C-3- and C-4-alkylated polyhydroxypyrrolidines: Enantiospecific syntheses and glycosidase inhibitory activity

Article abstract of DOI:10.1021/jo980078m

Short, efficient, and stereoselective syntheses of enantiomerically pure C-3- and C-4-alkylated analogues of (2R,3S,4R)-3,4-dihydroxy-2- (hydroxymethyl)pyrrolidine, a potent α-galactosidase inhibitor, from 4- hydroxy-L-proline are presented. Grignard addition or enolate alkylation of a N-(9-phenylfluoren-9-yl)-4-oxo-3-[(methoxymethyl)oxy]proline and epoxidation or hydroboration of a 4-methylene-3-[(methoxymethyl)oxy]proline proceeded with complete stereoselection and in excellent yields. The inhibitory activities of the synthesized pyrrolidines were measured and showed that the fit of A. niger α-galactosidase and the jack bean α-mannosidase around C-3 of the pyrrolidine ring (α face) must be very tight, while the fit around C- 4 (α face) is much looser. Positioning a methylene group between the hydroxyl at C-4 and the pyrrolidine ring completely abolishes the inhibitory activity (see analogue 5).

Full text of DOI:10.1021/jo980078m

J . Org. Chem. 1998, 63, 3411-3416
3411
C-3- a n d C-4-Alk yla ted P olyh yd r oxyp yr r olid in es: En a n tiosp ecific
Syn th eses a n d Glycosid a se In h ibitor y Activity
Mar´ıa-J esu´s Blanco and F. J avier Sardina*
Departamento de Quı´mica Orga´nica, Universidad de Santiago de Compostela,
15706 Santiago de Compostela, Spain
Received J anuary 15, 1998
Short, efficient, and stereoselective syntheses of enantiomerically pure C-3- and C-4-alkylated
analogues of (2R,3S,4R)-3,4-dihydroxy-2-(hydroxymethyl)pyrrolidine, a potent R-galactosidase
inhibitor, from 4-hydroxy-L-proline are presented. Grignard addition or enolate alkylation of a
N-(9-phenylfluoren-9-yl)-4-oxo-3-[(methoxymethyl)oxy]proline and epoxidation or hydroboration of
a 4-methylene-3-[(methoxymethyl)oxy]proline proceeded with complete stereoselection and in
excellent yields. The inhibitory activities of the synthesized pyrrolidines were measured and showed
that the fit of A. niger R-galactosidase and the jack bean R-mannosidase around C-3 of the pyrrolidine
ring (R face) must be very tight, while the fit around C-4 (R face) is much looser. Positioning a
methylene group between the hydroxyl at C-4 and the pyrrolidine ring completely abolishes the
inhibitory activity (see analogue 5).
In tr od u ction
halfchair conformation of the glycosyl cation.3c While
these approaches have provided several highly potent
inhibitors and have helped to delineate the enzymic
structural requirements around the glycosidic center,
progress in our knowledge of the detailed interactions of
the enzymic active sites with the inhibitors have not
ensued. This state of affairs has prompted us to under-
take a program of synthesis and evaluation of analogues
of polyhydroxylated pyrrolidines and indolizidines in
which the steric parameters of specific parts of the
inhibitors are systematically varied by incorporation of
an extra methylene group into the molecules.5 We have
chosen the potent R-galactosidase inhibitor 1 as our first
model compound,6 since it is one of the simplest glycosi-
dase inhibitors known, and have designed compounds
2-5 (Figure 1) as probes to find out how tight the
inhibitor-enzyme interaction is around carbons 3 and 4
of the pyrrolidine ring.
We have recently reported an efficient approach to the
synthesis of polyhydroxylated pyrrolidines and indoliz-
idines;6 this methodology, conveniently modified, should
allow the introduction of carbon appendages at C-3 and
C-4 of the pyrrolidine ring of 1. Our plans to synthesize
the enantiomerically pure compounds 2-5 rest on the
functionalization of the versatile ketol 7, prepared in four
steps from trans-4-hydroxy-L-proline (6, 72% overall
yield).6 Attack of carbon nucleophiles on the carbonyl
group at C-4 should yield precursors of 2, 3, and 5, while
alkylation of the enolate of an O-protected derivative of
Glycoproteins located in the outer envelope of cells are
key players in a variety of biological recognition events,
such as cell-cell communication and cell growth, as well
as in some stages of the bacterial and viral infection
cycles. These glycoproteins are synthesized by attaching
a common oligosaccharide unit to the nascent protein
chain; this oligosaccharide conjugate then undergoes a
series of hydrolysis and glycosilation reactions to provide
the final, mature form of the protein. Among all the
types of enzymes that process glycoproteins, glycosidases
play a very important role: that of trimming monosac-
charide units from the oligosaccharide already bound to
the protein chain.1 Inhibition of this pruning process has
led to interesting biological responses, namely, antiviral,
anti-HIV, anticancer, antifeedant, and immunoregulatory
activities.2
Polyhydroxylated pyrrolidine, piperidine, and indolizi-
dine alkaloids have attracted considerable attention
recently due to their ability to inhibit glycosidases. A
myriad of analogues of these compounds have been
synthesized in search of better inhibitors or more promis-
ing pharmacological agents.3 Since detailed information
about the structures of the target oligosaccharide-
processing enzymes is lacking, most analogues have been
designed to bear resemblance to the proposed reaction
intermediates: the glycosyl cations.3 The most recurrent
themes in the structural variations chosen to prepare
these analogues have been the following: (1) to permute
the configurations of the hydroxyl-bearing carbons3a,b,d
and (2) to flatten the heterocyclic ring to mimic the
(4) (a) Fleet, G. W. J .; Nicholas, S. J .; Smith, P. W.; Evans, S. V.;
Fellows, L. E.; Nash, R. J . Tetrahedron Lett. 1985, 26, 3127. (b) Austin,
G. N.; Baird, P. D.; Fleet, G. W. J .; Peach, J . M.; Smith, P. W.; Watkin,
D. J . Tetrahedron 1987, 43, 3095.
(1) Warren, L. Bound Carbohydrates in Nature; Cambridge Uni-
versity Press: Cambridge, 1994.
(5) Only a handful of analogues bearing extra methylene groups
have been prepared. Pyrrolidines: (a) Bols, M.; Persson, M. P.; Butt,
W. M.; J ørgensen, M.; Christensen, P.; Hansen, L. T. Tetrahedron Lett.
1996, 37, 2097. (b) Burley, I.; Hewson, A. T. Tetrahedron Lett. 1994,
35, 7099. Piperidines: (c) Khanna, I. K.; Weier, R. M.; J ulien, J .;
Mueller, R. A.; Lankin, D. C.; Swenton, L. Tetrahedron Lett. 1996, 37,
1355. (d) Hansen, A.; Tagmose, T. M.; Bols, M. Tetrahedron 1997, 53,
697. (e) Reference 4a. Indolizidines: (f) Pearson, W. H.; Hembre, E. J .
J . Org. Chem. 1996, 61, 5546.
(2) For reviews of glycosidase inhibitors see: (a) Elbein, A. D. Am.
Rev. Biochem. 1987, 56, 497. (b) Elbein, A. D. FASEB J . 1991, 5, 3055.
(c) Winchester, B.; Fleet, G. W. J . Glycobiology 1992, 2, 199.
(3) (a) Winkler, D. A. J . Med. Chem. 1996, 39, 4332. (b) Asano, N.;
Oseki, K.; Kizu, H.; Matsui, K. J . Med. Chem. 1994, 37, 3701. (c)
Papandreou, G.; Tong, M. K.; Ganem, B. J . Am. Chem. Soc. 1993, 115,
11682. (d) Winchester, B.; Al Daher, S.; Carpenter, N. C.; Cenci di Bello,
I.; Choi, S. S.; Fairbanks, A. J .; Fleet, G. W. J . Biochem. J . 1993, 290,
743, and references therein.
(6) (a) Blanco, M. J .; Sardina, F. J . Tetrahedron Lett. 1994, 35, 8493.
(b) Blanco, M. J .; Sardina, F. J . J . Org. Chem. 1996, 61, 4748.
S0022-3263(98)00078-4 CCC: $15.00 © 1998 American Chemical Society
Published on Web 04/22/1998

3412 J . Org. Chem., Vol. 63, No. 10, 1998
Sardina
evident when we attempted to alkylate ketone 8 with
MeLi (THF, -78 °C) to introduce the desired methyl
group at C-4: the alkylation product (9) was obtained in
a modest 50% yield, the material balance being starting
ketone 8. When this methylation reaction was quenched
with D2O, the recovered 8 showed a high deuterium
incorporation at C-3, showing that the MeLi was acting
partly as a base. Surprisingly, no products coming from
epimerization at C-3 could be detected in this reaction.
Aparently, the enolate of 8 had formed regioselectively
and had been protonated stereoselectively during the
quench. We carried out Molecular Mechanics (MM3) and
semiempirical (AM1 and PM3) calculations on 3,4-disub-
stituted N-Pf-∆3,4-dehydroproline model systems (analo-
gous to the enolate intermediate) to account for these
results. Our calculations showed that the CO2Me group
in these molecules is locked in a pseudoaxial position due
to the interaction with the Pf and the OMOM groups,
thus blocking the â face of the pyrrolidine ring. The
fluorenyl ring shields the hydrogens at C-5 from abstrac-
tion by the base.7
F igu r e 1.
Sch em e 1
This facile regioselective enolization-stereoselective
reprotonation boded well for the preparation of the
3-methyl analogue 4, but it cast serious doubts on the
viability of our approach to the C-4-alkylated analogues
(2 and 5). After some experimentation, we found two
solutions for this enolization problem. Thus, addition of
LiClO4 (a Lewis acid, to activate the carbonyl group, 500
mol %) to a THF solution of ketone 8, prior to addition of
MeLi (-78 °C), afforded 9 in 80% yield. A better
procedure simply involved alkylation of 8 with the less
basic MeMgBr (120 mol %, THF, -78 to -25 °C, 2 h),
which provided the desired tertiary alcohol 9 in 94%
yield. It is likely that the success of the addition of
MeMgBr to ketone 8 also depends on chelation of the
nucleophile with the carbonyl and MOM groups.8
The configuration at C-4 was established by NOE
experiments that showed that H-2, H-3, and the methyl
group at C-4 were all syn to each other. The reaction
was completely stereoselective; we could not detect the
C-4 epimer of 9 in the crude reaction mixture. MM and
semiempirical calculations on 3-substituted N-Pf-4-
oxoprolines showed that these molecules adopt two
almost isoenergetic, minimum energy conformations, in
which two substituents (one of them being always the
Pf group) are pseudoequatorial and the remaining (pseudo-
axial) group (OMOM or CO2Me) effectively blocks the â
face of the pyrrolidine ring, thus explaining the observed
stereochemistry at C-4 in 9.
7 should lead to a precursor of 4. A key question to be
addressed during the syntheses will be the provision of
regio- and stereocontrol.
With 9 in hand, we turned our attention to the
reduction of the ester group, which proved more difficult
than anticipated. LiEt3BH failed to achieve the desired
transformation,6 but LiBH4 (300 mol %, THF) at room
temperature did reduce ester 9 to give an unstable triol
that was immediately subjected to hydrogenolysis in an
acidic medium [H2, 52 psi, Pd/C (10%), 30% w/w, MeOH/
HCl] to remove all protecting groups. In this way, the
Resu lts a n d Discu ssion
We chose 4-methylpyrrolidine 2 as our initial target
(Scheme 1). Since all of the C-C bond-forming reactions
that we planned to use required strong basic media, we
proceeded to protect the hydroxyl group of 7 with MOMCl
and imidazole (500 mol % each) in DMF at room tem-
perature to secure the key intermediate, ketone 8, in 96%
yield. When bases stronger than imidazole (i-Pr2NEt or
N-methylmorpholine) were employed for this protection
reaction, small amounts of the C-3 epimer of the ketone
8 were detected in the crude product along with the
desired 8. The ease of abstraction of H-3 was even more
(7) Molecular Mechanics calculations were carried out with the
Accumodel 1.0 program (MicroSimulations). Semiempirical calculations
were carried out with the MacSpartan Plus 1.0 program (Wavefunc-
tion). Several dozens of starting conformations of each molecule were
optimized to find the lowest energy conformer (MM). The structures
within 3 kcal of the lowest energy conformers were further optimized
by semiempirical (AM1 and PM3) calculations.
(8) Chen, X.; Hortelano, E. R.; Eliel, E. L.; Frye, S. V. J . Am. Chem.
Soc. 1992, 114, 1778.

C-3- and C-4-Alkylated Polyhydroxypyrrolidines
J . Org. Chem., Vol. 63, No. 10, 1998 3413
Sch em e 2
F igu r e 2.
inhibitor analogue 2‚HCl was obtained in 92% combined
yield (60% overall yield, eight steps from 6).
Once the C-4-R-methyl analogue 2 had been secured,
our attention turned to the preparation of the C-3-methyl
analogue 4, which required the regiospecific alkylation
of ketone 8 at C-3. With regard to this transformation,
Lubell and co-workers had shown that 3-alkyl-4-oxopro-
lines can be regioselectively alkylated at C-3, although
the question of the stereoselectivity of the reaction was
not addressed.9 Our own results (vide supra) showed
that ketone 8 underwent proton abstraction at C-3 when
treated with bases, but despite this fact, attempts to
methylate the enolate of 8 generated with LHMDS,
NaHMDS, or LDA led only to decomposition products.
Fortunately, and somewhat surprisingly, treatment of 8
with n-BuLi (110 mol %, THF/HMPA 9/1, -78 °C, 1 h)
led to the exclusive formation of an enolate that could
be alkylated with MeI (300 mol %, -78 to 0 °C). Due to
the instability of the resulting 3-methyl ketone, the crude
product was subjected to carbonyl reduction with NaBH4
(250 mol %, MeOH/THF 1/1, -78 °C) to give 10a (62%
combined yield) as the sole reaction product. We did not
detect other diastereoisomers in the crude reaction
mixture. NOE experiments carried out on the acetate
10b (Ac2O, py, 91% yield) allowed us to assign the
configurations shown for the newly introduced stereo-
genic centers. Once again, we attribute the remarkable
degree of stereoselection observed in both the alkylation
and reduction steps to the conformations induced in the
reacting molecules by the Pf group. Figure 2 shows the
minimum energy conformation of the enol form of 8 (a
model for the corresponding enolate). As can be seen
from Figure 2, the carboxy methyl group is pseudoaxial
and blocks the upper face of the reacting C3, thus forcing
approach of the electrophile toward the R face (marked
by an arrow).
In view of the fact that the additions to compounds
with an sp2 center at C-4 had proceeded from the R face
with complete stereoselection, we decided to explore the
utility of alkene 11 as a common intermediate toward
analogues 3 and 5: an epoxidation-reduction sequence
should lead to a precursor of 3, while hydroboration-
oxidation-reduction should provide access to 5 (Scheme
2).
Attempts to obtain 11 by Wittig reaction on 8 were
plagued by low conversions, due to competing ketone
enolization; but when the methylenation was performed
with the mild reagent developed by Tour and co-workers10
(Cp2ZrCl2, 120 mol %, Zn,11 800 mol %, CH2Br2 120 mol
%, THF, rt, 3 h), the desired alkene was obtained in 88%
yield.
Epoxidation of 11 with m-CPBA (120 mol %, Na2CO3,
240 mol %, CHCl3, 0 °C), followed by treatment of the
resulting unstable epoxide with LiEt3BH (150 mol %,
THF, rt), led to tertiary alcohol 12 (67% combined yield)
as the sole reaction product. Hydroboration-oxidation
of 11 (BH3‚SMe2, H2O2/NaOH) provided the hydroxy-
methylene compound 13 (81% yield) regio- and stereo-
selectively. NOE experiments performed on 12 and 13
allowed us to assign the configurations at C-4 as shown.
It is remarkable that the electrophilic additions to alkene
11 show the same degree of stereoselection as the
nucleophilic additions to ketone 8, thus attesting to the
beneficial conformational effects introduced by the N-Pf
group on the proline ring.
Finally, reduction of the ester groups of 12 and 13 and
removal of the protecting groups were carried out as for
alcohol 9 to give the desired analogues 3‚HCl (90% yield;
Reduction of the methoxycarbonyl group of 10a (LiBH4,
300 mol %, THF, rt, 24 h) followed by hydrogenolysis in
an acidic medium [H2, 52 psi, Pd/C (10%), 30% w/w,
MeOH/HCl], as above, led to the C-3-methyl analogue
4‚HCl in 90% combined yield (38% overall yield, nine
steps from 6).
(10) Tour, J . M.; Bedworth, P. V.; Wu, R. Tetrahedron Lett. 1989,
30, 3927.
(11) Extreme care in the purification of the Zn used in this reaction
is essential for its success: Fieser, L. F.; Fieser, M. Reagents for
Organic Synthesis; Wiley: New York, 1967; Vol 1, p 1292.
(9) Sharma, R.; Lubell, W. D. J . Org. Chem. 1996, 61, 202.

3414 J . Org. Chem., Vol. 63, No. 10, 1998
Sardina
Ta ble 1. Con cen tr a tion Requ ir em en ts for 50%
the double bond of exomethyleneproline 11 also pro-
ceeded stereoselectively from the R face and paved the
way to analogues 3 and 5. The inhibitory activities of
2-5 were measured and showed that the fit of A. niger
R-galactosidase and the jack bean R-mannosidase around
C-3 of the pyrrolidine ring (R-face) must be very tight,
while the fit around C-4 (R-face) is much looser. Posi-
tioning a methylene group between the OH at C-4 and
the pyrrolidine ring completely abolishes the inhibitory
activity of analogue 5. The origin of this effect might be
conformational in origin since the pseudoaxial-pseu-
doequatorial dispositions of the OH groups at C-4 and
C-3 are inverted in 5 with respect to 1.
In h ibition (µM)a
compd
R-gal
R-man
â-gal
â-glu
1
2
3
4
5
0.19
0.80
NI
12
NI
17
26
NI
250
NI
140
210
NI
270
890
450
500
300
NI
600
a
NI ) less than 50% inhibitioin at 1 mM. R-gal ) R-galactosi-
dase (A. niger). R-man ) R-mannosidase (jack bean). â-gal )
â-galactosidase (A. niger). â-glu ) â-glucosidase (almonds).
36% overall yield, 10 steps from 6) and 5‚HCl (quant; 49%
overall yield, nine steps from 6).
An extension of these studies to other analogues of 1
as well as to analogues of the R-mannosidase inhibitor
swainsonine is currently underway and will be reported
in due course.
Glycosid a se In h ibition Stu d ies
The four pyrrolidine analogues 2-5 were screened for
inhibitory activity against a number of common glycosi-
dases that accept p-nitrophenyl glycosides as sub-
strates.12 The results, compared to those of the natural
inhibitor 1, are shown in Table 1.
Exp er im en ta l Section
Gen er a l Meth od s. For general methods, see ref 6b.
â-Glucosidase (from almonds), R-galactosidase (from A. niger),
â-galactosidase (from A. niger), R-mannosidase (from jack
bean), and all p-nitrophenyl glycoside substrates were pur-
chased from Sigma Chemical Co.
The most interesting results are those obtained with
the R-galactosidase and the R-mannosidase enzymes. As
can be easily seen from Table 1, methyl substitution at
C-4 (2) or C-3 (4) adversely affects the inhibitory potency
of the analogues, but substitution at C-4 is much more
well tolerated than at C-3: 2 retains 25% of the activity
of the parent compound against R-galactosidase and 65%
against R-mannosidase, while 4 is only 2% as active as
the parent compound against R-galactosidase and 7%
against R-mannosidase. Positioning the C-4 hydroxyl
group further away from the ring (as in 5) or substitution
at C-4 coupled with inversion of stereochemistry (as in
3) are catastrophic for the inhibitory activity. All the
compounds tested are only weakly active against â-glu-
cosidase and â-galactosidase.
To better relate the data of Table 1 to the structures
of compunds 2-5, we performed semiempirical calcula-
tions to determine if the most stable conformations of
2-5 closely matched that of the parent model 1. The
calculations were performed on the protonated forms of
the inhibitors (the significant ones at physiological pH)
and showed that the lowest energy conformation of 1 is
an envelope in which the CH2OH and the OH at C-3 are
pseudoequatorial while the OH at C-4 is pseudoaxial. The
most stable conformations of 2-4 do not differ signifi-
cantly from that of 1, while in 5 both CH2OH groups
occupy pseudoequatorial positions and the OH at C-3 is
pseudoaxial (a conformation of 5 that closely matches the
most stable conformation of 1 is 1.8 kcal higher in energy
than the global minimum).
(2S,3S)-3-(Meth oxym eth oxy)-4-oxo-N-(9′-ph en ylflu or en -
9′-yl)p yr r olid in e-2-ca r boxylic Acid Meth yl Ester (8).
Imidazole (573 mg, 8.33 mmol, 500 mol %) and ClMOM (0.63
mL, 8.33 mmol, 500 mol %) were added to a solution of the
keto alcohol 7 (665 mg, 1.67 mmol) in DMF (7 mL). The
resulting solution was stirred at room temperature for 18 h
to give a red-brown mixture that was diluted with ether (5
mL), cooled at 0 °C, and treated with aqueous saturated
NaHCO3 (2 mL). The reaction mixture was partitioned
between aqueous saturated NaHCO3 (10 mL) and ether (15
mL); the organic phase was washed with H2O (10 mL), H3PO4
(10%, 10 mL), and H2O (10 mL). The aqueous phase was
reextracted with ether (10 mL), and the combined organic
layers were dried and concentrated to give a residue that was
purified by column chromatography (EtOAc/hexanes 1:3) to
give 709 mg (96%) of 8 as a white foam: [R]23 ) -158.2° (c
D
1.1, CHCl3); IR (NaCl) 2933, 1773, 1750, 1454 cm-1; 1H NMR
(CD2Cl2) δ 3.06 (s, 3H), 3.24 (s, 3H), 3.57 (d, J ) 17.2 Hz, 1H),
3.86 (d, J ) 17.3 Hz, 1H), 3.93 (d, J ) 7.7 Hz, 1H), 4.47 (d, J
) 7.8 Hz, 1H), 4.52 (d, J ) 6.7 Hz, 1H), 7.24-7.48 (m, 13H);
13C NMR (CD2Cl2) δ 51.5, 52.6, 56.2, 61.7, 75.6, 78.3, 97.1,
120.7, 120.8, 125.9, 127.3, 127.4, 128.1, 128.4, 128.6, 128.4,
128.6, 129.2, 129.5, 129.6, 140.5, 141.9, 145.6, 147.3, 171.1,
209.3. Anal. Calcd for C27H25O5N: C, 73.1; H, 5.7; N, 3.2.
Found: C, 73.0; H, 5.7; N, 3.1.
(2S,3S,4R)-4-Hyd r oxy-4-m eth yl-3-(m eth oxym eth oxy)-
N-(9′-p h en ylflu or en -9′-yl)p yr r olid in e-2-ca r boxylic Acid
Meth yl Ester (9). Methylmagnesium bromide (0.122 mL,
0.366 mmol, 120 mol %, 3.0 M in ether) was added dropwise
to a solution of 8 (135 mg, 0.305 mmol) in THF (3 mL) at -78
°C. The resulting mixture was stirred for 2.5 h from -78 to
-25 °C. The reaction was quenched at -25 °C with H3PO4
(10%, 0.25 mL) and then partitioned between H2O (45 mL)
and CH2Cl2 (3 × 50 mL). The organic phase was washed with
brine, dried, and concentrated. The residue was purified by
column chromatography (EtOAc/hexane 1:1.5) to afford 132
Con clu sion
We have developed short, efficient, and stereoselective
syntheses of alkylated analogues of the potent R-galac-
tosidase inhibitor 1, starting from 4-hydroxyproline. The
3-alkoxy-4-oxoproline 8 was the common, key intermedi-
ate in the preparation of pyrrolidines 2-5. Addition of
nucleophiles to the ketone group of N-Pf-4-oxoprolines
and alkylation of the enolate of 8 proceeded with complete
stereoselection from the R face of the molecule to provide
access to analogues 2 and 4. Electrophilic additions to
mg (94%) of 9 as a white foam: [R]28 ) 242.6° (c 1.7, CHCl3);
D
1
IR (NaCl) 3510, 1745, 1448 cm-1; H NMR (CDCl3) δ 1.16 (s,
3H), 2.86 (d, J ) 9.4 Hz, 1H), 3.18 (d, J ) 9.5 Hz, 1H), 3.19 (d,
J ) 8.5 Hz, 1H), 3.21 (s, 3H), 3.31 (s, 3H), 3.65 (d, J ) 9.5 Hz,
1H), 4.37 (d, J ) 6.8 Hz, 1H), 4.40 (d, J ) 6.8 Hz, 1H), 4.63
(bs, 1H), 7.12-7.81 (m, 13H); 13C NMR (CDCl3) δ 20.9, 52.1,
56.0, 59.0, 62.3, 75.9, 76.1, 80.3, 96.4, 120.4, 120.7, 127.3, 127.4,
127.7, 128.0, 128.2, 128.9, 129.0, 129.4, 139.8, 141.7, 142.4,
145.0, 148.2, 175.9. Anal. Calcd for C28H29O5N: C, 73.2; H,
6.4; N, 3.1. Found: C, 73.0; H, 6.3; N, 3.0.
(2R,3S,4R)-3,4-Dih yd r oxy-2-(h yd r oxym eth yl)-4-m eth -
ylp yr r olid in e Hyd r och lor id e (2‚HCl). LiBH4 (13 mg, 0.561
(12) Tropea, J . E.; Molyneaux, R. J .; Kaushal, G. P.; Pan, Y. T.;
Mitchell, M.; Elbein, A. D. Biochemistry 1989, 28, 2027.

C-3- and C-4-Alkylated Polyhydroxypyrrolidines
J . Org. Chem., Vol. 63, No. 10, 1998 3415
mmol, 300 mol %) was added to a solution of 9 (86 mg, 0.187
mmol) in THF (1.5 mL) at room temperature. After 24 h, the
reaction mixture was diluted with ether (2 mL), and H3PO4
(10%, 0.75 mL) was added dropwise. The reaction mixture
was partitioned between H2O (25 mL) and ether (30 mL). The
organic phase was washed with brine, dried, and evaporated.
The crude product was dissolved in deoxygenated MeOH (2
mL), and Pd/C (24 mg, 30 wt %, 10%) and HCl (c) (57 µL, 0.564
mmol, 300 mol %) were added. The flask was purged with
argon and then evacuated (water aspirator) and pressurized
(H2) three times. The reaction mixture was mechanically
shaken under 52 psi of H2 for 20 h. The catalyst was removed
by filtration through a pad of Celite, and the filtrate was
evaporated. The residue was washed with toluene to give a
clear yellow oil that was recrystallized from methanol/EtOAc
to afford 31 mg of 2‚HCl (92%, two steps) as a white solid:
mp 140-143 °C; [R]20D ) 23.1° (c 0.75, MeOH); IR (KBr) 3390,
127.0, 127.8, 128.5, 128.8, 129.1, 139.6, 142.5, 143.4, 146.2,
148.0, 169.9, 171.4. Anal. Calcd for C30H31O6N: C, 71.8; H,
6.2; N, 2.8. Found: C, 72.0; H, 6.3; N, 2.9.
(2R,3S,4R)-3,4-Dih yd r oxy-2-(h yd r oxym eth yl)-3-m eth -
ylp yr r olid in e Hyd r och lor id e (4‚HCl). By following the
same procedure described above for 2‚HCl, 15 mg (90%, two
steps) of 4‚HCl were obtained as a white solid: mp 156-158
°C; [R]20 ) 14.1° (c 0.8, MeOH); IR (KBr) 3390, 3100, 2930,
D
2800, 2450, 1550 cm-1; 1H NMR (D2O) δ 1.56 (s, 3H), 3.00 (dd,
J ) 12.2, 5 Hz, 1H), 3.39 (m, 2H), 3.60 (m, 1H), 3.79 (dd, J )
4.1, 12.0 Hz, 1H), 3.94 (t, J ) 5 Hz, 1H); 13C NMR (D2O ref
dioxane) δ 21.3, 51.1, 59.8, 67.5, 77.6, 79.6. Anal. Calcd for
C6H14O3NCl: C, 42.5; H, 8.3; N, 8.3. Found: C, 42.3; H, 8.5;
N, 8.2.
(2S ,3R )-4-M e t h y le n e -3-(m e t h o x y m e t h o x y )-N -(9′-
p h en ylflu or en -9′-yl)p yr r olid in e-2-ca r boxylic Acid Meth -
yl Ester (11). CH2Br2 (0.10 mL, 1.53 mmol, 220 mol %) was
added to a suspension of 8 (307 mg, 0.69 mmol), Zn (362 mg,
5.54 mmol, 800 mol %), and Cp2ZrCl2 (243 mg, 0.83 mmol, 120
mol %) in THF (1.7 mL). The resulting gray suspension was
stirred at room temperature for 3 h, turning into a yellow
suspension. The reaction was quenched by adding H2O
dropwise until gas evolution ceased. The reaction mixture was
partitioned between ether (30 mL) and H2O (20 mL). The
aqueous phase was reextracted with ether (20 mL), and the
combined organic layers were washed with brine, dried, and
evaporated. The residue was purified by column chromatog-
raphy (EtOAc/hexane 1/4) to give 269 mg (88%) of 11 as a white
1
3100, 2935, 2800, 2450, 1550 cm-1; H NMR (CD3OD) δ 1.21
(s, 3H), 3.01 (d, J ) 12.2 Hz, 1H), 3.18 (d, J ) 12.2 Hz, 1H),
3.73 (m, 3H), 3.98 (d, J ) 4.1 Hz, 1H); 13C NMR (CD3OD) δ
22.9, 54.1, 59.8, 64.1, 75.6, 77.2. Anal. Calcd for C6H14O3-
NCl: C, 42.5; H, 8.3; N, 8.3. Found: C, 42.2; H, 8.5; N, 8.2.
(2S,3S,4R)-4-Hyd r oxy-3-m eth yl-3-(m eth oxym eth oxy)-
N-(9′-p h en ylflu or en -9′-yl)p yr r olid in e-2-ca r boxylic Acid
Meth yl Ester (10a ). A solution of 8 (66 mg, 0.149 mmol) in
THF (2.1 mL) was added dropwise (15 min) to a solution of
n-BuLi (0.14 mL, 0.16 mmol, 105 mol %, 1.1 M in hexanes)
and HMPA (70 µL) in THF (0.7 mL) at -78 °C; the resulting
solution was stirred for 1.2 h at -78 °C, and then MeI (46 µL,
0.75 mmol, 500 mol %) was added and the stirring continued
for 4.5 h from -78 to 0 °C. The reaction was quenched by
addition of H3PO4 (10%, 0.25 mL). The resulting suspension
was partitioned between H2O (30 mL) and ether (45 mL). The
organic phase was washed with saturated Na2S2O3 (30 mL)
and then with H2O (30 mL). The organic phase was dried,
filtered, and concentrated. Due to the instability of the
resulting tertiary alcohol, the crude mixture was dissolved in
THF/MeOH (1/1, 1.2 mL), cooled at -78 °C, and treated with
NaBH4 (15 mg, 0.372 mmol, 250 mol %). The resulting
suspension was stirred for 4 h at -78 °C. The reaction was
quenched with H3PO4 (10%, 0.25 mL). The crude mixture was
partitioned between H2O (20 mL) and ether (2 × 30 mL). The
organic phase was washed with brine, dried, and concentrated.
The residue was purified by column chromatography (EtOAc/
hexane 1/4) to give 42 mg (62%, two steps) of 10a as a white
foam: [R]28 ) -24.8° (c 1.0, CHCl3); IR (NaCl) 1741, 1448,
D
1
743 cm-1; H NMR (CDCl3) δ 3.21 (s, 3H), 3.26 (s, 3H), 3.52
(d, J ) 13.7 Hz, 1H), 3.61 (d, J ) 8.1 Hz, 1H), 3.94 (d, J )
13.6 Hz, 1H), 4.44 (d, J ) 6.8 Hz, 1H), 4.96 (bs, 1H), 5.04 (bs,
1H), 7.15-7.62 (m, 13H); 13C NMR (CDCl3) δ 50.9, 51.0, 55.7,
63.6, 76.1, 76.7, 95.7, 106.6, 119.9, 120.0, 125.9, 126.6, 127.3
(2C), 127.5 (2C), 127.9, 128.4 (2C), 128.5, 128.7, 140.5, 142.6,
146.7, 147.3, 172.4. Anal. Calcd for C28H27O4N: C, 76.2; H,
6.2; N, 3.2. Found: C, 76.0; H, 6.2; N, 3.1.
(2S,3S,4S)-4-Hyd r oxy-4-m et h yl-3-(m et h oxym et h oxy)-
N-(9′-p h en ylflu or en -9′-yl)p yr r olid in e-2-ca r boxylic Acid
Meth yl Ester (12). A solution of m-CPBA (70%, 54 mg, 0.22
mmol, 120 mol %) in CHCl3 (0.75 mL) was added dropwise to
a suspension of 11 (80 mg, 0.18 mmol) and Na2CO3 (37 mg,
0.44 mmol, 240 mol %) in CHCl3 (1 mL) at 0 °C. The reaction
mixture was stirred at 0 °C for 15 h and then quenched with
an aqueous solution of Na2SO3 (10%, 0.5 mL). The resulting
mixture was partitioned between aqueous saturated NaHCO3
(20 mL) and CH2Cl2 (2 × 25 mL). The combined organic layers
were washed with brine, dried, and evaporated. Due to the
instability of the resulting epoxide, the reaction mixture was
used in the next step without further purification. To a
solution of the crude epoxide in THF (1.5 mL) at 0 °C was
added dropwise LiEt3BH (0.32 mL, 0.27 mmol, 150 mol %, 0.9
M in THF). After 5 min, the bath was removed, and the
resulting mixture was stirred at room temperature for 15 h.
The reaction was quenched with H3PO4 (10%, 0.2 mL). The
suspension was partitioned between H2O (20 mL) and CH2-
Cl2 (2 × 30 mL). The combined organic layers were washed
with brine, dried, and evaporated. The resulting oily residue
was purified by short column chromatography (EtOAc/hexane
1:3) to afford 57 mg (67%) of 12: [R]29D ) 166.5° (c 1.3, CHCl3);
IR (NaCl) 3520, 1744, 1448 cm-1; 1H NMR (CD2Cl2) δ 1.43 (s,
3H), 3.25 (s, 3H), 3.40 (s, 3H), 3.41 (d, J ) 8.4 Hz, 2H), 3.87
(d, J ) 9.5 Hz, 1H), 4.27 (d, J ) 9.5 Hz, 1H), 4.51 (d, J ) 6.6
Hz, 1H), 7.13-7.73 (m, 13H); 13C NMR (CD2Cl2) δ 25.9, 52.3,
55.9, 61.0, 66.1, 68.2, 76.6, 77.8, 96.1, 120.4, 120.5, 121.1, 125.6,
127.5, 127.6, 127.9, 128.3, 128.9, 129.1, 129.3, 129.7, 130.0,
140.6, 141.1, 147.1, 177.7. Anal. Calcd for C28H29O5N: C, 73.2;
H, 6.4; N, 3.1. Found: C, 73.0; H, 6.2; N, 3.0.
foam: [R]27 ) 161.7° (c 0.6, CHCl3); IR (NaCl) 3500, 1744,
D
1451 cm-1; 1H NMR (CD2Cl2) δ 0.83 (s, 3H), 2.74 (s, 1H), 3.21
(s, 3H), 3.30 (s, 3H), 3.40 (d, J ) 11.4 Hz, 1H), 3.72 (dd, J )
3.3, 11.7 Hz, 1H), 4.43 (d, J ) 11.9 Hz, 1H), 4.51 (d, J ) 6.9
Hz, 1H), 4.65 (d, J ) 6.9 Hz, 1H), 7.08-7.82 (m, 13 H); 13C
NMR (CD2Cl2) δ 23.4, 52.0, 55.3, 55.8, 70.1, 75.8, 75.9, 83.6,
93.0, 120.4, 120.8, 126.9, 127.4, 127.6, 128.0, 128.1, 128.2,
128.8, 129.0, 129.2, 129.4, 139.8, 141.7, 142.4, 145.2, 148.5,
176.2. Anal. Calcd for C28H29O5N: C, 73.2; H, 6.4; N, 3.1.
Found: C, 73.5; H, 6.4; N, 3.1.
(2S,3S,4R)-4-Acetoxy-3-m eth yl-3-(m eth oxym eth oxy)-N-
(9′-p h en ylflu or en -9′-yl)p yr r olid in e-2-ca r b oxylic Acid
Meth yl Ester (10b). Pyridine (0.2 mL) was added to a
solution of 10a (20 mg, 0.044 mmol) in Ac2O (0.7 mL). The
resulting solution was stirred at room temperature for 20 h,
and then aqueous saturated NaHCO3 (0.2 mL) was added. The
reaction mixture was partitioned between aqueous saturated
NaHCO3 (10 mL) and EtOAc (15 mL). The organic phase was
washed with saturated CuSO4 (10 mL) and H2O (10 mL). The
aqueous phase was reextracted with EtOAc (10 mL). The
combined organic layers were washed with brine, dried, and
evaporated. The residue was purified by column chromatog-
raphy (EtOAc/hexane 1/4) to afford 20 mg (91%) of 10b as a
white foam: [R]27 ) 193.1° (c 0.55, CHCl3); IR (NaCl) 2935,
(2S,3R,4S)-4-(Hyd r oxym eth yl)-3-(m eth oxym eth oxy)-N-
(9′-p h en ylflu or en -9′-yl)p yr r olid in e-2-ca r b oxylic Acid
Meth yl Ester (13). BH3‚SMe2 (0.18 mL, 0.18 mmol, 37 mol
%; a solution of BH3‚SMe2 1.0 M in THF was prepared
beforehand) was added to a solution of 11 (220 mg, 0.50 mmol)
in THF (1 mL) at 0 °C. After 5 min at 0 °C, the bath was
removed, and the reaction mixture was stirred at room
D
1
1746, 1451 cm-1; H NMR (C6D6) δ 1.10 (s, 3H), 1.72 (s, 3H),
3.14 (s, 1H), 3.17 (s, 3H), 3.26 (s, 3H), 3.59 (dd, J ) 6.2, 11.2
Hz, 1H), 3.71 (dd, J ) 6.8, 11.2 Hz, 1H), 4.56 (d, J ) 7.5 Hz,
1H), 4.79 (d, J ) 7.5 Hz, 1H), 4.87 (t, J ) 6.5 Hz, 1H), 6.91-
7.85 (m, 13H); 13C NMR (C6D6) δ 20.3, 21.0, 50.5, 51.9, 55.0,
70.7, 77.3 (2C), 83.0, 92.4, 119.8, 120.2, 126.3, 126.5, 126.7,

3416 J . Org. Chem., Vol. 63, No. 10, 1998
Sardina
temperature for 3 h. The reaction was quenched by adding
H2O dropwise until H2 ceased to be given off. The resulting
mixture was cooled to 0 °C, and NaOH (2 M, 0.13 mL, 0.25
mmol, 50 mol %) and H2O2 (30%, 55 µL, 0.54 mmol, 110 mol
%) were added dropwise (the internal temperature should be
kept below 40 °C). After 5 min of stirring at 0 °C, the bath
was removed and the resulting mixture was heated at 50 °C
for 1 h. The reaction mixture was poured into ice and washed
with ether (40 mL). The organic phase was washed with H2O
(2 × 30 mL); the aqueous phase was reextracted with ether (2
× 30 mL), and the combined organic layers were washed with
brine, dried, and evaporated to give a residue that was purified
by column chromatography (EtOAc/hexane 1:1.5), providing
NMR (CDCl3) δ 2.24 (m, 1H), 2.71 (m, 1H), 2.84 (dd, J ) 3.7,
10.8 Hz, 1H), 3.17 (m, 3H), 3.38 (s, 3H), 3.60 (m, 2H), 4.02 (t,
J ) 5.1 Hz, 1H), 4.64 (d, J ) 6.3 Hz, 1H), 4.68 (d, J ) 6.5 Hz,
1H), 7.14-7.73 (m, 13H); 13C NMR (CDCl3) δ 44.5, 52.6, 56.2,
60.3, 61.4, 64.5, 77.5, 83.0, 98.8, 119.8, 120.0, 125.8, 126.5,
127.1, 127.3, 127.8, 128.0, 128.4, 128.5, 128.8, 139.0, 142.0,
144.3, 146.9, 148.8. Anal. Calcd for C27H29O4N: C, 75.2; H,
6.8; N, 3.3. Found: C, 75.0; H, 6.7; N, 3.2. A solution of the
above compound (20 mg, 0.05 mmol) in deoxygenated MeOH
(0.5 mL) was hydrogenated in an acidic medium following the
same procedure as used for 2‚HCl. In this way, 8 mg (100%)
of 5‚HCl was obtained as a white solid: mp 161-163 °C; [R]20
D
) 22.3° (c 0.8, MeOH); IR (KBr) 3390, 2940, 1548, 1451 cm-1
;
186 mg (81%) of 13 as a white foam: [R]28 ) 252.2° (c 1.7,
1H NMR (CD3OD) δ 3.18 (t, J ) 11.1 Hz, 1H), 3.46 (dd, J )
11.1, 7.5 Hz, 1H), 3.62 (m, 1H), 3.71 (dd, J ) 10.9, 6.7 Hz,
1H), 3.83 (dd, J ) 10.9, 6.8 Hz, 1H), 3.94 (m, 3H), 4.40 (t, J )
2.9 Hz, 1H); 13C NMR (CD3OD) δ 46.8, 47.4, 59.3, 60.2, 68.2,
71.2. Anal. Calcd for C6H14O3NCl: C, 39.2; H, 7.7; N, 7.6.
Found: C, 38.9; H, 7.9; N, 7.6.
D
CHCl3); IR (NaCl) 3519, 1753, 1448 cm-1; 1H NMR (CD2Cl2) δ
2.24 (m, 1H), 3.14 (m, 3H), 3.21 (s, 3H), 3.26 (s, 3H), 3.76 (m,
2H), 4.12 (t, J ) 7.5 Hz, 1H), 4.33 (d, J ) 6.7 Hz, 1H), 4.41 (d,
J ) 6.7 Hz, 1H), 7.00-7.69 (m, 13H); 13C NMR (CD2Cl2) δ 44.3,
50.1, 51.2, 56.0, 60.2, 64.7, 76.8, 80.0, 97.0, 119.6, 120.1, 126.4,
127.0, 127.3, 127.4, 127.8, 127.9, 128.3, 128.4, 128.8, 139.1,
142.2, 142.5, 145.0, 147.5, 173.2. Anal. Calcd for C28H29O5N:
C, 73.2; H, 6.4; N, 3.1. Found: C, 72.8; H, 6.5; N, 3.0.
(2R,3S,4S)-3,4-Dih yd r oxy-2-(h yd r oxym et h yl)-4-m et h -
ylp yr r olid in e Hyd r och lor id e (3‚HCl). By following the
same procedure as 2‚HCl, 12 afforded 25 mg (90%, two steps)
En zym a tic Assa ys. Enzyme inhibition was assayed colo-
rimetrically by monitoring the release of p-nitrophenol from
the appropriate p-nitrophenyl glycoside substrate according
to the procedure described in ref 12. All reaction mixtures
contained 20 µmol of sodium acetate buffer (pH ) 5), 2 µmol
of p-nitrophenyl glycoside, and the appropriate enzyme in a
final volume of 0.4 mL. Incubations were performed at 37 °C
for 15 min, and the reactions were stopped by the addition of
2.5 mL of 0.4 M glycine (pH ) 10.4). The p-nitrophenol
liberated in the reaction was determined by measuring the
UV absorbance of the mixture at 410 nm.
of 3‚HCl as a white solid: mp 162-164 °C; [R]20 ) 34.1° (c
D
0.8, MeOH); IR (KBr) 3390, 3110, 2930, 2800, 2450, 1550 cm-1
;
1H NMR (CD3OD) δ 1.41 (s, 3H), 3.01 (d, J ) 12.1 Hz, 1H),
3.12 (d, J ) 12.0 Hz, 1H), 3.69 (m, 3H), 3.89 (d, J ) 4.1 Hz,
1H); 13C NMR (CD3OD) δ 22.8, 52.3, 59.8, 64.3, 75.5, 77.0.
Anal. Calcd for C6H14O3NCl: C, 42.5; H, 8.3; N, 8.3. Found:
C, 42.3; H, 8.5; N, 8.2.
Ack n ow led gm en t . Financial support from the
CICYT (Spain, Grant No. SAF96-0251) and the Xunta
de Galicia (Grant No. XUGA 20912B96) is gratefully
acknowledged as well as predoctoral fellowships from
the Xunta de Galicia (M.J .B.). We also thank Prof.
Rafael Suau (University of Ma´laga, Spain) for the
elemental analyses and Dr. J . P. Sestelo for a generous
gift of purified Zn.
(2R,3R,4S)-2,4-Bis(h yd r oxym eth yl)-3-h yd r oxyp yr r oli-
d in e Hyd r och lor id e (5‚HCl). By following the same proce-
dure as used for 2‚HCl, a solution of 13 (80 mg, 0.17 mmol) in
THF (2 mL) was treated with LiBH4 (12 mg, 0.52 mmol, 300
mol %). The crude product was purified by short column
chromatography (EtOAc/hexane 1/1) to afford 73 mg (97%) of
(2R,3R,4S)-2,4-bis(h yd r oxym eth yl)-3-(m eth oxym eth oxy)-
N-(9′-p h en ylflu or en -9′-yl)p yr r olid in e as a white foam:
1
[R]22 ) 268.0° (c 0.7, CHCl3); IR (NaCl) 3604, 1451 cm-1; H
J O980078M
D