Identification of Dihydrofuro[3,4-d]pyrimidine Derivatives as Novel HIV-1 Non-Nucleoside Reverse Transcriptase Inhibitors with Promising Antiviral Activities and Desirable Physicochemical Properties

Dongwei Kang; Heng Zhang; Zhao Wang; Tong Zhao; Tiziana Ginex; Francisco Javier Luque; Yang Yang; Gaochan Wu; Da Feng; Fenju Wei; Jian Zhang; Erik De Clercq; Christophe Pannecouque; Chin Ho Chen; Kuo-Hsiung Lee; N Arul Murugan; Thomas A Steitz; Peng Zhan; Xinyong Liu

doi:10.1021/acs.jmedchem.8b01656

. Author manuscript; available in PMC: 2020 Aug 21.

Published in final edited form as: J Med Chem. 2019 Jan 18;62(3):1484–1501. doi: 10.1021/acs.jmedchem.8b01656

Identification of Dihydrofuro[3,4-d]pyrimidine Derivatives as Novel HIV-1 Non-Nucleoside Reverse Transcriptase Inhibitors with Promising Antiviral Activities and Desirable Physicochemical Properties

Dongwei Kang ^†, Heng Zhang ^†, Zhao Wang ^†, Tong Zhao ^†, Tiziana Ginex ^‡, Francisco Javier Luque ^‡, Yang Yang ^§, Gaochan Wu ^†, Da Feng ^†, Fenju Wei ^†, Jian Zhang ^†, Erik De Clercq ^∥, Christophe Pannecouque ^∥, Chin Ho Chen ^⊥, Kuo-Hsiung Lee ^#,^∇, N Arul Murugan ^○, Thomas A Steitz ^§, Peng Zhan ^†,^*, Xinyong Liu ^†,^*

PMCID: PMC7441537 NIHMSID: NIHMS1618663 PMID: 30624934

Abstract

To address drug resistance to HIV-1 non-nucleoside reverse transcriptase inhibitors (NNRTIs), a series of novel diarylpyrimidine (DAPY) derivatives targeting “tolerant region I” and “tolerant region II” of the NNRTIs binding pocket (NNIBP) were designed utilizing a structure-guided scaffold-hopping strategy. The dihydrofuro[3,4-d]pyrimidine derivatives 13c2 and 13c4 proved to be exceptionally potent against a wide range of HIV-1 strains carrying single NNRTI-resistant mutations (EC₅₀ = 0.9–8.4 nM), which were remarkably superior to that of etravirine (ETV). Meanwhile, both compounds exhibited comparable activities with ETV toward the virus with double mutations F227L+V106A and K103N+Y181C. Furthermore, the most active compound 13c2 showed favorable pharmacokinetic properties with an oral bioavailability of 30.96% and a half-life of 11.1 h, which suggested that 13c2 is worth further investigation as a novel NNRTI to circumvent drug resistance.

Graphical Abstract

graphic file with name nihms-1618663-f0001.jpg

INTRODUCTION

In the life cycle of HIV-1, reverse transcriptase (RT) is responsible for the reverse transcription of single-stranded RNA to double-stranded DNA and represents one of the most successful targets for the development of anti-HIV-1 therapeutics due to the well-characterized mechanisms of action and abundant structure information.^1,2 RT inhibitors could be divided into nucleoside/nucleotide RT inhibitors (NRTIs/NtRTIs) and non-nucleoside RT inhibitors (NNRTIs).³ Especially, NNRTIs are widely used in highly active antiretroviral therapy (HAART) regimens, owing to their potent antiviral activity, high selectivity, and favorable pharmacokinetics. So far, six NNRTIs have been approved by U.S. Food and Drug Administration (FDA).⁴ Among them, nevirapine (NVP), delavirdine (DLV) and efavirenz (EFV) are the first-generation NNRTIs.⁵ However, toxicity and rapid emergence ofdrug resistance limited their clinical application. In particular, the K103N and Y181C mutations are prevalent in clinical HIV-1 isolates.^6,7 Even in naïve patients, low frequencies of K103N and Y181C variants can lead to an increased risk of virologic failure.⁸ Diarylpyrimidine (DAPY) derivatives represent a promising class of second-generation NNRTIs, and two representative compounds etravirine (1, ETV) and rilpivirine (2, RPV) (see Figure 1) were approved by U.S. FDA in 2008 and 2011, respectively.⁵ Although they could effectively suppress most of the NNRTIs-resistant mutants selected by the treatment of the first-generation NNRTIs, drug resistance and adverse effects continue to emerge in patients receiving second-generation NNRTIs regimens.^9,10 It underlines the demand to seek novel inhibitors with improved tolerability and resistance profiles.

Figure 1. — Chemical structures of U.S. FDA approved NNRTI drugs and our previously reported thiophene[3,2-d]pyrimidine leads 3 and 4.

In the effort to discover structurally distinct, best-in-class NNRTIs, our group previously identified piperidine-substituted thiophene[3,2-d]pyrimidine derivatives 3 (K-5a2) and 4 (25a) (Figure 1), which showed extraordinarily potent anti-HIV-1 activities and improved antiresistance profiles compared with ETV.^11-13 However, 3 exhibited weaker efficacy toward the particularly challenging double-mutation variant strain RES056 (K103N+Y181C) (EC₅₀ = 30.6 nM) compared with ETV (EC₅₀ = 17.0 nM). Although 4 achieved superior activity against RES056, it displayed a sharply increased cytotoxicity (CC₅₀ = 2.30 μM) than 3 (CC₅₀ > 227 μM). Apparently, the cyanovinyl group in the left wing of 4 accounts for the increased cytotoxicity considering that this chemical group may be sufficiently electrophilic to act as a “Michael acceptor”, resulting in potential covalent modification of nucleic acids, proteins, or other biological entities.¹⁴ Besides, lipophilic aromatic rings introduced to DAPY NNRTIs for better RT-binding affinities result in their poor aqueous solubility (the solubility of 3, 4, and ETV is less than 1 μg/mL), likely due to the strong intermolecular π−π stacking interactions.¹⁵ Therefore, development of novel DAPY NNRTIs with enhanced potency and solubility is still highly necessary.

Our previous work on thiophene[3,2-d]pyrimidine DAPY inhibitors explored the structure–activity relationships (SARs) of the “tolerant region I” of NNRTI binding pocket (NNIBP) (Figure 2) and identified that aryl groups with multiple hydrogen bond donors or acceptors were preferred.¹¹ As an extension of the previous work on the scaffold of thiophene[3,2-d]pyrimidine, in this study we have kept the “privileged structure” of 3 unchanged, and explored elaborated structural modifications of the piperidine linker utilizing scaffold hopping strategy. Preliminary results prove that the piperidine linker plays a critical role in maintaining potent antiviral activities.

Figure 2. — Further optimization of the piperidine linker and center thiophene[3,2-d]pyrimidine scaffold of the lead 3 (**K-5a2**) via scaffold hopping and crystal packing analysis.

On the other hand, subsequent structural optimization has been focused on the center core of the lead guided by scaffold hopping and crystal packing analysis. In particular, the thiophene[3,2-d]pyrimidine platform has been replaced by a set of six alicyclic-fused pyrimidine rings (Figure 2) with the hope that the additional alicyclic rings could change the crystal packing of the original aromatic ring structure and disrupt the possible intermolecular π−π stacking interactions, thus decreasing the lattice energy and improving the solubility.^16,17 In addition, the exposed oxygen atom of dihydrofuro[3,4-d]pyrimidine is able to develop additional hydrogen bond interactions with amino acid residues in the NNIBP as a hydrogen bond receptor, which could contribute to improve the resistance profiles of compounds. Overall, the combination of those structural modification has led to dihydrofuro[3,4-d]pyrimidine derivatives as novel HIV-1 NNRTIs, which demonstrated not only significantly improved drug resistance profiles but also enhanced solubility and bioavailability.

CHEMISTRY

All derivatives were synthesized by well-established methods as described in our previous article.¹² Representative synthetic routes of the series 8 that modify the piperidine linker are illustrated in Scheme 1. The previous prepared intermediate 5 was reacted with tert-butyl 4-aminoazepane-1-carboxylate to afford 6a, which gave the key intermediate 7a after removal of the Boc protecting group. Nucleophilic addition of 7a with substituted benzyl chloride (or bromine) or 4-picolyl chloride hydrochloride afforded target compounds 8a1–5. In an analogous way, 8b1–5, 8c1–5, 8d1–5, and 8e1–5 were prepared (Scheme S1 in Supporting Information).

Scheme 1. — ^aReagents and conditions: (i) *tert*-butyl 4-aminoazepane-1-carboxylate, DMF, K₂CO₃, 120 °C; (ii) TFA, DCM, rt; (iii) substituted benzyl chloride (or bromide) or 4-picolyl chloride hydrochloride, DMF, K₂CO₃, rt.

The synthetic routes of series 13a, 13b, 13c, 13d, 13e, and 13f were depicted in Schemes 2 and 3. 2,4-Dichloro-substituted pyrimidines 9a–f were selected as the starting materials and were reacted with 4-hydroxy-3,5-dimethylbenzonitrile by nucleophilic substitution to produce 10a–f. Then another successive nucleophilic addition of 4-(tert-butoxycarbonyl)-aminopiperidine with 10a–f generated intermediates 11a–f, which were converted to target compounds 13a1–6, 13b1–5, 13c1–6, 13d1–6, 13e1–6, and 13f1–5 followed by a similar proceduce in Scheme 1.

Scheme 2. — ^aReagents and conditions: (i) 3,5-dimethyl-4-hydroxybenzonitrile, DMF, K₂CO₃, rt; (ii) N-(*tert*-butoxycarbonyl)-4-aminopiperidine, DMF, K₂CO₃, 120 °C; (iii) TFA, DCM, rt; (iv) substituted benzyl chloride (or bromide) or 4-picolyl chloride hydrochloride, DMF, K₂CO₃, rt.

Scheme 3. — ^aReagents and conditions: (i) 3,5-dimethyl-4-hydroxybenzonitrile, DMF, K₂CO₃, rt; (ii) N-(*tert*-butoxycarbonyl)-4-aminopiperidine, DMF, K₂CO₃, 120 °C; (iii) TFA, DCM, rt; (iv) substituted benzyl chloride (or bromide) or 4-picolyl chloride hydrochloride, DMF, K₂CO₃, rt.

RESULTS AND DISCUSSION

The target compounds 8a1–5, 8b1–5, 8c1–5, 8d1–5, and 8e1–5 were evaluated against wild-type (WT) HIV-1 (NL4-3) replication in TZM-bl cell lines, and compounds 13a1–6, 13b1–5, 13c1–6, 13d1–6, 13e1–6, and 13f1–5 were evaluated against WT HIV-1 (IIIB), NNRTI-resistant strain K103N+Y181C (RES056), and a HIV-2 strain (ROD) in the MT4 cell line. Etravirine (ETV) was selected as control drug. The values of EC₅₀ (anti-HIV potency), CC₅₀ (cytotoxicity), SI (selectivity index, CC₅₀/EC₅₀ ratio) of the synthesized compounds are summarized in Tables 1-5. In addition, the RF (fold-resistance factor, EC₅₀ against mutant strains/EC₅₀ against WT strain) values of some potent compounds are shown in Table 5.

Table 1.

Anti-HIV-1 Activity and Cytotoxicity of 8a1–5, 8b1–5, 8c1–5, 8d1–5, and 8e1–5


compd	Ar	NL4-3
compd	Ar	EC₅₀ (nM)^a	CC₅₀ (nM)^b	SI^c
8a1	4-SO₂NH₂-Ph	2.20 ± 0.67	>222	>100
8a2	4-CONH₂-Ph	>237	>237	X1^d
8a3	3-CONH₂-Ph	10.3 ± 5.05	>237	23
8a4	4-SO₂CH₃-Ph	9.98 ± 4.16	>222	22
8a5	pyridine-4-yl	31.7 ± 11.0	>257	8
8b1	4-SO₂NH₂-Ph	>217	>217	X1^d
8b2	4-CONH₂-Ph	>232	>232	X1^d
8b3	3-CONH₂-Ph	>232	>232	X1^d
8b4	4-SO₂CH₃-Ph	>217	>217	X1^d
8b5	pyridine-4-yl	>251	>251	X1^d
8c1	4-SO₂NH₂-Ph	8.69 ± 2.74	>233	26
8c2	4-CONH₂-Ph	10.4 ± 2.70	>250	24
8c3	3-CONH₂-Ph	41.3 ± 14.4	>250	6
8c4	4-SO₂CH₃-Ph	13.9 ± 4.81	>234	16
8c5	pyridine-4-yl	16.0 ± 4.24	>273	17
8d1	4-SO₂NH₂-Ph	104 ± 36.8	>233	2.2
8d2	4-CONH₂-Ph	55.7 ± 16.6	>250	4.5
8d3	3-CONH₂-Ph	>250	>250	X1^d
8d4	4-SO₂CH₃-Ph	50.7 ± 12.8	>234	4.6
8d5	pyridine-4-yl	16.7 ± 4.88	>273	16
8e1	4-SO₂NH₂-Ph	4.53 ± 1.30	>240	53
8e2	4-CONH₂-Ph	4.76 ± 1.50	>257	54
8e3	3-CONH₂-Ph	8.95 ± 2.64	>257	29
8e4	4-SO₂CH₃-Ph	207 ± 48.6	>240	1.1
8e5	pyridine-4-yl-Ph	2.21 ± 0.61	>282	128
K-5a2 (3)		1.16 ± 0.43	>227	196

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EC₅₀: concentration of compound that causes 50% inhibition of viral infection and determined in at least triplicate against HIV-1 virus in TZM-bl cell lines. NL4-3 is wild-type HIV-1 viral strain.

CC₅₀: concentration that is cytopathic to 50% of cells. The highest concentration of the tested compounds was 125 ng/mL.

SI: selectivity index, the ratio of CC₅₀/EC₅₀.

X1: X1 means a value of SI > 1 or SI ≤ 1.

Table 5.

SI and RF Values of 13a1–2, 13c1–4, 13d1 and 13f1

compd	SI (RF)^a
compd	L100I	K103N	Y181C	Y188L	E138K	F227L+V106A
13a1	2880 (1.2)	2835 (1.3)	2071 (1.7)	1645 (2.2)	1298 (2.7)	1220 (2.9)
13a2	9576 (3.3)	28912 (1.1)	17281 (1.8)	6594 (4.8)	9515 (3.3)	3999 (7.9)
13c1	8941 (1.1)	9220 (1.0)	5087 (1.9)	1810 (5.3)	3471 (2.7)	2270 (4.2)
13c2	>104167 (1.5)	>277778 (0.6)	>56818 (2.8)	>29762 (5.3)	>35714 (4.4)	>13158 (11.9)
13c3	9050 (1.8)	15221 (1.0)	6318 (2.5)	3940 (4.0)	3986 (4.0)	4349 (3.7)
13c4	9035 (1.8)	31212 (0.5)	7630 (2.1)	4769 (3.3)	6023 (2.6)	3576 (4.4)
13d1	>39683 (5.3)	>21186 (10)	>16447 (12.9)	>16667 (12.7)	>5543 (38.4)	>6579 (32.3)
13f1	≥11488 (4.7)	≥28500 (1.9)	≥14250 (3.8)	≥18525 (3.0)	≥17435 (3.1)	≥6645 (8.3)
ETV	>755 (1.1)	>1379 (0.6)	>315 (2.3)	>225 (3.3)	>471 (1.6)	>233 (3.2)

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RF is the ratio of EC_{50(resistant viral strain)}/EC_{50(wild-type viral strain)}.

First, we assessed the anti-HIV-1 activities of the novel derivatives 8a1–5, 8b1–5, 8c1–5, 8d1–5, and 8e1–5 in which the piperidine linker of the lead 3 was replaced by nitrogen-containing cyclic analogues (Table 1). Most derivatives in subseries-8a, subseries-8c, subseries-8d, and subseries-8e were active against WT HIV-1 strain with moderate to high potencies (EC₅₀ = 2.20–207 nM), while the compounds in subseries-8b displayed dramatically impaired anti-HIV potency even at the maximum tested concentration (1 μg/mL). Compounds in subseries-8e featuring an azetidine linker yielded the most active potency with EC₅₀ values ranging from 2.21 to 8.95 nM with an exception of 8e4 (EC₅₀ = 207 nM). Among these derivatives, 8a1 and 8e5 proved to be the most effective inhibitors (EC₅₀ = 2.20 and 2.21 nM, respectively), comparable to that of 3 (EC₅₀ = 1.16 nM).

The preliminary SARs indicated that the linker of the right wing affected the activity significantly. Comparison of the designed five linkers indicates that the piperidine linker in the lead 3 was crucial for the potent antiviral activities. Furthermore, this result revealed that the cytotoxicity was not directly related to the modification of the piperidine linker.

To design more potent NNRTIs with improved drug resistance profiles and optimal physiochemical properties, a scaffold-hopping approach was applied to the center core (exploiting “tolerant region II”). The activities of five-membered ring fused pyrimidine derivatives 13a1–6, 13b1–5, 13c1–5, and 13d1–6, characterized by different scaffolds, are shown in Table 2. All the compounds displayed promising activity against WT HIV-1 with EC₅₀ values ranging from 1.6 to 37.2 nM. The compounds in subseries-13c and subseries-13d exhibited the most potent activity against WT HIV-1. Compounds 13c2, 13c3, 13d1, 13d4, and 13d6 turned out to be the most potent inhibitors, with EC₅₀ values ranging from 1.1 to 2.0 nM, being about 2.5- to 4.6-fold more potent than the reference drug ETV (EC₅₀ = 5.1 nM). In addition, 13a1,2, 13a4, 13b2, 13c1, 13c4, and 13d3 also showed higher potency than ETV with EC₅₀ values of 2.3–4.9 nM. Importantly, these potent compounds exhibited extremely high SI values (SI > 10 000) except for 13a1 (SI = 4584) and 13c1 (SI = 9518). Moreover, 13a1–4 and 13c1–4 showed promising activity (EC₅₀ = 28.8–46.6 nM) against the double mutant strain RES056, being similar to or more potent than that of ETV (EC₅₀ = 45.4 nM). 13a1 was the most potent inhibitor with an EC₅₀ value of 28.8 nM, about 1.5-fold more potent than ETV. In addition, the results also suggest that some compounds exhibited micromole activity against HIV-2 (ROD), such as 13a1 (EC₅₀ = 5.1 μM) and 13c4 (EC₅₀ = 5.1 μM), which could be considered as potential lead compounds for further optimization to find novel HIV-2 inhibitors with new mechanisms of action.

Table 2.

Anti-HIV (IIIB, RES056, and ROD) Activity and Cytotoxicity of 13a1–6, 13b1–5, 13c1–6, and 13d1–6


compd	Ar	EC₅₀(nM)^a		EC₅₀ (μM)^a		SI^c
compd	Ar	IIIB	RES056	ROD	CC₅₀ (μM)^b	IIIB	RES056
13a1	4-SO₂NH₂-Ph	4.3 ± 0.7	28.8 ± 1.2	6.55 ± 0.82	19.9 ± 10.1	4584	692
13a2	4-CONH₂-Ph	4.8	43.6 ± 5.0	>165	165 ± 36.9	34162	3804
13a3	4-SO₂CH₃-Ph	5.9 ± 1.3	46.6 ± 7.8	13.5 ± 9.00	11.9 ± 5.57	2025	257
13a4	pyridine-4-yl	2.6 ± 1.0	46.0 ± 1.9	15.7 ± 12.2	≥106	≥40104	≥2305
13a5	4-NO₂-Ph	8.0 ± 1.7	495 ± 1.3	>6.18	6.18 ± 1.74	770	12
13a6	3-CONH₂-Ph	27.7 ± 21.5	629 ± 64.5	>223	≥223	≥8048	≥355
13b1	4-SO₂NH₂-Ph	37.2 ± 26.6	734 ± 337.2	≥26.7	26.6 ± 1.96	715	36
13b2	4-SO₂Me-Ph	3.8 ± 1.3	170 ± 32	≥22.7	45.3 ± 31.1	11690	259
13b3	4-NO₂-Ph	11.5 ± 0.4	1143 ± 108	>27.1	27.1 ± 2.55	2341	24
13b4	4-NH₂-Ph	8.4 ± 2.8	704 ± 273	108 ± 53.3	>256.9	>30377	>364
13b5	4-NHSO₂Me-Ph	11.2 ± 0.6	5240 ± 1500	>257	>256.9	>19670	>42
13c1	4-SO₂NH₂-Ph	2.8 ± 1.0	38.0 ± 7.5	≥8.37	27.5 ± 7.49	9518	725
13c2	4-CONH₂-Ph	1.6 ± 0.4	41.5 ± 9.9	12.7 ± 0.72	>250	>156250	>6039
13c3	4-SO₂CH3-Ph	1.9 ± 0.1	41.6 ± 4.9	>27.0	31.3 ± 7.2	15946	752
13c4	pyridine-4-yl	2.3 ± 0.2	43.1 ± 1.2	5.12 ± 1.85	37.6 ± 11.0	15895	871
13c5	4-NO₂-Ph	7.4 ± 0.9	511 ± 165	>11.1	≥11.1	≥1497	≥22
13c6	3-CONH₂-Ph	7.8 ± 3.4	1033 ± 51.1	209 ± 29.7	>250	>31780	>243
13d1	4-SO₂NH₂-Ph	1.1 ± 0.5	25.8 ± 1.19	58.3 ± 4.28	>234	>208333	>9091
13d2	4-CONH₂-Ph	6.1 ± 2.7	173 ± 110	139 ± 24.3	>251	>40984	>1448
13d3	4-SO₂CH₃-Ph	4.9 ± 0.8	187 ± 0.79	41.8 ± 5.76	7.45 ± 0.93	1495	40
13d4	pyridine-4-yl	1.8 ± 0.7	54.7 ± 6.22	138 ± 25.7	>274	>147059	>5020
13d5	4-NO₂-Ph	14.5 ± 3.6	941 ± 368	>8.73	8.73 ± 3.53	599	9
13d6	3-CONH₂-Ph	2.0 ± 0.5	26786	>205	205 ± 1.13	124410	9
ETV		5.1 ± 0.8	45.4 ± 15.5		>4.59	>889	>101

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EC₅₀: concentration of compound required to achieve 50% protection of MT-4 cell cultures against HIV-1-induced cytopathic, as determined by the MTT method.

CC₅₀: concentration required to reduce the viability of mock-infected cell cultures by 50%, as determined by the MTT method.

SI: selectivity index, the ratio of CC₅₀/EC₅₀.

The activities of derivatives 13e1–6 and 13f1–5 bearing tetrahydroquinazoline and 7,8-dihydro-6H-thiopyrano[3,2-d]-pyrimidine center cores, respectively, are summarized in Table 3. 13f1 (EC₅₀ = 2.7 nM) and 13f2 (EC₅₀ = 3.0 nM) exhibited prominent potency against WT HIV-1, comparable to that of ETV. Although most compounds of subseries 13e showed single-digital activity toward WT HIV-1, their potencies against RES056 were inferior to that of ETV. Among them, the most effective inhibitor 13f1 (EC₅₀ = 40.6 nM) exhibited similar activity as ETV.

Table 3.

Anti-HIV (IIIB, RES056, and ROD) Activity and Cytotoxicity of 13e1–6 and 13f1–6


compd	Ar	EC₅₀(nM)		EC₅₀ (μM)		SI
compd	Ar	IIIB	RES056	ROD	CC₅₀ (μM)	IIIB	RES056
13e1	4-SO₂NH₂-Ph	6.0 ± 2.3	667 ± 556	>228	>228	>37879	>342
13e2	4-CONH₂-Ph	6.0 ± 2.0	291 ± 185	>233	>233	>38709	>799
13e3	4-SO₂CH₃-Ph	8.0 ± 3.1	66.1 ± 4.52	≥163	>266	>33333	>4032
13e4	pyridine-4-yl	8.6 ± 0.7	172 ± 20.2	21.7 ± 0.96	75.8 ± 45.1	8748	440
13e5	4-NO₂-Ph	77.4 ± 46.5	6896 ± 1104	>29.1	29.1 ± 7.19	376	4
13e6	3-CONH₂-Ph	6.5 ± 3.3	3148 ± 315	>158	158 ± 35.8	24415	50
13f1	4-SO₂NH₂-Ph	2.7 ± 2.6	40.6 ± 6.7	>148	≥148.2	≥54889	≥3650
13f2	4-CONH₂-Ph	3.0 ± 2.8	93.0 ± 77.3	>44.3	≥44.3	>14767	>476
13f3	4-SO₂CH₃-Ph	3.9 ± 2.0	98.3 ± 59.6	>3.40	≥3.4	≥872	≥34.6
13f4	4-NO₂-Ph	8.6 ± 4.7	604 ± 83.9	>4.00	4.0 ± 0.3	465	6.6
13f5	3-CONH₂-Ph	5.1 ± 0.4	1049 ± 149	>5.80	>4.59	>889	>101
ETV		5.1 ± 0.8	45.4 ± 15.5		>4.59	>889	>101

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Analysis of the SARs of these six subseries derivatives demonstrated that the hydrophilic substituents harboring hydrogen bond donors or acceptors (i.e., Ar = 4-SO₂NH₂-Ph, 4-SO₂CH₃-Ph, 4-CO₂NH₂-Ph, and pyridine-4-yl) were preferred. On the contrary, the hydrophobic substituents (i.e., Ar = 4-NO₂-Ph) were detrimental to its potency against WT HIV-1 and especially against mutated strains. 13a5, 13b3, 13c5, 13d5, 13e5, and 13f5 only exerted moderate activity with EC₅₀ values of 495, 1143, 511, 941, 6896, and 604 nM toward RES056, respectively. Pairwise comparison of the activities (compounds 13a1 vs 13a6, 13c1 vs 13c6, 13d1 vs 13d6, 13e1 vs 13e6, and 13f1 vs 13f5) confirmed the critical role of the para-substitution in improving the activity against RES056 over meta-substitution. Furthermore, the fused heterocycle on the pyrimidine was found to have a major influence on their anti-HIV-1 activity, especially against RES056. For instance, 13a1–4 (EC₅₀ = 28.8–46.6 nM) with dihydrothieno[3,2-d]pyrimidine and 13c1–4 (EC₅₀ = 38.0–43.1 nM) with dihydrofuro[3,4-d]pyrimidine scaffold exhibited prominent potency against RES056, while the activity of subseries 13b with dihydrothieno[3,4-d]pyrimidine scaffold considerably decreased (EC₅₀ = 170–5240 nM).

Subsequently, the efficacy of the compounds with potent activities toward WT and RES056 HIV-1 (13a1,2, 13c1–4, 13d1, and 13f1) were evaluated in MT-4 cells against mutant HIV-1 strains harboring single amino acid mutations L100I, K103N, Y181C, Y188L, E138K and double mutation F227L+V106A in the RT (Table 4 and Table 5). The results demonstrated that 5,7-dihydrofuro[3,4-d]pyrimidine derivatives 13c1–4 were potent inhibitors of the whole viral panel of NNRTI-resistant strains. Especially, 13c2 and 13c4 exhibited higher potency against K103N mutant strain than WT HIV-1 strain (RF = 0.6 and 0.5, respectively), with EC₅₀ values of 0.9 and 1.2 nM, about 2.7- and 3.6-fold lower than that of ETV (EC₅₀ = 3.3 nM). In the case of L100I, Y181C, Y188L, and E138K, 13c2, 13c3, and 13c4 provided single-digit nanomolar activities (EC₅₀ = 0.9–8.4 nM), being superior to that of ETV. For F227L+V106A, 13c3 (EC₅₀ = 7.2 nM) showed the highest potency and was 2.7-fold more potent than ETV (EC₅₀ = 19.7 nM). Moreover, 13a1, 13c1, 13c2, and 13c4 also inhibited F227L+V106A (EC₅₀ = 13.7, 12.7, 19.0, and 10.5 nM) to a greater extent than ETV. However, dihydrothieno[3,2-d]-pyrimidine derivative 13a2 and dihydrofuro[3,4-d]pyrimidine derivative 13d1 exhibited weaker efficacy toward most of the mutations compared with ETV. Specifically, 13d1 inhibited the two most prevalent single mutations K103N and E138K with much lower activity (EC₅₀ = 11.0 and 42.3 nM) and higher relative factor (RF) values (10 and 38.4) in comparison to ETV (EC₅₀ = 3.3 and 9.7 nM, RF = 0.6 and 1.6) (Table 5).

Table 4.

Activity against Mutant HIV-1 Strains of 13a1–2, 13c1–4, 13d1 and 13f1

compd	EC₅₀ (nM)
compd	L100I	K103N	Y181C	Y188L	E138K	F227L+V106A
13a1	5.8 ± 0.7	5.9 ± 0.1	8.0 ± 0.3	10.1 ± 0.7	12.8 ± 0.2	13.7 ± 0.6
13a2	15.2 ± 1.4	5.0 ± 1.0	8.4 ± 0.9	22.1 ± 0.55	15.3 ± 2.7	36.5 ± 1.0
13c1	3.0 ± 1.1	2.9 ± 0.5	5.4 ± 0.0	15.2 ± 0.3	7.9 ± 0.6	12.7 ± 7.6
13c2	2.4 ± 0.2	0.9 ± 0.1	4.4 ± 1.1	8.4 ± 3.6	7.0 ± 2.5	19.0 ± 2.2
13c3	3.4 ± 0.9	2.0 ± 1.0	4.9 ± 0.1	7.9 ± 0.9	7.8 ± 2.6	7.2 ± 0.1
13c4	4.1 ± 0.6	1.2 ± 0.1	4.9 ± 0.1	7.8 ± 1.5	6.2 ± 1.0	10.5 ± 0.6
13d1	5.9 ± 0.1	11.0 ± 8.7	14.2 ± 0.2	14.0 ± 3.9	42.3 ± 10.4	35.6 ± 8.4
13f1	12.9 ± 2.9	5.2 ± 1.4	10.4 ± 1.9	8.0 ± 2.0	8.5 ± 0.8	22.3 ± 4.8
ETV	6.0 ± 1.5	3.3 ± 0.6	14.5 ± 8.2	20.4 ± 8.6	9.7 ± 6.9	19.7 ± 7.3

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Some representative derivatives were further evaluated for their ability to inhibit recombinant WT HIV-1 RT enzyme to confirm the binding target (data shown in Table 6). Except for 13b1, 13d5, and 13d6, most compounds demonstrated high binding-affinity to WT HIV-1 RT (IC₅₀ = 0.051–0.175 μM). Generally, the derivatives with hydrophobic substituent and meta-substituent group have reduced inhibitory activities, exemplified by these pairwise comparisons: 13a1–4 vs 13a5, 13c1–4 vs 13c5,6, 13d1–4 vs 13d5,6, and 13e1–4 vs 13e5,6. Unexpectedly, the compounds containing 4-CONH₂-Ph substituent (13a2, 13b2, 13c2, 13d2, and 13e2) offered the highest RT inhibitory activities in each subseries, which were inconsistent with their anti-HIV-1 potency. This discrepancy, which has been reported in our previous articles, may be caused by the variations in the HIV-1 RT-substrate binding affinities and polymerase processivity on different nucleic acid templates.^11,18 Nonetheless, these results suggest that the newly synthesized derivatives behave as typical NNRTIs.

Table 6.

Inhibitory Activity against WT HIV-1 RT

compd	IC₅₀ (μM)	compd	IC₅₀ (μM)	compd	IC₅₀ (μM)
13a1	0.074 ± 0.002	13b5	0.140 ± 0.013	13d4	0.073 ± 0.007
13a2	0.066 ± 0.005	13c1	0.109 ± 0.003	13d5	0.241 ± 0.009
13a3	0.130 ± 0.033	13c2	0.051 ± 0.001	13d6	0.241 ± 0.009
13a4	0.112 ± 0.023	13c3	0.077 ± 0.009	13e1	0.084 ± 0.015
13a5	0.175 ± 0.047	13c4	0.056 ± 0.003	13e2	0.051 ± 0.004
13a6	0.094 ± 0.009	13c5	0.092 ± 0.024	13e3	0.089 ± 0.011
13b1	0.410 ± 0.112	13c6	0.091 ± 0.007	13e4	0.072 ± 0.003
13b2	0.091 ± 0.027	13d1	0.091 ± 0.029	13e5	0.094 ± 0.004
13b3	0.129 ± 0.057	13d2	0.073 ± 0.015	13e6	0.101 ± 0.016
13b4	0.115 ± 0.008	13d3	0.157 ± 0.022	ETV	0.011 ± 0.000

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Molecular Modeling Studies.

Classical molecular dynamics (MD) simulations were performed to examine the binding mode of 13c2, chosen on the basis of the potent inhibition against NNRTI-resistant strains (Table 4), bound to the NNRTIs binding site in WT HIV-RT. The ligand–protein complex was built up using the X-ray crystallographic structure of the HIV-1 WT RT/K-5a2 complex (PDB code 6C0J) as template.¹³ In the setup of the simulated system, attention was paid to preserve the hydrogen bond between the pyrimidine-bound NH group of 13c2 and the carbonyl oxygen of K101, and the network of hydrogen bonds between the protonated piperidine nitrogen, the backbone carbonyl oxygen of P236 and K103, and a water molecule (W936 in the crystallographic structure).

Three independent MD simulations were run, and the analysis consistently supported the stability of the ligand in the complex (see Figure S1 in Supporting Information), as noted in the resemblance of representative structures of the initial and final binding modes for 13c2 (Figure 3) and the low root-mean square deviation between the energy-minimized average structures sampled along the last 5 ns of the three trajectories (RMSD < 1.4 Å). The benzonitrile moiety is stably accommodated in a hydrophobic pocket formed by Y181, Y188, and W229. The pyrimidine-bound NH of 13c2 is hydrogen-bonded to the carbonyl oxygen of K101 (C═O–HN distance of 2.0 ± 0.2 Å; Supporting Information Figure S2). A network of water-mediated protein–ligand and protein–protein interactions can be observed around the protonated piperidine nitrogen of 13c2. Whereas in the initial structure (Figure 3A) the nitrogen atom is hydrogen-bonded to a water molecule (1.8 Å; see Table S1), which forms additional interactions with the backbone oxygen of K103 (2.4 Å; Table S1) and the NH amide group of P236 (1.9 Å; in Table S1), such a water molecule is displaced along the trajectory, allowing the protonated nitrogen to form a direct hydrogen bond with the backbone oxygen of K103 (1.8 ± 0.2 Å; Figure 3B and Supporting Information Figure S2) while preserving its interactions of the water molecule with K103 and P236 (see Table S1).

Figure 3. — Initial (A) and final (B) binding mode for ligand **13c2** into the NNIBP (PDB code 6C0J). The displacement of the water molecule occurred during MD simulations, enabling the formation of a direct hydrogen bond between the backbone carbonyl oxygen of K103 and the protonated pyrimidine nitrogen.

The presence of an ordered water molecule around the protonated piperidine nitrogen of 13c2 was confirmed by water density analysis (Figure S2). In this regard, a density isocontour corresponding to the water oxygen atom was visible near the piperidine nitrogen (Figure S2A). The water molecule is slightly shifted relative to the position of the crystallographic water (Figure S2) due to the gradual approach of residues K102–S105 to the ligand.

No stable direct interactions were formed between residues in the “tolerance region I” and the terminal benzamide group of 13c2. This is in contrast with the X-ray structure of the complex with K-5a2, where the sulfonamide group forms hydrogen bonds with the carbonyl oxygen of K104 and the amide NH group of V106. The lack of direct interactions may be ascribed to the lower flexibility of the benzamide moiety in 13c2 upon bioisosteric replacement of the benzosulfonamide group in K-5a2 and to the intrinsic flexibility of residues that shape the pocket filled by the benzamide unit, such as Q222-E224 (Supporting Information Figure S1), in agreement with the high B-factor values observed for these residues in the crystallographic structure.

As noted above, 13c2 can inhibit single (L100I, K103N, Y181C, Y188L, E138K) and double (F227+V106A) mutated HIV-RT variants (see Figure S3 for the location of these mutations). Five mutations (L100I, Y181C, Y188L, F227L, and V106) are located in the hydrophobic pocket that accommodates the benzonitrile and benzamide moieties of the ligand. The inhibitory activity of 13c2 can be justified by the fact that the mutated residues do not reduce the volume filled by these chemical fragments and the preservation of the hydrophobic nature of the pockets. This is supported by the existence of crystallographic complexes of 25a (a ligand similar to K-5a2) with the double mutant F227+V106A (PDB code 6DUF) and with the single mutant Y181I (PDB code 6DUH) of HIV-RT.13 Finally, the K103N mutation (Figure S3) is expected to be compatible with binding of 13c2 since local protein–ligand interactions only involve the backbone amide group of residue 103.

Solubility and Lipophilic Efficiency.

A potential drug candidate should possess a balance between drug-like properties and potency.¹⁹ Thus, derivatives 13a1 and 13c1–4 were chosen for assessment of certain physicochemical properties. Considering that solubility is important for in vivo dosing formulation and bioavailability, the solubility studies were performed on selected compounds at three different pH values (7.4, 7.0, and 2.0) and fasted state simulated intestinal fluid (FaSSIF). As depicted in Table 7, all the selected compounds displayed excellent solubility at pH 2.0 (S = 164–223 μg/mL). Notably, the solubility of 13a1, 13c1, 13c2, and 13c4 (S = 2.54–14.6 μg/mL) is much higher than that of 3 and ETV at pH 7.0 and pH 7.4. Furthermore, 13c2 and 13c3 also showed better solubility in FaSSIF (S = 32.5 and 23.6 μg/mL, respectively) compared to that of ETV (S = 13.2 μg/mL). These results indicated that the strategy of replacing the aromatic thiophene[3,2-d]pyrimidine with nonaromatic fused scaffold has achieved remarkable improvement in compound solubility.

Table 7.

Physicochemical Parameters of 13a1 and 13c1–4

compd	aqueous solubility (μg/mL)^a				log P^c	LE^d	LLE^e	LELP^f
compd	pH 7.4	pH 7.0	pH 2.0	FaSSIF^b	log P^c	LE^d	LLE^e	LELP^f
13a1	2.54	8.26	192	10.0	3.73	0.30	4.63	12.4
13c1	5.68	10.3	174	8.42	2.67	0.31	5.99	8.60
13c2	6.59	13.3	198	32.5	3.09	0.32	5.61	9.64
13c3	<1	1.37	164	23.6	2.85	0.31	5.89	9.08
13c4	3.28	14.6	223	8.57	3.07	0.34	5.48	8.95
3	≪1	0.05	190	1.24	4.46	0.31	4.39	14.2
ETV	<1	<1	127	13.2	4.19	0.40	4.10	10.4

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Measured by HPLC.

Fasted state simulated intestinal fluid.

Predicted by the software of ACS/Lab 6.0.

Calculated by the formula −ΔG/HA (non-hydrogen atom), in which normalizing binding energy ΔG = −RT ln K_d, presuming K_d ≈ EC_50(IIIB).

Calculated by the formula pEC₅₀ −log P.

Calculated by the formula log P/LE.

On the other hand, lipophilic efficiency (LE), ligand lipophilic efficiency (LLE), and ligand efficiency dependent lipophilicity (LELP) are important metrics in medicinal chemistry that have been increasingly applied to guide lead optimization toward drug candidates with critical balance between in vitro efficacy and in vivo drug-like properties.²⁰ Five NNRTI candidates, 13a1, 13c1, 13c2, 13c3, and 13c4, were assessed for their lipophilic parameters (Table 7). Inspiringly, 13c1, 13c2, 13c3, and 13c4 with dihydrofuro[3,4-d]pyrimidine scaffold satisfy the criteria of all the three ligand lipophilic efficiency indices (LE > 0.3, LLE > 5, LELP < 10).²¹ The results of solubility and lipophilic efficiency demonstrated that the presence of polar dihydrofuran substituents in “tolerant region II” allowed for tuning of lipophilicity to achieve both the desired antiviral activities and optimal physiochemical properties. However, 13a1 with dihydrothieno[3,2-d]pyrimidine scaffold showed a lower LLE value of 4.63 and a higher LELP value of 12.4.

CYP Enzymatic Inhibitory Activity.

Moreover, the most soluble compound 13c2 was evaluated for its ability to inhibit CYP drug-metabolizing enzymes in vitro. As reported in Table 8, 13c2 displayed no significant inhibition of CYP1A2 (IC₅₀ > 50.0 μM), CYP2C9 (IC₅₀ = 46.6 μM), CYP2C19 (IC₅₀ > 50.0 μM), CYP2D6 (IC₅₀ > 50.0 μM), and CYP3A4M (IC₅₀ > 50.0 μM), indicating an unlikely adverse effect on liver.

Table 8.

Effects of 13c2 on Inhibition of CYP1A2, CYP2C9, CYP2C19, CYP2D6, and CYP3A4M

CYP isozyme	standard inhibitor	IC₅₀ (μM)	compd	IC₅₀ (μM)
1A2	α-naphthoflavone	0.194	13c2	>50.0
2C9	sulfaphenazole	0.660	13c2	46.6
2C19	(+)-N-3-benzylnirvanol	0.256	13c2	>50.0
2D6	quinidine	0.156	13c2	>50.0
3A4M	ketoconazole	0.0366	13c2	>50.0

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Determination of Plasma Protein Binding Rate.

Furthermore, 13c2 was determined for its binding ability with human and Wistar rat plasma proteins with dialysis equilibrium method, and warfarin was selected as control drug. As shown in Table 9, the plasma protein binding rates of 13c2 were different in human and rate and the plasma protein binding rate was about 93.0% and 91.8%, respectively. The result demonstrated that the compound could bind strongly to plasma and there is a low concentration of free drug.

Table 9.

Plasma Protein Binding Rate of 13c2

compd	protein-binding rate		recovery rate
compd	human plasma protein	rat plasma protein	human plasma protein	rat plasma protein
13c2^a	93.0%	91.8%	86.3%	77.9%
warfarin^b	98.5%	99.1%	97.5%	107%

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The concentration of 13c2 is 2 μM.

The concentration of warfarin is 1 μM.

In Vivo Pharmacokinetics Study.

In view of its improved potency against resistance-associated variants and promising physiochemical properties, the pharmacokinetics of compound 13c2 was evaluated after single iv and po administration in Wistar rats. After a single 2 mg/kg iv dose of 13c2, the mean clearance rate (CL) and half-time (t_1/2) was 3.69 L h⁻¹ kg⁻¹ and 1.09 h (Table 10 and Figure 4). Absorption of 13c2 was assessed after being dosed at 20 mg/kg; it reached maximum concentration (T_max) at 1.33 h with a maximum concentration (C_max) of 400 ng/mL. Notably, the t_1/2 of 13c2 was up to 11.1 h. The oral bioavailability (F) was 30.96%, which was sufficiently high enough for a drug candidate.

Table 10.

Pharmacokinetic Profile of 13c2^a

subject	T_1/2 (h)	T_max (h)	C_max (ng/mL)	AUC_0−t (h·ng/mL)	AUC_0−∞ (h·ng/mL)	CL (L h⁻¹ kg⁻¹)	F (%)
13c2 (iv)^b	1.09 ± 0.13	0.033	460 ± 65.9	500 ± 17.5	543 ± 20.5	3.69
13c2 (po)^c	11.1 ± 0.61	1.33 ± 0.22	400 ± 76.1	1546 ± 114	1658 ± 130		30.96

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PK parameters (mean ± SD, n = 5).

Dosed intravenously at 2 mg/kg.

Dosed orally at 20 mg/kg.

Figure 4. — Plasma **13c2** concentration–time profiles in rats following oral administration (20 mg·kg⁻¹) and intravenous administration (2 mg·kg⁻¹).

Safety Assessment.

Assessment of Acute Toxicity.

A single-dose toxicity trial of 13c2 was carried out in Kunming mice. No death occurred after intragastric administration of 13c2 at a dose of 800 mg/kg. Furthermore, there was no significant behavior abnormalities compared to the control group in 2 weeks. All these results support the potential of 13c2 as a novel NNRTI drug candidate with high efficiency, low toxicity, and good oral bioavailability.

Assessment of Subacute Toxicity.

The subacute toxicity experiments of 13c2 were carried out to further evaluate their in vivo safety. No behavioral abnormalities were observed during the treatment period of po administered mice treated with 50 mg kg⁻¹ day⁻¹ of 13c2 in 2 weeks. Furthermore, organs toxicity experiments were examined by hematoxylin–eosin (HE) staining. ETV was selected as positive drug. As depicted in Figure 5, there is alveolar interstitial thickening and alveolar hemorrhage (the blue arrow) in the lung and proximal convoluted tubule edema (the red circle) in the kidney after treatment with ETV. After treatment with 13c2, the results demonstrated that the heart, liver, spleen, lungs, and kidneys of the mice were not damaged.

Figure 5. — HE of different organs of normal mice with ETV (50 mg/kg) and **13c2** (50 mg/kg). Scale bars represent 50 μm.

Assessment of hERG Activity.

Compounds with a high affinity for the hERG potassium channel could induce QT interval prolongation, which is frequently related to potentially risk for cardiotoxicity. So we tested the hERG inhibition activity of 13c2 with in vitro manual patch-clamp electrophysiology, and terfenadine was selected as reference drug (Table S2 and Table S3).12 The result (Figure 6) showed that compound 13c2 (IC₅₀ = 0.83 μM) had much weaker inhibition against the potassium channel, much lower than that of terfenadine (IC₅₀ = 0.086 μM).

Figure 6. — Activity of **13c2** and terfenadine against hERG potassium channel in HEK293 cells.

CONCLUSION

Starting from our previously described thiophene[3,2-d]-pyrimidine derivative 3, a structure-guided scaffold hopping was performed to design structurally diverse derivatives and to explore the chemical space in the “tolerant region I” and “tolerant region II” of the RT NNIBP. SARs of the lead structure were further extended. Encouragingly, the desired antiviral activities and physiochemical properties were achieved with the dihydrofuro[3,4-d]pyrimidine scaffold, which was found to lead to a stable binding mode in MD simulations of the ligand-bound complex. Especially, 13c2 and 13c4 proved to be the exceptionally potent inhibitors, exhibiting EC₅₀ values of 0.9–7.0 nM against single NNRTI-resistant strains L100I, K103N, Y181C, Y188L, and E138K in MT-4 cells and bringing approximately 1.3- to 3.6 fold improvement in potency compared with ETV (EC₅₀ = 3.3–20.4 nM). For double mutant HIV-1 strains F227L+V106A and RES056, both inhibitors displayed comparable activities with those of ETV. Furthermore, 13c2 also had much lower cytotoxicity (CC₅₀ > 250 μM), dramatically higher SI values, and low resistance FC values of 0.6–5.3 for single mutant strains. The results of MD simulation studies demonstrated that the bulkier molecular surface contributed to the increased binding affinity and resistance profiles compared with ETV. The CYP enzymatic inhibition test proved that 13c2 has relatively low adverse effect on the liver. The hERG inhibition activity of 13c2 (IC₅₀ = 0.83 μM) was a major improvement over terfenadine (IC₅₀ = 0.086 μM). Notably, 13c2 exhibited appealing PK profiles, with a low/moderate clearance in rats. The bioavailability of 13c2 is projected to be 30.96%. Taken together, 13c2 is a promising anti-HIV-1 drug candidate with potent antiviral activities and desirable drug-like properties.

EXPERIMENTAL SECTION

Chemistry.

All melting points were determined on a micro melting point apparatus (RY-1G, Tianjin TianGuang Optical Instruments) and are uncorrected. Proton nuclear magnetic resonance (¹H NMR) and carbon nuclear magnetic resonance (¹³C NMR) spectra were recorded in DMSO-d₆ on a Bruker AV-400 spectrometer with tetramethylsilane (TMS) as the internal standard. Coupling constants are given in hertz, and chemical shifts are reported in δ values (ppm) from TMS; signals are abbreviated as s (singlet), d (doublet), t (triplet), q (quarter), and m (multiplet). A G1313A standard LC autosampler (Agilent) was used to collect samples for measurement of mass spectra. The temperature of the reaction mixture was monitored with a mercury thermometer. All reactions were routinely monitored by thin layer chromatography (TLC) on silica gel GF254 for TLC (Merck), and spots were visualized with iodine vapor or by irradiation with UV light (λ = 254 nm). After completion of each reaction, the mixture was brought to ambient temperature via air-jet cooling. Flash column chromatography was performed on columns packed with silica gel (200–300 mesh), purchased from Qingdao Haiyang Chemical Company. Solvents were purified and dried by means of standard methods when necessary. Organic solutions were dried over anhydrous sodium sulfate and concentrated with a rotary evaporator under reduced pressure. Other reagents were obtained commercially and were used without further purification. Sample purity was analyzed on a Shimadzu SPD-20A/20AV HPLC system with a Inertsil ODS-SP, 5 μm C18 column (150 mm × 4.6 mm). HPLC conditions: methanol/water with 0.1% formic acid 80:20; flow rate 1.0 mL/min; UV detection from 210 to 400 nm; temperature, ambient; injection volume, 20 μL. 4-((2-Chlorothieno-[3,2-d]pyrimidin-4-yl)oxy)-3,5-dimethylbenzonitrile (5) was prepared as previously reported.11 Purity of all tested compounds was >95%.

4-((2-(Azepan-4-ylamino)thieno[3,2-d]pyrimidin-4-yl)oxy)-3,5-dimethylbenzonitrile (7a).

A solution of 5 (1.0 g, 3.17 mmol), tert-butyl 4-aminoazepane-1-carboxylate (0.81 g, 3.80 mmol), and anhydrous K₂CO₃ (0.87 g, 6.33 mmol) in 15 mL of DMF was heated at 120 °C under magnetic stirring for 8 h. After completion (monitored by TLC), the mixture was cooled to room temperature and 50 mL of ice water was added. The reaction mixture was continuously stirred for another 30 min and the resulting precipitate was collected and dried to give the intermediate 6a, which was used directly without purification. To a solution of 6a (0.49 g, 1.0 mmol) in dichloromethane (DCM) (3 mL) was added trifluoroacetic acid (TFA) (1.10 mL, 15 mmol) at room temperature. After the mixture was stirred for 3 h (monitored by TLC), it was alkalized to pH 9 with saturated sodium bicarbonate solution and washed with saturated salt water (15 mL). The aqueous phase was extracted with DCM (3 × 5 mL). The combined organic phase was dried over Na₂SO₄. The filtrate was concentrated and purified by column chromatography on silica gel to get 7a as a white solid with 71% yield. Mp: 86–88 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 8.23 (d, J = 5.3 Hz, 1H, C₆-thienopyrimidine-H), 7.73 (s, 2H, C₃,C₅-Ph-H), 7.26 (d, J = 5.4 Hz, 1H, C₇-thienopyrimidine-H), 6.81 (s, 1H, NH), 2.96–2.93 (m, 2H), 2.12 (s, 6H), 1.95–1.71 (m, 7H), 1.60–1.52 (m, 2H). ESI-MS: m/z 406.4 [M + 1]⁺. C₂₂H₂₃N₅OS (405.16). HPLC purity: 98.26%.

4-((2-((8-Azabicyclo[3.2.1]octan-3-yl)amino)thieno[3,2-d]-pyrimidin-4-yl)oxy)-3,5-dimethylbenzonitrile (7b).

Recrystallized from ethyl acetate (EA)/petroleum ether (PE) as a white solid, 62% yield. Mp: 131–133 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 8.22 (d, J = 5.4 Hz, 1H, C₆-thienopyrimidine-H), 7.75 (s, 2H, C₃,C₅-Ph-H), 7.25 (d, 1H, C₇-thienopyrimidine-H), 6.89 (s, 1H, NH), 3.96–3.94 (m, 1H), 2.68–2.66 (m, 2H), 2.12 (s, 6H), 1.92–1.14 (m, 8H). ESI-MS: m/z 394.5 [M + 1]⁺. C₂₁H₂₃N₅OS (393.16). HPLC purity: 99.28%.

(R)-3,5-Dimethyl-4-((2-(pyrrolidin-3-ylamino)thieno[3,2-d]-pyrimidin-4-yl)oxy)benzonitrile (7c).

Recrystallized from EA/PE as a white solid, 57% yield. Mp: 168–170 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 8.21 (d, J = 5.4 Hz, 1H, C₆-thienopyrimidine-H), 7.73 (s, 2H, C₃,C₅-Ph-H), 7.25 (d, J = 5.4 Hz, 1H, C₇-thienopyrimidine-H), 7.19 (s, 1H, NH), 2.95–2.84 (m, 2H), 2.32–2.28 (m, 2H), 2.08 (s, 6H), 1.65–1.54 (m, 2H). ESI-MS: m/z 366.5 [M + 1]⁺. C₁₉H₁₉N₅OS (365.13). HPLC purity: 95.73%.

(S)-3,5-Dimethyl-4-((2-(pyrrolidin-3-ylamino)thieno[3,2-d]-pyrimidin-4-yl)oxy)benzonitrile (7d).

Recrystallized from EA/PE as a white solid, 62% yield. Mp: 151–153 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 8.22 (d, J = 5.4 Hz, 1H, C₆-thienopyrimidine-H), 7.71 (s, 2H, C₃,C₅-Ph-H), 7.25 (d, J = 5.3 Hz, 1H, C₇-thienopyrimidine-H), 7.16 (s, 1H, NH), 4.32 (s, 1H), 2.97–2.91 (m, 2H), 2.38–2.31 (m, 2H), 2.08 (s, 6H), 1.81–1.57 (m, 2H). ESI-MS: m/z 366.5 [M + 1]⁺. C₁₉H₁₉N₅OS (365.13). HPLC purity: 99.83%.

4-((2-(Azetidin-3-ylamino)thieno[3,2-d]pyrimidin-4-yl)oxy)-3,5-dimethylbenzonitrile (7e).

Recrystallized from EA/PE as a white solid, 57% yield. Mp: 132–134 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 8.22 (d, J = 5.4 Hz, 1H, C₆-thienopyrimidine-H), 7.73 (s, 2H, C₃,C₅-Ph-H), 7.25 (d, J = 5.4 Hz, 1H, C₇-thienopyrimidine-H), 7.13 (s, 1H, NH), 4.36 (s, 1H), 3.46–3.43 (m, 2H), 2.98–2.93 (m, 2H), 2.11 (s, 6H). ESI-MS: m/z 352.4 [M + 1]⁺. C₁₈H₁₇N₅OS (351.12). HPLC purity: 99.47%.

General Procedure for the Preparation of Final Compounds 8a1–5.

To a solution of 7a (0.5 mmol) in DMF (8 mL) were added anhydrous K₂CO₃ (0.6 mmol) and substituted benzyl chloride (bromide) (0.55 mmol). After being stirred at ambient temperature for 4–7 h (monitored by TLC), the mixture was poured into water (20 mL) and extracted with ethyl acetate (3 × 10 mL). The organic phase was washed with saturated sodium chloride (2 × 10 mL) and then dried over Na₂SO₄. The filtrate was concentrated, purified by column chromatography, and recrystallized from ethyl acetate/petroleum ether to afford the target compounds 8a1–5.

4-((4-((4-(4-Cyano-2,6-dimethylphenoxy)thieno[3,2-d]-pyrimidin-2-yl)amino)azepan-1-yl)methyl)benzenesulfonamide (8a1).

Recrystallized from EA/PE as a white solid, 59% yield. Mp: 239–241 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 8.21 (d, J = 5.4 Hz, 1H, C₆-thienopyrimidine-H), 7.87 (d, J = 8.0 Hz, 2H, C₃,C₅-Ph′-H), 7.73 (s, 2H, C₃,C₅-Ph″-H), 7.60 (d, J = 8.0 Hz, 2H, C₂,C₆-Ph′-H), 7.31 (s, 2H, SO₂NH₂), 7.24 (d, J = 5.4 Hz, 1H, C₇-thienopyrimidine-H), 6.80 (s, 1H, NH), 3.56 (s, 2H, N-CH₂), 2.99–2.97 (m, 2H), 2.12 (s, 6H), 1.87–1.75 (m, 7H), 1.60–1.57 (m, 2H). ¹³C NMR (100 MHz, DMSO-d₆) δ 161.6, 160.7, 156.4, 150.7, 143.2, 133.2, 132.9, 129.3, 126.1, 123.5, 119.0, 109.0, 58.1, 55.6, 43.3, 35.7, 26.1, 16.2. ESI-MS: m/z 575.6 [M + 1]⁺. C₂₉H₃₀N₆O₃S₂ (574.18). HPLC purity: 95.46%.

4-((4-((4-(4-Cyano-2,6-dimethylphenoxy)thieno[3,2-d]-pyrimidin-2-yl)amino)azepan-1-yl)methyl)benzamide (8a2).

Recrystallized from EA/PE as a white solid, 63% yield. Mp: 175–177 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 8.20 (d, J = 5.4 Hz, 1H, C₆-thienopyrimidine-H), 7.87 (d, J = 8.2 Hz, 2H, C₃,C₅-Ph′-H), 7.73 (s, 2H, C₃,C₅-Ph″-H), 7.51 (d, J = 8.1 Hz, 2H, C₂,C₆-Ph′-H), 7.40 (s, 2H, CONH₂), 7.25 (d, J = 5.4 Hz, 1H, C₇-thienopyrimidine-H), 6.78 (s, 1H, NH), 3.54 (s, 2H, N-CH₂), 3.07–2.88 (m, 2H), 2.11 (s, 6H), 1.99–1.77 (m, 7H), 1.72–1.63 (s, 2H). ¹³C NMR (100 MHz, DMSO-d₆) δ 168.4, 161.5, 156.3, 153.7, 150.6, 143.2, 134.7, 133.2, 132.9, 128.4, 126.1, 119.3, 108.6, 62.5, 57.8, 53.4, 36.2, 26.5, 16.2. ESI-MS: m/z 539.6 [M + 1]⁺. C₃₀H₃₀N₆O₂S (538.22). HPLC purity: 95.01%.

3-((4-((4-(4-Cyano-2,6-dimethylphenoxy)thieno[3,2-d]-pyrimidin-2-yl)amino)azepan-1-yl)methyl)benzamide (8a3).

Recrystallized from EA/PE as a white solid, 62% yield. Mp: 143–146 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 8.20 (d, J = 5.4 Hz, 1H, C₆-thienopyrimidine-H), 7.94 (s, 1H, C₂-Ph′-H), 7.75–7.73 (m, 3H), 7.49 (d, J = 7.7 Hz, 1H, C₆-Ph′-H), 7.39 (d, J = 7.6 Hz, 1H, C₅-Ph′-H), 7.33–7.32 (m, 2H), 7.25 (d, J = 5.4 Hz, 1H, C₇-thienopyrimidine-H), 6.78 (s, 1H, NH), 3.55 (s, 2H, N-CH₂), 3.09–2.85 (m, 2H), 2.11 (s, 6H), 1.99–1.78 (m, 7H), 1.64 (s, 2H). ¹³C NMR (100 MHz, DMSO-d₆) δ 168.5, 160.7, 156.3, 150.7, 143.2, 134.6, 133.2, 132.9, 128.4, 126.1, 119.2, 108.6, 61.8, 57.8, 53.4, 36.2, 26.5, 16.2. ESI-MS: m/z 539.6 [M + 1]⁺. C₃₀H₃₀N₆O₂S (538.22). HPLC purity: 96.12%.

3,5-Dimethyl-4-((2-((1-(4-(methylsulfonyl)benzyl)azepan-4-yl)amino)thieno[3,2-d]pyrimidin-4-yl)oxy)benzonitrile (8a4).

Recrystallized from EA/PE as a white solid, 67% yield. Mp: 118–120 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 8.21 (d, J = 5.4 Hz, 1H, C₆-thienopyrimidine-H), 7.87 (d, J = 8.0 Hz, 2H, C₃,C₅-Ph′-H), 7.73 (s, 2H, C₃,C₅-Ph″-H), 7.61 (d, J = 8.0 Hz, 2H, C₂,C₆-Ph′-H), 7.25 (d, J = 5.4 Hz, 1H, C₇-thienopyrimidine-H), 6.80 (s, 1H, NH), 3.55 (s, 2H, N-CH₂), 3.20 (s, 3H, SO₂CH₃), 2.98 (s, 2H), 2.12 (s, 6H), 1.90–1.81 (m, 7H), 1.67–1.63 (m, 2H). ¹³C NMR (100 MHz, DMSO-d₆) δ 165.8, 160.7, 153.4, 147.1, 139.5, 133.2, 132.9, 129.3, 127.3, 123.5, 119.0, 109.0, 58.1, 55.6, 44.1, 43.3, 35.7, 26.1, 16.2. ESI-MS: m/z 574.5 [M + 1]⁺. C₃₀H₃₁N₅O₃S₂ (573.19). HPLC purity: 95.78%.

3,5-Dimethyl-4-((2-((1-(pyridin-4-ylmethyl)azepan-4-yl)amino)thieno[3,2-d]pyrimidin-4-yl)oxy)benzonitrile (8a5).

Recrystallized from EA/PE as a white solid, 63% yield. Mp: 246–248 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 8.50 (d, J = 5.0 Hz, 2H, C₃,C₅-Py-H), 8.21 (d, J = 5.4 Hz, 1H, C₆-thienopyrimidine-H), 7.73 (s, 2H, C₃,C₅-Ph′-H), 7.31 (d, J = 5.1 Hz, 2H, C₂,C₆-Py-H), 7.25 (d, J = 5.4 Hz, 1H, C₇-thienopyrimidine-H), 6.87 (s, 1H, NH), 3.55 (s, 2H, N–CH₂), 2.98–2.96 (s, 2H), 2.12 (s, 6H), 1.90–1.77 (m, 7H), 1.65–1.54 (m, 2H). ¹³C NMR (100 MHz, DMSO-d₆) δ 165.8, 160.7, 153.4, 151.6, 149.9, 139.5, 133.2, 132.9, 129.4, 127.3, 123.5, 119.0, 109.0, 58.1, 55.6, 43.4, 35.7, 26.1, 16.2. ESI-MS: m/z 497.6 [M + 1]⁺. C₂₈H₂₈N₆OS (496.20). HPLC purity: 98.69%.

4-((3-((4-(4-Cyano-2,6-dimethylphenoxy)thieno[3,2-d]-pyrimidin-2-yl)amino)-8-azabicyclo[3.2.1]octan-8-yl)methyl)benzenesulfonamide (8b1).

Recrystallized from EA/PE as a white solid, 76% yield. Mp: 115–117 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 8.20 (d, J = 5.4 Hz, 1H, C₆-thienopyrimidine-H), 7.78 (d, J = 7.9 Hz, 2H, C₃,C₅-Ph′-H), 7.73 (s, 2H, C₃,C₅-Ph″-H), 7.48 (d, J = 8.0 Hz, 2H, C₂,C₆-Ph′-H), 7.31 (s, 2H, SO₂NH₂), 7.24–7.23 (m, 1H, C₇-thienopyrimidine-H), 6.89 (s, 1H, NH), 4.09–3.93 (m, 1H), 3.61 (s, 2H, N-CH₂), 2.72–2.65 (m, 2H), 2.12 (s, 6H), 1.92–1.14 (m, 8H). ¹³C NMR (100 MHz, DMSO-d₆) δ 161.7, 160.2, 156.4, 154.7, 150.8, 143.0, 133.2, 132.9, 129.1, 126.0, 119.0, 109.0, 61.9, 53.8, 34.3, 32.9, 25.0, 16.2. ESI-MS: m/z 563.5 [M + 1]⁺. C₂₈H₃₀N₆O₃S₂ (562.18). HPLC purity: 98.43%.

4-((3-((4-(4-Cyano-2,6-dimethylphenoxy)thieno[3,2-d]-pyrimidin-2-yl)amino)-8-azabicyclo[3.2.1]octan-8-yl)methyl)benzamide (8b2).

Recrystallized from EA/PE as a white solid, 65% yield. Mp: 103–105 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 8.20 (d, J = 5.3 Hz, 1H, C₆-thienopyrimidine-H), 7.79 (d, J = 8.1 Hz, 2H, C₃,C₅-Ph′-H), 7.73 (s, 2H, C₃,C₅-Ph″-H), 7.49 (d, J = 8.0 Hz, 2H, C₂,C₆-Ph′-H), 7.30 (s, 2H, CONH₂), 7.24–7.23 (m, 1H, C₇-thienopyrimidine-H), 6.89 (s, 1H, NH), 4.01–3.93 (m, 1H), 3.62 (s, 2H, N-CH₂), 2.72–2.70 (m, 2H), 2.12 (s, 6H), 1.89–1.21 (m, 8H). ¹³C NMR (100 MHz, DMSO-d₆) δ 168.5, 161.7, 156.4, 154.7, 150.8, 143.0, 133.2, 132.9, 129.7, 126.2, 119.0, 109.0, 61.9, 53.8, 32.9, 25.0, 16.2. ESI-MS: m/z 527.6 [M + 1]⁺. C₂₉H₃₀N₆O₂S (526.22). HPLC purity: 97.15%.

3-((3-((4-(4-Cyano-2,6-dimethylphenoxy)thieno[3,2-d]-pyrimidin-2-yl)amino)-8-azabicyclo[3.2.1]octan-8-yl)methyl)benzamide (8b3).

Recrystallized from EA/PE as a white solid, 61% yield. Mp: 162–164 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 8.20 (d, J = 5.4 Hz, 1H, C₆-thienopyrimidine-H), 7.79 (s, 1H, C₂-Ph′-H), 7.74–7.73 (m, 3H), 7.44 (d, J = 7.4 Hz, 1H, C₆-Ph′-H), 7.39 (d, J = 7.5 Hz, 1H, C₅-Ph′-H), 7.34 (s, 2H, CONH₂), 7.24–7.23 (m, 1H, C₇-thienopyrimidine-H), 6.88 (s, 1H, NH), 4.06–4.04 (m, 1H), 3.57 (s, 2H, N-CH₂), 2.75–2.68 (m, 2H), 2.12 (s, 6H), 1.90–1.58 (s, 8H). ¹³C NMR (100 MHz, DMSO-d₆) δ 168.4, 156.8, 153.4, 150.2, 143.1, 140.2, 134.6, 133.2, 132.9, 131.7, 128.4, 128.2, 126.3, 119.0, 62.3, 40.5, 33.0, 25.0, 16.2. ESI-MS: m/z 527.6 [M + 1]⁺. C₂₉H₃₀N₆O₂S (526.22). HPLC purity: 98.98%.

3,5-Dimethyl-4-((2-((8-(4-(methylsulfonyl)benzyl)-8-azabicyclo[3.2.1]octan-3-yl)amino)thieno[3,2-d]pyrimidin-4-yl)oxy)benzonitrile (8b4).

Recrystallized from EA/pE as a white solid, 72% yield. Mp: 114–116 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 8.21 (d, J = 5.4 Hz, 1H, C₆-thienopyrimidine-H), 7.90 (d, J = 8.0 Hz, 2H, C₃,C₅-Ph′-H), 7.73 (s, 2H, C₃,C₅-Ph″-H), 7.58 (d, J = 8.0 Hz, 2H, C₂,C₆-Ph′-H), 7.24–7.23 (m, 1H, C₇-thienopyrimidine-H), 6.90 (s, 1H, NH), 4.20–3.95 (m, 1H), 3.62 (s, 2H, N-CH₂), 3.22 (s, 3H, SO₂CH₃), 2.72–2.67 (m, 2H), 2.12 (s, 6H), 1.92–1.35 (m, 8h). ¹³C NMR (100 MHz, DMSO-d₆) δ 163.2, 156.8, 153.4, 149.2, 145.0, 139.4, 138.0, 137.3 136.4, 133.8, 133.2, 132.9, 131.0, 123.7, 113.8, 67.0, 40.5, 37.8, 33.0, 25.0, 16.2. ESI-MS: m/z 562.6 [M + 1]⁺. C₂₉H₃₁N₅O₃S₂ (561.19). HPLC purity: 98.26%.

3,5-Dimethyl-4-((2-((8-(pyridin-4-ylmethyl)-8-azabicyclo-[3.2.1]octan-3-yl)amino)thieno[3,2-d]pyrimidin-4-yl)oxy)benzonitrile (8b5).

Recrystallized from EA/pE as a white solid, 70% yield. Mp: 96–98 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 8.50 (d, J = 5.1 Hz, 2H, C₃,C₅-Py-H), 8.21 (d, J = 5.3 Hz, 1H, C₆-thienopyrimidine-H), 7.73 (s, 2H, C₃,C₅-Ph′-H), 7.30 (d, J = 4.9 Hz, 2H, C₂,C₆-Py-H), 7.24–7.23 (s, 1H, C₇-thienopyrimidine-H), 6.89 (s, 1H, NH), 4.07–4.04 (m, 1H), 3.58 (s, 2H, N-CH₂), 2.72–2.67 (m, 2H), 2.12 (s, 6H), 1.95–1.08 (m, 8H). ¹³C NMR (100 MHz, DMSO-d₆) δ160.8, 156.7, 154.0, 151.2, 149.9, 133.2, 132.9, 126.5, 123.8, 119.0, 109.0, 61.3, 39.6, 34.2, 33.0, 25.0, 16.2. ESI-MS: m/z 485.7 [M + 1]⁺. C₂₇H₂₈N₆OS (484.20). HPLC purity: 98.44%.

(R)-4-((3-((4-(4-Cyano-2,6-dimethylphenoxy)thieno[3,2-d]-pyrimidin-2-yl)amino)pyrrolidin-1-yl)methyl)benzenesulfonamide (8c1).

Recrystallized from EA/PE as a white solid, 54% yield. Mp: 138–140 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 8.21 (d, J = 5.4 Hz, 1H, C₆-thienopyrimidine-H), 7.77 (d, J = 8.0 Hz, 2H, C₃,C₅-Ph′-H), 7.73 (s, 2H, C₃,C₅-Ph″-H), 7.46 (d, J = 7.9 Hz, 2H, C₂,C₆-Ph′-H), 7.31 (s, 2H, SO₂NH₂), 7.25 (d, J = 5.4 Hz, 1H, C₇-thienopyrimidine-H), 7.19 (s, 1H, NH), 3.60 (s, 2H, N-CH₂), 3.02–2.80 (m, 2H), 2.30–2.29 (m, 2H), 2.08 (s, 6H), 1.79–1.19 (m, 2H). ¹³C NMR (100 MHz, DMSO-d₆) δ 153.4, 143.7, 143.1, 133.2, 133.0, 132.9, 129.2, 126.0, 119.0, 109.0, 60.4, 59.3, 52.9, 30.9, 16.2. ESI-MS: m/z 535.6 [M + 1]⁺. C₂₆H₂₆N₆O₃S₂ (534.15). HPLC purity: 98.49%.

(R)-4-((3-((4-(4-Cyano-2,6-dimethylphenoxy)thieno[3,2-d]-pyrimidin-2-yl)amino)pyrrolidin-1-yl)methyl)benzamide (8c2).

Recrystallized from EA/PE as a white solid, 50% yield. Mp: 119–121 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 8.21 (d, J = 5.4 Hz, 1H, C₆-thienopyrimidine-H), 7.82 (d, J = 7.8 Hz, 2H, C₃,C₅-Ph′-H), 7.69 (s, 2H, C₃,C₅-Ph″-H), 7.34 (d, J = 8.0 Hz, 2H, C₂,C₆-Ph′-H), 7.25 (s, 2H, CONH₂), 7.24 (d, J = 5.4 Hz, 1H, C₇-thienopyrimidine-H), 7.16 (s, 1H, NH), 4.30 (s, 1H), 3.56 (s, 2H, N-CH₂), 2.89–2.80 (m, 2H), 2.32–2.25 (m, 2H), 2.08 (s, 6H), 1.92–1.19 (m, 2H). ¹³C NMR (101 MHz, DMSO-d₆) δ 168.2, 153.4, 133.2, 132.9, 128.7, 127.8, 119.0, 109.0, 60.4, 52.9, 39.6, 39.4, 30.9, 16.2. ESI-MS: m/z 499.3 [M + 1]⁺. C₂₆H₂₆N₆O₃S₂ (498.18). HPLC purity: 97.20%.

(R)-3-((3-((4-(4-Cyano-2,6-dimethylphenoxy)thieno[3,2-d]-pyrimidin-2-yl)amino)pyrrolidin-1-yl)methyl)benzamide (8c3).

Recrystallized from EA/PE as a white solid, 53% yield. Mp: 125–126 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 8.20 (d, J = 5.4 Hz, 1H, C₆-thienopyrimidine-H), 7.78–7.76 (m, 2H), 7.69 (s, 2H, C₃,C₅-Ph″-H), 7.43–7.38 (m, 2H), 7.34 (s, 2H, CONH₂), 7.25 (d, J = 5.4 Hz, 1H, C₇-thienopyrimidine-H), 7.14 (s, 1H, NH), 4.31 (s, 1H), 3.56 (s, 2H, N-CH₂), 2.84–2.80 (m, 2H), 2.32–2.27 (m, 2H), 2.08 (s, 6H), 1.86–1.52 (m, 2H). ¹³C NMR (100 MHz, DMSO-d₆) δ 168.3, 162.4, 153.4, 134.6, 133.2, 132.9, 131.9, 128.5, 128.2, 126.4, 109.0, 60.4, 59.8, 52.9, 39.6, 39.4, 30.9, 16.1. ESI-MS: m/z 499.3 [M + 1]⁺. C₂₆H₂₆N₆O₃S₂ (498.18). HPLC purity: 96.75%.

(R)-3,5-Dimethyl-4-((2-((1-(4-(methylsulfonyl)benzyl)pyrrolidin-3-yl)amino)thieno[3,2-d]pyrimidin-4-yl)oxy)benzonitrile (8c4).

Recrystallized from EA/PE as a white solid, 59% yield. Mp: 125–127 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 8.21 (d, J = 5.4 Hz, 1H, C₆-thienopyrimidine-H), 7.88 (d, J = 8.0 Hz, 2H, C₃,C₅-Ph′-H), 7.69 (s, 2H, C₃,C₅-Ph″-H), 7.55 (d, J = 8.0 Hz, 2H, C₂,C₆-Ph′-H), 7.25 (d, J = 5.6 Hz, 1H, C₇-thienopyrimidine-H), 7.14 (s, 1H, NH), 4.32 (s, 1H), 3.65 (s, 2H, N-CH₂), 3.21 (s, 3H, SO₂CH₃), 2.88–2.85 (m, 2H), 2.31–2.27 (m, 2H), 2.08 (s, 6H), 1.87–1.51 (m, 2H). ¹³C NMR (100 MHz, DMSO-d₆) δ 162.4, 160.7, 153.4, 139.8, 133.2, 132.9, 129.6, 127.3, 127.0, 119.0, 109.0, 60.4, 59.2, 52.9, 44.0, 39.6, 39.4, 31.0, 16.1. ESI-MS: m/z 534.6 [M + 1]⁺. C₂₇H₂₇N₅O₃S₂ (533.16). HPLC purity: 96.16%.

(R)-3,5-Dimethyl-4-((2-((1-(pyridin-4-ylmethyl)pyrrolidin-3-yl)amino)thieno[3,2-d]pyrimidin-4-yl)oxy)benzonitrile (8c5).

Recrystallized from EA/PE as a white solid, 55% yield. Mp: 174–176 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 8.50 (d, J = 4.9 Hz, 2H, C₃,C₅-Py-H), 8.21 (d, J = 5.4 Hz, 1H, C₆-thienopyrimidine-H), 7.69 (s, 2H, C₃,C₅-Ph′-H), 7.29 (d, J = 5.1 Hz, 2H, C₂,C₆-Py-H), 7.25 (d, J = 5.5 Hz, 1H, C₇-thienopyrimidine-H), 7.14 (s, 1H, NH), 4.32 (s, 1H), 3.54 (s, 2H, N-CH₂), 2.87–2.86 (m, 2H), 2.30–2.29 (m, 2H), 2.08 (s, 6H), 1.72–1.65 (m, 2H). ¹³C NMR (100 MHz, DMSO-d₆) δ 162.4, 153.4, 149.9, 148.5, 133.2, 132.9, 123.9, 119.0, 109.0, 60.4, 58.7, 53.0, 31.0, 16.2. ESI-MS: m/z 457.4 [M + 1]⁺. C₂₅H₂₄N₆OS (456.17). HPLC purity: 97.20%.

(S)-4-((3-((4-(4-Cyano-2,6-dimethylphenoxy)thieno[3,2-d]-pyrimidin-2-yl)amino)pyrrolidin-1-yl)methyl)benzenesulfonamide (8d1).

Recrystallized from EA/PE as a white solid, 53% yield. Mp: 129–131 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 8.21 (d, J = 5.4 Hz, 1H, C₆-thienopyrimidine-H), 7.78 (d, J = 8.2 Hz, 2H, C₃,C₅-Ph′-H), 7.70 (s, 2H, C₃,C₅-Ph″-H), 7.46 (d, J = 7.6 Hz, 2H, C₂,C₆-Ph′-H), 7.31 (s, 2H, SO₂NH₂), 7.25 (d, J = 5.3 Hz, 1H, C₇-thienopyrimidine-H), 7.17 (s, 1H, NH), 4.32 (s, 1H), 3.60 (s, 2H, N-CH₂), 3.10–2.89 (m, 2H), 2.41–2.23 (m, 2H), 2.08 (s, 6H), 1.89–1.54 (m, 2H). ¹³C NMR (100 MHz, DMSO-d₆) δ 153.4, 143.1, 133.2, 132.9, 132.7, 129.3, 129.2, 126.1, 126.0, 123.9, 119.0, 109.0, 60.3, 59.3, 52.9, 30.9, 16.2. ESI-MS: m/z 535.5 [M + 1]⁺. C₂₆H₂₆N₆O₃S₂ (534.15). HPLC purity: 97.93%.

(S)-4-((3-((4-(4-Cyano-2,6-dimethylphenoxy)thieno[3,2-d]-pyrimidin-2-yl)amino)pyrrolidin-1-yl)methyl)benzamide (8d2).

Recrystallized from EA/PE as a white solid, 49% yield. Mp: 130–132 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 8.20 (d, J = 5.4 Hz, 1H, C₆-thienopyrimidine-H), 7.83 (d, J = 8.1 Hz, 2H, C₃,C₅-Ph′-H), 7.72, 7.65 (m, 2H, C₃,C₅-Ph″-H), 7.36–7.29 (m, 2H), 7.25 (d, J = 5.4 Hz, 1H, C₇-thienopyrimidine-H), 7.19(s, 1H, NH), 4.29 (s, 1H), 3.57 (s, 2H, N-CH₂), 2.85–2.67 (m, 2H), 2.32–2.25 (m, 2H), 2.08 (s, 6H), 1.72–1.52 (s, 2H). ¹³C NMR (100 MHz, DMSO-d₆) δ 168.2, 153.4, 133.3, 133.2, 132.9, 128.7, 128.6, 127.9, 127.8, 119.0, 109.0, 60.4, 59.5, 52.9, 39.6, 30.9, 16.2. ESI-MS: m/z 499.4 [M + 1]⁺. C₂₆H₂₆N₆O₃S₂ (498.18). HPLC purity: 98.11%.

(S)-3-((3-((4-(4-Cyano-2,6-dimethylphenoxy)thieno[3,2-d]-pyrimidin-2-yl)amino)pyrrolidin-1-yl)methyl)benzamide (8d3).

Recrystallized from EA/PE as a white solid, 53% yield. Mp: 109–111 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 8.20 (d, J = 5.4 Hz, 1H, C₆-thienopyrimidine-H), 7.81–7.74 (m, 2H), 7.69 (s, 2H, C₃,C₅-Ph″-H), 7.42–7.41 (m, 2H), 7.34 (s, 2H, CONH₂), 7.25 (d, J = 5.4 Hz, 1H, C₇-thienopyrimidine-H), 7.16 (s, 1H, NH), 4.30 (s, 1H), 3.56 (s, 2H, N-CH₂), 2.85–2.81 (m, 2H), 2.30–2.26 (m, 2H), 2.08 (s, 6H), 1.72–1.68 (m, 2H). ¹³C NMR (100 MHz, DMSO-d₆) δ 168.3, 160.8, 134.6, 133.2, 132.9, 131.9, 128.5, 128.2, 126.4, 119.0, 109.0, 60.4, 59.8, 52.9, 39.4, 30.9, 16.1. ESI-MS: m/z 499.5 [M + 1]⁺. C₂₆H₂₆N₆O₃S₂ (498.18). HPLC purity: 95.73%.

(S)-3,5-Dimethyl-4-((2-((1-(4-(methylsulfonyl)benzyl)pyrrolidin-3-yl)amino)thieno[3,2-d]pyrimidin-4-yl)oxy)benzonitrile (8d4).

Recrystallized from EA/PE as a white solid, 61% yield. Mp: 95–98 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 8.20 (d, J = 5.3 Hz, 1H, C₆-thienopyrimidine-H), 7.87 (d, J = 8.0 Hz, 2H, C₃,C₅-Ph′-H), 7.69 (s, 2H, C₃,C₅-Ph″-H), 7.55 (d, J = 7.9 Hz, 2H, C₂,C₆-Ph′-H), 7.25 (d, J = 5.5 Hz, 1H, C₇-thienopyrimidine-H), 7.14 (s, 1H, NH), 4.32, (s, 1H), 3.65 (s, 2H, N-CH₂), 3.22 (s, 3H, SO₂CH₃), 2.87–2.79 (m, 2H), 2.30–2.28 (m, 2H), 2.08 (s, 6H), 1.79–1.48 (m, 2H). ¹³C NMR (100 MHz, DMSO-d₆) δ 162.7, 160.7, 153.4, 139.8, 133.2, 132.9, 129.6, 127.4, 127.0, 119.0, 109.1, 60.4, 59.2, 53.0, 44.0, 39.4, 30.8, 16.1. ESI-MS: m/z 534.0 [M + 1]⁺. C₂₇H₂₇N₅O₃S₂ (533.16). HPLC purity: 95.81%.

(S)-3,5-Dimethyl-4-((2-((1-(pyridin-4-ylmethyl)pyrrolidin-3-yl)amino)thieno[3,2-d]pyrimidin-4-yl)oxy)benzonitrile (8d5).

Recrystallized from EA/PE as a white solid, 54% yield. Mp: 184–486 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 8.51 (d, J = 4.9 Hz, 2H, C₃,C₅-Py-H), 8.21 (d, J = 5.4 Hz, 1H, C₆-thienopyrimidine-H), 7.69 (s, 2H, C₃,C₅-Ph″-H), 7.29 (d, J = 4.9 Hz, 2H, C₂,C₆-Py-H), 7.25 (d, J = 5.5 Hz, 1H, C₇-thienopyrimidine-H), 7.14 (s, 1H, NH), 4.32 (s, 1H), 3.54 (s, 2H, N-CH₂), 2.86–2.84 (m, 2H), 2.30–2.28 (m, 2H), 2.08 (s, 6H), 1.82–1.63 (m, 2H). ¹³C NMR (100 MHz, DMSO-d₆) δ 162.4, 153.5, 149.9, 148.5, 133.1, 132.9, 124.0, 119.1, 109.0, 60.3, 58.7, 53.1, 31.2, 16.2. ESI-MS: m/z 457.4 [M + 1]⁺. C₂₅H₂₄N₆OS (456.17). HPLC purity: 99.42%.

4-((3-((4-(4-Cyano-2,6-dimethylphenoxy)thieno[3,2-d]-pyrimidin-2-yl)amino)azetidin-1-yl)methyl)benzenesulfonamide (8e1).

Recrystallized from EA/PE as a white solid, 63% yield. Mp: 130–1321 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 8.22 (d, J = 5.4 Hz, 1H, C₆-thienopyrimidine-H), 7.76 (d, J = 8.2 Hz, 2H, C₃,C₅-Ph′-H), 7.73 (s, 2H, C₃,C₅-Ph″-H), 7.42 (d, J = 8.0 Hz, 2H, C₂,C₆-Ph′-H), 7.30 (s, 2H, SO₂NH₂), 7.26 (d, J = 5.4 Hz, 1H, C₇-thienopyrimidine-H), 7.13 (s, 1H, NH), 4.37 (s, 1H), 3.60 (s, 2H, N-CH₂), 3.46–3.43 (m, 2H), 2.98–2.96 (m, 2H), 2.11 (s, 6H). ¹³C NMR (100 MHz, DMSO-d₆) δ 153.3, 143.2, 143.0, 133.2, 133.0, 128.7, 126.0, 125.9, 119.0, 109.1, 62.5, 61.2, 40.4, 16.2. ESI-MS: m/z 521.3 [M + 1]⁺. C₂₅H₂₄N₆O₃S₂ (520.14). HPLC purity: 96.26%.

4-((3-((4-(4-Cyano-2,6-dimethylphenoxy)thieno[3,2-d]-pyrimidin-2-yl)amino)azetidin-1-yl)methyl)benzamide (8e2).

Recrystallized from EA/PE as a white solid, 66% yield. Mp: 143–145 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 8.22 (d, J = 5.4 Hz, 1H, C₆-thienopyrimidine-H), 7.81 (d, J = 8.0 Hz, 2H, C₃,C₅-Ph′-H), 7.72 (s, 2H, C₃,C₅-Ph″-H), 7.48 (s, 2H, CONH₂), 7.30 (d, J = 7.0 Hz, 2H, C₂,C₆-Ph′-H), 7.26 (d, J = 5.4 Hz, 1H, C₇-thienopyrimidine-H), 7.12 (s, 1H, NH), 4.39 (s, 1H), 3.58 (s, 2H, N-CH₂), 3.47–2.46 (m, 2H), 2.93–2.91 (m, 2H), 2.11 (s, 6H). ¹³C NMR (101 MHz, DMSO-d₆) δ 168.2, 162.5, 153.3, 143.2, 133.2, 133.0, 128.2, 127.9, 125.9, 119.0, 109.1, 62.5, 61.2, 40.4, 16.2. ESI-MS: m/z 485.5 [M + 1]⁺. C₂₆H₂₄N₆O₂S (484.17). HPLC purity: 95.66%.

3-((3-((4-(4-Cyano-2,6-dimethylphenoxy)thieno[3,2-d]-pyrimidin-2-yl)amino)azetidin-1-yl)methyl)benzamide (8e3).

Recrystallized from EA/PE as a white solid, 62% yield. Mp: 210–212 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 8.21 (d, J = 5.3 Hz, 1H, C₆-thienopyrimidine-H), 7.82–7.76 (m, 2H), 7.68 (s, 2H, C₃,C₅-Ph″-H), 7.43–7.42 (m, 2H), 7.37 (s, 2H, CONH₂), 7.26 (d, J = 5.4 Hz, 1H, C₇-thienopyrimidine-H), 7.13 (s, 1H, NH), 4.38 (s, 1H), 3.57 (s, 2H, N-CH₂), 3.46–3.43(m, 2H), 2.96–2.94 (m, 2H), 2.08 (s, 6H). ¹³C NMR (100 MHz, DMSO-d₆) δ 168.2, 162.5, 153.3, 143.2, 133.1, 132.8, 128.2, 128.0, 125.9, 119.0, 109.1, 62.5, 61.2, 40.4, 16.2. ESI-MS: m/z 485.4 [M + 1]⁺. C₂₆H₂₄N₆O₂S (484.17). HPLC purity: 98.24%.

3,5-Dimethyl-4-((2-((1-(4-(methylsulfonyl)benzyl)azetidin-3-yl)amino)thieno[3,2-d]pyrimidin-4-yl)oxy)benzonitrile (8e4).

Recrystallized from EA/PE as a white solid, 55% yield. Mp: 175–177 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 8.21 (d, J = 5.3 Hz, 1H, C₆-thienopyrimidine-H), 7.77 (d, J = 8.0 Hz, 2H, C₃,C₅-Ph′-H), 7.73 (s, 2H, C₃,C₅-Ph″-H), 7.43 (d, J = 7.9 Hz, 2H, C₂,C₆-Ph′-H), 7.25 (d, J = 5.5 Hz, 1H, C₇-thienopyrimidine-H), 7.14 (s, 1H, NH), 4.36 (s, 1H), 3.61 (s, 2H, N-CH₂), 3.46–3.43 (m, 2H), 3.22 (s, 3H, SO₂CH₃), 2.98–2.95 (m, 2H), 2.08 (s, 6H). ¹³C NMR (100 MHz, DMSO-d₆) δ 153.2, 143.3, 143.1, 133.2, 133.0, 128.7, 126.0, 125.7, 119.0, 109.1, 62.7, 61.3, 40.5, 16.2. ESI-MS: m/z 520.4 [M + 1]⁺. C₂₆H₂₅N₅O₃S₂ (519.14). HPLC purity: 95.01%.

3,5-Dimethyl-4-((2-((1-(pyridin-4-ylmethyl)azetidin-3-yl)amino)thieno[3,2-d]pyrimidin-4-yl)oxy)benzonitrile (8e5).

Recrystallized from EA/PE as a white solid, 61% yield. Mp: 199–201 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 8.52 (d, J = 4.9 Hz, 2H, C₃,C₅-Py-H), 8.21 (d, J = 5.3 Hz, 1H, C₆-thienopyrimidine-H), 7.71 (s, 2H, C₃,C₅-Ph″-H), 7.29 (d, J = 4.9 Hz, 2H, C₂,C₆-Py-H), 7.25 (d, J = 5.5 Hz, 1H, C₇-thienopyrimidine-H), 7.15 (s, 1H, NH), 4.32 (s, 1H), 3.54 (s, 2H, N-CH₂), 3.45–3.43 (m, 2H), 2.97–2.96 (m, 2H), 2.08 (s, 6H). ¹³C NMR (100 MHz, DMSO-d₆) δ 162.4, 153.5, 149.9, 148.5, 133.1, 132.9, 124.0, 119.1, 109.0, 62.7, 61.2, 40.4, 16.2. ESI-MS: m/z 443.5 [M + 1]⁺. C₂₄H₂₂N₆OS (442.16). HPLC purity: 95.78%.

General Procedure for the Preparation of Compounds 10a–f. 4-((2-Chloro-6,7-dihydrothieno[3,2-d]pyrimidin-4-yl)oxy)-3,5-dimethylbenzonitrile (10a).

The synthetic method was similar to that described for IIIA-2 except that the starting material was 2,4-dichloro-6,7-dihydrothieno[3,2-d]pyrimidine (9a). Recrystallized from EA/PE as a white solid, 86% yield. Mp: 272–274 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 7.67 (s, 2H, C₃,C₅-Ph-H), 3.37–3.35 (m, 2H), 3.14 (t, J = 8.2 Hz, 2H, S-CH₂), 2.06 (s, 6H). HRMS: m/z 318.0411 [M + 1]⁺. C₁₅H₁₂ClN₃OS (317.0390). HPLC purity: 99.62%.

4-((2-Chloro-5,7-dihydrothieno[3,4-d]pyrimidin-4-yl)oxy)-3,5-dimethylbenzonitrile (10b).

The synthetic method was similar to that described for IIIA-2 except that the starting material was 2,4-dichloro-5,7-dihydrothieno[3,4-d]pyrimidine (9b). Recrystallized from EA/PE as a white solid, 88% yield. Mp: 268–270 °C. ¹H NMR (400 MHz, DMSO-d₆): δ 7.72 (s, 2H, C₃,C₅-Ph-H), 4.12 (s, 2H, S-CH₂), 4.07 (s, 2H, S-CH₂), 2.10 (s, 6H). ESI-MS: m/z 318.2 [M + 1]⁺. C₁₅H₁₂ClN₃OS (317.04). HPLC purity: 99.20%.

4-((2-Chloro-5,7-dihydrofuro[3,4-d]pyrimidin-4-yl)oxy)-3,5-dimethylbenzonitrile (10c).

The synthetic method was similar to that described for IIIA-2 except that the starting material was 2,4-dichloro-5,7-dihydrofuro[3,4-d]pyrimidine (9c). Recrystallized from EA/PE as a white solid, 88% yield. Mp: 180–183 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 7.69 (s, 2H, C₃,C₅-Ph-H), 4.97 (s, 2H, C₅-dihydrofuropyrimidine), 4.80 (s, 2H, C₇-dihydrofuropyrimidine), 2.10 (s, 6H). HRMS: m/z 302.0687 [M + 1]⁺. C₁₅H₁₂ClN₃O₂ (301.0618). HPLC purity: 98.96%.

4-((2-Chloro-6,7-dihydro-5H-cyclopenta[d]pyrimidin-4-yl)oxy)-3,5-dimethylbenzonitrile (10d).

The synthetic method was similar to that described for IIIA-2 except that the starting material was 2,4-dichloro-6,7-dihydro-5H-cyclopenta[d]pyrimidine (9d). Recrystallized from EA/PE as a white solid, 97% yield. Mp: 254–256 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 7.73 (s, 2H, C₃,C₅-Ph-H), 3.04 (t, J = 7.8 Hz, 2H), 2.92 (t, J = 7.7 Hz, 2H), 2.13 (p, J = 7.7 Hz, 2H), 2.09 (s, 6H). HRMS: m/z 300.0895 [M + 1]⁺. C₁₆H₁₄ClN₃O (299.0825). HPLC purity: 99.20%.

4-((2-Chloro-5,6,7,8-tetrahydroquinazolin-4-yl)oxy)-3,5-dimethylbenzonitrile (10e).

The synthetic method was similar to that described for IIIA-2 except that the starting material was 2,4-dichloro-5,6,7,8-tetrahydroquinazoline (9e). Recrystallized from EA/PE as a white solid, 88% yield. Mp: 175–177 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 7.65 (s, 2H, C₃,C₅-Ph-H), 2.56 (d, J = 16.0 Hz, 4H), 2.06 (s, 6H), 1.72 (t, J = 3.3 Hz, 4H). HRMS: m/z 314.1053 [M + 1]⁺. C₁₇H₁₆ClN₃O (313.0982). HPLC purity: 98.65%.

4-((2-Chloro-7,8-dihydro-6H-thiopyrano[3,2-d]pyrimidin-4-yl)oxy)-3,5-dimethylbenzonitrile (10f).

The synthetic method was similar to that described for IIIA-2 except that the starting material was 2,4-dichloro-7,8-dihydro-6H-thiopyrano[3,2-d]pyrimidine (9f). Recrystallized from EA/PE as a white solid, 66% yield. Mp: 254–255 °C. ¹H NMR (400 MHz, DMSO-d₆) δ: 7.73 (s, 2H), 3.16 (d, J = 5.6 Hz, 2H), 2.92 (t, J = 6.2 Hz, 2H), 2.23–2.13 (m, 2H), 2.07 (s, 6H). ESI-MS: m/z 332.4 [M + 1]⁺. HPLC purity: 98.14%.

General Procedure for the Preparation of Compounds 12a–f.

The synthetic method was similar to that described for 7a except that the starting material 10a–f (1.0 mmol) was reacted with tert-butyl 4-aminopiperidine-1-carboxylate (1.2 mmol), respectively.

3,5-Dimethyl-4-((2-(piperidin-4-ylamino)-6,7-dihydrothieno[3,2-d]pyrimidin-4-yl)oxy)benzonitrile (12a).

Recrystallized from EA/PE as a white solid, 58% yield. Mp: 135–137 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 7.67 (s, 2H, C₃,C₅-Ph-H), 7.09 (s, 1H, NH), 3.36–3.35 (m, 3H), 3.13 (t, J = 8.0 Hz, 2H, S-CH₂), 2.71–2.62 (m, 2H), 2.06 (s, 6H), 1.83–1.45 (m, 6H). HRMS: m/z 382.1692 [M + 1]⁺. C₂₀H₂₃N₅OS (381.1623). HPLC purity: 95.82%.

3,5-Dimethyl-4-((2-(piperidin-4-ylamino)-5,7-dihydrothieno[3,4-d]pyrimidin-4-yl)oxy)benzonitrile (12b).

Recrystallized from EA/PE as a white solid, 96% yield. Mp: 268–2701 °C. ¹H NMR (400 MHz, DMSO-d₆): δ 7.72 (s, 2H, C₃,C₅-Ph-H), 6.89 (s, 1H, NH), 4.12 (s, 2H, S-CH₂), 4.08 (s, 2H, S-CH₂), 3.72–3.70 (m, 1H), 2.74–2.72 (m, 2H), 2.10 (s, 6H), 1.97–1.75 (m, 4H), 1.47–1.42 (m, 2H). ESI-MS: m/z 382.2 [M + 1]⁺. C₂₀H₂₃N₅OS (381.16). HPLC purity: 99.47%.

3,5-Dimethyl-4-((2-(piperidin-4-ylamino)-5,7-dihydrofuro-[3,4-d]pyrimidin-4-yl)oxy)benzonitrile (12c).

Recrystallized from EA/PE as a white solid, 61% yield. Mp: 122–124 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 7.67 (s, 2H, C₃,C₅-Ph-H), 7.08 (s, 1H, NH), 4.96 (s, 2H, C₅-dihydrofuropyrimidine), 4.77 (s, 2H, C₇-dihydrofuropyrimidine), 3.63–3.61 (m, 1H), 2.67 (s, 2H), 2.09 (s, 6H), 1.97–1.32 (m, 6H). HRMS: m/z 366.1843 [M + 1]⁺. C₂₀H₂₃N₅O₂ (365.1852). HPLC purity: 96.12%.

3,5-Dimethyl-4-((2-(piperidin-4-ylamino)-6,7-dihydro-5H-cyclopenta[d]pyrimidin-4-yl)oxy)benzonitrile (12d).

Recrystallized from EA/PE as a white solid, 67% yield. Mp: 135–137 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 7.64 (s, 2H, C₃,C₅-Ph-H), 6.90 (s, 1H, NH), 3.65 (s, 1H), 2.76–2.72 (m, 4H), 2.69–2.59 (m, 2H), 2.07 (s, 6H), 2.02–1.97 (m, 2H), 1.81–1.21 (m, 6H). HRMS: m/z 364.2132 [M + 1]⁺. C₂₁H₂₅N₅O (363.2059). HPLC purity: 95.78%.

3,5-Dimethyl-4-((2-(piperidin-4-ylamino)-5,6,7,8-tetrahydroquinazolin-4-yl)oxy)benzonitrile (12e).

Recrystallized from EA/PE as a white solid, 59% yield. Mp: 193–196 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 7.65 (s, 2H, C₃,C₅-Ph-H), 6.82 (s, 1H, NH), 3.59 (s, 1H), 2.72–2.70 (m, 2H), 2.57 (d, J = 16.0 Hz, 4H), 2.06 (s, 6H), 1.76 (t, J = 3.3 Hz, 4H), 1.70–1.23 (m, 6H). HRMS: m/z 378.2283 [M + 1]⁺. C₂₂H₂₇N₅O (377.2216). HPLC purity: 97.14%.

3,5-Dimethyl-4-((2-(piperidin-4-ylamino)-7,8-dihydro-6H-thiopyrano[3,2-d]pyrimidin-4-yl)oxy)benzonitrile (12f).

Recrystallized from EA/PE as a white solid, 62% yield. Mp: >250 °C. ESI-MS: m/z 396.4 [M + 1]⁺. C₂₁H₂₅N₅OS (395.1780). HPLC purity: 97.93%.

General Procedure for the Preparation of Final Compounds 13a1–6, 13b1–6, 13c1–6, 13d1–6, 13e1–6, and 13f1–6.

The synthetic method was similar to that described for 8a1–5 except that the starting material 12a–f (1.0 mmol) was reacted with substituted benzyl chloride (bromide) (1.2 mmol), respectively.

4-((4-((4-(4-Cyano-2,6-dimethylphenoxy)-6,7-dihydrothieno[3,2-d]pyrimidin-2-yl)amino)piperidin-1-yl)methyl)benzenesulfonamide (13a1).

Recrystallized from EA/PE as a white solid, 58% yield. Mp: 201–203 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 7.77 (d, J = 8.0 Hz, 2H, C₃,C₅-Ph′-H), 7.67 (s, 2H, C₃,C₅-Ph″-H), 7.45 (d, J = 8.0 Hz, 2H, C₂,C₆-Ph′-H), 7.31 (s, 2H, SO₂NH₂), 7.09 (s, 1H, NH), 3.45 (s, 2H, N-CH₂), 3.37–3.35 (m, 3H), 3.14 (t, J = 8.2 Hz, 2H, S-CH₂), 2.71–2.60 (m, 2H), 2.06 (s, 6H), 1.80–1.20 (m, 6H). ¹³C NMR (100 MHz, DMSO-d₆) δ 162.0, 160.6, 143.3, 143.1, 133.1, 129.4, 126.0, 119.1, 108.6, 61.9, 52.6, 31.5, 29.3, 16.2. HRMS: m/z 551.1890 [M + 1]⁺. C₂₇H₃₀N₆O₃S₂ (550.1821). HPLC purity: 99.71%.

4-((4-((4-(4-Cyano-2,6-dimethylphenoxy)-6,7-dihydrothieno[3,2-d]pyrimidin-2-yl)amino)piperidin-1-yl)methyl)benzamide (13a2).

Recrystallized from EA/PE as a white solid, 67% yield. Mp: 245–248 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 7.92 (s, 1H), 7.82 (d, J = 8.0 Hz, 2H, C₃,C₅-Ph′-H), 7.67 (s, 2H, C₃,C₅-Ph″-H), 7.34–7.32 (m, 3H), 6.95 (s, 1H, NH), 3.43 (s, 2H, N-CH₂), 3.39–3.30 (m, 3H), 3.14 (t, J = 8.1 Hz, 2H, S-CH₂), 2.67 (s, 2H), 2.06 (s, 6H), 1.78–1.19 (m, 6H). ¹³C NMR (101 MHz, DMSO-d₆) δ 168.2, 160.6, 142.5, 133.4, 133.1, 132.6, 128.8, 127.8, 108.6, 62.2, 52.6, 36.9, 31.5, 29.3, 16.2. HRMS: m/z 515.2219 [M + 1]⁺. C₂₈H₃₀N₆O₂S (514.2151). HPLC purity: 96.63%.

3,5-Dimethyl-4-((2-((1-(4-(methylsulfonyl)benzyl)piperidin-4-yl)amino)-6,7-dihydrothieno[3,2-d]pyrimidin-4-yl)oxy)benzonitrile (13a3).

Recrystallized from EA/PE as a white solid, 70% yield. Mp: 142–144 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 7.87 (d, J = 7.9 Hz, 2H, C₃,C₅-Ph′-H), 7.67 (s, 2H, C₃,C₅-Ph″-H), 7.54 (d, J = 8.0 Hz, 2H, C₂,C₆-Ph′-H), 7.10 (s, 1H, NH), 3.50 (s, 2H, N-CH₂), 3.40–3.29 (m, 3H), 3.20 (s, 3H, SO₂CH₃), 3.15 (t, J = 8.0 Hz, 2H, S-CH₂), 2.67 (s, 2H), 2.07 (s, 6H), 1.77–1.21 (m, 6H). ¹³C NMR (100 MHz, DMSO-d₆) δ 160.6, 145.4, 139.8, 133.1, 132.7, 129.7, 127.4, 119.1, 108.6, 61.8, 52.6, 44.0, 31.6, 29.3, 16.2. HRMS: m/z 550.1946 [M + 1]⁺. C₂₈H₃₁N₅O₃S₂ (549.1868). HPLC purity: 97.62%.

3,5-Dimethyl-4-((2-((1-(pyridin-4-ylmethyl)piperidin-4-yl)amino)-6,7-dihydrothieno[3,2-d]pyrimidin-4-yl)oxy)benzonitrile (13a4).

Recrystallized from EA/PE as a white solid, 54% yield. Mp: 140–142 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 8.49 (d, J = 5.4 Hz, 2H, C₃,C₅-Ph′-H), 7.66 (s, 2H, C₃,C₅-Ph″-H), 7.28 (d, J = 5.1 Hz, 2H, C₂,C₆-Ph′), 7.07 (s, 1H, NH), 3.43 (s, 2H, N-CH₂), 3.39–3.25 (m, 3H), 3.15 (t, J = 8.1 Hz, 2H, S-CH₂), 2.67 (s, 2H), 2.07 (s, 6H), 1.79–1.23 (m, 6H). ¹³C NMR (100 MHz, DMSO-d₆) δ 162.0, 160.6, 153.9, 149.9, 148.1, 133.1, 132.6, 124.1, 108.6, 61.2, 52.6, 36.9, 31.5, 29.3, 16.2. HRMS: m/z 473.2118 [M + 1]⁺. C₂₆H₂₈N₆OS (472.2045). HPLC purity: 99.33%.

3,5-Dimethyl-4-((2-((1-(4-nitrobenzyl)piperidin-4-yl)amino)-6,7-dihydrothien[3,2-d]pyrimidin-4-yl)oxy)benzonitrile (13a5).

Recrystallized from EA/PE as a white solid, 62% yield. Mp: 158–160 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 8.19 (d, J = 8.3 Hz, 2H, C₃,C₅-Ph′-H), 7.66 (s, 2H, C₃,C₅-Ph″-H), 7.55 (d, J = 8.3 Hz, 2H, C₂,C₆-Ph′-H), 7.07 (s, 1H, NH), 3.54 (s, 2H, N-CH₂), 3.39–3.25 (m, 3H), 3.15 (t, J = 8.0 Hz, 2H, S-CH₂), 2.68 (s, 2H), 2.07 (s, 6H), 1.82–1.20 (m, 6H). ¹³C NMR (100 MHz, DMSO-d₆) δ 162.0, 160.6, 147.5, 146.9, 133.1, 132.7, 130.0, 123.8, 119.0, 108.6, 61.6, 52.6, 39.4, 36.9, 31.6, 29.3, 16.2. HRMS: m/z 517.2016 [M + 1]⁺. C₂₇H₂₈N₆O₃S (516.1944). HPLC purity: 99.92%.

3-((4-((4-(4-Cyano-2,6-dimethylphenoxy)-6,7-dihydrothieno[3,2-d]pyrimidin-2-yl)amino)piperidin-1-yl)methyl)benzamide (13a6).

Recrystallized from EA/PE as a white solid, 47% yield. Mp: 215–217 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 7.95 (s, 1H, C₂-Ph′-H), 7.75 (dd, J = 11.0, 3.9 Hz, 2H, C₅,C₆-Ph′-H), 7.66 (s, 2H, C₃,C₅-Ph″-H), 7.46–7.25 (m, 3H), 7.06 (s, 1H, NH), 3.43 (s, 2H, N-CH₂), 3.39–3.25 (m, 3H), 3.15 (t, J = 8.1 Hz, 2H, S-CH₂), 2.67 (s, 2H), 2.07 (s, 6H), 1.81–1.24 (m, 6H).¹³C NMR (100 MHz, DMSO-d₆) δ 168.4, 162.0, 160.6, 139.2, 134.6, 133.1, 132.7, 132.0, 128.4, 126.4, 119.0, 108.6, 62.4, 52.6, 36.9, 31.6, 29.3, 16.2. HRMS: m/z 515.2229 [M + 1]⁺. C₂₈H₃₀N₆O₂S (514.2151). HPLC purity: 99.95%.

4-((4-((4-(4-Cyano-2,6-dimethylphenoxy)-5,7-dihydrothieno[3,4-d]pyrimidin-2-yl)amino)piperidin-1-yl)methyl)benzenesulfonamide (13b1).

Recrystallized from EA/PE as a white solid, 79% yield. Mp: 144–146 °C. ¹H NMR (400 MHz, DMSO-d₆): δ 7.78 (d, J = 8.2 Hz, 2H, C₃,C₅-Ph′-H), 7.72 (s, 2H, C₃,C₅-Ph″-H), 7.46 (d, J = 8.1 Hz, 2H, C₂,C₆-Ph′-H), 7.30 (s, 2H, SO₂NH₂), 6.89 (s, 1H, NH), 4.12 (s, 2H, S-CH₂), 4.08 (s, 2H, S-CH₂), 3.72 (s, 1H), 3.42 (s, 2H, N-CH₂), 2.72–2.74 (m, 2H), 2.10 (s, 6H), 1.79–2.04 (m, 4H), 1.41–1.47 (m, 2H). ¹³C NMR (100 MHz, DMSO-d₆): δ 165.7, 161.7, 160.3, 153.7, 143.0, 135.3, 134.2, 126.0, 123.9, 123.7, 119.2, 109.7, 61.2, 52.8, 48.3, 31.7, 16.2. HRMS: m/z 551.1890 [M + 1]⁺. C₂₇H₃₀N₆O₃S₂ (550.1821). HPLC purity: 98.77%.

3,5-Dimethyl-4-((2-((1-(4-(methylsulfonyl)benzyl)piperidin-4-yl)amino)-5,7-dihydrothieno[3,4-d]pyrimidin-4-yl)oxy)benzonitrile (13b2).

Recrystallized from EA/PE as a white solid, 71% yield. Mp: 181–183 °C. ¹H NMR (400 MHz, DMSO-d₆, ppm): δ 7.84 (d, 2H, J = 8.2 Hz, C₃,C₅-Ph′-H), 7.71 (s, 2H, C₃,C₅-Ph″-H), 7.52 (d, 2H, J = 8.2 Hz, C₂,C₆-Ph′-H), 6.88 (s, 1H, NH), 4.12 (s, 2H, S-CH₂), 4.08 (s, 2H, S-CH₂), 3.75–3.78 (m, 1H), 3.57 (s, 2H, N-CH₂), 3.06 (s, 3H, SO₂CH₃), 2.71–2.75 (m, 2H), 2.10 (s, 6H), 1.96–2.09 (m, 4H), 1.39–1.41 (m, 2H). ¹³C NMR (100 MHz, DMSO-d₆): δ 165.1, 162.3, 159.4, 153.9, 145.2, 139.5, 134.3, 133.0, 126.8, 123.3, 118.9, 109.7, 62.3, 52.6, 48.5, 44.7, 32.0, 16.6. HRMS: m/z 550.1942 [M + 1]⁺. C₂₈H₃₁N₅O₃S₂ (549.1868). HPLC purity: 95.16%.

3,5-Dimethyl-4-((2-((1-(4-nitrobenzyl)piperidin-4-yl)amino)-5,7-dihydrothieno[3,4-d]pyrimidin-4-yl)oxy)benzonitrile (13b3).

Recrystallized from EA/PE as a white solid, 82% yield. Mp: 180–182 °C. ¹H NMR (400 MHz, DMSO-d₆, ppm): δ 7.84 (d, 2H, J = 8.3 Hz, C₃,C₅-Ph′-H), 7.72 (s, 2H, C₃,C₅-Ph″-H), 7.57 (d, J = 8.4 Hz, 2H, C₂,C₆-Ph′-H), 6.90 (s, 1H, NH), 4.14 (s, 2H, S-CH₂), 4.05 (s, 2H, S-CH₂), 3.75–3.76 (m, 1H), 3.59 (s, 2H, N-CH₂), 2.73–2.75 (m, 2H), 2.11 (s, 6H), 1.80–1.85 (m, 2H), 1.41–1.44 (m, 4H). ¹³C NMR (100 MHz, DMSO-d₆): δ 162.8, 160.7, 153.0, 147.6, 147.1, 141.3, 136.4, 133.7, 132.9, 123.8, 119.3, 109.8, 61.6, 52.9, 48.2, 31.7, 16.2. HRMS: m/z 517.2015 [M + 1]⁺. C₂₇H₂₈N₆O₃S (516.1944). HPLC purity: 95.28%.

4-((2-((1-(4-Aminobenzyl)piperidin-4-yl)amino)-5,7-dihydrothieno[3,4-d]pyrimidin-4-yl)oxy)-3,5-dimethylbenzonitrile (13b4).

Recrystallized from EA/PE as a white solid, 55% yield. Mp: 143–145 °C. ¹H NMR (400 MHz, DMSO-d₆ ppm): δ 7.80 (d, 2H, J = 8.3 Hz, C₃,C₅-Ph′-H), 7.71 (s, 2H, C₃,C₅-Ph″-H), 7.54 (d, J = 8.4 Hz, 2H, C₂,C₆-Ph′-H), 6.92 (s, 1H, NH), 4.83 (s, 2H, NH₂), 4.12 (s, 2H, S-CH₂), 4.04 (s, 2H, S-CH₂), 3.73–3.76 (m, 1H), 3.58 (s, 2H, N-CH₂), 2.71–2.73 (m, 2H), 2.10 (s, 6H), 1.82–1.87 (m, 2H), 1.71–1.73 (m, 2H), 1.45–1.48 (m, 2H). ¹³C NMR (100 MHz, DMSO-d₆): δ 162.0, 160.7, 153.1, 147.3, 146.7, 140.3, 136.9, 133.4, 132.2, 123.1, 119.3, 109.5, 61.6, 52.4, 48.5, 31.7, 16.2. HRMS: m/z 487.2272 [M + 1]⁺. C₂₇H₃₀N₆OS (486.2202). HPLC purity: 96.38%.

N-(4-((4-((4-(4-Cyano-2,6-dimethylphenoxy)-5,7-dihydrothieno[3,4-d]pyrimidin-2-yl)amino)piperidin-1-yl)methyl)phenyl)methanesulfonamide (13b5).

Recrystallized from EA/PE as a white solid, 47% yield. Mp: 186–187 °C. ¹H NMR (400 MHz, DMSO-d₆) δ: 9.68 (s, 1H), 7.67 (s, 2H, C₃,C₅-Ph″-H), 7.20 (d, J = 8.2 Hz, 2H, C₃,C₅-Ph′-H), 7.13 (d, J = 8.1 Hz, 2H, C₂,C₆-Ph′-H), 6.97 (s, 1H), 4.13 (s, 2H, S-CH₂), 4.07 (s, 2H, S-CH₂), 3.66 (s, 1H), 3.35 (s, 2H, N-CH₂), 2.96 (s, 3H, SO₂CH₃), 2.63–2.73 (m, 2H), 2.08 (s, 6H, 2 × CH₃), 1.23–1.89 (m, 6H). ¹³C NMR (100 MHz, DMSO) δ: 164.4, 162.1, 153.7, 151.0, 137.5, 133.1, 132.5, 130.1, 120.4, 120.2, 119.1, 116.1, 113.4, 112.2, 108.6, 93.6, 62.0, 52.3, 48.4, 31.6, 16.3. HRMS: m/z 566.2133 [M + 1]⁺. C₂₈H₃₂N₆O₃S₂ (564.1977). HPLC purity: 98.01%.

4-((4-((4-(4-Cyano-2,6-dimethylphenoxy)-5,7-dihydrofuro-[3,4-d]pyrimidin-2-yl)amino)piperidin-1-yl)methyl)benzenesulfonamide (13c1).

Recrystallized from EA/PE as a white solid, 66% yield. Mp: 191–193 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 7.77 (d, J = 8.0, Hz, 2H, C₃,C₅-Ph′-H), 7.67 (s, 2H, C₃,C₅-Ph″-H), 7.45 (d, J = 8.0 Hz, 2H, C₂,C₆-Ph′-H), 7.31 (s, 2H, SO₂NH₂), 7.08 (s, 1H, NH), 4.96 (s, 2H, C₅-dihydrofuropyrimidine), 4.77 (s, 2H, C₇-dihydrofuropyrimidine), 3.63 (s, 1H), 3.47 (s, 2H, N-CH₂), 2.69 (s, 2H), 2.09 (s, 6H), 1.94–1.18 (m, 6H). ¹³C NMR (100 MHz, DMSO-d₆) δ 162.9, 162.0, 143.3, 143.1, 133.1, 129.4, 126.9, 126.2, 126.0, 119.0, 61.9, 52.6, 31.5, 16.2. HRMS: m/z 535.2126 [M + 1]⁺. C₂₇H₃₀N₆O₄S (534.2049). HPLC purity: 97.68%.

4-((4-((4-(4-Cyano-2,6-dimethylphenoxy)-5,7-dihydrofuro-[3,4-d]pyrimidin-2-yl)amino)piperidin-1-yl)methyl)benzamide (13c2).

Recrystallized from EA/PE as a white solid, 57% yield. Mp: 238–240 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 7.85 (s, 2H, CONH₂), 7.75 (d, J = 7.9 Hz, 2H, C₃,C₅-Ph′-H), 7.60 (s, 2H, C₃,C₅-Ph″-H), 7.26 (d, J = 8.1 Hz, 2H, C₂,C₆-Ph′-H), 7.00 (s, 1H, NH), 4.89 (s, 2H, C₅-dihydrofuropyrimidine), 4.70 (s, 2H, C₇-dihydrofuropyrimidine), 3.60 (s, 1H), 3.42 (s, 2H, N-CH₂), 2.74–2.53 (m, 2H), 2.02 (s, 6H), 1.93–1.11 (m, 6H). ¹³C NMR (100 MHz, DMSO-d₆) δ 168.2, 162.9, 162.5, 142.5, 133.4, 133.1, 132.5, 128.8, 127.8, 119.0, 108.7, 62.2, 52.6, 31.6, 16.2. HRMS: m/z 499.2457 [M + 1]⁺. C₂₈H₃₀N₆O₃ (498.2397). HPLC purity: 99.27%.

3,5-Dimethyl-4-((2-((1-(4-(methylsulfonyl)benzyl)piperidin-4-yl)amino)-5,7-dihydrofuro[3,4-d]pyrimidin-4-yl)oxy)benzonitrile (13c3).

Recrystallized from EA/PE as a white solid, 72% yield. Mp: 209–211 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 7.87 (d, J = 8.0 Hz, 2H, C₃,C₅-Ph′-H), 7.67 (s, 2H, C₃,C₅-Ph″-H), 7.54 (d, J = 8.0 Hz, 2H, C₂,C₆-Ph′-H), 7.08 (s, 1H, NH), 4.96 (s, 2H, C₅-dihydrofuropyrimidine), 4.77 (s, 2H, C₇-dihydrofuropyrimidine), (s, 1H), 3.51 (s, 2H, N-CH₂), 3.20 (s, 3H, SO₂CH₃), 2.76–2.62 (m, 2H), 2.09 (s, 6H), 1.90–1.15 (m, 6H). ¹³C NMR (100 MHz, DMSO-d₆) δ 162.9, 162.5, 145.4, 139.8, 133.1, 129.7, 127.4, 108.7, 69.3, 61.8, 52.6, 44.0, 31.5, 16.2. HRMS: m/z 534.2173 [M + 1]⁺. C₂₈H₃₁N₅O₄S (533.2097). HPLC purity: 99.34%.

3,5-Dimethyl-4-((2-((1-(pyridin-4-ylmethyl)piperidin-4-yl)amino)-5,7-dihydrofuro[3,4-d]pyrimidin-4-yl)oxy)benzonitrile (13c4).

Recrystallized from EA/PE as a white solid, 73% yield. Mp: 155–157 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 8.50 (d, J = 5.9 Hz, 2H, C₃,C₅-Ph′-H), 7.67 (s, 2H, C₃,C₅-Ph″-H), 7.28 (d, J = 5.1 Hz, 2H, C₂,C₆-Ph′-H), 7.09 (s, 1H, NH), 4.96 (s, 2H, C₅-dihydrofuropyrimidine), 4.77 (s, 2H, C₇-dihydrofuropyrimidine), 3.68 (s, 1H), 3.45 (s, 2H, N-CH₂), 2.77–2.59 (m, 2H), 2.09 (s, 6H), 1.91–1.22 (m, 6H). ¹³C NMR (100 MHz, DMSO-d₆) δ 162.9, 149.9, 148.1, 133.1, 124.1, 108.7, 69.4, 61.2, 52.6, 31.4, 16.2. HRMS: m/z 457.2345 [M + 1]⁺. C₂₆H₂₈N₆O₂ (456.2274). HPLC purity: 97.81%.

3,5-Dimethyl-4-((2-((1-(4-nitrobenzyl)piperidin-4-yl)amino)-5,7-dihydrofuro[3,4-d]pyrimidin-4-yl)oxy)benzonitrile (13c5).

Recrystallized from EA/PE as a white solid, 61% yield. Mp: 199–201 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 8.12 (d, J = 8.6 Hz, 2H, C₃,C₅-Ph′-H), 7.60 (s, 2H, C₃,C₅-Ph″-H), 7.49 (d, J = 8.4 Hz, 2H, C₂,C₆-Ph′-H), 7.02 (s, 1H, NH), 4.89 (s, 2H, C₅-dihydrofuropyrimidine), 4.70 (s, 2H, C₇-dihydrofuropyrimidine), 3.61 (s, 1H), 3.47 (s, 2H, N-CH₂), 2.63 (s, 2H), 2.02 (s, 6H), 1.80–1.14 (m, 6H). ¹³C NMR (100 MHz, DMSO-d₆) δ 162.9, 146.9, 133.1, 130.0, 123.8, 61.6, 52.6, 39.6, 39.4, 31.5, 16.2. HRMS: m/z 501.2247 [M + 1]⁺. C₂₇H₂₈N₆O₄ (500.2172). HPLC purity: 95.77%.

3-((4-((4-(4-Cyano-2,6-dimethylphenoxy)-5,7-dihydrofuro-[3,4-d]pyrimidin-2-yl)amino)piperidin-1-yl)methyl)benzamide (13c6).

Recrystallized from EA/PE as a white solid, 43% yield. Mp: 225–227 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 7.88 (s, 2H, CONH₂), 7.70 (s, 1H, C₂-Ph′-H), 7.69–7.66 (m, 1H), 7.60 (s, 2H, C₃,C₅-Ph″-H), 7.35–7.30 (m, 2H, C₂,C₆-Ph′-H), 7.01 (s, 1H, NH), 4.89 (s, 2H, C₅-dihydrofuropyrimidine), 4.70 (s, 2H, C₇-dihydrofuropyrimidine), 3.60 (s, 1H), 3.36 (s, 2H, N-CH₂), 2.63 (s, 2H), 2.02 (s, 6H), 1.92–1.16 (m, 6H). ¹³C NMR (100 MHz, DMSO-d₆) δ 168.4, 163.0, 162.5, 139.2, 134.6, 133.1, 132.0, 128.4, 126.4, 119.0, 108.7, 69.4, 62.4, 55.3, 52.6, 39.6, 31.6, 16.2. HRMS: m/z 499.2456 [M + 1]⁺. C₂₈H₃₀N₆O₃ (498.2379). HPLC purity: 97.74%.

4-((4-((4-(4-Cyano-2,6-dimethylphenoxy)-6,7-dihydro-5H-cyclopenta[d]pyrimidin-2-yl)amino)piperidin-1-yl)methyl)benzenesulfonamide (13d1).

Recrystallized from EA/PE as a white solid, 80% yield. Mp: 207–209 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 7.77 (d, J = 8.3 Hz, 2H, C₃,C₅-Ph′-H), 7.64 (s, 2H, C₃,C₅-Ph″-H), 7.45 (d, J = 8.1 Hz, 2H, C₂,C₆-Ph′-H), 7.30 (s, 2H, SO₂NH₂), 6.90 (s, 1H, NH), 3.65 (s, 1H), 3.45 (s, 2H), 2.76 (dt, J = 22.3, 7.7 Hz, 4H), 2.69–2.59 (m, 2H), 2.07 (s, 6H), 2.02 (d, J = 7.4 Hz, 2H), 1.81–1.21 (m, 6H). ¹³C NMR (100 MHz, DMSO-d₆) δ 162.0, 143.3, 143.1, 133.1, 132.6, 129.4, 126.0, 119.1, 108.3, 62.0, 52.6, 39.6, 31.6, 25.9, 22.0, 16.3. HRMS: m/z 533.2330 [M + 1]⁺. C₂₈H₃₂N₆O₃S (532.2257). HPLC purity: 97.64%.

4-((4-((4-(4-Cyano-2,6-dimethylphenoxy)-6,7-dihydro-5H-cyclopenta[d]pyrimidin-2-yl)amino)piperidin-1-yl)methyl)benzamide (13d2).

Recrystallized from EA/PE as a white solid, 68% yield. Mp: 225–227 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 7.90 (s, 2H, CONH₂), 7.81 (d, J = 7.9 Hz, 2H, C₃,C₅-Ph′-H), 7.64 (s, 2H, C₃,C₅-Ph″-H), 7.32 (d, J = 8.0 Hz, 2H, C₂,C₆-Ph′-H), 6.89 (s, 1H, NH), 3.65 (s, 1H), 3.48–3.36 (m, 2H), 2.76 (dt, J = 22.1, 7.7 Hz, 4H), 2.70–2.58 (m, 2H), 2.07 (s, 6H), 2.02 (d, J = 7.4 Hz, 2H), 1.70–1.23 (m, 6H). ¹³C NMR (100 MHz, DMSO-d₆) δ 168.2, 162.0, 143.1, 142.5, 133.4, 133.1, 129.5, 128.8, 127.8, 119.5, 108.9, 62.2, 52.7, 39.0, 31.2, 25.9, 22.0, 16.3. HRMS: m/z 497.2657 [M + 1]⁺. C₂₉H₃₂N₆O₂ (496.2587). HPLC purity: 95.88%.

3,5-Dimethyl-4-((2-((1-(4-(methylsulfonyl)benzyl)piperidin-4-yl)amino)-6,7-dihydro-5H-cyclopenta[d]pyrimidin-4-yl)oxy)benzonitrile (13d3).

Recrystallized from EA/PE as a white solid, 69% yield. Mp: 170–172 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 7.89–7.85 (m, 2H), 7.65 (s, 2H, C₃,C₅-Ph″-H), 7.54 (d, J = 8.1 Hz, 2H), 6.80 (s, 1H, NH), 3.66 (s, 1H), 3.50 (s, 2H, N-CH₂), 2.89 (s, 3H), 2.78 (dd, J = 14.7, 7.3 Hz, 4H), 2.71–2.61 (m, 2H), 2.07 (s, 6H), 2.03 (d, J = 7.5 Hz, 2H), 1.81–1.23 (m, 6H). ¹³C NMR (100 MHz, DMSO-d₆) δ 162.7, 162.0, 145.4, 139.8, 133.1, 132.6, 129.7, 127.4, 127.3, 108.3, 62.6, 61.9, 52.6, 44.1, 44.0, 36.2, 31.6, 31.2, 25.9, 22.0, 16.3. HRMS: m/z 532.2377 [M + 1]⁺. C₂₉H₃₃N₅O₃S (531.2304). HPLC purity: 96.53%.

3,5-Dimethyl-4-((2-((1-(pyridin-4-ylmethyl)piperidin-4-yl)amino)-6,7-dihydro-5H-cyclopenta[d]pyrimidin-4-yl)oxy)benzonitrile (13d4).

Recrystallized from EA/PE as a white solid, 72% yield. Mp: 173–175 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 8.57 (d, J = 7.9 Hz, 2H, C₃,C₅-Ph′-H), 7.64 (s, 2H, C₃,C₅-Ph″-H), 7.28 (d, J = 8.0 Hz, 2H, C₂,C₆-Ph′-H), 6.83 (s, 1H, NH), 3.60 (s, 1H), 3.48–3.42 (m, 2H), 2.76 (dt, J = 21.6, 7.2 Hz, 4H), 2.70–2.62 (m, 2H), 2.07 (s, 6H), 2.02, (d, J = 7.4 Hz, 2H), 1.85–1.21 (m, 6H). ¹³C NMR (100 MHz, DMSO-d₆) δ 168.2, 162.4, 142.9, 142.1, 133.7, 133.2, 129.5, 127.8, 1120.3, 108.7, 62.2, 52.6, 39.0, 31.6, 25.9, 22.0, 16.3. HRMS: m/z 455.2557 [M + 1]⁺. C₂₇H₃₀N₆O (454.2481). HPLC purity: 96.17%.

3,5-Dimethyl-4-((2-((1-(4-nitrobenzyl)piperidin-4-yl)amino)-6,7-dihydro-5H-cyclopenta[d]pyrimidin-4-yl)oxy)benzonitrile (13d5).

Recrystallized from EA/PE as a white solid, 71% yield. Mp: 200–212 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 8.17 (d, J = 8.1 Hz, 2H, C₃,C₅-Ph′-H), 7.65 (s, 2H, C₃,C₅-Ph″-H), 7.54 (d, J = 8.1 Hz, 2H), 6.80 (s, 1H, NH), 3.72 (s, 1H), 3.50 (s, 2H, N-CH₂), 2.78 (dd, J = 14.4, 7.2 Hz, 4H), 2.71–2.64 (m, 2H), 2.07 (s, 6H), 2.03 (d, J = 7.5 Hz, 2H), 1.81–1.23 (m, 6H). ¹³C NMR (100 MHz, DMSO-d₆) δ 159.7, 147.5, 146.9, 145.4, 139.8, 133.1, 132.6, 130.2, 127.4, 123.8, 108.3, 62.1, 61.7, 52.6, 44.1, 36.2, 31.6, 31.2, 25.9, 21.8, 16.2. HRMS: m/z 499.2456 [M + 1]⁺. C₂₈H₃₀N₆O₃ (498.2379). HPLC purity: 99.14%.

3-((4-((4-(4-Cyano-2,6-dimethylphenoxy)-6,7-dihydro-5H-cyclopenta[d]pyrimidin-2-yl)amino)piperidin-1-yl)methyl)benzamide (13d6).

Recrystallized from EA/PE as a white solid, 59% yield. Mp: 234–236 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 7.96 (s, 2H, CONH₂), 7.79–7.75 (m, 1H), 7.65 (s, 2H, C₃,C₅-Ph″-H), 7.39–7.35 (m, 3H), 6.80 (s, 1H, NH), 3.72 (s, 1H), 3.50 (s, 2H, N-CH₂), 2.78 (dd, J = 14.3, 7.2 Hz, 4H), 2.71–2.63 (m, 2H), 2.07 (s, 6H), 2.03 (d, J = 7.5 Hz, 2H), 1.80–1.18 (m, 6H). ¹³C NMR (100 MHz, DMSO-d₆) δ 162.3, 143.2, 142.5, 133.4, 133.1, 129.4, 128.8, 127.6, 119.5, 108.9, 62.2, 52.7, 39.0, 31.3, 25.9, 22.0, 16.3. ESI-MS: m/z 497.2657 [M + 1]⁺. C₂₉H₃₂N₆O₂ (496.2587). HPLC purity: 95.93%.

4-((4-((4-(4-Cyano-2,6-dimethylphenoxy)-5,6,7,8-tetrahydroquinazolin-2-yl)amino)piperidin-1-yl)methyl)benzenesulfonamide (13e1).

Recrystallized from EA/PE as a white solid, 63% yield. Mp: 221–224 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 7.77 (d, J = 8.0 Hz, 2H, C₃,C₅-Ph′-H), 7.65 (s, 2H, C₃,C₅-Ph″-H), 7.45 (d, J = 7.9 Hz, 2H, C₂,C₆-Ph′-H), 7.32 (s, 2H, SO₂NH₂), 6.82 (s, 1H, NH), 3.59 (s, 1H), 3.45 (s, 2H, N-CH₂), 2.72–2.69 (m, 2H), 2.57 (d, J = 16.0 Hz, 4H), 2.06 (s, 6H), 1.76 (t, J = 3.3 Hz, 4H), 1.70–1.01 (m, 6H). ¹³C NMR (100 MHz, DMSO-d₆) δ 166.0, 143.3, 143.1, 133.1, 132.5, 129.4, 126.0, 108.2, 62.0, 52.6, 39.4, 31.7, 22.5, 21.3, 16.2. HRMS: m/z 547.2485 [M + 1]⁺. C₂₉H₃₄N₆O₃S (546.2413). HPLC purity: 98.01%.

4-((4-((4-(4-Cyano-2,6-dimethylphenoxy)-5,6,7,8-tetrahydroquinazolin-2-yl)amino)piperidin-1-yl)methyl)benzamide (13e2).

Recrystallized from EA/PE as a white solid, 62% yield. Mp: 230–232 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 7.90 (s, 2H, CONH₂), 7.81 (d, J = 7.7 Hz, 2H, C₃,C₅-Ph′-H), 7.64 (s, 2H, C₃,C₅-Ph″-H), 7.31 (d, J = 7.9 Hz, 2H, C₂,C₆-Ph′-H),, 6.80 (s, 1H, NH), 3.57 (s, 1H), 3.42 (s, 2H, N-CH₂), 2.70–2.69 (m, 2H), 2.57 (d, J = 17.9 Hz, 4H), 2.06 (s, 6H), 1.84–1.71 (m, 4H), 1.69–1.12 (m, 6H). ¹³C NMR (100 MHz, DMSO-d₆) δ 166.4, 143.7, 143.1, 133.2, 132.5, 130.0, 126.0, 108.3, 62.1, 51.9, 39.9, 31.7, 22.5, 21.3, 16.2. HRMS: m/z 511.2816 [M + 1]⁺. C₃₀H₃₄N₆O₂ (510.2743). HPLC purity: 98.81%.

3,5-Dimethyl-4-((2-((1-(4-(methylsulfonyl)benzyl)piperidin-4-yl)amino)-5,6,7,8-tetrahydroquinazolin-4-yl)oxy)benzonitrile (13e3).

Recrystallized from EA/PE as a white solid, 62% yield. Mp: 203–206 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 7.87 (d, J = 7.9 Hz, 2H, C₃,C₅-Ph′-H), 7.65 (s, 2H, C₃,C₅-Ph″-H), 7.54 (d, J = 8.0 Hz, 2H, C₂,C₆-Ph′-H), 6.83 (s, 1H, NH), 3.57 (s, 1H), 3.49 (s, 2H, N-CH₂), 3.20 (s, 3H, SO₂CH₃), 2.66 (s, 2H), 2.57 (d, J = 15.6 Hz, 4H), 2.06, (s, 6H), 1.84–1.71 (m, 4H), 1.70–1.17 (m, 6H). ¹³C NMR (100 MHz, DMSO-d₆) δ 166.0, 145.4, 139.7, 133.1, 132.6, 129.7, 127.4, 61.9, 52.6, 44.0, 31.7, 22.5, 21.3, 16.2. HRMS: m/z 546.2530 [M + 1]⁺. C₃₀H₃₅N₅O₃S (545.2461). HPLC purity: 97.14%.

3,5-Dimethyl-4-((2-((1-(pyridin-4-ylmethyl)piperidin-4-yl)amino)-5,6,7,8-tetrahydroquinazolin-4-yl)oxy)benzonitrile (13e4).

Recrystallized from EA/PE as a white solid, 51% yield. Mp: 181–184 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 8.59 (d, J = 8.0 Hz, 2H, C₃,C₅-Ph′-H), 7.65 (s, 2H, C₃,C₅-Ph″-H), 7.27 (d, J = 5.2 Hz, 2H, C₂,C₆-Ph′-H), 6.81 (s, 1H, NH), 3.57 (s, 1H), 3.42 (s, 2H, N-CH₂), 2.64, (s, 2H), 2.57 (d, J = 15.2 Hz, 4H), 2.06 (s, 6H), 1.76 (p, J = 2.9 Hz, 4H), 1.69–1.14 (m, 6H). ¹³C NMR (100 MHz, DMSO-d₆) δ 166.0, 159.4, 149.9, 148.1, 133.1, 132.5, 124.0, 61.2, 52.6, 32.0, 31.6, 22.5, 21.3, 16.2. HRMS: m/z 469.2710 [M + 1]⁺. C₂₈H₃₂N₆O (468.2638). HPLC purity: 99.89%.

3,5-Dimethyl-4-((2-((1-(4-nitrobenzyl)piperidin-4-yl)amino)-5,6,7,8-tetrahydroquinazolin-4-yl)oxy)benzonitrile (13e5).

Recrystallized from EA/PE as a white solid, 49% yield. Mp: 225–227 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 8.19 (d, J = 8.2 Hz, 2H, C₃,C₅-Ph′-H), 7.64 (s, 2H, C₃,C₅-Ph″-H), 7.55 (d, J = 8.3 Hz, 2H, C₂,C₆-Ph′-H), 6.83 (s, 1H, NH), 3.75 (s, 1H), 3.53 (s, 2H, N-CH₂), 2.66 (s, 2H), 2.57 (d, J = 15.7 Hz, 4H), 2.06 (s, 6H), 1.86–1.69 (m, 4H), 1.70–1.09 (m, 6H). ¹³C NMR (100 MHz, DMSO-d₆) δ 166.0, 147.5, 146.9, 133.1, 132.5, 130.0, 123.8, 108.2, 61.6, 52.6, 31.7, 22.5, 21.3, 16.2. HRMS: m/z 513.2605 [M + 1]⁺. C₂₉H₃₂N₆O₃ (512.2536). HPLC purity: 99.51%.

3-((4-((4-(4-Cyano-2,6-dimethylphenoxy)-5,6,7,8-tetrahydroquinazolin-2-yl)amino)piperidin-1-yl)methyl)benzamide (13e6).

Recrystallized from EA/PE as a white solid, 57% yield. Mp: 251–253 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 7.96 (s, 2H, CONH₂), 7.79–7.72 (m, 1H), 7.65 (s, 2H, C₃,C₅-Ph″-H), 7.39–7.36 (m, 3H), 6.80 (s, 1H, NH), 3.61 (s, 1H), 3.42 (s, 2H, N-CH₂), 2.67 (s, 2H), 2.57 (d, J = 14.9 Hz, 4H), 2.06 (s, 6H), 1.76 (p, J = 3.0 Hz, 4H), 1.70–1.42 (m, 6H). ¹³C NMR (100 MHz, DMSO-d₆) δ 168.4, 166.0, 139.2, 134.6, 133.1, 132.6, 132.0, 128.4, 126.4, 62.4, 52.6, 31.7, 22.5, 21.3, 16.2. HRMS: m/z 511.2812 [M + 1]⁺. C₃₀H₃₄N₆O₂ (510.2743). HPLC purity: 99.73%.

4-((4-((4-(4-Cyano-2,6-dimethylphenoxy)-7,8-dihydro-6H-thiopyrano[3,2-d]pyrimidin-2-yl)amino)piperidin-1-yl)methyl)benzenesulfonamide (13f1).

Recrystallized from EA/PE as a white solid, 84% yield. Mp: 193–195 °C. ¹H NMR (400 MHz, DMSO-d₆) δ: 7.77 (d, J = 8.3 Hz, 2H), 7.66 (s, 2H), 7.45 (d, J = 8.0 Hz, 2H), 7.30 (s, 2H), 6.88 (s, 1H), 3.45 (s, 2H), 3.13–2.95 (m, 2H), 2.91–2.57 (m, 4H), 2.16–2.08 (m, 2H), 2.06 (s, 6H), 1.99–0.81 (m, 6H). ¹³C NMR (100 MHz, DMSO) δ: 163.2, 157.9, 154.2, 143.3, 143.1, 133.0, 132.6, 129.4, 126.0, 119.1, 108.5, 61.9, 52.6, 49.0, 31.8, 31.6, 26.3, 23.4, 16.1. ESI-MS: m/z 565.5 [M + 1]⁺. C₂₈H₃₂N₆O₃S₂ (564.1977). HPLC purity: 95.37%.

4-((4-((4-(4-Cyano-2,6-dimethylphenoxy)-7,8-dihydro-6H-thiopyrano[3,2-d]pyrimidin-2-yl)amino)piperidin-1-yl)methyl)benzamide (13f2).

Recrystallized from EA/PE as a white solid, 74% yield. Mp: 261–263 °C. ¹H NMR (400 MHz, DMSO-d₆) δ: 7.91 (s, 1H), 7.81 (d, J = 8.1 Hz, 2H), 7.66 (s, 2H), 7.33 (d, J = 7.9 Hz, 2H), 7.30 (s, 1H), 6.86 (br, 1H), 3.43 (s, 2H), 3.08–2.94 (m, 2H), 2.69 (t, J = 6.2 Hz, 4H), 2.17–2.10 (m, 2H), 2.09 (s, 1H), 2.06 (s, 6H), 1.60 (s, 4H), 1.31 (s, 2H). ¹³C NMR (100 MHz, DMSO) δ: 168.2, 163.2, 158.0, 154.2, 142.6, 142.5, 133.4, 133.0, 132.6, 128.8, 127.8, 119.1, 108.5, 79.6, 62.1, 52.6, 31.8, 31.6, 31.1, 26.3, 23.4, 16.1. ESI-MS: m/z 529.5 [M + 1]⁺, 551.6 [M + 23]⁺. C₂₉H₃₂N₆O₂S (528.2307). HPLC purity: 96.74%.

3,5-Dimethyl-4-((2-((1-(4-(methylsulfonyl)benzyl)piperidin-4-yl)amino)-7,8-dihydro-6H-thiopyrano[3,2-d]pyrimidin-4-yl)oxy)benzonitrile (13f3).

Recrystallized from EA/PE as a white solid, 66% yield. Mp: 265–267 °C. ¹H NMR (400 MHz, DMSO-d₆) δ: 7.87 (d, J = 8.3 Hz, 2H), 7.66 (s, 2H), 7.54 (d, J = 8.2 Hz, 2H), 6.89 (br, 1H), 3.50 (s, 2H), 3.20 (s, 3H), 3.10–2.95 (m, 2H), 2.69 (t, J = 6.2 Hz, 4H), 2.11 (m, 2H), 2.09 (s, 1H), 2.06 (s, 6H), 1.64 (s, 4H), 1.32 (s, 2H). ¹³C NMR (100 MHz, DMSO) δ: 163.2, 158.0, 157.9, 154.2, 145.4, 139.8, 133.0, 132.6, 129.7, 127.3, 119.1, 108.5, 61.8, 52.6, 48.6, 44.0, 31.8, 31.6, 26.3, 23.4, 16.1. ESI-MS: m/z 564.5 [M + 1]⁺, 586.5 [M + 23]⁺. C₂₉H₃₃N₅O₃S₂ (563.2025). HPLC purity: 96.93%.

3,5-Dimethyl-4-((2-((1-(4-nitrobenzyl)piperidin-4-yl)amino)-7,8-dihydro-6H-thiopyrano[3,2-d]pyrimidin-4-yl)oxy)benzonitrile (13f4).

Recrystallized from EA/PE as a white solid, 52% yield. Mp: 190–192 °C. ¹H NMR (400 MHz, DMSO-d₆) δ: 8.18 (d, J = 8.7 Hz, 2H), 7.66 (s, 2H), 7.55 (d, J = 8.6 Hz, 2H), 6.88 (s, 1H), 3.53 (s, 2H), 3.11–2.92 (m, 2H), 2.69 (t, J = 6.2 Hz, 4H), 2.19–2.10 (m, 2H), 2.09 (s, 1H), 2.06 (s, 6H), 1.59 (s, 4H), 1.33 (s, 2H). ¹³C NMR (100 MHz, DMSO) δ: 163.2, 157.9, 154.2, 147.5, 146.9, 133.0, 132.6, 130.0, 123.8, 119.1, 108.5, 61.6, 52.6, 31.8, 31.6, 31.1, 26.3, 23.4, 19.0, 16.1. ESI-MS: m/z 531.5 [M + 1]⁺. C₂₈H₃₀N₆O₃S (530.2100). HPLC purity: 99.57%.

3-((4-((4-(4-Cyano-2,6-dimethylphenoxy)-7,8-dihydro-6H-thiopyrano[3,2-d]pyrimidin-2-yl)amino)piperidin-1-yl)methyl)benzamide (13f5).

Recrystallized from EA/PE as a white solid, 71% yield. Mp: 245–247 °C. ¹H NMR (400 MHz, DMSO-d₆) δ: 7.95 (s, 1H), 7.76 (d, J = 11.6 Hz, 2H), 7.66 (s, 2H), 7.39 (d, J = 7.3 Hz, 2H), 7.33 (s, 1H), 6.86 (s, 1H), 3.43 (s, 2H), 3.02 (s, 2H), 2.77–2.58 (m, 4H), 2.19–2.09 (m, 2H), 2.09 (s, 1H), 2.06 (s, 6H), 1.63 (s, 4H), 1.32, (s, 2H). ¹³C NMR (100 MHz, DMSO) δ: 168.4, 163.2, 157.9, 154.2, 139.2, 134.6, 133.0, 132.6, 132.0, 128.4, 126.4, 119.1, 108.5, 62.4, 52.6, 48.8, 31.8, 31.6, 31.1, 26.3, 23.4, 16.1. ESI-MS: m/z 529.4 [M + 1]⁺. C₂₉H₃₂N₆O₂S (528.2307). HPLC purity: 95.88%.

Supplementary Material

Supporting Information

NIHMS1618663-supplement-Supporting_Information.pdf^{(610.3KB, pdf)}

Molecular Formula Strings

NIHMS1618663-supplement-Molecular_Formula_Strings.csv^{(7.8KB, csv)}

ACKNOWLEDGMENTS

We gratefully acknowledge financial support from the Key Project of NSFC for International Cooperation (Grant 81420108027), the National Natural Science Foundation of China (NSFC Grants 81273354 and 81573347), Young Scholars Program of Shandong University (YSPSDU Grant 2016WLJH32), Key Research and Development Project of Shandong Province (Grant 2017CXGC1401), KU Leuven (Grant GOA 10/014), and National Institutes of Health (Grant AI033066). We acknowledge the Spanish Ministerio de Economía y Competitividad (Grant SAF2017-88107-R) and the Generalitat de Catalunya (Grant 2017SGR1746) for financial support and the Barcelona Supercomputing Center (Grant BCV-2018-2-0006) and Consorci de Serveis Universitaris de Catalunya (Molecular Recognition) for computational resources. We acknowledge Prof. Thomas A. Steitz and express our gratitude and remembrance to him.

ABBREVIATIONS USED

AIDS: acquired immune deficiency syndrome
CC₅₀: 50% cytotoxicity concentration
DAPY: diarylpyrimidine
DLV: delavirdine
EFV: efavirenz
ETV: etravirine
EC₅₀: effective concentration causing 50% inhibition of viral cytopathogenicity
HAART: highly active antiretroviral therapy
HIV: human immunodeficiency virus
hERG: human ether-à-go-go related gene
MD: molecular dynamics
NNIBP: NNRTI binding pocket
NNRTI: non-nucleoside reverse transcriptase inhibitor
NRTI: nucleoside reverse transcriptase inhibitor
NVP: nevirapine
RF: fold-resistance
RMSD: root-mean-square displacement
RMSF: root-mean-square fluctuation
RPV: rilpivirine
RT: reverse transcriptase
SAR: structure–activity relationship
SI: selectivity index
WT: wild-type

Footnotes

Supporting Information

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jmedchem.8b01656.

Experimental procedures and characterizations of derivatives, in vitro assay of anti-HIV activities in TZM-bl cells, in vitro assay of anti-HIV activities in MT-4 cells, recombinant HIV-1 reverse transcriptase (RT) inhibitory assays, computational modeling, cytochrome P450 inhibition assay, plasma protein binding assay, pharmacokinetic methods, acute toxicity experiment, and assay procedures for hERG activity (PDF)

Molecular formula strings and some data (CSV)

The authors declare no competing financial interest.

The authors declare that all experimental work complied with the institutional guidelines on animal studies (care and use of laboratory animals).

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supporting Information

NIHMS1618663-supplement-Supporting_Information.pdf^{(610.3KB, pdf)}

Molecular Formula Strings

NIHMS1618663-supplement-Molecular_Formula_Strings.csv^{(7.8KB, csv)}

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PERMALINK

Identification of Dihydrofuro[3,4-d]pyrimidine Derivatives as Novel HIV-1 Non-Nucleoside Reverse Transcriptase Inhibitors with Promising Antiviral Activities and Desirable Physicochemical Properties

Dongwei Kang

Heng Zhang

Zhao Wang

Tong Zhao

Tiziana Ginex

Francisco Javier Luque

Yang Yang

Gaochan Wu

Da Feng

Fenju Wei

Jian Zhang

Erik De Clercq

Christophe Pannecouque

Chin Ho Chen

Kuo-Hsiung Lee

N Arul Murugan

Thomas A Steitz

Peng Zhan

Xinyong Liu

Abstract

Graphical Abstract

INTRODUCTION

Figure 1.

Figure 2.

CHEMISTRY

Scheme 1. Synthesis of 8a1–5a.

Scheme 2. Synthesis of 13a1–6, 13b1–6, 13c1–6, and 13d1–6a.

Scheme 3. Synthesis of 13e1–6 and 13f1–6a.

RESULTS AND DISCUSSION

Table 1.

Table 5.

Table 2.

Table 3.

Table 4.

Table 6.

Molecular Modeling Studies.

Figure 3.

Solubility and Lipophilic Efficiency.

Table 7.

CYP Enzymatic Inhibitory Activity.

Table 8.

Determination of Plasma Protein Binding Rate.

Table 9.

In Vivo Pharmacokinetics Study.

Table 10.

Figure 4.

Safety Assessment.

Assessment of Acute Toxicity.

Assessment of Subacute Toxicity.

Figure 5.

Assessment of hERG Activity.

Figure 6.

CONCLUSION

EXPERIMENTAL SECTION

Chemistry.

4-((2-(Azepan-4-ylamino)thieno[3,2-d]pyrimidin-4-yl)oxy)-3,5-dimethylbenzonitrile (7a).

4-((2-((8-Azabicyclo[3.2.1]octan-3-yl)amino)thieno[3,2-d]-pyrimidin-4-yl)oxy)-3,5-dimethylbenzonitrile (7b).

(R)-3,5-Dimethyl-4-((2-(pyrrolidin-3-ylamino)thieno[3,2-d]-pyrimidin-4-yl)oxy)benzonitrile (7c).

(S)-3,5-Dimethyl-4-((2-(pyrrolidin-3-ylamino)thieno[3,2-d]-pyrimidin-4-yl)oxy)benzonitrile (7d).

4-((2-(Azetidin-3-ylamino)thieno[3,2-d]pyrimidin-4-yl)oxy)-3,5-dimethylbenzonitrile (7e).

General Procedure for the Preparation of Final Compounds 8a1–5.

4-((4-((4-(4-Cyano-2,6-dimethylphenoxy)thieno[3,2-d]-pyrimidin-2-yl)amino)azepan-1-yl)methyl)benzenesulfonamide (8a1).

4-((4-((4-(4-Cyano-2,6-dimethylphenoxy)thieno[3,2-d]-pyrimidin-2-yl)amino)azepan-1-yl)methyl)benzamide (8a2).

3-((4-((4-(4-Cyano-2,6-dimethylphenoxy)thieno[3,2-d]-pyrimidin-2-yl)amino)azepan-1-yl)methyl)benzamide (8a3).

3,5-Dimethyl-4-((2-((1-(4-(methylsulfonyl)benzyl)azepan-4-yl)amino)thieno[3,2-d]pyrimidin-4-yl)oxy)benzonitrile (8a4).

3,5-Dimethyl-4-((2-((1-(pyridin-4-ylmethyl)azepan-4-yl)amino)thieno[3,2-d]pyrimidin-4-yl)oxy)benzonitrile (8a5).

4-((3-((4-(4-Cyano-2,6-dimethylphenoxy)thieno[3,2-d]-pyrimidin-2-yl)amino)-8-azabicyclo[3.2.1]octan-8-yl)methyl)benzenesulfonamide (8b1).

4-((3-((4-(4-Cyano-2,6-dimethylphenoxy)thieno[3,2-d]-pyrimidin-2-yl)amino)-8-azabicyclo[3.2.1]octan-8-yl)methyl)benzamide (8b2).

3-((3-((4-(4-Cyano-2,6-dimethylphenoxy)thieno[3,2-d]-pyrimidin-2-yl)amino)-8-azabicyclo[3.2.1]octan-8-yl)methyl)benzamide (8b3).

3,5-Dimethyl-4-((2-((8-(4-(methylsulfonyl)benzyl)-8-azabicyclo[3.2.1]octan-3-yl)amino)thieno[3,2-d]pyrimidin-4-yl)oxy)benzonitrile (8b4).

3,5-Dimethyl-4-((2-((8-(pyridin-4-ylmethyl)-8-azabicyclo-[3.2.1]octan-3-yl)amino)thieno[3,2-d]pyrimidin-4-yl)oxy)benzonitrile (8b5).

(R)-4-((3-((4-(4-Cyano-2,6-dimethylphenoxy)thieno[3,2-d]-pyrimidin-2-yl)amino)pyrrolidin-1-yl)methyl)benzenesulfonamide (8c1).

(R)-4-((3-((4-(4-Cyano-2,6-dimethylphenoxy)thieno[3,2-d]-pyrimidin-2-yl)amino)pyrrolidin-1-yl)methyl)benzamide (8c2).

(R)-3-((3-((4-(4-Cyano-2,6-dimethylphenoxy)thieno[3,2-d]-pyrimidin-2-yl)amino)pyrrolidin-1-yl)methyl)benzamide (8c3).

(R)-3,5-Dimethyl-4-((2-((1-(4-(methylsulfonyl)benzyl)pyrrolidin-3-yl)amino)thieno[3,2-d]pyrimidin-4-yl)oxy)benzonitrile (8c4).

(R)-3,5-Dimethyl-4-((2-((1-(pyridin-4-ylmethyl)pyrrolidin-3-yl)amino)thieno[3,2-d]pyrimidin-4-yl)oxy)benzonitrile (8c5).

(S)-4-((3-((4-(4-Cyano-2,6-dimethylphenoxy)thieno[3,2-d]-pyrimidin-2-yl)amino)pyrrolidin-1-yl)methyl)benzenesulfonamide (8d1).

(S)-4-((3-((4-(4-Cyano-2,6-dimethylphenoxy)thieno[3,2-d]-pyrimidin-2-yl)amino)pyrrolidin-1-yl)methyl)benzamide (8d2).

Scheme 1. Synthesis of 8a1–5^a.

Scheme 2. Synthesis of 13a1–6, 13b1–6, 13c1–6, and 13d1–6^a.

Scheme 3. Synthesis of 13e1–6 and 13f1–6^a.