Design and Synthesis of Natural Product Inspired Libraries Based on the Three-Dimensional (3D) Cedrane Scaffold: Toward the Exploration of 3D Biological Space

Article abstract of DOI:10.1021/acs.jmedchem.8b00194

A chemoinformatic method was developed to extract nonflat scaffolds embedded in natural products within the Dictionary of Natural Products (DNP). The cedrane scaffold was then chosen as an example of a nonflat scaffold that directs substituents in three-dimensional (3D) space. A cedrane scaffold that has three orthogonal handles to allow generation of 1D, 2D, and 3D libraries was synthesized on a large scale. These libraries would cover more than 50% of the natural diversity of natural products with an embedded cedrane scaffold. Synthesis of three focused natural product-like libraries based on the 3D cedrane scaffold was achieved. A phenotypic assay was used to test the biological profile of synthesized compounds against normal and Parkinson's patient-derived cells. The cytological profiles of the synthesized analogues based on the cedrane scaffold revealed that this 3D scaffold, prevalidated by nature, can interact with biological systems as it displayed various effects against normal and Parkinson's patient-derived cell lines.

Full text of DOI:10.1021/acs.jmedchem.8b00194

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Article
Design and Synthesis of Natural Product Inspired Libraries Based on the
D Cedrane Scaffold: Towards the Exploration of 3D Biological Space
3
fatemeh Mazraati Tajabadi, Rebecca H Pouwer, Miaomiao Liu, Yousef Dashti, Marc Campitelli,
Mariyam Murtaza, George D Mellick, Stephen A Wood, Ian D. Jenkins, and Ronald J. Quinn
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.8b00194 • Publication Date (Web): 13 Jul 2018
Downloaded from http://pubs.acs.org on July 15, 2018
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Design and Synthesis of Natural Product Inspired Libraries Based on
the 3D Cedrane Scaffold: Towards the Exploration of 3D Biological
Space
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Fatemeh Mazraati Tajabadi, Rebecca H. Pouwer, Miaomiao Liu, Yousef Dashti,
Marc R. Campitelli, Mariyam Murtaza, George D. Mellick, Stephen A. Wood, Ian
D. Jenkins, Ronald J. Quinn*
Griffith Institute for Drug Discovery, Griffith University, Brisbane, QLD 4111, Australia
ABSTRACT
A chemoinformatic method was developed to extract nonꢀflat scaffolds embedded in
natural products within the Dictionary of Natural Products (DNP). The cedrane scaffold
was then chosen as an example of a nonꢀflat scaffold that directs substituents in threeꢀ
dimensional (3D) space. A cedrane scaffold that has three orthogonal handles to allow
generation of 1D, 2D and 3D libraries was synthesised on a largeꢀscale. These libraries
would cover more than 50% of the natural diversity of natural products with an
embedded cedrane scaffold. Synthesis of three focused natural productꢀlike libraries
based on the 3D cedrane scaffold was achieved. A phenotypic assay was used to test the
biological profile of synthesized compounds against normal and Parkinson’s patientꢀ
derived cells. The cytological profiles of the synthesized analogues based on the
cedrane scaffold revealed that this 3D scaffold, preꢀvalidated by nature, can interact
with biological systems as it displayed various effects against normal and Parkinson’s
patientꢀderived cell lines.
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INTRODUCTION
Natural products are an important source of inspiration for the development of
1
therapeutically useful compounds. Many marketed drugs have natural origins with
32% of developed therapeutic drugs approved by the US Food and Drug Administration
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(
FDA), between 1981 and 2014 being natural products or natural productꢀderived. In
the ongoing search for novel drug candidates, the extraordinary chemical complexity of
3
those natural products that contain high numbers of sp hybridized carbons and chiral
3
ꢀ7
8, 9
centres may be exploited to access neglected chemical space and avoid flatland.
From a biomolecular interaction perspective, natural products have met at least one
10ꢀ14
biosynthetic enzyme, providing an embedded biological imprint.
High chemical
diversity and embedded biosynthetic molecular recognition may explain the high
success rate of natural products in hitting biological targets. The so far explored
chemical universe, however, is suffering from insufficient overlap/correlation between
biological and chemical space. With some essential biological targets such as
phosphatases and proteinꢀprotein interactions, it has proved difficult to find small
15
molecule modulators of function. Several synthetic approaches have been introduced
inspired by nature to insert more complexity and diversity into the chemical collections.
1
6, 17
16, 18
These include diversityꢀoriented synthesis,
targetꢀoriented synthesis,
functionꢀ
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20, 21
oriented synthesis, and biologyꢀoriented synthesis.
Current commercial compounds tend to be flat, mainly composed of planar aromatic
3
22
and heteroaromatic scaffolds (Fsp < 0.45). The vast majority of scaffolds utilized in
synthetic approaches are flat (22% nonꢀflat) while the trend is reversed in natural
22
23
products with more than 75% nonꢀflat scaffolds. According to Hert et al. 83% of the
ring scaffolds among natural products are absent from commercial molecules. While the
cost effectiveness and simple synthesis of aromatic systems may justify the popularity
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of planar chemical collections, investing in the development of nonꢀflat scaffolds that
are preꢀvalidated in nature may provide a generation of diverse natural productꢀbased
libraries that have the potential to cover neglected areas of biological space.
Herein, we describe an approach to identify nonꢀflat scaffolds from the DNP that have
high potential in directing pendant groups in 3D space. A chemoinformatic process was
developed to overlay and cluster natural products based on their common core in order
to visualize and assess the ability of a scaffold to direct the orientation of substituents in
3D space. Amongst the identified 3D natural product scaffolds, cedrane (1) was chosen
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for further studies. The cedrane scaffold is highly saturated (Fsp =1) with 3 chiral
centres and directs the orientation of pendent groups in three dimensional space.
Furthermore, a range of biological activities have been reported for natural products and
24ꢀ31
synthetic compounds containing the cedrane scaffold.
A series of compounds based
on the cedrane scaffold were synthesized to assess their biological activity using a
3
2
phenotypic assay against a Parkinson’s patientꢀderived cell line. In this cellꢀbased
multidimensional profiling method, the effects of compounds on six independent
cellular compartments on a patientꢀderived cell model were investigated. The assay
utilised nonꢀtransformed/nonꢀimmortalized primary human olfactory neurosphereꢀ
derived (hONS) cells with a normal karyotype, derived from a patient with idiopathic
33, 34
Parkinson’s disease (PD).
This aims to study a disease state and enables
investigation of multiple targets and pathways. For disorders such as Parkinson’s and
Alzheimer’s diseases, for which targetꢀbased strategies have failed to produce
successful drug candidates for clinical development, this strategy may identify novel
35
drug targets.
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RESULTS AND DISCUSSION
Identification of 3D Scaffolds Embedded in Natural Products
A multistep analysis was performed on the secondary metabolites represented in the
DNP to identify 3D scaffolds embedded in natural products (Figure 1). Initially all ring
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systems in the DNP were abstracted according to the Bemis and Murcko method.
Then, in order to preserve important chemical functionality (e.g. the carbonyl oxygen
present in a lactone ring), all contiguous ring systems were extracted with retention of
exocyclic double bonds to terminal atoms. Metalꢀcontaining ring systems were excluded
from consideration, resulting in the identification of 18,128 ring systems. To exclude
large, nonꢀdrugꢀlike ring systems (e.g. polycyclic ring systems and macrocycles) an
additional molecular weight filter (MW < 350 Da) was applied which resulted in 15,822
natural product derived scaffolds. In order to increase the drugꢀlikeness of the extracted
scaffold library, the following property filters were applied: number of aromatic rings <
3; number of rings < 4; number of bonds < 20; S ≤ 2; O ≤ 4; N ≤ 4. This resulted in
6,102 natural product derived scaffolds. This scaffold subset was investigated to
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identify nonꢀflat scaffolds (Fsp > 0.45) located within the central core of a natural
product, with the ability to direct substituents in 3D space.
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Figure 1. Summarized process used for identification of nonꢀflat (3D) natural product scaffolds from
DNP.
Natural product ring systems meeting these criteria were then ranked based on
synthetic viability and biological activity of the parent natural products. For this
proof of concept study, the cedrane scaffold
1 (Figure 2) was selected as a
suitable candidate for further study. There are 161 natural products reported in
the DNP that contain the scaffold
1
, providing a reasonable sample size for
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molecular modelling. In addition, scaffold
1
is highly saturated (Fsp = 1),
bearing 3 chiral centres and natural products containing scaffold
1
have a range
of biological activities (Figure 2). For instance, elisapterosin B (
2) has shown
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strong in vitro antiꢀtuberculosis activity, takaneone B (3) has been reported as a
2
5
cytotoxic compound against K562/Adr multidrugꢀresistant (MDR) cancer cells,
2
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taxuspine H (
4
) showed microtubule depolymerisation and cytotoxicity towards
2
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Vꢀ13 cells, phloroglucinol derivative revealed activity against Bacillus cereus
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,
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Staphylococcus epidermidis and Micrococcus luteus
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and merochlorin A (6)
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showed in vitro activity against Methicillinꢀresistant Staphylococcus aureus
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Figure 2. Cedrane scaffold 1 and structure of some bioactive compounds that contain scaffold 1.
Assessment of Scaffoldꢀdirected Occupation of 3D Space
All 161 natural products reported in the DNP that contain scaffold
1 were overlaid and
clustered based on their common core using molecular modelling in order to visualize
and assess the ability of the scaffold to direct the substituents in 3D space. Of the 161
natural products that contain scaffold
1, eight compounds have defined absolute
configuration. To perform clustering, firstly all 161 natural product structures
containing the cedrane skeleton were drawn in MaestroꢀSchrödinger software (version
9
.7). For the natural products with relative configuration (153 molecules), a mirror
structure of each molecule was also drawn. Multiꢀminimization was performed on all
structures using MacroModel. The two most complete and wellꢀparameterized force
fields, MMFFs (Merck Molecular Force Field) and OPLS (Optimized Potentials for
Liquid Simulations)ꢀ2005, were used in energy calculations. At the primary stages of
calculations, it was found that MMFFs, in a few cases, provided two different global
minima for basically identical structures. The quality comparison of three parameters
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(stretch, bend and torsion parameters) revealed that OPLS 2005 with a larger “high
quality” torsion parameter (241 vs 221) and less “low quality” torsion parameter (0 vs
21) is more appropriate for the energy calculations in this study (Table S1). Therefore,
OPLS 2005 force field was employed for minimization followed by conformational
search using the same force field to retain only the lowest energy conformer of each
structure.
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A similar order of atom numbers was applied for the cedrane scaffold in each natural
product; therefore, renumbering based on “scaffold heavy atoms” was a necessary step
prior to clustering. As there was no defined function in Maestro to change numbering
order, Chem & Bio 3D was used to sort the atom number order of the scaffold in all
natural products. For the natural products with relative configuration the true
enantiomer structure was established through Xꢀcluster using atomic RMSD as a
distance criterion. It provides similarity comparison between pairs of structures
following rigidꢀbody superposition. The similarity matrix provided by Xꢀclustering
allows a many by many superimposition of scaffold heavy atom of each relative
structure on one or more absolute structures. The corresponding r.m.s. (root mean
square) values were determined and compared. Using this matrix, the relative structures
with higher r.m.s. values resulted from overlaying on the absolute structures were
omitted from consideration. Finally, all 161 structures were superimposed based on the
cedrane scaffold using the average linkage method, which resulted in 9 clusters (Figure
3). The same criterion of atomic RMS (clustering level: 0.21) was used in Xꢀclustering
which clustered the NPs containing the cedrane core based on the geometry of the
cedrane core and their distance between atoms within the core. This groups structures
that have substituents on the core scaffold protruding in the same relative orientation in
space.
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Figure 3. Clustering 161 natural products containing the cedrane scaffold based on the geometry of the
cedrane core and their distance between atoms within the core gave 9 clusters. Three major clusters,
cluster 6 with 69 natural product hits, clusters 1 and 3 each with 35 NPs are delineated with two examples
of NP molecules present in their group. Clusters 1, 3 and 6 plus cluster 5 composed of 14 hits were the 4
major clusters that cover > 95% (153 out of 161) of natural products bearing the cedrane scaffold. Minor
clusters were clusters 2, 4, 7, 8 and 9 with 3, 2, 2, 1 and 1 hit (s), respectively, representing only 8
molecules from 161 NPs in the DNP.
The numbers of natural products represented in clusters 1 to 9 were 35, 3, 35, 2, 14, 69,
2, 1 and 1, respectively. Cluster 6 with 69 natural product hits, clusters 1 and 3 each
with 35 natural products and cluster 5 composed of 14 hits were the 4 major clusters
that cover > 95% (153 out of 161) of natural products containing scaffold 1. The other 5
clusters only represent 8 natural products.
The results of overlaying clustered natural products for the 3 major clusters are depicted
in Figure 4. Natural products in cluster 6 (69 hits), tend to be functionalized in positions
1, 3, 5, 9, and 11. Figure 5 demonstrates that the cedrane core orientates the substituents
from 69 overlaid natural product molecules into relatively limited areas of space as
shown by the highlighted arrows. Cluster 1 (35 hits) natural products are functionalized
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in positions 1, 2, 3, 4 and 5 with a tendency towards large substituents. Natural products
in cluster 3 (35 hits) bear substituents in positions 1, 2, 3, 4, 5, 6 and 10 of the cedrane
scaffold with positions 3 and 4 mainly linked through a functionalized 6 membered
fused ring. Thus, all ten positions of scaffold 1 are substituted in Nature.
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Synthesis of the Cedrane Scaffold and Selection of Diversifying Reagents
According to the molecular modelling results, synthesizing all possible diꢀ
substituted libraries based on the cedrane scaffold is desirable to explore 3D
biological space around the scaffold. A comprehensive literature survey revealed
that 23 out of 45 possible disubstituted combinations for 2D libraries (Figure S1)
are achievable using methods reported in the literature. To generate oneꢀ and
multiꢀdimensional libraries containing a cedrane core, a scaffold with three
orthogonally reactive chemical handles was synthesised on a 5g scale (Scheme
38ꢀ40
1).
Briefly, the methylation of 7 with iodomethane was promoted by
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Figure 4. Six different views of natural products containing the cedrane scaffold in the 3 major clusters. Characterization of each cluster: Clusters 1 (35 hits in DNP)
functionalized in positions 1, 2, 3, 4 and 5. Cluster 3 (35 hits) functionalized in positions 1, 2, 3, 4, 5, 6 and 10 where mostly positions 3 and 4 are attached to each other
through a functionalized 6 member fused ring. Cluster 6, the largest cluster, which covers 69 hits, is heavily decorated in positions 1, 3, 5, 9, and 11.
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6
7
8
9
0
Figure 5. Sixtyꢀnine natural product molecules in cluster 6 overlaid based on the cedrane scaffold 1.
Highlighted arrows show the 3D orientation of the precursors into a relatively limited space by cedrane
scaffold.
Scheme 1. Synthetic plan adopted from literature to make cedrane scaffold with three orthogonally reactive
a
chemical handles.
OCH
3
OH
OMe
OCH
3
(
i)
(ii)
(iii)
8
5%
70%
52%
H CO
H
3
Me
Me
Me
CN
7
8
9
10
11α,β
(iv) 72%
H
O
OH
H
1
8
O
O
2
9
6
1
0
(v)
5%
(
vi)
OH
O
+
H CO
3
3
7
6
5%
7
HO
11
H CO
3
3
H CO
4
HO
13a
O
13b
14
5
O
12
O
a
(
i) MeI, K2CO3, acetone, reflux, (ii) Na, NH (liq), H O, EtOH, (iii) acrylonitrile, 120 ˚C, 16h (iv) a:
3 2
LDA/THF, ꢀ78 ˚C; b: O ꢀ78 ˚C; c: Na SO , 0ꢀ25 ˚C, (v) 2ꢀ(2ꢀbromoethyl)ꢀ1,3ꢀdioxane, Mg, Br(CH ) Br, (vi)
2
2
3
2 2
TsOH, acetone, reflux.
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9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
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4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
3
8
potassium carbonate in refluxing acetone to give 8 in 85% yield. Then, Birch reduction of 8
yielded 9 (70%). Cycloaddition of acrylonitrile and the conjugated diene 10, generated in situ
39
from 9, gave a mixture of nitriles 11a,b. Deprotonation of 11 with lithium diisopropylamide
followed by treatment with oxygen, and subsequent reduction with sodium sulfite gave the
ketone 12 in 72% yield. Ketone 12 was then treated with 2ꢀ(2ꢀbromoethyl)ꢀ1,3ꢀdioxane and
magnesium turnings in THF to obtain a readily separable mixture of endo (13a) and exo
alcohols (13b) in 62:38 ratio in good yield (75%). In the final step, the mixture of alcohols 13
underwent pinacol rearrangement to yield ketone 14, upon treatment with pꢀTsOH (1.3 eq) in
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
4
0
refluxing acetone for 16 h. This strategy provided 5 g of scaffold with three orthogonally
reactive chemical handles allowing decoration at positions 3, 5 and 9 to generate 1D, 2D and
3
D libraries that sample the diversity of the natural products. The overall approach for library
generation is outlined in Scheme 2. Esterification of the αꢀhydroxyl group at C3 of 14
afforded Chemset 1 (15aꢀ15r). In order to prepare the second library, the C3 hydroxyl group
of 14 was inverted through a Mitsunobu reaction to give intermediate 16 (Scheme 6) which
was followed by hydrolysis to yield 17. Subsequent esterification of the resulting alcohol
with the same groups utilized in the first library synthesis generated Chemset 2. Finally, the
members of Chemset 1 were subject to sodium borohydride reduction to yield Chemset 3.
To select the diversifying reagents and avoid a totally random diversification in the generated
libraries, an Rꢀgroup analysis was performed on the 161 cedrane scaffold containing natural
products reported in the DNP to determine the patterns of functionalization by Nature in the
1
0 available positions (Figure S2). This selected diversification will also give us a chance to
further examine the hypothesis regarding the similarities of the active sites of proteins
1
0, 11
involved in biosynthetic pathways and biological targets
through the analysis of
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biological responses. The Rꢀgroup analysis identified mostly esterification at position 3.
Carboxylic acids AꢀR used for the esterification of the cedrane scaffold to generate the 3
libraries (Chemset 1, 2 and 3) are presented in Figure 6. Carboxylic acids AꢀE correspond
directly to known natural product C3 substituents. The remaining acid reagents FꢀR were
selected for structural diversity
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Scheme 2. Overall approach to generate 1D focused libraries of Chemset 1 and Chemset 2 along with 2D
library of Chemset 3 based on the cedrane scaffold.
41
and drugꢀlikeness. Specifically, carboxylic acids that contained inappropriately reactive
functional groups and elements (e.g. boronic acids, nitro, sulfate, sulfur, halogens…) were
not considered. In order to assess the drugꢀlike properties of the 60 synthesized compounds
their physicochemical properties were analyzed and checked against Lipinski’s Rule of 5
(Figure 7 and Table S3). All synthesized molecules had between 0ꢀ2 HBD and 2ꢀ3 HBA, and
as depicted in Figure 7, molecular weight and calculated Log P of the synthesized
compounds are in the acceptable drugꢀlike range.
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1
1
1
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1
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1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
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5
5
5
5
5
6
O
O
O
O
O
OH
OH
OH
OH
OH
A
B
C
D
E
O
O
O
O
O
OH
OH
F
OH
OH
F
F
F
G
H
I
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
O
F
O
F
O
O
F
O
3
F C
OH
OH
OH
OH
OH
F
J
K
L
M
CF3
N
O
O
O
O
OH
OH
OH
OH
O
N
O
O
O
Q
O
P
R
Figure 6. Diversifying reagents AꢀR used for library generation.
Figure 7. Boxplot of the molecular weight and Log P of the Chemsets 1 (green), 2 (red), and 3 (blue).
Synthesis of 1D and 2D Libraries at Positions 3 and 9 of the Cedrane Scaffold
Synthesis of Chemset 1 was achieved via esterification of the αꢀalcohol at C3 with carboxylic
acids AꢀR to afford esters 15 (Scheme 3). Esterification was generally carried out via the
corresponding acid chlorides, followed by coupling with the hydroxyl group of the cedrane
scaffold in the presence of triethylamine (procedure (i)). The reactions proceeded in
moderate to good yields (Table 1) with the exception of esters 15o and 15p, which were
obtained in only 10% yield. Ester 15a was obtained in 87% yield through acetylation of the
hydroxyl group using acetic acid anhydride in the presence of 4ꢀdimethylaminopyridine
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(
DMAP) (procedure (ii)). Esterification of the hydroxyl group of the cedrane scaffold with
acid reagent R was carried out similarly by conversion to the corresponding acid chloride
with thionyl chloride followed by coupling with 14 in the presence of DMAP and pyridine as
solvent (procedure (iii)) to afford the ester 15r in 75% yield.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
a
Scheme 3. Synthetic procedures used to generate Chemset 1
a
(
i) a: (COCl) , DCM, DMF (1ꢀ2 drops), rt, 2 h; b: Acid chloride, Et N, DCM, DMAP (cat. amount), rt, 4ꢀ24 h,
2 3
(
ii) (CH CO) O, DMAP, Et N, rt, 20 h, (iii) SOCl , DMAP, pyridine, 17 h.
3 2 3 2
NMR analysis of the product of the esterification of the αꢀOH on C3 with 3,3ꢀ
dimethylacrylic acid (acid reagent C in Figure 6) revealed that the expected ester product 15c
was not produced. Instead, the β,γꢀunsaturated ester 15c', resulting from deconjugative
rearrangement of the sideꢀchain, was produced (Scheme 4). A mechanism for the formation
4
2
of compound 15c' via a ketene intermediate is suggested in Scheme 5.
Scheme 4. Production of unexpected rearranged product 15c' and synthesis of diastereomers 21α and 21β from
a
compound 15c'.
a
(
i) See scheme 3, (v) a) NaBH , MeOH, 0 °C, 1h; b) HCl (2M), 10 min.
4
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Scheme 5. Mechanism of formation of 15c'.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
a
Table 1. Isolated yields for compounds 15 in Chemset 1
Compound
15a
R
A
B
C
D
E
F
G
H
I
Procedure
Yield
87%
70%
56%
63%
61%
58%
61%
57%
42%
Compound
15j
R
J
K
L
M
N
O
P
Q
R
Procedure
Yield
56%
56%
30%
45%
42%
10%
10%
42%
75%
(ii)
(i)
(i)
(i)
(i)
(i)
(i)
(i)
(i)
(i)
(i)
(i)
(i)
(i)
(i)
(i)
(i)
(iii)
1
5b
15k
b
1
5c'
5d
5e
15l
1
15m
15n
15o
15p
15q
1
1
5f
5g
5h
1
1
15i
15r
a
b
See Scheme 3 for conditions (i)ꢀ(iii). See Scheme 4 for the structure of 15c'.
Chemset 2 was generated using the inverted alcohol 17 with the aim of introducing the same
substituents as Chemset 1 in order to study the effect of stereochemistry and 3D orientation
on biological activity. The Mitsunobu reaction was employed to generate the inverted
cedrane scaffold 17 from 14 according to Scheme 6. Alcohol 14 was treated with pꢀ
nitrobenzoic acid in the presence of diisopropyl azodicarboxylate (DIAD),
triphenylphosphine (PPh ) and toluene as solvent at 80 °C for 2 h. The generated ester 16
3
was then hydrolyzed using potassium hydroxide to give inverted alcohol 17. Esterification of
17 with carboxylic acids A, B, D, FꢀR (Figure 6) afforded esters 18a, 18b, 18d, 18fꢀ18r in
moderate to good yields with the exception of compounds 18o and 18p with 12% yields
(Table 2).
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a
Scheme 6. Synthesis of Chemset 2
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
a
(i) a: (COCl) , DCM, DMF (1ꢀ2 drops), rt, 2 h; b: Acid chloride, Et N, DCM, DMAP (cat. amount), rt, 4–24 h;
2
3
(ii) Ac O, DMAP, Et N, rt, 20 h; (iii) a: SOCl , DMAP, dry pyridine, 17 h; (iv) PPh (2.5 eq), DIAD (2.5 eq),
2
3
2
3
toluene, 80 °C, 2 h; (vi) KOH, EtOH, 50 °C, 3 h.
In general procedure (i) was used to produce the corresponding acid chloride from the
carboxylic acid, followed by coupling of the acid chloride and βꢀhydroxyl group in the
presence of triethylamine. However, phenylacetic acid derivatives (18fꢀ18h) and ester 18i
were not produced by procedure (i). Instead, a Mitsunobu reaction, which was used to
produce compound 16, was used to produce derivatives 18fꢀ18i directly from 14 in good
yield (procedure iv in Scheme 6).
a
Table 2. Isolated yields for compounds 18 in Chemset 2
Compound
8a
R
A
B
D
F
G
H
I
Procedure
(ii)
Yield
85%
56%
68%
72%
68%
75%
69%
68%
Compound
18k
R
K
L
M
N
O
P
Procedure
Yield
76%
39%
51%
54%
12%
12%
42%
77%
1
(i)
(i)
(i)
(i)
(i)
(i)
(i)
(iii)
1
1
8b
8d
(i)
(i)
18l
18m
18n
18o
18p
18q
18f
(iv)
(iv)
(iv)
(iv)
(i)
1
1
8g
8h
1
1
8i
8j
Q
R
J
18r
a
See Scheme 6 for conditions (i)ꢀ(iv).
We next sought to generate a 2D library by further elaboration of Chemset 1 through
reduction of the C9 carbonyl group of esters 15 to afford the corresponding hydroxy esters
1
9ꢀ30 (Scheme 7).
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
a
Scheme 7. Synthesis of Chemset 3
O
HO
HO
(v)
+
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
RO
RO
RO
15
ꢀC9
ꢀC9
1
2
9; R: hydrogen
0; R: B
25; R: H
26; R: I
27; R: J
28; R: L
29; R: N
30; R: Q
21; R: C
2
2
2; R: D
3; R: E
24; R: G
a
(v) a: NaBH , MeOH, 0 ⁰ C, 1 h; b: HCl (0.35 mol), 10 min.
4
Sodium borohydride reduction of the C9ꢀcarbonyl occurred in high yield (Table 3), however,
this nonꢀstereoselective reduction yielded both diastereomers. The configuration of the
hydroxyl group generated in position 9 was determined by calculation of the dihedral angles
(
Figures S3 and S4) and confirmed by comparison of the spectral data with those of reported
43, 44
values.
Molecular modelling on compound 19α predicted a doublet of doublets for the βꢀ
H on C9 with J values of 8.8 Hz and 7.1 Hz while the J values for compound 19β were
predicted to be 4.5 Hz and 2.0 Hz. These predicted values correlate well with the observed
values (dd; 10.3 Hz and 5.8 Hz for the compounds 19α and broad singlet; 2.6 Hz and 1.2 Hz
for the compound 19β). Separation of the diastereomeric pairs was carried out using silica gel
column chromatography. Further purification using HPLC was also employed in some cases
in order to achieve acceptable purity for biological screening.
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a,b
Table 3. Isolated yields for compounds in Chemset 3
Compound
R
Procedure
Ratio (α:β)
62:38
42:58
42:58
39:61
40:60
45:55
41:59
65:35
44:56
39:61
43:57
56:44
Yield
87%
95%
92%
94%
94%
89%
91%
95%
96%
93%
89%
94%
1
2
2
22
23
24
25
26
27
9
0
1
Hydrogen
(v)
(v)
(v)
(v)
(v)
(v)
(v)
(v)
B
C
D
E
G
H
I
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
J
(v)
28
29
30
L
N
Q
(v)
(v)
(v)
a
b
See Scheme 5 for condition (v). See Scheme 4 for the structure of 21α and 21β.
Biological Activity of the Synthesised Compounds in a PD Phenotypic Assay
A phenotypic assay was used to test the biological profile of all synthesized compounds
against normal and Parkinson’s patientꢀderived hONS cells. Cells were treated with 10 ꢁM of
each compound for 24 h in a 96ꢀwell plate format. Cytological parameters were assessed by
staining with fluorescent probes targeting various cellular pathways and organelles
implicated in PD. These included mitochondria, early endosomes, lysosomes, microtubuleꢀ
based cytoskeleton, and autophagosomes. The cellular markers including Dapi (nucleus),
MitoTracker (mitochondria), LysoTracker (lysosome), anti αꢀtubulin antibody (microtubule,
cytosckeleton), anti EEA1 antibody (early endosomes) and anti LC3b antibody
(
autophagosomes) were used due to their significance in the biology of PD. In order to
quantify the biological effects, 38 parameters from the combination of different fluorescent
32
probes were used (Table S4). Comparative analyses were performed on the effect of
compounds on the normal vs Parkinson’s patientꢀderived cells followed by comparative
analysis between of the effect of Chemsets against Parkinson’s patientꢀderived cell line.
None of the compounds used in the assay were found to have cytotoxicity effects based on
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1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
the total number of cells remaining after 24 hours of treatment with compound. The stability
of synthesized cedrane scaffold 14 and two ester derivatives 15o and 28β was assessed under
the phenotypic assay conditions (Figures S5ꢀS7). The LCꢀMS results showed no evidence of
hydrolysis of compounds 15o or 28β and the cedrane scaffold 14 was stable.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Comparative analysis of the biological activity of synthesized compounds against
healthy and Parkinson’s patientꢀderived cell lines
Chemset 1: Most of the compounds in Chemset 1 showed a slightly higher negative effect
on nucleus intensity marker of normal cell line compared to Parkinson’s patientꢀderived cell,
of which the 3ꢀfuroic ester derivative 15p showed the highest effect (Figure 8). The majority
of compounds showed a larger negative effect on αꢀtubulin markers of the normal cell line.
Phenylacetic ester derivatives 15g displayed the highest effect on αꢀtubulin markers PD
patient while 15h displayed the highest effect on αꢀ tubulin markers in the normal cell lines.
For markers related to mitochondria the effects on PD patient cell line were stronger and
positive for most of the derivatives, and compounds 15n, with trifluoromethyl group, and
1
5q, with heteroatoms on the side chain, displayed the highest positive effects. The 3ꢀfuroic
ester derivative 15p had the most significant positive effect on mitochondria of healthy cell
line. The core scaffold 14, its derivatives with aliphatic side chains (compounds 15a, 15b,
1
5c', 15d and 15e) and compounds 15h, 15i, 15j and 15k displayed higher negative effects
on LC3b markers of the healthy control cell line, of which compound 15e exhibited the
strongest effect. The effects of most of the compounds on lysosome markers were generally
negative for healthy control and positive for PD patient cell lines. All derivatives in this
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Figure 8. Heatmap depicting the cytological profile of compounds 14ꢀ15r of the Chemset 1 at 10 ꢁM on 38 parameters of normal (orange) and PD derived
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blue) cell lines. The effects of compounds were plotted as log2 ratio to the DMSO control. Yellow shows a decrease versus control and purple shows an increase
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versus control. Individual compounds are presented on the yꢀaxis with individual features on the xꢀaxis. 1. Nucleus marker intensity mean /µm 2. Nucleus
morphology area 3. Nucleus morphology roundness 4. Nucleus morphology width (µm) 5. Nucleus morphology length (µm) 6. Nucleus morphology ratio width
to length 7. Nucleus marker texture index SER spot 1 px 8. Cell morphology area 9. Cell morphology roundness index 10. Cell morphology width (µm) 11. Cell
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morphology length (µm) 12. Cell morphology ratio width to length 13. αꢀTubulin marker intensity mean 14. αꢀTubulin marker intensity outer region mean/µm
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5. αꢀTubulin marker intensity inner region mean/µm 16. Mitochondria marker intensity mean 17. Mitrochondria marker intensity outer region mean/µm 18.
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Mitrochondria marker intensity inner region mean/µm 19. LC3b marker intensity mean 20. LC3b marker intensity outer region mean/µm 21. LC3b marker
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intensity inner region mean/µm 22. Tubulin marker texture SER spot 1 px 23. Mitochondria marker texture SER spot 1 px 24. LC3b marker texture SER spot 1
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px 25. Lysosome marker texture SER spot 1 px 26. Lysosome marker intensity mean 27. Lysosome marker intensity outer region mean/µm 28. Lysosome
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marker intensity inner region mean/µm 29. Relative spot signal on cytoplasm 30. Number of spots on cytoplasm 31. Number of spots per area of cytoplasm 32.
Relative spot signal on outer region 33. Number of spots on outer region 34. Number of spots per area of outer region 35. Relative spot signal on inner region 36.
Number of spots on inner region 37. Number of spots per area of inner region 38. EEA1 marker texture SER spot 1 px.
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chemset except 15e showed a strong negative effect on the number and intensities of spot
signal on cytoplasm markers of PD patient cell line.
Chemset 2: All compounds in Chemset 2 had negative effects on nucleus intensity marker of
both cell lines, however, the effect was generally 1ꢀ3 fold higher for the normal cell line
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(Figure S8). The only compound that violated this pattern was compound 18b which showed
the highest observed negative effect against PD patient cell line. Comparison of the activity
of compounds on αꢀtubulin markers of normal cell line vs PD Patient cell line showed that
they are not following a general pattern. Compounds 18b, 18k and 18l displayed a high
positive effect on αꢀtubulin markers of PD patient cell line and compounds 18o, 18p, 18q and
18r had high negative effects on αꢀtubulin markers of normal cell line. The markers related
to lysosome have been positively affected by most of the compounds but the effect was
stronger for PD cell line. All derivatives in this Chemset had shown a stronger (2ꢀ4 fold)
negative effect on number of spot signal on cytoplasm marker of PD patient cell line only.
The strongest effect on this marker was observed for the core scaffold, compound 17.
Chemset 3 (αꢀC9 and βꢀC9): Comparative analysis of the biological activity of the
synthesized compounds in Chemset 3 (αꢀC9 and βꢀC9) against control cell line vs PD patientꢀ
derived cell line (Figure S9) revealed the following general pattern: 1) Most of the
compounds did not have significant effects on nucleus and cell morphology markers of both
cell lines; 2) There are stronger effects (2ꢀ3 fold) for most of the compounds on αꢀtubulin,
mitochondria, LC3b markers of normal cell line; 3) Most of the compounds showed a
stronger effect on lysosome markers of PD patient cell line; 4) Most of the compounds
showed a stronger negative effect (2ꢀ5 fold) on the number of spots on cytoplasm marker of
PD patient cell line. In addition, the mꢀfluoro phenylacetic ester derivative, compound 25α,
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exhibited significant negative effects on αꢀtubulin and LC3b markers and positive effects on
number and intensities of EEA1 marker of only normal cell line. Amongst the α series of
Chemset 3, compound 29α displayed considerably strong effects on the vast majority of the
biological markers of both normal and PD patient cell lines. Compound 29α showed the
strongest negative effects on nucleus intensity, αꢀtubulin, mitochondria, LC3b and lysosome
markers and also strongest positive effects on cell morphology area, number of spots on
cytoplasm as well as number of spots on outer and inner regions. In β series, compound 27β
displayed considerably strong effects on the vast majority of the biological markers of the
normal and PD patient cell lines. It showed the strongest negative effects on nucleus
intensity, αꢀtubulin, mitochondria, LC3b, lysosome and relative spot signal on inner region
markers and strongest positive effects on cell morphology area, number of spots on
cytoplasm and also number of spots on outer and inner regions.
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Comparative analysis of the biological profile of synthesised compounds against
Parkinson’s patientꢀderived cell line
Chemset 1 v Chemset 2: The biological effect of compounds in Chemset 1 and their
diastereomers in Chemset 2 on the biological markers (38 parameters) of PD patient cell line
are compared in Figure 9. Comparative analysis of the biological activity of Chemsets 1 and
2
revealed the following observations for both Chemsets: 1) Small negative effect on nuclear
and cell morphology markers; 2) Moderate positive effects on αꢀtubulin, mitochondria, LC3b
and lysosome markers; 3) Negative effect for most of compounds in both Chemsets on
number and intensities of EEA1 markers.
In addition to the above mentioned similarities some interesting differences between the
biological effects of compounds in Chemset 1 and their stereoisomers in Chemset 2 were
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Figure 9. Heatmap depicting the comparative cytological profile of compounds 14ꢀ15r of Chemset 1 and 17ꢀ18r of the Chemset 2 at 10 ꢁM on 38 parameters of
Parkinson’s patientꢀderived cell lines. The effects of compounds were plotted as log2 ratio to the DMSO control. Yellow shows a decrease versus control and
purple shows an increase versus control. Individual compounds are presented on the yꢀaxis (compounds with same derivatives are shown with the same colour)
with individual features on the xꢀaxis. For the features descriptions refer to Figure 8.
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noticed. The core scaffold 14 displayed 4ꢀfold stronger effect on αꢀtubulin marker texture
SER spot 1 px while its diastereomer in Chemest 2, compound 17, showed 3 and 5 times
higher effects on lysosome and number of spot on inner region, respectively. Compound 15a
exhibited a negative effect on relative spot signal on inner region, whereas compound 18a,
had the reverse positive effect. Compound 18b had a 5ꢀfold stronger effect on nucleus
marker intensity mean compared to its diastereomer in Chemset 1, compound 15b.
Furthermore, compound 15b displayed moderate positive effect on αꢀtubulin and
mitochondria markers while compound 18b showed small negative effects. There was a 2ꢀ
fold positive effect on αꢀtubulin, mitochondria and LC3b markers for compound 18d and a
double size negative effect on number and intensities of EEA1 markers for compound 15d.
The mꢀflouro phenylacetic ester 15g compared to its diastereomer 18g had a stronger effect
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(2ꢀ5 times more) on markers related to αꢀtubulin and number and intensities of EEA1 but the
effect was reversed for relative spot signal on inner region and number of spots per area of
inner region markers for which compound 18g had stronger effect. Compound 18h showed 3
times stronger effects on lysosome marker while its diastereomer, derivative 15h, had a 6ꢀ
fold stronger positive effect on relative spot signal on inner region. Compound 18k exhibited
a 2ꢀfold effect compared to 15k on markers related to αꢀtubulin, mitochondria and LC3b.
Derivative 18l compared to its diastereomer in Chemset 1, derivative 15l, had a 3 times
stronger effect on αꢀtubulin and LC3b whereas 15l showed double size effect on relative spot
signal on cytoplasm and number of spots on cytoplasm. While compound 15n showed a
stronger effect on LC3b marker, its distereomer 18n had stronger effects on relative spot
signal on inner region, number of spots on inner region and number of spots per area of inner
region markers. The 2ꢀfuroic ester derivative in Chemset 1, compound 15o, displayed 3ꢀfold
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stronger effect on αꢀtubulin whereas the diastereomer 18o showed a 3ꢀfold stronger effect on
mitochondria marker. The 3ꢀfuroic ester derivatives 15p and 18p, exhibited different size
effects on many parameters. Markers related to cell morphology area and length, LC3b,
mitochondria, numbers of spots on cytoplasm and inner region have been affected by
compound 18p two fold more strongly than compound 15p. The ester 15q in comparison to
its diastereomer 18q had stronger effects (4 times more) on markers related to mitochondria.
There was a weak negative effect on αꢀtubulin markers caused by compound 18r while the
effect on these markers was positive for compound 15r. The observed different pattern of
biological profile between diastereomers in Chemsets 1 and 2 highlights the important role of
both stereochemistry and substituted groups at Cꢀ3 of the scaffold on biological activity.
Chemsets 1 v Chemset 3 [(αꢀC9) and (βꢀC9)]: The effect of compounds in Chemset 1 and
their diastereomers in Chemset 3 [(αꢀC9) and (βꢀC9)] on the 38 biological markers of
Parkinson’s patientꢀderived cell line are depicted in Figure 10. Comparison of the biological
activity of Chemsets 1 and 3 showed the following trends: 1) Except for compounds 27β and
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9α, no considerable effects on parameters related to nucleolus and cell morphology was
observed for all compounds; 2) Positive effect on mitochondria markers was noticed for all
compounds except for compounds 24β, 26α, 27β and 29α; 3) Four times stronger positive
effect was observed for compounds in Chemset 1 compared to their diasteromers in Chemset
3
[(αꢀC9) and (βꢀC9)]; 4) All compounds had negative effect on mitochondria and LC3B
texture SER spot 1 px markers; 5) Except for compounds 27β and 29α, positive effect on
lysosome markers was observed for all compounds; 6) Negative effect on number of spots on
cytoplasm and inner region markers was observed for all compounds except for compounds
27β and 29α.
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In addition, the following differences between the biological effects of compounds in
Chemest 1 and their stereoisomers in Chemest 3 were observed. The reduction of carbonyl
group in the core scaffold 14 led to an increase in the effect caused by 19α and 19β on
nucleus intensity mean, lysosome and number of spots on inner region markers. On the other
hand, distereomers 19α and 19β showed 4 times lower effect on mitochondria markers
compared to the oxidized form, compound 14. Compound 19α showed a moderate positive
effect on relative spot signal on outer region while the observed effect for its diastereomer
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9β was negative. Compound 15b, compared to its oxidized form, compounds 20α and 20β,
showed 3 times larger effect on mitochondria, relative spot signal on cytoplasm and inner
region markers. The biological markers related to αꢀtubulin and LC3b have been affected
equally by 15b and 20β and that was 3 times stronger compared to what observed for
compound 20α. Compound 15c' compared to its reduced forms in Chemset 3, distereomers
21α and 21β, showed stronger effects on number and intensities of EEA1 markers.
Compound 21α displayed moderate negative effects on αꢀtubulin, LC3b and large positive
effect on relative spot signal on outer and inner regions which is opposite to the effect
observed for the diasteroemers 15c' and 21β. There was an increased effect for the reduced
forms of compound 15d, derivatives 22α and 22β, on nucleus intensity mean marker, while
for the markers related to αꢀtubulin, LC3b, number of spots on inner region, number of spots
per area of inner region and also EEA1 marker texture SER spot 1 px, the observed effect for
the oxidized form, compound 15d, was 1ꢀ4 times stronger. Compounds 15d and 22β
displayed negative effect on relative spot signal on cytoplasm, number and relative spot
signal on outer and inner region but the effect observed for derivative 22α on these
parameters was positive.
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Figure 10. Heatmap depicting the comparative cytological profile of compounds 14ꢀ15q of Chemset 1, 19αꢀ30α of the Chemset 3 (αꢀC9) as well as 19ꢀ30β of
the Chemset 3 (βꢀC9) at 10 ꢁM on 38 parameters of Parkinson’s patientꢀderived cell lines. The effects of compounds were plotted as log2 ratio to the DMSO
control. Yellow shows a decrease versus control and purple shows an increase versus control. Individual compounds are presented on the yꢀaxis with individual
features on the xꢀaxis. For the features descriptions refer to Figure 8. Compounds with same derivatives are shown with the same colour.
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