Monoenomycin: A simplified trienomycin A analogue that manifests anticancer activity

Article abstract of DOI:10.1021/ml200108y

Macrocyclic natural products are a powerful class of leadlike chemical entities. Despite commonly violating Lipinski's "rule of 5", these compounds often demonstrate superior druglike physicochemical and pharmacokinetic attributes when compared to their acyclic counterparts. However, the elaborate structural architectures of such molecules require rigorous synthetic investigation that complicates analogue development and their application to drug discovery programs. To circumvent these limitations, a conformation-based approach using limited structure-activity relationships and molecular modeling was implemented to design simplified analogues of trienomycin A, in which the corresponding analogues could be prepared in a succinct manner to rapidly identify essential structural components necessary for biological activity. Trienomycin A is a member of the ansamycin family of natural products that possesses potent anticancer activity. These studies revealed a novel trienomycin A analogue, monoenomycin, which manifests potent anticancer activity.

Full text of DOI:10.1021/ml200108y

LETTER
pubs.acs.org/acsmedchemlett
Monoenomycin: A Simplified Trienomycin A Analogue That Manifests
Anticancer Activity
Gary E. L. Brandt and Brian S. J. Blagg*
Department of Medicinal Chemistry, The University of Kansas, 1251 Wescoe Hall Drive, Malott 4070, Lawrence, Kansas 66045-7563,
United States
S Supporting Information
b
ABSTRACT: Macrocyclic natural products are a powerful class
of leadlike chemical entities. Despite commonly violating
Lipinski's “rule of 5”, these compounds often demonstrate
superior druglike physicochemical and pharmacokinetic attri-
butes when compared to their acyclic counterparts. However,
the elaborate structural architectures of such molecules require
rigorous synthetic investigation that complicates analogue
development and their application to drug discovery programs. To circumvent these limitations, a conformation-based approach
using limited structureÀactivity relationships and molecular modeling was implemented to design simplified analogues of
trienomycin A, in which the corresponding analogues could be prepared in a succinct manner to rapidly identify essential structural
components necessary for biological activity. Trienomycin A is a member of the ansamycin family of natural products that possesses
potent anticancer activity. These studies revealed a novel trienomycin A analogue, monoenomycin, which manifests potent
anticancer activity.
KEYWORDS: Trienomycin A, conformation, anticancer, ansamycin synthesis, structureÀactivity relationship
he macrocycle is an important structural motif amongst
bioactive natural products and imparts favorable physico-
ansamycin family that possess a p-quinone or p-hydroquinone
moiety within the aromatic bridge, trienomycin A contains a
nonredox active phenol. In addition, trienomycin A displays a bio-
logical profile that contrasts the activity manifested by other
ansamycins. For example, mycotrienin II, which is structurally
identical to trienomycin A with the exception of a p-hydroqui-
none moiety, is a potent antifungal agent as well as a promising
anticancer agent.3,6 In contrast, trienomycin A manifests potent
anticancer activity (IC50 of 128 nM against HeLa cervical cancer
cells)7 but displays no antifungal activity, nor does it display
any significant antimicrobial, antiviral, or immunosuppressant
activities.3,5,7À9
A previous report indicated that while trienomycin A exhibits
potent anticancer and antitumor activities, it was found to be
significantly less toxic to nontransformed cells.8 Thus, the unique
biological activity manifested by trienomycin A poses this natural
product as an attractive lead compound for cancer chemother-
apeutic development. Unfortunately, limited structureÀactivity
relationships (SAR) for trienomycin A have been reported, and
the mechanism of action remains unknown. Furthermore, sig-
nificant quantities of the natural product are not available, and
only one total synthesis has been reported, which produces the
natural product in 31 linear steps.10 Although elegant in nature,
this route does not afford a succinct method for evaluation of
T
chemical and pharmacological attributes. Macrocyclic geometry
maintains a delicate balance between flexibility and rigidity that
commonly affects solubility, permeability, and binding to biolo-
gical targets. Despite their inherent druglike properties, the com-
plicated architecture exhibited by macrocyclic natural products
provides a significant challenge toward their utility in drug
development. While the discovery of novel chemical methodol-
ogies has led to the de novo synthesis of numerous macrocyclic
natural products, approaches toward simplified analogues remain
underinvestigated. In this letter, a strategy utilizing molecular
modeling combined with a conformation-based approach was
used to develop simplified ansamycin analogues that maintain
biological activity.
Mycobacteria producetheansamycinfamilyofnaturalproducts,
which manifest diverse biological activities. Structural character-
istics of this family include a macrocyclic polyketide bridged by
an aromatic core. Several ansamycins display significant clinical
attributes, including rifampicin, which manifests antimicrobial
activity for the treatment tuberculosis,1 and geldanamycin, deriv-
atives of which have entered phase III clinical trials for the treat-
ment of cancer.2 In general, the ansamycin family exhibits a high
degree of broad inhibitory activities that include antiviral, antifungal,
and immunosuppressant activities.3,4
Trienomycin A (Figure 1, 1) is a member of the ansamycin
family that was first isolated from Streptomyces sp. no. 83-16 by
Umezawa and co-workers.5 In contrast to other members of the
Received: April 25, 2011
Accepted: July 21, 2011
Published: July 21, 2011
r
2011 American Chemical Society
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Figure 1. Structures of rifampicin, geldanamycin, trienomycin A, and mycotrienin II.
SAR. Therefore, a generalized method for the production of
synthetically useful trienomycin A analogues employing a con-
formation-based approach was pursued.
using SYBYL. The three-dimensional geometries of these
compounds were then analyzed using the Surflex Ligand Simi-
larity tool (default parameter settings).11 Rigid superposition
maintained low energy conformations and provided informative
differences in ligand similarity (Figure 2). Semisynthetic deriva-
tives 2À4 exhibited significant conformational perturbations in
macrocycle geometry and placement of NCxDA side chain, but
methyl-phenyl ether 5 retained the native geometry. Because
these semisynthetic derivatives maintain most of the hydrogen-
bonding capabilities of trienomycin A, we hypothesized that
orientation of the NCxDA side chain and projection of the
phenol are critical to the biological activity manifested by
trienomycin A.
Previously reported semisynthetic modifications to the natural
product including acetylation of the 13-OH (2), saturation of the
triene motif (3), and deletion of the N-cyclohexylcarbonyl D-Ala
(NCxDA) side chain (4) resulted in almost complete ablation of
anticancer activity (Figure 3).3 However, methylation of the free
phenol did produce an analogue equipotent to the natural product
(Figure 2, 5). Without apparent SAR trends and the lack of
activity for semisynthetic derivatives, no obvious hypothesis
for further exploration was available. Therefore, an alternate
approach was pursued based on the overall conformation of the
macrocyclic compounds and the lowest energy conformations of
such analogues.
The efficiency by which simplified analogues of trienomycin A
could overlay with the natural product was evaluated in silico,
also via Surflex calculations. Sequential removal of functionality
and subsequent comparison of the energy-minimized derivatives
The natural product (1) and four semisynthetic derivatives
(2À5) were constructed in the lowest energy conformations
Figure 2. (A) Overlay of energy-minimized trienomycin A and select semisynthetic derivatives using the Ligand Similarity tool (rigid superposition)
from the Surflex software suite. (B) Relative energies and similarity score from the Ligand Similarity tool.
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LETTER
to the natural product were analyzed. The 13-OH, 12-CH3, and
3-OCH3 functionalities appeared to exhibit little effect on the
overall conformation, which suggested that they could be omitted
without significantly affecting biological activity (Figure 3).
Because monoene A (7) exhibited the highest similarity
score to trienomycin A, it was the target for chemical synth-
esis. Synthesis of 7 was envisioned retrosynthetically to occur
through a Wittigolefination between triphenylphosphonium iodide
salt 12 and ketone 13,12 followed by Mitsunobu coupling of the
D-Ala side chain 1413 and subsequent ring-closing metathesis
(Figure 5).14 This synthetic route would also provide access to
monoene E (11), which exhibited the lowest similarity score and
could be used as a negative control to evaluate our conformation-
based approach.
Synthesis of Wittig salt 12 commenced with nucleophilic
aromatic substitution of methyl 3,5-dinitrobenzoate 15 by lithium
methoxide to provide methyl-phenyl ether 16,15 which was then
reduced to aldehyde17 by diisobutylaluminum hydride.16 Aldehyde
17 was subjected to HornerÀWadsworthÀEmmons conditions
to furnish 18,17 which was subjected to diimide mediated reduc-
tion to provide 19.18 The ethyl ester of 19 was reduced to the
alcohol using diisobutylaluminum hydride to yield 20, the nitro
moiety of which was reduced to the aniline 21 by palladium on
carbon under a hydrogen atmosphere. Selective amidation of com-
pound 21 with non-8-enoic acid was accomplished using (1-cyano-
2-ethoxy-2-oxoethylidenaminooxy)dimethylamino-morpholino-
carbenium hexafluorophosphate (COMU) to afford amido-alcohol
22.19 Until this point, no chromatographic separation was required,
enabling the preparation of large quantities. Amido-alcohol 22
was then iodinated and converted to Wittig salt 12 (Scheme 1).
Synthesis of ketone 13 was similarly straightforward. Oxidation
of ketol 23 to the ketoaldehyde and treatment of the dicarbonyl
species with 0.5 equiv of Brown's allylborane furnished optically
active ketol 24.20 Synthesis of ketone 13 was completed following
silyl ether formation (Scheme 2).
Figure 3. (A) Structure of theoretical trienomycin A analogue lacking
the 13-OH, 12-CH3, and 3-OMe functional groups. (B) Overlay of
trienomycin A and the theoretical derivative lacking the 13-OH, 12-
CH3, and 3-OCH3 functional groups (6). The theoretical analogue
exhibited an energy value of 21.527 kcal/mol and a similarity score of
0.7935 with the trienomycin A (1).
The triene motif was evaluated next, with the aim of generat-
ing the most synthetically accessible derivatives. Twelve energy-
minimized “monoene” derivatives, containing all possible olefin
isomers and the methyl ether of the reported derivative, were
evaluated based upon their ability to adopt a conformation that
allowed both the phenol and the NCxDA side chain to occupy
the same conformational space as the natural product. Olefin
geometry was critical in these structures and was responsible
for orienting both the NCxDA side chain and the phenol into
conformations that were similar to the natural product. Most
of the monoene derivatives exhibited significant differences in
macrocycle geometry as compared to trienomycin A. How-
ever, derivatives 7À10 manifested a macrocycle geometry
similar to the natural product and produced relatively high
similarity scores (Figure 4). Monoene A, which contains trans-
olefins at both C8 and C14, occupied the closest three-dimen-
sional orientation to trienomycin A and exhibited the highest
similarity score (0.7815).
N-Cyclohexylcarbonyl D-alanine acid 14 was constructed in
one step by treating D-alanine with cyclohexylcarbonyl chloride
in the presence of tribasic potassium phosphate in tetrahydrofur-
an (Scheme 3).
With the synthons in hand, Wittig olefination reaction be-
tween 12 and 13, followed by silyl deprotection, and Mitsunobu
Figure 4. (A) Labeled monoene derivative key. (B) Comparison of monoene derivatives and trienomycin A. (C) Relative energies and similarity score
from the Ligand Similarity tool.
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Figure 5. Retrosynthetic analysis of compound 7, monoene A.
Scheme 1. Synthesis of Wittig Salt 12a
a Reagents and conditions: (a) LiOMe, MeOH, reflux, 97%. (b) DIBAl-H, DCM, À78 °C, 93%. (c) Triethylphosphonoacetate, NaH, THF, 0 °C, 94%.
(d) Potassium azodicarboxylate, AcOH, DME, 50 °C, 97%. (e) DIBAl-H, PhMe, 0 °C. (f) 10% Pd/C, H2, EtOAc. (g) Non-8-enoic acid, COMU,
DIPEA, DMF, 93% over two steps. (h) PPh3, imidazole, I2, DCM, 83%. (i) PPh3, CAN, 90%.
Scheme 2. Synthesis of Ketone 13a
Scheme 3. Synthesis of N-Cyclohexylcarbonyl D-Alanine
Acid 14a
a Reagents and conditions: (a) PCC, DCM. (b) Allyl magnesium
bromide, (+)-B-methoxydiisopinocampheylborane, Et2O, ee > 95%,
82% (BRSM) over two steps. (c) TBSCl, imidazole, DMF, 79%.
a Reagents and conditions: (a) K3PO4, THF, 76%.
coupling between 25 and 14 led to metathesis precursors 26
(trans) and 27 (cis) in a 1:1 mixture, which were separable by
chromatography. Originally, it was desired to obtain the macrocyclic
alcohol after ring-closing metathesis, which could subsequently
undergo diversification at a later stage. However, the macrocyclic
alcohol failed to efficiently undergo esterification via Mitsunobu
conditions or direct acylation, due to steric constraints resulting
from the macrocyclic conformation. Therefore, the NCxDA side
was appended prior to cyclization. Each isomer was subjected to
ring-closing metathesis with Grubbs first generation catalyst to
yield products 7 and 11 as a 2:1 and 5:1 (trans:cis) mixture of
olefin isomers, respectively (Scheme 4). The isomeric mixtures
were evaluated for antiproliferative activity against MCF-7 and
HeLa cancer cells. As predicted in our conformation-based design
approach, compound 7 retained antiproliferative activity (IC50
0.47 μM) while compound 11 was inactive.
=
The anticancer activity exhibited by compound 7 prompted
investigations into the amino acid side chain. Specifically, the
effects of increasing steric bulk in lieu of the methyl substituent
and alterations of the amide to include small alkyl groups were
pursued. The various side chains were synthesized from the corre-
sponding amino acids and appropriate acyl chlorides similar to
compound 14. Once synthesized, the side chains were coupled
with compound 25 using Mitsunobu conditions and subsequently
cyclized with Grubbs first generation catalyst (Figure 6, 28À34).
These compounds were evaluated for antiproliferative activity
against HeLa and MCF-7 cancer cells (Table 1). From the data
presented, deviation in steric bulk, with functionalities that exhibit
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LETTER
Scheme 4. Synthesis of Monoene Derivatives of Trienomycin Aa
a Reagents and conditions: (a) KHMDS, toluene, 78%. (b) TBAF, THF, 97%. (c) Diethyl azodicarboxylate, PPh3, 14, 37% (26), 39% (27). (d) Grubbs
first generation catalyst, DCM, 83%.
Table 1. Anti-proliferative Effects of Trienomycin A Com-
pound Library against HeLA and MCF-7 Cancer Cell Linesa
IC50 (μM)
compound
HeLa
MCF-7
7
0.47 ( 0.04
0.53 ( 0.02
11
>100
13 ( 0.5
1.7 ( 0.03
>100
12 ( 0.5
1.9 ( 0.05
28
29
30
9.1 ( 0.09
4.6 ( 0.09
1.0 ( 0.04
5.1 ( 0.07
31
32
>100
>100
33
1.6 ( 0.09
2.5 ( 0.9
0.72 ( 0.5
1.7 ( 0.4
34
geldanamycin
>5
0.053 ( 0.001
a IC50 values based on MTS assay run in triplicate (see the Supporting
Information).
smaller or larger groups than methyl, is not tolerated at the R-
position of the D-Ala side chain. Similarly, decreasing steric bulk
of the alkyl amide decreases biological activity.
In conclusion, molecular modeling was used to predict simplified
derivatives of trienomycin A that were energetically predisposed
to adopt a conformation similar to the natural product. The
analogues manifested potent antiproliferative activities against
MCF-7 and HeLa cancer cell lines and provided the first SAR
for these natural product analogues. Because trienomycin A is
Figure 6. Structures of monoenomycin analogues 28À34.
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synthetically complex, simplified derivatives provide a method
that enables rapid elucidation of SAR and provide a potential tool
by which the biological target may be discovered through
subsequent studies. Further investigation into the SAR and
mechanism of action for monoenomycin is ongoing and will be
reported in due course.
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’ ASSOCIATED CONTENT
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gen, XXVII aluminiumtrialkyle und dialkyl-aluminiumhydride aus alumi-
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Supporting Information. Full experimental procedures
b
and characterization for all compounds. This material is available
free of charge via the Internet at http://pubs.acs.org.
’ AUTHOR INFORMATION
Corresponding Author
*E-mail: bblagg@ku.edu.
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Funding Sources
We acknowledge NIH/NCI for financial support, CA109265.
G.E.L.B. acknowledges the Madison and Lila Self Graduate
Fellowship and the American Foundation for Pharmaceutical
Education for financial support and Dr. Geraldine Calvet for
preliminary studies.
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