Asymmetric Total Synthesis of (-)-(3 R)-Inthomycin C

Article abstract of DOI:10.1021/acs.orglett.8b01370

A short (10 step) and efficient (15% overall yield) synthesis of the natural product (-)-(3R)-inthomycin C is reported. The key steps comprise three C-C bond-forming reactions: (i) a vinylogous Mukaiyama aldol, (ii) an olefin cross-metathesis reaction, and (iii) an asymmetric Mukaiyama-Kiyooka aldol. This route is notable for its brevity and has the advantage of lacking stoichiometric tin-promoted cross-coupling reactions present in previous approaches. Initial investigations on the biological activity of (-)-(3R)-inthomycin C and structural analogues on human cancer cell lines are also described for the first time.

Full text of DOI:10.1021/acs.orglett.8b01370

Letter
Cite This: Org. Lett. XXXX, XXX, XXX−XXX
pubs.acs.org/OrgLett
Asymmetric Total Synthesis of (−)-(3R)‑Inthomycin C
†
†
†
́†,§
Sandra Balcells, Maxwell B. Haughey, Johannes C. L. Walker, Laia Josa-Cullere,
‡
,†
Christopher Towers, and Timothy J. Donohoe*
†
Department of Chemistry, University of Oxford, Chemistry Research Laboratory, Mansfield Road, Oxford OX1 3TA, U.K.
‡
Department of Oncology, University of Oxford, Old Road Campus Research Building, Roosevelt Drive Oxford OX3 7DQ, U.K.
*
S Supporting Information
ABSTRACT: A short (10 step) and efficient (15% overall yield) synthesis of the
natural product (−)-(3R)-inthomycin C is reported. The key steps comprise three
C−C bond-forming reactions: (i) a vinylogous Mukaiyama aldol, (ii) an olefin
cross-metathesis reaction, and (iii) an asymmetric Mukaiyama−Kiyooka aldol. This
route is notable for its brevity and has the advantage of lacking stoichiometric tin-
promoted cross-coupling reactions present in previous approaches. Initial
investigations on the biological activity of (−)-(3R)-inthomycin C and structural
analogues on human cancer cell lines are also described for the first time.
1
he inthomycins are a small family of polyene natural
inhibition of cellulose biosynthesis in Acetobacter xylinum.
T
products isolated from Streptomyces sp. containing a
Inthomycins A and B also show in vitro inhibitory activity
against human prostate cancer growth by suppressing tumor-
methylene-interrupted oxazolyl-triene motif common to a
broader class of antibiotic natural products known as the
oxazolomycins. Differing in the geometry of their respective
triene motifs, the inthomycins have attracted considerable
attention from synthetic chemists due to their interesting
biological activity and closely related structures.
5
,6
stromal cell interactions. To our knowledge, there have been
no reported studies on the biological activity of inthomycin C.
To date, there have been four reported asymmetric total
7
8
9
syntheses of inthomycin C by Taylor, Ryu, Hatakeyama and
10
Hale. Additionally, a formal synthesis of racemic material has
been reported by Maulide, and an asymmetric formal
synthesis has been reported by Reddy. Recently, Hale and
11
Isolated by Omura in 1990 from Streptomyces sp. OM-5714,
inthomycin A (4Z,6Z,8E) was the first member of the
inthomycin family to be discovered. In 1991, Henkel and
Zeeck isolated its geometrical isomers, inthomycin B
12
1
Hatakeyama jointly confirmed the absolute configuration of
13
(
−)-(3R)-inthomycin C by collaboration. It is noteworthy
(
4Z,6E,8E) and inthomycin C (4E,6E,8E), Scheme 1, from
that all published syntheses of (−)-(3R)-inthomycin C (ranging
between 11−16 steps) rely on a Stille cross-coupling reaction to
form the C7−C8 bond and differ in their approach toward the
2
̈
Streptomyces sp. Go 2 and also reisolated inthomycin A.
Inthomycin A displays moderate antifungal activity against
cellulose-containing Phytophthora parasitica and Phytophthora
capsici. Potent herbicidal activity has also been demonstrated
14
synthesis of the β-hydroxyl carbonyl moiety.
3
As part of our ongoing efforts to prepare oxazolomycin
3
4
against radish seedlings and velvet leaf in addition to the
15,16
B,
we required an efficient synthesis of (−)-(3R)-
inthomycin C and sought to differentiate our approach by
avoiding the use of the stoichiometric organotin reagents on
which all previous syntheses rely. Initial investigations on the
biological activity of synthetic (−)-(3R)-inthomycin C and
structural analogues on human cancer cell lines are also
described for the first time.
Scheme 1. Retrosynthesis of (−)-(3R)-Inthomycin C
Our retrosynthetic analysis of (−)-(3R)-inthomycin C was
based around three key C−C bond-forming reactions (Scheme
1
). In common with previous work, we planned to use an
asymmetric aldol reaction between 2 and 3 to set the C-3
stereocenter. As an alternative disconnection to the triene
motif, we envisaged that aldehyde 2 could then be prepared via
the cross metathesis (CM) of alkenes 4 and 5, followed by an
elimination reaction, which should afford the thermodynami-
cally most stable (4E,6E,8E) isomer. The final C−C bond-
Received: April 30, 2018
©
XXXX American Chemical Society
A
DOI: 10.1021/acs.orglett.8b01370
Org. Lett. XXXX, XXX, XXX−XXX

Organic Letters
Letter
forming reaction to investigate would be a (γ-selective)
vinylogous aldol reaction between a crotonaldehyde acetal
derivative (6) and dienyl silyl ether 7 to form alkene 5.
We began by preparing the two cross-metathesis alkene
precursors 4 and 5 (Scheme 2). Tiglic aldehyde 8 was
Table 1. Optimization of the Cross-Metathesis Reaction To
Form 12
Scheme 2. Synthesis of the Cross-Metathesis Precursors
yield
a
4
5
catalyst
temp time of 12
entry (equiv) (equiv) (10 mol %) solvent
(°C)
(h)
(%)
1
2
3
4
5
6
7
3
1
1
1
1
1
1
1
HG-II
HG-II
HG-II
HG-II
HG-II
HG-II
G-II
CH Cl2
40
40
80
40
40
40
40
28
26
23
25
23
48
51
17
32
26
41
48
41
57
2
1.5
2
CH Cl2
2
DCE
3.3
5
CH Cl2
2
b
b
b
CH Cl2
2
10
CH Cl2
2
5
CH Cl2
2
a
b
Isolated yields. Syringe pump addition of 4 over 7 h.
With compound 12 in hand, we then looked toward the
development of conditions for the elimination of methanol and
formation of the triene motif (Table 2). As shown below,
Table 2. Optimization of the Elimination Reaction
converted into its silyl enol ether 7 (geometry proven by
NOE), and this was reacted with acetal 6 (itself prepared in one
step from crotonaldehyde 9). As expected, the vinylogous
Mukaiyama aldol reaction of acetal 6 and enol ether 7 took
place with high selectivity for the γ-position of the enol ether.
Pleasingly, 5 was isolated as the desired E-alkene isomer, as
proven by extensive NOE measurements.
b
temp
time
(h)
yield 2
entry
reagents (equiv)
(°C)
(%)
1
2
DBU (2)
rt
6
0
0
17
AcOH (12)
rt to
18
1
00
3
4
TBSOTf (1.1), Et N (1.5)
0 to rt
0 to rt
22
20
25
3
TBSOTf (1.5), Et N (2); then
trace
Separately, and following precedent from Merck, oxazole 10
was lithiated and then protected at the C-2 position with a
3
CSA (1.5)
18,19
5
6
TBSOTf (2), Et
Pyr HBr (2)
N (3); then
3
0 to rt
0 to rt
0 to rt
55
29
21
6
57
74
TIPS group (11).
Subsequent lithiation of 11 then took
place at the C-5 position to furnish allyl TIPS oxazole 4 after
transmetalation and quenching in situ with allyl bromide.
With 4 and 5 in hand, we turned to the second of our key
C−C bond-forming reactions: the cross-metathesis reaction to
form 12 (Table 1). This summary of the optimization results
using Hoveyda−Grubbs II (HG-II) catalyst reveals that an
excess of the less reactive alkene 5 relative to 4 was necessary in
order to achieve good yields of 12 (compare entries 1, 2, and
TBSOTf (1.1), Et N (1.5); then
3
Sc(OTF) (2)
3
c
7
TBSOTf (1.8), Et N (2.8); then
3
Sc(OTf) (2.3)
3
a
Although the identity of the minor isomer could not be proven
unequivocally, J values indicate a probable (4E,6E,8Z) geometry.
Isolated product obtained as a mixture of isomers, typically 8:1.
3
HH
b
c
Freshly distilled Et N.
3
2
0,21
4).
When an excess of 4 was used in the CM, significant
quantities of the homodimerization product derived from 4
were observed (this product was shown to be almost inactive in
a subsequent CM reaction). Screening showed dichloro-
methane at a temperature of 40 °C to be optimal, with
dichloroethane at 80 °C giving no improvement in yield (entry
preliminary attempts at elimination employing Brønsted base
or acid were unsuccessful (entries 1 and 2). However, we
were pleased to observe the formation of 2 in 25% yield when
22
TBSOTf and Et N were employed (entry 3). Only trace
3
amounts of product were observed when Brønsted acids were
3). A syringe pump was used to allow the slow addition of 4
added after silyl enol ether formation using TBSOTf and Et N
3
and in so doing reduced its concentration even further,
resulting in an additional increase in yield to 48% (entry 5).
Finally, the highest yield of 12 (57%) was obtained by the use
of Grubbs II (9−11) catalyst under the previously optimized
(entries 4 and 5). Crucially, the addition of the oxophilic Lewis
acid Sc(OTf) to the TBSOTf/Et N mixture afforded 2 in 57%
3
3
yield by successfully promoting the elimination of methanol
from the silyl enol ether intermediate (entry 6). This reaction
was found to be particularly sensitive to water, and the use of
conditions (entry 7). Note that the trans stereochemistry of 12
3
was confirmed by the olefinic coupling constant ( J = 15.4
freshly distilled Et N and a greater excess of the reagents
HH
3
Hz).
further increased the yield to 74% (entry 7). The all-E
B
DOI: 10.1021/acs.orglett.8b01370
Org. Lett. XXXX, XXX, XXX−XXX

Organic Letters
Letter
geometry of 2 was confirmed by olefinic 3JHH coupling constant
values. In each instance, 2 was formed as a mixture of
diastereoisomers, typically 8:1 in favor of the desired all-E
isomer. It was possible to enrich the purity of 2 up to 22:1 dr by
silica column chromatography, although large quantities could
not be isolated with this ratio. However, 2 could be reliably
enriched to ratios of around 14.3:1 dr on scale, and it was this
material that we used to continue the synthesis.
Scheme 3. Completion of the Synthesis
We next turned our attention toward the asymmetric
induction of the (3R) stereocenter. A wide range of asymmetric
aldol reaction catalysts and conditions were investigated using
13 as a model aldehyde substrate. Of those conditions tested,
the most promising were those of Mukaiyama−Kiyooka, as
7
employed previously by Taylor. Under these reaction
conditions, chiral oxazaborolidinone 15 is formed in situ,
which promotes the asymmetric reaction of a silyl enol ether
(Table 3). Application of these reaction conditions afforded 14
Table 3. Optimization of the Asymmetric Aldol Conditions
completed the formal synthesis of (−)-(3R)-inthomycin C and
intersected a common intermediate with previous workers in
the field. Analytical and spectroscopic data of compound 17
7
−11
b
b
yield of 14
yield of 16
were in close agreement with those previously reported.
c
entry
1
procedure/variation
(%)
er
(%)
Given the recent debate concerning the absolute stereo-
chemistry of (−)-(3R)-inthomycin C, the (R)-and (S)-MTPA
esters of 17 were then prepared and analysis proved
unambiguously the (R) configuration (see the Supporting
standard; see conditions
73
94:6
19
a
below
2
3
4
5
6
3 h before addition of 13
addition of 3 1 h after 13
62
53
51
91
81
92:8
93:7
91:9
65:35
93:7
29
45
6
23
Information for details).
BH ·THF (1 equiv)
BH ·DMS (2 equiv)
syringe pump addition of 13
over 3 h
3
Ester hydrolysis and acetylation of 17 afforded acid 18 in
87% yield. Finally, amidation via in situ acid chloride formation,
followed by treatment with lithium hydroxide, afforded a 11.1:1
mixture of isomers of (−)-(3R)-inthomycin C in 77% yield
0
3
14
a
N-Ts-L-valine (2.2 equiv), BH THF (2 equiv), CH Cl , 0-rt, 30 min,
78 °C, 13, then 1.2 equiv 3, 2 h; (ii) 1 M (aq), HCl THF, rt, 1 h.
Isolated yields. Determined by HPLC (chiral stationary phase) with
reference to a racemic standard.
8
3
2
2
over two steps. Pleasingly, measurement of the specific
−
b
c
rotation for synthetic (−)-(3R)-inthomycin C obtained in
this work [α] −8.2 (c 1.0, CHCl ) was in close agreement
D
3
7
with all values reported previously, with the exception of one.
However, thorough collaborative work by the groups of Hale
and Hatakeyama has concluded that the previous positive [α]D
value obtained was erroneous due to the presence of a urea
in 73% yield with 94:6 er in addition to a 19% yield of the
reduction byproduct 16, (entry 1). Alternative amino acids
were also screened at this stage. However, N-Ts-L-valine was
shown to be optimal. Changes in timings designed to combat
possible incomplete catalyst formation (entry 2) or incomplete
complexation of 13 and 15 (entry 3) both led to decreased
yields of 14. In an attempt to minimize the unwanted reduction
process leading to 16, we investigated reducing the equivalents
of borane (entry 4) and alternative borane sources (entry 5).
However, these did not improve the reaction. However,
addition of 13 by syringe pump over a 3 h period was found
to result in the optimal yield of 14 while maintaining the high
enantioselectivity of the reaction (entry 7). Treatment of the
crude reaction mixture with 1 M aqueous HCl was found to be
necessary to deprotect any TMS protected 14 formed during
the reaction. The (R) stereochemistry of 14 was confirmed
unambiguously via the preparation and analysis of both (R)-and
13
contaminant.
In collaboration with a preclinical validation facility at the
Cancer Research UK Oxford Centre, several (−)-(3R)-
inthomycin C precursors, including the natural product itself,
were assayed for viability/cytotoxicity using human cancer cell
lines (chosen from HeLa, H460, MCF-7, KMS-12BM, and
SKOV-3). Despite IC50 values >50 μM being obtained in each
case, further investigation revealed a surprising proteasome
inhibition activity for ester 17. IC50 values for proteasome
inhibition in H460 and KMS-12BM cells were determined as
11 and 32 μM, respectively. Subsequent experiments
demonstrated that the inhibitory effect of 17 was reversible in
nature. To the best of our knowledge, there have been no
previous reports of similar compounds acting as proteasome
inhibitors and given the importance of proteasome inhibition in
the field of oncology, we anticipate that this will prove an
23
(
S)-MTPA esters.
Pleasingly, subjection of 2 to the optimized Mukaiyama−
24
exciting area of investigation in the future.
Kiyooka conditions, followed by TIPS deprotection, afforded
adduct 17 in 63% yield and with 94:6 er (Scheme 3). This
In conclusion, we have reported the total synthesis of
(−)-(3R)-inthomycin C in 15% yield (94:6 er) over 10 steps
C
DOI: 10.1021/acs.orglett.8b01370
Org. Lett. XXXX, XXX, XXX−XXX

Organic Letters
Letter
from commercially available oxazole. The (3R) stereochemistry
was confirmed unambiguously by MTPA ester derivatization,
(12) Athawale, P. R.; Kashinath, K.; Reddy, D. S. ChemistrySelect
2
016, 1, 495.
13
(13) Hale, K. J.; Hatakeyama, S.; Urabe, F.; Ishihara, J.; Manaviazar,
S.; Grabski, M.; Maczka, M. Org. Lett. 2014, 16, 3536.
further supporting recent work by Hale and Hatakeyama.
key advantage of this route is its concise nature and scalability
in total ca. 250 mg of 18 was prepared). This study was the
A
(
14) For early synthetic work on the inthomycins which describes the
Stille disconnection, see: Henaff, N.; Whiting, A. Org. Lett. 1999, 1,
137.
15) Donohoe, T. J.; Chiu, J. Y. K.; Thomas, R. E. Org. Lett. 2007, 9,
21.
(16) Donohoe, T. J.; O’Riordan, T. J. C.; Peifer, M.; Jones, C. R.;
(
́
first to investigate the biological activity of (−)-(3R)-
inthomycin C and preceding intermediates in human cancer
cell lines, and a surprising proteasome inhibition response was
discovered for ester 17. Work is currently underway in our
laboratories toward the total synthesis of oxazolomycin B and
will be disclosed in due course.
1
(
4
Miles, T. J. Org. Lett. 2012, 14, 5460.
(17) (a) Denmark, S. E.; Heemstra, J. R.; Beutner, G. L. Angew.
Chem., Int. Ed. 2005, 44, 4682. (b) Paterson, I.; Smith, J. D.; Ward, R.
A. Tetrahedron 1995, 51, 9413.
ASSOCIATED CONTENT
■
(
18) Miller, R. A.; Smith, R. M.; Karady, S.; Reamer, R. A.
Tetrahedron Lett. 2002, 43, 935.
19) Miller, R. A.; Smith, R. M.; Marcune, B. J. Org. Chem. 2005, 70,
074.
20) Chatterjee, A. K.; Choi, T.-L.; Sanders, D. P.; Grubbs, R. H. J.
*
S
Supporting Information
(
9
(
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.or-
glett.8b01370.
Am. Chem. Soc. 2003, 125, 11360.
Full experimental details, copies of spectral data (PDF)
(21) Chatterjee, A. K.; Sanders, D. P.; Grubbs, R. H. Org. Lett. 2002,
4
(
, 1939.
22) Ishida, A.; Mukaiyama, T. Bull. Chem. Soc. Jpn. 1977, 50, 1161.
AUTHOR INFORMATION
■
*
(23) Hoye, T. R.; Jeffrey, C. S.; Shao, F. Nat. Protoc. 2007, 2, 2451.
(24) Manasanch, E. E.; Orlowski, R. Z. Nat. Rev. Clin. Oncol. 2017,
4, 417.
Corresponding Author
1
E-mail: timothy.donohoe@chem.ox.ac.uk.
ORCID
Timothy J. Donohoe: 0000-0001-7088-6626
Notes
The authors declare no competing financial interest.
§
(
L.J.-C.) To whom correspondence regarding the biological
assays should be addressed.
ACKNOWLEDGMENTS
■
S.B. and L.J-C. received funding for this project from the
People Programme (Marie Curie Actions) of the European
Union’s Seventh Framework Programme (FP7/2007-2013)
under REA Grant Agreement No. 316955. C.T. received
funding from Cancer Research UK (CRUK, C38302/A13012)
through the Preclinical Validation Lab, CRUK Oxford Cancer
Research Centre.
REFERENCES
■
(
1) Omura, S.; Tanaka, Y.; Kanaya, I.; Shinose, M.; Takahashi, Y. J.
Antibiot. 1990, 43, 1034.
(
2) Henkel, T.; Zeeck, A. Liebigs Ann. Chem. 1991, 1991, 367.
(
3) Tanaka, Y.; Kanaya, I.; Takahashi, Y.; Shinose, M.; Tanaka, H.;
Omura, S. J. Antibiot. 1993, 46, 1208.
(
4) Omura, S. Gene 1992, 115, 141.
(
5) Kawada, M.; Inoue, H.; Usami, I.; Ikeda, D. Cancer Sci. 2009, 100,
1
(
50.
6) Kawada, M.; Yoshimoto, Y.; Minamiguchi, K.; Kumagai, H.;
Someno, T.; Masuda, T.; Ishizuka, M.; Ikeda, D. Anticancer Res. 2004,
4, 1541.
7) Webb, M. R.; Addie, M. S.; Crawforth, C. M.; Dale, J. W.; Franci,
X.; Pizzonero, M.; Donald, C.; Taylor, R. J. K. Tetrahedron 2008, 64,
778.
8) Senapati, B. K.; Gao, L.; Lee, S. I.; Hwang, G.-S.; Ryu, D. H. Org.
Lett. 2010, 12, 5088.
9) Yoshino, M.; Eto, K.; Takahashi, K.; Ishihara, J.; Hatakeyama, S.
Org. Biomol. Chem. 2012, 10, 8164.
10) Hale, K. J.; Grabski, M.; Manaviazar, S.; Maczka, M. Org. Lett.
014, 16, 1164.
11) Souris, C.; Freb
Maulide, N. Org. Lett. 2013, 15, 3242.
2
(
4
(
(
(
2
(
́
ault, F.; Patel, A.; Audisio, D.; Houk, K. N.;
D
DOI: 10.1021/acs.orglett.8b01370
Org. Lett. XXXX, XXX, XXX−XXX