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

Article abstract of DOI:10.1002/anie.202106654

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.

Full text of DOI:10.1002/anie.202106654

A Journal of the Gesellschaft Deutscher Chemiker
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Accepted Article
Title: Combination of pseudo-natural product design and formal natural
product ring distortion yields stereochemically and biologically
diverse pseudo-sesquiterpenoid alkaloids
Authors: Herbert Waldmann, Jie Liu, Jana Flegel, Felix Otte, Axel Pahl,
Sonja Sievers, and Carsten Strohmann
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To be cited as: Angew. Chem. Int. Ed. 10.1002/anie.202106654
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10.1002/anie.202106654
Angewandte Chemie International Edition
Combination of pseudo-natural product design and formal natural product ring distortion
yields stereochemically and biologically diverse pseudo-sesquiterpenoid alkaloids
Jie Liu[a,b], Jana Flegel[a,b], Felix Otte[c], Axel Pahl[a,d], Sonja Sievers[a,d], Carsten Strohmann[c],
Herbert Waldmann[a,b] *
[a] Max Planck Institute of Molecular Physiology, Department of Chemical Biology
Otto-Hahn-Strasse 11, 44227 Dortmund (Germany)
[b] Technical University Dortmund, Faculty of Chemistry, Chemical Biology
Otto-Hahn-Strasse 6, 44221 Dortmund (Germany)
[c] Technical University Dortmund, Faculty of Chemistry, Inorganic Chemistry
Otto-Hahn-Strasse 6, 44221 Dortmund (Germany)
[d] Compound Management and Screening Center, Dortmund (Germany)
* corresponding author, email: Herbert.waldmann@mpi-dortmund.mpg.de
Keywords: Cycloaddition, Stereodivergent synthesis, Ring distortion, Pseudo-natural products
1
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Graphical Abstract:
Combination of the conceptually complementary formal ring distortion- and pseudo-natural
product strategies yields novel natural product-inspired bioactive pseudo-sesquiterpenoid
alkaloids.
2
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Abstract
Structurally complex and diverse natural products (NPs) display diverse biological activities and
inspire the synthesis of new bioactive compound classes. We describe the synthesis and
biological evaluation of a new natural product-inspired compound class obtained by combination
of 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 (SLs) whose different 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. Cheminformatic analysis and morphological profiling in the cell painting assay revealed
that the resulting pseudo-sesquiterpenoid alkaloids are both chemically and biologically diverse,
and that 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
Design and synthesis of structurally complex and chemically diverse compound collections,
encoding diverse biological activities are at the heart of chemical biology and drug discovery, and
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] for instance by late-stage functionalization of NPs[3], function-
oriented synthesis (FOS)[4] and biology-oriented synthesis (BIOS)[5]. However, these strategies
yield compound collections which explore chemical and biological space closely related to the
parent NPs. For 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 pseudo-natural
products (pseudo-NPs)[8],[9] have recently been introduced (Fig. 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, natural 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-
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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 a priori endowed with bioactivity. On the one hand
fragment-sized NPs would be subjected to ring-distortion transformations, which requires that
scaffolds of natural products themselves need to be distorted by chemical transformations. This
strategy could be complemented by an approach which can be regarded as formal adaptation of
the ring distortion principle. In this 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. the 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 Fig. 1.
Here we report a first exploration of this approach. We employed several fragment-sized[11] α-
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 acid-
derived azomethine ylides resulted in the formation of stereoisomeric spiro-pyrrolidine fragments,
characteristic for pyrrolidine alkaloids (Fig. 1). Cheminformatic analysis of the resulting pseudo-
sesquiterpenoid alkaloids and biological characterization in a high content morphological assay
monitoring a wide 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 a novel chemotype inhibiting
Hedgehog-dependent osteoblast differentiation.
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Figure 1. Combination of the (formal) ring distortion strategy with the pseudo-NP principle
for the synthesis of novel chemically diverse NP-inspired compound classes with diverse
biological performance. a. Previously reported synthetic strategies toward NP-inspired
compound collections. b. (Formal) ring distortion of sesquiterpene lactones (SLs) led to diverse
scaffolds which were recombined with pyrrolidine fragments.
Results
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. Sesquiterpene lactones (SLs) are a diverse 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 a formal sense it is imaginable that these fragments
could be converted into each other by means of ring-distortion. For instance, santonin 1 contains
a cyclohexadienone ring, whereas in structurally related parthenolide 2 C5 and C10 are not
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connected, and transannulation of parthenolide yields tricyclic micheliolide 3, which can also be
seen as a rearranged scaffold of santonin 1 (Figure 1b).
Sesquiterpene lactones frequently embody or can be equipped with a reactive α-methylene-γ-
lactone which is a major determinant of bioactivity due to covalent reaction with biological
nucleophiles[13]. We reasoned that conversion of this reactive functional group into a new NP
fragment by means of an appropriate complexity-generating reaction would enable both,
exploration of novel chemical space and new bioactivity. As complexity-generating reaction, the
asymmetric 1,3-dipolar cycloaddition with azomethine ylides was chosen. This cycloaddition is a
[15]
very reliable and powerful method for the incorporation of the pyrrolidine moiety,[14]
which
occurs in numerous alkaloids, like kainic acid[16], cocaine[17] and nicotine[18] (Figure 1b). For the
asymmetric steering of these cycloadditions several efficient catalyst systems are available, but
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 a
stereochemically diverse collection, i.e. to generate all possible stereoisomers of the newly
formed pyrrolidine ring in a stereocomplementary 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. Thus, 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].
For the development of suitable reaction conditions, santonin 1 was selected and converted into
the corresponding α-methylene-γ-lactone 7 (see Supplementary Fig. 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
10a (Tab. 1, entry 1). Triphenyl phosphine (PPh3) as ligand induced a small decrease in endo
selectivity (Tab. 1, entry 2), and (R)-Fesulphos L2[24] yielded moderate endo selectivity (Tab. 1,
entry 5). We previously reported that hydrogen bonding between the ligand and dipolarophile can
improve endo selectivity.[25] Accordingly, use of amine-substituted phosphine L3 was explored
and the corresponding catalyst indeed catalyzed the dipolar cycloaddition with good endo/exo-
and excellent facial selectivity (Tab. 1, entry 6). When THF was used as the solvent, ligand L3
(condition A) and its enantiomer (condition B) gave excellent endo-Si selectivity (Tab. 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.
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However, in the presence of bulky phosphine ligand BINAP L1 the endo/exo selectivity was
reversed, and the (R)-configured phosphine favored Si facial attack (11a:12a = 71:2) (Tab. 1,
entry 3). In comparison, the enantiomeric (S)-BINAP (ent-L1) induced only low Re facial selectivity
(11a:12a=19:45) which suggests that the (S)-configured phosphine ligand forms a mismatched
pair with the substrate (Tab. 1, entry 4).
Based on these findings, bulky phosphine ligands were used for exo selective formation of the
cycloadducts. Thus, in the presence of (R)-DTBM Segphos L4 as ligand in DCE as solvent, the
reaction proceeded with almost quantitative exo-Si selectivity (Tab. 1, entry 9; condition C).
However, the enantiomeric ligand did not lead to satisfying exo-Re facial selectivity (Tab. 1, entry
10). Exploration of several ligands revealed that (S)-DTBM Biphep L5 induced the highest
diastereoselectivity (Tab. 1, entries 11-13; Supplementary Tab. S1). In these transformations
choice of the right solvent is crucial to obtain high selectivity. In THF the endo-Re attack is favored
(Tab. 1, entry 11), however, the selectivity is switched to exo-Si when chloroform is employed as
the solvent (Tab. 1, entry 12). The solvent 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 a unified catalyst system.[26] Finally, lowering the temperature and increasing the amount of
the Schiff base did not substantially influence yield and selectivity (Tab. 1, entry 13; condition D).
The chirality of the substrate has only a minor influence on the endo selectivity, such that with
both enantiomers of L3 endo-cycloadducts are formed with high selectivity (Tab. 1, entries 7 and
8). However, in the exo-selective transformations, the stereogenic character of the substrate
introduces facial bias, probably due to the quaternary center embedded in the santonin scaffold
(Supplementary Fig. S1). When the dipole approaches the dipolarophile from the Si face, the
methyl group attached to the quaternary center points away from the phosphine ligands, such
that substrate and chiral ligand form a matched pair leading to excellent selectivity. Attack of the
azomethine ylide to the Re-face of the dipolarophile, on the contrary, is mismatched due to
unfavorable interaction of the methyl group with the ylide, resulting in low endo/exo selectivity.
Gratifyingly, tuning the solvents and using powerful ligands realized such a transformation.
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Table 1. Development of the stereodivergent synthesis of santonin-pyrrolidines
d.r.
Entry Ligand
Base
Solvent Temp
Yield
(9a:10a:11a:12a)
1
2
3
4
5
6
7
8
9
10
11¶
12¶
13*
-
PPh3
L1
ent-L1
L2
L3
L3
ent-L3
L4
ent-L4
L5
Et3N
Et3N
Et3N
Et3N
Et3N
Et3N
Et3N
Et3N
Et3N
Et3N
Cs2CO3
CH2Cl2
CH2Cl2
CH2Cl2
CH2Cl2
CH2Cl2
CH2Cl2
THF
THF
DCE
CH2Cl2
THF
r.t.
r.t.
r.t.
r.t.
r.t.
r.t.
r.t.
0 oC
r.t.
r.t.
r.t.
r.t.
0 oC
74%
79%
93%
78%
88%
72:23:04:01
68:18:11:03
21:06:71:02
22:14:19:45
60:05:09:26
89:00:11:00
91:02:07:00
03:95:01:01
00:00:100:00
28:01:03:68
88:00:00:12
13:00:02:86
04:00:00:96
91%
82% (73%)†
79% (95%)†
92% (89%)†
68%
53%
77%
72% (88%)†
L5
L5
Cs2CO3 CHCl3
Cs2CO3 CHCl3
Dehydrosantonin 7 (0.10 mmol, 1.0 equiv.), Schiff base 8a (0.15 mmol, 1.5 equiv.) and base (0.02
mmol, 0.20 equiv.) were reacted in solvent (0.10 M, 1 mL) with 5 mol% AgOAc and 6 mol% ligand
for 24-48 hours. Yield for all isomers and d.r. (9a:10a:11a:12a) were determined from the reaction
mixture by means of 1H NMR with CH2Br2 as the internal standard. †The main isomer was purified
by column chromatography; isolated yields of main isomers are shown in brackets. ¶0.50 equiv.
Cs2CO3 was used. *2.0 equiv. Schiff base 8a was used.
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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). The transformation tolerates diverse substituents in the para- and meta- position of the phenyl
group. In general, condition B delivers higher yield and diastereoselectivity compared to condition
A. However, ortho-substituted Schiff bases did not afford satisfying results (11m-11o) under
condition C because 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. In total, 61 stereochemically diverse compounds were efficiently
synthesized. The absolute configurations of 9b, 10f and 12e were unambiguously confirmed by
X-ray crystallographic analysis (see the Supporting Information, Figures S3-S5).
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Figure 2. Substrate scope for the stereodivergent synthesis of santonin-pyrrolidines.
Condition A: 0.10 mmol dehydrosantonin and 0.15 mmol Schiff base, 0.02 mmol Et3N in 1 mL
THF with 5 mol% AgOAc and 6 mol% L3 at r.t. for 24-48 hours; Condition B: as condition A but
ent-L3 was used at 0oC; Condition C: as condition A except L4 was used in 1 mL DCE; Condition
D: 0.10 mmol dehydrosantonin and 0.20 mmol Schiff base, 0.05 mmol Cs2CO3 in 1 mL CHCl3
with 5 mol% AgOAc and 6 mol% L5 at 0 oC for 24-48 hours. N.d.: not determined
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 Fig. S2), i.e. oxime formation, C-H activation[27] and
Heck reaction[28] to give analogs 13, 14 and 15 respectively (Figure 3). Under acidic conditions,
dehydrosantonin underwent a ring distortion reaction and rearranged to a phenol with
stereoinversion of C6[29] which was O-alkylated to yield benzyl ether 16. Aromatization of rings is
considered a genuine 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. Parthenolide 2 can be
viewed as a ring-opened analog of santonin. Transannular 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 α-methylene-δ-lactones 17 and 18 via a two-step procedure, resulting in a ring
contraction[19d] (see Figure 3; Supplementary Figure S2).
Thus, for the synthesis of a collection of pseudo-sesquiterpenoid alkaloids, three structurally
related natural sesquiterpene lactones were subjected to different ring distortion reactions.
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Figure 3. Sesquiterpene lactone-derived compound classes employed in the synthesis of
the pseudo-sesquiterpenoid alkaloids. (a) Ring distortion of commercially available
sesquiterpene lactones. (b). Structures of the nine lactones with six different scaffolds employed
in the synthesis of the sesquiterpenoid alkaloid collection.
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As shown in Figure 4 the dipolar cycloaddition also displays appreciable scope for different
sesquiterpene lactones. Changes in ring A and B of dehydrosantonin 7 are well tolerated, and the
oxime derivatives afforded the desired cycloadducts 20-23 and 24-27 in excellent yields and
selectivity. Cycloadditions 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 (44-47).[32] However, the reaction was highly sensitive to
modification of the γ-lactone. Compound 15 with a phenyl ring attached to the methylene group
showed low reactivity under the conditions for the exo-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 B and D. The ring size of the lactone has a clear impact
on the course of the reaction. Artemisinin derivatives 17 and 18 exclusively yielded endo products
(40-41; 42-43).
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Figure 4. Substrate scope for diverse sesquiterpenes. All reactions were carried out under
the conditions described above (Fig. 2). 1,3-dipolar cycloadditions with 15, 16, 17 and 18 were
conducted on 0.05 mmol scale.
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Cheminformatic and analysis of the sesquiterpene lactones and pseudo-sesquiterpenoid
alkaloids
For characterization 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. The NP likeness score compares the frequency
of occurrence of a given fragment in natural products with the frequency of occurrence in
commercial compounds. Scores >0 indicate that the fragments are more common in natural
products. Not unexpectedly, the synthesized sesquiterpene lactones directly obtained from
natural products showed high scores. In comparison the scores recorded for the pseudo-
sesquiterpenoid alkaloids are lower, since their scaffolds result from fragment combinations and
are novel (Supplementary Fig. S6). However, the scores calculated for the pseudo-
sesquiterpenoid alkaloids are represented in the area characteristic for NPs (Fig. 5a). This result
differs from the corresponding findings for pseudo natural product libraries, obtained 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 a high degree of NP character in the combination with other NP-fragments.
For analysis of compound class similarity, a representative group of the pseudo-sesquiterpenoid
alkaloids depicted in Fig. 4 was analyzed by computing Tanimoto similarity of the Morgan
fingerprints[37] (for the structures of the compounds included in the analysis see Supplementary
Fig. S7). As shown in Fig. 5b, sesquiterpene lactones 2, 3, 7 and 13-18 display low cross similarity
(Fig. 5b, region A), and cross-similarity to the pseudo-sesquiterpene alkaloids was low as well
(Fig. 5b, region B). The intraclass chemical similarity of the pseudo-sesquiterpenoid alkaloids 9a
and 20-42 was higher (Fig. 5b, region 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. In order to
analyze the incorporation of differently configured stereocenters in the compound collection, we
[38]
calculated principal moments of inertia (PMI)
(Fig. 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, the top-right represents spheres and
the bottom corner denotes disc-shaped compounds. As shown in Fig. 5c, the pseudo-
sesquiterpenoid alkaloids (red dots) are broadly distributed in the PMI plot. On the contrary, most
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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 a shift to the
sphere-disc part of the PMI plot, representative for a higher degree in stereogenic character.
Notably, even different stereoisomers occupy significantly different space in the plot, as
exemplified for stereoisomers 9c-12c (identified by blue arrows) in Fig. 5c. This analysis
demonstrates that for the compound class analyzed here combination of ring distortion and
pseudo NP synthesis yielded a stereochemically diverse compound collection that is structurally
distinct from the guiding natural products which were subjected to ring distortion.
a.
b.
c.
Figure 5. Characterization of the pseudo-sesquiterpenoid alkaloids by means of
cheminformatic. a, Comparison of the NP likeness scores calculated for the pseudo-
sesquiterpenoid alkaloids (Pseudo SAs) with the scores recorded for the NPs in the ChEMBL
database and in Drugbank. b, Tanimoto cross chemical similarity comparison of the sub-libraries.
c, Analysis of the molecular shapes of the sesquiterpene lactones and the pseudo-
sesquiterpenoid alkaloids by means of a PMI plot.
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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 a characteristic profile. The number of
significantly changed features in a profile relative to a DMSO 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% (Fig. 6a). We note that the α-methylene-γ-lactone is considered a promiscuous covalent
binder, potentially targeting multiple nucleophiles in proteins, and this reactivity is no longer
present in the pseudo-NPs. Thus, 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) was 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 Tab. S5) and analysis of the fingerprints revealed that the induction was
concentration-dependent (Supplementary Fig. S8). Five compounds showed >5% induction at 10
µM and at 50 µM 49 compounds induced significant morphological changes. To investigate 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
(Fig. 6b) which revealed that the two compound classes collectively display distinctly different
morphological profiles. This discovery is consistent with the notion that the α-methylene lactone
can serve as an electrophile to covalently bind biological nucleophiles. Combination with the
pyrrolidine fragment abolishes this mode of action. Even in comparison with saturated
sesquiterpene lactones, a relatively low cross biosimilarity was observed (Supplementary Fig.
9a,b).
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The influence of the ring distortion was gleaned from the cross biosimilarity analysis for pseudo-
sesquiterpenoid alkaloids synthesized from diverse SLs (Supplementary Fig. S9c). For
compounds representing modifications based on santonin (compounds 20, 24 and aromatized
scaffold 30), performance was relatively similar with cross biosimilarities >70% (Fig. 6c;
Supplementary Fig. S9c). Notably 40 and 42 which shared the highest cross chemical similarity
displayed distinctly different biological performance (Fig. 6c). However, 32, 36 and 40 showed
high cross biosimilarity (>75%; red oval) even though they are structurally different (Fig. 6c;
Supplementary Fig. S9c). The corresponding sesquiterpene lactones 2, 3 and 17 displayed highly
different biological performance (Supplementary Fig. S9d). After combination with the same
pyrrolidine fragment, the resulting biological performance does not differentiate the influence of
the SL scaffolds anymore. Pyrrolidine 44 itself did not induce any significant morphological
changes even at 50 µM concentration (Supplementary Tab. S5). Therefore, the convergence of
biological performance was not induced by a dominant effect of the pyrrolidine fragment.
In order to analyze the influence of different stereoisomers the pseudo-sesquiterpenoid alkaloids
derived from dehydrosantonin 1 were analyzed. The data revealed a separation between classes
9-12 (Fig. 4f; Supplementary Fig. S10). The finding that cell painting can differentiate
stereoisomers has been reported before[40]. As mentioned above, compounds 32, 36 and 40
displayed relatively high cross biosimilarity regardless of different sesquiterpene scaffolds. Very
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 Fig. S9e,f).
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10.1002/anie.202106654
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a.
b.
c.
d.
Figure 6. Characterization of the SLs and pseudo-sesquiterpenoid alkaloids by means of
high-content morphological profiling in the cell painting assay. a. Cell painting assay
fingerprint profiles for SL derivatives at 10 µM. Biosimilarity refers to the bioactivity similarity of
the cell painting assay fingerprint profiles. The induction value is calculated based on the
18
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10.1002/anie.202106654
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fraction of parameters (in %) that underwent significant changes (median absolute deviation
[MAD] value) upon compound treatment of at least ± threefold of the median determined for the
DMSO controls. b,c,d: Principal component analysis (PCA) of cell painting profiles. Unless
otherwise noted, all analyses were based on active samples at all concentrations. b.
Comparison between pseudo-sesquiterpenoid alkaloids (PSA) and SLs. Expl. Var. PC1=40.9%,
PC2=13.3%, PC3=7.9%. c. Comparison of pseudo-sesquiterpenoid alkaloids synthesized from
diverse sesquiterpene scaffolds under 30 µM concentration. Compounds in red circle displayed
>75% cross biological similarity. Expl. Var. PC1=50.0%, PC2=16.4%, PC3=11.3%. d.
Comparison of stereoisomeric santonin-pyrrolidines 9-12. Expl. Var. PC1=22.4%, PC2=16.3%,
PC3=13.6%.
Identification of a pseudo 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,
including autophagy, T-cell signaling as well as signal transduction through the Wnt- and the
Hedgehog (Hh) pathways. These investigations revealed a novel 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 a Hh-
dependent osteoblast differentiation assay using C3H/10T1/2 cells[43]. The pathway 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 Hh-dependent osteoblast differentiation with half-
maximal inhibitory concentration (IC50) of 1.5 ± 0.9 μM. Very notably, stereoisomeric pseudo-
sesquiterpenoid alkaloids 20-22 showed no inhibitory activity at 10 µM concentration. Under these
conditions, also compounds 47 and 48 which are partial structures of the new inhibitor chemotype
and pseudo-sesquiterpenoid 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 a more general sense.
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10.1002/anie.202106654
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Figure 7. Pseudo-sesquiterpenoid alkaloid 23 defines a new chemotype inhibiting Hedgehog
dependent differentiation of multipotent murine mesenchymal progenitor stem cells into
osteoblasts. a. Structures of compound 23 and its analogs. b. Hh-dependent osteoblast
differentiation assay. Data are mean values ±SD and representative of four biological replicates,
each performed in three technical replicates. c. IC50 values of compounds depicted in (a).
Conclusion
We describe the combination of the pseudo-natural product principle with chemical ring distortion
of NPs and a formal adaptation of the complexity-to-diversity strategy, to yield novel bioactive
natural product-inspired compound classes. In this new strategy, in general, on the one hand
diverse scaffolds are formed from fragment-sized NPs by subjecting them to ring-distortion
reactions. On the 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 a ring distortion
reaction, e.g. 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. Subsequently, chemical fragment recombination through complexity-generating
transformations affords complex and diverse compound collections. We exemplify the new
principle by the synthesis of a collection of pseudo-sesquiterpenoid alkaloids obtained from
fragment-sized sesquiterpenes by means of stereocomplementary 1,3-dipolar cycloadditions with
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10.1002/anie.202106654
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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 a novel 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, natural
product-inspired, structurally complex compound classes, which are not accessible by currently
described biosynthesis pathways. However, we note that silent, unexploited biosynthesis
pathways exist[45], and that biosynthesis pathways can be reassembled, e.g. in systems biology
approaches.[46] It is, therefore, 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, the isolation of the natural product vlasoulamine A was reported[47], whose scaffold
is structurally related to pseudo-sesquiterpenoid alkaloids 36.
Acknowledgements
This work was supported by the Max Planck Society (to H.W.). The authors thank Prof. Dr.
Andrey Antonchick and Prof. Dr. Christian Merten for insightful discussion about the reaction
mechanism.
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