Synthesis and biological evaluation of a phosphonate analog of the natural acetyl cholinesterase inhibitor cyclophostin

Article abstract of DOI:10.1021/jo801453v

(Chemical Equation Presented) Two diastereomers of a phosphonate analog 6 of the AChE inhibitor cyclophostin were synthesized. The substitution reaction of phosphono allylic carbonate 10a with methyl acetoacetate gave the vinyl phosphonate 9a. Attempted hydrogenation/debenzylation gave an unexpected enolether lactone. Alternatively, selective hydrogenation, demethylation, cyclization and debenzylation gave the phosphonate analog of cyclophostin as a separable mixture of diastereomers 6. The trans phosphonate isomer was more active than the cis isomer against AChE from two sources.

Full text of DOI:10.1021/jo801453v

Synthesis and Biological Evaluation of a Phosphonate Analog of the
Natural Acetyl Cholinesterase Inhibitor Cyclophostin
Saibal Bandyopadhyay,† Supratik Dutta,‡ Christopher D. Spilling,*,† Cynthia M. Dupureur,‡
and Nigam P. Rath‡
Department of Chemistry and Biochemistry and Center for Nanoscience, UniVersity of Missouri-St. Louis,
1 UniVersity BouleVard, St. Louis, Missouri 63121
cspill@umsl.edu
ReceiVed July 7, 2008
Two diastereomers of a phosphonate analog 6 of the AChE inhibitor cyclophostin were synthesized. The
substitution reaction of phosphono allylic carbonate 10a with methyl acetoacetate gave the vinyl
phosphonate 9a. Attempted hydrogenation/debenzylation gave an unexpected enolether lactone.
Alternatively, selective hydrogenation, demethylation, cyclization and debenzylation gave the phosphonate
analog of cyclophostin as a separable mixture of diastereomers 6. The trans phosphonate isomer was
more active than the cis isomer against AChE from two sources.
Introduction
Cyclophostin 1, a novel bicyclic organophosphate, was
isolated from a fermentation solution of Streptomyces laVendulae
(strain NK901093) during a search for natural insecticides.1 The
natural product 1 showed potent inhibition of acetyl cholinest-
erase (AChE) from housefly (CSMA strain) and the brown plant
hopper with reported IC50 of 7.6 × 10-10 M. The structure of
cyclophostin was first assigned by spectroscopic 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 butyrolactone ring. There are chiral centers
at both C3a and the phosphorus atom. The absolute configura-
tions of the chiral centers were determined to be 3aR, 6S by
the anomalous scattering method.
FIGURE 1. Cyclophostin and related structures.
The unusual bicylic enolphosphate is found in some related
natural compounds 2 and 3 and the enolphosphate moiety
adjacent to a carbonyl is also found in the synthetic insecticides
monocrotphos 4 and phosphamidan 5.2-4 The unnamed tet-
rahydrofuran fused enolphosphates 2a and 2b were isolated
during an earlier search for insecticides and were shown to be
AChE inhibitors.3 The cyclipostins 3 possess a core structure
similar to that of cyclophostin but differ in the phosphate ester.4
The cyclipostins 3 are phosphate esters of long chain lypophilic
alcohols of various lengths and structures and all are potent
inhibitors of hormone sensitive lipase.4
AChE has been identified as a therapeutic target for myas-
thenia gravis,5 glaucoma,6 and Alzheimer’s disease7 and is well-
† Department of Chemistry and Biochemistry.
‡ Center for Nanoscience.
(1) Kurokawa, T.; Suzuki, K.; Hayaoka, T.; Nagakawa, T.; Izawa, T.;
Kobayashi, M.; Harada, M. J. Antibiot. 1993, 46, 1315–1318.
(2) He, W. H. Phosphorus, Sulfur Silicon Relat. Elem. 2008, 183, 266.
(3) Neumann, R.; Peter, H. H. Experientia 1987, 43, 1235.
(4) (a) Wink, J.; Schmidt, F. R.; Seibert, G.; Aretz, W. J. Antibiot. 2002, 55,
472. (b) V`ırtesy, L.; Beck, B.; Bro¨nstrup, M.; Ehrlich, K.; Kurz, M.; Muˆller, G.;
Schummer, D.; Seibert, G. J. Antibiot. 2002, 55, 480.
(5) Garica-Carrasco, M.; Esca´rcega, R. O.; Fuentes-Alexandro, S.; Riebeling,
C.; Cervera, R. Autoimmunity ReV. 2007, 6, 373.
(6) Kaur, J.; Zhang, M.-Q. Curr. Med. Chem. 2000, 3, 273.
(7) (a) Li, W. M.; Kan, K. K.; Carlier, P. R.; Pang, Y. P.; Han, Y. F. Curr.
Alzheimer Res. 2007, 4, 386. (b) Lleo´, A.; Greenberg, S. M.; Growdon, J. H.
Annu. ReV. Med. 2006, 57, 513.
8386 J. Org. Chem. 2008, 73, 8386–8391
10.1021/jo801453v CCC: $40.75 2008 American Chemical Society
Published on Web 09/27/2008

Natural Acetyl Cholinesterase Inhibitor Cyclophostin
TABLE 1. Palladium Catalyzed Allylic Substitution Reactions
with Acetoacetates and Malonates
carbonate
R
NuH
base
product
yield
10a
10a
10b
10a
Bn
Bn
TBS
Bn
CH3COCH2CO2Me
CH3COCH2CO2t-Bu
CH3COCH2CO2Me
t-BuO2CCH2CO2Me
none
none
none
BSA
9a
9b
9c
9d
85%
56%
64%
81%
FIGURE 2. Proposed reaction with the active site serine and a
phosphonate analog of cyclophostin.
known as the target for insecticides and “nerve gas” chemical
warfare agents. The exact mode of inhibition of AChE by
cyclophostin has not been reported. Because other phosphate
inhibitors of AChE are known to form a covalent bond between
the phosphorus and the serine residue of enzyme active site, it
is likely that the mode of inhibition by cyclophostin involves a
similar kind of interaction (Figure 2). It is also probable that
the enolphosphate acts as a leaving group on reaction with the
active site serine.
Phosphonate analogs of biologically active phosphates have
been shown to be an extremely useful tool in investigating
mechanistic detail of various enzymatic systems.8 This success
is usually attributed to the nonhydrolyzability of a P-C bond
(phosphonate analog) when compared to the P-O bond of the
corresponding phosphate leading to enhanced compound lifetime
in vivo. It should be possible to replace the noncritical oxygen
at position 5 in cyclophostin (and the cyclipostins) with a
methylene and still retain the AChE inhibitory activity (Figure
2), whereas loss of the enol oxygen (position 7) should eliminate
activity. Herein, we report the synthesis of the first phosphonate
analog 6 of cyclophostin.
4-Benzyloxy- and 4-(tert-butyldimethyl)silyloxy-cis-2-buten-
1-ol 11a and 11b were oxidized using PCC in CH2Cl2 to give
the known aldehydes 12a and 12b, respectively.13 The Et3N
catalyzed Pudovik reaction of the R,ꢀ-unsaturated aldehydes
with dimethyl phosphite gave the racemic hydroxy phosphonates
13a and 13b, which were converted into the corresponding
carbonate derivatives 10a and 10b by reaction with methyl
chloroformate in pyridine.
The palladium catalyzed substitution reaction of phosphono
allylic carbonates 10a and 10b with acetoacetates and malonates
was investigated. The reaction of methyl acetoacetate with
phosphonate 10a gave the vinyl phosphonate 9a in 85% yield
(Table 1). The formation of the vinyl phosphonate 9a was
always accompanied by diene formation, especially after
prolonged reaction times. However, the use of freshly distilled
methyl acetoacetate and careful monitoring of the reaction (31P
NMR spectroscopy) minimized diene formation. The phospho-
nate 10b reacted with methyl acetoacetate similarly to give the
vinyl phosphonate 9c in 64% yield. The reaction of tert-butyl
acetoacetate with phosphonate 10a was slower than with the
methyl acetoacetate, and a moderate amount of diene formation
was always observed. The palladium (0) catalyzed malonate
susbstitution reaction of phosphono allylic carbonate 10a was
again comparatively slow and required the presence of a weak
base like BSA. In each case, the products 9a-d were formed as
a mixture of diastereomers.
The attempted concomitant hydrogenation and debenzylation
of vinyl phosphonate 9a with hydrogen over palladium on
carbon unexpectedly gave the lactone methylenolether 14a
independent of the solvent used (Scheme 3). More surprisingly,
a similar product was also observed with t-butyl acetoacetate
substituted vinyl phosphonate 9b giving the t-butyl enolether
substituted butyrolactone 14b in quantitative yield. The TBS
protected phosphonate 9c was uneventfully hydrogenated to give
the saturated phosphonate 15. Attempted deprotection and
lactonization of phosphonate 15 with HF/Py also produced the
lactone methylenolether 14a. All attempts to cleave either the
methyl or t-butyl enolether failed to produce desired 2-acetyl
butyrolactone system.
Results and Discussion
A retrosynthetic analysis of the bicyclic phosphonate 6
suggested that either the lactone or the enol phosphonate bond
could be formed first giving rise to intermediates 7 and 8,
respectively. The common intermediate 9 could be formed via
a palladium catalyzed substitution reaction of the carbonate
derivative 10 of an allylic hydroxy phosphonate with an
acetoacetate ester. We reported the use of a similar strategy for
the synthesis of the lignan enterolactone.9
It is now well established that allylic hydroxy phosphonate
derivatives can be used as intermediates in the synthesis of
γ-substituted vinyl phosphonates by 1,3-transposition of
functionality.10-12 Following the original work of Zhu and Lu,11
we reported that the facile addition of soft nucleophiles to
optically pure carbonate derivatives proceeded with complete
transfer of chirality.9,12
(8) For examples, see: (a) Hilderbrand, R. L. The Role of Phosphonates in
LiVing Systems; CRC Press: Boca Raton, FL, 1983. (b) Blackburn, G. M.; Taylor,
G. E.; Tattershall, R. H.; Thatcher, G. R. J.; McLennan, A. Phosphonate
Analogues of Biological Phosphates. In Biophosphates and their Analogues -
Synthesis Structure, Metabolism and ActiVity; Bruzik, K. S., Stec, W. J., Eds.;
Elsevier Science Publishers B. V.: Amsterdam, The Netherlands, 1987; p 451.
(c) Engel, R. Chem. ReV. 1977, 77, 349. (d) Pompliano, D. L.; Rands, E.; Schaber,
M. D.; Mosser, S. D.; Anthony, N. J.; Gibbs, J. B. Biochemistry 1992, 31, 3800.
(e) Magnin, D. R.; Biller, S. A.; Chen, Y.; Dickson, J. K., Jr.; Fryszman, O. M.;
Lawrence, R. M.; Logan, J. V. H.; Sieber-McMaster, E. S.; Sulsky, R. B.; Traeger,
S. C.; Hsieh, D. C.; Lan, S.-J.; Rinehart, J. K.; Harrity, T. W.; Jolibois, K. G.;
Kunselman, L. K.; Rich, L. C.; Slusarchyk, D. A.; Ciosek, C. P., Jr. J. Med.
Chem. 1996, 39, 657. (f) Berkowitz, D. B.; Bose, M.; Pfannenstiel, T. J.; Doukov,
T. J. Org. Chem. 2000, 65, 4498. (g) Hakogi, T.; Monden, Y.; Iwama, S.;
Katsumura, S. Org. Lett. 2000, 2, 2627.
An alternate strategy (Scheme 4) involving the construction
of the seven membered enolphosphonate ring prior to the
butyrolactone was pursued. Selective hydrogenation of vinyl
¨
¨
(10) (a) Ohler, E.; Kanzler, S. Liebigs Ann. Recueil. 1997, 1437. (b) Ohler,
E.; Kanzler, S. Phosphorus, Sulfur Silicon Relat. Elem. 1996, 112, 71.
(11) (a) Zhu, J.; Lu, X. Chem. Commun. 1987, 1318. (b) Zhu, J.; Lu, X.
Tetrahedron Lett. 1987, 28, 1897.
(12) (a) He, A.; Yan, B.; Thanavaro, A.; Spilling, C. D.; Rath, N. P J. Org.
Chem. 2004, 69, 8643. (b) Rowe, B. J.; Spilling, C. D J. Org. Chem. 2003, 68,
9502.
(13) (a) Giner, J.-L. Tetrahedron Lett. 1998, 39, 2479. (b) Haruki, E.;
Arakawa, M.; Matsumura, N.; Otsuji, Y.; Imoto, E. Chem. Lett. 1974, 1, 427.
(9) Yan, B.; Spilling, C. D. J. Org. Chem. 2004, 69, 2859.
J. Org. Chem. Vol. 73, No. 21, 2008 8387

Bandyopadhyay et al.
SCHEME 1. Retrosynthetic Analysis for the Phosphonate Analog of Cyclophostin
SCHEME 2. Preparation of the Allylic Hydroxy
Phosphonates
SCHEME 3. Hydrogenation and Debenzylation of the Vinyl
Phosphonates
phosphonate 9a using hydrogen over palladium on carbon
poisoned with pyridine cleanly generated methyl acetoacetate
substituted saturated phosphonate 16. The phosphonate was
selectively monodemethylated using one equivalent of sodium
iodide in refluxing acetonitrile. The sodium salt was protonated
with Amberlite resin (IR 120) to generate the corresponding
phosphonic acid 17. Enol phosphonate ring formation was
successfully achieved by reaction of the phosphonic acid 17
with EDC, HOBt and Hunig’s base in CH2Cl2 giving mono-
cyclic enolphosphonate 8 as a 1:1.4 mixture of diastereomers.
Finally, selective debenzylation of 8 with hydrogen over
palladium on carbon resulted in clean hydrogenolysis to give
the primary alcohol which cyclized to the butyrolactone without
over reduction of enolphosphonate. The phosphonate analogue
of cyclophostin 6 was obtained as a mixture of two diastere-
oisomers having characteristic peaks in the 31P NMR spectrum
at 21 (6a) and 25 (6b) ppm. Mixture of diastereomers was
separated using silica gel chromatography. The diastereoisomer
6b is a crystalline solid and was further purified by crystalliza-
tion from EtOAc and hexane. An X-ray crystal structure (Figure
3) showed the relative stereochemistry of the C3a and the
phosphorus atom to be the same as the natural product (H and
methoxy are cis).
SCHEME 4. Synthesis of the Phosphonate Analog
SCHEME 5. Synthesis of an Enol Phosphate Analog of
Cyclophostin
To examine the effect of the bicylic ring structure on the
AChE activity, a simple enolphosphate analog 19 was prepared
for comparison (Scheme 5). Phosphorylation of 2-acetyl buty-
rolactone 18 was accomplished by reaction with dimethyl
chlorophosphate using a reported procedure.14 Alternatively, the
enol phosphate 19 was formed as a mixture of geometrical
isomers by reaction of 2-acetyl butyrolactone 18 with dimethyl
phosphoric acid and DCC.
The new compounds were examined for inhibitory activity
against AChE from human and electric eel using an Elman
assay.15 Surprisingly, the phosphonate analog 6a with the
“unnatural” relative stereochemistry (Table 2, Entry 1) showed
the most potent activity against both human and electric eel
acetylcholinesterase (AChE). Interestingly, compound 6a is
more effective against AChE from human than eel. This type
FIGURE 3. Projection view of the phosphonate 6b with 50% thermal
ellipsoids.
8388 J. Org. Chem. Vol. 73, No. 21, 2008

Natural Acetyl Cholinesterase Inhibitor Cyclophostin
TABLE 2. Inhibition Data for AChE from Human and Eel
mL) and the solution was cooled to 0 °C. Pyridine (7.6 mL, 88
mmol) and DMAP (0.10 g, 0.8 mmol) were added to the solution,
followed by the slow addition of methyl chloroformate (9.1 mL,
120 mmol). After the complete addition of methyl chloroformate,
the reaction mixture was slowly allowed to warm up to room
temperature and then it was stirred until the reaction was complete
(TLC, 24 h). The reaction mixture was washed with H2O (2×) and
saturated CuSO4 (2×), and then the organic layer was dried over
MgSO4. The solvent was evaporated in vacuo and the crude product
was purified by column chromatography (SiO2, EtOAc: Hexane,
60: 40) to give 10a as a colorless oil (10.4 g, 61% in two steps).
IR (neat) 1758 cm-1; 1H NMR (CDCl3) δ 7.31-7.36 (5H, m), 6.09
(1H, m), 5.89 (1H, m), 5.55 (1H, ddd, JHH ) 1.1, 6.6 Hz, JHP ) 14
Hz), 4.52 (2H, s), 4.08 (2H, m), 3.84 (3H, s), 3.83 (6H, d, JHP
)
10.7 Hz); 13C NMR (CDCl3) δ 154.9 (d, JCP ) 9.3 Hz), 138.2,
133.1 (d, JCP ) 12 Hz), 128.6, 127.95, 127.92, 122.8 (d, JCP ) 3.9
Hz), 72.5 (d, JCP ) 169 Hz), 72.6, 69.5 (d, JCP ) 1.5 Hz), 55.7,
54.2 (d, JCP ) 7.0 Hz), 54.0 (d, JCP ) 6.5 Hz); 31P NMR (CDCl3)
δ 19.7; HRMS (FAB, NBA, MH+) calcd. for C15H22O7P: 345.1103.
Found: 345.1108; Anal. Calcd for C15H21O7P: C, 52.33; H, 6.15.
Found: C, 52.35; H, 6.02.
Dimethyl [1-(methoxycarbonyloxy)-4-(t-butyldimethylsilyloxy)-
2-butenyl]phosphonate 10b. To a mixture of dimethyl phosphite
(3.8 mL, 41 mmol) and aldehyde 12b13b (4.73 g, 23.6 mmol) was
added Et3N (1.5 mL, 11 mmol). The reaction mixture was stirred
overnight then the volatiles were evaporated in vacuo to give the
crude hydroxy phosphonate 13b (6.6 g). The crude hydroxyphos-
phonate 13b (6.6 g, 21 mmol) was dissolved in anhydrous CH2Cl2
(60 mL) and the solution was cooled to 0 °C. Pyridine (2.9 mL, 36
mmol) and DMAP (0.41 g, 3.4 mmol) were added to the solution,
followed by the slow addition of methyl chloroformate (3.74 mL,
48.4 mmol). After the addition of methyl chloroformate was
complete, the reaction mixture was slowly allowed to warm up to
room temperature then it was stirred until the reaction was complete
(TLC, 24 h). The reaction mixture was washed with H2O (2×) and
saturated CuSO4 (2×), and then the organic layer was dried over
MgSO4. The solvent was evaporated in vacuo and the crude product
was purified by column chromatography (SiO2, EtOAc: Hexane,
60: 40) to give 10b as a colorless oil (4.09 g, 46% in two steps).
IR (neat) 1757 cm-1; 1H NMR (CDCl3) δ 6.04 (1H, m), 5.85 (1H,
m), 5.54 (1H, ddd, JHH ) 1.2, 7.1 Hz, JHP ) 13.4 Hz), 4.23 (2H,
m), 3.83 (3H, s), 3.82 (6H, d, JHP ) 10.7 Hz), 0.91 (9H, s), 0.07
(6H, s); 13C NMR (CDCl3) δ 154.7 (d, JCP ) 9.3 Hz), 136.0 (d,
JCP ) 10 Hz), 119.8 (d, JCP ) 3.4 Hz), 71.0 (d, JCP ) 169 Hz),
62.6, 55.3, 53.8 (d, JCP ) 6.8 Hz), 25.9, 18.3, -5.4; 31P NMR
of species dependent difference in inhibition has been observed
with phosphate inhibitors like soman, tabun, and VX.16 The
phosphonate analog 6b with natural relative stereochemistry was
10-fold less potent against AChE from both sources when
compared to the diastereomer 6a (Table 2, Entry 2). However,
the phosphonate analogs were significantly less potent than the
natural product cyclophostin 1 (reported for insect AchE only).
The fused butyrolactone did not appear to be necessary for
activity since the monocyclic diastereomers 8a and 8b (Table
2, Entries 3 and 4) showed IC50 similar to those of the bicycles
6a and 6b. However, the more active monocycle 8b had the
opposite relative stereochemistry to the more active bicycle 6a.
Furthermore, the simple enolphosphate derivative 19 of acetyl
butyrolactone was inactive (Table 2, Entry 5). Not surprisingly,
the methyl enolether 14a was also inactive (Table 2, Entry 6).
In summary, two diastereomers of a phosphonate analog of
cyclophostin were synthesized. The trans isomer was more
active than cis isomer against AChE from two sources. Because
the natural product has the cis configuration, the unnatural
isomer may well prove more potent. We are currently pursuing
a synthesis of both isomers of the natural product.
(CDCl3)
δ
20.0; HRMS (FAB, NBA, MNa+) calcd. for
C14H29O7PSiNa: 391.1319. Found: 391.1321.
(E)-Methyl 2-Acetyl-3-(benzyloxymethyl)-5-(dimethoxyphos-
phoryl)pent-4-enoate 9a. Pd2(dba)3 (0.049 g, 0.053 mmol) and
dppe (0.063 g, 0.16 mmol) were dissolved in anhydrous THF (15
mL). The reaction mixture was stirred at room temperature for 3-4
min under argon. Freshly distilled methyl acetoacetate (0.58 mL,
5.4 mmol) was added followed by a solution of phosphonate 10a
(0.93 g, 2.7 mmol) in anhydrous THF (5 mL). The resulting reaction
mixture was heated at 70 °C in a preheated oil bath for 2.5 h. The
reaction mixture was allowed to cool to room temperature and was
then partitioned between brine and Et2O. After separation, the
aqueous layer was re-extracted with Et2O and the combined organic
layers were dried over anhydrous Na2SO4. The solvent was
evaporated in Vacuo and the crude product was purified by
chromatography (SiO2, EtOAc: Hexane, 60: 40) to give of 9a as a
colorless oil (1.2:1 mixture of diastereomers, 0.89 g, 86%). IR (neat)
1742, 1717 cm-1; 1H NMR (CDCl3) δ 7.30 (5H, m), 6.73 (1H, m),
5.75 (1H, m), 4.43 (2H, m), 3.87 (1H, dd, JHH ) 3.5, 8.9 Hz), 3.67
(9H, m), 3.56 (1H, m), 3.48 (1H, m), 3.31 (1H, m), 2.22 (1.5H, s),
2.21 (1.5H, s); 13C NMR (CDCl3) δ 201.3, 201.1, 168.4, 168.3,
150.8 (d, JCP ) 3.9 Hz), 150.7 (d, JCP ) 4.8 Hz), 137.7, 137.6,
128.5, 128.4, 127.9, 127.81, 127.78, 119.1 (d, JCP ) 185 Hz), 118.9
(d, JCP ) 185 Hz), 73.7, 70.0, 69.8, 59.9, 59.8, 52.6, 52.5 (d, JCP
Experimental Section
Dimethyl [1-(methoxycarbonyloxy)-4-(benzyloxy)-2-bute-
nyl]phosphonate 10a. To a mixture of dimethyl phosphite (8.2
mL, 89 mmol) and aldehyde 12a13a (9.2 g, 52 mmol) was added
Et3N (3.1 mL, 22 mmol). The reaction mixture was stirred overnight
and then the volatiles were evaporated in vacuo to give crude
hydroxy phosphonate 13a (16.9 g). The crude hydroxy phosphonate
13a (16.9 g, 59.1 mmol) was dissolved in anhydrous CH2Cl2 (60
(14) Buechel, K. H.; Roechling, H.; Korte, F. Leibigs Ann. 1965, 685, 10.
(15) Ellman, G. J.; Courtney, K. D., Jr.; Featherstone, R. M. Biochem.
Pharmacol. 1961, 7, 88.
(16) Luo, C.; Tong, M.; Chilukuri, N.; Brecht, K.; Maxwell, D. M.; Saxena,
A. Biochemistry 2007, 46, 11771.
J. Org. Chem. Vol. 73, No. 21, 2008 8389

Bandyopadhyay et al.
) 6.9 Hz), 52.4 (d, JCP ) 5.6 Hz), 43.6 (d, JCP ) 22 Hz), 43.5 (d,
JCP ) 22 Hz), 30.7, 30.6; 31P NMR (CDCl3) δ 20.6, 20.5 ppm;
HRMS (EI, MH+) calcd. for C18H26O7P: 385.1416. Found: 385.1418.
(E)-Tert-butyl 2-Acetyl-3-(benzyloxymethyl)-5-(dimethoxy-
phosphoryl)pent-4-enoate 9b. Pd2(dba)3 (0.13 g, 0.14 mmol) and
dppe (0.17 g, 0.43 mmol) were dissolved in anhydrous THF (15
mL). The reaction mixture was stirred at room temperature for 3-4
min under argon. tert-Butyl acetoacetate (0.96 mL, 5.8 mmol) was
added followed by a solution of phosphonate 10a (0.50 g, 1.5 mmol)
in anhydrous THF (5 mL). The resulting reaction mixture was
heated at 70 °C in a preheated oil bath for 2 h. The reaction mixture
was allowed to cool to room temperature and was then partitioned
between brine and Et2O. After separation, the aqueous layer was
re-extracted with Et2O and the combined organic layers were dried
over anhydrous Na2SO4. The solvent was evaporated in Vacuo and
the crude product was purified by column chromatography (SiO2,
EtOAc: Hexane, 60: 40) to give 9b as a colorless oil (1.4:1 mixture
IR (neat) 1748, 1730 cm-1; 1H NMR (CDCl3) δ 7.30 (5H, m), 6.79
(1H, m), 5.76 (1H, m), 4.46 (2H, m), 3.79 (1H, m), 3.67 (6H, d,
JHP ) 10.9 Hz), 3.66 (3H, s), 3.58 (2H, m), 3.22 (1H, m), 1.42
(9H, s); 13C NMR (CDCl3) δ 168.9, 168.7, 167.0, 166.8, 150.8 (d,
JCP ) 5.3 Hz), 138.0, 128.60, 128.59, 127.96, 127.91, 119.0 (d,
JCP ) 187 Hz), 82.8, 73.5, 70.2, 70.0, 53.6, 53.5, 52.59 (d, JCP
)
5.7 Hz), 52.57 (d, JCP ) 9 Hz), 44.2 (d, JCP ) 22 Hz), 44.0 (d, JCP
) 22 Hz), 28.1; 31P NMR (CDCl3) δ 20.7; HRMS (FAB, NBA,
MNa+) calcd. for C21H31O8PNa: 465.1654. Found: 465.1653.
Dimethyl 2-(4-(1-Methoxyethylidene)-5-oxotetrahydrofuran-
3-yl)ethylphosphonate 14a. Vinyl phosphonate 9a (0.4 g, 1.0
mmol) was dissolved in CH2Cl2 (3 mL). The reaction flask was
flushed with argon for 10 min and then 5% Pd on C (0.10 g) was
added. The resulting reaction mixture was flushed with argon for
another 5 min and then with hydrogen. The reaction mixture was
stirred under hydrogen (balloon) for 6 h and then filtered through
Celite. The Celite was washed with CH2Cl2 and the solvent was
evaporated in Vacuo to give 14a (0.28 g, 100%) as an oil. IR (neat)
1
of diastereomers, 0.38 g, 62%). IR (neat) 1736, 1712 cm-1; H
1
1693, 1638 cm-1; H NMR (CDCl3) δ 4.42 (1H, dd, JHH ) 9.6,
NMR (CDCl3) δ 7.29 (5H, m), 6.74 (1H, m) 5.74 (1H, m), 4.45
(2H, m), 3.78 (2H, m), 3.69 (6H, d, JHP ) 12 Hz), 3.48 (1H, m),
3.26 (1H, m), 2.20 (3H, s), 1.43 (9H, s); 13C NMR (CDCl3) δ
201.81, 201.76, 167.2, 167.0, 151.5 (d, JCP ) 4.9 Hz), 151.3 (d,
JCP ) 5.0 Hz), 138.0, 137.8, 128.6, 128.0, 127.94, 127.87, 118.8
(d, JCP ) 185 Hz), 118.7 (d, JCP ) 186 Hz), 82.8, 73.5, 70.2, 61.2,
9.6 Hz), 4.14 (1H, dd, JHH ) 4.5, 9.5 Hz), 3.75 (3H, d, JHP ) 10.8
Hz), 3.74 (3H, d, JHP ) 10.8 Hz), 3.72 (3H, s), 3.26 (1H, m), 2.20
(3H, d, JHH ) 1.1 Hz), 1.68 (4H, m); 13C NMR (CDCl3) δ 170.1,
166.4, 105.2, 75.2, 52.6 (d, JCP ) 6.4 Hz), 51.0, 42.4 (d, JCP ) 18
Hz), 26.1 (d, JCP ) 4.5 Hz), 21.5 (d, JCP ) 141 Hz), 14.5; 31P
NMR (CDCl3) δ 35.2; HRMS (EI, M+) calcd. for C11H19O6P:
278.0919. Found: 278.0923; Anal. Calcd for C11H19O6P·3H2O: C,
46.2; H, 7.00 Found: C, 46.13; H, 7.00.
61.0, 52.5 (d, JCP ) 5.7 Hz), 43.9 (d, JCP ) 22 Hz), 43.6 (d, JCP
)
22 Hz) 30.3, 30.1, 28.03, 28.01; 31P NMR (CDCl3) δ 20.80, 20.76;
HRMS (FAB, NBA, MH+) calcd. for C21H31O7P: 426.1876. Found.
371:1323 (M-tert-Bu); Anal. Calcd for C21H31O7P·H2O: C, 58.32;
H, 3.38. Found: C, 58.32; H, 3.37.
Dimethyl 2-(4-(1-Tert-butoxyethylidene)-5-oxotetrahydrofu-
ran-3-yl)ethylphosphonate 14b. Vinyl phosphonate 9b (0.10 g,
0.23 mmol) was dissolved in MeOH (3 mL). The reaction flask
was flushed with argon for 10 min. Then 5% Pd on C (0.05 g) was
added and the reaction mixture was flushed with argon for another
5 min and then with hydrogen. The reaction mixture was stirred
under hydrogen (balloon) for 6 h and then filtered through Celite.
The Celite was washed with CH2Cl2 and the solvent was evaporated
in Vacuo to give 14b (0.07 g, quantitative) as an oil. IR (neat) 1689,
1638 cm-1; 1H NMR (CDCl3) δ 4.37 (1H, dd, JHH ) 9.7, 9.7 Hz),
4.07 (1H, dd, JHH ) 4.6, 9.5 Hz), 3.72 (3H, d, JHP ) 10.8 Hz),
3.72 (3H, d, JHP ) 10.8 Hz), 3.20 (1H, m), 2.15 (3H, d, JHH ) 1.0
Hz) 1.75 (4H, m), 1.47 (9H, s); 13C NMR (CDCl3) δ 168.8, 165.5,
106.5, 79.9, 74.9, 52.6 (d, JCP ) 6.5 Hz), 42.7 (d, JCP ) 18 Hz),
28.6, 26.0 (d, JCP ) 4.4 Hz), 21.4 (d, JCP ) 141 Hz), 14.5; 31P
NMR (CDCl3) δ 35.4; HRMS (EI, M+) calcd. for C14H25O6P:
320.1389. Found: 320.1389.
(E)-Methyl 2-Acetyl-3-((tert-butyldimethylsilyloxy)methyl)-
5-(dimethoxyphosphoryl)pent-4-enoate 9c. Pd2(dba)3 (0.09 g, 0.09
mmol) and dppe (0.11 g, 0.28 mmol) were dissolved in anhydrous
THF (20 mL). The reaction mixture was stirred at room temperature
for 3-4 min under argon. Freshly distilled methyl acetoacetate (0.82
mL, 7.6 mmol) was added followed by a solution of phosphonate
10b (1.4 g, 3.8 mmol) in anhydrous THF (5 mL). The resulting
reaction mixture was heated at 70 °C in a preheated oil bath for
2 h. The reaction mixture was allowed to cool to room temperature
and was then partitioned between brine and Et2O. After separation,
the aqueous layer was re-extracted with Et2O and the combined
organic layers were dried over anhydrous Na2SO4. The solvent was
evaporated in Vacuo and the crude product was purified by column
chromatography (SiO2, EtOAc: Hexane, 60: 40) to give 9c as a
colorless oil (1.2:1 mixture of diastereomers, 0.99 g, 64%). IR (neat)
1740, 1715 cm-1; 1H NMR (CDCl3) δ 6.71 (1H, m), 5.65 (1H, m),
3.88 (1H, dd, JHH ) 4.2, 9.0 Hz), 3.70 (10H, m), 3.60 (1H, m),
3.17 (1H, m), 2.27 (1.5H, s), 2.22 (1.5H, s), 0.87 (9H, s), 0.02
(6H, s); 13C NMR (CDCl3) δ 201.7, 201.5, 168.7, 168.6, 151.0 (d,
JCP ) 4.8 Hz), 119.3 (d, JCP ) 185 Hz), 119.0 (d, JCP ) 185 Hz),
63.4, 63.3, 59.6, 59.5, 52.7 (d, JCP ) 5.4 Hz), 52.6 (d, JCP ) 5.1
Hz), 52.5, 45.9 (d, JCP) 21 Hz), 45.7 (d, JCP) 21 Hz), 30.6, 30.4,
26.0, 18.5, -5.4; 31P NMR (CDCl3) δ 20.6, 20.5; HRMS (FAB,
NBA, MH+) calcd. for C17H34O7PSi: 409.1812. Found: 409.1817.
(E)-1-Tert-butyl 3-Methyl 2-(1-(benzyloxy)-4-(dimethoxy-
phosphoryl)but-3-en-2-yl)malonate 9d. Pd2(dba)3 (0.21 g, 0.23
mmol) and dppe (0.27 g, 0.68 mmol) were dissolved in anhydrous
THF (30 mL). The reaction mixture was stirred at room temperature
for 3-4 min under argon. A mixture of tert-Butyl methyl malonate
(3.4 mL, 20 mmol) and BSA (4.85 mL, 19.8 mmol) in THF (5
mL) was added followed by a solution of phosphonate 10a (3.08
g, 8.91 mmol) in anhydrous THF (5 mL). The resulting reaction
mixture was heated at 70 °C in a preheated oil bath for 6 h. The
reaction mixture was allowed to cool to room temperature and was
then partitioned between brine and Et2O. After separation, the
aqueous layer was re-extracted with Et2O and the combined organic
layers were dried over anhydrous Na2SO4. The solvent was
evaporated in Vacuo and the crude product was purified by column
chromatography (SiO2, EtOAc: Hexane, 70: 30) to give 9d as a
colorless oil (unresolved mixture of diastereomers, 3.21 g, 81%).
Methyl
2-Acetyl-3-(tert-butyldimethylsilyloxymethyl)-5-
(dimethoxyphosphoryl)pentanoate 15. The vinyl phosphonate 9c
(0.3 g, 0.7 mmol) was dissolved in MeOH (3 mL) and the reaction
flask was flashed with argon for 5 min. 10% Pd on C (0.08 g) was
added and the reaction flask was flushed with argon for another 5
min and then with hydrogen. The reaction mixture was stirred under
hydrogen (balloon) for 3 h, and then through Celite. The Celite
was washed with CH2Cl2 and the solvent was evaporated in Vacuo
to give the saturated phosphonate 15 (1:1 mixture of diastereoiso-
mers, 0.3 g, 100%) as a colorless oil. IR (neat) 1743, 1716 cm-1
;
1H NMR (CDCl3) δ 3.72 (9H, m), 3.49 (3H, m), 2.34 (1H, m),
2.27 (1.5H, s), 2.26 (1.5H, s), 1.74 (4H, m), 0.88 (9H, s), 0.02
(6H, s); 13C NMR (CDCl3) δ 202.9, 202.8, 169.50, 169.48, 62.0,
60.9, 60.8, 52.6, 52.5 (d, JCP ) 6.5 Hz), 40.93 (d, JCP ) 17 Hz),
40.90 (d, JCP ) 17 Hz), 30.5, 30.1, 26.0, 22.7 (d, JCP ) 140 Hz),
22.6 (d, JCP ) 140 Hz), 22.0 (d, JCP ) 6.5 Hz), 21.9 (d, JCP ) 6.6
Hz), 18.4, -5.5 (m); 31P NMR (CDCl3) δ 34.6; HRMS (FAB, NBA,
MH+) calcd. for C17H36O7PSi: 411.1968. Found: 411.1969.
Methyl 2-Acetyl-3-(benzyloxymethyl)-5-(dimethoxyphospho-
ryl)pentanoate 16. The vinyl phosphonate 9a (1.5 g, 3.9 mmol)
was dissolved in MeOH (3 mL) and the reaction flask was flushed
with argon for 5 min. 5% Pd on C (0.68 g) and pyridine (0.05 mL)
were added. The resulting reaction mixture was flushed with argon
for another 5 min and then hydrogen. The mixture was stirred under
hydrogen (balloon) for 6 h, and then filtered through Celite. The
8390 J. Org. Chem. Vol. 73, No. 21, 2008

Natural Acetyl Cholinesterase Inhibitor Cyclophostin
Celite was washed with CH2Cl2 (100 mL) and the solvent was
evaporated in Vacuo to give the saturated phosphonate 16 (1:1
3.83 (3H, d,, JHP )11 Hz), 3.81 (1H, app m), 3.40 (1H, m), 2.42
(3H, s), 2.05 (4H, m); 13C NMR (CDCl3) δ 170.2, 160.3 (d, JCP
9.8 Hz), 113.9, 69.9, 52.9 (d, JCP ) 6.8 Hz), 38.5, 26.1 (d, JCP
)
)
mixture of diastereomers, 1.52 g, 100%). IR (neat) 1740, 1712 cm-1
;
1H NMR (CDCl3) δ 7.33 (5H, m), 4.43 (1H, s), 4.42 (1H, s), 3.72
(10H, m), 3.45 (2H, m), 2.49 (1H, m), 2.25 (1.5H, s), 2.22 (1.5H,
s), 1.70 (4H, m);13C NMR (CDCl3) δ 202.5, 202.2, 169.11, 169.07,
137.8, 137.7, 128.1, 128.2, 127.6, 127.5, 73.0, 69.2, 69.1, 60.7,
52.2, 52.1 (d, JCP ) 6.6 Hz), 38.8 (d, JCP ) 17 Hz), 29.9, 29.7,
134 Hz), 26.8 (d, JCP ) 7.7 Hz), 18.6; 31P NMR (CDCl3) δ 21.9
ppm; HRMS (EI, M+) calcd. for C9H13O5P: 232.0501. Found:
232.0503.
Dimethyl 1-(2-Oxodihydrofuran-3(2H)-ylidene)ethyl Phos-
phate 19. A solution of 2-acetylbutyrolactone (0.37 g, 2.9 mmol)
and Hunig’s base in anhydrous CH2Cl2 (5 mL) was added to a
solution of dimethylphosphoric acid (0.30 g, 2.4 mmol) and DCC
(0.74 g, 3.6 mmol) in anhydrous CH2Cl2 (10 mL). Then DMAP
(0.12 g, 0.98 mmol) was added and the reaction mixture was stirred
overnight. The reaction mixture was diluted with CH2Cl2 and
washed with water. The aqueous layer was extracted with CH2Cl2
(2×) and the combined organic layers were dried over MgSO4.
The solvent was evaporated in vacuo and the residue was purified
by column chromatography (SiO2, EtOAc:Hexane, 1:1) to yield
the enolphosphate 19 (0.36 g, 64%) as a colorless oil. IR (neat)
1751, 1689 cm-1; 1H NMR (CDCl3) δ 4.31 (2H, t, JHH ) 7.5 Hz),
3.87 (6H, d, JHP ) 11 Hz), 3.04 (2H, m), 2.53 (3H, d, JHP ) 4.4
Hz); 13C NMR (CDCl3) δ 170.7, 157.3 (d, JCP ) 7.1 Hz), 111.7
(d, JCP ) 9.1 Hz), 64.7, 55.2 (d, JCP ) 6.2 Hz), 26.1, 16.7; 31P
NMR (CDCl3) δ -4.7; HRMS (EI, M+) calcd. for C8H13O6P:
236.0450 Found: 236.0447.
Quantitation of Antiacetylcholinesterase Activity. The action
of the new compounds against recombinant human and electric eel
acetylcholinesterase (AChE) were determined by using Ellman’s
assay.15 The lyophilized AChE was solubilized in 20 mM Tris HCl
buffer, pH 7.5 and 1% BSA. The compounds were solubilized in
isopropanol and preincubated with the enzyme for 30 min at room
temperature and the residual activity of the enzyme was determined
with 0.5 mM acetylthiocholine iodide and 0.3 mM 5,5′-dithiobis-
2-nitrobenzoic acid in 100 mM sodium phosphate, pH 8.0 at 37
°C. The absorbance of thionitrobenzoate anion at 412 nm was
monitored every 2 s for 50 s. Reaction rates (average of triplicate)
in absorbance/sec were converted to umol/min by using thioni-
trobenzoate anion extinction coefficient 14,150 M-1cm-1. In the
assay, the concentration of isopropanol was always maintained
below 10%. The activity of the enzyme was not altered by the
presence of 10% of isopropanol. The enzyme activity at each
concentration (0 - 320 µM) of test compound was expressed as a
percent of the activity in the absence of compound to get % residual
activity. Residual activity (%) was plotted against the compound
concentration (uM) and the inhibitory action was calculated as an
IC50, defined as the concentration of compound (µM) required to
inhibit 50% of enzymatic activity.
22.20 (d, JCP ) 140 Hz), 22.24 (d, JCP ) 4.4 Hz), 22.1 (d, JCP
)
140 Hz); 31P NMR (CDCl3) δ 34.6, 34.5; HRMS (FAB, NBA, MH+)
calcd. for C15H28O7P: 387.1573. Found: 387.1555.
Synthesis of Monocyclic Phosphonate Analog 8. To a solution
of phosphonate 16 (1.13 g, 2.91 mmol) in CH3CN (1.5 mL) was
added NaI (0.44 g, 2.9 mmol). The resulting mixture was heated
at reflux overnight. The solvent was removed in Vacuo to obtain
the monosodium salt as a white solid (1.43 g, crude). To a
suspension of the sodium salt in acetone (5 mL) was added
Amberlite IR 120 resin. The resulting mixture was shaken on an
orbital shaker until the sodium salt completely dissolved and the
color of the solution became amber. The resin was removed by
filtration and washed with acetone to give the phosphonic acid 17
as an amber colored solution in acetone. The acetone was
evaporated in Vacuo to obtain the crude monophosphonic acid as
a red viscous liquid (1.03 g). To a solution of the monophosphonic
acid (1.03 g, 2.75 mmol) in freshly distilled CH2Cl2 (14 mL) was
added EDC (0.67 g, 3.5 mmol), HOBt (0.54 g, 4.0 mmol) and
Hunig’s base (0.66 mL, 3.9 mmol). After stirring the resulting
solution for 24 h the solvent was evaporated in Vacuo. The resulting
crude product was dissolved in EtOAc (150 mL) and washed with
0.5 M HCl (2x) and saturated NaHCO3 (2x). The organic layer
was dried over MgSO4 and the solvent was evaporated in Vacuo.
The crude product was purified by column chromatography (SiO2,
EtOAc: Hexane, 70: 30) to give a 1:1.4 diastereomeric mixture of
8 as a colorless oil (0.73 g, 73% in 3 steps). Further chromato-
graphic separation (SiO2, EtOAc: Hexane, 50: 50) gave the pure
1
diastereomer 8a. IR (neat) 1717, 1643 cm-1; H NMR (CDCl3) δ
7.32 (5H, m), 4.50 (2H, m), 3.80 (3H, d, JHP ) 11 Hz), 3.73 (3H,
s), 3.65 (1H, d, JHH ) 9.2 Hz), 3.53 (1H, dd, JHH ) 6.5, 9.3 Hz),
3.38 (1H, m), 2.24 (3H, s), 2.07 (4H, m); 13C NMR (CDCl3) δ
168.6, 157.4 (d, JCP ) 7.5 Hz), 138.3, 128.6, 127.9, 127.8, 120.2
(JCP ) 5.0 Hz), 73.1, 68.9, 52.3 (JCP ) 7.0 Hz), 52.2, 37.7, 22.12
(JCP ) 134 Hz), 22.5 (JHP ) 7.1 Hz), 21.6; 31P NMR (CDCl3) δ
26.6. HRMS (FAB, NBA, MH+) calcd. for C17H23O6P: 354.1232.
Found: 355.1315.
Phosphonate Analog of Cyclophostin 6. The monocyclic
enolphosphonate 8 (1.06 g, 2.99 mmol) was dissolved in MeOH
(6 mL). The reaction flask was flushed with argon for 10 min. 10%
Pd on C (0.50 g) was added and the reaction mixture was flushed
with argon for another 5 min followed by hydrogen. The resulting
mixture was stirred under hydrogen (balloon) for 3 h. The reaction
mixture was filtered through Celite. The Celite was washed with
CH2Cl2 and the solvent was evaporated in Vacuo to give the bicyclic
phosphonate 6 (1.4:1mixture of diastereoisomers, 0.70 g, 100%)
as a colorless oil. Chromatographic separation (SiO2, EtOAc:
Hexane, 50: 50) gave cis-diastereomer 6b was white crystalline
solid which was further purified by crystallizing from EtOAc/hexane
and the trans-diastereomer 6a as a low melting solid.
Acknowledgment. The project described was supported by
grant number R01-GM076192 from the National Institute of
General Medicine. We thank National Science Foundation
(CHE-0313736) for funding the early studies on the reactions
of allylic hydroxy phosphonates employed in this synthesis, and
for grants to purchase the NMR spectrometer (CHE-9974801),
the mass spectrometer (CHE-9708640) and the X-ray diffrac-
tometer (CHE-0420497) used in this work. We thank Mr. Joe
Kramer and Prof. R. E. K. Winter for mass spectra.
cis-Phosphonate Analog of Cyclophostin 6b: (mp 119 °C) IR
1
(neat) 1747, 1673 cm-1; H NMR (CDCl3) δ 4.5 (1H, dd, JHH
)
Supporting Information Available: General experimental,
1H and 13C NMR spectra for compounds 10a, 10b, 9a, 9b, 9c,
9d, 14a, 14b, 15, 16, 8, 8a, 6b, and 6a. X-Ray data for
compounds 6b. This material is available free of charge via the
Internet at http://pubs.acs.org.
9.5, 9.0 Hz), 3.87 (3H, d, JHP ) 11 Hz), 3.83 (1H, m), 3.34 (1H,
m), 2.46 (3H, s), 2.34 (1H, m), 2.04 (3H, m); 13C NMR (CDCl3)
δ 170.2, 161.1 (d, JCP ) 6.6 Hz), 114.8 (d, JCP ) 3.8 Hz), 70.1,
52.8 (d, JCP ) 7.2 Hz), 39.1, 26.46 (d, JCP ) 136 Hz), 26.51 (d,
JCP ) 6.9 Hz),18.92, 18.90; 31P NMR (CDCl3) δ 25.4.
trans-Phosphonate Analog of Cyclophostin 6a: IR (neat) 1749,
1672 cm-1; 1H NMR (CDCl3) δ 4.5 (1H, dd,, JHH ) 9.0, 6.0 Hz),
JO801453V
J. Org. Chem. Vol. 73, No. 21, 2008 8391