Structure–Activity Relationship (SAR) Study of Spautin-1 to Entail the Discovery of Novel NEK4 Inhibitors

Lung cancer is one of the most frequently diagnosed cancers accounting for the highest number of cancer-related deaths in the world. Despite significant progress including targeted therapies and immunotherapy, the treatment of advanced lung cancer remains challenging. Targeted therapies are highly efficacious at prolonging life, but not curative. In prior work we have identified Ubiquitin Specific Protease 13 (USP13) as a potential target to significantly enhance the efficacy of mutant EGFR inhibition. The current study aimed to develop lead molecules for the treatment of epidermal growth factor receptor (EGFR)-mutant non-small cell lung cancer (NSCLC) by developing potent USP13 inhibitors initially starting from Spautin-1, the only available USP13 inhibitor. A SAR study was performed which revealed that increasing the chain length between the secondary amine and phenyl group and introducing a halogen capable of inducing a halogen bond at position 4’ of the phenyl group, dramatically increased the activity. However, we could not confirm the binding between Spautin-1 (or its analogues) and USP13 using isothermal titration calorimetry (ITC) or thermal shift assay (TSA) but do not exclude binding under physiological conditions. Nevertheless, we found that the anti-proliferative activity displayed by Spautin-1 towards EGFR-mutant NSCLC cells in vitro was at least partially associated with kinase inhibition. In this work, we present N-[2-(substituted-phenyl)ethyl]-6-fluoro-4-quinazolinamines as promising lead compounds for the treatment of NSCLC. These analogues are significantly more effective towards EGFR-mutant NSCLC cells than Spautin-1 and act as potent never in mitosis A related kinase 4 (NEK4) inhibitors (IC50~1 µM) with moderate selectivity over other kinases.


Introduction
The World Health Organization estimated that 1.76 million people died of lung cancer in 2018, which represents almost 20% of all cancer-related deaths [1]. The predominant subtype of lung cancer is NSCLC, which accounts for 80-85% of all lung cancer patients [2]. In NSCLC patients with little or no smoking history, mutations in the EGFR, a major regulator of cell proliferation and apoptosis, are frequently observed [3]. The exon 19 in frame deletion (EGFR del E746-A750) and the exon 21 point mutation (L858R) represent together 90% of all activating mutations [4]. Despite the promising initial response of EGFR-mutant NSCLC patients towards first (reversible), second (irreversible), and third The importance of posttranslational modifications in tumorigenesis is widely accepted [6][7][8]. Insight in these processes can lead to new therapeutic strategies to improve current treatments [8]. Ubiquitination is, besides phosphorylation, an important posttranslational modification linked to cancer. It consists of the reversible attachment of one or multiple ubiquitin moieties to target proteins, altering their stability, activity, interactors and/or localization [9,10]. It is a three-step process catalyzed by the consecutive action of the E1 activating enzyme, the E2 conjugating enzyme and the E3 ligase [9,11]. Conversely, deubiquitinating enzymes (DUBs) reverse this process by selectively cleaving ubiquitin or poly-ubiquitin chains from ubiquitinated proteins [12].
Despite the differential role of USP13 in tumorigenesis, this enzyme is considered as a potential therapeutic target. Recently, we have reported that inhibition of USP13 destabilizes EGFR and that co-inhibition of USP13 and EGFR suppresses oncogenic signaling in NSCLC cells [25]. These findings motivated us to initiate the development of more potent USP13 inhibitors, starting from the only USP13 inhibitor described in the literature to date (Spautin-1, 5aa, Figure 2) [26], by exploring the chemical space around Spautin-1 and determining the importance of the quinazoline core by performing a "N-screening". The The importance of posttranslational modifications in tumorigenesis is widely accepted [6][7][8]. Insight in these processes can lead to new therapeutic strategies to improve current treatments [8]. Ubiquitination is, besides phosphorylation, an important posttranslational modification linked to cancer. It consists of the reversible attachment of one or multiple ubiquitin moieties to target proteins, altering their stability, activity, interactors and/or localization [9,10]. It is a three-step process catalyzed by the consecutive action of the E1 activating enzyme, the E2 conjugating enzyme and the E3 ligase [9,11]. Conversely, deubiquitinating enzymes (DUBs) reverse this process by selectively cleaving ubiquitin or poly-ubiquitin chains from ubiquitinated proteins [12].
Despite the differential role of USP13 in tumorigenesis, this enzyme is considered as a potential therapeutic target. Recently, we have reported that inhibition of USP13 destabilizes EGFR and that co-inhibition of USP13 and EGFR suppresses oncogenic signaling in NSCLC cells [25]. These findings motivated us to initiate the development of more potent USP13 inhibitors, starting from the only USP13 inhibitor described in the literature to date (Spautin-1, 5aa, Figure 2) [26], by exploring the chemical space around Spautin-1 and determining the importance of the quinazoline core by performing a "N-screening". The quino-quinoline [27][28][29][30], isoquinoline [31,32], cinnoline [33][34][35], and quinoxaline [36] cores wer considered, all privileged scaffolds which have shown their effectiveness as kinase inhib itors. Herein, we elaborate upon N- [2-(substituted-phenyl)ethyl]-6-fluoro-4-quinazo linamines as promising lead compounds for the treatment of NSCLC. Based on an EGFR mutant NSCLC cell viability screening, these analogues were found to be significantl more potent when compared to Spautin-1. We attempted to demonstrate binding of th spautin-1 analogues to USP13 using isothermal titration calorimetry (ITC) or thermal shi assay (TSA) but were unsuccessful. Nevertheless, the N- [2-(substituted-phenyl)ethyl]-6 fluoro-4-quinazolinamines showed good activity and moderate selectivity towards NEK4 a kinase previously linked to NSCLC. Park and co-workers reported, for instance, th overexpression of NEK4 in lung cancer and suggested that NEK 4 suppresses Tumor ne crosis factor-Related Apoptosis Inducing Ligand (TRAIL)-induced apoptosis, which i turn results into tumor resistance. In contrast, downregulation of NEK4 sensitizes tumo cells to TRIAL-induced apoptosis via decreased levels of survivin, an anti-apoptotic pro tein [37]. In 2018, along the same lines, Ding and co-workers published that NEK4 is positive regulator of epithelial-to-mesenchymal transition (EMT) which plays an im portant role in lung cancer metastasis [38]. To our best knowledge, there are no potent an selective NEK4 inhibitors described in the literature. Some previously reported kinase in hibitors showed good off-target activity for NEK4 but lacked selectivity. Some example are: BAY 61-3606 (6, SYK inhibitor), Abbott compound 17 (7,MAP3K8 inhibitor), and Ab bott 1141 (8, multikinase inhibitor) (structures depicted in Figure 3) [39][40][41][42]. In light of the above, we hypothesize that the activity of our analogues toward Herein, we elaborate upon N-[2-(substituted-phenyl)ethyl]-6-fluoro-4-quinazolinamines as promising lead compounds for the treatment of NSCLC. Based on an EGFRmutant NSCLC cell viability screening, these analogues were found to be significantly more potent when compared to Spautin-1. We attempted to demonstrate binding of the spautin-1 analogues to USP13 using isothermal titration calorimetry (ITC) or thermal shift assay (TSA) but were unsuccessful. Nevertheless, the N-[2-(substituted-phenyl)ethyl]-6fluoro-4-quinazolinamines showed good activity and moderate selectivity towards NEK4, a kinase previously linked to NSCLC. Park and co-workers reported, for instance, the overexpression of NEK4 in lung cancer and suggested that NEK 4 suppresses Tumor necrosis factor-Related Apoptosis Inducing Ligand (TRAIL)-induced apoptosis, which in turn results into tumor resistance. In contrast, downregulation of NEK4 sensitizes tumor cells to TRIAL-induced apoptosis via decreased levels of survivin, an anti-apoptotic protein [37]. In 2018, along the same lines, Ding and co-workers published that NEK4 is a positive regulator of epithelial-to-mesenchymal transition (EMT) which plays an important role in lung cancer metastasis [38]. To our best knowledge, there are no potent and selective NEK4 inhibitors described in the literature. Some previously reported kinase inhibitors showed good off-target activity for NEK4 but lacked selectivity. Some examples are: BAY 61-3606 (6, SYK inhibitor), Abbott compound 17 (7, MAP3K8 inhibitor), and Abbott 1141 (8, multikinase inhibitor) (structures depicted in Figure 3) [39][40][41][42]. quinoline [27][28][29][30], isoquinoline [31,32], cinnoline [33][34][35], and quinoxaline [36] cores were considered, all privileged scaffolds which have shown their effectiveness as kinase inhibitors.
Herein, we elaborate upon N- [2-(substituted-phenyl)ethyl]-6-fluoro-4-quinazolinamines as promising lead compounds for the treatment of NSCLC. Based on an EGFRmutant NSCLC cell viability screening, these analogues were found to be significantly more potent when compared to Spautin-1. We attempted to demonstrate binding of the spautin-1 analogues to USP13 using isothermal titration calorimetry (ITC) or thermal shift assay (TSA) but were unsuccessful. Nevertheless, the N- [2-(substituted-phenyl)ethyl]-6fluoro-4-quinazolinamines showed good activity and moderate selectivity towards NEK4, a kinase previously linked to NSCLC. Park and co-workers reported, for instance, the overexpression of NEK4 in lung cancer and suggested that NEK 4 suppresses Tumor necrosis factor-Related Apoptosis Inducing Ligand (TRAIL)-induced apoptosis, which in turn results into tumor resistance. In contrast, downregulation of NEK4 sensitizes tumor cells to TRIAL-induced apoptosis via decreased levels of survivin, an anti-apoptotic protein [37]. In 2018, along the same lines, Ding and co-workers published that NEK4 is a positive regulator of epithelial-to-mesenchymal transition (EMT) which plays an important role in lung cancer metastasis [38]. To our best knowledge, there are no potent and selective NEK4 inhibitors described in the literature. Some previously reported kinase inhibitors showed good off-target activity for NEK4 but lacked selectivity. Some examples are: BAY 61-3606 (6, SYK inhibitor), Abbott compound 17 (7, MAP3K8 inhibitor), and Abbott 1141 (8, multikinase inhibitor) (structures depicted in Figure 3) [39][40][41][42]. In light of the above, we hypothesize that the activity of our analogues towards EGFR-mutant NSCLC cells is at least in part related to inhibition of NEK4. As far as we In light of the above, we hypothesize that the activity of our analogues towards EGFRmutant NSCLC cells is at least in part related to inhibition of NEK4. As far as we know, Scheme 1. Synthesis of Spautin-1 analogues 5 starting from substituted 2-aminobenzamides 9 or 2-aminobenzoic acids 10.
Further exploratory diversification was introduced using palladium-catalyzed crosscoupling reactions. In literature described strategies were applied to introduce a phenyl group as in 13, using the Suzuki reaction, and an alkyne substituent to reach compounds of type 14, via the Sonogashira reaction (Scheme 2) [47,48]. These larger substituents provided insight in the available chemical space around the quinazoline core. These conversions made use of the I>>F selectivity during Pd-catalyzed transformations. Scheme 2. Palladium-catalyzed cross coupling reactions: Suzuki and Sonogashira reactions.
As a ring-contracted analogue, the synthesis of a Spautin-1 analogue bearing the benzimidazole core 17, was pursued (Scheme 3). The benzimidazole system is a well-established scaffold in medicinal chemistry, but in the current study, it can be regarded as a Scheme 1. Synthesis of Spautin-1 analogues 5 starting from substituted 2-aminobenzamides 9 or 2-aminobenzoic acids 10.
Further exploratory diversification was introduced using palladium-catalyzed crosscoupling reactions. In literature described strategies were applied to introduce a phenyl group as in 13, using the Suzuki reaction, and an alkyne substituent to reach compounds of type 14, via the Sonogashira reaction (Scheme 2) [47,48]. These larger substituents provided insight in the available chemical space around the quinazoline core. These conversions made use of the I>>F selectivity during Pd-catalyzed transformations. know, these are the first inhibitors described in the literature targeting NEK4, which also show promising single agent anticancer activity towards EGFR-mutant NSCLC cells.
Further exploratory diversification was introduced using palladium-catalyzed crosscoupling reactions. In literature described strategies were applied to introduce a phenyl group as in 13, using the Suzuki reaction, and an alkyne substituent to reach compounds of type 14, via the Sonogashira reaction (Scheme 2) [47,48]. These larger substituents provided insight in the available chemical space around the quinazoline core. These conversions made use of the I>>F selectivity during Pd-catalyzed transformations. Scheme 2. Palladium-catalyzed cross coupling reactions: Suzuki and Sonogashira reactions.
As a ring-contracted analogue, the synthesis of a Spautin-1 analogue bearing the benzimidazole core 17, was pursued (Scheme 3). The benzimidazole system is a well-established scaffold in medicinal chemistry, but in the current study, it can be regarded as a Scheme 2. Palladium-catalyzed cross coupling reactions: Suzuki and Sonogashira reactions.
As a ring-contracted analogue, the synthesis of a Spautin-1 analogue bearing the benzimidazole core 17, was pursued (Scheme 3). The benzimidazole system is a well-established scaffold in medicinal chemistry, but in the current study, it can be regarded as a quinazoline analogue in which one of both rings is compacted. In consequence, the substituents will be oriented differently as compared to the topological orientation in its homologue. The substituted benzimidazole was synthesized starting from 5-fluoro-2-nitroaniline 15. In our hands, the highest isolated yield for the nucleophilic substitution reaction towards 16 was obtained in DMF using Cs 2 CO 3 as a base. Finally, the benzimidazole core was obtained after reduction of the nitro-group using an excess of iron in acetic acid, followed by the cyclisation using trimethyl orthoformate and a catalytic amount of p-toluene sulfonic acid yielding 17 [49,50]. quinazoline analogue in which one of both rings is compacted. In consequence, the substituents will be oriented differently as compared to the topological orientation in its homologue. The substituted benzimidazole was synthesized starting from 5-fluoro-2-nitroaniline 15. In our hands, the highest isolated yield for the nucleophilic substitution reaction towards 16 was obtained in DMF using Cs2CO3 as a base. Finally, the benzimidazole core was obtained after reduction of the nitro-group using an excess of iron in acetic acid, followed by the cyclisation using trimethyl orthoformate and a catalytic amount of p-toluene sulfonic acid yielding 17 [49,50].
Scheme 3. Synthesis of a ring contracted spautin-1 analogue bearing the benzimidazole core.
Thereafter, a "N-screening" was performed to determine how important the quinazoline scaffold is for the biological activity. First, the quinazoline core was replaced by the quinoline and isoquinoline core to determine the importance of each individual nitrogen. In one step, compound 19 was obtained from the commercially available 4-chloro-6-fluoroquinoline 18 through a microwave-assisted nucleophilic aromatic substitution (Scheme 4). The previously described conditions of Felts et al. were not efficient in this case, because of which we opted for a solvolytic reaction [51]. The isoquinoline core was obtained following the procedure reported by Zhao and co-workers (Scheme 5) [52]. Starting from 5-fluoro-2-methylbenzamide 20, 7-fluoro-isoquinolinone 22 was obtained in two steps. First 20 was allowed to react with N,N-dimethylformamide dimethyl acetal yielding intermediate 21, which was subsequently cyclized under basic conditions. Next, 1-chloro-7-fluoroisoquinoline 23 was synthesized using phosphorus oxychloride, which was subsequently converted to the Spautin-1 analogue 24 via a nucleophilic aromatic substitution.  Thereafter, a "N-screening" was performed to determine how important the quinazoline scaffold is for the biological activity. First, the quinazoline core was replaced by the quinoline and isoquinoline core to determine the importance of each individual nitrogen. In one step, compound 19 was obtained from the commercially available 4-chloro-6-fluoroquinoline 18 through a microwave-assisted nucleophilic aromatic substitution (Scheme 4). The previously described conditions of Felts et al. were not efficient in this case, because of which we opted for a solvolytic reaction [51]. quinazoline analogue in which one of both rings is compacted. In consequence, the substituents will be oriented differently as compared to the topological orientation in its homologue. The substituted benzimidazole was synthesized starting from 5-fluoro-2-nitroaniline 15. In our hands, the highest isolated yield for the nucleophilic substitution reaction towards 16 was obtained in DMF using Cs2CO3 as a base. Finally, the benzimidazole core was obtained after reduction of the nitro-group using an excess of iron in acetic acid, followed by the cyclisation using trimethyl orthoformate and a catalytic amount of p-toluene sulfonic acid yielding 17 [49,50].
Scheme 3. Synthesis of a ring contracted spautin-1 analogue bearing the benzimidazole core.
Thereafter, a "N-screening" was performed to determine how important the quinazoline scaffold is for the biological activity. First, the quinazoline core was replaced by the quinoline and isoquinoline core to determine the importance of each individual nitrogen. In one step, compound 19 was obtained from the commercially available 4-chloro-6-fluoroquinoline 18 through a microwave-assisted nucleophilic aromatic substitution (Scheme 4). The previously described conditions of Felts et al. were not efficient in this case, because of which we opted for a solvolytic reaction [51]. The isoquinoline core was obtained following the procedure reported by Zhao and co-workers (Scheme 5) [52]. Starting from 5-fluoro-2-methylbenzamide 20, 7-fluoro-isoquinolinone 22 was obtained in two steps. First 20 was allowed to react with N,N-dimethylformamide dimethyl acetal yielding intermediate 21, which was subsequently cyclized under basic conditions. Next, 1-chloro-7-fluoroisoquinoline 23 was synthesized using phosphorus oxychloride, which was subsequently converted to the Spautin-1 analogue 24 via a nucleophilic aromatic substitution.  The isoquinoline core was obtained following the procedure reported by Zhao and coworkers (Scheme 5) [52]. Starting from 5-fluoro-2-methylbenzamide 20, 7-fluoro-isoquinolinone 22 was obtained in two steps. First 20 was allowed to react with N,N-dimethylformamide dimethyl acetal yielding intermediate 21, which was subsequently cyclized under basic conditions. Next, 1-chloro-7-fluoroisoquinoline 23 was synthesized using phosphorus oxychloride, which was subsequently converted to the Spautin-1 analogue 24 via a nucleophilic aromatic substitution. quinazoline analogue in which one of both rings is compacted. In consequence, the substituents will be oriented differently as compared to the topological orientation in its homologue. The substituted benzimidazole was synthesized starting from 5-fluoro-2-nitroaniline 15. In our hands, the highest isolated yield for the nucleophilic substitution reaction towards 16 was obtained in DMF using Cs2CO3 as a base. Finally, the benzimidazole core was obtained after reduction of the nitro-group using an excess of iron in acetic acid, followed by the cyclisation using trimethyl orthoformate and a catalytic amount of p-toluene sulfonic acid yielding 17 [49,50].
Scheme 3. Synthesis of a ring contracted spautin-1 analogue bearing the benzimidazole core.
Thereafter, a "N-screening" was performed to determine how important the quinazoline scaffold is for the biological activity. First, the quinazoline core was replaced by the quinoline and isoquinoline core to determine the importance of each individual nitrogen. In one step, compound 19 was obtained from the commercially available 4-chloro-6-fluoroquinoline 18 through a microwave-assisted nucleophilic aromatic substitution (Scheme 4). The previously described conditions of Felts et al. were not efficient in this case, because of which we opted for a solvolytic reaction [51]. The isoquinoline core was obtained following the procedure reported by Zhao and co-workers (Scheme 5) [52]. Starting from 5-fluoro-2-methylbenzamide 20, 7-fluoro-isoquinolinone 22 was obtained in two steps. First 20 was allowed to react with N,N-dimethylformamide dimethyl acetal yielding intermediate 21, which was subsequently cyclized under basic conditions. Next, 1-chloro-7-fluoroisoquinoline 23 was synthesized using phosphorus oxychloride, which was subsequently converted to the Spautin-1 analogue 24 via a nucleophilic aromatic substitution.
Alternatively, the two nitrogens in the central scaffold were preserved but positioned differently. The cinnoline core was accessed via a Sonogashira coupling, followed by a cyclisation through in situ formation of nitrous acid (Scheme 6) [53]. The obtained 6-fluorocinnoline-4-ol 27 was then converted to 4-chloro-6-fluorocinnoline 28 using thionyl chloride in the presence of a catalytic amount of DMF. Finally, a nucleophilic aromatic substitution was performed yielding the desired product 29 in moderate yield. Scheme 6. Synthesis of a Spautin-1 analogue bearing the cinnoline core.
An alternative to the cinnoline core is the quinoxaline scaffold. 7-Fluoro-3,4-dihydro-1H-quinoxalin-2-one 32 was obtained from 4-fluoro-2-nitroaniline 30, which was allowed to react with ethyl bromoacetate, yielding 31 (Scheme 7). Afterwards the nitro group was reduced, which immediately resulted in the formation of the cyclized product. After stirring the mixture for another 48 h in the absence of hydrogen gas, the desired core structure 32 was obtained. Similarly to abovementioned schemes, an analogous two-step strategy was used to couple the 4-fluorobenzylamine yielding 34.

NSCLC Cell Viability Screening
First, the synthesized Spautin-1 analogues were subjected to an EGFR-mutant NSCLC cell viability screening to determine which compounds were the most promising in terms of reducing the viability of EGFR-mutant NSCLC cells in vitro. A reduction of Scheme 7. Synthesis of a Spautin-1 analogue bearing the quinoxaline core.

NSCLC Cell Viability Screening
First, the synthesized Spautin-1 analogues were subjected to an EGFR-mutant NSCLC cell viability screening to determine which compounds were the most promising in terms of reducing the viability of EGFR-mutant NSCLC cells in vitro. A reduction of cell viability in the presence of the analogues indicates induction of apoptosis and/or an inhibition of proliferation. The viability assay also helped to get insights in the structure-activity relationships (SARs) of the prepared analogues. As shown below, the analogues were subdivided in nine series in order to efficiently discover SAR trends.
At first, a "F-screening" was performed to determine the importance of the fluorine substituent positioning ( Figure 4). Remarkably, two compounds showed a significant decrease in tumor cell numbers compared to Spautin-1 (5aa), namely the analogue with a fluorine at the ortho position of the benzyl group 5ae and the analogue with a fluorine at position 8 of the quinazoline core 5ah. The other analogues were equally 5ag or less potent 5ad and 5af as compared to Spautin-1. cell viability in the presence of the analogues indicates induction of apoptosis and/or an inhibition of proliferation. The viability assay also helped to get insights in the structureactivity relationships (SARs) of the prepared analogues. As shown below, the analogues were subdivided in nine series in order to efficiently discover SAR trends. At first, a "F-screening" was performed to determine the importance of the fluorine substituent positioning ( Figure 4). Remarkably, two compounds showed a significant decrease in tumor cell numbers compared to Spautin-1 (5aa), namely the analogue with a fluorine at the ortho position of the benzyl group 5ae and the analogue with a fluorine at position 8 of the quinazoline core 5ah. The other analogues were equally 5ag or less potent 5ad and 5af as compared to Spautin-1. In the second series, multiple substituents, which differ in lipophilicity and electronic properties, were introduced at position 6 of the quinazoline core ( Figure 5). It was observed that electron-donating substituents 5aj, 5ak had a positive effect on the activity, compared to Spautin-1 5aa, while the effect of electron withdrawing groups 5ai, 5al, 5am was less consistent. A large hydrophobic phenyl group 13b slightly improved the activity and confirmed that large substituents are probably allowed at this site of the central scaffold. Interestingly, a 4-hydroxybutynyl substituent at this position 14b, previously described in EGFR TKIs [48], led to the strongest growth inhibition of the EGFR-mutant NSCLC cells. In the second series, multiple substituents, which differ in lipophilicity and electronic properties, were introduced at position 6 of the quinazoline core ( Figure 5). It was observed that electron-donating substituents 5aj, 5ak had a positive effect on the activity, compared to Spautin-1 5aa, while the effect of electron withdrawing groups 5ai, 5al, 5am was less consistent. A large hydrophobic phenyl group 13b slightly improved the activity and confirmed that large substituents are probably allowed at this site of the central scaffold. Interestingly, a 4-hydroxybutynyl substituent at this position 14b, previously described in EGFR TKIs [48], led to the strongest growth inhibition of the EGFR-mutant NSCLC cells.  The fluorine at the para position of the benzyl group was replaced by a variety of groups (Cl, OMe, CF3, H) ( Figure 6). Notably, a methoxy 5ao, trifluoromethyl 5ap group and H atom 5aq significantly improved the activity while the chlorinated analogue 5an was equally potent to Spautin-1 (5aa). Nevertheless, the electron donating methoxy group resulted in the strongest reduction in the viability of the lung cancer cells in vitro. To further improve the activity, the aliphatic chain length was shortened or extended, by altering the number of methylene groups (-CH2-) between the secondary amine and the phenyl group ( Figure 7). Remarkably, both a reduction 5ar and extension 5as-5au resulted in slightly and significantly more potent analogues, respectively. The extended chain length clearly resulted in an improved activity, while the increased activity of the reduced chain length can be potentially linked to inhibition of EGFR (cfr. examples in Figure 1) [54,55]. We hypothesize that the additional methylene groups increase the flexibility and in this way improve the activity. The fluorine at the para position of the benzyl group was replaced by a variety of groups (Cl, OMe, CF 3 , H) ( Figure 6). Notably, a methoxy 5ao, trifluoromethyl 5ap group and H atom 5aq significantly improved the activity while the chlorinated analogue 5an was equally potent to Spautin-1 (5aa). Nevertheless, the electron donating methoxy group resulted in the strongest reduction in the viability of the lung cancer cells in vitro.  The fluorine at the para position of the benzyl group was replaced by a variety of groups (Cl, OMe, CF3, H) ( Figure 6). Notably, a methoxy 5ao, trifluoromethyl 5ap group and H atom 5aq significantly improved the activity while the chlorinated analogue 5an was equally potent to Spautin-1 (5aa). Nevertheless, the electron donating methoxy group resulted in the strongest reduction in the viability of the lung cancer cells in vitro. To further improve the activity, the aliphatic chain length was shortened or extended, by altering the number of methylene groups (-CH2-) between the secondary amine and the phenyl group ( Figure 7). Remarkably, both a reduction 5ar and extension 5as-5au resulted in slightly and significantly more potent analogues, respectively. The extended chain length clearly resulted in an improved activity, while the increased activity of the reduced chain length can be potentially linked to inhibition of EGFR (cfr. examples in Figure 1) [54,55]. We hypothesize that the additional methylene groups increase the flexibility and in this way improve the activity. To further improve the activity, the aliphatic chain length was shortened or extended, by altering the number of methylene groups (-CH 2 -) between the secondary amine and the phenyl group (Figure 7). Remarkably, both a reduction 5ar and extension 5as-5au resulted in slightly and significantly more potent analogues, respectively. The extended chain length clearly resulted in an improved activity, while the increased activity of the reduced chain length can be potentially linked to inhibition of EGFR (cfr. examples in Figure 1) [54,55]. We hypothesize that the additional methylene groups increase the flexibility and in this way improve the activity.

Compound R1 R2
Cl 3 (a) Despite the fact that analogues with n = 0 might act as EGFR TKIs, a small series of analogues was tested ( Figure 8). Remarkably, a fluorine at the 3' or 2' position (5av, 5aw) of the phenyl group significantly improved the activity as compared to the fluorine at the 4' position 5ar. Moreover, for n = 1, a fluorine substituent at the 2' position also gave the best result (Figure 4, 5ae). However, in the n = 1 series, a fluorine at the 3' position resulted in a less active compound, as compared to Spautin-1, which indicates that a fluorine in proximity of the quinazoline core is beneficial. An electron-donating group at position R1, on the other hand, providing 5ay, resulted in a weaker reduction in tumor cell viability as compared to 5av. Remarkably, a phenyl group at the 3' position of the phenyl group 13a was allowed while the 1-hydroxy-3-butynyl group 14a completely abolished the activity. Despite the fact that analogues with n = 0 might act as EGFR TKIs, a small series of analogues was tested ( Figure 8). Remarkably, a fluorine at the 3' or 2' position (5av, 5aw) of the phenyl group significantly improved the activity as compared to the fluorine at the 4' position 5ar. Moreover, for n = 1, a fluorine substituent at the 2' position also gave the best result (Figure 4, 5ae). However, in the n = 1 series, a fluorine at the 3' position resulted in a less active compound, as compared to Spautin-1, which indicates that a fluorine in proximity of the quinazoline core is beneficial. An electron-donating group at position R 1 , on the other hand, providing 5ay, resulted in a weaker reduction in tumor cell viability as compared to 5av. Remarkably, a phenyl group at the 3' position of the phenyl group 13a was allowed while the 1-hydroxy-3-butynyl group 14a completely abolished the activity.
Cl 3 (a) Despite the fact that analogues with n = 0 might act as EGFR TKIs, a small series of analogues was tested ( Figure 8). Remarkably, a fluorine at the 3' or 2' position (5av, 5aw) of the phenyl group significantly improved the activity as compared to the fluorine at the 4' position 5ar. Moreover, for n = 1, a fluorine substituent at the 2' position also gave the best result (Figure 4, 5ae). However, in the n = 1 series, a fluorine at the 3' position resulted in a less active compound, as compared to Spautin-1, which indicates that a fluorine in proximity of the quinazoline core is beneficial. An electron-donating group at position R1, on the other hand, providing 5ay, resulted in a weaker reduction in tumor cell viability as compared to 5av. Remarkably, a phenyl group at the 3' position of the phenyl group 13a was allowed while the 1-hydroxy-3-butynyl group 14a completely abolished the activity. Nonetheless, the most promising compounds have an extended chain between the secondary amine and the phenyl group. Highly comparable results were obtained for all analogues, except for analogue 5bd, which was significantly less potent at a test concentration of 5 µM (Figure 9). Nonetheless, the most promising compounds have an extended chain between the secondary amine and the phenyl group. Highly comparable results were obtained for all analogues, except for analogue 5bd, which was significantly less potent at a test concentration of 5 µM (Figure 9).
Cl(4') 5az Cl(3') 5ba Cl(2') 5bb Cl(3',4') 5bc OMe(4') 5bd H(4') 5be Me(4') 5bf Br(4') (a) To distinguish these analogues more precisely, IC50 values were determined to unravel differences in activity (see Figure S1). These data confirmed that the phenethylamine bearing analogues were definitely more potent than Spautin-1 5aa ( Figure 10). Additionally, a chlorine 5at at the para position of the phenyl group of the phenethylamine seemed beneficial for the potency compared to the fluorinated counterpart 5as but further extension of the chain length, as in 5au, lowered the potency by a factor of seven. Subsequently, the influence of the substitution pattern of the phenyl group on the activity was evaluated which revealed that the para substituted analogue 5at is about 1.3 times more potent as compared to the meta 5az and disubstituted 5bb, and about 1.1 times more potent than the ortho substituted analogue 5ba. Replacing the chlorine 5at by a methoxy group 5bc resulted in a slightly more active analogue. Remarkably, a methyl group at the para position was disadvantageous 5be, while the brominated analogue 5bf resulted in a further increase in activity as compared to 5at and 5bc. The observed trend in the IC50 values (F > Cl > Br) makes us belief that the formation of a halogen bond in the active site plays a significant role in the activity [56,57].

DMSO
Spautin-1 5as 5at 5az 5ba 5bb 5bc 5bd 5be 5bf To distinguish these analogues more precisely, IC 50 values were determined to unravel differences in activity (see Figure S1). These data confirmed that the phenethylamine bearing analogues were definitely more potent than Spautin-1 5aa ( Figure 10). Additionally, a chlorine 5at at the para position of the phenyl group of the phenethylamine seemed beneficial for the potency compared to the fluorinated counterpart 5as but further extension of the chain length, as in 5au, lowered the potency by a factor of seven. Subsequently, the influence of the substitution pattern of the phenyl group on the activity was evaluated which revealed that the para substituted analogue 5at is about 1.3 times more potent as compared to the meta 5az and disubstituted 5bb, and about 1.1 times more potent than the ortho substituted analogue 5ba. Replacing the chlorine 5at by a methoxy group 5bc resulted in a slightly more active analogue. Remarkably, a methyl group at the para position was disadvantageous 5be, while the brominated analogue 5bf resulted in a further increase in activity as compared to 5at and 5bc. The observed trend in the IC 50 values (F > Cl > Br) makes us belief that the formation of a halogen bond in the active site plays a significant role in the activity [56,57].
Finally, a N-screening was performed to determine the importance of the quinazoline core ( Figure 11). Interestingly, the nitrogen atom at the 3-position does not seem to be important for the activity, as the Spautin-1 analogue bearing the quinoline core (i.e., 19) showed a higher potency as compared to the quinazoline core 5aa. This is in contrast to the nitrogen at the 1-position, which appears to be crucial for the activity since its absence resulted in the inactive analogue 24 while the cinnoline 29 and quinoxaline 34 bearing Spautin-1 analogues showed some activity towards EGFR mutant NSCLC cells; however, they were less active compared to Spautin-1. Finally, a N-screening was performed to determine the importance of the quinazoline core ( Figure 11). Interestingly, the nitrogen atom at the 3-position does not seem to be important for the activity, as the Spautin-1 analogue bearing the quinoline core (i.e., 19) showed a higher potency as compared to the quinazoline core 5aa. This is in contrast to the nitrogen at the 1-position, which appears to be crucial for the activity since its absence resulted in the inactive analogue 24 while the cinnoline 29 and quinoxaline 34 bearing Spautin-1 analogues showed some activity towards EGFR mutant NSCLC cells; however, they were less active compared to Spautin-1. Remarkably, the Spautin-1 analogue bearing the benzimidazole core 17 turned out to be less active in the viability screening ( Figure 12). We hypothesized that the lack of the secondary amine was responsible for the dramatically decreased activity.  Finally, a N-screening was performed to determine the importance of the quinazoline core ( Figure 11). Interestingly, the nitrogen atom at the 3-position does not seem to be important for the activity, as the Spautin-1 analogue bearing the quinoline core (i.e., 19) showed a higher potency as compared to the quinazoline core 5aa. This is in contrast to the nitrogen at the 1-position, which appears to be crucial for the activity since its absence resulted in the inactive analogue 24 while the cinnoline 29 and quinoxaline 34 bearing Spautin-1 analogues showed some activity towards EGFR mutant NSCLC cells; however, they were less active compared to Spautin-1. Remarkably, the Spautin-1 analogue bearing the benzimidazole core 17 turned out to be less active in the viability screening ( Figure 12). We hypothesized that the lack of the secondary amine was responsible for the dramatically decreased activity. Remarkably, the Spautin-1 analogue bearing the benzimidazole core 17 turned out to be less active in the viability screening ( Figure 12). We hypothesized that the lack of the secondary amine was responsible for the dramatically decreased activity.
As a control reaction, the secondary amine present in most of the Spautin-1 analogues described above, was alkylated ( Figure 13), since it has been reported that this secondary amine is crucial for the activity of EGRF-TKIs, [55] but also kinase inhibitors in general [58][59][60]. Methylation of the secondary amine dramatically reduced the activity of the analogues (5aa vs. 5bg and 5at vs. 5bh) and as such, indicated that this secondary amine was also crucial for the activity in this study. As a control reaction, the secondary amine present in most of the Spautin-1 analogues described above, was alkylated ( Figure 13), since it has been reported that this secondary amine is crucial for the activity of EGRF-TKIs, [55] but also kinase inhibitors in general [58][59][60]. Methylation of the secondary amine dramatically reduced the activity of the analogues (5aa vs. 5bg and 5at vs. 5bh) and as such, indicated that this secondary amine was also crucial for the activity in this study. Next, the binding between the spautin-1 analogues and USP13 was evaluated to confirm that the reduced viability of the EGFR mutant NSCLC cells was caused by inhibition of USP13.

ITC/TSA
In order to confirm the binding event between spautin-1 and USP13, an isothermal titration calorimetry (ITC) experiment was performed on recombinant USP13. No binding curve could be fitted on the obtained data (see Figure S2), thus no binding was detected. The addition of DMSO further complicated the experiment, resulting in buffer mismatches and aggregation of the protein. Since the purification yield of protein was low, it  As a control reaction, the secondary amine present in most of the Spautin-1 analogues described above, was alkylated ( Figure 13), since it has been reported that this secondary amine is crucial for the activity of EGRF-TKIs, [55] but also kinase inhibitors in general [58][59][60]. Methylation of the secondary amine dramatically reduced the activity of the analogues (5aa vs. 5bg and 5at vs. 5bh) and as such, indicated that this secondary amine was also crucial for the activity in this study. Figure 13. (a) Alkylation of the secondary amine; (b) viability assay (5µM) with Spautin-1 as reference.
Next, the binding between the spautin-1 analogues and USP13 was evaluated to confirm that the reduced viability of the EGFR mutant NSCLC cells was caused by inhibition of USP13.

ITC/TSA
In order to confirm the binding event between spautin-1 and USP13, an isothermal titration calorimetry (ITC) experiment was performed on recombinant USP13. No binding curve could be fitted on the obtained data (see Figure S2), thus no binding was detected. The addition of DMSO further complicated the experiment, resulting in buffer mismatches and aggregation of the protein. Since the purification yield of protein was low, it Next, the binding between the spautin-1 analogues and USP13 was evaluated to confirm that the reduced viability of the EGFR mutant NSCLC cells was caused by inhibition of USP13.

ITC/TSA
In order to confirm the binding event between spautin-1 and USP13, an isothermal titration calorimetry (ITC) experiment was performed on recombinant USP13. No binding curve could be fitted on the obtained data (see Figure S2), thus no binding was detected. The addition of DMSO further complicated the experiment, resulting in buffer mismatches and aggregation of the protein. Since the purification yield of protein was low, it was decided to shift to another less consuming method for the analysis of compound binding.
An additional screening was performed by means of a thermal shift assay (TSA). Ligand interactions usually increase protein stability, resulting in a shift of the melting temperature with a few degrees. This assay would thus allow to determine the melting temperature (T m ) and to detect a thermal shift upon binding of the ligand to the USP13 protein. Therefore, the protein was incubated with Spautin-1 5aa or 5bc and a melting curve was determined for each sample (see Figure S3). Fitting a Boltzmann-sigmoidal curve to this data allows to determine the corresponding melting temperature, shown in Table 1. To determine a temperature difference, the reference condition was the protein in buffer with the corresponding percentage of DMSO, since addition of DMSO seemed to increase aggregation during the ITC experiment. However, for both compounds no consistent thermal shift was seen after incubation with the ligands. While binding could not be observed from this dataset, a potential impact on any binding resulting from producing the protein recombinantly in Escherichia coli can also not be excluded. Table 1. Melting temperatures, Tm ( • C), and the thermal shift, ∆Tm ( • C), are given for each sample analyzed in a thermal shift assay. The melting temperatures were derived by fitting a Boltzmannsigmoidal equation to the data. To determine a temperature difference (∆Tm), the reference condition was the protein in buffer with the corresponding percentage of DMSO.

Kinase Screening
Since we could not confirm the interaction between Spautin-1 analogues and USP13, a kinase screening was performed using a set of four compounds (Spautin-1 and three analogues) to discover off-targets and identify kinases that are potentially responsible for the reduction in cell viability observed in our screen ( Table 2). The KINOMEscan from Eurofins was performed (see Table S1). This screen consists of a competitive binding assay in which the DNA-tagged kinase is incubated with one of the compounds in the presence of immobilized active site binding ligands. Binding of the compounds to the kinase hampers the binding of the kinase to the ligands and this inhibition was quantified by qPCR. The residual kinase activity, which represents the percentage of kinase attached to the immobilized ligands, was determined for 428 kinases at a standard screening concentration of 10 µM of the compounds dissolved in DMSO [61].
Remarkably, Spautin-1 5aa showed moderate EGFR TK inhibitory activity at the standard screening concentration (Table 2), however we did not observe a reduction in pEGFR at 10 µM in EGFR mutant NSCLC cells [25]. We reasoned that the concentration used in the kinase screening does not represent the actual intracellular concentration upon treatment with the same concentration on a cellular basis. As expected, reducing the chain length, as in 5ay, augments the EGFR TK inhibitory activity (Table 2), which probably also induced cell death in the viability screening. Conversely, extending the chain length (e.g., 5as and 5at), reduced the EGFR TK inhibitory activity, which implies that the improved activity, observed during the EGFR-mutant NSCLC cell viability screening, was not caused by inhibition of EGFR. Interestingly, the newly synthesized analogues (5ay, 5as and 5at) inhibited NEK4 more efficiently than Spautin-1 (5aa). Moreover, it has been reported that NEK4 is frequently overexpressed in lung cancer [37]. We confirmed the expression of NEK4 in our PC9 cell line model based on the publicly RNA-seq data available through the Cancer Cell Line Encyclopedia (CCLE) [62,63]. The available data represents RNA-seq expression data of a total of 1103 cell lines, including 205 lung cancer cell lines. Based on this data (see Table S2), we conclude that NEK4 is expressed throughout most NSCLC cell lines. Moreover, we found that PC9 expresses 19.3% more mRNA than the A549 NSCLC cell line, in which NEK4 has been functionally characterized and its expression confirmed through western blotting [38]. Collectively, this suggests that NEK4 inhibition possibly causes growth inhibition of EGFR mutant NSCLC cells.     For the above stated reasons, we decided to determine the IC 50 (NEK4) values for Spautin-1 and 5 diverse analogues (NEK4 Human Other Protein Kinase Enzymatic Radiometric Assay by Eurofins) (see Figure S4) [64]. Interestingly, a reduced (5ay) and extended (5at, 5au) chain length between the secondary amine and the phenyl group improved the inhibitory activity for NEK4 (Figure 14), which is in line with the viability data. However, only for n ≥ 2 a dramatic decrease in NSCLC cell viability was observed and the activity of 5au (n = 3) was significantly better as compared to 5ay (n = 0) (Figures 7 and 8) which is contrary to the IC 50 values. Therefore, we postulate that inhibition of NEK4 only, does not fully explain the results of the viability screening. The introduction of a 1-hydroxy-3-butynyl group at position 6 of the quinazoline core 14b resulted in a two-fold improved NEK4 inhibitory activity compared to Spautin-1, which suggests that the reduced cell viability was potentially caused by inhibition of NEK4. Remarkably, the Spautin-1 analogue bearing the quinoline core 19 was clearly less potent for NEK4 which is also in contrast to the viability data. PC9 cell line model based on the publicly RNA-seq data available through the Cancer Cell Line Encyclopedia (CCLE) [62,63]. The available data represents RNA-seq expression data of a total of 1103 cell lines, including 205 lung cancer cell lines. Based on this data (see Table S2), we conclude that NEK4 is expressed throughout most NSCLC cell lines. Moreover, we found that PC9 expresses 19.3% more mRNA than the A549 NSCLC cell line, in which NEK4 has been functionally characterized and its expression confirmed through western blotting [38]. Collectively, this suggests that NEK4 inhibition possibly causes growth inhibition of EGFR mutant NSCLC cells.
For the above stated reasons, we decided to determine the IC50 (NEK4) values for Spautin-1 and 5 diverse analogues (NEK4 Human Other Protein Kinase Enzymatic Radiometric Assay by Eurofins) (see Figure S4) [64]. Interestingly, a reduced (5ay) and extended (5at, 5au) chain length between the secondary amine and the phenyl group improved the inhibitory activity for NEK4 (Figure 14), which is in line with the viability data. However, only for n ≥ 2 a dramatic decrease in NSCLC cell viability was observed and the activity of 5au (n = 3) was significantly better as compared to 5ay (n = 0) (Figures 7 and 8) which is contrary to the IC50 values. Therefore, we postulate that inhibition of NEK4 only, does not fully explain the results of the viability screening. The introduction of a 1-hydroxy-3-butynyl group at position 6 of the quinazoline core 14b resulted in a two-fold improved NEK4 inhibitory activity compared to Spautin-1, which suggests that the reduced cell viability was potentially caused by inhibition of NEK4. Remarkably, the Spautin-1 analogue bearing the quinoline core 19 was clearly less potent for NEK4 which is also in contrast to the viability data.  Even though the IC 50 (NEK4) values are not fully in line with the viability data, we have strong indications that our compounds bear great potential for the treatment of EGFR mutant NSCLC. The reduced viability is presumably a multifactorial effect of which USP13 and EGFR inhibition cannot be excluded. Further research is needed due to the lack of experimental data on NEK4 and EGFR inhibition at the doses used on a cellular level. The kinase screening also revealed a low remaining activity for CLK1 and CLK4 (Table 2), to our best knowledge, two kinases which have not been linked to lung cancer. However, these can probably be considered as moderate off-targets at the relatively high concentration of inhibitors used (10 µM).

Discussion
In this work, a SAR-study of spautin-1 was performed to discover lead compounds for the treatment of EGFR mutant NSCLC. The most important SAR trends can be summarized as follow: The secondary amine is crucial for the activity, which has been previously reported for kinase inhibitors in general [55,[58][59][60]; an increased chain length (n ≥ 2) between the secondary amine and phenyl group dramatically increases the activity, in contrast to EGFR-TKIs [55]; however, the highest potency was observed for n = 2; a halogen capable of inducing an halogen bond at position 4' of the phenyl group is beneficial for the activity. A N-screening was performed to determine the importance of the quinazoline core, which lead to the conclusion that the nitrogen atom at the 3-position does not seem to be important for the activity, as the analogue bearing the quinoline core showed a high potency. On the contrary, the nitrogen at the 1-position appears to be crucial for the activity, while the cinnoline and quinoxaline bearing Spautin-1 analogues showed limited activity towards EGFR mutant NSCLC cells. In line with these results, quinoline containing kinase inhibitors have been widely reported and some of these inhibitors were even approved by the FDA [27][28][29][30]. Altogether, the extensive SAR study provided compounds with IC 50 values as low as 210 nM, as determined in the applied cell viability assay, opening up a gateway towards more potent lead structures.
Surprisingly, we were unable to confirm binding between spautin-1, or the analogues, and USP13. Inability to detect binding using TSA or ITC does not exclude a binding under physiological conditions. Previously published assays did not show direct binding either and it might be that Spautin-1 inhibits USP13 via an indirect way [26]. On the other hand, these assays used USP13 purified from eukaryotic cells, while our experiments were performed with recombinant human USP13 [26]. Possible differences in post-translational modifications, protein folding, protein stability and solubility of recombinant USP13 may create a bias which cannot be controlled for, as spautin-1 is the only known small molecule that may bind USP13. In search of off-targets of the investigated analogues, a broad kinase screening unraveled NEK4 as a potential target. The latter kinase is often overexpressed in lung cancer and the expression in our PC9 cell line model was confirmed [37]. The highest activity towards NEK4 was observed for the N-[2-(substituted-phenyl)ethyl]-4quinazolinamines, which were also responsible for the lowest EGFR-mutant NSCLC cell viability. For this reason, we hypothesize that the decreased viability of NSCLC cells was at least partially caused by the inhibition of NEK4. As far as we know, N-[2-(substitutedphenyl)ethyl]-4-quinazolinamines have been reported as acetyl-and butyrylcholinesterase inhibitors (AChE IC 50 = 6.2 µM; BuChE IC 50 = 14.1 µM) for the treatment of Parkinson disease and inhibitors of NFκB [65,66]. These compounds showed moderate potency towards EGFR as compared to the anilino-and benzylamino derivatives [55], but were never related to NEK4.
In summary, we report N-[2-(substituted-phenyl)ethyl]-6-fluoro-4-quinazolinamines as promising single agent lead compounds for the future treatment of EGFR mutant NSCLC. To the best of our knowledge, the N-[2-(substituted-phenyl)ethyl]-6-fluoro-4quinazolinamines are the first potent and relatively selective NEK4 inhibitors reported in literature to date. More research is ongoing to further increase the potency and selectivity of these lead compounds (e.g., replacing the quinazoline core by the quinoline core) and get insight in the mechanism of action. Such optimized inhibitors may eventually be combined with FDA approved EGFR-TKIs to obtain a stronger initial response, increase the progression-free survival and improve the quality of life in EGFR-mutant NSCLC patients [26].

Chemistry
Unless stated otherwise, all commercial materials were used without further purification and were purchased from fluorochem Ltd. (14 Graphite Way, Hadfield, Derbyshire SK13 1QH United Kingdom) or Merck (2000 Galloping Hill Road, Kenilworth, NJ 07033 U.S.A.). Anhydrous N,N-dimethylformamide was obtained by storing under argon atmosphere on activated 4Å molecular sieves for 24 h prior to use. Non-commercial starting materials were prepared based on literature procedures and are described below. 1 H and 13 C NMR spectra were recorded using different spectrometers. A Bruker Avance II 500 spectrometer (Bruker Scientific LLC, 40 Manning Road, Manning Park, Billerica, MA 01821, USA) was used at 500 MHz ( 1 H NMR) and 126 MHz ( 13 C NMR) at ambient temperature. To obtain spectra at 250 MHz and 63 MHz, the Bruker Avance DRX 250 was used. The chemical shifts were reported in delta (δ) units in parts per million (ppm) relative to the signal of the deuterated solvent. For the CDCl 3 , the singlet in 1 H NMR was calibrated at 7.26 ppm and the 13 C NMR at the central line of the triplet at 77.16 ppm. For DMSO-d 6 , the calibration was performed at 2.50 ppm for the 1 H NMR and 39.52 ppm for the 13 C NMR, respectively. The assignments were made using one dimensional (1D) 1 H and 13 C spectra and two-dimensional (2D) HSQC, HMBC and COSY spectra. Multiplicities were described as singlet (s), doublet (d), triplet (t), quartet (q), multiplet (m), broad (br), or a combination thereof. The corresponding coupling constants (J values) were reported in Hertz (Hz). Analytical RP-HPLC analyses were carried out on a Chromaster system (VWR Hitachi, Researchpark Haasrode 2020, Geldenaaksebaan 464, 3001 Leuven) equipped with a Hitachi 5430 DAD, 5310 column oven, 5260 autosampler, and 5160 pump. Chromolith High Resolution RP-18e from Merck (150 Å, 1.1 µm, 50 × 4.6 mm, 3 mL/min flow rate) columns were used for analysis using UV detection at 214 nm. Solvents A and B are 0.1% TFA in water and 0.1% TFA in acetonitrile, respectively. Gradients are 1 to 100% B/A over 4 min. Mass spectra were recorded with a LC-MS triplequadrupole system. HPLC unit was a Waters 600 system combined to a Waters 2487 UV detector at 215 nm (Waters Corporation, 34 Maple Street, Milford, MA 01757, USA) and as stationary phase a Vydac MS RP C18-column (150 mm × 2.1 mm, 3 µm, 300 Å). The solvent system was 0.1% formic acid in water (A) and 0.1% formic acid in acetonitrile (B) with a gradient going from 3% to 100% B over 20 min with a flow rate of 0.3 mL/min. The MS unit, coupled to the HPLC system, was a Micromass QTOF-micro system. For the high resolution mass spectroscopy, the same MS system was used with reserpine (2.10-3 mg/mL) solution in H 2 O:CH 3 CN (1:1) as reference. Automated flash chromatography was performed using Grace ® Reveleris X2 system equipped with ELSD and UV detector (254 nm or 280 nm). The used normal phase column for the systems were Interchim ® Puriflash ® Silica HC 25 µM Method 1: The synthesis was performed according to a literature procedure [43]. Into a flame-dried 10 mL microwave vial, the substituted-2-aminobenzamide 9 (3.0 mmol, 1.0 equiv.) was dissolved in anhydrous DMF (1.5 mL). Subsequently trimethyl orthoformate (3.3 mmol, 1.1 equiv.) was added and the mixture was heated for 2-3 h at 170 • C.
Then the mixture was allowed to cool to room temperature. The precipitate was collected by filtration, washed with CH 2 Cl 2 and dried in vacuo yielding the desired compound in good purity. The product was used in the next step without further purification.
Then the mixture was allowed to cool to room temperature. The precipitate was collected by filtration, washed with CH 2 Cl 2 and dried in vacuo yielding the desired compound in good purity. The product was used in the next step without further purification.
Afterwards the solvent was removed under reduced pressure and the crude mixture was purified using preparative HPLC (AcN + 0.1% TFA/H 2 O + 0.1% TFA).

Kinase Screening
Eurofins performed a "Full KP Panel [Km ATP], KinaseProfile" which contains 429 radiometric kinase activity assays. The remaining kinase activity (%) was determined after treatment with the compounds at a concentration of 10 µM (Cfr. supporting information).

ITC
Pure and untagged USP13 protein was produced for the purpose of analysing ligand binding. The pGEX4T2-USP13 vector encoding for an N-terminal GST tag fused to fulllength human USP13 was transformed into E. coli BL21(DE3) cells for expression. Cells were grown at 37 • C in Terrific Broth (TB) medium, supplemented with 100 µg/mL Ampicillin, until the OD595 reached 0.7-0.8. Expression was then induced with 1 mM Isopropyl β-D-1-thiogalactopyranoside (IPTG). After 6 h induction at 28 • C, cells were collected by centrifugation and resuspended in PBS buffer. The cells were lysed with a cell disruptor (Constant Systems) and centrifuged to remove cell debris. The cell lysate was added to glutathione sepharose resin beads (17-0756-01, GE healthcare) equilibrated in PBS. Washing steps with PBS were performed to remove all other proteins before proceeding. To cleave the GST-tag, 10U/mL Thrombin was added to the beads and incubated at 4 • C on a shaker at slow rocking speed for 6 h. USP-13 was subsequently eluted in PBS and further purified on size exclusion chromatography, using a Superdex 200 16/900 column (GE Healthcare Life Sciences) in protein buffer (20 mM Tris-HCl (pH = 8), 50 mM NaCl, 0.1 mM EDTA), supplemented with 0.5 mM DTT and 5% glycerol for storage purposes.
An isothermal titration calorimetry (ITC) experiment was performed with the Micro-Cal iTC200 (GE Healthcare) at 25 • C. Spautin-1 was dissolved in DMSO and diluted in the protein buffer (25 mM Tris pH 7.5; 150 mM NaCl; 0.1 mM EDTA) to an end concentration of 300 µM with 5% DMSO. The compound was added to the syringe. The sample cell was filled with 30 µM of the protein in buffer to which 5% DMSO was added to avoid buffer mismatch. A control titration of buffer-buffer and compound-buffer was performed according to the same protocol. Data analysis was done with the Origin software accompanying the ITC instrument (Origin 7, OriginLab Corporation, Northampton, MA, USA).

TSA
The melting temperature (T m ) of the protein sample can be determined by following the fluorescence of SYPRO orange with a CFX Connect real-time PCR instrument (Bio-Rad, Hercules, CA, USA). Samples contained 0.4 mg/mL USP13 protein in 25 mM Tris Ph 7.5; 150 mM NaCl; 0.1 mM EDTA; 0.5 mM DTT. Compounds were added to the protein and incubated for 30 min before adding 20× SYPRO Orange Protein Gel Stain (Thermo Fisher Scientific, Waltham, MA, USA). Control measurements were done for the protein in the corresponding percentages of DMSO.
Fluorescence was measured while increasing the temperature from 10 • C to 85 • C in 0.5 • C/30 s increments. The melting temperature for each protein sample could be determined from the relative fluorescence versus temperature curve by deleting postpeak quenching data and subsequently fitting the Boltzmann-sigmoidal equation, using Graphpad Prism software version 8.4.2 (GraphPad Software, 2365 Northside Dr. Suite 560, San Diego, CA 92108).
Due to lack of protein material, the measurements were only performed once in a screening setup.

Conflicts of Interest:
The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.