A Tryptoline Ring-Distortion Strategy Leads to Complex and Diverse Biologically Active Molecules from the Indole Alkaloid Yohimbine

Article abstract of DOI:10.1002/chem.201604795

High-throughput screening (HTS) is the primary driver to current drug-discovery efforts. New therapeutic agents that enter the market are a direct reflection of the structurally simple compounds that make up screening libraries. Unlike medically relevant natural products (e.g., morphine), small molecules currently being screened have a low fraction of sp³ character and few, if any, stereogenic centers. Although simple compounds have been useful in drugging certain biological targets (e.g., protein kinases), more sophisticated targets (e.g., transcription factors) have largely evaded the discovery of new clinical agents from screening collections. Herein, a tryptoline ring-distortion strategy is described that enables the rapid synthesis of 70 complex and diverse compounds from yohimbine (1); an indole alkaloid. The compounds that were synthesized had architecturally complex and unique scaffolds, unlike 1 and other scaffolds. These compounds were subjected to phenotypic screens and reporter gene assays, leading to the identification of new compounds that possessed various biological activities, including antiproliferative activities against cancer cells with functional hypoxia-inducible factors, nitric oxide inhibition, and inhibition and activation of the antioxidant response element. This tryptoline ring-distortion strategy can begin to address diversity problems in screening libraries, while occupying biologically relevant chemical space in areas critical to human health.

Full text of DOI:10.1002/chem.201604795

A Journal of
Accepted Article
Title: A Tryptoline Ring Distortion Strategy Leads to Complex and
Diverse Biologically Active Molecules from the Indole Alkaloid
Yohimbine
Authors: Nicholas Paciaroni, Ranjala Ratnayake, James Matthews,
Verrill Norwood, Austin Arnold, Long Dang, Hendrik Luesch,
and Robert William Huigens III
This manuscript has been accepted after peer review and appears as an
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of the final Version of Record (VoR). This work is currently citable by
using the Digital Object Identifier (DOI) given below. The VoR will be
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content of this Accepted Article.
To be cited as: Chem. Eur. J. 10.1002/chem.201604795
Link to VoR: http://dx.doi.org/10.1002/chem.201604795
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A Tryptoline Ring Distortion Strategy Leads to Complex and
Diverse Biologically Active Molecules from the Indole Alkaloid
Yohimbine
Nicholas G. Paciaroni,[a, b] Ranjala Ratnayake,[a, b] James H. Matthews,[a, b] Verrill M. Norwood IV,[a, b]
Austin C. Arnold,[a] Long H. Dang,[a, b, c] Hendrik Luesch,[a, b] Robert W. Huigens III*[a, b]
Abstract: High-throughput screening (HTS) is the primary driver to
current drug discovery efforts. New therapeutic agents that enter the
market are a direct reflection of the structurally simple compounds that
make up screening libraries. Unlike medically relevant natural
products (e.g., morphine), small molecules currently being screened
have low fraction sp3 character and few, if any, stereogenic centers.
Although simple compounds have been useful in drugging certain
biological targets (e.g., protein kinases), more sophisticated targets
(e.g., transcription factors) have largely evaded the discovery of new
clinical agents from screening collections. Here, we describe a
tryptoline ring distortion strategy that enabled the rapid synthesis of
70 complex and diverse compounds from yohimbine 1, an indole
alkaloid. The compounds that were synthesized had architecturally
complex and unique scaffolds, unlike 1 and other scaffolds. These
compounds were subjected to phenotypic screens and reporter gene
assays leading to the identification of new compounds that possess
various biological activities, including: antiproliferative activities
against cancer cells with functional hypoxia inducible factors, nitric
oxide inhibition, inhibition and activation of the antioxidant response
element (ARE). This tryptoline ring distortion strategy can begin to
address diversity problems in our screening libraries while occupying
biologically relevant chemical space in areas critical to human health.
compounds involved screening collections of natural products or
crude extracts.1 These were highly fruitful expeditions that gave
us bioactive entities, such as morphine, vancomycin and taxol,
which all possess exquisite biological activities due to their
complex molecular structures interacting with their corresponding
biological target in a highly specific and selective fashion. Due to
advances in molecular biology, solid-phase synthesis and
screening technologies, the 1990’s led to a paradigm shift from
screening natural products/extracts in whole-cell (phenotype)
screens to large collections of relatively simple organic
compounds (containing low sp3-character and essentially no
stereochemistry) and target-based, high throughput screens
(HTS).2,3
HTS has become the primary driver in drug discovery and has led
to several FDA-approved drugs, including: gefitinib, erlotinib,
sorafenib, tipranavir, tolvaptan and lapatinib.2 Despite success in
drugging certain protein targets (tyrosine kinases), current
screening libraries have been ineffective at targeting more
sophisticated biological targets (i.e., protein-protein interactions4-
6, transcription factors7,8) and disease areas (i.e., antibiotic-
resistant bacteria3). The inability of current screening libraries to
produce compounds that effectively drug “undruggable” biological
targets are attributed to an underrepresentation of chemical
diversity, or chemical space.3,9-11 As such, complex and diverse
small molecules are of significant interest to drug discovery.
Introduction
Through the 1980’s, natural products were the primary source of
new drug molecules as efforts to discover new biologically active
Several elegant synthesis strategies have been developed to
address chemical diversity deficiencies through the rapid
synthesis of complex molecules for biological screening; however,
the two most well-known approaches include: diversity-oriented
synthesis9,12-17 (DOS; pioneered by Schreiber and co-workers)
and biology-oriented synthesis18-23 (BIOS; led by Waldmann and
co-workers). Additionally, alternative and elegant syntheses of
natural product-inspired scaffolds has been an area of significant
interest in recent years.24-26 These strategies are aimed at
capturing various structural features of natural products (i.e.,
fused ring systems, stereochemical complexity) into small
molecules that can be rapidly accessed for biological screening.
DOS and BIOS have led to several interesting biological
discoveries in diverse disease areas and are promising
approaches to the identification of therapeutic leads and probe
molecules to interrogate biological systems.20,22,23,27-29
[a]
[b]
[c]
N. G. Paciaroni, Dr. R. Ratnayake, Dr. J. H. Matthews, V. M.
Norwood IV, A. C. Arnold, Prof. L. H. Dang, Prof. H. Luesch, Prof. R.
W. Huigens III
Department of Medicinal Chemistry, University of Florida
Gainesville, Florida 32610, United States
E-mail: rhuigens@cop.ufl.edu
N. G. Paciaroni, Dr. R. Ratnayake, Dr. J. H. Matthews, V. M.
Norwood IV, Prof. L. H. Dang, Prof. H. Luesch, Prof. R. W. Huigens
III
Center for Natural Product Drug Discovery and Development
(CNPD3), University of Florida
Gainesville, Florida 32610, United States
Prof. L. H. Dang
Department of Medicine, University of Florida
Gainesville, Florida 32610, United States
Supporting information for this article is given via a link at the end of
the document.
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Figure 1. A) Biologically active indole-containing and indole-related molecules. B) Tryptoline ring distortion strategy applied to the indole alkaloid Yohimbine. C)
Complex and diverse small molecules rapidly synthesized from Yohimbine.
An alternative “ring distortion” approach utilizes available natural
products containing a complex and fused ring system as synthetic
starting points. Through rapid synthetic sequences from the
selected natural product, the strategic goal is to dramatically alter
the complex ring system inherent to the natural product thus
generating complex small molecules with unique and diverse
molecular architectures.30 Ring distortion involves the altering of
complex ring systems through various chemical reactions that
enable ring cleavage, expansion, rearrangement and fusion.30-32
The Hergenrother lab has carried out ring distortion campaigns
on gibberellic acid30, adrenosterone30, quinine30, abietic acid31
and pleuromutilin.32 These have been largely successful due to
the utility of strategically useful ketone- and alkene-based
chemistries in addition to complex molecular frameworks that
enable dramatic ring rearrangement reactions for ring distortion.
trees and the Rauwolfia root that acts as an α2-adrenergic
receptor antagonist.33 This alkaloid is highly abundant and sold
over-the-counter for its fat-burning/stimulatory and aphrodisiac
properties. We became interested in 1 as a starting point for ring
distortion due to its highly complex ring system fused to an indole
nucleus. Multiple synthetic reactions of 1 as a starting material
have been reported in the literature, including: oxidation of the
secondary alcohol34, allylation of the 3-position of the indole
nucleus35, alkylation of the indole nitrogen36, hydroxylation of the
indole nitrogen37,38, reduction of the 2,3-positions of the indole
heterocycle37,38, and ethylene glycol protection of the 2,3-
positions of the indole nucleus.39 Our ring distortion strategy
involved two known transfromations, including: cyanogen
bromide ring cleavage with alcohols40 and transformation of 1 into
a spirooxindole-containing scaffold.41 Yohimbine 1 provides an
outstanding platform to develop an innovative ring distortion
strategy involving the basic reactivity of the indole heterocycle to
rapidly generate complex and diverse small molecules for
biological screening in diverse disease areas (i.e., cancer,
inflammation, drug-resistant bacteria).
Indole-containing compounds span an array of chemical space
from complex natural products to very simple compounds (Fig 1A,
indoles 1-9). Interestingly, simple and complex indole-containing
compounds demonstrate diverse biological activities due to the
numerous biological targets that bind various indoles, making the
indole nucleus a privileged scaffold that occupies an interesting
and biologically relevant chemical space. Yohimbine 1 is a
complex indole alkaloid isolated from the bark of Pausinystalia
Results and Discussion
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Scheme 1. Summary of ring distortion from 1. Reagents and Conditions: a) BrCN (3.0 eq.), DMF, µw, 100°C, 2.75 min, 45%; b) BrCN (3.0 eq.), ROH:CHCl3 (3:1),
rt, 16 h, 14 – 63% (11 examples); c) Copper(I) Iodide (0.35 eq.), K2CO3 (1.5 eq.), DMEDA (0.7 eq.), MeCN, µw, 160 ᵒC, 13 min, 70 – 73%; d) NCS (1.1 eq.), CH2Cl2,
-41 ᵒC, 0.75 h, crude; e) 0.5 M NaOMe/MeOH, rt, 2.5 h, 66%; f) 10% TFA (aq), reflux, 1 h, 58%; g) BrCN (3.0 eq.), DMF, rt to 60 ᵒC, 22 h, 52%. Notes: DMEDA =
N,N’-dimethylethylenediamine, NCS = N-chlorosuccinimide. *Indicates combined percent yield and isolated yields of ether diastereomers.
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Tryptoline-Based Ring Distortion Strategy. We devised a ring
distortion strategy based on two structural features connected
through the tryptoline 10 sub-structure of 1, which include: 1.) the
centrally positioned basic nitrogen with three unequivocal C-N
bonds (bonds B, C and D; Fig 1B), ideal for dramatic and diverse
ring cleavage reactions to afford 11-13 and 2.) oxidative
rearrangement of the 2,3-disubstituted indole nucleus to give ring-
rearranged product Y7b (bond A; Fig 1B). Our goals were to
develop short synthetic sequences that involve ring distortion
reactions directly from 1 to enable optimal choreographing of ring
cleavage, scrambling, fusion or rearrangement reactions in
tandem with subsequent diversification reactions (i.e., click
chemistry). This tryptoline ring distortion strategy was developed
to generate several complex and diverse scaffolds totaling 70
small molecules in rapid synthetic sequences from 1 (Fig 1C).
576 milligrams, which is sufficient for additional ring distortion
reactions, analogue synthesis and biological screening.
We took advantage of the lack of diastereocontol of cyanogen
bromide/alcohol ring cleavage to generate diverted ring fusion
products Y4a and Y5a (Scheme 1B), which have unique complex
molecular architectures. Yohimbine was initially subjected to
cyanogen bromide ring cleavage in chloroform:2-iodobenzyl
alcohol to give a 1.2:1 diastereomeric mixture of Y6r and Y6s
(47% combined yield). The diastereomeric mixture was readily
separated and each diastereomer was taken forward and
subjected to copper(I) iodide-catalyzed intramolecular C-N
coupling between the aryl iodide and indole nitrogen to give
diverted ring fusion products Y4a (70%) and Y5a (73%) on 163-
222 milligram scale.
Ring Distortion of the Indole Alkaloid Yohimbine.
Indole heterocycles can undergo oxidation with subsequent alkyl
migration, translating to a ring rearrangement of 1. An oxidative
rearrangement such as this allows us to take advantage of the
intrinsic reactivity of the indole ring, which was a critical aspect of
our tryptoline ring distortion strategy. A three-step, oxidative-
rearrangement of yohimbine to afford ring-rearranged
spirooxindole Y7b (Scheme 1C) was previously reported.41
Although initial attempts to repeat the literature chlorination
reaction with hypochlorite failed in our hands, this was remedied
by using N-chlorosuccinimide (NCS) to afford clean chlorination
at the 3-position of the indole of 1. This was followed by treatment
with sodium methoxide (NaOMe) to induce stereospecific alkyl
migration and expulsion of the 3-chloro group. Final treatment
with trifluoroacetic acid (TFA) afforded ring-rearranged product
Y7b (38%/3 steps). We were able to generate 748 milligrams of
Y7b in one run through this three step route during these
investigations without focused scale-up efforts.
Initially, we targeted the C-N bonds of yohimbine’s central amine
for regioselective ring cleavage reactions. Using cyanogen
bromide (BrCN), we were able to activate the basic nitrogen of 1
to give intermediate 14 (Scheme 1), from which we generated
numerous diverted ring-distorted compounds.
Two of the
unequivocal C-N bonds of 1 could be selectively cleaved with
cyanogen bromide through careful selection of solvent.
Cyanogen bromide C-N ring cleavage of 1 proceeds through an
SN2 pathway in dimethylformamide (DMF; Scheme 1A). The
liberated bromide anion generated upon formation of 14 attacks
the most accessible “b” carbon adjacent to the activated central
nitrogen of 14 in only 3 minutes using microwave irradiation
resulting in Y1a as the only observed product. We were able to
successfully carry this reaction out on a 985 milligram scale (45%
yield), which was crucial for subsequent analogue synthesis.
We subjected Y7b to cyanogen bromide ring cleavage in DMF to
afford Y2a as a single product in 52% yield (233 milligrams;
Scheme 1C; X-ray in Supporting Information). The propensity of
bromide to attack the “b” carbon of Y7b can be rationalized by an
SN2 attack of the least sterically encumbered carbon atom of the
respective cyanamide intermediate (not shown). Despite many
attempts, ring cleavage at the “D” bond (Fig 1B) of 1 was evaded;
however, future developments are aimed to address this to
generate interesting structures similar to 13.
Contrasting to the SN2 pathway, a 3:1 chloroform:alcohol solvent
mixture results in an SN1-like pathway in which the alcohol is a
nucleophile and attacks the “a” carbon of 14. This reaction has
been previously described40; however, we extended this reaction
by utilizing diverse alcohol solvent/nucleophiles, which react with
intermediates 14 and 15 to yield diastereomeric mixtures of ethers
16 (“SN2 product”; inverted stereochemistry at “a”) and 17 (“SN1
product”; retention of stereochemistry at “a”). Diastereomers
produced from this reaction can be readily separated via
chromatography and tested in biological screens. Interestingly,
less sterically encumbered alcohols (i.e., methanol) gave “SN2
products” (i.e., 16) as the major diastereomer (3.4:1 to 50:1 drs;
Scheme 1A), which can be rationalized by considering that 14 and
15 can permit less hindered nucleophiles to undergo “back-side
attack” at carbon “a” of structure 14. Alcohols with more steric
bulk (i.e., 2-iodobenzyl alcohol) gave ~1:1 mixtures of
diastereomeric products, which can be explained through an SN1-
like mechanism involving nucleophilic attack of both faces on
resonance-stabilized carbocation 15. This ring-cleavage reaction
proceeded in 14-63% yield (11 examples yielded 19 ethers) and
the major diastereomers were isolated on scales between 6.9 and
The rapid, tryptoline-based ring distortion pathways developed
during these investigations were highly efficient in the generation
of several new scaffolds in 1 to 4 steps from 1. During these
investigations, multiple C-N bonds to the central nitrogen atom of
1 were selectively cleaved with cyanogen bromide, an oxidative
rearrangement of the indole heterocycle of 1 was utilized and C-
N bond coupling reactions were used in ring fusion reactions to
generate diverted complex scaffolds. Overall, this tryptoline ring-
distortion platform enabled the rapid synthesis of novel complex
and diverse chemotypes that are important to biological screening
and drug discovery efforts.
Diversification of Ring Distorted Compounds.
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Scheme 2. Library synthesis to afford sub-libraries of yohimbine ring-distorted compounds. Reagents and Conditions: a) BrCN (3.0 eq.), THF:H2O (2.5:1), rt, 23 h,
57%; b) BrCN (3.0 eq), alcohol:CHCl3 (1:3), rt (or reflux for Y6j-Y6o), 6.5 h (major diastereomer yields: 7 – 55%; minor diastereomer yields: 7 – 21%); c) BrCN (3.0
eq.), DMF, µw, 100 ᵒC, 2.75 min, 56%; d) NaN3 (3.0 eq.), DMF, rt to 75 ᵒC, 22 h, 91%; e) BrCN (3.0 eq), DMF, rt to 60 ᵒC, 22 h, 52%; f) NaN3 (3.0 eq), DMF, rt to
80 ᵒC, 16.5 h, 99%; g) Aryl iodide (1.1 eq.), DMEDA (0.70 eq.), CuI (0.35 eq.), MeCN, µw, 160 ᵒC, 13 min, 20 – 37%; h) alkyl halide (1.9 eq.), K2CO3 (3.8 eq.), DMF,
rt, 15 h, 44 – 80%; i) NaH (4.3 eq.), THF, 0 ᵒC; then benzyl bromide (2.0 eq.), THF, 0 ᵒC to rt, 16 h, 29 – 66%; j) alkyne (3.0 eq.), anhydrous CuSO4 (0.5 eq.), sodium
ascorbate (1.5 eq.), tBuOH:H2O (1:2), CH2Cl2, rt, 2 h, Y1 yields: 19 – 78%, Y2 yields: 50 – 78%. BrCN = cyanogen bromide, DMEDA = N,N’-dimethylethylenediamine.
Following tryptoline ring distortion of 1, our goals were to decorate
newly synthesized complex and diverse scaffolds using more
traditional diversification strategies. This allows for the synthesis
of sub-libraries for each scaffold enabling further exploration of
chemical space during biological screening endeavors.
Diversification of yohimbine-derived architectures found in
Scheme 1 was ideal as these complex small molecules are
endowed with multiple functional groups serving as potential
synthetic handles for library generation.
We found ring-rearranged Y7b to be very useful in diverted library
synthesis due to the amide and hydroxyl functional groups readily
available as synthetic handles. Ten mono- or dialkylated products
of Y7b were synthesized with available alkyl halides (Y7c-Y7l,
47% average yield; Scheme 2). Y7b was also diversified using
copper(I)-catalyzed C-N coupling between the amide nitrogen
and nine diverse (hetero)aryl iodides (29% average yield).
Other synthetic modifications were made to yohimbine-derived
small molecules, including: 1) lithium aluminum hydride
reductions of cyanamide/ester functional groups and 2) acylation
(Supporting Information). These efforts resulted in 70 complex
and diverse compounds synthesized from yohimbine using rapid
and diverted synthetic pathways. During these investigations, we
were able to synthesize each of the analogues in these sub-
libraries on a 6.0 to 576 milligram scale, which is sufficient for
extensive biological investigations.
Reactions that lead to ring distortion AND chemical diversification
(sub-libraries) in the same step are highly useful in this approach.
For example, subjecting 1 to cyanogen bromide and 11 different
alcohols (including water; Scheme 1A) led to 19 total ring-cleaved
ethers (Y6a-Y6s; Scheme 2). The library potential for this
reaction was enhanced as two diastereomers are typically
produced from this reaction. In 8 of the 11 examples, both
diastereomers were isolated (pure) for biological screening and
additional synthesis efforts.
Biological Investigations of Yohimbine-Derived Small
Molecules.
Due to the synthetic utility of cyanogen bromide in ring cleavage
reactions, multiple ring-cleaved compounds contained primary
bromides (Y1a and Y2a) which were subjected to sodium azide
displacement (SN2) to yield azides Y1b and Y2b, respectively
(Scheme 2). Each azide was then subjected to copper(I)-
catalyzed click reactions with a diverse panel of available terminal
alkynes to generate two diverse triazole sub-libraries (Y1c-Y1h, 6
examples, 61% average yield; Y2c-Y2p, 14 examples, 69%
average yield).
We screened our yohimbine ring distortion library in several
phenotypic and focused pathway-specific screens related to
cancer and inflammation as well as pathogenic bacteria (Fig 2A).
Specifically, we aimed to identify selective antiproliferative agents
that preferentially act on cancer cells with functional hypoxia-
inducible factors (HIF) pathways.
HIF transcription factor
mediated signaling is known to be activated in cancer and linked
to angiogenesis, cell growth, survival and metastasis. We
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Figure 2. A) Heat map matrix summarizing primary biological screening results of the yohimbine ring distortion library (biological activity scale key: red = inhibition;
yellow = no change; green = activation), including the structures of six validated hit compounds. Rows correspond to compounds tested. Columns correspond to
primary biological activity screens (1-10) and concentrations tested (200, 100, 10 and 1 µM). 1) Parental HCT116 and 2) HCT116HIF-1α−/−HIF-2α−/− cell viability; 3)
RAW264.7 cell viability; 4) RAW264.7 NO production; 5) LNCaP cell viability; 6) LNCaP cell ARE activity; 7) MDA-MB-231-ARE-luc cell viability; 8) MDA-MB-231-
ARE-luc cell ARE activity; 9) S. aureus and 10) A. baumannii growth. B)−E) Validation experiments of hit compounds. B) HIF-dependent anticancer activity. Cell
viability of parental HCT116 and HCT116HIF-1α−/−HIF-2α−/− cells was determined after 48 h exposure using MTT assay (p-values: ≤ 0.01 **, ≤ 0.05 *; pairwise student t-
test comparing cell viability between parental HCT116 and HCT116HIF-1α−/−HIF-2α−/− cells at a given concentration). C) NO production and cell viability. Production of
NO and cell viability of RAW264.7 cells were determined after 24 h exposure using Griess reagent and MTT assay, respectively (p-values: ≤ 0.01 **, ≤ 0.05 *;
pairwise student t-test comparing relative NO production and viability of RAW264.7 cells at a given concentration). D) MDA-231-ARE-luc cell ARE activity and E)
LNCaP cell ARE activity. ARE inhibition in MDA-MB-231-ARE-luc and ARE activation in LNCaP cells after 24 h exposure were validated by effects on endogenous
NQO1 transcript levels measured by RT-qPCR. ACTB expression was used as an internal control for normalization. All validation data are presented relative to
vehicle control (0.5% DMSO). Brusatol (125 nM) and sulforaphane (10 µM) served as positive controls for MDA-MB-231-ARE-luc ARE inhibition and LNCaP ARE
activation assays, respectively.
out as the most selective antiproliferative agent upon dose-
employed isogenic HCT116 colorectal cancer cell lines, including
parental HCT116 and HCT116 double knockout cells lacking HIF-
1α and HIF-2α,42,43 and screened our library for differential
antiproliferative activity. Of the 70 compounds tested, Y7g stood
response analysis (Fig 2B), suggesting that this compound could
be a starting point for the development of selective and more
potent HIF inhibitors. Chronic inflammation is a known factor in
the etiology of colorectal cancer and, intriguingly, the same
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compound (Y7g) also showed the most selective anti-
inflammatory activity in RAW264.7 macrophage cells based on
the inhibition of nitric oxide (NO) production (Fig 2C). Cell viability
was almost unaffected in this cell type, further supporting the
cancer cell selectivity of Y7g.
compounds we identified in a relatively small library of 70
compounds.
Indole alkaloids possess an array of complex molecular
architectures that is able to produce diverse biological activities
as this class of natural products has intrigued scientists for
decades.50-54 Total synthesis efforts date back to Woodward’s
elegant construction of (±)-reserpine52 reported in 1956 and
continue to inspire the development of new synthetic methods and
approaches53 today, including Jacobsen’s 2013 total synthesis of
(+)-reserpine.54
As alternative synthesis approaches have emerged to drive
biological discoveries, indoles have remained a considerable
interest as unnatural indole small molecules have demonstrated
impressive biological activities in diverse disease areas of
importance to human health. Synthetic efforts have led to the
discovery of indoline compounds capable of resensitizing
methicillin-resistant Staphylococcus aureus to methicillin55,56
while indole-inspired BIOS has led to the identification of inhibitors
of protein phosphatases, which are important drug targets for
diabetes and cancer.19 BIOS efforts have also led to the
discovery of spirooxindole small molecules that influence
neuronal network formation and demonstrate neurite outgrowth-
promoting activities.22
We then assessed the potential of the library compounds to
modulate the activity of another key transcription factor, NF-E2-
related factor 2 (Nrf2), which acts on the antioxidant response
element (ARE) in the promoter region upstream of antioxidant and
phase II detoxification enzymes. Activators of the Nrf2-ARE
pathway have potential as cancer preventive agents because of
their ability to increase antioxidant status of the cell and protect
from oxidative damage that could lead to cancer. Inhibitors in turn
may increase the susceptibility of cancer cells to
chemotherapeutic agents and overcome drug resistance since
Nrf2 is commonly activated in cancer.44 To screen for Nrf2
inhibitors, we used MDA-MB-231 breast cancer cells (a model for
invasive, triple negative breast cancer) stably transfected with the
ARE-luciferase reporter, where the promoter region is derived
from the human NQO1 gene.45 As a control, we monitored cell
viability to ensure the identification of false positives in the
reporter gene assay that are a result of concomitant reduced cell
viability. We selected Y3e, Y6q and Y1f for validation by RT-
qPCR, assessing the effects of the compounds on the
transcription of the endogenous Nrf2 target gene, NQO1, in
comparison with brusatol.46 All three compounds downregulated
NQO1 transcription (Fig 2D).
To identify activators of Nrf2 we transiently transfected LNCaP
androgen-sensitive prostate cancer cells with ARE-luciferase
reporter based on the human NQO1 promoter47,48 and then
challenged them with our yohimbine library. As a control, we
monitored LNCaP viability in parallel (Fig 2E). While many
compounds activated the ARE at 100 µM potentially as a stress
response, Y5b and Y6u were also active at lower concentrations
when we validated the hits by RT-qPCR for levels of endogenous
NQO1. These compounds were only slightly less potent than the
positive control, sulforaphane.
We also screened our yohimbine library against Staphylococcus
aureus (Gram-positive pathogen) and Acinetobacter baumannii
(Gram-negative); however, only a few compounds demonstrated
weak antibacterial activities (i.e., partial growth inhibition) against
A. baumannii at 200 µM. We did find the antibacterial screen
insightful to cross-reference activity profiles of validated hit
compounds with selective antiproliferative, NO inhibition and
Nrf2-ARE modulation. Of the six validated hit compounds
identified during these biological investigations (Y7g, Y6q, Y3e,
Y1f, Y5b, Y6u; Figure 2), only Y1f and Y6q demonstrated slight
(~20%) growth inhibition activities at 200 µM against S. aureus.
Interestingly, we note that 1 does not possess any of these
activities inherently, illustrating the potential for yohimbine, and
other natural products, to reach beyond their own biological
Figure 3. A) Analysis of the stereochemical complexity of the yohimbine ring
distortion library (Y Series) compared to FDA Drugs and a ChemBridge library.
B) Comparison of the Fsp3 character of the yohimbine library (Y Series), FDA
Drugs and a ChemBridge library.
capabilities using this ring distortion approach.
Overall,
yohimbine ring-distorted small molecules cover a biologically
relevant chemical space as evident by the diverse array of active
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Supporting Information. All experimental details concerning procedures
for chemical synthesis/biological screens/validation of biologically active
hit compounds, full characterization data (1H, 13C NMR, HRMS, melting
point) for all new compounds with 2-D NMR spectra and X-ray data for
select compounds can be found in the Supporting Information associated
with this manuscript.
Here, we utilized the complex indole alkaloid, yohimbine 1, as a
starting point for diverted ring distortion by combining the inherent
reactivity of the indole nucleus with the tryptoline scaffold as a
complementary approach to DOS and BIOS, termed “Complexity-
to-Diversity” (CtD). New ring-distorted compounds from 1 were
advanced through further derivatization and subjected to initial
biological screens leading to the discovery of Y7g which
demonstrated dual HIF-dependent anticancer and anti-
inflammatory activities while five additional ring distorted
compounds (Y6q, Y3e, Y1f, Y5b, Y6u; Figure 2) were found to
selectively modulate the ARE pathway in cancer cell lines.
Acknowledgements
The authors would like to acknowledge the College of Pharmacy
at the University of Florida for start-up funds (R.W.H.) and the NIH
(R01CA172310, H.L.; R50CA211487, R.R.) to support this work.
We thank Professors Donna Zhang for MDA-MB-231-ARE-luc
cells and Jeffrey Johnson for ARE-luc plasmid. We also thank Dr.
Khalil Abboud and The Center for X-Ray Crystallography at the
University of Florida for obtaining an X-ray of Y2a and Y6s. All
high resolution mass spectra (HRMS) were obtained from the
Chemistry Department at the University of Florida.
We compared the yohimbine ring distortion library to
a
ChemBridge (commercial) Library31 and the 100 top-selling, FDA-
approved drugs from 2013 by analyzing the number of
stereocenters per compound and fraction sp3 (Fsp3) character for
compounds in each library (Fig 3). Each compound from our
yohimbine-derived library averages 5.54±0.53 stereocenters,
which is considerably higher than the FDA-approved drugs
(2.58±2.61 stereocenters per compound) and the ChemBridge
Library (0.24 stereocenters per compound). The Fsp3 character
for the yohimbine-derived library is 0.50±0.08 which is similar to
the FDA-approved drugs (Fsp3 = 0.49±0.21) and higher than the
ChemBridge library (Fsp3 = 0.24).
Keywords: drug discovery • tryptoline • ring distortion •
yohimbine • biological screening
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Nicholas G. Paciaroni, Ranjala
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A Tryptoline Ring Distortion Strategy
Leads to Complex and Diverse
Biologically Active Molecules from
the Indole Alkaloid Yohimbine
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