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Discovery of the Soluble Guanylate Cyclase Stimulator Vericiguat (BAY 1021189) for the Treatment of Chronic Heart Failure
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Discovery of the Soluble Guanylate Cyclase Stimulator Vericiguat (BAY 1021189) for the Treatment of Chronic Heart Failure
发现可溶性鸟苷酸环化酶激动剂维利吉乌特(BAY 1021189)用于治疗慢性心力衰竭
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Drug Discovery, Bayer AG, Aprather Weg 18a, 42113 Wuppertal, Germany
*E-mail: markus.follmann@bayer.com. Phone: +49202365272.
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Journal of Medicinal Chemistry

Cite this: J. Med. Chem. 2017, 60, 12, 5146–5161
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https://doi.org/10.1021/acs.jmedchem.7b00449
Published May 30, 2017

Copyright © 2017 American Chemical Society. This publication is licensed under these Terms of Use.

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Abstract 摘要

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The first-in-class soluble guanylate cyclase (sGC) stimulator riociguat was recently introduced as a novel treatment option for pulmonary hypertension. Despite its outstanding pharmacological profile, application of riociguat in other cardiovascular indications is limited by its short half-life, necessitating a three times daily dosing regimen. In our efforts to further optimize the compound class, we have uncovered interesting structure–activity relationships and were able to decrease oxidative metabolism significantly. These studies resulting in the discovery of once daily sGC stimulator vericiguat (compound 24, BAY 1021189), currently in phase 3 trials for chronic heart failure, are now reported.
首次引入的同类可溶性鸟苷酸环化酶(sGC)激动剂瑞伐西单抗最近被作为一种新型肺动脉高压治疗选择。尽管其药理特性卓越,但由于其半衰期短,瑞伐西单抗在其他心血管适应症中的应用受到限制,需要每日三次的给药方案。在我们进一步优化该化合物类别的努力中,我们发现了有趣的构效关系,并显著降低了氧化代谢。这些研究导致了每日一次 sGC 激动剂维西单抗(化合物 24,BAY 1021189)的发现,目前该药物正在接受慢性心力衰竭的 3 期临床试验,现在予以报道。

Copyright © 2017 American Chemical Society
版权©2017 美国化学学会

Introduction 引言

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The NO–sGC–cGMP axis belongs to the key signal transduction pathways involved in regulating the cardiovascular system. (1, 2) Central to this pathway is soluble guanylate cyclase (sGC), an intracellular enzyme present in the smooth muscle cells of blood vessels and in platelets but also in various other cell types like cardiomyocytes. sGC displays a high affinity for, and is activated by, the first messenger signaling molecule nitric oxide (NO). Biosynthesis of NO is mediated by three distinct isoforms of nitric oxide synthases, endothelial nitric oxide synthase (eNOS), neuronal nitric oxide synthase (nNOS), and the inducible isoform of nitric oxide synthase (iNOS), all synthesizing NO from l-arginine. Within endothelial cells, NO is produced by eNOS and diffuses rapidly to the underlying smooth muscle cells where it binds to its target enzyme sGC. This binding leads to the stimulation of sGC, resulting in increased production of intracellular second messenger cGMP. cGMP interacts with three types of intracellular proteins such as cGMP-dependent protein kinases, cGMP-regulated ion channels, and phosphodiesterases. Further downstream, these transduction cascades mediate various physiological and tissue-protective effects including smooth muscle relaxation and inhibition of smooth muscle proliferation, leukocyte recruitment, and platelet function. The pathogenesis of various diseases, especially those of the cardiovascular system, has been related to insufficient bioavailability of NO and thus impaired stimulation of sGC and therefore decreased cGMP production. (3-7)
NO-sGC-cGMP 通路是调节心血管系统的重要信号转导通路之一。(1,2)该通路的核心是可溶性鸟苷酸环化酶(sGC),这是一种存在于血管平滑肌细胞和血小板中的细胞内酶,也存在于各种其他细胞类型中,如心肌细胞。sGC 对第一信使信号分子一氧化氮(NO)具有高亲和力,并由其激活。一氧化氮的生物合成由三种不同的氧化酶同型异构体介导,即内皮型一氧化氮合酶(eNOS)、神经元型一氧化氮合酶(nNOS)和诱导型一氧化氮合酶(iNOS),它们均从 L-精氨酸合成一氧化氮。在内皮细胞中,一氧化氮由 eNOS 产生并迅速扩散到其下方的平滑肌细胞,在那里它与靶酶 sGC 结合。这种结合导致 sGC 的刺激,从而增加细胞内第二信使 cGMP 的产生。cGMP 与三种类型的细胞内蛋白相互作用,如 cGMP 依赖性蛋白激酶、cGMP 调节的离子通道和磷酸二酯酶。 下游进一步,这些转导级联介导多种生理和组织保护作用,包括平滑肌松弛、抑制平滑肌增殖、白细胞募集和血小板功能。各种疾病的发病机制,尤其是心血管系统疾病,与一氧化氮的生物利用度不足有关,从而损害 sGC 的刺激,因此导致 cGMP 产生减少。 (3-7)
sGC itself is a cytosolic, heterodimeric protein composed of an α- and a β-subunit with a prosthetic heme group located in the β-subunit (heme-binding domain). (8-13) Mechanistically, NO stimulates sGC by binding to the Fe2+ of the heme group, which induces cleavage of an Fe2+–histidine (His105) bond, likely resulting in conformational reorganizations which propagate into the catalytic subunit, increasing cGMP production. cGMP interacts with three types of intracellular proteins, namely cGMP-dependent protein kinases, cGMP-regulated ion channels, and phosphodiesterases. Further downstream, these transduction cascades mediate various physiological and tissue-protective effects, including smooth muscle relaxation and inhibition of smooth muscle proliferation, leukocyte recruitment, and platelet function. (1)
sGC 本身是一种细胞质中的异源二聚体蛋白,由 α-亚基和 β-亚基组成,β-亚基中含有一个位于其上的辅因子血红素(血红素结合域)。(8-13)从机制上讲,NO 通过与血红素中的 Fe2+ 结合来刺激 sGC,这会导致 Fe2+-组氨酸(His105)键的断裂,这很可能是导致构象重组,进而传播到催化亚基,增加 cGMP 的产生。cGMP 与三种类型的细胞内蛋白相互作用,即 cGMP 依赖性蛋白激酶、cGMP 调节的离子通道和磷酸二酯酶。进一步下游,这些转导级联反应介导各种生理和器官保护作用,包括平滑肌松弛、抑制平滑肌增殖、白细胞募集和血小板功能。(1)
sGC can exist in two different states, a native heme-containing or reduced form of sGC, which is the endogenous receptor for NO, and a heme-free form of sGC. (1, 14) Under conditions of oxidative stress, which is thought to be causal in the pathogenesis of many cardiovascular diseases, reactive oxygen species (ROS) are produced. ROS are capable of oxidizing the heme iron of sGC (Fe2+ → Fe3+), resulting ultimately in heme loss from oxidized sGC. As a consequence, the heme-free form of sGC is no longer binding NO, is not responsive to NO, and thus termed dysfunctional. During the last 20 years, two distinct compound classes have been discovered at Bayer that are capable of activating sGC in a NO-independent manner, the heme-dependent sGC stimulators and the heme-independent sGC activators. sGC stimulators display a dual mode of action: they synergize with endogenous NO and, on top of this, are also able to directly stimulate the native form of the enzyme independently of NO, resulting in increased cGMP production. (15) In contrast, sGC activators are capable of activating the dysfunctional heme-free sGC, resulting in increased cGMP production even under conditions of reduced NO bioavailability. (16)
sGC 可以存在于两种不同的状态,即含有天然血红素的 sGC 或还原形式的 sGC,它是 NO 的内源性受体,以及不含血红素的 sGC。在氧化应激条件下,氧化应激被认为是许多心血管疾病发病机制的原因,会产生活性氧(ROS)。ROS 能够氧化 sGC 中的血红素铁(Fe2+ → Fe3+),最终导致氧化 sGC 失去血红素。因此,不含血红素的 sGC 不再结合 NO,对 NO 无反应,因此被称为功能障碍。在过去 20 年里,拜耳公司发现了两种不同的化合物类别,它们能够以 NO 非依赖性方式激活 sGC,即血红素依赖性 sGC 激动剂和血红素非依赖性 sGC 激活剂。sGC 激动剂具有双重作用模式:它们与内源性 NO 协同作用,并且在此基础上,还能直接刺激酶的天然形式,独立于 NO,从而增加 cGMP 的产生。 (15) 相比之下,sGC 激活剂能够激活功能失调的无铁血红素 sGC,即使在 NO 生物利用度降低的条件下,也能增加 cGMP 的产生。 (16)
The discovery of the sGC stimulators at Bayer, along with the efforts of several other pharmaceutical companies to identify sGC stimulators and activators, has recently been reviewed. (2) Riociguat (1) is the first sGC stimulator to have made a successful transition from animal experiments to controlled clinical studies in patients. (17) In 2013, 1 gained market approval for two life-threatening diseases: pulmonary arterial hypertension (PAH) (18) and chronic thromboembolic pulmonary hypertension (CTEPH). (19, 20) However, application of 1 in other cardiovascular indications, such as heart failure, is limited by its short half-life. (21)
贝耶尔公司发现了 sGC 激动剂,以及多家制药公司努力寻找 sGC 激动剂和激活剂的努力,最近得到了综述。 (2) Riociguat(1)是第一个成功从动物实验过渡到患者临床试验的 sGC 激动剂。 (17) 2013 年,1 号药物获得了市场批准,用于治疗两种危及生命的疾病:肺动脉高压(PAH)(18) 和慢性血栓栓塞性肺动脉高压(CTEPH)(19, 20)。然而,由于 1 号药物的半衰期较短,其在其他心血管适应症,如心力衰竭中的应用受到限制。 (21)
Herein, we report our recent efforts in optimizing the pharmacokinetic profile of sGC stimulators, culminating in the discovery of vericiguat (compound 24, BAY 1021189), which has been evaluated in phase 2 studies (SOCRATES trials) in heart failure patients with reduced (HFrEF) and preserved (HFpEF) ejection fraction. (22, 23) Currently, 24 is being investigated in a phase 3 outcome study (VICTORIA trial) in heart failure patients with HFrEF in codevelopment with MSD (vericiguat code name: MK-1242). (24) That study is aimed at assessing whether 24, on top of standard of care, is able to decrease mortality and morbidity in such HFrEF patients.

Results and Discussion

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Optimization Strategy

Our goal was to identify orally bioavailable sGC stimulators with a longer duration of action than 1 in order to support a profile allowing for a once daily oral dosing and less oxidative metabolism in order to lower interaction potentials. 1 is a very potent sGC stimulator in vitro and in vivo; however, it has a moderate half-life in different animal species (25) and this pharmacokinetic profile translated into a three times daily dosing regimen in patients. N-Demethylation to compound 9, as described by Gnoth et al. (26) in 2015, is the major biotransformation of 1 and is mainly catalyzed by CYP1A1 and also by CYP3A4, CYP3A5, and CYP2J2. (21, 26-31) Our strategy was to further optimize the metabolic stability of 1 and hence reduce blood clearance to achieve a longer half-life. We began by optimizing the substituents on the 5-carbamoyl residue on the pyrimidine ring, aiming to achieve metabolically more stable derivatives while maintaining good potency. Later, in a second optimization step, we focused on variations of the central pyrazolopyridine scaffold.

Chemistry

Scheme 1 outlines the strategy for the synthesis of the 5-carbamoyl variations. Starting from triamine 1c, which is available in two steps from amidine 1a and phenyldiazenyl-substituted malonodinitrile 1b, selective carbamate formation at the 5-amino group was achieved either by treatment with the corresponding chloroformate or by addition of the respective alcohol preactivated with triphosgene to yield compounds 914. (25) Deprotonation of the carbamate NH group and subsequent treatment with the corresponding halide or trichloromethanesulfonate electrophile allowed further derivatization to the corresponding N-alkylated carbamates (1, 2, 48). Alternatively, the alkyl side chain on the 5-amino group could first be introduced via reductive amination and the product 1d subsequently treated with methyl chloroformate, giving access to the hydroxyethyl derivative 3. For the cyclic 5-(2-oxo-1,3-oxazolidin-3-yl) analogues 1518, the previously employed conditions for the selective formation of carbamates provided 1eg bearing a chloro- or bromo-substituted alkyl chain, allowing for a base-induced intramolecular cyclization (NaHMDS).

Scheme 1

Scheme 1. Synthesis of Compounds 118a

Scheme aReagents and conditions: (a) NaOMe, DMF, 110 °C; (b) H2 (65 bar), Raney nickel, DMF; (c) R2OCOCl, pyridine, or (i) triphosgene, pyridine, (ii) R2OH, pyridine; (d) R1X (X = halide or trichloromethanesulfonate), LiHMDS; (e) glycolaldehyde, NaBH3CN, AcOH, MeOH, 0 °C to rt; (f) methyl chloroformate, pyridine, rt; (g) NaHMDS, THF, 0 °C to rt.

The synthesis of the compounds with a core variation is outlined in Schemes 25. For the 1H-pyrazolo[4,3-b]pyridine derivative 19, the synthesis started from 3,5-dichloropyridine-2-carbonyl chloride (2a), which was converted into 3-(2-fluorobenzyl)-1H-pyrazolo[4,3-b]pyridine (2b) in four steps. The fluoro derivative 2d was synthesized in a similar manner from the corresponding difluoropyridine analogue 2c. Both key intermediates 2b and 2d were then reacted with 2-chloro-5-nitropyrimidine-4,6-diamine, (32) followed by reduction of the pyrimidine 5-nitro group and methyl carbamate formation, to give the 1H-pyrazolo[4,3-b]pyridine core analogues 19 and 20 (Scheme 2).

Scheme 2

Scheme 2. Synthesis of Compounds 19 and 20a

Scheme aReagents and conditions: (a) methyl 2-(2-fluorophenyl)acetate, LiHMDS; (b) aq NaCl solution, DMSO, microwave, 150 °C; (c) hydrazine hydrate, pyridine, reflux; (d) H2, 10% Pd/C, Et3N, EtOH, THF; (e) NaH, DMF, 80 °C; (f) H2, 10% Pd/C, pyridine; (g) methyl chloroformate, pyridine.

A different route was used for the preparation of 1H-pyrazolo[3,4-c]pyridazine 21 (Scheme 3), which began with the cyclization of methyl 2-aminothiophene-3-carboxylate (3a) with hydrazine hydrate to give 1H-pyrazolo[3,4-c]pyridazin-3-ol (3b). (33) Subsequent bromination afforded intermediate 3c, which was alkylated with 2-fluorobenzyl bromide to yield 3d. Then, conversion of the bromide into the cyanide 3e and Pinner-type reaction of the cyano group with sodium methoxide and ammonium chloride afforded key intermediate 3f. Further modifications, analogous to the formation of carbamate 9 from 1a (Scheme 1), yielded the methyl carbamate derivative 21.

Scheme 3

Scheme 3. Synthesis of Compound 21a

Scheme aReagents and conditions: (a) hydrazine hydrate; (b) POBr3, sulfolane, 150 °C; (c) 2-fluorobenzyl bromide, Cs2CO3, DMF, rt; (d) CuCN, DMSO, 150 °C; (e) NaOMe, NH4Cl, AcOH, MeOH; (f) 1b, DMF, Et3N, 100 °C; (g) H2 (1 bar), 10% Pd/C, DMF; (h) methyl chloroformate, pyridine, DCM.

The preparation of imidazo[1,5-a]pyrimidine 22 (Scheme 4) started with condensation of the readily available thioimide 4a and amine 4b to access aminoimidazole 4c. Further orthoester condensation then gave the bicyclic imidazopyrimidine derivative 4d bearing an ethyl ester functionality. Subsequent saponification, amidation, and dehydration yielded nitrile 4g as a key intermediate. Pinner-type reaction with sodium methoxide and ammonium chloride delivered the corresponding amidine 4h, which could be converted into the final compound 22 in analogy to the previous examples.

Scheme 4

Scheme 4. Synthesis of Compound 22a

Scheme aReagents and conditions: (a) MeI, K2CO3, acetone; (b) TBAI, KOt-Bu, DCM, then aq HCl, Et2O; (c) dioxane, 60 °C; (d) 1,1,3,3-tetramethoxypropane, aq HCl, MeOH, EtOH, reflux; (e) NaOH; (f) HATU, NH4Cl, Hünig’s base, DMF; (g) POCl3, 120 °C; (h) NaOMe, NH4Cl, MeOH; (i) 1b, DMF, Et3N, 100 °C; (j) H2 (1 bar), 10% Pd/C, DMF, 0 °C; (k) methyl chloroformate, pyridine.

The synthesis of the related imidazo[1,5-b]pyridazine derivative 23 (Scheme 5) started from methyl 5-aminolevulinate hydrochloride (5a). Addition of hydrazine hydrate yielded the dihydropyridazinone 5b, which was reacted with the readily available acid chloride 5c under basic conditions. The resulting amide was oxidized with bromine to provide the corresponding pyridazinone 5d, which underwent an intramolecular condensation in the presence of phosphoryl chloride to give the bicyclic intermediate 5e. Dechlorination of the pyridazine core catalyzed by palladium on charcoal and subsequent bromination of the imidazo core with N-bromosuccinimide gave access to 5f. Further modifications, as outlined previously, then yielded amidine 5g as a key intermediate which was converted into the final target compound 23.

Scheme 5

Scheme 5. Synthesis of Compound 23a

Scheme aReagents and conditions: (a) hydrazine hydrate, Et3N, reflux; (b) Et3N, MeCN; (c) Br2, AcOH, 50 °C; (d) POCl3, DCE, reflux; (e) H2, 5% Pd/C, Et3N, EtOAc; (f) NBS, DCM; (g) CuCN; (h) NaOMe, NH4Cl, AcOH; (i) 1b, DMF, Et3N, 100 °C; (j) H2 (1 bar), 10% Pd/C, DMF, MeOH; (k) methyl chloroformate, pyridine.

The synthesis of the 1H-pyrazolo[3,4-b]pyridine derivative (24) (Scheme 6), involved initial activation of 2,2,3,3-tetrafluoro-1-propanol (6a) with trifluoromethanesulfonic anhydride and treatment with morpholine to yield 6b after aqueous workup and distillation. At 135 °C, 6b was alkylated with methylmethanesulfonate, leading to quaternized derivative 6c which was converted into 6d using sodium hydroxide. Further reaction with morpholine and triethylamine yielded 6e after crystallization from toluene. Acrylaldehyde derivative 6e was reacted with ethyl 5-amino-1-(2-fluorobenzyl)-1H-pyrazole-3-carboxylate (25) in ethanol, yielding ethyl ester intermediate 6f. Ester 6f was then converted in three steps into the corresponding amidine 6i via amide 6g and nitrile 6h in analogy to the chemistry outlined in Scheme 4. The synthesis of 24 was completed as described for compound 9 (Scheme 1) via the diazenyl derivative 6j and triamine 6k.

Scheme 6

Scheme 6. Synthesis of 24a

Scheme aReagents and conditions: (a) Tf2O, 70 °C, then morpholine, 5 °C, then 40 °C; (b) MeSO3Me, 135 °C, then 100 °C; (c) 45% aq NaOH, 50 °C; (d) morpholine, Et3N, reflux; (e) MsOH, LiCl, EtOH, reflux; (f) formamide, NaOMe, MeOH, EtOH, 95–125 °C; (g) POCl3, sulfolane, 107 °C; (h) NaOMe, NH4Cl, MeOH, EtOH, 65 °C; (i) 1b, DMF, Et3N, 100 °C; (j) H2 (60 bar), 5% Pd/C, DMF, 60 °C; (k) methyl chloroformate, i-PrOH, MeOH, then Et3N, 50 °C.

SAR and DMPK Optimization

The SAR of the series of novel N-substituted methyl carbamates 18 was explored using a cGMP formation assay with sGC-overexpressing Chinese hamster ovary (CHO) cells. (34, 35) In addition, the metabolic stability of the compounds was assessed in vitro by incubation with rat hepatocytes. Initially, different carbamate N-substituents were tested with the aim of achieving higher metabolic stability than 1 while maintaining high potency (Table 1). Increasing steric bulk as in the ethyl derivative 2 had no beneficial effect on either potency or metabolic stability. The introduction of polarity (e.g., a hydroxyethyl functionality, as in 3) led to a significant loss of potency relative to 1, along with decreased metabolic stability. The next strategy, the introduction of fluorine atoms at the terminal position of the N-substituent, was designed to block metabolism. Interestingly, both the 2,2-difluoroethyl and 2,2,2-trifluoroethyl derivatives, 4 and 5, exhibited high potency in the cGMP assay but had a 2–4.5-fold higher clearance in rat hepatocytes. N-Fluorobenzyl substitution revealed that the ortho-fluoro derivative 6 is significantly more potent (MEC = 0.1 μM) than the meta and para isomers, 7 and 8 (MEC = 0.2 and 0.7 μM, respectively). Nevertheless, all the benzylic derivatives had a high clearance in rat hepatocytes. These results and the testing of many further derivatives (data not shown) suggested that improving the metabolic stability by altering the N-substitution might be very difficult to achieve. Hence, optimization efforts were focused on the main metabolite of 1, the N-desmethyl derivative 9. This compound is less potent than 1 but displayed a somewhat higher metabolic stability in the rat hepatocyte assay, with a clearance of 0.1 L/h/kg. When tested in vivo in rats (iv dosing), compound 9 had a moderate clearance of 1.2 L/h/kg and a short half-life of only 1.2 h.
Table 1. Properties of the N-Substituted Methyl Carbamates 18
Table a

MEC: Minimal effective concentration to achieve stimulation of cGMP formation (≥3-fold increase in basal luminescence) in a recombinant sGC-overexpressing cell line. (34)

Therefore, variations in the carbamate alkoxy group while leaving the N-position unsubstituted were examined next, with the goal of improving the half-life and other properties relative to compound 9 (Table 2).
Table 2. Properties of the N–H Alkyl Carbamates 914
Table a

MEC: Minimal effective concentration to achieve stimulation of cGMP formation (≥3-fold increase in basal luminescence) in a recombinant sGC-overexpressing cell line. (34)

Table b

n.d.: not determined.

The introduction of steric bulk in the form of an isopropyl residue (compound 10) led to a strong decrease in potency relative to 9 as well as decreased metabolic stability. Interestingly, there was increased potency with cyclobutyl derivative 11; however, 11 was also unfavorable with respect to rat hepatocyte clearance. The oxetanyl group is well-known in medicinal chemistry for improving metabolic stability (36, 37) over related alkyl derivatives. Remarkably, the exchange of cyclobutyl for oxetanyl (compound 12) led to a 14-fold higher stability in rat hepatocytes, but sGC stimulation was weaker than with derivative 9 or 11. Because the permeability across Caco-2 cell monolayers was also very low (Papp A–B = 2 nm/s), combined with a high efflux ratio of 74, compound 12 was not pursued further. Additional efforts to improve the overall profile by the introduction of fluorine or steric bulk (compounds 13 and 14) led to a slight increase in potency (MEC = 0.2 vs 0.3 μM for 9); however, metabolic stability was dramatically reduced.
The oxazolidinone derivatives 1518, as conformationally fixed versions of the foregoing carbamates, were also studied to assess the influence of the constrained nature on sGC stimulation (Table 3). The parent oxazolidinone 15 was as equipotent as carbamate 9 and exhibited slightly decreased stability in rat hepatocytes. Intravenous dosing of compound 15 to rats resulted in a moderate clearance of 1.9 L/h/kg and a short half-life of about 1.0 h, and thus 15 was not profiled further. Other attempts to increase metabolic stability by the introduction of steric bulk were unsuccessful, probably due to the more lipophilic (38) character of the resulting compounds 1618.
Table 3. Properties of the Oxazolidinones 1518
Table a

MEC: Minimal effective concentration to achieve stimulation of cGMP formation (≥3-fold increase in basal luminescence) in a recombinant sGC-overexpressing cell line. (34)

Table b

n.d.: not determined.

In summary, although further optimization of the carbamate motif of 1 could not be achieved, a number of interesting and novel SAR observations were made, with the N-unsubstituted carbamate 9 being identified as the most promising path forward. Thus, the focus of the optimization strategy was shifted away from the carbamate moiety and our efforts were directed to the central scaffold and the identification of alternative cores that could lead to superior overall pharmacokinetic profiles of the corresponding compounds.
Our studies with respect to central scaffold modifications are summarized in Table 4. Efforts were concentrated on novel core systems which had not been tested before in combination with 4,6-diaminopyrimidin-5-ylcarbamates.
Table 4. Properties of the Core Variation Compounds 1924
Table a

MEC: Minimal effective concentration to achieve stimulation of cGMP formation (≥3-fold increase in basal luminescence) in a recombinant sGC-overexpressing cell line. (34)

Table b

n.d.: not determined.

First, changes were made to the pyrazolo portion of the molecule, leading to the 1H-pyrazolo[4,3-b]pyridine derivative 19. In addition to the benefit of a shorter synthetic route than that for compound 9, 19 proved to be a moderately potent sGC stimulator with an MEC of 1.2 μM and had high metabolic stability when tested in rat hepatocytes. The introduction of substituents at the 6-position of the 1H-pyrazolo[4,3-b]pyridine core was, in general, not well tolerated, leading to a dramatic loss of potency, with the exception of the 6-fluoro derivative 20, which exhibited an MEC of 0.5 μM and good metabolic stability. To our surprise, compounds 19 and 20 had very different in vivo clearances when compared in a rat pharmacokinetic experiment (iv dosing): fluoro derivative 20 had a low clearance of 0.3 vs 1.0 L/h/kg for compound 19. As metabolite identification did not point to metabolism occurring at the pyridine core, the rationale for this 3-fold reduction in blood clearance remains unclear.
Novel 1H-pyrazolo[3,4-c]pyridazine 21 proved to be a reasonably potent sGC stimulator but exhibited a high clearance of 3.8 L/h/kg when tested in vivo in rats and thus was not further profiled, along with imidazo[1,5-a]pyrimidine 22 for similar reasons. In contrast, imidazo[1,5-b]pyridazine 23 exhibited potent sGC stimulator properties (MEC = 0.7 μM), good metabolic stability in rat hepatocytes, and a low to moderate clearance of 0.9 L/h/kg after intravenous dosing to rats. Finally, revisiting 1H-pyrazolo[3,4-b]pyridine 9 with an additional fluorine at the 5-position resulted in the potent sGC stimulator 24 (MEC = 0.3 μM) with good metabolic stability in rat hepatocytes and a surprisingly low clearance of 0.3 L/h/kg after intravenous dosing to rats (Table 4). Thus, the blood clearance of derivative 9 in rats (1.2 L/h/kg) was reduced 4-fold by fluorination at position 5.
Finally, compounds 20, 23, and 24 were selected for further pharmacokinetic profiling across species (Table 5). Fluoropyrazolo[3,4-b]pyridine derivative 24 exhibited the best overall pharmacokinetic profile by far, with a low clearance and long half-life in rats and dogs after intravenous dosing, as well as high oral bioavailability. In addition, 24 had no inhibitory effects on major CYP isoforms (1A2, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6, and 3A4), as indicated by IC50 values of >50 μM. Metabolite profiling in human hepatocytes was characterized by a low turnover, with glucuronide 25 (major) and debenzylated compound 26 (minor) being the only metabolites identified (Figure 1). Thus, the main biotransformation pathway shifted from predominantly phase I oxidative CYP-mediated metabolism (1) to primarily phase II UGT-mediated conjugation with glucuronic acid.
Table 5. In Vivo Pharmacokinetic Properties of 20, 23, and 24 in Comparison with 1
compdspeciesVss [L/kg]CLb [L/h/kg]t1/2 [h]bioavailability [%]
20rat0.50.31.526
 dog1.00.24.156
23rat0.30.90.537
 dog2.00.9 (Clp)a1.8n.d.b
24rat1.00.33.465
 dog1.40.26.275
1rat1.21.31.446
 dog0.70.3 (Clp)a2.479
a

Clp: plasma clearance.

b

n.d.: not determined.

Figure 1

Figure 1. Biotransformation products of 24 in human hepatocytes.

Pharmacology

24 was extensively profiled preclinically in vitro, on the isolated sGC enzyme and on isolated vessels, ex vivo in isolated hearts, and in vivo in a rat model of cardiovascular disease associated with cardiorenal syndrome.

Highly Purified Recombinant sGC

Studies on the in vitro effects of 24 on highly purified sGC revealed that 24 (0.01 μM to 100 μM) stimulates recombinant sGC concentration dependently, by 1.7-fold to 57.6-fold (Figure 2). When combined with the NO donor diethylamine/nitric oxide complex (DEA/NO), 24 and DEA/NO had a synergistic effect on the enzyme activity over a wide range of concentrations. At highest concentrations of 24 (100 μM) and DEA/NO (100 nM), the specific activity of sGC was 341.6-fold above baseline (Figure 2). Moreover, the effects of 24 in the presence of the sGC inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) were evaluated. The sGC stimulation induced by 24 was nearly completely blocked by ODQ and reduced to 18-fold (Figure 2). Thus, 24 exhibits all the characteristics of a potent and selective sGC stimulator, stimulating sGC NO-independently and in synergy with NO. In addition, 24 predominantly acts on the heme-containing nonoxidized form of sGC.

Figure 2

Figure 2. Effects of 24 and NO on the stimulation of highly purified sGC and blocking effects of the sGC inhibitor ODQ.

sGC-Overexpressing Cells

The stimulation of sGC by 24 was examined with a recombinant CHO cell line overexpressing rat sGC. 24 stimulated the sGC reporter cell line concentration dependently, with an EC50 of 1005 ± 145 nM. In the presence of the NO donor S-nitroso-N-acetyl-d,l-penicillamine (SNAP) (30 and 100 nM), the EC50 value shifted to 39.0 ± 5.1 and 10.6 ± 1.7 nM, respectively. In the presence of ODQ, pretreatment of the sGC reporter cell line with 10 μM ODQ for 3 h resulted in a significantly reduced efficacy of 24, with an EC50 of 256 ± 40 nM being observed.

Isolated Vessels and Tolerance

24 inhibited phenylephrine-induced contractions of rabbit saphenous artery rings, rabbit aortic rings, and canine femoral vein rings concentration dependently, with IC50 values of 798, 692, and 3072 nM, respectively. In addition, 24 inhibited the U46619-induced contractions of porcine coronary artery rings concentration dependently, with an IC50 of 956 nM.
Chronic administration of organic nitrates leads to the rapid development of nitrate tolerance. Thus, the vasorelaxant effect of 24 on isolated saphenous artery rings taken from normal and nitrate-tolerant rabbits was examined. Treatment with isosorbide dinitrate for 3–4 days resulted in a marked inhibition of glycerol trinitrate (GTN) mediated vasodilation. GTN inhibited phenylephrine-induced contractions with an IC50 of 1.9 nM in control vessels and an IC50 of 9.6 nM in tolerant vessels, confirming the presence of nitrate tolerance. In contrast to GTN, 24 is a potent inhibitor of phenylephrine-induced contractions both in normal and nitrate-tolerant saphenous artery rings, with IC50 values of 5.6 and 5.8 nM, respectively.

Langendorff-Perfused Hearts

In rat heart Langendorff preparations, ex vivo, 24 reduced the coronary perfusion pressure in a concentration-dependent manner (Figure 3). Up to the highest concentration tested, 24 had no effect on heart rate, left ventricular diastolic pressure, and contractility.

Figure 3

Figure 3. Effects of 24 on rat heart Langendorff preparations.

Long-Term Study with L-NAME-Treated Renin Transgenic Rats

The potential impact on cardiovascular health of the stimulation of sGC was evaluated by determining the long-term effects of 24 on hemodynamic and hormonal parameters in renin transgenic rats (RenTG) carrying the additional mouse renin gene (mRenR2)27. These RenTG(mRenR2)27 rats were additionally treated with the NO synthase inhibitor Nω-nitro-l-arginine methyl ester (L-NAME) in the drinking water. This well-established rodent disease model is characterized by hypertension, heart and kidney failure, and increased mortality. (39) The RenTG(mRenR2)27/L-NAME-supplemented rats were chronically treated with either placebo (2 mL/kg qd), which served as the control group, or 24 (3 or 10 mg/kg qd).

Effects on Blood Pressure and on the Heart

Chronic oral treatment with 3 or 10 mg/kg 24 qd resulted in a significant attenuation of blood pressure increase during the course of the study. However, the overall rise of blood pressure increase was not halted in the 3 and 10 mg/kg treatment groups (Figure 4).

Figure 4

Figure 4. Increase in systolic blood pressure in mmHg during the course of the study with L-NAME-treated renin transgenic rats.

In addition, a significant and dose-dependent reduction of heart hypertrophy, in both the right and left ventricle, was found in the 3 and 10 mg/kg treatment groups compared to the placebo group (Figure 5). Furthermore, plasma ANP levels decreased significantly in both treatment groups, also suggesting a functional improvement of the heart.

Figure 5

Figure 5. Right and left ventricle weight normalized on body weight (left/middle) and plasma atrial natriuretic peptide levels [in pg/mL] at the study end, after 3 weeks of treatment.
图 5. 右心室和左心室重量按体重标准化(左/中)以及研究结束时,经过 3 周治疗后血浆心房利钠肽水平[以 pg/mL 计]。

Effects on the Kidneys 肾脏影响

With respect to kidney damage, 24 treatment at 3 or 10 mg/kg led to a significant reduction in kidney injury molecule Kim-1 and osteopontin expression which are used as biomarkers for renal injury and dysfunction (data not shown). In addition, proteinuria was significantly and dose dependently decreased in the treatment groups, also suggesting a functional improvement of the kidneys (Figure 6).
关于肾脏损伤,3 毫克/千克或 10 毫克/千克的 24 小时治疗显著降低了肾脏损伤分子 Kim-1 和骨桥蛋白的表达,这些指标被用作肾损伤和功能障碍的生物标志物(数据未显示)。此外,治疗组的蛋白尿显著且剂量依赖性地减少,这也表明肾脏功能有所改善(图 6)。

Figure 6 图 6

Figure 6. Effects on proteinuria at the study end after 3 weeks of treatment.
图 6. 治疗结束后 3 周对蛋白尿的影响。

Effects on Mortality 死亡率影响

Treatment with 24 resulted in a significant and dose-dependent increase in survival rates. In the 3 and 10 mg/kg qd treatment groups, the rat survival rate was 70% and 90%, respectively, at the study end. In contrast, the survival rate in the placebo group was only 25% after 21 days (Figure 7).
治疗 24 小时后,生存率显著且呈剂量依赖性增加。在 3 毫克/千克和 10 毫克/千克每日一次的治疗组中,研究结束时大鼠的生存率分别为 70%和 90%。相比之下,安慰剂组的生存率在 21 天后仅为 25%(见图 7)。

Figure 7 图 7

Figure 7. Kaplan–Meier survival curves.
图 7. Kaplan-Meier 生存曲线。

These in vivo data strongly suggest that the sGC stimulator 24 can maintain heart and kidney function in a model of hypertension-induced end-organ damage, with substantially reduced overall mortality, strongly suggesting a beneficial role of 24 for the treatment of cardiovascular diseases associated with cardiorenal syndrome. (40)
这些体内数据强烈表明,sGC 激动剂 24 可以在高血压诱导的终末器官损伤模型中维持心脏和肾脏功能,显著降低总体死亡率,强烈表明 24 在治疗与心肾综合征相关的心血管疾病中具有积极作用。(40)

Conclusion 结论

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In summary, we have identified 24 as a potent, orally available stimulator of sGC. Our optimization work starting from 1 led to the identification of compounds with superior in vitro and in vivo pharmacokinetic and metabolic profiles. 24, the compound with the best overall profile from these preclinical studies, was selected as clinical candidate and proved to have a pharmacokinetic profile in humans suitable for once daily dosing. Additional in vivo studies in animal models of hypertension, heart failure, and kidney disease have revealed dose-dependent antifibrotic and organ-protective properties in line with the sGC stimulator mode of action. 24 is currently being investigated in a phase 3 clinical trial in HFrEF patients.
总结来说,我们已确定 24 号化合物是一种强效、可口服的 sGC 激动剂。从 1 号化合物开始的优化工作,使我们鉴定出具有优越的体外和体内药代动力学及代谢特征的化合物。在这些临床前研究中,24 号化合物因其最佳的整体特征被选为临床候选药物,并在人体中表现出适合每日一次给药的药代动力学特征。在高血压、心力衰竭和肾脏疾病动物模型中的进一步体内研究揭示了 24 号化合物具有剂量依赖性的抗纤维化和器官保护作用,这与 sGC 激动剂的作用模式一致。目前,24 号化合物正在 HFrEF 患者中进行 3 期临床试验。

Experimental Section 实验部分

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Chemistry 化学

General Procedures 通用程序

Unless otherwise noted, all nonaqueous reactions were carried out under an argon atmosphere with commercial-grade reagents and solvents. All final products were at least 95% pure, as determined by analytical HPLC.
除非另有说明,所有非水溶液反应均在氩气气氛下,使用商业级试剂和溶剂进行。所有最终产品纯度至少为 95%,通过分析高效液相色谱法确定。
1H NMR spectra were recorded on Bruker Avance spectrometers operating at 400 or 500 MHz. Chemical shifts (δ) are reported in ppm relative to TMS as an internal standard, and coupling constants (J) are given in hertz (Hz). Spin multiplicities are reported as s = singlet, br s = broad singlet, d = doublet, dd = doublet of doublets, t = triplet, q = quartet, m = multiplet. LC-MS analysis was performed using the respective method a–d, as noted. Method a: instrument, Micromass TOF-MUX interface 4× parallel injection with Waters 600 HPLC; column, Phenomenex Synergi 2 μm Hydro-RP Mercury 20 mm × 4 mm; mobile phase A, H2O (1 L) + 50% formic acid (0.5 mL); mobile phase B, MeCN (1 L) + 50% formic acid (0.5 mL); gradient, 0.0 min 90% A → 2.5 min 30% A → 3.0 min 5%A → 4.5 min 5% A; oven, 20 °C; flow, 1 mL/min; UV detection, 210 nm. Method b: instrument, Micromass Quattro Premier with Waters Acquity UPLC; column, Thermo Hypersil GOLD 1.9 μm, 50 mm × 1 mm; mobile phase A, H2O (1 L) + 50% formic acid (0.5 mL); mobile phase B, MeCN (1 L) + 50% formic acid (0.5 mL); gradient, 0.0 min 90% A → 0.1 min 90% A → 1.5 min 10% A → 2.2 min 10% A; oven, 50 °C; flow, 0.33 mL/min; UV detection, 210 nm. Method c: instrument, Waters Micromass Quattro Micro with Agilent 1100 series HPLC; column, Thermo Hypersil GOLD 3 μm, 20 mm × 4 mm; mobile phase A, H2O (1 L) + 50% formic acid (0.5 mL); mobile phase B, MeCN (1 L) + 50% formic acid (0.5 mL); gradient, 0.0 min 100% A → 3.0 min 10% A → 4.0 min 10% A; ove,: 50 °C; flow, 2 mL/min; UV detection, 210 nm. Method d: instrument, Waters Acquity SQD UPLC system; column, Waters Acquity UPLC HSS T3 1.8 μm, 50 mm × 1 mm; mobile phase A, H2O (1 L) + 99% formic acid (0.25 mL); mobile phase B, MeCN (1 L) + 99% formic acid (0.25 mL); gradient, 0.0 min 90% A → 1.2 min 5% A → 2.0 min 5% A; oven, 50 °C; flow, 0.4 mL/min; UV detection, 208–400 nm. 

Materials 材料

Intermediates 1ac, (25)3b, (33)4h, (41) and compounds 1, (25)2, (42)48, (43)9, (44)10, (44)1114, (45)1518, (43)19, (46)20, (47) and 23 (48) were synthesized according to the methods described previously.
中间体 1a-c、(25)3b、(33)4h、(41)以及化合物 1、(25)2、(42)4-8、(43)9、(44)10、(44)11-14、(45)15-18、(43)19、(46)20、(47)和 23(48)均按照先前描述的方法合成。

Methyl {4,6-Diamino-2-[1-(2-fluorobenzyl)-1H-pyrazolo[3,4-b]pyridin-3-yl]pyrimidin-5-yl}(2-hydroxyethyl)carbamate (3)
甲基{4,6-二氨基-2-[(1-氟苯基)-1H-吡唑并[3,4-b]吡啶-3-基]嘧啶-5-基}(2-羟基乙基)甲酰胺(3)

1c (200 mg, 0.57 mmol) and AcOH (33 μL, 0.57 mmol) were dissolved in MeOH (20 mL), and the mixture was cooled to 0 °C. Then a solution of glycolaldehyde (34 mg, 0.57 mmol) in MeOH (20 mL) was added dropwise. After complete addition, the mixture was stirred for a further 15 min at 0 °C. Then NaBH3CN (50 mg, 0.80 mmol) was added portionwise. The mixture was stirred at rt for 20 h and then concentrated under reduced pressure. The residue was diluted with EtOAc and washed with concd aq NH4Cl solution. The organic layer was concentrated under reduced pressure, and the residue was purified by HPLC (MeCN/H2O gradient) to give 2-({4,6-diamino-2-[1-(2-fluorobenzyl)-1H-pyrazolo[3,4-b]pyridin-3-yl]pyrimidin-5-yl}amino)ethanol (1d) as a yellow solid. Yield 165 mg (73%). 1H NMR (400 MHz, DMSO-d6): δ = 9.04 (dd, J = 8.1, 1.7 Hz, 1H), 8.58 (dd, J = 4.7, 1.7 Hz, 1H), 7.27–7.39 (m, 2H), 7.22 (dd, J = 10.0, 8.8 Hz, 1H), 7.08–7.16 (m, 2H), 6.09 (br s, 4H), 5.77 (s, 2H), 4.77 (t, J = 5.1 Hz, 1H), 3.47 (td, J = 5.6, 5.1 Hz, 2H), 3.27 (t, J = 6.4 Hz, 1H), 2.83 (dt, J = 6.4, 5.6 Hz, 2H). LC-MS (method a): tR (min) = 1.27. MS (EI+): m/z = 395 [M + H]+. 
1d (265 mg, 0.67 mmol) was dissolved in pyridine (5.0 mL), and the solution was cooled to 0 °C. Methyl chloroformate (83 mg, 0.87 mmol) was added, and the mixture was stirred at rt overnight. Then, the mixture was concentrated under reduced pressure, and the residue was purified by preparative HPLC (H2O/MeCN/1% TFA 56:30:14) to give 3 as a white solid. Yield 37 mg (12%). 1H NMR (500 MHz, DMSO-d6): δ = 9.04 (dd, J = 8.0, 1.5 Hz, 1H), 8.59 (dd, J = 4.5, 1.5 Hz, 1H), 7.34–7.38 (m, 1H), 7.32 (dd, J = 8.0, 4.5 Hz, 1H), 7.22 (dd, J = 10.0, 8.8 Hz, 1H), 7.09–7.15 (m, 2H), 6.07 (br s, 4H), 5.78 (s, 2H), 4.14 (t, J = 5.7 Hz, 2H), 3.70 (s, 3H), 3.54 (t, J = 7.0 Hz, 1H), 3.00 (dt, J = 7.0, 5.7 Hz, 2H). LC-MS (method a): tR (min) = 1.49. MS (EI+): m/z = 453 [M + H]+. 

3-Bromo-1H-pyrazolo[3,4-c]pyridazine (3c)
3-溴-1H-吡唑并[3,4-c]吡啶杂氮(3c)

1H-Pyrazolo[3,4-c]pyridazin-3-ol (33) (3b; 18.00 g, 132.24 mmol) was dissolved in sulfolane (176 mL), and POBr3 (39.81 g, 138.85 mmol) was then added to the solution. The mixture was stirred at 150 °C for 3 h. After being cooled to rt, the mixture was poured onto ice–water and extracted with EtOAc (3×). The combined organic extracts were washed with H2O and brine, dried over Na2SO4, filtered, and concentrated until a precipitate formed. The solids were collected by filtration and dried under reduced pressure. Yield 16.27 g (62%). 1H NMR (400 MHz, DMSO-d6): δ = 14.90 (br s, 1H), 9.23 (d, J = 5.6 Hz, 1H), 8.10 (d, J = 5.6 Hz, 1H). LC-MS (method b): tR (min) = 1.15. MS (ESI+): m/z = 199 [M + H]+.
1H-Pyrazolo[3,4-c]pyridazin-3-ol(33)(3b;18.00 克,132.24 摩尔)溶于磺化物(176 毫升),然后向溶液中加入 POBr3(39.81 克,138.85 摩尔)。混合物在 150°C 下搅拌 3 小时。冷却至室温后,将混合物倒入冰水中,用乙酸乙酯(EtOAc)萃取(3 次)。合并有机萃取物,用水和盐水洗涤,经无水硫酸钠干燥,过滤,浓缩至有沉淀形成。通过过滤收集固体,并在减压下干燥。产率 16.27 克(62%)。1H NMR(400 MHz,DMSO-d6):δ = 14.90(br s,1H),9.23(d,J = 5.6 Hz,1H),8.10(d,J = 5.6 Hz,1H)。LC-MS(方法 b):tR(分钟)= 1.15。MS(ESI+):m/z = 199 [M + H]+。

3-Bromo-1-(2-fluorobenzyl)-1H-pyrazolo[3,4-c]pyridazine (3d)
3-溴-1-(2-氟苯基)-1H-吡唑并[3,4-c]吡啶嗪(3d)

3c (16.27 g, 81.75 mmol) and Cs2CO3 (31.97 g, 98.10 mmol) were mixed in DMF (150 mL). A solution of 2-fluorobenzyl bromide (17.00 g, 89.93 mmol) in DMF (50 mL) was then added dropwise, and the mixture was stirred at rt overnight. The mixture was then diluted with EtOAc (500 mL) and washed with H2O (3×) and brine (1×). The organic phase was separated, dried over MgSO4, filtered, and concentrated. The residue was purified by flash column chromatography on silica gel (DCM/MeOH gradient). The obtained crude product was triturated with MTBE and DCM, collected by filtration, washed with MTBE, and dried under reduced pressure. Yield 11.36 g (45%). 1H NMR (400 MHz, DMSO-d6): δ = 9.30 (d, J = 5.6 Hz, 1H), 8.15 (d, J = 5.6 Hz, 1H), 7.35–7.44 (m, 2H), 7.16–7.28 (m, 2H), 5.96 (s, 2H). LC-MS (method c): tR (min) = 1.11. MS (ESI+): m/z = 307 [M + H]+.
3c(16.27 克,81.75 毫摩尔)和 Cs2CO3(31.97 克,98.10 毫摩尔)在 150 毫升 DMF 中混合。随后,将 2-氟苄基溴(17.00 克,89.93 毫摩尔)的 DMF 溶液(50 毫升)逐滴加入,混合物在室温下搅拌过夜。然后,用 500 毫升乙酸乙酯稀释混合物,并用水(3 次)和盐水(1 次)洗涤。将有机相分离,用 MgSO4 干燥,过滤,浓缩。将残留物通过硅胶柱色谱(DCM/MeOH 梯度)纯化。得到的粗产品与 MTBE 和 DCM 研磨,过滤收集,用 MTBE 洗涤,减压干燥。产率 11.36 克(45%)。1H NMR(400 MHz,DMSO-d6):δ = 9.30(d,J = 5.6 Hz,1H),8.15(d,J = 5.6 Hz,1H),7.35–7.44(m,2H),7.16–7.28(m,2H),5.96(s,2H)。LC-MS(方法 c):tR(分钟)= 1.11。MS(ESI+):m/z = 307 [M + H]+。

1-(2-Fluorobenzyl)-1H-pyrazolo[3,4-c]pyridazine-3-carbonitrile (3e)
1-(2-氟苯基)-1H-吡唑并[3,4-c]吡啶-3-基腈(3e)

3d (1.00 g, 3.26 mmol) and CuCN (0.32 g, 3.58 mmol) were treated with anhyd DMSO (10 mL), and the mixture was stirred at 150 °C for 9 h. The mixture was brought to rt, then poured onto ice–water and treated with 25% aq NH4OH until all solids were dissolved (blue solution). The mixture was then extracted with EtOAc (3×). The combined organic extracts were dried over Na2SO4, filtered, and concentrated. The residue was purified by flash column chromatography on silica gel (cyclohexane/EtOAc 4:1) to give the crude product which was used without further purification. Yield 0.57 g (51%, purity 74%). 1H NMR (400 MHz, DMSO-d6): δ = 9.45 (d, J = 5.9 Hz, 1H), 8.50 (d, J = 5.9 Hz, 1H), 7.39–7.51 (m, 2H), 7.18–7.30 (m, 2H), 6.11 (s, 2H). LC-MS (method d): tR (min) = 0.94. MS (ESI+): m/z = 254 [M + H]+.
3d(1.00 克,3.26 毫摩尔)和 CuCN(0.32 克,3.58 毫摩尔)与无水 DMSO(10 毫升)混合,在 150°C 下搅拌 9 小时。将混合物冷却至室温,倒入冰水中,用 25%的氨水溶液处理至所有固体溶解(蓝色溶液)。然后将混合物用乙酸乙酯(3 次)萃取。将合并的有机层干燥后用无水硫酸钠过滤,浓缩。得到的残留物通过硅胶柱色谱(环己烷/乙酸乙酯 4:1)纯化,得到粗产品,未进一步纯化即使用。产率 0.57 克(51%,纯度 74%)。1H NMR(400 MHz,DMSO-d6):δ = 9.45(d,J = 5.9 Hz,1H),8.50(d,J = 5.9 Hz,1H),7.39–7.51(m,2H),7.18–7.30(m,2H),6.11(s,2H)。LC-MS(方法 d):tR(分钟)= 0.94。MS(ESI+):m/z = 254 [M + H]+。

1-(2-Fluorobenzyl)-1H-pyrazolo[3,4-c]pyridazine-3-carboximidamide (3f)
1-(2-氟苯基)-1H-吡唑并[3,4-c]吡啶-3-羧基脒(3f)

3e (1.19 g, 4.70 mmol) and NaOMe (0.96 g, 17.86 mmol) were treated with anhyd MeOH (31 mL), and the mixture was stirred at rt for 2 h. NH4Cl (0.30 g, 5.64 mmol) and AcOH (1.05 mL) were then added, and the mixture was stirred under reflux overnight. The mixture was then concentrated, and the residue was taken up in EtOAc and 1 M aq NaOH. The organic phase was separated, washed with 1 M aq NaOH, dried over Na2SO4, filtered, and concentrated. The solid residue was triturated with MTBE, collected by filtration, washed with DCM/MeOH (50:1), and dried under reduced pressure. Yellow solid; yield 0.43 g (34%). 1H NMR (400 MHz, DMSO-d6): δ = 9.26 (d, J = 5.1 Hz, 1H), 8.48 (d, J = 5.1 Hz, 1H), 7.38 (d, J = 5.9 Hz, 1H), 7.11–7.32 (m, 3H), 6.71 (br s, 2H), 5.99 (s, 2H). LC-MS (method c): tR (min) = 0.52. MS (ESI+): m/z = 271 [M + H]+. 

2-[1-(2-Fluorobenzyl)-1H-pyrazolo[3,4-c]pyridazin-3-yl]-5-[(E)-phenyldiazenyl]pyrimidine-4,6-diamine (3i)
2-[(2-氟苯基)-1H-吡唑并[3,4-c]吡啶嗪-3-基]-5-[(E)-苯基偶氮基]嘧啶-4,6-二胺(3i)

Aniline (0.202 mL, 2.22 mmol) was dissolved in H2O (2.0 mL), and the solution was cooled to 0 °C. Aqueous HCl (37%, 0.38 mL) was then added dropwise, followed by a solution of NaNO2 (153 mg, 2.22 mmol) in H2O (0.5 mL). The mixture was stirred for an additional 15 min, and a solution of NaOAc (231 mg, 2.82 mmol) in H2O (0.5 mL) was then added dropwise followed by a solution of malononitrile (147 mg, 2.22 mmol) in EtOH (3.0 mL). The mixture was then stirred for an additional 2 h at 0 °C. The resulting precipitate was collected by filtration, washed with H2O (3×), and then dissolved in DMF (5.0 mL). In a separate flask, 3f (599 mg, 2.22 mmol) was dissolved in DMF (5.0 mL), then Et3N (0.309 mL, 2.22 mmol) was added dropwise. The mixture was warmed to 85 °C, and the aforementioned DMF solution was added. The resulting mixture was stirred at 100 °C overnight then cooled to 0 °C and treated with H2O. The formed solids were collected by filtration, washed with H2O and MeOH, and dried to give 2-[1-(2-fluorobenzyl)-1H-pyrazolo[3,4-c]pyridazin-3-yl]-5-[(E)-phenyldiazenyl]pyrimidine-4,6-diamine as a yellow solid. Yield 0.68 g (68%). 1H NMR (400 MHz, DMSO-d6): δ = 9.35 (d, J = 5.4 Hz, 1H), 9.01 (d, J = 5.6 Hz, 1H), 8.54 (br s, 2H), 7.89–8.07 (m, J = 7.8 Hz, 4H), 7.45–7.54 (m, 2H), 7.31–7.44 (m, 3H), 7.16–7.30 (m, 2H), 6.06 (s, 2H). LC-MS (method d): tR (min) = 1.08. MS (ESI+): m/z = 441 [M + H]+. 

2-[1-(2-Fluorobenzyl)-1H-pyrazolo[3,4-c]pyridazin-3-yl]pyrimidine-4,5,6-triamine (3h)
2-[1-(2-氟苯基)-1H-吡唑并[3,4-c]吡啶并[3,4-c]三胺]-4,5,6-三胺(3h)

3i (300 mg, 0.68 mmol) was suspended in DMF (15 mL) and 10% Pd/C (58 mg) was added. The mixture was stirred in an H2 atmosphere at ambient pressure overnight. The solids were collected by filtration and washed with MeOH and DCM, and the filtrate was concentrated under reduced pressure. The residue was triturated with DCM, and the solids were collected by filtration and dried under reduced pressure at 50 °C to give 2-[1-(2-fluorobenzyl)-1H-pyrazolo[3,4-c]pyridazin-3-yl]pyrimidine-4,5,6-triamine. The crude product was used without further purification. Yield 198 mg (74%, purity 90%). LC-MS (method d): tR (min) = 1.08. MS (ESI+): m/z = 352 [M + H]+.
3i(300 毫克,0.68 毫摩尔)悬浮于 15 毫升 DMF 中,加入 10% Pd/C(58 毫克)。混合物在常压下的 H2 气氛中搅拌过夜。通过过滤收集固体,并用甲醇和二氯甲烷洗涤,然后在减压下浓缩滤液。将残渣与二氯甲烷研磨,通过过滤收集固体,并在 50°C 下减压干燥,得到 2-[1-(2-氟苯基)-1H-吡唑并[3,4-c]吡啶-3-基]嘧啶-4,5,6-三胺。粗产品未经进一步纯化即使用。产率 198 毫克(74%,纯度 90%)。液相色谱-质谱(方法 d):保留时间(分钟)= 1.08。质谱(ESI+):相对分子质量 m/z = 352 [M + H]+。

2-[8-(2-Fluorobenzyl)imidazo[1,5-a]pyrimidin-6-yl]-5-[(E)-phenyldiazenyl]pyrimidine-4,6-diamine (4i)
2-[8-(2-氟苯基咪唑[1,5-a]嘧啶-6-基)]-5-[(E)-苯基偶氮基]嘧啶-4,6-二胺(4i)

8-(2-Fluorobenzyl)imidazo[1,5-a]pyrimidine-6-carboximidamide (41) (4h; 70% purity, 1.30 g, 3.40 mmol) was mixed with DMF (24.6 mL) and Et3N (0.52 mL, 3.74 mmol). At 85 °C, a solution of [(E)-phenyldiazenyl]malononitrile (1b; 694 mg, 4.08 mmol) in DMF (12.0 mL) was added dropwise and the mixture was stirred for an additional 15 h at 100 °C. The resulting mixture was then poured into H2O (300 mL), and the resulting precipitate was collected by filtration, washed with H2O, and dried. Then, MeCN (160 mL) was added and the remaining solids were collected by filtration and dried again to give 2-[8-(2-fluorobenzyl)imidazo[1,5-a]pyrimidin-6-yl]-5-[(E)-phenyldiazenyl]pyrimidine-4,6-diamine. Yield 419 mg (28%). 1H NMR (400 MHz, DMSO-d6): δ = 10.23–10.31 (m, 1H), 9.42 (br s, 1H), 8.60–8.66 (m, 1H), 8.12 (d, J = 7.6 Hz, 2H), 7.51–7.58 (m, 2H), 7.44–7.50 (m, 1H), 7.23–7.36 (m, 3H), 7.15–7.23 (m, 1H), 7.08–7.15 (m, 1H), 4.44 (s, 2H). LC-MS (method c): tR (min) = 1.14. MS (ESI+): m/z = 440 [M + H]+. 

2-[8-(2-Fluorobenzyl)imidazo[1,5-a]pyrimidin-6-yl]pyrimidine-4,5,6-triamine (4j)
2-[8-(2-氟苯基)咪唑并[1,5-a]嘧啶-6-基]嘧啶-4,5,6-三胺(4j)

4i (100 mg, 0.228 mmol) was dissolved in DMF (11.4 mL) and MeOH (2.9 mL). The solution was cooled with ice, and then 10% Pd/C (20 mg) was added. The mixture was stirred at 0 °C under 1 atm of H2 for 48 h. The resulting mixture was filtered through Celite, and the solids were washed with MeOH. The combined filtrate was concentrated under reduced pressure to give crude 2-[8-(2-fluorobenzyl)imidazo[1,5-a]pyrimidin-6-yl]pyrimidine-4,5,6-triamine, which was purified by preparative reversed-phase HPLC (H2O + 0.1% NH3/MeCN gradient). Brown solid; yield 36 mg (45%). 1H NMR (400 MHz, DMSO-d6): δ = 10.13 (br d, J = 7.4 Hz, 1H), 8.20 (br d, J = 3.9 Hz, 1H), 7.19–7.31 (m, 2H), 7.04–7.17 (m, 2H), 6.81 (dd, J = 3.7, 7.6 Hz, 1H), 5.89 (s, 4H), 4.27 (s, 2H), 4.06 (s, 2H). LC-MS (method d): tR (min) = 0.74. MS (ESI+): m/z = 351 [M + H]+.
4i(100 毫克,0.228 毫摩尔)溶解于 DMF(11.4 毫升)和 MeOH(2.9 毫升)中。溶液冷却至冰点,然后加入 10% Pd/C(20 毫克)。混合物在 0°C、1 个大气压的 H2 下搅拌 48 小时。所得混合物通过 Celite 过滤,固体用 MeOH 洗涤。合并滤液,减压浓缩,得到粗品 2-[8-(2-氟苯基)咪唑[1,5-a]嘧啶-6-基]嘧啶-4,5,6-三胺,经制备型反相高效液相色谱(H2O + 0.1% NH3/MeCN 梯度)纯化。棕色固体;产率 36 毫克(45%)。1H NMR(400 MHz,DMSO-d6):δ = 10.13(br d,J = 7.4 Hz,1H),8.20(br d,J = 3.9 Hz,1H),7.19–7.31(m,2H),7.04–7.17(m,2H),6.81(dd,J = 3.7,7.6 Hz,1H),5.89(s,4H),4.27(s,2H),4.06(s,2H)。LC-MS(方法 d):tR(分钟)= 0.74。MS(ESI+):m/z = 351 [M + H]+。

4-(2,2,3,3-Tetrafluoropropyl)morpholine (6b)
4-(2,2,3,3-四氟丙基)吗啉(6b)

Tf2O (252.5 g, 0.895 mol) was heated to 40 °C and, at this temperature, 2,2,3,3-tetrafluoro-1-propanol (6a; 130.0 g, 0.984 mol) was metered in while cooling. After the addition was completed, the mixture was heated to 70–75 °C and stirred for 2 h. Then the mixture was cooled to 20 °C, and the reaction solution of 2,2,3,3-tetrafluoropropyl trifluoromethanesulfonate was used without further purification.
Tf2O(252.5 克,0.895 摩尔)被加热至 40°C,在此温度下,2,2,3,3-四氟-1-丙醇(6a;130.0 克,0.984 摩尔)被计量加入,同时冷却。加入完成后,混合物被加热至 70-75°C,搅拌 2 小时。然后混合物被冷却至 20°C,未进一步纯化的 2,2,3,3-四氟丙基三氟甲烷磺酸反应溶液被使用。
Morpholine (158.5 g, 1.82 mol) was cooled to 5 °C. At 5–10 °C, the reaction solution of the triflate (189.5 g, max 0.455 mol) was added dropwise while cooling and then the mixture was stirred at 5–10 °C for 30 min. Then, the mixture was heated to 40 °C and stirred for 1 h. After cooling to 20 °C, H2O (160 mL) and toluene (160 mL) were added and the phases were separated. The organic phase was washed with H2O (160 mL) and concentrated on a rotary evaporator at 50 °C/50 mbar. The residue (81.0 g) was distilled at 67–68 °C/18 mbar to give 6b. Yield 77.0 g (84%). 1H NMR (400 MHz, CDCl3): δ = 5.83–6.22 (m, 1H), 3.61–3.78 (m, 4H), 2.89 (tt, J = 14.0, 1.7 Hz, 2H), 2.53–2.70 (m, 4H).
Morpholine(158.5 克,1.82 摩尔)被冷却至 5°C。在 5-10°C 时,将三氟甲磺酸酯(189.5 克,最大 0.455 摩尔)逐滴加入并继续冷却,然后在 5-10°C 下搅拌 30 分钟。之后,将混合物加热至 40°C,搅拌 1 小时。冷却至 20°C 后,加入 160 毫升水和 160 毫升甲苯,分离两相。用 160 毫升水洗涤有机相,并在 50°C/50 mbar 下用旋转蒸发器浓缩。得到的残留物(81.0 克)在 67-68°C/18 mbar 下蒸馏,得到 6b。产率 77.0 克(84%)。1H NMR(400 MHz,CDCl3):δ = 5.83-6.22(m,1H),3.61-3.78(m,4H),2.89(tt,J = 14.0,1.7 Hz,2H),2.53-2.70(m,4H)。

4-Methyl-4-(2,2,3,3-tetrafluoropropyl)morpholin-4-ium Methanesulfonate (6c)
4-甲基-4-(2,2,3,3-四氟丙基)吗啉-4-ium 甲磺酸盐(6c)

Methylmethanesulfonate (143.7 g, 1.31 mol) was heated to 135 °C and, at this temperature, 6b (250.0 g, 1.24 mol) was added dropwise. The mixture was stirred at 100 °C for 22 h, then cooled to 85 °C and i-PrOH (375 mL) was added. After cooling to 0–5 °C, the mixture was stirred for a further 30 min. The product was collected by suction filtration, washed with i-PrOH (3 × 125 mL), and dried at 45 °C under a gentle N2 stream in a vacuum drying cabinet. Yield 336.8 g (87%). 1H NMR (400 MHz, D2O): δ = 6.13–6.48 (m, 1H), 4.33–4.51 (m, 2H), 4.01–4.24 (m, 4H), 3.68–3.93 (m, 4H), 3.55 (s, 3H), 2.81 (s, 3H).
甲基甲烷磺酸(143.7 克,1.31 摩尔)加热至 135°C,在此温度下,6b(250.0 克,1.24 摩尔)逐滴加入。混合物在 100°C 下搅拌 22 小时,然后冷却至 85°C,加入 375 毫升-PrOH。冷却至 0-5°C 后,继续搅拌 30 分钟。产品通过吸滤收集,用-PrOH(3×125 毫升)洗涤,并在 45°C 下,在真空干燥柜中用温和的 N2 气流干燥。产率 336.8 克(87%)。1H NMR(400 MHz,D2O):δ = 6.13–6.48(m,1H),4.33–4.51(m,2H),4.01–4.24(m,4H),3.68–3.93(m,4H),3.55(s,3H),2.81(s,3H)。

4-Methyl-4-(2,3,3-trifluoroprop-1-enyl)morpholin-4-ium Methanesulfonate (6d)
4-甲基-4-(2,3,3-三氟丙-1-烯基)吗啉-4-ium 甲磺酸盐(6d)

First, 45% aq NaOH (16.9 g, 189.9 mmol) was metered into a solution of 6c (53.8 g, 172.7 mol) in H2O (40 mL) at 50–55 °C, and the mixture was stirred at 50 °C for 1 h then cooled to 20 °C. Then the precipitated salts were removed by suction filtration and washed with H2O (5 mL). The aqueous solution of product 6d (102.1 g, max 172.7 mmol) was used in the next stage. For analytical purposes, a sample was concentrated and dried. 1H NMR (400 MHz, D2O): δ = 6.74–6.83 (m, 1H), 6.39–6.69 (m, 1H), 4.12–4.20 (m, 2H), 3.97–4.09 (m, 4H), 3.76–3.85 (m, 2H), 3.59 (s, 3H), 2.81 (s, 3H).
首先,将 45%的 NaOH 溶液(16.9 克,189.9 毫摩尔)加入 6c 溶液(53.8 克,172.7 摩尔)中,该溶液在 40 毫升水中,温度控制在 50-55°C,混合物在 50°C 下搅拌 1 小时,然后冷却至 20°C。随后,通过吸滤去除沉淀的盐,并用 5 毫升水洗涤。产品 6d 的溶液(102.1 克,最大 172.7 毫摩尔)用于下一阶段。为了分析目的,取样品浓缩并干燥。1H NMR(400 MHz,D2O):δ = 6.74-6.83(m,1H),6.39-6.69(m,1H),4.12-4.20(m,2H),3.97-4.09(m,4H),3.76-3.85(m,2H),3.59(s,3H),2.81(s,3H)。

2-Fluoro-3-(morpholin-4-yl)acrylaldehyde (6e)
2-氟-3-(4-吗啉基)丙烯醛(6e)

A mixture of morpholine (30.2 g, 345.3 mmol) and Et3N (52.5 g, 518.0 mmol) was heated to 75 °C, and the aqueous solution of 6d (max 172.7 mmol) was added dropwise at 75–80 °C. The mixture was stirred under reflux for 2 h, cooled to 23 °C, and washed with DCM (100 mL). The aqueous phase was washed twice with DCM/Et3N (100:15, 115 mL), and the combined organic phases were washed with satd aq K2CO3 solution (85 mL) and concentrated under reduced pressure at 45–50 °C. Toluene (120 mL) was added, then toluene (60 mL) was distilled off. The suspension was stirred at rt overnight, and the product was collected by suction filtration and dried at 50 °C under a gentle N2 stream in a vacuum drying cabinet. Yield 19.2 g (68%). 1H NMR (500 MHz, CDCl3): δ = 8.59 (d, J = 18.9 Hz, 1H), 6.16 (d, J = 27.1 Hz, 1H), 3.72–3.83 (m, 4H), 3.51–3.60 (m, 4H).
一种由 30.2 克(345.3 毫摩尔)的哌啶和 52.5 克(518.0 毫摩尔)的 Et3N 混合物在 75°C 下加热,然后逐滴加入最多 172.7 毫摩尔的 6d 水溶液,温度保持在 75-80°C。混合物在回流条件下搅拌 2 小时,冷却至 23°C,并用 100 毫升二氯甲烷(DCM)洗涤。水相用二氯甲烷/三乙胺(100:15,115 毫升)洗涤两次,合并后的有机相用饱和碳酸钾溶液(85 毫升)洗涤,并在 45-50°C 下减压浓缩。加入 120 毫升甲苯,然后蒸馏掉 60 毫升甲苯。将悬浮液在室温下搅拌过夜,通过吸滤收集产物,并在 50°C 下用氮气流在真空干燥柜中干燥。产率 19.2 克(68%)。1H NMR(500 MHz,CDCl3):δ = 8.59(d,J = 18.9 Hz,1H),6.16(d,J = 27.1 Hz,1H),3.72-3.83(m,4H),3.51-3.60(m,4H)。

Ethyl 5-Fluoro-1-(2-fluorobenzyl)-1H-pyrazolo[3,4-b]pyridine-3-carboxylate (6f)
乙基 5-氟-1-(2-氟苯基)-1H-吡唑并[3,4-b]吡啶-3-甲酸酯(6f)

Ethyl 5-amino-1-(2-fluorobenzyl)-1H-pyrazole-3-carboxylate (49) (22.3 g, 84.8 mmol) was initially charged into EtOH (59.5 mL), and MsOH (11.0 mL, 169.6 mmol), LiCl (9.0 g, 212.1 mmol), and 6e (15.0 g, 84.8 mmol) were added at rt. The mixture was stirred under reflux for 4.5 h. After cooling to rt, the product was collected by suction filtration, washed with EtOH (2 × 4.5 mL), and stirred with H2O (325 mL) for 1 h. The solids were collected by suction filtration, washed with H2O (2 × 11.5 mL), and dried at 50 °C under a gentle N2 stream in a vacuum drying cabinet. Yield 21.8 g (81%). 1H NMR (400 MHz, CDCl3): δ = 8.51 (dd, J = 2.7, 1.7 Hz, 1H), 8.15 (dd, J = 7.7, 2.8 Hz, 1H), 7.19–7.33 (m, 1H), 6.93–7.16 (m, 3H), 5.88 (s, 2H), 4.52 (q, J = 7.2 Hz, 2H), 1.48 (t, J = 7.2 Hz, 3H). MS (ESI+): m/z = 318 [M + H]+. 

5-Fluoro-1-(2-fluorobenzyl)-1H-pyrazolo[3,4-b]pyridine-3-carboxamide (6g)
5-氟-1-(2-氟苯基)-1H-吡唑并[3,4-b]吡啶-3-甲酰胺(6g)

EtOH (10 mL), formamide (14.9 mL, 441.2 mmol), and 30% NaOMe in MeOH (3.6 g, 19.8 mmol) were added to 6f (7.0 g, 22.1 mmol). The reaction mixture was heated to 95–100 °C, and the volatile solvents were distilled off by a downward distillation within 1 h. The mixture was stirred at 125 °C for 1.5 h, H2O (30 mL) was added, and the resulting mixture was cooled to rt and stirred for 1 h. The precipitated solids were collected by suction filtration, washed with H2O (3 × 8.5 mL), and dried at 45 °C under a gentle N2 stream in a vacuum drying cabinet. Yield 6.2 g (97%). 1H NMR (400 MHz, DMSO-d6): δ = 8.72 (dd, J = 2.7, 1.7 Hz, 1H), 8.28 (dd, J = 8.3, 2.8 Hz, 1H), 7.87 (br s, 1H), 7.60 (br s, 1H), 7.34–7.40 (m, 1H), 7.12–7.26 (m, 3H), 5.87 (s, 2H). MS (ESI+): m/z = 289 [M + H]+.
乙醇(10 mL)、甲酰胺(14.9 mL,441.2 mmol)和 30% NaOMe 的甲醇溶液(3.6 g,19.8 mmol)加入 6f(7.0 g,22.1 mmol)。反应混合物加热至 95-100°C,在 1 小时内通过向下蒸馏去除挥发性溶剂。混合物在 125°C 下搅拌 1.5 小时,加入 30 mL 水,然后将混合物冷却至室温并搅拌 1 小时。通过吸滤收集析出的固体,用 8.5 mL 水洗涤 3 次,并在真空干燥柜中在 45°C 下用温和的氮气流干燥。产率 6.2 g(97%)。1H NMR(400 MHz,DMSO-d6):δ = 8.72(dd,J = 2.7,1.7 Hz,1H),8.28(dd,J = 8.3,2.8 Hz,1H),7.87(br s,1H),7.60(br s,1H),7.34-7.40(m,1H),7.12-7.26(m,3H),5.87(s,2H)。MS(ESI+):m/z = 289 [M + H]+。

5-Fluoro-1-(2-fluorobenzyl)-1H-pyrazolo[3,4-b]pyridine-3-carbonitrile (6h)
5-氟-1-(2-氟苯基)-1H-吡唑并[3,4-b]吡啶-3-基腈(6h)

6g (17.3 g, 60.0 mmol) was heated to 103–107 °C in sulfolane (40.5 mL) and MeCN (5.4 mL). POCl3 (6.9 g, 45.0 mmol) was slowly added dropwise while stirring, the dropping funnel was rinsed with MeCN (2.8 mL), and then the mixture was stirred at 107 °C for 1.5 h until conversion was complete (HPLC). Then the mixture was cooled to rt and sulfolane/MeCN (5:1, 2.8 mL) and then H2O (17.8 mL) were added dropwise. The mixture was stirred for 0.5 h, a solution of aq NH3 (28%, 9.4 g) in H2O (22.7 mL) was added dropwise, and the resulting mixture was stirred for a further 2 h. The precipitated solids were collected by suction filtration, washed with H2O (3 × 20.5 mL), and dried at 50 °C under a gentle N2 stream in a vacuum drying cabinet. Yield 14.7 g (92%). 1H NMR (400 MHz, DMSO-d6): δ = 8.87 (dd, J = 2.6, 1.7 Hz, 1H), 8.52 (dd, J = 8.1, 2.6 Hz, 1H), 7.17–7.42 (m, 4H), 5.87 (s, 2H). MS (ESI+): m/z = 271 [M + H]+.
6g(17.3g,60.0mmol)在 103~107°C 下用磺酰烷(40.5mL)和甲腈(5.4mL)加热。在搅拌下缓慢滴加 POCl3(6.9g,45.0mmol),滴液漏斗用甲腈(2.8mL)冲洗,然后在 107°C 下搅拌 1.5 小时,直至转化完全(HPLC)。然后冷却至室温,加入磺酰烷/甲腈(5:1,2.8mL)和 H2O(17.8mL),滴加。搅拌 0.5 小时,滴加 28%的氨水溶液(9.4g,22.7mL),继续搅拌 2 小时。通过吸滤收集沉淀固体,用 H2O(3×20.5mL)洗涤,在 50°C 下在真空干燥柜中用温和的 N2 气流干燥。产率 14.7g(92%)。1H NMR(400MHz,DMSO-d6):δ=8.87(dd,J=2.6,1.7Hz,1H),8.52(dd,J=8.1,2.6Hz,1H),7.17–7.42(m,4H),5.87(s,2H)。MS(ESI+):m/z=271 [M + H]+。

5-Fluoro-1-(2-fluorobenzyl)-1H-pyrazolo[3,4-b]pyridine-3-carboximidamide Hydrochloride (6i)
5-氟-1-(2-氟苯基)-1H-吡唑并[3,4-b]吡啶-3-羧基脒盐酸盐(6i)

6h (406.0 g, 1.50 mol) was suspended in EtOH (2.08 L). Then, 30% NaOMe in MeOH (54.1 g, 0.30 mol) was added and the mixture was stirred at rt overnight. NH4Cl (88.4 g, 1.65 mol) was added, and the mixture was heated to 65 °C and stirred at 65 °C for 3.5 h. The solvents were distilled off, and the residue was stirred with EtOAc (1.60 L) overnight. The precipitated solids were collected by suction filtration, washed with EtOAc (2 × 140 mL), and dried at 50 °C under a gentle N2 stream in a vacuum drying cabinet. Yield 441.4 g (91%). 1H NMR (400 MHz, DMSO-d6): δ = 9.35 (br s, 3H), 8.86 (dd, J = 2.5, 1.5 Hz, 1H), 8.48 (dd, J = 8.8, 2.6 Hz, 1H), 7.36–7.43 (m, 1H), 7.29–7.35 (m, 1H), 7.22–7.28 (m, 1H), 7.15–7.20 (m, 1H), 5.90 (s, 2H). MS (ESI+): m/z = 288 [M + H]+.
6 小时(406.0 克,1.50 摩尔)的原料在乙醇(2.08 升)中悬浮。然后,加入 30%的 NaOMe 甲醇溶液(54.1 克,0.30 摩尔),混合物在室温下搅拌过夜。加入氯化铵(88.4 克,1.65 摩尔),将混合物加热至 65°C,并在 65°C 下搅拌 3.5 小时。蒸馏掉溶剂,将残留物与乙酸乙酯(1.60 升)搅拌过夜。通过抽滤收集析出的固体,用乙酸乙酯(2×140 毫升)洗涤,并在 50°C 下,在真空干燥柜中用温和的氮气流干燥。产率 441.4 克(91%)。1H NMR(400 MHz,DMSO-d6):δ = 9.35(br s,3H),8.86(dd,J = 2.5,1.5 Hz,1H),8.48(dd,J = 8.8,2.6 Hz,1H),7.36–7.43(m,1H),7.29–7.35(m,1H),7.22–7.28(m,1H),7.15–7.20(m,1H),5.90(s,2H)。MS(ESI+):m/z = 288 [M + H]+。

2-[5-Fluoro-1-(2-fluorobenzyl)-1H-pyrazolo[3,4-b]pyridin-3-yl]-5-[(E)-phenyldiazenyl]pyrimidine-4,6-diamine (6j)
2-[(5-氟-1-(2-氟苯基)-1H-吡唑并[3,4-b]吡啶-3-基)]-5-[(E)-苯基偶氮基]嘧啶-4,6-二胺(6j)

Concd HCl (262 g, 2.59 mol) and H2O (117.5 mL) were added dropwise at 0–5 °C to H2O (1.525 L) and aniline (117.5 g, 1.26 mol). Then, a solution of NaNO2 (87.1 g, 1.26 mol) in H2O (222.5 mL) was added dropwise within 1 h, the dropping funnel was rinsed with H2O (60 mL), and the mixture was stirred at 0–5 °C for 15 min. Thereafter, at this temperature, a solution of NaOAc (131.4 g, 1.60 mol) in H2O (665 mL) was added dropwise within 45 min, the dropping funnel was rinsed with H2O (60 mL), and a solution of malononitrile (83.4 g, 1.26 mol) in EtOH (233 mL) was added dropwise within 1 h. The dropping funnel was rinsed with EtOH (68.5 mL), and the mixture was further stirred at 0–5 °C for 2 h. The yellow solids were collected by suction filtration and washed with H2O (3 × 625 mL) and cold toluene (488 mL). The still-moist residue was dissolved in DMF (872 g), which gave a DMF solution of [(E)-phenyldiazenyl]malononitrile (1.117 kg). 6i (30.0 g, 92.7 mmol) was suspended in DMF (72 mL). The mixture was heated to 100 °C, and a mixture of Et3N (14.2 mL, 101.9 mmol) and the DMF solution of [(E)-phenyldiazenyl]malononitrile (150 g) was added dropwise at this temperature within 30 min. The dropping funnel was rinsed with DMF (30 mL), and the mixture was further stirred at 100 °C for 20 h. Then, it was cooled to 95–90 °C, H2O (24 mL) was added dropwise within 10 min, and the resulting mixture was cooled to 0–5 °C within 1.5 h and stirred for 1 h. The solids were collected by suction filtration, washed with H2O (60 mL)/DMF (63 mL), twice with H2O (50 mL)/MeOH (63 mL), and then with MeOH (63 mL), suction-dried, and then dried at 50 °C under a gentle N2 stream in a vacuum drying cabinet. Yield 35.5 g (84%). 1H NMR (400 MHz, DMSO-d6): δ = 9.03 (dd, J = 8.8, 2.8 Hz, 1H), 8.65–8.77 (m, 1H), 8.50 (br s, 2H), 8.02 (d, J = 7.6 Hz, 2H), 7.86–7.98 (m, 2H), 7.44–7.57 (m, 2H), 7.32–7.44 (m, 2H), 7.11–7.31 (m, 3H), 5.84 (s, 2H). LC-MS (method d): tR (min) = 1.15. MS (ESI+): m/z = 458 [M + H]+.
浓盐酸(262 克,2.59 摩尔)和水(117.5 毫升)在 0-5°C 下逐滴加入 1.525 升水中和苯胺(117.5 克,1.26 摩尔)。然后,在 1 小时内逐滴加入 87.1 克亚硝酸钠(1.26 摩尔)的水溶液(222.5 毫升),滴液漏斗用 60 毫升水冲洗,混合物在 0-5°C 下搅拌 15 分钟。之后,在此温度下,在 45 分钟内逐滴加入 131.4 克醋酸钠(1.60 摩尔)的水溶液(665 毫升),滴液漏斗用 60 毫升水冲洗,并逐滴加入 83.4 克丙二腈(1.26 摩尔)的乙醇溶液(233 毫升),在 1 小时内完成。滴液漏斗用 68.5 毫升乙醇冲洗,混合物在 0-5°C 下继续搅拌 2 小时。黄色固体通过吸滤收集,用 3×625 毫升水和 488 毫升冷甲苯洗涤。仍然潮湿的残留物溶解在 872 克二甲基甲酰胺中,得到[(E)-苯基叠氮基]丙二腈的二甲基甲酰胺溶液(1.117 千克)。6i(30.0 克,92.7 毫摩尔)悬浮在 72 毫升二甲基甲酰胺中。将混合物加热至 100°C,并在 30 分钟内逐滴加入 14.2 毫升三乙胺(101.9 毫摩尔)和[(E)-苯基叠氮基]丙二腈的二甲基甲酰胺溶液(150 克)。 滴定漏斗用 DMF(30 mL)冲洗,混合物在 100°C 下搅拌 20 小时。然后,将其冷却至 95-90°C,在 10 分钟内逐滴加入 24 mL 水,所得混合物在 1.5 小时内冷却至 0-5°C,并搅拌 1 小时。通过吸滤收集固体,用 H2O(60 mL)/DMF(63 mL)洗涤,然后用 H2O(50 mL)/甲醇(63 mL)洗涤两次,接着用甲醇(63 mL)洗涤,吸滤干燥,然后在 50°C 下,在真空干燥柜中用温和的氮气流干燥。产率 35.5 g(84%)。1H NMR(400 MHz,DMSO-d6):δ = 9.03(dd,J = 8.8,2.8 Hz,1H),8.65-8.77(m,1H),8.50(br s,2H),8.02(d,J = 7.6 Hz,2H),7.86-7.98(m,2H),7.44-7.57(m,2H),7.32-7.44(m,2H),7.11-7.31(m,3H),5.84(s,2H)。LC-MS(方法 d):tR(min)= 1.15。MS(ESI+):m/z = 458 [M + H]+。

2-[5-Fluoro-1-(2-fluorobenzyl)-1H-pyrazolo[3,4-b]pyridin-3-yl]pyrimidine-4,5,6-triamine (6k)
2-[5-氟-1-(2-氟苯基)-1H-吡唑并[3,4-b]吡啶-3-基]嘧啶-4,5,6-三胺(6k)

6j (182.0 g, 0.39 mol) was initially charged into DMF (1.82 L), and then 5% Pd/C (50% water-moist, 4.2 g) was added. Hydrogenation was effected at 60 °C and H2 pressure of 60 bar while stirring overnight. The mixture was filtered through kieselguhr, and the solids were washed thoroughly with DMF (150 mL) and then with MeOH (150 mL). The filtrate was concentrated at 60–70 °C to a weight of 425 g of distillation residue. The residue was heated to 75–80 °C, MeOH (300 mL) was added dropwise at this temperature, and the mixture was stirred for 15 min. The mixture was cooled to rt within 1 h, then H2O (1.29 L) was added dropwise and the mixture was stirred overnight. The solids were collected by suction filtration, washed with H2O (2 × 500 mL), suction-dried, and then dried at 50 °C under a gentle N2 stream in a vacuum drying cabinet. Yield 159.7 g. For analytical purposes, a sample was purified by chromatography on silica gel (DCM/MeOH 9:1). 1H NMR (400 MHz, DMSO-d6): δ = 8.85 (dd, J = 9.0, 2.9 Hz, 1H), 8.62 (dd, J = 2.8, 1.7 Hz, 1H), 7.32–7.39 (m, 1H), 7.10–7.26 (m, 3H), 5.86 (br s, 4H), 5.75 (s, 2H), 4.04 (br s, 2H). MS (ESI+): m/z = 369 [M + H]+.
6j(182.0 克,0.39 摩尔)最初加入 1.82 升 DMF 中,然后加入 5% Pd/C(50%水湿,4.2 克)。在 60°C 和 60 bar 的 H2 压力下搅拌过夜进行加氢。混合物通过硅藻土过滤,固体用 DMF(150 毫升)和甲醇(150 毫升)彻底洗涤。滤液在 60-70°C 浓缩至 425 克蒸馏残渣。残渣加热至 75-80°C,在此温度下逐滴加入 300 毫升甲醇,混合物搅拌 15 分钟。混合物在 1 小时内冷却至室温,然后逐滴加入 1.29 升水,混合物搅拌过夜。固体通过吸滤收集,用水(2×500 毫升)洗涤,吸滤干燥,然后在真空干燥柜中在 50°C 下用温和的 N2 气流干燥。产率 159.7 克。为分析目的,样品通过硅胶柱层析(DCM/MeOH 9:1)进行纯化。1H NMR(400 MHz,DMSO-d6):δ = 8.85(dd,J = 9.0,2.9 Hz,1H),8.62(dd,J = 2.8,1.7 Hz,1H),7.32-7.39(m,1H),7.10-7.26(m,3H),5.86(br s,4H),5.75(s,2H),4.04(br s,2H)。MS(ESI+):m/z = 369 [M + H]+。

Methyl {4,6-Diamino-2-[1-(2-fluorobenzyl)-1H-pyrazolo[3,4-c]pyridazin-3-yl]pyrimidin-5-yl}carbamate Formic Acid Salt (21)
甲基{4,6-二氨基-2-[(1-氟苯基)-1H-吡唑并[3,4-c]吡啶-3-基]嘧啶-5-基}甲酰胺甲酸盐(21)

The crude intermediate (3h: 198 mg, 0.51 mmol) was treated with pyridine (15.0 mL), and the mixture was cooled to 0 °C. Next, a solution of methyl chloroformate (43.6 μL, 0.57 mmol) in DCM (1.0 mL) was slowly added and the mixture was stirred at rt for 3 d. Additional methyl chloroformate (0.1 equiv) in DCM (1.0 mL) was added at 0 °C, and the mixture was stirred for a further 30 min at rt. The reaction mixture was then concentrated, and the residue was treated with MeCN. The resulting suspension was filtered, and the filtrate was concentrated. The residue was purified by preparative reversed-phase HPLC (H2O + 0.05% formic acid/MeOH gradient) to give 21 as a white solid. Yield 72 mg (33%, purity 97%). 1H NMR (400 MHz, DMSO-d6): δ = 9.28 (d, J = 5.4 Hz, 1H), 8.88 (d, J = 5.6 Hz, 1H), 8.15 (s, 1H), 7.95–8.06 (m, 1H), 7.34–7.43 (m, 1H), 7.12–7.33 (m, 4H), 6.17–6.32 (m, 4H), 6.01 (s, 2H), 3.62 (s, 3H). LC-MS (method d): tR (min) = 0.66. MS (ESI+): m/z = 410 [M + H]+.
粗中间体(3h:198 mg,0.51 mmol)与吡啶(15.0 mL)反应,混合物冷却至 0°C。随后,将甲基氯甲酸酯(43.6 μL,0.57 mmol)的 DCM(1.0 mL)溶液缓慢加入,混合物在室温下搅拌 3 天。在 0°C 下,再向混合物中加入 0.1 当量的甲基氯甲酸酯(DCM 1.0 mL),并在室温下继续搅拌 30 分钟。然后将反应混合物浓缩,将残留物用 MeCN 处理。得到的悬浮液经过滤,滤液浓缩。残留物通过制备型反相高效液相色谱(H2O + 0.05%甲酸/MeOH 梯度)纯化,得到 21 号化合物,为白色固体。产率 72 mg(产率 33%,纯度 97%)。1H NMR(400 MHz,DMSO-d6):δ = 9.28(d,J = 5.4 Hz,1H),8.88(d,J = 5.6 Hz,1H),8.15(s,1H),7.95–8.06(m,1H),7.34–7.43(m,1H),7.12–7.33(m,4H),6.17–6.32(m,4H),6.01(s,2H),3.62(s,3H)。LC-MS(方法 d):tR(min)= 0.66。MS(ESI+):m/z = 410 [M + H]+。

Methyl {4,6-Diamino-2-[8-(2-fluorobenzyl)imidazo[1,5-a]pyrimidin-6-yl]pyrimidin-5-yl}carbamate (22)
甲基{4,6-二氨基-2-[8-(2-氟苯基)咪唑[1,5-a]嘧啶-6-基]嘧啶-5-基}甲酰胺(22)

4j (45 mg, 0.128 mmol) was dissolved in pyridine (3.46 mL), and the solution was cooled to 0 °C. Then, methyl chloroformate (12.9 μL, 0.167 mmol) was added and the mixture was stirred for an additional 5 min at 0 °C and for 19 h at rt. The resulting mixture was concentrated under reduced pressure to a volume of 1.5 mL, and the residue was purified by preparative reversed-phase HPLC (H2O + 0.1% NH3/MeCN gradient) to give 22 as a yellow solid. Yield 24 mg (45%). 1H NMR (400 MHz, DMSO-d6): δ = 10.22 (d, J = 7.15 Hz, 1H), 8.28 (d, J = 3.8 Hz, 1H), 7.97 (br s, 0.65 H), 7.67 (br s, 0.35 H), 7.20–7.29 (m, 2H), 7.11–7.18 (m, 1H), 7.05–7.11 (m, 1H), 6.89 (dd, J = 3.7, 7.3 Hz, 1H), 6.24 (br s, 4H), 4.30 (s, 2H), 3.61 (br s, 3H). LC-MS (method d): tR (min) = 0.73. MS (ESI+): m/z = 409 [M + H]+.
4j(45 mg,0.128 mmol)溶解于吡啶(3.46 mL)中,溶液冷却至 0°C。然后加入甲基氯甲酸酯(12.9 μL,0.167 mmol),在 0°C 下搅拌 5 分钟,并在室温下搅拌 19 小时。将得到的混合物在减压下浓缩至 1.5 mL,并通过制备型反相高效液相色谱(H2O + 0.1% NH3/MeCN 梯度)纯化,得到 22 号化合物,为黄色固体。产率 24 mg(45%)。1H NMR(400 MHz,DMSO-d6):δ = 10.22(d,J = 7.15 Hz,1H),8.28(d,J = 3.8 Hz,1H),7.97(br s,0.65 H),7.67(br s,0.35 H),7.20–7.29(m,2H),7.11–7.18(m,1H),7.05–7.11(m,1H),6.89(dd,J = 3.7,7.3 Hz,1H),6.24(br s,4H),4.30(s,2H),3.61(br s,3H)。LC-MS(方法 d):tR(min)= 0.73。MS(ESI+):m/z = 409 [M + H]+。

Methyl {4,6-Diamino-2-[5-fluoro-1-(2-fluorobenzyl)-1H-pyrazolo[3,4-b]pyridin-3-yl]pyrimidin-5-yl}carbamate (24)
甲基{4,6-二氨基-2-[5-氟-1-(2-氟苯基)-1H-吡唑并[3,4-b]吡啶-3-基]嘧啶-5-基}甲酰胺(24)

6k (77% by weight, 4.0 g, 8.36 mmol) in i-PrOH (37.9 mL) was heated to 35 °C, and then methyl chloroformate (0.84 mL, 10.87 mmol) was added dropwise. The mixture was stirred at 35–40 °C for 20 h, heated to 50 °C, and MeOH (9.5 mL) was added. Then, Et3N (1.9 mL) was added dropwise within 0.5 h, the dropping funnel was rinsed with MeOH (1.3 mL), and the resulting mixture was stirred at 50 °C for 1 h. Thereafter, the mixture was cooled to rt and stirred at rt for 1 h. The solids were collected by suction filtration, washed with EtOH (3 × 8 mL), suction-dried, and then dried at 50 °C under a gentle N2 stream in a vacuum drying cabinet. Yield 3.4 g of crude product. The crude product (3.0 g) was stirred in DMSO (8 mL) for 5 min, EtOAc (13.0 mL) and activated carbon (50 mg) were added, and the mixture was heated under reflux (84 °C) for 15 min. The suspension was hot-filtered, and the filter residue was washed with EtOAc (1.9 mL). EtOAc (60 mL) and EtOH (16 mL) were heated to 60 °C, the combined filtrates were added dropwise, and the resulting mixture was stirred at 60 °C for 1.5 h. The suspension was cooled to rt within 25 min, stirred for a further 1.5 h, cooled further to 0–5 °C, and stirred for a further 1 h. The solids were collected by suction filtration, washed with EtOAc (2 × 6.4 mL), suction-dried, and then dried at 50 °C under a gentle N2 stream in a vacuum drying cabinet to give 24. Yield 2.2 g (70%). 1H NMR (400 MHz, DMSO-d6): δ = 8.89 (dd, J = 9.0, 2.8 Hz, 1H), 8.66 (m, 1H), 7.99 and 7.67 (2 br s, 1H), 7.32–7.40 (m, 1H), 7.19–7.26 (m, 1H), 7.10–7.19 (m, 2H), 6.22 (br s, 4H), 5.79 (s, 2H), 3.62 (br s, 3H). LC-MS (method d): tR (min) = 0.79. MS (ESI+): m/z = 427 [M + H]+.
6k(质量分数 77%,4.0 克,8.36 毫摩尔)在-PrOH(37.9 毫升)中加热至 35°C,然后逐滴加入甲基氯甲酸酯(0.84 毫升,10.87 毫摩尔)。混合物在 35-40°C 下搅拌 20 小时,加热至 50°C,加入甲醇(9.5 毫升)。接着,在 0.5 小时内逐滴加入三乙胺(1.9 毫升),用甲醇(1.3 毫升)冲洗分液漏斗,所得混合物在 50°C 下搅拌 1 小时。之后,将混合物冷却至室温,在室温下搅拌 1 小时。通过吸滤收集固体,用乙醇(3×8 毫升)洗涤,吸滤干燥,然后在真空干燥柜中在 50°C 下用温和的氮气流干燥。产率 3.4 克粗产品。将粗产品(3.0 克)在 DMSO(8 毫升)中搅拌 5 分钟,加入乙酸乙酯(13.0 毫升)和活性炭(50 毫克),混合物在回流(84°C)下加热 15 分钟。热过滤悬浮液,用乙酸乙酯(1.9 毫升)洗涤滤渣。将乙酸乙酯(60 毫升)和乙醇(16 毫升)加热至 60°C,将混合滤液逐滴加入,所得混合物在 60°C 下搅拌 1.5 小时。将悬浮液在 25 分钟内冷却至室温,再搅拌 1 小时。5 小时,进一步冷却至 0-5°C,并搅拌 1 小时。通过吸滤收集固体,用乙醚(2×6.4 mL)洗涤,吸滤干燥,然后在 50°C 下,在真空干燥柜中用温和的氮气流干燥,得到 24.产率 2.2 g(70%)。1H NMR(400 MHz,DMSO-d6):δ = 8.89(dd,J = 9.0,2.8 Hz,1H),8.66(m,1H),7.99 和 7.67(2 br s,1H),7.32-7.40(m,1H),7.19-7.26(m,1H),7.10-7.19(m,2H),6.22(br s,4H),5.79(s,2H),3.62(br s,3H)。LC-MS(方法 d):tR(min)= 0.79。MS(ESI+):m/z = 427 [M + H]+。

Biology 生物

General 通用

Animal experiments were conducted in accordance with the German animal welfare laws, approved by local authorities, and in accordance with the ethical guidelines of Bayer AG.
动物实验是在遵守德国动物福利法、经当地当局批准以及符合拜耳公司伦理指南的情况下进行的。

CYP Inhibition Assay CYP 抑制测定法

The inhibitory potency of 24 was assessed in vitro by means on formation of metabolites from standard probes mediated by CYP isoforms (for details, please refer to the Supporting Information) based on assay conditions described. (50) To investigate time-dependency, preincubation experiments on CYP3A4 were performed. (51)
24 的抑制活性通过体外实验评估,该实验基于标准探针在 CYP 同工酶介导下形成代谢物的条件进行(具体细节请参阅补充材料)。为了研究时间依赖性,对 CYP3A4 进行了预孵育实验。(50)为了研究时间依赖性,对 CYP3A4 进行了预孵育实验。(51)

In Vitro Clearance Determinations with Rat and Human Hepatocytes
体外清除度测定:大鼠和人肝细胞

Incubations with hepatocytes were performed at 37 °C, pH 7.4, in a total volume of 1.5 mL using a modified Janus robotic system (PerkinElmer). The incubation mixtures contained 1 × 106 cells/mL (corrected, according to the viability of the cells, determined via microscopy after staining with trypan blue), 1 μM substrate, and Williams’ medium E (Sigma, product no. W1878). The final MeCN concentration was ≤1%. Aliquots of 125 μL were withdrawn from the incubation mixture after 2, 10, 20, 30, 50, 70, and 90 min and dispensed in a 96-well plate, containing MeCN (250 μL) to stop the reaction. After centrifugation at 1000g, supernatants were analyzed by LC-MS/MS (AB Sciex Triple Quad 5500).
在 37°C、pH 7.4 的条件下,使用改良的 Janus 机器人系统(PerkinElmer)进行肝细胞的培养。培养混合物中含有 1×10^6 个细胞/mL(根据细胞活力校正,通过显微镜染色后用台盼蓝染色确定),1 μM 底物和 Williams’ E 培养基(Sigma,产品编号 W1878)。最终 MeCN 浓度≤1%。在 2、10、20、30、50、70 和 90 分钟后,从培养混合物中取出 125 μL 的样品,并将其分装到含有 MeCN(250 μL)的 96 孔板中,以终止反应。在 1000g 的离心后,上清液通过 LC-MS/MS(AB Sciex Triple Quad 5500)进行分析。
The calculation of in vitro clearance values from half-life data using hepatocytes, reflecting substrate depletion, was performed using the following equations: CL′intrinsic [mL/(min·kg)] = (0.693/in vitro t1/2 [min]) (liver weight [g liver/kg body mass]) (cell no. [1.1 × 108]/liver weight [g])/(cell no. [1 × 106]/incubation volume [mL]). The CLblood was estimated using the nonrestricted well-stirred model: CLblood well-stirred [L/(h·kg)] = (QH [L/(h·kg)]·CL′intrinsic [L/(h·kg)])/(QH[L/(h·kg)] + CL′intrinsic [L/(h·kg)]). For calculations, the following values were used: human specific liver weight of 21 g/kg body mass, hepatic blood flow of 1.32 L/(h·kg), cell number in the liver was estimated to be 1.1 × 108 cells/g liver; rat specific liver weight of 32 g/kg body mass, hepatic blood flow of 4.2 L/(h·kg), cell number in the liver was estimated to be 1.1 × 108 cells/g liver. (52)
使用肝细胞从半衰期数据计算体外清除值,反映底物耗竭,采用以下公式进行:CL′intrinsic [mL/(min·kg)] = (0.693/in vitro t1/2 [min]) × (肝重 [g 肝/kg 体重]) × (细胞数 [1.1 × 10^8]/肝重 [g]) / (细胞数 [1 × 10^6]/培养体积 [mL])。CLblood 使用非限制性全混模型进行估算:CLblood well-stirred [L/(h·kg)] = (QH [L/(h·kg)] × CL′intrinsic [L/(h·kg)]) / (QH[L/(h·kg)] + CL′intrinsic [L/(h·kg)])。计算中使用了以下数值:人体特异肝重为 21 g/kg 体重,肝血流量为 1.32 L/(h·kg),肝脏中的细胞数估计为 1.1 × 10^8 个细胞/g 肝;大鼠特异肝重为 32 g/kg 体重,肝血流量为 4.2 L/(h·kg),肝脏中的细胞数估计为 1.1 × 10^8 个细胞/g 肝。 (52)

Caco-2 Permeability Assay
Caco-2 渗透性测定

The in vitro permeation of test compounds across a Caco-2 cell monolayer, a well-established in vitro system to predict the permeability from the gastrointestinal tract, was tested according to Artursson and Karlsson. (53) Caco-2 cells (ACC 169, DSMZ, German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany) were seeded on 24-well insert plates and were allowed to grow for 14–16 d. For permeability studies, the test compounds were dissolved in DMSO and diluted to the final test concentration of 2 μM with transport buffer [Hanks’ Buffered Salt Solution, Gibco/Invitrogen, further supplemented with glucose (final concentration 19.9 mM) and HEPES (final concentration 9.8 mM)]. For determination of the apical to basolateral permeability (Papp A–B), the test compound solution was added to the apical side of the cell monolayer and transport buffer to the basolateral side of the monolayer. For determination of the basolateral to apical permeability (Papp B–A), the test compound solution was added to the basolateral side of the cell monolayer and transport buffer to the apical side of the monolayer. Samples were taken from the donor compartment at the beginning of the experiment to confirm mass balance. After an incubation of 2 h at 37 °C, samples were taken from both compartments. Samples were analyzed by LC-MS, and the apparent permeability coefficients were calculated. The efflux ratio was calculated as Papp B–A/Papp A–B. Lucifer yellow permeability was assayed for each cell monolayer to ensure cell monolayer integrity, and the permeability of atenolol (low permeability marker) and sulfasalazine (marker for active excretion) was determined for each batch as a quality control.
在体外渗透实验中,测试化合物通过 Caco-2 细胞单层(一种用于预测胃肠道渗透性的成熟体外系统)的渗透性,按照 Artursson 和 Karlsson 的方法进行测试。(53)Caco-2 细胞(ACC 169,来源于德国微生物和细胞培养物收藏中心 DSMZ,位于德国不伦瑞克)被接种在 24 孔板中,并允许其生长 14-16 天。在渗透性研究中,测试化合物溶解于 DMSO 中,并稀释至最终测试浓度 2 μM,使用转运缓冲液[汉克斯缓冲盐溶液,Gibco/Invitrogen,进一步补充葡萄糖(最终浓度 19.9 mM)和 HEPES(最终浓度 9.8 mM)]。为了确定细胞单层的顶端到基底侧渗透性(Papp A-B),将测试化合物溶液添加到细胞单层的顶端,转运缓冲液添加到基底侧。为了确定细胞单层的基底侧到顶端渗透性(Papp B-A),将测试化合物溶液添加到细胞单层的基底侧,转运缓冲液添加到顶端。 实验开始时从供体室取样,以确认质量平衡。在 37°C 下孵育 2 小时后,从两个室中取样。样品通过液相色谱-质谱联用(LC-MS)进行分析,并计算了表观渗透系数。通过计算 Papp B-A/Papp A-B 得到外排比。对每个细胞单层进行了 Lucifer yellow 渗透性测定,以确保细胞单层完整性,并确定了每个批次中 atenolol(低渗透性标志物)和 sulfasalazine(活性排泄标志物)的渗透性,以进行质量控制。

Pharmacokinetic Parameters after Intravenous and Oral Application in Rats and Dogs
**药代动力学参数在大鼠和狗体内静脉和口服给药后的研究**

For in vivo pharmacokinetic experiments, male Wistar rats and female beagle dogs were used. Intravenous application was carried out with a species-specific plasma/DMSO formulation in rats and with a H2O/PEG 400/EtOH formulation in dogs. Oral application in both species was by gavage with a H2O/PEG 400/EtOH formulation. For simplification of blood drawing in rats, a silicone catheter was implanted into the right vena jugularis externa. The surgery was performed at least 1 d before substance application under isoflurane anesthesia and additional administration of an analgetic (atropine/rimadyl 3:1, 0.1 mL sc). Blood drawing (usually more than 10 time points) was done in a time window that included at least two time points after 24 h (postsubstance application). Blood was passed into heparinized tubes. Afterward, blood plasma was obtained by centrifugation at 1000g. Where necessary, the plasma was stored at −20 °C until further analysis.
在体内药代动力学实验中,使用了雄性 Wistar 大鼠和雌性比格犬。在老鼠身上,采用特定物种的血浆/DMSO 制剂进行静脉给药;在狗身上,采用 H2O/PEG 400/EtOH 制剂。两种动物均通过灌胃方式使用 H2O/PEG 400/EtOH 制剂进行口服给药。为了简化老鼠的采血,将硅胶导管植入右侧外颈静脉。手术在给药前至少 1 天进行,使用异氟醚麻醉,并额外给予镇痛剂(阿托品/瑞马唑 3:1,0.1 mL 皮下注射)。采血(通常超过 10 个时间点)在包括给药后至少 24 小时的两个时间点的时间窗口内进行。血液注入肝素化试管中。之后,通过 1000g 离心获得血浆。如有必要,将血浆储存在-20°C 直至进一步分析。
An internal standard was added to the sample, calibration, and qualifier solutions. The internal standard could also have been a compound from a different chemical class than the analyte of interest. Afterward, protein precipitation was performed by using an excess of MeCN. A buffer solution was added with a composition based on the mobile phases used in subsequent liquid chromatography. After centrifugation at 1000g, the supernatant was analyzed by LC-MS using different C18 reversed-phase columns and various mobile phase compositions. Quantification of the substance was conducted by using peak height or area calculated from extracted ion chromatograms of specific selected ion-monitoring experiments or high-resolution LC-MS experiments.
在样品、校准液和质控液中添加了内部标准。该内部标准也可以是与分析物不属于同一化学类的化合物。随后,通过使用过量的甲醇氰化物(MeCN)进行蛋白质沉淀。随后加入了一种缓冲溶液,其组成基于后续液相色谱中使用的流动相。在 1000g 的离心力下离心后,上清液通过使用不同的 C18 反相色谱柱和不同的流动相组成,由液相色谱-质谱联用(LC-MS)进行分析。通过使用从特定选择的离子监测实验或高分辨率 LC-MS 实验中提取的离子色谱图计算出的峰高或面积进行物质的定量分析。
From the plasma concentration–time course, the pharmacokinetic parameters CL (clearance), t1/2 (terminal half-life), VSS (volume of distribution at steady state), and F (bioavailability after oral administration) were calculated by using a validated internal pharmacokinetic calculation software.
从血浆浓度-时间曲线中,通过使用经过验证的内部药代动力学计算软件,计算了药代动力学参数 CL(清除率)、t1/2(终末半衰期)、VSS(稳态分布容积)和 F(口服后的生物利用度)。
Because substance quantification was done in plasma, the blood/plasma distribution needed to be analyzed to calculate a blood clearance value. Therefore, a defined amount of the substance was added to blood in heparinized tubes and incubated for 20 min by gently swinging. The plasma was obtained by centrifugation at 1000g. The cblood/cplasma value was calculated after measurement of the substance concentration in plasma and blood by using peak height or area calculated from extracted ion chromatograms of specific selected ion-monitoring experiments or high-resolution LC-MS experiments.
因为物质定量是在血浆中进行的,所以需要分析血液/血浆分布来计算血液清除率。因此,将一定量的物质添加到肝素化试管中的血液中,并轻轻摇动孵育 20 分钟。通过 1000g 的离心获得血浆。通过测量血浆和血液中物质浓度,并使用从特定选择的离子监测实验或高分辨率 LC-MS 实验中提取的离子色谱图计算出的峰高或面积来计算 cblood/cplasma 值。

Highly Purified sGC 高度纯化的 sGC

Enzyme activity was measured by the formation of [32P]-cGMP from α-[32P]-GTP, modified according to Hoenicka et al. (54) and Schermuly et al. (55) The modifications included using GTP, Mn2+/Mg2+, and cGMP at concentrations of 200 μM, 3 mM, and 1 mM, respectively. Enzyme concentrations were chosen carefully to achieve a substrate turnover of less than 10%, thus avoiding substrate or cofactor depletion. The characterization of the purified enzyme was performed at a protein concentration of 0.2 μg/mL. All measurements were performed in duplicate and were repeated five times. For enzyme characterization, the specific activity of sGC was expressed as x-fold stimulation vs specific basal activity. The highest DMSO concentration in the assay was 1% (v/v) and did not elicit any effect per se on cGMP production.
酶活性通过α-[32P]-GTP 形成[32P]-cGMP 来测定,方法参照 Hoenicka 等(54)和 Schermuly 等(55)的研究,并进行了修改。修改内容包括使用 GTP、Mn2+/Mg2+和 cGMP,其浓度分别为 200 μM、3 mM 和 1 mM。酶浓度选择得非常谨慎,以实现底物转换率低于 10%,从而避免底物或辅因子耗竭。纯化酶的特性分析在蛋白质浓度为 0.2 μg/mL 时进行。所有测量均重复两次,并重复五次。对于酶的特性分析,sGC 的比活性以倍数表示,与特定基础活性相比。实验中 DMSO 的最高浓度为 1%(体积比),本身对 cGMP 的产生没有影响。

Recombinant sGC-Overexpressing Cell Line
重组 sGC 高表达细胞系

The cellular activity of the test compounds was determined using a recombinant sGC-overexpressing cell line, as previously described. (34) Briefly, cells were plated in a volume of 25 μL on white 384-well Greiner Bio-One microplates and were cultured for 1 or 2 d in medium. Medium was removed, and cells were loaded for 3 h with calcium-free Tyrode-containing coelenterazine. Serial dilutions of the test compounds in a volume of 10 μL in calcium-free Tyrode were applied to the cells for 6 min. Thereafter, 35 μL of Tyrode-containing calcium (final concentration: 3 mM) was added to the cells and the emitted light was measured for 40 s using a CCD camera in a light-tight box. The minimal effective concentration (MEC) was determined as the concentration where a ≥3-fold increase in the basal luminescence value was observed.
细胞活性测试化合物的活性通过重组 sGC 过表达细胞系进行测定,方法如前所述。(34)简要来说,细胞以 25 μL 的体积接种于白色 384 孔 Greiner Bio-One 微孔板中,并在培养基中培养 1 或 2 天。移除培养基后,细胞用无钙 Tyrode 培养基和海葵素共培养 3 小时。随后,将测试化合物在 10 μL 无钙 Tyrode 培养基中进行系列稀释,并作用于细胞 6 分钟。之后,向细胞中加入含钙 Tyrode 培养基(终浓度:3 mM)35 μL,并使用密封箱中的 CCD 相机测量发射的光 40 秒。最小有效浓度(MEC)被定义为观察到基线荧光值增加≥3 倍时的浓度。

Isolated Vessels and Tolerance
孤立船舶与容忍

The relaxing effects of 24 on aortas, saphenous arteries, coronary arteries, and veins, as well as the investigation on aortic rings taken from either normal or nitrate-tolerant rabbits, were performed as previously described. (39)
24 对主动脉、隐静脉、冠状动脉和静脉的放松作用,以及从正常或耐受硝酸盐的兔子身上取出的主动脉环的研究,均按先前所述进行。(39)

Rat Heart Langendorff Preparation
大鼠心脏 Langendorff 制备

Male Wistar rats (200–250 g) were anesthetized using Narcoren (100 mg/kg ip). The heart was rapidly excised and connected to a Langendorff perfusion system (FMI GmbH, Seeheim-Ober Beerbach, Germany). The heart was perfused at a constant flow rate of 10 mL/min with Krebs–Henseleit buffer solution equilibrated with 95% O2 and 5% CO2. The perfusion solution contained (in mmol/L): NaCl 118, KCl 3, NaHCO3 22, KH2PO4 1.2, MgSO4 1.2, CaCl2 1.8, glucose 10, and sodium pyruvate 2. A pressure transducer registered the perfusion pressure in the system. The left ventricular pressure was measured using a second pressure transducer connected to a water-filled balloon which was inserted into the left ventricle via the left atrium. The end diastolic pressure was initially set to 8–10 mmHg by adjusting the volume of the balloon. The hearts were spontaneously beating. The signals from the pressure transducer were amplified, registered, and used for the calculation of the heart frequency and + dP/dtmax by a personal computer. 24 was dissolved in a mixture of 10% DMSO and 90% saline and infused for 20 min with increasing concentration steps into the aortic cannula at a rate of 1% of the total flow rate. All values are presented as relative changes of baseline values before compound application.
雄性 Wistar 大鼠(体重 200-250 克)使用纳可伦(100 毫克/千克,ip)进行麻醉。迅速取出心脏并将其连接到 Langendorff 灌流系统(德国 FMI GmbH,Seeheim-Ober Beerbach)。心脏以每分钟 10 毫升的恒定流速用 Krebs-Henseleit 缓冲液灌流,该缓冲液与 95%氧气和 5%二氧化碳平衡。灌流液成分(以毫摩尔/升计):NaCl 118,KCl 3,NaHCO3 22,KH2PO4 1.2,MgSO4 1.2,CaCl2 1.8,葡萄糖 10,和钠丙酮酸 2。一个压力传感器记录系统中的灌流压力。使用连接到水充气囊的第二压力传感器测量左心室压力,该气囊通过左心房插入左心室。初始将舒张末期压力设置为 8-10 毫米汞柱,通过调整气囊的体积来实现。心脏自发跳动。压力传感器的信号经过放大、记录,并由个人计算机用于计算心率及+ dP/dtmax。 24%的溶液溶解在 10%的二甲基亚砜和 90%的盐水中,以总流量的 1%的速率,通过增加浓度梯度,在主动脉导管中持续输注 20 分钟。所有数值均以化合物应用前的基础值相对变化表示。

Chronic Treatment Study with L-NAME-Treated Renin Transgenic Rats
慢性 L-NAME 处理肾素转基因大鼠治疗研究

Fifty male renin transgenic rats carrying an additional mouse renin gene [RenTG(mRRen2)27] at the age of 8 weeks were used. L-NAME was chronically administered via the drinking water (50 mg/L) in all study groups. Animals were randomly allocated to three study groups: placebo (control) (n = 20), 24 low dose, and 24 high dose (3 and 10 mg/kg per day, respectively, administered po by gavage qd, n = 15 per group). Blood pressure was measured via the tail-cuff method once before the start of the study (day 0) to exclude preexisting differences between the groups and on day 7, 14, and 21. Body weight and survival were assessed on day 1, 8, and 15 and at the study end. At the end of the study (day 22), all animals were anesthetized, blood was collected, and animals were sacrificed; blood was taken in order to assess plasma parameters, and the heart was dissected into the left and right ventricles and was weighed to assess potential heart hypertrophy. Creatinine, urea, and renin activity in plasma were determined after extraction, as previously described. (39, 56)
在 8 周龄时,使用了 50 只携带额外小鼠肾素基因[RenTG(mRRen2)27]的雄性肾素转基因大鼠。所有研究组均通过饮用水(50 mg/L)慢性给予 L-NAME。动物被随机分配到三个研究组:安慰剂(对照组)(n = 20)、24 只低剂量组和 24 只高剂量组(分别每天口服灌胃 3 mg/kg 和 10 mg/kg,每组 n = 15)。血压在研究开始前(第 0 天)和第 7 天、14 天和 21 天通过尾动脉血压计法测量,以排除各组之间存在的先存差异。体重和存活情况在第 1 天、第 8 天、第 15 天和研究结束时进行评估。在研究结束时(第 22 天),所有动物均被麻醉,采集血液,并进行安乐死;血液采集用于评估血浆参数,心脏被解剖成左心室和右心室,并称重以评估潜在的心脏肥大。血浆中的肌酐、尿素和肾素活性在提取后按先前描述的方法测定。(39,56)

Statistics 统计

The unpaired t test was used to detect significant differences between the groups of interest. Results (mean ± SEM) were considered significant when the probability error (P) was less than 0.05, 0.01,and 0.001 for *, **, and ***, respectively.
配对样本 t 检验用于检测感兴趣组之间的显著差异。结果(均值±标准误)认为当概率误差(P)小于 0.05、0.01 和 0.001 时,分别表示为*、**和***,认为结果具有统计学意义。

Supporting Information 支持信息

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点击复制章节链接
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The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jmedchem.7b00449.
支持信息可在 ACS 出版物网站上免费获取,网址为 DOI:10.1021/acs.jmedchem.7b00449。

  • 1H NMR spectra of selected compounds 3, 21, 22, and 24; CYP-inhibition results of 24 (PDF)
    1H NMR 光谱图选自化合物 3、21、22 和 24;化合物 24 的 CYP 抑制结果(PDF)

  • Molecular formula strings (CSV)
    分子式字符串(CSV)

Discovery of the Soluble Guanylate Cyclase Stimulator Vericiguat (BAY 1021189) for the Treatment of Chronic Heart Failure

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S1  S1 S1
Discovery of the Soluble Guanylate Cyclase Stimulat
发现可溶性鸟苷酸环化酶激动剂
or Vericiguat (BAY 1021189) for the
或维利吉单抗(BAY 1021189)用于
Treatment of Chronic Heart Failure
慢性心力衰竭的治疗
Markus Follmann,* Jens Ackerstaff, Gorden Redlich,
马库斯·福尔曼、* 詹斯·阿克斯塔夫、戈登·雷德里希,
Frank Wunder, Dieter Lang, Armin
弗兰克·温德,迪特·朗,阿明
Kern, Peter Fey, Nils Griebenow, Walter Kroh, Eva-M
凯恩,彼得·费伊,尼尔·格里本诺,瓦尔特·克罗,伊娃-玛
aria Becker-Pelster, Axel Kretschmer,
贝克尔-佩尔斯特,阿克斯·克雷施默
Volker Geiss, Volkhart Min-Jian Li, Alexander Strau
Volker Geiss,Volkhart Min-Jian Li,Alexander Strau
b, Joachim Mittendorf, Rolf Jautelat,
b, 耶奥希姆·米特多夫,罗尔夫·贾特拉特,
Hartmut Schirok, Karl-Heinz Schlemmer, Klemens Lust
哈特穆特·施里罗克,卡尔-海因茨·施莱默,克莱门斯·卢斯特
ig, Michael Gerisch, Andreas Knorr,
""" ig,迈克尔·格里斯奇,安德烈亚斯·诺尔, """
Hanna Tinel, Thomas Mondritzki, Hubert Trübel, Pete
汉娜·蒂内尔,托马斯·蒙德里茨基,胡伯特·特鲁贝尔,皮特
r Sandner, and Johannes-Peter Stasch
桑德纳,约翰内斯-彼得·斯塔什
Bayer AG, Drug Discovery, Aprather Weg 18a, 42113 W
拜耳股份公司,药物发现,阿帕瑟尔韦格 18a,42113 W 区
uppertal, Germany  乌普塔尔,德国
*Corresponding author:  对应作者:
Dr. Markus Follmann
马库斯·福尔曼博士
E-mail:  电子邮件:
markus.follmann@bayer.com
Supporting information  支持信息
1.
NMR spectra of selected compounds
核磁共振谱图选定的化合物
3
,
21
,
22
,
24
2.
Table S6 表 S6
: CYP-inhibition results of vericiguat
维立西格他尔的 CYP 抑制结果
24
S2
1
H NMR of compound
化合物核磁共振氢谱
3
3
F
N
N
N
H
2
O
O
N
O
H
NH
2
N
N
N
S3
1
H NMR of compound
21
21
N
N
N
NH
2
O
O
N
H
N
H
2
N
N
N
F

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Author Information 作者信息

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  • Corresponding Author 对应作者
    • Markus Follmann - Drug Discovery, Bayer AG, Aprather Weg 18a, 42113 Wuppertal, GermanyOrcidhttp://orcid.org/0000-0003-1246-3603 Email: markus.follmann@bayer.com
      马库斯·福尔曼 - 药物发现,拜耳公司,德国乌珀塔尔市阿帕瑟尔韦格 18a 号,42113; Orcid http://orcid.org/0000-0003-1246-3603;邮箱:markus.follmann@bayer.com
  • Authors 作者
    • Jens Ackerstaff - Drug Discovery, Bayer AG, Aprather Weg 18a, 42113 Wuppertal, Germany
    • Gorden Redlich - Drug Discovery, Bayer AG, Aprather Weg 18a, 42113 Wuppertal, Germany
    • Frank Wunder - Drug Discovery, Bayer AG, Aprather Weg 18a, 42113 Wuppertal, Germany
    • Dieter Lang - Drug Discovery, Bayer AG, Aprather Weg 18a, 42113 Wuppertal, Germany
    • Armin Kern - Drug Discovery, Bayer AG, Aprather Weg 18a, 42113 Wuppertal, Germany
    • Peter Fey - Drug Discovery, Bayer AG, Aprather Weg 18a, 42113 Wuppertal, Germany
    • Nils Griebenow - Drug Discovery, Bayer AG, Aprather Weg 18a, 42113 Wuppertal, Germany
    • Walter Kroh - Drug Discovery, Bayer AG, Aprather Weg 18a, 42113 Wuppertal, Germany
    • Eva-Maria Becker-Pelster - Drug Discovery, Bayer AG, Aprather Weg 18a, 42113 Wuppertal, Germany
    • Axel Kretschmer - Drug Discovery, Bayer AG, Aprather Weg 18a, 42113 Wuppertal, Germany
    • Volker Geiss - Drug Discovery, Bayer AG, Aprather Weg 18a, 42113 Wuppertal, Germany
    • Volkhart Li - Drug Discovery, Bayer AG, Aprather Weg 18a, 42113 Wuppertal, Germany
    • Alexander Straub - Drug Discovery, Bayer AG, Aprather Weg 18a, 42113 Wuppertal, Germany
    • Joachim Mittendorf - Drug Discovery, Bayer AG, Aprather Weg 18a, 42113 Wuppertal, Germany
    • Rolf Jautelat - Drug Discovery, Bayer AG, Aprather Weg 18a, 42113 Wuppertal, Germany
    • Hartmut Schirok - Drug Discovery, Bayer AG, Aprather Weg 18a, 42113 Wuppertal, Germany
    • Karl-Heinz Schlemmer - Drug Discovery, Bayer AG, Aprather Weg 18a, 42113 Wuppertal, Germany
    • Klemens Lustig - Drug Discovery, Bayer AG, Aprather Weg 18a, 42113 Wuppertal, Germany
    • Michael Gerisch - Drug Discovery, Bayer AG, Aprather Weg 18a, 42113 Wuppertal, Germany
    • Andreas Knorr - Drug Discovery, Bayer AG, Aprather Weg 18a, 42113 Wuppertal, Germany
    • Hanna Tinel - Drug Discovery, Bayer AG, Aprather Weg 18a, 42113 Wuppertal, Germany
    • Thomas Mondritzki - Drug Discovery, Bayer AG, Aprather Weg 18a, 42113 Wuppertal, Germany
    • Hubert Trübel - Drug Discovery, Bayer AG, Aprather Weg 18a, 42113 Wuppertal, Germany
    • Peter Sandner - Drug Discovery, Bayer AG, Aprather Weg 18a, 42113 Wuppertal, Germany
    • Johannes-Peter Stasch - Drug Discovery, Bayer AG, Aprather Weg 18a, 42113 Wuppertal, Germany
  • Author Contributions

    The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.

  • Notes
    The authors declare the following competing financial interest(s): All authors are or have been employees of Bayer AG.

Acknowledgment

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Picture used for abstract/Table of Contents graphic taken from “Unseen Extremes: Mapping the World’s Greatest Mountains” by courtesy of Deutsches Zentrum für Luft- und Raumfahrt eV.

Abbreviations Used

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sGC

soluble guanylate cyclase

cGMP

cyclic guanosine 5′-monophosphate

HMDS

hexamethyldisilazane

MEC

minimum effective concentration

ClogD

calculated logarithm of distribution coefficient

Clb

blood clearance

References

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This article references 56 other publications.

  1. 1
    Evgenov, O. V.; Pacher, P.; Schmidt, P. M.; Hasko, G.; Schmidt, H. H. H. W.; Stasch, J.-P. NO-independent stimulators and activators of soluble guanylate cyclase: discovery and therapeutic potential Nat. Rev. Drug Discovery 2006, 5, 755 768 DOI: 10.1038/nrd2038
  2. 2
    Follmann, M.; Griebenow, N.; Hahn, M. G.; Hartung, I.; Mais, F.-J.; Mittendorf, J.; Schaefer, M.; Schirok, H.; Stasch, J.-P.; Stoll, F.; Straub, A. The chemistry and biology of soluble guanylate cyclase stimulators and activators Angew. Chem., Int. Ed. 2013, 52, 9442 9462 DOI: 10.1002/anie.201302588
  3. 3
    Cerra, M. C.; Pellegrino, D. Cardiovascular cGMP-generating systems in physiological and pathological conditions Curr. Med. Chem. 2007, 14, 585 599 DOI: 10.2174/092986707780059715
  4. 4
    Gladwin, M. T. Deconstructing endothelial dysfunction: soluble guanylyl cyclase oxidation and the NO resistance syndrome J. Clin. Invest. 2006, 116, 2330 2332 DOI: 10.1172/JCI29807
  5. 5
    Hoenicka, M.; Schmid, C. Cardiovascular effects of modulators of soluble guanylyl cyclase activity Cardiovasc. Hematol. Agents Med. Chem. 2008, 6, 287 301 DOI: 10.2174/187152508785909555
  6. 6
    Priviero, F. B.; Webb, R. C. Heme-dependent and independent soluble guanylate cyclase activators and vasodilation J. Cardiovasc. Pharmacol. 2010, 56, 229 233 DOI: 10.1097/FJC.0b013e3181eb4e75
  7. 7
    Packer, C. S. Soluble guanylate cyclase (sGC) down-regulation by abnormal extracellular matrix proteins as a novel mechanism in vascular dysfunction: implications in metabolic syndrome Cardiovasc. Res. 2006, 69, 302 303 DOI: 10.1016/j.cardiores.2005.12.006
  8. 8
    Mayer, B.; Koesling, D. cGMP signalling beyond nitric oxide Trends Pharmacol. Sci. 2001, 22, 546 548 DOI: 10.1016/S0165-6147(00)01889-7
  9. 9
    Nioche, P.; Berka, V.; Vipond, J.; Minton, N.; Tsai, A. L.; Raman, C. S. Femtomolar sensitivity of a NO sensor from Clostridium botulinum Science 2004, 306, 1550 1553 DOI: 10.1126/science.1103596
  10. 10
    Pellicena, P.; Karow, D. S.; Boon, E. M.; Marletta, M. A.; Kuriyan, J. Crystal structure of an oxygen-binding heme domain related to soluble guanylate cyclases Proc. Natl. Acad. Sci. U. S. A. 2004, 101, 12854 12859 DOI: 10.1073/pnas.0405188101
  11. 11
    Wedel, B.; Humbert, P.; Harteneck, C.; Foerster, J.; Malkewitz, J.; Bohme, E.; Schultz, G.; Koesling, D. Mutation of His-105 in the β1 subunit yields a nitric oxide-insensitive form of soluble guanylyl cyclase Proc. Natl. Acad. Sci. U. S. A. 1994, 91, 2592 2596 DOI: 10.1073/pnas.91.7.2592
  12. 12
    Zabel, U.; Hausler, C.; Weeger, M.; Schmidt, H. H. Homodimerization of soluble guanylyl cyclase subunits. Dimerization analysis using a glutathione S-transferase affinity tag J. Biol. Chem. 1999, 274, 18149 18152 DOI: 10.1074/jbc.274.26.18149
  13. 13
    Zabel, U.; Weeger, M.; La, M.; Schmidt, H. H. W. Human soluble guanylate cyclase: functional expression and revised isoenzyme family Biochem. J. 1998, 335, 51 57 DOI: 10.1042/bj3350051
  14. 14
    Murad, F. Shattuck Lecture. Nitric oxide and cyclic GMP in cell signaling and drug development N. Engl. J. Med. 2006, 355, 2003 2011 DOI: 10.1056/NEJMsa063904
  15. 15
    Stasch, J. P.; Hobbs, A. J. NO-independent, haem-dependent soluble guanylate cyclase stimulators Handb. Exp. Pharmacol. 2009, 191, 277 308 DOI: 10.1007/978-3-540-68964-5_13
  16. 16
    Schmidt, H. H.; Schmidt, P. M.; Stasch, J. P. NO- and haem-independent soluble guanylate cyclase activators Handb. Exp. Pharmacol. 2009, 191, 309 339 DOI: 10.1007/978-3-540-68964-5_14
  17. 17
    Stasch, J.-P.; Evgenov, O. V. Soluble guanylate cyclase stimulators in pulmonary hypertension Handb. Exp. Pharmacol. 2013, 218, 279 313 DOI: 10.1007/978-3-662-45805-1_12
  18. 18
    Ghofrani, H.-A.; Galie, N.; Grimminger, F.; Gruenig, E.; Humbert, M.; Jing, Z.-C.; Keogh, A. M.; Langleben, D.; Kilama, M. O.; Fritsch, A.; Neuser, D.; Rubin, L. J. Riociguat for the treatment of pulmonary arterial hypertension N. Engl. J. Med. 2013, 369, 330 340 DOI: 10.1056/NEJMoa1209655
  19. 19
    Ghofrani, H.-A.; D’Armini, A. M.; Grimminger, F.; Hoeper, M. M.; Jansa, P.; Kim, N. H.; Mayer, E.; Simonneau, G.; Wilkins, M. R.; Fritsch, A.; Neuser, D.; Weimann, G.; Wang, C. Riociguat for the treatment of chronic thromboembolic pulmonary hypertension N. Engl. J. Med. 2013, 369, 319 329 DOI: 10.1056/NEJMoa1209657
  20. 20
    Hambly, N.; Granton, J. Riociguat for the treatment of pulmonary hypertension Expert Rev. Respir. Med. 2015, 9, 679 695 DOI: 10.1586/17476348.2015.1106316
  21. 21
    Frey, R.; Mueck, W.; Unger, S.; Artmeier-Brandt, U.; Weimann, G.; Wensing, G. Single-dose pharmacokinetics, pharmacodynamics, tolerability, and safety of the soluble guanylate cyclase stimulator BAY 63–2521: an ascending-dose study in healthy male volunteers J. Clin. Pharmacol. 2008, 48, 926 934 DOI: 10.1177/0091270008319793
  22. 22
    Gheorghiade, M.; Greene, S. J.; Butler, J.; Filippatos, G.; Lam, C. S.; Maggioni, A. P.; Ponikowski, P.; Shah, S. J.; Solomon, S. D.; Kraigher-Krainer, E.; Samano, E. T.; Müller, K.; Roessig, L.; Pieske, B. Effect of vericiguat, a soluble guanylate cyclase stimulator, on natriuretic peptide levels in patients with worsening chronic heart failure and reduced ejection fraction: the SOCRATES-REDUCED randomized trial JAMA, J. Am. Med. Assoc. 2015, 314, 2251 2262 DOI: 10.1001/jama.2015.15734
  23. 23
    Pieske, B.; Butler, J.; Filippatos, G.; Lam, C.; Maggioni, A. P.; Ponikowski, P.; Shah, S.; Solomon, S.; Kraigher-Krainer, E.; Samano, E. T.; Scalise, A. V.; Mueller, K.; Roessig, L.; Gheorghiade, M. Rationale and design of the SOluble guanylate Cyclase stimulatoR in heArT failurE Studies (SOCRATES) Eur. J. Heart Failure 2014, 16, 1026 38 DOI: 10.1002/ejhf.135
  24. 24

    Clinicaltrials.gov. Identifier: NCT02861534 (accessed August 5, 2016).

  25. 25
    Mittendorf, J.; Weigand, S.; Alonso-Alija, C.; Bischoff, E.; Feurer, A.; Gerisch, M.; Kern, A.; Knorr, A.; Lang, D.; Muenter, K.; Radtke, M.; Schirok, H.; Schlemmer, K.-H.; Stahl, E.; Straub, A.; Wunder, F.; Stasch, J.-P. Discovery of riociguat (BAY 63–2521): a potent, oral stimulator of soluble guanylate cyclase for the treatment of pulmonary hypertension ChemMedChem 2009, 4, 853 865 DOI: 10.1002/cmdc.200900014
  26. 26
    Gnoth, M. J.; Hopfe, P. M.; Czembor, W. Determination of riociguat and its major human metabolite M-1 in human plasma by stable-isotope dilution LCMS/MS Bioanalysis 2015, 7, 193 205 DOI: 10.4155/bio.14.257
  27. 27
    Becker, C.; Frey, R.; Thomas, D.; Reber, M.; Weimann, G.; Arens, E. R.; Mück, W.; Unger, S.; Dietrich, H. Pharmacokinetic interaction of riociguat with ketoconazole, clarithromycin, and midazolam Pulm. Circ. 2016, 6, S49 57 DOI: 10.1086/685016
  28. 28
    Frey, R.; Becker, C.; Unger, S.; Schmidt, A.; Wensing, G.; Mück, W. Assessment of the effects of renal impairment and smoking on the pharmacokinetics of a single oral dose of the soluble guanylate cyclase stimulator riociguat (BAY 63–2521) Pulm. Circ. 2016, 6, S15 26 DOI: 10.1086/685017
  29. 29
    Saleh, S.; Frey, R.; Becker, C.; Unger, S.; Wensing, G.; Mück, W. Bioavailability, pharmacokinetics, and safety of riociguat given as an oral suspension or crushed tablet with and without food Pulm. Circ. 2016, 6, S66 74 DOI: 10.1086/685020
  30. 30
    Zhao, X.; Wang, Z.; Wang, Y.; Zhang, H.; Blode, H.; Yoshikawa, K.; Becker, C.; Unger, S.; Frey, R.; Cui, Y. Pharmacokinetics of the soluble guanylate cyclase stimulator riociguat in healthy young Chinese male non-smokers and smokers: results of a randomized, double-blind, placebo-controlled study Clin. Pharmacokinet. 2016, 55, 615 624 DOI: 10.1007/s40262-015-0337-4
  31. 31
    Highlights of Prescribing Information: Riociguat (Adempas); U.S. Food and Drug Administration: Silver Spring, MD, 2014; http://www.accessdata.fda.gov/drugsatfda_docs/label/2014/204819s002lbl.pdf (accessed April 4, 2014).
  32. 32
    Bitterli, P.; Erlenmeyer, H. Some derivatives of triazolopyrimidine Helv. Chim. Acta 1951, 34, 835 840 DOI: 10.1002/hlca.19510340311
  33. 33
    Reinecke, M. G.; Woodrow, T. A.; Brown, E. S. Pyrazolo[3,4-c]pyridazines from hydrazine and aminothiophenecarboxylates J. Org. Chem. 1992, 57, 1018 1021 DOI: 10.1021/jo00029a046
  34. 34
    Wunder, F.; Stasch, J.-P.; Hütter, J.; Alonso-Alija, C.; Hüser, J.; Lohrmann, E. A cell-based cGMP assay useful for ultra-high-throughput screening and identification of modulators of the nitric oxide/cGMP pathway Anal. Biochem. 2005, 339, 104 112 DOI: 10.1016/j.ab.2004.12.025
  35. 35
    Hillisch, A.; Heinrich, N.; Wild, H. Computational chemistry in the pharmaceutical industry: from childhood to adolescence ChemMedChem 2015, 10, 1958 1962 DOI: 10.1002/cmdc.201500346
  36. 36
    Burkhard, J. A.; Wuitschik, G.; Rogers-Evans, M.; Müller, K.; Carreira, E. M. Oxetanes as versatile elements in drug discovery and synthesis Angew. Chem., Int. Ed. 2010, 49, 9052 9067 DOI: 10.1002/anie.200907155
  37. 37
    Wuitschik, G.; Carreira, E. M.; Wagner, B.; Fischer, H.; Parrilla, I.; Schuler, F.; Rogers-Evans, M.; Müller, K. Oxetanes in drug discovery: structural and synthetic insights J. Med. Chem. 2010, 53, 3227 3246 DOI: 10.1021/jm9018788
  38. 38
    Leeson, P. D.; Young, R. J. Molecular property design: does everyone get it? ACS Med. Chem. Lett. 2015, 6, 722 725 DOI: 10.1021/acsmedchemlett.5b00157
  39. 39
    Sharkovska, Y.; Kalk, P.; Lawrenz, B.; Godes, M.; Hoffmann, L. S.; Wellkisch, K.; Geschka, S.; Relle, K.; Hocher, B.; Stasch, J.-P. Nitric oxide-independent stimulation of soluble guanylate cyclase reduces organ damage in experimental low-renin and high-renin models J. Hypertens. 2010, 28, 1666 1675 DOI: 10.1097/HJH.0b013e32833b558c
  40. 40
    Dubin, R. F.; Shah, S. J. Soluble guanylate cyclase stimulators: a novel treatment option for heart failure associated with cardiorenal syndromes? Curr. Heart Failure Rep. 2016, 13, 132 139 DOI: 10.1007/s11897-016-0290-z
  41. 41
    Follmann, M.; Stasch, J.-P.; Redlich, G.; Griebenow, N.; Lang, D.; Wunder, F.; Huebsch, W.; Lindner, N.; Vakalopoulos, A.; Tersteegen, A. Preparation of annelated pyrimidine derivatives useful in the treatment and prophylaxis of cardiovascular diseases. WO2013030288, 2013.
  42. 42
    Follmann, M.; Stasch, J.-P.; Redlich, G.; Ackerstaff, J.; Griebenow, N.; Knorr, A.; Wunder, F.; Li, V. M.-J.; Baerfacker, L.; Weigand, S. Bicyclic aza-heterocycles as guanylate cyclase inhibitors and their preparation and use in the treatment of cardiovascular diseases. WO 2012028647, 2012.
  43. 43
    Follmann, M.; Stasch, J.-P.; Redlich, G.; Ackerstaff, J.; Griebenow, N.; Knorr, A.; Wunder, F.; Li, V. M.-J.; Schirok, H.; Jautelat, R. Substituted methyl pyrimidin-5-yl carbamates as guanylate cyclase inhibitors and their preparation and use in the treatment of cardiovascular diseases. WO 2012010578, 2012.
  44. 44
    Alonso-Alija, C.; Bischoff, E.; Muenter, K.; Stasch, J.-P.; Stahl, E.; Weigand, S.; Feurer, A. Preparation of [(pyrazolopyridinyl)pyrimidinyl]carbamates stimulating soluble guanylate cyclase for treating cardiovascular diseases and/or sexual dysfunction. WO 2003095451, 2003.
  45. 45
    Follmann, M.; Stasch, J.-P.; Redlich, G.; Ackerstaff, J.; Griebenow, N.; Wunder, F.; Li, V. M.-J.; Mittendorf, J.; Jautelat, R. Carbamate-substituted diaminopyrimidines as guanylate cyclase inhibitors and their preparation and use in the treatment of cardiovascular diseases. WO 2012010576, 2012.
  46. 46
    Schirok, H.; Mittendorf, J.; Stasch, J.-P.; Wunder, F.; Stoll, F.; Schlemmer, K.-H. Azabicyclic derivatives as stimulators of guanylate cyclase, their preparation, pharmaceutical compositions, and use for the treatment of cardiovascular disorders. WO 2008031513, 2008.
  47. 47
    Follmann, M.; Stasch, J.-P.; Redlich, G.; Ackerstaff, J.; Griebenow, N.; Knorr, A.; Wunder, F.; Li, V. M.-J.; Mittendorf, J.; Schlemmer, K.-H.; Jautelat, R. Substituted 6-fluoro-1H-pyrazolo[4,3-b]pyridines as guanylate cyclase inhibitors and their preparation and use in the treatment of cardiovascular diseases. WO 2012059549, 2012.
  48. 48
    Follmann, M.; Stasch, J.-P.; Redlich, G.; Straub, A.; Ackerstaff, J.; Griebenow, N.; Knorr, A.; Wunder, F.; Li, V. M.-J. Substituted imidazopyridazines as guanylate cyclase inhibitors and their preparation and use in the treatment of cardiovascular diseases. WO 2012152630, 2012.
  49. 49
    Straub, A.; Feurer, A.; Alonso-Alija, C.; Stasch, J.-P.; Perzborn, E.; Huetter, J.; Dembowsky, K.; Stahl, E. Substituted pyrazole derivatives condensed with six-membered heterocyclic rings as cardiovascular agents and their preparation. WO 2000006569, 2000.
  50. 50
    Walsky, R. L.; Obach, R. S. Validated assays for human cytochrome P450 activities Drug Metab. Dispos. 2004, 32, 647 660 DOI: 10.1124/dmd.32.6.647
  51. 51
    Obach, R. S.; Walsky, R. L.; Venkatakrishnan, K. Mechanism-based inactivation of human cytochrome p450 enzymes and the prediction of drug-drug interactions Drug Metab. Dispos. 2007, 35, 246 255 DOI: 10.1124/dmd.106.012633
  52. 52
    Obach, R. S. Predicting clearance in humans from in vitro data Curr. Top. Med. Chem. 2011, 11, 334 339 DOI: 10.2174/156802611794480873
  53. 53
    Artursson, P.; Karlsson, J. Correlation between oral drug absorption in humans and apparent drug permeability coefficients in human intestinal epithelial (Caco-2) cells Biochem. Biophys. Res. Commun. 1991, 175, 880 885 DOI: 10.1016/0006-291X(91)91647-U
  54. 54
    Hoenicka, M.; Becker, E.-M.; Apeler, H.; Sirichoke, T.; Schröder, H.; Gerzer, R.; Stasch, J.-P. Purified soluble guanylyl cyclase expressed in a baculovirus/Sf9 system: stimulation by YC-1, nitric oxide, and carbon monoxide J. Mol. Med. (Heidelberg, Ger.) 1999, 77, 14 23 DOI: 10.1007/s001090050292
  55. 55
    Schermuly, R. T.; Stasch, J.-P.; Pullamsetti, S. S.; Middendorff, R.; Müller, D.; Schlüter, K.-D.; Dingendorf, A.; Hackemack, S.; Kolosionek, E.; Kaulen, C.; Dumitrascu, R.; Weissmann, N.; Mittendorf, J.; Klepetko, W.; Seeger, W.; Ghofrani, H. A.; Grimminger, F. Expression and function of soluble guanylate cyclase in pulmonary arterial hypertension Eur. Respir. J. 2008, 32, 881 891 DOI: 10.1183/09031936.00114407
  56. 56
    Geschka, S.; Kretschmer, A.; Sharkovska, Y.; Evgenov, O. V.; Lawrenz, B.; Hucke, A.; Hocher, B.; Stasch, J.-P. Soluble guanylate cyclase stimulation prevents fibrotic tissue remodeling and improves survival in salt-sensitive Dahl rats PLoS One 2011, 6, e21853 DOI: 10.1371/journal.pone.0021853

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  8. Michael G. Hahn, Thomas Lampe, Sherif El Sheikh, Nils Griebenow, Elisabeth Woltering, Karl-Heinz Schlemmer, Lisa Dietz, Michael Gerisch, Frank Wunder, Eva-Maria Becker-Pelster, Thomas Mondritzki, Hanna Tinel, Andreas Knorr, Armin Kern, Dieter Lang, Joerg Hueser, Tibor Schomber, Agnes Benardeau, Frank Eitner, Hubert Truebel, Joachim Mittendorf, Vijay Kumar, Focco van den Akker, Martina Schaefer, Volker Geiss, Peter Sandner, Johannes-Peter Stasch. Discovery of the Soluble Guanylate Cyclase Activator Runcaciguat (BAY 1101042). Journal of Medicinal Chemistry 2021, 64 (9) , 5323-5344. https://doi.org/10.1021/acs.jmedchem.0c02154
  9. Wei Zheng, Zhen Wang, Xiangrui Jiang, Qingjie Zhao, Jingshan Shen. Targeted Drugs for Treatment of Pulmonary Arterial Hypertension: Past, Present, and Future Perspectives. Journal of Medicinal Chemistry 2020, 63 (24) , 15153-15186. https://doi.org/10.1021/acs.jmedchem.0c01093
  10. Vincent Le Fouler, Yu Chen, Vincent Gandon, Vincent Bizet, Christophe Salomé, Thomas Fessard, Fang Liu, K. N. Houk, Nicolas Blanchard. Activating Pyrimidines by Pre-distortion for the General Synthesis of 7-Aza-indazoles from 2-Hydrazonylpyrimidines via Intramolecular Diels–Alder Reactions. Journal of the American Chemical Society 2019, 141 (40) , 15901-15909. https://doi.org/10.1021/jacs.9b07037
  11. Lei Tian, Xiuding Yang, Sichang Hao, Jinrong Hu, Kairui Kang, Xiaolin Xie, Yue Wu, Taotao Qiang, Chengyuan Liang. Verquvo, a first-in-class soluble guanylate cyclase stimulator for the treatment of heart failure. 2025, 515-536. https://doi.org/10.1016/B978-0-443-23932-8.00028-5
  12. Peter Sandner, Markus Follmann, Eva Becker‐Pelster, Michael G. Hahn, Christian Meier, Cecilia Freitas, Lothar Roessig, Johannes‐Peter Stasch. Soluble GC stimulators and activators: Past, present and future. British Journal of Pharmacology 2024, 181 (21) , 4130-4151. https://doi.org/10.1111/bph.15698
  13. Lauro Figueroa-Valverde, Marcela Rosas-Nexticapa, Magdalena Alvarez-Ramirez, Emilio Aguilar-Sanchez, Maria Virginia Mateu-Armad, Enrique Bonilla-Zavaleta. Interaction of some chalcone derivatives with calcium channels using a theoretical model. Brazilian Journal of Science 2024, 3 (11) , 1-15. https://doi.org/10.14295/bjs.v3i11.658
  14. S. Zhazykbayeva, H. Budde, M. Kaçmaz, Y. Zemedie, H. Osman, R. Hassoun, K. Jaquet, I. Akin, I. El-Battrawy, M. Herwig, N. Hamdani. Exploring PKG signaling as a therapeutic avenue for pressure overload, ischemia, and HFpEF. Expert Opinion on Therapeutic Targets 2024, 3 , 1-17. https://doi.org/10.1080/14728222.2024.2400093
  15. Yunhan Jiang, Pingxian Liu, Zhiqiang Qiu, Meng Zhou, Mengdi Cheng, Tao Yang. The U.S. FDA approved cardiovascular drugs from 2011 to 2023: A medicinal chemistry perspective. European Journal of Medicinal Chemistry 2024, 275 , 116593. https://doi.org/10.1016/j.ejmech.2024.116593
  16. Lingling Wu, Mario Rodriguez, Karim El Hachem, W. H. Wilson Tang, Chayakrit Krittanawong. Management of patients with heart failure and chronic kidney disease. Heart Failure Reviews 2024, 29 (5) , 989-1023. https://doi.org/10.1007/s10741-024-10415-9
  17. Jin Rao, Xiangyu Chen, Yudi Liu, Xuefu Wang, Pengchao Cheng, Zhinong Wang. Assessment of adverse events of the novel cardiovascular drug vericiguat: a real-world pharmacovigilance study based on FAERS. Expert Opinion on Drug Safety 2024, , 1-9. https://doi.org/10.1080/14740338.2024.2382226
  18. Vaibhav Tavare, Prof. Sharvari Chavan. A Review Article on Analytical Method on Vericiguat. International Journal of Advanced Research in Science, Communication and Technology 2024, , 680-684. https://doi.org/10.48175/IJARSCT-19180
  19. Xiya Li, Xiaoyang Zhou, Ling Gao. Diabetes and Heart Failure: A Literature Review, Reflection and Outlook. Biomedicines 2024, 12 (7) , 1572. https://doi.org/10.3390/biomedicines12071572
  20. Achim Fritsch, Michaela Meyer, Robert O. Blaustein, Maria E. Trujillo, Eunkyung Kauh, Lothar Roessig, Michael Boettcher, Corina Becker. Clinical Pharmacokinetic and Pharmacodynamic Profile of Vericiguat. Clinical Pharmacokinetics 2024, 63 (6) , 751-771. https://doi.org/10.1007/s40262-024-01384-1
  21. Yuichi Hattori, Kohshi Hattori, Kuniaki Ishii, Masanobu Kobayashi. Challenging and target-based shifting strategies for heart failure treatment: An update from the last decades. Biochemical Pharmacology 2024, 224 , 116232. https://doi.org/10.1016/j.bcp.2024.116232
  22. Penglei Chen, Yixiang Wang, Xin Liu, Jiaqi Yu, Xuwei Zheng. Cost–Utility Analysis of Vericiguat in Heart Failure with Reduced Ejection Fraction After Worsening Heart Failure Events in China. American Journal of Cardiovascular Drugs 2024, 24 (3) , 445-454. https://doi.org/10.1007/s40256-024-00637-5
  23. Po-Cheng Chang, Hui-Ling Lee, Hung-Ta Wo, Hao-Tien Liu, Ming-Shien Wen, Chung-Chuan Chou, . Vericiguat suppresses ventricular tachyarrhythmias inducibility in a rabbit myocardial infarction model. PLOS ONE 2024, 19 (4) , e0301970. https://doi.org/10.1371/journal.pone.0301970
  24. Dionysios-Panagiotis Kintos, Konstantinos Salagiannis, Antonis Sgouros, Sotiris S. Nikolaropoulos, Stavros Topouzis, Manolis A. Fousteris. Identification of new multi-substituted 1H-pyrazolo[3,4-c]pyridin-7(6H)-ones as soluble guanylyl cyclase (sGC) stimulators with vasoprotective and anti-inflammatory activities. Bioorganic Chemistry 2024, 144 , 107170. https://doi.org/10.1016/j.bioorg.2024.107170
  25. 素梅 王. The Mechanism of Soluble Guanylate Cyclase-Veliciguat for Heart Failure. Advances in Clinical Medicine 2024, 14 (01) , 622-629. https://doi.org/10.12677/ACM.2024.141086
  26. Pengfei Xu, Lijun Zhu, Desheng Zhang, Zilong Li, Raoling Ge, Qiang Tian. Design and synthesis of fluorine aromatic scaffolds containing drugs approved by the US FDA from 2002 to 2022. Results in Chemistry 2024, 7 , 101446. https://doi.org/10.1016/j.rechem.2024.101446
  27. Ram B. Singh, Jan Fedacko, Oleg Medvedev, Lyudemila Shogenova, Yogesh K. Gaur, Nikolay Tselov, Krasimira Hristova. Vericiguat as part of a multidrug approach following a worsening heart failure event. 2024, 397-407. https://doi.org/10.1016/B978-0-12-822972-9.00037-7
  28. Tao Chen, Bin Kong, Wei Shuai, Yang Gong, Jingjing Zhang, He Huang. Vericiguat alleviates ventricular remodeling and arrhythmias in mouse models of myocardial infarction via CaMKII signaling. Life Sciences 2023, 334 , 122184. https://doi.org/10.1016/j.lfs.2023.122184
  29. Doaa M. Mustafa, Nancy Magdy, Noha F. El Azab. Different spectrophotometric methods for simultaneous quantitation of Vericiguat and its alkaline degradation product: a comparative study with greenness profile assessment. Scientific Reports 2023, 13 (1) https://doi.org/10.1038/s41598-023-50097-1
  30. Louise F. Dow, Alfie M. Case, Megan P. Paustian, Braeden R. Pinkerton, Princess Simeon, Paul C. Trippier. The evolution of small molecule enzyme activators. RSC Medicinal Chemistry 2023, 14 (11) , 2206-2230. https://doi.org/10.1039/D3MD00399J
  31. Siju Bi, Wenshuang Diao, Ting Zhou, Kuaile Lin, Weicheng Zhou. Development of a new synthetic route of the key intermediate of riociguat. Synthetic Communications 2023, 53 (21) , 1844-1853. https://doi.org/10.1080/00397911.2023.2252539
  32. Frank Wunder, Johannes‐Peter Stasch, Andreas Knorr, Thomas Mondritzki, Damian Brockschnieder, Eva‐Maria Becker‐Pelster, Peter Sandner, Hanna Tinel, Gorden Redlich, Ingo V. Hartung, Alexandros Vakalopoulos, Markus Follmann. Identification and characterization of the new generation soluble guanylate cyclase stimulator BAY‐747 designed for the treatment of resistant hypertension. British Journal of Pharmacology 2023, 180 (19) , 2500-2513. https://doi.org/10.1111/bph.16142
  33. Figueroa‐Valverde Lauro, López‐Ramos Maria, Rosas‐Nexticapa Marcela, Alvarez‐Ramirez Magdalena, Díaz‐Cedillo Francisco, Lopez‐Gutierrez Tomas. Evaluation of biological activity of some pyridine derivatives on perfusion pressure and their interaction with the M 2 muscarinic receptor. Vietnam Journal of Chemistry 2023, 61 (5) , 594-604. https://doi.org/10.1002/vjch.202300005
  34. Jan R. Kraehling, Agnes Benardeau, Tibor Schomber, Laura Popp, Julia Vienenkoetter, Heidrun Ellinger-Ziegelbauer, Mira Pavkovic, Elke Hartmann, Krystyna Siudak, Alexius Freyberger, Ina Hagelschuer, Ilka Mathar, Joerg Hueser, Michael G. Hahn, Volker Geiss, Frank Eitner, Peter Sandner. The sGC Activator Runcaciguat Has Kidney Protective Effects and Prevents a Decline of Kidney Function in ZSF1 Rats. International Journal of Molecular Sciences 2023, 24 (17) , 13226. https://doi.org/10.3390/ijms241713226
  35. Chiara Mozzini, Mauro Pagani. The Heart Failure Knights. Current Problems in Cardiology 2023, 48 (9) , 101834. https://doi.org/10.1016/j.cpcardiol.2023.101834
  36. Aimi Yokoi, Toru Kawada, Shohei Yokota, Midori Kakuuchi, Hiroki Matsushita, Akitsugu Nishiura, Meihua Li, Kazunori Uemura, Joe Alexander, Ryou Tanaka, Keita Saku, . Impact of vericiguat on baroreflex-mediated sympathetic circulatory regulation: An open-loop analysis. PLOS ONE 2023, 18 (8) , e0286767. https://doi.org/10.1371/journal.pone.0286767
  37. Andreas Friebe, Jan R. Kraehling, Michael Russwurm, Peter Sandner, Achim Schmidtko. The 10th International Conference on cGMP 2022: recent trends in cGMP research and development—meeting report. Naunyn-Schmiedeberg's Archives of Pharmacology 2023, 396 (8) , 1669-1686. https://doi.org/10.1007/s00210-023-02484-8
  38. Girish Chandra, Durg Vijay Singh, Gopal Kumar Mahato, Samridhi Patel. Fluorine-a small magic bullet atom in the drug development: perspective to FDA approved and COVID-19 recommended drugs. Chemical Papers 2023, 77 (8) , 4085-4106. https://doi.org/10.1007/s11696-023-02804-5
  39. Ghulam Shabir, Aamer Saeed, Wajeeha Zahid, Fatima Naseer, Zainab Riaz, Nafeesa Khalil, Muneeba, Fernando Albericio. Chemistry and Pharmacology of Fluorinated Drugs Approved by the FDA (2016–2022). Pharmaceuticals 2023, 16 (8) , 1162. https://doi.org/10.3390/ph16081162
  40. Bao-Anh Tran, Erini S. Serag-Bolos, Joel Fernandez, Aimon C. Miranda. Vericiguat: The First Soluble Guanylate Cyclase Stimulator for Reduction of Cardiovascular Death and Heart Failure Hospitalization in Patients With Heart Failure Reduced Ejection Fraction. Journal of Pharmacy Practice 2023, 36 (4) , 905-914. https://doi.org/10.1177/08971900221087096
  41. Melinton Miguel López Ruano. Uso de vericiguat en pacientes con falla cardiaca y fracción de eyección reducida. Revista Ciencia Multidisciplinaria CUNORI 2023, 7 (2) , 141-151. https://doi.org/10.36314/cunori.v7i2.233
  42. Anna Gorący, Jakub Rosik, Joanna Szostak, Bartosz Szostak, Szymon Retfiński, Filip Machaj, Andrzej Pawlik. Improving mitochondrial function in preclinical models of heart failure: therapeutic targets for future clinical therapies?. Expert Opinion on Therapeutic Targets 2023, 27 (7) , 593-608. https://doi.org/10.1080/14728222.2023.2240021
  43. Burkert Pieske, Elisabeth Pieske‐Kraigher, Carolyn S.P. Lam, Vojtěch Melenovský, Karen Sliwa, Yuri Lopatin, Juan Luis Arango, M. Cecilia Bahit, Christopher M. O'Connor, Mahesh J. Patel, Lothar Roessig, Daniel A. Morris, Martin Kropf, Cynthia M. Westerhout, Yinggan Zheng, Paul W. Armstrong, . Effect of vericiguat on left ventricular structure and function in patients with heart failure with reduced ejection fraction: The VICTORIA echocardiographic substudy. European Journal of Heart Failure 2023, 25 (7) , 1012-1021. https://doi.org/10.1002/ejhf.2836
  44. Eric H. Mace, Melissa J. Kimlinger, Frederic T. Billings, Marcos G. Lopez. Targeting Soluble Guanylyl Cyclase during Ischemia and Reperfusion. Cells 2023, 12 (14) , 1903. https://doi.org/10.3390/cells12141903
  45. Urjosee Sahana, Markus Wehland, Ulf Simonsen, Herbert Schulz, Daniela Grimm. A Systematic Review of the Effect of Vericiguat on Patients with Heart Failure. International Journal of Molecular Sciences 2023, 24 (14) , 11826. https://doi.org/10.3390/ijms241411826
  46. Doaa M. Mustafa, Nancy Magdy, Noha F. El Azab. The first validated stability-indicating HPLC/DAD method for quantitation of Vericiguat in its pharmaceutical formulation; Application to degradation kinetic studies. Talanta 2023, 259 , 124498. https://doi.org/10.1016/j.talanta.2023.124498
  47. Wei-Lin Liang, Bo Liang. Soluble Guanylate Cyclase Activators and Stimulators in Patients with Heart Failure. Current Cardiology Reports 2023, 25 (6) , 607-613. https://doi.org/10.1007/s11886-023-01884-9
  48. Jay Patel, Negin Rassekh, Gregg C. Fonarow, Prakash Deedwania, Farooq H. Sheikh, Ali Ahmed, Phillip H. Lam. Guideline-Directed Medical Therapy for the Treatment of Heart Failure with Reduced Ejection Fraction. Drugs 2023, 83 (9) , 747-759. https://doi.org/10.1007/s40265-023-01887-4
  49. Yu. N. Belenkov, M. V. Kozhevnikova. Soluble guanylate cyclase: restoration of the NO–sGC–cGMP signaling pathway activity. A new opportunity in the treatment of heart failure. Kardiologiia 2023, 63 (5) , 68-76. https://doi.org/10.18087/cardio.2023.5.n2422
  50. Guofang Ma, Yuefang Pan, Chaoyi Qu, Feng Li. The efficacy of vericiguat for heart failure: A meta-analysis of randomized controlled trials. Medicine 2023, 102 (21) , e33807. https://doi.org/10.1097/MD.0000000000033807
  51. Stefania Angela Di Fusco, Alessandro Alonzo, Alberto Aimo, Andrea Matteucci, Rita Cristina Myriam Intravaia, Stefano Aquilani, Manlio Cipriani, Leonardo De Luca, Alessandro Navazio, Serafina Valente, Michele Massimo Gulizia, Domenico Gabrielli, Fabrizio Oliva, Furio Colivicchi. ANMCO position paper on vericiguat use in heart failure: from evidence to place in therapy. European Heart Journal Supplements 2023, 25 (Supplement_D) , D278-D286. https://doi.org/10.1093/eurheartjsupp/suad106
  52. Carla Rizzo, Sara Amata, Ivana Pibiri, Andrea Pace, Silvestre Buscemi, Antonio Palumbo Piccionello. FDA-Approved Fluorinated Heterocyclic Drugs from 2016 to 2022. International Journal of Molecular Sciences 2023, 24 (9) , 7728. https://doi.org/10.3390/ijms24097728
  53. Ahmed K. Siddiqi, Stephen J. Greene, Marat Fudim, Robert J Mentz, Javed Butler, Muhammad Shahzeb Khan. Vericiguat for the treatment of heart failure with reduced ejection fraction. Expert Review of Cardiovascular Therapy 2023, 21 (4) , 245-257. https://doi.org/10.1080/14779072.2023.2189101
  54. Jianhua Ma, Sheng Guo, Huan Jiang, Bo Li. Efficacy and safety of vericiguat in heart failure: a meta-analysis. Journal of International Medical Research 2023, 51 (3) https://doi.org/10.1177/03000605231159333
  55. Bo-ang Hu, Yu-lin Li, Hai-tao Han, Bin Lu, Xu Jia, Lu Han, Wei-xuan Ma, Ping Zhu, Zhi-hao Wang, Wei Zhang, Ming Zhong, Lei Zhang. Stimulation of soluble guanylate cyclase by vericiguat reduces skeletal muscle atrophy of mice following chemotherapy. Frontiers in Pharmacology 2023, 14 https://doi.org/10.3389/fphar.2023.1112123
  56. Shuo Yuan, Dan-Shu Wang, Hui Liu, Sheng-Nan Zhang, Wei-Guang Yang, Meng Lv, Yu-Xue Zhou, Sai-Yang Zhang, Jian Song, Hong-Min Liu. New drug approvals for 2021: Synthesis and clinical applications. European Journal of Medicinal Chemistry 2023, 245 , 114898. https://doi.org/10.1016/j.ejmech.2022.114898
  57. Rohit Patel, Yiling Fu, Ser Khang, Agnes M. Benardeau, Scott C. Thomson, Volker Vallon. Responses in Blood Pressure and Kidney Function to Soluble Guanylyl Cyclase Stimulation or Activation in Normal and Diabetic Rats. Nephron 2023, 147 (5) , 281-300. https://doi.org/10.1159/000526934
  58. 东平 杨. Research Progress of Ferroptosis and Its Role in the Medication for Heart Failure. Advances in Clinical Medicine 2023, 13 (02) , 2267-2271. https://doi.org/10.12677/ACM.2023.132317
  59. Jingrui He, Ziyi Li, Gagan Dhawan, Wei Zhang, Alexander E. Sorochinsky, Greg Butler, Vadim A. Soloshonok, Jianlin Han. Fluorine-containing drugs approved by the FDA in 2021. Chinese Chemical Letters 2023, 34 (1) , 107578. https://doi.org/10.1016/j.cclet.2022.06.001
  60. Qi Lou, Luyifei Li, Guangzhong Liu, Tiankai Li, Li Zhang, Yanxiang Zang, Chengchuang Zhan, Hong Wang, Weimin Li. Vericiguat reduces electrical and structural remodeling in a rabbit model of atrial fibrillation. Journal of Cardiovascular Pharmacology and Therapeutics 2023, 28 https://doi.org/10.1177/10742484231185252
  61. Eamon P. Mulvaney, Fabiana Renzo, Rui Adão, Emilie Dupre, Lucia Bialesova, Viviana Salvatore, Helen M. Reid, Glória Conceição, Julien Grynblat, Aida Llucià-Valldeperas, Jean-Baptiste Michel, Carmen Brás-Silva, Charles E. Laurent, Luke S. Howard, David Montani, Marc Humbert, Anton Vonk Noordegraaf, Frédéric Perros, Pedro Mendes-Ferreira, B. Therese Kinsella. The thromboxane receptor antagonist NTP42 promotes beneficial adaptation and preserves cardiac function in experimental models of right heart overload. Frontiers in Cardiovascular Medicine 2022, 9 https://doi.org/10.3389/fcvm.2022.1063967
  62. Nadine Haase, Sarah M. Kedziora, Dominik Nikolaus Müller, Ralf Dechend. Lösliche Guanylatzyklase(sGC)-Stimulation mit Vericiguat. Die Kardiologie 2022, 16 (6) , 466-478. https://doi.org/10.1007/s12181-022-00576-y
  63. Barry H. Greenberg. Emerging Treatment Approaches to Improve Outcomes in Patients with Heart Failure. Cardiology Discovery 2022, 2 (4) , 231-240. https://doi.org/10.1097/CD9.0000000000000060
  64. Guangchen Li, Yifu Cheng, Chi Han, Chun Song, Niu Huang, Yunfei Du. Pyrazole-containing pharmaceuticals: target, pharmacological activity, and their SAR studies. RSC Medicinal Chemistry 2022, 13 (11) , 1300-1321. https://doi.org/10.1039/D2MD00206J
  65. Eleonora Diamanti, María Méndez, Tatjana Ross, Christian A. Kuttruff, Julien Lefranc, Franca‐Maria Klingler, Franz von Nussbaum, Manfred Jung, Matthias Gehringer. Frontiers in Medicinal Chemistry 2022 Goes Virtual. ChemMedChem 2022, 17 (21) https://doi.org/10.1002/cmdc.202200419
  66. Dionysios-Panagiotis Kintos, Konstantinos Salagiannis, Vasiliki Vazoura, Theresa Wittrien, Athanasios Papakyriakou, Sotiris S. Nikolaropoulos, Soenke Behrends, Stavros Topouzis, Manolis A. Fousteris. Design, synthesis and biological evaluation of new 3,4-dihydroquinoxalin-2(1H)-one derivatives as soluble guanylyl cyclase (sGC) activators. Heliyon 2022, 8 (11) , e11438. https://doi.org/10.1016/j.heliyon.2022.e11438
  67. Dmitriy Yu. Vandyshev, Khidmet S. Shikhaliev, Mikhail Yu. Krysin, Tatiana N. Ilyinova, Daria A. Mangusheva, Oleg E. Sidorenko, Renata R. Iminova. New regio/chemoselective synthesis of hydrogenated imidazo[1,5-b]pyridazines. Mendeleev Communications 2022, 32 (5) , 637-639. https://doi.org/10.1016/j.mencom.2022.09.023
  68. Irene Paula Popa, Mihai Ștefan Cristian Haba, Minela Aida Mărănducă, Daniela Maria Tănase, Dragomir N. Șerban, Lăcrămioara Ionela Șerban, Radu Iliescu, Ionuț Tudorancea. Modern Approaches for the Treatment of Heart Failure: Recent Advances and Future Perspectives. Pharmaceutics 2022, 14 (9) , 1964. https://doi.org/10.3390/pharmaceutics14091964
  69. Xiao-Yan Jia, Yong-Ming Liu, Yong-Fei Wang, Jin-Yang An, Ke-Ling Peng, Hua Wang. Bibliometric study of soluble guanylate cyclase stimulators in cardiovascular research based on web of science from 1992 to 2021. Frontiers in Pharmacology 2022, 13 https://doi.org/10.3389/fphar.2022.963255
  70. Masashi Tawa, Tomio Okamura. Factors influencing the soluble guanylate cyclase heme redox state in blood vessels. Vascular Pharmacology 2022, 145 , 107023. https://doi.org/10.1016/j.vph.2022.107023
  71. Karel Urbánek. Vericiguat: pharmacological profile. Klinická farmakologie a farmacie 2022, 36 (2) , 77-80. https://doi.org/10.36290/far.2022.013
  72. Alberto Aimo, Vincenzo Castiglione, Giuseppe Vergaro, Giorgia Panichella, Michele Senni, Carlo Mario Lombardi, Michele Emdin. The place of vericiguat in the landscape of treatment for heart failure with reduced ejection fraction. Heart Failure Reviews 2022, 27 (4) , 1165-1171. https://doi.org/10.1007/s10741-021-10146-1
  73. Vojtěch Melenovský. (Vericiguat - soluble guanylate cyclase stimulator, in therapy of heart failure). Cor et Vasa 2022, 64 (3) , 316-319. https://doi.org/10.33678/cor.2022.034
  74. Garyfallia I. Makrynitsa, Aikaterini I. Argyriou, Aikaterini A. Zompra, Konstantinos Salagiannis, Vassiliki Vazoura, Andreas Papapetropoulos, Stavros Topouzis, Georgios A. Spyroulias. Mapping of the sGC Stimulator BAY 41-2272 Binding Site on H-NOX Domain and Its Regulation by the Redox State of the Heme. Frontiers in Cell and Developmental Biology 2022, 10 https://doi.org/10.3389/fcell.2022.925457
  75. Michael Boettcher, Hans‐Dirk Düngen, Frank Donath, Gerd Mikus, Nikos Werner, Petra A. Thuermann, Mahir Karakas, Nina Besche, Tanja Koch, Matthias Gurniak, Corina Becker. Vericiguat in Combination with Short‐Acting Nitroglycerin in Patients With Chronic Coronary Syndromes: The Randomized, Phase Ib, VENICE Study. Clinical Pharmacology & Therapeutics 2022, 111 (6) , 1239-1247. https://doi.org/10.1002/cpt.2574
  76. J.R. González-Juanatey, M. Anguita-Sánchez, A. Bayes-Genís, J. Comín-Colet, A. García-Quintana, A. Recio-Mayoral, J.L. Zamorano-Gómez, J.M. Cepeda-Rodrigo, L. Manzano. Vericiguat en insuficiencia cardíaca: de la evidencia científica a la práctica clínica. Revista Clínica Española 2022, 222 (6) , 359-369. https://doi.org/10.1016/j.rce.2021.12.005
  77. J.R. González-Juanatey, M. Anguita-Sánchez, A. Bayes-Genís, J. Comín-Colet, A. García-Quintana, A. Recio-Mayoral, J.L. Zamorano-Gómez, J.M. Cepeda-Rodrigo, L. Manzano. Vericiguat in heart failure: From scientific evidence to clinical practice. Revista Clínica Española (English Edition) 2022, 222 (6) , 359-369. https://doi.org/10.1016/j.rceng.2021.12.006
  78. Wiebke Janssen, Thomas Schwarz, Ulf Bütehorn, Wolfram Steinke, Steffen Sandmann, Dieter Lang, Armin Kern, Frank Hucke, Michael Gerisch. Pharmacokinetics and mass balance of vericiguat in rats and dogs and distribution in rats. Xenobiotica 2022, 52 (5) , 453-462. https://doi.org/10.1080/00498254.2022.2082899
  79. Bibhuti B. Das. Therapeutic Approaches in Heart Failure with Preserved Ejection Fraction (HFpEF) in Children: Present and Future. Pediatric Drugs 2022, 24 (3) , 235-246. https://doi.org/10.1007/s40272-022-00508-z
  80. Juan Xia, Nan Hui, Lei Tian, Chengyuan Liang, Jie Zhang, Jifang Liu, Jun Wang, Xiaodong Ren, Xiaolin Xie, Kun Wang. Development of vericiguat: The first soluble guanylate cyclase (sGC) stimulator launched for heart failure with reduced ejection fraction (HFrEF). Biomedicine & Pharmacotherapy 2022, 149 , 112894. https://doi.org/10.1016/j.biopha.2022.112894
  81. Nicole Campbell, Julie Kalabalik-Hoganson, Kathleen Frey. Vericiguat: A Novel Oral Soluble Guanylate Cyclase Stimulator for the Treatment of Heart Failure. Annals of Pharmacotherapy 2022, 56 (5) , 600-608. https://doi.org/10.1177/10600280211041384
  82. Barbara Petzuch, Agnès Bénardeau, Lucas Hofmeister, Jutta Meyer, Elke Hartmann, Mira Pavkovic, Ilka Mathar, Peter Sandner, Heidrun Ellinger-Ziegelbauer. Urinary miRNA Profiles in Chronic Kidney Injury—Benefits of Extracellular Vesicle Enrichment and miRNAs as Potential Biomarkers for Renal Fibrosis, Glomerular Injury, and Endothelial Dysfunction. Toxicological Sciences 2022, 187 (1) , 35-50. https://doi.org/10.1093/toxsci/kfac028
  83. Markus Follmann, Corina Becker, Lothar Roessig, Peter Sandner, Johannes‐Peter Stasch. Discovery and Development of the Soluble Guanylate Cyclase Stimulator Vericiguat for the Treatment of Chronic Heart Failure. 2022, 27-50. https://doi.org/10.1002/9781119627784.ch3
  84. Molly Weisert, Jennifer A. Su, Jondavid Menteer, Robert E. Shaddy, Paul F. Kantor. Drug Treatment of Heart Failure in Children: Gaps and Opportunities. Pediatric Drugs 2022, 24 (2) , 121-136. https://doi.org/10.1007/s40272-021-00485-9
  85. Davide Benedetto Tiz, Luana Bagnoli, Ornelio Rosati, Francesca Marini, Luca Sancineto, Claudio Santi. New Halogen-Containing Drugs Approved by FDA in 2021: An Overview on Their Syntheses and Pharmaceutical Use. Molecules 2022, 27 (5) , 1643. https://doi.org/10.3390/molecules27051643
  86. Aušra Mongirdienė, Laurynas Skrodenis, Leila Varoneckaitė, Gerda Mierkytė, Justinas Gerulis. Reactive Oxygen Species Induced Pathways in Heart Failure Pathogenesis and Potential Therapeutic Strategies. Biomedicines 2022, 10 (3) , 602. https://doi.org/10.3390/biomedicines10030602
  87. Eddie L Myers. Bicyclic 5–6 Systems: Other Four Heteroatoms 2:2. 2022, 500-564. https://doi.org/10.1016/B978-0-12-818655-8.00056-1
  88. Antonio García-Quintana, Alejandro Recio-Mayoral, José María Cepeda-Rodrigo, José Luis Zamorano, José Ramón González-Juanatey. Papel del vericiguat en la etiopatogenia global de la insuficiencia cardiaca con fracción de eyección reducida. Posicionamiento actual. Revista Española de Cardiología Suplementos 2022, 22 , 8-14. https://doi.org/10.1016/S1131-3587(22)00002-4
  89. Hayah Kassis-George, Nathan J Verlinden, Sheng Fu, Manreet Kanwar. Vericiguat in Heart Failure with a Reduced Ejection Fraction: Patient Selection and Special Considerations. Therapeutics and Clinical Risk Management 2022, Volume 18 , 315-322. https://doi.org/10.2147/TCRM.S357422
  90. Barry Greenberg. Medical Management of Patients With Heart Failure and Reduced Ejection Fraction. Korean Circulation Journal 2022, 52 (3) , 173. https://doi.org/10.4070/kcj.2021.0401
  91. Lin Luo, Xu Yang, Kai Tang, Jianli Wu, Dejin Li, Jiuju Ran, Li Zhang, Dan Wang, Dan Zhao, Min Yu, Anfang Chen, Maya Saranathan. Efficacy of three novel drugs in the treatment of heart failure: A network meta-analysis. Medicine 2022, 101 (29) , e29415. https://doi.org/10.1097/MD.0000000000029415
  92. Hauke Ruehs, Dagmar Klein, Matthias Frei, Joachim Grevel, Rupert Austin, Corina Becker, Lothar Roessig, Burkert Pieske, Dirk Garmann, Michaela Meyer. Population Pharmacokinetics and Pharmacodynamics of Vericiguat in Patients with Heart Failure and Reduced Ejection Fraction. Clinical Pharmacokinetics 2021, 60 (11) , 1407-1421. https://doi.org/10.1007/s40262-021-01024-y
  93. Dmitriy Yu. Vandyshev, Evgeniya A. Kosheleva, Vladimir A. Polikarchuk, Daria A. Mangusheva, Gleb L. Denisov, Khidmet S. Shikhaliev. Regioselective synthesis of novel imidazo[1,5-b]pyridazine derivatives from diaminoimidazoles and α-acylacrylonitriles. Mendeleev Communications 2021, 31 (6) , 821-823. https://doi.org/10.1016/j.mencom.2021.11.017
  94. David J. Cordwin, Theodore J. Berei, Kristen T. Pogue. The Role of sGC Stimulators and Activators in Heart Failure With Reduced Ejection Fraction. Journal of Cardiovascular Pharmacology and Therapeutics 2021, 26 (6) , 593-600. https://doi.org/10.1177/10742484211042706
  95. Zh. D. Kobalava, P. V. Lazarev. Nitric oxide — soluble guanylate cyclase — cyclic guanosine monophosphate signaling pathway in the pathogenesis of heart failure and search for novel therapeutic targets. Cardiovascular Therapy and Prevention 2021, 20 (6) , 3035. https://doi.org/10.15829/1728-8800-2021-3035
  96. Jean-Sébastien Hulot, Jean-Noël Trochu, Erwan Donal, Michel Galinier, Damien Logeart, Pascal De Groote, Yves Juillière. Vericiguat for the treatment of heart failure: mechanism of action and pharmacological properties compared with other emerging therapeutic options. Expert Opinion on Pharmacotherapy 2021, 22 (14) , 1847-1855. https://doi.org/10.1080/14656566.2021.1937121
  97. Andrew J. S. Coats, Heli Tolppanen. Drug Treatment of Heart Failure with Reduced Ejection Fraction: Defining the Role of Vericiguat. Drugs 2021, 81 (14) , 1599-1604. https://doi.org/10.1007/s40265-021-01586-y
  98. Amr Abdin, Michael Böhm. Renal function and vericiguat in heart failure patients: light at the end of the tunnel!. European Journal of Heart Failure 2021, 23 (8) , 1322-1324. https://doi.org/10.1002/ejhf.2280
  99. Amr Abdin, Johann Bauersachs, Norbert Frey, Ingrid Kindermann, Andreas Link, Nikolaus Marx, Mitja Lainscak, Jonathan Slawik, Christian Werner, Jan Wintrich, Michael Böhm. Timely and individualized heart failure management: need for implementation into the new guidelines. Clinical Research in Cardiology 2021, 110 (8) , 1150-1158. https://doi.org/10.1007/s00392-021-01867-2
  100. Shalini Murali Krishnan, Johannes Nordlohne, Lisa Dietz, Alexandros Vakalopoulos, Petra Haning, Elke Hartmann, Roland Seifert, Jörg Hüser, Ilka Mathar, Peter Sandner. Assessing the Use of the sGC Stimulator BAY-747, as a Potential Treatment for Duchenne Muscular Dystrophy. International Journal of Molecular Sciences 2021, 22 (15) , 8016. https://doi.org/10.3390/ijms22158016
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Journal of Medicinal Chemistry

Cite this: J. Med. Chem. 2017, 60, 12, 5146–5161
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https://doi.org/10.1021/acs.jmedchem.7b00449
Published May 30, 2017

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  • Abstract

    Scheme 1

    Scheme 1. Synthesis of Compounds 118a

    Scheme aReagents and conditions: (a) NaOMe, DMF, 110 °C; (b) H2 (65 bar), Raney nickel, DMF; (c) R2OCOCl, pyridine, or (i) triphosgene, pyridine, (ii) R2OH, pyridine; (d) R1X (X = halide or trichloromethanesulfonate), LiHMDS; (e) glycolaldehyde, NaBH3CN, AcOH, MeOH, 0 °C to rt; (f) methyl chloroformate, pyridine, rt; (g) NaHMDS, THF, 0 °C to rt.

    Scheme 2

    Scheme 2. Synthesis of Compounds 19 and 20a

    Scheme aReagents and conditions: (a) methyl 2-(2-fluorophenyl)acetate, LiHMDS; (b) aq NaCl solution, DMSO, microwave, 150 °C; (c) hydrazine hydrate, pyridine, reflux; (d) H2, 10% Pd/C, Et3N, EtOH, THF; (e) NaH, DMF, 80 °C; (f) H2, 10% Pd/C, pyridine; (g) methyl chloroformate, pyridine.

    Scheme 3

    Scheme 3. Synthesis of Compound 21a

    Scheme aReagents and conditions: (a) hydrazine hydrate; (b) POBr3, sulfolane, 150 °C; (c) 2-fluorobenzyl bromide, Cs2CO3, DMF, rt; (d) CuCN, DMSO, 150 °C; (e) NaOMe, NH4Cl, AcOH, MeOH; (f) 1b, DMF, Et3N, 100 °C; (g) H2 (1 bar), 10% Pd/C, DMF; (h) methyl chloroformate, pyridine, DCM.

    Scheme 4

    Scheme 4. Synthesis of Compound 22a

    Scheme aReagents and conditions: (a) MeI, K2CO3, acetone; (b) TBAI, KOt-Bu, DCM, then aq HCl, Et2O; (c) dioxane, 60 °C; (d) 1,1,3,3-tetramethoxypropane, aq HCl, MeOH, EtOH, reflux; (e) NaOH; (f) HATU, NH4Cl, Hünig’s base, DMF; (g) POCl3, 120 °C; (h) NaOMe, NH4Cl, MeOH; (i) 1b, DMF, Et3N, 100 °C; (j) H2 (1 bar), 10% Pd/C, DMF, 0 °C; (k) methyl chloroformate, pyridine.

    Scheme 5

    Scheme 5. Synthesis of Compound 23a

    Scheme aReagents and conditions: (a) hydrazine hydrate, Et3N, reflux; (b) Et3N, MeCN; (c) Br2, AcOH, 50 °C; (d) POCl3, DCE, reflux; (e) H2, 5% Pd/C, Et3N, EtOAc; (f) NBS, DCM; (g) CuCN; (h) NaOMe, NH4Cl, AcOH; (i) 1b, DMF, Et3N, 100 °C; (j) H2 (1 bar), 10% Pd/C, DMF, MeOH; (k) methyl chloroformate, pyridine.

    Scheme 6

    Scheme 6. Synthesis of 24a

    Scheme aReagents and conditions: (a) Tf2O, 70 °C, then morpholine, 5 °C, then 40 °C; (b) MeSO3Me, 135 °C, then 100 °C; (c) 45% aq NaOH, 50 °C; (d) morpholine, Et3N, reflux; (e) MsOH, LiCl, EtOH, reflux; (f) formamide, NaOMe, MeOH, EtOH, 95–125 °C; (g) POCl3, sulfolane, 107 °C; (h) NaOMe, NH4Cl, MeOH, EtOH, 65 °C; (i) 1b, DMF, Et3N, 100 °C; (j) H2 (60 bar), 5% Pd/C, DMF, 60 °C; (k) methyl chloroformate, i-PrOH, MeOH, then Et3N, 50 °C.

    Figure 1

    Figure 1. Biotransformation products of 24 in human hepatocytes.

    Figure 2

    Figure 2. Effects of 24 and NO on the stimulation of highly purified sGC and blocking effects of the sGC inhibitor ODQ.

    Figure 3

    Figure 3. Effects of 24 on rat heart Langendorff preparations.

    Figure 4

    Figure 4. Increase in systolic blood pressure in mmHg during the course of the study with L-NAME-treated renin transgenic rats.

    Figure 5

    Figure 5. Right and left ventricle weight normalized on body weight (left/middle) and plasma atrial natriuretic peptide levels [in pg/mL] at the study end, after 3 weeks of treatment.

    Figure 6

    Figure 6. Effects on proteinuria at the study end after 3 weeks of treatment.

    Figure 7

    Figure 7. Kaplan–Meier survival curves.

  • References


    This article references 56 other publications.

    1. 1
      Evgenov, O. V.; Pacher, P.; Schmidt, P. M.; Hasko, G.; Schmidt, H. H. H. W.; Stasch, J.-P. NO-independent stimulators and activators of soluble guanylate cyclase: discovery and therapeutic potential Nat. Rev. Drug Discovery 2006, 5, 755 768 DOI: 10.1038/nrd2038
    2. 2
      Follmann, M.; Griebenow, N.; Hahn, M. G.; Hartung, I.; Mais, F.-J.; Mittendorf, J.; Schaefer, M.; Schirok, H.; Stasch, J.-P.; Stoll, F.; Straub, A. The chemistry and biology of soluble guanylate cyclase stimulators and activators Angew. Chem., Int. Ed. 2013, 52, 9442 9462 DOI: 10.1002/anie.201302588
    3. 3
      Cerra, M. C.; Pellegrino, D. Cardiovascular cGMP-generating systems in physiological and pathological conditions Curr. Med. Chem. 2007, 14, 585 599 DOI: 10.2174/092986707780059715
    4. 4
      Gladwin, M. T. Deconstructing endothelial dysfunction: soluble guanylyl cyclase oxidation and the NO resistance syndrome J. Clin. Invest. 2006, 116, 2330 2332 DOI: 10.1172/JCI29807
    5. 5
      Hoenicka, M.; Schmid, C. Cardiovascular effects of modulators of soluble guanylyl cyclase activity Cardiovasc. Hematol. Agents Med. Chem. 2008, 6, 287 301 DOI: 10.2174/187152508785909555
    6. 6
      Priviero, F. B.; Webb, R. C. Heme-dependent and independent soluble guanylate cyclase activators and vasodilation J. Cardiovasc. Pharmacol. 2010, 56, 229 233 DOI: 10.1097/FJC.0b013e3181eb4e75
    7. 7
      Packer, C. S. Soluble guanylate cyclase (sGC) down-regulation by abnormal extracellular matrix proteins as a novel mechanism in vascular dysfunction: implications in metabolic syndrome Cardiovasc. Res. 2006, 69, 302 303 DOI: 10.1016/j.cardiores.2005.12.006
    8. 8
      Mayer, B.; Koesling, D. cGMP signalling beyond nitric oxide Trends Pharmacol. Sci. 2001, 22, 546 548 DOI: 10.1016/S0165-6147(00)01889-7
    9. 9
      Nioche, P.; Berka, V.; Vipond, J.; Minton, N.; Tsai, A. L.; Raman, C. S. Femtomolar sensitivity of a NO sensor from Clostridium botulinum Science 2004, 306, 1550 1553 DOI: 10.1126/science.1103596
    10. 10
      Pellicena, P.; Karow, D. S.; Boon, E. M.; Marletta, M. A.; Kuriyan, J. Crystal structure of an oxygen-binding heme domain related to soluble guanylate cyclases Proc. Natl. Acad. Sci. U. S. A. 2004, 101, 12854 12859 DOI: 10.1073/pnas.0405188101
    11. 11
      Wedel, B.; Humbert, P.; Harteneck, C.; Foerster, J.; Malkewitz, J.; Bohme, E.; Schultz, G.; Koesling, D. Mutation of His-105 in the β1 subunit yields a nitric oxide-insensitive form of soluble guanylyl cyclase Proc. Natl. Acad. Sci. U. S. A. 1994, 91, 2592 2596 DOI: 10.1073/pnas.91.7.2592
    12. 12
      Zabel, U.; Hausler, C.; Weeger, M.; Schmidt, H. H. Homodimerization of soluble guanylyl cyclase subunits. Dimerization analysis using a glutathione S-transferase affinity tag J. Biol. Chem. 1999, 274, 18149 18152 DOI: 10.1074/jbc.274.26.18149
    13. 13
      Zabel, U.; Weeger, M.; La, M.; Schmidt, H. H. W. Human soluble guanylate cyclase: functional expression and revised isoenzyme family Biochem. J. 1998, 335, 51 57 DOI: 10.1042/bj3350051
    14. 14
      Murad, F. Shattuck Lecture. Nitric oxide and cyclic GMP in cell signaling and drug development N. Engl. J. Med. 2006, 355, 2003 2011 DOI: 10.1056/NEJMsa063904
    15. 15
      Stasch, J. P.; Hobbs, A. J. NO-independent, haem-dependent soluble guanylate cyclase stimulators Handb. Exp. Pharmacol. 2009, 191, 277 308 DOI: 10.1007/978-3-540-68964-5_13
    16. 16
      Schmidt, H. H.; Schmidt, P. M.; Stasch, J. P. NO- and haem-independent soluble guanylate cyclase activators Handb. Exp. Pharmacol. 2009, 191, 309 339 DOI: 10.1007/978-3-540-68964-5_14
    17. 17
      Stasch, J.-P.; Evgenov, O. V. Soluble guanylate cyclase stimulators in pulmonary hypertension Handb. Exp. Pharmacol. 2013, 218, 279 313 DOI: 10.1007/978-3-662-45805-1_12
    18. 18
      Ghofrani, H.-A.; Galie, N.; Grimminger, F.; Gruenig, E.; Humbert, M.; Jing, Z.-C.; Keogh, A. M.; Langleben, D.; Kilama, M. O.; Fritsch, A.; Neuser, D.; Rubin, L. J. Riociguat for the treatment of pulmonary arterial hypertension N. Engl. J. Med. 2013, 369, 330 340 DOI: 10.1056/NEJMoa1209655
    19. 19
      Ghofrani, H.-A.; D’Armini, A. M.; Grimminger, F.; Hoeper, M. M.; Jansa, P.; Kim, N. H.; Mayer, E.; Simonneau, G.; Wilkins, M. R.; Fritsch, A.; Neuser, D.; Weimann, G.; Wang, C. Riociguat for the treatment of chronic thromboembolic pulmonary hypertension N. Engl. J. Med. 2013, 369, 319 329 DOI: 10.1056/NEJMoa1209657
    20. 20
      Hambly, N.; Granton, J. Riociguat for the treatment of pulmonary hypertension Expert Rev. Respir. Med. 2015, 9, 679 695 DOI: 10.1586/17476348.2015.1106316
    21. 21
      Frey, R.; Mueck, W.; Unger, S.; Artmeier-Brandt, U.; Weimann, G.; Wensing, G. Single-dose pharmacokinetics, pharmacodynamics, tolerability, and safety of the soluble guanylate cyclase stimulator BAY 63–2521: an ascending-dose study in healthy male volunteers J. Clin. Pharmacol. 2008, 48, 926 934 DOI: 10.1177/0091270008319793
    22. 22
      Gheorghiade, M.; Greene, S. J.; Butler, J.; Filippatos, G.; Lam, C. S.; Maggioni, A. P.; Ponikowski, P.; Shah, S. J.; Solomon, S. D.; Kraigher-Krainer, E.; Samano, E. T.; Müller, K.; Roessig, L.; Pieske, B. Effect of vericiguat, a soluble guanylate cyclase stimulator, on natriuretic peptide levels in patients with worsening chronic heart failure and reduced ejection fraction: the SOCRATES-REDUCED randomized trial JAMA, J. Am. Med. Assoc. 2015, 314, 2251 2262 DOI: 10.1001/jama.2015.15734
    23. 23
      Pieske, B.; Butler, J.; Filippatos, G.; Lam, C.; Maggioni, A. P.; Ponikowski, P.; Shah, S.; Solomon, S.; Kraigher-Krainer, E.; Samano, E. T.; Scalise, A. V.; Mueller, K.; Roessig, L.; Gheorghiade, M. Rationale and design of the SOluble guanylate Cyclase stimulatoR in heArT failurE Studies (SOCRATES) Eur. J. Heart Failure 2014, 16, 1026 38 DOI: 10.1002/ejhf.135
    24. 24

      Clinicaltrials.gov. Identifier: NCT02861534 (accessed August 5, 2016).

    25. 25
      Mittendorf, J.; Weigand, S.; Alonso-Alija, C.; Bischoff, E.; Feurer, A.; Gerisch, M.; Kern, A.; Knorr, A.; Lang, D.; Muenter, K.; Radtke, M.; Schirok, H.; Schlemmer, K.-H.; Stahl, E.; Straub, A.; Wunder, F.; Stasch, J.-P. Discovery of riociguat (BAY 63–2521): a potent, oral stimulator of soluble guanylate cyclase for the treatment of pulmonary hypertension ChemMedChem 2009, 4, 853 865 DOI: 10.1002/cmdc.200900014
    26. 26
      Gnoth, M. J.; Hopfe, P. M.; Czembor, W. Determination of riociguat and its major human metabolite M-1 in human plasma by stable-isotope dilution LCMS/MS Bioanalysis 2015, 7, 193 205 DOI: 10.4155/bio.14.257
    27. 27
      Becker, C.; Frey, R.; Thomas, D.; Reber, M.; Weimann, G.; Arens, E. R.; Mück, W.; Unger, S.; Dietrich, H. Pharmacokinetic interaction of riociguat with ketoconazole, clarithromycin, and midazolam Pulm. Circ. 2016, 6, S49 57 DOI: 10.1086/685016
    28. 28
      Frey, R.; Becker, C.; Unger, S.; Schmidt, A.; Wensing, G.; Mück, W. Assessment of the effects of renal impairment and smoking on the pharmacokinetics of a single oral dose of the soluble guanylate cyclase stimulator riociguat (BAY 63–2521) Pulm. Circ. 2016, 6, S15 26 DOI: 10.1086/685017
    29. 29
      Saleh, S.; Frey, R.; Becker, C.; Unger, S.; Wensing, G.; Mück, W. Bioavailability, pharmacokinetics, and safety of riociguat given as an oral suspension or crushed tablet with and without food Pulm. Circ. 2016, 6, S66 74 DOI: 10.1086/685020
    30. 30
      Zhao, X.; Wang, Z.; Wang, Y.; Zhang, H.; Blode, H.; Yoshikawa, K.; Becker, C.; Unger, S.; Frey, R.; Cui, Y. Pharmacokinetics of the soluble guanylate cyclase stimulator riociguat in healthy young Chinese male non-smokers and smokers: results of a randomized, double-blind, placebo-controlled study Clin. Pharmacokinet. 2016, 55, 615 624 DOI: 10.1007/s40262-015-0337-4
    31. 31
      Highlights of Prescribing Information: Riociguat (Adempas); U.S. Food and Drug Administration: Silver Spring, MD, 2014; http://www.accessdata.fda.gov/drugsatfda_docs/label/2014/204819s002lbl.pdf (accessed April 4, 2014).
    32. 32
      Bitterli, P.; Erlenmeyer, H. Some derivatives of triazolopyrimidine Helv. Chim. Acta 1951, 34, 835 840 DOI: 10.1002/hlca.19510340311
    33. 33
      Reinecke, M. G.; Woodrow, T. A.; Brown, E. S. Pyrazolo[3,4-c]pyridazines from hydrazine and aminothiophenecarboxylates J. Org. Chem. 1992, 57, 1018 1021 DOI: 10.1021/jo00029a046
    34. 34
      Wunder, F.; Stasch, J.-P.; Hütter, J.; Alonso-Alija, C.; Hüser, J.; Lohrmann, E. A cell-based cGMP assay useful for ultra-high-throughput screening and identification of modulators of the nitric oxide/cGMP pathway Anal. Biochem. 2005, 339, 104 112 DOI: 10.1016/j.ab.2004.12.025
    35. 35
      Hillisch, A.; Heinrich, N.; Wild, H. Computational chemistry in the pharmaceutical industry: from childhood to adolescence ChemMedChem 2015, 10, 1958 1962 DOI: 10.1002/cmdc.201500346
    36. 36
      Burkhard, J. A.; Wuitschik, G.; Rogers-Evans, M.; Müller, K.; Carreira, E. M. Oxetanes as versatile elements in drug discovery and synthesis Angew. Chem., Int. Ed. 2010, 49, 9052 9067 DOI: 10.1002/anie.200907155
    37. 37
      Wuitschik, G.; Carreira, E. M.; Wagner, B.; Fischer, H.; Parrilla, I.; Schuler, F.; Rogers-Evans, M.; Müller, K. Oxetanes in drug discovery: structural and synthetic insights J. Med. Chem. 2010, 53, 3227 3246 DOI: 10.1021/jm9018788
    38. 38
      Leeson, P. D.; Young, R. J. Molecular property design: does everyone get it? ACS Med. Chem. Lett. 2015, 6, 722 725 DOI: 10.1021/acsmedchemlett.5b00157
    39. 39
      Sharkovska, Y.; Kalk, P.; Lawrenz, B.; Godes, M.; Hoffmann, L. S.; Wellkisch, K.; Geschka, S.; Relle, K.; Hocher, B.; Stasch, J.-P. Nitric oxide-independent stimulation of soluble guanylate cyclase reduces organ damage in experimental low-renin and high-renin models J. Hypertens. 2010, 28, 1666 1675 DOI: 10.1097/HJH.0b013e32833b558c
    40. 40
      Dubin, R. F.; Shah, S. J. Soluble guanylate cyclase stimulators: a novel treatment option for heart failure associated with cardiorenal syndromes? Curr. Heart Failure Rep. 2016, 13, 132 139 DOI: 10.1007/s11897-016-0290-z
    41. 41
      Follmann, M.; Stasch, J.-P.; Redlich, G.; Griebenow, N.; Lang, D.; Wunder, F.; Huebsch, W.; Lindner, N.; Vakalopoulos, A.; Tersteegen, A. Preparation of annelated pyrimidine derivatives useful in the treatment and prophylaxis of cardiovascular diseases. WO2013030288, 2013.
    42. 42
      Follmann, M.; Stasch, J.-P.; Redlich, G.; Ackerstaff, J.; Griebenow, N.; Knorr, A.; Wunder, F.; Li, V. M.-J.; Baerfacker, L.; Weigand, S. Bicyclic aza-heterocycles as guanylate cyclase inhibitors and their preparation and use in the treatment of cardiovascular diseases. WO 2012028647, 2012.
    43. 43
      Follmann, M.; Stasch, J.-P.; Redlich, G.; Ackerstaff, J.; Griebenow, N.; Knorr, A.; Wunder, F.; Li, V. M.-J.; Schirok, H.; Jautelat, R. Substituted methyl pyrimidin-5-yl carbamates as guanylate cyclase inhibitors and their preparation and use in the treatment of cardiovascular diseases. WO 2012010578, 2012.
    44. 44
      Alonso-Alija, C.; Bischoff, E.; Muenter, K.; Stasch, J.-P.; Stahl, E.; Weigand, S.; Feurer, A. Preparation of [(pyrazolopyridinyl)pyrimidinyl]carbamates stimulating soluble guanylate cyclase for treating cardiovascular diseases and/or sexual dysfunction. WO 2003095451, 2003.
    45. 45
      Follmann, M.; Stasch, J.-P.; Redlich, G.; Ackerstaff, J.; Griebenow, N.; Wunder, F.; Li, V. M.-J.; Mittendorf, J.; Jautelat, R. Carbamate-substituted diaminopyrimidines as guanylate cyclase inhibitors and their preparation and use in the treatment of cardiovascular diseases. WO 2012010576, 2012.
    46. 46
      Schirok, H.; Mittendorf, J.; Stasch, J.-P.; Wunder, F.; Stoll, F.; Schlemmer, K.-H. Azabicyclic derivatives as stimulators of guanylate cyclase, their preparation, pharmaceutical compositions, and use for the treatment of cardiovascular disorders. WO 2008031513, 2008.
    47. 47
      Follmann, M.; Stasch, J.-P.; Redlich, G.; Ackerstaff, J.; Griebenow, N.; Knorr, A.; Wunder, F.; Li, V. M.-J.; Mittendorf, J.; Schlemmer, K.-H.; Jautelat, R. Substituted 6-fluoro-1H-pyrazolo[4,3-b]pyridines as guanylate cyclase inhibitors and their preparation and use in the treatment of cardiovascular diseases. WO 2012059549, 2012.
    48. 48
      Follmann, M.; Stasch, J.-P.; Redlich, G.; Straub, A.; Ackerstaff, J.; Griebenow, N.; Knorr, A.; Wunder, F.; Li, V. M.-J. Substituted imidazopyridazines as guanylate cyclase inhibitors and their preparation and use in the treatment of cardiovascular diseases. WO 2012152630, 2012.
    49. 49
      Straub, A.; Feurer, A.; Alonso-Alija, C.; Stasch, J.-P.; Perzborn, E.; Huetter, J.; Dembowsky, K.; Stahl, E. Substituted pyrazole derivatives condensed with six-membered heterocyclic rings as cardiovascular agents and their preparation. WO 2000006569, 2000.
    50. 50
      Walsky, R. L.; Obach, R. S. Validated assays for human cytochrome P450 activities Drug Metab. Dispos. 2004, 32, 647 660 DOI: 10.1124/dmd.32.6.647
    51. 51
      Obach, R. S.; Walsky, R. L.; Venkatakrishnan, K. Mechanism-based inactivation of human cytochrome p450 enzymes and the prediction of drug-drug interactions Drug Metab. Dispos. 2007, 35, 246 255 DOI: 10.1124/dmd.106.012633
    52. 52
      Obach, R. S. Predicting clearance in humans from in vitro data Curr. Top. Med. Chem. 2011, 11, 334 339 DOI: 10.2174/156802611794480873
    53. 53
      Artursson, P.; Karlsson, J. Correlation between oral drug absorption in humans and apparent drug permeability coefficients in human intestinal epithelial (Caco-2) cells Biochem. Biophys. Res. Commun. 1991, 175, 880 885 DOI: 10.1016/0006-291X(91)91647-U
    54. 54
      Hoenicka, M.; Becker, E.-M.; Apeler, H.; Sirichoke, T.; Schröder, H.; Gerzer, R.; Stasch, J.-P. Purified soluble guanylyl cyclase expressed in a baculovirus/Sf9 system: stimulation by YC-1, nitric oxide, and carbon monoxide J. Mol. Med. (Heidelberg, Ger.) 1999, 77, 14 23 DOI: 10.1007/s001090050292
    55. 55
      Schermuly, R. T.; Stasch, J.-P.; Pullamsetti, S. S.; Middendorff, R.; Müller, D.; Schlüter, K.-D.; Dingendorf, A.; Hackemack, S.; Kolosionek, E.; Kaulen, C.; Dumitrascu, R.; Weissmann, N.; Mittendorf, J.; Klepetko, W.; Seeger, W.; Ghofrani, H. A.; Grimminger, F. Expression and function of soluble guanylate cyclase in pulmonary arterial hypertension Eur. Respir. J. 2008, 32, 881 891 DOI: 10.1183/09031936.00114407
    56. 56
      Geschka, S.; Kretschmer, A.; Sharkovska, Y.; Evgenov, O. V.; Lawrenz, B.; Hucke, A.; Hocher, B.; Stasch, J.-P. Soluble guanylate cyclase stimulation prevents fibrotic tissue remodeling and improves survival in salt-sensitive Dahl rats PLoS One 2011, 6, e21853 DOI: 10.1371/journal.pone.0021853
  • Supporting Information

    Supporting Information


    The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jmedchem.7b00449.

    • 1H NMR spectra of selected compounds 3, 21, 22, and 24; CYP-inhibition results of 24 (PDF)

    • Molecular formula strings (CSV)


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