Discovery of a Dual SENP1 and SENP2 Inhibitor

SUMOylation is a reversible post–translational modification (PTM) involving covalent attachment of small ubiquitin-related modifier (SUMO) proteins to substrate proteins. Dysregulation of SUMOylation and deSUMOylation results in cellular malfunction and is linked to various diseases, such as cancer. Sentrin-specific proteases (SENPs) were identified for the maturation of SUMOs and the deconjugation of SUMOs from their substrate proteins. Hence, this is a promising target tackling the dysregulation of the SUMOylation process. Herein, we report the discovery of a novel protein-protein interaction (PPI) inhibitor for SENP1-SUMO1 by virtual screening and subsequent medicinal chemistry optimization of the hit molecule. The optimized inhibitor ZHAWOC8697 showed IC50 values of 8.6 μM against SENP1 and 2.3 μM against SENP2. With a photo affinity probe the SENP target was validated. This novel SENP inhibitor represents a new valuable tool for the study of SUMOylation processes and the SENP-associated development of small molecule-based treatment options.


Introduction
SUMOylation is a reversible post-translational modification that targets a variety of proteins by attachment of small ubiquitin-like modifiers (SUMOs) [1,2]. SUMO proteins are similar to ubiquitin and are considered members of the ubiquitin-like protein family. It has been shown that SUMOylation and deSUMOylation has a major influence on cellular processes such as transcription, DNA damage response or cell division [3][4][5][6]. SUMOylation is regulated by a cascade of reactions catalyzed by SUMO-specific activating enzyme E1, conjugation enzyme E2, and ligase enzyme E3, whereas deSUMOylation is regulated by the family of Sentrin-specific proteases (SENPs) [7]. SENPs are cysteine proteases, which are responsible for the activation of proSUMOs to SUMOs and removal of small ubiquitin-like modifiers (SUMOs) from the post-translational modified proteins.
All SUMOs are expressed as proSUMOs. At the C-terminus, during the maturation of proSUMOs to SUMOs, two to eleven amino acids are proteolytically cleaved by SENP endopeptidases. In total, there are six different human SENPs (1, 2, 3, 5, 6, and 7), which have a sequence similarity between 20 and 60%.
SUMOylation is of great importance for the regulated cell cycle and genome stability. If SENP1 expression is incorrectly regulated, serious diseases, such as prostate [8,9], bladder [10], multiple myeloma [11], pancreatic [12] or neuroblastoma cancer [13] can occur. SENP1 is therefore a clinically highly relevant antitumor target and of growing interest for developing novel treatment options for cancer patients [14,15]. Other research showed that SENP1 deSUMOylates and stabilizes hypoxia-inducible factor 1α (HIF-1α) during hypoxia [16].

Virtual Screening
We envisaged a virtual screening on the interface between SENP1 and SUMO1 ( code: 2G4D) proteins to identify new structural elements for the development of n SENP1 inhibitors (Figure 2A). An in-house diversity library containing 10,240 compo was screened against the SENP1-SUMO1 interface using AutoDock Vina [27] and res in 598 virtual hits with a binding score of <−7.3 kcal/mol ( Figure 2B). After visual in tion of the compounds in the hypothesized binding mode to SENP1, 50 promising pounds were shortlisted for screening at 50 µM by a fluorescence-based assay agains catalytic domain of SENP1, using SUMO1-AMC as substrate. At this concentration compounds showed a SENP1 activity decrease of >50% ( Figure 2C,D). Two of the hit ecules had solubility issues at the tested concentration and were not further investig IC50 values were determined of the remaining three hit molecules with the most prom compound 11 (ZHAWOC8697) showing an IC50 of 5.1 µM ( Figure 2D).
The resynthesized hit molecule 11 and its derivatives 23 and 24 were tested for the inhibition of the catalytic activity of SENP1 (Table 1). Pleasingly, the inhibition of the resynthesized hit compound 11 was confirmed with an IC 50 of 8.6 µM. Interestingly, the gem-dimethyl (23) and non-substituted (24) DHQ derivatives showed reduced inhibition by a factor of 1.5 and 10, respectively (Table 1). This raises the question, as to whether this is due to the reduced compound lipophilicity or the steric restriction of the amide bond [29] between the DHQ amine and the bulky carboxylic amide phenyl tetrazole substituent. MD calculations for the rotational barrier of this amide bond were performed by umbrella sampling using Amber 17. The results indicate that with the gem-dimethyl and non-substituted dihydroquinoxalinone derivatives two preferred orientations of the amide linker with an angle of 44 • and 136 • or 22 • and 158 • are possible, whereas the spirocyclic compound has just one energy minimum at 120 • due to steric clash with the bulky spirocyclopentane DHQ scaffold ( Figure S1). The synthesis of the phenyl tetrazole building block 21 commenced with a [2 + 3] dipolar cycloaddition of benzonitrile 17 with sodium azide to generate tetrazole 18. Subsequently the phenyl tetrazole 18 was N-alkylated with methyl chloroacetate to the desired 1H- (19) and 2H-regioisomer (20), followed by ester hydrolysis to obtain compounds 21 and 22. The 2H-tetrazole 21 was converted to an acid chloride with oxalyl chloride and directly coupled to either the spirocyclic (15), gem-dimethyl (16) or non-substituted (25) DHQ derivative to isolate the hit molecule 11 and its close analogues 23 and 24 (Scheme 1).
The resynthesized hit molecule 11 and its derivatives 23 and 24 were tested for the inhibition of the catalytic activity of SENP1 (Table 1). Pleasingly, the inhibition of the resynthesized hit compound 11 was confirmed with an IC50 of 8.6 µ M. Interestingly, the gem-dimethyl (23) and non-substituted (24) DHQ derivatives showed reduced inhibition by a factor of 1.5 and 10, respectively (Table 1). This raises the question, as to whether this is due to the reduced compound lipophilicity or the steric restriction of the amide bond [29] between the DHQ amine and the bulky carboxylic amide phenyl tetrazole substituent. MD calculations for the rotational barrier of this amide bond were performed by umbrella sampling using Amber 17. The results indicate that with the gem-dimethyl and non-substituted dihydroquinoxalinone derivatives two preferred orientations of the amide linker with an angle of 44° and 136° or 22° and 158° are possible, whereas the spirocyclic compound has just one energy minimum at 120° due to steric clash with the bulky spirocyclopentane DHQ scaffold ( Figure S1). Table 1. IC50 values of hit compound 11 and the corresponding modifications of the spiro DHQ scaffold 23 and 24; (a) n = 5; (b) n = 2.

Fragments
To investigate this theory, structurally relevant fragments were tested in the SENP1 inhibition assay at concentrations of up to 1 mM. The spirocyclic compound 15 had an IC50 of 116 μM, whereas the gem-dimethyl DHQ derivative 16 showed only around 50% inhibition at 500 µM ( Table 2). The sterically hindered amide bond is not present in those fragments, allowing us to conclude that the different orientations of the amide bond do not result in different ligand binding affinities. The synthesis of the phenyl tetrazole building block 21 commenced with a [2 + 3] dipolar cycloaddition of benzonitrile 17 with sodium azide to generate tetrazole 18. Subsequently the phenyl tetrazole 18 was N-alkylated with methyl chloroacetate to the desired 1H- (19) and 2H-regioisomer (20), followed by ester hydrolysis to obtain compounds 21 and 22. The 2H-tetrazole 21 was converted to an acid chloride with oxalyl chloride and directly coupled to either the spirocyclic (15), gem-dimethyl (16) or non-substituted (25) DHQ derivative to isolate the hit molecule 11 and its close analogues 23 and 24 (Scheme 1).
The resynthesized hit molecule 11 and its derivatives 23 and 24 were tested for the inhibition of the catalytic activity of SENP1 (Table 1). Pleasingly, the inhibition of the resynthesized hit compound 11 was confirmed with an IC50 of 8.6 µ M. Interestingly, the gem-dimethyl (23) and non-substituted (24) DHQ derivatives showed reduced inhibition by a factor of 1.5 and 10, respectively (Table 1). This raises the question, as to whether this is due to the reduced compound lipophilicity or the steric restriction of the amide bond [29] between the DHQ amine and the bulky carboxylic amide phenyl tetrazole substituent. MD calculations for the rotational barrier of this amide bond were performed by umbrella sampling using Amber 17. The results indicate that with the gem-dimethyl and non-substituted dihydroquinoxalinone derivatives two preferred orientations of the amide linker with an angle of 44° and 136° or 22° and 158° are possible, whereas the spirocyclic compound has just one energy minimum at 120° due to steric clash with the bulky spirocyclopentane DHQ scaffold ( Figure S1). Table 1. IC50 values of hit compound 11 and the corresponding modifications of the spiro DHQ scaffold 23 and 24; (a) n = 5; (b) n = 2.

Fragments
To investigate this theory, structurally relevant fragments were tested in the SENP1 inhibition assay at concentrations of up to 1 mM. The spirocyclic compound 15 had an IC50 of 116 μM, whereas the gem-dimethyl DHQ derivative 16 showed only around 50% inhibition at 500 µM ( Table 2). The sterically hindered amide bond is not present in those fragments, allowing us to conclude that the different orientations of the amide bond do not result in different ligand binding affinities. The synthesis of the phenyl tetrazole building block 21 commenced with a [2 + 3] dipolar cycloaddition of benzonitrile 17 with sodium azide to generate tetrazole 18. Subsequently the phenyl tetrazole 18 was N-alkylated with methyl chloroacetate to the desired 1H- (19) and 2H-regioisomer (20), followed by ester hydrolysis to obtain compounds 21 and 22. The 2H-tetrazole 21 was converted to an acid chloride with oxalyl chloride and directly coupled to either the spirocyclic (15), gem-dimethyl (16) or non-substituted (25) DHQ derivative to isolate the hit molecule 11 and its close analogues 23 and 24 (Scheme 1).
The resynthesized hit molecule 11 and its derivatives 23 and 24 were tested for the inhibition of the catalytic activity of SENP1 (Table 1). Pleasingly, the inhibition of the resynthesized hit compound 11 was confirmed with an IC50 of 8.6 µ M. Interestingly, the gem-dimethyl (23) and non-substituted (24) DHQ derivatives showed reduced inhibition by a factor of 1.5 and 10, respectively (Table 1). This raises the question, as to whether this is due to the reduced compound lipophilicity or the steric restriction of the amide bond [29] between the DHQ amine and the bulky carboxylic amide phenyl tetrazole substituent. MD calculations for the rotational barrier of this amide bond were performed by umbrella sampling using Amber 17. The results indicate that with the gem-dimethyl and non-substituted dihydroquinoxalinone derivatives two preferred orientations of the amide linker with an angle of 44° and 136° or 22° and 158° are possible, whereas the spirocyclic compound has just one energy minimum at 120° due to steric clash with the bulky spirocyclopentane DHQ scaffold ( Figure S1). Table 1. IC50 values of hit compound 11 and the corresponding modifications of the spiro DHQ scaffold 23 and 24; (a) n = 5; (b) n = 2.

Fragments
To investigate this theory, structurally relevant fragments were tested in the SENP1 inhibition assay at concentrations of up to 1 mM. The spirocyclic compound 15 had an IC50 of 116 μM, whereas the gem-dimethyl DHQ derivative 16 showed only around 50% inhibition at 500 µM ( Table 2). The sterically hindered amide bond is not present in those fragments, allowing us to conclude that the different orientations of the amide bond do not result in different ligand binding affinities. 12 The synthesis of the phenyl tetrazole building block 21 commenced with a [2 + 3] dipolar cycloaddition of benzonitrile 17 with sodium azide to generate tetrazole 18. Subsequently the phenyl tetrazole 18 was N-alkylated with methyl chloroacetate to the desired 1H- (19) and 2H-regioisomer (20), followed by ester hydrolysis to obtain compounds 21 and 22. The 2H-tetrazole 21 was converted to an acid chloride with oxalyl chloride and directly coupled to either the spirocyclic (15), gem-dimethyl (16) or non-substituted (25) DHQ derivative to isolate the hit molecule 11 and its close analogues 23 and 24 (Scheme 1).
The resynthesized hit molecule 11 and its derivatives 23 and 24 were tested for the inhibition of the catalytic activity of SENP1 (Table 1). Pleasingly, the inhibition of the resynthesized hit compound 11 was confirmed with an IC50 of 8.6 µ M. Interestingly, the gem-dimethyl (23) and non-substituted (24) DHQ derivatives showed reduced inhibition by a factor of 1.5 and 10, respectively (Table 1). This raises the question, as to whether this is due to the reduced compound lipophilicity or the steric restriction of the amide bond [29] between the DHQ amine and the bulky carboxylic amide phenyl tetrazole substituent. MD calculations for the rotational barrier of this amide bond were performed by umbrella sampling using Amber 17. The results indicate that with the gem-dimethyl and non-substituted dihydroquinoxalinone derivatives two preferred orientations of the amide linker with an angle of 44° and 136° or 22° and 158° are possible, whereas the spirocyclic compound has just one energy minimum at 120° due to steric clash with the bulky spirocyclopentane DHQ scaffold ( Figure S1). Table 1. IC50 values of hit compound 11 and the corresponding modifications of the spiro DHQ scaffold 23 and 24; (a) n = 5; (b) n = 2.

Fragments
To investigate this theory, structurally relevant fragments were tested in the SENP1 inhibition assay at concentrations of up to 1 mM. The spirocyclic compound 15 had an IC50 of 116 μM, whereas the gem-dimethyl DHQ derivative 16 showed only around 50% inhibition at 500 µM ( Table 2). The sterically hindered amide bond is not present in those fragments, allowing us to conclude that the different orientations of the amide bond do not result in different ligand binding affinities. 77.6 (57.8-104) (b)

Fragments
To investigate this theory, structurally relevant fragments were tested in the SENP1 inhibition assay at concentrations of up to 1 mM. The spirocyclic compound 15 had an IC 50 of 116 µM, whereas the gem-dimethyl DHQ derivative 16 showed only around 50% inhibition at 500 µM ( Table 2). The sterically hindered amide bond is not present in those fragments, allowing us to conclude that the different orientations of the amide bond do not result in different ligand binding affinities.  Various modifications of fragment 15 were synthesized to gain a better understanding of the important features and aiding inhibitor optimization. Compound 26 indicates that the secondary amine is not crucial and can be replaced by an ether resulting in comparable activity ( Table 2). Increasing the spirocyclic ring size to increase the lipophilic character was beneficial for the cyclooctane derivative 28 (IC50: 51 µ M), whereas a slightly reduced activity of the cyclohexyl analogue 27 (IC50: 261 µ M) was measured. The spiro cyclobutyl compound 29 had a slightly increased potency compared to 15 (Table 2). This is a surprising observation, that the spirocyclic ring size is equally important to the lipophilicity. Incorporation of an oxetane (30) or tetrahydropyran (31) into the spirocyclic compound resulted in a comparable inhibition profile similar to the aliphatic 4-(29) or 6membered (27) derivatives (Table 2). In addition, with the heterospirocyclic compounds, the 6-membered spirocyclic compound 31 is less active compared to the 4-membered analogue 30. The inverse amide 32 was self-fluorescent at concentrations of up 10 µM and hence no IC50 could be determined. Reduction of the amide bond of 15 to the amine 33 resulted in a loss of activity indicating that this hydrogen bond acceptor is crucial ( Table  2). Modifications of the aromatic part by introducing a pyridine (34) resulted in a complete loss of activity up to 500 µ M. Meanwhile, replacing the benzene moiety to a thiophene (35 and 36) gave a three-fold affinity increase, which is also a solid improvement in terms of ligand efficiency from 0.37 to 0.44 ( Table 2). The attachment of substituents to the aromatic core of the dihydroquinoxalinone, either with an electron-donating methoxy group (37,39) or an electron-withdrawing group such as methyl ester (38,40) or nitro (41)  Various modifications of fragment 15 were synthesized to gain a better understanding of the important features and aiding inhibitor optimization. Compound 26 indicates that the secondary amine is not crucial and can be replaced by an ether resulting in comparable activity ( Table 2). Increasing the spirocyclic ring size to increase the lipophilic character was beneficial for the cyclooctane derivative 28 (IC50: 51 µ M), whereas a slightly reduced activity of the cyclohexyl analogue 27 (IC50: 261 µ M) was measured. The spiro cyclobutyl compound 29 had a slightly increased potency compared to 15 (Table 2). This is a surprising observation, that the spirocyclic ring size is equally important to the lipophilicity. Incorporation of an oxetane (30) or tetrahydropyran (31) into the spirocyclic compound resulted in a comparable inhibition profile similar to the aliphatic 4-(29) or 6membered (27) derivatives (Table 2). In addition, with the heterospirocyclic compounds, the 6-membered spirocyclic compound 31 is less active compared to the 4-membered analogue 30. The inverse amide 32 was self-fluorescent at concentrations of up 10 µM and hence no IC50 could be determined. Reduction of the amide bond of 15 to the amine 33 resulted in a loss of activity indicating that this hydrogen bond acceptor is crucial ( Table  2). Modifications of the aromatic part by introducing a pyridine (34) resulted in a complete loss of activity up to 500 µ M. Meanwhile, replacing the benzene moiety to a thiophene (35 and 36) gave a three-fold affinity increase, which is also a solid improvement in terms of ligand efficiency from 0.37 to 0.44 ( Table 2). The attachment of substituents to the aromatic core of the dihydroquinoxalinone, either with an electron-donating methoxy group (37,39) or an electron-withdrawing group such as methyl ester (38,40) or nitro (41), was well  Various modifications of fragment 15 were synthesized to gain a better understanding of the important features and aiding inhibitor optimization. Compound 26 indicates that the secondary amine is not crucial and can be replaced by an ether resulting in comparable activity ( Table 2). Increasing the spirocyclic ring size to increase the lipophilic character was beneficial for the cyclooctane derivative 28 (IC50: 51 µ M), whereas a slightly reduced activity of the cyclohexyl analogue 27 (IC50: 261 µ M) was measured. The spiro cyclobutyl compound 29 had a slightly increased potency compared to 15 (Table 2). This is a surprising observation, that the spirocyclic ring size is equally important to the lipophilicity. Incorporation of an oxetane (30) or tetrahydropyran (31) into the spirocyclic compound resulted in a comparable inhibition profile similar to the aliphatic 4-(29) or 6membered (27) derivatives (Table 2). In addition, with the heterospirocyclic compounds, the 6-membered spirocyclic compound 31 is less active compared to the 4-membered analogue 30. The inverse amide 32 was self-fluorescent at concentrations of up 10 µM and hence no IC50 could be determined. Reduction of the amide bond of 15 to the amine 33 resulted in a loss of activity indicating that this hydrogen bond acceptor is crucial ( Table  2). Modifications of the aromatic part by introducing a pyridine (34) resulted in a complete loss of activity up to 500 µ M. Meanwhile, replacing the benzene moiety to a thiophene (35 and 36) gave a three-fold affinity increase, which is also a solid improvement in terms of ligand efficiency from 0.37 to 0.44 ( Table 2). The attachment of substituents to the aromatic core of the dihydroquinoxalinone, either with an electron-donating methoxy group (37,39) or an electron-withdrawing group such as methyl ester (38,40) or nitro (41), was well  Various modifications of fragment 15 were synthesized to gain a better understanding of the important features and aiding inhibitor optimization. Compound 26 indicates that the secondary amine is not crucial and can be replaced by an ether resulting in comparable activity ( Table 2). Increasing the spirocyclic ring size to increase the lipophilic character was beneficial for the cyclooctane derivative 28 (IC50: 51 µ M), whereas a slightly reduced activity of the cyclohexyl analogue 27 (IC50: 261 µ M) was measured. The spiro cyclobutyl compound 29 had a slightly increased potency compared to 15 (Table 2). This is a surprising observation, that the spirocyclic ring size is equally important to the lipophilicity. Incorporation of an oxetane (30) or tetrahydropyran (31) into the spirocyclic compound resulted in a comparable inhibition profile similar to the aliphatic 4-(29) or 6membered (27) derivatives (Table 2). In addition, with the heterospirocyclic compounds, the 6-membered spirocyclic compound 31 is less active compared to the 4-membered analogue 30. The inverse amide 32 was self-fluorescent at concentrations of up 10 µM and hence no IC50 could be determined. Reduction of the amide bond of 15 to the amine 33 resulted in a loss of activity indicating that this hydrogen bond acceptor is crucial ( Table  2). Modifications of the aromatic part by introducing a pyridine (34) resulted in a complete loss of activity up to 500 µ M. Meanwhile, replacing the benzene moiety to a thiophene (35 and 36) gave a three-fold affinity increase, which is also a solid improvement in terms of ligand efficiency from 0.37 to 0.44 ( Table 2). The attachment of substituents to the aromatic core of the dihydroquinoxalinone, either with an electron-donating methoxy group (37,39) or an electron-withdrawing group such as methyl ester (38,40) or nitro (41), was well  Various modifications of fragment 15 were synthesized to gain a better understanding of the important features and aiding inhibitor optimization. Compound 26 indicates that the secondary amine is not crucial and can be replaced by an ether resulting in comparable activity ( Table 2). Increasing the spirocyclic ring size to increase the lipophilic character was beneficial for the cyclooctane derivative 28 (IC50: 51 µ M), whereas a slightly reduced activity of the cyclohexyl analogue 27 (IC50: 261 µ M) was measured. The spiro cyclobutyl compound 29 had a slightly increased potency compared to 15 (Table 2). This is a surprising observation, that the spirocyclic ring size is equally important to the lipophilicity. Incorporation of an oxetane (30) or tetrahydropyran (31) into the spirocyclic compound resulted in a comparable inhibition profile similar to the aliphatic 4-(29) or 6membered (27) derivatives (Table 2). In addition, with the heterospirocyclic compounds, the 6-membered spirocyclic compound 31 is less active compared to the 4-membered analogue 30. The inverse amide 32 was self-fluorescent at concentrations of up 10 µM and hence no IC50 could be determined. Reduction of the amide bond of 15 to the amine 33 resulted in a loss of activity indicating that this hydrogen bond acceptor is crucial ( Table  2). Modifications of the aromatic part by introducing a pyridine (34) resulted in a complete loss of activity up to 500 µ M. Meanwhile, replacing the benzene moiety to a thiophene (35 and 36) gave a three-fold affinity increase, which is also a solid improvement in terms of ligand efficiency from 0.37 to 0.44 ( Table 2). The attachment of substituents to the aromatic core of the dihydroquinoxalinone, either with an electron-donating methoxy group (37,39) or an electron-withdrawing group such as methyl ester (38,40) or nitro (41), was well  Various modifications of fragment 15 were synthesized to gain a better understanding of the important features and aiding inhibitor optimization. Compound 26 indicates that the secondary amine is not crucial and can be replaced by an ether resulting in comparable activity ( Table 2). Increasing the spirocyclic ring size to increase the lipophilic character was beneficial for the cyclooctane derivative 28 (IC50: 51 µ M), whereas a slightly reduced activity of the cyclohexyl analogue 27 (IC50: 261 µ M) was measured. The spiro cyclobutyl compound 29 had a slightly increased potency compared to 15 (Table 2). This is a surprising observation, that the spirocyclic ring size is equally important to the lipophilicity. Incorporation of an oxetane (30) or tetrahydropyran (31) into the spirocyclic compound resulted in a comparable inhibition profile similar to the aliphatic 4-(29) or 6membered (27) derivatives (Table 2). In addition, with the heterospirocyclic compounds, the 6-membered spirocyclic compound 31 is less active compared to the 4-membered analogue 30. The inverse amide 32 was self-fluorescent at concentrations of up 10 µM and hence no IC50 could be determined. Reduction of the amide bond of 15 to the amine 33 resulted in a loss of activity indicating that this hydrogen bond acceptor is crucial ( Table  2). Modifications of the aromatic part by introducing a pyridine (34) resulted in a complete loss of activity up to 500 µ M. Meanwhile, replacing the benzene moiety to a thiophene (35 and 36) gave a three-fold affinity increase, which is also a solid improvement in terms of ligand efficiency from 0.37 to 0.44 ( Table 2). The attachment of substituents to the aromatic core of the dihydroquinoxalinone, either with an electron-donating methoxy group (37,39) or an electron-withdrawing group such as methyl ester (38,40) or nitro (41), was well  Various modifications of fragment 15 were synthesized to gain a better understanding of the important features and aiding inhibitor optimization. Compound 26 indicates that the secondary amine is not crucial and can be replaced by an ether resulting in comparable activity ( Table 2). Increasing the spirocyclic ring size to increase the lipophilic character was beneficial for the cyclooctane derivative 28 (IC50: 51 µ M), whereas a slightly reduced activity of the cyclohexyl analogue 27 (IC50: 261 µ M) was measured. The spiro cyclobutyl compound 29 had a slightly increased potency compared to 15 (Table 2). This is a surprising observation, that the spirocyclic ring size is equally important to the lipophilicity. Incorporation of an oxetane (30) or tetrahydropyran (31) into the spirocyclic compound resulted in a comparable inhibition profile similar to the aliphatic 4-(29) or 6membered (27) derivatives (Table 2). In addition, with the heterospirocyclic compounds, the 6-membered spirocyclic compound 31 is less active compared to the 4-membered analogue 30. The inverse amide 32 was self-fluorescent at concentrations of up 10 µM and hence no IC50 could be determined. Reduction of the amide bond of 15 to the amine 33 resulted in a loss of activity indicating that this hydrogen bond acceptor is crucial ( Table  2). Modifications of the aromatic part by introducing a pyridine (34) resulted in a complete loss of activity up to 500 µ M. Meanwhile, replacing the benzene moiety to a thiophene (35 and 36) gave a three-fold affinity increase, which is also a solid improvement in terms of ligand efficiency from 0.37 to 0.44 ( Table 2). The attachment of substituents to the aromatic core of the dihydroquinoxalinone, either with an electron-donating methoxy group (37,39) or an electron-withdrawing group such as methyl ester (38,40) or nitro (41)  Various modifications of fragment 15 were synthesized to gain a better understanding of the important features and aiding inhibitor optimization. Compound 26 indicates that the secondary amine is not crucial and can be replaced by an ether resulting in comparable activity ( Table 2). Increasing the spirocyclic ring size to increase the lipophilic character was beneficial for the cyclooctane derivative 28 (IC50: 51 µ M), whereas a slightly reduced activity of the cyclohexyl analogue 27 (IC50: 261 µ M) was measured. The spiro cyclobutyl compound 29 had a slightly increased potency compared to 15 (Table 2). This is a surprising observation, that the spirocyclic ring size is equally important to the lipophilicity. Incorporation of an oxetane (30) or tetrahydropyran (31) into the spirocyclic compound resulted in a comparable inhibition profile similar to the aliphatic 4-(29) or 6membered (27) derivatives (Table 2). In addition, with the heterospirocyclic compounds, the 6-membered spirocyclic compound 31 is less active compared to the 4-membered analogue 30. The inverse amide 32 was self-fluorescent at concentrations of up 10 µM and hence no IC50 could be determined. Reduction of the amide bond of 15 to the amine 33 resulted in a loss of activity indicating that this hydrogen bond acceptor is crucial ( Table  2). Modifications of the aromatic part by introducing a pyridine (34) resulted in a complete loss of activity up to 500 µ M. Meanwhile, replacing the benzene moiety to a thiophene (35 and 36) gave a three-fold affinity increase, which is also a solid improvement in terms of ligand efficiency from 0.37 to 0.44 ( Table 2). The attachment of substituents to the aromatic core of the dihydroquinoxalinone, either with an electron-donating methoxy group (37,39) or an electron-withdrawing group such as methyl ester (38,40) or nitro (41), was well  Various modifications of fragment 15 were synthesized to gain a better understanding of the important features and aiding inhibitor optimization. Compound 26 indicates that the secondary amine is not crucial and can be replaced by an ether resulting in comparable activity ( Table 2). Increasing the spirocyclic ring size to increase the lipophilic character was beneficial for the cyclooctane derivative 28 (IC50: 51 µ M), whereas a slightly reduced activity of the cyclohexyl analogue 27 (IC50: 261 µ M) was measured. The spiro cyclobutyl compound 29 had a slightly increased potency compared to 15 (Table 2). This is a surprising observation, that the spirocyclic ring size is equally important to the lipophilicity. Incorporation of an oxetane (30) or tetrahydropyran (31) into the spirocyclic compound resulted in a comparable inhibition profile similar to the aliphatic 4-(29) or 6membered (27) derivatives (Table 2). In addition, with the heterospirocyclic compounds, the 6-membered spirocyclic compound 31 is less active compared to the 4-membered analogue 30. The inverse amide 32 was self-fluorescent at concentrations of up 10 µM and hence no IC50 could be determined. Reduction of the amide bond of 15 to the amine 33 resulted in a loss of activity indicating that this hydrogen bond acceptor is crucial ( Table  2). Modifications of the aromatic part by introducing a pyridine (34) resulted in a complete loss of activity up to 500 µ M. Meanwhile, replacing the benzene moiety to a thiophene (35 and 36) gave a three-fold affinity increase, which is also a solid improvement in terms of ligand efficiency from 0.37 to 0.44 ( Table 2). The attachment of substituents to the aromatic core of the dihydroquinoxalinone, either with an electron-donating methoxy group (37,39) or an electron-withdrawing group such as methyl ester (38,40) or nitro (41)  Various modifications of fragment 15 were synthesized to gain a better understanding of the important features and aiding inhibitor optimization. Compound 26 indicates that the secondary amine is not crucial and can be replaced by an ether resulting in comparable activity ( Table 2). Increasing the spirocyclic ring size to increase the lipophilic character was beneficial for the cyclooctane derivative 28 (IC50: 51 µ M), whereas a slightly reduced activity of the cyclohexyl analogue 27 (IC50: 261 µ M) was measured. The spiro cyclobutyl compound 29 had a slightly increased potency compared to 15 (Table 2). This is a surprising observation, that the spirocyclic ring size is equally important to the lipophilicity. Incorporation of an oxetane (30) or tetrahydropyran (31) into the spirocyclic compound resulted in a comparable inhibition profile similar to the aliphatic 4-(29) or 6membered (27) derivatives (Table 2). In addition, with the heterospirocyclic compounds, the 6-membered spirocyclic compound 31 is less active compared to the 4-membered analogue 30. The inverse amide 32 was self-fluorescent at concentrations of up 10 µM and hence no IC50 could be determined. Reduction of the amide bond of 15 to the amine 33 resulted in a loss of activity indicating that this hydrogen bond acceptor is crucial ( Table  2). Modifications of the aromatic part by introducing a pyridine (34) resulted in a complete loss of activity up to 500 µ M. Meanwhile, replacing the benzene moiety to a thiophene (35 and 36) gave a three-fold affinity increase, which is also a solid improvement in terms of ligand efficiency from 0.37 to 0.44 ( Table 2). The attachment of substituents to the aromatic core of the dihydroquinoxalinone, either with an electron-donating methoxy group (37,39) or an electron-withdrawing group such as methyl ester (38,40) or nitro (41)  Various modifications of fragment 15 were synthesized to gain a better understanding of the important features and aiding inhibitor optimization. Compound 26 indicates that the secondary amine is not crucial and can be replaced by an ether resulting in comparable activity ( Table 2). Increasing the spirocyclic ring size to increase the lipophilic character was beneficial for the cyclooctane derivative 28 (IC50: 51 µ M), whereas a slightly reduced activity of the cyclohexyl analogue 27 (IC50: 261 µ M) was measured. The spiro cyclobutyl compound 29 had a slightly increased potency compared to 15 (Table 2). This is a surprising observation, that the spirocyclic ring size is equally important to the lipophilicity. Incorporation of an oxetane (30) or tetrahydropyran (31) into the spirocyclic compound resulted in a comparable inhibition profile similar to the aliphatic 4-(29) or 6membered (27) derivatives (Table 2). In addition, with the heterospirocyclic compounds, the 6-membered spirocyclic compound 31 is less active compared to the 4-membered analogue 30. The inverse amide 32 was self-fluorescent at concentrations of up 10 µM and hence no IC50 could be determined. Reduction of the amide bond of 15 to the amine 33 resulted in a loss of activity indicating that this hydrogen bond acceptor is crucial ( Table  2). Modifications of the aromatic part by introducing a pyridine (34) resulted in a complete loss of activity up to 500 µ M. Meanwhile, replacing the benzene moiety to a thiophene (35 and 36) gave a three-fold affinity increase, which is also a solid improvement in terms of ligand efficiency from 0.37 to 0.44 ( Table 2). The attachment of substituents to the aromatic core of the dihydroquinoxalinone, either with an electron-donating methoxy group (37,39) or an electron-withdrawing group such as methyl ester (38,40) or nitro (41)  Various modifications of fragment 15 were synthesized to gain a better understanding of the important features and aiding inhibitor optimization. Compound 26 indicates that the secondary amine is not crucial and can be replaced by an ether resulting in comparable activity ( Table 2). Increasing the spirocyclic ring size to increase the lipophilic character was beneficial for the cyclooctane derivative 28 (IC50: 51 µ M), whereas a slightly reduced activity of the cyclohexyl analogue 27 (IC50: 261 µ M) was measured. The spiro cyclobutyl compound 29 had a slightly increased potency compared to 15 (Table 2). This is a surprising observation, that the spirocyclic ring size is equally important to the lipophilicity. Incorporation of an oxetane (30) or tetrahydropyran (31) into the spirocyclic compound resulted in a comparable inhibition profile similar to the aliphatic 4-(29) or 6membered (27) derivatives (Table 2). In addition, with the heterospirocyclic compounds, the 6-membered spirocyclic compound 31 is less active compared to the 4-membered analogue 30. The inverse amide 32 was self-fluorescent at concentrations of up 10 µM and hence no IC50 could be determined. Reduction of the amide bond of 15 to the amine 33 resulted in a loss of activity indicating that this hydrogen bond acceptor is crucial ( Table  2). Modifications of the aromatic part by introducing a pyridine (34) resulted in a complete loss of activity up to 500 µ M. Meanwhile, replacing the benzene moiety to a thiophene (35 and 36) gave a three-fold affinity increase, which is also a solid improvement in terms of ligand efficiency from 0.37 to 0.44 ( Table 2). The attachment of substituents to the aromatic core of the dihydroquinoxalinone, either with an electron-donating methoxy group (37,39) or an electron-withdrawing group such as methyl ester (38,40) or nitro (41)  Various modifications of fragment 15 were synthesized to gain a better understanding of the important features and aiding inhibitor optimization. Compound 26 indicates that the secondary amine is not crucial and can be replaced by an ether resulting in comparable activity ( Table 2). Increasing the spirocyclic ring size to increase the lipophilic character was beneficial for the cyclooctane derivative 28 (IC50: 51 µ M), whereas a slightly reduced activity of the cyclohexyl analogue 27 (IC50: 261 µ M) was measured. The spiro cyclobutyl compound 29 had a slightly increased potency compared to 15 (Table 2). This is a surprising observation, that the spirocyclic ring size is equally important to the lipophilicity. Incorporation of an oxetane (30) or tetrahydropyran (31) into the spirocyclic compound resulted in a comparable inhibition profile similar to the aliphatic 4-(29) or 6membered (27) derivatives (Table 2). In addition, with the heterospirocyclic compounds, the 6-membered spirocyclic compound 31 is less active compared to the 4-membered analogue 30. The inverse amide 32 was self-fluorescent at concentrations of up 10 µM and hence no IC50 could be determined. Reduction of the amide bond of 15 to the amine 33 resulted in a loss of activity indicating that this hydrogen bond acceptor is crucial ( Table  2). Modifications of the aromatic part by introducing a pyridine (34) resulted in a complete loss of activity up to 500 µ M. Meanwhile, replacing the benzene moiety to a thiophene (35 and 36) gave a three-fold affinity increase, which is also a solid improvement in terms of ligand efficiency from 0.37 to 0.44 ( Table 2). The attachment of substituents to the aromatic core of the dihydroquinoxalinone, either with an electron-donating methoxy group (37,39) or an electron-withdrawing group such as methyl ester (38,40) or nitro (41)  Various modifications of fragment 15 were synthesized to gain a better understanding of the important features and aiding inhibitor optimization. Compound 26 indicates that the secondary amine is not crucial and can be replaced by an ether resulting in comparable activity ( Table 2). Increasing the spirocyclic ring size to increase the lipophilic character was beneficial for the cyclooctane derivative 28 (IC50: 51 µ M), whereas a slightly reduced activity of the cyclohexyl analogue 27 (IC50: 261 µ M) was measured. The spiro cyclobutyl compound 29 had a slightly increased potency compared to 15 (Table 2). This is a surprising observation, that the spirocyclic ring size is equally important to the lipophilicity. Incorporation of an oxetane (30) or tetrahydropyran (31) into the spirocyclic compound resulted in a comparable inhibition profile similar to the aliphatic 4-(29) or 6membered (27) derivatives (Table 2). In addition, with the heterospirocyclic compounds, the 6-membered spirocyclic compound 31 is less active compared to the 4-membered analogue 30. The inverse amide 32 was self-fluorescent at concentrations of up 10 µM and hence no IC50 could be determined. Reduction of the amide bond of 15 to the amine 33 resulted in a loss of activity indicating that this hydrogen bond acceptor is crucial ( Table  2). Modifications of the aromatic part by introducing a pyridine (34) resulted in a complete loss of activity up to 500 µ M. Meanwhile, replacing the benzene moiety to a thiophene (35 and 36) gave a three-fold affinity increase, which is also a solid improvement in terms of ligand efficiency from 0.37 to 0.44 ( Table 2). The attachment of substituents to the aromatic core of the dihydroquinoxalinone, either with an electron-donating methoxy group (37,39) or an electron-withdrawing group such as methyl ester (38,40) or nitro (41)  Various modifications of fragment 15 were synthesized to gain a better understanding of the important features and aiding inhibitor optimization. Compound 26 indicates that the secondary amine is not crucial and can be replaced by an ether resulting in comparable activity ( Table 2). Increasing the spirocyclic ring size to increase the lipophilic character was beneficial for the cyclooctane derivative 28 (IC50: 51 µ M), whereas a slightly reduced activity of the cyclohexyl analogue 27 (IC50: 261 µ M) was measured. The spiro cyclobutyl compound 29 had a slightly increased potency compared to 15 (Table 2). This is a surprising observation, that the spirocyclic ring size is equally important to the lipophilicity. Incorporation of an oxetane (30) or tetrahydropyran (31) into the spirocyclic compound resulted in a comparable inhibition profile similar to the aliphatic 4-(29) or 6membered (27) derivatives (Table 2). In addition, with the heterospirocyclic compounds, the 6-membered spirocyclic compound 31 is less active compared to the 4-membered analogue 30. The inverse amide 32 was self-fluorescent at concentrations of up 10 µM and hence no IC50 could be determined. Reduction of the amide bond of 15 to the amine 33 resulted in a loss of activity indicating that this hydrogen bond acceptor is crucial ( Table  2). Modifications of the aromatic part by introducing a pyridine (34) resulted in a complete loss of activity up to 500 µ M. Meanwhile, replacing the benzene moiety to a thiophene (35 and 36) gave a three-fold affinity increase, which is also a solid improvement in terms of ligand efficiency from 0.37 to 0.44 ( Table 2). The attachment of substituents to the aromatic core of the dihydroquinoxalinone, either with an electron-donating methoxy group (37,39) or an electron-withdrawing group such as methyl ester (38,40) or nitro (41)  Various modifications of fragment 15 were synthesized to gain a better understanding of the important features and aiding inhibitor optimization. Compound 26 indicates that the secondary amine is not crucial and can be replaced by an ether resulting in comparable activity ( Table 2). Increasing the spirocyclic ring size to increase the lipophilic character was beneficial for the cyclooctane derivative 28 (IC50: 51 µ M), whereas a slightly reduced activity of the cyclohexyl analogue 27 (IC50: 261 µ M) was measured. The spiro cyclobutyl compound 29 had a slightly increased potency compared to 15 (Table 2). This is a surprising observation, that the spirocyclic ring size is equally important to the lipophilicity. Incorporation of an oxetane (30) or tetrahydropyran (31) into the spirocyclic compound resulted in a comparable inhibition profile similar to the aliphatic 4-(29) or 6membered (27) derivatives (Table 2). In addition, with the heterospirocyclic compounds, the 6-membered spirocyclic compound 31 is less active compared to the 4-membered analogue 30. The inverse amide 32 was self-fluorescent at concentrations of up 10 µM and hence no IC50 could be determined. Reduction of the amide bond of 15 to the amine 33 resulted in a loss of activity indicating that this hydrogen bond acceptor is crucial ( Table  2). Modifications of the aromatic part by introducing a pyridine (34) resulted in a complete loss of activity up to 500 µ M. Meanwhile, replacing the benzene moiety to a thiophene (35 and 36) gave a three-fold affinity increase, which is also a solid improvement in terms of ligand efficiency from 0.37 to 0.44 ( Table 2). The attachment of substituents to the aromatic core of the dihydroquinoxalinone, either with an electron-donating methoxy group (37,39) or an electron-withdrawing group such as methyl ester (38,40) or nitro (41), was well  Various modifications of fragment 15 were synthesized to gain a better understanding of the important features and aiding inhibitor optimization. Compound 26 indicates that the secondary amine is not crucial and can be replaced by an ether resulting in comparable activity ( Table 2). Increasing the spirocyclic ring size to increase the lipophilic character was beneficial for the cyclooctane derivative 28 (IC50: 51 µ M), whereas a slightly reduced activity of the cyclohexyl analogue 27 (IC50: 261 µ M) was measured. The spiro cyclobutyl compound 29 had a slightly increased potency compared to 15 (Table 2). This is a surprising observation, that the spirocyclic ring size is equally important to the lipophilicity. Incorporation of an oxetane (30) or tetrahydropyran (31) into the spirocyclic compound resulted in a comparable inhibition profile similar to the aliphatic 4-(29) or 6membered (27) derivatives (Table 2). In addition, with the heterospirocyclic compounds, the 6-membered spirocyclic compound 31 is less active compared to the 4-membered analogue 30. The inverse amide 32 was self-fluorescent at concentrations of up 10 µM and hence no IC50 could be determined. Reduction of the amide bond of 15 to the amine 33 resulted in a loss of activity indicating that this hydrogen bond acceptor is crucial ( Table  2). Modifications of the aromatic part by introducing a pyridine (34) resulted in a complete loss of activity up to 500 µ M. Meanwhile, replacing the benzene moiety to a thiophene (35 and 36) gave a three-fold affinity increase, which is also a solid improvement in terms of ligand efficiency from 0.37 to 0.44 ( Table 2). The attachment of substituents to the aromatic core of the dihydroquinoxalinone, either with an electron-donating methoxy group (37,39) or an electron-withdrawing group such as methyl ester (38,40)   Various modifications of fragment 15 were synthesized to gain a better understanding of the important features and aiding inhibitor optimization. Compound 26 indicates that the secondary amine is not crucial and can be replaced by an ether resulting in comparable activity ( Table 2). Increasing the spirocyclic ring size to increase the lipophilic character was beneficial for the cyclooctane derivative 28 (IC50: 51 µ M), whereas a slightly reduced activity of the cyclohexyl analogue 27 (IC50: 261 µ M) was measured. The spiro cyclobutyl compound 29 had a slightly increased potency compared to 15 (Table 2). This is a surprising observation, that the spirocyclic ring size is equally important to the lipophilicity. Incorporation of an oxetane (30) or tetrahydropyran (31) into the spirocyclic compound resulted in a comparable inhibition profile similar to the aliphatic 4-(29) or 6membered (27) derivatives (Table 2). In addition, with the heterospirocyclic compounds, the 6-membered spirocyclic compound 31 is less active compared to the 4-membered analogue 30. The inverse amide 32 was self-fluorescent at concentrations of up 10 µM and hence no IC50 could be determined. Reduction of the amide bond of 15 to the amine 33 resulted in a loss of activity indicating that this hydrogen bond acceptor is crucial ( Table  2). Modifications of the aromatic part by introducing a pyridine (34) resulted in a complete loss of activity up to 500 µ M. Meanwhile, replacing the benzene moiety to a thiophene (35 and 36) gave a three-fold affinity increase, which is also a solid improvement in terms of ligand efficiency from 0.37 to 0.44 ( Table 2). The attachment of substituents to the aromatic core of the dihydroquinoxalinone, either with an electron-donating methoxy group (37,39) or an electron-withdrawing group such as methyl ester (38,40) or nitro (41), was well  Various modifications of fragment 15 were synthesized to gain a better understanding of the important features and aiding inhibitor optimization. Compound 26 indicates that the secondary amine is not crucial and can be replaced by an ether resulting in comparable activity ( Table 2). Increasing the spirocyclic ring size to increase the lipophilic character was beneficial for the cyclooctane derivative 28 (IC50: 51 µ M), whereas a slightly reduced activity of the cyclohexyl analogue 27 (IC50: 261 µ M) was measured. The spiro cyclobutyl compound 29 had a slightly increased potency compared to 15 (Table 2). This is a surprising observation, that the spirocyclic ring size is equally important to the lipophilicity. Incorporation of an oxetane (30) or tetrahydropyran (31) into the spirocyclic compound resulted in a comparable inhibition profile similar to the aliphatic 4-(29) or 6membered (27) derivatives (Table 2). In addition, with the heterospirocyclic compounds, the 6-membered spirocyclic compound 31 is less active compared to the 4-membered analogue 30. The inverse amide 32 was self-fluorescent at concentrations of up 10 µM and hence no IC50 could be determined. Reduction of the amide bond of 15 to the amine 33 resulted in a loss of activity indicating that this hydrogen bond acceptor is crucial ( Table  2). Modifications of the aromatic part by introducing a pyridine (34) resulted in a complete loss of activity up to 500 µ M. Meanwhile, replacing the benzene moiety to a thiophene (35 and 36) gave a three-fold affinity increase, which is also a solid improvement in terms of ligand efficiency from 0.37 to 0.44 ( Table 2). The attachment of substituents to the aromatic core of the dihydroquinoxalinone, either with an electron-donating methoxy group (37,39) or an electron-withdrawing group such as methyl ester (38,40) or nitro (41), was well NA * [b] NA Various modifications of fragment 15 were synthesized to gain a better understanding of the important features and aiding inhibitor optimization. Compound 26 indicates that the secondary amine is not crucial and can be replaced by an ether resulting in comparable activity (Table 2). Increasing the spirocyclic ring size to increase the lipophilic character was beneficial for the cyclooctane derivative 28 (IC 50 : 51 µM), whereas a slightly reduced activity of the cyclohexyl analogue 27 (IC 50 : 261 µM) was measured. The spiro cyclobutyl compound 29 had a slightly increased potency compared to 15 (Table 2). This is a surprising observation, that the spirocyclic ring size is equally important to the lipophilicity. Incorporation of an oxetane (30) or tetrahydropyran (31) into the spirocyclic compound resulted in a comparable inhibition profile similar to the aliphatic 4-(29) or 6-membered (27) derivatives (Table 2). In addition, with the heterospirocyclic compounds, the 6-membered spirocyclic compound 31 is less active compared to the 4-membered analogue 30. The inverse amide 32 was self-fluorescent at concentrations of up 10 µM and hence no IC 50 could be determined. Reduction of the amide bond of 15 to the amine 33 resulted in a loss of activity indicating that this hydrogen bond acceptor is crucial ( Table 2). Modifications of the aromatic part by introducing a pyridine (34) resulted in a complete loss of activity up to 500 µM. Meanwhile, replacing the benzene moiety to a thiophene (35 and 36) gave a three-fold affinity increase, which is also a solid improvement in terms of ligand efficiency from 0.37 to 0.44 ( Table 2). The attachment of substituents to the aromatic core of the dihydroquinoxalinone, either with an electron-donating methoxy group (37,39) or an electron-withdrawing group such as methyl ester (38,40) or nitro (41), was well tolerated overall but showed a remarkable dependence on the point of attachment, suggesting defined interactions. Thus, the best affinities in this series were measured with electronwithdrawing groups in the 7-and 8-position (38, 41).

Modifications
Subsequently, the influence of modifications of the phenyl substituent on the heteroaromatic moiety was investigated with respect to the inhibitory effect. For easier synthetic accessibility, the tetrazole was replaced by a 1,2,3 triazole. The synthesis for those derivatives commenced by an N-acylation of DHQ fragment 15 with 2-chloroacetic chloride to obtain intermediate 42 (Scheme 2). Substitution of the chlorine with azide by an S N 2 reaction yielded the azide derivative 43, which was used to react with different alkyne substrates by copper catalyzed azide-alkyne coupling to obtain the desired triazole derivatives 44-53 in moderate yields.

Modifications
Subsequently, the influence of modifications of the phenyl substituent on the heteroaromatic moiety was investigated with respect to the inhibitory effect. For easier synthetic accessibility, the tetrazole was replaced by a 1,2,3 triazole. The synthesis for those derivatives commenced by an N-acylation of DHQ fragment 15 with 2-chloroacetic chloride to obtain intermediate 42 (Scheme 2). Substitution of the chlorine with azide by an SN2 reaction yielded the azide derivative 43, which was used to react with different alkyne substrates by copper catalyzed azide-alkyne coupling to obtain the desired triazole derivatives 44-53 in moderate yields. In the fluorescence-based assay, the triazole derivative 44 showed a three-fold activity decrease compared to tetrazole 11 with an IC50 of 23 µM (Table 3). Despite the reduced activity, the structure activity relationship (SAR) can be well compared to the tetrazole derivatives within this compound series. In general, a flat SAR was observed. Exchanging the phenyl ring (44) by a saturated cyclohexyl (45) maintained the inhibition profile. A subtle activity decrease was measured with a benzyl (46) or 3-benzoic acid (47) substituent (Table 3). Having a substituent on the para position of the phenyl ring, such as the electron-deficient methyl ester (48) or electron-rich methoxy (49), resulted in a slightly increased inhibition of SENP1 with an IC50 of 7.5 and 13 µ M, respectively (Table 3). Replacement of the phenyl ring with a basic 2-pyridine analogue (50) showed only slight inhibition at 200 µM (Table 3). This is not surprising as SENP1 is a lysine rich protein, hence, electronic repulsion of the ligand with the protein may explain this activity decrease. Replacement of the phenyl group with a cyclopropyl (51), carboxylic acid (52) or ethyl methyl ether (53) diminished the SENP1 activity. In addition, other phenyl tetrazole replacements (Supplementary compounds S1-S7) such as phenols, anilines and indolines were tested, however, all of them had drastically reduced inhibition (Table S1). Additionally, the 1H-phenyl tetrazole DHQ derivative (54) was synthesized by coupling 15 with 22 in an analogous manner to the procedure described in Scheme 1 and tested, which resulted in a five-fold activity reduction compared to the 2H derivative (11). Compound 55 was synthesized by an amide coupling of thieno [3,4-b]pyrazine (35) with the 2H-tetrazole (21) and is equipotent to the DHQ derivative (11). In the fluorescence-based assay, the triazole derivative 44 showed a three-fold activity decrease compared to tetrazole 11 with an IC 50 of 23 µM (Table 3). Despite the reduced activity, the structure activity relationship (SAR) can be well compared to the tetrazole derivatives within this compound series. In general, a flat SAR was observed. Exchanging the phenyl ring (44) by a saturated cyclohexyl (45) maintained the inhibition profile. A subtle activity decrease was measured with a benzyl (46) or 3-benzoic acid (47) substituent (Table 3). Having a substituent on the para position of the phenyl ring, such as the electrondeficient methyl ester (48) or electron-rich methoxy (49), resulted in a slightly increased inhibition of SENP1 with an IC 50 of 7.5 and 13 µM, respectively ( Table 3). Replacement of the phenyl ring with a basic 2-pyridine analogue (50) showed only slight inhibition at 200 µM (Table 3). This is not surprising as SENP1 is a lysine rich protein, hence, electronic repulsion of the ligand with the protein may explain this activity decrease. Replacement of the phenyl group with a cyclopropyl (51), carboxylic acid (52) or ethyl methyl ether (53) diminished the SENP1 activity. In addition, other phenyl tetrazole replacements (Supplementary compounds S1-S7) such as phenols, anilines and indolines were tested, however, all of them had drastically reduced inhibition (Table S1). Additionally, the 1H-phenyl tetrazole DHQ derivative (54) was synthesized by coupling 15 with 22 in an analogous manner to the procedure described in Scheme 1 and tested, which resulted in a five-fold activity reduction compared to the 2H derivative (11). Compound 55 was synthesized by an amide coupling of thieno [3,4-b]pyrazine (35) with the 2H-tetrazole (21) and is equipotent to the DHQ derivative (11).

Mode of Action and Target Verification
To confirm 11 is active on the native substrates and not just on the SUMO1-AMC test substrate in the fluorogenic assay, a protocol for gel-based assay for the recombinant human proSUMO substrate was adopted from Mikolajczyk et al. [31]. For proSUMO1, the terminal four amino acids are cleaved by SENP1 in the maturation process of the protein, whereas for proSUMO3 there are 12 amino acids. In the case of proSUMO1, the protein

Mode of Action and Target Verification
To confirm 11 is active on the native substrates and not just on the SUMO1-AMC test substrate in the fluorogenic assay, a protocol for gel-based assay for the recombinant human proSUMO substrate was adopted from Mikolajczyk et al. [31]. For proSUMO1, the terminal four amino acids are cleaved by SENP1 in the maturation process of the protein, whereas for proSUMO3 there are 12 amino acids. In the case of proSUMO1, the protein 50 (43-57) 55 Table 3. IC50 values of 11 and its analogues; (a) n = 5.

Mode of Action and Target Verification
To confirm 11 is active on the native substrates and not just on the SUMO1-AMC test substrate in the fluorogenic assay, a protocol for gel-based assay for the recombinant human proSUMO substrate was adopted from Mikolajczyk et al. [31]. For proSUMO1, the terminal four amino acids are cleaved by SENP1 in the maturation process of the protein, whereas for proSUMO3 there are 12 amino acids. In the case of proSUMO1, the protein 10 (8-13) 56 Table 3. IC50 values of 11 and its analogues; (a) n = 5.

Mode of Action and Target Verification
To confirm 11 is active on the native substrates and not just on the SUMO1-AMC test substrate in the fluorogenic assay, a protocol for gel-based assay for the recombinant human proSUMO substrate was adopted from Mikolajczyk et al. [31]. For proSUMO1, the terminal four amino acids are cleaved by SENP1 in the maturation process of the protein, whereas for proSUMO3 there are 12 amino acids. In the case of proSUMO1, the protein 24 (20-29) 57 Table 3. IC50 values of 11 and its analogues; (a) n = 5.

Mode of Action and Target Verification
To confirm 11 is active on the native substrates and not just on the SUMO1-AMC test substrate in the fluorogenic assay, a protocol for gel-based assay for the recombinant human proSUMO substrate was adopted from Mikolajczyk et al. [31]. For proSUMO1, the terminal four amino acids are cleaved by SENP1 in the maturation process of the protein, whereas for proSUMO3 there are 12 amino acids. In the case of proSUMO1, the protein 6.6 (5.7-7.7)

Mode of Action and Target Verification
To confirm 11 is active on the native substrates and not just on the SUMO1-AMC test substrate in the fluorogenic assay, a protocol for gel-based assay for the recombinant human proSUMO substrate was adopted from Mikolajczyk et al. [31]. For proSUMO1, the terminal four amino acids are cleaved by SENP1 in the maturation process of the protein, whereas for proSUMO3 there are 12 amino acids. In the case of proSUMO1, the protein bands on the SDS gel could not be separated. However, with proSUMO3 a good separation of the matured protein with a 15% SDS polyacrylamide gel was achieved. For this reason, the inhibitory endopeptidase activity of 11 on SENP1 was determined with the proSUMO3 substrate. It could be clearly demonstrated that a full inhibition of SENP1 was observed at an inhibitor concentration of 200 µM, whereas at lower concentrations a dose-dependent response was observed ( Figure 3A). This confirms that the inhibitor does not only show activity on the SUMO-AMC test substrate, but also on native substrates, which underlines its biological significance and makes it a useful tool for further biological studies.
The photo affinity probe 56 consisting of the spiro DHQ scaffold coupled to a minimalist terminal alkyne-containing diazirine photo crosslinker [32] was synthesized to confirm the inhibitor-protein interaction (Scheme S1). With the fluorescent SENP1-SUMO1-AMC bioassay this compound showed an IC 50 value of 24 µM (Table 3). In comparison, the n-octyl derivative 57, which does not include the photo labile group, showed similar IC 50 data, indicating the diazirine group did not interfere in the fluorescent assay. To assess the interaction of SENP1 with the photo affinity probe, 56 was incubated with purified SENP1 and crosslinked via UV irradiation at 350 nM. The protein ligand mixture was coupled by CuAAc with the fluorescent TAMRA-azide dye and separated by SDS page electrophoresis. A fluorescent band was visible indicating the covalent binding of the probe to SENP1, confirming its interaction ( Figure 3B,C). A competition experiment with the photo probe 56 and 11 under the same conditions showed that the fluorescently labelled band of SENP1 was significantly decreased, indicating a selective binding to the protein ( Figure 3D,E).
bands on the SDS gel could not be separated. However, with proSUMO3 a good separation of the matured protein with a 15% SDS polyacrylamide gel was achieved. For this reason, the inhibitory endopeptidase activity of 11 on SENP1 was determined with the proSUMO3 substrate. It could be clearly demonstrated that a full inhibition of SENP1 was observed at an inhibitor concentration of 200 µ M, whereas at lower concentrations a dosedependent response was observed ( Figure 3A). This confirms that the inhibitor does not only show activity on the SUMO-AMC test substrate, but also on native substrates, which underlines its biological significance and makes it a useful tool for further biological studies.
The photo affinity probe 56 consisting of the spiro DHQ scaffold coupled to a minimalist terminal alkyne-containing diazirine photo crosslinker [32] was synthesized to confirm the inhibitor-protein interaction (Scheme S1). With the fluorescent SENP1-SUMO1-AMC bioassay this compound showed an IC50 value of 24 µM (Table 3). In comparison, the n-octyl derivative 57, which does not include the photo labile group, showed similar IC50 data, indicating the diazirine group did not interfere in the fluorescent assay. To assess the interaction of SENP1 with the photo affinity probe, 56 was incubated with purified SENP1 and crosslinked via UV irradiation at 350 nM. The protein ligand mixture was coupled by CuAAc with the fluorescent TAMRA-azide dye and separated by SDS page electrophoresis. A fluorescent band was visible indicating the covalent binding of the probe to SENP1, confirming its interaction ( Figure 3B,C). A competition experiment with the photo probe 56 and 11 under the same conditions showed that the fluorescently labelled band of SENP1 was significantly decreased, indicating a selective binding to the protein ( Figure 3D,E). A mode of inhibition study with 11 was performed by kinetic measurement with SENP1 and SUMO1-AMC employing five different substrate concentrations (Figure 4). The IC50 values decreased slightly by lowering the SUMO1-AMC substrate concentration from 1000 nM to 62.5 nM, indicating a slight binding preference to the SUMO1-SENP1 complex ( Figure 4A). On analysis of the mixed inhibition model using GraphPad Prism on the global Michaelis-Menten plot, a KI of 10.6 µM and alpha of 0.23 was observed (Figure 4B). The alpha value indicates that a slight preference for the SUMO1-SENP1 complex is demonstrated. A mode of inhibition study with 11 was performed by kinetic measurement with SENP1 and SUMO1-AMC employing five different substrate concentrations (Figure 4). The IC 50 values decreased slightly by lowering the SUMO1-AMC substrate concentration from 1000 nM to 62.5 nM, indicating a slight binding preference to the SUMO1-SENP1 complex ( Figure 4A). On analysis of the mixed inhibition model using GraphPad Prism on the global Michaelis-Menten plot, a K I of 10.6 µM and alpha of 0.23 was observed ( Figure 4B). The alpha value indicates that a slight preference for the SUMO1-SENP1 complex is demonstrated.

Target Selectivity
The selectivity of the most promising compounds 11, 55 and the most potent published SENP1 inhibitor 7 was evaluated against SUMO2 and SUMO3 as well as SENP2 and the two DUB proteins, UCHL1 and Ataxin 3 ( Table 4). The compounds 11 and 55 showed a moderate selectivity for the SUMO1 substrate over the SUMO2 and SUMO3 substrates, whereas 7 resulted in no significant differences. Surprisingly, this compound series had a slightly increased activity on the SENP2 protein. SENP2 has been identified by others to modulate atherosclerosis [33], neurodegenerative diseases [34], fatty acid metabolism [35] and adipogenesis [36]. With this wide range of functions, our discovered inhibitors could also be used to explore further the understudied SENP2 biology. Table 4. IC50 values of 11 (ZHAWOC8697), 55 and published compound 7 [21]; n = 2.

Target Selectivity
The selectivity of the most promising compounds 11, 55 and the most potent published SENP1 inhibitor 7 was evaluated against SUMO2 and SUMO3 as well as SENP2 and the two DUB proteins, UCHL1 and Ataxin 3 ( Table 4). The compounds 11 and 55 showed a moderate selectivity for the SUMO1 substrate over the SUMO2 and SUMO3 substrates, whereas 7 resulted in no significant differences. Surprisingly, this compound series had a slightly increased activity on the SENP2 protein. SENP2 has been identified by others to modulate atherosclerosis [33], neurodegenerative diseases [34], fatty acid metabolism [35] and adipogenesis [36]. With this wide range of functions, our discovered inhibitors could also be used to explore further the understudied SENP2 biology.  SENP1 from co-crystal structure (PDB-code: 2G4D [37]) was used as receptor. The receptor was first prepared by deleting the solvent molecules and SUMO1 protein. In a second step, AutoDockTools package (version 1.5.6) was employed to add the hydrogen atoms and the corresponding charges to the atoms and generate the necessary receptor.pdbqt. The structure preparation of our in-house diversity library was performed with OpenBabel [38] (version 3.1.1) and the python script "prepare_ligand4.py" from the Autodock-Tools [39,40] package and included 3D structure generation, the addition of hydrogens and partial charges using Gasteiger charges. With AutodockTools, the search space (x = −35.8, y = −25.6, z = 22.4) and grid size (40,40,40) were defined and the "exhaustiveness" of the search parameter was set to 9. For the number of binding modes, the default setting of 9 was used.

Molecular Dynamics Simulations
To validate the conformational preferences of the DHQ tetrazole amide bond, umbrella sampling simulations were carried out for compounds 11, 23 and 24 calculating the potential of mean force (PMF) as a function of the torsion angles. Prior to umbrella sampling, each small molecule was solvated (TIP3P) and equilibrated at 300 K for 100 ps. The C-N bond was varied from 0 to 180 • using umbrella windows spaced 3 • apart. Each window was then subjected to a 50 ps incrementally restrained equilibration prior to a 100 ps restrained simulation at constant temperature (25 • C) and pressure (1 atm). The torsions of the central residue were restrained using a harmonic penalty function with a force constant of 200 kcal/mol rad 2 for each window with 3 • intervals about each torsion angle. From each set, the unbiased potential of mean force was reconstructed using the weighted histogram analysis method (WHAM) [41,42].

General Information
All reagents and solvents were purchased from Sigma-Aldrich, Alfa Aesar, Enamine, or Fluorochem and used as received. All NMR spectra were recorded on a Bruker AVANCE III HD 500 One Bay spectrometer with a magnetic field of 11.75 T and a 5 mm SmartProbe BB(F)-H-D. For 1 H-NMR spectra, a frequency of 500 MHz resulted. Chemical shifts are reported in ppm from tetramethylsilane as internal standard in CDCl 3 or from [D 6 ]DMSO as an internal standard (δ = 2.50). Data are reported as follows: chemical shift, multiplicity (s = singlet, d = doublet, t = triplet, q = quartet, quint = quintet, br = broad, m = multiplet), coupling constants (Hz), integration. For 13 C-NMR spectra, a frequency of 126 MHz resulted. Chemical shifts are reported in ppm from tetramethylsilane as internal standard in CDCl 3 or from DMSO-d 6 (δ = 39.52). Purity was assayed by HPLC (Interchim Strategy C-18 column, 4.6 mm × 250 mm) with a gradient of 5−100% methanol in 0.2% aqueous acetic acid with UV detection at λ = 254 nm. All final compounds were obtained with ≥95% purity.

General Procedure A for the Bargellini Reaction
To an ice-cooled suspension of diamine 14 (1 eq.), ketone (2.5 eq.), chloroform (2 eq.) and benzyltriethylammonium chloride (0.05 eq.) in dichloromethane (V DCM = n Diamin 2 mol/L) was added an aq. 33% NaOH solution (5 eq.) over 45 min. The reaction mixture was allowed to warm to ambient temperature and stirred overnight. EtOAc (100 mL) was added to the reddish suspension and the organic layer was washed with brine (2 × 50 mL), dried (Na 2 SO 4 ), filtered and concentrated in vacuo.

Conclusions
Building on a virtual screening campaign, a series of novel SENP inhibitors could be established by fragment-based and synthetic modifications of the screening hit. It was also shown that our inhibitor 11 (ZHAWOC8697) is not only able to inhibit the cleavage of the SUMO1-AMC test substrate, but more importantly the maturation of the native proSUMO protein. The SENP1-inhibitor interaction was further validated with the photo affinity probe 56. Under our experimental conditions, compound 11 is equipotent compared to the most potent SENP1 inhibitors known to us, published by Chen et al. [21] and Lindemann et al. [25]. Moreover, the selectivity profile of the developed inhibitor toward other DUB proteins is comparable to that of the aforementioned inhibitors. In addition, 11 also inhibits SENP2 very effectively. Thus, this compound represents a valuable small molecule tool to study SENP1/2-SUMO interactions in a biological context and to develop small molecule drugs for the treatment of SENP1/2-associated diseases.