Total Synthesis of the GRP78-Downregulatory Macrolide (+)-Prunustatin A, the Immunosuppressant (+)-SW-163A, and a JBIR-04 Diastereoisomer That Confirms JBIR-04 Has Nonidentical Stereochemistry to (+)-Prunustatin A

Article abstract of DOI:10.1021/acs.orglett.6b01235

A unified total synthesis of the GRP78-downregulator (+)-prunustatin A and the immunosuppressant (+)-SW-163A based upon [1 + 1 + 1 + 1]-fragment condensation and macrolactonization between O(4) and C(5) is herein described. Sharpless asymmetric dihydroxylation was used to set the C(2) stereocenter present in both targets. In like fashion, coupling of the (+)-prunustatin A macrolide amine with benzoic acid furnished a JBIR-04 diastereoisomer whose NMR spectra did not match those of JBIR-04, thus confirming that it has different stereochemistry than (+)-prunustatin A.

Full text of DOI:10.1021/acs.orglett.6b01235

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Letter
pubs.acs.org/OrgLett
Total Synthesis of the GRP78-Downregulatory Macrolide
(+)-Prunustatin A, the Immunosuppressant (+)-SW-163A, and a JBIR-
04 Diastereoisomer That Confirms JBIR-04 Has Nonidentical
Stereochemistry to (+)-Prunustatin A
Soraya Manaviazar, Peter Nockemann, and Karl J. Hale*
School of Chemistry & Chemical Engineering and Centre for Cancer Research and Cell Biology (CCRCB), Queen’s University
Belfast, Stranmillis Road, Belfast BT9 5AG, Northern Ireland, United Kingdom
S
* Supporting Information
ABSTRACT: A unified total synthesis of the GRP78-down-
regulator (+)-prunustatin A and the immunosuppressant
(+)-SW-163A based upon [1 + 1 + 1 + 1]-fragment condensation
and macrolactonization between O(4) and C(5) is herein
described. Sharpless asymmetric dihydroxylation was used to
set the C(2) stereocenter present in both targets. In like fashion,
coupling of the (+)-prunustatin A macrolide amine with benzoic
acid furnished a JBIR-04 diastereoisomer whose NMR spectra
did not match those of JBIR-04, thus confirming that it has
different stereochemistry than (+)-prunustatin A.
(+)-Prunustatin A is a chemically alluring β-keto ester macrolide
first discovered by Shin-ya and co-workers1 in fermentation
broths of Streptomyces violaceoniger 4521-SVS3, an actinomycete
found in soil of the Okinawan island of Kumejima. While initially
a full stereostructure for (+)-prunustatin A could not be
proposed, later chemical degradation and fragment correlation
studies by Shin-ya did eventually reveal the full absolute
stereostructure to be as that shown in Scheme 1.2
From a therapeutic perspective, (+)-prunustatin A is of
considerable pharmaceutical interest because of its pronounced
downregulatory effects on GRP78/BIP (78 kDa glucose-
regulated protein) expression in glucose-deprived HT1080
human fibrosarcoma cells at very low drug concentrations
(IC50 = 11.5 nM), with total inhibition of GRP78 expression
occurring at the 80 nM level and full cancer cell apoptosis
occurring at the slightly higher drug concentration of 100 nM.1
Importantly, (+)-prunustatin A is non-cytotoxic toward HT1080
cells under normal conditions, where it functions as a cytostatic
agent even at concentrations as high as 500 nM. This remarkable
property of (+)-prunustatin A to selectively induce apoptosis
within highly stressed, glucose-deprived, cancer cells suggests
that it might potentially be useful to combat hypoxic human
tumors while leaving normal healthy tissue undamaged.
This has prompted a number of groups to devise elegant total
syntheses of (+)-prunustatin A to increase the supply, with the
teams of Kawanishi3 and Usuki4 scoring particularly notable
successes in this regard.
Apart from the potential value of GRP78 inhibitors for treating
drug-resistant cancers, such molecules could possibly sensitize
drug-resistant bacteria to the effects of existing antibiotics (e.g.,
Gram-negative Neisseria gonorrheae and Neisseria meningitides
strains),5 and they might also prove useful for counteracting
many lethal viral infections. In the latter regard, many viruses rely
on GRP78-regulated machinery to create functionally active
virions (e.g., the Ebola, Lassa, and Marburg hemorrhagic RNA
viruses).5 Because of this, we became interested in developing a
new synthetic route to (+)-prunustatin A to expedite its future
clinical development and that of its powerful reduced
immunosuppressant congener, (+)-SW-163A.6 In this Letter
we report our success in these endeavors.
Following several abortive attempts to synthesize (+)-prunus-
tatin A by strategies involving macrocyclization between O(7)
and C(8), which each furnished prunustatin A diastereomers, we
eventually decided to pursue a new synthetic plan wherein the
C(1) keto group would be installed before ring closure between
(O)4 and C(5). In our newly proposed strategem (Scheme 1), a
late-stage macrolactonization would now be effected on seco-acid
3.3 The resulting macrolide would then be converted into
amine−lactone 2, which would be coupled to acid 1. The
resulting product would then be O-debenzylated. Compound 3
would itself be acquired from 4 by O-deallylation and O-
Upregulated GRP78 expression within hypoxic solid tumors is
now thought to contribute significantly toward them becoming
refractory toward treatment with drugs and radiotherapy. There
is thus a very good medical case for clinically establishing whether
(+)-prunustatin A will be of value for treating such cancers.
However, preliminary screening of (+)-prunustatin A against
xenografted tumors in mice has not been possible to date because
of the dearth of material that is presently available for testing.
Received: April 29, 2016
© XXXX American Chemical Society
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silylation. Of course, a good option for constructing the (E)-
olefin in 14 would be a Julia−Kocienski olefination8 between
tetrazolylsulfone 15 and known β-aldehydo ester 16.9
Scheme 1. Our Retrosynthetic Planning for Prunustatin A
Our new (+)-prunustatin A campaign began with a repetition
of the synthesis of β-aldehydo ester 16,9 which was typically
prepared in 67% yield from 17 by pyridinium chlorochromate
(PCC) oxidation in CH2Cl2 (Scheme 2). Aldehyde 16 was then
Scheme 2. Our Synthesis of Acid 9
desilylation, with tetraester 4 being assembled from the sterically
hindered acid 9 by two successive esterifications, the first
involving 8 and 9 and the second involving 5 and 6. Keto acid
5 would be derived from triester 7 by O-debenzylation at C(1),
oxidation of the alcohol to the ketone, and O-deallylation. Acid 9
was envisioned to emanate from alcohol 12 by esterification with
protected L-lactic acid derivative 11 allied with selective O-
desilylation and oxidation of the primary alcohol to the acid. The
main issue associated with constructing alcohol 12 would be
correct positioning of the various protecting groups to enable the
alcohol at C(2) to be selectively presented to 11. For this, we
hoped to take advantage of a Sharpless asymmetric dihydrox-
ylation (AD) reaction7 on (E)-alkene 14 accompanied by in situ
lactonization to give the corresponding β-hydroxybutyrolactone
with high ee. If successful, such an approach would nicely allow
protection of the secondary hydroxyl at C(1) as an O-p-
methoxybenzyl (OPMB) ether, as in compound 13, to allow the
remaining features to be elaborated by reduction and selective O-
condensed with the anion derived from 15 to give (E)-alkene 14
in 64% yield with total stereocontrol. While initially we accessed
19 in near optically pure condition via Sharpless AD with AD-
mix-β,7a we found this process to be inconveniently slow,
needing 7 days to reach completion. We therefore evaluated less
hindered Sharpless ligands for this purpose. A considerable
improvement in the reaction rate was found when the AD was
conducted with catalytic potassium osmate (1 mol %) and the
DHQD-MEQ ligand7b (13 mol %), which afforded the
lactonized product 19 in 95% yield with 100% ee after only 30
h of stirring at 0 °C, which represented a considerable
B
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operational improvement and was also much cheaper to carry
out.
Scheme 3. Completion of Our Total Synthesis of
(+)-Prunustatin A and the Immunosuppressant SW-163A
The alcohol in 19 was then protected as an OPMB ether with
NaH and PMBCl in DMF, and the product, lactone 13, was
reduced with LiAlH4 to obtain diol 20 in 85% yield. The less
hindered primary hydroxyl of 20 was next regioselectively
protected as an O-triethylsilyl (OTES) ether to allow the all-
important ester bond to be grafted onto O(2). For this, (S)-lactic
acid derivative 21 was first converted into the Yamaguchi10 2,4,6-
trichlorobenzoic acid mixed anhydride 22, and this was reacted
with 12 in C6H6 at rt for 17 h in the presence of 4-
(dimethylamino)pyridine (DMAP) (3.25 equiv). This proved
to be the optimal method for esterifying this system, furnishing
10 in 93% yield. Having reliably fulfilled its alcohol-differ-
entiating role, the primary OTES ether was selectively cleaved
from 10 by catalytic pyridinium p-toluenesulfonate (PPTS) (0.1
equiv) in MeOH over 45 min at rt. A two-stage oxidation
thereafter converted alcohol 23 into carboxylic acid 9. In this
sequence, a Ley−Griffith catalytic n-Pr4NRuO4/N-methylmor-
pholine N-oxide (NMO) oxidation11 first furnished aldehyde 24
in near quantitative yield, and a Pinnick oxidation subsequently
provided 9 in 89% yield.12
We next focused our attention on converting L-isoleucine (25)
into L-isoleucic acid (26) and the latter into O-allyl ester 8
(Scheme 3).13 To access the former, we followed the
diazotization procedure of Plenkiewicz and Poterala,13a which
worked very well in our hands, and generated the HNO2 in situ
from 1 M aqueous H2SO4 and NaNO2. It delivered the crystalline
acid 26 in 76% yield on a large scale from 25 (53 g).
Chemoselective O-allylation13b was next achieved with
K2CO3/allyl bromide/Bu4NI in DMF at rt; the product ester 8
was isolated in 82% yield. It was then coupled to acid 9 using
excess N-(3-(dimethylamino)propyl)-N-ethyl carbodiimide hy-
drochloride (EDCI) and DMAP as the acid activators; triester 7
was formed in 72% yield after 18 h of stirring at rt in CH2Cl2.
DDQ was now used to chemoselectively remove the PMB
group from O(1) without disturbing the potentially sensitive O-
allyl ester. The resulting alcohol 27 was oxidized to the ketone
with n-Pr4NRuO4/NMO in MeCN, and the O-allyl ester was
detached with PhSiH3 and Pd(0). The acid 5 so produced was
then coupled to partially protected L-threonine derivative 6 using
2-methyl-6-nitrobenzoic anhydride (MNBA)14 and DMAP in
CH2Cl2, affording the desired product 4 in 74% yield.
Subsequently, 4 was O-deallylated with PhSiH3 and catalytic
Pd(PPh3)4 in CH2Cl2,15 and the product acid was O-desilylated
with HF·pyridine complex in a mixture of pyridine/THF. Both
deprotections proceeded cleanly to provide the required seco-
acid 3 in 82% yield over the two steps. The latter was then
macrolactonized on a 0.3 g scale under high-dilution conditions
by addition of a solution of 3 in CH2Cl2/THF (1:1) over 9 h to a
solution of DMAP (2 equiv) and MNBA14 (1.3 equiv) in dry
THF at rt, attaining a final reaction concentration of ca. 0.00048
M with respect to 3. The reactants were then allowed to stir at rt
for 39 h to bring about the desired ring closure. Macrolide 29 was
isolated pure in 42% yield after SiO2 flash chromatography. The
structure of 29 was unambiguously confirmed by single-crystal X-
ray analysis (see the Supporting Information). Importantly, the
the seco-acid solution to the MNBA/DMAP solution, when
either a syringe pump or slow cannulation was used to deliver the
THF/CH2Cl2 solution of 3. Not only did the hot vapor from the
reaction mixture consistently oppose a carefully controlled slow
addition of the solution of 3 into the reaction flask, but also, the
heating process caused much more variable reaction outcomes,
with the attendant formation of more complex mixtures. Our
very best yield of 29 from adhering to the 50 °C cyclization
1
400 MHz H NMR spectrum of 29 in CDCl3 matched that of
Kawanishi.3
Although we did attempt to repeat the 50 °C macro-
lactonization protocol of Yamakoshi and Kawanishi3,16 on 3 at
the reaction concentration of 0.0012 M that they reported, we
found it extremely difficult to control the rate of the addition of
C
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protocol in ref 3 was 49%, but this was not the norm. Because of the
significant technical difficulties and reaction variations that
attend this method,3 we recommend that other workers use the
much more consistent ultrahigh-dilution rt cyclization procedure that
we have described in the Supporting Information. However, even
under our rt conditions, intermolecular dimerization of 3 still
continues to be significant, but generally less so than under the 50
°C reaction conditions.3,16
SQUEEZE-processed crystallographic data for 29 (CIF)
Original crystallographic data for 29 (CIF)
Crystallographic data for 13 (CIF)
Crystallographic data for 19 (CIF)
AUTHOR INFORMATION
Corresponding Author
■
*k.j.hale@qub.ac.uk
In order to complete our synthesis of (+)-prunustatin A, the
Boc group of 29 was detached with neat CF3CO2H in CH2Cl2,
and the crude TFA salt 30 was coupled with 13,4,18 using EDCI,
N-ethylmorpholine (NEM), and 1-hydroxybenzotriazole
(HOBt). The desired product 31 was isolated in 63% yield
after SiO2 flash chromatography; it was identical to the same
compound prepared by Usuki.4 Compound 31 was then
deprotected by catalytic hydrogenation with 10% Pd/C in
EtOAc/MeOH (1:1) at 1 atm; synthetic (+)-prunustatin A was
isolated in 65% yield after SiO2 chromatography (0.74% overall).
Its spectroscopic values closely matched those reported by Shin-
ya,1,2 Kawanishi,3 and Usuki,4 thus confirming that the natural
product had indeed been synthesized. NaBH4 reduction of
(+)-prunustatin A in EtOH also furnished the immunosuppres-
sant (+)-SW-163A,6 in accord with Shin-ya’s 2007 limited
report.2
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank QUB and the ACS for helping to fund this work. We
are also extremely grateful to Dr. Eiji Kawanishi of Mitsubishi
Tanabe Pharma for very kindly supplying us with PDF copies of
his spectra for (+)-prunustatin A, SW-163A, and macrolide 29.
REFERENCES
■
(1) Umeda, Y.; Chijiwa, S.; Furihata, K.; Furihata, K.; Sakuda, S.;
Nagasawa, H.; Watanabe, H.; Shin-ya, K. J. Antibiot. 2005, 58, 206.
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Given that we had unambiguously proven the stereochemistry
of 29, we next deprotected its Boc group and coupled 30 to
PhCO2H in order to secure what we hoped was going to be the
structurally related natural product JBIR-0417 (Scheme 4),
Scheme 4. Our Attempted Synthesis of JBIR-04
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Chem. Soc. Jpn. 1979, 52, 1989.
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Marsden, S. P. Synthesis 1994, 1994, 639.
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whose absolute stereostructure has not been assigned to date.
Unfortunately, our spectroscopic comparisons of 32 with JBIR-
04 soon confirmed that JBIR-04 has different absolute
stereochemistry than (+)-prunustatin A, which perhaps explains
why its GRP78-downregulatory effects are 200 times lower.
In conclusion, we have devised unified, highly stereoselective
total syntheses of (+)-prunustatin A, SW-163A, and JBIR-04
diastereoisomer 32.18 The latter synthesis also revealed that the
absolute stereochemistry of JBIR-04 differs from that found in
(+)-prunustatin A. We expect that our new synthetic pathway to
these molecules will prove useful for fashioning analogues,
including biotinylated ones, which would have potential value for
new drug target retrieval by affinity chromatography.
(16) Because ref 3 reports no experimental procedures at all, we
reproduced as best we could the 50 °C macrocyclization in THF/
CH2Cl2 outlined in that paper.
(17) Izumikawa, M.; Ueda, J.; Chijiwa, S.; Takagi, M.; Shin-ya, K. J.
Antibiot. 2007, 60, 640.
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.orglett.6b01235.
(18) Recent syntheses of respirantin and kitastatin: (a) Pettit, G. R.;
Smith, T. H.; Feng, S.; Knight, J. C.; Tan, R.; Pettit, R. K.; Hinrichs, P. A.
J. Nat. Prod. 2007, 70, 1073. (b) Beveridge, R. E.; Batey, R. A. Org. Lett.
2014, 16, 2322.
■
S
Full experimental procedures for all steps, copies of the IR,
HMRS, and 1H/13C NMR spectra of every intermediate,
and X-ray plots and crystallographic data for 19, 13, and 29
(including CCDC accession numbers) (PDF)
D
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