μM, and 44 compounds disrupted complex formation by >60%, which corresponds to three standard deviations from the mean (or 3σ), affording a hit rate of 1.34% (Figure S1 and Table S1). Of the 44 hits, 38 contained a similar substituted N2-phenylquinazoline-2,4-diamine core, while the other hits contained benzimidazoles and indazoles (Figure S2). Importantly, these 44 compounds are druglike as they have similar physicochemical properties to the rest of the library and to known drugs in DrugBank (Figure 2A). On average, the 44 hit compounds have a molecular weight of 327 ± 35 g/mol, a c Log P of 3.2 ± 0.9, TPSA of 76 ± 15 Å, and 2.5 ± 0.9 hydrogen bond donors. The in vitro potencies (IC₅₀s) of the 44 compounds were then measured in the TR-FRET assay, affording seven compounds (1–7) with IC₅₀s < 50 μM (highlighted in red in Figures 2B,C and Table S1). Interestingly, all seven of these compounds contained the substituted N2-phenylquinazoline-2,4-diamine core.

Analysis of Hit Compounds

The binding of 1–7 to an RNA containing a single 2 × 2 CU/UC was further studied by NMR spectroscopy. Compounds 1, 2, and 6 showed significant aggregation in NMR experiments and were excluded from further analysis. In contrast, waterLOGSY spectra showed that 3, 4, 5, and 7 bound to the RNA, based on positive phase signals in the presence of RNA (Figures S3–S6). To confirm these interactions, one-dimensional (1D) NMR spectra of 3, 4, 5, and 7 bound to the RNA containing one 2×2 CU/UC internal loop were analyzed. Aromatic protons of the unbound RNA were assigned via a two-dimensional (2D) nuclear over-Hauser effect spectroscopy (NOESY) spectrum (Figure S7). Significant shifts in resonances corresponding to H6 and H8 protons of the RNA were observed in the presence of these four compounds, indicating binding to the RNA (Figure S8).

After confirming binding to r(CCUG) repeats by NMR spectroscopy, the compounds were evaluated for their ability to improve the deregulation of the IR exon 11 alternative splicing in DM2 patient-derived fibroblasts. All four compounds were well tolerated in the patient-derived fibroblasts, as no significant toxicity was observed up to a 20 μM dose (Figure S9). As previously mentioned, IR exon 11 is frequently excluded in DM2 patient-derived fibroblasts as compared to wild-type cells. Interestingly, the most potent compound, 7, rescued splicing by ∼50% at 5 μM (Figure 2D).

Synthesis and In Vitro Analysis of Derivatives of 7

To improve the potency of 7, a library of derivatives that replace the functional groups on the N2-phenylquinazoline-2,4-diamine core was synthesized and then evaluated for disrupting the r(CCUG)₁₂–MBNL1 complex in vitro. As an overview of medicinal chemistry effort, analogues were synthesized to explore the quinazoline core (54–61), the aniline moiety (45–53), and combinations thereof (62–64) (Figures 3A and S10). Synthetically, compounds were prepared starting from 2,4-dichloroquinazoline or 2,4-dichloropyrimidine analogue, followed by the addition of 3-aminopropan-1-ol in a 4-position selective manner. The aniline derivatives at the N2 position were then installed in a microwave-assisted reaction (Scheme 1, Supporting Information). Of the nine compounds in which N2–2,3-dihydrobenzo[b][1,4]dioxine was replaced with various aniline analogues, three compounds containing 2-methylbenzooxazole (49), 2-methylbenzothiazole (50), and 1-methyl-1H-indazole (51) substituents had IC₅₀s that were ∼2–8 times lower than 7 (IC₅₀ = 48.2 ± 0.07 μM) (Figures 3A, S11, and Table S1). Eight derivatives (54–61) with various substitution patterns of quinazolines and fused pyrimidines were then evaluated, and one compound containing 6-methylquinazoline (58) was identified with an IC₅₀ of 12.7 ± 0.2 μM, about 4 times more potent than 7 (Figures 3A, S11, and Table S1).

Figure 3.

Lead optimization of 7 and activity of analogues in vitro and in DM2 patient-derived fibroblasts. (A) Synthetic optimization scheme of compound 7. (B) Structures of derivatives of 7 where “R” indicates the position(s) that were varied. IC₅₀ values for disrupting the r(CCUG)₁₂–MBNL1 complex for each derivative (n = 3). (C) Structures of compounds with IC₅₀s < 5 μM. (D) Rescue of IR exon 11 mis-splicing by nontoxic compounds 63, 79, and 80 in DM2 patient-derived fibroblasts (n = 3). ** P < 0.01, *** P < 0.001, as determined by a one-way analysis of variance (ANOVA) relative to untreated (“0”). (E) Binding affinity curve of 63 and r(CCUG)₁₂ (K_d = 192 ± 20 nM). (F) Binding affinity curve of 63 and a base pair control RNA (K_d > 20 μM). Error bars represent standard deviation (SD) for all panels.

To combine features of the derivates with improved IC₅₀s, three additional compounds were synthesized that contained 6-methylquinazoline and 2-methylbenzooxazole (62), 2-methylbenzothiazole (63), and 1-methyl-1H-indazole (64) (Figures 3A, S11, and Table S1). Compounds 62 and 63 had slightly lower IC₅₀s than corresponding analogues without a methyl substituent at the 6 position (49 and 50, respectively), and 63 was the most potent compound synthesized thus far with an IC₅₀ of 2.2 ± 0.5 μM (Figures 3A, S11, and Table S1).

To explore further SAR around the benzothiazole moiety of 63, 18 analogues with extensions built onto benzothiazole were synthesized (Schemes 2 and 3 and Figures 3A and S10). Six of these analogues were synthesized via Suzuki coupling using a bromobenzothiazole intermediate of 63 (Intermediate D, Scheme 2). Notably, an aromatic ring was added to derivatives 65–70 to potentially add π-stacking interactions with r(CCUG)^exp’s internal loops and thereby enhance binding interactions. The other 12 analogues were synthesized via thermal amine replacement toward the corresponding bromobenzothiazole derivative of 63 (Intermediate D, Scheme 3). This series of compounds was designed to add functional groups that could potentially interact with the RNA such as those with a positive charge (71, 74, 79, and 82), additional hydrogen bond donors and acceptors (71, 72, 74–82), or those with aromatic rings to form π–π interactions (75, 76, 77, and 81). Within these analogues, seven compounds (71, 72, 73, 74, 78, 79, and 82; Figure 3C) had IC₅₀s < 5 μM and were chosen for further investigation, along with 63 (Figure 3B). Interestingly, four compounds (71–74) have IC₅₀s < 500 nM, which represents a 100-fold improvement in in vitro potency compared to the starting compound, 7.

Evaluation of the Cellular Activity of Compounds

The eight compounds with in vitro IC₅₀s < 5 μM were studied in DM2 patient-derived fibroblasts, both for their effects on cell viability and for rescue of IR exon 11 splicing. For the latter, we compared the percentage of exon 11 excluded upon compound treatment to untreated DM2 patient-derived fibroblasts and untreated WT fibroblasts, as measured by RT-PCR analysis. The percent rescue of a compound that restores the splicing pattern to that observed in WT cells is set to 100. Compounds 64, 65, 71, 72, 73, 74, and 82 displayed significant toxicity at 5 μM (Figure S11) and were not further evaluated. Of compounds with no toxicity at 5 μM (63, 78, and 79), 63 and 78 significantly improved IR splicing at 5 μM, while 63 also rescued splicing, by ∼30%, at 1 μM (Figures 3D and S12). Compound 79 did not significantly rescue splicing at any concentration studied (Figures 3D and S12). Interestingly, this derivative (79) is structurally similar to 64; however, the addition of the di-N-methylated functional group ablated its cellular activity, which could be due to reduced cellular uptake or changes to subcellar localization. At 1 μM dose, 63 rescues IR splicing more potently than 7 (33 ± 5 vs 12 ± 5; p = 0.026), and thus 63 is the most potent compound identified in these studies for improving DM2-associated splicing defects in cells.

The affinity and specificity of 63 for r(CCUG)^exp were measured by microscale thermophoresis (MST). Compound 63 binds to r(CCUG)₁₂ with a K_d,app of 192 ± 20 nM, while no binding was observed to a base-paired RNA that does not contain internal loops (K_d > 20 μM) (Figure 3E,F).

As 63 selectively bound to r(CCUG)₁₂ over a base-paired control, its ability to improve other DM2-associated defects was assessed in DM2 patient-derived fibroblasts. Compound 63 (5 μM) reduced the number of r(CCUG)^exp–MBNL1 nuclear foci per cell from 7 ± 1 in untreated patient-derived fibroblasts to 5 ± 0.5 in treated cells, or an ∼30% reduction, as determined by RNA fluorescence in situ hybridization (FISH) and MBNL1 immunofluorescence (Figure 4A,B). This decrease in the number of nuclear foci correlates with the improvement in IR receptor splicing observed at 5 μM (Figure 3D) as MBNL1 is no longer sequestered in foci and can resume its normal function. Importantly, 63 did not affect MAP4K4 exon 22a splicing, a NOVA-dependent splicing event (Figure S13). (25)

Figure 4.

Compound 63 rescues disease-associated defects in DM2 patient-derived fibroblasts. (A) Representative RNA FISH and MBNL1 immunofluorescence images of r(CCUG)^exp–MBNL1 foci. (B) Quantification of the number of nuclear foci/cell (n = 3, 40 nuclei counted/replicate). *** P < 0.001, as determined by a Student’s t-test. (C) Analysis of CNBP intron 1 levels upon treatment with 63 via RT-qPCR (n = 3).* P < 0.05, *** P < 0.001, as determined by a one-way ANOVA. (D) Analysis of CNBP mature mRNA levels upon treatment with 63 via RT-qPCR (n = 3). Error bars represent standard deviation (SD) for all panels.

As mentioned above, r(CCUG)^exp causes the aberrant retention of the intron in which it is harbored in the mature mRNA. We have previously shown that small molecules that bind r(CCUG)^exp can rescue defects in intron retention by facilitating removal of the retained intron, which is subsequently degraded. (10) Thus, we evaluated 63’s ability to rescue r(CCUG)^exp-mediated intron retention. Indeed, 63 significantly reduced the abundance of CNBP intron 1 in DM2 patient-derived fibroblasts at doses of 5 and 1 μM (Figure 4C), as determined by RT-qPCR. Importantly, 63 did not affect the levels of CNBP mature mRNA (Figure 4D). The effects on intron 1 levels were also specific to mutant CNBP containing r(CCUG)^exp, as no effects were observed in wild-type fibroblasts (Figure S14). Furthermore, 63 did not affect IR exon 11 inclusion in wild-type fibroblasts (Figure S14). Thus, 63 specifically improved r(CCUG)^exp-mediated defects that are dysregulated in DM2 including nuclear foci, IR splicing, and intron retention.

Implications for Targeted Degradation of r(CCUG)^exp

Importantly, all of the bioactive compounds identified herein possess druglike properties. The optimized compound, 63, has a low molecular weight (379.48 g/mol) and has a similar number of hydrogen bond donors (n = 3) to known drugs in DrugBank (n = 5). Furthermore, 63 possesses a favorable c Log P (3.75) and TPSA (83 Å) and satisfies the Rule of 5 criteria for druglikeness. Through medicinal chemistry optimization of screening hits, we identified compounds with >10-fold improvement in in vitro activity for disruption of an r(CCUG)₁₂–MBNL1 complex. The most bioactive compound from lead optimization showed ∼5-fold improvement in its ability to improve DM2-associated splicing defects. Importantly, this lead-optimized compound (63) also rescued r(CCUG)^exp-containing intron 1 retention, resulting in degradation of the expanded repeat but not the mature transcript.

Endogenous decay of r(CCUG)^exp activated by a small molecule could occur in the nucleus or the cytoplasm. If the small molecule bound the mutant allele in the cytoplasm, any MBNL1 bound to the cytoplasmic repeat expansion would be liberated and would return to the nucleus. Since r(CCUG)^exp–MBNL1 foci are not observed in the cytoplasm in the absence of compound in our imaging studies (Figure 4A), such a mechanism is unlikely. Alternatively, the small molecule could bind the repeat expansion in the nucleus, freeing MBNL1, increasing intron 1’s accessibility to the splicing machinery, reducing nuclear foci, and rescuing splicing defects. The liberated intron would then be shuttled through endogenous degradation pathways. In such a mechanism, intron 1 levels would be reduced while levels of the mature CNBP transcript would be unchanged. Collectively, our data (Figures 3 and 4) suggest that the latter, rather than the former, is the primary mode of action for 63.

Targeted degradation is an emerging field for affecting the biological function of disease-causing RNAs. Thus far, targeted RNA degradation can be achieved through: (i) direct cleavage of RNAs through conjugation of an RNA-binding module to bleomycin or derivatives of bleomycin; (13,26) (ii) recruitment of a ribonuclease through ribonuclease targeting chimeras (RIBOTACs), comprising an RNA-binding module and a compound that recruits RNaseL; (14,27) and (iii) small molecules that degrade repeat-containing introns, likely by increasing the accessibility of the disease-causing RNA to endogenous decay mechanisms. (10,12) As the former two approaches require chimeric compounds, the molecular weights of such modalities are larger and can negatively alter physicochemical properties. Targeted degradation through endogenous RNA decay mechanisms, however, can be accomplished with monomeric small molecules. Herein, we show that it is indeed possible to degrade r(CCUG)^exp with druglike monomeric small molecules. Targeted degradation of RNA repeat expansions with monomeric compounds can have broad applications as other repeat expansions, such as r(G₄C₂)^exp that causes c9ALS/FTD or r(CUG)^exp that causes FECD, which are both found in retained introns. (4,28) Furthermore, targeted degradation of RNA through RNA quality control mechanisms may be broadly applicable with monomeric, druglike small molecules that recognize RNA structures.

Conclusions

We identified novel compounds that bind r(CCUG)^exp and improve DM2-associated defects in patient-derived cells by high-throughput screening of a druglike RNA-focused small molecule library. Lead chemical optimization of small molecule binders afforded a compound with 20-fold improvement in vitro activity and 5-fold improvement in cellular activity. The lead-optimized small molecule selectively improves various DM2-associated defects including retention of r(CCUG)^exp-containing intron 1, which is decayed in cells upon treatment with the compound. The physicochemical properties of the compounds identified herein indicate that druglike monomeric small molecules can indeed selectively bind RNA structures. The ability to selectively degrade introns containing RNA repeat expansions with druglike small molecules will have broad implications in drug discovery efforts toward targeting RNAs.

EXPERIMENTAL SECTION

General Synthetic Procedures

Reagents and solvents were purchased from commercial sources and used without further purification. Microwave (MW)-assisted reactions were performed by an Initiator + (Biotage).

Compounds were purified: (i) by Isolera One flash chromatography system (Biotage) using prepacked C18 columns (spherical 20–35 μm, Agela Technologies) or prepacked silica gel columns (spherical 20–35 μm, Agela Technologies) or (ii) by high-performance liquid chromatography (HPLC) (Waters 2489 and 1525) using a SunFire Prep C18 OBD 5 μm column (19 mm × 150 mm) with a 5 mL/min flow. HPLC purity analysis was performed using a SunFire C18 3.5 μm column (4.6 mm × 150 mm) with a linear gradient (0–100% methanol (MeOH) + 0.1% (v/v) trifluoracetic acid (TFA) and water + 0.1% (v/v) TFA) over 60 min at a flow rate of 1 mL/min. The purity of all derivatives was evaluated via analytical HPLC and was >95% in all cases.

NMR spectra were collected on a 400 UltraShield (Bruker) (400 MHz for ¹H and 100 MHz for ¹³C) or Ascend 600 (Bruker) (600 MHz for ¹H and 150 MHz for ¹³C). Chemical shifts are reported in ppm relative to tetramethylsilane (TMS) for ¹H and residual solvent for ¹³C as internal standards. Coupling constants (J values) are expressed in hertz.

High-resolution mass spectra were recorded on an Agilent 1260 Infinity LC system coupled to an Agilent 6230 TOF (HR-ESI) with a Poroshell 120 EC-C18 column (Agilent, 50 mm × 4.6 mm, 2.7 μm). Liquid chromatography–mass spectrometry (LCMS) analysis was performed by Agilent 1260 Infinity LC system coupled to an Agilent 6130 quadrupole LC/MS (ESI) with a ZORBAX SB-C18 column (Agilent, 50 mm × 2.1 mm, 1.8 μm). Compounds 1–44 were purchased from ChemBridge and used without further purification.

General Synthetic Procedure for 7, 45–64 (General Synthetic Procedure 1)

For step 1, a mixture of dichloroquinazoline (A) (0.469 mmol, 1.0 equiv) and 3-aminopropan-1-ol (1.52 mmol, 3.2 equiv) in isopropanol (IPA) (0.40 M) was heated at 85 °C for 30 min under MW irradiation. The reaction was cooled to room temperature, and product formation was confirmed by LCMS. The reaction mixture was then concentrated in vacuo, washed with H₂O (4 mL × 2), and dried to afford intermediate B. The material was used in the next reaction without further purification. Then in step 2, a mixture of B (79.5 μmol, 1.0 equiv) and a corresponding aniline (C) (159 μmol, 2.0 equiv) in ethanol (EtOH) was heated at 150 °C for 30–60 min under MW irradiation. The reaction was cooled to room temperature, and product formation was confirmed by LCMS. The reaction mixture was then concentrated in vacuo and purified by column chromatography [Agela Technologies, Silica, 20 g, 0–30% MeOH in dichloromethane (DCM)] to afford compounds 45–64.

Synthesis of 7

Following general synthetic procedure 1 using 2,4-dichloroquinazoline (93.3 mg, 0.469 mmol) in step 1 (obtained monochloro intermediate; 105.0 mg, 94%) and 2,3-dihydrobenzo[b][1,4]dioxin-6-amine (24.0 mg, 159 μmol) in step 2 (18.9 mg, 79.5 μmol of monochloro intermediate) afforded 3-((2-((2,3-dihydrobenzo[b][1,4]dioxin-6-yl)amino)quinazolin-4-yl)amino)propan-1-ol (7) (12.2 mg, 44%). ¹H NMR (400 MHz, MeOD) δ 8.08 (d, J = 8.2 Hz, 1H), 7.78 (d, J = 7.4 Hz, 1H), 7.52 (d, J = 8.2 Hz, 1H), 7.45 (d, J = 7.4 Hz, 1H), 7.11–6.86 (m, 3H), 4.28 (s, 4H), 3.76 (t, J = 7.1 Hz, 2H), 3.67 (t, J = 6.1 Hz, 2H), and 2.00–1.90 (m, 2H). ¹³C NMR (150 MHz, DMSO-d₆) δ 160.4, 154.7, 145.4, 143.5, 140.0, 134.2, 133.1, 123.9, 123.3, 121.7, 117.2, 114.4, 111.4, 110.1, 64.7, 64.4, 59.0, 38.9, and 32.1; HR-MS (ESI): calcd for C₁₉H₁₉N₄O₃^– [M – H]⁻; 351.1463; found, 351.1463.

Synthesis of 45

Following general synthetic procedure 1 using 2,4-dichloroquinazoline (93.3 mg, 0.469 mmol) in step 1 (obtained monochloro intermediate; 105.0 mg, 94%) and benzo[d][1,3]dioxol-5-amine (21.8 mg, 159 μmol) in step 2 (18.9 mg, 79.5 μmol of monochloro intermediate) afforded 3-((2-((4-methyl-3,4-dihydro-2H-benzo[b][1,4]oxazin-7-yl)amino)quinazolin-4-yl)amino)propan-1-ol (45) (16.6 mg, 62%). ¹H NMR (400 MHz, DMSO-d₆) δ 10.4 (br s, 1H), 9.89 (br s, 1H), 8.48–8.37 (m, 1H), 7.83–7.73 (m, 1H), 7.55–7.47 (m, 1H), 7.45–7.29 (m, 2H), 7.05–6.97 (m, 1H), 6.97–6.91 (m, 1H), 6.06 (s, 2H), 4.75–4.57 (m, 1H), 3.65–3.59 (m, 2H), 3.53–3.47 (m, 2H), and 1.87–1.78 (m, 2H); HR-MS (ESI): calcd for C₁₈H₁₇N₄O₃^– [M – H]⁻; 337.1306; found, 337.1321.

Synthesis of 46

Following general synthetic procedure 1 using 2,4-dichloroquinazoline (93.3 mg, 0.469 mmol) in step 1 (obtained monochloro intermediate; 105.0 mg, 94%) and 2,2-difluorobenzo[d][1,3]dioxol-5-amine (27.5 mg, 159 μmol) in step 2 (18.9 mg, 79.5 μmol of monochloro intermediate) afforded 3-((2-((2,2-difluorobenzo[d][1,3]dioxol-5-yl)amino)quinazolin-4-yl)amino)propan-1-ol (46) (12.4 mg, 42%). ¹H NMR (400 MHz, DMSO-d₆) δ 10.5 (br s, 1H), 9.77 (br s, 1H), 8.40–8.33 (m, 1H), 7.91–7.75 (m, 2H), 7.60–7.56 (m, 1H), 7.51–7.41 (m, 2H), 7.37–7.31 (m, 1H), 4.79–4.43 (m, 1H), 3.69–3.58 (m, 2H), 3.57–3.46 (m, 2H), and 1.87–1.78 (m, 2H); HR-MS (ESI): calcd for C₁₈H₁₅F₂N₄O₃^– [M – H]⁻; 373.1118; found, 373.1119.

Synthesis of 47

Following general synthetic procedure 1 using 2,4-dichloroquinazoline (93.3 mg, 0.469 mmol) in step 1 (obtained monochloro intermediate; 105.0 mg, 94%) and 5-methyl-2,3-dihydrobenzo[b][1,4]dioxin-6-amine (26.3 mg, 159 μmol) in step 2 (18.9 mg, 79.5 μmol of monochloro intermediate) afforded 3-((2-((5-methyl-2,3-dihydrobenzo[b][1,4]dioxin-6-yl)amino)quinazolin-4-yl)amino)propan-1-ol (47) (29.0 mg, 100%). ¹H NMR (400 MHz, DMSO-d₆) δ 9.86–8.69 (m, 2H), 8.24 (d, J = 7.8 Hz, 1H), 7.72–7.65 (m, 1H), 7.46 (d, J = 7.6 Hz, 1H), 7.35–7.27 (m, 1H), 6.96 (d, J = 8.6 Hz, 1H), 6.75 (d, J = 8.6 Hz, 1H), 4.58–4.54 (m, 1H), 4.33–4.28 (m, 2H), 4.27–4.22 (m, 2H), 3.59–3.49 (m, 2H), 3.49–3.43 (m, 2H), 2.06 (s, 3H), and 1.80–1.73 (m, 2H); HR-MS (ESI): calcd for C₂₀H₂₁N₄O₃^– [M – H]⁻; 365.1619; found, 365.1622.

Synthesis of 48

Following general synthetic procedure 1 using 2,4-dichloroquinazoline (93.3 mg, 0.469 mmol) in step 1 (obtained monochloro intermediate; 105.0 mg, 94%) and 4-methyl-3,4-dihydro-2H-benzo[b][1,4]oxazin-7-amine (26.1 mg, 159 μmol) in step 2 (18.9 mg, 79.5 μmol of monochloro intermediate) afforded 3-((2-((4-methyl-3,4-dihydro-2H-benzo[b][1,4]oxazin-7-yl)amino)quinazolin-4-yl)amino)propan-1-ol (48) (6.3 mg, 22%). ¹H NMR (400 MHz, MeOD) δ 7.86–7.82 (m, 1H), 7.86–7.82 (m, 1H), 7.57–7.52 (m, 1H), 7.39–7.35 (m, 1H), 7.20 (d, J = 2.4 Hz, 1H), 7.16–7.11 (m, 1H), 7.01 (dd, J = 8.6, 2.5 Hz, 1H), 6.70 (d, J = 8.7 Hz, 1H), 4.31–4.27 (m, 1H), 3.72–3.65 (m, 4H), 3.21–3.17 (m, 2H), 2.84 (s, 3H), and 1.97–1.88 (m, 2H); HR-MS (ESI): calcd for C₂₀H₂₂N₅O₂^– [M – H]⁻; 364.1779; found, 364.1786.

Synthesis of 49

Following general synthetic procedure 1 using 2,4-dichloroquinazoline (93.3 mg, 0.469 mmol) in step 1 (obtained monochloro intermediate; 105.0 mg, 94%) and 2,3-dihydrobenzo[b][1,4]dioxin-6-amine (23.5 mg, 159 μmol) in step 2 (18.9 mg, 79.5 μmol of monochloro intermediate) afforded 3-((2-((2-methylbenzo[d]oxazol-6-yl)amino)quinazolin-4-yl)amino)propan-1-ol (49) (8.2 mg, 30%). ¹H NMR (400 MHz, MeOD) δ 8.29 (d, J = 1.8 Hz, 1H), 7.94 (dd, J = 8.2, 1.0 Hz, 1H), 7.68–7.62 (m, 1H), 7.54–7.45 (m, 2H), 7.43 (dd, J = 8.6, 2.0 Hz, 1H), 7.29–7.23 (m, 1H), 3.75 (t, J = 7.0 Hz, 2H), 3.69 (t, J = 6.2 Hz, 2H), 2.63 (s, 3H), and 2.00–1.92 (m, 2H). ¹³C NMR (150 MHz, DMSO-d₆) δ 163.1, 160.6, 151.2, 138.6, 135.8, 133.6, 128.5, 126.0, 124.3, 123.5, 122.7, 118.8, 117.0, 112.0, 101.6, 59.1, 38.7, 32.3, and 14.6; HR-MS (ESI): calcd for C₁₉H₁₈N₅O₂^– [M – H]⁻; 348.1466; found, 348.1470.

Synthesis of 50

Following general synthetic procedure 1 using 2,4-dichloroquinazoline (93.3 mg, 0.469 mmol) in step 1 (obtained monochloro intermediate; 105.0 mg, 94%) and 2-methylbenzo[d]thiazol-6-amine (26.1 mg, 159 μmol) in step 2 (18.9 mg, 79.5 μmol of monochloro intermediate) afforded 3-((2-((2-methylbenzo[d]thiazol-6-yl)amino)quinazolin-4-yl)amino)propan-1-ol (50) (7.7 mg, 27%). ¹H NMR (400 MHz, MeOD) δ 8.56 (d, J = 2.0 Hz, 1H), 7.93–7.88 (m, 1H), 7.70 (d, J = 8.8 Hz, 1H), 7.66–7.63 (m, 1H), 7.63–7.57 (m, 1H), 7.49–7.45 (m, 1H), 7.24–7.17 (m, 1H), 3.74 (t, J = 7.0 Hz, 2H), 3.70 (t, J = 6.2 Hz, 2H), 2.79 (s, 3H), and 2.00–1.92 (m, 2H). ¹³C NMR (150 MHz, DMSO-d₆) δ 163.8, 160.6, 157.4, 151.4, 147.6, 139.5, 136.3, 133.0, 125.8, 123.2, 122.0, 121.9, 118.7, 112.2, 110.1, 59.2, 38.5, 32.4, and 20.1; HR-MS (ESI): calcd for C₁₉H₁₈N₅OS^– [M – H]⁻; 364.1238; found, 364.1245.

Synthesis of 51

Following general synthetic procedure 1 using 2,4-dichloroquinazoline (93.3 mg, 0.469 mmol) in step 1 (obtained monochloro intermediate; 105.0 mg, 94%) and 1-methyl-1H-indazol-6-amine (23.4 mg, 159 μmol) in step 2 (18.9 mg, 79.5 μmol of monochloro intermediate) afforded 3-((2-((1-methyl-1H-indazol-6-yl)amino)quinazolin-4-yl)amino)propan-1-ol (51) (25.4 mg, 92%). ¹H NMR (400 MHz, MeOD) δ 8.31 (br s, 1H), 7.97–7.93 (m, 1H), 7.89 (d, J = 0.9 Hz, 1H), 7.67–7.61 (m, 2H), 7.52–7.48 (m, 1H), 7.28–7.21 (m, 2H), 3.10 (s, 3H), 3.80 (t, J = 6.9 Hz, 2H), 3.69 (t, J = 6.1 Hz, 2H), and 2.02–1.94 (m, 2H); HR-MS (ESI): calcd for C₁₉H₁₉N₆O^– [M – H]⁻; 347.1626; found, 347.1630.

Synthesis of 52

Following general synthetic procedure 1 using 2,4-dichloroquinazoline (93.3 mg, 0.469 mmol) in step 1 (obtained monochloro intermediate; 105.0 mg, 94%) and 1-methyl-1H-indazol-3-amine (23.4 mg, 159 μmol) in step 2 (18.9 mg, 79.5 μmol of monochloro intermediate) afforded 3-((2-((1-methyl-1H-indazol-3-yl)amino)quinazolin-4-yl)amino)propan-1-ol (52) (5.2 mg, 19%). ¹H NMR (400 MHz, DMSO-d₆) δ 12.8 (br s, 0.5H), 11.5 (br s, 0.5H), 9.87 (br s, 1H), 8.51 (d, J = 8.1 Hz, 1H), 8.02 (d, J = 8.1 Hz, 1H), 7.90–7.82 (m, 1H), 7.79–7.72 (m, 1H), 7.68 (d, J = 8.6 Hz, 1H), 7.55–7.45 (m, 2H), 7.23–7.15 (m, 1H), 4.72–4.56 (m, 1H), 4.10 (3H, s), 3.75–3.56 (m, 2H), 3.52–3.39 (m, 2H), and 1.88–1.70 (m, 2H); HR-MS (ESI): calcd for C₁₉H₁₉N₆O^– [M – H]⁻; 347.1626; found, 347.1635.

Synthesis of 53

Following general synthetic procedure 1 using 2,4-dichloroquinazoline (93.3 mg, 0.469 mmol) in step 1 (obtained monochloro intermediate; 105.0 mg, 94%) and N-methyl-2,3-dihydrobenzo[b][1,4]dioxin-6-amine (26.3 mg, 159 μmol) in step 2 (18.9 mg, 79.5 μmol of monochloro intermediate) afforded 3-((2-((2,3-dihydrobenzo[b][1,4]dioxin-6-yl)(methyl)amino)quinazolin-4-yl)amino)propan-1-ol (53) (7.5 mg, 26%). ¹H NMR (400 MHz, DMSO-d₆) δ 9.63 (br s, 1H), 8.36 (d, J = 8.2 Hz, 1H), 7.75–7.69 (m, 2H), 7.40–7.34 (m, 1H), 7.04 (d, J = 2.2 Hz, 1H), 7.00–6.91 (m, 2H), 4.65–4.58 (m, 1H), 4.32–4.28 (m, 4H), 3.58–3.49 (m, 2H), 3.52 (s, 3H), 3.48–3.41 (m, 2H), and 1.81–1.70 (m, 2H); HR-MS (ESI): calcd for C₂₀H₂₁N₄O₃^– [M – H]⁻; 365.1619; found, 365.1636.

Synthesis of 54

Following general synthetic procedure 1 using 2,4-dichloro-5,6,7,8-tetrahydroquinazoline (95.2 mg, 0.469 mmol) in step 1 (obtained monochloro intermediate; 86.3 mg, 76%) and 2,3-dihydrobenzo[b][1,4]dioxin-6-amine (24.0 mg, 159 μmol) in step 2 (19.2 mg, 79.5 μmol of monochloro intermediate) afforded 3-((2-((2,3-dihydrobenzo[b][1,4]dioxin-6-yl)amino)-5,6,7,8-tetrahydroquinazolin-4-yl)amino)propan-1-ol (54) (15.4 mg, 54%). ¹H NMR (400 MHz, DMSO-d₆) δ 9.40 (br s, 1H), 7.60 (br s, 1H), 7.30 (d, J = 2.2 Hz, 1H), 7.01 (dd, J = 8.8, 2.2 Hz, 1H), 6.77 (d, J = 8.8 Hz, 1H), 4.54 (t, J = 4.7 Hz, 1H), 4.26–4.17 (m, 4H), 3.51–3.42 (m, 4H), 2.54–2.46 (m, 2H), 2.28–2.21 (m, 2H), and 1.79–1.66 (m, 6H); HR-MS (ESI): calcd for C₁₉H₂₃N₄O₃^– [M – H]⁻; 355.1778; found, 355.1773.

Synthesis of 55

Following general synthetic procedure 1 using 2,4-dichloro-5,7-dihydrofuro[3,4-d]pyrimidine (89.6 mg, 0.469 mmol) in step 1 (obtained monochloro intermediate; 88.8 mg, 82%) and 2,3-dihydrobenzo[b][1,4]dioxin-6-amine (24.0 mg, 159 μmol) in step 2 (18.2 mg, 79.5 μmol of monochloro intermediate) afforded 3-((2-((2,3-dihydrobenzo[b][1,4]dioxin-6-yl)amino)-5,7-dihydrofuro[3,4-d]pyrimidin-4-yl)amino)propan-1-ol (55) (7.8 mg, 29%). ¹H NMR (400 MHz, DMSO-d₆) δ 8.86 (br s, 1H), 7.45 (d, J = 2.5 Hz, 1H), 7.12 (dd, J = 8.8, 2.5 Hz, 1H), 7.02 (t, J = 5.2 Hz, 1H), 6.69 (d, J = 8.8 Hz, 1H), 4.80 (br s, 2H), 4.68–4.63 (m, 2H), 4.45 (t, J = 4.6 Hz, 1H), 4.23–4.13 (m, 4H), 3.53–3.38 (m, 4H), and 1.77–1.68 (m, 2H); HR-MS (ESI): calcd for C₁₇H₁₉N₄O₄^– [M – H]⁻; 343.1412; found, 343.1418.

Synthesis of 56

Following general synthetic procedure 1 using 4,6-dichloro-3-methyl-1H-pyrazolo[3,4-d]pyrimidine (95.2 mg, 0.469 mmol) in step 1 (obtained monochloro intermediate; 44.0 mg, 39%) and 2,3-dihydrobenzo[b][1,4]dioxin-6-amine (24.0 mg, 159 μmol) in step 2 (19.2 mg, 79.5 μmol of monochloro intermediate) afforded 3-((6-((2,3-dihydrobenzo[b][1,4]dioxin-6-yl)amino)-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-4-yl)amino)propan-1-ol (56) (12.5 mg, 44%). ¹H NMR (400 MHz, DMSO-d₆) δ 12.4 (br s, 1H), 8.79 (br s, 1H), 7.62–7.41 (m, 1H), 7.20–7.05 (m, 1H), 6.91 (br s, 1H), 6.79–6.65 (m, 1H), 4.84–4.49 (m, 1H), 4.28–4.13 (m, 4H), 3.64–3.50 (m, 4H), 2.45 (br s, 3H), and 1.85–1.74 (m, 2H); HR-MS (ESI): calcd for C₁₇H₁₉N₆O₃^– [M – H]⁻; 355.1524; found, 355.1537.

Synthesis of 57

Following general synthetic procedure 1 using 2,4-dichloro-8-methylquinazoline (100.0 mg, 0.469 mmol) in step 1 (obtained monochloro intermediate; 104.6 mg, 89%) and 2,3-dihydrobenzo[b][1,4]dioxin-6-amine (24.0 mg, 159 μmol) in step 2 (20.0 mg, 79.5 μmol of monochloro intermediate) afforded 3-((2-((2,3-dihydrobenzo[b][1,4]dioxin-6-yl)amino)-8-methylquinazolin-4-yl)amino)propan-1-ol (57) (4.2 mg, 14%). ¹H NMR (400 MHz, MeOD) δ 7.95 (d, J = 7.9 Hz, 1H), 7.34 (d, J = 7.3 Hz, 1H), 7.39–7.35 (m, 1H), 7.29–7.24 (m, 1H), 6.99 (d, J = 8.7, 2.6 Hz, 1H), 6.89 (d, J = 8.7 Hz, 1H), 4.31–4.25 (m, 4H), 3.78–3.74 (m, 2H), 3.67 (t, J = 6.2 Hz, 2H), 2.51 (s, 3H), and 1.99–1.91 (m, 2H); HR-MS (ESI): calcd for C₂₀H₂₁N₄O₃^– [M – H]⁻; 365.1619; found, 365.1628.

Synthesis of 58

Following general synthetic procedure 1 using 2,4-dichloro-6-methylquinazoline (100.0 mg, 0.469 mmol) in step 1 (obtained monochloro intermediate; 103.8 mg, 88%) and 2,3-dihydrobenzo[b][1,4]dioxin-6-amine (24.0 mg, 159 μmol) in step 2 (20.0 mg, 79.5 μmol of monochloro intermediate) afforded 3-((2-((2,3-dihydrobenzo[b][1,4]dioxin-6-yl)amino)-6-methylquinazolin-4-yl)amino)propan-1-ol (58) (9.0 mg, 31%). ¹H NMR (400 MHz, MeOD) δ 7.90 (br s, 1H), 7.65–7.60 (m, 1H), 7.41 (d, J = 8.5 Hz, 1H), 7.06 (br s, 1H), 6.95–6.88 (m, 2H), 4.27 (s, 4H), 3.75 (t, J = 7.1 Hz, 2H), 3.67 (t, J = 6.2 Hz, 2H), 2.47 (s, 3H), 1.99–1.89 (m, 2H). ¹³C NMR (150 MHz, DMSO-d₆) δ 160.2, 152.4, 143.7, 140.9, 138.7, 136.4, 134.1, 131.3, 123.8, 118.6, 117.5, 115.6, 111.3, 110.7, 64.7, 64.5, 58.8, 39.3, 31.9, and 21.2; HR-MS (ESI): calcd for C₂₀H₂₁N₄O₃^– [M – H]⁻; 365.1619; found, 365.1622.

Synthesis of 59

Following general synthetic procedure 1 using 2,4-dichloro-6-fluoroquinazoline (101.8 mg, 0.469 mmol) in step 1 (obtained monochloro intermediate; 93.8 mg, 78%) and 2,3-dihydrobenzo[b][1,4]dioxin-6-amine (24.0 mg, 159 μmol) in step 2 (20.3 mg, 79.5 μmol of monochloro intermediate) afforded 3-((2-((2,3-dihydrobenzo[b][1,4]dioxin-6-yl)amino)-6-fluoroquinazolin-4-yl)amino)propan-1-ol (59) (8.0 mg, 27%). ¹H NMR (400 MHz, DMSO-d₆) δ 9.59 (br s, 1H), 8.92 (br s, 1H), 8.19–8.08 (m, 1H), 7.65–7.56 (m, 1H), 7.56–7.48 (m, 1H), 7.42–7.28 (m, 1H), 7.16–7.05 (m, 1H), 6.82 (d, J = 8.7 Hz, 1H), 4.64–4.50 (m, 1H), 4.29–4.18 (m, 4H), 3.65–3.56 (m, 2H), 3.56–3.49 (m, 2H), and 1.87–1.78 (m, 2H); HR-MS (ESI): calcd for C₁₉H₁₈FN₄O₃^– [M – H]⁻; 369.1368; found, 369.1375.

Synthesis of 60

Following general synthetic procedure 1 using 2,4-dichloro-7-fluoroquinazoline (101.8 mg, 0.469 mmol) in step 1 (obtained monochloro intermediate; 46.1 mg, 38%) and 2,3-dihydrobenzo[b][1,4]dioxin-6-amine (24.0 mg, 159 μmol) in step 2 (20.3 mg, 79.5 μmol of monochloro intermediate) afforded 3-((2-((2,3-dihydrobenzo[b][1,4]dioxin-6-yl)amino)-7-fluoroquinazolin-4-yl)amino)propan-1-ol (60) (3.0 mg, 10%). ¹H NMR (400 MHz, MeOD) δ 8.21–8.12 (m, 1H), 7.31–7.19 (m, 2H), 7.11–7.00 (m, 1H), 6.98–6.87 (m, 2H), 4.28 (s, 4H), 3.75 (t, J = 7.1 Hz, 2H), 3.67 (t, J = 6.1 Hz, 2H), and 1.99–1.90 (m, 2H); HR-MS (ESI): calcd for C₁₉H₁₈FN₄O₃^– [M – H]⁻; 369.1368; found, 369.1383.

Synthesis of 61

Following general synthetic procedure 1 using 2,4-dichloro-8-fluoroquinazoline (101.8 mg, 0.469 mmol) in step 1 (obtained monochloro intermediate; 76.5 mg, 64%) and 2,3-dihydrobenzo[b][1,4]dioxin-6-amine (24.0 mg, 159 μmol) in step 2 (20.3 mg, 79.5 μmol of monochloro intermediate was used) afforded 3-((2-((2,3-dihydrobenzo[b][1,4]dioxin-6-yl)amino)-8-fluoroquinazolin-4-yl)amino)propan-1-ol (61) (11.5 mg, 39%). ¹H NMR (400 MHz, DMSO-d₆) δ 9.21 (br s, 1H), 8.38 (br s, 1H), 7.91 (d, J = 8.2 Hz, 1H), 7.73–7.56 (m, 1H), 7.51–7.40 (m, 1H), 7.30–7.21 (m, 1H), 7.17–7.07 (m, 1H), 6.75 (d, J = 8.7 Hz, 1H), 4.64–4.47 (m, 1H), 4.30–4.15 (m, 4H), 3.66–3.48 (m, 4H), and 1.90–1.78 (m, 2H); HR-MS (ESI): calcd for C₁₉H₁₈FN₄O₃^– [M – H]⁻; 369.1368; found, 369.1383.

Synthesis of 62

Following general synthetic procedure 1 using 2,4-dichloro-6-methylquinazoline (100.0 mg, 0.469 mmol) in step 1 (obtained monochloro intermediate; 103.8 mg, 88%) and 2-methylbenzo[d]oxazol-6-amine (23.5 mg, 159 μmol) in step 2 (20.0 mg, 79.5 μmol of monochloro intermediate) afforded 3-((6-methyl-2-((2-methylbenzo[d]oxazol-6-yl)amino)quinazolin-4-yl)amino)propan-1-ol (62) (6.2 mg, 21%). ¹H NMR (400 MHz, MeOD) δ 8.11 (d, J = 1.8 Hz, 1H), 7.84 (br s, 1H), 7.60–7.55 (m, 2H), 7.44–7.38 (m, 2H), 3.75 (t, J = 7.0 Hz, 2H), 3.67 (t, J = 6.1 Hz, 2H), 2.64 (s, 3H), 2.46 (s, 3H), and 2.00–1.92 (m, 2H). ¹³C NMR (150 MHz, DMSO-d₆) δ 163.8, 160.3, 154.1, 151.1, 136.9, 135.8, 133.2, 128.6, 126.0, 123.3, 121.5, 119.1, 117.9, 111.3, 102.9, 58.9, 39.0, 32.0, 21.3, and 14.6; HR-MS (ESI): calcd for C₂₀H₂₀N₅O₂^– [M – H]⁻; 362.1623; found, 362.1624.

Synthesis of 63

Following general synthetic procedure 1 using 2,4-dichloro-6-methylquinazoline (100.0 mg, 0.469 mmol) in step 1 (obtained monochloro intermediate; 103.8 mg 88%) and 2-methylbenzo[d]thiazol-6-amine (26.1 mg, 159 μmol) in step 2 (20.0 mg, 79.5 μmol of monochloro intermediate) afforded 3-((6-methyl-2-((2-methylbenzo[d]thiazol-6-yl)amino)quinazolin-4-yl)amino)propan-1-ol (63) (15.4 mg, 56%). ¹H NMR (400 MHz, MeOD) δ 8.31 (d, J = 2.0 Hz, 1H), 7.91–7.85 (m, 2H), 7.62–7.58 (m, 2H), 7.41 (d, J = 8.5 Hz, 1H), 3.76 (t, J = 7.1 Hz, 2H), 3.66 (t, J = 6.1 Hz, 2H), 2.83 (s, 3H), 2.47 (s, 3H), and 1.98–1.90 (m, 2H). ¹³C NMR (150 MHz, DMSO-d₆) δ 164.7, 160.3, 155.4, 148.5, 145.9, 138.0, 136.2, 135.2, 132.2, 123.5, 122.8, 122.0, 119.6, 111.6, 59.1, 38.8, 32.3, 21.3, and 20.1; HR-MS (ESI): calcd for C₂₀H₂₂N₅OS^– [M – H]⁻; 378.1394; found, 378.1403.

Synthesis of 64

Following general synthetic procedure 1 using 2,4-dichloro-6-methylquinazoline (100.0 mg, 0.469 mmol) in step 1 (obtained monochloro intermediate; 103.8 mg, 88%) and 1-methyl-1H-indazol-6-amine (23.4 mg, 159 μmol) in step 2 (20.0 mg, 79.5 μmol of monochloro intermediate) afforded 3-((6-methyl-2-((1-methyl-1H-indazol-6-yl)amino)quinazolin-4-yl)amino)propan-1-ol (64) (7.9 mg, 27%). ¹H NMR (400 MHz, MeOD) δ 8.27 (br s, 1H), 7.89 (d, J = 0.9 Hz, 1H), 7.78 (br s, 1H), 7.67–7.63 (m, 1H), 7.53–7.48 (m, 1H), 7.41 (d, J = 8.5 Hz, 1H), 7.24–7.20 (m, 1H), 4.04 (s, 3H), 3.79 (t, J = 6.9 Hz, 2H), 3.69 (t, J = 6.1 Hz, 2H), 2.45 (s, 3H), and 2.02–1.93 (m, 2H); HR-MS (ESI): calcd for C₂₀H₂₁N₆O^– [M – H]⁻; 361.1782; found, 361.1778.

Synthesis of intermediate D

Following general synthetic procedure 1 using 2,4-dichloro-6-methylquinazoline (1.50 g, 7.04 mmol) in step 1 (obtained monochloro intermediate; 1.56 g, 88%) and 2-bromobenzo[d]thiazol-6-amine (500 mg, 2.18 mmol) in step 2 (1.10 g, 4.37 mmol of monochloro intermediate) afforded 3-((2-((2-bromobenzo[d]thiazol-6-yl)amino)-6-methylquinazolin-4-yl)amino)propan-1-ol (D) (1.00 g, quant). ¹H NMR (400 MHz, MeOD) δ 8.32–8.25 (m, 1H), 8.02–7.94 (m, 2H), 7.69 (dd, J = 8.5, 1.5 Hz, 1H), 7.64 (dd, J = 8.8, 2.2 Hz, 1H), 7.44 (d, J = 8.5 Hz, 1H), 3.80–3.74 (m, 2H), 3.65 (t, J = 6.1 Hz, 2H), 2.49 (s, 3H), and 1.99–1.89 (m, 2H); HR-MS (ESI): calcd for C₁₉H₁₈BrN₅OS⁺ [M + H]⁺; 444.0488; found, 444.04777.

General Synthetic Procedure for 65–70 (General Synthetic Procedure 2)

A mixture of 3-((2-((2-bromobenzo[d]thiazol-6-yl)amino)-6-methylquinazolin-4-yl)amino)propan-1-ol (D) (20.0 mg, 45.0 μmol, 1.0 equiv), Ar-boronic acid (67.5 μmol, 1.5 equiv), Pd(PPh₃)₄ (5.2 mg, 4.5 μmol, 0.1 equiv), and K₃PO₄ (19.1 mg, 90.0 μmol, 2.0 equiv) in dioxane/H₂O (2/1, 0.50 mL) was heated at 120 °C overnight. The reaction was cooled to room temperature, and product formation was confirmed by LCMS. The reaction mixture was then concentrated in vacuo and purified by column chromatography as described above (Agela Technologies, Silica, 20 g, 0–20% MeOH in DCM) and HPLC to afford compounds 65–70.

Synthesis of 65

Following general synthetic procedure 2 using D (20.0 mg, 45.0 μmol) and (4-fluorophenyl)boronic acid (9.4 mg, 67.5 μmol) afforded 3-((2-((2-(4-fluorophenyl)benzo[d]thiazol-6-yl)amino)-6-methylquinazolin-4-yl)amino)propan-1-ol (65) (5.0 mg, 24%). ¹H NMR (400 MHz, DMSO-d₆) δ 12.6 (br s, 1H), 10.6 (br s, 1H), 9.55 (br s, 1H), 8.51 (br s, 1H), 8.19–8.06 (m, 4H), 7.73–7.65 (m, 2H), 7.54–7.40 (m, 3H), 4.79–4.49 (m, 1H), 3.72–3.63 (m, 2H), 3.57–3.49 (m, 2H), 2.43 (s, 3H), and 1.90–1.80 (m, 2H); HR-MS (ESI): calcd for C₂₅H₂₃FN₅OS⁺ [M + H]⁺; 460.1602; found, 460.1613.

Synthesis of 66

Following general synthetic procedure 2 using D (20.0 mg, 45.0 μmol) and (3-fluorophenyl)boronic acid (9.4 mg, 67.5 μmol) afforded 3-((2-((2-(3-fluorophenyl)benzo[d]thiazol-6-yl)amino)-6-methylquinazolin-4-yl)amino)propan-1-ol (66) (5.0 mg, 24%). ¹H NMR (400 MHz, DMSO-d₆) δ 12.7 (br s, 1H), 10.6 (br s, 1H), 9.58 (br s, 1H), 8.57 (br s, 1H), 8.15–8.07 (m, 2H), 7.96–7.87 (m, 2H), 7.75–7.61 (m, 3H), 7.53–7.42 (m, 2H), 4.77–4.56 (m, 1H), 3.72–3.63 (m, 2H), 3.57–3.49 (m, 2H), 2.43 (s, 3H), and 1.90–1.80 (m, 2H); HR-MS (ESI): calcd for C₂₅H₂₃FN₅OS⁺ [M + H]⁺; 460.1602; found, 460.1621.

Synthesis of 67

Following general synthetic procedure 2 using D (20.0 mg, 45.0 μmol) and o-tolylboronic acid (9.2 mg, 67.5 μmol) afforded 3-((6-methyl-2-((2-(o-tolyl)benzo[d]thiazol-6-yl)amino)quinazolin-4-yl)amino)propan-1-ol (67) (9.4 mg, 46%). ¹H NMR (400 MHz, DMSO-d₆) δ 12.6 (br s, 1H), 10.6 (br s, 1H), 9.60 (br s, 1H), 8.53 (br s, 1H), 8.16–8.07 (m, 2H), 7.83 (d, J = 7.3 Hz, 1H), 7.75–7.63 (m, 2H), 7.53–7.38 (m, 4H), 4.75–4.52 (m, 1H), 3.73–3.63 (m, 2H), 3.58–3.50 (m, 2H), 2.65 (s, 3H), 2.43 (s, 3H), and 1.91–1.80 (m, 2H); HR-MS (ESI): calcd for C₂₆H₂₆N₅OS⁺ [M + H]⁺; 456.1853; found, 456.1872.

Synthesis of 68

Following general synthetic procedure 2 using D (20.0 mg, 45.0 μmol) and (1H-pyrazol-3-yl)boronic acid (7.6 mg, 67.5 μmol) afforded 3-((2-((2-(1H-pyrazol-3-yl)benzo[d]thiazol-6-yl)amino)-6-methylquinazolin-4-yl)amino)propan-1-ol (68) (18.5 mg, 95%). ¹H NMR (400 MHz, DMSO-d₆) δ 13.4 (s, 1H), 12.5 (br s, 1H), 10.6 (br s, 1H), 9.66 (br s, 1H), 8.42 (br s, 1H), 8.19–7.93 (m, 3H), 7.71–7.60 (m, 2H), 7.54–7.46 (m, 1H), 6.93 (s, 1H), 4.73–4.57 (m, 1H), 3.72–3.62 (m, 2H), 3.57–3.49 (m, 2H), 2.43 (s, 3H), and 1.89–1.82 (m, 2H); HR-MS (ESI): calcd for C₂₂H₂₂N₇OS⁺ [M + H]⁺; 432.1601; found, 432.1613.

Synthesis of 69

Following general synthetic procedure 2 using D (20.0 mg, 45.0 μmol) and (4-carbamoylphenyl)boronic acid (11.1 mg, 67.5 μmol) afforded 4-(6-((4-((3-hydroxypropyl)amino)-6-methylquinazolin-2-yl)amino)benzo[d]thiazol-2-yl)benzamide (69) (12.5 mg, 57%). ¹H NMR (400 MHz, DMSO-d₆) δ 12.9 (br s, 1H), 10.7 (br s, 1H), 9.56 (br s, 1H), 8.59 (br s, 1H), 8.22–8.02 (m, 6H), 7.76–7.64 (m, 2H), 7.57 (s, 1H), 7.49 (d, J = 8.4 Hz, 1H), 6.93 (s, 1H), 4.78–4.52 (m, 1H), 3.76–3.62 (m, 2H), 3.60–3.51 (m, 2H), 2.43 (s, 3H), and 1.91–1.81 (m, 2H); HR-MS (ESI): calcd for C₂₆H₂₅N₆O₂S⁺ [M + H]⁺; 485.1754; found, 485.1778.

Synthesis of 70

Following general synthetic procedure 2 using D (20.0 mg, 45.0 μmol) and (1H-indol-5-yl)boronic acid (10.9 mg, 67.5 μmol) afforded 3-((2-((2-(1H-indol-5-yl)benzo[d]thiazol-6-yl)amino)-6-methylquinazolin-4-yl)amino)propan-1-ol (70) (19.6 mg, 91%). ¹H NMR (400 MHz, DMSO-d₆) δ 12.6 (br s, 1H), 11.5 (s, 1H), 10.6 (br s, 1H), 9.60 (br s, 1H), 8.43 (br s, 1H), 8.32 (d, J = 1.7 Hz, 1H), 8.13 (s, 1H), 8.03 (d, J = 8.7 Hz, 1H), 7.87 (dd, J = 8.5, 1.8 Hz, 1H), 7.71–7.61 (m, 2H), 7.59–7.48 (m, 3H), 6.64–6.61 (m, 1H), 4.83–4.44 (m, 1H), 3.72–3.63 (m, 2H), 3.57–3.51 (m, 2H), 2.43 (s, 3H), and 1.90–1.81 (m, 2H); HR-MS (ESI): calcd for C₂₇H₂₈N₆OS⁺ [M + H]⁺; 481.1805; found, 481.1829.

General Synthetic Procedure for 71–82 (General Synthetic Procedure 3)

A mixture of 3-((6-bromo-2-((2-methylbenzo[d]thiazol-6-yl)amino)quinazolin-4-yl)amino)propan-1-ol (D) (15.0 mg, 33.8 μmol, 1.0 equiv), N,N-diisopropylethylamine (DIPEA; 35.4 μL, 203 μmol, 6.0 equiv) and amine (203 μmol, 6.0 equiv) in 3-pentanol (0.10 M) was heated in a microwave vial at 120 °C overnight. The reaction was cooled to room temperature, and product formation was confirmed by LCMS. The reaction mixture was then concentrated in vacuo and purified by column chromatography (Biotage SNAP cartridge, KP-NH, 11g, 2%–30% MeOH in DCM) to afford compounds 71–82.

Synthesis of 71

Following general synthetic procedure 3 using D (15.0 mg, 33.8 μmol) and 1-benzylpiperidin-4-amine (38.6 mg, 203 μmol) afforded 3-((2-((2-((1-benzylpiperidin-4-yl)amino)benzo[d]thiazol-6-yl)amino)-6-methylquinazolin-4-yl)amino)propan-1-ol (71) (13.8 mg, 74%). ¹H NMR (400 MHz, MeOD) δ 8.11 (d, J = 1.9 Hz, 1H), 7.71–7.67 (m, 1H), 7.46–7.25 (m, 9H), 3.81–3.64 (m, 5H), 3.56 (s, 2H), 2.97–2.86 (m, 2H), 2.42 (s, 3H), 2.28–2.19 (m, 2H), 2.13–2.04 (m, 2H), 1.96–1.89 (m, 2H), and 1.69–1.55 (m, 2H); HR-MS (ESI): calcd for C₃₁H₃₆N₇OS⁺ [M + H]⁺; 554.2697; found, 554.2722.

Synthesis of 72

Following general synthetic procedure 3 using D (15.0 mg, 33.8 μmol) and morpholine (17.7 mg, 203 μmol) afforded 3-((6-methyl-2-((2-morpholinobenzo[d]thiazol-6-yl)amino)quinazolin-4-yl)amino)propan-1-ol (72) (7.3 mg, 48%).¹H NMR (400 MHz, MeOD) δ 8.26 (d, J = 1.6 Hz, 1H), 7.71–7.67 (m, 1H), 7.48–7.40 (m, 3H), 7.33 (d, J = 8.5 Hz, 1H), 3.84–3.79 (m, 4H), 3.74–3.64 (m, 4H), 3.60–3.54 (m, 4H), 2.42 (s, 3H), and 2.00–1.89 (m, 2H). ¹³C NMR (150 MHz, DMSO-d₆) δ 167.3, 160.2, 157.0, 149.7, 146.5, 136.9, 134.5, 131.0, 130.7, 125.6, 122.4, 118.9, 118.3, 111.9, 110.6, 66.0 (2C), 59.2, 48.7 (2C), 38.4, 32.4, and 21.3; HR-MS (ESI): calcd for C₂₃H₂₇N₆O₂S⁺ [M + H]⁺; 451.1911; found, 451.1929.

Synthesis of 73

Following general synthetic procedure 3 using D (15.0 mg, 33.8 μmol) and 2-methylpropan-1-amine (14.8 mg, 203 μmol) afforded 3-((2-((2-(isobutylamino)benzo[d]thiazol-6-yl)amino)-6-methylquinazolin-4-yl)amino)propan-1-ol (73) (11.6 mg, 79%). ¹H NMR (400 MHz, MeOD) δ 8.11 (d, J = 2.0 Hz, 1H), 7.70–7.67 (m, 1H), 7.45–7.38 (m, 2H), 7.35–7.31 (m, 2H), 3.74–3.65 (m, 4H), 3.23 (d, J = 7.0 Hz, 2H), 2.42 (s, 3H), 2.03–1.89 (m, 3H), 1.02 (s, 3H), and 1.00 (s, 3H). ¹³C NMR (150 MHz, DMSO-d₆) δ 165.0, 160.2, 157.1, 149.8, 147.1, 136.2, 134.4, 130.7, 130.6, 125.6, 122.4, 117.9, 117.7, 111.8, 110.8, 59.3, 52.1, 38.4, 32.5, 28.2, 21.3, and 20.6 (2C); HR-MS (ESI): calcd for C₂₃H₂₇N₆OS^– [M – H]⁻; 435.1973; found, 435.1989.

Synthesis of 74

Following general synthetic procedure 3 using D (15.0 mg, 33.8 μmol) and 2-morpholinoethan-1-amine (26.4 mg, 203 μmol) afforded 3-((6-methyl-2-((2-((2-morpholinoethyl)amino)benzo[d]thiazol-6-yl)amino)quinazolin-4-yl)amino)propan-1-ol (74) (12.6 mg, 75%).¹H NMR (400 MHz, MeOD) δ 8.14–8.09 (m, 1H), 7.68 (br s, 1H), 7.45–7.29 (m, 4H), 3.77–3.63 (m, 8H), 3.57 (d, J = 6.4 Hz, 2H), 2.66 (d, J = 6.4 Hz, 2H), 2.60–2.49 (m, 4H), 2.41(s, 3H), and 1.98–1.88 (m, 2H). ¹³C NMR (150 MHz, DMSO-d₆) δ 164.8, 160.2, 157.1, 149.8, 147.0, 136.3, 134.4, 130.8, 130.6, 125.6, 122.4, 118.0, 117.7, 111.9, 110.8, 66.6(2C), 59.3, 57.6, 53.8(2C), 41.5, 38.4, 32.5, 21.3; HR-MS (ESI): calcd for C₂₅H₃₂N₇O₂S⁺ [M + H]⁺; 494.2333; found, 494.2357.

Synthesis of 75

Following general synthetic procedure 3 using D (15.0 mg, 33.8 μmol) and benzo[d][1,3]dioxol-5-ylmethanamine (29.1 mg, 203 μmol) afforded 3-((2-((2-((benzo[d][1,3]dioxol-5-ylmethyl)amino)benzo[d]thiazol-6-yl)amino)-6-methylquinazolin-4-yl)amino)propan-1-ol (75) (8.9 mg, 51%). ¹H NMR (400 MHz, MeOD) δ 7.89 (s, 1H), 7.83 (d, J = 2.0 Hz, 1H), 7.60 (dd, J = 8.5, 1.4 Hz, 1H), 7.48 (d, J = 8.5 Hz, 1H), 7.40 (d, J = 8.5 Hz, 1H), 7.37–7.32 (m, 1H), 6.91–6.86 (m, 2H), 6.79 (d, J = 11.8 Hz, 1H), 5.93 (s, 2H), 4.54 (s, 2H), 3.74 (t, J = 7.1 Hz, 2H), 3.65 (t, J = 6.1 Hz, 2H), 2.46 (s, 3H), 1.98–1.88 (m, 2H); HR-MS (ESI): calcd for C₂₇H₂₇N₆O₃S⁺ [M + H]⁺; 515.1860; found, 515.1883.

Synthesis of 76

Following general synthetic procedure 3 using D (15.0 mg, 33.8 μmol) and 2-phenoxyethan-1-amine (27.8 mg, 203 μmol) afforded 3-((6-methyl-2-((2-((2-phenoxyethyl)amino)benzo[d]thiazol-6-yl)amino)quinazolin-4-yl)amino)propan-1-ol (76) (6.3 mg, 37%).¹H NMR (400 MHz, MeOD) δ 7.95–7.89 (m, 2H), 7.68–7.63 (m, 1H), 7.56–7.51 (m, 1H), 7.48–7.41 (m, 2H), 7.31–7.24 (m, 2H), 6.99–6.90 (m, 3H), 4.25 (t, J = 5.2 Hz, 2H), 3.90 (t, J = 5.2 Hz, 2H), 3.76 (t, J = 7.1 Hz, 2H), 3.65 (t, J = 6.1 Hz, 2H), 2.47 (s, 3H), and 1.98–1.88 (m, 2H); HR-MS (ESI): calcd for C₂₇H₂₇N₆O₃S⁺ [M + H]⁺; 515.1860; found, 515.1883.

Synthesis of 77

Following general synthetic procedure 3 using D (15.0 mg, 33.8 μmol) and 2-(benzyloxy)ethan-1-amine (30.7 mg, 203 μmol) afforded 3-((2-((2-((2-(benzyloxy)ethyl)amino)benzo[d]thiazol-6-yl)amino)-6-methylquinazolin-4-yl)amino)propan-1-ol (77) (7.7 mg, 44%). ¹H NMR (400 MHz, MeOD) δ 7.98–7.91 (m, 2H), 7.68–7.63 (m, 1H), 7.52–7.46 (m, 2H), 7.44 (d, J = 8.5 Hz, 1H), 7.36–7.21 (m, 5H), 4.59 (s, 2H), 3.79–3.70 (m, 6H), 3.65 (t, J = 6.1 Hz, 2H), 2.48 (s, 3H), and 1.98–1.88 (m, 2H); HR-MS (ESI): calcd for C₂₈H₃₁N₆O₂S⁺ [M + H]⁺; 515.2224; found, 515.2246.

Synthesis of 78

Following general synthetic procedure 3 using D (15.0 mg, 33.8 μmol) and 1-amino-2-methylpropan-2-ol (18.1 mg, 203 μmol) afforded 3-((2-((2-((2-hydroxy-2-methylpropyl)amino)benzo[d]thiazol-6-yl)amino)-6-methylquinazolin-4-yl)amino)propan-1-ol (78) (14.0 mg, 91%). ¹H NMR (400 MHz, MeOD) δ 8.12 (d, J = 2.1 Hz, 1H), 7.68 (br s, 1H), 7.44–7.37 (m, 2H), 7.35–7.30 (m, 2H), 3.73–3.64 (m, 4H), 3.44 (s, 2H), 2.41 (s, 3H), 1.98–1.87 (m, 2H), and 1.27 (s, 6H); HR-MS (ESI): calcd for C₂₃H₂₉N₆O₂S⁺ [M + H]⁺; 453.2067; found, 453.2069.

Synthesis of 79

Following general synthetic procedure 3 using D (15.0 mg, 33.8 μmol) and N1,N1-dimethylethane-1,2-diamine (17.9 mg, 203 μmol) afforded 3-((2-((2-((2-(dimethylamino)ethyl)amino)benzo[d]thiazol-6-yl)amino)-6-methylquinazolin-4-yl)amino)propan-1-ol (79) (12.9 mg, 84%). ¹H NMR (400 MHz, MeOD) δ 8.14 (d, J = 2.0 Hz, 1H), 7.68 (br s, 1H), 7.44–7.38 (m, 2H), 7.38–7.30 (m, 2H), 3.73–3.65 (m, 4H), 3.56 (t, J = 6.7 Hz, 2H), 2.64 (t, J = 6.7 Hz, 2H), 2.42 (s, 3H), 2.32 (s, 6H), and 1.98–1.89 (m, 2H); HR-MS (ESI): calcd for C₂₃H₃₀N₇OS⁺ [M + H]⁺; 452.2227; found, 452.2242.

Synthesis of 80

Following general synthetic procedure 3 using D (15.0 mg, 33.8 μmol) and 2,2′-azanediylbis(ethan-1-ol) (21.3 mg, 203 μmol) afforded 2,2′-((6-((4-((3-hydroxypropyl)amino)-6-methylquinazolin-2-yl)amino)benzo[d]thiazol-2-yl)azanediyl)bis(ethan-1-ol) (80) (5.2 mg, 33%). ¹H NMR (400 MHz, MeOD) δ 8.10–8.02 (m, 1H), 7.95–7.90 (m, 1H), 7.65 (dd, J = 8.5, 1.3 Hz, 1H), 7.56–7.50 (m, 2H), 7.44 (d, J = 8.5 Hz, 1H), 3.95–3.88 (m, 4H), 3.88–3.82 (m, 4H), 3.76 (t, J = 7.2 Hz, 2H), 3.66 (t, J = 6.1 Hz, 2H), 2.47 (s, 3H), and 1.98–1.88 (m, 2H); HR-MS (ESI): calcd for C₂₃H₂₉N₆O₃S⁺ [M + H]⁺; 469.2016; found, 469.2031.

Synthesis of 81

Following general synthetic procedure 3 using D (15.0 mg, 33.8 μmol) and 4-(2-aminoethyl)benzenesulfonamide (40.7 mg, 203 μmol) afforded 4-(2-((6-((4-((3-hydroxypropyl)amino)-6-methylquinazolin-2-yl)amino)benzo[d]thiazol-2-yl)amino)ethyl)benzenesulfonamide (81) (7.5 mg, 39%). ¹H NMR (400 MHz, MeOD) δ 7.94–7.91 (m, 1H), 7.91–7.82 (m, 3H), 7.67 (dd, J = 8.5, 1.4 Hz, 1H), 7.53–7.40 (m, 5H), 3.82–3.71 (m, 4H), 3.65 (t, J = 6.1 Hz, 2H), 3.11 (t, J = 7.0 Hz, 2H), 2.48 (s, 3H), and 1.99–1.89 (m, 2H); HR-MS (ESI): calcd for C₂₇H₃₀N₇O₃S₂⁺ [M + H]⁺; 564.1846; found, 564.1856.

Synthesis of 82

Following general synthetic procedure 3 using D (15.0 mg, 33.8 μmol) and 1-methylpiperazine (20.3 mg, 203 μmol) afforded 3-((6-methyl-2-((2-(4-methylpiperazin-1-yl)benzo[d]thiazol-6-yl)amino)quinazolin-4-yl)amino)propan-1-ol (82) (2.9 mg, 18%). ¹H NMR (400 MHz, MeOD) δ 8.25 (d, J = 1.7 Hz, 1H), 7.69 (br s, 1H), 7.47–7.39 (m, 3H), 7.37–7.30 (m, 1H), 3.75–3.58 (m, 8H), 2.65 (t, J = 10.2 Hz, 4H), 2.42 (s, 3H), 2.36 (s, 3H), and 1.98–1.88 (m, 2H); HR-MS (ESI): calcd for C₂₄H₃₀N₇OS⁺ [M + H]⁺; 464.2227; found, 464.2235.

High-Throughput Screen of the RNA-Focused Small-Molecule Library

Measurements for disruption of the r(CCUG)₁₂–MBNL1 complex were completed using a previously reported TR-FRET assay with minor modifications. (23,24) Briefly, 5′-biotinylated r(CCUG)₁₂ was folded in 1 × Folding Buffer (20 mM HEPES, pH 7.5, 110 mM KCl, and 10 mM NaCl) at 60 °C for 5 min then cooled to room temperature. The buffer was adjusted to 1 × Assay Buffer (20 mM HEPES, pH 7.5, 110 mM KCl, 10 mM NaCl, 2 mM MgCl₂, 2 mM CaCl₂, 5 mM DTT, 0.1% BSA, and 0.5% Tween-20). Then, MBNL1-His₆ was added, and the samples were incubated at room temperature for 15 min and added to a white 384-well plate using an Aurora FRD-IB reagent dispenser. Compounds from the RNA-focused small molecule library were then delivered to each well using a Beckman Biomek NXP liquid handler with a pin tool, and the samples were incubated for another 15 min at room temperature. The final concentration of r(CCUG)₁₂ was 80 nM, while the final concentration of MBNL1 was 60 nM. A solution of streptavidin-XL665 and anti-His₆-Tb antibody was then added to a total volume of 10 μL using an Aurora FRD-IB reagent dispenser, with final concentrations of 40 nM and 0.44 ng/μL, respectively. The sample plate was incubated for 30 min at room temperature and TR-FRET was measured on a Molecular Devices SpectraMax M5 plate reader using an excitation wavelength of 345 and a 420 nm cutoff. The ratios of fluorescence intensity at 545 and 665 nm were calculated, and ratios in the absence of a compound and in the absence of RNA were used to calculate percent disruption.

In Vitro IC₅₀ Measurements

The IC₅₀s for disruption of r(CCUG)₁₂–MBNL1 by hit compounds from the RNA-focused small-molecule library were completed as described above except compounds were added manually to wells at varying concentrations to afford a dose response. The resulting curves were fit to eq 1 to determine IC₅₀ values

$$y=B+\frac{A-B}{1+{\left(\frac{\mathrm{I}{\mathrm{C}}_{50}}{x}\right)}^{\text{hillslope}}}$$ (1)

where y is the ratio of fluorescence intensities at 545 and 665 nm (F545/F665), x is the concentration of a compound, B is F545/F665 value at max FRET effect (solution has RNA and protein but no compound added); A is F545/F665 value at min FRET effect (solution has antibodies but no RNA, protein, or compound); and IC₅₀ is the concentration of a compound where half of the protein is displaced by a compound.

NMR Sample Preparation

A self-complementary RNA construct, r(5′-GACAGCCUGCUGUC-3′)_, was purchased from GE Healthcare Dharmacon, Inc., deprotected according to the manufacturer’s recommended protocol, and desalted with PD-10 columns (GE Healthcare, cat: 17–0851-01) also per the manufacturer’s protocol. RNA samples were dissolved in NMR Buffer [10 mM KH₂PO₄/K₂HPO₄ and 0.05 mM ethylenediamine tetraacetic acid (EDTA) (pH 6.0)] and folded by heating to 95 °C for 3 min and slowly cooling to room temperature.

NMR Spectroscopy

NMR spectra were acquired at 25 °C on Bruker Avance III 600 and 700 MHz spectrometers equipped with cryoprobes. One-dimensional NMR spectra were acquired on samples containing 100 μM of RNA alone in 100% D₂O. Compounds were each titrated into separate samples at 0.5, 1.0, 1.5, and 2.0 compound: RNA molar ratios. WaterLOGSY (water-ligand observed via gradient spectroscopy) spectra (29) were acquired on samples containing 300 μM of each compound alone or in the presence of 15 μM of RNA at a 20:1 compound: RNA ratio in H₂O, to which D₂O was added to 5% by volume. WaterLOGSY spectra were phased to give negative NOEs for nonbinders. Two-dimensional NOESY and DQF-COSY spectra were acquired on samples containing 400 μM of RNA alone in 100% D₂O. Two-dimensional NMR spectra were processed with nmrPipe (30) and assigned with SPARKY. (31)

Cell Lines and Cell Culture

Compounds were tested in two cell lines: (i) DM2 patient-derived fibroblasts (generous gift from University of Florida, Center for NeuroGenetics) and (ii) fibroblasts from a healthy donor (wild-type; WT GM07492; Coriell Institute). Cells were maintained at 37 °C with 5% CO₂. DM2 fibroblasts were cultured in Dulbecco’s modified Eagle’s medium (DMEM)/high glucose (HyClone) supplemented with 20% (v/v) fetal bovine serum (FBS) (Sigma) and 1% (v/v) antibiotic–antimycotic solution (Corning). Wild-type fibroblasts were cultured in MEM (Corning) supplemented with 10% (v/v) FBS, 1% (v/v) Glutagro (Corning), and 1% (v/v) antibiotic–antimycotic solution.

Evaluation of Cell Viability

Compound toxicity in DM2 fibroblasts was evaluated using a CellTiter-Glo Kit (Promega) as described in the manufacturer’s protocol. Briefly, after 48 h treatment in 96-well plates, 100 μL of the CellTiter-Glo reagent was added to each well. The plate was then incubated at room temperature for 10 min, and luminescence was measured using BioTek FLX-800 luminescence plate reader (n = 6 replicates; 1 independent experiment).

Evaluation of Pre-mRNA Splicing via RT-PCR

Pre-mRNA splicing was analyzed as previously described. (10) Briefly, cells were grown in 6-well plates and treated with the desired compound in growth medium at ∼40% confluency. After 48 h, the cells were lysed, and total RNA was harvested using a Zymo Quick RNA Miniprep Kit. Approximately 1 μg of total RNA was reverse transcribed using a qScript cDNA synthesis kit (20 μL of total reaction volume, Quanta BioSciences); 2 μL of the RT reaction was used for PCR using GoTaq DNA polymerase (Promega). RT-PCR products were observed after 35 cycles of 95 °C for 30 s, 58 °C for 30 s, 72 °C for 1 min, and a final extension at 72 °C for 5 min. Products were separated on a 2% agarose gel (110 V for 1 h in 1 × TBE buffer), visualized by staining with ethidium bromide, and imaged using a Typhoon 9410 variable mode imager. Gels were quantified using ImageJ. Percent rescue was calculated by dividing the difference between treated and untreated DM2 samples by the difference between untreated DM2 and WT samples (eq 2).

$$\%\text{rescue}=\frac{\%\text{exon}\text{exclusion}\text{DM2}-\%\text{exon}\text{exclusion}\text{treated}}{\%\text{exon}\text{exclusion}\text{DM2}-\%\text{exon}\text{exclusion}\text{WT}}\times 100$$ (2)

Evaluation of CNBP Abundance via RT-qPCR

CNBP abundance was evaluated as previously described. (10) Briefly, cells were grown in 6-well plates and treated with the desired compound in growth medium at 40% confluency. After 48 h, the cells were lysed, and total RNA was harvested using a Zymo Quick RNA Miniprep Kit per the manufacturer’s protocol. Approximately 1 μg of total RNA was reverse transcribed using a qScript cDNA synthesis kit (20 μL of total reaction volume, Quanta BioSciences); 2 μL of the reverse transcription (RT) reaction was used for each primer pair (Table S2) for quantitative (q)PCR with SYBR Green Master Mix, performed on a QuantStudio 5, 384-well Block Real-Time PCR System (Applied Biosciences). Relative abundance was determined by normalizing to GAPDH (n = 6 replicates per concentration; 2 independent experiments).

Evaluation of Nuclear Foci via RNA Fluorescence In Situ Hybridization (FISH)

RNA FISH was used to determine the small molecules’ effects on the number of nuclear foci as previously described. (10) The number of foci was counted in 40 nuclei/replicate (120 total nuclei counted); n = 3 replicates; 1 independent experiment.

Affinity Measurements

Binding affinity measurements were performed via microscale thermophoresis (MST) on a Monolith NT.115 system (NanoTemper Technologies) with Cy5-labeled (CCUG)₁₂ (5′-Cy5-GCG(CCUG)₁₂CGC; Dharmacon) and Cy5-labeled base pair control (BP; 5′-Cy5-GCG(CCUG)₇(GCAG)₅CGC; Dharmacon), which were deprotected according to the manufacturer’s protocol and then desalted using a PD-10 column (GE LifeSciences) per the manufacturer’s recommended procedure. RNA (5 nM) was prepared in 8 mM Na₂HPO₄, pH 7.0, 185 mM NaCl, and 1 mM EDTA and folded by heating at 60 °C for 5 min and slowly cooling to room temperature. Compound dilutions (1:1) were prepared using 1 × MST Buffer (8 mM Na₂HPO₄, pH 7.0, 185 mM NaCl, 1 mM EDTA) and 0.1% (v/v) Tween-20 was added. After cooling, the RNA solution was added to the compound solution (0.05% (v/v) Tween-20 final concentration). Samples were incubated for 30 min at room temperature and then loaded into standard capillaries (NanoTemper Technologies). The following parameters were used to acquire thermophoretic data: 5–20% LED, 80% MST power, laser-on time = 30 s, laser-off time = 5 s. Fluorescence was detected using excitation wavelengths of 605–645 nm and emission wavelengths of 680–685 nm. The resulting data were analyzed by thermophoresis analysis and fitted using a quadratic binding equation in the MST analysis software (NanoTemper Technologies). Dissociation constants were then determined using eq 3. The reported K_d values are an average of two independent sets of experiments.

$$\text{fraction}\text{bound}=\frac{c+c_{\text{T}}+K_{\text{d}}-\sqrt{{\left(c+C_{\text{T}}+K_{\text{d}}\right)}^2+4c{c}_{\text{T}}}}{2c_{\text{T}}}$$ (3)

where c is the ligand concentration, c_T is the concentration of the RNA, and K_d is the dissociation constant.

SMILES and Physicochemical Properties

Marvin 20.8.0 (ChemAxon; https://www.chemaxon.com) was used to generate SMILES and to calculate physicochemical properties.

ABBREVIATIONS

CNBP

CCHC-type zinc finger nucleic acid binding protein

DCM

dichloromethane

DIPEA

N,N-diisopropylethylamine

DMF

dimethylformamide

DMSO

dimethyl sulfoxide

DM2

myotonic dystrophy type 2

HCl

hydrochloric acid

ESI

electrospray ionization

EtOH

ethanol

FISH

fluorescence in situ hybridization

HPLC

high-performance liquid chromatography

IPA

isopropanol

IR

insulin receptor

LCMS

liquid chromatography mass spectrometry

MBNL1

muscleblind-like 1

MeOH

methanol

MW

microwave

NMR

nuclear magnetic resonance

TFA

trifluoroacetic acid

TR-FRET

time-resolved fluorescence resonance energy transfer