The synthesis and antimicrobial activity of heterocyclic derivatives of totarol

Article abstract of DOI:10.1021/ml3001775

The synthesis and antimicrobial activity of heterocyclic analogues of the diterpenoid totarol are described. An advanced synthetic intermediate with a ketone on the A-ring is used to attach fused heterocycles, and a carbon-to-nitrogen atom replacement is made on the B-ring by de novo synthesis. A-ring analogues with an indole attached exhibit, for the first time, enhanced antimicrobial activity relative to the parent natural product. Preliminary experiments demonstrate that the indole analogues do not target the bacterial cell division protein FtsZ as had been hypothesized for totarol.

Full text of DOI:10.1021/ml3001775

Letter
pubs.acs.org/acsmedchemlett
The Synthesis and Antimicrobial Activity of Heterocyclic Derivatives
of Totarol
Michelle B. Kim,† Terrence E. O'Brien,† Jared T. Moore,† David E. Anderson,† Marie H. Foss,‡
Douglas B. Weibel,‡,§ James B. Ames,† and Jared T. Shaw*,†
†Department of Chemistry, University of California, One Shields Avenue, Davis, California 95616, United States
§
‡Department of Biochemistry and Department of Biomedical Engineering, University of Wisconsin, Madison, Wisconsin 53706,
United States
S
* Supporting Information
ABSTRACT: The synthesis and antimicrobial activity of hetero-
cyclic analogues of the diterpenoid totarol are described. An
advanced synthetic intermediate with a ketone on the A-ring is
used to attach fused heterocycles, and a carbon-to-nitrogen atom
replacement is made on the B-ring by de novo synthesis. A-ring
analogues with an indole attached exhibit, for the first time,
enhanced antimicrobial activity relative to the parent natural
product. Preliminary experiments demonstrate that the indole
analogues do not target the bacterial cell division protein FtsZ as had been hypothesized for totarol.
KEYWORDS: totarol, antimicrobial activity, small molecule inhibitors, FtsZ, structure−activity relationships and optimization
ethicillin-resistant Staphylococcus aureus (MRSA) is now
M
the leading cause of bacterial infections affecting
numerous hospitalized patients worldwide.1 Despite the rise
in antibiotic resistance, the discovery of new small molecule
antibiotics has decreased. The demand for a new antimicrobial
agent that targets a new protein function is necessary, but in
recent years, there have been few antibiotics based on new
structural classes that have been discovered.
The phenolic diterpenoid totarol is a major constituent
isolated from the sap of Podocarpus totara, a tree that is native
to New Zealand.2 Totarol exhibits antiplasmodial,3 antifungal,4
and antimicrobial activity against several organisms, including
Propionibacterium acnes5,6 and MRSA.5,7 The mechanism of
action by which totarol derives its antimicrobial activity has not
been fully elucidated. That said, it has been speculated that this
compound may inhibit bacterial respiratory transport,8 disrupt
phospholipid membranes,9 or function as an efflux-pump
inhibitor.10 Totarol was recently reported to inhibit bacterial
cytokinesis by targeting the protein FtsZ.11
Previous structure−activity relationship (SAR) studies have
focused on modifications of the B- and C-rings without any
observed increase in antimicrobial activity (Figure 1).12−14 Our
interests have been directed toward SAR studies on
heterocyclic analogues of totarol, which have not yet been
explored. In addition to A-ring analogues that might exhibit
enhanced activity (Figure 1C), we envisioned that a more
soluble form of totarol would emerge by replacing one of the B-
ring carbons with nitrogen. This report describes the synthesis
and preliminary evaluation of these two basic modifications to
totarol.
Figure 1. (A) Structure of (+)-totarol (1). (B) Previous SAR of
(+)-totarol; areas modified in the study are highlighted in blue (refs
12−14). (C) A-ring analogues described in this study. (D) B-ring
derivative containing a heterocycle installed de novo.
an advanced intermediate (+)-2 (Figure 2). Intermediate (+)-2
was synthesized from two commercially available fragments,
dimethoxybenzonitrile 3 and geraniol 4, through an asymmetric
nine-step sequence.15 In the first series of analogues, methyl
totarolone (+)-2 was converted into structurally diverse
analogues at positions C2 and C3. Claisen condensation of 2
with ethyl formate formed an intermediate used in the
preparation of several azoles. Substituting the ketone to an
oxime allowed us to expand the A-ring to a lactam by
Beckmann rearrangement or reduced to the primary amine.
Received: July 1, 2012
The concise, enantioselective synthesis of (+)-totarol
developed in our lab enabled us to modify the A-ring through
Accepted: August 28, 2012
Published: August 28, 2012
© 2012 American Chemical Society
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Letter
oxime (+)-10 with thionyl chloride.21 The resultant lactam was
demethylated to (+)-12 in 46% yield over two steps. Two
additional derivatives were obtained sequentially by removing
the methyl ether on (+)-10 with n-PrSH and AlBr3 to oxime
(+)-11 and then reducing oxime (+)-11 to amine (+)-13 with
LiAlH4 (Scheme 2).22
Scheme 2. Synthesis of Diverse Totarol A-ring Analogues
from Ketone 2
Figure 2. Advanced intermediate (+)-2, synthesized from nitrile 3 and
geraniol (4) in 10 steps, as a key intermediate for A-ring analogues.
Eight enantiomerically enriched derivatives were synthesized
by exploiting the A-ring ketone functionality on (+)-2. A
formate group was installed by a Claisen condensation as a
means to synthesize heterocyclic derivatives on the A-ring of
(+)-2 (Scheme 1).16 Treating (+)-2 with sodium hydride,
Scheme 1. Synthesis of Azole-Fused Totarol Analogues
Carbon-to-nitrogen substitution of the B-ring was also
investigated as a means to enhance solubility and, potentially,
activity toward FtsZ.23 Although both oxygen- and nitrogen-
containing derivatives were initially investigated, preparation of
the oxygenated core structure could not be accomplished by a
cyclization reaction analogous to what was employed for
totarol.24 The synthesis of nitrogen-containing derivatives
proved successful using a catalytic cyclization reaction recently
discovered in our laboratory.25 Azatotarol 16 was synthesized
by replacing a benzylic carbon with nitrogen prior to the
cyclization. Complex tetrahydroisoquinoline 14 (Scheme 3)
resulted from a diastereoselective heteropolycyclization reac-
tion of a geranylated aniline derivative via an episulfonium
intermediate.26 Compound 14 was converted to “azatotarol”
(16) by removal of both the thioether and the nitro-
benzenesulfonyl (Ns, nosyl) groups by reduction with Raney
nickel and subsequent deprotection. N-Acetylated azatotarol 17
methanol, and ethyl formate produced intermediate (+)-5 in
73% yield. Azoles (+)-6 and (+)-7 were prepared as their
methyl ethers (not shown) by treatment of (+)-5 with
hydrazine monohydrate or phenylhydrazine hydrochloride,
respectively.17 Demethylation was achieved using AlBr3 and
n-PrSH to give pyrazole (+)-6 in 19% and phenyl pyrazole
(+)-7 in 40% yield over two steps. Isoxazole (+)-8 resulted
from heating (+)-5 in the presence of hydroxylamine
hydrochloride18 and subsequent deprotection.
Scheme 3. Synthesis of Totarol B-Ring Analogues from
Cyclization Product 14
The A-ring ketone of 2 was used in several additional
modifications. Acetic acid-mediated Fischer indole conditions
with (+)-2 and phenylhydrazine hydrochloride followed by
methyl deprotection yielded “indolototarol” (+)-9 in 20% yield
over two steps.19 A-ring analogues with hydrogen-bonding
capability were also prepared. Methyl totarolone (+)-2 was
converted to methyl oxime (+)-10 in 83% yield.20 A ring
expansion of the A-ring to the corresponding caprolactam was
achieved by Beckmann rearrangement induced by treating
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ACS Medicinal Chemistry Letters
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required a different order of events, specifically nosyl group
cleavage of intermediate 14 to yield the free amine 15 in 64%
yield. This compound was acylated with acetyl chloride and
treated with Raney nickel to remove the thiophenyl ether. Final
deisopropylation was achieved using BCl3 to give 17 in 43%
yield over the three steps.
The first round of totarol analogues was evaluated for
antibacterial activity by measuring minimum inhibitory
concentrations (MICs, Table 1) for B. subtilis 168. Although
indole synthesis provided significant improvements in yield.29
Optimization reactions were carried out using microwave
irradiation, and the initial improvement in the reaction of
phenylhydrazine was traced to this more efficient heating
method and the use of ethanol as solvent. Use of these
optimized conditions with the more problematic p-CF3
phenylhydrazine demonstrated that the addition of Sb2(SO4)3
was crucial to the success of the reaction. (Table 2, entries 5
and 6). Little product formation was observed without the
additive, whereas a 61% yield of indole 19 was observed in the
presence of 1 equiv of Sb2(SO4)3. Although the role of
Sb2(SO4)3 in this reaction is unclear, subsequent Fischer indole
reactions were consistently carried out using 1 equiv of this
additive to ensure optimal yields.
The second round of A-ring indole analogues was prepared
using racemic starting material ( )-2 (Table 3). The racemic
synthesis of methyl totarolone ( )-2 was achieved in a seven-
step synthesis that is both more expedient and higher yielding
than the enantioselective route. In addition to the ease of
synthesis, examination of a racemic mixture can reveal instances
where the opposite enantiomer has enhanced activity provided
that separate enantiomers are easily accessed once activity is
observed. Our optimized indole synthesis involved treating
( )-2 with substituted phenylhydrazine salts and heating in a
microwave reactor in the presence of 1 equiv of Sb2(SO4)3. The
resulting ( )-indole analogues were taken directly into
established demethylation conditions using n-PrSH and AlBr3
to give the desired products over two steps.
Indolototarol derivatives ( )-21−29 were assessed for
antimicrobial activity against B. subtilis 168 (Table 4). In this
experiment, we observed minor variation from the first round in
that totarol's MIC (2.5 μM) was slightly lower than (+)-9 (4
μM). The o-ethyl ( )-28 and p-ethyl ( )-25 indoles were also
comparable to totarol with MIC values of 1.0 and 2.5 μM,
respectively. Three indoles with electron-withdrawing sub-
stituents, that is, m,m-dichloro ( )-21, p-CF3 ( )-22, and p-
CN ( )-23, exhibited the best activity at 0.78 μM. The
hydroxyl and sulfonamide groups of ( )-29 and ( )-26
reduced the activity slightly and showed MICs of 6.25 and 5.5
μM, respectively. Previous studies by Evans et al. demonstrated
that O-methylation completely removes all antimicrobial
activity.12 Consistent with this trend, compound ( )-30, the
methyl ether precursor of ( )-23, is completely inactive.
Totarol11 and vancomycin30 were used as positive controls, and
their MICs corresponded with literature values.
Table 1. Preliminary MIC Measurements for A-Ring
Derivatives against B. subtilis 168
compound
MIC
compound
MIC
1, totarol
10
>160
>160
>160
40
11
12
13
16
17
>160
>160
>160
20
5
6
7
8
9
>256
2.5
most analogues showed little or no inhibition of growth, three
compounds showed notable activity. A-ring analogue isoxazole
(+)-8 and indole (+)-9 were approximately 4-fold less and 4-
fold more potent, with MICs of 40 and 2.5 μM, respectively
(Table 1). “Azatotarol” analogue 16 showed modest growth
inhibition (20 μM), while N-acylated analogue 17 was inactive.
These results are consistent with the relatively stark structural
changes that were made to the structure of totarol and were
successful in identifying one compound (9) with enhanced
activity. These studies are also notable in demonstrating that
the whole of totarol's activity is not simply due to the presence
of a free phenolic hydroxyl group.
The promising antimicrobial activity of the indolototarol
derivative prompted us to further functionalize the indole with
the goal of developing a more potent antimicrobial compound.
Unfortunately, the yields of indoles from standard Fischer
conditions using hindered ketone 16 varied greatly depending
on the electronic nature of the hydrazine. Standard protic and
Lewis acidic conditions did not offer a general solution.27,28
The Fischer indole synthesis on our hindered ketone required
us to investigate conditions on a model system. A model system
of dimethyl-β-tetralone 18 was employed to find optimal
reaction conditions (Table 2). After examining several different
conditions, a recently reported Sb2(SO4)3-catalyzed Fischer
Indole analogues (+)-9 and ( )-23 were investigated for
their ability to inhibit the GTPase activity of FtsZ, one of the
putative targets of totarol (Figure 3). An enzyme-coupled assay
monitoring the hydrolysis of GTP (guanosine triphosphate)
was used to assess the inhibitory concentration resulting in 50%
enzyme activity (IC50). Totarol (1) exhibited an IC50 of 24 μM,
which compares favorably to the reported value. To our
surprise, (+)-9 and ( )-23 showed no activity in the assay up
to 64 μM. Because of this drastic decrease in activity between
totarol and indolototarol analogues in this enzymatic assay, the
validity of totarol as an FtsZ inhibitor was brought into
question. Studies conducted by Shoichet have demonstrated
that small organic molecules can act as nonspecific inhibitors by
forming large colloidal aggregates in aqueous media.31,32 In
their investigations, aggregates were disrupted by including
0.01% of a detergent into the enzymatic assay, resulting in a loss
of activity against the target protein in the cases of nonspecific
inhibition. The addition of 0.01% of triton X-100, a nonionic
Table 2. Optimization of Fischer Indole Synthesis with
Ketone 18 as a Model System
entry
R
additive
mol %
yield (%)
1
2
3
4
5
6
H
Sb2(SO4)3
MgSO4
ZnCl2
10
10
10
78
79
80
85
NR
61
H
H
H
CF3
CF3
Sb2(SO4)3
100
820
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ACS Medicinal Chemistry Letters
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Table 3. Sb2(SO4)3-Mediated Synthesis of Indoles 21−29
a
a
compound
R1
R2
R3
Cl
yield (%)
compound
R1
R2
R3
yield (%)
21
22
23
24
25
Cl
H
H
H
H
H
H
H
H
H
34
20
40
66
41
26
27
28
29
H
H
SO2NH2
33
66
11
33
CF3
CN
F
CH3
Et
CH3
H
H
H
H
OH
H
Et
a
Over two steps after purification.
Table 4. MIC of Indolototarol Derivatives against B. subtilis
compound
R1
R2
R3
MIC
compound
R1
R2
R3
MIC
9
21
22
23
24
25
26
H
Cl
H
H
H
H
H
H
H
H
H
H
H
H
H
4.0
0.78
0.78
0.78
80
27
CH3
Et
CH3
H
H
H
H
11
1.0
Cl
28
CF3
CN
F
29
OH
H
6.3
30
>100
2.5
1, totarol
vancomycin
Et
2.5
0.63
SO2NH2
5.5
detergent, into the enzyme-coupled GTPase assay eliminated
the inhibitory activity of totarol. This experiment suggests that
the activity of totarol is caused by aggregates of the molecule
and not inhibition of FtsZ.
by totarol results from unselective inhibition by aggregates that
form in solution.33 Given the compelling antimicrobial activity
of ( )-23, current studies are directed at identifying the target
of this compound through the selection and sequencing of
resistant strains of B. subtilis.
ASSOCIATED CONTENT
■
S
* Supporting Information
Synthetic experimental details, analytical data of compounds,
and biological assay protocols. This material is available free of
charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Author
*E-mail: jtshaw@ucdavis.edu.
Author Contributions
The manuscript was written through contributions of all
authors. All authors have given approval to the final version of
the manuscript.
Figure 3. Enzyme-coupled GTPase assay of totarol and analogues for
B. subtilis FtsZ.
Funding
We have described the synthesis of heterocyclic analogues of
totarol enabling the discovery, for the first time, of a compound
with enhanced antimicrobial activity. By examining structurally
diverse scaffolds available from our synthetic intermediate, we
were able to identify the indole moiety as a scaffold for further
exploration. Three indolototarol derivatives were found to have
increased antimicrobial activity as compared to the parent
compound totarol. The lack of activity in the enzymatic assay
for FtsZ inhibition prompted us to demonstrate that inhibition
This work was supported by the NIH/NIAID (R01AI08093)
and by NIH/NIGMS (T32-GM008799), which provided
J.T.M. with predoctoral fellowship support. J.T.M. and T.E.O.
were also supported by Bradford Borge fellowships from UC
Davis. M.B.K. and T.E.O. acknowledge the United States
Department of Education for GAANN fellowships. T.E.O. was
also supported by a fellowship from the Alfred P. Sloan
foundation, and J.B.A. acknowledges support from NIH/NEI
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ACS Medicinal Chemistry Letters
Letter
(15) Kim, M. B.; Shaw, J. T. Synthesis of antimicrobial natural
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Org. Lett. 2010, 3324−3327.
(EY012347). D.B.W. acknowledges support from the NIH
(1DP2OD008735-01).
Notes
(16) Krawczuk, P. J.; Schone, N.; Baran, P. S. A Synthesis of the
̈
The authors declare no competing financial interest.
Carbon Skeleton of Maoecrystal V. Org. Lett. 2009, 11, 4774−4776.
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cholest-4α-methyl-7-en-3β-ol derivatives. Steroids 2006, 71, 476−483.
(18) Parra-Delgado, H.; Compadre, C. M.; Ramirez-Apan, T.;
Munoz-Fambuena, M. J.; Compadre, R. L.; Ostrosky-Wegman, P.;
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ACKNOWLEDGMENTS
■
We thank Doug Mende from Mende Biotech Limited for their
large donation of totarol isolated from the natural source.
ABBREVIATIONS
■
MIC, minimum inhibitory concentration; IC50, inhibitory
concentration at 50%; GTP, guanosine triphosphate
(20) Csuk, R.; Schwarz, S.; Siewert, B.; Kluge, R.; Strohl, D. Synthesis
̈
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