Combination of Pseudo‐Natural Product Design and Formal Natural Product Ring Distortion Yields Stereochemically and Biologically Diverse Pseudo‐Sesquiterpenoid Alkaloids

Abstract We describe the synthesis and biological evaluation of a new natural product‐inspired compound class obtained by combining the conceptually complementary pseudo‐natural product (pseudo‐NP) design strategy and a formal adaptation of the complexity‐to‐diversity ring distortion approach. Fragment‐sized α‐methylene‐sesquiterpene lactones, whose scaffolds can formally be viewed as related to each other or are obtained by ring distortion, were combined with alkaloid‐derived pyrrolidine fragments by means of highly selective stereocomplementary 1,3‐dipolar cycloaddition reactions. The resulting pseudo‐sesquiterpenoid alkaloids were found to be both chemically and biologically diverse, and their biological performance distinctly depends on both the structure of the sesquiterpene lactone‐derived scaffolds and the stereochemistry of the pyrrolidine fragment. Biological investigation of the compound collection led to the discovery of a novel chemotype inhibiting Hedgehog‐dependent osteoblast differentiation


Introduction
Thed esign and synthesis of structurally complex and chemically diverse compound collections,e ncoding diverse biological activities are at the heart of chemical biology and drug discovery,a nd novel approaches are in high demand. [1] Natural product (NP) structures harness biological relevance established in evolution and have served as inspiration for the discovery of novel bioactive compound classes [2] fori nstance by late-stage functionalization of NPs, [3] function-oriented synthesis (FOS) [4] and biology-oriented synthesis (BIOS). [5] However,t hese strategies yield compound collections which explore chemical and biological space closely related to the parent NPs.F or wider exploration of NP-inspired chemical and biological space,ring distortion by means of the complexity-to-diversity approach [6,7] and the development of pseudonatural products (pseudo-NPs) [8,9] have recently been introduced ( Figure 1a).
Ring distortion uses available natural products as starting points which are then subjected to different transformations resulting in chemical modulation of the underlying NP scaffolds to yield compound classes with different ring structure and bioactivity. [7] In the design and synthesis of pseudo-NPs,n atural products are viewed as combinations of fragments into which they can be disassembled by means of cheminformatic methods, [10] and they can themselves be fragment-sized. Chemical recombination of NP-fragments from different sources in unprecedented arrangements by means of complexity-generating transformations will generate novel NP-inspired compound classes with unexpected or new bioactivities.
We envisioned that combination of these two strategies would yield novel natural product-inspired compound classes with diverse scaffolds and ap riori endowed with bioactivity. On the one hand fragment-sized NPs would be subjected to ring-distortion transformations,w hich requires that scaffolds of natural products themselves need to be distorted by chemical transformations.T his strategy could be complemented by an approach which can be regarded as formal adaptation of the ring distortion principle.I nt his alternative approach NPs are directly employed whose scaffolds can formally be regarded as formed by means of ring distortion reaction for instance in biosynthesis cascades (i.e.t he ring distortion reaction would already have occurred in nature). Subsequent chemical combination with structurally unrelated NP-fragments through complexity-generating transformations would afford compound collections with pronounced scaffold-and stereochemical complexity as outlined in Figure 1.
Here we report af irst exploration of this approach. We employed several fragment-sized [11] a-methylene-sesquiterpene lactones (SLs) whose different scaffolds can formally be viewed as related to each other by ring distortion (i.e., we took advantage of the natural diversity in this terpene class), or subjected them to ring distortion reactions.Stereocomplementary asymmetric 1,3-dipolar cycloadditions of dehydrolactones obtained from the natural products with amino acidderived azomethine ylides resulted in the formation of stereoisomeric spiro-pyrrolidine fragments,c haracteristic for pyrrolidine alkaloids (Figure 1). Cheminformatic analysis of the resulting pseudo-sesquiterpenoid alkaloids and biological characterization in ah igh content morphological assay monitoring aw ide range of bioactivity and in several assays monitoring different signaling pathways revealed that the novel NP-inspired pseudo-sesquiterpenoid alkaloids are both chemically and biologically performance diverse and define an ovel chemotype inhibiting Hedgehog-dependent osteoblast differentiation.

Development of Stereodivergent 1,3-Dipolar Cycloadditions with Sesquiterpene Lactones
In order to explore the combination of ring distortion or the adapted approach as described above with pseudo-NP design, we planned to combine different sesquiterpene lactone scaffolds with pyrrolidine fragments.S esquiterpene lactones (SLs) are adiverse class of biologically active natural products with multiple bioactivities [12] and many are fragment-sized. [11] Their scaffolds can be considered structurally related, and in af ormal sense it is imaginable that these fragments could be converted into each other by means of ring-distortion. Fori nstance,s antonin 1 contains ac yclohexadienone ring, whereas in structurally related parthenolide 2 C5 and C10 are not connected, and transannulation of parthenolide yields tricyclic micheliolide 3,w hich can also be seen as ar earranged scaffold of santonin 1 (Figure 1b). Sesquiterpene lactones frequently embody or can be equipped with ar eactive a-methylene-g-lactone which is am ajor determinant of bioactivity due to covalent reaction with biological nucleophiles. [13] We reasoned that conversion of this reactive functional group into an ew NP fragment by means of an appropriate complexity-generating reaction would enable both, exploration of novel chemical space and new bioactivity.A sc omplexity-generating reaction, the asymmetric 1,3-dipolar cycloaddition with azomethine ylides was chosen. This cycloaddition is avery reliable and powerful method for the incorporation of the pyrrolidine moiety, [14,15] which occurs in numerous alkaloids,l ike kainic acid, [16] cocaine [17] and nicotine [18] (Figure 1b). Fort he asymmetric steering of these cycloadditions several efficient catalyst systems are available,b ut it has rarely been applied for the functionalization of natural products. [19]  Since stereogenic character and content parallel bioactivity [20] it was planned to synthesize astereochemically diverse collection, that is,t og enerate all possible stereoisomers of the newly formed pyrrolidine ring in astereocomplementary synthesis approach. This task was considered particularly challenging because the complex chiral environment and diverse functional groups of NPs may complicate catalyst-and substrate-mediated control of stereoselectivity.T hus, while stereoselective synthesis of one isomer frequently is achievable, [21] stereodivergent synthesis of all isomers is much more demanding because of match-mismatch effects [22] and limited availability of catalysts in stereoisomeric forms. [23] Fort he development of suitable reaction conditions,santonin 1 was selected and converted into the corresponding a-methylene-g-lactone 7 (see Supplementary Figure S2 and Table 1). [19c] Use of AgOAc in the absence of any ligand as catalyst for the dipolar cycloadditions with the azomethine ylide generated from Schiff base 8a by deprotonation predominantly yielded endo-isomers 9a and 10 a ( Table 1, entry 1). Tr iphenyl phosphine (PPh 3 )a sl igand induced as mall decrease in endo selectivity (Table 1, entry 2), and (R)-Fesulphos L2 [24] yielded moderate endo selectivity (Table 1, entry 5). We previously reported that hydrogen bonding between the ligand and dipolarophile can improve endo selectivity. [25] Accordingly,u se of amine-substituted phosphine L3 was explored and the corresponding catalyst indeed catalyzed the dipolar cycloaddition with good endo/exo-a nd excellent facial selectivity (Table 1, entry 6). When THF was used as the solvent, ligand L3 (condition A) and its enantiomer (condition B) gave excellent endo-Si selectivity (Table 1, entry 7; condition A, and entry 8; condition B). Thus,under these conditions the endo selectivity is controlled by the chiral catalyst and not the chirality of the dipolarophile.
[b] The main isomer was purified by column chromatography;isolated yields of main isomers are shown in brackets.
[c] 0.50 equiv Cs 2 CO 3 was used. [d] 2.0 equiv Schiff base 8a was used. Table S1). In these transformations choice of the right solvent is crucial to obtain high selectivity.InTHF the endo-Re attack is favored (Table 1, entry 11), however,t he selectivity is switched to exo-Si when chloroform is employed as the solvent (Table 1, entry 12). Thes olvent effects on the stereochemical course of 1,3-dipolar cycloadditions using azomethine ylides were also observed in our recent report about dynamic catalytic highly enantioselective 1,3-dipolar cycloaddition wherein thermodynamic and kinetic reaction control enabled the efficient synthesis of stereochemically diverse compound libraries using aunified catalyst system. [26] Finally,l owering the temperature and increasing the amount of the Schiff base did not substantially influence yield and selectivity (Table 1, entry 13;c ondition D). Thec hirality of the substrate has only am inor influence on the endo selectivity,such that with both enantiomers of L3 endo-cycloadducts are formed with high selectivity (Table 1, entries 7a nd 8). However,i nt he exo-selective transformations,t he stereogenic character of the substrate introduces facial bias,p robably due to the quaternary center embedded in the santonin scaffold (Supplementary Figure S1). When the dipole approaches the dipolarophile from the Si face,t he methyl group attached to the quaternary center points away from the phosphine ligands,s uch that substrate and chiral ligand form am atched pair leading to excellent selectivity. Attack of the azomethine ylide to the Re-face of the dipolarophile,o nt he contrary,i sm ismatched due to unfavorable interaction of the methyl group with the ylide,resulting in low endo/exo selectivity.G ratifyingly,t uning the solvents and using powerful ligands realized such atransformation.

Asymmetric Synthesis of Pseudo-Sesquiterpenoid Alkaloids
With the conditions for the stereodivergent synthesis of the four diastereomeric cycloadducts in hand, the scope of the stereoselective cycloaddition to dehydrosantonin 7 was explored ( Figure 2). Thet ransformation tolerates diverse substituents in the para-and meta-position of the phenyl group. In general, condition Bd elivers higher yield and diastereoselectivity compared to condition A. However, ortho-substituted Schiff bases did not afford satisfying results (11 m-11 o) under condition Cb ecause of low conversion. Azomethine ylides derived from aliphatic aldehydes were not explored because they are not reactive in this transformation, presumably due to tautomerization of the imines.I nt otal, 61 stereochemically diverse compounds were efficiently synthesized. Thea bsolute configurations of 9b, 10 f and 12 e were unambiguously confirmed by X-ray crystallographic analysis (see the Supporting Information, Figures S3-S5).
Encouraged by the wide scope of the asymmetric cycloaddition for the dipoles,the scope for different dipolarophiles was explored. To this end, santonin was subjected to different substitutions of the scaffold (Supplementary Figure S2), that is,o xime formation, C À Ha ctivation [27] and Heck reaction [28] to give analogs 13, 14 and 15 respectively (Figure 3). Under acidic conditions,d ehydrosantonin underwent ar ing distortion reaction and rearranged to aphenol with stereoinversion of C6 [29] which was O-alkylated to yield benzyl ether 16. Aromatization of rings is considered agenuine ring distortion reaction as introduced by Hergenrother et al. [6b, 30] In order to further explore the ring distortion concept, we included sesquiterpenoid lactones whose scaffolds can be formally viewed as related to santonin by ring distortion.

Research Articles
Parthenolide 2 can be viewed as ar ing-opened analog of santonin. Tr ansannular ring closure between the alkene and epoxide in parthenolide 2 results in formation of 5/7/5 tricyclic micheliolide 3 [31] (see Figure 3; Supplementary Figure S2). We also included artemisinin and its deoxy-derivative and converted them to a-methylene-d-lactones 17 and 18 via at wo-step procedure,r esulting in ar ing contraction [19d] (see Figure 3; Supplementary Figure S2).
Thus,f or the synthesis of ac ollection of pseudo-sesquiterpenoid alkaloids,t hree structurally related natural sesquiterpene lactones were subjected to different ring distortion reactions.
As shown in Figure 4t he dipolar cycloaddition also displays appreciable scope for different sesquiterpene lactones.Changes in ring Aand Bofdehydrosantonin 7 are well tolerated, and the oxime derivatives afforded the desired cycloadducts 20-23 and 24-27 in excellent yields and selectivity.C ycloadditions to the formally ring-opened analog 2 and the rearranged scaffold 3 yielded very satisfying results (32-35; 36-39). Even cycloaddition to the simplest lactone 19 afforded excellent stereoselectivity (44)(45)(46)(47). [32] However,t he reaction was highly sensitive to modification of the g-lactone.
Compound 15 with ap henyl ring attached to the methylene group showed low reactivity under the conditions for the exo-

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Research Articles selective reaction. Inversion of the stereocenter at C6 (16) confined the reaction to the Si face to yield cycloadducts 30 and 31 as the main isomers even under conditions Ba nd D. Thering size of the lactone has aclear impact on the course of the reaction. Artemisinin derivatives 17 and 18 exclusively yielded endo products (40-41; 42-43).

Cheminformatic and Analysis of the Sesquiterpene Lactones and Pseudo-Sesquiterpenoid Alkaloids
Forc haracterization of the 94 synthesized sesquiterpene lactones and pseudo-sesquiterpenoid alkaloids the NP-likeness score [33] was computed using the open-source software RDKit [34] and compared to NPs in the ChEMBL database [35] and compounds in the DrugBank, [36] which characterize marketed and experimental drugs.T he NP likeness score compares the frequency of occurrence of agiven fragment in natural products with the frequency of occurrence in commercial compounds.S cores > 0i ndicate that the fragments are more common in natural products.Not unexpectedly,the synthesized sesquiterpene lactones directly obtained from natural products showed high scores.I nc omparison the scores recorded for the pseudo-sesquiterpenoid alkaloids are lower, since their scaffolds result from fragment combinations and are novel (Supplementary Figure S6). However,t he scores calculated for the pseudo-sesquiterpenoid alkaloids are represented in the area characteristic for NPs (Figure 5a). This result differs from the corresponding findings for pseudo natural product libraries,o btained by combination of different NP-fragments which are more shifted to the area characteristic for the compounds in Drugbank. [8a] This preliminary finding indicates that ring distortion of fragment-sized NPs appears to retain ah igh degree of NP character in the combination with other NP-fragments.
Foranalysis of compound class similarity,arepresentative group of the pseudo-sesquiterpenoid alkaloids depicted in Figure 4w as analyzed by computing Tanimoto similarity of the Morgan fingerprints [37] (for the structures of the compounds included in the analysis see Supplementary Figure S7). As shown in Figure 5b,sesquiterpene lactones 2, 3, 7 and 13-18 display low cross similarity (Figure 5b,r egion A), and cross-similarity to the pseudo-sesquiterpene alkaloids was low as well (Figure 5b,region B). Theintraclass chemical similarity of the pseudo-sesquiterpenoid alkaloids 9a and 20-42 was higher (Figure 5b,r egion C) probably because they share the same pyrrolidine fragment. Similarity was particularly high (89 %) for compounds 40 and 42 sharing almost the same scaffold. NP likeness score and Tanimoto cross similarity do not distinguish stereoisomers.I no rder to analyze the incorporation of differently configured stereocenters in the compound collection, we calculated principal moments of inertia (PMI) [38] (Figure 5c). PMI plots are used to describe the molecular shape of lowest-energy conformations and, therefore,they differentiate stereoisomers.The top-left of the plot represents rod-shaped compounds,t he top-right represents spheres and the bottom corner denotes disc-shaped compounds.A ss hown in Figure 5c,t he pseudo-sesquiterpenoid alkaloids (red dots) are broadly distributed in the PMI plot. On the contrary,m ost of the sesquiterpene lactones (black dots) resided along the rod-disc side of the plot. Obviously, the recombination of the natural product scaffolds with the pyrrolidine moiety led to ashift to the sphere-disc part of the PMI plot, representative for ah igher degree in stereogenic character.N otably,e ven different stereoisomers occupy significantly different space in the plot, as exemplified for stereoisomers 9c-12 c (identified by blue arrows) in Figure 5c.T his analysis demonstrates that for the compound class analyzed here combination of ring distortion and pseudo NP synthesis yielded as tereochemically diverse compound collection that is structurally distinct from the guiding natural products which were subjected to ring distortion.

Biological Performance Analysis of the Sesquiterpene Lactones and Pseudo-Sesquiterpenoid Alkaloids
Bioactivity of the sesquiterpene lactones 2, 3, 7 and 13-18 and the pseudo-sesquiterpenoid alkaloids 9-12 and 20-47 was analyzed by means of the cell painting assay which monitors phenotypic changes in cells by employing six fluorescent dyes that selectively stain various compartments. [39] Imaging via multi-channel fluorescence microscopy identifies morphological changes condensed into 579 features to generate ac haracteristic profile.T he number of significantly changed features in ap rofile relative to aD MSO control defines an induction value (see SI for more details). Biosimilarities of phenotypic profiles are calculated from the correlation distances between two profiles.Differences in morphological features can be observed among SL derivatives 2, 3, 7 and 14-18,whose biosimilarities compared to dehydrosantonin 7 are below 60 % ( Figure 6a). We note that the a-methylene-glactone is considered ap romiscuous covalent binder,p otentially targeting multiple nucleophiles in proteins,a nd this reactivity is no longer present in the pseudo-NPs.T hus, already in the design of the pseudo-NP library described here, it could be expected that the biological performance of the synthesized pseudo-NPs would be significantly different from the profiles for the guiding NPs indicating novel bioactivity (see also below).
Principal component analysis (PCA)w as used to condense the phenotypic profiles of several compounds into three-dimensional plots visualizing differences between compound classes by cluster formation. This approach has previously been used by Schreiber et al. [40] and by us [1b,9b] to demonstrate differences in cellular responses to stereo-and regioisomers.
Human osteosarcoma cells (U-2 OS) were incubated with compounds at different concentrations (Supplementary Table S5) and analysis of the fingerprints revealed that the induction was concentration-dependent( Supplementary Figure S8). Five compounds showed > 5% induction at 10 mM and at 50 mM4 9c ompounds induced significant morphological changes.T oinvestigate the impact of fragment combination on bioactivity pattern, the sesquiterpene lactones (red dots) were compared with the corresponding pseudo-sesquiterpenoid alkaloids (blue dots) by means of PCA (Figure 6b) which revealed that the two compound classes collectively display distinctly different morphological profiles.T his discovery is consistent with the notion that the a-methylene lactone can serve as an electrophile to covalently bind biological nucleophiles.C ombination with the pyrrolidine fragment abolishes this mode of action. Even in comparison with saturated sesquiterpene lactones,arelatively low cross biosimilarity was observed (Supplementary Figure 9a,b).
Theinfluence of the ring distortion was gleaned from the cross biosimilarity analysis for pseudo-sesquiterpenoid alkaloids synthesized from diverse SLs ( Table S5). Therefore,t he convergence of biological performance was not induced by adominant effect of the pyrrolidine fragment.
In order to analyze the influence of different stereoisomers the pseudo-sesquiterpenoida lkaloids derived from dehydrosantonin 1 were analyzed. Thed ata revealed as eparation between classes 9-12 (Figure 4f;S upplementary Figure S10). Thef inding that cell painting can differentiate stereoisomers has been reported before. [40] As mentioned above,c ompounds 32, 36 and 40 displayed relatively high cross biosimilarity regardless of different sesquiterpene scaffolds.V ery interestingly when different stereoisomers of these compounds (33 vs. 41; 35 vs. 39)were compared, the influence of ring distortion was again visible leading to highly different biological performance (Supplementary Figure S9e,f).

Identification of aPseudo-Sesquiterpenoid Alkaloid Inhibitor of Hedgehog-Dependent Osteoblast Differentiation
In order to get more detailed insight into the bioactivity of the sesquiterpenoid alkaloids we subjected them to several cell-based assays monitoring different cellular pathways and programs,i ncluding autophagy,T -cell signaling as well as signal transduction through the Wnt and the Hedgehog (Hh) pathways.T hese investigations revealed an ovel inhibitor of Hh-dependent osteoblast differentiation.
Hh signaling is essential for embryonic and post-embryonic development and regeneration. [41] It has been linked to cancers like basal cell carcinoma and medulloblastoma, [42] and novel Hh inhibitor classes are in high demand. To identify inhibitors of Hh signaling, we performed aH h-dependent osteoblast differentiation assay using C3H/10T1/2 cells (Figure 7). [43] Thepathway agonist purmorphamine was employed to induce osteoblast differentiation which leads to the expression of alkaline phosphatase as readout for pathway activity. [44] This assay revealed that compound 23 inhibits Hhdependent osteoblast differentiation with half-maximal inhibitory concentration (IC 50 )o f1 .5 AE 0.9 mM. Very notably, stereoisomeric pseudo-sesquiterpenoid alkaloids 20-22 showed no inhibitory activity at 10 mMconcentration. Under these conditions,also compounds 47 and 48 which are partial structures of the new inhibitor chemotype and pseudosesquiterpenoid alkaloids 35 and 39 were inactive.The results indicate that combination of ring distortion and the pseudo-NP strategy may enable the discovery of novel bioactive chemical matter in amore general sense.

Conclusion
We describe the combination of the pseudo-natural product principle with chemical ring distortion of NPs and aformal adaptation of the complexity-to-diversity strategy,to yield novel bioactive natural product-inspired compound classes.I nt his new strategy,i ng eneral, on the one hand diverse scaffolds are formed from fragment-sized NPs by subjecting them to ring-distortion reactions.O nt he other hand, fragment-sized NPs are directly employed (i.e.without separate ring distortion reaction) whose scaffolds can be regarded as formed by means of aring distortion reaction, for example,through biosynthesis pathways.The second option is to apply ring distortion strategy which requires that chemical transformations should be used to distort the scaffolds of natural products.S ubsequently,c hemical fragment recombination through complexity-generating transformations affords complex and diverse compound collections.W ee xemplify the new principle by the synthesis of ac ollection of pseudo-sesquiterpenoid alkaloids obtained from fragmentsized sesquiterpenes by means of stereocomplementary 1,3dipolar cycloadditions with azomethine ylides resulting in the highly stereoselective formation of pyrrolidine fragments. Cheminformatic and biological characterization revealed that the compound collection is stereochemically diverse and that this diversity is reflected in diverse biological performance, including the discovery of an ovel chemotype inhibiting Hedgehog-dependent differentiation of multipotent murine mesenchymal progenitor stem cells into osteoblasts.
Combination of these two complementary principles will give efficient access to new,n atural product-inspired, structurally complex compound classes,w hich are not accessible by currently described biosynthesis pathways.H owever,w e note that silent, unexploited biosynthesis pathways exist, [45] and that biosynthesis pathways can be reassembled, for example,i ns ystems biology approaches. [46] It is,t herefore, possible that natural product scaffolds exist in nature which resemble the structures of the new NP-inspired compound classes resulting from our approach thereby further validating it. In agreement with this notion, very recently and while we developed the principle,t he isolation of the natural product vlasoulamine Awas reported, [47] whose scaffold is structurally related to pseudo-sesquiterpenoid alkaloids 36.