Note: Descriptions are shown in the official language in which they were submitted.
WO 93/10127 PCI`/US92/09845
2123128
ETHOD_FOR MAKING A PROLINEBORO~AFL ESTER
Field of the Invention
The invention relates to a method for making optically
active prolineboronate esters. These are useful as
intermediates in the production of peptides which
incorporate prolineboronic acid instead of proline.
These peptides, in turn, are useful for inhibiting
various biologically important proteases.
Backqround of the Invention
Interest in boronic acid analogs of ~-amino acids,
as well as peptid~s incorporating a boronic acid
analog of an ~-amino acid instead of the C-terminal
residue, has been generated by reports that these
compounds are efficient inhibitors of many serine
proteases. See, for example, Matteson et al. ~J. Am.
Chem. Soc. 103, 5241 ~1981)], Kettner et al. ~J.
Biol. Chem. 259, 15106 (1984)]; and, Kinder et al. ~-
~J. Med. Chem. 27, 1919 (1985)~. `
Several workers, including Koehler et al.
[Biochemistry, 10, 2477 (1971)] and Rawn et al.
[Biochemistry, 13, 3124 (1974~] have hypothesized
that the empty p-orbital centered at boron in these
compounds interacts with an active-site hydroxyl group
of the enzyme, to form a tetrahedral adduct that
mimics the transition state of enzymatic hydrolysis.
It is thought that the boronic acid analog binds more
tightly to the enzyme than does the substrate itself,
thereby inhibiting enzymatic action upon the substrate.
WO93/10127 PCT/US92~09~5
.
2123128 2
The boronic acid analogs of ~-amino acids and
peptides incorporating them are currently of use in
research because they are able to shed light on the
biological functions of the enzymes they inhi~it.
Further, as explained below, they are also
therapeutically useful.
Peptides which incorporate the ~-aminoboronic acid
analog of proline (BoroPro) at the C-terminus are of'
special interest because they have been shown to be
potent inhibitors of certain post-proline cleaving
enzymes. For example, Bachovchin et al. [J. Biol.
Chem. 265, 3738 (1990)] have reported that such
peptides are inhibitors of IgA proteinases from
certain bacteria. These enzymes are strongly
implicated in bacterial virulence. Flentke et al.
[Proc. Natl. Acad. Sci. USA 88, 1556 (1991)] have
reported that such peptides inhibit dipeptidyl
peptidase IV (DP-IV), which in turn causes inhibition -
of antigen-induced proliferation and IL-2 production
in T-cells. The latter effects are known to result in
suppression of the immune response. Suppression of
the immune response is, in turn, useful in the
treatment of, for example, organ transplant rejection,
graft versus host disease, and various autoimmune
diseases.
Previous synthetic routes to ~-amino boronic acids
rely on the procedure published in 1981 by Matteson et
al., supra, which follows the sequence of
hydroboration, (asymmetric) homologation with
chloromethyllithium, and aminolysis. Matteson et al.
[Organometallics 3, 1284 (1984)] have described the
use of this technique to synthesize the boronic acid
WO93/10127 PCT/US92/09~5
` .:
212~128
analogues of N-acetylalanine, N-acetylvaline,
N~acetylleucine, and N-acetylphenylalanine; some of -
these have been obtained with good ~9
diastereomeric ratios.
The application of the Matteson procedure to the
synthesis of BoroPro has been demonstrated by
Bachovachin, supra, and Flentke, supra, but the
extensive modifications required for the construction
of the pyrrolidine ring render it unappealing.
Furthermore, conditions for the preparation of a
single enantiomer of BoroPro, either by asymmetric
synthesis or by resolution, have not been reported.
,.
Ef~orts to explore further the biochemistry of the
post-proline clea~ing enzymes, particularly DP-IV, and
the possible therapeutic uses of BoroPro-based enzyme
inhi~itors, have been hampered by the lack of an
efficient route to prolineboronic acid.
~ ,
The need for a better source of BoroPro led us to
explore alternate routes to this compound, especially
the compound in its optically active form, and has
resulted in the present invention.
'~''.
~ SummarV of the Invention
, .
A first broad aspect of the present invention
comprises three closely related methods for the
synthesis of prolineboronic acid esters. Two of these
syntheses commence from pyrrole. The third commences
from pyrrolidine. Included within the scope of this
first aspect of the invention are certain novel
intermediates. Prolineboronic acid has a chiral
WO93/10127 PC~/US92~09~5
21~3128
center ~ to the boron atom. A second broad aspect
of the invention comprises a method for resolving the
enantiomers of prolineboronic acid, if desired.
According to this method, the prolineboronic ester is
formed through reaction with a chiral alcohol, with
the use of pinanediol being particularly preferred.
Resolution of enantiomers is facilitated by separating
the diastereomPric mixture resulting from the
introduction of additional chiral centers. The '
resulting prolineboronic acid esters can be easily
coupled to activated carboxylic acid groups, such as
are typically used in peptide synthesis, to yield
peptides having a prolineboronic acid ester, instead
of an amino acid, at the C-terminus. The ester
protecting group can be removed to yield the free
boronic acid peptide. When the est2r protecting group
is pinanediol, it is not easily removed by known per
se techniques. A third aspect of the invention
comprises several methods for removing the pinanediol
protecting group.
Br ef Descriptlon_of the Drawinq
Figure l illustrates a reaction scheme which is a
preferred embodiment of the invention.
Detailed Description of the Invention
A first synthesis according to the invention commences
with pyrrole, which is reacted with an activated
derivative of carbonic acid, in order to protect the
nitrogen atom with a group of the formula -COOR,
wherein R is Cl 6alkyl, C3 6cycloalkyl, benzyl,
phenyl, phenyl substituted with one or more Cl 6
alkyl groups, or trimethylsilylethyl, in order to
yield a compound of the formula I
WQ~3~10127 PCT/US92/09~5
,
212312~
R0 ~ (I)
In the preferred protecting groups, R is tert-butyl,
benzyl, trimethylsilylethyl, phenyl, methyl or ethyl.
The most preferred protecting group is
tert-butyloxycarbonyl, or Boc. The protecting group
is applied using well known techniques. A specific
synthesis for 1-Boc-pyrrole has been described by
Grehn et al. [Angew. Chem. Int. Ed. Engl. 23, 296
(1~84)~.
The compound of formula I is next treated with a
lithiating agent to yield a compound of the formula II
N Li
RO~o (II)
'
wherein R is defined as before.
Lithiation of the compound of formula I can ~e
accomplished by treatment with lithium
tetramethylpiperidide in a known per se manner; such
as that described by Hasan et al. ~J. Org. Chem. 46,
157 (1981)], or with other hindered lithium amides
such as lithium diisopropyl amide or lithium
dicyclohexylamide, or with n-butyl lithium in the
WO93~10127 PCT/US92/09~5
,.. ~, :
21~3128 6
presence of tetramethyl ethylenediamine. This
reaction is conveniently carried out in an inert
solvent, preferably an ether such as THF, diethyl ~.
ether, dimethoxyethane, or methyl t-butyl ether at a
temperature between -78C and -40C. Alternatively,
pyrrole can be brominated at the 2-position, in a ;:
known per se manner, such as that described by Chen et
al., [Org. Syn., 70, l5l (l99l)], and the resulting
product can be protected and then li.thiated, using
other less expensive lithiating agents, such as
n-butyl lithium, using known per se techniques.
.
The intermediate of formula II, which is not isolated,
is next reacted with a trialkyl borate wherein each
al~yl group may be straight, branched or cyclic and
contains l to 6 carbon atoms, preferably trimethyl or
triethyl borate, followed by acid-catalyzed
hydrolysis, using a weak acid such as citric or acetic
acid, or potassium hydrogen sulfate, in order to yield
a protected pyrrole-2-boronic acid of formula III
~ \ (III)
L OH
~: RO O
wherein R is defined as before.
The intermediate of formula III is next reduced, using
catalytic hydrogenation, to form a protected
prolineboronic acid of the formula IV
WO93/10127 PCT/US92/0
212~128
B/ H
~ O~
wherein R is defined as before.
1~
The catalytic hydrogenation of the intermediate of
formula III may be carried out in an organic solvent,
such as ethyl acetate or tetrahydrofuran, using a
catalyst such as 5~ platinum on carbon, platinum
oxide, rhodium on carbon, rhodium on alumina,
palladium on carbon, or Raney nickel, either at
atmospheric pressure, or at about 50 psi.
An alternative synthesis of the boronic acid of
formula IV uses pyrrolidine which is treated with a
suitable acylating agent, to yield a protected
compound of the formula VIII
WO93/10~7 P~T/US~2/0984~
2123~8 8
¦ (VIII)
R o/~o
wherein R is defined as before.
~ ,
The protecting group is chosen to allow activation of
the pyrrolidine to lithiation adjacent to the
nitrogen, and it should contain a bulky moiety which
hinders attack on the carbonyl by the lithiating
agent. It is preferred to use a carbamoyl protecting
group of the formula -COOR, for example, groups
wherein R is tert-butoxy or 2,4,6-tri-tert-
butylphenoxy. However, certain acyl or aroyl groups
can also be used, for example tert-butylcarbonyl or
triphenylmethylcarbonyl. Other suitable activating :;
groups are outlined in Beak et al., [Chem. Rev., 84, -
471-523, (1984)]. The most preferred protecting group
is tert-butyloxycarbonyl, or Boc. The protecting
group may be applied to pyrrolidine by well known
techniques.
The compound of formula VIII is next treated with a
lithiating agent to yield a compound of the formula IX
WO93/1~127 PCT/US92/0~5
2123128 :~
~ (IX)
RQ"~0
wherein R is defined as before.
Lithiation of the compound of formula VIII can be
accomplished by treatment with sec-butyl lithium and
tetramethyl ethylenediamine in a known per se manner,
such as that described by Beak et al. [Tet. Lett.
30, 1197 ~1989)]. This reaction is conveniently
arried out in an inert organic solvent, preferably an
ether such as diethyl ether, methyl tert-butyl ether
or THF at a temperature between about -78C and 0C, ~-~
preferably -78~ to 40C. Lithiation may be achieved
with a reactive alkyl lithium such as sec-butyl
lithium or tert-butyl lithium, pr~ferably in the
presence of a coordinating additive such as
tetramethyl ethylenediamine, hexamethyl phosphoramide ~-
or N,N'-dimethylpropyleneurea ~DMPU).
.
The intermediate of formula IX, which is not isolated,
is next rea~ted with a trialkyl borate wherein each
alkyl group may be straight, branched or cyclic and
contains 1 to 6 carbon atoms, preferably trimethyllor
triethyl borate, followed by hydrolysis with water-and
extraction into aqueous alkali such as sodium
hydroxide or potassium hydroxide to aid purification.
Acidification of the alkali solution to about pH 3 and
extraction yields the protected prolineboronic acid of
formula IV.
W~ g3J10127 Pcr/uss2/~s~4s
-' 10
~1~31~.8
In order to form a boronic acid ester, the free
boronic acid intermediate of formula IV, is next
esterified by reaction with a diol of the formula V,
HO-X-OH (V)
wherein X is a linking group, to yield a compound of'
the formula VI
~N~ B~
\O ~ ~VI)
RO'-~'~
wherein X is the same linking group mentioned above
and R is defined as before. The ester group thus
formed is intended to function only as a removable
protecting group. The structures, syntheses, and
methods for attachment and removal of such ester
protecting groups are generally known in the chemical
art. Accordingly, those skilled in the chemical art
will appreciate that the structure of the linking
group X is not critical. The linking group X can be,
by way of non-limiting examples, a saturated 2- to
3-membered hydrocarbon chain; a saturated 2- to
3-membered hydrocarbon chain which constitutes part of
a C5 12 carbocyclic system which may optionally
contain unsaturations or ring fusions; a 2- to
3-membered hydrocarbon chain which constitutes part of
an aromatic ring system; or, a group of the formula
WO93~10127 PCT/US92/~9~5
"
11 .
2 t23~ 28 -
-(CH2)n-NH-(CH2)m~, wherein n and m are each 2
or 3; wherein such groups may be unsubstituted or
substituted by one or more C1 3alkyl or phenyl ~
groups. ~.
Accordingly, suitable diols of formula V are, for
example, ethylene glycol, pinacol, catechol,
pinanediol, butan-2,3-diol, 2,2-dimethyl propan
1,3-diol, diethanolamine and 1,2-diphenylethan-
1,2-diol.
With the boronic acid moiety protected by the ester .
group, the protecting group on the nitrogen is next
removed using known per se techniques, such as those
described by Greene in "Protective Groups in Organic
Synthesis" (J. Wiley & Sons, 1981), to yield the
hydrochloride of the desired prolineboronic acid ester
of the formula VII.
:: A
\ (VII)
H 0''
; ~:
,
For example, when the protecting group is Boc, it may
be easily removed with dry hydrogen chloride in ethyl
acetate.
It is preferred to perform the esterification of the ;~
compound of formula IV with a chiral, non-racemic diol
such as (lS,2S,3R,5S~ )-pinanediol,
1,2-diphenylethan-1,2-diol or butan-2,3,-diol, because :
so doing introduces additional chiral centers into the
molecule. This permits resolution of the chiral
WO93/10127 PCT/US92~09845 ~
2123128 12 ~
center ~ to the boron atom, using known per se
methods for separation of diastereomers, such as HPLC,
or fractional crystallization. This is illustrated in
the reaction scheme of Figure l, where the isomers of
the compound of formula VI in which the boronic acid
is protected with (lS,2S,3R,5S)~ pinanediol can be
separated by HPLC to give compounds VIa and VIb.
Alternatively, the isomers of the hydrochloride of the
compound of formula VII, with the same boron
protecting group, may be separated by fractional
crystallization in a solvent such as ethyl acetate, or
a dichloromethanetethyl acetate mixture, isopropanol,
or ethanol to give compound VIIb as a single isomer
with the ~ configuration at the carbon attached to
boron. ~-
.
A further advantage of using pinanediol is that the
boronate esters so formed are more stable than those
derived from other diols, for example, pinacol, with
which significant loss of the protecting group is
often observed during chromatography. This is useful
in both purification and isomer separation by -
chromatography on silica gel, since better recovery of
the desired material is achieved.
In a slight modification of the synthesis described
above, a derivatized pyrrole of formula III can be
directly esterified with a diol of the formula V. iThe
resulting ester of the formula IIIA
WO93/101~7 PCT/US92/0~
2123128
~ \ ~x (IIIA)
R0
can be reduced in the same manner as the compound of
formula III, yielding the protected prolineboronic
acid ester of formula VI. In other words, the order
of the steps in which the pyrrole ring is reduced and
the boronic acid group is esterified may be reversed.
The prolineboronic acid esters thus produced are
easily coupled to activated carboxylic acids such as .
those typically used in peptide synthesis, for example
a nitrogen-protected amino acid to yield a compound of
the formula X
~ O
H N ~ X ( X )
R 2~0~f~N~ O
0 R
wherein -COOR2 is an amino protecting group of the
sort commonly used in peptide synthesis, so that R2
is, for example, tert-butyl, benzyl, or
fluorenylmethyl, and R3 is the side chain of a
naturally occurring amino acid, optionally with
appropriate protecting groups of the sort commonly
used in peptide synthesis.
W0~3/10127 PCT/US92/09~
21231~ 14
Compounds of formula X contain protecting groups both
on the boronate and on the amino acid nitrogen. It
may be necessary to remove either or both protecting
groups for biological activity or for further chemical
manipulation. The protecting groups may be removed in
either order. Various methods for removing these
protecting groups are described below.
Removal of the nitrogen protecting group may be
achieved by known methods to yield a compound of the
formula XI. -
~\y~-~/o\
H2N ~ ~ X (XI)
'I :
R
.
The ester group which protects the boronic acid moiety
is stable to neutral and acidic organic media but many
boronate esters are cleaved rapidly under mildly basic
(pH - 7.5) aqueous conditions to yield the boronic
acid. In the case of boronate esters of pinanediol,
however, hydrolysis is known to be difficult, and
special conditions are required for removal of the
pinanediol. See for example Matteson et al. rJ. Am.
Chem. Soc., 102, 7590 (1980)] and Brown et al. [J.
Organometallic Chem., 385, lS (1988)]. These
methods are not suitable for the removal of pinanediol
from a compound of formula VI, X, or XI. We have
found several methods for removal of pinanediol from a
boronate such as compound VI, X or XI. Thus, removal
W093/10127 PCT/US92/09~5
2123128
of the pinanediol may be achieved under mild
conditions using an oxidizing agent capable of
cleaving l,2 diols to remove the pinanediol from the
equilibrium and hence drive it in the direction of the
free boronic acid. For example, treatment of
compounds of formula VI and X, in which the protecting
group is pinanediol, with sodium metaperiodate in
a~ueous ammonium acetate and acetone at ambient '
temperature yields compounds of formula IV and XII,
respectively.
\
V ~ N ~ O OH (XII)
O R3 '
This reaction is conveniently carried out in water,
optionally with an added buffer such as ammonium
acetate or disodium hydrogen phosphate, at a pH
between 3 and lO, preferably 6 to 8, and a temperature
of O to 80 C, preferably 20 to 40 C, in the presence
of a water miscible organic cosolvent such as acetone,
methanol, ethanol, THF, or acetonitrile. Suitably the
oxidizing agent is a non-nucleophilic oxidant capable
of cleaving l,2-diols,such as periodic acid or its
salts or permanganate salts. Under these conditions
oxidative cleavage of the carbon-boron bond is not
observed. It will be appreciated that this method is
applicable to any boronic acid protected with
pinanediol. Furthermore, it is applicable to any
boronate protecting group which is a l,2-diol,
,
WO93/10127 PCT/US92/~
- 16
2123128
although it is particularly useful for protecting
groups where simple aqueous hydrolysis is slow or
incomplete.
In the case of a pinanediol boronate ester of a
compound containing an unprotected amine, such as a
compound of formula XI, the method described above may
also be employed, but a second new method is
prererable for compounds of this type. This me'hod
consists of applying an aqueous solution of a compound
of formula XI, at pH 4 or lower, to a column of a
cation exchange resin, and eluting the column with
water or dilute acetic acid to remove the pinanediol.
This remo~es the pinanediol from the equilibrium, and
thus drives the reaction in the direction of
hydrolysis. The column is then eluted with dilute
aqueous ammonium hydroxide to remove the product,
which after evaporation and acidification is obtained
as a salt of the compound of formula XIII.
.~
. .
\ (XIII)
H2N ~ OH
Suitably, a strongly acidic cation exchange resin, for
example a sulfonic acid type of resin is used, such
as, for example, Dowex 50. The pinanediol eluted
from the resin may be recovered from the water
solution and reused. This is most conveniently
achieved by passing the water solution through a
W093/10127 PCT/US92/09~5
,.
17
2123128
column of a nonionic polymeric adsorbent, such as
Amberlite XAD-200, which adsorbs pinanediol :
almost quantitatively. The pinanediol is removed from
the column by elution with methanol or ethanol. The
two operations of ion exchange and pinanediol
adsorption may be combined in a single process in ;
which water is recycled from one column to the other
using a pump. This has the advantage of requiring
much smaller amounts of water, and allows the process
to be continued long enough to achieve a high
conversion to the product.
Persons skilled in the art will appreciate that the
above-described method for removing pinanediol using a
cation exchange resin is only appropriate for
compounds containing a basic functional group, such as
an unprotected amine.
A third method ls also applicable to pinanediol esters
of compounds containing an unprotected amine, such as
a compound of the formula XI. This method consists of
transesterification of the pinanediol boronate with
another boronic acid of the formula R4-B(OH)2, in
a two-phase system. R4 represents a Cl 12
hydrocarbon group, which may be composed of straight,
branched or cyclic alkyl chains and phenyl rings.
R4 is preferably phenyl. One of the phases is water
adjusted to a pH below 7, preferably pH 1-4, and the
other is a hydrocarbon organic solvent such as hexane,
petroleum ether, or toluene. Thus, treatment of a
compound of formula XI with phenylboronic acid in a
mixture of water at pH l and hexane, followed by
separation of the phases, produces the pinanediol
ester of phenylboronic acid in the organic phase,
WO~3/10~27 PCT/US92/09845
,f"~ '~
21~12~ 18
which may be recovered simply by evaporation, and a
solution of the free boronic acid of formula XIII in
the aqueous phase, which may be isolated using an ion
exchange resin in a similar manner to that described
above. In this system the only component which is
soluble in the organic ph~se is the pinanediol
phenylboronate, thus removing the pinanediol from the
equilibrium. The compound of formula XI, and the
compound of formula XIII which is formed, both remain'
in the aqueous layer, since neither are soluble in `
hydrocarbon solvents. The reaction may be carried out
with any boronic acid with a hydrocarbon sidechain,
providing its pinanediol ester is soluble in
hydrocarbon solvents.
The fully deprotected compound of formula XIII may
also ~e prepared by removal of the nitrogen protecting
group from a compound of the formula XII using known
methods. It will be appreciated that compounds of
formula X, XI, XII, and XIII generally posess two
chiral centers. One is adjacent to the boron atom,
and the other is present in the amino acid moiety,
except when that moiety is glycine. It will be
further appreciated that the pure single
diastereoisomers of these compounds are more desirable
for biological use than mixtures of diastereoisomers.
Accordingly it is important to be able to produce
these compounds as pure single isomers. In principal,
since amino acids are generally available as single
enatiomers, this may be achieved by separating the
mixture of diastereoisomers formed by coupling an
optically pure amino acid with racemic prolineboronic
acid, using known techniques. Nevertheless, it has
Wo93/1~127 PCT/USg~/09B45
19
2~ 23 1 2~ -
been found that, except in the special case of valine,
such separations are often difficult and time
consuming. Thus it is preferable to use a form of
prolineboronic acid which is a single isomer at the
chiral center adjacent to boron, since no isomer
separation is then necessary after coupling to an
optically pure amino acid. The present invention
provides an easy means for resolving the
enantiomers of prolineboronic acid.
The following examples further illustrate the
invention.
.
Example 1
~ Dimethylethoxycarbonyl)-pyrrole-2-boronic acid
To a solution of tetramethylpiperidine (8.8 mL,
52 mmol) in THF (275 mL~ at -7~ C under an argon
atmosphere was added a 2M solution of butyllithium in
hexanes (26 mL, 52 mmol~. After 15 min,
l-tl,1-dimethylethoxycarbonyl)-pyrxole (8.35 g,
SOmmol) in TH~ (10 mL) was added and the solution was
stirred for 4 h at -78 C. Triethylborate ~30 mL,
17Ç mmol) was then added ~nd the mixture was allowed
to warm to room temperature over 3 h. After an
additional 12 h the reaction mixture was diluted with
ether (500 mL) and washed with lM aqueous KHS04 (3 x
100 mL) followed by lM aqueous NaHC03 ~1 x 100 mL)..
Drying over MgS04 and rotary evaporation produced a
brown solid which was purified by flash chromatography
over silica gel (1:9 EtOAc:Hexane) to yield 8.7 g
(82~) of a white crystalline solid (mp 101.0 -
101,5 C).
... , .. . ~ ,
WO93/~01~7 PCT/U~92/09~5
~ 1 2 8 20
lH NMR ~CDC13) ~ 1.65 (s, 9 H), 6.26 (t, J = 3.3
Hz, 1 H), 7.10 (dd, J = 1.6, 3.2 Hz, lH), 7.15 (s, 2
H), 7.44 (dd, J = 1.6, 3.2 Hz, lH); 13C NMR
(CDC13) 27.9, 85.5, 112.0, 127.Q, 128.7, 152.0; ~-
CIMS m/z (% rel int) 212 (MH+, 11), 156 (100), 138
(68); Anal. Calcd for CgH14BN04: C, 51.23, H~
6.69, N, 6.64. Found: C, 51.22, H, 6.51, N, 6.67.
Example 2
~ DimethylethoxYcarbonyl)-~yrrolidine-2-
boronic acid
A solut'on of 6.15 g (24 mmol) of
dimethylethoxycarbonyl)-pyrrole-2-boronic acid,
produced as in Example 1, in EtOAc (100 m~) was
hydrogenated over 5~ Pt / C (ca. 500 mg) at 50 psi for
24 to 48h. The resulting suspension was filtered
through a pad of Celite and concentrated. This
material was chromatographed on silica gel using
sequential elutions of 9:1 hexanes:EtOAc then
acetone. The acetone fractions were concentrated to ~-
produce 6.05 g (97%) of the desired compound as a
clear glass that crystallized upon removal of trace
solvents (mp 100-101C~.
H NMR (CDC13): ~ 1.42 (s, 9 H), 1.6 - 2.15 (m,
S H), 3.1 - 3.6 (m, 2 H); C NMR (CDC13) ~ !
25.1, 25.7, 28.4, 45.6, 46.2, 78.6, 154.5; CIMS m/z (%
rel int) 116 (100), 70 (46); Anal. Calcd for
CgH18BNO4: C, 50.27, H, 8.44, N, 6.51.
Found: C, 50.52, H, 8.22, N, 6.58.
WO93/10127 PCT/U~92/09845
, .
21
2123128
ExamPle 3
(lS,2S,3Rt5S3-Pinanediol l-(l 1-dimethylethoxY-
carbonYl)-pYrrolidine-2S-boronate and
LlS.2s ! 3R,5S)-Pinanediol 1-(1 1-dimethvl-
ethoxycarbonyll~ yrrolidine-2R-boronate
I
A solution of l-(l,1-dimethylethoxycarbonyl)-
pyrrolidine-2-boronic acid, produced as in Example 2,
(1.52 g, 7.1 mmol) and (lS,2S,3R,5S)-(+)-pinanediol
~1.36 g, 8.0 mmol) was stirred at room temperature in
ether (2S mL3 for 2 h. Concentration and flash
ch~omatography over silica gel (85:15 hexanes:EtOAc)
produced 2.1 g (85%) of a 1:1 mixture of the two
diastereomers. These were separated by HPLC over a
300 X 3.9 mm column of microporasil A eluting with
methyl tert-butyl ether:hexanes (1:9) and using u.v.
detection at 220 nm. The isomer
(lS,2S,3R,5S)-pinanediol l-(1,1-dimethyl-
ethoxycarbonyl)-pyrrolidine-2S-boronate eluted first
under these con~itions.
S-isomer: lH NMR (C6D63: ~ 0.55 (s, 3 H), 1.09
(s, 3 H), 1.52 (s, 9 H), 1.60 (s, 3 H), 1.2 - 2.2 (m,
8 H), 3.1 - 3.5 (m, 3 H), 4.11 tm, 0.3 H), 4.33 tm,
0.7 H); C NMR (C6D6): 6 23.9, 26.6, 27.1,
27.3, 28.4, 28.6, 28.8, 36.0, 38.2, 39.9, 46.1, Sl.9, ~-
78.3, 78.5, 85.7, 154.9.
WO93/10127 PCT/~S92/~9~S
~123~28 22
R-isomer: lH NMR (C6D6): ~ 0.52 (s, 3 H), 1.08 -~
(s, 3 H), 1.52 (s, 9 H), 1.61 ~s, 3 H), 1.2 - 2.2 (m,
8 H), 3.1 - 3.6 (m, 3 H), 4.01 (m, 0.3 H), 4.25 (m,
0.7 H~; 13C NMR (C6D6) 23.9, 26.6, 27.1, 27.3,
28.4, 28.7, 28.9, 35.8, 38.2, 39.6, 46.2, 51.B, 78.1,
78.5, 85.7, 154.5.
It will be recognized that ~lR,2R,3S,5R)-pinanediol-
l-(1,1-dimethylethoxycarbonyl)-pyrrolidine-2S-borona~e
and (lR,2R,3S,5R)-pinanediol-
~-(1,1-dimethylethoxycarbonyl)-pyrrolidine-2R-boronate
could be produced in an analogous manner, starting
with (lR,2R,3S,5R)-(-)-pinanediol.
Example_4 -~
rlS.2S.3R~5S)-Pinanediol ~yrrolidine-2S-boronate ~-
hvdrochloride
A solution of (lS,~S,3R,5S)-pinanediol- -
1-(1,1-dimethylethoxycarbonyl)-pyrrolidine-2S-boronate,
produced as in Example 3 (28.5 mg, 0.08 mmol) was
stirred in a solution of dry HCl in EtOAc
(approximately 3M). After 2 h the solution was
concentrated twice from EtOAc to produce 21.2 mg (91~)
of the desired hydrochloride as a white solid (mp
204C (dec)).
1H NMR (CDCl3): ~ 0.83 (s, 3 H), 1.14 (d, J =
llHz, 1 H), 1.29 (s, 3 H), 1.45 (s, 3 H), 1.85 - 2.15
~m, 6 H), 2.17 - 2.50 (m, 3 H), 3.18-3.25 (m, 1 H),
3.45 (bs, 2 H~, 4.42 (dd, J = 1.8, 8.6Hz, 1 H), 8.80
WO93/10127 P~T/US~2/09~S
. . ~.,
23
2123l28
(bs, lH~, 10.56 (bs, lH); C NMR (CDC13): ~
23.9, 24.5, 26.5, 27.0, 27.2, 28.4, 34.9, 38.2, 39.4,
45.8, 51.2, 79.0, 87.6; CIMS m/z (~ rel int) 250
(MH+, 100); HRMS (EI) for C14H24BNO calcd
249.1900, found ~49.1899.
It will be recognized that (lR,2R,3S,5R)-pinanediol- ~
pyrrolidine-2S-boronate hydrochloride could be made in ~;-
an analogous manner.
Example 5
(lS 2S 3R,5S)-Pinanediol pyrrolidine-2R-boronate
hy~rochloride
A solution of ~lS,2S,3R,5S)-pinanediol-
l-(l,l-dimethylethoxycarbonyl)-pyrrolidine-2R-boronate,
produced as in Example 3 (18.3 mg, 0.05 mmol) was
treated with dry HCl in EtOAc as above. Work~up
produced 14.3 mg (96~) of the desired hydrochloride as
a white solid (mp 248 oC (dec)).
1H NMR (CDC13): ~ 0.83 (s, 3 H), 1.14 (d, J =
llHz, 1 H), 1.29 (s, 3 H~, 1.45 (s, 3 H), 1.85 - 2.15
(m, 6 H), 2.17 - 2.50 (m, 3 H), 3.18-3.25 (m, 1 H),
3.45 (bs, 2 H), 4.42 (dd, J = 1.8, 8.6Hz, 1 H), 8.80
(bs, 1 H), 10.56 (bs, 1 H); 13C NMR (CDCl ~ ~
23.9, 24.5, 26.5, 27.0, 27.2, 28.S, 34.9, 38.1, 39.4,
45.8, 51.2, 79.0, 87.8; CIMS m/z (% rel int) 250
(MH~, 100); HRMS (EI) for C14H24BN02 calcd
249.1900, found 249.1903.
WO 93~10127 PCI`/USg2/Og845 -
212312~
24
It will be recognized that (lR,2R,3S,5R)-pinanediol
pyrrolidine-2R-boronate hydrochloride could be made in
an analogous manner.
Example 6
(lS~2S~3R,5s)-Pinanediol
dimethylethoxvcarbonvl)-pyrrole-2-boronate
A solution of 1-(1,1-dimethylethoxycarbonyl)-
pyrrole-2-boronic acid, produced as in Example
(1.36 g, 6.45 mmol) and (lS,2S,3R,5S)-(+)-pinanediol
(1.10 g, 6.45 mmol) was stirred in 20 mL of ether for
4 h. Rotary evaporation followed by flash
chromatography over silica gel (95:5 hexane:EtOAc) `~
produced 1.83 g (82%) of the desired product as a
clear oil.
H NM~ (CDC13): ~ 0.90 (s, 3 H), 1-30 (s, 3 H~,
1.41 (dj J = 11 Hz, 1 H), 1.50 (s, 3 H), 1.50 (s, 3
H), 1.59 (s, 9 H), 1.96 (m, 2 H), 2.21 (t, J = 6Hz, 1
H3, 2.16 - 2.40 (m, 2 H), 4.45 (dd, J = 2, 8Hz, 1 H), ~;
6.20 (t, J = 3Hz, 1 H), 6.65 (d, J = 3Hz, 1 H), 7.40
(d, J = 3Hz, 1 H); 13C NMR (CDC13): ~ 24.0,
26.4, 27.2, 28.1, 28.6, 35.5, 38.3, 39.8, 51.8, 79.8,
83.6, 83.9, 111.6, 123.2, 124.7, 150.0; CIMS m/z (%
rel int) 346 (MH+, 4), 246 (81), 153 (100), 135 (56).
It will be recognized that (lR,2R,3S,5R)-pinanediol-
1-(1,1-dilnethylethoxycarbonyl)-pyrrole-2-boronate
could be made in an analogous manner.
WO93/10127 PCT/US92~0~5
. ~ .
2123:~28
Example 7 -
~ DimethYlethoxvcarbonyl~-PYrrolidine-2-boronic
acid from 1~ dimethylethoxvcarbonyl)-pYrrolidine
To a solution of 1-(1,1-dimethylethoxycarbonyl)-
pyrrolidine (17.1 g, 100 mmol) in diethyl ether
(200 mL) at -78 C under an atmosphere of nitrogen was
added 1.3M sec-butyllithium in cyclohexane (92.3 mL,
120 mmol) whilst maintaining the temperature below
-60 C. After addition was complete, the reaction
mixture was stirred at -7~ C for 4 h.
Trimethylborate (31.1 g, 300 mmol) was added and the
mixture was allowed to warm to room temperature over
3h; After an additional 12h, water (150 mL) was added
followed by 2M NaOH (200 mL~. The aqueous phase was
isolated and the organic phase was reextracted with 2M
NaOH (150 mL). The combined basic extracts were
acidified to pH 3 using 2M HCl and extracted using
EtOAc (5 x 200 mL). The combined organic extracts were
dried (Na2SO4) and concentrated to afford the
desired product as a white crystalline solid (15.49g,
72%), identical with the material produced in Example
Example 8
flS,2S,3R,5S)-Pinanediol
1-(1 1-dimethYlethoxycarbonvl)-pyrrolidine-2RS-boronate
To a stirred solution of the material obtained in
Example 7 (15.49 g, 72.0 mmol) in chloroform (250 mL)
was added (lS,2S,3R,5S)-(+)-pinanediol (12.77 g,
WO93/10127 PCT/US~2/09~5
21~31~ 26
75 mmol~. After stirring at room temperature under a
nitrogen atmosphere for 16 h, the solvent was removed
and the residue purified via flash chromatography over
silica gel (hexane/EtOAc 9:1, 4:1) to give the desired
product as a 1:1 mixture of diastereomers as an oil
(23.62 g, 67.7% based on l-(l,1-dimethyl-
ethoxycarbonyl)-pyrrolidine). This was identical with ~-
the mixture of isomers produced in Example 3.
H NMR (CDCl3): ~ 0.85 (s, 3H), 1.12-1.21 (m,
lH), 1.29 (s, 3H), 1.41 (s, 3H), 1.45 (s, 9H),
1.81-2.20 (m, 8H~, 2.28-2.39 ~m, lH), 3.04-3.18 (m,
lH), 3.34-3.45 ~m, 2H), 4.28-4.38 (m, lH); 13C NMR
(CDCl3): ~ 23.7, 26.2, 27.1, 28.5, 35.5, 38.2,
39;6, 46.1, 78.0, 78.8, 85.7, 85.8, 154.7; CIMS m/z (~
rel int) 350 (MH+j 100), 294 (72), 250 (30).
ExamPle g
Analvtical Method for the Diastereoisomers of
(lS 2S,3R,5S)-Pinanediol PYrrolidine-2-boronate
hydrochloride.
A reagent solution of Q.2 M phenyl isothiocyanate in
dichloromethane-triethylamine (9:1~ was prepared. The
sample to be analyzed (1-5 mg) was treated with
10 ~L of the reagent solution per ~mole of analyte
and the clear solution was allowed to stand at room
temperature for 15 min. A 1 ~L sample of the
solution was then diluted in 1.00 mL of HPLC-grade
acetonitrile and 10 ~L of this solution was analyzed
by ~PLC (column: YMC AQ-303 S-5 120A, 4.6 x 250 mm;
WO93/10127 PCT/US92/09~5
- ~12~ l ~8
mobile phase: 65% MeCN - 35~ 25 mM ammonium phosphate,
pH 7.5; flow rate l mL/min; detection by W at 254
nm). The phenylthiourea derivative of the R isomer of
proline boronic acid elutes at about 6.4 min, its
epimer elutes at about 7.8 min, and unreacted phenyl
isothiocyanate, which serves as an internal standard,
elutes at 12.2 min. .
Example 10
(ls~2s~3R~ssL~pinanediol p~rrolidine-2RS-boronate
hvdrochloride
A stirred solution of ~lS,2S,3R,5S)-pinanediol
1-(1,1-dimethylethoxycarbonyl)-pyrrolidine-2RS-boronate
~224 g, 0.64 mol) in diethyl ether (900 mL) was cooled
in ice and dry HCl gas passed into the solution for
35 min at 10-18 C. The solution was stirred at room
temperature overnight, cooled again in ice and the
precipitate filtered off. The solid was washed with
cold ether (400 mL) followed by petroleum
ether/diethyl ether 9:1 (200 mL~ and dried under
~acuum to give the desired hydrochloride as a white
solid (113 g, 62~) (mp 228-234C). Analysis of this
material by HPLC as described in Example 9 showed it
to be a 60:40 mixture of R:S isomers of the boronic
acid.
WOg3~1~127 PCT/US92/09~5
212~1~8
1H NMR ~CDC13): ~ 0.83 (s, 3 H), 1.14 (d, J =
llHz, 1 H), 1.29 (s, 3 H), 1.45 (s, 3 H), 1.85 - 2.15
(m, 6 H), 2.17 - 2.50 (m, 3 H), 3.18-3.25 (m, 1 H),
3.45 (bs, 2 H), 4.42 (dd, J = 1.8, 8.6Hz, 1 H), 8~0
(bs, 1 H), 10.56 (bs, 1 H); C NMR lCDCi3)
23.9, 24.5, 26.5, 27.0, 27.2, 28.4, 34.9, 38.2, 39.4,
45.8, 51.2, 79.0, 87.6r CIMS m/z (% rel int) 250
(MH~, 100); Anal. Calcd for C14H24BNO2. HCl: C,
58.87, H, 8.B2, N, 4.90, Cl, 12.41. Found: C, 58.40,~
H, 8.86, N, 4.81, Cl, 12.39.
A similar reaction on a 29 g, 101 mmol scale using
ethereal HCl (approx. 4.5M, 200 mL~ and EtOAc (150 mL)
as solvent yielded the hydrochloride (11.1 g, 47%) as
a B1:19-mixture of R:S isomers
Example 11
-. :
(lS,2S,3R~ inanediol Pvrrolidine-2R-boronate
hYdrochloride bY fractional crvstallization.
~ '
Method ~: ;
The 60:40 isomeric mixture obtained in Example 10 `
(1.18 g 4.13 mmol) was dissolved in CH2C12 (65 mL)
with slight warming, and the solution filtered. The
filtrate was diluted with EtOAc (65 mL) and
crystallization began within a minute. The suspension
was stirred for 1-2 h at room temperature and the
first crop of solid was collected and the
;~- diastereomeric ratio determined as described in
Example 9 (540 mg, 46%, R:S ratio 97.1:2.9). Solvent
WO~3~0127 PCT/US92/09
29
2123128
was distilled from the filtrate until most of the
CH2C12 was removed, then the residual EtOAc solution
was stirred at room temperature overnight to afford a
second crop of off-white solid (346 mg, 29~, R:S ratio
39.2:60.8). The first crop was recrystallized from
isopropyl alcohol (10 mL) to afford 430 mg (80%
recovery) of material >99% 2-R isomer. (mp 269-272 C
(dec)) ~]25D +0~70O (c=1.15, MeOH)
H NMR ~CDC13): ~ 0.83 (s, 3 H), 1.14 (d, J =
llHz, 1 H), 1.29 (s, 3 H), 1.45 (s, 3 H), 1.85 - 2.15
(m, 6 H), 2.17 - 2.50 (m, 3 H), 3.18-3.25 (m, 1 H),
3.45 ~bs, 2 H), 4.42 (dd, J = 1.8, 8.5Hz, 1 H), 8.80
(bs, 1 H), 10.56 (bs, 1 H); l C NMR (CDC13) ~
23;9, 24.5, 26.5, 27.0, ~7.2, 28.5, 34.9, 38.1, 39.4,
45.8, 51.2, 79.0, 87.8; CIMS m/z (% rel int) 250
(MH+, 100); Anal. Calcd for C14H~4BN~2. HCl: C,
58.87, H, 8.82, N, 4.90, Cl, 12.41. Found: C, 58.64,
H, ~.79, N, 4.90, Cl, 12.66.
Method B:
r
A suspension of (lS,2S,3R,5S)-pinanediol-
pyrrolidine-2RS-boronate hydrochloride as a 1:1
mixture of isomers (850 mg, 2.98 mmol) in EtOAc
(60 mL) was heated under reflux with stirring for
4 h. The mixture was filtered hot and the collected
solid dried to give material enriched in the R isomer
(541 mg 64%~, R:S ratio 2:1. Evaporation of the
filtrate yielded material enriched in the S isomer
~217 mg), R:S = 1:4. The filtered solid (500 mg) was
treated in the same way with EtOAc (45 mL) for 1.5 h
and again filtered hot to yield a solid (366 mg, 73%),
R:S = 7:1. This material was again treated with EtOAc
~V093/10127 PCT/US92~0~5
', ,,
~123~2~ :~
(38 mL) for 1.5 h. The solid isolated (287 mg, 78%)
now had R:S ratio 97:3. The spectral properties were
the same as those of material obtained by method A.
'':
Example 12 - ~-
1-(l,i-DimethylethoxycarbonYl)-pyrrolidine-2-boronic
acid from (lS,2S,3R,5S) pinanediol
1-(1,l-dimethylethox~carbonYl)-Pyrrolidine-2-boronate
To a solution of (lS,2S,3R,5S)-pinanediol-
~ dimethylethoxycarbonyl)-pyrrolidine-2RS-boronate ~-
from Example 8 (1.9 g, S.44 mmol) in acetone (80 mL)
wa~ added O.lM ammonium acetate solution (80 mL) and
sodium metaperiodate (3.49 g,16.33 mmol). The
reaction mixture was stirred at room temperature for
40 h, then the acetone was evaporated and the residue
was treated with 2M NaOH solution. This a~ueous phase ;
was washed with CH2C12 (2 x 80 mL), acidified with
2M HCl to pH 3 and extracted with CH2C12 (4 x 80
mL). The combined organic extracts were dried
(Na2S04) and concentrated to afford the desired
product as a white foamy solid (890 mg, 76%),
identical by NMR with the material prepared in Example ~;
2. The boronic acid was derivatized with pinacol for
purposes of analysis.
'
W O 93/10127 P(~r/US92/09845
~1231 2~
Example 13
Pinacol ~ dimethYlethoxycarbonyl)- :
Pyrrolidine-2Rs-boronate
To a stirred solution of the boronic acid from Example
12 (890 mg, 4.14 mmol) in chloroform was added pinacol
(489 mg, 4.14 mmol). After stirring for 16h at room
temperature, the solvent was removed and the residue
was purified via chromatography over silica gel
(hexane/EtOAc, 4:1) to give the desired product as a
white solid (1.04 g, 85%) (mp 73-75 C).
H NMR (CDCl3): ~ 1.18 (s, 6H), 1.21 (s, 6H),
1.38 ~s, 9H), 1.5.-2.00 (m, 4H), 2.9$ (br s, lH), 3.27
(m, 2H~; 13C NMR (CDCl3): ~ 24.3, 24.5, 24.7,
24.~, 25.3, 27.0~ 27.6, 28.4, 2~.6, 43.6, 45.8, 46.3,
78~8, 83.2, 154.4, 154.8; CIMS m/z (% rel int) 29~
(18), 242 (100, MH+ - tBu), 198 (30, MH+ - Boc); Anal.
Calcd for C15H28BNO4: C, 60.62, H,9.50, N, 4.71.
Found: C, 60.94, H, 9.65, N, 4.88.
ExamPle 14
N~ DimetbvlethoxYcarbonYl)-L-valvlpyrrolidine-2R-
boronate (lS 2S 3R 5S)-Pinanediol ester
A solution of t-BOC-L-Valine (351.7 g, 1.62 mol) in
CH2Cl2 (1.6 L) was cooled with an ice bath and a
solution of dicyclohexylcarbodiimide (161.8 g,
0.784 mol) in CH2C12 (0.75 L) was added within
40 min at 0-2 C. After addition the solution was
WO~3/10127 PCT/US92/09~5 `
; ; ~
2123128 32
stirred for 3.5 h at 0-5 C. The white precipitate was
filtered off and washed with CH2Cl2 (0.2 L). The
resulting clear solution was added at 18-20 C
(waterbath cooling) to a solution of -~
(lS,2S,3R,5S)-pinanediol pyrrolidine-2RS-boronate
hydrochloride (210 g, 0.73S mol), prepared as in
Example lO, in CH2Cl2 (2.0 L) containing
N-methylmorpholine (164 g, 1.62 mol). The mixture was
allowed to stir at~room temperature overnight. The
cloudy solution was filtered through a 16 cm dia. x
2 cm high bed of silica gel (200-425 mesh) and washed
wlth CH2Cl2 (1.5 ~). The solvent was evaporated
to yield a highly viscous oil (542 g). This oil was
dissolved in ethyl acetate (0.7 L~ and the mixture
cooled in an ice bath. Crystals formed, which were
filtered off at low temperature, and washed with cold
ethyl acetate (0.1 L). The wet filter cake was
transferred into petroleum ether (0.65 L) and stirred
at room temperature for 1 h. The white solid was
filtered, washed with cold petroleum ether (0.1 L),
and dried to constant weight to yield the title
compound as a white solid ~113.4 g) (mp 128-13~ C).
ll the mother Iiquors were combined and concentrated
to a volume of approx. 0.8 L. After standing for 2
days in the freezer a solid formed, which was filtered
off, and treated with petroleum ether as above to
yield a beige solid (50.4 g). This was a mixture of
impurities and the unwanted diastereoisomer. The
mother liquor from above was concentrated and the
residue purified over a silica gel column (14 cm dia.
x 60cm) using héxane/ethyl acetate (85:15) (14 L). ~;
Appropriate fractions were collected, treated with -
petroleum ether, the solid collected by filtration and
f
';
WO93/10~27 P~T/US92/~9845
.....
~,~
33
212312~
dried, to yield more desired product ~18.5 g). The
other diastereoisomer was also obtained (7.5 g) (mp
82-83 C). A second column was performed on the
combined mixed fractions and mother li~uors to yield
additional pure compound (13.5 g), total combined
yield 145.4 g t44.3%) of desired diastereoisomer
(mp 128-130 C).
H NMR (CDCl3): ~ 0.83 (s, 3 H), 0.91 (d, J =
6.7Hz, 3 H), 0.97 (d, J = 6.7Hz, 3 H), 1.27 (s, 3 H),
1.35-1~45 (m, 1 H), 1.39 (s, 3 H), 1.41 (s, 9 H),
1.72-2.14 (m, 9 H), 2.26-2.36 (m, 1 H), 3.15 (dd, J =
6.7, lO.lHz, 1 H), 3.43-3.51 (m, 1 H), 3.70-3.81 (m,
l H), 4.19-4.28 tm, 2 H), 5.29 (d, J - 9.2~z, 1 H);
3~C NMR (CDCl3): ~ 17.3, 19.2, 24.0, 26.3, 27.1
27.2, 27.4, 28.4, 28.6, 31.4, 33.9, 35.5, 38.2, 39.6,
46.7, 51.2, 56.6, 77.8, 79.2, 85.8, 155.9, 170.2; CIMS
m/z (~ rel int) 449 ~MH~, 100), 393 (50); Anal. Calcd
for C24H41BN2O~: C, 64.28, H, 9.22, N, 6.25.
Found: C, 64.58, H, 9.33, N, 6.52.
- ....
Example 15
L-valylpyrrolidine-2R-boronate
(lS,2S,3R 5S)-pinanediol ester hydroqen maleate
N-(1,1-Dimethylethoxycarbonyl)-L-valylpyrrolidine-2~-
boronate (lS,2S,3R,5S)-pinanediol ester (248 mg,
0.553 mmol) was added to a stirred solution of dry
hydrogen chloride in ethyl acetate. After 1.5 h the
solvent was evaporated to leave the deprotected
hydrochloride. The residue was partitioned between
'
WO~3~10127 P~T/US92/0984~
212~128 34
CH2Cl2 and sodium carbonate solution, and the
organic layer dried over magnesium sulfate. The
organic layer contains the free base of the title
compound, which exists as a cyclic form containing a
nitrogen-boron bond, but reverts to the open form on
adding acid. The organic solution was filtered into a
solution of maleic acid (64 mg, 0.553 mmol) in
methanol ~5 mL), and the solvent evaporated to leave a
crystalline residue (258 mg), ~hich was recrystallized
from ethyl acetate to give the title compound ~193 mg,
75%) (mp 145-146 C).
- H NMR ~CDCl3): ~ 0.84 (s, 3 H), 1-08 ~d, J =
6~9Hz, 3 H), 1.13 (d, J = 6.9Hz, 3 H), 1.26-1.31 (m,
2 H), 1.23 (s, 3 H), 1.38 ~s, 3 H), 1.72-2.15 (m,
7 H), 2.2~-2.38 (m, 2 H), 3.28 (dd, J = 6.9, 9.4 Hz,
1 H), 3.38-3.47 (m, 1 H), 3.73-3.78 (m, 1 H), 4.14 (d,
J = 5.1Hz, 1 H), 4.26 (d, J = 7.1Hz, 1 H), 6.25 (s,
2 H), 7.5-9.0 (v. br, 4 H); C NMR (CDCl3): ~ ~
17.0, 18.4, 24.0, 26.3, 27.0, 27.1, 28.7, 30.0, 35.4, -
38.2, 39.5, ~7.3, 51.2, 56.6, 78.1, 86.2, 135.5,
166.3, 169.5; CIMS m/z (% rel int) 349 (MH+, 100), 197
(18); Anal. Calcd for C23H37BN207: C, 59.49,
: H, 8.03, N, 6.03. Found: C, 59.50, H, 8.13, N, 6.03.
.
.
WO9~/10127 ~CT/US92/09~5
35
2123128
Example 16
L-valYlpvrrolidine-2R-boronic acid methanesulfonate
a) cyclo-L-valylpyrrolidine-2R-boronic acid ~
A solution of the maleate salt obtained in Example 15 -
(5.0 g, 10.8 mmol) in dilute acetic acid (1.0%, 60 mL)
was loaded on to a column (3.5 cm deep x 4 cm dia.) of
Dowex 50X2-200 ion exchange resin in the H+ form. The .
column was then eluted with acetic acid (1.0%, 14 L~,
water (42 L) and ammonium hydroxide solution (1:100
dilution of commercial 0.880 solution). Pinanediol
could be recovered from the neutral and acidic
: fractions. The product was found in early basic
fractions, which were collected and washed with
CH2Cl2 (2 x 100 mL). The organic extracts were
dried and concentrated to afford recovered free base
of thP starting material (741 mg, 20%), along with
some pinanediol. The aqueous phase was lyophilized to
afford the title compound, which exists in a cyclic .
form with a nitrogen-boron bond, as a white solid
(1.52g, 66%) (mp 120-130C). -
,
: H NMR (D20) ~ 0 97 (d, J = 7.0 Hz, 3 H), 1.06
(d, J = 7.0 Hz, 3 H), 1.59-1.80 (m, 2 H), 1.95-2.03
: (m, 2 H), 2.41-2.51 (m, 1 H), 2.62-2.69 (m, 1 H),
3.23-3.32 (m, 1 H), 3.Sl-3.58 (m with overlapping
doublet, J = 4.2 Hz, 2 H); 13C NMR (D20): ~
19.0, 21.7, 27.3, 30.7, 29.9, 49.6, 61.0, 170.3; IR
(cm 1) 3400-3314, 3221-3108, 2961-2872, 1637,
1452-1369; CIMS m/z (~ rel int) 375 (9o,
WO93~0127 PCT/US92/09845
2 1 ~ 8
36
M2H+-3H20), 197 (100, MH+-H20)i Anal. Calcd for
C9HlgBN203: C, 50.50, H~ 8.95~ N, 13-09-
Found: C, 50.43, H, 8~76, N, 12.93.
b) L-valylpyrrolidine-2R-boronic acid methanesulfonate
To a stirred suspension of the cyclized boronic acid
obtained above (5.17 g, 24.16 mmol) in acetonitrile
(190 mL) under nitrogen was added a solution of ' ~:
methanesulfonic acid (2.32 g, 24.16 mmol) in `.
acetonitrile (10 mL) dropwise over five minutes and
the mixture stirred at room temperature for 2 h. The ~;
solid was collected by filtration, washed well with
acetonitrile and diethyl ether and dried to afford the
title compound as a white solid (6.14g, 82%)
(mp 179-180 C). Crystallization of this ~aterial from
dimethylformamide/acetonitrile ga~e a 70~ recovery of
material in a single crop (mp 181-182 C).
- ;
H NMR (D20, phosphate, pH2): ~ 0.99 (d, J = 6.8
Hz, 3H), 1.09 (d, J = 6.9Hz, 3H), 1.69-1.75 (m' lH),
1.90-1.99 (m, lH), 2.10-2.14 (m, 2H), 2.28-2.35 ~m,
lH), 2.80 (s, 3H), 3.07 (dd, J = 7.0 and 11.2 Hz, lH),
3.46-3.51 (m, lH), 3.75 ~t, J = 9.0 Hz, lH), 4.14 (d,
J = 5.1 Hz, lH); the cis amide rotamer (ca. 3%) is
also observed at 3.53-3.55 (m) and 3.83 (d, J =
6.2 Hz); 13C NMR: ~ 16.2, 18.4, 26.9, 27.1, 29.0,
38.8, 47.9, 49.0, 57.2, 167.2; peaks due to the cis
amide rotamer are observed at 16.8, 24.3, 29.9, 57.8, -
167.5; IR (cm ) 3387, 3000 (br~, 2972, 2655, 1646,
1370, 1197; CIMS m/z (% rel int, ethylene glycol
adduct) 241 (MH+ 100); Anal. Calcd for
C1oH23BN206S: C, 38.72, H, 7.47, N, 9.03.
Found: C,38.65, H, 7.45, N, 8.44.
WO93/10127 PCT/US92/09845
37
21231 23
Exam~le 17
N~ DimethvlethoxycarbonYl)-L-valylPvrrolidine-2R-
boronic acid .
To a stirred solution of N-(l,l-dimethyl-
ethoxycarbonyl)-L-valylpyrrolidine-2R-boronate
~lS,2S,3R,SS)-pinanediol ester, prepared as in Example
14, (1.0 g, 2.3 mmol) in acetone (75 mL) was added
ammonium acetate solution (60 ml, 0.1 M) and sodium
metaperiodate (1.48 g, 6.91 mmol). The reaction
mixture was stirred at room temperature for 48 h, then
the acetone was evaporated. The residue was treated
with 2M sodium hydroxide solution (100 mL), and washed
with CH2Cl2 (2 x 50 mL). The aqueou~ layer was
carefully acidified with 2M hydrochloric acid to pH 3
and extracted with CH2Cl2 (4 x 70 mL). The
combined organic extracts of the acid solution were
dried over sodium sulphate and concentrated to afford
the desired product as a white ~oamy solid (700mg,
97%). Further purification vi~ chromatography over
silica gel (CH2Cl2/methanol, 9:1) gave the boronic
acid again as a white solid (449 mg, 62%) (mp 82-92C).
H NMR (CDCl3~: ~ 0.95 (d, J = 5.7 Hz, 6H), 1.42
(s, 9H), 1.55-1.80 (m, lH) 1.80-2.20 (m, 4H),
2.89-3.07 (m, lH), 3.30-3.55 (m, lH), 3.55-3.65 (m, :
lH), 4.10-4.30 (m, lH), 5.34 (d, J = 9 2 Hz, lH);
1 C NMR (~DCl3): ~ 18.0, 19.1, 26.3, 27.7, 28.3,
31.2, 46.1, 52.0, 55.7, 79.5, lS5.6, 170.8; IR
(cm 1) 3395-3319, 2971-2875, 1711, 1619, 1400, 1174;
CIMS m/z (% rel int, ethylene glycol adduct)
341(MH , 100), 285(MH -tBu, 67), 241(MH -BOC,
21).
WO93/10127 PCT/~S92/09~5
.
- 2123128 38
Exam~le 18
L-valYlp~rrolidine-2R-boronic acid hydrochloride
N-(1,1-Dimethylethoxycarbonyl)-L-valylpyrrolidine-2R ~-
boronic acid, obtained in Example 17 (250 mg, 0.796
mmol) was stirred with HCl/ether (4.5M, 20 mL) at room
temperature under nitrogen for 1.5 h. The solvent was
then evaporated and the residue triturated with
diethyl ether (3 x 10 mL) and each time the ether was
decanted. The residue was dried to yield the title
compound as a white powdery solid (172 mg, 86%) (mp
211-213C).
lH`NMR ~D2O, phosphate pH 2): ~ 0.99 (d, J = -~
6.9 Hz, 3 H), 1.09 (d, J = 7.0 Hz, 3 H), 1.67-1.76 (m,
1 H), 1.87-2.01 ~m, 1 H), 2.09-2.15 (m, 2 H), .~:
.28-2.35 (m, 1 H), 3.07 (dd, J = 7.0 and 11.4 Hz,
1 H), 3.48 (dt, 3 = 6.7 and 10.3 Hz, 1 H), 3.73 (dt,
J = 1.7 and 10.2 Hz, 1 H), 4.14 (d, J = 5.2 Hz, 1 H);
3C NMR: ~ 16.0, 18.3, 26.9, 27.1, 28.9, 47.9, .
48.9, 57.2, 167.3; IR (cm ) 3400-2800, 3368,
2970l2880, 1635, 1475-1378, 1400; CIMS m/z (% rel int,
e~hylene glycol adduct) 241 (MH , 100).
'
WOg3~10127 PC~/US92/09
39
.
xample 19 2123128
cyclo-L-Valvlpyrrolidine-2R-boronic acid by
transesterification with ~henylboronic acid
A solution of L-valylpyrrolidine-2R-boronate -
(lS,2S,3R,5S~-pinanediol ester hydrochloride, prepared -
as in Example 15 ~500 mg, 1.3 mmol) in lM hydrochloric
acid (10 mL) containing hexane (20 mL) and
phenylboronic acid (500 mg, 2.6 mmol) was stirred
vigorously for lh at room temperature. The hexane was
removed by decantation, then more hexane (20 ml~ was
added and the mixture stirred for a further 30 min.
The layers were separated and the combined hexane
l~yers dried over sodium sulphate and concentrated to
give pinanediol phenylboronate (331 mg, 99%) as a
..
white crystalline solid. The aqueous layer was then
passed through a column of Dowex 50 ion exchange
resin. The column was eluted with water (200 mL),
followed by ammonium hydroxide solution (1:100
dilution of commercial 0.880 solution, 50 ml).
Isolation of the basic fractions followed by
lyophilization gave the free boronic acid (230 mg,
83~) as a white powder, identical by NMR with the
material obtained in Example 16a.
.
. .
