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Article 5-Methoxybenzothiophene-2-Carboxamides as Inhibitors of Clk1/4: Optimization of Selectivity and Cellular Potency

Ahmed K. ElHady 1,2 , Dalia S. El-Gamil 1, Po-Jen Chen 3,4, Tsong-Long Hwang 3,5,6,7 , Ashraf H. Abadi 1, Mohammad Abdel-Halim 1 and Matthias Engel 8,*

1 Department of Pharmaceutical Chemistry, Faculty of Pharmacy and Biotechnology, German University in Cairo, Cairo 11835, Egypt; [email protected] (A.K.E.); [email protected] (D.S.E.-G.); [email protected] (A.H.A.); [email protected] (M.A.-H.) 2 School of Life and Medical Sciences, University of Hertfordshire Hosted by Global Academic Foundation, New Administrative Capital, Cairo 11865, Egypt 3 Graduate Institute of Natural Products, College of Medicine, Chang Gung University, Taoyuan 333, Taiwan; [email protected] (P.-J.C.); [email protected] (T.-L.H.) 4 Department of Cosmetic Science, Providence University, Taichung 433, Taiwan 5 Research Center for Chinese Herbal Medicine, Graduate Institute of Health Industry Technology, College of Human Ecology, Chang Gung University of Science and Technology, Taoyuan 333, Taiwan 6 Department of Anesthesiology, Chang Gung Memorial Hospital, Taoyuan 333, Taiwan 7 Department of Chemical Engineering, Ming Chi University of Technology, New Taipei City 243, Taiwan 8 Pharmaceutical and Medicinal Chemistry, Saarland University, Campus C2.3, D-66123 Saarbrücken, Germany * Correspondence: [email protected]; Tel.: +49-681-302-70312; Fax: +49-681-302-70308



 Abstract: Clks have been shown by recent studies to be promising targets for cancer therapy, as Citation: ElHady, A.K.; El-Gamil, they are considered key regulators in the process of pre-mRNA splicing, which in turn affects every D.S.; Chen, P.-J.; Hwang, T.-L.; Abadi, aspect of tumor biology. In particular, Clk1 and -4 are overexpressed in several human tumors. Most A.H.; Abdel-Halim, M.; Engel, M. of the potent Clk1 inhibitors reported in the literature are non-selective, mainly showing off-target 5-Methoxybenzothiophene-2- activity towards Clk2, Dyrk1A and Dyrk1B. Herein, we present new 5-methoxybenzothiophene- Carboxamides as Inhibitors of Clk1/4: 2-carboxamide derivatives with unprecedented selectivity. In particular, the introduction of a 3,5- Optimization of Selectivity and difluoro benzyl extension to the methylated amide led to the discovery of compound 10b (cell-free Cellular Potency. Molecules 2021, 26, 1001. https://doi.org/10.3390/ IC50 = 12.7 nM), which was four times more selective for Clk1 over Clk2 than the previously published molecules26041001 flagship compound 1b. Moreover, 10b showed an improved growth inhibitory activity with T24 cells (GI50 = 0.43 µM). Furthermore, a new binding model in the ATP pocket of Clk1 was developed based Academic Editors: Simona Collina on the structure-activity relationships derived from new rigidified analogues. and Andrea Trabocchi Keywords: Clk1 inhibitor; pre-mRNA splicing; anticancer Received: 10 January 2021 Accepted: 8 February 2021 Published: 13 February 2021 1. Introduction Publisher’s Note: MDPI stays neutral Pre-mRNA splicing represents a critically important step for various processes such as with regard to jurisdictional claims in development and differentiation. Recently, there has been increased interest in mutations published maps and institutional affil- of pre-mRNA splicing factors and their roles in oncogenesis of hematological as well iations. as solid cancers [1]. High-frequency mutations of SRSF2 (serine/arginine-rich splicing factor 2) have been described in patients with myelodysplastic syndromes (MDS), chronic myelomonocytic leukemia and acute myeloid leukemia (AML), in addition to various mutations that have been found in splicing-related genes in lung, breast and pancreatic Copyright: © 2021 by the authors. cancers. Therefore, pharmacological modulation of pre-mRNA splicing as a novel strategy Licensee MDPI, Basel, Switzerland. in combating cancer has gained attention in the last decade [1]. This article is an open access article Clks (cdc-like kinases) belong to the most essential regulators in the process of pre- distributed under the terms and mRNA splicing, which is achieved through their ability to phosphorylate and activate conditions of the Creative Commons serine/arginine-rich (SR) proteins. Upon phosphorylation, SR proteins are released from Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ nuclear speckles into the nucleoplasm, where they modulate the selection of splice sites 4.0/). during pre-mRNA processing [2]. Targeting Clks has great potential to serve as a novel

Molecules 2021, 26, 1001. https://doi.org/10.3390/molecules26041001 https://www.mdpi.com/journal/molecules Molecules 2021, 26, 1001 2 of 21

treatment for cancer, as alternative splicing (pre-mRNA splicing modulation) has been found to affect nearly every aspect of tumor biology, including metabolism, apoptosis, cell cycle control, metastasis, invasion and angiogenesis (reviewed in [3]). The Clk enzyme family consists of four members (Clk1, 2, 3 and 4) and belongs to the CMGC kinase branch of the human kinome. Both Clk1 and -4 have been shown to be frequently overexpressed kinases in a variety of human tumors [4], though Clk4 is much less investigated than Clk1. In a previous study, we identified compound 1b (compound 21b in [2]) as a potent and selective Clk1/4 inhibitor, with a slightly diminished potency against Clk2 [2]. Compound 1b exhibited an unprecedented selectivity over Dyrk1A, a highly homologous kinase with functions in brain development but also in alternative splicing [5]. Unwanted co-inhibition of Dyrk1A should be avoided, since it has been reported to induce cardiac hypertrophy as a result of its role in antagonizing calcineurin signaling [6]; in addition, depending on the cellular context, Dyrk1A may have tumor- suppressive properties due to its role as a negative regulator of cell cycle progression at least in some cell types [7]. In our previous study, 1b demonstrated great ability in inhibiting the growth of various types of tumor cells through selective depletion of cancer-relevant proteins, an effect that is mediated through the modulation of pre-mRNA splicing, which in turn leads to the nonsense-mediated decay of the mRNAs of genes that are essential for cell growth and survival [2]. Besides Clk1 and -4, Clk2 has also been identified as an anticancer target, especially in breast cancer [8]. Thus, depending on the tumor type, selective Clk1/4 or Clk2 inhibitors might be beneficial, whereas non-selective pan-Clk inhibitors might cause side effects due to the possibly strong impact on alternative splicing in all tissues. Fused heteroaryl tricyclic scaffolds are among the most frequently reported Clk1 inhibitors, for example, those reported by Besson and co-workers [9], as well as those reported by the Moreau group and others [10–12]. Some of these tricyclic inhibitors display a significant growth inhibitory activity against various tumor cell lines; however, co- inhibition of related kinases besides Clk1 cannot be excluded, as selectivity data for some common off-targets are incomplete, e.g., regarding Dyrk1B, Dyrk2, haspin, CK2, STK17 and MLCK2 [13]. The best chemical inhibitors described thus far inhibit Clk1, 2 and 4 with about the same potency; Nassima et al. succeeded in developing two highly potent imidazo[1,2- b]pyridazines (A, B in Figure1) with IC 50 values of 28 and 23 nM, respectively; both dis- played equal potency against Clk2 and Clk4 kinases [14]. Moreover, Sunitinib (C, Figure1 ), which is clinically approved for the treatment of imatinib-resistant gastrointestinal stromal tumor (GIST), renal carcinoma, and pancreatic neuroendocrine tumors [15], was reported by Murar et al. to be a highly potent Clk1 inhibitor with an IC50 of 22 nM, also showing high activity against Clk2 and Clk4, with IC50 values of 20 and 29 nM, respectively [16]. In addition, two imidazole[1,2-a]pyridine derivatives (Figure1D,E) were identified by Araki et al. through high-throughput screening to be highly potent dual Clk1/Clk2 inhibitors, with an IC50 of 1.1 nM for both compounds against Clk1, and 2.4 and 2.1 nM, respectively, against Clk2; however, the inhibition of Clk4 was not tested for these derivatives [17]. The lack of isotype selectivity can be explained on the basis of the high homology between the isoenzymes: Both Clk1 and -4 share a 78.4% sequence identity [18], with both kinases harboring fully identical amino acid residues in and around their ATP binding pockets [2]. Moreover, Prak et al. also described a high degree of similarity between the catalytic domain of Clk1 and Clk2; indeed, the inhibitors reported in this study [19] and those cited above were equipotent against both kinases. Hence, it appears a challenging task to develop inhibitors specific for a certain isoform among the Clk enzyme family. Herein we report the optimization of our previously reported benzo[b]thiophen-2- carboxamides [2] through systematic synthetic modifications of the amide linker and the benzyl extensions to the amide function, followed by a biological evaluation. Molecules 2021, 26, 1001 3 of 23 Molecules 2021, 26, 1001 3 of 21

1b A B Clk1 IC50 = 6.9 nM Clk1 IC50 = 28 nM Clk1 IC50 = 23 nM Clk2 IC50 = 19.1 nM Clk2 IC50 ≈ 28 nM a Clk2 IC50 ≈ 23 nM a Clk4 IC50 = 2.3 nM Clk4 IC50 ≈ 28 nM a Clk4 IC50 ≈ 23 nM a

C D E Clk1 IC50 = 22 nM Clk1 IC50 = 1.1 nM Clk1 IC50 = 1.1 nM Clk2 IC50 = 20 nM Clk2 IC50 = 2.4 nM Clk2 IC50 = 2.1 nM Clk4 IC50 = 29 nM Clk4 IC50 (not tested) Clk4 IC50 (not tested)

FigureFigure 1. Potent1. Potent Clk1 Clk1 inhibitors inhibitors described described previouslypreviously inin thethe literature;literature; 5-methoxybenzothiophene-2-carboxamide (1b ()1b ) imidazo[1,2-b]pyridazines (A,B); Sunitinib (C); imidazo [1,2-a]pyridines (D,E). aa No precise IC50s were provided; the imidazo[1,2-b]pyridazines (A,B); Sunitinib (C); imidazo [1,2-a]pyridines (D,E). No precise IC50s were provided; the authors stated that Clk2 and Clk4 were inhibited with potencies equal to that of Clk1. authors stated that Clk2 and Clk4 were inhibited with potencies equal to that of Clk1. The lack of isotype selectivity can be explained on the basis of the high homology 2. Results between the isoenzymes: Both Clk1 and -4 share a 78.4% sequence identity [18], with both kinasesTo improve harboring the fully potency identical of the amino previous acid residues 5-methoxybenzothiophene-2-carboxamide in and around their ATP binding (1bpockets, Figure [2].2) Moreover, and its selectivity Prak et al. over also the desc mostribed common a high off-targetsdegree of similarity for Clk1 inhibitorsbetween the [ 13 ], variouscatalytic structural domain of modifications Clk1 and Clk2; were indeed, applied, the whichinhibitors included reported modifications in this study at [19] the and amide linkerthose as cited well above as introduction were equipotent of various against structural both kinases. extensions Hence, at the it appears amide function a challenging (summa- rizedtask into develop Figure2). inhibitors These aimed specific at thefor stabilizationa certain isoform of the among biologically the Clk enzyme active conformation family. of theHerein benzyl we moiety report for the Clk1 optimization inhibition. of Inou addition,r previously the reported conformationally benzo[b]thiophen-2- constrained analoguescarboxamides were [2] also through planned systematic in order tosynthetic verify andmodifications possibly refine of the the amide previously linker and proposed the bindingbenzyl extensions mode. In parallel,to the amide many function, new mono- followed and by di-substituted a biological evaluation. benzyl extensions were included, taking advantage of the favorable effect of the fluorine substituent in 1b, while introducing2. Results additional groups for the interaction with the receptor but also for establish- Molecules 2021, 26, 1001 ing intramolecularTo improve the H-bonds potency (e.g., of the in previous13a and 14a5-methoxybenzothiophene-2-carboxamide(Scheme1), between the amide and4 of 23 the

2-methoxy(1b, Figure substituent). 2) and its selectivity over the most common off-targets for Clk1 inhibitors [13], various structural modifications were applied, which included modifications at the amide linker as well as introduction of various structural extensions at the amide function (summarized in Figure 2). These aimed at the stabilization of the biologically active N Nconformation of the benzyl moiety for Clk1 inhibition. In addition, the conformationally constrained analogues were also planned in order to verify and possibly refine the N Cl previously proposed binding mode. In parallel, many newN mono- and di-substituted benzyl extensions were included, taking advantage of the favorable effect of the fluorine HN Nsubstituent in 1b, while introducing additional groups for the interaction with the F receptor but also for establishing intramolecularF H-bondsH3C (e.g., in 13a and 14a (Scheme 1), between the amide and the 2-methoxy substituent). O H3C H3CO N F F F S O 1b

Figure 2. The planned structural modifications for the new series of compounds. Figure 2. The planned structural modifications for the new series of compounds.

2.1. Chemistry A three-step synthesis was employed to access the 5-methoxybenzothiophene-2- carboxamides (Scheme 1). Ethyl thioglycolate was reacted with 2-fluoro-5- methoxybenzaldehyde in the presence of potassium carbonate to produce the 5- methoxybenzothiophene-2-carboxylic acid ethyl ester (I) in a good yield. (I) was subjected to alkaline ester hydrolysis to produce the 5-methoxybenzothiophene carboxylic acid (II), which was then coupled with different amines in the presence of HBTU and trimethylamine to produce the final 5-methoxybenzothiophene-2-carboxamides (3a–15a). Alkylated amide derivatives (1c, 1d, 3b, 4b, 7b–15b) were synthesized through the deprotonation of the secondary amide using potassium bis(trimethylsilyl)amide (KHMDS) at 0 °C; this was followed by the addition of methyl iodide, ethyl iodide or allyl bromide to the trapped anion (Scheme 2). MoleculesMolecules2021 2021, 26,, 26 1001, 1001 5 of 423 of 21

◦ Scheme 1. Synthesis of compounds 3a–15a. Reagents and conditions: (i) K2CO3 in DMF at 0 C, 1.2 eq. ethyl thioglycolate, Scheme 1. Synthesis of compounds 3a–15a. Reagents and conditions: (i) K2CO3 in DMF at 0 °C, 1.2 eq. ethyl thioglycolate, ◦ ◦ 20 min,20 min, 1 eq. 1 eq. 2-fluoro-5-methoxybenzaldehyde 2-fluoro-5-methoxybenzaldehyde inin DMF, 70 °C,C, 4 4 h, h, % % yield yield = =47%. 47%. (ii) (ii) 4 eq. 4 eq. KOH KOH in ethanol/water, in ethanol/water, 80 °C,80 3 C, 3 hh,, % % yieldyield = 90%. 90%. (iii) (iii) 2 eq. 2 eq.HBTU, HBTU, 4 eq. 4triethylamine, eq. triethylamine, 4 eq. of 4the eq. appropriate of the appropriate amine in DCM, amine room in DCM,temperature, room temperature,overnight, overnight,% yield = % 18–97%. yield = 18–97%.

2.1. Chemistry A three-step synthesis was employed to access the 5-methoxybenzothiophene-2-carboxamides (Scheme1). Ethyl thioglycolate was reacted with 2-fluoro-5-methoxybenzaldehyde in the pres- ence of potassium carbonate to produce the 5-methoxybenzothiophene-2-carboxylic acid ethyl ester (I) in a good yield. (I) was subjected to alkaline ester hydrolysis to pro- duce the 5-methoxybenzothiophene carboxylic acid (II), which was then coupled with different amines in the presence of HBTU and trimethylamine to produce the final 5- methoxybenzothiophene-2-carboxamides (3a–15a). Alkylated amide derivatives (1c, 1d, 3b, 4b, 7b–15b) were synthesized through the deprotonation of the secondary amide using potassium bis(trimethylsilyl)amide (KHMDS) at 0 ◦C; this was followed by the addition of methyl iodide, ethyl iodide or allyl bromide to the trapped anion (Scheme2). Molecules 2021, 26, 1001 6 of 23 Molecules 2021, 26, 1001 5 of 21

Scheme 2. Synthesis of compounds 1c, 1d, 3b, 4b, 7b–15b. Reagents and conditions: (i) 3 eq. KHMDS, 0 ◦C, 1 h, 1.5 eq. Scheme 2. Synthesis of compounds 1c, 1d, 3b, 4b, 7b–15b. Reagents and conditions: (i) 3 eq. KHMDS, 0 °C, 1 h, 1.5 eq. CH3I, or CH3CH2I or allyl bromide, room temperature, 24 h, %yield = 14–79%. CH3I, or CH3CH2I or allyl bromide, room temperature, 24 h, %yield = 14–79%. 2.2. Biological Evaluation and Development of a Binding Model 2.2. Biological Evaluation and Development of a Binding Model 2.2.1. In Vitro Clk1/Clk2 Inhibitory Activity 2.2.1. In Vitro Clk1/Clk2 Inhibitory Activity All the newly synthesized derivatives (compounds 1c, 1d, 3a–16a, 3b, 4b, and 7b–15b) All the newly synthesized derivatives (compounds 1c, 1d, 3a–16a, 3b, 4b, and 7b– were tested for their ability to inhibit Clk1 and Clk2 in vitro. With Clk1, the compounds 15b) were tested for their ability to inhibit Clk1 and Clk2 in vitro. With Clk1, the were initially screened at a concentration of 100 nM in duplicates; with Clk2 the initial compounds were initially screened at a concentration of 100 nM in duplicates; with Clk2 screening dose increased to 250 nM. IC50s were determined for compounds that displayed the initial screening dose increased to 250 nM. IC50s were determined for compounds that a percentage of inhibition higher than 50% in the initial screening, through testing a range displayed a percentage of inhibition higher than 50% in the initial screening, through oftesting five concentrations a range of five concentrations with at least two with replicates at least two per replicates concentration per concentration (Tables1 and (Tables2). The Clk11 and and 2). Clk2The Clk1 inhibitory and Clk2 activities inhibitory of compounds activities of1a compounds, 2a, 1b, 2b 1afrom, 2a, our 1b, previous2b from our study areprevious included study in Tableare included1 for comparison. in Table 1 for comparison. Molecules 2021Molecules, 26, 1001 2021, 26, 1001 7 of 23 7 of 23 Molecules 2021Molecules , 26, 1001 2021 , 26, 1001 7 of 23 7 of 23 MoleculesMolecules 2021 Molecules , 26 2021, 1001, 26 2021 , 1001, 26 , 1001 7 of 23 7 of 237 of 23 Molecules 2021 , Molecules26, 1001 2021, 26, 1001 7 of 23 7 of 23 Molecules 2021 , 26 , 1001 7 of 23 Molecules 2021, 26, 1001 6 of 21 Table 1. Clk1/Clk2Table 1. inhibitory Clk1/Clk2 activity inhibitory of the activity mono-substituted of the mono-substituted benzyl derivatives. benzyl derivatives. Table 1. Clk1/Clk2TableTable 1.1. inhibitory Clk1/Clk2Clk1/Clk2 activity inhibitoryinhibitory of the activityactivity mono-substituted ofof thethe mono-substitutedmono-substituted benzyl derivatives. benzylbenzyl derivatives.derivatives. TableTable 1.1. Clk1/Clk2Clk1/Clk2Table 1. inhibitoryinhibitory Clk1/Clk2 activityactivity inhibitory ofof thethe activity mono-substitutedmono-substituted of the mono-substituted benzylbenzyl derivatives.derivatives. benzyl derivatives. TableTable 1. Clk1/Clk2Table 1.Table Clk1/Clk2 1. inhibitory 1.Clk1/Clk2 Clk1/Clk2 inhibitory activity inhibitory inhibitory activity of the activity activity ofmono-substituted the of mono-substituted ofthe the mono-substituted mono-substituted benzyl benzyl derivatives. benzyl derivatives.benzyl derivatives. derivatives. TableTable 1. Clk1/Clk2 1. Clk1/Clk2Table inhibitory 1. Clk1/Clk2 inhibitory Oactivity inhibitory activity of the activity ofmono-substituted the mono-substituted of the mono-substituted benzyl benzylderivatives. benzyl derivatives. derivatives. O OO R R OO O O RR R O O R R RR S S O SS O SS OO S O S SO SSO O O Clk1% Clk1% Clk2% Clk2% S O O Clk1% Clk1% Clk2 % Clk2 % Cpd. Clk1% Clk1% Clk2% Clk2%IC50 (nM) Cpd.50 Clk1% Clk1%IC 50 Clk250 % Clk2IC %50 (nM) 50 Cpd. Cpd. Clk1% Clk1% b Clk2% Clk2%IC50 (nM) ICCpd.50 (nM) Cpd. Clk1% Clk1%IC 50 ICClk250 % Clk2IC %50 (nM) IC50 (nM) Cpd. Cpd.R Inhibition Clk1%Clk1% at Clk1% IC50 (nM) b 50InhibitionClk2%b Clk2% at Clk2% IC50 (nM) ICCpd.50 (nM) Cpd.R Inhibition Clk1%Clk1% at Clk1% IC 50 InhibitionIC50Clk2 %Clk2 at %Clk2 IC %50 (nM) IC50 (nM) Cpd. Cpd.R Clk1%InhibitionR Clk1% Inhibition atInhibition ICClk1%50 (nM) at b ICb 50Inhibition (nM)Clk2% b Inhibition atInhibition Clk2%IC5050 b(nM) at ICCpd.50 (nM)b Cpd.R RInhibition Clk1%Clk1% atInhibition InhibitionClk1%IC 5050at b InhibitionIC50Clk2 b % atInhibitionClk2 Clk2IC %50 Inhibition50 b(nM)% at IC50 (nM) b Cpd.Cpd. No. Cpd. Cpd.R InhibitionR Clk1% atInhibition ICClk1%50IC (nM) at (nM) b IC50Inhibition (nM)Clk2% b atInhibition Clk2%IC50 b(nM) atIC IC 50 (nM)(nM)ICCpd.50 (nM)b Cpd. Cpd. Cpd. No.R R Inhibition Clk1% atInhibition Clk1%IC at50 b IC50IC InhibitionIC(nM)50Clk2 b % atInhibition Clk2IC50 b% (nM)atIC 50 (nM)IC50 IC(nM)b (nM) No.Cpd. Cpd.No.Cpd.R InhibitionR atInhibitiona IC50 (nM) 50at b IC50Inhibition (nM)b b atInhibition a IC50 b(nM) at 50 ICICCpd.No. b(nM)50b (nM) Cpd.No.Cpd.R RInhibition atInhibition (nM)ICa at50b (nM)InhibitionIC50 IC bb50 atInhibition IC50 b(nM) at a ICIC b(nM)50b (nM)50 No.Cpd. No.No.Cpd.R InhibitionRat 100 nMa atInhibition IC50 (nM)a at 50 b IC50Inhibition (nM)at b 250ba atInhibition nM IC50 ba(nM) at ICCpd.No.b50 (nM) No.No.Cpd. R R Inhibitiona atInhibitionat 100 nM(nM)ICa at50b (nM)(nM)InhibitionICb50 a atInhibition atIC 25050 nMba(nM) at ICb50 (nM) No. No.R R InhibitionR100 nMInhibition a atInhibition 100 ICat nM 50 (nM) a atIC b(nM) IC Inhibition (nM)50 250 InhibitionnM ba atInhibition 250 at nM ab at No.b No. R R RInhibition100 nMInhibition a atInhibition 100 at nM (nM) a at b (nM)Inhibition250b nMInhibition a atInhibition 250 at nM ab at b No. No.R Inhibition100R nM a at Inhibition100IC nM50 (nM) a at ICInhibition50250 (nM) nM a at Inhibition250 nMab at b No.b No. R InhibitionR100 nM a at Inhibition100 nM(nM) a bat b(nM)Inhibition250b nM a at Inhibition250 nM ab at b b No. No. No.No. 100 nM a 100 nM a 250 nM a 250 nMab No. b No. No.No. 100 nM a 100 nM(nM) a b (nM) (nM) (nM)250 nMb a 250 nM ab b No. No. 100 nM a 100a nM a 250 nMa 250a nMa No. No. 100 nM a 100a nM(nM) a (nM)250 nM a 250a nM a 100 nM100 a nM100 nM a 250 nM250a nM250 nM a 100 nM100 a nM100 nM a 250 nM250 a nM250 nM a HN 100HN nM 100 nM 250 nM 250 nM 100 nM 100 nM 250 nM 250 nM HN HN HN HN c c cHN HN HN HN 1a 1a c 1a c HN HN HN33.933.9 33.9n.d. n.d.n.d.29.8 29.829.8n.d. n.d. n.d.4a 4a4a HN HN 54 5454 114 ± 5.4 114 114 ±± 5.4 n.i.5.4 n.i. n.i. n.d. n.d. n.d. 1a c 1a c HN 33.9HN 33.9n.d. n.d.29.8 29.8n.d. n.d.4a F 4a HN F HN 54 54 114 ± 5.4 114 ± 5.4 n.i. n.i. n.d. n.d. 1a c 1a c HN 33.9HN 33.9 n.d. n.d. 29.8 29.8 n.d. n.d.4a F 4a HN FF HN HN 54 54 114 ± 5.4 114 ± 5.4 n.i. n.i. n.d. n.d. 1a1a c c 1a c 33.933.9 33.9 n.d.n.d. n.d. 29.829.8 29.8 n.d.n.d. n.d.4a4a F 4a HN F HN5454 54 114 114 ±± 5.45.4 114 ± 5.4 n.i. n.i. n.i. n.d. n.d. n.d. 1a c 1aF 1a c F 33.9 33.9 33.9 n.d. n.d. n.d. 29.8 29.8 29.8 n.d. n.d. n.d.4a 4a F 4a HN F 54 54 54 114 ± 5.4 114 ± 1145.4 ± 5.4 n.i. n.i. n.i. n.d. n.d. n.d. 1a c F 1a F 33.9 33.9n.d. n.d.29.8 29.8n.d. 4an.d. F 4aF F F 54 11454 ± 5.4 114 ± n.i.5.4 n.i. n.d. n.d. F F F F F F F F F F F N F N N NN N N c c c NN N N N 1b c 1b c N N N80 80 6.9 ± 0.12± 6.9 ± 0.12 -- 19.1-- ± 0.5 ± 19.14b ± 0.5 4b N N n.i. n.i. n.d. n.d. n.i. n.i. n.d. n.d. 1b 1b c 1b c N 80N80 80 6.9 ± 6.9 0.120.12 6.9 ± 0.12 -- – 19.1-- ± 0.5 19.1 19.10.54b ± 0.5 F 4b4b N F N n.i. n.i.n.i. n.d. n.d. n.d. n.i. n.i. n.i. n.d. n.d. n.d. 1b c 1b c N 80N 80 6.9 ± 0.12 6.9 ± 0.12 -- -- 19.1 ± 0.5 19.14b ± 0.5 F 4b N F N N n.i. n.i. n.d. n.d. n.i. n.i. n.d. n.d. 1b c c 1b c 80 80 6.9 ± 0.12 6.9 ± 0.12 -- -- 19.1 ± 0.5 19.14b ± 0.5 F 4b N F Nn.i. n.i. n.d. n.d. n.i. n.i. n.d. n.d. 1b c 1bF 1b c F 80 80 80 6.9 ± 0.12 6.9 ± 0.12 6.9 ± 0.12 ------19.1 ± 19.10.5 ± 19.10.54b ± 0.5 4b F 4b F F n.i. n.i. n.i. n.d. n.d. n.d. n.i. n.i. n.i. n.d. n.d. n.d. 1b F 1b F 80 6.980 ± 0.12 6.9 ± 0.12 -- 19.1 -- ± 0.5 19.14b ± 0.5 F 4b F n.i. n.i.n.d. n.d.n.i. n.i.n.d. n.d. F F F F F F F F F N N N N N N N NN 1c 1c N N N50.2 50.2100 100 42.4 42.4n.d. n.d.5a 5a NN N n.i. n.i. n.d. n.d. n.i. n.i. n.d. n.d. 1c 1c 1c N 50.250.2N 50.2 100 100100 42.4 42.442.4 n.d. n.d. n.d.5a 5a5a N N N Nn.i. n.i.n.i. n.d. n.d. n.d. n.i. n.i. n.i.n.d. n.d. n.d. 1c 1c 1c 50.2 50.2 50.2 100 100 100 42.4 42.4 42.4 n.d. n.d. n.d.5a 5a 5a N N n.i. n.i. n.i. n.d. n.d. n.d. n.i. n.i. n.i. n.d. n.d. n.d. 1c F 1c F 50.2 50.2100 10042.4 42.4n.d. 5an.d. 5a n.i. n.i.n.d. n.d.n.i. n.i.n.d. n.d. F F F F F F F N N 1d N NNn.i. n.d. n.i. n.d. 6a n.i. n.d. n.i. n.d. 1d 1d NN Nn.i. n.i. n.d. n.d. n.i. n.i. n.d. n.d.6a 6a n.i. n.i. n.d. n.d. n.i. n.i. n.d. n.d. 1d1d 1d N N Nn.i.n.i.N n.i. n.d. n.d.n.d. n.i. n.i.n.i. n.d. n.d. n.d.6a 6a6a n.i. n.i.n.i. n.d. n.d. n.d. n.i. n.i. n.i.n.d. n.d. n.d. 1d 1d 1d N n.i.N n.i. n.i. n.d. n.d. n.d. n.i. n.i. n.i. n.d. n.d. n.d.6a 6a 6a N N n.i. n.i. n.i. n.d. n.d. n.d. n.i. n.i. n.i. n.d. n.d. n.d. 1d F 1d F n.i. n.i.n.d. n.d.n.i. n.i.n.d. 6an.d. 6aN N n.i. n.i.n.d. n.d.n.i. n.i.n.d. n.d. F FF N N N FF F N N F F F F F F HN HN HN c HN HN HN HN c c HN HNHN HN HN 2a c F 2a c HN HN23.5 23.5n.d. n.d.n.d. n.d.n.d. n.d.7a 7a HN HN HN 34.7 34.7n.d. n.d. 55 55 n.d. n.d. 2ac c F 2a c HN F HN HN23.5 23.5 n.d. n.d. n.d. n.d. n.d. n.d.7a 7a HN HN34.7 34.7 n.d. n.d. 55 55 n.d. n.d. 2a c F c 2a c HN F HN23.5HN 23.5 n.d. n.d. n.d. n.d. n.d. n.d.7a 7a HN 34.7 34.7 n.d. n.d. 55 55 n.d. n.d. 2a 2a c 2aF 2a c HNc F 23.523.5HN 23.5 23.5 n.d. n.d. n.d. n.d. n.d. n.d.n.d. n.d. n.d. n.d. n.d. n.d.7a 7a 7a7a 34.7 34.7 34.734.7 n.d. n.d. n.d. n.d. 55 55 55 55 n.d. n.d. n.d. n.d. 2a c F 2a2aF c F 23.5 23.523.5 n.d. n.d.n.d. n.d. n.d.n.d. n.d. n.d.7an.d. Cl 7a7a Cl 34.7 34.734.7 n.d. n.d.n.d. 55 55 55 n.d. n.d. n.d. 2a F 2a F 23.5 23.5n.d. n.d.n.d. n.d.n.d. 7an.d. Cl 7a CCll 34.7 34.7n.d. n.d. 55 55 n.d. n.d. F F Cl Cl Cl Cl Cl Cl Cl Cl N Cl N N N N NN c c N N NN N 2b c 2b cc N 28.7N 28.7n.d. n.d.n.d. n.d.n.d. 7bn.d. 7b N N N 88.5 88.5 6.2 ± 0.1 6.2 ± 0.1 73.4 73.4 104 ± 4.2 104 ± 4.2 2b c F 2b2b c N F 28.7N 28.728.7 n.d. n.d.n.d. n.d. n.d.n.d. n.d. n.d.7bn.d. 7b7b N N88.5 88.588.5 6.2 ± 0.1 6.2 6.2 ±± 0.10.1 73.4 73.4 73.4 104 ± 4.2 104 104 ±± 4.24.2 2bc c F c 2b c N F N 28.7N 28.7 n.d. n.d. n.d. n.d. n.d. n.d.7b 7b N N88.5 88.5 6.2 ± 0.1 6.2 ± 0.1 73.4 73.4 104 ± 4.2 104 ± 4.2 2b c F 2b c c N F 28.7N 28.7 n.d. n.d. n.d. n.d. n.d. n.d.7b 7b 88.5 88.5 6.2 ± 0.1 6.2 ±± 0.1 73.4 73.4 104 ± 4.2 104 ± 4.2 ± 2b 2b c 2bF 2b c F 28.728.7 28.7 28.7 n.d. n.d. n.d. n.d. n.d. n.d.n.d. n.d. n.d. n.d. n.d. n.d.7b 7b Cl7b7b Cl 88.5 88.5 88.588.5 6.2 ± 0.1 6.2 ± 0.1 6.2 6.2 ± 0.1 73.40.1 73.4 73.4 104 73.4 ± 4.2 104 ± 1044.2 ± 1044.2 4.2 2b F 2bF F F 28.7 28.7n.d. n.d.n.d. n.d.n.d. 7bn.d. Cl 7b Cl 88.5 88.5 6.2 ± 0.1 6.2 ± 73.40.1 10473.4 ± 4.2 104 ± 4.2 F F Cl Cl Cl Cl Cl Cl Cl Cl Cl Cl HN HN HN HN 3a 3a HN HN28.7 28.7n.d. n.d. n.i. n.i. n.d. n.d.8a 8a HN HNn.i. n.i. n.d. n.d. n.i. n.i. n.d. n.d. 3a F 3a HN F HN28.7 28.7n.d. n.d. n.i. n.i. n.d. n.d.8a H3CO 8a HHN3CO HNn.i. n.i. n.d. n.d. n.i. n.i. n.d. n.d. 3a F 3a HN F HN HN28.7 28.7 n.d. n.d. n.i. n.i. n.d. n.d.8a H3CO8a HHN3CO HN HNn.i. n.i. n.d. n.d. n.i. n.i. n.d. n.d. 3a3a F 3a HN F 28.728.7HN 28.7 n.d.n.d. n.d. n.i. n.i. n.i. n.d. n.d. n.d.8a8a H3CO8a HHN3CO n.i.n.i.HN n.i. n.d.n.d. n.d. n.i.n.i. n.i. n.d.n.d. n.d. 3a 3aFF 3a HN F 28.7HN 28.7 28.7 n.d. n.d. n.d. n.i. n.i. n.i. n.d. n.d. n.d.8a 8aHH3 COCO8a HHN3CO n.i.HN n.i. n.i. n.d. n.d. n.d. n.i. n.i. n.i. n.d. n.d. n.d. 3a 3a F F3a F 28.728.7 28.7n.d. n.d. n.d. n.i. n.i. n.i. n.d. n.d. 8a n.d. H3CO8a H8a3CO H3CO n.i. n.i.n.i.n.d. n.d.n.d.n.i. n.i. n.i.n.d. n.d. n.d. 3a F 3a F 28.7 28.7n.d. n.d. n.i. n.i. n.d. 8a n.d. H3CO 8a 3 H3CO n.i. n.i.n.d. n.d.n.i. n.i.n.d. n.d. F F H3CO H3CO

N N N N 3b 3b N n.i.N n.i. n.d. n.d. n.i. n.i. n.d. 8bn.d. 8b N 45.5N 45.5n.d. n.d.23.7 23.7n.d. n.d. 3b F 3b N F n.i.N n.i. n.d. n.d. n.i. n.i. n.d. 8bn.d. H3CO 8b H3NCO 45.5N 45.5n.d. n.d.23.7 23.7n.d. n.d. 3b F 3b N F N n.i.N n.i. n.d. n.d. n.i. n.i. n.d. n.d.8b H3CO8b H3NCO N 45.5N 45.5 n.d. n.d. 23.7 23.7 n.d. n.d. 3b3b F 3b N F n.i.n.i.N n.i. n.d.n.d. n.d. n.i.n.i. n.i. n.d.n.d. n.d.8b8b H3CO8b H3NCO 45.545.5N 45.5 n.d.n.d. n.d. 23.723.7 23.7 n.d.n.d. n.d. 3b 3bFF 3b N F n.i.N n.i. n.i. n.d. n.d. n.d. n.i. n.i. n.i. n.d. n.d. n.d.8b 8bHH3 COCO8b H3NCO 45.5N 45.5 45.5 n.d. n.d. n.d. 23.7 23.7 23.7 n.d. n.d. n.d. 3b F F3b F n.i. n.i.n.d. n.d.n.i. n.i.n.d. 8bn.d. H3CO H8b3CO H3CO 45.5 45.5n.d. n.d.23.7 23.7n.d. n.d. 3b 3b F 3b F n.i.n.i. n.i.n.d. n.d. n.d.n.i. n.i.n.i.n.d. n.d. 8bn.d. H3CO8b 8b 3 H3CO 45.5 45.545.5n.d. n.d.n.d.23.7 23.7 23.7n.d. n.d. n.d. F F H3CO H3CO

a Data shown are the mean of at least two independent experiments, SD ≤ 10%. b IC50 ± SD, n.d., not determined; n.i., no inhibition. c Compounds 1a, 1b, 2a and 2b were reported previously in Ref. [2]. Molecules 2021MoleculesMolecules, 26, 1001 2021 2021, ,26 26, ,1001 1001 8 of 23 88 of of 23 23 Molecules Molecules 2021Molecules , 262021, 1001, 26 2021 , 1001, 26 , 1001 8 of 238 of 23 8 of 23 Molecules 2021Molecules , 26, 1001 2021 , 26, 1001 8 of 23 8 of 23 Molecules 2021 Molecules, 26, 1001 2021 , 26, 1001 8 of 23 8 of 23

a a b b c c a Dataa showna Dataare the shown mean are of theat least mean two of independentat least two independent experiments, experiments, SD ≤ 10%. b IC50SD b≤ ±10%. SD, b n.d., IC50 not ± SD, determ n.d.,ined; not determn.i., noined; inhibition. n.i., no c Compoundsinhibition.c c Compounds 1a, 1b, 2a and 1a 2b, 1b were, 2a and 2b were a Data Datashown shown Data are the shown are mean the are mean of the at leastofmean at twoleast of atindependent two least independent two independent experiments, experiments, experiments, SD ≤ SD10%. ≤ 10%.b SDIC50 ≤ 10%.IC50± SD, ± n.d., IC50SD, not n.d.,± SD, determ not n.d., determined; not determ ined;n.i., no n.i.,ined; inhibition. no n.i., inhibition. no c inhibition.Compounds Compounds Compounds 1a, 1b 1a, 2a, 1b and , 1a2a ,2b 1band were, 2a 2b and were 2b were a Data showna Data are the shown mean are of the at leastmean two of atindependent least two independent experiments, experiments, SD ≤ 10%. b SDIC50 ≤ 10%.± SD, b n.d.,IC50 not ± SD, determ n.d.,ined; not determ n.i., noined; inhibition. n.i., no c inhibition.Compounds c Compounds 1a, 1b, 2a and 1a ,2b 1b were, 2a and 2b were Molecules 2021areported aData, 26, 1001 shown previouslyareported Data are the shown in meanpreviously Ref. are [2].of the at leastinmean Ref. two of[2]. atindependent least two independent experiments, experiments, SD ≤ 10%. b SDbIC50 ≤ 10%.± SD, b n.d.,IC50 not ± SD, determ n.d.,ined; not determ n.i., noined; inhibition. n.i., no c inhibition.Compoundsc c Compounds 1a, 1b, 2a and 1a ,2b 1b were, 2a and 2b were 7 of 21 reported Data Datareported shown shown previouslyreported Data arepreviously are the shownthe in previouslymean meanRef. inare [2].of Ref. ofthe at at in least[2].mean least Ref. two two[2].of atindependent independent least two independent experiments, experiments, experiments, SD SD ≤ ≤10%. 10%. SDIC50 IC50 ≤ 10%.± ±SD, SD, n.d., IC50n.d., not ±not SD, determ determ n.d.,ined; notined; determ n.i., n.i., no noined; inhibition. inhibition. n.i., no inhibition.Compounds Compounds Compounds 1a 1a, 1b, 1b, 2a, 2a and and1a ,2b 1b2b were, were2a and 2b were reported previouslyreported inpreviously Ref. [2]. in Ref. [2]. reportedreported previously previouslyreported inpreviously in Ref. Ref. [2]. [2]. in Ref. [2]. Table 2. Clk1/Clk2Table 2.inhibitory Clk1/Clk2 activity inhibitory of the activity di-substituted of the di-substituted benzyl derivatives. benzyl derivatives. TableTable 2. Clk1/Clk2 2.Table Clk1/Clk2 2. inhibitory Clk1/Clk2 inhibitory activity inhibitory activity of the activity of di-substituted the di-substitutedof the di-substituted benzyl benzyl derivatives. benzyl derivatives. derivatives. Table 2. Clk1/Clk2Table 2. inhibitory Clk1/Clk2 activity inhibitory of the activity di-substituted of the di-substituted benzyl derivatives. benzyl derivatives. TableTableTable 2. 2. Clk1/Clk2 2.Clk1/Clk2TableClk1/Clk2 2. inhibitory Clk1/Clk2inhibitory inhibitory activity inhibitoryactivity activity of of the activitythe ofdi-substituted di-substituted the of di-substituted the di-substituted benzyl benzyl benzylderivatives. derivatives. benzyl derivatives. derivatives. O O R R O O O R R R O O R R OO O RR R S O S O S SO OS O S O S O SS OO S O Clk1%Clk1% Clk1%Clk1% Clk2%Clk2% Clk2%Clk2% Clk1%Clk1% Clk1%Clk1% Clk2%Clk2% Clk2%Clk2% Cpd. Cpd.Cpd. Clk1%Clk1% Clk1%IC50 IC50IC50Clk2%Clk2% Clk2%IC50 Cpd.IC50IC50 Cpd.Cpd. Clk1% Clk1%Clk1%IC50 IC50IC50Clk2% Clk2%Clk2%IC50 IC50IC50 Cpd.Cpd. Cpd.R RInhibition Clk1% Inhibitionat Clk1%IC50 atIC50 b InhibitionIC50Clk2% Inhibitionat Clk2%IC50 atIC50 b Cpd. IC50Cpd. Cpd.R RInhibition Clk1% Inhibitionat Clk1%IC50 atIC50 InhibitionIC50Clk2% b Inhibitionat Clk2%IC50 atIC50 IC50 b Cpd.Cpd. No. Cpd.R R RInhibitionInhibition Clk1%Inhibition at Inhibition at Clk1%IC50 at IC50 (nM) atb InhibitionIC50InhibitionClk2%Inhibition b Inhibition at at Clk2% atIC50 IC50 (nM)atb Cpd.IC50 Cpd. b Cpd. No.R R R Inhibition Clk1%InhibitionInhibitionInhibition at Clk1% at IC50 at atb IC50InhibitionIC50 (nM)Clk2%Inhibition b Inhibition atInhibition Clk2% at IC50 atb IC50IC50 (nM) b Cpd.No. Cpd.No.R RInhibition a Inhibition at (nM)IC50 atb bInhibition(nM)IC50b aInhibition at (nM)IC50 atb Cpd.bNo.(nM)IC50 b Cpd.No.R RInhibition Inhibition at (nM)IC50a atb bInhibition(nM)IC50b Inhibition at (nM)IC50a atb b (nM)IC50b No.Cpd. No. Cpd.No.R RInhibition aInhibition at (nM)IC50a at(nM)b Inhibition (nM)IC50b aInhibition at (nM)IC50a at(nM)b No. (nM)Cpd.IC50 No.b Cpd.No.R RInhibition aInhibition at (nM)IC50a at(nM)b Inhibition (nM)IC50b aInhibition at (nM)IC50a at(nM)b (nM)IC50b No. No.R RInhibition100 100 nM nMaInhibition at 100a nM(nM)a at b Inhibition(nM)250250b nMnMaInhibition at 250a nM(nM)a at b No.(nM) b No.R RInhibition 100 nMaInhibition at100 100a nM nM(nM)a at b Inhibition(nM)250b nMaInhibition at 250250a nM(nM)a at b (nM)b No. No. R R Inhibition100 nM100a Inhibition nM at100 nM(nM)a atb (nM)Inhibition250b nM250a Inhibition nM at250 nM(nM)a atb No.(nM) b No. R R Inhibition100 nM100a Inhibition nM at100 nM(nM)a atb (nM)Inhibition250b nM250a Inhibition nM at250 nM(nM)a atb (nM)b No.No. No. 100 nMa 100 nM(nM)(nM)a b (nM)250b nMa 250 nM(nM)(nM)a b No.(nM)No. b No. 100 nMa 100 nM(nM)(nM)a b (nM)250b nMa 250 nM(nM)(nM)a b (nM)b 100 nMa a 100 nMa 250 nMa a 250 nMa 100 nMa a 100 nMa 250 nMa a 250 nMa HN HN100 nM 100 nM 250 nM 250 nM N 100N nM 100 nM 250 nM 250 nM HN HN HN N N N 16.8 ± 16.816.8 ±± F HN F HN H3CO NH3CO N 16.8 ±16.8 ± 16.8 ± 9a 9a F 9a HN F HNn.i.n.i. n.i. n.d. n.d. n.d.25.8 25.8 25.8n.d. n.d. 12bn.d. H12b3CO12b HN3CO N82.8 82.882.8 16.8 ± 1.239.7 39.7 39.7n.d. n.d. n.d. 9a F 9a F HNHNF HN n.i. n.i. n.d. n.d.25.8 25.8n.d. 12bn.d. H3CO12bH3CO HN3NCO N82.8 82.816.8 ± 16.8 39.7± 39.7n.d. n.d. 9a 9a F 9a F n.i. n.i. n.i. n.d. n.d. n.d.25.8 25.8 25.8n.d. n.d. 12bn.d. 12b H3 CO12b H3CO 82.8 82.8 82.816.8 ± 39.7 39.7 39.7n.d. n.d. n.d. 9a F 9a F n.i. n.i. n.d. n.d.25.8 25.8n.d. 12bn.d. H3CO12b H3CO 82.8 82.816.8 1.2 ± 16.81.2 39.7± 39.7n.d. n.d. 9a F 9a F n.i. n.i. n.d. n.d.25.8 25.8n.d. 12bn.d. H3CO12b H3CO 82.8 82.8 1.2 1.2 1.2 39.7 39.7n.d. n.d. 9a9a F 9a F F F n.i. n.i. n.i. n.d. n.d. n.d.25.8 25.8 25.8n.d. n.d. 12b12bn.d. H3CO12bF H3CO F 82.882.8 82.8 1.2 1.2 39.739.7 39.7n.d. n.d. n.d. F F F F F F 1.2 1.2 F F F F 1.2 1.2 F F F F F F N N HN HN N N N HN HN HN F N F N HN HN 9b F 9bF N F N 8.5 8.5 n.d. n.d.16.9 16.9n.d. 13an.d. 13a HN HN 12.5 12.5n.d. n.d.26.9 26.9n.d. n.d. 9b9b 9b F 9b NF N8.58.5 8.5 8.5 n.d. n.d. n.d. n.d.16.9 16.9 16.9 16.9n.d. n.d. n.d. 13an.d. 13a 13a 13a HN HN 12.5 12.5 12.512.5n.d. n.d. n.d.26.9 26.9 26.9 26.9n.d. n.d. n.d. n.d. 9b F 9b F 8.5 8.5 n.d. n.d.16.9 16.9n.d. 13an.d. 13a 12.5 12.5n.d. n.d.26.9 26.9n.d. n.d. 9b F9b F 8.5 8.5 n.d. n.d.16.9 16.9n.d. 13an.d. F 13aOCH F OCH3 12.5 12.5n.d. n.d.26.9 26.9n.d. n.d. 9b 9b F FF 8.5 8.5 n.d. n.d. 16.9 16.9 n.d. 13an.d. F 13aOCH 3 F OCH3 12.5 12.5 n.d. n.d. 26.9 26.9 n.d. n.d. F F F F OCHF 3 OCH F 3 OCH 3 F F F OCH3 F OCH3 F F F F F OCH3 F OCH3 F F F F F F OCH3 F OCH3 F HN F HN N N F HN FHN HN 53.5 ± 53.5 ± N N N F F HN F HN 53.5 ±53.5 ± 53.5 ± N N 10a 10a HN HN 62.2 62.253.5 ± 53.5 ±30 30n.d. 13bn.d. 13b N N 17.6 17.6n.d. n.d.17 17n.d. n.d. 10a10a 10a 10a HNHN HN62.262.2 62.2 62.2 53.553.5 ± 3.6 ± 53.5 ±30 30 30 30 n.d. n.d. n.d. 13bn.d. 13b 13b 13b N N 17.6 17.6 17.617.6n.d. n.d. n.d. 17 17 17 17 n.d. n.d. n.d. n.d. 10a 10a 62.2 62.253.553.5 3.6 ± ± 53.53.63.6 ± 30 30 n.d. 13bn.d. 13b 17.6 17.6n.d. n.d. 17 17 n.d. n.d. 10a 10a 62.2 62.2 3.6 3.6 3.6 30 30 n.d. 13bn.d. F 13bOCH F OCH3 17.6 17.6n.d. n.d. 17 17 n.d. n.d. 10a F 10a FF 62.2 62.2 3.6 3.6 30 30 n.d. 13bn.d. F 13bOCHF 3 OCH F OCH3 17.6 17.6 n.d. n.d. 17 17 n.d. n.d. F F F 3.6 3.6 F OCH3 F 3 OCH 3 F F 3.6 3.6 F OCH3 F OCH3 F FF F F OCHOCH3 FF OCH3 F F F F F F 3 F 3 F N F NN F HN F HNHN F F N F N N 12.7 ± 12.712.7 ±± F F HN FHN HN 10b 10b N N 88.8 88.812.7 ±12.7 ± 12.7 74.7± 74.7 125 ± 7 14a 125 ± 7 14a HN HN 43.2 43.2n.d. n.d.24.5 24.5n.d. n.d. 10b 10b 10b NN N 88.8 88.8 88.812.7 ± 12.7 74.7± 74.7 74.7 125 ± 125 7 ± 7 12514a ±14a 7 14a HNHN HN 43.2 43.2 43.2n.d. n.d. n.d.24.5 24.5 24.5n.d. n.d. n.d. 10b10b 10b 88.888.8 88.8 12.712.712.7 ±0.90.9 ± ± 12.70.90.9 74.7± 74.7 74.7 125 125 ±± 7 7 12514a ± 7 14a14a 43.2 43.243.2n.d. n.d.24.5 24.5 24.5n.d. n.d. n.d. 10b 10b 88.8 88.8 0.9 0.9 0.9 74.7 74.7 125 ± 7 12514a ± 7 14aOCH OCH3 43.2 43.2n.d. n.d.24.5 24.5n.d. n.d. 10b F10b FF 88.8 88.8 0.9 0.9 74.7 74.7 125 ± 7 12514a ± 7 14aOCH 3 OCH3 43.2 43.2 n.d. n.d. 24.5 24.5 n.d. n.d. F F F 0.9 0.9 OCH3 OCH 3 OCH 3 F F 0.90.9 0.9 OCH3 OCH3 F FF F OCH3 F OCH3 F F F F F FOCH3 F OCH3 F HN F HNHN F N F NN F F HN FHN HN F F N F N N 11a 11a HN HN 31.8 31.8n.d. n.d. 3 3 n.d. 14b n.d. 14b N N 49.2 49.2n.d. n.d.44 44n.d. n.d. 11a 11a 11a HNHN HN 31.8 31.8 31.8n.d. n.d. n.d. 3 3 3 n.d. n.d. 14b n.d. 14b 14b NN N 49.2 49.2 49.2n.d. n.d. n.d. 44 44 44 n.d. n.d. n.d. 11a11a 11a 31.831.8 31.8 n.d.n.d. n.d. 3 3 n.d. n.d. 14b n.d. 14b14b 49.2 49.249.2n.d. n.d. 44 44 44 n.d. n.d. n.d. 11a 11a 31.8 31.8n.d. n.d. 3 3 n.d. 14b n.d. 14bOCH OCH3 49.2 49.2n.d. n.d. 44 44 n.d. n.d. 11a 11aF FF 31.8 31.8 n.d. n.d. 3 3 n.d. 14b n.d. 14bOCH 3 OCH3 49.2 49.2 n.d. n.d. 44 44 n.d. n.d. F F F OCH3 OCH 3 OCH 3 F F OCH3 OCH3 F F F F OCH3 OCH3 F FF F F OCH3 OCH3 F N F NN HN HNHN F F N F N N 33.6 ± 33.633.6 ±± HN HN HN 11b 11b N N 71.2 71.233.6 ±33.6 ± 33.6 52.7± 52.7 103 ± 5 15a 103 ± 5 15a HN HN 42 42n.d. n.d.20 20n.d. n.d. 11b 11b 11b NN N 71.2 71.2 71.233.6 ± 33.6 52.7± 52.7 52.7 103 ± 103 5 ± 5 10315a ±15a 5 15a HNHN HN 42 42 42 n.d. n.d. n.d. 20 20 20 n.d. n.d. n.d. 11b 11b 71.2 71.233.633.6 1.8 ± ± 33.61.81.8 52.7± 52.7 103 ± 5 10315a ± 5 15a 42 42 n.d. n.d. 20 20 n.d. n.d. 11b11b11b 11bF F 71.271.2 71.2 71.2 33.6 ±1.81.8 1.8 1.8 52.7 52.752.7 52.7 103 103 103 ± ± 5 5 10315a15a ± 5 15aF 15a F 42 42 4242 n.d.n.d. n.d. 2020 20 20 n.d.n.d. n.d. n.d. F F F 1.8 1.8 F F F F F 1.8 1.8 F F F F F F F F HN HN N N HN HN HN N N N 10.9 ± 10.910.9 ±± 12a H3CO12a HHNH3COCO HNn.i. n.i.n.d. n.d. 27 27 n.d. 15b n.d. 15b N N 87.8 87.810.9 ±10.9 ± 10.9 72.5± 72.5 167 ± 9 167 ± 9 12a H3CO12aH3CO HHN3CO HNn.i. n.i.n.d. n.d. 27 27 n.d. 15b n.d. 15b N N 87.8 87.810.9 ± 10.9 72.5± 72.5 167 ± 9 167 ± 9 12a 12aH 3CO12a H3HNCO HNn.i. n.i. n.i. n.d. n.d. n.d. 27 27 27 n.d. n.d. 15b n.d. 15b 15b N N 87.8 87.8 87.810.9 ± 10.9 72.5± 72.5 72.5 167 ± 167 9 ± 9 167 ± 9 12a H3CO12a H3CO n.i. n.i. n.d. n.d. 27 27 n.d. 15b n.d. 15b 87.8 87.810.9 0.6 ± 10.90.60.6 72.5± 72.5 167 ± 9 167 ± 9 12a12a 3H3CO12aF H3CO F n.i. n.i.n.d. n.d. 27 27 n.d. 15b n.d. 15bF 15b F 87.8 87.8 0.6 0.6 0.6± 72.5 72.5 167 ± 9 167 ± ±9 12a 12aF F F n.i.n.i. n.i. n.d. n.d. n.d. 27 27 27 n.d. n.d. 15b n.d. F 15bF F 87.8 87.887.8 0.6 10.90.60.6 72.5 72.5 72.5 167 ± 9 167 167 ± 9 9 F F F F 0.60.6 0.6 F a F a Fb Fb F a Data F a showna Dataare the shown mean are of theat least mean two of independentat least two independent experiments, experiments, SD ≤ 10%. b F IC50SD b≤ ±10%. SD, Fb n.d., IC50 not ± SD,determined; n.d., not determined;n.i., no inhibition. n.i., no inhibition. a Data Datashown shownData are the shown are mean the are mean of the at least ofmean at twoleast of atindependent two least independent two independent experiments, experiments, experiments, SD ≤ 10%.SD ≤ 10%.b SDIC50 ≤ 10%.±IC50 SD, ± n.d., IC50SD, notn.d., ± SD, determined; not n.d., determined; not determined; n.i., no n.i., inhibition. no n.i., inhibition. no inhibition. a Data showna Data are the shown mean are of the at least mean two of atindependent least two independent experiments, experiments, SD ≤ 10%. b SDIC50 ≤ 10%.± SD, bn.d., IC50 not ± SD, determined; n.d., not determined; n.i., no inhibition. n.i., no inhibition. a Dataa showna Data are the shown mean are of the at least mean two of atindependent least two independent experiments, experiments, SD ≤ 10%. b bSDIC50 ≤ 10%.± SD, bn.d., IC50 not ± SD, determined; n.d., not determined; n.i., no inhibition. n.i., no inhibition. Data showna DataData shownare shownthe aremean theare meanofthe at meanleast of at two leastof at independent twoleast independent two independent experiments, experiments, experiments, SD SD≤ 10%.≤ 10%. SD IC50b ≤IC50 10%. ± SD,± SD, IC50n.d., n.d., ±not SD, not determined; n.d., determined; not determined; n.i., n.i., no no inhibition. inhibition. n.i., no inhibition. Molecules 2021, 26, 1001 8 of 21

2.2.2. Development of a Binding Model Using Rigidified Analogues The strongly rigidified analogues 5a and 6a were synthesized to probe the biologically active conformation of our inhibitor class on the basis of our previously presented binding model of 1b [2]. The dihedral angle of the benzyl moiety was fixed at two different degrees, which would direct the benzene ring either to a small hydrophobic cavity in the lid of the ATP pocket (in 6a) or more planar toward the pocket exit (in 5a), where favorable CH-π interactions might additionally boost the binding affinity. However, to our surprise, 5a and 6a were both inactive or only weakly active, respectively. This result suggested that the binding orientation predicted for 1b, where the carbonyl interacted with Lys191, might not be preferred by all 5-methoxybenzothiophene-2-carboxamide analogues, because in such case either 5a or 6a should have exhibited a clear inhibitory potency. Already in previous docking runs, an inverted binding mode had occasionally been observed, where the ligand was flipped by 180◦ and the methoxy group interacted with Lys191. Hence, we decided to dock the most active and some inactive compounds of the present series and evaluated the consistency with the SAR in both potential binding modes. Indeed, we found that the SAR observed with the new compounds were all consistent with the inverted binding mode, where the amide carbonyl interacts with the Leu244-NH in the hinge region. Assuming this to be the generally preferred orientation of our ligands, the lack of activity observed with 5a could eventually be explained by a predicted clash of the dihydro-isoquinoline moiety with the Leu167 side chain (Figure S1, Supplementary Material). With respect to the racemic probe compound 6a, only the (S)-enantiomer could be placed in the ATP binding pocket at all; however, this was at the cost of several inter- and intramolecular steric clashes, explaining the very low activity (Figure S2B, Supplementary Material). Apparently, the original binding mode, with H-bonding between the carbonyl and Lys191, has other energetic disadvantages, resulting in a low binding affinity and precluding inhibition. Furthermore, the originally published binding mode would not be in accordance with the loss of potency found with 1d, 3a (S), 9b and 13b, while this could be explained unambiguously by the flipped binding orientation. Altogether, the SAR obtained with the entire set of the new, challenging modifications made it possible to derive a new binding model for the compounds presented here (cf. Figures3 and4), which will be employed in the following as a basis for the discussion.

2.2.3. Variation of the N-alkylation and Methylation of the Benzyl Position In our previous study, it was reported that N-methylation of the amide greatly en- hanced the inhibitory activity against Clk1 [2] (cf. 1a vs. 1b). This overall tendency was confirmed with all unmethylated/ N-methylated compound pairs of the present series, too. However, when the methyl was enlarged to ethyl and allyl (compounds 1c and 1d, respectively, Table1), no further enhancement, but rather a marked drop of activity was noted, suggesting that the available space around the methyl group is rather limited. This observation is in accordance with the binding models for the potent compounds 7b and 10b, which were derived later (cf. Figures3 and4). The p-fluoro analogues having the methyl group added to the benzylic carbon spacer seemed to be more potent than the unmethylated and N-methylated congeners (compare 2a and 2b with 4a, Table1). However, this was only true for the ( R) enantiomer (4a), since the lower activity of the corresponding racemate (3a) indicated that the (S) enantiomer was inactive, probably due to a steric collision of the methyl group with Leu244 (cf. binding model in Figure S2A). In contrast, there is more space for the methyl in the (R) enantiomer (4a), as it will point away from Leu244. On the other hand, combining a methyl group at the benzylic carbon spacer with an N- methylation completely abolished the inhibitory activity, irrespective of the stereochemistry (compounds 3b and 4b, Table1). It can be assumed that the additional methyl group at the benzylic carbon spacer is forced to adopt an anti or gauche conformation relative to the N-methyl, thus strongly increasing the steric demands which would prevent binding due to similar reasons as with 3a (S) (cf. Figure3). Molecules 2021, 26, 1001 10 of 23

Molecules 2021, 26, 1001 9 of 21 conformation relative to the N-methyl, thus strongly increasing the steric demands which would prevent binding due to similar reasons as with 3a (S) (cf. Figure 3). 2.2.4. Variation at the Benzyl Amide Function 2.2.4.In theVariation next optimization at the Benzyl step,Amide various Function new moieties were tested as an extension to the amide function,In the next includingoptimization mono- step, various and di-substituted new moieties were benzyl tested derivatives. as an extensionCompound to the 7b, havingamide the function, bulkier including and more mono- lipophilic and di-substitutedm-chloro benzyl substituent derivatives. on the Compound phenyl ring, 7b, was equipotenthaving the to compoundbulkier and 1bmore; however, lipophilic7b mwas-chloro more substituent selective foron Clk1the phenyl overClk2 ring, (selectivity was equipotent to compound 1b; however, 7b was more selective for Clk1 over Clk2 factor = 16) than 1b (selectivity factor = 2.7). The high potency of 7b could be explained (selectivity factor = 16) than 1b (selectivity factor = 2.7). The high potency of 7b could be based on our new binding model, predicting that CH-π interactions were established with explained based on our new binding model, predicting that CH-π interactions were Leu167established and Leu246 with Leu167 via the andm-chloro Leu246 phenylvia the moiety,m-chloro and phenyl that moiety, halogen and bonds that halogen were formed involvingbonds were the formed chlorine, involving Ser247 the and chlorine, Asp250 Ser247 (Figure and3). Asp250 (Figure 3).

FigureFigure 3. The 3. predicted The predicted binding binding mode mode of compoundof compound7b 7b(magenta) (magenta) dockeddocked in the the ATP ATP bind bindinging pocket pocket of ofClk1 Clk1 (PDB (PDB code code of of the coordinates: 1Z57) using MOE. 7b was anchored inside the pocket through H-bonds with Leu244 and Lys191 the coordinates:(indicated in 1Z57) black), using CH- MOE.π interactions7b was anchoredwith Leu167 inside and theVal175 pocket (red), through an edge-to-face H-bonds CH- withπ Leu244interaction and with Lys191 Phe241, (indicated in black),hydrophobic CH-π interactions interactions withbetween Leu167 the N and-methyl, Val175 Leu167 (red), and an Val175 edge-to-face residues (vio CH-let),π interaction and halogen withbonds Phe241, (green) between hydrophobic interactionsthe chlorine between and the the carbonylN-methyl, groups Leu167 of Ser247 and as Val175 well as residues Asp250. (violet),Interactions and are halogen indicated bonds by dashed (green) lines, between and distances the chlorine and thebetween carbonyl the groupsheavy atoms of Ser247 are given as well in Å. as Asp250. Interactions are indicated by dashed lines, and distances between the heavy atoms are given in Å.

In our previous study, it was reported that using an electron withdrawing fluoro sub- stituent at the phenyl ring was more favorable for the potency than an electron donating methyl or methoxy substituent at the meta-position; however, a closer investigation in the present study revealed that the effects were dependent on the substitution positions: fluo- rine was only favorable in the meta-position of the phenyl ring, while in the para-position, a methoxy substituent was superior to fluorine (compare 2b with 8b). The latter finding was in accordance with our binding model, where the predicted CH-π interactions involving Leu167 and Leu246 basically preferred an electron-rich phenyl ring; it is, therefore, likely Molecules 2021, 26, 1001 10 of 21

that fluorine only becomes favorable if it can undergo additional interactions, such as an H-bond acceptor to Ser247 (cf. Figure4). Based on these considerations, we decided to test the effect of disubstituted benzyl extensions, by combining fluorine with an electron donat- ing group but also by introducing a second fluorine at different positions. With respect to the di-fluoro substitutions, the presence of one fluorine at the meta-position was essential for Clk1 inhibitory activity (compare 9b with 10b and 11b, Table2). Installing both fluorine atoms at the meta-position was optimum among the di-fluorinated rings regarding Clk1 inhibitory activity (10b). The importance of the m-fluoro substituent could be explained by comparing the binding models of 10b (Figure4) with that of 9b (Supplementary Materials, Figure S3) in the Clk1 ATP binding pocket; despite forming a CH-π interaction with Leu167, 9b failed to establish an H-bond interaction with Leu244 in the hinge region, and it was evident that the H-bond with Ser247 can only form with fluorine in the meta-position, like in 10b (Figure4). Of note, despite being slightly less potent (IC 50 = 12.7 nM) than 1b, 10b was more selective for Clk1 over Clk2 (selectivity factor = 10). Regarding the combinations of fluorine with an electron withdrawing group on the phenyl ring, the 3-fluoro-4-methoxy (12b) and the 3-fluoro-5-methyl (15b) disubstituted benzyl rings were most beneficial for the Clk1 inhibitory activity. 15b also exhibited a high selectivity for Clk1 over Clk2 (15-fold), Molecules 2021, 26, 1001 12 of 23 although it was slightly less potent than 7b against purified Clk1 (IC50s = 10.9 nM and 6.2 nM, respectively).

FigureFigure 4. The 4. predictedThe predicted binding binding mode mode of of compound compound10b 10b (cyan).(cyan). 10b10b waswas docked docked in inthe the ATP ATP binding binding pocket pocket of Clk1 of (PDB Clk1 (PDB code of the coordinates: 1Z57) using MOE. 10b was anchored inside the pocket through H-bonds (indicated in black) with code of the coordinates: 1Z57) using MOE. 10b was anchored inside the pocket through H-bonds (indicated in black) with Leu244, Lys191 as well as a weaker H-bond interaction between the fluorine and Ser247. In addition, CH-π interactions Leu244,(red) Lys191 were aspredicted well as between a weaker the H-bond benzyl moiety interaction and Leu167/ betweenLeu264, the fluorine as well as and between Ser247. the In benzothiophene addition, CH- coreπ interactions and (red) wereVal175/Val324. predicted An between edge-to-face the benzyl CH-π moietyinteraction and with Leu167/Leu264, Phe241 and hydrophobi as well asc interactions between the (violet) benzothiophene between the coreN- and Val175/Val324.methyl, Leu167 An edge-to-face and Val175 were CH- πalsointeraction predicted. with All interactio Phe241ns and are hydrophobic indicated by dashed interactions lines, (violet)and distances between between the N the-methyl, Leu167heavy and atoms Val175 are were given also in Å. predicted. All interactions are indicated by dashed lines, and distances between the heavy atoms are given in Å. 2.2.5. Inhibition of Tumor Cell Growth In Vitro In our previous study, the bladder carcinoma cell line T24 was identified as a sensitive system for testing the cellular activity of Clk1/4 inhibitors, where 1b showed a GI50 value of 0.63 µM [2]. This was in accordance with the Oncomine data from several studies (Oncomine.org) which had revealed Clk1 mRNA overexpression in bladder cancer. Therefore, we decided to evaluate the cellular activity of the most potent and selective Clk1 inhibitors among the new series (7b, 10b, 12b and 15b) against the T24 cell line (Table 3).

Table 3. Growth inhibition of the most potent Clk1 inhibitors against the T24 cancer cell line.

Cpd. GI50 (μM) Clk1 Inhibition IC50 Selectivity Factor for Clk1 over No. a (nM) Clk2 1b 0.63 6.9 2.7 7b 8.3 6.2 16 10b 0.43 12.7 10 12b 12.12 16.8 ˃15 15b 7.92 10.9 15 a Data shown are the mean of two independent experiments, S.D. ≤ 12%. Molecules 2021, 26, 1001 11 of 21

2.2.5. Inhibition of Tumor Cell Growth In Vitro In our previous study, the bladder carcinoma cell line T24 was identified as a sensitive system for testing the cellular activity of Clk1/4 inhibitors, where 1b showed a GI50 value of 0.63 µM[2]. This was in accordance with the Oncomine data from several studies (On- comine.org) which had revealed Clk1 mRNA overexpression in bladder cancer. Therefore, we decided to evaluate the cellular activity of the most potent and selective Clk1 inhibitors among the new series (7b, 10b, 12b and 15b) against the T24 cell line (Table3).

Table 3. Growth inhibition of the most potent Clk1 inhibitors against the T24 cancer cell line.

Clk1 Inhibition IC Selectivity Factor for Cpd. No. GI (µM) a 50 50 (nM) Clk1 over Clk2 1b 0.63 6.9 2.7 7b 8.3 6.2 16 10b 0.43 12.7 10 12b 12.12 16.8 >15 15b 7.92 10.9 15 a Data shown are the mean of two independent experiments, S.D. ≤ 12%.

In this assay, 10b emerged as the most potent inhibitor of T24 cell growth, exhibiting higher potency than 1b, despite being somewhat less potent in the cell-free assay. There was no strict correlation between the cell-free vs. cellular potencies, suggesting that pharmacokinetic properties, such as cellular uptake, played a major role for the efficacy in cells. Obviously, the mono- and difluoro-substitution in 1b and 10b strongly improved the physicochemical properties of the 5-methoxybenzothiophene-2-carboxamide scaffold so that the compounds became more available to the cellular Clk1. Since the selectivity of 10b had increased compared with the reference compound 1b (cf. Table4), it was less likely that the inhibition of additional kinases was responsible for the higher cellular activity, though it cannot be totally ruled out.

Table 4. Selectivity profiling of compound 10b.

10b Kinase % Inhibition at 1 µM a CDK5/p25 3 75% @ 0.75 µM DYRK1A b IC50 = 355 ± 20 nM DYRK1B 70 DYRK2 19 DYRK3 3 Clk1 80 Clk3 48 Clk4 100 PIM1 6 CK1 γ2 2 CK2 α1 5 Haspin 0 MLCK2 3 STK17A (DRAK1) 25 SRPK1 4 SRPK2 3 a Screenings were performed as a service at ThermoFisher Scientific using ATP concentrations at the Km value. b Data represent mean values of duplicates that differed by less than 7%. Dyrk1a screening and IC50 determination were performed in house at an ATP concentration of 15 µM; IC50 value ± SD.

2.2.6. Kinase Selectivity Profiling Having identified 10b as the most potent inhibitor in T24 cells, it was important to further analyze the selectivity of this compound against a larger panel of kinases that were Molecules 2021, 26, 1001 12 of 21

frequently reported as off-targets for Clk1 inhibitors [13,20–24], due to the highly similar ATP binding pockets. The results are shown in Table4. The co-inhibition of Clk4 by 10b was expected, since Clk1 and Clk4 share the highest sequence identity among the Clk family members. In view of their fully identical ATP binding pocket [2], selective inhibition of Clk1 vs. Clk4 and vice versa might not be achievable by ATP competitive compounds. Among the tested non-Clk kinases, only Dyrk1A and Dyrk1B were appreciably inhibited; however, the IC50 determination for Dyrk1A revealed that 10b was 28 times more potent for Clk1; this selectivity factor was higher than that reported for many highly potent Clk1 inhibitors in the literature: e.g., the quinazoline derivatives 34 and 35 described in [25], with selectivity factors of 2.8 and 2.7 respectively; the aminopyrimidine derivatives 15 and 17 reported in Ref. [26], with selectivity factors of 3.25 and 2, respectively; the azaindole derivative 10c reported in Ref. [27], with a selectivity factor of 3.2; TG003 reported in [28], which showed higher potency against Dyrk1A than against Clk1 (IC50s of 12 and 20 nM, respectively); and KH-CB19 reported in [29], with a selectivity factor of 2.8.

3. Materials and Methods 3.1. Chemistry Solvents and reagents were obtained from commercial suppliers and used as received. Melting points were determined on a Stuart SMP3 melting point apparatus. A Bruker DRX 500 spectrometer was used to obtain the 1H-NMR and 13C-NMR spectra. The chemical shifts are referenced to the residual protonated solvent signals. All final compounds had a percentage purity of at least 95%, and this could be verified using HPLC coupled with mass spectrometry. Mass spectra (HPLC−ESIMS) were obtained using a TSQ quantum (Thermo Electron Corp., Waltham, MA, USA) instrument prepared with a triple quadrupole mass detector (Thermo Finnigan, Waltham, MA, USA) and an ESI source. All samples were injected using an autosampler (Surveyor, Thermo Finnigan) by an injection volume of 10 µL. The MS detection was determined using a source CID of 10 V and carried out at a spray voltage of 4.2 kV, a nitrogen sheath gas pressure of 4.0 × 105 Pa, a capillary temperature of 400 ◦C, a capillary voltage of 35 V, and an auxiliary gas pressure of 1.0 × 105 Pa. The stationary phase used was an RP C18 NUCLEODUR 100-3 (125 mm × 3 mm) column (Macherey & Nagel, Düren, Germany). The solvent system consisted of water containing 0.1% TFA (A) and 0.1% TFA in acetonitrile (B). The HPLC method used a flow rate of 400 µL/min. The percentage of B started at 5%, increased up to 100% during 7 min, was kept at 100% for 2 min, and was flushed back to 5% in 2 min and was kept at 5% for 2 min. Melting points were determined using a BUCHI B-540 melting point apparatus and are uncorrected. The high-resolution mass analyses were performed using a Orbitrap Q exactive mass spectrometer, equipped with a heated ESI source and an quadrupole-orbitrap coupled mass detector and an Ultimate3000 HPLC (Thermo Finnigan, San Jose, CA, USA). The MS detection was carried out at a spray voltage of 3.5 kV, a nitrogen sheath gas pressure of 4.0 × 105 Pa, an auxiliary gas pressure of 1.0 × 105 Pa and a capillary temperature of 300 ◦C. All samples were injected by autosampler with an injection volume of 15 µL. A RP Nucleoshell Phenyl-hexyl® (100-3, 2.7 µm) column (Macherey-Nagel GmbH, Dühren, Germany) was used as stationary phase. The solvent system consisted of 0.1% formic acid (A) and 0.1% formic acid in acetonitrile (B). HPLC-Method: Flow rate 550 µL/min. The percentage of B started at an initial of 5%, increased up to 40% during 10 min, then to 99% during the next 4 min, kept at 99% for 2 min and flushed back to the initial 5%. Xcalibur software was used for data acquisition and plotting.

3.1.1. General Synthetic Procedures Procedure A, Synthesis of 5-Methoxybenzo[b]thiophene-2-carboxylic Acid Ethyl Ester (I)

To an ice-cooled suspension of (7.2 g, 2 eq.) of K2CO3 in DMF (20 mL) was added ethyl thioglycolate (31.2 mmol, 1.2 eq.). The reaction mixture was stirred for 20 min at 0 ◦C under nitrogen, this was followed by the gradual addition of 2-fluoro-5-methoxybenzaldehyde Molecules 2021, 26, 1001 13 of 21

(26 mmol, 1 eq.) in DMF (10 mL). The mixture was heated to reflux at 70 ◦C for 4 h. Afterwards, the suspension was cooled to room temperature and poured over 100 mL of 2 M HCl, the aqueous layer was extracted by DCM (5 × 10 mL), and the combined organic layers were thoroughly washed with water and brine, dried over anhydrous MgSO4, evaporated under reduced pressure and the resulting residue was purified by column chromatography (CC) using a solvent system of (petroleum ether/ DCM 3:2), giving the pure carboxylic acid ethyl ester as a yellow solid in a yield of 2.9 g (47%); 1H-NMR (500 MHz, DMSO-d6) δ 8.09 (s, 1H), 7.93 (d, J = 8.9 Hz, 1H), 7.53 (d, J = 2.5 Hz, 1H), 7.17 (dd, J = 8.9, 2.6 Hz, 1H), 4.34 (q, J = 7.1 Hz, 2H), 3.82 (s, 3H), 1.32 (t, J = 7.1 Hz, 3H); 13C-NMR (126 MHz, DMSO-d6) δ 161.98, 157.48, 139.64, 133.92, 133.80, 130.35, 123.75, 118.04, 107.07, 61.36, 55.33, 14.11.

Procedure B, Synthesis of 5-Methoxybenzo[b]thiophene-2-carboxylic Acid (II) The ethyl ester (I) was suspended in a mixture of 50 mL of ethanol and 25 mL of water; this was followed by the addition of 4 eq. KOH. The mixture was heated to reflux at 80 ◦C for 3 h. Afterwards, the solvent was removed under reduced pressure, and the resulting aqueous layer was cooled to 0 ◦C, followed by the gradual addition of conc. HCl until the pH was adjusted to 1. The aqueous layer was then extracted using DCM (5 × 50 mL), the combined organic extracts were filtered over anhydrous MgSO4, and evaporated under reduced pressure giving the carboxylic acid derivative as a white solid in a yield of 2.3 g 1 (90%). H-NMR (500 MHz, DMSO-d6) δ 8.01 (d, J = 0.6 Hz, 1H), 7.91 (d, J = 8.9 Hz, 1H), 7.52 (d, J = 2.5 Hz, 1H), 7.15 (dd, J = 8.9, 2.6 Hz, 1H), 3.82 (s, 3H); 13C-NMR (126 MHz, DMSO-d6) δ 163.51, 157.38, 139.84, 135.64, 133.82, 129.90, 123.73, 117.74, 106.93, 55.33.

Procedure C, General Procedure for the Synthesis of 5-Methoxybenzo[b]thiophene-2-carboxylic Acid Amide Derivatives 5-Methoxybenzo[b]thiophene-2-carboxylic acid (II) (0.1 g, 0.5 mmol) was dissolved in DCM (30 mL), then 0.3 g of HBTU and 2 mL of TEA were added. The reaction mixture was stirred for 30 min, following this, the appropriate amine (4 eq.) was added dropwise, and the reaction mixture was stirred at room temperature overnight. The solvent was evaporated under reduced pressure. The residue was partitioned between 50 mL of ethyl acetate and 20 mL of water then the aqueous layer was extracted with three 20 mL portions of ethyl acetate. The combined organic extracts were filtered over anhydrous MgSO4, the solvent was removed under reduced pressure, and the product was purified by CC to give the 5-methoxybenzo[b]thiophene-2-carboxylic acid amide derivatives in different yields.

Procedure D, General Procedure for the Synthesis of 5-Methoxybenzo[b]thiophene-2-carboxylic Acid Alkyl Amide Derivatives The respective amide derivative was dissolved in dry THF and stirred at 0 ◦C, this was followed by the gradual addition of 3 eq. of potassium bis(trimethylsilyl)amide (0.5 M solution in THF), the reaction mixture was left to stir for 1 h, after which methyliodide or ethyliodide or allylbromide (1.5 eq.) were added. The reaction mixture was left to stir for 24 h at room temperature, afterwards, the solvent was removed under reduced pressure, then a small amount of water was added, and extraction was carried out using DCM (5 × 30 mL). The organic layers were combined, dried over anhydrous MgSO4, the solvent was removed under reduced pressure and the product was purified by CC or by salting out to give the 5-methoxybenzo[b]thiophene-2-carboxylic acid alkyl amide derivatives in different yields.

3.1.2. Experimental Details and Synthesis of the Compounds N-(3-fluorobenzyl)-5-methoxybenzothiophene-2-carboxamide (1a). The experimental details and synthesis of the compound have been previously reported in Ref. [2], annotated as compound 21a. Molecules 2021, 26, 1001 14 of 21

N-Methyl-N-(3-fluorobenzyl)-5-methoxybenzothiophene-2-carboxamide (1b). The experi- mental details and synthesis of the compound have been previously reported in Ref. [2], annotated as compound 21b. N-Ethyl-N-(3-fluorobenzyl)-5-methoxybenzothiophene-2-carboxamide (1c). The compound was synthesized according to procedure D using compound 1a and ethyliodide to give a yellow oil: yield (21%). The product was purified by CC (DCM); 1H-NMR (500 MHz, CDCl3) δ 7.69 (d, J = 8.8 Hz, 1H), 7.43 (s, 1H), 7.35 (dd, J = 12.9, 6.4 Hz, 1H), 7.20 (s, 1H), 7.10 (d, J = 7.5 Hz, 1H), 7.07–6.98 (m, 3H), 4.79 (s, 2H), 3.85 (s, 3H), 3.55 (q, J = 7.1 Hz, 2H), 1.25 13 1 (t, J = 7.1 Hz, 3H); C-NMR (126 MHz, CDCl3) δ 165.5, 163.6 (d, JC−F = 246.9 Hz), 158.2, 2 3 140.1, 138.6, 133.2, 130.8, 125.1, 123.71 (d, JC−F = 20.4 Hz), 123.4, 122.5 (d, JC−F = 4.0 Hz), 2 117.0, 114.9 (d, JC−F = 21.0 Hz), 114.7, 106.5, 55.9, 53.8, 30.1, 1.4; (ESI-MS) m/z = 344.16 [M + H]+. N-Allyl-N-(3-fluorobenzyl)-5-methoxybenzothiophene-2-carboxamide (1d). The compound was synthesized according to procedure D using compound 1a and allylbromide to give a yellow oil: yield (78.9%). The product was purified by CC (DCM/Petroleum ether 2:1); 1H- NMR (500 MHz, CDCl3) δ 7.67 (d, J = 8.8 Hz, 1H), 7.47 (d, J = 8.6 Hz, 1H), 7.32 (d, J = 6.4 Hz, 1H), 7.18 (s, 1H), 7.08 (d, J = 7.4 Hz, 1H), 7.05–6.96 (m, 3H), 5.88 (dtd, J = 15.7, 10.5, 5.4 Hz, 1H), 5.32 (d, J = 9.8 Hz, 1H), 5.24 (d, J = 16.1 Hz, 1H), 4.75 (s, 2H), 4.10 (d, J = 5.3 Hz, 13 1 2H), 3.83 (s, 3H); C-NMR (126 MHz, CDCl3) δ 165.3, 163.1 (d, JC−F = 247.0 Hz), 157.8, 3 3 143.2, 139.7, 139.4 (d, JC−F = 6.9 Hz), 137.9, 132.9, 132.5 (d, JC−F = 7.0 Hz), 130.4 (d, 2 2 JC−F = 10.3 Hz), 124.9, 123.8, 123.0, 118.3, 116.8, 114.6 (d, JC−F = 21.2 Hz), 106.2, 55.5, 30.3, 29.4; (ESI-MS) m/z = 356.1 [M + H]+. N-(4-fluorobenzyl)-5-methoxybenzothiophene-2-carboxamide (2a). The experimental details and synthesis of the compound have been previously reported in Ref. [2] 2, annotated compound as 22a. N-Methyl-N-(3-fluorobenzyl)-5-methoxybenzothiophene-2-carboxamide (2b). The experi- mental details and synthesis of the compound have been previously reported in Ref. [2], annotated as compound 22b. (RS)-N-(1-(4-fluorophenyl)ethyl)-5-methoxybenzothiophene-2-carboxamide (3a). The com- pound was synthesized according to procedure C using (RS)-1-(4-fluoro-phenyl)-ethylamine to give a white solid: yield (43.7%); The product was purified by CC (DCM); mp 163.3– ◦ 1 164.3 C; H-NMR (500 MHz, DMSO-d6) δ 9.08 (d, J = 8.0 Hz, 1H), 8.11 (d, J = 0.6 Hz, 1H), 7.91–7.84 (m, 1H), 7.46–7.41 (m, 3H), 7.19–7.13 (m, 2H), 7.10 (dd, J = 8.9, 2.5 Hz, 1H), 5.14 13 (quint, J = 7.1 Hz, 1H), 3.83 (s, 3H), 1.49 (d, J = 7.1 Hz, 3H); C-NMR (126 MHz, DMSO-d6) 1 4 δ 161.0 (d, JC-F = 242.1 Hz), 160.7, 157.3, 140.9, 140.6 (d, JC-F = 3.0 Hz), 140.2, 132.6, 128.0 (d, 3 2 JC-F = 8.1 Hz), 124.7, 123.5, 116.5, 114.9 (d, JC-F = 21.2 Hz), 106.7, 55.3, 48.0, 22.1; (ESI-MS) m/z = 330.16 [M + H]+. (RS)-N-(1-(4-fluorophenyl)ethyl)-N-methyl-5-methoxybenzothiophene-2-carboxamide (3b). The compound was synthesized according to procedure D using compound 3a and methyliodide to give a pale yellow solid: yield (40.6%); the product was purified by ◦ 1 CC (DCM); mp 123.7 C; H-NMR (500 MHz, DMSO-d6, 373 Kelvin) δ 7.84 (d, J = 8.8 Hz, 1H), 7.65 (s, 1H), 7.43 (d, J = 2.4 Hz, 1H), 7.41–7.35 (m, 2H), 7.22–7.15 (m, 2H), 7.09 (dd, J = 8.8, 2.5 Hz, 1H), 5.72 (q, J = 6.9 Hz, 1H), 3.84 (s, 3H), 2.87 (s, 3H), 1.61 (d, J = 7.0 Hz, 3H); 13 1 C-NMR (126 MHz, DMSO-d6, 373 Kelvin) δ 163.44, 161.02 (d, JC-F = 243.8 Hz), 157.22, 4 3 139.50, 138.33, 136.03 (d, JC-F = 3.1 Hz), 131.53, 128.39 (d, JC-F = 8.1 Hz), 124.56, 122.59, 2 115.76, 114.70 (d, JC-F = 21.4 Hz), 106.83, 55.11, 52.81, 29.74, 16.06; (ESI-MS) m/z = 344.18 [M + H]+. (R)-N-(1-(4-fluorophenyl)ethyl)-5-methoxybenzothiophene-2-carboxamide (4a). The com- pound was synthesized according to procedure C using (R)-1-(4-fluoro-phenyl)-ethylamine to give a white solid: yield (44.4%); the product was purified by CC (DCM); mp 150 -151 ◦ 1 C; H-NMR (500 MHz, DMSO-d6) δ 9.08 (d, J = 8.0 Hz, 1H), 8.11 (d, J = 0.5 Hz, 1H), 7.88 (d, J = 8.9 Hz, 1H), 7.46–7.41 (m, 3H), 7.19–7.13 (m, 2H), 7.10 (dd, J = 8.9, 2.5 Hz, 1H), 5.14 13 (quint, J = 7.1 Hz, 1H), 3.83 (s, 3H), 1.49 (d, J = 7.1 Hz, 3H); C-NMR (126 MHz, DMSO-d6) 1 4 δ 161.1 (d, JC-F = 242.1 Hz), 160.8, 157.4, 141.0, 140.7 (d, JC-F = 3.0 Hz), 140.3, 132.7, 128.1 Molecules 2021, 26, 1001 15 of 21

3 2 (d, JC-F = 8.1 Hz), 124.8, 123.6, 116.5, 115.0 (d, JC-F = 21.2 Hz), 106.8, 55.4, 48.1, 22.12; (ESI-MS) m/z = 330.15 [M + H]+. (R)-N-(1-(4-fluorophenyl)ethyl)-N-methyl-5-methoxybenzothiophene-2-carboxamide (4b). The compound was synthesized according to procedure D using compound 4a and methylio- dide to give a pale yellow solid: yield (26%); the product was purified by CC (DCM); ◦ 1 mp 76.8 C; H-NMR (500 MHz, DMSO-d6, 373 Kelvin) δ 7.84 (d, J = 8.8 Hz, 1H), 7.64 (s, 1H), 7.43 (d, J = 2.4 Hz, 1H), 7.41–7.36 (m, 2H), 7.22–7.14 (m, 2H), 7.09 (dd, J = 8.8, 2.5 Hz, 1H), 5.72 (q, J = 7.0 Hz, 1H), 3.85 (s, 3H), 2.87 (s, 3H), 1.62 (d, J = 7.0 Hz, 3H); 13 1 C-NMR (126 MHz, DMSO-d6, 373 Kelvin) δ 163.4, 161.0 (d, JC-F = 244.0 Hz), 157.2, 139.4, 4 3 138.3, 136.01 (d, JC-F = 3.1 Hz), 131.5, 128.3 (d, JC-F = 8.2 Hz), 124.5, 122.5, 115.7, 114.6 2 + (d, JC-F = 21.4 Hz), 106.8, 55.1, 52.8, 29.6, 16.0; (ESI-MS) m/z = 344.18 [M + H] . (3, 4-dihydro-1H-isoquinolin-2-yl)-(5-methoxy-benzo[b]thiophen-2-yl)-methanone (5a). The compound was synthesized according to procedure C using 1,2,3,4-tetrahydro-isoquinoline to give a yellow oil: yield (27.8%); the product was purified by CC (DCM/Ethyl acetate 1 99:1); H-NMR (500 MHz, DMSO-d6, 373 Kelvin) δ 7.86 (d, J = 8.8 Hz, 1H), 7.69 (s, 1H), 7.47 (d, J = 2.5 Hz, 1H), 7.23–7.18 (m, 4H), 7.11 (dd, J = 8.8, 2.5 Hz, 1H), 4.85 (s, 2H), 3.91 13 (t, J = 6.0 Hz, 2H), 3.86 (s, 3H), 2.96 (t, J = 6.0 Hz, 2H); C-NMR (126 MHz, DMSO-d6, 373 Kelvin) δ 162.6, 157.2, 139.5, 138.0, 134.1, 132.7, 131.6, 127.9, 126.1, 125.8, 125.7, 124.7, 122.6, 115.8, 106.9, 55.1, 46.2, 42.9, 28.0; (ESI-MS) m/z = 324.21 [M + H]+. (RS)-(5-methoxy-benzo[b]thiophen-2-yl)-(2-phenyl-pyrrolidin-1-yl)-methanone (6a). The compound was synthesized according to procedure C using (RS)-2-phenyl-pyrrolidine to give a beige solid: yield (18.4%); the product was purified by CC (DCM/Ethyl acetate ◦ 1 99:1); mp 142.5 C; H-NMR (500 MHz, DMSO-d6, 373 Kelvin) δ 7.80 (d, J = 8.8 Hz, 1H), 7.71 (s, 1H), 7.37 (s, 1H), 7.34–7.29 (m, 2H), 7.26 (d, J = 7.2 Hz, 2H), 7.24–7.19 (m, 1H), 7.08 (dd, J = 8.8, 2.5 Hz, 1H), 5.35 (dd, J = 7.0, 4.0 Hz, 1H), 4.06–3.91 (m, 2H), 3.83 (s, 3H), 2.41 (dq, J = 12.3, 7.7 Hz, 1H), 2.01–1.93 (m, 2H), 1.90–1.84 (m, 1H); 13C-NMR (126 MHz, DMSO-d6, 373 Kelvin) δ 161.2, 157.1, 143.1, 139.9, 139.7, 131.9, 127.7, 126.0, 125.3, 125.0, 122.5, 116.0, 106.8, 61.4, 55.1, 48.7, 33.9, 22.9; (ESI-MS) m/z = 338.2 [M + H]+. N-(3-chlorobenzyl)-5-methoxybenzothiophene-2-carboxamide (7a). The compound was synthesized according to procedure C using 3-chlorobenzylamine to give a white solid: yield (94%); the product was purified by CC (DCM/Petroleum ether 4:1); mp 158-160 ◦C; 1 H-NMR (500 MHz, DMSO-d6) δ 9.35 (t, J = 6.0 Hz, 1H), 8.04 (s, 1H), 7.89 (d, J = 8.9 Hz, 1H), 7.44 (d, J = 2.4 Hz, 1H), 7.38 (dd, J = 10.3, 4.7 Hz, 2H), 7.32 (t, J = 8.7 Hz, 2H), 7.10 (dd, 13 J = 8.9, 2.5 Hz, 1H), 4.49 (d, J = 6.0 Hz, 2H), 3.83 (s, 3H); C-NMR (126 MHz, DMSO-d6) δ 161.6, 157.4, 141.9, 140.5, 140.3, 133.0, 132.7, 130.3, 127.1, 126.8, 126.0, 124.8, 123.6, 116.8, 106.8, 55.4, 42.1; (ESI-MS) m/z = 332.07 [M + H]+. N-Methyl-N-(3-chlorobenzyl)-5-methoxybenzothiophene-2-carboxamide (7b). The com- pound was synthesized according to procedure D using compound 7a and methyl iodide to give a yellow solid: yield (38%); the product was purified by salting out, the product was dissolved in 3 ml DCM, 30 ml Petroleum ether were then added, this was followed by filtration over anhydrous MgSO4, 7b was recovered from the filtrate after removing ◦ 1 the solvent under reduced pressure; mp 145-147 C; H-NMR (500 MHz, CDCl3) δ 7.63 (d, J = 8.7 Hz, 1H), 7.38 (s, 1H), 7.28–7.22 (m, 3H), 7.14 (s, 2H), 6.99 (dd, J = 8.9, 1.9 Hz, 1H), 13 4.70 (s, 2H), 3.78 (s, 3H), 3.08 (s, 3H); C-NMR (126 MHz, CDCl3) δ 159.4, 158.2, 152.2, 142.1, 140.5, 140.10, 139.1, 135.2, 134.2, 133.3, 130.6, 128.3, 125.9, 123.4, 117.2, 106.6, 55.9, + 30.1, 23.0; (ESI-MS) m/z = 346.13 [M + H] ; HRMS (ESI): calcd for (C18H17ClNO2S) 346.0663, found 346.0653 [M + H]+. N-(4-methoxybenzyl)-5-methoxybenzothiophene-2-carboxamide (8a). The compound was synthesized according to procedure C using 4-methoxybenzylamine to give a white solid: yield (97.3%); the product was purified by CC (DCM); mp 176-178 ◦C; 1H-NMR (500 MHz, DMSO-d6) δ 9.22 (t, J = 6.0 Hz, 1H), 8.02 (s, 1H), 7.88 (d, J = 8.9 Hz, 1H), 7.42 (d, J = 2.5 Hz, 1H), 7.28–7.25 (m, 2H), 7.09 (dd, J = 8.9, 2.5 Hz, 1H), 6.92–6.88 (m, 2H), 13 4.41 (d, J = 6.0 Hz, 2H), 3.82 (s, 3H), 3.73 (s, 3H); C-NMR (126 MHz, DMSO-d6) δ 161.2, Molecules 2021, 26, 1001 16 of 21

158.1, 157.2, 140.9, 140.1, 132.5, 131.1, 128.6, 124.4, 123.4, 116.5, 113.6, 106.6, 55.2, 54.9, 42.0; (ESI-MS) m/z = 328.02 [M + H]+. N-Methyl-N-(4-methoxybenzyl)-5-methoxybenzothiophene-2-carboxamide (8b). The com- pound was synthesized according to procedure D using compound 8a and methyliodide to give an orange oil: yield (14.4%); the product was purified by CC (DCM); 1H-NMR (500 MHz, Acetone-d6) δ 7.83 (d, J = 8.9 Hz, 1H), 7.63 (s, 1H), 7.39 (s, 1H), 7.30 (d, J = 8.2 Hz, 2H), 7.08 (dd, J = 8.9, 2.5 Hz, 1H), 6.95 (d, J = 8.6 Hz, 2H), 4.75 (s, 2H), 3.85 (s, 3H), 3.80 (s, 13 3H), 3.13 (s, 3H); C-NMR (126 MHz, Acetone-d6) δ 160.2, 158.9, 143.0, 141.2, 129.9, 125.1, 123.9, 123.8, 117.7, 117.4, 114.9, 114.6, 107.2, 55.8, 55.5, 55.5, 43.5; (ESI-MS) m/z = 342.14 [M + H]+. N-(2,4-difluorobenzyl)-5-methoxybenzothiophene-2-carboxamide (9a). The compound was synthesized according to procedure C using 2,4-difluorobenzylamine to give a yellowish white solid: yield (91%); the product was purified by CC (DCM/Petroleum ether 5:1); mp ◦ 1 166-168 C; H-NMR (500 MHz, DMSO-d6) δ 9.27 (t, J = 5.8 Hz, 1H), 8.04 (d, J = 0.6 Hz, 1H), 7.89 (d, J = 8.9 Hz, 1H), 7.48–7.44 (m, 1H), 7.43 (d, J = 2.4 Hz, 1H), 7.24 (ddd, J = 10.5, 9.4, 2.6 Hz, 1H), 7.11–7.06 (m, 2H), 4.49 (d, J = 5.7 Hz, 2H), 3.83 (s, 3H); 13C-NMR (126 MHz, 1 3 1 3 DMSO-d6) δ 161.6 (dd, JC−F, JC−F = 176.8, 12.3 Hz), 161.5, 159.6 (dd, JC−F, JC−F = 179.0, 3 3 12.3 Hz), 157.2, 140.3, 140.1, 132.6, 130.9 (dd, JC−F, JC−F = 9.9, 5.9 Hz), 124.7, 123.4, 122.0 2 4 2 4 (dd, JC−F, JC−F = 15.0, 3.6 Hz), 116.6, 111.2 (dd, JC−F, JC−F = 21.1, 3.6 Hz), 106.6, 103.5 2 3 + (t, JC−F = 25.8 Hz), 55.2, 36.0 (d, JC−F = 3.8 Hz); (ESI-MS) m/z = 334.08 [M + H] . N-Methyl-N-(2,4-difluorobenzyl)-5-methoxybenzothiophene-2-carboxamide (9b). The com- pound was synthesized according to procedure D using compound 9a and methyliodide to give a yellow solid: yield (40.6%); the product was purified by CC (DCM/Petroleum ◦ 1 ether 4:1); mp 116-118 C; H-NMR(500 MHz, DMSO-d6) δ 7.88 (d, J = 8.9 Hz, 1H), 7.75 (s, 1H), 7.43 (p, J = 8.4 Hz, 2H), 7.29 (ddd, J = 10.6, 9.4, 2.6 Hz, 1H), 7.15–7.11 (m, 1H), 7.09 (dd, 13 J = 8.9, 2.5 Hz, 1H), 4.77 (s, 2H), 3.81 (s, 3H), 3.19 (s, 3H); C NMR(126 MHz, DMSO-d6) 1 3 1 3 δ 162.0 (dd, JC−F, JC−F = 160.8, 12.0 Hz), 160.0 (dd, JC−F, JC−F = 162.4, 12.2 Hz), 157.4, 3 3 139.9, 138.3, 131.9, 131.0 (dd, JC−F, JC−F = 11.3, 2.0 Hz), 126.1, 124.9, 123.2, 120.2 (dd, 2 4 2 4 JC−F, JC−F = 15.4, 3.2 Hz), 116.6, 111.70 (dd, JC−F, JC−F = 21.2, 3.3 Hz), 106.7, 104.1 (t, 2 3 + JC−F = 25.4 Hz), 55.3, 45.1 (d, JC−F = 7.6 Hz), 29.2; (ESI-MS) m/z = 348.06 [M + H] . N-(3,5-difluorobenzyl)-5-methoxybenzothiophene-2-carboxamide (10a). The compound was synthesized according to procedure C using 3,5-difluorobenzylamine to give a white solid: yield (88.1%); the product was purified by CC (DCM/Petroleum ether 4:1.5); mp ◦ 1 170-172 C; H NMR(500 MHz, DMSO-d6) δ 9.37 (t, J = 6.0 Hz, 1H), 8.05 (d, J = 0.5 Hz, 1H), 7.90 (d, J = 8.9 Hz, 1H), 7.45 (d, J = 2.5 Hz, 1H), 7.16–7.09 (m, 2H), 7.08–7.03 (m, 2H), 13 1 4.51 (d, J = 6.0 Hz, 2H), 3.83 (s, 3H); C-NMR (126 MHz, DMSO-d6) δ 162.4 (dd, JC−F, 3 3 JC−F = 246.2, 13.1 Hz), 161.8, 157.4, 144.1 (t, JC−F = 8.8 Hz), 140.3, 140.2, 132.7, 125.0, 123.6, 2 4 2 116.8, 110.2 (dd, JC−F, JC−F = 19.5, 5.8 Hz), 106.8, 102.3 (t, JC−F = 25.8 Hz), 55.4, 42.0; (ESI-MS) m/z = 334.09 [M + H]+. N-Methyl-N-(3,5-difluorobenzyl)-5-methoxybenzothiophene-2-carboxamide (10b). The com- pound was synthesized according to procedure D using compound 10a and methyliodide to give a white solid: yield (72.3%), the product was purified by CC (DCM/Petroleum ◦ 1 ether 4:1); mp 107–109 C; H-NMR (500 MHz, DMSO-d6) δ 7.89 (d, J = 8.9 Hz, 1H), 7.43 (s, 1H), 7.18 (t, J = 9.4 Hz, 1H), 7.09 (d, J = 7.8 Hz, 1H), 7.04 (d, J = 6.7 Hz, 2H), 4.75 (s, 2H), 3.81 13 1 3 (s, 3H), 3.25 (s, 3H); C NMR(126 MHz, DMSO-d6) δ 165.0, 162.0 (dd, JC−F, JC−F = 247.5, 2 4 12.6 Hz), 161.7, 157.6, 139.9, 138.9, 133.9, 129.2, 123.8, 118.0, 112.0 (dd, JC−F, JC−F = 27.0, 2 + 13.8 Hz), 107.8 (t, JC−F = 25.8 Hz), 107.2, 55.4, 54.9, 45.8; (ESI-MS) m/z = 348.1 [M + H] ; + HRMS (ESI): calcd for (C18H16F2NO2S) 348.0864, found 348.0855 (M+ H) . N-(2,5-difluorobenzyl)-5-methoxybenzothiophene-2-carboxamide (11a). The compound was synthesized according to procedure C using 2,5-difluorobenzylamine to give a white solid: yield (89.6%); the compound was purified by CC (DCM/Petroleum ether 4:1); mp 143-145 ◦ 1 C; H-NMR (500 MHz, DMSO-d6) δ 9.30 (t, J = 5.8 Hz, 1H), 8.06 (d, J = 0.6 Hz, 1H), 7.89 (d, J = 8.9 Hz, 1H), 7.44 (d, J = 2.5 Hz, 1H), 7.27 (td, J = 9.2, 4.5 Hz, 1H), 7.23–7.14 (m, 2H), 7.11 13 (dd, J = 8.9, 2.5 Hz, 1H), 4.51 (d, J = 5.8 Hz, 2H), 3.83 (s, 3H); C-NMR (126 MHz, DMSO-d6) Molecules 2021, 26, 1001 17 of 21

1 4 1 4 δ 161.7, 158.1 (dd, JC−F, JC−F = 240.0, 2.1 Hz), 157.4, 156.1 (dd, JC−F, JC−F = 240.7, 2.0 2 3 Hz), 140.3, 140.2, 132.7, 128.0 (dd, JC−F, JC−F = 17.6, 7.6 Hz), 125.0, 123.6, 116.8, 116.7 2 3 2 3 2 (dd, JC−F, JC−F = 24.2, 9.0 Hz), 115.8 (dd, JC−F, JC−F = 24.8, 4.8 Hz), 115.3 (dd, JC−F, 3 + JC−F = 24.0, 8.6 Hz), 106.8, 55.4, 36.5; (ESI-MS) m/z = 334.07 [M + H] . N-Methyl-N-(2,5-difluorobenzyl)-5-methoxybenzothiophene-2-carboxamide (11b). The com- pound was synthesized according to procedure D using compound 11a and methylio- dide to give a yellowish white solid: yield (49.3%); the compound was purified by CC ◦ 1 (DCM/Petroleum ether 3:1); mp 91-93 C; H-NMR (500 MHz, DMSO-d6) δ 7.88 (d, J = 8.9 Hz, 1H), 7.81 (s, 1H), 7.44 (d, J = 2.2 Hz, 1H), 7.30 (td, J = 9.2, 4.5 Hz, 1H), 7.20 (tdd, J = 8.9, 6.6, 3.2 Hz, 2H), 7.09 (dd, J = 8.8, 2.4 Hz, 1H), 4.78 (s, 2H), 3.81 (s, 3H), 3.24 13 1 (s, 3H); C-NMR (126 MHz, DMSO-d6) δ 163.8, 158.3 (d, JC−F = 240.2 Hz), 157.4, 156.4 1 2 3 2 (d, JC−F = 231.8 Hz), 139.9, 138.3 (dd, JC−F, JC−F = 7.2, 4.5 Hz), 131.9, 126.0 (dd, JC−F, 3 2 3 JC−F = 17.3, 7.5 Hz), 123.2, 122.8, 117.0 (dd, JC−F, JC−F = 24.8, 8.4 Hz), 116.6, 115.7 (dd, 2 3 + JC−F, JC−F = 24.4, 7.0 Hz), 106.7, 55.3, 45.7, 38.6; (ESI-MS) m/z = 348.05 [M + H] ; HRMS + (ESI): calcd for (C18H16F2NO2S) 348.0864, found 348.0855 [M + H] . N-(3-fluoro-4-methoxybenzyl)-5-methoxybenzothiophene-2-carboxamide (12a). The com- pound was synthesized according to procedure C using 3-fluoro-4-methoxybenzylamine to give a white solid: yield (86.4%); the compound was purified by CC (DCM); mp 170–172 ◦C; 1 H-NMR (500 MHz, DMSO-d6) δ 9.26 (t, J = 6.0 Hz, 1H), 8.02 (d, J = 0.6 Hz, 1H), 7.89 (dt, J = 8.9, 0.5 Hz, 1H), 7.43 (d, J = 2.4 Hz, 1H), 7.19–7.11 (m, 3H), 7.10 (dd, J = 8.9, 2.5 Hz, 13 1H), 4.41 (d, J = 6.0 Hz, 2H), 3.83 (s, 3H), 3.81 (s, 3H); C-NMR (126 MHz, DMSO-d6) 1 2 δ 161.5, 157.4, 151.3 (d, JC−F = 243.7 Hz), 146.0 (d, JC−F = 10.6 Hz), 140.7, 140.3, 132.7, 3 2 132.2 (d, JC−F = 5.8 Hz), 124.7, 123.6, 123.6, 116.67, 115.0 (d, JC−F = 18.2 Hz), 113.7 (d, 3 + JC−F = 1.6 Hz), 106.7, 56.0, 55.4, 41.8; (ESI-MS) m/z = 346.07 [M + H] . N-Methyl-N-(3-fluoro-4-methoxybenzyl)-5-methoxybenzothiophene-2-carboxamide (12b). The compound was synthesized according to procedure D using compound 12a and methylio- dide to give a white solid: yield (37.3%); the compound was purified by CC (DCM/Petroleum ◦ 1 ether 3:1); mp 107–109 C); H-NMR (500 MHz, DMSO-d6) δ 7.88 (d, J = 8.9 Hz, 1H), 7.75 (s, 1H), 7.42 (d, J = 1.7 Hz, 1H), 7.18 (t, J = 8.6 Hz, 2H), 7.09 (dd, J = 8.8, 2.3 Hz, 2H), 4.67 (s, 2H), 13 3.83 (s, 3H), 3.80 (s, 3H), 3.15 (s, 3H); C-NMR (126 MHz, DMSO-d6) δ 163.6, 157.4, 151.4 (d, 1 2 3 JC−F = 244.6 Hz), 146.4 (d, JC−F = 10.5 Hz), 140.0, 138.4, 131.8, 129.9 (d, JC−F = 4.7 Hz), 3 2 126.1, 123.8 (d, JC−F = 2.3 Hz), 123.2, 116.5, 115.3 (d, JC−F = 12.7 Hz), 114.0, 106.6, 56.0, + 55.3, 50.4, 36.9; (ESI-MS) m/z = 360.11 [M + H] ; HRMS (ESI): calcd for (C19H19FNO3S) 360.1064, found 360.1054 (M+ H)+. N-(3-fluoro-2-methoxybenzyl)-5-methoxybenzothiophene-2-carboxamide (13a). The com- pound was synthesized according to procedure C using 3-fluoro-2-methoxybenzylamine to give a white solid: yield (93.6%); the compound was purified by CC (DCM/Petroleum ◦ 1 ether 4:1); mp 136-138 C); H-NMR (500 MHz, DMSO-d6) δ 9.21 (t, J = 5.8 Hz, 1H), 8.06 (d, J = 0.5 Hz, 1H), 7.89 (d, J = 8.9 Hz, 1H), 7.43 (d, J = 2.4 Hz, 1H), 7.18 (ddd, J = 11.6, 8.0, 1.8 Hz, 1H), 7.15–7.12 (m, 1H), 7.12–7.06 (m, 2H), 4.52 (d, J = 5.8 Hz, 2H), 3.90 (d, J = 1.5 Hz, 13 1 3H), 3.83 (s, 3H); C-NMR (126 MHz, DMSO-d6) δ 161.6, 157.4, 154.9 (d, JC−F = 245.0 Hz), 2 4 144.8 (d, JC−F = 10.9 Hz), 140.7, 140.3, 133.8 (d, JC−F = 1.8 Hz), 132.7, 124.7, 124.0 (d, 3 3 2 JC−F = 2.8 Hz), 123.9 (d, JC−F = 8.0 Hz), 123.6, 116.7, 115.5 (d, JC−F = 19.0 Hz), 106.8, 61.2 4 + (d, JC−F = 5.2 Hz), 55.4, 37.4; (ESI-MS) m/z = 346.06 [M + H] . N-Methyl-N-(3-fluoro-2-methoxybenzyl)-5-methoxybenzothiophene-2-carboxamide (13b). The compound was synthesized according to procedure D using compound 13a and methylio- dide to give a white oil: yield (75%); the compound was purified by CC (DCM/Petroleum 1 ether 4:1); H-NMR (500 MHz, DMSO-d6) δ 7.88 (d, J = 8.9 Hz, 1H), 7.79 (s, 1H), 7.43 (s, 1H), 7.26–7.20 (m, 1H), 7.13 (dt, J = 12.2, 6.0 Hz, 1H), 7.08 (dd, J = 10.1, 4.4 Hz, 2H), 4.78 (s, 2H), 13 3.84 (s, 3H), 3.81 (s, 3H), 3.22 (s, 3H); C-NMR (126 MHz, DMSO-d6) δ 163.6, 157.4, 154.9 1 2 (d, JC−F = 245.1 Hz), 145.1 (d, JC−F = 14.5 Hz), 139.9, 138.6, 131.8, 131.6, 126.0, 124.1 (d, 3 3 2 JC−F = 7.9 Hz), 123.8 (d, JC−F = 6.4 Hz), 123.2, 116.5, 116.1 (d, JC−F = 14.3 Hz), 106.6, 61.1 4 + (d, JC−F = 2.7 Hz), 55.3, 49.9, 37.5; (ESI-MS) m/z = 360.14 [M + H] . Molecules 2021, 26, 1001 18 of 21

N-(5-fluoro-2-methoxybenzyl)-5-methoxybenzothiophene-2-carboxamide (14a). The com- pound was synthesized according to procedure C using 5-fluoro-2-methoxybenzylamine to give a white solid: yield (82.5%); the compound was purified by CC (DCM/Petroleum ether ◦ 1 4:1); mp 172-174 C; H-NMR (500 MHz, DMSO-d6) δ 9.19 (t, J = 5.8 Hz, 1H), 8.09 (s, 1H), 7.90 (d, J = 8.9 Hz, 1H), 7.45 (d, J = 2.4 Hz, 1H), 7.11 (dd, J = 8.9, 2.5 Hz, 1H), 7.07 (dd, J = 8.5, 3.1 Hz, 1H), 7.01 (dd, J = 9.0, 4.0 Hz, 2H), 4.44 (d, J = 5.8 Hz, 2H), 3.83 (s, 3H), 3.82 (s, 3H); 13 1 C-NMR (126 MHz, DMSO-d6) δ 161.8, 157.4, 156.3 (d, JC−F = 235.5 Hz), 152.8, 140.6, 140.3, 3 2 2 132.7, 128.7 (d, JC−F = 6.9 Hz), 124.8, 123.6, 116.7, 114.2 (d, JC−F = 24.3 Hz), 113.7 (d, JC−F 3 + = 22.6 Hz), 111.8 (d, JC−F = 8.2 Hz), 106.8, 56.0, 55.4, 37.5; (ESI-MS) m/z = 346.07 [M + H] . N-Methyl-N-(5-fluoro-2-methoxybenzyl)-5-methoxybenzothiophene-2-carboxamide (14b). The compound was synthesized according to procedure D using compound 14a and methylio- dide to give a white oil: yield (75%); the compound was purified by CC (DCM/Petroleum 1 ether 4:1); H-NMR (500 MHz, DMSO-d6) δ 7.88 (d, J = 8.8 Hz, 1H), 7.51 (s, 1H), 7.43 (s, 1H), 7.14–7.06 (m, 2H), 7.04 (dd, J = 9.0, 4.5 Hz, 2H), 4.67 (s, 2H), 3.81 (s, 6H), 3.26 13 1 (s, 3H); C-NMR (126 MHz, DMSO-d6) δ 157.4, 156.4 (d, JC−F = 236.1 Hz), 153.3, 139.9, 3 2 138.6, 131.8, 130.6, 126.5 (d, JC−F = 5.8 Hz), 126.2, 123.2, 116.5, 114.40 (d, JC−F = 24.0 Hz), 3 2 114.1 (d, JC−F = 4.8 Hz), 112.1 (d, JC−F = 8.1 Hz), 106.6, 55.9, 55.3, 54.9, 44.9; (ESI-MS) m/z = 360.1 [M + H]+. N-(3-fluoro-5-methybenzyl)-5-methoxybenzothiophene-2-carboxamide (15a). The compound was synthesized according to procedure C using 3-fluoro-5-methylbenzylamine to give a white solid: yield (93.9%); the compound was purified by CC (DCM/Petroleum ether 4:1); 1 mp; H-NMR (500 MHz, DMSO-d6) δ 9.30 (t, J = 6.0 Hz, 1H), 8.04 (d, J = 0.5 Hz, 1H), 7.89 (d, J = 8.9 Hz, 1H), 7.44 (d, J = 2.4 Hz, 1H), 7.10 (dd, J = 8.9, 2.5 Hz, 1H), 7.00–6.98 (m, 1H), 6.95–6.90 (m, 2H), 4.46 (d, J = 6.0 Hz, 2H), 3.83 (s, 3H), 2.30 (s, 3H); 13C-NMR (126 MHz, 1 3 DMSO-d6) δ 162.2 (d, JC−F = 242.8 Hz), 161.6, 157.4, 141.9 (d, JC−F = 7.7 Hz), 140.6, 140.3, 3 2 140.3, 132.7, 124.8, 123.9 (d, JC−F = 2.2 Hz), 123.6, 116.7, 114.1 (d, JC−F = 20.9 Hz), 111.0 2 4 4 (d, JC−F = 21.7 Hz), 106.7, 55.4, 42.2 (d, JC−F = 1.6 Hz), 20.8 (d, JC−F = 1.7 Hz); (ESI-MS) m/z = 330.07 [M + H]+. N-Methyl-N-(3-fluoro-5-methybenzyl)-5-methoxybenzothiophene-2-carboxamide (15b). The compound was synthesized according to procedure D using compound 15a and methylio- dide to give a white oil: yield (57.5%); the compound was purified by CC (DCM/Petroleum 1 ether 4:1); H-NMR (500 MHz, CDCl3) δ 7.68 (d, J = 8.8 Hz, 1H), 7.45 (s, 1H), 7.18 (s, 1H), 7.03 (dd, J = 8.9, 2.2 Hz, 1H), 6.89 (d, J = 14.2 Hz, 1H), 6.81 (d, J = 9.1 Hz, 2H), 4.72 (s, 2H), 3.83 13 1 (s, 3H), 3.12 (s, 3H), 2.34 (s, 3H); C-NMR (126 MHz, CDCl3) δ 163.4 (d, JC−F = 246.7 Hz), 3 3 161.6, 158.0, 141.3, 140.0, 139.1 (d, JC−F = 7.5 Hz), 138.4 (d, JC−F = 5.6 Hz), 133.2, 124.7 (d, 4 2 2 JC−F = 3.8 Hz), 123.5, 123.3, 116.9, 115.5 (d, JC−F = 21.0 Hz), 111.4 (d, JC−F = 23.7 Hz), 4 4 106.4, 55.8, 29.9, 26.0 (d, JC−F = 4.1 Hz), 21.6 (d, JC−F = 1.5 Hz); (ESI-MS) m/z = 344.12 + + [M + H] ; HRMS (ESI): calcd for (C19H19FNO2S) 344.1115, found 344.1105 (M+ H) .

3.2. Biological Assays 3.2.1. Protein Kinases and Inhibition Assays Active human Clk1 and Clk2 were purchased from Life Technologies (Catalog No. PV3315 and PV4279, respectively). RS repeat substrate peptide for Clk1 (GRSRSRSRSRSRSRSR) was custom synthesized at the Department of Medical Biochemistry and Molecular Biology, Saarland University, Homburg, Germany. Kinase Inhibition assays for Clk1 and Clk2 were performed as described previously, in the presence of 15 µM ATP [20]. The calculated IC50 values are representative of at least two independent determinations performed in duplicates. The larger panel of kinases shown in Table4 was screened by the SelectScreen Kinase Profiling Service, Thermo Fisher Scientific, Paisley, UK.

3.2.2. Antiproliferative Assay The T24 bladder carcinoma cell line was cultured in Dulbecco’s Modified Eagles Medium (DMEM, Sigma-Aldrich). The DMEM medium was supplemented with 1% pen/strep solution (Invitrogen) and 5% fetal calf serum (FCS, Sigma-Aldrich, St. Louis, Molecules 2021, 26, 1001 19 of 21

MO, USA). T24 cells were seeded in a 96-well plate (5000 cells per well) already containing DMSO as a control (0.2% final concentration) or the test compounds dissolved in DMSO. ◦ The cells were grown for five days at 37 C in a humidified incubator containing 5% CO2, without further change of medium, before the detection was carried out as described in [30].

3.3. Molecular Modeling All procedures were performed using the Molecular Operating Environment (MOE) software package (version 2016, Chemical Computing Group, Montreal, Canada). For the docking simulations, PDB entry 1Z57 (Clk1 co-crystallized with hymenialdisine) [18] was used. Molecular docking simulations with the 3a (S), 5a, 6a, 7b, 9b and 10b ligands were performed using the MMFF94x force field, and the Amber10:EHT force field was used for compound 7b; the placement method was set to “Alpha PMI” (number of return poses set to 2000), and refinement was set to “induced fit” to enable free side chain movements. Using these settings, docking runs were performed in two different ways: (i) the binding pocket was defined by selecting nearby residues to the co-crystallized ligand (hymenialdisine); (ii) after an initial run using condition i, the MOE LigX routine was applied (settings: receptor strength = 5, ligand strength = 5000) for energy minimization, and a pharmacophore was defined based on a suitable pose by selecting both the methoxy oxygen close to the NH of the leucine and the carbonyl oxygen near the conserved lysine as acceptor points (this binding orientation prevailed among the poses with the compound docked deep in the pocket). In the pharmacophore definition window, the radius of the pharmacophore points was raised to 1.2 Å. The subsequent docking was performed using the pharmacophore- supported placement. The number of retained poses was set to 500 each time. Only the poses with the top 10 scoring values were further evaluated for plausibility. The final selected poses were optimized again using the LigX routine, using the same settings as described above.

4. Conclusions In the present study, various strategies were employed to increase the potency and/or selectivity of our previously reported N-methylbenzothiophene-2-carboxamides as in- hibitors of Clk1 [2]. While keeping the methylated amide function, introduction of the m-chlorobenzyl as well as di-substituted benzyl extensions led to an enhanced selectivity for Clk1 over Clk2 without loss of the high potency toward Clk1, when compared with the former flagship compound 1b. To the best of our knowledge, 10b, 7b, 12b and 15b show the highest selectivity for Clk1/4 over Clk2, as reported previously for Clk1 inhibitors. In addition, compound 10b was more potent in cells than 1b, exhibiting the highest anti- proliferative activity against T24 bladder carcinoma cells. The overall selectivity profile of 10b against the most common off-targets for Clk1 inhibitors was comparable to that of 1b, with the important difference that 10b was four times more selective for Clk2 inhibi- tion. The latter could be considered a significant achievement, because more selective Clk inhibitors might reduce the side effects that can be expected following the application of pan Clk inhibitors; in such cases, the alternative splicing in all tissues might be strongly compromised due to the simultaneous blocking of all Clk activities. In terms of a targeted tumor therapy, it seems more appropriate to inhibit only the Clk isoforms that are over- expressed in the respective tumor type. An analysis of the Oncomine mRNA expression database (oncomine.org) revealed that for dual Clk1/Clk4 inhibitors, the most promising oncologic indications are glioblastoma and lymphoma, in which mRNA overexpression compared to healthy tissue was detected for both isoforms.

Supplementary Materials: The following are available online, Figure S1: Molecular docking of compound 5a in the ATP binding pocket of Clk1, Figure S2: Molecular docking of compounds 3a (S) and 6a in the ATP binding pocket of Clk1, Figure S3: Molecular docking of compound 9b in the ATP binding pocket of Clk1. Molecules 2021, 26, 1001 20 of 21

Author Contributions: Conceptualization, M.E. and M.A.-H.; methodology, A.K.E., P.-J.C. and D.S.E.-G.; software, A.K.E.; validation, A.K.E., P.-J.C. and D.S.E.-G.; formal analysis, A.K.E., P.-J.C. and D.S.E.-G.; investigation, A.K.E., P.-J.C. and D.S.E.-G.; resources, M.E., T.-L.H. and M.A.-H.; data curation, A.K.E., P.-J.C. and D.S.E.-G.; writing—original draft preparation, A.K.E., M.E. and M.A.-H.; writing—review and editing, all authors.; visualization, all authors.; supervision, M.E., T.-L.H. and A.H.A.; project administration, M.E.; funding acquisition, M.E., T.-L.H. and M.A.-H. All authors have read and agreed to the published version of the manuscript. Funding: The work in this paper was partially supported by the DAAD with funds of the Federal Ministry of Education and Research, Germany, in the frame of the project “German University in Cairo (GUC): Towards excellence and international visibility in research and teaching”. Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: Data is contained within the article and in the Supplementary Materials. Conflicts of Interest: The authors declare no conflict of interest. Sample Availability: Samples of the compounds are available from the authors.

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