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Bioorganic Chemistry
Pseudo Natural Products—Chemical Evolution of
Natural Product Structure
George Karageorgis, Daniel J. Foley, Luca Laraia, Susanne Brakmann, and
Herbert Waldmann*
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Keywords:
biological activity ·
chemical biology ·
fragment-based design ·
natural products ·
natural selection
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How to cite:
International Edition: doi.org/10.1002/anie.202016575
German Edition: doi.org/10.1002/ange.202016575
2 2021 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH Angew. Chem. Int. Ed. 2021,60,2–21
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1. Introduction
Small molecules are powerful tools for the dissection of
complex biological processes due to their ability to acutely
modulate their biological targets in a tuneable manner, and
are the dominant chemical entities[1] in our arsenal to treat
disease.[2] The discovery of novel small molecules with
suitable properties to interrogate biological phenomena in
a time-resolved manner is underpinned by the ability to
design and prepare new molecular scaffolds. However, the
vastness of chemical space[3] renders its complete exploration
by means of synthesis impossible, hampering our ability to
efficiently discover new bioactive molecular scaffolds.[4]
To address these challenges, several complementary
approaches have been developed. For example, diversity-
oriented synthesis (DOS)[5] is an approach aimed towards the
preparation of compound libraries whereby creation of
molecular diversity is an embedded part of the synthetic
strategy (Figure 1 A). DOS exploits intermolecular building-
block coupling, followed by intramolecular functional group
pairings[6,7] resulting in molecular scaffolds with high stereo-
chemical content and fractions of sp3-hybridised centres. DOS
has led to the discovery of a range of bioactive small
molecules,[8] which have inspired drug discovery pro-
grammes[9] or have been used as tools to investigate biological
processes.[10]
Natural products (NPs) are a rich and continuously
explored resource in the search for bioactive small mole-
cules.[11] Biology-oriented synthesis (BIOS) takes inspiration
from NP structures to guide the synthesis of biologically
relevant compound collections. BIOS employs a hierarchical
classification of NP scaffolds, generated by a computational
algorithm,[12] to select simplified, NP-derived and -inspired
scaffolds which retain their ability to modulate biological
systems (Figure 1 B,C,E)[13] yet are more synthetically tract-
able. BIOS exploits the gaps in chemical space not covered by
NPs and facilitates the preparation of derivatives. However,
the partial retention of the guiding NP-scaffold limits the
chemical space that can be explored. Additionally, BIOS
scaffolds may also inherit the same kind of bioactivity as the
guiding NPs, thus limiting the explora-
tion of biological space.[14]
To overcome these limitations and
take advantage of the biological rele-
vance of NPs, the design of novel
scaffolds can benefit from the efficient
sampling of chemical space offered by
fragment-based compound design.[15]
This argument is supported by the fact
that NPs may already be fragment-
sized,[16] or can be converted into frag-
ment-sized ring-systems,[17] and the
properties of NPs are retained in these
NP-derived fragments.[18] Thus, com-
bining NP-derived fragments in unpre-
cedented ways can provide access to
molecular scaffolds which inherit the
biological characteristics of NPs, yet lie
in biologically relevant regions of
chemical space not attainable by nature. We have termed
these compounds “pseudo-natural products” (pseudo-NPs),
as these novel scaffolds would not be accessible through
current naturally occurring biosynthetic pathways and can be
regarded as non-biogenic fusions of NP-derived fragments
(Figure 1B,D).[19,20] The term “pseudo-natural product” has
previously been used sparingly, for example, to describe cyclic
peptides,[21,22] and products of altered or intercepted biosyn-
thetic pathways.[23,24] We have demonstrated how alternate
scaffold connectivity patterns can be used to design new
pseudo-NP scaffolds which occupy different regions of
chemical space.[20] This design process can be thought as
a chemical counter-part to naturally occurring (biologically-
driven) evolution of compound structure, which relies on
a simple optimisation algorithm comprising diversification
and selection, as introduced below.
Pseudo-natural products (PNPs) combine natural product (NP)
fragments in novel arrangements not accessible by current biosynthesis
pathways. As such they can be regarded as non-biogenic fusions of
NP-derived fragments. They inherit key biological characteristics of
the guiding natural product, such as chemical and physiological
properties, yet define small molecule chemotypes with unprecedented
or unexpected bioactivity. We iterate the design principles under-
pinning PNP scaffolds and highlight their syntheses and biological
investigations. We provide a cheminformatic analysis of PNP collec-
tions assessing their molecular properties and shape diversity. We
propose and discuss how the iterative analysis of NP structure, design,
synthesis, and biological evaluation of PNPs can be regarded as
a human-driven branch of the evolution of natural products, that is,
a chemical evolution of natural product structure.
[*] Dr. G. Karageorgis, Dr. D. J. Foley, Dr. L. Laraia,
Prof. Dr. H. Waldmann
Max-Planck Institute of Molecular Physiology
Otto-Hahn Strasse 11, 44227, Dortmund (Germany)
Dr. D. J. Foley
Current address: School of Physical and Chemical Sciences,
University of Canterbury
Private Bag 4800, Christchurch 8140 (New Zealand)
Dr. L. Laraia
Current address: Department of Chemistry
Technical University of Denmark, kemitorvet 207
2800 Kgs. Lyngby (Denmark)
Prof. Dr. S. Brakmann, Prof. Dr. H. Waldmann
Faculty of Chemistry and Chemical Biology
TU Dortmund University
Otto-Hahn Strasse 4a, 44227, Dortmund (Germany)
E-mail: herbert.waldmann@mpi-dortmund.mpg.de
Supporting information and the ORCID identification number(s) for
the author(s) of this article can be found under:
https://doi.org/10.1002/anie.202016575.
2021 The Authors. Angewandte Chemie International Edition
published by Wiley-VCH GmbH. This is an open access article under
the terms of the Creative Commons Attribution License, which
permits use, distribution and reproduction in any medium, provided
the original work is properly cited.
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In this Minireview we describe the development of the
pseudo-NP concept, the principles of pseudo-NP-library
design, and their relationship to the biological evolution of
NP structure. We describe syntheses of pseudo-NP compound
collections and their investigation in different biological
settings, showing that pseudo-NPs can harbour novel bioac-
tivity not shared by the guiding NPs.
2. Design Principles for Pseudo-Natural Products
In general, new pseudo-NP scaffolds preferably have
a high degree of three-dimensional character, as chirality and
stereogenic content contribute to biological relevance and
bioactivity.[25–27] To provide structurally distinct pseudo-NPs,
NP-derived fragments with complementary heteroatom con-
tent may be combined (e.g. N and O). Combination of
fragments sourced from different organisms or biosynthetic
pathways may increase the novelty of the resulting scaffold,
and its biological relevance, by combining features from
discrete and otherwise unrelated areas of chemical space.
NP-derived fragments can be connected in entirely novel
arrangements not found in nature (see below), or, to retain
certain biologically relevant components in the resulting
pseudo-NP, through specific structural patterns already en-
countered in NPs. These connectivity patterns can be classed
into two sets; those where the connected fragments share
common atoms (Figure 2, Panel A, 1–3), and those where the
fragments are connected through intervening atoms (Fig-
ure 2, Panel A, 4–8). For example, two fragments can be
combined through a common edge and sharing two common
atoms as in the abstract scaffold 1. This connectivity pattern
can be observed in alkaloids containing an indole and
a chromane fragment, such as 9[28] (Figure 2, Panel B).
George Karageorgis was born in Nicosia,
Cyprus, and graduated from the Aristotle
University of Thessaloniki, Greece, with
a B.Sc. Chemistry degree in 2010. He
obtained a M.Sc. in Chemical Biology from
the University of Leeds in 2011, and then
joined Prof. A. Nelson’s group in Leeds as
a PhD student where he worked on the
development of activity-directed synthesis.
He earned an Alexander von Humboldt
Fellowship and joined Prof. H. Waldmann’s
group in 2015 at the Max Planck Institute
in Dortmund, working on the design and
syntheses of biologically relevant small mol-
ecules with novel molecular scaffolds.
Dan Foley carried out his PhD with Profs.
Steve Marsden and Adam Nelson at Univ.
Leeds (2015), then completed an EPRSC
Doctoral Prize Fellowship (2015–2017). He
carried out further postdoctoral studies
(2017–2018) with Prof. Herbert Waldmann
at the MPI of Molecular Physiology, where
he held a Marie Skłodowska-Curie Fellow-
ship. Dan recently joined the faculty at the
University of Canterbury, New Zealand,
where his research focuses on the develop-
ment of new synthetic methods of value to
molecular discovery and medicinal
chemistry.
Luca Laraia studied chemistry at Imperial
College London, before moving to the Uni-
versity of Cambridge to carry out his Cancer
Research UK-funded PhD in chemical
biology with Prof. David R. Spring and Prof.
Ashok R. Venkitaraman. After graduating in
2014 he moved to the Max Planck Institute
of Molecular Physiology (Dortmund, Ger-
many), first as an Alexander von Humboldt
postdoctoral fellow and then as project
leader in the chemical biology department
with Prof. Herbert Waldmann. He moved
to DTU in November 2017 to take up an
Assistant Professorship in chemical biology.
Susanne Brakmann studied Chemistry at
the TU Braunschweig (diploma, 1988) and
received her Ph.D. from the University of
Karlsruhe (with Reinhold Tacke, 1991). In
1992, she moved to the MPI for Biophysical
Chemistry in Gçttingen as a postdoctoral
researcher with Manfred Eigen. During the
period of 1999 to 2000, she applied her
knowledge in an employment at Evotec
Biosystems AG and in 2001, moved to the
University of Leipzig as a junior research
group leader. She received her habilitation
from the TU Braunschweig in 2004 before
she joined the TU Dortmund’s Faculty of
Chemistry and Chemical Biology.
Herbert Waldmann obtained his PhD in
organic chemistry in 1985 under the super-
vision of Horst Kunz. After a postdoctoral
period with George Whitesides at Harvard
University, he returned to the University of
Mainz and completed his habilitation in
1991. He was appointed as Director at MPI
Dortmund and professor of Biochemistry at
TU Dortmund University in 1999. His
research focuses on new principles for the
design and syntheses of natural-product-
inspired compound classes and their
biological evaluation.
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Alternatively, fragments can be connected through a single
common atom resulting in a spiro-fusion (Figure 2, Panel A,
2), as in the case of the NP ()-horsfiline, 10.[29] If two
fragments are connected through three consecutive common
atoms, such a pattern would result in a bridged-fusion
(Figure 2, Panel A, 3). An example of this connectivity
pattern can be seen in the NP sespenine, 11.[30] The bipodal
connection can be observed in pseudo-natural product
scaffold 12,[31] and an example of a bridged tripodal con-
nection is found in structures such as scaffold 13.[32]
Exploiting different connectivity patterns to connect NP-
fragments gives rise to pseudo-NP scaffolds which can be used
to probe distinct regions of biologically relevant chemical
space (Figure 2, Panel C, Design Principle 1).[20] For example,
scaffolds 14 and 15 are pyrrotropanes stemming from the use
of two different connectivity patterns; an edge-fusion and
Figure 1. Approaches for the design and preparation of novel biologically relevant molecular scaffolds. A) DOS employs a build-couple-pair
approach leading to diverse scaffolds that can be considered NP-like. B) Natural products are secondary metabolites that provide inspiration for
the discovery of bioactive small molecules. C) BIOS draws inspiration from NPs, preparing analogues of NP-derived scaffolds with reduced
structural complexity. D) Pseudo-natural products emerge from unprecedented combinations of NP-derived fragments. E) Differences between
(left to right) NPs, NP-derived scaffolds (i.e. scaffold is identical to NP scaffold), NP-inspired scaffolds (i.e. scaffold is closely related to NP-
scaffold[13]), and pseudo-NP scaffolds.
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Figure 2. Design principles for pseudo-NP scaffolds. In general, pseudo-NP scaffolds should have high stereogenic content and three-dimensional
character, complementary heteroatom content, and combine fragments from different sources. Parts of structures have been greyed for clarity.
Fragments are coloured for distinction. Black dots represent connectivity atoms. A) Examples of connectivity patterns illustrated with abstract
structures. B) Examples of the connectivity patterns in natural and pseudo-natural product scaffolds. C) Design principles for pseudo-NPs.
Pseudo-NP scaffolds arise by combining different fragments using different connectivity patterns, or by combining the same fragments and the
same connectivity patterns through different common atoms. It is also possible to combine more than two fragments.
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a spiro-fusion, respectively. In addition, combinations of the
same NP-fragments, using the same connectivity pattern, can
also result in further regioisomeric pseudo-NP scaffolds by
changing the connectivity points between the connecting
fragments (Figure 2, Panel C, Design Principle 2). An exam-
ple can be seen when comparing pyrroquinolines 16 and 17.
Additionally, these connectivity patterns have been identi-
fied, and can be exploited for the combination of more than
two NP-derived fragments at a time (Figure 2, Panel C,
Design Principle 3). Taken together, these design principles
can be used to reveal unexplored areas of chemical space with
potentially high bioactivity value.
3. Synthesis of Pseudo-NP Libraries
3.1. Chemical Synthesis of Pseudo-NPs by NP Fragment Fusion
A significant strategic approach for the preparation of
pseudo-natural product libraries is to develop and apply novel
synthetic chemistries that enable the de novo fusion of ring
systems from natural products in fusion patterns unprece-
dented in nature, ideally combining ring systems that are
normally not found together.[20] Here we provide some
examples of pseudo-NPs that meet the design criteria
described in the section above.
Edge-fusion of NP fragments: In work from our group,
pyrano-furo-pyridones 21–22 and 24–25, combining 2-pyri-
done and (dihydro)pyran fragments, were prepared by
annulation reactions (Scheme 1a).[33] These included Pd-
catalysed Tsuji–Trost cascades (!21–22), Pd-catalysed Tsu-
ji–Trost oxa-Michael cascades ( !24), and quinine-mediated
Michael transacetalisation cascades (!25). Subsequent
modifications provided a library of >160 compounds. Indo-
morphans 28, combining indole- and morphan-alkaloid frag-
ments, were prepared from known bicyclic ketones 26, which
were subjected to Fischer indolisations.[34] The resulting
compounds were decorated to provide a screening collection
of >40 compounds (Scheme 1 b). Yu and Liu described the
synthesis of benzodiazepine-fused isoindolinones 32, using
a mesoporous silica nanoparticle-catalysed multi-component
reaction (Scheme 1 c).[35]
Spiro-fusion of NP fragments: The spirocyclic indiridoids
35, combining characteristic substructures of iridoid terpenes,
oxindole, and dihydropyran fragments, were prepared using
an AuI-catalysed reaction cascade involving a 6-endo-dig ene–
yne cyclisation followed by ring opening and rearrangement
(Scheme 1 d). Variation of the Au catalyst and the substitution
pattern on the starting material gave rise to differentially
fused scaffolds (not shown).[36]
Bridged-fusion of NP fragments: Chromane and tetrahy-
dropyrimidinone fragments were combined to give chromo-
pynones 36, which were synthesised in a one-pot procedure
involving a Biginelli reaction (Scheme 2a). Indotropanes 37
combined tropane and indole fragments by harnessing a CuI-
catalysed enantioselective intermolecular 1,3-dipolar cyclo-
addition (Scheme 2b). Indoles were also fused with piper-
idones to prepare indopipenones 38, using a one-pot process
that included the use of an enantioselective Pictet–Spengler
reaction (Scheme 2 c).[37]
Combination of the same NP fragments using different
connectivity patterns (Scheme 2d):A 155-membered pyrro-
quinoline pseudo-NP collection was generated by combining
the tetrahydroquinoline and pyrrolidine fragments in eight
different molecular connectivity/regioisomeric arrange-
ments.[38] Notably, scaffolds 40,42, and 45 have the same
fused scaffold at the graph level,[39] but vary the position of the
pyrrolidine nitrogen, whilst 47 has spirocyclic connectivity,
and 49 merges the scaffolds in a bridged fashion. A unifying
synthetic approach harnessing Ag-catalysed 1,3-dipolar cyclo-
additions of azomethine ylides with electron deficient alkenes
delivered the majority of the pyrroquinoline scaffolds (40,42,
and 45). Bridged bicyclic compounds 49 were prepared via
Povarov-type dimerisation of enamines, generated from ani-
lines and methyl pyruvate (!48), followed by acid-mediated
lactamisation. Oxidised versions of scaffolds 40,42, and 45,in
which the pyrrolidine ring was aromatised to the correspond-
ing pyrrole were also prepared, either by direct oxidation of
40 or 42 with DDQ, or, to give the oxidised versions of
scaffold 45, by developing a novel reaction in which azome-
thine ylides were reacted with quinolinium salts (not shown).
3.2. Pseudo-NPs from Existing NPs
Natural products themselves can be fragment-sized[16] and
can therefore serve as starting points for new pseudo-NPs
(Scheme 3).[40,41] This design principle was employed in
unprecedented fusions of the highly NP-prevalent indole or
chromanone ring systems with readily accessible NP-frag-
ments derived from commercially available Cinchona alka-
loids quinine (QN) and quinidine (QD), griseofulvin (G), and
sinomenine (S) (structures not shown).[40,41] Firstly, ketone
fragments (50,51,56,59, and 62) were derived from the
natural products in short synthetic sequences (3 steps). The
ketones were harnessed in a range of annulation reactions,
including edge-fusion by indolisations (blue arrows; com-
pounds 52–53,57–58, and 63–64), and spiro-fusions by either
oxa-Pictet–Spengler reactions (green arrow; compounds 65
and 66), or Kabbe condensations (pink arrows; compounds
54–55 and 60–61). Indolisation was achieved either by Pd-
catalysed Heck-type annulation (conditions a) or Fischer
indolisation (conditions c). The indolisations produced sepa-
rable regioisomers from ketone 62 (!63 and 64). Compounds
derived from the Kabbe condensation in each case provided
two separable diastereomers at the spirocyclic point of
fragment connection (e.g. 54,55 and 60,61). Overall, a library
of 244 compounds was prepared.
Synthesis of pseudo-NPs using microorganisms: Li de-
scribed the synthesis of novel pseudo-natural products from
an ortho-quinone methide 69, produced by P. crustosum PRB-
2, a clavatol-producing fungus. P. crustosum PRB-2 was
directly incubated with alternate indole- and aniline nucleo-
philes, which reacted with the ortho-quinone methide 69,
a Michael acceptor, to produce a total of 15 compounds
including 70–74 (Scheme 4).[42]
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4. Cheminformatic Analysis
To examine the relationship between pseudo-NPs and
NPs the natural product likeness score (NP-Score)[43] was
calculated for selected pseudo-NP classes reported by us so
far (see SI, Figures S1–S6). Briefly, this cheminformatic tool
compares connection pathways between atoms (up to six)
found in NPs and a reference set of synthetic molecules. For
comparison, we also determined the NP-Score of the set of
experimental and approved drugs in DrugBank[44] (Figure 3,
top left, orange) and the set of NPs in the ChEMBL
repository[45] (Figure 3, top left, green). Most of the ChEMBL
NPs display a NP-Score between 0 and 4, while the molecules
in DrugBank display a much wider NP-Score distribution
between 4 and 3. There is significant overlap between these
two sets, as a range of molecules in the DrugBank set are
either inspired by NPs or are NPs themselves.[46–48] Pseudo-
NPs (Figure 3, top left, blue) however, display a narrower NP-
Score distribution between 2 and 2.[20] This observation may
appear counterintuitive at first glance, as pseudo-NPs consist
Scheme 1. De novo synthesis of edge- and spiro-fused pseudo-NPs. a–c) Synthesis of edge-fused pseudo-NPs, including a) pyrano-furo-pyridones
21–22 and 24–25, by Pd- or quinine-catalysed cascades; b) indomorphans 26, by Fischer indolisations; c) isoindolinones 32, by a mesoporous
silica nanoparticle-catalysed multi-component reaction. d) Synthesis of spiro-fused indiridoids 35 by an AuI-catalysed cascade.
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Scheme 2. De novo synthesis of bridged-fused pseudo-natural products and combination of pyrrolidine and tetrahydroquinoline NP fragments in
alternative fusion modes. a–c) Synthesis of bridge-fused pseudo-NPs, including a) chromopynones 36, via a multicomponent reaction ;
b) indotropanes 37, using a a CuI-catalysed enantioselective 1,3-dipolar cycloaddition; c) indopipenones 38, via an enantioselective Pictet–
Spengler reaction. d) Combination of the same NP fragments in different connectivity patterns, using Ag-catalysed 1,3-dipolar cycloadditions as
a general unifying approach. Bridged bicycles 49 were prepared via a Povarov-type reaction.
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Scheme 3. Synthesis of 244 pseudo-NPs from natural product fragments using indolisation reactions (blue arrows, conditions a and c); oxa-
Pictet–Spengler reactions (green arrows, condition d) ; and Kabbe reactions (pink arrows, condition c). * 1 step from 56.
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Scheme 4. Synthesis of pseudo-natural products by incubation of nucleophiles with P. crustosum PRB-2. Exploiting a microorganism to source the
reactive ortho-quinone methide 69 provided access to a total of 15 diverse compounds including chemotypes 70–74.
Figure 3. Cheminformatic analyses of pseudo-NPs. Top left : NP-score distributions of pseudo-NPs (blue line), approved and experimental drugs
in DrugBank, and NPs in the ChEMBL repository. Top right: Plot of molecular weight against lipophilicity of each molecule: 76% of compounds
fall within the “rule-of-five” space denoted by the dashed black line. Bottom left: PMI plot demonstrating the high degree of three-dimensional
character of pseudo-NPs, as most of the molecules lie away from the rod-disc-like axis. Bottom right: PMI plot of selected natural products and
non-naturally occurring bioactive compounds, demonstrating a similar breadth and distribution with pseudo-NPs (see SI, Scheme S7 for chemical
structures).
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of NP-derived fragments or fragment-sized NPs. The lower
collective NP-Score of pseudo-NPs can be explained by the
fact that their design is based on unprecedented combinations
of NP-derived fragments. Thus, the particular connection
pathways between atoms in pseudo-NPs are different to an
extent from those in NPs. As a result, pseudo-NPs can be
regarded as novel molecular matter, extending beyond the
mere sum of their constituent parts.
Additionally, the NP-Score distribution of pseudo-NPs
has significant overlap with the NP-Score distribution of
DrugBank compounds. Further analysis of the molecular
properties of pseudo-NPs, in particular molecular weight and
calculated lipophilicity, showed that most pseudo-NPs (67 %)
fall within the “rule-of-five” space[49] (Figure 3, top right).
This observation extends to additional metrics such as total
polar surface area[50] and fraction of sp3-hybridised carbons[26]
(Table S1), suggesting that pseudo-NPs may be inherently
endowed with desirable physiochemical properties. Further-
more, the shape diversity of the pseudo-NP collection was
assessed by calculating the molecules three principal mo-
ments of inertia (PMI).[51] This assessment enables the direct
evaluation of a molecules shape in three-dimensional space.
The results (Figure 3, bottom left) show that pseudo-NPs have
a high degree of three-dimensional character, and their
distribution in the triangular plot is not congested along the
rod-disc-like axis as observed with compound collections
deriving from combinatorial design approaches.[52] The dis-
tribution of pseudo-NPs in the PMI plot is also similar to a set
of selected NPs and non-naturally occurring bioactive com-
pounds (Figure 3, bottom right, see SI, Scheme S7 for
structures), further demonstrating the high degree of shape
diversity among different pseudo-NP collections.
5. Biological Evaluation of Pseudo-NP Libraries
To determine the utility of pseudo-NPs systematically,
broad biological screening is recommended. In this context,
unbiased phenotypic screens offer an advantage over target-
based screens due to their greater coverage of biological
space. This occurs because typically any given phenotype can
be affected by the modulation of multiple macromolecular
targets. All pseudo-NPs we have produced have been
screened in cell-based assays monitoring glucose uptake,
autophagy, Wnt and hedgehog signalling pathway activity,
and induction of reactive oxygen species (ROS). Importantly,
modulators of these therapeutically relevant phenotypes were
prevalent amongst pseudo-NPs. For example, chromopy-
none[19] and indomorphan[34] pseudo-NPs such as 77 and 75
(Figure 4) were identified as highly potent inhibitors of
glucose uptake by targeting the glucose transporters GLUT1
and GLUT3. Both are unprecedented chemotypes for GLUT
inhibition. Furthermore, the 7-azaindole-fused quinine de-
rivatives[40] such as 76 (Figure 4) were highly potent inhibitors
of starvation- and rapamycin-induced autophagy. Important-
ly, the appropriate combination of NP fragments was essential
for GLUT and autophagy inhibition, as none of the individual
fragments possessed this activity. Finally indotropanes includ-
ing Myokinasib (Figure 4, 80), inhibited correct cytokinesis by
acting as ATP-competitive inhibitorsof the myosin light chain
kinase 1.[53] Crucially, Myokinasib represents an unprecedent-
ed and unexpected bioactivity profile, as it is the first MLCK1
inhibitor reported, and a novel kinase inhibitory chemotype.
In addition to screens monitoring specific cellular pheno-
types, a high-content multiparametric imaging approach
termed the cell painting assay (CPA)[54] can be used to
determine bioactivity in a broad sense (Figure 5). By staining
Figure 4. Structures of bioactive pseudo-NPs and their molecular targets. Pseudo-NPs display a diverse range of biological activities ranging from
metabolic (GLUT inhibition) to anti-microbial (MptpA) related targets. GLUT =glucose transporter; VPS34=vacuolar protein sorting 34;
MLCK1=myosin light chain kinase 1.
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different organelles with fluorescent dyes and imaging in five
fluorescent channels, a multitude of parameters related to
cellular morphology can be probed simultaneously.[55,56] These
can be used to generate fingerprints specific to a given
treatment condition; for example, incubation with a com-
pound. Compounds that induce a significant change com-
pared to controls, as assessed by a so-called “induction value”
or alternatively by the Mahalanobis distance, are classed as
bioactive.
For each of the different connectivity/regioisomeric
arrangements of the pyrroquinolines (Scheme 2 d), distinct
phenotypic outcomes were observed in the CPA.[38] This work
showed that differential combination of a confined set of NP-
fragments can provide scaffolds that exhibit diverse biological
Figure 5. Morphological profiling using the cell painting assay. Cells are incubated with test compounds before being fixed and stained with dyes
for different cellular components. Automated image acquisition and analysis allows morphological fingerprints to be generated for each small
molecule. These can be compared to generate target or mode-of-action hypotheses, as well as clustering of bioactive molecules. Adapted from
Ziegler et al.[56] .
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activity patterns. Morphological screening of the pseudo-NP
library described in Scheme 3 in the CPA revealed that a large
proportion of compounds were bioactive. Notably, and as
expected for pseudo-NPs, the griseofulvin- and Cinchona
alkaloid-derived compounds were shown to have significantly
different bioactivity profiles compared to their parent natural
products. Investigation of the scaffolds and their bioactivity
profiles by principal component analysis revealed that, in
general, alternative combinations of the different fragments
generated disparate biological effects, as indicated by their
different phenotypic profiles. The fragments from which these
pseudo-NPs were constructed, therefore, do not dominate
bioactivity of the new combination, and can be considered
favourable choices for the design and synthesis of further
pseudo-NP classes with novel fragment combinations. Nota-
bly, however, compounds derived from the sinomenine NP-
fragment generally displayed highly similar phenotypic pro-
files, suggesting a dominating biological effect by the im-
bedded morphine-type scaffold, such that the sinomenine
fragment may not be a favourable choice for additional
fragment combinations aiming at different bioactivity. In-
deed, these insights enabled prospective design of pseudo-NP
collections with alternative bioactivity profiles.
As well as identifying bioactivity in a general sense, the
CPA has the additional advantage of potentially identifying
molecular targets or predicting compound mode-of-action by
comparing the fingerprint produced by a novel small mole-
cule to those of an annotated reference set. Furthermore, non-
dominant fragments can be identified in this manner, which
can provide guidance for the design of subsequent pseudo-NP
classes with potentially new biological activity (see Section 5).
Crucially, and unlike chemoproteomic target identification
strategies, the CPA can identify targets and modes-of-action
that occur as a result of the modulation of a non-protein
target. For example, it is an excellent tool for identifying
compounds that interfere with lysosomal activity,[57] and metal
ion chelators.[58] In the context of pseudo-NPs, the CPA was
able to identify the target of pyrano-furo-pyridones as
mitochondrial complex I due to the high biosimilarity of
these compounds with the reported complex I inhibitor
aumitin.[59] The target of the azaindole-fused quinine, Aza-
quindole-1, was identified as the lipid kinase VPS34 due to
the high biosimilarity of this compound to the selective
VPS34 inhibitor SAR405.
6. Chemical Evolution of NP Structure
NPs are biologically relevant because they can interact
with proteins which, for example, serve as receptors, or
enzymes, and have co-evolved together with specifically
binding small molecules. New or altered NPs emerge by
natural evolution. They are the product of coordinated
enzymatic cascades which, in turn, result from regulated gene
clusters. During organismal replication, alterations such as
gene recombination, duplication, or mutation occur within
these clusters which lead to modified enzymes and thereby to
altered NPs. A typical scenario for the consequences of
recombination and mutation is that, in a certain organism
which executes the synthetic strategy for a new NP, binding
between a NP and a specific protein is enabled or improved,
and consequently exerts a positive reproductive effect for
exactly this organism. Natural evolution can therefore be
regarded as a gigantic “process of learning by matter” which is
based on a simple algorithm as well as the requirement that
every target molecule, cell, or organism can be described by
the information which is passed on to descendants. The genetic
information of every living organism or, their genotype,is
“encoded” by the linear copolymers of DNA and RNA, and it
is “expressed” in proteins which form the entirety of proper-
ties of this genotype, the phenotype and target of selection.
The evolutionary algorithm has long been exploited for
the “directed evolution of biomolecules” such as RNA or
proteins,[60] and specifically the tailoring of naturally occuring
enzymes for specific purposes.[61] However, the principle of
evolution can also accelerate the development of NPs and
lead to pseudo-NPs. When considering evolutionary optimi-
sation of NPs, we need to rethink the terms genotype and
phenotype: Each small molecular structure encodes chemical
information that is the sum of informational contributions
stored within the chemical microenvironment of every
individual atom of a molecule.[62] From this perspective, NP-
derived fragments consist of connected informational units
similar to DNA or RNA and represent individual genotypes.
Their chemical information is densely packed and encom-
passes high fractions of sp3-hybridized atoms, high stereogenic
content, high heteroatom content, and low aromaticity.[62, 63]
Each genotype determines a three-dimensional structure with
specific physico-chemical and biochemical characteristics,
that is, the genotype is expressed as a phenotype. The
phenotype, in turn, can form complementary interactions
with other molecules and, consequently, serve as the target of
selection and evolutionary optimisation. A similar genotype–
phenotype dichotomy is known for RNA.
The way in which previously unknown NPs that combine
known fragments in a new or uncommon way can arise, was
recently shown in a landmark study: For this, genetic material
encoding enzymatic cascades from different sources and
cDNA libraries of diverse organisms was recombined in
a microbial host and submitted to selective constraints
requiring the action of target proteins in a survival assay
(Figure 6).[64,65] From surviving cells, 74 novel chemical
structures were isolated, more than 75% of which had not
been described so far. Their detailed inspection revealed that
a fraction of these compounds emerged from hitherto
unknown combinations of known NP-derived substructures
(i.e. combination of substructures for the first time), as well as
new combinations of known natural product-derived sub-
structures (i.e. combinations of substructures in new con-
nectivities).[65] This finding suggests that novel combinations
of NP fragments in nature can be induced, and that the
current repertoire of known NPs, in principle, can be
complemented by existing, but maybe currently not actively
or differently used biosynthetic pathways. By analogy, NP
fragments could be employed as “inheritable building blocks”
in a new, evolutionary strategy towards bioactive compound
discovery. In this strategy, the novel combination of NP-
fragments by biosynthetic steps as described above would be
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replaced by synthetic fragment combinations leading to
pseudo-natural products. These pseudo-NPs currently have
not been identified from natural sources, but the example
described above suggests that, in principle, they may be
amenable to biosynthesis. Also, it is possible that they may
indeed exist in nature but have not been identified yet. In fact,
after the pyrroquinoline scaffold 38 had been synthesised in
the context of a pseudo-NP program,[38] the scaffold was
reported to occur in nature in the NP Albogrisin.[66]
Evolutionary optimisation requires that genotypes are
varied by recombination and/or mutation, and their pheno-
types are sorted under specific constraints. The best-perform-
ing phenotypes (“the fittest”) are selected because they
confer an advantage, for example, improved molecular
recognition or effector features. Since the fittest descendants
have a genotype (=hereditary information) that was not
present before, evolution can also be understood as a process
during which new information is continually generated.
As shown below (Figure 7), the design and synthesis of
pseudo-NPs starts from a pool of biologically relevant
fragments, corresponding to a set of genotypes. Their
variation (mutation) is achieved using synthetic strategies
which consist of fragment assembly by recombination (Fig-
ure 2; connectivity patterns) and further derivatisation. The
resulting compound library is a pool of new genotypes which
express new phenotypes, represented by their (three-dimen-
sional) structures which confer the ability to recognise and
bind target proteins. When exposed to a biological system
such as a cell culture, some phenotypes eventually reveal new
biological activity, for example, by perturbing vital processes
or modifying biomarkers. Selection takes place when these
new pseudo-NPs are identified, isolated and characterised,
the latter serving for recognition and understanding of the
newly generated chemical information. Following natures
example, the complete process may be repeated in a circular
manner, where the output of a previous cycle serves as the
input for a subsequent cycle (Figure 7). In this sense, the
process of: i) designing, ii) preparing, and iii) biologically
characterising pseudo-NPs, resulting in new information
(both chemical and biological), which subsequently initiates
iterative process cycles, can be regarded as a chemical
evolution of natural product structure.
Figure 6. Scheme of the “synthetic biology combinatorial genetics” approach developed by Evolva.[65] Briefly, the procedure starts with the
collection and cloning of genetic material encoding biosynthetic pathways together with cDNA libraries from diverse natural sources. After
recombination and expression in a microbial host, additional or altered enzymes will supplement or modify existing pathways, thereby enabling
the synthesis of new or modified natural products. Their presence and possible action in vivo can be challenged in cellular assays in which
surviving clones may reveal a “fitter” or, simply, altered behaviour. Clones are sorted according to selective criteria and submitted to a range of
preparative and analytic procedures for obtaining and identifying small molecules.
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7. Discussion
The discovery of bioactive small molecules that can
modulate biological processes in a selective and time-resolved
manner, as well as their efficient preparation, can have great
impact on the understanding of biology and disease, and thus
provide new therapeutic opportunities.[2] Historically, NPs
have provided ample inspiration for the design of new
biologically relevant compounds.[46]
Several approaches have been developed exploiting NP
structures in distinct ways. More specifically, biology-oriented
synthesis (BIOS), exploits substructures of NP scaffolds to
prepare compound collections that inherit the biological
relevance of NPs.[12,13] Alternatively, “complexity-to-diversi-
ty” (CtD), exploits low-to-medium molecular weight NPs
amenable to chemoselective processes as starting materials
for the preparation of diverse and biologically relevant
molecular scaffolds.[68–70] However, both of these approaches
may be limited in the extent of the exploration of chemical
space that they can offer. Additionally, the observed biolog-
ical activity of compound classes directly delineated from
existing NPs may not differ significantly from that of the
guiding NP structure, and thus limits the range of biological
space that can be interrogated by them.
In an effort to mitigate these limitations a new approach
has been developed, involving the fusion of NP-derived
fragments in unprecedented combinations, affording novel
biologically relevant molecular structures termed pseudo-
natural products (pseudo-NPs). This approach builds on the
biological relevance of NPs and the efficient exploration of
chemical space offered by combinations of fragment-sized
compounds. In this context the original “rule-of-three”
definition of fragments was relaxed since it may not be
entirely valid for natural products, and as a filter for fragment
likeness AlogP 3.5, MW 120–350 Da, 3 hydrogen bond
donors, 6 hydrogen bond acceptors, and 6 rotatable
Figure 7. Focused chemical evolution. (1) The cyclic procedure starts with the generation of a “pool of genotypes” which is synthesised from NP
fragments (=inheritable chemical information) using synthetic strategies that allow for the combinatorial application of a set of feasible
connectivity patterns as well as further derivatisations. (2) Structural properties of the resulting compound library, such as content of sp3-
hybridised atoms, stereocentres, heteroatoms, and aromaticity, account for the expression of “phenotypes”—the potential to interact with specific
structural motifs of proteins. (3) The compound library then is applied to a cellular screening platform (e.g., yeast cells) which is optically
monitored for structural changes, e.g., by fluorescence imaging. Data are combined, analysed, and sorted according to selective constraints.
(4) Molecules causing a desired change in the cellular system are isolated and identified (NMR, MS) and submitted to further structural
characterisation, for example, by co-crystallisation with putative target proteins (example used here: 4PYP[67]). The outcome of this round is the
beginning of a new cycle which includes the “new chemical information” that was received.
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bonds were chosen. Earlier proof-of-concept studies have
demonstrated the viability of the approach and produced a set
of guidelines for the design of new structures.[19,20] For
example, two NP-derived fragments can be connected
through different connectivity patterns (Figure 2, Panel A),
to produce different pseudo-NP classes, each of which has
been shown to occupy different regions of chemical space
(Figure 2, Panel C, Design Principle 1).[20] Additional pseudo-
NP classes can be designed by maintaining the same
connectivity pattern through different connection points on
one or both fragments (Figure 2, Panel C, Design Principle 2).
These guidelines demonstrate the great potential for the
preparation of novel molecular scaffolds, which may be
inherently biological relevant, limited only by human imag-
ination and the availability of synthetic methods.
The preparation of pseudo-NP classes such as pyrroquino-
lines (Scheme 2i) demonstrated in practice that the combi-
nation of a set of common NP-derived fragments in different
connectivity patterns (exploiting Design Principle 1) can yield
chemically and biologically diverse libraries. Additionally,
this work also demonstrated that these regioisomeric pseudo-
NP classes display distinct biological effects. Furthermore,
combinations of different fragments in complementary ar-
rangements (exploiting Design Principle 2) can produce
chemically and biologically diverse compound libraries
(Scheme 3).[40,41] As such, pseudo-NPs constitute novel chem-
ical matter and possess more than just the additive properties
of their individual constituting fragments. This observation is
also strongly supported by the biological activity observed for
different pseudo-NP classes. For example, the activity of
pseudo-NPs, such as the chromopynones,[19] pyrano-furo-
pyridones,[33] indomorphans,[34] and azaquindoles[40] was not
shared by either of the individual NP-derived fragments.
This inherited biological relevance from NPs, to NP-
derived fragments, to pseudo-NPs, suggests a continuation of
biologically relevant chemical space. In nature, entry points to
this space created through evolution convey an advantage to
the host organism and usually are not re-optimised. Instead,
they are used in different arrangements. Thus, such biolog-
ically relevant portions of chemical space may only be
accessible by exploiting features encoded and subsequently
passed-on through the incorporation of structural motifs
generated by secondary metabolite biosynthesising proteins,
which themselves share evolutionary conserved structural
features.[13] This hypothesis is supported by the fact that NPs
interact with multiple proteins during their biosynthesis and
must be thus endowed with several biologically relevant
structural motifs. Additionally, recent reports make a case for
a more prevalent role of active transport in the cellular uptake
of small molecules.[71] Work by Kell and co-workers advocates
that small molecule cellular transport occurs through specific
interactions with membrane proteins.[72–74] Such a mechanism
would require the existence of distinct biologically relevant
structural motifs to be present in bioactive molecules,
independent of their ultimate molecular properties such as
molecular weight or lipophilicity.
The finding that novel combinations of NP fragments can
be induced by recombination of existing biosynthesis path-
ways suggests that the current repertoire of known NPs, in
principle, can be extended through synthetic biology tech-
niques. This may be achieved, for instance, by changes to
natural biosynthesis to make use of different existing
biosynthesis pathways, including “silent” pathways that are
not usually active. By analogy, synthetic chemical recombi-
nation of NP fragments as “inheritable building blocks”
defines an evolutionary strategy for the discovery of novel
natural product-inspired compound classes a priori endowed
with biological relevance. In this strategy the novel combina-
tion of NP-fragments by biosynthetic steps is replaced by
unprecedented synthetic NP-fragment combinations. Thus,
the iterative design, synthesis, and biological investigation of
pseudo-NPs can be regarded as a chemical evolution of
natural product structure, and as a human and chemically-
driven branch of the evolution of NPs (Figure 7).
In order to investigate the generality of these principles,
we also conducted a literature search for further examples of
molecules that may satisfy their classification as pseudo-NPs.
This search revealed pseudo-NPs prepared independently by
different research groups. Notable examples include the
pyrrofuranolactones 12,[31] carbazopyrrolones 13,[32] as well
as the more recent penindolones 71–74,[42] piperazopyridones
81,[75] and tetracyclic-fused isoindolinones 32 (Figure 8).[35]
Some of these molecules have displayed biological activity.
For example, penindolones were found to inhibit membrane
fusion of the Influenza A virus,[42] and derivatives of piper-
azopyridones were identified as TRPV6 channel inhibitors.[75]
Other applications include the preparation of pseudo-NP
dyes from betelamic acid.[76] These examples indicate that the
concept might have been intuitively employed previously,
without the intellectual framework and guidance of the design
principles delineated here.
8. Outlook
Pseudo-NPs stem from combinations of NP-derived frag-
ments leading to molecular scaffolds that may not be
accessible by natural biosynthesis. These structures combine
the ability of fragments to rapidly explore chemical space with
the biological relevance of NPs, resulting in a molecular
discovery strategy that can uncover unexplored regions of
biologically relevant chemical space potentially harbouring
compounds with unprecedented biological activity. Through
the case studies highlighted above it is evident that pseudo-
NPs represent a validated general design approach for
molecular discovery, and can generate numerous opportuni-
ties for chemical biology and medicinal chemistry research.
The design of pseudo-NPs takes into account general
molecular properties, such as Fsp3(fraction of sp3-hybridised
carbon atoms) and heteroatom content, as well as the more
specific principle of joining individual fragments based on
connectivity patterns observed in known NPs. As such, this
chemocentric approach may be regarded as a chemically
driven branch of the evolution of NPs. These design principles
have been exploited by the scientific community as shown by
selected examples above, yet not in a methodical manner.
Potentially numerous compound classes which fall under the
definition of pseudo-NPs may already be known and can be
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analysed and assessed for biological activity. This evaluation
would benefit from the application of multi-parametric
screening methods and may highlight applications of pseu-
do-NPs in challenging areas such as developing antibiotics to
combat multidrug resistant bacteria.[60]
Finally, pseudo-NP design and preparation are under-
pinned by organic synthesis. The tools and methodologies
developed for the preparation of NPs are certainly applicable
in pseudo-NPs as well. However, new synthetic method-
ologies,[77] for example, photochemical CH functionalisa-
tions,[78,79] and creative scaffold-forming multicomponent
reactions[80,81] will continue to have great impact on our
ability to prepare and study further examples of these exciting
and potentially highly beneficial compound classes.
Acknowledgements
The development and experimental validation of the pseudo-
natural concept reflects the work of numerous former and
present members of our research group whose names are
found in the corresponding publications cited in this Minire-
view. Their work at the heart of chemical biology research is
testimony to their intellectual and experimental talent and
their ability to embrace the methods and cultures of
chemistry, biology, and computer science in truly interdisci-
plinary endeavours. Our research was supported by the Max-
Planck-Gesellschaft, the Alexander von Humboldt-Stiftung
and the Fonds der Chemischen Industrie. Open access
funding enabled and organized by Projekt DEAL.
Conflict of interest
The authors declare no conflict of interest. G.K. is now an
employee of AstraZeneca, U.K.
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Manuscript received: December 14, 2020
Revised manuscript received: January 27, 2021
Accepted manuscript online: March 1, 2021
Version of record online: &&
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,
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Bioorganic Chemistry
G. Karageorgis, D. J. Foley, L. Laraia,
S. Brakmann,
H. Waldmann*
&&&& —&&&&
Pseudo Natural Products—Chemical
Evolution of Natural Product Structure
Pseudo-Natural Products provide new
opportunities in the discovery of bioactive
small molecules and can be regarded as
a human-driven chemical evolution of
natural product structure.
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