The first total synthesis of (±)-cyclophostin and (±)-cyclipostin P: Inhibitors of the serine hydrolases acetyl cholinesterase and hormone sensitive lipase

Article abstract of DOI:10.1021/ol200991x

Cyclophostin, a structurally unique and potent naturally occurring acetyl cholinesterase (AChE) inhibitor, and its unnatural diastereomer were prepared in 6 steps and 15% overall yield from hydroxymethyl butyrolactone. The unnatural diastereomer of cyclop

Full text of DOI:10.1021/ol200991x

ORGANIC
LETTERS
2011
Vol. 13, No. 12
3094–3097
The First Total Synthesis of (()-
Cyclophostin and (()-Cyclipostin P:
Inhibitors of the Serine Hydrolases
Acetyl Cholinesterase and Hormone
Sensitive Lipase
Raj K. Malla, Saibal Bandyopadhyay, Christopher D. Spilling,* Supratik Dutta, and
Cynthia M. Dupureur
Department of Chemistry and Biochemistry, University of Missouri;St. Louis,
One University Boulevard, St. Louis, Missouri 63121, United States
cspill@umsl.edu
Received April 14, 2011
ABSTRACT
Cyclophostin, a structurally unique and potent naturally occurring acetyl cholinesterase (AChE) inhibitor, and its unnatural diastereomer were
prepared in 6 steps and 15% overall yield from hydroxymethyl butyrolactone. The unnatural diastereomer of cyclophostin was converted into
cyclipostin P, a potent naturally occurring hormone sensitive lipase (HSL) inhibitor, using a one pot dealkylationꢀalkylation process. The
inhibition [IC50] of human AChE by cyclophostin and its diastereomer are reported, as well as constituent binding (KI) and reactivity (k2) constants.
Cyclophostin 1 (Figure 1) is a novel bicyclic organopho-
sphate isolated from a fermentation solution of Strepto-
myces lavendulae (strain NK901093).1 The natural product
1 showedpotent inhibition ofacetylcholinesterase (AChE)
from the housefly (CSMA strain) and the brown plant
hopper with a reported IC50 of 7.6 ꢁ 10ꢀ10 M. The
structure of cyclophostin was first assigned by spectro-
scopic methods and then confirmed by single crystal X-ray
diffraction studies as a bicyclic structure with a seven-
membered cyclic enol-phosphate triester fused to a butyro-
lactonering. Thereare chirality centersatbothC3a and the
phosphorus atom (6). The absolute configurations were
determined to be 3aR,6S.2
cyclipostins 2.3 The cyclipostins 2 possess a core structure
similar to that of cyclophostin, but differ in the phosphate
ester. The cyclipostins 2 are phosphate esters of long chain
lypophilic alcohols of various lengths and structures, and
all are potent inhibitors of hormone sensitive lipase (HSL).
In addition, cyclophostin and the cyclipostins are probably
biosynthetically related to A-factor 3 and the virginiae
butanolides,4 and the PLA2 inhibitor 4.5
The significance of cyclophostin and the cyclipostins is
due in part to their unique structures and the enzymes they
(3) (a) Wink, J.; Schmidt, F.-R.; Seibert, G.; Aretz, W. J. Antibiot.
ꢀ
€
2002, 55, 472. (b) Vertesy, L.; Beck, B.; Bronstrup, M.; Ehrlich, K.;
Kurz, M.; Muller, G.; Schummer, D.; Seibert, G. J. Antibiot. 2002, 55,
480.
The unusual bicyclic enolphosphate is also found in the
family of structurally related natural products, named the
(4) (a) Sakuda, S.; Tanaka, S.; Mizuno, K.; Sukcharoen, O.; Nihira,
T.; Yamada, Y. J. Chem. Soc., Perkin Trans. 1 1993, 2309. (b) Kato, J.;
Funa, N.; Watanabe, H.; Ohnishi, Y.; Horinouchi, S. Proc. Natl. Acad.
Sci. U.S.A. 2007, 104, 2378. (c) Ohnishi, Y.; Kameyama, S.; Onaka, H.;
Horinouchi, S. Mol. Microbiol. 1999, 34, 102. (d) Miyake, K.; Horinouchi,
S.; Yoshida, M.; Chiba, N.; Mori, K.; Nogawa, N.; Morikawa, N.; Beppu,
T. J. Bacteriol. 1989, 171, 4298.
(1) Kurokawa, T.; Suzuki, K.; Hayaoka, T.; Nakagawa, T.; Izawa,
T.; Kobayashi, M.; Harada, N. J. Antibiot. 1993, 46, 1315.
(2) The configuration for cyclophostin namedþin reꢀf 1, 3aR,6S, is
actually incorrect. A common error is to treat P ;O as PdO and
hence assign the priority incorrectly. Natural cyclophostin is actually
3aR,6R. See: Cahn, R. S.; Ingold, C.; Prelog, V. Angew. Chem., Int. Ed.
1966, 5, 385. The configuration is correctly named in chemical abstracts.
(5) Campbell, M. M.; Fox, J. L.; Sainsbury, M.; Liu, Y. Tetrahedron
1989, 45, 4551.
r
10.1021/ol200991x
Published on Web 05/17/2011
2011 American Chemical Society

Scheme 1. Cyclophostin Retrosynthetic Analysis
Figure 1. Cyclophostin and related structures.
inhibit. Interest in AChE has re-emerged due its role as a
therapeutic target for Alzheimer’s disease,6 myasthenia
gravis,7 and glaucoma,8 whereas HSL is a therapeutic
target for type II diabetes.9
(route A) or conversely condensation of the primary
alcohol with an enolphosphoric acid via intermediate 10
(route B). Both intermediates can be formed by C-acyla-
tion of derivatives of hydroxymethyl lactone 6. The lactone 6
and various derivatives are available in the racemic
modification11 and either enantiomer.12 It was thought
necessary to protect the hydroxyl of lactone 6 prior to
C-acetylation to avoid complications arising from cycliza-
tion of the acetyl lactone 11 to the hemiketal 12.12b The most
expedient route would be to introduce the phosphate early in
the synthesis, simultaneously protecting the hydroxyl group.
The racemic hydroxy lactone 6 was prepared using
published methods.11 The hydroxyl was phosphorylated
using dimethyl bromophosphate, prepared in situ by reac-
tion of trimethyl phosphite with bromine, to give the
phosphate 7 (Scheme 2). The phosphorylated butyrolac-
tone 7 was deprotonated with 1 equiv of LiHMDS in THF,
and the resulting enolate was acylated with acetyl chlo-
ride.4a,5 Initially, mixtures of the acetyl lactone 14 and the
enolacetate 13 were observed, so an excess of acetyl
chloride was added to ensure the complete acylation giving
enolacetate 13in 65% yield asa mixture oftwo geometrical
isomers. The geometrical isomers could be separated, but
were generally carried through to the next step as a
mixture. Deacetylation of enolacetates 13 was achieved
using a catalytic amount of DMAP in MeOH to give the
acetyl lactone 14 in 62% yield.
We recently reported the synthesis of a phosphonate
analog 5a of cyclophostin.10 The activity of the phospho-
nates was g100-fold less than the value reported for
cyclophostin. The trans isomer (H and OMe) 5b was 10-
fold more active (IC50 of 3 μM human AChE) than the cis
isomer 5a (IC50 of 30 μM human AChE). Since the natural
product has the cis (H and OMe) configuration, the
unnatural isomer may well prove more potent. To accu-
rately compare the activities of cyclophostin, the phospho-
nate analog, and their diastereomers with a detailed kinetic
analysis, we needed reasonable quantities of the natural
product. Furthermore, we proposed that cyclophostin
would be an excellent precursor for the synthesis of the
family of cyclipostins. Herein, we report the first synthesis
of (() cyclophostin and conversion into (() cyclipostin P.
A retrosynthetic analysis (Scheme 1) of the bicyclic
phosphate 1 suggested that the cyclic enolphosphate could
be formed by either condensation of the acetyl group
(as the enol) with a phosphoric acid via intermediate 8
(6) Li, W. M.; Kan, K. K.; Carlier, P. R.; Pang, Y. P.; Han, Y. F.
Curr. Alzheimer Res. 2007, 4, 386.
ꢁ
(7) Garcia-Carrasco, M.; Escarcega, R. O.; Fuentes-Alexandro, S.;
Riebeling, C.; Cervera, R. Autoimmun. Rev. 2007, 6, 373.
(8) Kaur, J.; Zhang, M.-Q. Curr. Med. Chem. 2000, 3, 273.
(9) (a) Holm, C.; Osterlund, T.; Laurell, H.; Conteras, J. A. Annu.
Rev. Nutr. 2000, 20, 365. (b) Yeaman, S. J. Biochem. J. 2004, 379, 11.
(10) Bandyopadhyay, S.; Dutta, S.; Spilling, C. D.; Dupureur, C. M.;
Rath, N. P. J. Org. Chem. 2008, 73, 8386.
(11) For examples, see: (a) Gadir, S. A.; Smith, Y.; Taha, A. A.;
Thaller, V. J. Chem. Res (S) 1986, 222. (b) Yoda, H.; Mizutani, M.;
Takabe, K. Synlett 1998, 855. (c) Sengoku, T.; Suzuki, T.; Kakimoto, T.;
Takahashi, M.; Yoda, H. Tetrahedron 2009, 65, 2415.
Scheme 2. Synthesis of Primary Phosphate
(12) For examples, see: (a) Banfi, L.; Basso, A.; Guanti, G.; Zannetti,
M. T. Tetrahedron: Asymmetry 1997, 8, 4079. (b) Parsons, P. J.;
Lacrouts, P.; Buss, A. D. Chem. Commun. 1995, 437. (c) Crawforth,
J. M.; Fawcett, J.; J. Rawlings, B. J. Chem. Soc., Perkin Trans. 1 1998,
1721. (d) Posner, G. H.; Wietzberg, M.; Jew, S.-S. Synth. Commun. 1987,
17, 611. (e) Takabe, K.; Mase, N.; Matsumura, H.; Hasegawa, T.; Iida,
Y.; Kuribayashi, H.; Adachi, K.; Yoda, H.; Ao, M. Bioorg. Med. Chem.
Lett. 2002, 12, 2295. (f) Mori, K.; Chiba, N. Liebigs Ann. Chem. 1989,
957. (g) Takabe, K.; Tanaka, M.; Sugimoto, M.; Yamada, T.; Yoda, H.
Tetrahedron: Asymmetry 1992, 3, 1385.
Org. Lett., Vol. 13, No. 12, 2011
3095

workup, a significant amount of mass was lost in the
organic soluble crude product. The peak at ꢀ8.0 had
disappeared, and only the peaks at ꢀ7.6 and ꢀ11.7 ppm
remained in the crude product. Chromatographic separa-
tion of the product mixture using preparative TLC yielded
cyclophostin 1 and its unnatural diastereomer 17 in very
low yield (<10% combined yield). The identity of cyclo-
phostin was confirmed by comparing the 1H and 31P NMR
Scheme 3. Formation of Cyclopropanes
data with those reported for the natural product.1
A
The successful C-acylation of the lactone 7 was both
gratifying and somewhat surprising. It is well-known that
the enolates derived from γ-phosphoryloxy carboxylates
cyclize to form cyclopropanes.13 Indeed, when the acyclic
γ-phosphoryloxy carboxylate 15 was treated with
LiHMDS and acetyl chloride (Scheme 3), the only isolable
product was the cyclopropane 16. In contrast, a solution of
the enolate of butyrolactone 7 was stable in the absence of
an external electrophile up to ꢀ20 °C for 1 h. At higher
temperatures decomposition to intractable products was
observed.
crystalline tetracyclic byproduct 18 was also isolated, and
the structure was confirmed by an X-ray diffraction
study.16
Scheme 5. Synthesis and Cyclization of the Enolphosphate
Scheme 4. Attempted Cyclization of the Primary Phosphate
The lack of success in finding reaction conditions for the
cyclization of 8 led us to examine an alternate route B
(Scheme 5). The hydroxyl of lactone 6 was protected as a
p-methoxybenzyl (pmb) ether 9 by the copper(II) triflate
catalyzed reaction with p-methoxybenzyloxy trichloro-
acetimidate.17 Deprotonation with NaHMDS and acyla-
tion with acetic anhydride furnished the acetyl lactone 19
directly. Selective phosphorylation of 2-acetyl butyrolac-
tone 19 was accomplished by reaction with dimethyl
chlorophosphate using a procedure reported to give the
E-enol phosphate.18,19
The pmb ether was removed using DDQ in wet CH2Cl2,
and the enolphosphate 21 was selectively monodemethy-
lated using 1 equiv of sodium iodide in acetone at 45 °C.
The sodium salt was protonated with Amberlite IR 120
resin to generate the corresponding phosphoric acid 10.
Reaction of the phosphoric acid 10 with DCC and DMAP
in CH2Cl2 successfully formed the cyclic enolphosphates 1
With the desired 2-acetyl butyrolactone intermediate 14
in hand, the final step of the cyclophostin synthesis was
explored (Scheme 4). The intermediate 14 was demethy-
lated using 1 equiv of sodium iodide in refluxing acetoni-
trile solution to give the corresponding sodium salt in
quantitative yield.14 The sodium salt was protonated using
Amberlite (sulfonic acid) resin to yield the phosphoric acid
8. Attempted cyclization via intramolecular condensation
of the monophosphoric acid moiety with the enol form of
the acetyl group using EDC, HOBt, and Hunig’s base and
related reactions were not successful.15 These condi-
tions had been used previously inthe synthesisofthe phos-
phonate analog.10 Other reaction conditions were exam-
ined. Reaction with TsCl and pyridine gave a crude
product with signals at ꢀ7.6, ꢀ8.0, and ꢀ11.7 ppm in the
31P NMR spectrum (among others). After aqueous
(13) For examples, see: (a) Jacks, T. E.; Nibbe, H.; Wiemer, D. F.
J. Org. Chem. 1993, 58, 4584. (b) Singh, A. K.; Rao, M. N.; Simpson,
J. H.; Li, W.-S.; Thornton, J. E.; Kuehner, D. E.; Kacsur, D. J. Org.
Process Res. Dev. 2002, 6, 618. (c) Krawczyk, H.; Wasek, K.; Kedzia, J.;
Wojciechowski, J.; Wolf, W. M. Org. Biomol. Chem. 2008, 6, 308.
(14) The reaction of phosphates with NaI is generally performed in
acetone, but other solvent including MeCN also work well. See: Zervas,
L.; Dilaris, I. J. Am. Chem. Soc. 1955, 77, 5354.
(16) The details of the structure determination have been deposited at
the Cambridge Crystallographic Data Center as CCDC 812559.
(17) (a) Rai, A. N.; Basu, A. Tetrahedron Lett. 2003, 44, 2267. (b)
Nakajima, N.; Horita, K.; Abe, R.; Yonemitsu, O. Tetrahedron Lett.
1988, 29, 4139.
(18) Dutta, S.; Malla, R. K.; Bandyopadhyay, S.; Spilling, C. D.;
Dupureur, C. M. Bioorg. Med. Chem. 2010, 18, 2265.
(19) (a) White, J. D.; Kawasaki, M. J. Org. Chem. 1992, 57, 5292. (b)
Lichtenenthaler, F. W. Chem. Rev. 1961, 61, 607.
(15) (a) Smith, M.; Drummond, G. I.; Khorana, H. G. J. Am. Chem.
Soc. 1961, 83, 698. (b) Smith, M.; Moffatt, J. G.; Khorana, H. G. J. Am.
Chem. Soc. 1958, 80, 6204. (c) Khorana, H. G.; Tener, G. M.; Wright,
R. S.; Moffatt, J. G. J. Am. Chem. Soc. 1957, 79, 430.
3096
Org. Lett., Vol. 13, No. 12, 2011

and 17 as a 1:1 mixture. The diastereomers were separated
using silica gel chromatography to give natural cyclophos-
tin 1 and its diastereomer 17 in 55% combined yield.
Cyclophostin and its diastereomer are potential precur-
sors for the synthesis of the family of cyclipostins by ester
exchange. We opted to explore a novel one pot process for
this conversion (Scheme 6). The unnatural diastereomer 17
was treated with hexadecyl bromide (10 equiv) and cata-
lytic tetrabutylammonium iodide (TBAI) in refluxing di-
oxane to give cyclipostin P 2 and its diastereomer 22 with
95% conversion and a 1:1 ratio. The diastereomers were
separated by column chromatography to give a 77%
combined yield. The reaction of either 2 or 22 with
hexadecyl bromide and catalytic TBAI in refluxing diox-
ane resulted in a mixture of both 2 and 22 in a 1:1 ratio.
There was no reaction in the absence of TBAI.
Table 1. Inhibition of AChE by Cyclophostin and Related
Compounds
no.
IC50
KI
k2, minꢀ1
k-2, minꢀ1
5a
5b
1
30 μM
3 μM
45 nM
40 nM
140 ( 60 μM
24 ( 6.0 μM
140 ( 72 nM
210 ( 140 nM
0.4 ( 0.1
0.3 ( 0.06
0.7 ( 0.02
0.9 ( 0.5
4e-3
0.02
0
17
0
To dissect this behavior, residual enzyme activities were
obtained as a function of inhibitor concentration and
incubation time for 1 and 17 and analyzed using the
equation shown in Table 1 as described previously.18 Inter-
estingly, the chemical (rate) constants k2 and kꢀ2 vary little
among the four compounds, but the KI, which represents
the noncovalent binding event, reflects 100ꢀ1000-fold
stronger binding by the phosphates than the phosphonates.
Thus while chemical intuition would suggest that the carbon
replacement of the endocyclic oxygen would cause a differ-
ence in reactivity, these data indicate that this substitution
instead has profoundly adverse effects on binding. We
speculate that this is due to a combination of additional
hydrogen bonds (to the O) and subtle conformational
differences in the bicyclic ring system. This observation
points to the exquisite ability of enzymes to discriminate
compounds in their active sites.
Scheme 6. Conversion of Cyclophostin into Cyclipostin P
It is proposed that the iodide cleaves the methyl (or
alkyl) phosphate ester bond to give a phosphate anion
which is then alkylated by the hexadecyl bromide. The
formation of the anion would account for the scrambling
of the stereochemistry at phosphorus.
In conclusion, cyclophostin and its diastereomer have
been prepared in six steps and 15% overall yield from lactone
6. The diastereomer of cyclophostin was converted into
cyclipostin P using a one pot process. Syntheses of additional
examples of the cyclipostins and corresponding biological
studies are ongoing and will be reported in due course.
(()-Cyclophostin and its diastereomer were examined
for inhibitory activity against human AChE using a mod-
ified Ellman assay.20 Compounds 1 and 17 are potent
inhibitors of human AChE: both have IC50’s of ∼40 nM
(Table 1). This is only slightly weaker than the previously
reported values for the natural product with AChE iso-
lated from the insect.1 This could be a reflection of either
the difference in AChE source species or the selectivity
of the single (natural) enantiomer versus the racemic
(synthetic) mixture. There are two additional comparisons
of interest. One is that although they are diastereomers, the
IC50’s for 1 and 17 are very similar. The other is that these
IC50’s represent a 103ꢀ104 improvement in potency rela-
tive to the corresponding (()-phosphonate analogs 5.10,18
Acknowledgment. This work was supported by Grant
R01-GM076192 from the National Institute of General
Medical Sciences. We thank Prof. R. K. Winter and
Mr. Joe Kramer, the Department of Chemistry and Bio-
chemistry, University of Missouri;St. Louis, for mass
spectra and Prof. Paul Hanson, University of Kansas, for
bringing cyclophostin and the cyclipostins toour attention.
Supporting Information Available. Detailed experimen-
tal procedures and full spectroscopic data for all new
compounds. The material is available free of charge via
the Internet at http://pubs.acs.org.
(20) Ellman, G. J.; Courtney, K. D., Jr.; Featherstone, R. M. Bio-
chem. Pharmacol. 1961, 7, 88–95.
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