U.S. patent application number 16/985815 was filed with the patent office on 2021-02-11 for thermally sensitive protecting groups for cysteine, and manufacture and use thereof.
This patent application is currently assigned to University of Windsor. The applicant listed for this patent is University of Windsor. Invention is credited to Anthony Emanuel CHIFOR, Mohadeseh DASHTI-POUR, John HAYWARD, Daniel MEISTER, Sarah NASRI, Seyedeh Maryamdokht TAIMOORY, John Frederick TRANT.
Application Number | 20210040105 16/985815 |
Document ID | / |
Family ID | 1000005065098 |
Filed Date | 2021-02-11 |
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United States Patent
Application |
20210040105 |
Kind Code |
A1 |
TRANT; John Frederick ; et
al. |
February 11, 2021 |
Thermally Sensitive Protecting Groups for Cysteine, and Manufacture
and Use Thereof
Abstract
In a preferred embodiment, there is provided a protecting group
for protecting the thiol side chain of a cysteine residue, the
protecting group comprising a Diels-Alder cycloadduct of a furan
and a maleimide, and optionally, a linker interposed between the
thiol side chain and the Diels-Alder cycloadduct.
Inventors: |
TRANT; John Frederick;
(LaSalle, CA) ; MEISTER; Daniel; (Windsor, CA)
; NASRI; Sarah; (LaSalle, CA) ; CHIFOR; Anthony
Emanuel; (LaSalle, CA) ; HAYWARD; John;
(Windsor, CA) ; TAIMOORY; Seyedeh Maryamdokht;
(LaSalle, CA) ; DASHTI-POUR; Mohadeseh; (Windsor,
CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
University of Windsor |
Windsor |
|
CA |
|
|
Assignee: |
University of Windsor
|
Family ID: |
1000005065098 |
Appl. No.: |
16/985815 |
Filed: |
August 5, 2020 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
62883332 |
Aug 6, 2019 |
|
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07K 1/067 20130101;
C07D 491/18 20130101 |
International
Class: |
C07D 491/18 20060101
C07D491/18; C07K 1/06 20060101 C07K001/06 |
Claims
1. A protecting group for protecting a cysteine residue, the
protecting group having structural formula 1 or 2: ##STR00024##
wherein R is an electron withdrawing group or a leaving group; X
and Y are independently of each other oxygen, sulfur, nitrogen or
phosphorus; R.sub.1 and R.sub.2 are independently of each other
hydrogen, hydroxyl, halo, alkyl, alkenyl, alkynyl, cycloalkyl,
heterocycloalkyl, aryl, aralkyl, heteroaryl, heteroaralkyl, formyl,
haloformyl, carbonyl, carboxyl, alkoxy, alkoxycarbonyl,
(alkoxycarbonyl)oxy, carbamoyl, amino, amido, imino, imido, azo,
cyanato, isocyanato, cyano, nitro, sulfanyl, thiocyanato or
phosphono, each of which is optionally substituted; and n is an
integer between 1 and 12, inclusive.
2. The protecting group of claim 1, wherein R.sub.1 and R.sub.2 are
independently of each other hydrogen, hydroxyl, halo, alkyl,
cycloalkyl, heterocycloalkyl, aryl, heteroaryl, formyl, carbonyl,
carboxyl, alkoxy, amino or nitro, each of which is optionally
substituted.
3. The protecting group of claim 1, wherein R.sub.1 is hydrogen,
hydroxyl, halo, alkyl, formyl, carbonyl, carboxyl, alkoxy,
alkoxycarbonyl, amino or nitro, each of which is optionally
substituted, and R.sub.2 is hydrogen, alkyl, cycloalkyl,
heterocycloalkyl, aryl or heteroaryl, each of which is optionally
substituted.
4. The protecting group of claim 1, wherein R.sub.1 is hydrogen,
nitro, halo or alkoxy, and R.sub.2 is alkyl, aryl or heteroaryl,
each of which is optionally substituted, or wherein R.sub.1 is
hydrogen, nitro, bromo, chloro, fluoro, methoxy or ethoxy, and
R.sub.2 is methyl, ethyl, propyl, butyl, phenyl, p-methoxyphenyl,
p-nitrophenyl or benzyl.
5. The protecting group of claim 1, wherein X and Y are
independently of each other oxygen or nitrogen, or wherein X is
nitrogen and Y is oxygen.
6. The protecting group of claim 1, wherein n is an integer between
1 and 4, inclusive.
7. The protecting group of claim 1, wherein R is hydroxyl, alkyl,
alkenyl or halo, or wherein R is hydroxyl, chloro, methyl or
allyl.
8. A protected cysteine having structural formula 9 or 10:
##STR00025## wherein X and Y are independently of each other
oxygen, sulfur, nitrogen or phosphorus; R.sub.1 and R.sub.2 are
independently of each other hydrogen, hydroxyl, halo, alkyl,
alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, aralkyl,
heteroaryl, heteroaralkyl, formyl, haloformyl, carbonyl, carboxyl,
alkoxy, alkoxycarbonyl, (alkoxycarbonyl)oxy, carbamoyl, amino,
amido, imino, imido, azo, cyanato, isocyanato, cyano, nitro,
sulfanyl, thiocyanato or phosphono, each of which is optionally
substituted; R.sub.3 and R.sub.4 are independently each other
hydrogen or a protecting group; and n is an integer between 1 and
12, inclusive.
9. The protected cysteine of claim 8, wherein R.sub.1 and R.sub.2
are independently of each other hydrogen, hydroxyl, halo, alkyl,
cycloalkyl, heterocycloalkyl, aryl, heteroaryl, formyl, carbonyl,
carboxyl, alkoxy, amino or nitro, each of which is optionally
substituted.
10. The protected cysteine of claim 8, wherein R.sub.1 is hydrogen,
hydroxyl, halo, alkyl, formyl, carbonyl, carboxyl, alkoxy,
alkoxycarbonyl, amino or nitro, each of which is optionally
substituted, and R.sub.2 is hydrogen, alkyl, cycloalkyl,
heterocycloalkyl, aryl or heteroaryl, each of which is optionally
substituted.
11. The protected cysteine of claim 8, wherein R.sub.1 is hydrogen,
nitro, halo or alkoxy, and R.sub.2 is alkyl, aryl or heteroaryl,
each of which is optionally substituted or wherein R.sub.1 is
hydrogen, nitro, bromo, chloro, fluoro, methoxy or ethoxy, and
R.sub.2 is methyl, ethyl, propyl, butyl, phenyl, p-methoxyphenyl,
p-nitrophenyl or benzyl.
12. The protected cysteine of claim 8, wherein X and Y are
independently of each other oxygen or nitrogen or wherein X is
nitrogen and Y is oxygen.
13. The protected cysteine of claim 8, wherein n is an integer
between 1 and 4, inclusive.
14. The protected cysteine of claim 8, wherein R.sub.3 and R.sub.4
are independently each other hydrogen, alkyl, allyl,
tert-Butyloxycarbonyl (Boc), fluorenylmethyloxycarbonyl (Fmoc),
tert-butyldimethylsilyl (TBS), methoxymethyl (MOM), ethoxymethyl
(EOM), p-methoxybenzyl or p-nitrobenzyl or wherein R.sub.3 is
hydrogen or allyand R.sub.4 is Fmoc.
15. A compound having structural formula 1 or 2: ##STR00026##
wherein R is an electron withdrawing group or a leaving group; X
and Y are independently of each other oxygen, sulfur, nitrogen or
phosphorus; R.sub.1 and R.sub.2 are independently of each other
hydrogen, hydroxyl, halo, alkyl, alkenyl, alkynyl, cycloalkyl,
heterocycloalkyl, aryl, aralkyl, heteroaryl, heteroaralkyl, formyl,
haloformyl, carbonyl, carboxyl, alkoxy, alkoxycarbonyl,
(alkoxycarbonyl)oxy, carbamoyl, amino, amido, imino, imido, azo,
cyanato, isocyanato, cyano, nitro, sulfanyl, thiocyanato or
phosphono, each of which is optionally substituted; and n is an
integer between 1 and 12, inclusive.
16. The compound of claim 15, wherein R.sub.1 and R.sub.2 are
independently of each other hydrogen, hydroxyl, halo, alkyl,
cycloalkyl, heterocycloalkyl, aryl, heteroaryl, formyl, carbonyl,
carboxyl, alkoxy, amino or nitro, each of which is optionally
substituted.
17. The compound of claim 15, wherein R.sub.1 is hydrogen,
hydroxyl, halo, alkyl, formyl, carbonyl, carboxyl, alkoxy,
alkoxycarbonyl, amino or nitro, each of which is optionally
substituted, and R.sub.2 is hydrogen, alkyl, cycloalkyl,
heterocycloalkyl, aryl or heteroaryl, each of which is optionally
substituted.
18. The compound of claim 15, wherein R.sub.1 is hydrogen, nitro,
halo or alkoxy, and R.sub.2 is alkyl, aryl or heteroaryl, each of
which is optionally substituted or wherein R.sub.1 is hydrogen,
nitro, bromo, chloro, fluoro, methoxy or ethoxy, and R.sub.2 is
methyl, ethyl, propyl, butyl, phenyl, p-methoxyphenyl,
p-nitrophenyl or benzyl.
19. The compound of claim 15, wherein X and Y are independently of
each other oxygen or nitrogen or wherein X is nitrogen and Y is
oxygen.
20. The compound of claim 15, wherein n is an integer between 1 and
4, inclusive.
21. The compound of claim 15, wherein R is hydroxyl, alkyl, alkenyl
or halo or wherein R is hydroxyl, chloro, methyl or allyl.
Description
RELATED APPLICATIONS
[0001] This application claims the benefit under 35 USC 119(e) to
U.S. Provisional Application Ser. No. 62/883,332 filed Aug. 6,
2019, the entire contents of which are incorporated herein by
reference.
SCOPE OF THE INVENTION
[0002] The present invention relates to a protected cysteine
residue having a protecting group bonded to a thiol side chain
thereof, and which is configured to permit deprotection at a
preselected temperature. The present invention also relates to a
method for producing the protected cysteine residue, and a method
for synthesizing a peptide containing a plurality of cysteines and
using the protected cysteine residues to sequentially generate a
plurality of specific disulfide bonds.
BACKGROUND OF THE INVENTION
[0003] Peptide synthesis involves formation of amide bonds between
multiple amino acids by the condensation reaction of the carboxyl
group of one amino acid with the amino group of another amino acid.
To synthesize a peptide of specific amino acid sequence, solid
phase peptide synthesis may be used to form a peptide chain using
successive reaction and introduction of preselected amino acids to
a solid insoluble and porous support, which includes a polymeric
resin bead having a reactive linker group for attaching a growing
peptide chain. The reactive linker group may include an amino
group, and the amino acids to be introduced to the solid support
may be protected on the N-terminus (and possibly the side chain as
needed), using known protecting groups, such as
tert-Butyloxycarbonyl (Boc) or fluorenylmethyloxycarbonyl (Fmoc)
protecting group.
[0004] Solid phase peptide synthesis may involve repeated cycles of
coupling and N-terminal deprotection reactions, with washing of the
resin bead between each cycle. Specifically, a first N-terminus
protected amino acid is coupled to the amino group of the reactive
linker group in the solid support, and the amino acid is
deprotected to leave the amino functional group of the amino acid
available to form an amide bond with a second N-terminus protected
amino acid. The cycles are repeated with a preselected sequence of
amino acids, and until the peptide of the desired length and
sequence is formed. The peptide as formed is then cleaved from the
solid support, isolated and purified, and may be subject to further
treatment.
[0005] Aside from the primary peptide sequence which may be
obtained with solid phase peptide synthesis and which will fold
into defined secondary structures, disulfide bonds play an
important role in the organization of proteins and peptides, such
as determining the ternary structure. Formation of correct
disulfide bonds between correct cysteine residues may thus
facilitate formation of correct three-dimensional structure,
especially in short peptides. Disulfide bonds may however present
challenges in synthesizing peptides and proteins by way of
biotransformations; while the linear sequence may be readily
programmed into DNA, the information regarding which disulfide
bonds should be formed is often regulated by additional factors and
is part of the post-translational modifications.
[0006] It has been recognized that a synthesized peptide intended
to function as a natural peptide of the same amino acid sequence
and with specific disulfide bonds may require further treatment
during and after peptide synthesis to ensure that the synthesized
peptide possesses the same natural disulfide bonds. Previously,
orthogonal protection of the thiol side chains (involved in forming
disulfide bonds) of cysteine residues have been used to effect
selective deprotection and disulfide bond formation after peptide
synthesis. Specifically, a crude peptide is formed with cysteine
residues having their respective thiol side chains protected with a
protecting group, such as acetamidomethyl, tert-butyl,
3-nitro-2-pyridine sulfenyl, 2-pyridine-sulfenyl or trityl, and
which remains on the thiol side chains after peptide synthesis. By
having each pair of specific cysteine residues intended to form a
disulfide bond having orthogonal protection independent from other
pairs of cysteine residues, it has been possible to successively
deprotect different pairs of cysteine residues and introduce
regioselectivity to disulfide bond formation.
[0007] Peptide synthesis may thus employ multiple pairs of cysteine
protecting groups, in cases where multiple cysteine bonds are
desired. A bioactive peptide ordinarily possesses both the linear
amino acid sequence and the specific disulfide bonds made in the
naturally-occurring material, which if present contribute to the
three dimensional structure. For example, if there are 8 cysteines
in a short peptide, these would make 4 disulfide bonds, and there
would be 70 (8!4) possible structures containing 4 disulfide bonds
that can be generated, where only 1 is the desirable compound. Such
need for increasing degrees of orthogonality between protecting
groups increases the complexity of the peptide, and requires
specialist and fine-tuning of peptide synthesis conditions, as well
as iterative cycles of reaction and purification to isolate the
desired compound and remove the unwanted reagents. This is a slow,
costly and inefficient process, which may lead to low yields and
complex purification problems, and which may effectively prohibit
scalability to produce larger amounts of the desired peptide.
[0008] An improvement may reside with use of a common class of
protecting groups that are removed under the same trigger and
differ from one another only by the threshold level of that
trigger. This may preferably involve only a single reagent
introduced to the reaction mixture, with slow increase of its
concentration. An existing example involves use of acid-sensitive
end-caps, although such approach has two complications: the acidic
conditions used for deprotection are not compatible with the
conditions required to dimerize the cysteine residues, and the
dynamic range between the acid-sensitivity of the protecting groups
is insufficient to provide the required levels of selectivity, with
chemistry relegated between a pH of 0 and 5, sufficient for no more
than three different acid-sensitive protecting groups (e.g.
triggered at pH=0, 2.5 and 5). However, 2.5 pH units may not
provide sufficient discrimination in reaction rates to ensure
complete selectivity, and being triggered at pH 5 may result in
background cleavage at neutral pH and may also limit synthetic
operations available to the chemist making the peptide.
[0009] It has thus been appreciated that known regioselective
formation of disulfide bonds may be associated with greater costs,
time and complexity and reduced yield and selectivity, often
stemming from the need to develop customized residues and
protecting groups, and in the absence of any general approach to
providing orthogonal protection.
SUMMARY OF THE INVENTION
[0010] It is a non-limiting object of the present invention to
provide a protecting group for protecting a cysteine residue and
permitting more regioselective formation of multiple disulfide
bonds during peptide synthesis, and which includes a Diels-Alder
cycloadduct of an optionally substituted furan and an optionally
substituted maleimide, and optionally a cyclization spacer for
placement between the thiol side chain and the Diels-Alder
cycloadduct.
[0011] It is another non-limiting object of the present invention
to provide a protecting group for protecting a cysteine residue for
use in peptide synthesis, and which may permit deprotection in
response to a single physical change, such as the temperature,
without necessary requiring use of multiple reagents.
[0012] It is another non-limiting object of the present invention
to provide a protecting group for protecting a cysteine residue for
use in peptide synthesis, and which may permit configuration to
obtain a family of different protecting groups selected for
deprotection at different temperatures, so as to allow the peptide
synthesis in a single pot without necessary requiring isolation and
purification steps between formation of multiple disulfide
bonds.
[0013] It is another non-limiting object of the present invention
to provide a protected cysteine residue for preparing a synthetic
peptide or protein having a three dimensional structure, and which
may permit ready incorporation into synthetic peptide synthesis to
form two or more preselected disulfide bonds to facilitate
achieving the three dimensional structure.
[0014] It is another non-limiting object of the present invention
to provide a process for synthesizing a peptide or protein having
two of more preselected disulfide bonds, and which includes
increasing a reaction temperature to effect sequential deprotection
of protected cysteine residue pairs to form the preselected
disulfide bonds, without necessarily requiring use deprotection
reagents or separation or purification steps.
[0015] It is another non-limiting object of the present invention
to provide a process for synthesizing a peptide having two of more
preselected disulfide bonds, and which may permit formation of
functioning peptides with reduced production of by-products having
disulfide bonds other than the preselected disulfide bonds, and
which may be adopted for a larger scale production of a peptide of
commercial value, such as insulin or the conotoxins, or a library
of disulfide-containing research peptides.
[0016] In one simplified aspect, the present invention provides a
protecting group for protecting the thiol side chain of a cysteine
residue, the protecting group comprising a Diels-Alder cycloadduct,
either the endo or exo diastereomer, or a mixture of the two, of a
furan and a maleimide, and optionally, a linker interposed between
the thiol side chain and the Diels-Alder cycloadduct. It is to be
appreciated that the furan, the maleimide and the linker are
optionally substituted.
[0017] In one aspect, the present invention provides a compound
having structural formula 1 or 2:
##STR00001##
wherein R is an electron withdrawing group or a leaving group; X
and Y are independently of each other oxygen, sulfur, nitrogen or
phosphorus; R.sub.1 and R.sub.2 are independently of each other
hydrogen, hydroxyl, halo, alkyl, alkenyl, alkynyl, cycloalkyl,
heterocycloalkyl, aryl, aralkyl, heteroaryl, heteroaralkyl, formyl,
haloformyl, carbonyl, carboxyl, alkoxy, alkoxycarbonyl,
(alkoxycarbonyl)oxy, carbamoyl, amino, amido, imino, imido, azo,
cyanato, isocyanato, cyano, nitro, sulfanyl, thiocyanato or
phosphono, each of which is optionally substituted; and n is an
integer between 1 and 12, inclusive.
[0018] In another aspect, the present invention provides a
protecting group for protecting a cysteine residue, the protecting
group having structural formula 1 or 2:
##STR00002##
wherein R is an electron withdrawing group or a leaving group; X
and Y are independently of each other oxygen, sulfur, selenium,
nitrogen or phosphorus; R.sub.1 and R.sub.2 are independently of
each other hydrogen, hydroxyl, halo, alkyl, alkenyl, alkynyl,
cycloalkyl, heterocycloalkyl, aryl, aralkyl, heteroaryl,
heteroaralkyl, formyl, haloformyl, carbonyl, carboxyl, alkoxy,
dialkoxy, trialkoxy, alkoxycarbonyl, (alkoxycarbonyl)oxy,
carbamoyl, amino, amido, ammonio, imino, imido, azido, azo,
cyanato, isocyanato, nitroxy, cyano, isocyano, nitrosooxy, nitro,
nitrosyl, (carbamoyl)oxy, sulfanyl, disulfanyl, alkylsulfanyl,
sulfinyl, sulfonyl, sulfoamido, sulfino, thiocyanate,
isothiocyanato, thioyl, methanethioyl, mercaptocarbonyl,
hydroxyl(thiocarbonyl), dithiocarboxy, phosphanyl or phosphono; and
n is an integer between 1 and 12, inclusive. It is to be
appreciated that R, R.sub.1 and R.sub.2 are optionally
substituted.
[0019] In yet another aspect, the present invention provides a
protecting group for protecting a cysteine residue, the protecting
group having structural formula 1 or 2:
##STR00003##
wherein R is an electron withdrawing group or a leaving group; X
and Y are independently of each other oxygen, sulfur, nitrogen or
phosphorus; R.sub.1 and R.sub.2 are independently of each other
hydrogen, hydroxyl, halo, alkyl, alkenyl, alkynyl, cycloalkyl,
heterocycloalkyl, aryl, aralkyl, heteroaryl, heteroaralkyl, formyl,
haloformyl, carbonyl, carboxyl, alkoxy, alkoxycarbonyl,
(alkoxycarbonyl)oxy, carbamoyl, amino, amido, imino, imido, azo,
cyanato, isocyanato, cyano, nitro, sulfanyl, thiocyanato or
phosphono, each of which is optionally substituted; and n is an
integer between 1 and 12, inclusive.
[0020] In one embodiment, R is an activated ester or acid; X is
sulfur, oxygen or nitrogen; Y is oxygen or sulfur; R.sub.1 and
R.sub.2 are independently of each other alkyl, aryl, a halogen, an
ether, a thioether, a dialkylamine or trialkylammonium, an ester or
an acid derivative thereof, or a ketone; and n is an integer
between 1 and 9, inclusive.
[0021] In one embodiment, R.sub.1 and R.sub.2 are independently of
each other hydrogen, hydroxyl, halo, alkyl, alkenyl, alkynyl,
cycloalkyl, heterocycloalkyl, aryl, aralkyl, heteroaryl,
heteroaralkyl, formyl, haloformyl, carbonyl, carboxyl, alkoxy,
alkoxycarbonyl, (alkoxycarbonyl)oxy, carbamoyl, amino, imino,
imido, azo, cyanato, isocyanato, nitroxy, cyano, isocyano, nitro,
sulfanyl, alkylsulfanyl, sulfinyl, sulfino, thiocyanate or
isothiocyanato. In one embodiment, R.sub.1 and R.sub.2 are
independently of each other hydrogen, hydroxyl, halo, alkyl,
cycloalkyl, heterocycloalkyl, aryl, heteroaryl, formyl, carbonyl,
carboxyl, alkoxy, alkoxycarbonyl, carbamoyl, amino, nitro or
alkylsulfanyl.
[0022] In one embodiment, R.sub.1 is hydrogen, hydroxyl, halo,
alkyl, formyl, carbonyl, carboxyl, alkoxy, alkoxycarbonyl, amino or
nitro. In one embodiment, R.sub.1 is hydrogen, nitro, halo or
alkoxy, preferably, hydrogen, nitro, bromo, chloro, fluoro, nitro,
methoxy or ethoxy, or more preferably, hydrogen, nitro, bromo or
methoxy. In one embodiment, R.sub.2 is alkyl or aryl. In one
embodiment, R.sub.2 is methyl, ethyl, propyl, butyl or phenyl. In
one embodiment, R.sub.2 is p-methoxyphenyl, p-nitrophenyl or
benzyl.
[0023] In one embodiment, R.sub.1 and R.sub.2 are independently of
each other hydrogen, hydroxyl, halo, alkyl, cycloalkyl,
heterocycloalkyl, aryl, heteroaryl, formyl, carbonyl, carboxyl,
alkoxy, amino or nitro, each of which is optionally substituted. In
one embodiment, R.sub.1 is hydrogen, hydroxyl, halo, alkyl, formyl,
carbonyl, carboxyl, alkoxy, alkoxycarbonyl, amino or nitro, each of
which is optionally substituted, and R.sub.2 is hydrogen, alkyl,
cycloalkyl, heterocycloalkyl, aryl or heteroaryl, each of which is
optionally substituted. In one embodiment, R.sub.1 is hydrogen,
nitro, halo or alkoxy, and R.sub.2 is alkyl, aryl or heteroaryl,
each of which is optionally substituted. In one embodiment, R.sub.1
is hydrogen, nitro, bromo, chloro, fluoro, methoxy or ethoxy, and
R.sub.2 is methyl, ethyl, propyl, butyl, phenyl, p-methoxyphenyl,
p-nitrophenyl or benzyl.
[0024] In one embodiment, X and Y are independently of each other
oxygen or nitrogen. In one embodiment, X is nitrogen and Y is
oxygen.
[0025] In one embodiment, n is an integer between 1 and 4,
inclusive.
[0026] In one embodiment, R being the electron withdrawing group or
the leaving group is as defined below. In one embodiment, R is
hydroxyl, alkyl, alkenyl or halo. In one embodiment, R is hydroxyl,
chloro, methyl or allyl.
[0027] In one embodiment, R.sub.1 is substituted aryl, preferably
4-methoxyphenyl, 4-nitrophenyl, 4-fluorophenyl,
4-trifluoromethylphenyl, 4-cyanophenyl, 4-chlorophenyl,
4-bromophenyl, 4-iodophenyl, 4-di-N,N-methylaminophenyl,
4-(methoxycarbonyl)phenyl, 3-methoxyphenyl, 3-nitrophenyl,
3-fluorophenyl, 3-trifluoromethylphenyl, 3-cyanophenyl,
3-chlorophenyl, 3-bromophenyl, 3-iodophenyl,
3-di-N,N-methylaminophenyl or 3-(methoxycarbonyl)phenyl. In one
embodiment, R.sub.2 is propargyl, alkylazido, 4-azidophenyl,
4-alkynylphenyl or 4-propargyphenyl. In one embodiment, R is OH,
Cl, 4-nitrophenoxyl, Br, succinyl or other leaving group.
[0028] It is to be appreciated that compound 1 or 2 preferably
comprises a Diels-Alder cycloadduct portion which may exist in an
endo or exo stereoisomeric form. It is to be also appreciated that
compound 1 or 2 may include a mixture of the endo and exo
stereoisomers in different proportions. In one embodiment, compound
1 or 2 contains, or is purified to contain, a greater portion of
the endo or exo stereoisomer, preferably the endo stereoisomer. In
one embodiment, compound 1 or 2 contains 90 weight % or more,
preferably 95 weight % or more, more preferably 97% or more, or
most preferably 99% or more of the endo or exo stereoisomer, or
preferably the endo stereoisomer, based on the total weight of
compound 1 or 2.
[0029] In another aspect, the present invention provides a method
for preparing a protecting group, preferably compound 1 or compound
2, the method including conducting a Diels-Alder reaction between a
furan of structural formula 5 and a maleimide of structural formula
6 to produce a cycloadduct of structural formula 7:
##STR00004##
wherein R.sub.1, R.sub.2 and Y are as defined herein in respect of
compound 1 or 2. It is to be appreciated that Y included in the
furan of formula 5 or the cycloadduct of formula 7 may additionally
include one or more hydrogens or other substituents to satisfy the
octet rule. Preferably, the furan of formula 5 is a hydroxymethyl
furan, i.e., Y is oxygen bonded to a hydrogen (hydroxyl).
[0030] In one embodiment, the Diels-Alder reaction is conducted at
a reaction temperature between about 0.degree. C. and about
160.degree. C., preferably between about 0.degree. C. and about
120.degree. C. or more preferably between about 0.degree. C. and
about 90.degree. C. in a solvent for a reaction time of between
about 10 seconds and about 96 hours, preferably between about 5
minutes and about 72 hours or more preferably between about 15
minutes and about 48 hours. In one embodiment, the solvent is one
or more of benzene, acetonitrile, chloroform, dichloromethane,
tetrahydrofuran, DMSO, DMF, toluene, xylene, hydrocarbon solvents,
dichloroethane, tetrachloroethane, dioxane, methanol and
isopropanol.
[0031] It is to be appreciated that the cycloadduct of formula 7
may include endo and exo cycloadducts. In one embodiment, the
method further comprises separating the endo and exo cycloadducts,
preferably using thin layer chromatography, column chromatography,
high performance liquid chromatography (HPLC), cyclotron or
crystallization from a crystallization solvent. In one embodiment,
said separating the endo and exo cycloadducts comprises separating
the endo and exo cycloadducts to obtain the endo cycloadduct.
[0032] In one embodiment, the method further comprises activating Y
with an activating reagent, preferably to obtain an activated
compound or compound 2. In one embodiment, the activated compound
comprises an activated ester or acid coupled to Y, or preferably,
an acyl halide, carboxyl, succinamyl (2,5-dioxo-1-pyrrolidinyl)
carbonic ester or alkoxycarbonyl coupled to Y, wherein said acyl
halide comprises --F, --Cl, --Br or --I, or preferably, the acyl
halide is chloroformyl or bromoformyl. In one embodiment, the
activating reagent comprises phosgene, diphosgene, triphosgene,
4-nitrophenylchloroformate or carbonyl diimidazole.
[0033] In one embodiment, the method further comprises coupling a
linker to the activated compound or compound 2 to obtain compound
1, wherein the linker is preferably a compound of structural
formula 8:
##STR00005##
wherein X, R and n are as defined above in respect of compound 1.
In one embodiment, one or both of X and R are alkylamino.
[0034] In an alternative embodiment, the linker comprises
optionally substituted straight chain or branched alkyl having a
pair of functionalized ends, wherein one said end is functionalized
with a nucleophilic heteroatom and the other said end is
functionalized with carboxyl or protected or masked carboxyl, and
wherein the alkyl of the linker comprises 3 to 10 methylene groups
between the functionalized ends. In one embodiment, the
nucleophilic heteroatom is amino, hydroxyl or thiol. In one
embodiment, the linker is a cyclization spacer.
[0035] In yet another aspect, the present invention provides a
cysteine residue for use in peptide synthesis, preferably solid
phase peptide synthesis or more preferably Fmoc or Boc solid phase
peptide synthesis, the cysteine residue having structural formula
3:
##STR00006##
wherein R.sub.4 is Fmoc or Boc, and R.sub.3 is a protecting group
compatible with coupling of the protecting group on the thiol side
chain.
[0036] In one embodiment, R.sub.3 is allyl, tert-butyldimethylsilyl
or TBS, methoxymethyl or MOM, ethoxymethyl or EOM, methyl,
p-methoxybenzyl or p-nitrobenzyl. In one embodiment, R.sub.3 is not
a thermally labile or hydrogenolysis-labile protecting group. In
one embodiment, the cysteine residue is selected for use with
compound 1 or 2.
[0037] In one preferred embodiment, compound 3 having as R.sub.4
Fmoc and as R.sub.3 allyl is prepared from a commercially available
compound 4 through sequential allyl protection and trityl
deprotection;
##STR00007##
[0038] It is to be appreciated that the cysteine residue may
include an L-cysteine, a D-cysteine or a combination thereof.
[0039] In yet another aspect, the present invention provides a
protected cysteine residue for peptide synthesis, the protected
cysteine residue having structural formula 9 or 10:
##STR00008##
wherein R.sub.1 to R.sub.4, X, Y and n are as defined herein.
[0040] In yet another aspect, the present invention provides a
protected cysteine having structural formula 9 or 10:
##STR00009##
wherein X and Y are independently of each other oxygen, sulfur,
nitrogen or phosphorus; R.sub.1 and R.sub.2 are independently of
each other hydrogen, hydroxyl, halo, alkyl, alkenyl, alkynyl,
cycloalkyl, heterocycloalkyl, aryl, aralkyl, heteroaryl,
heteroaralkyl, formyl, haloformyl, carbonyl, carboxyl, alkoxy,
alkoxycarbonyl, (alkoxycarbonyl)oxy, carbamoyl, amino, amido,
imino, imido, azo, cyanato, isocyanato, cyano, nitro, sulfanyl,
thiocyanato or phosphono, each of which is optionally substituted;
R.sub.3 and R.sub.4 are independently each other hydrogen or a
protecting group; and n is an integer between 1 and 12,
inclusive.
[0041] In one embodiment, R.sub.3 and R.sub.4 are independently
each other hydrogen, alkyl, allyl, tert-Butyloxycarbonyl (Boc),
fluorenylmethyloxycarbonyl (Fmoc), tert-butyldimethylsilyl (TB S),
methoxymethyl (MOM), ethoxymethyl (EOM), p-methoxybenzyl or
p-nitrobenzyl. In one embodiment, R.sub.3 is hydrogen or allyl and
R.sub.4 is Fmoc.
[0042] In one embodiment, R is an electron withdrawing group
selected to facilitate coupling of the protecting group to the
thiol side chain of the cysteine residue. In one embodiment, the
electron withdrawing group is selected to draw electrons from the
adjacent carbonyl or the carbon atom thereof. In one embodiment,
the electron withdrawing group is selected to draw electrons from
the adjacent carbonyl to facilitate a substitution reaction with
the thiol side chain, wherein in the substitution reaction, the
electron withdrawing group is replaced by the thiol side chain. In
one embodiment, the electron withdrawing group is a group selected
to reduce electron density of the moiety to which it is attached
(relative to the density of the moiety without the substituent). In
one embodiment, the electron withdrawing group is nitro, haloalkyl,
halo, formyl, haloformyl, alkanoyl, alkylsulfonyl, cyano,
alkylsulfinyl, carboxyl, alkoxycarbonyl, sulfonamido, amido,
CONR.sup.10R.sup.20, wherein R.sup.10 and R.sup.20 are
independently of each other hydrogen, alkyl, aryl, arylalkyl,
heterocycloalkyl or cycloalkyl.
[0043] In one embodiment, R is a leaving group selected to
facilitate coupling of the protecting group to the thiol side chain
of the cysteine residue. In one embodiment, the leaving group is a
species or moiety selected to detach from the protecting group
during a reaction, such as a substitution reaction. In one
embodiment, the leaving group is dinitrogen, triflate, halogen,
hydroxyl, amino, alkoxy, acyloxy (preferably --OAc,
--OC(O)CF.sub.3), sulfonate (preferably mesyl or tosyl), acetamide
(preferably --NHC(O)Me), carbamate (preferably N(Me)C(O)Ot-Bu),
phosphonate (preferably --OP(O)(OEt).sub.2) or alcohol.
[0044] In one embodiment, X is sulfur, oxygen or nitrogen, and Y is
oxygen or sulfur, preferably, X is nitrogen and Y is oxygen.
[0045] In one embodiment, the term "alkyl" refers to a
straight-chained or branched hydrocarbon group containing 1 to 12
carbon atoms. The alkyl may include lower alkyl, referring to a
Cl-C6 alkyl chain. Examples of the alkyl group include methyl,
ethyl, n-propyl, isopropyl, tert-butyl, and n-pentyl. The Alkyl
group may be optionally substituted with one or more
substituents.
[0046] In one embodiment, the term "alkenyl" refers to an
unsaturated hydrocarbon chain that may be a straight chain or
branched chain, containing 2 to 12 carbon atoms and at least one
carbon-carbon double bond. The Alkenyl group may be optionally
substituted with one or more substituents. In one embodiment, the
term "alkynyl" refers to an unsaturated hydrocarbon chain that may
be a straight chain or branched chain, containing 2 to 12 carbon
atoms and at least one carbon-carbon triple bond. The alkynyl
groups may be optionally substituted with one or more substituents.
The sp.sup.2 or sp carbons of the alkenyl or alkynyl group may
optionally be the point of attachment of the group.
[0047] In one embodiment, the term "alkylene" refers to an alkyl
group that has two points of attachment, and may preferably include
(C1-C6) alkylene. In one embodiment, the alkylene is methylene,
ethylene, n-propylene or isopropylene.
[0048] In one embodiment, the term "amino" refers to a functional
group having a nitrogen atom bonded to two hydrogen atoms, where
one or both of the hydrogen atoms may optionally be substituted,
preferably but not limited to, alkyl or aryl, i.e., the amino
includes primary, secondary, tertiary or quaternary amino. For
instance, the amino includes alkylamino, dialkylamino or
trialkylamino.
[0049] In one embodiment, the term "cycloalkyl" refers to a
hydrocarbon 3-8 membered monocyclic or 7-14 membered bicyclic ring
system having at least one non-aromatic ring. The Cycloalkyl group
is optionally substituted with one or more substituents, and may be
cyclopropyl, cyclopentyl, cyclohexyl, cyclobutyl, cycloheptyl,
cyclooctyl, cyclononyl or cyclodecyl.
[0050] In one embodiment, the term "heterocycloalkyl" refers to a
nonaromatic 5-8 membered monocyclic, 8-12 membered bicyclic or
11-14 membered tricyclic ring system comprising 1-3 heteroatoms if
monocyclic, 1-6 heteroatoms if bicyclic or 1-9 heteroatoms if
tricyclic, said heteroatoms being O, N, S, B, P or Si. The
heterocycloalkyl is optionally substituted with one or more
substituents. In one embodiment, the heterocycloalkyl is
piperidinyl, piperazinyl, 2-oxopiperazinyl, 2-oxopiperidinyl,
2-oxopyrrolidinyl, 4-piperidonyl, tetrahydropyranyl,
tetrahydrothiopyranyl, tetrahydrothiopyranyl sulfone, morpholinyl,
thiomorpholinyl, thiomorpholinyl sulfoxide, thiomorpholinyl
sulfone, 1,3-dioxolane, tetrahydrofuranyl, tetrahydrothienyl or
thiirene.
[0051] In one embodiment, the term "aryl" refers to a hydrocarbon
monocyclic, bicyclic or tricyclic aromatic ring system, and which
is optionally substituted with one or more substituents. In one
embodiment, the aryl is phenyl, naphthyl, anthracenyl, fluorenyl,
indenyl or azulenyl.
[0052] In one embodiment, the term "aralkyl" refers to aryl
attached to another group by a (C1-C6)alkylene group. The aralkyl
is optionally substituted, either on the aryl portion or the
alkylene portion of the aralkyl, with one or more substituent. In
one embodiment, the aralkyl is benzyl, 2-phenyl-ethyl or
naphth-3-yl-methyl.
[0053] In one embodiment, the term "heteroaryl" refers to an
aromatic 5-8 membered monocyclic, 8-12 membered bicyclic or 11-14
membered tricyclic ring system having 1-4 ring heteroatoms if
monocyclic, 1-6 heteroatoms if bicyclic or 1-9 heteroatoms if
tricyclic, where the heteroatoms are independently O, N or S, and
the remainder ring atoms are carbon. The heteroaryl is optionally
substituted with one or more substituents. In one embodiment, the
heteroaryl is pyridyl, 1-oxo-pyridyl, furanyl, benzo[1,3]dioxolyl,
benzo[1,4]clioxinyl, thienyl, pyrrolyl, oxazolyl, oxadiazolyl,
imidazolyl, thiazolyl, isoxazolyl, quinolinyl, pyrazolyl,
isothiazolyl, pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl,
triazolyl, thiadiazolyl, isoquinolinyl, indazolyl, benzoxazolyl,
benzofuryl, indolizinyl, imidazopyridyl, tetrazolyl,
benzimidazolyl, benzothiazolyl, benzothiadiazolyl, benzoxadiazolyl,
indolyl, tetrahydroindolyl, azaindolyl, imidazopyridyl,
quinazolinyl, purinyl, pyrrolo[2,3]pyrimidinyl,
pyrazolo[3,4]pyrimidinyl, and benzo[b]thienyl,
3H-thiazolo[2,3-c][1,2,4]thiadiazolyl,
imidazo[1,2-d]-1,2,4-thiadiazolyl,
imidazo[2,1b]-1,3,4-thiadiazolyl,
1H,2H-furo[3,4-d]-1,2,3-thiadiazolyl,
1H-pyrazolo[5,1-c]-1,2,4-triazolyl, pyrrolo[3,4-d]-1,2,3-triazolyl,
cyclopentatriazolyl or pyrrolo[2,1b]oxazolyl.
[0054] In one embodiment, the term "heteroaralkyl" or
"heteroarylalkyl" means a heteroaryl group attached to another
group by a (C1-C6)alkylene. The heteroaralkyl may be optionally
substituted, either on the heteroaryl portion or the alkylene
portion of the heteroaralkyl, with one or more substituent. In one
embodiment, the heteroaralkyl is 2-(pyridin-4-yl)-propyl,
2-(thien-3-yl)-ethyl or imidazol-4-yl-methyl.
[0055] In one embodiment, the term "alkoxy" refers to an -O-alkyl
radical.
[0056] In one embodiment, the term "ester" refers to a
--C(O)OR.sup.30, wherein R.sup.30 is preferably alkyl or aryl.
[0057] In one embodiment, the term "halogen" or "halo" is --F,
--Cl, --Br or --I. In one embodiment, the term "haloalkyl" is an
alkyl group in which one or more hydrogen radicals are replaced by
halogen, and may include perhaloalkyl. In one embodiment, the
haloalkyl is trifluoromethyl, difluoromethyl, bromomethyl,
1,2-dichloroethyl, 4-iodobutyl or 2-fluoropentyl.
[0058] In one embodiment, the term "substituent" or "substituted"
means that a hydrogen radical is replaced with a group that does
not substantially adversely affect the stability or activity of the
compound. The term "substituted" refers to one or more
substituents, which may be the same or different, each replacing a
hydrogen atom. In one embodiment, the substituent is halogen,
hydroxyl, amino, alkylamino, arylamino, dialkylamino, diarylamino,
cyano, nitro, mercapto, oxo, carbonyl, thio, imino, formyl,
carbamido, carbamyl, carboxyl, thioureido, thiocyanato, sulfoamido,
sulfonylalkyl, sulfonylaryl, alkyl, alkenyl, alkoxy,
mercaptoalkoxy, aryl, heteroaryl, cyclyl, heterocyclyl, wherein
alkyl, alkenyl, alkyloxy, aryl, heteroaryl, cyclyl and heterocyclyl
are optionally substituted with alkyl, aryl, heteroaryl, halogen,
hydroxyl, amino, mercapto, cyano, nitro, oxo, thioxo or imino.
[0059] It is to be appreciated that specific moieties recited in
the definitions of the above variable groups, including, but not
limited to, R and R.sub.1 to R.sub.4, may be optionally
substituted.
BRIEF DESCRIPTION OF THE DRAWINGS
[0060] Reference may now be had to the following detailed
description taken together with the accompanying drawings in
which:
[0061] FIG. 1 is a reaction mechanism illustrating a process for
synthesizing a peptide in accordance with a preferred embodiment of
the present invention, and which involves solid-phase synthesis of
the peptide with a protected cysteine residue and subsequent
deprotection of the protected cysteine residue to produce a free
cysteine available to form a disulfide bond;
[0062] FIG. 2 is a scheme illustrating a process for synthesizing a
peptide in accordance with a preferred embodiment of the present
invention, and which involves formation of two disulfide bonds at
60.degree. C. and 100.degree. C.;
[0063] FIG. 3 shows on the upper end, the example of a reverse
Diels-Alder reaction for compound 188, and on the lower end a
series of .sup.1H NMR spectra taken of an initial sample of
compound 188 after specific timepoints (selected timepoints only).
Specific protons in the starting material and furna product are
highlighted; the relative integration of the two indicated signals
can be used to quantify the conversion of the reaction. This was
cross-checked with three other signals and all measurements give
the same values of conversion; and
[0064] FIG. 4 shows a deprotection curve showing release of a
preferred protecting group from a protected cysteine residue 188,
with the Y-axis represents the percent release of the cysteine and
the X-axis represents the time in minutes. The Y values do not
reach 100% due to the high concentration of the experiment, and the
restoration of the cyloadduct following release. Reaction is
essentially complete after 40 minutes. This figure corresponds to
the experimental series provided in FIG. 3.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0065] In one aspect, the invention provides a set of cysteine
building blocks for solid-phase peptide synthesis designed for the
selective formation of disulfide bonds after synthesis. They can be
incorporated into normal peptide synthesis using either Fmoc or Boc
strategies (accounting for >99% of all syntheses) without
necessarily requiring any need for modification or
customization.
[0066] It has been recognized disulfide bonds may be essential for
biological activity of peptides and proteins, and may assist
organizing the three-dimensional shape. The peptide synthesis gives
us the linear order of amino acids but does not in and of itself
help us make the three-dimensional shapes. To make the
three-dimensional shapes, you need to make the disulfide bonds
after the peptide synthesis. This may require selectively
deprotecting each pair of cysteines in turn, and thus requiring
many different pairs of protecting group strategies if you have
multiple disulfide bonds, as well as special reagents and very
expensive, customized residues that need to be developed, tested,
and evaluated for each specific case, as there are no general
solutions currently.
[0067] This means everything must be redesigned every time,
although it has been proposed that escalating conditions can be
used to control selective deprotection. Acid-sensitive protecting
groups are potentially the best candidates, with the idea being
that a protecting group that cleaves at pH 5 will cleave 100-times
faster than one that cleaves at 3, which in turn will cleave
100-times faster than one that cleaves at 1. It has been recognized
however that the problem with this approach is that 100-times is
often insufficient, and in this case you are limited to three pairs
of protecting groups; this means that this approach has not worked
very well. Finally, there may be a lot of unintentional
deprotection during synthesis because neutral conditions will still
result in cleavage. The publication "Direct palladium-mediated
on-resin disulfide formation from Allocam protected peptides",
Organic & Biomolecular Chemistry, 15.14 (2017): 2914-2918 to
Stockdill reports using allylation chemistry to selectively
deprotect cysteines, but the required levels of discrimination were
not possible to achieve, and the reaction requires a lot of some
very expensive reagents (metallic palladium) that need to be
removed from the reaction mixture prior to biological use.
[0068] Therefore, it has been appreciated that a preferred approach
may allow for a one-pot sequential and selective formation of the
required disulfide bonds with no or less reagents needed, and no or
less purifications or changes in the reaction mixture composition
over the course of the reaction. It has been recognized that one
possibility for a reagent-free approach may use heat. Most stable
chemical bonds may not be highly heat-sensitive, however,
retro-cycloadditions can be highly temperature sensitive, including
Diels-Alder cycloadducts formed between dienes and dienophiles.
[0069] The applicant has recognized that reactions between furans
and maleimides may be promising due to near-physiological
temperatures which may be involved in the retro-Diels-Alder
reaction. It has been envisioned that this may be due to a
combination of a highly favored forward reaction with a very low
lying lowest unoccupied molecular orbital or LUMO for the
maleimide, and a favorable reverse reaction as furan is aromatic.
It has also been envisioned that the precise thermodynamics and the
temperature required to overcome the reversible energy barrier may
be modulated through adjusting the electronics of the transition
states of the reaction, and by changing the substituents on the
furan and maleimide.
[0070] It has been envisioned that a process for synthesizing a
peptide using the protected cysteine residue of the invention may
be practiced "reagent-free" or with a reduced number of required
reagents, and with use of heat as the stimulus for deprotection and
formation of disulfide bonds. To get around the background cleavage
issue, it may be possible to raise the initial temperature to
60.degree. C., which may provide background cleavage at ambient
temperature (23.degree. C.) at about 2000 times slower than at
60.degree. C. The following preferred non-limiting protected
cysteine residue may decompose at different temperatures to allow
for disassembly, while being reasonably stable at room temperature
or at least sufficiently stable to handle and use for peptide
synthesis:
##STR00010##
wherein R.sub.1 is hydrogen, nitro, bromine or methoxy, and R.sub.2
is p-methoxyphenyl, p-nitrophenyl or benzyl. The above preferred
non-limiting cysteine residue is protected with a protecting group
having two components, or namely a Diels-Alder cycloadduct of furan
and maleimide and a linker or cyclization spacer interposed between
the thiol side chain and the Diels-Alder adduct.
[0071] It has been recognized that some background cleavage may
remain if one set of protecting groups cleave at 60.degree. C., and
the second at 80.degree. C. So, the protecting group of the current
invention most preferably incorporates the Diels-Alder cycloadduct
coupled with a second gating mechanism, or namely a cyclization
spacer. It has been recognized that smaller rings (for instance,
down to 5 atoms) form faster than larger rings. So, a low
temperature trigger coupled with a 5-membered spacer may liberate a
cysteine much faster than a mid-temperature trigger with a
6-membered spacer (the 5-membered ring closes about 100-times
faster than a 6-membered ring, which in turn is 100 times faster
than a 7, which is about 100-times faster than an 8, which is 1000
times faster than a 9; differentiation between ring sizes may cease
to significantly matter much beyond this point). Consequently, even
if the higher temperature thermally-active protecting group falls
off at a lower temperature than expected, the liberated cyclization
spacer is less likely to cyclize, and the cysteine will not be
easily liberated. This dual gating may permit each pair of
cysteines to be liberated or deprotected in turn, and will then
dimerize to provide the disulfide bond before the next pair of
cysteines is liberated.
[0072] The applicant has appreciated that consequently, the
deprotection reaction may proceed without the need for any change
in conditions, except for a gradual increase in temperature. The
products of the reactions may be innocuous, and may include for
example a maleimide, a furan and a cyclic lactam, which may be
readily separated from the peptide by precipitation of the
resulting peptide or an aqueous-organic extraction to remove the
organic-soluble byproducts from the reaction mixture. A sacrificial
amount of hindered thiol may be required to scavenge the maleimide
if maleimide-thiol reactions are a possible complication in
specific cases, although the high dilution of these reaction
mixtures renders this an uncommon prospect as the two cysteines in
the molecule are held in close proximity.
[0073] It has been envisioned that while slow reaction rates of the
different cyclization spacers may potentially lead to problems,
this may be counteracted by slowly increasing temperature, i.e., as
the temperature rises, the rates of reactions increase, and a
spacer that is slow to cyclize at 60.degree. C. will cyclize far
faster at 80 or 100 .degree. C. With different combinations of the
Diels-Alder cycloadduct with varying substituents and the
cyclization spacer with varying lengths, it may be possible to
devise a series of protecting groups that will be thermally
triggerable at 15 to 20.degree. C. increments starting at 45
.degree. C., and allow for the formation of 5 different systems,
preferably triggerable at 45.degree. C., 60.degree. C., 75.degree.
C., 90.degree. C. and 105.degree. C., within reasonable temperature
ranges. It is expected the higher temperatures may denature large
proteins, and the current invention may preferably encompass
processes for synthesizing peptides, such as insulin and the
non-addictive conotoxin pain-killers, where the higher temperatures
are not expected to be significantly problematic. The applicant has
recognized that the higher temperature ranges are often used during
peptide synthesis in the microwave reactors attached to many modern
peptide synthesis machines. Finally, the cyclization could occur
before cleavage from the solid-support, further simplifying
purification and improving yields.
[0074] Reference is made to FIG. 1 which illustrates a reaction
pathway for deprotection of the protected cysteine residue after
peptide synthesis. First, a crude peptide was synthesized with
Fmoc-protected amino acids, including an Fmoc-protected cysteine
shown in FIG. 1 with a protecting group having a 7-oxanorbornene
construct with R.sub.1 and R.sub.2, coupled with a cyclization
spacer having the variable length n. While not wishing to be bound
by a theory, it has been appreciated that the 2-methylsubstituted
furan ring forming part of the protecting group may possess natural
instability which may be masked when incorporated as part of the
7-oxanorbornene construct with maleimide, and with thermal
decomposition of that construct, the unstable 2-methylsubstituted
furan ring is removed from the cyclization spacer.
[0075] Reference is made to FIG. 2 which illustrates a possible
reaction pathway for formation of two disulfide bonds sequentially
between a pair of protected thiol side chains "SR.sub.1" and then
between a pair of thiol side chains "SR.sub.2". For the sequential
formation of the two disulfide bonds, a reaction mixture containing
synthesized peptides incorporating cysteine residues having pairs
of SR.sub.1 and SR.sub.2 is heated first to about 60.degree. C. to
effect deprotection of the pair of SR.sub.1 and form a disulfide
bond therebetween. Then the temperature is raised to 100.degree. C.
to do the same with the two SR.sub.2.
[0076] Reference is made to FIG. 3 which demonstrates that at XXX
.degree. C., the endcap is essentially completely removed from the
cysteine in YYY minutes and that this change can be readily
monitored by nuclear magnetic resonance.
[0077] Reference is made to FIG. 4 which demonstrates a typical
experiment, like that in FIG. 3, showing the end-cap removal and
release of free cysteine as a function of time. This particular
example relates to the spectra provided in FIG. 3, compound 188
releasing the end-cap at YYY .degree. C.
[0078] In another preferred embodiment, there is provided a
thermally-sensitive protecting group provided with a cycloadduct of
a 5-substituted furfural alcohol and an N-alkyl or
N-aryl-substituted maleimide, and which may be configured to permit
different thermal sensitive triggers from 40 .degree. C. to 120
.degree. C., and which may permit attachment to a protected
cysteine amino acid for a solid-phase synthesis, such as Fmoc
solid-phase synthesis. Again, heat may be an ideal trigger for
selective deprotection and disulfide bond formation, without
necessarily requiring use of a reagent, and which may be applicable
to conditions with a greater dynamic range, lower background
cleavage under standard operations, and reduced interference with
ideal reaction conditions for disulfide bond formation.
[0079] Preferably, the protecting group is compound having
structural formula 1 or 2 as shown below:
##STR00011##
wherein R is an activated ester or acid or forms part thereof; X is
sulfur, oxygen or nitrogen; Y is oxygen or sulfur; R.sub.1 and
R.sub.2 are alkyl, aryl, a halogen, an ether, a thioether, a
dialkylamine, trialkylammonium, an ester or an acid derivative
thereof, or a ketone; and n is an integer from 1 to 9. It is to be
appreciated that compound 2 is provided without a linker included
with compound 1 for direct attachment to a cysteine residue.
[0080] Compounds 1 and 2 were made through the Diels-Alder reaction
between the appropriate hydroxymethyl furan and maleimide using
temperatures of reaction between 0 and 90.degree. C., and solvents,
such as benzene, acetonitrile, chloroform, dichloromethane,
tetrahydrofuran, DMSO, DMF, toluene, xylene, hydrocarbon solvents,
dichloroethane, tetrachloroethane, dioxane, methanol and/or
isopropanol with a reaction times between 15 minutes and 48 hours.
The endo and exo cycloadducts were separated using column
chromatography, HPLC or crystallization from an appropriate
solvent.
[0081] The group Y was then activated using a carbonate equivalent
agent to make a chloroformate or activated carbonate. Preferred
reagents include phosgene, diphosgene, triphosgene,
4-nitrophenylchloroformate, carbonyl diimidazole and others alike.
The foregoing steps could provide for the active protecting group
precursor 2.
[0082] In one embodiment, the activated carbonate thus obtained was
then treated with an alkyl linker with both ends of the chain
functionalized--one with a nucleophilic heteroatom (amine, hydroxyl
or thiol) and the other with a carboxylic acid or protected
carboxylic acid, or masked carboxylic acid. The number of methylene
groups or substituted methylene groups between the two terminal
functionalities may be 3 to 10, inclusive.
[0083] Alternatively, the activated carbonate derivative of the
chloroformate was not further functionalized, in which case the
activated carbonate was used directly with the cysteine
residue.
General Procedures and Materials
[0084] Solvents were purchased from Caledon Labs (Caledon,
Ontario), Sigma-Aldrich (Oakville, Ontario) or VWR Canada
(Mississauga, Ontario). Other chemicals were purchased from
Sigma-Aldrich, AK Scientific, Oakwood Chemicals, Alfa Aesar or
Acros Chemicals and were used without further purification unless
otherwise noted. Anhydrous toluene, tetrahydrofuran (THF), diethyl
ether and N,N-dimethylformamide (DMF) were obtained from an
Innovative Technology (Newburyport, USA) solvent purification
system based on aluminium oxide columns. CH.sub.2C.sub.12,
pyridine, acetonitrile, N,N-diisopropylethylamine (DIPEA) and
NEt.sub.3 were freshly distilled from CaH.sub.2 prior to use.
Purified water was obtained from a Millipore deionization system.
All heated reactions were conducted using oil baths on IKA RET
Basic stir plates equipped with a P1000 temperature probe. Thin
layer chromatography was performed using EMD aluminum-backed silica
60 F254-coated plates and were visualized using either UV-light
(254 nm), KMnO.sub.4, vanillin, Hanessian's stain, or Dragendorff's
stain. Preparative TLC was done using glass-backed silica plates
(Silicycle) of either 250, 500, 1000 or 2000 .mu.m thickness
depending on application. Column chromatography was carried out
using standard flash technique with silica (Siliaflash-P60, 230-400
mesh Silicycle) under compressed air pressure. Standard work-up
procedure for all reactions undergoing an aqueous wash involved
back extraction of every aqueous phase, a drying of the combined
organic phases with anhydrous magnesium sulphate, filtration either
using vacuum and a sintered-glass frit or through a glass-wool plug
using gravity, and concentration under reduced pressure on a rotary
evaporator (Buchi or Synthware). .sup.1H NMR spectra were obtained
at 300 MHz or 500 MHz, and .sup.13C NMR spectra were obtained at 75
or 125 MHz on Bruker instruments. NMR chemical shifts (.delta.) are
reported in ppm and are calibrated against residual solvent signals
of CHCl.sub.3 (.delta.7.26), DMSO-d.sub.5 (.delta.2.50),
acetone-d.sub.5 (.delta.2.05), or methanol-d.sub.3 (.delta.3.31).
HRMS were conducted on a Waters XEVO G2-XS TOF instrument with an
ASAP probe in CI mode. Peptide synthesis was accomplished using a
modified Focus XC-6RV from AAPPTEC controlled by a PC loaded with
Focus-XC software. Lyophilization was accomplished using a Sharp
Freeze -80.degree. C/6L lyophilizer from AAPPTEC equipped with a
10-sample manifold. HPLC purification was conducted using
analytical analysis on either a Waters HPLC with a 2489 UV/Vis
detector and 1525 Binary HPLC pump; or a Varian ProStar HPLC
equipped with two 218 pumps, a 320 UV detector, 330 photodiode
array detector, 351 RI detector, and a 410 autosampler. Preparative
HPLC was conducted using an Interchim puriFlash 5.25 multi HPLC
with four individual solvent pumps.
Synthesis of Cycloadducts:
General Procedure for the Synthesis of Maleamic Acids 5a, 6a,
7a:
[0085] All compounds, or namely N-(4-methoxyphenyl)maleamic acid
5a, N-(4-nitrophenyl)maleamic acid 6a and N-benzylmaleamic acid 7a
were synthesized using methodologies described in Sortino, M. et
al., N-Phenyl and N-phenylalkyl-maleimides acting against Candida
spp.: Time-to-kill, stability, interaction with maleamic acids.
Bioorgan. Med. Chem. 2008, 16 (1), 560-568. DOI:
http://dx.doi.org/10.1016/j.bmc.2007.08.030, the entire content of
which is incorporated herein by reference, with yields ranging from
90% to 91%. Briefly, maleic anhydride (S1) (500 mg, 5.2 mmol) and
equimolar amounts of the required amine were combined in CHCl.sub.3
(6 mL) and stirred for 45 minutes as a precipitate formed. This
precipitate was then filtered and washed with cold (4 .degree. C.)
water. The analytical data for these maleamic acids have been
previously published, and the data was consistent with the
published spectra, as shown in Sortino noted above and in Sortino,
M. et al., Antifungal, cytotoxic and SAR studies of a series of
N-alkyl, N-aryl and N-alkylphenyl-1,4-pyrrolediones and related
compounds. Bioorgan. Med. Chem. 2011, 19 (9), 2823-2834. DOI:
http://dx.doi.org/10.1016/j.bmc.2011.03.038, the entire content of
which is incorporated herein by reference.
General Procedure for Synthesis of Maleimides 5, 6 and 7
[0086] The maleamic acids (5a, 6a or 7a, 4.7 mmol) were dissolved
in 5 mL of acetic anhydride along with sodium acetate (100 mg, 1.2
mmol). The mixture was heated for 2-4 h at 100 .degree. C. (exact
reaction time depended on the substituent), until the reaction was
determined to be complete by TLC. The solution was then cooled,
diluted with water, then extracted repeatedly with ethyl acetate.
The combined organics were dried with magnesium sulfate, and
filtered and concentrated in the usual fashion. The solid residue
was then redissolved in a minimum amount of THF, and precipitated
through the dropwise addition of ice-cold ether. The solid was
recovered, resuspended in additional minimal THF, and precipitated
by addition into cold water. A final filtration provided the
desired maleimides in 73% to 80% yield. Spectroscopic data is
consistent with previous reports, as noted in Sortino, M. et al.,
Antifungal, cytotoxic and SAR studies of a series of N-alkyl,
N-aryl and N-alkylphenyl-1,4-pyrrolediones and related compounds.
Bioorgan. Med. Chem. 2011, 19 (9), 2823-2834. DOI:
http://dx.doi.org/10.1016/j.bmc.2011.03.038.
N-benzyl-Maleimide 7
[0087] Prepared as per the general procedure above using 1 g of
maleic anhydride (2-fold scale of the general protocol). Synthesis
of 7a proceeded for 45 minutes, providing the maleimic acid in 93%
crude yield; the ring closing to 7 required only 1 hour. The crude
mixture was first purified by flash chromatography (7:3
hexanes-ethyl acetate), and the fractions containing the product
were combined, concentrated, and then recrystallized from
2-propanol and water to provide an 80% yield of the title compound,
as white crystals in 75% overall yield after vacuum drying.
[0088] White crystals. .sup.1H NMR (300 MHz, CDCl.sub.3)
.delta..sub.ppm: 7.33-7.22 (5H, m), 6.69 (2H, s), 4.66 (2H, s).
Spectral data are consistent with previously published spectra in
Sortino, M. et al., N-Phenyl and N-phenylalkyl-maleimides acting
against Candida spp.: Time-to-kill, stability, interaction with
maleamic acids. Bioorgan. Med. Chem. 2008, 16 (1), 560-568. DOI:
http://dx.doi.org/10.1016/j.bmc.2007.08.030.
N-(p-methoxyphenyl)-Maleimide 5
[0089] Prepared as per the general procedure above using 12.0 g of
maleic anhydride (12-fold scale of the general protocol). Synthesis
of 5a proceeded for 45 minutes, providing the maleimic acid in 90%
crude yield as a yellow powder. The ring closing (using 130 mL of
acetic anhydride and 5.8 g of sodium acetate) provided a dark
yellow amorphous solid, that after recrystallization as in the
general protocol, was recovered as bright yellow needles in 73%
yield; 66% yield overall from the maleic anhydride. Yellow needles.
.sup.1H NMR (300 MHz, CDCl.sub.3) .delta..sub.ppm: 7.27-7.23 (m,
2H), 7.03-6.98 (m, 2H), 6.85 (s, 2H), 3.80 (s, 3H). Spectral data
are consistent with previously published spectra in Lee, H. S. et
al., Substituent chemical shifts of N-arylsuccinanilic acids,
N-arylsuccinimides, N-arylmaleanilic acids, and N-arylmaleimides.
Magn. Reson. Chem. 2009, 47 (9), 711-715. DOI: 10.1002/mrc.2450,
the entire content of which is incorporated herein by
reference.
N-(p-nitrophenyl)-Maleimide 6
[0090] Prepared as per the general procedure above using 10.0 g of
maleic anhydride (10-fold scale of the general protocol). Synthesis
of 6a proceeded for 2 hours, providing the maleimic acid in 91%
crude yield as brown crystals. The ring closing (using 115 mL of
acetic anhydride and 5.1 g of sodium acetate) provided a dark
yellow amorphous solid, that after recrystallization as in the
general protocol, was recovered as a pale yellow powder in 78%
yield; 71% yield overall from the maleic anhydride. Yellow powder,
.sup.1H NMR (300 MHz, CDCl.sub.3) .delta..sub.ppm: 8.35-8.32 (m,
2H), 7.70-7.60 (m, 2H,), 6.94 (s, 2H). Spectral data are consistent
with previously published spectra in Lee, H. S. et al., Substituent
chemical shifts of N-arylsuccinanilic acids, N-arylsuccinimides,
N-arylmaleanilic acids, and N-arylmaleimides. Magn. Reson. Chem.
2009, 47 (9), 711-715. DOI: 10.1002/mrc.2450.
5-Nitrofurfural diacetate, S3
[0091] Prepared according to a modified version of the protocol
noted in Jin, H. et al., Lead optimization and anti-plant
pathogenic fungi activities of daphneolone analogues from Stellera
chamaejasme L. Pestic. Biochem. Physiol. 2009, 93 (3), 133-137.
DOI: https://doi.org/10.1016/j.pestbp.2009.01.002, the entire
content of which is incorporated herein by reference. A mixture of
8.6 mL concentrated HNO3 and 0.06 mL concentrated H.sub.2SO.sub.4
was slowly added into 90 mL of acetic anhydride while stirring at a
temperature of 0.degree. C. This was followed by the slow addition
of 10.4 mL of furfural, S2, into the acid mixture with stirring and
temperature remaining at 0.degree. C. The mixture was left to stir
at this same temperature for 1 hour. At this time, 80 mL of water
was added and the mixture was left to stir at room temperature for
an additional 30 minutes, over which time a white precipitate
formed. A 10% NaOH solution (10 g of NaOH in 100 mL of water) was
then added to the mixture until the pH rose to 2.5. The mixture was
then heated in a water bath at 55 .degree. C. for 1 hour. After
cooling, the precipitate was filtered and washed with water prior
to being recrystallized from anhydrous ethanol and dried to provide
5.2 g of white crystals in a 75% yield. The material was then used
without further purification. R.sub.f=0.37 (6:4, hexanes-ethyl
acetate).
5-Nitrofurfural, S4
[0092] Prepared according to a modified version of the protocol
noted in Jin, H. et al., Lead optimization and anti-plant
pathogenic fungi activities of daphneolone analogues from Stellera
chamaejasme L. Pestic. Biochem. Physiol. 2009, 93 (3), 133-137.
DOI: https://doi.org/10.1016/j.pestbp.2009.01.002. Previously
prepared 5-nitrofufural diacetate (S3, 5.2 g, 21.4 mmol) was added
to 52 mL of 50% H.sub.2SO.sub.4 and the resulting mixture was
heated using a heat gun (Wagner model #283022 HT 775, 540.degree.
C.) for 2 minutes. After cooling, the hydrolysate was extracted via
ethyl acetate and the organic layer was washed with water, dried
with magnesium sulfate and then filtered and concentrated. A simple
distillation provided, after cooling of the distillate, 2.5 g of
the title compounds, S4, as a yellow-brownish solid in 83%
yield.
[0093] Yellow-brownish solid; R.sub.f=0.43 (1:1, hexanes-ethyl
acetate); .sup.1H NMR (300 MHz, CDCl.sub.3) .delta..sub.ppm: 9.85
(s, 1H), 7.43 (d, J=3.82 Hz, 1H), 7.36 (d, J=3.87 Hz, 1H). These
obtained values are in agreement with previously reported
spectroscopic data noted in Natarajan, P. et al.,
Silver(I)-Promoted ipso-Nitration of Carboxylic Acids by Nitronium
Tetrafluoroborate. J. Org. Chem. 2015, 80 (21), 10498-10504. DOI:
10.1021/acs.joc.5b02133, the entire content of which is
incorporated herein by reference.
5-Nitro-2-furanmethanol, 2:
[0094] Prepared according to a modified version of the protocol of
noted in Emami, S. et al., 7-Piperazinylquinolones with
methylene-bridged nitrofuran scaffold as new antibacterial agents.
Med. Chem. Res. 2013, 22 (12), 5940-5947. DOI:
10.1007/s00044-013-0581-9, the entire content of which is
incorporated herein by reference. 5-nitrofurfural (S4, 2.22 g, 15.7
mmol) was dissolved in 47 mL of absolute methanol and the solution
was cooled to 0.degree. C. Then NaBH.sub.4 (0.65 g, 0.017 mol) was
slowly added and the solution was stirred for another 30 minutes.
Once the reaction was complete, the solvent was removed under
reduced pressure and the residue was then dissolved in a minimum
amount of water. This solution was extracted with diethyl ether
(3.times.10 mL). The combined organic phases were then washed with
water, dried with magnesium sulfate, filtered and concentrated.
This provided 0.78 g of the title compound as a yellow oil in a
moderate 35% yield.
[0095] Yellow oil; R.sub.f=0.38 (1:1, hexanes-ethyl acetate);
.sup.1H NMR (300 MHz, CDCl.sub.3) .delta..sub.ppm: 7.28 (1 H, d,
J=3.71), 6.55 (1 H, d, J=3.58), 4.70 (2 H, s), 2.68 (1 H, bs).
These obtained values are in agreement with previously reported
spectroscopic data noted in Berry, J. M. et al.,
5-Nitrofuran-2-ylmethyl group as a potential bioreductively
activated pro-drug system. J Chem. Soc. Perkin. Trans. 1 1997, (8),
1147-1156. DOI: 10.1039/a607202j, the entire content of which is
incorporated herein by reference.
Methyl 5-bromo-2-furoate S6
[0096] Prepared according to the approach noted in Torii, S. et
al., Anodic Reaction of 2-Furoic Acids. II. Electrolysis of Methyl
5-Acetyl-2-furoate and Its Homologous in Protic Solvents. Bull.
Chem. Soc. Jpn. 1972, 45 (9), 2783-2787. DOI: 10.1246/bcsj.45.2783,
the entire content of which is incorporated herein by reference.
Bromine (6.07 g, 0.038 mol) was carefully added (dropwise over a
period of 15 minutes) to a solution of methyl furoate (S5, 3.2 g,
0.025 mol) stirred at 50.degree. C. under an argon atmosphere in a
flame-dried round-bottom flask. The resulting dark orange/brownish
solution was additionally stirred for another 15 minutes at
50.degree. C. The reaction mixture was then poured into cold water
(10 mL) and extracted with ethyl acetate (2.times.50 mL). The
combined extracts were washed with water (1.times.10 mL) and brine
(1.times.10 mL) prior to being dried with magnesium sulfate and
concentrated. The final product was purified by flash
chromatography (10:1, hexanes-ethyl acetate) to obtain 4.5 g of S6
in 85% yield. The spectral data was consistent with literature
reports as noted in Torii above. R.sub.f=0.17 (7:3, hexanes-ethyl
acetate).
5-Bromo-2-furanmethanol 3
[0097] Prepared according to a modified version of the approach
noted in Bi, J. et al., Application of furyl-stabilized sulfur
ylides to a concise synthesis of 8a-epi-swainsonine. Chem. Commun.
2008, (1), 120-122. DOI: 10.1039/b713447a, the entire content of
which is incorporated herein by reference. A stirred solution of
methyl 5-bromo-2-furoate (S6, 6.7 g, 4.6 mmol) in anhydrous THF
(111 mL) was cooled to 0.degree. C. LiAlH.sub.4 (1.4 g, 5.1 mmol)
was carefully added to the reaction mixture, which was then stirred
for a period of 15 minutes. Then, the reaction mixture was warmed
to room temperature over a period of 45 minutes prior to a standard
Fieser quench and filtration (see (a) Fieser, L. F. et al.,
Reagents for Organic Synthesis. Wiley: New York, 1976; p 1140; (b)
Amundsen, L. H. et al., Reduction of Nitriles to Primary Amines
with Lithium Aluminum Hydridel. J. Am. Chem. Soc. 1951, 73 (1),
242-244. DOI: 10.1021/ja01145a082, the entire contents of both of
which are incorporated herein by reference); the THF was mostly
removed via rotary evaporation. The resulting crude was diluted
with ethyl acetate (200 mL) and washed with water (50 mL) and brine
(50 mL). It was then dried over magnesium sulfate and concentrated
and the final product, 3.9 g of a colorless oil, was obtained after
flash chromatography (5:1, hexanes-ethyl acetate) in 65% yield.
Spectral data is consistent with the published data, as noted in Bi
above. R.sub.f0.50 (8:2, hexanes-ethyl acetate).
Methyl 5-methoxy-2-furoate S7
[0098] Prepared according to a modified version of the protocol of
Torii, S. et al., Anodic Reaction of 2-Furoic Acids. II.
Electrolysis of Methyl 5-Acetyl-2-furoate and Its Homologous in
Protic Solvents. Bull. Chem. Soc. Jpn. 1972, 45 (9), 2783-2787.
DOI: 10.1246/bcsj.45.2783. Methyl 5-bromo-2-furoate (S6, 2.2 g,
10.7 mmol) was added to a solution of sodium (0.6 g) and sodium
iodide (0.03 g) in 30 ml of absolute methanol. The solution was
refluxed for 7 hours, then poured into cold water (100 mL). The
mixture was then extracted thrice with diethyl ether, and the
combined organics were dried over magnesium sulfate, filtered and
concentrated. Chromatography of the resulting residue (8:2,
hexanes-ethyl acetate) provided 0.8 g of the title compound as an
oily product in 48% yield.
[0099] Clear oil; R.sub.f=0.50 (8:2, hexanes-ethyl acetate);
.sup.1H NMR (300 MHz, CDCl.sub.3) .delta..sub.ppm: 7.13 (d, J=3.69
Hz, 1H), 5.33 (d, J=3.60 Hz, 1H), 3.96 (s, 3H), 3.86 (s, 3H).
Spectral data is in agreement with published data noted in
Schwartz, D. A. et al., Synthetic approaches to haplophytine. 2.
Synthesis of
4-methylamino-1-(2-furanyl)-2-phenyl-2-(2-pivaloylamidophenyl)butan-1-one-
. Can. J. Chem. 1983, 61 (6), 1126-1131. DOI: 10.1139/v83-201, the
entire content of which is incorporated herein by reference.
5-methoxy-2-furanmethanol 4
[0100] Prepared according to a modified version of the protocol
noted in Manly, D. G. et al., Simple Furan Ethers. II: 2-Alkoxy-
and 2-Aryloxy-furans. J. Org. Chem. 1956, 21 (5), 516-519. DOI:
10.1021/jo01111a008, the entire content of which is incorporated
herein by reference. A solution of methyl 5-methoxy-2-furoate (S7,
0.45 g) in 2 mL of dry ether was slowly added to a fast-stirring
solution of LiAlH.sub.4 (0.14 g) in 4.5 mL of dry ether. After a
1.5 hour reflux, 0.3 mL of water was carefully added prior to 1.9
mL of sodium hydroxide. The reaction mixture was diluted with
ether, and the phases separated. The aqueous layer was then
extracted several times with ether, and the combined ether extracts
were dried and evaporated to provide 150 mg of the title compound
as a colorless liquid in 40% yield. No further purification was
required. Colourless oil; R.sub.f=0.21 (3:7, hexanes-ethyl
acetate); .sup.1H NMR (300 MHz, CDCl.sub.3) .delta..sub.ppm: 6.09
(d, J=3.30 Hz, 1H), 5.03 (d, J=3.30 Hz, 1H), 4.37 (s, 2H), 3.78 (s,
3H), 2.89 (bs, 1H). Spectral data is in agreement with published
data noted in Schwartz, D. A. et al., Synthetic approaches to
haplophytine. 2. Synthesis of
4-methylamino-1-(2-furanyl)-2-phenyl-2-(2-pivaloylamidophenyl)butan-1-one-
. Can. J. Chem. 1983, 61 (6), 1126-1131. DOI: 10.1139/v83-201.
rac-(3aR,4R,7S,7aS)-4-(hydroxymethyl)-2-(4-methoxyphenyl)-3a,4,7,7a-tetrah-
ydro-1H-4,7-epoxyisoindole-1,3(2H)-dione (15.sub.endo) and
rac-(3aS,4R,7S,7aR)-4-(hydroxymethyl)-2-(4-methoxyphenyl)-3a,4,7,7a-tetra-
hydro-1H-4,7-epoxyisoindole-1,3(2H)-dione (15.sub.exo)
[0101] N-(p-methoxyphenyl)-Maleimide 5 (0.5 g, 2.67 mmol) and
2-(hydroxymethyl) furan 1 (0.31 g, 3.2 mmol) were dissolved in
anhydrous acetonitrile under a nitrogen atmosphere in a flame-dried
flask equipped with a magnetic stirring-bar. The reaction was
stirred at 35.degree. C. for 18 hours. When TLC indicated there was
no longer starting material present, the solvent was removed, and
the reaction was concentrated under reduced pressure for 1 hour.
Endo and exo cycloadducts was separated by column (6:4 to 4:6,
hexanes-ethyl acetate). We obtained two fractions, one contained
690 mg of pure endo material (85% yield), while the second (<5%)
contained 26 mg of a mixture of the exo and endo isomers. Because
these compounds have an inherently unstable nature at ambient
temperatures, they are kept stored at -20.degree. C. until
required.
rac-(3aR,4R,7S,7aS)-4-(hydroxymethyl)-2-(4-methoxyphenyl)-3a,4,7,7a-tetrah-
ydro-1H-4,7-epoxyisoindole-1,3(2H)-dione (15.sub.endo)
[0102] R.sub.f=0.30 (2:8, hexanes-ethyl acetate); .sup.1H NMR (300
MHz, CD.sub.3CN): .delta..sub.ppm 7.17-7.12 (m, 4H), 6.71 (dd,
J=5.0, 1.5 Hz, 1H), 6.62 (dd, J=5.8, 1.6 Hz, 1H), 5.45 (d, J=6.7
Hz, 1H), 4.32 (dd, J=12.94, 5.67 Hz, 1H), 4.20 (dd, J=12.82, 6.37
Hz, 1H), 3.95 (s, 3H), 3.93-3.85 (m, 1H), 3.66 (d, J=7.67 Hz, 1H),
3.31 (t, J=5.88 Hz, 1H, OH); .sup.13C NMR (75 MHz, CD.sub.3CN):
.delta..sub.ppm 175.0, 174.7, 159.7, 135.7, 135.4, 128.0, 124.9,
114.0, 92.7, 79.5, 60.5, 55.3, 48.1, 45.7; HRMS (CI): Calculated
for [M].sup.+ (C.sub.16H.sub.15NO.sub.5): 301.0950. Found:
301.0944.
rac-(3aS,4R,7S,7aR)-4-(hydroxymethyl)-2-(4-methoxyphenyl)-3a,4,7,7a-tetrah-
ydro-1H-4,7-epoxyisoindole-1,3(2H)-dione (15.sub.exo)
[0103] R.sub.f=0.19 (1:9, hexanes-ethyl acetate); .sup.1H NMR (300
MHz, CDCl.sub.3): .delta..sub.ppm 7.07-6.89 (m, 4H), 6.65 (d,
J=5.71 Hz, 1H), 6.60-6.55 (m, 1H), 5.36 (d, J=1.65 Hz, 1H),
4.17-4.12 (m, 2H), 3.82 (s, 3H), 3.13 (d, J=6.57 Hz, 1H), 3.09 (d,
J=6.58 Hz, 1H), 2.84 (bs, 1H, OH).
rac-(3aR,4R,7S,7aS)-4-(hydroxymethyl)-2-(4-nitrophenyl)-3a,4,7,7a-tetrahyd-
ro-1H-4,7-epoxyisoindole-1,3(2H)-dione (16endo) and
rac-(3aS,4R,7S,7aR)-4-(hydroxymethyl)-2-(4-nitrophenyl)-3a,4,7,7a-tetrahy-
dro-1H-4,7-epoxyisoindole-1,3(2H)-dione (16.sub.exo)
[0104] N-(p-nitrophenyl)-Maleimide 6 (0.5 g, 2.30 mmol) and
2-(hydroxymethyl) furan 1 (0.273 g, 2.80 mmol) were dissolved in
anhydrous acetonitrile under a nitrogen atmosphere in a flame-dried
flask equipped with a magnetic stirring-bar. The reaction was
stirred at 40.degree. C. After 4.5 hours, a new polar spot formed
but starting material was still present and the reaction was left
to stir at the same temperature overnight. When TLC indicated there
was no longer starting material present, the solvent was removed,
and the reaction was concentrated under reduced pressure. A column
with (7:3 to 3:7, hexanes-ethyl acetate) was used to separate the
components. The pure endo product was separated (100 mg, 13%),
while a mixture of the endo and exo products (300 mg, 39% yield)
was obtained in a second fraction. Ultimately, there was a yield of
52% endo/exo cycloadduct mixture. Since this mixture has an
inherently unstable nature at ambient temperatures, it is kept
stored at -20.degree. C. until required.
rac-(3aR,4R,7S,7aS)-4-(hydroxymethyl)-2-(4-nitrophenyl)-3a,4,7
,7a-tetrahydro-1H-4,7-epoxyisoindole-1,3(2H)-dione
(16.sub.endo)
[0105] Light yellow solid; R.sub.f=0.30 (3:7, hexanes-ethyl
acetate); .sup.1H NMR (300 MHz, CD.sub.3CN): 8.29 (d, J=8.62 Hz,
2H), 7.42 (d, J=8.60 Hz, 2H), 6.60 (d, J=5.76 Hz, 1H), 6.49 (d,
J=5.69 Hz, 1H), 5.34 (d, J=5.50 Hz, 1H), 4.18 (dd, J=12.90, 5.80
Hz, 1H), 4.06 (dd, J=12.89, 6.30 Hz, 1H), 3.81 (dd, J=7.60, 5.61
Hz, 1H), 3.59 (d, J=7.75 Hz, 1H), 3.24 (t, J=6.08 Hz, 1H, OH);
.sup.13C NMR (75 MHz, CDCl.sub.3): .delta..sub.ppm 174.1, 173.8,
147.4, 137.6, 135.8, 135.6, 127.7, 124.4, 92.9, 79.6, 60.3, 48.3,
45.9; HRMS (CI): Calculated for [M].sup.+
(C.sub.15H.sub.12N.sub.2O.sub.6): 316.0695. Found: [M].sup.+
316.0697.
rac-(3aS,4R,7S,7aR)-4-(hydroxymethyl)-2-(4-nitrophenyl)-3a,4,7,7a-tetrahyd-
ro-1H-4,7-epoxyisoindole-1,3(2H)-dione (16.sub.exo)
[0106] White creamy solid; R.sub.f=0.25 (3:7, hexanes-ethyl
acetate); .sup.1H NMR (300 MHz, CDCl.sub.3): .delta..sub.ppm 8.32
(d, J=9.16 Hz, 2H), 7.58 (d, J=9.05 Hz, 2H), 6.67-6.60 (m, 2H),
5.43 (d, J=1.22 Hz, 1H), 4.19 (d, J=7.29 Hz, 2H), 3.24 (d, J=6.53
Hz, 1H), 3.19 (d, J=6.70 Hz, 1H), 2.54 (t, J=7.21 Hz, 1H, OH)
rac-(3aR,4R,7S,7aS)-2-benzyl-4-(hydroxymethyl)-3a,4,7,7a-tetrahydro-1H-4,7-
-epoxyisoindole-1,3(2H)-dione (17.sub.endo ) and
rac-(3aS,4R,7S,7aR)-2-benzyl-4-(hydroxymethyl)-3a,4,7,7a-tetrahydro-1H-4,-
7-epoxyisoindole-1,3(2H)-dione (17.sub.exo)
[0107] Made according to the protocol as noted in Fan, B. et al,
Thermo-responsive self-immolative nanoassemblies: Direct and
indirect triggering. Chem. Commun. 2017, 0, 12068-12071. DOI:
http://dx.doi.org/10.1039/c7cc06410a, the entire content of which
is incorporated herein by reference. N-benzyl maleimide (7, 2.0 g,
10.7 mmol) (see Sortino, M. et al., N-Phenyl and
N-phenylalkyl-maleimides acting against Candida spp.: Time-to-kill,
stability, interaction with maleamic acids. Bioorgan. Med. Chem.
2008, 16 (1), 560-568. DOI:
http://dx.doi.org/10.1016/j.bmc.2007.08.030) and 2-(hydroxymethyl)
furan (1, 931 .mu.L, 1.05 g, 10.7 mmol) were dissolved in anhydrous
acetonitrile under a nitrogen atmosphere in a flame-dried flask
equipped with a magnetic stirring-bar. The reaction was stirred at
35.degree. C. for 14 hours. When TLC indicated the reaction had
reached equilibrium, the solvent was removed, and the reaction was
concentrated under reduced pressure for 1 hour. Crude NMR indicated
a ratio of (1:0.4:0.3) of endo-exo-unreacted maleimide. The crude
material was then purified by flash chromatography (6:4 to 4:6,
hexanes-ethyl acetate) to provide 1.61g (53% yield) of the endo and
677 mg (22% yield) of the exo product for a combined 75% isolated
yield. Due to the inherent thermal instability, the material is
stored at -20.degree. C. until needed.
rac-(3aR,4R,7S,7aS)-2-benzyl-4-(hydroxymethyl)-3a,4,7,7a-tetrahydro-1H-4,7-
-epoxyisoindole-1,3(2H)-dione (17.sub.endo)
[0108] Clear oil; R.sub.f=0.27 (6:4, hexanes-ethyl acetate);
.sup.1H NMR (300 MHz, CDCl.sub.3): .delta..sub.ppm 7.31-7.26 (m,
5H), 6.15 (dd, J=5.8, 1.5 Hz, 1H), 6.06 (d, J=5.8 Hz, 1H), 5.26
(dd, J=5.5, 1.6 Hz, 1H), 4.47 (s, 2H), 4.25 (d, J=12.2 Hz, 1H),
4.15 (d, J=12.2 Hz, 1H), 3.63 (dd, J=7.6, 5.5 Hz, 1H), 3.40 (d,
J=7.6 Hz, 1H), 2.11 (s, 1H). Spectral data is consistent with the
published data noted in Fan, B. et al, Thermo-responsive
self-immolative nanoassemblies: Direct and indirect triggering.
Chem. Commun. 2017, 0, 12068-12071. DOI:
http://dx.doi.org/10.1039/c7cc06410a.
rac-(3aS,4R,7S,7aR)-2-benzyl-4-(hydroxymethyl)-3a,4,7,7a-tetrahydro-1H-4,7-
-epoxyisoindole-1,3(2H)-dione (17.sub.exo)
[0109] Colourless solid; R.sub.f=0.14 (6:4, hexanes-ethyl acetate);
.sup.1H NMR (300 MHz, CDCl.sub.3): .delta..sub.ppm 7.33-7.26 (m,
5H), 6.61 (d, J=5.7 Hz, 1H), 6.54 (dd, J=5.7, 1.5 Hz, 1H), 5.28 (d,
J=1.7 Hz, 1H), 4.66 (bs, 2H), 4.09 (dd, J=12.2, 8.8 Hz, 1H), 4.03
(dd, J=12.2, 6.3 Hz, 1H), 3.02 (d, J=6.5 Hz, 1H), 2.99 (d, J=6.5
Hz, 1H), 2.76 (bt, J=7.4 Hz, 1H, OH). Spectral data is consistent
with the published data noted in Fan, B. et al, Thermo-responsive
self-immolative nanoassemblies: Direct and indirect triggering.
Chem. Commun. 2017, 0, 12068-12071. DOI:
http://dx.doi.org/10.1039/c7cc06410a.
rac-(3aR,4R,7R,7aS)-4-(hydroxymethyl)-2-(4-methoxyphenyl)-7-nitro-3a,4,7,7-
a-tetrahydro-1H-4,7-epoxyisoindole-1,3(2H)-dione (25.sub.endo) and
rac-(3aS,4R,7R,7aR)-4-(hydroxymethyl)-2-(4-methoxyphenyl)-7-nitro-3a,4,7,-
7a-tetrahydro-1H-4,7-epoxyisoindole-1,3(2H)-dione (25exo)
[0110] N-(p-methoxyphenyl)-Maleimide 5 (0.349 g, 1.72 mmol) and
5-nitro-2 furanmethanol 2 (0.295 g, 2.06 mmol) were dissolved in
anhydrous acetonitrile under a nitrogen atmosphere in a flame-dried
flask equipped with a magnetic stirring-bar. The reaction was
stirred at 50-75.degree. C. for 8 hours, at which time TLC analysis
(3:7, hexanes-ethyl acetate) indicated the formation of a new,
polar spot (R.sub.f=0.13). The reaction would not proceed to
completion, and although we observed a new spot consistent with the
endo product form, it was negligible and was never able to be
isolated by chromatography. The reaction was stopped by cooling the
mixture, and the solvent was removed. Column chromatography (6:4 to
2:8 to 0.5:9.5, hexanes-ethyl acetate) was carried out, and
provided 405 mg of the exo product in 63% yield. The material was
kept at -20.degree. C. until required.
rac-(3aS,4R,7R,7aR)-4-(hydroxymethyl)-2-(4-methoxyphenyl)-7-nitro-3a,4,7,7-
a-tetrahydro-1H-4,7-epoxyisoindole-1,3(2H)-dione (25.sub.exo)
[0111] White spongy solid; R.sub.f=0.13 (3:7, hexanes-ethyl
acetate); .sup.1H NMR .sup.1H NMR (300 MHz, CDCl.sub.3):
.delta..sub.ppm 7.19 (d, J=9.03 Hz, 2H), 6.98 (d, J=8.98 Hz, 2H),
6.96 (d, J=5.31 Hz, 1H), 6.90 (d, J=5.58 Hz, 1H), 4.22 (d, J=2.65
Hz, 1H), 4.19 (d, J=1.56 Hz, 1H), 3.83 (s, 3H), 3.77 (d, J=6.64 Hz,
1H), 3.42 (d, J=6.64 Hz, 1H), 2.71 (t, J=7.46 Hz, 1H, OH); .sup.13C
NMR (300 MHz, MeOD): .delta..sub.ppm 172.8, 171.6, 160.2, 141.5,
134.8, 128.2, 124.7, 114.4, 112.3, 91.8, 59.2, 55.1, 52.6, 50.9.
HRMS (CI): Calculated for [M].sup.+
(C.sub.16H.sub.14N.sub.2O.sub.7): 346.0801, [M+H].sup.+
(C.sub.16H.sub.15N.sub.2O.sub.7): 347.0874. Found [M+H].sup.+:
347.0877.
rac-(3aR,4R,7R,7aS)-4-(hydroxymethyl)-7-nitro-2-(4-nitrophenyl)-3a,4,7,7a--
tetrahydro-1H-4,7-epoxyisoindole-1,3(2H)-dione (26.sub.endo)
[0112] N-(p-nitrophenyl)-Maleimide 2 (0.254 g, 1.16 mmol) and
5-nitro-2-furanmethanol 6 (0.200 g, 1.40 mmol) were dissolved in
anhydrous acetonitrile under a nitrogen atmosphere in a flame-dried
flask equipped with a magnetic stirring-bar. The reaction was
stirred at 40.degree. C. for 4 hours, then at 60.degree. C. for an
additional 4 hours, at which point only starting material was
observed by TLC (3:7, hexanes-ethyl acetate). The temperature was
accordingly increased to 80.degree. C., and was left to stir at
this temperature overnight (12 hours). TLC indicated moderate
conversion, and the solvent was removed, and the reaction mixture
was concentrated. Column purification was then performed (6:4 to
0.5:9.5, hexanes-ethyl acetate). 162 mg of the polar spot was
obtained in 31% yield (R.sub.f=0.16) and was determined to be exo
product. A negligible amount of endo material was observed by crude
NMR but was not able to be readily isolated from the starting
materials. Product was kept stored at -20.degree. C. until
required.
rac-(3aS,4R,7R,7aR)-4-(hydroxymethyl)-7-nitro-2-(4-nitrophenyl)-3a,4,7,7a--
tetrahydro-1H-4,7-epoxyisoindole-1,3(2H)-dione (26.sub.exo)
[0113] Off-white spongy solid; R.sub.f=0.16 (3:7, hexanes-ethyl
acetate); .sup.1H NMR (300 MHz, CD.sub.3CN): .delta..sub.ppm 8.34
(d, J=9.05 Hz, 2H), 7.54 (d, J=9.11 Hz, 2H), 6.93 (d, J=5.73 Hz,
1H), 6.90 (d, J=5.85 Hz, 1H), 4.26 (dd, J=13.21, 6.39 Hz, 1H), 4.05
(dd, J=13.32, 5.80 Hz, 1H), 3.94 (d, J=6.57 Hz, 1H), 3.46 (d,
J=6.70 Hz, 1H), 3.44 (t, J=5.93 Hz, 1H, OH); .sup.13C NMR (100 MHz,
CDCl.sub.3): .delta..sub.ppm 171.7, 170.7, 147.6, 141.3, 137.0,
134.8, 127.5, 124.6, 112.0, 91.7, 59.1, 52.7, 51.0. HRMS (CI):
Calculated for [M].sup.+ (C.sub.15H.sub.11N.sub.3O.sub.8):
361.0546; [M+H].sup.+ (C.sub.15H.sub.12N.sub.3O.sub.8): 362.0624.
Found [M].sup.+: 362.0614.
rac-(3aR,4R,7R,7aS)-2-benzyl-4-(hydroxymethyl)-7-nitro-3a,4,7,7a-tetrahydr-
o-1H-4,7-epoxyisoindole-1,3(2H)-dione (27.sub.endo) and
rac-(3aS,4R,7R,7aR)-2-benzyl-4-hydroxymethyl)-7-nitro-3a,4,7,7a-tetrahydr-
o-1H-4,7-epoxyisoindole-1,3(2H)-dione (27.sub.exo)
[0114] N-benzyl maleimide 7 (0.322 g, 1.72 mmol) and
5-nitro-2-furanmethanol 2 (0.295 g, 2.06 mmol) were dissolved in
anhydrous acetonitrile under a nitrogen atmosphere in a flame-dried
flask equipped with a magnetic stirring-bar. The reaction was
stirred at 65-70.degree. C. for 16 hours. At that point, TLC
analysis (2:8, hexanes-ethyl acetate) showed the formation a new
nonpolar spot (endo; R.sub.f=0.27) and the formation of a polar
spot (exo; R.sub.f=0.14). At this point, the reaction was
concentrated under reduced pressure, and purified by flash
chromatography (3:7 to 1:9, hexanes-ethyl acetate). The first
fraction contained 438 mg of product (69% yield) endo product and
129 mg (21% yield) of the exo product. They are both kept stored at
-20.degree. C. until required.
rac-(3aR,4R,7R,7aS)-2-benzyl-4-(hydroxymethyl)-7-nitro-3a,4,7,7a-tetrahydr-
o-1H-4,7-epoxyisoindole-1,3(2H)-dione (2.sub.endo)
[0115] White crystal; R.sub.f=0.27 (2:8, hexanes-ethyl acetate);
.sup.1H NMR (300 MHz, CDCl.sub.3): .delta..sub.ppm 7.35-7.27 (m,
5H), 6.46 (d, J=5.75 Hz, 1H), 6.23 (d, J=5.75 Hz, 1H), 4.55 (d,
J=13.96 Hz, 1H), 4.50 (d, J=13.94 Hz, 1H), 4.29 (dd, J=13.13, 6.18
Hz, 1H), 4.19 (dd, J=13.14, 6.94 Hz, 1H), 3.96 (d, J=7.95 Hz, 1H),
3.84 (d, J=7.95 Hz, 1H), 2.16 (t, J=6.60 Hz, 1H, OH); .sup.13C NMR
(300 MHz, CDCl.sub.3): .delta..sub.ppm 172.6, 170.7, 137.2, 134.9,
132.7, 129.3, 128.8, 128.6, 112.4, 91.7, 60.8, 57.6, 51.0, 48.1,
43.0. HRMS (CI): Calculated for [M].sup.+
(C.sub.16H.sub.14N.sub.2O.sub.6): 330.0852; [M+H].sup.+
(C.sub.16H.sub.15N.sub.2O.sub.6): 331.0930. Found [M+H].sup.+:
331.0939.
rac-(3aS,4R,7R,7aR)-2-benzyl-4-hydroxymethyl)-7-nitro-3a,4,7,7a-tetrahydro-
-1H-4,7-epoxyisoindole-1,3(2H)-dione (27.sub.exo)
[0116] White crystal; R.sub.f=0.14 (2:8, hexanes-ethyl acetate);
.sup.1H NMR (300 MHz, CDCl.sub.3): .delta..sub.ppm 7.38-7.27 (m,
5H), 6.89 (d, J=5.62 Hz, 1H), 6.83 (d, J=5.52 Hz, 1H), 4.71 (d,
J=14.23 Hz, 1H), 4.63 (d, J=14.31 Hz, 1H), 4.19-3.99 (m, 2H), 3.64
(d, J=6.48 Hz, 1H), 3.26 (d, J=6.47 Hz, 1H), 2.75-2.68 (m, 1H, OH);
.sup.13C NMR (300 MHz, CDCl.sub.3): .delta..sub.ppm 173.4, 170.6,
141.3, 135.4, 134.9, 129.1, 128.7, 128.6, 111.4, 9.4, 60.3, 51.7,
50.9, 43.5.
rac-(3aR,4S,7R,7aR)-4-bromo-7-(hydroxymethyl)-2-(4-methoxyphenyl)-3a,4,7,7-
a-tetrahydro-1H-4,7-epoxyisoindole-1,3(2H)-dione (35.sub.endo) and
rac-(3aS,4S,7R,7aS)-4-bromo-7-(hydroxymethyl)-2-(4-methoxyphenyl)-3a,4,7,-
7a-tetrahydro-1H-4,7-epoxyisoindole-1,3(2H)-dione (35.sub.exo)
[0117] N-(p-methoxyphenyl)-Maleimide 5 (0.8 g, 3.90 mmol) and
5-Bromo-2-furanmethanol 3 (0.60 g, 3.00 mmol) were dissolved in
anhydrous acetonitrile under a nitrogen atmosphere in a flame-dried
flask equipped with a magnetic stirring-bar. The reaction was
stirred at room temperature for 3 hours, then the progress of
reaction was monitored every 2 hours and the temperature was
increased from 35.degree. C. to 75.degree. C. over 6 hours, and
held at the highest temperature for an additional 10 hours. The
reaction was concentrated under reduced pressure and purified by
column (6:4 to 2:8, hexanes-ethyl acetate) to separate the starting
material from 851 mg of the non-polar endo (R.sub.f=0.50, 6:4,
hexanes-ethyl acetate) product (61% yield) and 63 mg of a polar exo
(R.sub.f=0.17, 6:4, hexanes-ethyl acetate) product in<5% yields.
The mass balance included some debrominated material, 15 and
starting materials. These products kept stored at -20.degree. C.
until required.
rac-(3aR,4S,7R,7aR)-4-bromo-7-(hydroxymethyl)-2-(4-methoxyphenyl)-3a,4,7,7-
a-tetrahydro-1H-4,7-epoxyisoindole-1,3(2H)-dione (35.sub.endo)
[0118] White solid. R.sub.f=0.50 (6:4, hexanes-ethyl acetate);
.sup.1H NMR (300 MHz, CD.sub.3CN): .delta..sub.ppm 7.06-6.89 (m,
4H), 6.64 (d, J=5.54 Hz, 1H), 6.53 (d, J=5.44 Hz, 1H), 4.14 (dd,
J=13.38, 6.13 Hz, 1H), 4.02 (dd, J=12.83, 6.25 Hz, 1H), 3.87 (d,
J=7.52 Hz, 1H), 3.79 (s, 3H), 3.74 (d, J=8.01 Hz, 1H), 3.35 (t,
J=5.99 Hz, 1H, OH); .sup.13C NMR (75 MHz, CD.sub.3CN):
.delta..sub.ppm 174.9, 174.0, 161.4, 140.9, 138.3, 129.7, 126.0,
115.9, 93.5, 89.8, 61.5, 58.2, 56.8, 46.7. HRMS (CI): Calculated
for [M].sup.+ (C.sub.16H.sub.14BrNO.sub.5): 379.0055. Found:
379.0046.
rac-(3aS,4S,7R,7
aS)-4-bromo-7-(hydroxymethyl)-2-(4-methoxyphenyl)-3a,4,7,7a-tetrahydro-1H-
-4,7-epoxyisoindole-1,3(2H)-dione (35.sub.exo)
[0119] White solid; R.sub.f=0.17 (6:4, hexanes-ethyl acetate);
.sup.1H NMR (300 MHz, CDCl.sub.3): .delta..sub.ppm 7.20 (d, J=8.64
Hz, 1H), 6.97 (d, J=8.67 Hz, 1H), 6.64 (d, J=7.74 Hz, 1H), 6.62 (d,
J=7.89 Hz, 1H), 4.01 (d, J=11.56 Hz, 1H), 3.84 (d, J=11.22 Hz, 1H),
3.83 (s, 3H), 3.36-3.33 (m, 2H); .sup.13C NMR (75 MHz, CDCl.sub.3):
.delta..sub.ppm 171.8, 170.5, 159.8, 142.1, 138.7, 127.6, 123.9,
114.5, 89.4, 89.0, 55.5, 54.8, 51.5, 27.4.
rac-(3aR,4S,7R,7aR)-4-bromo-7-(hydroxymethyl)-2-(4-nitrophenyl)-3a,4,7,7a--
tetrahydro-1H-4,7-epoxyisoindole-1,3(2H)-dione (36.sub.endo)
[0120] N-(p-nitrophenyl)-Maleimide 6 (990 mg, 5.6 mmol) and
5-Bromo-2-furanmethanol 3 (1.03 g, 4.7 mmol) were dissolved in
anhydrous acetonitrile under a nitrogen atmosphere in a flame-dried
flask equipped with a magnetic stirring-bar. The reaction was
stirred at 50.degree. C. for 72 hours until further conversion was
no longer noted by TLC (6:4 hexanes-ethyl acetate). The reaction
mixture was concentrated, and the residue purified by column
chromatography (6:4, hexanes-ethyl acetate) in two columns
successively (the first column provided product contaminated with
the starting materials). Crude 1H NMR had shown the presence of
both endo and exo derivatives, albeit in low conversion. The small
amount of exo was estimated to be well under 3% of the mass balance
and was not isolated. The chromatography provided 530 mg of the
title compound as a colourless solid in 26% yield.
rac-(3aR,4S,7R,7aR)-4-bromo-7-(hydroxymethyl)-2-(4-nitrophenyl)-3a,4,7,7a--
tetrahydro-1H-4,7-epoxyisoindole-1,3(2H)-dione (36.sub.endo)
[0121] White solid; R.sub.f=0.20 (6:4, hexanes-ethyl acetate);
.sup.1H NMR (300 MHz, CDCl.sub.3): .delta..sub.ppm 8.31 (d, J=8.75
Hz, 1H), 7.43 (d, J=8.48 Hz, 1H), 6.65 (d, J=5.46 Hz, 1H), 6.51 (d,
J=5.48 Hz, 1H), 4.36 (d, J=12.88 Hz, 1H), 4.24 (d, J=13.02 Hz, 1H),
4.00 (d, J=7.90 Hz, 1H), 3.91 (d, J=8.00 Hz, 1H); .sup.13C NMR (75
MHz, CDCl.sub.3): .delta..sub.ppm 171.9, 170.7, 147.4, 140.2,
136.0, 127.1, 126.8, 124.6, 91.9, 87.6, 61.0, 56.8, 48.4. HRMS
(CI): Calculated for [M].sup.+ (C.sub.15H.sub.11BrN.sub.2O.sub.6):
393.9800, [M+H].sup.+ (C.sub.15H.sub.12BrN.sub.2O.sub.6): 394.9879.
Found: 394.9872.
rac-(3aR,4S,7R,7aR)-2-benzyl-4-bromo-7-(hydroxymethyl)-3a,4,7,7a-tetrahydr-
o-1H-4,7-epoxyisoindole-1,3(2H)-dione (37.sub.endo) and
rac-(3aS,4S,7R,7aS)-2-benzyl-4-bromo-7-(hydroxymethyl)-3a,4,7,7a-tetrahyd-
ro-1H-4,7-epoxyisoindole-1,3(2H)-dione (37.sub.exo)
[0122] N-benzyl maleimide 7 (0.70 g, 3.8 mmol) and 5
Bromo-2-furanmethanol 3 (0.80 g, 4.5 mmol) were dissolved in
anhydrous acetonitrile under a nitrogen atmosphere in a flame-dried
flask equipped with a magnetic stirring-bar. The reaction was
stirred at room temperature for 7 hours, a TLC (7:3, hexanes-ethyl
acetate) showing starting material was left, so the temperature was
increased slowly (55.degree. C. to 70.degree. C.) and the progress
of reaction was monitored every 2 hours. The reaction was left for
an additional 14 hours at 70.degree. C. when TLC showed some
conversion. The solvent was removed, and the reaction mixture was
concentrated. After column chromatography purification of reaction
mixture (7:3, hexanes-ethyl acetate), 403 mg of an inseparable
mixture of the endo and exo (2:1) cycloadducts as a white solid was
obtained with an overall yield of 31%. In none of the more than 10
solvent mixtures examined was separation observed. The mixture was
kept stored at -20.degree. C. until required.
rac-(3aR,4S,7R,7aR)-2-benzyl-4-bromo-7-(hydroxymethyl)-3a,4,7,7a-tetrahydr-
o-1H-4,7-epoxyisoindole-1,3(2H)-dione (37.sub.endo)
[0123] R.sub.f=0.29 (7:3, hexanes-ethyl acetate); .sup.1H NMR (300
MHz, CDCl.sub.3): .delta..sub.ppm 7.37-7.27 (m, 5H), 6.12 (d,
J=5.51 Hz, 1H), 5.97 (d, J=5.24 Hz, 1H), 4.52-4.46 (m, 2H), 4.24
(dd, J=12.40, 1.86 Hz, 1H), 4.13 (dd, J=12.44, 2.95 Hz, 1H),
3.79-3.12 (m, 2H), 3.25-3.20 (m, 1H, OH); .sup.13C NMR (75 MHz,
CDCl.sub.3): .delta..sub.ppm 172.9, 172.2, 138.6, 135.2, 135.0,
129.1, 128.6, 128.2, 90.6, 89.1, 68.9, 56.0, 51.6, 47.96, 42.5.
rac-(3aS,4S,7R,7aS)-2-benzyl-4-bromo-7-(hydroxymethyl)-3a,4,7,7a-tetrahydr-
o-1H-4,7-epoxyisoindole-1,3(2H)-dione (37.sub.exo)
[0124] R.sub.f =0.28 (7:3, hexanes-ethyl acetate); .sup.1H NMR (300
MHz, CDCl.sub.3): .delta..sub.ppm 7.37-7.27 (m, 5H), 6.12 (d,
J=5.51 Hz, 1H), 5.97 (d, J=5.24 Hz, 1H ), 4.71 (d, J=14.35 Hz, 1H),
4.66 (d, J=14.37 Hz, 1H), 3.93 (d, J=11.41 Hz, 1H), 3.65 (dd,
J=14.15, 7.79 Hz, 1H), 3.77-3.73 (m, 1H); 3.25-3.20 (m, 1H, OH);
.sup.13C NMR (75 MHz, CDCl.sub.3): .delta..sub.ppm 172.0, 170.9,
139.2, 135.1, 135.0, 128.7, 128.3, 128.0, 90.5, 88.8, 69.1, 54.8,
48.04, 42.9, 27.3.
[0125] Endo/exo mixture HRMS (CI): Calculated for [M].sup.+
(C.sub.16H.sub.14BrNO.sub.4) 363.0106, [M+H].sup.+
(C.sub.16H.sub.15BrNO.sub.4): 364.0184. Found: 364.0176.
rac-(3aR,4R,7R,7aS)-4-(hydroxymethyl)-7-methoxy-2-(4-methoxyphenyl)-3a,4,7-
,7a-tetrahydro-1H-4,7-epoxyisoindole-1,3(2H)-dione (46.sub.endo)
and
rac-(3aR,4R,7R,7aS)-4-(hydroxymethyl)-7-methoxy-2-(4-methoxyphenyl)-3a,4,-
7,7a-tetrahydro-1H-4,7-epoxyisoindole-1,3(2H)-dione
(45.sub.exo)
[0126] N-(p-methoxyphenyl)-Maleimide 5 (0.32 g, 2.5 mmol) and
5-methoxy-2-furanmethanol 4 (0.43 g, 2.1 mmol) were dissolved in
anhydrous acetonitrile under a nitrogen atmosphere in a flame-dried
flask equipped with a magnetic stirring-bar. The reaction was
stirred at 45.degree. C. for 4 hours; TLC analysis (2:8,
hexanes-ethyl acetate) indicated that a new nonpolar spot
(R.sub.f=0.50) had formed with some starting material remaining.
The temperature was then increased to 55-60.degree. C. and the
reaction stirred for 14 hours. TLC showed a new polar spot
(R.sub.f=0.25) with no furan remaining. The solvent was then
removed under reduced pressure. Column purification (4:6 to
1.5:8.5, hexanes-ethyl acetate) was carried out to obtain 191 mg of
the endo product (25% yield) and 26 mg (4% yield) of the exo
product. Due to the fact that these compounds have an inherently
unstable nature at ambient temperatures, they are kept stored at
-20.degree. C. until required.
rac-(3aR,4R,7R,7aS)-4-(hydroxymethyl)-7-methoxy-2-(4-methoxyphenyl)-3a,4,7-
,7a-tetrahydro-1H-4,7-epoxyisoindole-1,3(2H)-dione
(45.sub.endo)
[0127] White solid; R.sub.f=0.50 (2:8, hexanes-ethyl acetate);
.sup.1H NMR (300 MHz, CD.sub.3CN): .delta..sub.ppm 7.03 (d, J=9.10
Hz, 2H), 6.98 (d, J=9.14 Hz, 2H), 6.63 (d, J=5.82 Hz, 1H), 6.58 (d,
J=5.83 Hz, 1H), 4.10 (dd, J=12.91, 5.81 Hz, 1H), 3.99 (dd, J=12.91,
6.13 Hz, 1H), 3.81 (s, 3H), 3.71 (d, J=7.87 Hz, 1H), 3.56 (s, 3H),
3.51 (d, J=7.90 Hz, 1H), 3.20 (t, J=6.02, Hz, 1H, OH); .sup.13C NMR
(125 MHz, CDCl.sub.3): .delta..sub.ppm 174.5, 173.8, 159.8, 137.7,
134.9, 128.2, 128.2, 124.9, 114.4, 86.8, 60.7, 55.3, 54.1, 50.2,
48.9. HRMS (CI): Calculated for [M].sup.+
(C.sub.17H.sub.17NO.sub.6): 331.1056, [M+H].sup.+: 332.1134. Found
[M+H].sup.+: 332.1127
rac-(3aR,4R,7R,7aS)-4-(hydroxymethyl)-7-methoxy-2-(4-methoxyphenyl)-3a,4,7-
,7a-tetrahydro-1H-4,7-epoxyisoindole-1,3(2H)-dione (45.sub.exo)
[0128] White solid; R.sub.f =0.25 (2:8, hexanes-ethyl acetate);
.sup.1H NMR (300 MHz, CDCl.sub.3): .delta..sub.ppm 7.21 (d, J=8.94
Hz, 2H), 6.97 (d, J=9.01 Hz, 2H), 6.79 (d, J=5.66 Hz, 1H), 6.56 (d,
J=5.66 Hz, 1H), 4.14 (d, J=12.65 Hz, 1H), 4.08 (d, J=12.71 Hz, 1H),
3.82 (s, 3H), 3.63 (s, 3H), 3.27 (d, J=6.59 Hz, 1H), 3.21 (d,
J=6.57 Hz, 1H).
rac-(3aR,4R,7R,7aS)-4-(hydroxymethyl)-7-methoxy-2-(4-nitrophenyl)-3a,4,7,7-
a-tetrahydro-1H-4,7-epoxyisoindole-1,3(2H)-dione (46.sub.endo) and
rac-(3aS,4R,7R,7aR)-4-(hydroxymethyl)-7-methoxy-2-(4-nitrophenyl)-3a,4,7,-
7a-tetrahydro-1H-4,7-epoxyisoindole-1,3(2H)-dione (46.sub.exo)
[0129] N-(p-nitrophenyl)-Maleimide 6 (458 mg, 2.1 mmol) and
5-methoxy-2-furanmethanol 4 (320 mg g, 2.5 mmol) were dissolved in
anhydrous acetonitrile under a nitrogen atmosphere in a flame-dried
flask equipped with a magnetic stirring-bar. The reaction was
stirred at 45.degree. C. for 4 hours and then 80.degree. C. for an
additional 8; TLC analysis (2:8, hexanes-ethyl acetate) indicated
that a new nonpolar spot (R.sub.f=0.50) and polar spot had formed
(R.sub.f=0.21). The solvent was then removed under reduced
pressure. Column purification (4:6 to 1.5:8.5, hexanes-ethyl
acetate) was carried out to obtain 400 mg of the endo product (52%
yield) and approximately 30 mg of the exo product (4% yield). Due
to the fact that these compounds have an inherently unstable nature
at ambient temperatures, they are kept stored at -20.degree. C.
until required.
rac-(3aR,4R,7R,7aS)-4-(hydroxymethyl)-7-methoxy-2-(4-nitrophenyl)-3a,4,7,7-
a-tetrahydro-1H-4,7-epoxyisoindole-1,3(2H)-dione (46.sub.endo)
[0130] White solid; R.sub.f=0.50 (2:8, hexanes-ethyl acetate);
.sup.1H NMR (300 MHz, CD.sub.3CN): .delta..sub.ppm 8.30 (d, J=9.08
Hz, 2H), 7.43 (d, J=9.08 Hz, 2H), 6.65 (d, J=5.87 Hz, 1H), 6.60 (d,
J=5.81 Hz, 1H), 4.12 (dd, J=12.95, 5.84 Hz, 1H), 4.01 (dd, J=12.90,
6.21 Hz, 1H), 3.80 (d, J=7.89 Hz, 1H), 3.59 (d, J=7.91 Hz, 1H),
3.57 (s, 3H), 3.24 (t, J=6.02 Hz, 1H, OH); .sup.13C NMR (125 MHz,
CDCl.sub.3): .delta..sub.ppm 173.5, 172.8, 147.5, 137.8, 137.6,
135.0, 127.7, 124.4, 114.5, 86.9, 60.6, 54.2, 50.5, 49.1; HRMS
(CI): Calculated for [M].sup.+ (C.sub.16H.sub.14N.sub.2O.sub.7):
346.0801, [M+H].sup.+ (C.sub.16H.sub.15N.sub.2O.sub.7): 347.0879.
Found: 347.0874.
rac-(3aS,4R,7R,7aR)-4-(hydroxymethyl)-7-methoxy-2-(4-nitrophenyl)-3a,4,7,7-
a-tetrahydro-1H-4,7-epoxyisoindole-1,3(2H)-dione (46.sub.exo)
[0131] R.sub.f=0.21 (2:8, hexanes-ethyl acetate); .sup.1H NMR (300
MHz, CD.sub.3CN): .delta..sub.ppm 8.48 (d, J=8.93 Hz, 2H), 7.69 (d,
J=8.92 Hz, 2H), 6.89 (d, J=5.68 Hz, 1H), 6.79 (d, J=5.49 Hz, 1H),
4.29 (d, J=12.03 Hz, 1H), 4.11 (d, J=12.77 Hz, 1H), 3.72 (s, 3H),
3.50-3.27 (m, 2H).
2-benzyl-4-(hydroxymethyl)-7-methoxyisoindoline-1,3-dione (47)
[0132] N-benzyl maleimide 7 (490 mg, 2.6 mmol) and
5-methoxy-2-furanmethanol 4 (400 mg g, 3.0 mmol) were dissolved in
anhydrous acetonitrile under a nitrogen atmosphere in a flame-dried
flask equipped with a magnetic stirring-bar. The reaction was
stirred at 40.degree. C. for 4 hours, and 50.degree. C. for 18
hours after which no reaction was observed. The reaction mixture
was then further heated to 65.degree. C. for an additional 4 hours
at which point a new spot was observed by TLC (R.sub.f =0.35, 2:8,
hexanes-ethyl acetate). The solvent was then removed under reduced
pressure. Column purification (4:6 to 1.5:8.5, hexanes-ethyl
acetate) was carried out to obtain 530 mg of a single product, the
title compound, in 60% yield as a white solid.
[0133] White solid. R.sub.f=0.35 (2:8, hexanes-ethyl acetate);
.sup.1H NMR (300 MHz, CDCl3): .delta..sub.ppm 7.47 (d, J=8.58 Hz,
1H), 7.39-7.31 (m, 2H), 7.28-7.13 (m, 3H), 7.05 (d, J=8.59 Hz, 1H),
4.78 (s, 2H), 4.74 (s, 2H), 3.93 (s, 3H); .sup.13C NMR (125 MHz,
CDCl.sub.3): .delta..sub.ppm 169.3, 166.5, 156.2, 136.2, 135.9,
132.9, 130.9, 128.73, 128.65, 127.8, 117.6, 117.5, 61.9, 56.4,
41.5. HRMS (CI): Calculated for [M].sup.+
(C.sub.17H.sub.15NO.sub.4): 297.1001, [M+H].sup.+
(C.sub.17H.sub.16NO.sub.4): 298.1079. Found: 298.1075.
Synthesis of Protecting Groups:
Synthesis of Benzvl-H Protecting Group JT70B
##STR00012##
[0134]
(2-benzyl-1,3-dioxo-2,3,3a,4,7,7a-hexahydro-1H-4,7-epoxyisoindol4-y-
l)methyl-carbonochloridate (103)
[0135]
2-Benzyl-4-(hydroxymethyl)-3a,4,7,7a-tetra-hydro-1H-4,7-epoxyisoind-
ole-1,3(2H)-dione (102) (0.25 g, 0.876 mmol) was dissolved in dry
toluene (5.0 ml) under nitrogen. Once cooled to 0.degree. C.,
N,N-diisopropylethylamine (0.46 ml) was added to the solution.
Triphosgene (139.9 mg) was subsequently added to the solution. The
reaction was stirred under nitrogen for 2 hours at 0.degree. C.,
and was then stirred for 12 hours at 25.degree. C. The reaction
mixture was extracted once with ethyl acetate (10 ml). The organic
fraction was washed once with a solution of ammonium chloride (15
ml), and once more with a solution of acidic brine (15 ml). The
organic phase was subsequently dried with anhydrous MgSO.sub.4 and
concentrated under reduced pressure to deliver the title compound
(0.221 g, 75% yield). Rf 0.71 in 3:7 hexane/ethyl acetate. .sup.1H
NMR (300 MHz, CDCl.sub.3) .delta. 7.42-7.24 (m, 10H), 7.24-7.15 (m,
2H), 6.23 (dd, J=5.8, 1.7 Hz, 1H), 6.04 (d, J=5.8 Hz, 1H),
5.42-5.26 (m, 1H), 5.13-4.97 (m, 1H), 4.91-4.63 (m, 2H), 4.52 (s,
2H), 3.70 (dd, J=7.7, 5.5 Hz, 1H), 3.41 (d, J=7.7 Hz, 1H). .sup.13C
NMR (300 MHz, CDCl.sub.3) .delta..sub.ppm 137.6, 137.2, 135.4,
135.1, 134.6, 129.1, 128.5, 125.3, 90.4, 81.1, 79.6, 68.3, 62.7,
61.9, 50.2, 48.5, 47.9, 46.8, 42.4, 29.8, 22.8, 21.7, 20.7,
16.0.
[0136] MS Calculated for C.sub.17H.sub.14ClNO.sub.5[M+H].sup.+:
348.7500. Found (ASAP): 348.0639.
##STR00013##
Methyl 4-aminobutanoate (104)
[0137] 4-aminobutanoic acid (5.0 g, 48.5 mmol) was dissolved in
methanol (75.0 ml) and allowed to stir at 0.degree. C. for 15
minutes. Thionyl chloride (5.27 ml) was slowly added dropwise and
the reaction was stirred at 0.degree. C. for 1.5 hours. The solvent
was evaporated at reduced pressure and the titled compound was
delivered (4.97 g, 87%). .sup.1H NMR (300 MHz, CDCl.sub.3)
.delta..sub.ppm 3.37 (s, 3H), 3.03 (t, J=7.3 Hz, 2H), 2.53 (t,
J=7.9 Hz, 2H), 1.99 (p, J=7.5 Hz, 2H).
##STR00014##
methyl
4-((((2-benzyl-1,3-dioxo-2,3,3a,4,7,7a-hexahydro-1H-4,7-epoxyisoin-
dol-4-yl)methoxy)carbonyl)amino)butanoate (105)
[0138]
(2-benzyl-1,3-dioxo-2,3,3a,4,7,7a-hexahydro-1H-4,7-epoxyisoindol-4--
yl)methyl-carbonchloridate (103) (500 mg, 1.63 mmol) was dissolved
in chloroform (2.0 ml) under nitrogen, and cooled to 0.degree. C.
N,N-diisopropylethylamine (0.851 ml) was then added to the
solution, followed by the addition of ethyl 4-aminobutanoate (104)
(228.8 mg, 1.956 mmol). The mixture was stirred under nitrogen at
room temperature for 6 hours. The reaction mixture was diluted with
10% hydrochloric acid (2 ml), and the resulting solution was
extracted once with chloroform (2 ml). The organic fraction was
washed once more with saturated sodium bicarbonate (2 ml) and once
more with brine (2 ml). The resulting organic fraction was dried
with anhydrous MgSO.sub.4 and concentrated under reduced pressure
in order to yield the titled compound as a waxy solid (302.1 mg,
43%) Rf 0.24 in 1:1 hexane/ethyl acetate. .sup.1H NMR (300 MHz,
DMSO) .delta. 7.39-7.12 (m, 5H), 6.59 (d, J=5.7 Hz, 1H), 6.45 (d,
J=5.7 Hz, 1H), 5.16 (d, J=1.8 Hz, 1H), 4.77 (d, J=12.8 Hz, 1H),
4.57 (s, 2H), 4.15 (d, J=12.7 Hz, 1H), 3.57 (s, 2H), 3.16 (d, J=6.4
Hz, 1H), 3.07 (s, 1H), 3.05-2.92 (m, 2H), 2.30 (t, J=7.4 Hz, 2H).
.sup.13C NMR (75 MHz, DMSO) .delta. 174.53, 173.05, 155.75, 135.77,
135.50, 134.54, 128.32, 127.81, 127.42, 89.78, 79.17, 78.78, 61.57,
51.23, 47.43, 46.17, 41.40, 30.51, 28.35, 24.65, 10.79.
[0139] MS Calculated for C.sub.22H.sub.24N.sub.2O.sub.7
[M+H].sup.+: 429.44. Found (ASAP): 429.1678.
##STR00015##
4-((((2-benzyl-1,3-dioxo-2,3,3a,4,7,7a-hexahydro-1H-4,7-epoxyisoindol-4-y-
l)methoxy)carbonyl)amino)butanoic acid (106)
[0140] Methyl
4-((((2-benzyl-1,3-dioxo-2,3,3a,4,7,7a-hexahydro-1H-4,7-epoxyisoindol
-4-yl)methoxy)carbonyl)amino)butanoate (105) (200 mg, 0.467 mmol)
was dissolved in 1,4-dioxane (0.92 ml) and 2N NaOH (4.00 ml). The
resulting solution was stirred at 45.degree. C. for 24 hours, and
subsequently cooled to room temperature. The reaction mixture was
diluted with ethyl acetate (5 ml) and then acidified to pH 1 with
the dropwise addition of 2N hydrochloric acid. Dichloromethane (5
ml) was added to the solution, and the organic and aqueous phases
were separated. The aqueous fraction was stored at 0.degree. C. for
12 hours, and the titled compound was obtained (100 mg, 53% yield)
appearing as white crystals which formed in the aqueous layer.
.sup.1H NMR (300 MHz, DMSO) .delta..sub.ppm 12.07 (s, 1H), 8.38 (s,
1H), 7.28 (m, 5H), 6.47 (d, J=4.9 Hz, 1H), 6.26 (d, J=6.0 Hz, 1H),
5.09 (s, 1H), 4.58 (d, J=12.3 Hz, 1H), 4.32 (m, 1H), 4.20 (m, 1H),
4.04 (d, 12.0 Hz, 1H), 2.98 (m, 2H), 2.81 (d, J=8.7 Hz, 1H), 2.64
(d, J=8.7 Hz, 2H), 1.60 (m, 2H).
##STR00016##
Ally 4-aminobutanoate (149)
[0141] At 0.degree. C. and under nitrogen atmosphere, acetyl
chloride (4.6 mL, 56.2 mmol) was added dropwise into a flame dried
flask of allyl alcohol (20 mL). It was stirred it for at least 30
minutes then add 4-GABA (2.0 g, 19.4 mmol) slowly. It was refluxed
overnight then cooled and evaporated in the morning under a
ventilated fume hood. The concentrated mixture was diluted with
ethyl acetate then quenched with saturated sodium bicarbonate. The
organic layer was separated, and another extraction by ethyl
acetate was performed. The organic layer was then washed with brine
then dried with magnesium sulfate. A silica column was used to
purify the crude at 4:6 methanol/ethyl acetate with the product
being the first to elute. The titled compound ranged from light to
dark brown oil (47%). Rf 0.7 in 4:6 hexane/ethyl acetate under
vanillin stain. .sup.1H NMR (300 MHz, CDCl3) .delta. 5.93-5.74 (m,
1H), 5.34-5.13 (m, 2H), 4.51 (dt, J=5.8, 1.4 Hz, 2H), 3.09 (t,
J=7.6 Hz, 2H), 2.49 (t, J=7.2 Hz, 2H), 2.07 (p, J=7.3 Hz, 2H).
##STR00017##
Allyl
4-((((2-benzyl-1,3-dioxo-2,3,3a,4,7,7a-hexahydro-1H-4,7-epoxyisoind-
ol-4-yl)methoxy)carbonyl)amino)butanoat (150)
[0142] The endcap (103) (722 mg, 2.08 mmol) was dissolved in 3 mL
of chloroform and cooled to 0.degree. C. DIPEA (1.7 ml) was
subsequently added to the solution and followed by addition of the
ally4-aminobutanoate (357 mg, 2.49 mmol). The mixture was stirred
under nitrogen in room temperature until starting material is
consumed. The reaction mixture was washed the with 10% HC1 (3 ml)
and was extracted with chloroform (3 ml) twice. The organic
fraction was subsequently washed with saturated sodium bicarbonate
(4 ml) and once more with brine (4 ml). The resulting organic
fraction was dried with anhydrous MgSO.sub.4 and concentrated under
reduced pressure and the crude mixture was purified by silica
column chromatography in order to yield the title compound as a
brown oil (39%) Rf 0.47 endo/0.29 exo in 1:1 hexane/ethyl
acetate.
[0143] .sup.1H NMR (300 MHz, CDCl3) .delta. 7.31-7.20 (m, 6H), 6.12
(dd, J=5.8, 1.6 Hz, 1H), 6.02 (d, J=5.8 Hz, 1H), 5.88 (ddt, J=17.2,
10.4, 5.8 Hz, 1H), 5.22 (ddt, J=11.7, 7.9, 1.4 Hz, 3H), 5.02 (s,
1H), 4.59-4.51 (m, 3H), 4.44 (s, 2H), 3.59 (dd, J=7.7, 5.5 Hz, 1H),
3.21 (q, J=6.6 Hz, 2H), 2.36 (d, J=14.6 Hz, 2H), 1.82 (t, J=7.1 Hz,
2H). .sup.13C NMR (76 MHz, CDCl3) .delta. 174.07, 173.86, 172.64,
155.63, 135.25, 135.17, 134.27, 131.97, 128.91, 128.36, 127.91,
118.25, 89.97, 79.50, 65.09, 62.32, 47.52, 46.45, 42.21, 40.34,
31.21, 30.78, 24.89.
[0144] MS Calculated for C.sub.24H.sub.26N.sub.2O.sub.7: 455.1818.
Found (ASAP): 455.1817.
##STR00018##
4-((((2-benzyl-1,3-dioxo-2,3,3a,4,7,7a-hexahydro-1H-4,7-epoxyisoindol-4-y-
l)methoxy)carbonyl)amino)butanoic acid (151)
[0145] Allyl
4-((((2-benzyl-1,3-dioxo-2,3,3a,4,7,7a-hexahydro-1H-4,7-epoxyisoindol
-4-yl)methoxy)carbonyl)amino)butanoate (150) (50 mg, 0.11 mmol) was
dissolved in THF (1.5 ml) in a flame dried round bottom flask under
nitrogen atmosphere. Then morpholine (0.09 ml, 1.1 mmol) and
tetrakis(triphenylphosphine)-palladium (11.6 mg, 0.01 mmol) were
subsequently added. The reaction was left stirring at room
temperature until starting material was consumed. The mixture was
filtered and evaporated then washed with 10% HCl (1 ml) and
extracted with DCM. A column starting at 3:7 to 1:1 hexane/ethyl
acetate to flush out the nonpolar components, then increased
polarity to bring the titled compound down with at least 7:3
hexane/ethyl acetate. The titled compound was obtained (32% yield)
appearing as yellow oil. Rf 0.19 in 1:1 hexane/ethyl acetate.
.sup.1H NMR (300 MHz, DMSO) .delta..sub.ppm 12.07 (s, 1H), 8.38 (s,
1H), 7.28 (m, 5H), 6.47 (d, J=4.9 Hz, 1H), 6.26 (d, J=6.0 Hz, 1H),
5.09 (s, 1H), 4.58 (d, J=12.3 Hz, 1H), 4.32 (m, 1H), 4.20 (m, 1H),
4.04 (d, 12.0 Hz, 1H), 2.98 (m, 2H), 2.81 (d, J=8.7 Hz, 1H), 2.64
(d, J=8.7 Hz, 2H), 1.60 (m, 2H).
Synthesis of Protected Cysteines:
##STR00019##
[0146] Ally2-((((9H-fluoren-9-yl)methoxy)carbonyl)
amino)-3-(tritylthio)propanoate (200)
[0147] Fmoc-Cys(Trt)-OH (100 mg, 0.17 mmol) was dissolved in
ethanol (1.25 ml) and an equimolar amount of cesium carbonate (55.4
mg, 0.17 mmol) was subsequently added to the solution. The ethanol
solvent was distilled off at reduced pressure, and the remaining
residue was taken up multiple times by benzene and evaporated to
dryness. The formed cesium salt was dissolved in dimethylformamide
(0.25 ml) and allyl bromide (0.34 ml, 4 mmol) was subsequently
added to the solution (225.2 mg, 1.86 mmol). The resulting mixture
was stirred at room temperature for 18 hours. The solvent was then
evaporated under reduced pressure, and the crude mixture was
purified by silica column chromatography in order to yield the
title compound (76.6 mg, 72% yield) Rf 0.44 in 1:4 hexane/ethyl
acetate.
[0148] .sup.1H NMR (300 MHz, DMSO) .delta. 7.90 (p, J=10.0, 9.1 Hz,
3H), 7.72 (d, J=7.5 Hz, 2H), 7.49-7.13 (m, 23H), 5.91-5.61 (m, 1H),
5.25-5.05 (m, 2H), 4.47 (d, J=5.2 Hz, 2H), 4.36-4.09 (m, 3H), 3.86
(td, J=9.2, 4.8 Hz, 1H), 2.67 (dd, J=12.8, 10.0 Hz, 1H). .sup.13C
NMR (75 MHz, DMSO) .delta. 170.29, 156.07, 144.42, 144.03, 143.96,
141.00, 132.34, 129.37, 128.40, 127.92, 127.34, 127.15, 125.51,
120.40, 117.71, 66.76, 66.04, 65.24, 53.79, 46.85, 32.96. (Gaussian
3.40 Hz apodization)
##STR00020##
Allyl-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-mercaptopropanoate
(201)
[0149] Ally
2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-(tritylthio)propanoate
(200) (100 mg, 0.16 mmol) was dissolved in DCM (2.0 ml) and stirred
at 0.degree. C. for 15 minutes. Triethylsilane (23.0 mg, 0.19 mmol)
was added to the mixture, followed by the addition of TFA (0.2 ml).
The mixture was stirred at room temperature for 1 hour. The solvent
was removed via nitrogen blowdown evaporation and the resultant
crude mixture was purified by silica column chromatography in order
to yield the title compound (37.1 mg, 61% yield) .sup.1H NMR (300
MHz, CDCl.sub.3) 6 7.86-7.67 (m, 2H), 7.59 (s, 2H), 7.50-7.23 (m,
4H), 5.99-5.66 (m, 1H), 5.42-5.17 (m, 1H), 4.68 (d, J=7.1 Hz, 2H),
4.39 (d, J=7.2 Hz, 2H), 4.30-4.18 (m, 1H), 3.09 (dq, J=28.8, 17.2,
9.8 Hz, 2H). .sup.13C NMR (76 MHz, CDCl3) .delta. 170.89, 170.59,
156.42, 156.30, 144.75, 144.28, 144.19, 144.05, 141.80, 134.49,
132.93, 131.81, 130.01, 129.93, 128.78, 128.54, 128.25, 127.60,
127.42, 127.28, 126.79, 126.63, 125.63, 125.34, 124.39, 120.50,
119.78, 119.66, 119.61, 119.28, 118.53, 117.71, 67.88, 67.10,
67.00, 66.96, 66.63, 56.93, 55.73, 53.96, 53.31, 47.65, 47.58,
47.54, 41.59, 35.17, 34.53, 33.15, 32.08, 31.21, 28.12, 27.42,
25.79, 23.15, 14.62. (Gaussian 3.7Hz appodization).
Coupling of Endcap Containing Various Linkers with Cysteines:
##STR00021##
Fmoc-Cys-(S-Benzyl-maleimide)-(O-allyl) (188)
[0150]
Allyl-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-mercaptopropa-
noate (94.3 mg) was dissolved in THF (2 ml) and cooled in ice bath
to 0.degree. C. 3 equimolar amount of DIPEA (0.06 ml) was
subsequently added to the solution, followed by the addition of
endcap (103) (leq, 50 mg). The mixture was stirred under nitrogen
at room temperature. The reaction mixture was diluted with 10%
hydrochloric acid (10 ml), and the resulting solution was extracted
with etheyl acetate (30 ml) two times. The organic fraction was
washed once more with saturated sodium bicarbonate (10 ml) and once
more with brine (20 ml). The resulting organic fraction was dried
with anhydrous MgSO.sub.4 and concentrated under reduced pressure
and the crude mixture was purified by silica column chromatography
in order to yield the title compound as solid (76.6 mg, 72% yield)
Rf 0.38 in 1:1 hexane/ethyl acetate. .sup.1H NMR (300 MHz, CDCl3)
.delta. 7.42-7.24 (m, 10H), 7.24-7.15 (m, 2H), 6.23 (dd, J=5.8, 1.7
Hz, 1H), 6.04 (d, J=5.8 Hz, 1H), 5.42-5.26 (m, 1H), 5.13-4.97 (m,
1H), 4.91-4.63 (m, 2H), 4.52 (s, 2H), 3.70 (dd, J=7.7, 5.5 Hz, 1H),
3.41 (d, J=7.7 Hz, 1H). .sup.13C NMR (76 MHz, CDCl3) .delta.
174.35, 170.93, 156.32, 144.32, 141.87, 136.65, 135.77, 134.78,
133.80, 131.91, 129.67, 129.47, 129.27, 129.12, 128.97, 128.75,
128.29, 127.65, 126.59, 125.71, 120.55, 119.73, 118.53, 89.51,
80.47, 68.75, 67.87, 67.04, 66.60, 60.96, 56.99, 54.00, 48.06,
47.65, 46.94, 43.04, 33.29, 31.31, 14.77.
##STR00022##
Fmoc-Cys-(S-Benzyl-maleimide)-OH (216)
[0151] Fmoc-Cys-(S-Benzyl-maleimide)-(O-allyl) (188) (50 mg, 0.072
mmol) was dissolved in THF (10 mL,1 mmol) and morpholine (0.062 mL,
0.72 mmol). The resulting solution was stirred under nitrogen at
room temperature overnight, and subsequently added
Pd(PPh.sub.3).sub.4 (4.15 mg, 0.0036 mmol). After consumption of
the starting material, the reaction mixture was diluted with 10%
hydrochloric acid (5 ml), and the resulting solution was extracted
with etheyl acetate (15 ml) two times and was washed once more with
brine (10 ml). The resulting organic fraction was dried with
anhydrous MgSO.sub.4 and concentrated under reduced pressure and
the crude mixture was purified by silica column chromatography in
order to yield the title compound as solid (20 mg, 40% yield) Rf
0.2 in 1:1 hexane/ethyl acetate. .sup.1H NMR (300 MHz, CDCl3)
.delta. 7.75-7.60 (m, 2H), 7.60-7.38 (m, 4H), 7.38-7.22 (m, 13H),
6.62 (d, J=5.7 Hz, 1H), 6.54 (dd, J=5.7, 1.7 Hz, 1H), 6.16 (dd,
J=5.8, 1.6 Hz, 1H), 6.07 (d, J=5.8 Hz, 1H), 5.34-5.20 (m, 2H), 4.67
(s, 2H), 4.48 (s, 2H), 4.34-3.98 (m, 4H), 3.64 (dd, J=7.7, 5.5 Hz,
1H), 3.41 (d, J=7.6 Hz, 1H), 3.05-2.93 (m, 2H). .sup.13C NMR (76
MHz, CDCl3) .delta. 175.83, 175.64, 174.89, 174.42, 138.44, 137.01,
135.40, 135.26, 134.60, 132.19, 132.06, 131.99, 131.95, 129.08,
128.71, 128.61, 128.52, 128.45, 128.14, 128.09, 127.94, 92.07,
91.46, 80.95, 79.63, 61.62, 60.85, 50.02, 48.22, 48.04, 46.18,
42.63, 42.40, 29.72. (Gussain 1.60)
##STR00023##
Fmoc-Cys-(S-4-aminobutyric-N-Benzyl-maleimide)-(O-allyl) (222)
[0152] Endcap (151) (67.7 mg, 0.216 ml) was dissolved in DMF (0.5
ml) and cooled to 0.degree. C. 3 equimolar amount of DIPEA (0.06
ml) was subsequently added to the solution, followed by the
addition of EDC (38.53 mg, 0.273 mmol) and HOBT (30.71 mg, 1.1 eq).
The mixture was stirred under nitrogen for 10 minutes. Then
Allyl-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-mercaptopropanoate
(201) (105.16 mg, 0.324 mmol) was added to the mixture and stirred
under nitrogen overnight. The reaction mixture was diluted with
ethyl acetate (20 ml) two times and then acidified with the 10%
hydrochloric acid. The organic fraction was washed once more with
saturated sodium bicarbonate (8 ml) and once more with brine (15
ml). The resulting organic fraction was dried with anhydrous
MgSO.sub.4 and concentrated under reduced pressure and the crude
mixture was purified by silica column chromatography in order to
yield the title compound as solid (11 mg, 16.2% yield) Rf 0.13 in
1:1 hexane/ethyl acetate. .sup.1H NMR (300 MHz, CDCl3) .delta. 7.76
(t, J=7.0 Hz, 3H), 7.69-7.50 (m, 3H), 7.50-7.22 (m, 8H), 6.03-5.82
(m, 4H), 5.76 (d, J=8.2 Hz, 1H), 5.66 (d, J=8.3 Hz, 1H), 5.48-5.04
(m, 9H), 4.63 (dt, J=22.9, 5.9 Hz, 12H), 4.41 (dq, J=12.8, 8.6, 6.4
Hz, 3H), 4.23 (dt, J=13.4, 7.1 Hz, 2H), 3.79 (d, J=4.4 Hz, 1H),
3.22-2.84 (m, 6H), 1.42 (p, J=5.7 Hz, 1H), 1.34-1.16 (m, 2H).
Evaluation of Thermal Sensitivity of End-Caps:
Details Regarding the Kinetic Studies
[0153] In other experiments, kinetic studies were carried out in an
NMR tube (see FIGS. 3 and 4). All samples were prepared in the same
way. Forward Diels-Alder reactions: 5.65 mg of furan (5.0 .mu.L,
0.057 mmol) and an equimolar amount of the maleimide was added
directly to 750 .mu.L of deuterated solvent (either acetonitrile-d3
or more often DMSO-d6 for any reaction 70.degree. C. or above). For
reverse Diels-Alder reactions, 16.0 mg of cycloadduct was added to
3.0 mL of deuterated solvent in a vial. The solution was then
partitioned between 4 NMR tubes and stored at -20.degree. C. until
used for an experiment. The experiments were carried out at the
indicated temperatures on a Bruker 300 .sup.1H NMR spectrometer
equipped with a variable temperature probe. A blank NMR tube
containing the solvent but no analyte was inserted into the probe
and the spectrometer was allowed to equilibrate to the indicated
temperature for 10 minutes. The tube was then switched for the
sample, and the experiment was run with a spectrum being collected
every minute for the first 11 minutes, and then every 5 minutes
thereafter for a total of four hours using the multi_zgvd script in
the Bruker Topspin suite (8 scans per spectrum). Following data
collection, the first and last spectra of the series were examined.
If there was no change (and no appearance of starting material or
product as determined by comparison of the spectra with those of
previously isolated samples), no further action was taken, and
conversion was determined to effectively be 0%. If any integration
was noted in the final spectrum at the frequency corresponding to
the peaks of the relevant reaction products, then all spectra in
the series were integrated over identical frequency ranges. The
data was exported to .txt files, imported into excel and converted
to reaction conversions. Initial rates were calculated based on the
first four data points. In all observed cases, this region was
effectively linear. The time required to obtain 25% conversion was
determined by a simple linear interpolation between the relevant
data points. This value is arbitrary, but a half-life was
considered to be a non accurate measurement as many of the
cycloreversions plateau before 50% conversion due to the
equilibrium kinetics of the system. The 25% conversion point was
generally observed in the early stages of the cycloreversion and
was selected as a meaningful surrogate for our own use of these
systems, and will potentially play the same role for other
researchers.
Synthesis of Linear Peptides:
[0154] The syntheses of the peptides were carried out using an
AAPTTEC FocusXC-6RV automated peptide synthesizer (6 reaction
vessels), equipped with an argon atmosphere, mechanical and
gas-bubbling shaking systems, and a reaction vessel heating and/or
cooling system controlled from an IBM PC using Focus XC software
(v. 3.03). Preloaded Fmoc-Gly-Wang or Fmoc-Ala-Wang resins were
swollen in DMF for 1 hour followed by filtration, and then were
subjected to 20% piperidine in DMF twice successively for 30 mins.
Peptide couplings were carried out according to standard protocols
for Fmoc solid phase synthesis using HCTU as coupling agent (see
Chan, W. C. et al., Basic procedures. In Fmoc Solid Phase Peptide
Synthesis: A Practical Approach, Chan, W. C.; White, P. D., Eds.
Oxford Univesity Press: Oxford, 2000; pp 41-76, the entire contents
of which are incorporated herein by reference). All residues were
coupled using 5 equivalents of amino acid per functionalized
position on the resin with 1 hour reaction times. All couplings
were carried out as double couplings. Following the coupling of
each residue deprotection of the Fmoc moiety was accomplished by
treatment with 20% piperidine in DMF twice successively for 30
mins. Before and after each coupling, the beads were shaken 4 times
with 4 mL of DMF followed by filtration. Following the synthesis
the beads were washed extensively with DMF (6.times.4 mL), MeOH
(6.times.4 mL), DCM (6.times.4 mL), hexanes (6.times.4 mL), and
finally by ethanol (3.times.6 mL) and then removed from the
synthesizer and stored in a desiccator under vacuum in the presence
of P.sub.2O.sub.5 until required. A small amount cleaved from the
resin using 92.5:5:2.5 (v/v/v) TFA: triisopropylsilane: water. The
peptide was purified using RP-HPLC (ramp from 0% to 18%
acetonitrile in water over 5 mins followed by isocratic flow for 25
mins) unless otherwise stated.
[0155] While the invention has been described with reference to
preferred embodiments, the invention is not or intended by the
applicant to be so limited. A person skilled in the art would
readily recognize and incorporate various modifications, additional
elements and/or different combinations of the described components
consistent with the scope of the invention as described herein.
* * * * *
References