Synthesis and biological evaluation of paleo-soraphens

Article abstract of DOI:10.1002/anie.201305331

Synthesis can provide molecules such as paleo-soraphens A and B (see scheme) that are genetically encoded but not obtained from the natural source. Although it is unclear whether this is part of an evolutionary process or the consequence of the chemical synthesis, the biological evaluation of these genetically encoded natural products can shed light on how natural products are structurally optimized with respect to their biological profile. Copyright

Full text of DOI:10.1002/anie.201305331

Angewandte
Chemie
DOI: 10.1002/anie.201305331
Natural Products Chemistry
Synthesis and Biological Evaluation of Paleo-Soraphens**
Hai-Hua Lu, Aruna Raja, Raimo Franke, Dirk Landsberg, Florenz Sasse, and Markus Kalesse*
Natural products are exploited as a rich source of biologically
active compounds and can be isolated as metabolites from
plants, fungi, animals, and microorganisms. In particular,
polyketides from fungi and bacteria have been the focus of
chemical and biological investigations. Since the biosynthesis
of modular polyketides follows a linear assembly, genetic
analysis can be used to rationalize the biosynthesis of
polyketides and even to predict the stereochemical out-
come.[1] Consequently, these genetic analyses have been used
to define the stereochemistry of natural products and thus
guide their total synthesis.[2] So far in natural product
synthesis the structures of isolated natural products have
served as targets. However, if one compares modular
polyketide synthases to the isolated natural products, it
becomes apparent that in a large variety of polyketides at
least one enzymatic activity is nonproductive. Whether this is
the consequence of an evolutionary process or a chemical
consequence of the substrate is still unknown. Nevertheless,
we were interested in natural products that exhibit the
structure predicted by the intact polyketide synthases to see if
and how their biological behavior would change as a conse-
quence of the altered structure.
Here, we present the synthesis and biological activity of
two soraphen derivatives that contain the structure derived
from its polyketide synthases. Soraphen[3] inhibits the eukary-
otic acetyl-coenzyme A carboxylase (ACC)[4] and conse-
quently was considered a potential antifungal and antitumor
compound.[5] Its biosynthetic origin was described by Ligon
et al.,[6] and Mꢀller and Flosset et al.[7] and they identified two
positions of the isolated natural product that differed from the
genetically expected outcome.
secondary alcohol was not produced either. Meanwhile, there
is still a double bond between C9 and C10. The presence of
this double bond can be rationalized by an isomerization of
the preceding a,b-unsaturated ester to the deconjugated b,g-
enoate. Ligon et al. proposed that this double bond is
a consequence of postketide transformations. In any case
the presence and position of this double bond is believed not
to be the direct consequence of the polyketide synthase
(Scheme 1).
The synthesis of soraphen A was completed independ-
ently by research groups led by Giese[8] and Trost,[9] and with
partial syntheses put forward by Sinnes and others.[10] Based
on reported difficulties in using the internal double bond as
a handle for a convergent synthesis, we decided to use
a macrolactonization and a Nozaki–Hiyama–Kishi reaction in
the endgame of our syntheses (Scheme 2). As depicted in
Scheme 3, our synthesis of the western segment 6 features
a strategic olefin cross-metathesis.[11]
Starting from the known chiral diol 1,[12] which in our case
was conveniently obtained by proline-catalyzed a-oxida-
tion[13] of 4-pentenal,[14] selective protection of the primary
hydroxy group and etherification of the remaining secondary
alcohol afforded alkene 2 (Scheme 3). This set the stage for
the cross-metathesis with the known (S)-1-phenyl-3-buten-1-
ol (3).[15] In the presence of 1.5 mol% of the Grubbs II
catalyst, the cross-metathesis reaction smoothly provided the
desired product in good yield. Subsequent reduction of the
double bond by either imide reduction[16] or heterogeneous
hydrogenation afforded chiral alcohol 4, which was then
protected as a PMB ether (5). Finally, TBS removal with CSA
and IBX-mediated oxidation[17] of the resulting primary
alcohol afforded the desired western segment 6. For the
synthesis of the eastern segment (Scheme 4), we envision that
the combination of syn-Evans aldol reaction[18] and anti-
Marshall reaction[19] with commercially available (R)-Roche
ester will greatly enhance the efficiency for constructing the
five contiguous stereocenters.
Ligon et al. pointed out that the expected trisubstituted
double bond (C2–C3), which should be established by
module 8, is missing due to the inactivity of the dehydratase
activity within that domain.[6] Additionally, the ketoreductase
must have been nonfunctional as well since the expected
Thus, syn-Evans aldol reaction of 7 with aldehyde 8[20]
afforded the corresponding adduct 9. Subsequent TBS
protection of the secondary hydroxy group and reductive
removal of the chiral auxiliary afforded alcohol 10, which was
transformed to the corresponding aldehyde 11.[21] Finally, an
anti-Marshall reaction generated two additional desired chiral
centers and the vinyl iodide segment 13 was completed
through a hydrozirconation–iodination sequence.
With both segments in hand, we began the endgame of the
synthesis with the Nozaki–Hiyama–Kishi coupling
(Scheme 5).[22] The two segments were joined to form the
desired adduct, albeit as a 2:1 mixture of the allylic alcohol
[*] Dr. H.-H. Lu, Dr. D. Landsberg, Prof. Dr. M. Kalesse
Institut fꢀr Organische Chemie und Biomolekulares
Wirkstoffzentrum (BMWZ), Leibniz Universitꢁt Hannover
Schneiderberg 1B, 30167 Hannover (Germany)
and
Helmholtz Centre for Infection Research (HZI)
Inhoffenstrasse 7, Braunschweig (Germany)
E-mail: Markus.Kalesse@oci.uni-hannover.de
A. Raja, Dr. R. Franke, Dr. F. Sasse
Department of Chemical Biology
Helmholtz-Zentrum fꢀr Infektionsforschung (HZI)
Inhoffenstrasse 7, Braunschweig (Germany)
[**] We thank the Alexander von Humboldt Foundation for a fellowship
to H.-H.L.
diastereoisomers. Hence, a sequence of oxidation and chela-
[23]
tion-controlled reduction with Zn(BH4)2
at low temper-
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201305331.
ature provided the desired alcohol 15 in excellent yield and
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provided the desired macrolactone
18 in 43% yield. Finally, removal of
the TBS groups was achieved with
HF–pyridine and furnished paleo-
soraphen A (19).
The synthesis of paleo-sora-
phen B follows the same route,
which diverges only at compound
16 (Scheme 5). However, reduction
of the double bond of 16 proved
nontrivial, and the Crabtree catalyst
20 was finally identified to achieve
the desired selective transformation
to give 21 in good yield. Other
reducing agents either caused no
conversion or gave poor selectivity
in the reduction of the double bond
between C9 and C10 relative to the
benzylic position at C17. Then,
starting from 21, we successfully
applied the same sequence as that
used for the synthesis of paleo-
soraphen A to afford paleo-sora-
phen B (22).
Scheme 1. Polyketide synthase of soraphen A and ketides as derived by fully functional domains.
Scheme 2. Retrosynthetic analysis of paleo-soraphens A and B.
Scheme 4. Synthesis of the eastern segment: a) Bu2BOTf, Et3N,
CH2Cl2, 85%; b) TBSOTf, 2,6-lutidine, CH2Cl2, 90%; c) LiBH4, THF,
88%; d) PivCl, Et3N, DMAP, CH2Cl2, 95%; e) (+)-CSA, MeOH–CH2Cl2,
08C to RT, 81%; f) (COCl)2, DMSO, Et3N, CH2Cl2, 61% over 3 steps;
g) (R)-but-3-yn-2-yl methanesulfonate, [Pd(dppf)Cl2], InI, THF–HMPA,
75%; h) TBSOTf, 2,6-lutidine, CH2Cl2, 95%; i) [Cp2ZrHCl], I2, CH2Cl2.
Bu2BOTf=di-n-butylboron triflate, TBSOTf=tert-butyldimethylsilyl tri-
flate, PivCl=pivaloyl chloride, [Cp2ZrHCl]=zirconocene hydrochloride.
Scheme 3. Synthesis of the western segment. a) TBSCl, imidazole,
DMF, RT; b) Me3OBF4, proton sponge, 4 ꢂ MS, CH2Cl2, 80% over 2
steps; c) (À)-Ipc2BOMe, allylMgBr, Et2O, À1008C, 75% (89% ee);
d) 1.5 mol% Grubbs II, CH2Cl2, reflux, 80%; e) NBSH, Et3N, CH2Cl2,
81%, RT or PtO2, H2 (1 atm), EtOAc, RT, 93%; f) KHMDS, TBAI,
PMBCl, THF, RT, 73%; g) (+)-CSA, MeOH-CH2Cl2, 08C to RT, 81%;
h) IBX, DMSO, RT, 100%. TBSCl=tert-butyldimethylsilyl chloride,
Grubbs II=(1,3-bis(2,4,6-trimethylphenyl)-2-imidazolidinylidene)
dichloro(phenylmethylene)(tricyclohexylphosphine)ruthenium,
NBSH=2-nitrobenzenesulfonylhydrazide, KHMDS=potassium bis(tri-
methylsilyl)amide, TBAI=tetra-n-butylammonium iodide, PMBCl=4-
methoxybenzylchloride, (+)-CSA=(+)-camphor-10-sulfonic acid,
IBX=2-iodoxybenzoic acid.
The biological activity of the paleo-soraphens was tested
with three fungal strains and compared with that of sor-
aphen A . Table 1 shows that the paleo-soraphens also have
antifungal activity but are much less active than soraphen A.
Cytotoxic effects on mammalian cells were tested with
a mouse fibroblast (L-929) and a cervical carcinoma cell
line (KB-3-1). While soraphen Awas found to be active in the
nm range (IC50 = 40 ngmLÀ1), the paleo-soraphens showed no
clear activity up to 1.2 mgmLÀ1, the highest test concentration.
It has already been established that soraphen A is
a eukaryotic ACC inhibitor but we wanted to check the
possibility that there was a shift in the mode of action of the
soraphens during evolution. We therefore characterized the
action of the two paleo-soraphens (at the highest possible
selectivity. Methylation and treatment with DIBAL-H
afforded primary alcohol 16 and subsequent transformations
led to compound 17. Application of the Shiina protocol[24]
concentration of 8.3 mgmLÀ1) and of soraphen A (83 ngmLÀ1
by impedance profiling. The basis of this method is that
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imation of the impedance data and the basic cubic spline
coefficients for statistical analysis (see the Supporting Infor-
mation). The results of a hierarchical cluster analysis showed
paleo-soraphen 19 in close proximity to soraphen A, while
compound 22 was found in an unrelated cluster together with
camptothecin (Figure 1).[25] This indicates that 22 unfolds its
mode of action through a mechanism that differs from that of
the other two soraphens (for details of the camptothecin
activity of compound 22 see Supporting Information).
Scheme 5. Endgame in the synthesis of paleo-soraphens A and B.
a) NiCl2, CrCl2, DMSO, RT; b) DMP, NaHCO3, CH2Cl2, 58% over
2 steps; c) Zn(BH4)2, Et2O, À558C, 90% (d.r. >25:1); d) Me3OBF4,
proton sponge, CH2Cl2, RT; e) DIBAL, THF, À508C, 83% over 2 steps;
=
f) DMP, NaHCO3, CH2Cl2, 92%; g) Ph3P C(CH3)CO2Et, CH2Cl2, RT,
95%; h) DDQ, pH 7 buffer, CH2Cl2, MeOH, 08C, 100%; i) LiOH,
THF–MeOH–H2O, RT, 94%; j) MNBA, DMAP, toluene, 4 ꢂ MS, 43%;
k) 70% HF, THF, 08C to RT, 89%; l) 10 mol% Crabtree cat. 20,
CH2Cl2, H2 (atm), 89%; m) DMP, NaHCO3, CH2Cl2, 67%;
Figure 1. Hierarchical cluster analysis of impedance kinetics. L-929
mouse fibroblasts were incubated with soraphen A, 19, and 22 and
compared with reference compounds. The impedance in each culture
was measured over a period of 5 days using an xCelligence system.
Basis coefficients of spline approximation curves are used for cluster
analysis. The results show compounds with similar modes of action in
close proximity.
=
n) Ph3P C(CH3)CO2Et, CH2Cl2, RT, 85%; o) DDQ, pH 7 buffer, CH2Cl2,
MeOH, 08C, 74%; p) LiOH, THF–MeOH–H2O, RT, 90%; q) MNBA,
DMAP, toluene, 4 ꢂ MS, 26%; r) 70% HF, THF, 08C to RT, 45%.
DMP=Dess–Martin periodinane, DIBAL=diisobutylaluminum hy-
dride, DDQ=2,3-dichloro-5,6-dicyano-1,4-benzoquinone, MNBA=2-
methyl-6-nitrobenzoic anhydride, DMAP=4-dimethylaminopyridine.
In conclusion, the synthesis reported herein provides two
potential evolutionary predecessors of the natural product
soraphen A. Our aim was to see whether the incorporated
modifications would lead to altered activities and thus
support the concept of evolutionary optimization. Indeed,
paleo-soraphen A still inhibits eukaryotic ACC, albeit with
reduced activity, which would support the concept of evolu-
tionary optimization. However, paleo-soraphen B shows
a completely different mode of action, namely topoisomerase
inhibition. At this point this observation might be pure
coincidence but it would also be consistent with the concept of
horizontal gene transfer (HGT) or biocombinatorial process-
es[26] which could lead to different activities and its subsequent
fine-tuning by disabling certain PKS activities. The fact that
the paleo-soraphens do not undergo Michael addition, which
would result in compounds that are structurally more closely
related to the parent soraphen, supports the assumption that
inactivation of the ketoreductase is probably not due to
spontaneous hemiacetal formation. Even though genetic
engineering could also provide these products,[27a] we are
convinced that this field would be very attractive for synthetic
chemists as they could target “genetic” compounds with
potentially new activities rather than synthesizing known
Table 1: Antifungal activity of paleo-soraphens and soraphen A.
Compound
Pythium
debaryanum
Botrytis
cinerea
Mucor
hiemalis
IC50 [mgmLÀ1 [a]
]
paleo-soraphen A (19)
paleo-soraphen B (22)
soraphen A
0.4
>40
0.045
>40
>40
0.0035
21
16
0.0006
[a] The IC50 was measured using a WST-1 test after incubation with
a serial dilution of the compounds for 2 days.
compounds with a similar mode of action induce similar time-
dependent impedance curves. L-929 mouse fibroblasts were
incubated with soraphen A, 19, and 22 in wells of a 96-well E-
Plate which had gold electrodes integrated in the bottom of
the wells. Impedance across the electrodes was measured in
real time (Figure S1 in the Supporting Information). The
resulting curves for soraphen A, 19, and 22 were compared to
a set of reference compounds covering a broad activity
spectrum. We used cubic smoothing splines for the approx-
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Received: June 20, 2013
Revised: September 9, 2013
Published online: November 7, 2013
Keywords: acetyl-CoA carboxylase · antitumor agents ·
.
evolution · paleo-soraphens · polyketides
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