U.S. patent application number 17/356339 was filed with the patent office on 2021-12-30 for conjugates of a pharmaceutical agent and a moiety capable of binding to a glucose sensing protein.
The applicant listed for this patent is Sanofi. Invention is credited to Stefan PETRY, Oliver PLETTENBURG, Norbert TENNAGELS, Ulrich WERNER.
Application Number | 20210401947 17/356339 |
Document ID | / |
Family ID | 1000005826001 |
Filed Date | 2021-12-30 |
United States Patent
Application |
20210401947 |
Kind Code |
A1 |
PETRY; Stefan ; et
al. |
December 30, 2021 |
CONJUGATES OF A PHARMACEUTICAL AGENT AND A MOIETY CAPABLE OF
BINDING TO A GLUCOSE SENSING PROTEIN
Abstract
The invention describes novel conjugates of formula (I) of a
pharmaceutical agent and a moiety capable of binding to a glucose
sensing protein allowing a reversible release of the pharmaceutical
agent depending on the glucose concentration.
Inventors: |
PETRY; Stefan; (Frankfurt am
Main, DE) ; PLETTENBURG; Oliver; (Frankfurt am Main,
DE) ; TENNAGELS; Norbert; (Frankfurt am Main, DE)
; WERNER; Ulrich; (Frankfurt am Main, DE) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Sanofi |
Paris |
|
FR |
|
|
Family ID: |
1000005826001 |
Appl. No.: |
17/356339 |
Filed: |
June 23, 2021 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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16306243 |
Nov 30, 2018 |
11090364 |
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PCT/EP2017/063447 |
Jun 2, 2017 |
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17356339 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07H 19/056 20130101;
A61K 38/28 20130101; C07H 15/04 20130101; C07H 13/10 20130101; C07H
15/26 20130101; C07H 15/12 20130101; C07K 14/62 20130101; C07H
13/08 20130101; A61K 47/549 20170801; A61P 3/08 20180101; C07H
19/04 20130101 |
International
Class: |
A61K 38/28 20060101
A61K038/28; A61K 47/54 20060101 A61K047/54; C07H 13/10 20060101
C07H013/10; C07K 14/62 20060101 C07K014/62; C07H 19/04 20060101
C07H019/04; C07H 19/056 20060101 C07H019/056; C07H 15/12 20060101
C07H015/12; C07H 15/04 20060101 C07H015/04; C07H 13/08 20060101
C07H013/08; C07H 15/26 20060101 C07H015/26; A61P 3/08 20060101
A61P003/08 |
Foreign Application Data
Date |
Code |
Application Number |
Jun 2, 2016 |
EP |
16305640.1 |
Claims
1-40. (canceled)
41. A compound of formula (Ia)
R--(O.dbd.C)-[L.sub.1].sub.m-[A.sub.1].sub.o-[L.sub.2].sub.p-[A.sub.2].su-
b.r-[L.sub.3].sub.q-S (Ia) wherein L.sub.1, L.sub.2, and L.sub.3
are independently a linker having a chain length of 1-20 atoms,
A.sub.1 and A.sub.2 are independently: a 5 to 6 membered monocyclic
ring, a 9 to 12 membered bicyclic ring, two 5 to 6 membered
monocyclic rings connected to each other, two 9 to 12 membered
bicyclic rings connected to each other, or a 5 to 6 membered
monocyclic ring and a 9 to 12 membered bicyclic ring connected to
each other wherein each ring is independently a saturated,
unsaturated, or aromatic carbocyclic or heterocyclic ring and
wherein each ring is optionally modified with at least one sub
stituent, S is a sugar moiety which binds to the insulin
independent glucose transporter GluT1, and wherein the sugar moiety
S comprises a terminal pyranose Si moiety which is attached via
position 2, 4, or 6 to the compound of formula (Ia), m, o, p, r,
and q are independently of each other 0 or 1, and wherein at least
one of r and o is 1, R is H, halogen, OH, O-alkyl-, an anhydride
forming group, an active ester forming group, 4-nitrophenylester,
succinate, or N-hydroxy benzotriazol, or a pharmaceutically
acceptable salt or solvate thereof.
42. (canceled)
43. The compound of formula (Ia) of claim 41, wherein L.sub.1,
L.sub.2, and L.sub.3 are independently of each other
(C.sub.1-C.sub.20) alkylene, (C.sub.2-C.sub.20) alkenylene, or
(C.sub.2-C.sub.20) alkynylene, wherein one or more C-atoms may be
replaced by heteroatoms or heteroatom moieties, particularly by O,
NH, N(C.sub.1-4) alkyl, S, SO, SO.sub.2, O--SO.sub.2, O--SO.sub.3,
O--PHO.sub.2, or O--PO.sub.3 and/or wherein one or more C-atoms may
be substituted with (C.sub.1-4) alkyl, (C.sub.1-4) alkyloxy, oxo,
carboxyl, halogen, F, Cl, Br, or I, or a phosphorus-containing
group.
44. The compound of formula (Ia) of claim 41, wherein L.sub.3 is
(C.sub.1-C.sub.6) alkylene, or (C.sub.1-4) alkylene, wherein one or
two C-atoms may be replaced by heteroatoms or heteroatom moieties,
particularly by O, NH, N(C.sub.1-4) alkyl, S, SO, SO.sub.2,
O--SO.sub.2, O--SO.sub.3, O--PHO.sub.2, or O--PO.sub.3 and/or
wherein one or more C-atoms may be substituted with (C.sub.1-4)
alkyl, (C.sub.1-4) alkyloxy, oxo, carboxyl, halogen, e.g. F, Cl,
Br, or I, or a phosphorus-containing group.
45. The compound of formula (Ia) of claim 41, wherein L.sub.3 is
C.dbd.O.
46. The compound of formula (Ia) of claim 41, wherein L.sub.2 is
selected from --CO--(CH.sub.2).sub.3--, --(CH.sub.2).sub.6--NH--,
--(CH.sub.2).sub.2--CO--(CH.sub.2--CH.sub.2--O).sub.2--(CH.sub.2).sub.2---
NH-- or --CH.sub.2--O--(CH.sub.2--CH.sub.2--O).sub.3--.
47. The compound of formula (Ia) of claim 41, wherein A.sub.1 and
A.sub.2 are independently of each other a heterocyclic ring,
wherein the ring may carry at least one sub stituent.
48. The compound of formula (Ia) of claim 41, wherein A.sub.1 and
A.sub.2 are independently of each other selected from a 5 to 6
membered monocyclic or a 9 to 12 membered bicyclic ring, wherein
the ring is heterocyclic with 1 to 4 ring atoms being selected from
N, O, and/or S, and wherein the ring may carry at least one
substituent.
49. The compound of formula (Ia) of claim 41, wherein A.sub.1 and
A.sub.2 are independently of each other a 5 to 6 membered
monocyclic ring, wherein the ring is a heteroalkyl ring,
particularly selected from pyrrolidinyl, pyrazolidinyl,
imidazolidinyl, triazolidinyl, piperazinyl, piperidinyl, or
morpholinyl, wherein the ring may carry at least one substituent,
or a 9 to 12 membered bicyclic ring wherein the ring is a
heteroalkyl ring with 1 to 4 ring atoms being selected from N, O,
and/or S, and wherein the ring may carry at least one
substituent.
50. The compound of formula (Ia) of claim 41, wherein A.sub.1 and
A.sub.2 are independently of each other 1,2,3-triazolyl.
51. The compound of formula (Ia) of claim 41, wherein A.sub.2 is
1,2,3-triazolyl.
52. The compound of formula (Ia) of claim 41, wherein A.sub.2 is
piperazinyl.
53. The compound of formula (Ia) of claim 41, wherein r=1 and
A.sub.2 is present and o=0 and A.sub.1 is absent.
54. The compound of formula (Ia) of claim 41, wherein r=1 and
A.sub.2 is present and o=1 and A.sub.1 is present.
55. The compound of formula (Ia) of claim 41, wherein (i) m=1, o=0,
p=0, and q=0 or 1, or wherein (ii) m=1, o=1, p=1, and q=0 or 1.
56. The compound of formula (Ia) of claim 41, wherein A.sub.2 is
piperazinyl, L.sub.2 is absent and A.sub.1 is cyclohexanyl.
57. The compound of formula (Ia) of claim 41, wherein A.sub.2 is
piperazinyl, L.sub.2 is absent and A.sub.1 is cyclohexanyl.
58. The compound of formula (Ia) of claim 41, wherein A.sub.2 is
piperazinyl, L.sub.2 is --CH.sub.2-- and A.sub.1 is
cyclohexanyl.
59. The compound of formula (Ia) of claim 41, wherein A.sub.2 is
piperazinyl, L.sub.2 is absent and A.sub.1 is phenyl.
60. The compound of formula (Ia) of claim 41, wherein A.sub.2
is1,2,3-triazolyl, L.sub.2 is absent and A.sub.1 is phenyl.
61. The compound of formula (Ia) of claim 41, wherein L3 is --CO--,
A.sub.1 is phenyl, L.sub.2 is --O-- and A.sub.1 is phenyl wherein
each ring may be unsubstituted or carry at least one substituent,
for example, 1 to 3 substituents selected from halogen, NO.sub.2,
CN, (C.sub.1-4) alkyl, (C.sub.1-4) alkoxy,
(C.sub.1-4)alkyl-(C.sub.3-7)cycloalkyl, (C.sub.3-7) cycloalkyl, OH,
benzyl, --O-benzyl, carboxyl, carboxyester, carboxamide, or mono
(C.sub.1-4) alkyl, or di (C.sub.1-4) alkyl carboxamide.
62. The compound of formula (Ia) of claim 41, wherein the group
-A.sub.2-L.sub.3- is selected from the group consisting of:
##STR00179##
63. The compound of formula (Ia) of claim 41, wherein the sugar
moiety S comprises a terminal pyranose moiety S1 having a backbone
structure of Formula (II) ##STR00180## wherein 1, 2, 3, 4, 5, and 6
denote the positions of the C-atoms in the pyranose moiety; wherein
is a single bond and is a single or a double bond; and R1 and R3
are H or a protecting group; which is attached via position 2, 4,
or 6 to the compound of formula (Ia).
64. The compound of formula (Ia) of claim 63, wherein the terminal
pyranose moiety Si is selected from glucose, galactose,
4-deoxyglucose, or 4,5-dehydroglucose derivatives, wherein the
terminal pyranose moiety S1 is attached via position 2, 4, or 6 to
the compound of formula (Ia); or S1 is mannose attached via
position 6.
65. The compound of formula (Ia) of claim 63, wherein the terminal
pyranose moiety S1 is of the Formula (IIIa) or (IIIb): ##STR00181##
wherein R1 is H or a protecting group, R2 is OR8, or NHR8 or an
attachment site to the compound of formula (Ia), wherein R8 is H or
a protecting group, R3 is H or a protecting group, R4 is H, OR8, or
NHR8 or an attachment site to the compound of formula (Ia), wherein
R8 is H or a protecting group, or R1 and R2 and/or R3 and R4 form
together with the pyranose ring atoms to which they are bound a
cyclic group, R5 and R6 are H or together with the carbon atom to
which they are bound form a carbonyl group, R7 is OR8, or NHR8 or
an attachment site to the compound of formula (Ia), wherein R8 is H
or a protecting group, and wherein one of R2, R4, and R7 is the
attachment site to the compound of formula (Ia).
66. The compound of formula (Ia) of claim 63, wherein R1 and R3 are
H.
67. The compound of formula (Ia) of claim 63, wherein R2 is OR8 or
an attachment site to the compound of formula (Ia), R4 is H, OR8,
or an attachment site to the compound of formula (Ia), and R7 is
OR8 or an attachment site to the compound of formula (Ia), and
wherein R8 is H or a protecting group.
68. The compound of formula (Ia) of claim 63, wherein position 6 of
the pyranose moiety S1 and particularly R7 is the attachment site
to the compound of formula (Ia).
69. The compound of formula (Ia) of claim 63, wherein the pyranose
moiety S1 is of formula (IVa), (IVb), (IVc), (IVd), or (IVe):
##STR00182## wherein R1, R2, R3, R5, R6, and R7 are defined as in
any one of claims 65-68 and wherein R4 is H, a protecting group, or
an attachment site to the compound of formula (Ia), or R4a is H, or
an attachment site to the compound of formula (Ia).
70. The compound of formula (Ia) of claim 41, wherein the sugar
moiety S is of Formula (V): --[X.sub.2--S.sub.2].sub.s--X.sub.1--S1
(V) wherein X.sub.1 is a bond or O, X.sub.2 is a bond, NH or O,
S.sub.2 is a mono- or disaccharide moiety, particularly comprising
at least one hexose or pentose moiety, more particularly at least
one pyranose or furanose moiety and S1 is a terminal pyranose
moiety as defined in any one of claims 63-68, and s is 0 or 1.
71. The compound of formula (Ia) of claim 70, wherein the
saccharide moiety S2 is a pyranose moiety, particularly selected
from glucose, galactose, 4-deoxyglucose and 4,5-dehydroglucose
derivatives, or a furanose moiety, particularly selected from
fructose derivatives.
72. The compound of formula (Ia) of claim 70, wherein the
saccharide moiety S2 is of Formula (VIa), (VIb), (VIc), (VId), or
(VIe): ##STR00183## wherein R11 is a bond to X.sub.1, R12 is OR8 or
NHR8 or an attachment site to X.sub.2, wherein R8 is H or a
protecting group, R13 is H or a protecting group, R14 is R8 or an
attachment site to X.sub.2, wherein R8 is H or a protecting group,
R14a is H or an attachment site to X.sub.2, R15 and R16 are H or
together with the carbon atom to which they are bound form a
carbonyl group, R17 is OR8 or an attachment site to X.sub.2,
wherein R8 is H or a protecting group, or R11 and R12 and/or R13
and R14 form together with the ring atoms to which they are bound a
cyclic group, and wherein one of R12, R14, and R17 is an attachment
site to X.sub.2.
73. The compound of formula (Ia) of claim 41, wherein the sugar
moiety S comprises a single terminal saccharide moiety.
Description
[0001] The invention describes novel conjugates of a pharmaceutical
agent and a moiety capable of binding to a glucose sensing protein
allowing a reversible release of the pharmaceutical agent depending
on the glucose concentration.
[0002] Over the last decades the number of patients suffering from
diseases, particulary from type 2 diabetes, has increased
dramatically. Despite education and treatment the growth rate is
exploding. The disease evolves slowly and in the beginning the
pancreas can compensate decreasing insulin sensitivity by an
increased release of insulin. At this stage oral antidiabetics like
insulin sensitizers and--releasers can support this compensation
mechanism, but cannot cure the disease. So after this period of
time external insulin has to be injected.
[0003] Several insulins are on the market, which are classified by
their duration of action. The intrinsic danger of hypoglycemia is
counteracted by very flat insulin profiles (so called basal
insulins), but is neither conceptionally addressed nor finally
overcome by these basal insulins.
[0004] The development of a real glucose sensing insulin
accomplishing a glucose dependent release from a depot simulating
the natural release by the pancreas is still one of the holy grails
in diabetes research. Such an insulin would generate a local (eg
intraparenteral) or moving depot (blood streem) from where it is
released in a glucose concentration dependent manner and finally
recaptured by the system on decreasing glucose concentrations.
[0005] The blood glucose concentration is under hormonal
regulation. While several hormones like glucagon, epinephrine,
norepinephrine, cortisol, and hormones from the thyroid gland
provoke elevated glucose levels, insulin is the only hormone which
lowers glucose levels. In addition the glucose level is of course
influenced by timing and composition of meals, physical stress, and
infections.
[0006] In healthy persons the fasting blood glucose level is around
5 mM (900 mg/L) and can after a meal increase to 40 mM for several
hours. In diabetic patients where blood glucose is out of control,
the level can vary between 1-30 mM and can unpredictable fluctuate
between the borders of hyperglycemia (>10 mM) and hypoglycemia
(<3 mM). Despite the possibility of exact blood glucose
measurement and titration of insulins, hypoglycemia is still a
serious problem. This problem can be solved by glucose sensitive
and--responsive delivery of pharmaceutical agents effecting the
glucose level.
[0007] Non glucose-sensitive depots to protect drugs (small
molecules and proteins like insulin) from degradation and elongate
their half life are used frequently in medicine. For insulin for
example a static subcutanous depot can be realized. Insulin is
stored as insoluble hexamers. From this depot soluble monomers are
released to the blood following law of mass equation.
[0008] An additional opportunity is the non-covalent binding of
modified insulins to albumin. Since unmodified insulin is not
binding to albumin, noncovalent hydrophobic binding is enabeled by
hydrophobic modification (eg by myristic acid). Coupling of fatty
acids to insulin enable protection of insulin from degradation and
dramatically increases half life by hours to days.
[0009] The release of insulin from such a circulating depot can be
described by the law of mass equation and is a function of the
amount of insulin, the albumin depot, and the affinity of the
insulin derivative to albumin. Since the depot is fixed, the amount
and affinity of insulin have to be adjusted. The release of basal
insulin can be controlled, but the release is glucose
independent.
[0010] Within the last decade efforts have been started to
establish glucose sensitive insulin depots. These efforts can be
summarized and assigned to three classical principles: [0011]
Chemical recognition of glucose by boronic acids [0012] Biochemical
recognition of glucose by carbohydrate binding proteins like such
as lectins (Concanavalin A, wheat germ agglutinin) [0013] Glucose
converting enzymes like glucose oxidase or hexokinase. Here binding
affinity can be used as a signal. More frequently associated pH
shift or change of charge is measured.
[0014] These principles can be used for glucose measurement or to
translate the signals into direct or indirect glucose release. Four
possibilities for realization are described below. [0015] Direct
modification of insulins [0016] "Glucose responsive" hydrogels,
these are synthetic pores, which are modified with a glucose
sensing molecule (boronic acid- or glucose oxidase based). These
gels are filled with insulins. In the presence of glucose they
expand, get leaky, and finally release insulin on increasing
glucose levels. [0017] "Device-approaches": In this case insulin
levels are only measured by a sensor. [0018] Closed loop
approaches: This describes a technical solution. A sensor measures
glucose levels. The signal is transmitted to an independent insulin
depot (eg a pump) which releases insulin triggered by the signal.
An independent insulin reservoir is triggered and releases insulin,
controlled by the sensor signal. An advantage may be a large
insulin depot which is not necessarily in the body.
[0019] Several patent applications, e.g. WO 2001/92334, WO
2011/000823, or WO 2003/048195 describe the use of boronic acid
modified insulin derivatives in combination with albumin for a
glucose sensitive insulin release. With this approach the floating
insulin/albumin depot shall be further developed to a glucose
sensing floating depot.
[0020] A different approach for a glucose sensing approach has been
described in WO 2010/088294, WO 2010/88300, WO 2010/107520, WO
2012/015681, WO 2012/015692, or WO 2015/051052. These documents
describe the concomitant administration of concanavalin A and a
glucose binding protein preferably recognizing mannose. Accordingly
mannose modified insulins can be released by mannose from a depot.
In addition an intrinsic mannose binding protein is described which
may be responsible for the binding of mannose without the need of
concanavalin.
[0021] Erythrocytes have been used as a vehicle for the transport
of drugs, e.g. for tumor starvation, enzyme replacement and
immunotherapy as described in WO 2015/121348, WO 2014/198788 and WO
2013/139906.
[0022] Liu et al. (Bioconjugate Chem. 1997, 8, 664-672) discloses a
glucose induced release of glucosylpoly(ethylene glycol) insulin
bound to a soluble conjugate of concanavalin A wherein the insulin
is linked at the B1 amino group with a poly(ethylene glycol) spacer
to the 1-position of the sugar.
[0023] WO2012/177701 discloses conjugates of .sup.68Ga-DOTA
labelled sugars for tissue specific disease imaging and
radiotherapy.
[0024] The use of erythrocytes as a classical depot, by binding
drugs to the surface of erythrocytes is described in WO
2013/121296. Here peptides are described, which bind to the surface
with a very high affinity (K.sub.D=6,2 nM). These peptides are used
for immunmodulation e.g. in transplantation medicine.
[0025] The present invention relates to a novel conjugate
comprising a pharmaceutical agent and a sugar moiety.
[0026] Further the present invention relates to a novel conjugate
comprising a pharmaceutical agent and a sugar moiety for use as a
pharmaceutical.
[0027] Further the present invention relates to a novel conjugate
comprising a pharmaceutical agent and a sugar moiety which binds to
the insulin dependent glucose transporter GluT1, which provides a
release of the pharmaceutical agent dependent on the glucose
concentration in blood. The insulin dependent glucose transporter
GluT1 is present on erythrocytes. Binding of glucose to GluT1 is
reversible based on the blood glucose concentration.
[0028] In one embodiment the conjugate of the invention is bound to
GluT1 at low glucose concentrations of e.g. 1-10 mM, which are
found under fasting conditions. Under these conditions, the stable
floating depot of the active agent is formed. After an increase in
the glucose concentration from e.g. 30 mM to 40 mM after a meal,
the free glucose competes for the GluT1 binding site and the
conjugate is released in a glucose concentration dependent manner
and the pharmaceutical agent is available to exert its effect. As
the glucose concentration decreases again, the conjugate molecules
are recaptured by GluT1. Thus, the presence of undesired high
amounts of free pharmaceutical agents is avoided.
[0029] The present invention relates to conjugates of formula
(I):
P-[L.sub.1].sub.m-[A.sub.1].sub.0-[L.sub.2].sub.p-[A.sub.2].sub.r-[L.sub-
.3].sub.q-S (I)
wherein P is a pharmaceutical agent, particularly a peptide,
L.sub.1, L.sub.2, and L.sub.3 are independently of each other a
linker having a chain length of 1-20 atoms, A.sub.1 and A.sub.2 are
independently of each other a 5 to 6 membered monocyclic ring or a
9 to 12 membered bicyclic ring, or two 5 to 6 membered monocyclic
and/or 9 to 12 membered bicyclic rings connected to each other,
wherein each ring is independently a saturated, unsaturated, or
aromatic carbocyclic or heterocyclic ring and wherein each ring may
carry at least one substituent, S is a sugar moiety which binds to
the insulin independent glucose transporter GluT1, and m, o, p, r,
and q are independently of each other 0 or 1, and wherein at least
one of r and o is 1, or a pharmaceutically acceptable salt or
solvate thereof.
[0030] The present invention relates also to conjugates of formula
(I) :
P-[L.sub.1].sub.m-[A.sub.1].sub.o-[L.sub.2].sub.p-[A.sub.2].sub.r-[L.sub-
.3].sub.q-S (I)
wherein P is an insulin or an insulinotropic peptide, L.sub.1,
L.sub.2, and L.sub.3 are independently of each other a linker
having a chain length of 1-20 atoms, A.sub.1 and A.sub.2 are
independently of each other a 5 to 6 membered monocyclic ring or a
9 to 12 membered bicyclic ring, or two 5 to 6 membered monocyclic
and/or 9 to 12 membered bicyclic rings connected to each other,
wherein each ring is independently a saturated, unsaturated, or
aromatic carbocyclic or heterocyclic ring and wherein each ring may
carry at least one substituent, S is a sugar moiety which binds to
the insulin independent glucose transporter GluT1, and comprises a
terminal pyranose S1 moiety which is attached via position 2, 4, or
6 to the conjugate of formula (I), s m, o, p, r, and q are
independently of each other 0 or 1, and wherein at least one of r
and o is 1, or a pharmaceutically acceptable salt or solvate
thereof.
[0031] Another aspect of the invention are compounds of formula
(Ia) and (Ib):
R--(O.dbd.C)-[L.sub.1].sub.m-[A.sub.1].sub.0-[L.sub.2].sub.p-[A.sub.2].s-
ub.r-[L.sub.3].sub.q-S (Ia)
[L.sub.1].sub.m-[A.sub.1].sub.o-[L.sub.2].sub.p-[A.sub.2].sub.r-[L.sub.3-
].sub.q-S (Ib)
wherein L.sub.1, L.sub.2, L.sub.3, A.sub.1, A.sub.2, S, m, o, p, r,
and q are defined as indicated above and R is H, halogen, OH,
O-alkyl-, an anhydride forming group or another active ester
forming group for coupling reactions, like 4-nitrophenylester,
succinate or N-hydroxy benzotriazol.
[0032] or pharmaceutically acceptable salts or solvates
thereof.
[0033] Compounds (Ia) and (Ib) are suitable as intermediates for
the synthesis of the conjugates of formula (I).
[0034] Another aspect of the present invention is the conjugate of
formula (I) as described above for the use in medicine,
particularly in human medicine.
[0035] Another aspect of the present invention is a pharmaceutical
composition comprising a conjugate of formula (I) as described
above as an active agent and a pharmaceutically acceptable
carrier.
[0036] Another aspect of the present invention is a method of
preventing and/or treating a disorder associated with, caused by,
and/or accompanied by a dysregulated glucose metabolism, comprising
administering a conjugate of formula (I) or a composition as
described above to a subject in need thereof, particularly a human
patient.
[0037] Another aspect of the present invention is a method of
preventing and/or treating diabetes type 1 or diabetes type 2.
[0038] The conjugates of formula (I) of the present invention
comprise a pharmaceutical agent P, which may a biomolecule, such as
a peptide. Preferably, the pharmaceutical agent has an effect of
directly or indirectly lowering the glucose concentration in blood.
For example, the pharmaceutical agent may be an insulin or an
insulinotropic peptide.
[0039] The term "insulin" according to the present invention
encompasses human insulin, porcine insulin, or analogs thereof,
e.g. prandial insulins with fast action or basal insulins with long
action. For example, the term "insulin" encompasses recombinant
human insulin, insulin glargine, insulin detemir, insulin
glulisine, insulin aspart, insulin lispro, etc. or an insulin
conjugated to a polyethylene, e.g. a low molecular weight PEG
having a molecular weight of 10 kDa or less. If P is an insulin, it
may be attached via an amino group to form the conjugate of formula
(I) , e.g. via an amino side chain, particularly via the amino side
chain of an insulin B29Lys residue or via the amino terminus of an
insulin B1Phe residue.
[0040] Further, the pharmaceutical agent may be an insulinotropic
peptide such as GLP-1, an exendin such as exendin-4, or a GLP-1
agonist such as lixisenatide, liraglutide.
[0041] The conjugate of formula (I) further comprises a sugar
moiety which binds to the insulin independent glucose transporter
GluT1, also known as solute carrier family 2, facilitated glucose
transporter member 1 (SLC2A1). The amino acid sequence of the human
protein is NP_006507, which is encoded by a nucleic acid sequence
NM_006516. GluT1 is an integral membrane protein which facilitates
diffusion of glucose into the erythrocyte. The highest expression
of GluT1 is found in erythrocytes.
[0042] For interaction with GluT1, the conjugate of formula (I)
comprises a moiety binding to GluT1 but preventing transport
through the erythrocyte membrane. A sugar moiety binding to GluT1
is preferably in an anomeric form, particularly in an anomeric
6-membered ring form such as a pyranose moiety. The sugar moiety
comprises an anomeric O atom as well as a hydroxy group or a
protected hydroxy group at position 3 and position 4 of a pyranose
backbone. In one embodiment, the sugar moiety S of the conjugate of
formula (I) comprises a terminal pyranose moiety which is attached
via position 2, position 4, or position 6 of the pyranose backbone
moiety.
[0043] Further, an aspect of the present invention is that
introduction of at least one cyclic residue A.sub.1 and/or A.sub.2
adjacent to the sugar moiety causes a substantial increase in the
affinity to GluT1 in comparison to glucose.
[0044] Thus, the present invention provides a pharmaceutical agent
in form of a conjugate of formula (I) which forms an
erythrocyte-based circulating depot that after administration
releases/delivers the agent as a function of glucose concentration.
Accordingly at low glucose concentrations (below 3 mM) no or only
low concentration of free unbound levels of the conjugate should be
detectable. On increasing blood glucose levels after a meal the
conjugate is released from the circulating depot into the blood
stream. The release is a consequence of a direct competition of
glucose with the conjugate of formula (I). Thus, release is
described by the law of mass equation and self adjusts to tiniest
changes in glucose levels. The same should be true for the
re-capturing process of the conjugate of formula (I) on decreasing
glucose levels.
[0045] These characteristics constitute an essential advantage in
comparison to the glucose sensing depots from the prior art.
[0046] By means of the present invention, the drawbacks of prior
art insulins with regard to glycemia are diminished or avoided. The
control of glucose recognition and associated release/retrapping
will be realized within a single molecule. This minimizes delays in
release/retrapping. Glucose sensitive binding and--release is
controlled by interaction with endogenous transport and recognition
processes. The biological recognition system based on GluT1
transport in erythrocytes is constantly regenerated by the
organism.
[0047] The present conjugate of formula (I) binds to the ubiquitary
glucose transporter GluT1, which has a binding affinity to glucose
in the same range as glucose oxidase, a protein frequently used in
glucose recognition. GluT1 is highly expressed in erythrocytes and
is responsible for the basal supply of these cells. The size of the
depot is large enough to accommodate the amount of pharmaceutical
agent needed without affecting the erythrocyte glucose supply.
[0048] The affinity of the present conjugate of formula (I) is
within an affinity window which guarantees binding at low (e.g.
<3 mM) glucose levels. With increasing glucose levels (e.g.
>10 mM) the conjugate of formula (I) is released accordingly.
With decreasing glucose levels the unbound conjugate of formula (I)
is recaptured by the transporter.
[0049] The release is following the law of mass equation and is
dependent on the size of the depot, the loading, and the affinity
of the conjugate of formula (I) to GluT1. Since the depot is fixed,
the free conjugate fraction is defined by the affinity to
GluT1.
[0050] In certain embodiments, the conjugate of formula (I) has an
affinity of 10-500 nM to the insulin independent glucose
transporter GluT1 as determined by affinity measurements for
example by a ligand displacement assay, by MST (microscale
thermophoresis) technology.
[0051] In the conjugate of formula (I) of the present invention,
the individual structural moieties P, A.sub.1, A.sub.2, and S may
be connected by linkers L.sub.1, L.sub.2, and L.sub.3. If present,
L.sub.1, L.sub.2, and L.sub.3 are linkers having a chain length of
1-20 atoms, particularly 3 to 10, or 3 to 6 atoms.
[0052] In some embodiments, L.sub.1, L.sub.2, and L.sub.3 are
independently of each other (C.sub.1-C.sub.20) alkylene,
(C.sub.2-C.sub.20) alkenylene, or (C.sub.2-C.sub.20) alkynylene,
wherein one or more C-atoms may be replaced by heteroatoms or
heteroatom moieties, particularly by O, NH, N(C.sub.1-4) alkyl, S,
SO.sub.2, O--SO.sub.2, O--SO.sub.3, O--PHO.sub.2 or O--PO.sub.3,
and/or wherein one or more C-atoms may be substituted with
(C.sub.1-4) alkyl, (C.sub.1-4) alkyloxy, oxo, carboxyl, halogen,
e.g. F, Cl, Br, or I, or a phosphorus-containing group. The
carboxyl group may be a free carboxylic acid group or a carboxylic
acid ester, e.g. C.sub.1-C.sub.4 alkyl ester or a carboxamide or
mono(C.sub.1-C.sub.4) alkyl or di(C.sub.1-C.sub.4) alkyl
carboxamide group. An example of a phosphorus-containing group is a
phosphoric acid or phosphoric acid (C.sub.1-.sub.4) alkyl ester
group.
[0053] In certain embodiments, the linker L.sub.3 has a chain
length of 1 to 15 atoms, particularly 1 to 6 or 1 to 4 atoms. For
example, L3 may be a (C.sub.1-C.sub.6) alkylene, particularly
(C.sub.1-4) alkylene group, wherein one or two C-atoms may be
replaced by heteroatoms or heteroatom moieties, particularly by O,
NH, N(C.sub.1-4) alkyl, S, SO.sub.2, O--SO.sub.2, O--SO.sub.3,
O--PHO.sub.2 or O--PO.sub.3, and/or wherein one or more C-atoms may
be substituted with (C.sub.1-4) alkyl, (C.sub.1-4) alkyloxy, oxo,
carboxyl, halogen, e.g. F, Cl, Br, or I, or a phosphorus-containing
group.
[0054] In one embodiment, the linker L.sub.3 is C.dbd.O.
[0055] In one embodiment, the linker L.sub.3 is absent.
[0056] In one embodiment, the linker L.sub.2 is
--CO--(CH.sub.2).sub.3--.
[0057] In one embodiment, the linker L.sub.2 is
--(CH.sub.2).sub.6--NH--.
[0058] In one embodiment, the linker L.sub.2 is
--(CH.sub.2).sub.2--CO--(CH.sub.2--CH.sub.2--O).sub.2--(CH.sub.2).sub.2---
NH--.
[0059] In one embodiment, the linker L.sub.2 is
--CH.sub.2--O--(CH.sub.2--CH.sub.2--O).sub.3--.
[0060] The conjugate of formula (I) of the present invention
comprises at least one cyclic group, particularly a cyclic group
A.sub.2 and optionally a further cyclic group A.sub.1. An aspect of
the present invention is that the presence of a cyclic group
adjacent to the sugar moiety S significantly enhances the binding
affinity of the sugar moiety S to the glucose transporter GluT1.
The cyclic groups A.sub.1 and A.sub.2 may be a 5 to 6 membered
monocyclic ring, a 9 to 12 membered bicyclic ring, or two 5 to 6
membered monocyclic and/or 9 to 12 membered bicyclic rings
connected to each other by a bond or 1-atom bridge, e.g. such as
--O-- or --CH.sub.2--. Each ring may be a saturated, unsaturated,
or aromatic carbocyclic or heterocyclic ring. Each ring may be
unsubstituted or carry at least one substituent, for example, 1 to
3 substituents selected from halogen, NO.sub.2, CN, (C.sub.1-4)
alkyl, (C.sub.1-4) alkoxy, (C.sub.1-4)alkyl-(C.sub.3-7)cycloalkyl,
(C.sub.3-7) cycloalkyl, OH, benzyl, --O-benzyl, carboxyl,
carboxyester, carboxamide, or mono (C.sub.1-4) alkyl, or di
(C.sub.1-4) alkyl carboxamide.
[0061] In a further embodyment, A.sub.2 and/or A.sub.1 are a
heterocyclic ring wherein 1 to 4 ring atoms, e.g. 1, 2, 3, or 4
ring atoms are selected from nitrogen, sulfur and/or oxygen and
wherein the ring may be unsubstituted or may carry at least one
substituent as described above. In an especially preferred
embodiment, A.sub.2 and A.sub.1, if present, are independently of
each other a 5 to 6 membered monocyclic ring, wherein the ring is a
heteroalkyl ring, particularly selected from pyrrolidinyl,
pyrazolidinyl, imidazolidinyl, triazolidinyl, piperazinyl,
piperidinyl, morpholinyl, wherein the ring may carry at least one
substituent, or a 9 to 12 membered bicyclic ring wherein the ring
is a heteroalkyl ring with 1 to 4 ring atoms being selected from N,
O, and/or S, and wherein the ring may carry at least one
substituent.
[0062] In an especially preferred embodiment, A.sub.2 and A.sub.1,
if present, are selected from pyrrolidinyl, pyrazolidinyl,
imidazolidinyl, triazolidinyl, piperazinyl, piperidinyl,
morpholinyl.
[0063] In a further embodiment A.sub.2 and/or A.sub.1 are
1,2,3-triazolyl.
[0064] In a further embodiment A.sub.2 is 1,2,3-triazolyl.
[0065] In a further embodiment A.sub.2 is piperazinyl.
[0066] A further group of embodiments are conjugates of formula (I)
wherein A.sub.2 is piperazinyl, L.sub.2 is absent and A.sub.1 is
cyclohexanyl.
[0067] A further group of embodiments are conjugates of formula (I)
wherein A.sub.2 is piperazinyl, L.sub.2 is absent and A.sub.1 is
cyclohexanyl.
[0068] A further group of embodiments are conjugates of formula (I)
wherein A.sub.2 is piperazinyl, L.sub.2 is --CH.sub.2-- and A.sub.1
is cyclohexanyl.
[0069] A further group of embodiments are conjugates of formula (I)
wherein A.sub.2 is piperazinyl, L.sub.2 is absent and A.sub.1 is
phenyl.
[0070] A further group of embodiments are conjugates of formula (I)
wherein A.sub.2 is 1,2,3-triazolyl, L.sub.2 is absent and A.sub.1
is phenyl.
[0071] A further group of embodiments are conjugates of formula (I)
wherein
L.sub.3 is --CO--, A.sub.1 is phenyl, L.sub.2 is --O-- and A.sub.1
is phenyl wherein each ring may be unsubstituted or carry at least
one substituent, for example, 1 to 3 substituents selected from
halogen, NO.sub.2, CN, (C.sub.1-4) alkyl, (C.sub.1-4) alkoxy,
(C.sub.1-4)alkyl-(C.sub.3-7)cycloalkyl, (C.sub.3-7) cycloalkyl, OH,
benzyl, --O-benzyl, carboxyl, carboxyester, carboxamide, or mono
(C.sub.1-4) alkyl, or di (C.sub.1-4) alkyl carboxamide.
[0072] A further group of embodiments are conjugates of formula (I)
wherein the group -A.sub.2-L.sub.3- is selected from
##STR00001##
[0073] In a further embodiment of the present invention, the
conjugate of formula (I) comprises a single cyclic group A.sub.2
and the second cyclic group A.sub.1 is absent. In such embodiments,
the conjugate of formula (I) may have a structure wherein m=1, o=0,
p=0, and q=0 or 1. In other embodiments, a second cyclic group
A.sub.1 is present. In such embodiments, the conjugates of formula
(I) may have a structure wherein m=1, o=1, p=1, and q=0 or 1.
[0074] The conjugate of formula (I) comprises a sugar moiety S
which binds to the insulin independent glucose transporter GluT1.
This sugar moiety S may comprise a terminal pyranose moiety which
is attached via position 2, 4, or 6 to the conjugate of formula
(I). In one embodiment the terminal pyranose moiety is attached via
position 6 to the conjugate of formula (I).
[0075] In some embodiments, the sugar moiety S may comprise a
terminal pyranose moiety S1 having a backbone structure of Formula
(II)
##STR00002##
wherein 1, 2, 3, 4, 5, and 6 denote the positions of the C-atoms in
the pyranose moiety, wherein is a single bond and is a single or a
double bond, and R1 and R3 are H or a protecting group, and wherein
S1 is attached via position 2, 4, or 6 to the conjugate of formula
(I) .
[0076] The protecting group may be any suitable protecting group
known in the art, e.g. an acyl group such as acetyl or benzoyl, an
alkyl group such as methyl, an aralkyl group such as benzyl, or
4-methoxybenzyl (PMB) including divalent protecting groups such as
isopropylidene or benzylidene.
[0077] In some embodiments, the terminal pyranose moiety may be
selected from glucose, galactose, 4-deoxyglucose, and
4,5-dehydroglucose derivatives, wherein the terminal pyranose
moiety is attached via position 2, 4, or 6 to the conjugate of
formula (I) or is mannose attached via position 6.
[0078] In another embodiment, the terminal pyranose moiety S1 is of
the Formula (IIIa) or (IIIb):
##STR00003##
wherein R1 is H or a protecting group such as methyl or acetyl, R2
is OR8, or NHR8 or an attachment site to the conjugate of formula
(I), wherein R8 is H or a protecting group such as acetyl or
benzyl, R3 is H or a protecting group such as acetyl or benzyl, R4
is H, OR8, or NHR8 or an attachment site to the conjugate of
formula (I), wherein R8 is H or a protecting group such as acetyl
or benzyl, or R1 and R2 and/or R3 and R4 form together with the
pyranose ring atoms to which they are bound a cyclic group, e.g. an
acetal, R5 and R6 are H or together form together with the carbon
atom to which they are bound a carbonyl group, R7 is OR8, or NHR8
or an attachment site to the conjugate of formula (I) , wherein R8
is H or a protecting group such as acetyl or benzyl, and wherein
one of R2, R4, and R7 is the attachment site to the conjugate of
formula (I).
[0079] In another embodiment of the terminal pyranose moiety S1 of
the formula (IIIa) and (IIIb), R1 and R3 are H. In further
embodiments of the terminal pyranose moiety S1 of the formula
(IIIa) and (IIIb), R2 is OR8, or an attachment site to the
conjugate of formula (I), R4 is H, OR8, or an attachment site to
the conjugate of formula (I), R7 is OR8 or an attachment site to
the conjugate of formula (I), and wherein R8 is H or a protecting
group.
[0080] In another embodiment of the terminal pyranose moiety S1 of
the formula (IIIa) and (IIIb), position 6 of the pyranose moiety
and particularly substituent R7 is the attachment site of the
terminal pyranose moiety S1 to the conjugate of formula (I).
[0081] In specific embodiments, the pyranose moiety S1 is of
formula (IVa), (IVb), (IVc), (IVd), or (IVe):
##STR00004##
wherein R1, R2, R3, R5, R6, and R7 are defined as indicated above,
wherein R4a is H or the attachment site to the conjugate of formula
(I), and wherein R4 is H, a protecting group, or the attachment
site to the conjugate of formula (I).
[0082] The sugar moiety S of the conjugate of formula (I) may
comprise one or more, e.g. 2, or 3 saccharide units. For example,
the sugar moiety has a structure of formula (V):
--[X.sub.2--S.sub.2].sub.s--X.sub.1--S1 (V)
wherein X.sub.1 is a bond or O, particularly a bond, X.sub.2 is a
bond, NH or O, particularly a bond, S2 is a mono- or disaccharide
moiety, particularly comprising at least one hexose or pentose
moiety, S1 is a terminal pyranose moiety as defined above, and s is
0 or 1.
[0083] The saccharide moiety S2 may be a pyranose moiety,
particularly selected from glucose, galactose, 4-deoxyglucose, and
4,5-dehydroglucose derivatives or a furanose moiety, particularly
selected from fructose derivates.
[0084] In specific embodiments, the saccharide moiety S2 is of
formula (VIa), (VIb), (VIc), (VId), or (VIe):
##STR00005##
wherein R11 is a bond to X.sub.1, R12 is OR8 or NHR8 or an
attachment site to X.sub.2, wherein R8 is H or a protecting group
such as acetyl or benzyl, R13 is H or a protecting group such as
acetyl or benzyl, R14 is R8 or an attachment site to X.sub.2,
wherein R8 is H or a protecting group such as acetyl, R14a is H or
an attachment site to X.sub.2, R15 and R16 are H or together form
together with the carbon atom to which they are bound a carbonyl
group, R17 is OR8 or an attachment site to X.sub.2, wherein R8 is H
or a protecting group such as acetyl or benzyl, or R11 and R12
and/or R13 and R14 form together with the ring atoms to which they
are bound a cyclic group such as an acetal, and wherein one of R12,
R14, R14a and R17 is an attachment site to X.sub.2.
[0085] In further embodiments, the conjugate of formula (I)
reversibly binds to the insulin independent glucose transporter
GluT1, dependent from the glucose concentration in the surrounding
medium, which is blood after administration. In a further
embodiment the conjugate of formula (I) of the present invention is
not transported through the cell membrane upon binding to GluT1. In
a further embodiment the sugar moiety S comprises a single terminal
saccharide moiety. In still further embodiments, the sugar moiety S
does not comprise a mannose unit, particularly a terminal mannose
unit.
Definitions
[0086] "Alkyl" means a straight-chain or branched carbon chain.
Alkyl groups may be unsubstituted or substituted, wherein one or
more hydrogens of an alkyl carbon may be replaced by a substituent
such as halogen. Examples of alkyl include methyl, trifluoromethyl,
ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl,
tert-butyl, n-pentyl, and n-hexyl.
[0087] "Alkylene" means a straight-chain or branched carbon chain
bonded to each side. Alkylene groups may be unsubstituted or
substituted.
[0088] "Aryl" refers to any substituent derived from a monocyclic
or polycyclic or fused aromatic ring, including heterocyclic rings,
e.g. phenyl, thiophene, indolyl, naphthyl, pyridyl, which may
optionally be further substituted.
[0089] "Acyl" means a chemical functional group of the structure
R--(C.dbd.O)--, wherein R is an alkyl, aryl, or aralkyl.
[0090] " Halogen" means fluoro, chloro, bromo, or iodo. Preferably,
halogen is fluoro or chloro.
[0091] A "5 to 7 membered monocyclic ring" means a ring with 5 or 7
ring atoms that may contain up to the maximum number of double
bonds (aromatic or non-aromatic ring which is fully, partially or
un-saturated) wherein at least one ring atom up to 4 ring atoms may
be replaced by a heteroatom selected from the group consisting of
sulfur (including --S(O)--, --S(O).sub.2--), oxygen and nitrogen
(including .dbd.N(O)--). Examples for 5 to 7 membered rings include
carbocycles such as cyclopentane, cyclohexane, and benzene, or
heterocycles such as furan, thiophene, pyrrole, pyrroline,
imidazole, imidazoline, pyrazole, triazole, pyrazoline, oxazole,
oxazoline, isoxazole, isoxazoline, thiazole, thiazoline,
isothiazole, isothiazoline, thiadiazole, thiadiazoline,
tetrahydrofuran, tetrahydrothiophene, pyrrolidine, imidazolidine,
pyrazolidine, oxazolidine, isoxazolidine, thiazolidine,
isothiazolidine, thiadiazolidine, sulfolane, pyran, dihydropyran,
tetrahydropyran, imidazolidine, pyridine, pyridazine, pyrazine,
pyrimidine, piperazine, piperidine, morpholine, tetrazole,
triazole, triazolidine, tetrazolidine, diazepame, azepine, or
homopiperazine.
[0092] "9 to 12 membered bicyclic ring" means a system of two rings
with 9 to 12 ring atoms, where at least one ring atom is shared by
both rings and that may contain up to the maximum number of double
bonds (aromatic or non-aromatic ring which is fully, partially or
un-saturated) wherein at least one ring atom up to 6 ring atoms may
be replaced by a heteroatom selected from the group consisting of
sulfur (including --S(O)--, --S(O).sub.2--), oxygen, and nitrogen
(including .dbd.N(O)--) and wherein the ring is linked to the rest
of the molecule via a carbon or nitrogen atom. Examples for 9 to 12
membered rings include carbocycles such as naphthalene and
heterocycles such as indole, indoline, benzofuran, benzothiophene,
benzoxazole, benzisoxazole, benzothiazole, benzisothiazole,
benzimidazole, benzimidazoline, quinoline, quinazoline,
dihydroquinazoline, quinoline, dihydroquinoline,
tetrahydroquinoline, decahydroquinoline, isoquinoline,
decahydroisoquinoline, tetrahydroisoquinoline, dihydroisoquinoline,
benzazepine, purine, or pteridine. The term 9 to 12 membered
heterobicycle also includes spiro structures of two rings like
1,4-dioxa-8-azaspiro[4.5]decane or bridged heterocycles like
8-aza-bicyclo[3.2.1]octane.
[0093] The term "protecting group" means a chemical protecting
group for protecting OH-groups, known in the art of sugar chemistry
as described in Theodora W. Greene, Peter G. M. Wuts, Protective
Groups in Organic Synthesis, 3rd Edition, John Wiley & Sonc,
Inc. 1999. Examples of a protecting group are: acetyl, benzyl, or
p-methoxybenzyl; or isopropylidene groups for protecting two
hydroxy groups.
[0094] The term "anhydride forming group" means a chemical group
which forms with the carbonyl group to which it is attached an
anhydride. An example is acetic anhydride which acetylates said
carbonyl group.
[0095] The term "active ester forming group" means a chemical group
which forms with the carbonyl group to which it is attached an
ester which activates said carbonyl group for a coupling reaction
with an amino group containing compound forming an amide group.
[0096] Examples of active ester forming groups are
4-Nitrophenylester, N-Hydroxybenzotriazol (HOBt),
1-Hydroxy-7-azabenzotriazol oder N-Hydroxysuccinimid (HOSu).
[0097] The term "pharmaceutically acceptable" means approved by a
regulatory agency such as the EMEA (Europe) and/or the FDA (US)
and/or any other national regulatory agency for use in animals,
and/or in humans.
[0098] The conjugate of formula (I) of the present invention is
suitable for use in medicine, e.g. in veterinary medicine or in
human medicine. Particularly, the conjugate of formula (I) is
suitable for human medicine. Due to the glucose dependent
release/recapture mechanism, the conjugate of formula (I) is
particularly suitable for use in the prevention and/or treatment of
disorders associated with, caused by, and/or accompanied by a
dysregulated glucose mechanism, for example for use in the
prevention and/or treatment of diabetes mellitus, particularly of
diabetes type 2 or type 1.
[0099] The invention also provides a pharmaceutical composition
comprising a conjugate of formula (I) as described above as an
active agent and a pharmaceutically acceptable carrier.
[0100] The term "pharmaceutical composition" indicates a mixture
containing ingredients that are compatible when mixed and which may
be administered. A pharmaceutical composition includes one or more
medicinal drugs. Additionally, the pharmaceutical composition may
include one or more pharmaceutically acceptable carriers such as
solvents, adjuvants, emollients, expanders, stabilizers, and other
components, whether these are considered active or inactive
ingredients.
[0101] The conjugates of formula (I) of the present invention, or
salts thereof, are administered in conjunction with an acceptable
pharmaceutical carrier as part of a pharmaceutical composition. A
"pharmaceutically acceptable carrier" is a compound or mixture of
compounds which is physiologically acceptable while retaining the
therapeutic properties of the substance with which it is
administered. Standard acceptable pharmaceutical carriers and their
formulations are known to one skilled in the art and described, for
example, in Remington: The Science and Practice of Pharmacy, (20th
ed.) ed. A. R. Gennaro A. R., 2000, Lippencott Williams &
Wilkins. One exemplary pharmaceutically acceptable carrier is
physiological saline solution.
[0102] Acceptable pharmaceutical carriers include those used in
formulations suitable for oral, rectal, nasal, or parenteral
(including subcutaneous, intramuscular, intravenous, intradermal,
and transdermal) administration. The compounds of the present
invention will typically be administered parenterally.
[0103] The term "pharmaceutically acceptable salt" means salts of
the conjugates of formula (I) of the invention which are safe and
effective for use in mammals. Pharmaceutically acceptable salts may
include, but are not limited to, acid addition salts and basic
salts. Examples of acid addition salts include chloride, sulfate,
hydrogen sulfate, (hydrogen) phosphate, acetate, citrate, tosylate,
or mesylate salts. Examples of basic salts include salts with
inorganic cations, e.g. alkaline or alkaline earth metal salts such
as sodium, potassium, magnesium, or calcium salts and salts with
organic cations such as amine salts. Further examples of
pharmaceutically acceptable salts are described in Remington: The
Science and Practice of Pharmacy, (20th ed.) ed. A. R. Gennaro A.
R., 2000, Lippencott Williams & Wilkins or in Handbook of
Pharmaceutical Salts, Properties, Selection and Use, e.d. P. H.
Stahl, C. G. Wermuth, 2002, jointly published by Verlag Helvetica
Chimica Acta, Zurich, Switzerland, and Wiley-VCH, Weinheim,
Germany.
[0104] The term "solvate" means complexes of the conjugates of
formula (I) of the invention or salts thereof with solvent
molecules, e.g. organic solvent molecules and/or water.
[0105] The compounds of the present invention will be administered
in a "therapeutically effective amount". This term refers to a
nontoxic but sufficient amount of the conjugate of formula (I) to
provide the desired effect. The amount of a conjugate of formula
(I) of the formula (I) necessary to achieve the desired biological
effect depends on a number of factors, for example the specific
conjugate of formula (I) chosen, the intended use, the mode of
administration, and the clinical condition of the patient. An
appropriate "effective" amount in any individual case may be
determined by one of ordinary skill in the art using routine
experimentation.
[0106] Pharmaceutical compositions of the invention are those
suitable for parenteral (for example subcutaneous, intramuscular,
intradermal, or intravenous), oral, rectal, topical, and peroral
(for example sublingual) administration, although the most suitable
mode of administration depends in each individual case on the
nature and severity of the condition to be treated and on the
nature of the conjugate of formula (I)) used in each case.
[0107] Suitable pharmaceutical compositions may be in the form of
separate units, for example capsules, tablets, and powders in vials
or ampoules, each of which contains a defined amount of the
conjugate of formula (I); as powders or granules; as solution or
suspension in an aqueous or nonaqueous liquid; or as an
oil-in-water or water-in-oil emulsion. It may be provided in single
dose injectable form, for example in the form of a pen. The
compositions may, as already mentioned, be prepared by any suitable
pharmaceutical method which includes a step in which the active
ingredient and the carrier (which may consist of one or more
additional ingredients) are brought into contact.
[0108] The conjugates of formula (I) of the present invention can
be widely combined with other pharmacologically active compounds,
such as all drugs mentioned in the Rote Liste 2016 e.g. with all
antidiabetics mentioned in the Rote Liste 2016, chapter 12.
[0109] The active ingredient combinations can be used especially
for a synergistic improvement in action. They can be applied either
by separate administration of the active ingredients to the patient
or in the form of combination products in which a plurality of
active ingredients are present in one pharmaceutical preparation.
When the active ingredients are administered by separate
administration of the active ingredients, this can be done
simultaneously or successively.
[0110] General methods for the synthesis of conjugates of formula
(I) and intermediates thereof are described in the following
schemes:
##STR00006##
[0111] OH-protected glucuronic acid compounds 4, where the
protecting groups (PG) are e.g. acetyl, benzyl, or p-methoxybenzyl,
or isopropylidene groups for protecting two hydroxy groups at the
same time, or the like, can be coupled with amines 5 using well
known amide coupling procedures e.g. using HATU, TBTU, BEP, TOTU,
or other activating methods for carboxylic acids in common known
solvents like dimethylformamide, tetrahydrofuran, dichlormethane,
acetonitrile, or the like. Dependent on the protecting group, the
deprotection to compounds 2aa takes place under different
conditions such as basic, acidic, hydrogenating or oxidative
conditions. For example, acetyl groups are cleaved under basic
conditions using sodium or lithium hydroxide in solvents like
methanol, water, tetrahydrofuran, or combinations thereof.
Isopropylidene groups are cleaved under acidic conditions, e.g.
using trifluoroacetic acid in water, hydrogenating conditions using
e.g. palladium on charcoal or other hydrogenating catalysts under
hydrogen atmosphere in solvents like methanol, ethanol, toluene,
acetic acid, tetrahydrofuran or the like, or oxidative conditions
like cerium ammonium nitrate or DDQ, like for p-methoxybenzyl.
[0112] Unsaturated compounds of formula 2ba can be synthesized like
shown in scheme 2:
##STR00007##
[0113] Starting from acetyl protected glucuronic acid,
deacetylation using acetic acid anhydride and triethylamine
generates compound 7, which can be coupled with compounds 5 using
coupling reagents for amide bond syntheses, like described above,
to give compounds 2ba.
[0114] A further method to synthesize compounds 2ba is shown in
scheme 3:
##STR00008##
[0115] Starting from 1,2,3,4-tetra acetyl protected glucose,
oxidation using Swern conditions leads to aldehyde 8. Reductive
amination of aldehyde 8 and amines 5 using reductive amination
conditions like sodium cyanoborohydride, sodium
triacetoxyborohydride or the like, in solvents like dichloroethane,
dichloromethane, methanol, and/or acetic acid leads to compounds
2ba.
[0116] Compounds 10, 11, and 12 can be synthesized as described in
scheme 4.
##STR00009##
[0117] Compounds of formula 10, 11, and 12 can be synthesized using
copper catalysed [3+2]-cycloaddition conditions, also known as
azide-alkyne or click cycloaddition. 1-, 2- or 6-azido-deoxyglucose
and alkynes 9, are reacted with CuSO.sub.4*5H.sub.2O,
tris(3-hydroxypropyltriazolylmethyl)amine (THPTA) and sodium
ascorbate.
[0118] Alkynes 9 can be synthesized as shown in scheme 5.
##STR00010##
[0119] Alkynes 9 can be synthesized using propargylamine under
different reaction conditions: alkylation conditions using bases
like diisopropylamine, triethylamine or the like, in presence of
different chlorides or halides like for 9a and 9c, or peptide
coupling conditions like for 9b.
[0120] Compounds 14, 17, and 18 can be synthesized as described in
scheme 6.
##STR00011##
[0121] Compounds 14 can be synthesized using known deprotection
methods. When protecting group PG is, for example, a benzyl group,
it can be deprotected under hydrogenation conditions as described
above. Alkynes 15 can be converted into alkylating reagents 16 with
R describing a leaving group like O-tosyl, O-mesyl, halogen or the
like. Compounds 14 and compounds 16 can be reacted to obtain
compounds 17 under alkylating conditions, e.g. using bases like
triethylamine, diisopropylamine, sodium hydride or the like in
aprotic solvents like dimethylformamide, tetrahydrofuran, toluene
or the like. Deprotection of compounds 17 to compounds 18 is
dependent of the used protecting groups, the conditions are as
described above.
[0122] Galactosyl derivatives 20 can be synthesized like described
in scheme 7.
##STR00012##
[0123] Reductive amination of isopropylidene protected
galactosyl-aldehyde and amines 5 using known reductive amination
conditions as described in scheme 3 lead to compounds 19.
Deprotection of the isopropylidene groups can be done as described
in scheme 1.
[0124] The synthesis of compounds 1 is described in scheme 8.
##STR00013##
[0125] The synthesis of compounds 22 can be done under reductive
amination conditions for compounds 21 and protected carbohydrate
aldehydes as described in scheme 3. Deprotection of compounds 22 to
compounds 23 can be done as described in scheme 1. Compounds 23 can
be coupled using copper-catalyzed azide-alkyne cycloaddition
conditions as described in scheme 4 to yield compounds 1.
[0126] The synthesis of compounds 24 is described in scheme 9.
##STR00014##
[0127] The synthesis of compounds 24 can be carried out by reaction
of compounds 25 with insulin under basic conditions, e.g. pH 10.
Therefore the insulin is dissolved in a dimethylformamide-water
mixture and brought to pH 10 by an organic base like triethylamine.
At low temperatures (e.g. 0.degree. C.) the activated
azido-dioxopyrrolidines 25 are added to yield compounds of formula
24.
Abbreviations:
[0128] BEP 2-bromo-1-ethyl pyridinium tetrafluoroborate
d Dublet
[0129] dd Double dublet ddd Double double dublet DDQ
2,3-dichloro-5,6-dicyano-1,4-benzochinone DMSO
dimethylsulfoxide
ELSD Evaporative Light Scattering Detector
[0130] Eq. Equivalent/s ES-API Electro spray atmospheric pressure
ionisation FCS Fetal calf serum HATU
1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium
3-oxide hexafluorophosphate HOBt 1-hydroxybenzotriazole HPLC High
pressure liquid chromatography
Hz Hertz
[0131] J coupling constant KRB Krebs-Ringer bicarbonate buffer LG
leaving group LC/MS Liquid chromatography/mass spectra m
multiplet
MEM Minimum-Essential-Medium
MHz Megahertz
[0132] MPLC Medium pressure liquid chromatography NEAA
Non-essential amino acids NMR Nuclear magnetic resonance PG
Protecting group q quadruplet s singulet t triplet td dublet of
triplets TBTU N,N,N',N'-Tetramethyl-O-(benzotriazol-1-yl)uronium
tetrafluoroborate TLC Thin layer chromatography THPTA
tris(3-hydroxypropyltriazolylmethyl)amine TOTU
O-[(ethoxycarbonyl)cyanomethylenamino]-N,N,N',N'-tetramethyluronium
tetrafluoroborate t.sub.R Retention time R.sub.f Relative to front
value UV Ultra violet v/v Volume by volume
[0133] Experimental Part
Chromatographic and Spectroscopic Methods
TLC/UV-Lamp
[0134] Thin layer chromatography (TLC) was done on glass plates
from Merck coated with silica gel 60 F254. Detection was done with
an UV-Lamp from Lamag at wavelengths of 254 nm and 366 nm.
[0135] Compounds which could not be detected by UV were stained by
different methods: (a) 10% H.sub.2SO.sub.4 in ethanol, b) 1%
KMnO.sub.4-solution, c) molybdatophosphoric acid-cerium(IV)sulfate
solution in sulfuric acid (6 mL concentrated sulfuric acid and 94
mL water, 2.5 g molybdatophosphoric acid, 1 g
cerium(IV)sulfate).
MPLC
[0136] Chromatography on normal phase was done on a CombiFlash.RTM.
Rf (Teledyne ISCO). The used gradients were given in the
description of the examples.
HPLC
[0137] Preparative reversed phase HPLC was done using
acetonitrile/water on an Agilent 1200 preparative HPLC machine and
an Agilent Prep-C18 column (10 .mu.m, 21.5.times.150 mm).
.sup.1H-NMR
[0138] For .sup.1H-NMR-spectra a Bruker ARX, 400 MHz device was
used.
.sup.13C-NMR
[0139] For .sup.13C-NMR- spectra a Bruker Avance, 600 MHz device
was used.
LC/MS
[0140] For retention time and mass detection a LC/MS-system from
Waters Acquity SDS with a Waters Acquity BEH C18 (1.7 .mu.m,
2.1.times.50 mm) column was used. The injection volume was 0.5
.mu.l. Molecular weights are given in gramm per mol [g/mol],
detected masses in mass per charge [m/e].
LC/MS-Method 1
[0141] 95% H.sub.2O (0.05% formic acid) to 95% acetonitrile (0.035%
formic acid) in 2 min, 95% acetonitrile till 2.60 min, 0.9 mL/min,
10.times.2 mm Phenomenex LunaC.sub.18 3 .mu.m.
LC/MS-Method 2
[0142] 93% H.sub.2O (0.05% trifluoroacetic acid) to 95%
acetonitrile (0.05% trifluoroacetic acid) in 1 min, 95%
acetonitrile till 1.45 min, 1.1 mL/min, 10.times.2.0 mm
LunaC.sub.18 3 .mu.m
LC/MS-Method 3
[0143] 99% H.sub.2O (0.05% trifluoroacetic acid) to 93% H.sub.2O
(0.05%) in 0.4 min, 95% acetonitrile (0.05% trifluoroacetic acid)
in 0.8 min, 95% acetonitrile till 1.8 min, 1.1 mL/min, 10.times.2.0
mm LunaC.sub.18 3 .mu.m
LC/MS-Method 4
[0144] 10% acetonitrile (0.1% formic acid) till 90% acetonitrile
(0.1% formic acid) in 10 min, 90% acetonitrile till 10.67 min, 10%
acetonitrile from 11 to 12 min, 0.5 mL/min, Aeris Widepore 3, 3
.mu.m, 100.times.2.1 mm, 40.degree. C.
Syntheses
Method A
[0145] Amide coupling with
1,2,3,4-tetra-O-acetyl-.beta.-D-glucuronic acid
[0146] To a solution of 0.55 mmol
1,2,3,4-tetra-O-acetyl-.beta.-D-glucuronic acid in 5 mL
dimethylformamide were added 1.4 eq. (0.77 mmol)
1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium
3-oxid hexafluorophosphate (HATU) and 1.4 eq. (0.77 mmol) amine.
The reaction mixture was stirred for 2-6 hours at room temperature.
Reaction control was done by TLC. As work up 5-10 mL
dichloromethane were added and the organic phase was washed with 1M
HCl, water, saturated aqueous NaHCO.sub.3 solution, and water. The
organic phases were dried with Na.sub.2SO.sub.4, filtered, and
evaporated. If needed the crude mixture was purified by MPLC.
EXAMPLE 1
[(2S,3R,4S,5S,6S)-2,3,5-Triacetoxy-6-(4-methylpiperazine-1-carbonyl)tetrah-
ydropyran-4-yl]acetate
##STR00015##
[0148] Example 1 was synthesized from
1,2,3,4-tetra-O-acetyl-.beta.-D-glucuronic acid and
1-methylpiperazine following the procedure described in synthesis
method A.
[0149] Purification: MPLC CombiFlash Rf (Teledyne ISCO): column:
RediSep Silica 12 g; flow rate: 30 mL/min; wavelength for
detection: 254 nm; eluent: (A) dichloromethane, (B) ethanol.
MPLC Gradient:
TABLE-US-00001 [0150] start % B end % B duration [min] 0 0 2.2 0
4.8 2.8 4.8 4.8 6.0 4.8 10.1 6.0 10.1 10.1 9.1
Yield: 137 mg (0.308 mmol, 55.8%), white solid. TLC: R.sub.f=0.250
(dichloromethane/ethanol, 19:1). LC/MS (ES-API): m/z=445.13
[M+H].sup.+; calculated: 445.44; t.sub.R (.lamda.=220 nm): 0.93 min
(LC/MS-method 1). .sup.1H-NMR (400 MHz, DMSO-d.sub.6): .delta.
=5.96 (d, J=8.3 Hz, 1H, CH), 5.37 (t, J=9.5 Hz, 1H, CH), 5.25 (t,
J=9.5 Hz, 1H, CH), 4.97 (m, 2H, 2.times.CH), 3.63 (m, 1H,
NCH.sub.2), 3.55 (m, 1H, CH.sub.2), 3.39 (m, 1H, NCH.sub.2), 3.23
(m, 1H, NCH.sub.2), 2.36 (m, 2H, NCH.sub.2), 2.16 (s, 3H,
CH.sub.3), 2.11 (m, 2H, NCH.sub.2), 2.08 (s, 3H, CH.sub.3), 2.02
(s, 3H, CH.sub.3), 1.93 (s, 3H, CH.sub.3), 1.90 (s, 3H, CH.sub.3)
ppm.
EXAMPLE 2
[(2S,3R,4S,5S,6S)-2,3,5-Triacetoxy-6-(4-ethylpiperazine-1-carbonyl)tetrahy-
dropyran-4-yl]acetate
##STR00016##
[0152] Example 2 was synthesized from
1,2,3,4-tetra-O-acetyl-.beta.-D-glucuronic acid and
1-ethylpiperazine following the procedure described in synthesis
method A.
[0153] Purification: MPLC CombiFlash Rf (Teledyne ISCO); column:
RediSep Silica 12 g; flow rate: 30 mL/min; wavelength for
detection: 220 nm; eluent: (A) dichloromethane, (B) ethanol.
MPLC Gradient
TABLE-US-00002 [0154] Start % B end % B duration [min] 0 0 2.2 0
4.8 2.8 4.8 4.8 6.0 4.8 10.1 6.0 10.1 10.1 9.1
Yield: 201 mg (0.438 mmol, 79.4%), white solid. TLC: R.sub.f=0.492
(dichloromethane/ethanol, 19:1). LC/MS (ES-API): m/z=459.12
[M+H].sup.+; calculated: 459.46; t.sub.R (.lamda.=220 nm): 0.98 min
(LC/MS-Method 1). .sup.1H-NMR (400 MHz, DMSO-d.sub.6): .delta.=5.97
(d, J=8.3 Hz, 1H, CH), 5.38 (t, J=9.5 Hz, 1H, CH), 5.25 (t, J=9.5
Hz, 1H, CH), 4.97 (m, 2H, 2.times.CH), 3.58 (m, 2H, NCH.sub.2),
3.40 (m, 1H, NCH.sub.2), =3.24 (m, 1H, NCH.sub.2), 2.41 (m, 2H,
NCH.sub.2), 2.32 (m, 2H, CH.sub.2), 2.16 (m, 2H, NCH.sub.2), 2.08
(s, 3H, CH.sub.3), 2.02 (s, 3H, CH.sub.3), 1.93 (s, 3H, CH.sub.3),
1.89 (s, 3H, CH.sub.3), 0.99 (t, J=7.1 Hz, 3H, CH.sub.3) ppm.
EXAMPLE 3
[(2S,3R,4S,5S,6S)-2,3,5-Triacetoxy-6-(4-n-propylpiperazine-1-carbonyl)tetr-
ahydropyran-4-yl]acetate (8)
##STR00017##
[0156] Example 3 was synthesized from
1,2,3,4-tetra-O-acetyl-.beta.-D-glucuronic acid and
1-n-propylpiperazine following the procedure described in synthesis
method A.
[0157] Purification: MPLC CombiFlash Rf (Teledyne ISCO); column:
RediSep Silica 12 g; flow rate: 30 mL/min; wavelength for
detection: 220 nm; eluent: (A) dichloromethane, (B) ethanol.
MPLC Gradient
TABLE-US-00003 [0158] start % B end % B Duration [min] 0 0 1.5 0
5.0 5.0 5.0 5.0 8.0
Yield: 144 mg (0.305 mmol, 55.2%), white solid. TLC: R.sub.f=0.417
(dichloromethane/ethanol, 19:1). LC/MS (ES-API): m/z=473.14
[M+H].sup.+; calculated: 473.89; t.sub.R (.lamda.=220 nm): 1.06 min
(LC/MS-Method 1). .sup.1H-NMR (400 MHz, DMSO-d.sub.6): .delta.=5.96
(d, J=8.3 Hz, 1H, CH), 5.38 (t, J=9.5 Hz, 1H, CH), 5.25 (t, J=9.5
Hz, 1H, CH), 4.98 (m, 2H, 2.times.CH), 3.58 (m, 2H, NCH.sub.2),
3.40 (m, 1H, NCH.sub.2), 3.24 (m, 1H, NCH.sub.2), 2.39 (m, 2H,
NCH.sub.2), 2.22 (m, 2H, NCH.sub.2), 2.15 (m, 2H, NCH.sub.2), 2.07
(s, 3H, CH.sub.3), 2.02 (s, 3H, CH.sub.3), 1.93 (s, 3H, CH.sub.3),
1.89 (s, 3H, CH.sub.3), 1.43 (m, 2H, CH.sub.2), 0.85 (t, J=7.3 Hz,
3H, CH.sub.3) ppm.
EXAMPLE 4
[(2S,3R,4S,5S,6S)-2,3,5-Triacetoxy-6-(4-n-butylpiperazine-1-carbonyl)tetra-
hydropyran-4-yl]acetate
##STR00018##
[0160] Example 4 was synthesized from
1,2,3,4-tetra-O-acetyl-.beta.-D-glucuronic acid and
1-n-butylpiperazine following the procedure described in synthesis
method A.
[0161] Purification: MPLC CombiFlash Rf (Teledyne ISCO); column:
RediSep Silica 12 g; flow rate: 30 mL/min; wavelength for
detection: 220 nm; eluent: (A) dichloromethane, (B) ethanol.
MPLC Gradient
TABLE-US-00004 [0162] start % B end % B duration [min] 0 0 1.0 0
10.1 20.0 10.1 10.1 3 10.1 0.0 0.0 0.0 0.0 1.0
Yield: 191 mg (0.393 mmol, 71.1%), white solid. TLC: R.sub.f=0.458
(dichloromethane/ethanol, 19:1). LC/MS (ES-API): m/z=487.19
[M+H].sup.+; calculated: 487.51; t.sub.R (.lamda.=220 nm): 1.14 min
(LC/MS-Method 1).
[0163] .sup.1H-NMR (400 MHz, DMSO-d.sub.6): .delta. =5.96 (d, J=8.4
Hz, 1H, CH), 5.38 (t, J=9.4 Hz, 1H, CH), 5.25 (t, J=9.4 Hz, 1H,
CH), 4.97 (m, 2H, 2.times.CH), 3.57 (m, 2H, NCH.sub.2), 3.40 (m,
1H, NCH.sub.2), 3.24 (m, 1H, NCH.sub.2), 2.40 (m, 2H, NCH.sub.2),
2.26 (m, 2H, NCH.sub.2), 2.15 (m, 2H, NCH.sub.2), 2.07 (s, 3H,
CH.sub.3), 2.02 (s, 3H, CH.sub.3), 1.93 (s, 3H, CH.sub.3), 1.90 (s,
3H, CH.sub.3), 1.39 (m, 2H, CH.sub.2), 1.28 (m, 2H, CH.sub.2), 0.87
(t, J=7.4 Hz, 3H, CH.sub.3) ppm.
EXAMPLE 5
[(2S,3R,4S,5S,6S)-2,3,5-Triacetoxy-6-(4-n-hexylpiperazine-1-carbonyl)tetra-
hydropyran-4-yl]acetate
##STR00019##
[0165] Example 5 was synthesized from
1,2,3,4-tetra-O-acetyl-.beta.-D-glucuronic acid and
1-n-hexylpiperazine following the procedure described in synthesis
method A.
[0166] Purification: MPLC CombiFlash Rf (Teledyne ISCO); column:
RediSep Silica 12 g; flow rate: 30 mL/min; wavelength for
detection: 220 nm; eluent: (A) dichloromethane, (B) ethanol.
MPLC Gradient
TABLE-US-00005 [0167] start % B end % B duration [min] 0 0 1.0 0
20.2 20.0 20.2 20.2 2.9 20.2 0.0 0.0 0.0 0.0 1.0
Yield: 226 mg (0.439 mmol, 79.6%), white solid. TLC: R.sub.f=0.489
(dichloromethane/ethanol, 19:1). LC/MS (ES-API): m/z=515.24
[M+H].sup.+; calculated: 515.57; t.sub.R (.lamda.=220 nm): 1.34 min
(LC/MS-Method 1).
[0168] .sup.1H-NMR (400 MHz, 26.9.degree. C., DMSO-d.sub.6):
.delta.=5.96 (d, J=8.3 Hz, 1H, CH), 5.37 (t, J=9.5 Hz, 1H, CH),
5.25 (t, J=9.5 Hz, 1H, CH), 4.97 (m, 2H, CH), 3.57 (m, 2H,
NCH.sub.2), 3.40 (m, 1H, CH.sub.2), 3.23 (m, 1H, NCH.sub.2), 2.39
(m, 2H, NCH.sub.2), 2.25 (t, J=7.1 Hz, 2H, NCH.sub.2), 2.15 (m, 2H,
NCH.sub.2), 2.16 (s, 3H, CH.sub.3), 2.07 (s, 3H, CH.sub.3), 2.02
(s, 3H, CH.sub.3), 1.93 (s, 3H, CH.sub.3), 1.89 (s, 3H, CH.sub.3),
1.40 (m, 2H, CH.sub.2), 1.26 (m, 6H, 3.times.CH.sub.2), 0.86 (t,
J=6.9 Hz, 2H, CH.sub.3) ppm.
EXAMPLE 6
[(2S,3R,4S,5S,6S)-2,3,5-Triacetoxy-6-(4-isopropylpiperazine-1-carbonyl)tet-
rahydropyran-4-yl]acetate
##STR00020##
[0170] Example 6 was synthesized from
1,2,3,4-tetra-O-acetyl-.beta.-D-glucuronic acid and
1-isopropylpiperazine following the procedure described in
synthesis method A.
[0171] Purification: MPLC CombiFlash Rf (Teledyne ISCO); column:
RediSep Silica 12 g; flow rate: 30 mL/min; wavelength for
detection: 220 nm; eluent: (A) dichloromethane, (B) ethanol.
MPLC Gradient
TABLE-US-00006 [0172] start % B end % B duration [min] 0 0 1.0 0
20.2 20.0 20.2 20.2 2.9 20.2 0.0 0.0 0.0 0.0 1.0
Yield: 129 mg (0.273 mmol, 49.5%), white solid. TLC: R.sub.f=0.412
(dichloromethane/ethanol, 19:1). LC/MS (ES-API): m/z=473.18
[M+H].sup.+; calculated: 473.47; t.sub.R (.lamda.=220 nm): 1.02 min
(LC/MS-Method 1).
[0173] .sup.1H-NMR (400 MHz, 26.9.degree. C.,
DMSO-d.sub.6.delta.=5.97 (d, J=8.3 Hz, 1H, CH), 5.38 (t, J=9.4 Hz,
1H, CH), 5.25 (t, J=9.4 Hz, 1H, CH), 4.97 (m, 2H, 2.times.CH), 3.56
(m, 2H, NCH.sub.2), 3.39 (m, 1H, CH.sub.2), 3.24 (m, 1H,
NCH.sub.2), 2.66 (m, 1H, CH), 2.43 (m, 2H, NCH.sub.2), 2.27 (m, 2H,
NCH.sub.2), 2.08 (s, 3H, CH.sub.3), 2.02 (s, 3H, CH.sub.3), 1.93
(s, 3H, CH.sub.3), 1.90 (s, 3H, CH.sub.3), 0.95 (dd, 6H, CH.sub.3)
ppm.
EXAMPLE 7
[(2S,3R,4S,5S,6S)-2,3,5-Triacetoxy-6-(4-tert-butylpiperazine-1-carbonyl)te-
trahydropyran-4-yl]acetate
##STR00021##
[0175] Example 7 was synthesized from
1,2,3,4-tetra-O-acetyl-.beta.-D-glucuronic acid and
1-tert-butylpiperazine following the procedure described in
synthesis method A.
[0176] Purification: MPLC CombiFlash Rf (Teledyne ISCO); column:
RediSep Silica 12 g; flow rate: 30 mL/min; wavelength for
detection: 220 nm; eluent: (A) dichloromethane, (B) ethanol.
MPLC Gradient
TABLE-US-00007 [0177] start % B end % B duration [min] 0 0 1.0 0
20.2 20.0 20.2 20.2 2.9 20.2 0.0 0.0 0.0 0.0 1.0
Yield: 139 mg (0.286 mmol, 51.8%), white solid. TLC: R.sub.f=0.464
(dichloromethane/ethanol, 19:1). LC/MS (ES-API): m/z=487.20
[M+H].sup.+; calculated: 487.51; t.sub.R: 1.06 min (LC/MS-Method
1). .sup.1H-NMR (400 MHz, DMSO-d.sub.6): .delta.=5.97 (d, J=8.3 Hz,
1H, CH), 5.38 (t, J=9.5 Hz, 1H, CH), 5.25 (t, J=9.5 Hz, 1H, CH),
4.97 (m, 2H, CH), 3.53 (m, 2H, NCH.sub.2), 3.39 (m, 1H, CH.sub.2),
3.26 (m, 1H, NCH.sub.2), 2.67 (m, 2H, NCH.sub.2), 2.32 (m, 2H,
NCH.sub.2), 2.07 (s, 3H, CH.sub.3), 2.02 (s, 3H, CH.sub.3), 1.93
(s, 3H, CH.sub.3), 1.90 (s, 3H, CH.sub.3), 0.99 (s, 9H,
3.times.CH.sub.3) ppm.
EXAMPLE 8
[(2S,3R,4S,5S,6S)-2,3,5-Triacetoxy-6-(4-allylpiperazine-1-carbonyl)tetrahy-
dropyran-4-yl]acetate
##STR00022##
[0179] Example 8 was synthesized from
1,2,3,4-tetra-O-acetyl-.beta.-D-glucuronic acid and
1-allylpiperazine following the procedure described in synthesis
method A.
[0180] Purification: MPLC CombiFlash Rf (Teledyne ISCO); column:
RediSep Silica 12 g; flow rate: 30 mL/min; wavelength for
detection: 220 nm; eluent: (A) dichloromethane, (B) ethanol.
MPLC Gradient
TABLE-US-00008 [0181] start % B end % B duration [min] 0 0 1.0 0
9.9 9.9
Yield: 184 mg (0.260 mmol, 70.8%), white solid. TLC: R.sub.f=0.479
(dichloromethane/ethanol, 19:1). LC/MS (ES-API): m/z=471.26 [M+H]+;
calculated: 471.19; t.sub.R: 1.02 min (LC/MS-Method .sup.1H-NMR
(400 MHz, DMSO-d.sub.6): .delta.=5.97 (d, J=8.1 Hz, 1H, CH), 5.81
(m, 1H, H2C.dbd.CH), 5.36 (t, J=9.4 Hz, 1H, CH), 5.24 (t, J=9.4 Hz,
1H, CH), 5.17 (m, 2H, HC=CH.sub.2), 4.98 (m, 2H, CH), 3.61 (m, 2H,
NCH.sub.2), 3.42 (m, 1H, NCH.sub.2), 3.23 (m, 1H, NCH.sub.2), 2.96
(m, 2H, NCH.sub.2), 2.41 (m, 2H, NCH.sub.2), 2.17 (m, 2H,
NCH.sub.2), 2.08 (s, 3H, CH.sub.3), 2.02 (s, 3H, CH.sub.3), 1.94
(s, 3H, CH.sub.3), 1.90 (s, 3H, CH.sub.3) ppm.
EXAMPLE 9
[(2S,3R,4S,5S,6S)-2,3,5-Triacetoxy-6-(4-cyclohexylpiperazine-1-carbonyl)te-
trahydropyran-4-yl]acetate
##STR00023##
[0183] Example 9 was synthesized from
1,2,3,4-tetra-O-acetyl-.beta.-D-glucuronic acid and
1-cyclohexylpiperazine following the procedure described in
synthesis method A.
[0184] Purification: MPLC CombiFlash Rf (Teledyne ISCO); column:
RediSep Silica 12 g; flow rate: 30 mL/min; wavelength for
detection: 220 nm; eluent: (A) dichloromethane, (B) ethanol.
MPLC Gradient
TABLE-US-00009 [0185] start % B end % B duration [min] 0 0 1.0 0
9.9 9.9
Yield: 227 mg (0.443 mmol, 80.2%), white solid. TLC: R.sub.f=0.610
(dichloromethane/ethanol, 19:1). LC/MS (ES-API): m/z=513.15
[M+H].sup.+; calculated: 513.24; t.sub.R: 1.22 min (LC/MS-Method
1). .sup.1H-NMR (400 MHz, 116.9.degree. C., DMSO-d.sub.6): 5.93 (d,
J=7.8 Hz, 1H, CH), 5.33 (m, 2H, 2.times.CH), 4.99 (m, 1H, CH), 4.88
(m, 1H, CH), 3.71 (m, 4H, NCH.sub.2), 3.04 (m, 6H, NCH.sub.2), 3.23
(m, 2H, NCH.sub.2), 2.36 (m, 2H, NCH.sub.2), 2.06 (s, 3H,
CH.sub.3), 1.99 (s, 3H, CH.sub.3), 1.93 (s, 3H, CH.sub.3), 1.90 (s,
3H, CH.sub.3), 1.82 (m, 2H, CH.sub.2), 1.63 (m, 1H, CH), 1.30 (m,
8H, CH.sub.2) ppm.
EXAMPLE 10
[(2S,3R,4S,5S,6S)-2,3,5-Triacetoxy-6-[4-(cyclohexylmethyl)piperazine-1-car-
bonyl]tetrahydropyran-4-yl] acetate
##STR00024##
[0187] Example 10 was synthesized from
1,2,3,4-tetra-O-acetyl-.beta.-D-glucuronic acid and
1-cyclohexylmethylpiperazine following the procedure described in
synthesis method A.
[0188] Purification: MPLC CombiFlash Rf (Teledyne ISCO); column:
RediSep Silica 12 g; flow rate: 30 mL/min; wavelength for
detection: 220 nm; eluent: (A) dichloromethane, (B) ethanol.
MPLC Gradient
TABLE-US-00010 [0189] start % B end % B duration [min] 0 0 1.0 0
20.0 20.0 20.0 20.0 3 20.0 0.0 0.0 0.0 0.0 1.0
Yield: 178 mg (0.338 mmol, 61.2%), white solid. TLC: R.sub.f=0.346
(ethylacetate/n-heptan, 2:1). LC/MS (ES-API): m/z=527.22
[M+H].sup.+; calculated: 527.25; t.sub.R: 1.31 min (LC/MS-Method
1). .sup.1H-NMR (400 MHz, DMSO-d.sub.6): .delta.=5.97 (d, J=8.3 Hz,
1H, CH), 5.39 (t, J=9.4 Hz, 1H, CH), 5.27 (t, J=9.4 Hz, 1H, CH),
4.96 (m, 2H, CH), 3.58 (m, 2H, NCH.sub.2), 3.40 (m, 1H, CH.sub.2),
3.25 (m, 1H, NCH.sub.2), 2.37 (m, 2H, NCH.sub.2), 2.13 (m, 2H,
NCH.sub.2), 2.19 (m, 2H, NCH.sub.2), 2.08 (s, 3H, CH.sub.3), 2.02
(s, 3H, CH.sub.3), 1.94 (s, 3H, CH.sub.3), 1.89 (s, 3H, CH.sub.3),
1.69 (m, 4H, 2.times.CH.sub.2), 1.47 (m, 1H, CH), 1.18 (m, 2H,
2.times.CH.sub.2), 0.82 (m, 2H, CH.sub.2) ppm.
EXAMPLE 11
[(2S,3R,4S,5S,6S)-2,3,5-Triacetoxy-6-[4-(2-cyclohexylethyl)piperazine-1-ca-
rbonyl]tetrahydropyran-4-yl]acetate
##STR00025##
[0191] Example 11 was synthesized from
1,2,3,4-tetra-O-acetyl-.beta.-D-glucuronic acid and
1-cyclohexylethylpiperazine following the procedure described in
synthesis method A.
[0192] Purification: MPLC CombiFlash Rf (Teledyne ISCO); column:
RediSep Silica 12 g; flow rate: 30 mL/min; wavelength for
detection: 220 nm; eluent: (A) dichloromethane, (B) ethanol.
MPLC Gradient
TABLE-US-00011 [0193] start % B end % B duration [min] 0 0 1.0 0
18.7 18.7
Yield: 183 mg (0.339 mmol, 61.3%), white solid. TLC: R.sub.f=0.511
(dichloromethane/ethanol, 19:1). LC/MS (ES-API): m/z=541.24
[M+H].sup.+; calculated: 541.27; t.sub.R: 1.41 min (LC/MS-Method
1). .sup.1H-NMR (400 MHz, 116.9.degree. C., DMSO-d.sub.6):
.delta.=5.93 (d, J=7.8 Hz, 1H, CH), 5.33 (m, 2H, 2.times.CH), 4.99
(m, 1H, CH), 4.88 (m, 1H, CH), 3.71 (m, 4H, NCH.sub.2), 3.04 (m,
6H, NCH.sub.2), 3.23 (m, 2H, NCH.sub.2), 2.36 (m, 2H, NCH.sub.2),
2.06 (s, 3H, CH.sub.3), 1.99 (s, 3H, CH.sub.3), 1.93 (s, 3H,
CH.sub.3), 1.90 (s, 3H, CH.sub.3), 1.71 (m, 1H, CH), 1.61 (m, 6H,
CH.sub.2), 1.24 (m, 4H, CH.sub.2), 1.02 (m, 2H, CH.sub.2) ppm.
EXAMPLE 12
[(2S,3R,4S,5S,6S)-2,3,5-Triacetoxy-6-(4-phenylpiperazine-1-carbonyl)tetrah-
ydropyran-4-yl]acetate
##STR00026##
[0195] Example 12 was synthesized from
1,2,3,4-tetra-O-acetyl-.beta.-D-glucuronic acid and
1-phenylpiperazine following the procedure described in synthesis
method A.
[0196] Purification: MPLC CombiFlash Rf (Teledyne ISCO); column:
RediSep Silica 12 g; flow rate: 30 mL/min; wavelength for
detection: 254 nm; eluent: (A) n-heptane, (B) ethylacetate.
Yield: 260 mg (0.514 mmol, 62.0%),white solid. TLC: R.sub.f=0.695
(dichloromethane/ethanol, 19:1). LC/MS (ES-API): m/z=507.14
[M+H].sup.+; calculated: 507.19; t.sub.R: 1.70 min (LC/MS-Method
1).
MPLC Gradient
TABLE-US-00012 [0197] start % B end % B duration [min] 0 0 1.3 0
100.0 13.7 100.0 100.0 3.7 100.0 0.0 0.0 0.0 0.0 1.3
.sup.1H-NMR (400 MHz, DMSO-d.sub.6): .delta.=7.24 (t, J=8.2 Hz, 2H,
ArH), 6.95 (d, J=8.2 Hz, 2H, ArH), 6.81 (t, J=7.4 Hz, 1H, ArH),
6.00 (d, J=8.4 Hz, 1H, CH), 5.40 (t, J=9.9 Hz, 1H, CH), 5.29 (t,
J=9.9 Hz, 1H, CH), 5.05 (d, J=9.6 Hz, 1H, CH), 5.00 (m, 1H, CH),
3.81 (m, 1H, NCH.sub.2), 3.75 (m, 1H, NCH.sub.2), 3.57 (m, 1H,
CH.sub.2), 3.39 (m, 1H, NCH.sub.2), 3.24 (m, 2H, NCH.sub.2), 2.82
(m, 2H, NCH.sub.2), 2.07 (s, 3H, CH.sub.3), 2.03 (s, 3H, CH.sub.3),
1.94 (s, 3H, CH.sub.3), 1.91 (s, 3H, CH.sub.3) ppm.
[0198] .sup.13C-NMR (150 MHz, DMSO-d.sub.6): .delta.=169.64 (s, C),
169.04 (s, C), 169.02 (s, C), 168.43 (s, C), 163.04 (s, C), 150.69
(s, C), 128.99 (s, CH), 119.47 (s, CH), 115.97 (s, CH), 90.94 (s,
CH), 72.02 (s, CH), 69.37 (s, CH), 69.15 (s, CH), 68.55 (s, CH),
49.14 (s, CH.sub.2), 48.07 (s, CH.sub.2), 44.78 (s, CH.sub.2),
41.41 (s, CH.sub.3), 20.44 (s, 2 CH.sub.3), 20.30 (s, 2 CH.sub.3)
ppm.
EXAMPLE 13
[(2S,3R,4S,5S,6S)-2,3,5-Triacetoxy-6-[4-[(E)-cinnamyl]piperazine-1-carbony-
l]tetrahydropyran-4-yl]acetate
##STR00027##
[0200] Example 13 was synthesized from
1,2,3,4-tetra-O-acetyl-.beta.-D-glucuronic acid and
trans-1-cinnamylpiperazine following the procedure described in
synthesis method A.
[0201] Purification: MPLC CombiFlash Rf (Teledyne ISCO); column:
RediSep Silica 12 g; flow rate: 30 mL/min; wavelength for
detection: 220 nm; eluent: (A) dichloromethane, (B) ethanol.
MPLC Gradient
TABLE-US-00013 [0202] start % B end % B duration [min] 0 0 1.0 0
9.9 9.9
Yield: 253 mg (0.463 mmol, 83.8%), white solid. TLC: R.sub.f=0.644
(dichloromethane/ethanol, 19:1). LC/MS (ES-API): m/z=547.27
[M+H].sup.+; calculated: 547.22; t.sub.R: 1.34 min (LC/MS-Method
1). .sup.1H-NMR (400 MHz, DMSO-d.sub.6): .delta.=7.44 (d, J=7.2 Hz,
2H, ArH), 7.32 (t, J=7.2 Hz, 2H, ArH), 7.23 (t, J=7.2 Hz, 2H, ArH),
6.54 (d, J=15.9 Hz, 1H, CH), 6.29 (m, 1H, CH), 5.96 (d, J=8.4 Hz,
1H, CH), 5.37 (t, J=9.5 Hz, 1H, CH), 5.25 (t, J=9.5 Hz, 1H, CH),
4.97 (m, 2H, CH), 3.61 (m, 2H, NCH.sub.2), 3.42 (m, 1H, CH.sub.2),
3.26 (m, 1H, NCH.sub.2), 3.12 (m, 2H, NCH.sub.2), 2.47 (m, 2H,
NCH.sub.2), 2.22 (m, 2H, NCH.sub.2), 2.06 (s, 3H, CH.sub.3), 2.02
(s, 3H, CH.sub.3), 1.93 (s, 3H, CH.sub.3), 1.89 (s, 3H, CH.sub.3)
ppm.
EXAMPLE 14
[(2S,3R,4S,5S,6S)-2,3,5-Triacetoxy-6-[4-(4-chlorophenyl)piperazine-1-carbo-
nyl]tetrahydropyran-4-yl]acetate
##STR00028##
[0204] Example 14 was synthesized from
1,2,3,4-tetra-O-acetyl-.beta.-D-glucuronic acid and
1-(4-chlorophenyl)-piperazine following the procedure described in
synthesis method A.
[0205] Purification: MPLC CombiFlash Rf (Teledyne ISCO); column:
RediSep Silica 12 g; flow rate: 30 mL/min; wavelength for
detection: 220 nm; eluent: (A) dichloromethane, (B) ethanol.
MPLC Gradient
TABLE-US-00014 [0206] start % B end % B duration [min] 0 0 2.4 0
5.0 7.9 5.0 5.0 3.7
Yield: 153 mg (0.282 mmol, 51.3%), white solid. TLC: R.sub.f=0.619
(ethylacetate/n-heptane, 2:1). LC/MS (ES-API): m/z=541.19
[M+H].sup.+; calculated: 541.15; t.sub.R: 1.80 min (LC/MS-Method
1). .sup.1H-NMR (400 MHz, DMSO-d.sub.6): .delta.=7.25 (d, J=9.0 Hz,
2H, ArH), 6.96 (d, J=9.0 Hz, 2H, ArH), 6.00 (d, J=8.3 Hz, 1H, CH),
5.40 (t, J=9.5 Hz, 1H, CH), 5.28 (t, J=9.5 Hz, 1H, CH), 5.01 (m,
2H, 2.times.CH), 3.76 (m, 2H, NCH.sub.2), 3.56 (m, 1H, NCH.sub.2),
3.37 (m, 1H, NCH.sub.2), 3.25 (m, 2H, NCH.sub.2), 2.92 (m, 2H,
NCH.sub.2), 2.07 (s, 3H, CH.sub.3), 2.03 (s, 3H, CH.sub.3), 1.94
(s, 3H, CH.sub.3), 1.90 (s, 3H, CH.sub.3) ppm.
EXAMPLE 15
[(2S,3R,4S,5S,6S)-2,3,5-Triacetoxy-6-[4-(2-chlorophenyl)piperazine-1-carbo-
nyl]tetrahydropyran-4-yl]acetate
##STR00029##
[0208] Example 15 was synthesized from
1,2,3,4-tetra-O-acetyl-.beta.-D-glucuronic acid and
1-(2-chlorophenyl)-piperazine following the procedure described in
synthesis method A.
[0209] Purification: MPLC CombiFlash Rf (Teledyne ISCO); column:
RediSep Silica 12 g; flow rate: 30 mL/min; wavelength for
detection: 254 nm; eluent: (A) n-heptane, (B) ethylacetate.
MPLC Gradient
TABLE-US-00015 [0210] start % B end % B duration [min] 0 0 1.3 0
100.0 13.7 100.0 100.0 3.7 100.0 0.0 0.0 0.0 0.0 1.3
Yield: 148 mg (0.274 mmol, 49.6%), white solid. TLC: R.sub.f=0.589
(dichloromethane/ethanol, 19:1). LC/MS (ES-API): m/z=541.10
[M+H].sup.+; calculated: 541.15; t.sub.R: 1.80 min (LC/MS-Method
1). .sup.1H-NMR (400 MHz, DMSO-d.sub.6): .delta.=7.44 (dd, J=1.4
Hz, 2H, ArH), 7.33 (m, 2H, ArH), 7.14 (dd, J=1.4 Hz, 2H, ArH), 7.07
(m, 2H, ArH), 5.98 (d, J=8.3 Hz, 1H, CH), 5.39 (t, J=9.4 Hz, 1H,
CH), 5.29 (t, J=9.4 Hz, 1H, CH), 5.01 (m, 2H, 2.times.CH), 3.84 (m,
1H, NCH.sub.2), 3.76 (m, 1H, NCH.sub.2), 3.57 (m, 1H, CH.sub.2),
3.38 (m, 1H, NCH.sub.2), 3.02 (m, 2H, NCH.sub.2), 2.80 (m, 2H,
NCH.sub.2), 2.08 (s, 3H, CH.sub.3), 2.03 (s, 3H, CH.sub.3), 1.94
(s, 3H, CH.sub.3), 1.92 (s, 3H, CH.sub.3) ppm.
EXAMPLE 16
[(2S,3R,4S,5S,6S)-2,3,5-Triacetoxy-6-[4-(4-bromophenyl)piperazine-1-carbon-
yl]tetrahydropyran-4-yl]acetate
##STR00030##
[0212] Example 16 was synthesized from
1,2,3,4-tetra-O-acetyl-.beta.-D-glucuronic acid and
1-(4-bromophenyl)-piperazine following the procedure described in
synthesis method A.
[0213] Purification: MPLC CombiFlash Rf (Teledyne ISCO); column:
RediSep Silica 12 g; flow rate: 30 mL/min; wavelength for
detection: 220 nm; eluent: (A) dichloromethane, (B) ethanol.
MPLC Gradient
TABLE-US-00016 [0214] start % B end % B duration [min] 0 0 1.0 0
20.2 20.0 20.2 20.2 2.9 20.2 0.0 0.0 0.0 0.0 1.0
Yield: 168 mg (0.287 mmol, 52.0%), white solid. TLC: R.sub.f=0.635
(ethylacetate/n-heptane, 2:1). LC/MS (ES-API): m/z=585.14
[M+H].sup.+; calculated: 585.10; t.sub.R: 1.82 min (LC/MS-Method
1). .sup.1H-NMR (400 MHz, DMSO-d.sub.6): .delta.=7.37 (d, J=9.0 Hz,
2H, ArH), 6.92 (d, J=9.0 Hz, 2H, ArH), 6.00 (d, J=8.3 Hz, 1H, CH),
5.40 (t, J=9.4 Hz, 1H, CH), 5.28 (t, J=9.4 Hz, 1H, CH), 5.01 (m,
2H, 2.times.CH), 3.76 (m, 2H, NCH.sub.2), 3.56 (m, 1H, NCH.sub.2),
3.37 (m, 1H, NCH.sub.2), 3.25 (m, 2H, NCH.sub.2), 2.93 (m, 2H,
NCH.sub.2), 2.07 (s, 3H, CH.sub.3), 2.03 (s, 3H, CH.sub.3), 1.94
(s, 3H, CH.sub.3), 1.90 (s, 3H, CH.sub.3) ppm.
EXAMPLE 17
[(2S,3R,4S,5S,6S)-2,3,5-Triacetoxy-6-[4-(4-methoxyphenyl)piperazine-1-carb-
onyl]tetrahydropyran-4-yl]acetate
##STR00031##
[0216] Example 17 was synthesized from
1,2,3,4-tetra-O-acetyl-.beta.-D-glucuronic acid and
1-(4-methoxyphenyl)-piperazine following the procedure described in
synthesis method A.
[0217] Purification: MPLC CombiFlash Rf (Teledyne ISCO); column:
RediSep Silica 12 g; flow rate: 30 mL/min; wavelength for
detection: 254 nm; eluent: (A) n-heptane, (B) ethylacetate.
MPLC Gradient
TABLE-US-00017 [0218] start % B end % B duration [min] 0 0 1.0 0
10.0 20.0 10.0 10.0 3.0 10.0 0.0 0.0 0.0 0.0 1.0
Yield: 192 mg (0.320 mmol, 58.0%), white solid. TLC: R.sub.f=0.508
(ethylacetate/n-heptane, 2:1). LC/MS (ES-API): m/z=537.14
[M+H].sup.+; calculated: 537.20; t.sub.R: 1.64 min (LC/MS-Method
1). .sup.1H-NMR (400 MHz, DMSO-d.sub.6): .delta.=6.91 (d, J=9.1 Hz,
2H, ArH), 6.83 (d, J=9.1 Hz, 2H, ArH), 5.99 (d, J=8.0 Hz, 1H, CH),
5.40 (t, J=9.9 Hz, 1H, CH), 5.28 (t, J=9.9 Hz, 1H, CH), 5.00 (m,
2H, 2.times.CH), 3.77 (m, 2H, NCH.sub.2), 3.69 (s, 3H, OCH.sub.3),
3.56 (m, 1H, CH.sub.2), 3.37 (m, 1H, NCH.sub.2), 3.08 (m, 2H,
NCH.sub.2), 2.80 (m, 2H, NCH.sub.2), 2.07 (s, 3H, CH.sub.3), 2.03
(s, 3H, CH.sub.3), 1.94 (s, 3H, CH.sub.3), 1.90 (s, 3H, CH.sub.3)
ppm.
EXAMPLE 18
[(2S,3R,4S,5S,6S)-2,3,5-Triacetoxy-6-[4-(3-methoxyphenyl)piperazine-1-carb-
onyl]tetrahydropyran-4-yl]acetate
##STR00032##
[0220] Example 18 was synthesized from
1,2,3,4-tetra-O-acetyl-.beta.-D-glucuronic acid and
1-(3-methoxyphenyl)-piperazine following the procedure described in
synthesis method A.
[0221] Purification: MPLC CombiFlash Rf (Teledyne ISCO); column:
RediSep Silica 12 g; flow rate: 30 mL/min; wavelength for
detection: 254 nm; eluent: (A) n-heptane, (B) ethylacetate.
MPLC Gradient
TABLE-US-00018 [0222] start % B end % B duration [min] 0 0 1.0 0
10.0 20.0 10.0 10.0 3.0 10.0 0.0 0.0 0.0 0.0 1.0
Yield: 120 mg (0.224 mmol, 40.6%), white solid. TLC: R.sub.f=0.571
(ethylacetate/n-heptane, 2:1). LC/MS (ES-API): m/z=537.12
[M+H].sup.+; calculated: 537.20; t.sub.R: 1.70 min (LC/MS-Method
1). .sup.1H-NMR (400 MHz, DMSO-d.sub.6): .delta.=7.12 (d, J=8.2 Hz,
1H, ArH), 6.83 (d, J=8.2 Hz, 1H, ArH), 6.47 (s, 1H, ArH), 6.41 (d,
J=8.2 Hz, 1H, ArH), 6.00 (d, J=8.0 Hz, 1H, CH), 5.39 (t, J=9.9 Hz,
1H, CH), 5.28 (t, J=9.9 Hz, 1H, CH), 5.00 (m, 2H, 2.times.CH), 3.78
(m, 2H, NCH.sub.2), 3.72 (s, 3H, OCH.sub.3), 3.56 (m, 1H,
CH.sub.2), 3.37 (m, 1H, NCH.sub.2), 3.24 (m, 2H, NCH.sub.2), 2.92
(m, 2H, NCH.sub.2), 2.08 (s, 3H, CH.sub.3), 2.02 (s, 3H, CH.sub.3),
1.96 (s, 3H, CH.sub.3), 1.90 (s, 3H, CH.sub.3) ppm.
EXAMPLE 19
[(2S,3R,4S,5S,6S)-2,3,5-Triacetoxy-6-[4-(2-methoxyphenyl)piperazine-1-carb-
onyl]tetrahydropyran-4-yl]acetate
##STR00033##
[0224] Example 19 was synthesized from
1,2,3,4-tetra-O-acetyl-.beta.-D-glucuronic acid and
1-(2-methoxyphenyl)-piperazine following the procedure described in
synthesis method A.
[0225] Purification: MPLC CombiFlash Rf (Teledyne ISCO); column:
RediSep Silica 12 g; flow rate: 30 mL/min; wavelength for
detection: 220 nm; eluent: (A) dichloromethane, (B) ethanol.
MPLC Gradient
TABLE-US-00019 [0226] start % B end % B duration [min] 0 0 2.5 0
5.0 8.5 5.0 5.0 3.9
Yield: 150 mg (0.280 mmol, 50.6%), white solid. TLC: R.sub.f=0.492
(ethylacetate/n-heptane, 2:1). LC/MS (ES-API): m/z=537.14
[M+H].sup.+; calculated: 537.20; t.sub.R: 1.67 min (LC/MS-Method
1). .sup.1H-NMR (400 MHz, DMSO-d.sub.6): .delta.=6.98 (t, J=4.1 Hz,
1H, ArH), 6.96 (t, J=4.1 Hz, 1H, ArH), 6.89 (d, J=2.2 Hz, 2H, ArH),
6.86 (d, J=2.2 Hz, 2H, ArH), 5.98 (d, J=8.4 Hz, 1H, CH), 5.40 (t,
J=9.5 Hz, 1H, CH), 5.29 (t, J=9.5 Hz, 1H, CH), 5.00 (m, 2H,
2.times.CH), 3.83 (m, 1H, NCH.sub.2), 3.79 (s, 3H, OCH.sub.3), 3.74
(m, 1H, NCH.sub.2), 3.70 (m, 1H, CH.sub.2), 3.54 (m, 1H,
NCH.sub.2), 3.03 (m, 2H, NCH.sub.2), 2.74 (m, 2H, NCH.sub.2), 2.07
(s, 3H, CH.sub.3), 2.03 (s, 3H, CH.sub.3), 1.94 (s, 3H, CH.sub.3),
1.91 (s, 3H, CH.sub.3) ppm.
EXAMPLE 20
[(2S,3R,4S,5S,6S)-2,3,5-Triacetoxy-6-[4-(1,3-benzodioxol-5-ylmethyl)pipera-
zine-1-carbonyl]tetrahydropyran-4-yl]acetate
##STR00034##
[0228] Example 20 was synthesized from
1,2,3,4-tetra-O-acetyl-.beta.-D-glucuronic acid and
1-(1,3-benzodioxol-5-ylmethyl)piperazine following the procedure
described in synthesis method A.
[0229] Purification: MPLC CombiFlash Rf (Teledyne ISCO); column:
RediSep Silica 12 g; flow rate: 30 mL/min; wavelength for
detection: 220 nm; eluent: (A) dichloromethane, (B) ethanol.
MPLC Gradient
TABLE-US-00020 [0230] start % B end % B duration [min] 0 0 1.0 0
10.0 20.0 10.0 10.0 3.0 10.0 0.0 0.0 0.0 0.0 1.0
Yield: 221 mg (0.391 mmol, 70.9%), white solid. TLC: R.sub.f=0.478
(dichloromethane/ethanol, 19:1). LC/MS (ES-API): m/z=565.14
[M+H].sup.+; calculated: 565.19; t.sub.R: 1.24 min (LC/MS-Method
1).
[0231] .sup.1H-NMR (400 MHz, DMSO-d6): .delta.=6.84 (m, 2H, ArH),
6.73 (dd, J=1.4 Hz, 1H, ArH), 5.99 (s, 2H, O--CH.sub.2--O), 5.95
(d, J=8.4 Hz, 1H, CH), 5.36 (t, J=9.5 Hz, 1H, CH), 5.24 (t, J=9.5
Hz, 1H, CH), 4.96 (m, 2H, 2.times.CH), 3.62 (m, 2H, NCH.sub.2),
3.55 (m, 1H, CH.sub.2), 3.39 (m, 1H, NCH.sub.2), 3.23 (m, 1H,
NCH.sub.2), 2.39 (m, 2H, NCH.sub.2), 2.21 (m, 2H, NCH.sub.2), 2.07
(s, 3H, CH.sub.3), 2.02 (s, 3H, CH.sub.3), 1.93 (s, 3H, CH.sub.3),
1.88 (s, 3H, CH.sub.3) ppm.
Method B
Deacetylation of Glucuronic Acid Amides
[0232] 20 mg of
[(2,3,5-triacetoxy-6-piperazine-1-carbonyl]tetrahydropyran-4-yl)acetates
were dissolved in 2 mL methanol/H.sub.2O/tetrahydrofuran (5:4:1)
and cooled to 0.degree. C. 20 .mu.l of a 2 M lithium hydroxide
solution in water were added and stirred for 2-12 hours at
0.degree. C. The reaction control was done by TLC and LC/MS. As
work-up procedure the reaction mixture was neutralized with 1M HCI,
and the organic solvents were evaporated. The residue was diluted
with water and lyophilized. The enantiomers were not separated. NMR
signals were listed for only one enantiomer.
[0233] sf-4482178
[0234] Example 21
(4-Methylpiperazin-1-yl)-[(2S,3S,4S,5R)-3,4,5,6-tetrahydroxytetrahydropyra-
n-2-yl]methanone
##STR00035##
[0236] Example 21 was synthesized from example 1 following the
deacteylation procedure described in synthesis method B.
Yield: 11.9 mg (43.07 .mu.mol, 95.7%), colorless oil. LC/MS
(ES-API): m/z=277.15 [M+H].sup.+; calculated: 277.13; t.sub.R
(ELSD): 0.21 min (LC/MS-Method 1). .sup.1H-NMR (400 MHz,
DMSO-d.sub.6): .delta.=6.51 (d, J=4.5 Hz, 1H, CH), 5.0-4.3 (m, 4H,
4.times.OH), 3.5-3.0 (m, 12H, 4.times.CH.sub.2, 4.times.CH), 2.21
(m, 3H, CH.sub.3) ppm.
EXAMPLE 22
(4-Ethylpiperazin-1-yl)-[(2S,3S,4S,5R)-3,4,5,6-tetrahydroxytetrahydropyran-
-2-yl]methanone
##STR00036##
[0238] Example 22 was synthesized from example 2 following the
deacteylation procedure described in synthesis method B.
Yield: 11.8 mg (40.65 .mu.mol, 93.2%), colorless oil. LC/MS
(ES-API): m/z=291.23 [M+H].sup.+; calculated: 291.15; t.sub.R
(ELSD): 0.20 min (LC/MS-Method 1). .sup.1H-NMR (400 MHz,
DMSO-d.sub.6): .delta. =6.48 (d, J=4.6 Hz, 1H, CH), 4.8-4.3 (m, 4H,
4.times.OH), 3.5-3.0 (m, 12H, 4.times.CH.sub.2, 4.times.CH), 2.20
(m, 5H, CH.sub.2CH.sub.3) ppm.
EXAMPLE 23
(4-n-Propylpiperazin-1-yl)-[(2S,3S,4S,5R)-3,4,5,6-tetrahydroxytetrahydropy-
ran-2-yl]methanone
##STR00037##
[0240] Example 23 was synthesized from example 3 following the
deacteylation procedure described in synthesis method B.
Yield: 12.4 mg (40.74 .mu.mol, 96.3%), colorless oil. LC/MS
(ES-API): m/z=305.21 [M+H].sup.+; calculated: 305.16; t.sub.R
(ELSD): 0.19 min (LC/MS-Method 1).
[0241] .sup.1H-NMR (400 MHz, DMSO-d.sub.6): .delta.=6.51 (d, J=4.4
Hz, 1H, CH), 4.9-4.1 (m, 4H, 4.times.OH), 3.6-3.0 (m, 12H,
4.times.CH.sub.2, 4.times.CH), 2.32 (m, 2H, NCH.sub.2), 1.49 (m,
2H, CH.sub.2), 0.89 (t, J=7.3 Hz, 3H, CH.sub.3) ppm.
EXAMPLE 24
(4-n-Butylpiperazin-1-yl)-[(2S,3S,4S,5R)-3,4,5,6-tetrahydroxytetrahydropyr-
an -2-yl]methanone
##STR00038##
[0243] Example 24 was synthesized from example 4 following the
deacteylation procedure described in synthesis method B.
Yield: 12.7 mg (39.89 .mu.mol, 97.0%), colorless oil. LC/MS
(ES-API): m/z=319.21 [M+H].sup.+; calculated: 319.18; t.sub.R
(ELSD): 0.23 min (LC/MS-Method 1).
[0244] .sup.1H-NMR (400 MHz, DMSO-d.sub.6): .delta.=6.52 (d, J=4.5
Hz, 1H, CH), 4.87 (dd, J=4.2 Hz, 1H, OH), 4.75 (dd, J=4.9 Hz, 1H,
OH), 4.29 (d, J=8.9 Hz, 1H, OH), 4.02 (d, J=9.4 Hz, 1H, OH),
3.6-3.15 (m, 12H, 4.times.CH2, 4.times.CH), 2.32 (m, 2H,
NCH.sub.2), 1.46 (m, 2H, CH.sub.2), 1.29 (m, 2H, CH.sub.2), 0.89
(t, J=7.1 Hz, 3H, CH.sub.3) ppm.
EXAMPLE 25
(4-n -H
exylpiperazin-1-yl)-[(2S,3S,4S,5R)-3,4,5,6-tetrahydroxytetrahydrop-
yran-2-yl]methanone
##STR00039##
[0246] Example 25 was synthesized from example 5 following the
deacteylation procedure described in synthesis method B.
Yield: 12.6 mg (36.37 .mu.mol, 93.6%), white solid. LC/MS (ES-API):
m/z=347.17 [M+H].sup.+; calculated: 347.21; t.sub.R1 (.lamda.=220
nm): 0.50 min; t.sub.R2 (.lamda.=220 nm): 0.53 min (LC/MS-Method
1).
[0247] .sup.1H-NMR (400 MHz, DMSO-d.sub.6): .delta.=6.50 (d, J=4.4
Hz, 1H, CH), 4.85 (dd, J=4.4 Hz, 1H, OH), 4.76 (dd, J=4.8 Hz, 1H,
OH), 4.27 (d, J=8.8 Hz, 1H, OH), 4.12 (d, J=9.3 Hz, 1H, OH),
3.6-3.1 (m, 12H, 4.times.CH.sub.2, 4.times.CH), 2.32 (m, 2H,
NCH.sub.2), 1.43 (m, 8H, 4.times.CH.sub.2), 0.90 (t, J=7.2 Hz, 3H,
CH.sub.3) ppm.
EXAMPLE 26
(4-Isopropylpiperazin-1-yl)-[(2S,3S,4S,5R)-3,4,5,6-tetrahydroxytetrahydrop-
yran -2-yl]methanone
##STR00040##
[0249] Example 26 was synthesized from example 6 following the
deacteylation procedure described in synthesis method B.
Yield: 12.2 mg (40.09 .mu.mol, 94.7%), colorless oil. LC/MS
(ES-API): m/z=305.23 [M+H].sup.+; calculated: 305.16; t.sub.R
(ELSD): 0.21 min (LC/MS-Method 1). .sup.1H-NMR (400 MHz,
DMSO-d.sub.6): .delta.=6.48 (d, J=4.6 Hz, 1H, CH), 4.8-4.1 (m, 4H,
4.times.OH), 3.5-3.1 (m, 12H, 4.times.CH.sub.2, 4.times.CH), 2.43
(m, 2H, NCH), 1.01 (m, 6H, 2.times.CH.sub.3) ppm.
EXAMPLE 27
(4-tert-Butylpiperazin-1-yl)-[(2S,3S,4S,5R)-3,4,5,6-tetrahydroxytetrahydro-
pyran-2-yl]methanone
##STR00041##
[0251] Example 27 was synthesized from example 7 following the
deacteylation procedure described in synthesis method B.
Yield: 12.5 mg (39.26 .mu.mol, 95.5%), white solid. LC/MS (ES-API):
m/z=319.25 [M+H].sup.+; calculated: 319.18; t.sub.R (ELSD): 0.22
min (LC/MS-Method 1). .sup.1H-NMR (400 MHz, DMSO-d.sub.6):
.delta.=6.49 (d, J=4.9 Hz, 1H, CH), 4.9-4.0 (m, 4H, 4.times.OH),
3.5-3.1 (m, 12H, 4.times.CH.sub.2, 4.times.CH), 1.04 (m, 9H,
3.times.CH.sub.3) ppm.
EXAMPLE 28
(4-Allylpiperazin-1-yl)-[(2S,3S,4S,5R)-3,4,5,6-tetrahydroxytetrahydropyran-
-2-yl]methanone
##STR00042##
[0253] Example 28 was synthesized from example 8 following the
deacteylation procedure described in synthesis method B.
Yield: 12.4 mg (41.02 .mu.mol, 96.5%), colorless oil. LC/MS
(ES-API): m/z=303.22 [M+H].sup.+; calculated: 303.15; t.sub.R
(ELSD): 0.21 min (LC/MS-Method 1). .sup.1H-NMR (400 MHz,
DMSO-d.sub.6): .delta.=6.29 (d, J=4.5 Hz, 1H, CH), 4.7-4.0 (m, 4H,
4.times.OH), 3.4-3.1 (m, 12H, 4.times.CH.sub.2, 4.times.CH), 2.41
(m, 5H, 2.times.CH.sub.2, CH) ppm.
EXAMPLE 29
(4-Cyclohexylpiperazin-1-yl)-[(2S,3S,4S,5R)-3,4,5,6-tetrahydroxytetrahydro-
pyran-2-yl]methanone
##STR00043##
[0255] Example 29 was synthesized from example 9 following the
deacteylation procedure described in synthesis method B.
Yield: 12.9 mg (37.46 .mu.mol, 96.0%), white solid. LC/MS (ES-API):
m/z=345.25 [M+H].sup.+; calculated: 345.19; t.sub.R1 (ELSD): 0.28
min (LC/MS-Method 1). .sup.1H-NMR (400 MHz, DMSO-d6): .delta.=6.51
(d, J=4.8 Hz, 1H, CH), 4.6-4.0 (m, 4H, 4.times.OH), 3.4-2.9 (m,
12H, 4.times.CH.sub.2, 4.times.CH), 1.71 (m, 4H, 2.times.CH.sub.2),
1.54 (m, 1H, CH), 1.19 (m, 6H, 3.times.CH.sub.2) ppm.
EXAMPLE 30
(4-Cyclohexylmethylpiperazin-1-yl)-[(2S,3S,4S,5R)-3,4,5,6-tetrahydroxytetr-
ahydropyran-2-yl]methanone
##STR00044##
[0257] Example 30 was synthesized from example 10 following the
deacteylation procedure described in synthesis method B.
Yield: 13.1 mg (36.55 .mu.mol, 96.2%), white solid. LC/MS (ES-API):
m/z=359.16 [M+H].sup.+; calculated: 359.21; t.sub.R1 (.lamda.=220
nm): 0.46 min; t.sub.R2 (.lamda.=220 nm): 0.48 min (LC/MS-Method
1).
[0258] .sup.1H-NMR (400 MHz, DMSO-d.sub.6): .delta.=6.47 (d, J=4.5
Hz, 1H, CH), 4.9-4.3 (m, 4H, 4.times.OH), 3.5-2.9 (m, 12H,
4.times.CH.sub.2, 4.times.CH), 2.07 (m, 2H, NCH.sub.2), 1.74 (m,
4H, 2.times.CH.sub.2), 1.52 (m, 1H, CH), 1.21 (m, 4H,
2.times.CH.sub.2), 0.87 (m, 2H, CH.sub.2) ppm.
EXAMPLE 31
(4-Cyclohexylethylpiperazin-1-yl)-[(2S,3S,4S,5R)-3,4,5,6-tetrahydroxytetra-
hydropyran-2-yl]methanone
##STR00045##
[0260] Example 31 was synthesized from example 11 following the
deacteylation procedure described in synthesis method B.
Yield: 12.2 mg (32.76 .mu.mol, 88.5%), white solid. LC/MS (ES-API):
m/z=373.17 [M+H].sup.+; calculated: 373.23; t.sub.R1 (.lamda.=220
nm): 0.81 min; t.sub.R2 (.lamda.=220 nm): 0.84 min (LC/MS-Method
1).
[0261] .sup.1H-NMR (400 MHz, DMSO-d.sub.6): .delta.=6.42 (d, J=4.8
Hz, 1H, CH), 4.6-4.1 (m, 4H, 4.times.OH), 3.6-3.0 (m, 12H,
4.times.CH.sub.2, 4.times.CH), 2.05 (m, 2H, NCH.sub.2), 1.72 (m,
4H, 2.times.CH.sub.2), 1.50 (m, 1H, CH), 1.23 (m, 4H,
2.times.CH.sub.2), 0.90 (m, 2H, CH.sub.2), 0.87 (m, 2H, CH.sub.2)
ppm.
EXAMPLE 32
(4-Phenylpiperazin-1-yl)-[(2S,3S,4S,5R)-3,4,5,6-tetrahydroxytetrahydropyra-
n-2-yl]methanone
##STR00046##
[0263] Example 32 was synthesized from example 12 following the
deacteylation procedure described in synthesis method B.
Yield: 11.8 mg (34.87 .mu.mol, 88.3%), white solid. LC/MS (ES-API):
m/z=339.13 [M+H].sup.+; calculated: 339.15; t.sub.R1 (.lamda.=220
nm): 0.90 min; t.sub.R2 (.lamda.=220 nm): 0.95 min (LC/MS-Method
1). .sup.1H-NMR (400 MHz, DMSO-d.sub.6): .delta.=7.23 (t, J=7.9 Hz,
2H, ArH), 6.95 (d, J=7.8 Hz, 2H, ArH), 6.81 (t, J=7.3 Hz, 1H, ArH),
6.66 (d, J=7.0 Hz, 1H, OH), 4.81 (d, J=4.7 Hz, 1H, OH), 4.74 (d,
J=5.3 Hz, 1H, OH), 4.62 (d, J=6.4 Hz, 1H, OH), 4.43 (t, J=7.3 Hz,
1H, CH), 4.09 (d, J=9.3 Hz, 1H, CH), 3.70-3.45 (m, 8H,
4.times.NCH.sub.2), 3.25-3.00 (m, 3H, 3.times.CH) ppm. .sup.13C-NMR
(150 MHz, DMSO-d.sub.6): .delta.=166.48 (s, CO), 150.76 (s, C),
129.06 (s, CH), 119.28 (s, CH), 115.78 (s, CH), 93.06 (s, CH),
74.52 (s, CH), 72.71 (s, CH), 71.33 (s, CH), 70.98 (s, CH), 48.93
(s, CH.sub.2), 48.22 (s, CH.sub.2), 44.59 (s, CH.sub.2), 41.15 (s,
CH.sub.2) ppm.
EXAMPLE 33
[4-[(E)-Cinnamyl]piperazin-1-yl]-[(2S,3S,4S,5R)-3,4,5,6-tetrahydroxytetrah-
ydropyran-2-yl]methanone
##STR00047##
[0265] Example 33 was synthesized from example 13 following the
deacteylation procedure described in synthesis method B.
Yield: 12.7 mg (33.56 .mu.mol, 91.7%), white solid. LC/MS (ES-API):
m/z=379.25 [M+H].sup.+; calculated: 379.19; t.sub.R1 (.lamda.=220
nm): 0.56 min; t.sub.R2 (.lamda.=220 nm): 0.60 min (LC/MS-Method
1). .sup.1H-NMR (400 MHz, DMSO-d.sub.6): .delta.=7.40 (d, J=7.0 Hz,
2H, ArH), 7.33 (t, J=7.0 Hz, 2H, ArH), 7.21 (t, J=7.0 Hz, 2H, ArH),
6.44 (d, J=4.6 Hz, 1H, CH), 4.6-4.1 (m, 4H, 4.times.OH), 3.65 (m,
2H, NCH.sub.2), 3.51 (m, 2H, 2.times.CH), 3.4-2.9 (m, 12H,
4.times.NCH.sub.2, 4.times.CH) ppm.
EXAMPLE 34
[4-(4-Chlorophenyl)piperazin-1-yl]-[(2S,3S,4S,5R)-3,4,5,6-tetrahydroxytetr-
ahydropyran-2-yl]methanone
##STR00048##
[0267] Example 34 was synthesized from example 14 following the
deacteylation procedure described in synthesis method B.
Yield: 13.6 mg (36.48 .mu.mol, 98.7%), white solid. LC/MS (ES-API):
m/z=373.16 [M+H].sup.+; calculated: 373.11; t.sub.R1 (.lamda.=220
nm): 1.20 min; t.sub.R2 (.lamda.=220 nm): 1.24 min (LC/MS-Method
1). .sup.1H-NMR (400 MHz, DMSO-d.sub.6): .delta.=7.40 (d, J=7.8 Hz,
4H, ArH), 6.49 (d, J=4.7 Hz, 1H, CH), 4.9-4.3 (m, 4H, 4.times.OH),
3.7-3.0 (m, 12H, 4.times.NCH.sub.2, 4.times.CH) ppm.
EXAMPLE 35
[4-(2-Chlorophenyl)piperazin-1-yl]-[(2S,3S,4S,5R)-3,4,5,6-tetrahydroxytetr-
ahydropyran-2-yl]methanone
##STR00049##
[0269] Example 35 was synthesized from example 15 following the
deacteylation procedure described in synthesis method B.
Yield: 13.3 mg (35.68 .mu.mol, 96.5%), white solid. LC/MS (ES-API):
m/z=373.06 [M+H].sup.+; calculated: 373.11; t.sub.R1 (.lamda.=220
nm): 1.23 min; t.sub.R2 (.lamda.=220 nm): 1.26 min (LC/MS-Method
1). .sup.1H-NMR (400 MHz, DMSO-d.sub.6): .delta.=7.45 (d, J=7.8 Hz,
1H, ArH), 7.31 (t, J=7.8 Hz, 1H, ArH), 7.14 (d, J=7.8 Hz, 1H, ArH),
7.03 (t, J=7.8 Hz, 1H, ArH), 6.52 (d, J=4.7 Hz, 1H, CH), 4.9-4.3
(m, 4H, 4.times.OH), 3.7-3.0 (m, 12H, 4.times.NCH.sub.2,
4.times.CH) ppm.
EXAMPLE 36
[4-(4-Bromophenyl)piperazin-1-yl]-[(2S,3S,4S,5R)-3,4,5,6-tetrahydroxytetra-
hydropyran-2-yl]methanone
##STR00050##
[0271] Example 36 was synthesized from example 16 following the
deacteylation procedure described in synthesis method B.
Yield: 12.9 mg (30.92 .mu.mol, 90.5%), white solid. LC/MS (ES-API):
m/z=416.11 [M+H].sup.+; calculated: 416.06; t.sub.R1 (.lamda.=220
nm): 1.25 min; t.sub.R2 (.lamda.=220 nm): 1.28 min (LC/MS-Method
1). .sup.1H-NMR (400 MHz, DMSO-d.sub.6): .delta.=7.69 (d, J=8.0 Hz,
4H, ArH), 6.52 (d, J=4.9 Hz, 1H, CH), 4.8-4.1 (m, 4H, 4.times.OH),
3.6-3.1 (m, 12H, 4.times.NCH.sub.2, 4.times.CH) ppm.
EXAMPLE 37
[4-(4-Methoxyphenyl)piperazin-1-yl]-[(2S,3S,4S,5R)-3,4,5,6-tetrahydroxytet-
rahydropyran-2-yl]methanone
##STR00051##
[0273] Example 37 was synthesized from example 17 following the
deacteylation procedure described in synthesis method B.
Yield: 13.4 mg (36.38 .mu.mol, 97.6%), white solid. LC/MS (ES-API):
m/z=369.11 [M+H].sup.+; calculated: 369.16; t.sub.R1 (.lamda.=220
nm): 0.74 min; t.sub.R2 (.lamda.=220 nm): 0.80 min (LC/MS-Method
1). .sup.1H-NMR (400 MHz, DMSO-d.sub.6): .delta.=6.91 (d, J=8.2 Hz,
2H, ArH), 6.83 (d, J=8.2 Hz, 2H, ArH), 6.53 (d, J=4.4 Hz, 1H, CH),
4.8-4.1 (m, 4H, 4.times.OH), 3.69 (s, 3H, OCH.sub.3), 3.6-2.9 (m,
12H, 4.times.NCH.sub.2, 4.times.CH) ppm.
EXAMPLE 38
[4-(3-Methoxyphenyl)piperazin-1-yl]-[(2S,3S,4S,5R)-3,4,5,6-tetrahydroxytet-
rahydropyran-2-yl]methanone
##STR00052##
[0275] Example 38 was synthesized from example 18 following the
deacteylation procedure described in synthesis method B.
Yield: 13.1 mg (35.56 .mu.mol, 95.4%), white solid. LC/MS (ES-API):
m/z=369.10 [M+H].sup.+; calculated: 369.16; t.sub.R1 (.lamda.=220
nm): 1.01 min; t.sub.R2 (.lamda.=220 nm): 1.04 min (LC/MS-Method
1). .sup.1H-NMR (400 MHz, DMSO-d.sub.6): .delta.=7.14 (d, J=8.2 Hz,
1H, ArH), 6.84 (d, J=8.2 Hz, 1H, ArH), 6.57 (s, 1H, ArH), 6.51 (d,
J=8.2 Hz, 1H, ArH), 6.43 (d, J=4.4 Hz, 1H, CH), 4.8-4.1 (m, 4H,
4.times.OH), 3.72 (s, 3H, OCH.sub.3), 3.60-3.45 (m, 4H,
4.times.CH), 3.25-3.00 (m, 8H, 4.times.NCH.sub.2) ppm.
EXAMPLE 39
[4-(2-Methoxyphenyl)piperazin-1-yl]-[(2S,3S,4S,5R)-3,4,5,6-tetrahydroxytet-
rahydropyran-2-yl]methanone
##STR00053##
[0277] Example 39 was synthesized from example 19 following the
deacteylation procedure described in synthesis method B.
Yield: 12.8 mg (34.75 .mu.mol, 93.2%), white solid. LC/MS (ES-API):
m/z=369.19 [M+H].sup.+; calculated: 369.16; t.sub.R1 (.lamda.=220
nm): 0.85 min; t.sub.R2 (.lamda.=220 nm): 0.90 min (LC/MS-Method
1). .sup.1H-NMR (400 MHz, DMSO-d.sub.6): .delta.=6.98 (m, 2H, ArH),
6.87 (d, J=7.2 Hz, 2H, ArH), 6.49 (d, J=4.4 Hz, 1H, CH), 4.8-4.1
(m, 4H, 4.times.OH), 3.70 (s, 3H, OCH.sub.3), 3.6-3.2 (m, 4H,
4.times.CH), 3.0-2.8 (m, 8H, 4.times.NCH.sub.2) ppm.
[0278] sf-4482178
[0279] Example 40
[4-(1,3-Benzodioxol-5-ylmethyl)piperazin-1-yl]-[(2S,3S,4S,5R)-3,4,5,6-tet-
rahydroxytetrahydropyran-2-yl]methanone
##STR00054##
[0280] Example 40 was synthesized from example 20 following the
deacteylation procedure described in synthesis method B.
Yield: 12.6 mg (31.79 .mu.mol, 89.7%), white solid. LC/MS (ES-API):
m/z=397.11 [M+H].sup.+; calculated: 397.15; t.sub.R1 (.lamda.=220
nm): 0.95 min; t.sub.R2 (.lamda.=220 nm): 0.99 min (LC/MS-Method
1). .sup.1H-NMR (400 MHz, DMSO-d.sub.6): .delta.=6.82 (m, 2H, ArH),
6.75 (dd, J=1.4 Hz, 1H, ArH), 6.00 (s, 2H, O--CH.sub.2--O), 6.49
(d, J=4.4 Hz, 1H, CH), 4.8-4.1 (m, 4H, 4.times.OH), 3.6-3.2 (m, 4H,
4.times.CH), 3.0-2.8 (m, 8H, 4.times.NCH.sub.2), 2.41 (m, 2H,
NCH.sub.2) ppm.
EXAMPLE 41
[(2S,3R,4S)-2,3-Diacetoxy-6-(4-butylpiperazine-1-carbonyl)-3,4-di
hydro-2H -pyran-4-yl]acetate
##STR00055##
[0282] Step 1:
(2S,3R,4S)-2,3,4-triacetoxy-3,4-dihydro-2H-pyran-6-carboxylic
acid
##STR00056##
[0283] A solution of 2 g (5.52 mmol)
1,2,3,4-tetra-O-acetyl-.beta.-D-glucuronic acid in 6 mL (63.59
mmol) acetic acid anhydride and 3 mL (22.08 mmol; 4 eq.)
triethylamine was stirred at room temperature for 8 hours. The
reaction mixture was diluted with water and lyophillized.
Yield: 1.6 g (5.38 mmol, 95.8%), colorless oil. LC/MS (ES-API): not
detectable. .sup.1H-NMR (400 MHz, CDCl.sub.3): .delta.=7.99 (s, 1H,
COOH), 6.33 (s, 1H, CH), 6.27 (m, 1H, CH), 5.19 (m, 1H, CH), 5.10
(m, 1H, CH), 2.05 (s, 3H, CH.sub.3), 2.04 (s, 3H, CH.sub.3), 2.03
(s, 3H, CH.sub.3) ppm.
Method C
[0284] Amide coupling with
(2S,3R,4S)-2,3,4-triacetoxy-3,4-dihydro-2H-pyran-6-carboxylic acid
(example 41, step 1)
[0285] To a solution of 0.66 mmol
(2S,3R,4S)-2,3,4-triacetoxy-3,4-dihydro-2H-pyran-6-carboxylic acid
(example 41, step 1) in 2 mL dimethylformamide were added 1.2 eq.
(0.79 mmol) HATU and 1.4 eq. (0.93 mmol) amine. The reaction
mixture was stirred at room temperature for 2 hours. The reaction
was controlled by TLC and LC/MS. After completion the reaction
mixture was extracted with 5-10 mL dichloromethane. The organic
phase was washed with 1 M aqueous HCl, water, saturated aqueous
NaHCO.sub.3 solution, and water. The organic phase was dried with
Na.sub.2SO.sub.4, filtered, and evaporated. The crude material was
purified by MPLC.
[0286] Step 2:
[(2S,3R,4S)-2,3-diacetoxy-6-(4-butylpiperazine-1-carbonyl)-3,4-dihydro-2H-
-pyran-4-yl]acetate
[0287] Example 41 was synthesized from example 41, step 1 and
1-n-butylpiperazine following the amide coupling procedure
described in synthesis method C.
[0288] Purification: MPLC CombiFlash Rf (Teledyne ISCO); column:
RediSep Silica 12 g; flow rate: 30 mL/min; wavelength for
detection: 220 nm; eluent: (A) dichloromethane, (B) ethanol.
MPLC Gradient
TABLE-US-00021 [0289] start % B end % B duration [min] 0 0 2.2 0
4.8 2.8 4.8 4.8 6.0 4.810.0 10.1 6.0 10.1 10.1 9.1
Yield: 141 mg (0.331 mmol, 50.8%), orange oil. LC/MS (ES-API):
m/z=427.20 [M+H].sup.+; calculated: 427.20; t.sub.R (.lamda.=220
nm): 0.57 min (LC/MS-Method 1). .sup.1H-NMR (400 MHz, CDCl.sub.3):
.delta.=6.28 (d, J=3.6 Hz, 1H, C=CH), 5.67 (d, J=3.8 Hz, 1H, CH),
5.25 (m, 1H, CH), 5.19 (m, 1H, CH), 3.73 (m, 4H,
2.times.NCH.sub.2), 2.60 (m, 4H, 2.times.NCH.sub.2), 2.15 (s, 3H,
CH.sub.3), 2.13 (s, 3H, CH.sub.3), 2.10 (s, 3H, CH.sub.3) ppm.
EXAMPLE 42
[(2S,3R,4S)-2,3-Diacetoxy-6-(4-tert-butylpiperazine-1-carbonyl)-3,4-dihydr-
o-2H-pyran-4-yl]acetate
##STR00057##
[0291] Example 42 was synthesized from example 41, step 1 and
1-tert-butylpiperazine following the amide coupling procedure
described in synthesis method C.
[0292] Purification: MPLC CombiFlash Rf (Teledyne ISCO); column:
RediSep Silica 12 g; flow rate: 30 mL/min; wavelength for
detection: 220 nm; eluent: (A) dichloromethane, (B) ethanol.
MPLC Gradient
TABLE-US-00022 [0293] start % B end % B duration [min] 0 0 2.2 0
4.8 2.8 4.8 4.8 6.0 4.810.0 10.1 6.0 10.1 10.1 9.1
Yield: 81 mg (0.190 mmol,28.7%), orange oil. LC/MS (ES-API):
m/z=427.15 [M+H].sup.+; calculated: 427.20; t.sub.R (.lamda.=220
nm): 0.53 min (LC/MS-Method 1). .sup.1H-NMR (400 MHz, CDCl.sub.3):
.delta.=6.29 (d, J=3.6 Hz, 1H, C.dbd.CH), 5.69 (d, J=3.8 Hz, 1H,
CH), 5.24 (m, 1H, CH), 5.20 (m, 1H, CH), 3.75 (m, 4H,
2.times.NCH.sub.2), 2.62 (m, 4H, 2.times.NCH.sub.2), 2.16 (s, 3H,
CH.sub.3), 2.13 (s, 3H, CH.sub.3), 2.10 (s, 3H, CH.sub.3), 1.27 (s,
9H, 3.times.CH.sub.3) ppm.
EXAMPLE 43
[(2S,3R,4S)-2,3-Diacetoxy-6-[4-(cyclohexylmethyl)piperazine-1-carbonyl]-3,-
4-dihydro-2H-pyran-4-yl]acetate
##STR00058##
[0295] Example 43 was synthesized from example 41, step 1 and
1-cyclohexylmethylpiperazine following the amide coupling procedure
described in synthesis method C.
[0296] Purification: MPLC CombiFlash Rf (Teledyne ISCO); column:
RediSep Silica 12 g; flow rate: 30 mL/min; wavelength for
detection: 220 nm; eluent: (A) dichloromethane, (B) ethanol.
MPLC Gradient
TABLE-US-00023 [0297] start % B end % B duration [min] 0 0 2.2 0
4.8 2.8 4.8 4.8 6.0 4.810.0 10.1 6.0 10.1 10.1 9.1
Yield: 145 mg (0.311 mmol, 47.0%), orange oil. LC/MS (ES-API):
m/z=467.20 [M+H].sup.+; calculated: 467.23; t.sub.R (.lamda.=220
nm): 0.64 min (LC/MS-Method 1). .sup.1H-NMR (400 MHz, CDCl.sub.3):
.delta.=6.29 (d, J=3.2 Hz, 1H, C.dbd.CH), 5.63 (d, J=3.2 Hz, 1H,
CH), 5.24 (m, 1H, CH), 5.19 (m, 1H, CH), 3.75 (m, 4H,
2.times.NCH.sub.2), 2.62 (m, 4H, 2.times.NCH.sub.2), 2.16 (s, 3H,
CH.sub.3), 2.13 (s, 3H, CH.sub.3), 2.11 (s, 3H, CH.sub.3), 1.73 (m,
1H, CH), 1.62 (m, 4H, 2.times.CH.sub.2), 1.13 (m, 4H,
2.times.CH.sub.2), 0.92 (m, 2H, CH.sub.2) ppm.
EXAMPLE 44
[(2S,3R,4S)-2,3-Diacetoxy-6-(4-phenylpiperazine-1-carbonyl)-3,4-dihydro-2H-
-pyran-4-yl]acetate
##STR00059##
[0299] Example 44 was synthesized from example 41, step 1 and
1-phenylpiperazine following the amide coupling procedure described
in synthesis method C.
[0300] Purification: MPLC CombiFlash Rf (Teledyne ISCO); column:
RediSep Silica 12 g; flow rate: 30 mL/min; wavelength for
detection: 220 nm; eluent: (A) dichloromethane, (B) ethanol.
MPLC Gradient
TABLE-US-00024 [0301] start % B end % B duration [min] 0 0 2.2 0
4.8 2.8 4.8 4.8 6.0 4.810.0 10.1 6.0 10.1 10.1 9.1
Yield: 135 mg (0.303 mmol, 45.5%), orange oil. LC/MS (ES-API):
m/z=447.13 [M+H].sup.+; calculated: 447.17; t.sub.R (.lamda.=220
nm): 0.81 min (LC/MS-Method 1).
[0302] .sup.1H-NMR (400 MHz, CDCl.sub.3): .delta.=7.31 (t, J=7.9
Hz, 2H, ArH), 7.00 (m, 3H, ArH), 6.32 (dd, J=0.9 Hz, 1H, C.dbd.CH),
5.67 (dd, J=0.8 Hz, 1H, CH), 5.27 (m, 1H, CH), 5.21 (m, 1H, CH),
3.76 (m, 4H, 2.times.NCH.sub.2), 3.22 (m, 4H, 2.times.NCH.sub.2),
2.15 (s, 3H, CH.sub.3), 2.13 (s, 3H, CH.sub.3), 2.10 (s, 3H,
CH.sub.3) ppm.
EXAMPLE 45
[(2S,3R,4S)-2,3-Diacetoxy-6-[(4-phenylpiperazin-1-yl)methyI]-3,4-dihydro-2-
H-pyran-4-yl]acetate
##STR00060##
[0304] Step 1:
[(2S,3R,4S)-2,3-diacetoxy-6-formyl-3,4-dihydro-2H-pyran-4-yl]acetate
##STR00061##
[0305] A solution of 2.87 mL dry DMSO (40.31 mmol; 2.6 eq.) in 3 mL
dry dichloromethane was dropped slowly to a solution of 1.61 mL
oxalylchloride (18.60 mmol; 1.2eq.) in 4 mL dry dichloromethane at
-70.degree. C. After stirring the reaction mixture at -70.degree.
C. for 30 minutes, a solution of 5.4 g
1,2,3,4-tetra-O-acetyl-.beta.-D-glucopyranose in 20 mL dry
dichloromethane was added. After stirring the reaction mixture at
-70.degree. C. for 30 minutes, 11 mL triethylamine were added
slowly. The reaction mixture was warmed to room temperature and
diluted with 20 mL water. After stirring for 10 minutes at room
temperature, the aqueous phase was separated and extracted with
dichloromethane. The combined organic phases were dried with
Na2SO4, filtered, and evaporated.
Yield: 3.13 g (10.94 mmol, 70.5%), colorless oil. TLC:
R.sub.f=0.509 (ethylacetate/n-heptane, 2:1). LC/MS (ES-API):
m/z=304.05 [M+H.sub.2O]; calculated: 287.07; t.sub.R (.lamda.=254
nm): 0.63 min (LC/MS-Method 1). .sup.1H-NMR (400 MHz,
DMSO-d.sub.6): .delta.=9.28 (s, 1H, CHO), 5.19 (d, J=4.2 Hz, 1H,
CH), 5.10 (m, 2H, 2.times.CH), 2.08 (s, 3H, CH.sub.3), 2.07 (s, 3H,
CH.sub.3), 2.04 (s, 3H, CH.sub.3) ppm.
Method D
Reductive amination with
[(2S,3R,4S)-2,3-diacetoxy-6-formyl-3,4-dihydro-2H-pyran-4-yl]acetate
(example 45, step 1)
[0306] To a solution of 0.52 mmol
[(25,3R,45)-2,3-diacetoxy-6-formyl-3,4-dihydro-2H-pyran-4-yl]
acetate (example 45, step 1) in 10 mL dichloroethane were added
0.57 mmol (1.1 eq.) amine and 0.74 mmol (1.41 eq.)
sodiumtriacetoxyboronhydride. The reaction mixture was stirred at
room temperature over night. The reaction was controlled by TLC and
LC/MS. The reaction mixture was filtered and evaporated. The crude
material was purified by MPLC.
Step 2:
[(2S,3R,4S)-2,3-diacetoxy-6-[(4-phenylpiperazin-1-yl)methyl]-3,4-d-
ihydro-2H-pyran-4-yl]acetate
[0307] Example 45 was synthesized from example 45, step 1 and
1-phenylpiperazine following the reductive amidation procedure
described in synthesis method D.
[0308] Purification: MPLC CombiFlash Rf (Teledyne ISCO); column:
RediSep Silica 12 g; flow rate: 30 mL/min; wavelength for
detection: 254 nm; eluent: (A) n-heptane, (B) ethylacetate.
Yield: 154 mg (0.356 mmol, 68.0%), yellow oil. TLC: R.sub.f=0.639
(ethylacetate/n-heptane, 2:1). LC/MS (ES-API): m/z=433.20
[M+H].sup.+; calculated: 433.19; t.sub.R (.lamda.=220 nm): 1.26 min
(LC/MS-Method 1).
MPLC Gradient
TABLE-US-00025 [0309] start % B end % B duration [min] 0.0 0.0 1.6
0.0 70.2 28.0
.sup.1H-NMR (400 MHz, DMSO-d.sub.6): .delta.=7.20 (t, J=8.0 Hz, 2H,
ArH), 6.92 (d, J=8.0 Hz, 2H, ArH), 6.76 (d, J=7.2 Hz, 1H, ArH),
5.96 (dd, J=3.6 Hz, J=1.0 Hz 1H, CH), 5.12 (d, J=4.2 Hz, 1H, CH),
5.04 (m, 2H, 2.times.CH), 3.12 (m, 4H, 2.times.NCH.sub.2), 3.02
(AB-system, q, J=14.3 Hz, 2H, CH.sub.2), 2.50 (m, 4H,
2.times.NCH.sub.2), 2.08 (s, 3H, CH.sub.3), 2.07 (s, 3H, CH.sub.3),
2.04 (s, 3H, CH.sub.3) ppm. .sup.13C-NMR (150 MHz, DMSO-d.sub.6):
.delta.=169.83 (s, C), 168.62 (s, C), 168.40 (s, C), 151.31
(s,2.times. C), 129.16 (s, CH), 118.98 (s, CH), 115.23 (s, CH),
97.54 (s, CH), 93.66 (s, C), 88.92 (s, CH), 66.82 (s, CH), 64.19
(s, CH), 52.04 (s, 2.times.CH), 47.96 (s,2.times. CH), 21.13 (s,
CH.sub.3), 20.85 (s, CH.sub.3), 20.74 (s, CH.sub.3) ppm.
EXAMPLE 46
[(2S,3R,4S)-2,3-Diacetoxy-6-[[4-[(E)-cinnamyl]piperazin-1-yl]methyl]-3,4-d-
ihydro-2H-pyran-4-yl]acetate
##STR00062##
[0311] Example 46 was synthesized from example 45, step 1 and
trans-1-cinnamylpiperazine following the reductive amidation
procedure described in synthesis method D.
[0312] Purification: MPLC CombiFlash Rf (Teledyne ISCO); column:
RediSep Silica 12 g; flow rate: 30 mL/min; wavelength for
detection: 220 nm; eluent: (A) dichloromethane, (B) ethanol.
Yield: 194 mg (0.411 mmol, 78.3%), yellow oil. TLC: R.sub.f=0.136
(ethylacetate/n-heptane, 2:1). LC/MS (ES-API): m/z=473.23
[M+H].sup.+; calculated: 473.22; t.sub.R (.lamda.=220 nm): 1.32 min
(LC/MS-Method 1).
MPLC Gradient
TABLE-US-00026 [0313] start % B end % B duration [min] 0.0 0.0 1.3
0.0 13.3 15.1
.sup.1H-NMR (400 MHz, DMSO-d.sub.6): .delta.=7.42 (d, J=7.3 Hz, 2H,
ArH), 7.31 (t, J=7.3 Hz, 2H, ArH), 7.23 (t, J=7.3 Hz, 1H, ArH),
6.52 (d, J=15.9 Hz, 1H, CH), 6.27 (m, 1H, CH), 6.18 (dd, J=1.2 Hz,
1H, CH), 5.08 (d, J=4.1 Hz, 1H, CH), 5.03 (m, 2H, 2.times.CH), 3.33
(m, 6H, 3.times.NCH.sub.2), 2.96 (q, J=14.2 Hz, 2H, CH.sub.2), 2.41
(m, 4H, 2.times.NCH.sub.2), 2.08 (s, 3H, CH.sub.3), 2.07 (s, 3H,
CH.sub.3), 2.04 (s, 3H, CH.sub.3) ppm.
EXAMPLE 47
[(2S,3R,4S)-2,3-Diacetoxy-6-[[4-(1,3-benzodioxol-5-ylmethyl)piperazin-1-yl-
]methyl]-3,4-dihydro-2H-pyran-4-yl]acetate
##STR00063##
[0315] Example 47 was synthesized from example 45, step 1 and
1-(1,3-benzodioxol-5-ylmethyl)piperazine following the reductive
amidation procedure described in synthesis method D.
[0316] Purification: MPLC CombiFlash Rf (Teledyne ISCO); column:
RediSep Silica 12 g; flow rate: 30 mL/min; wavelength for
detection: 220 nm; eluent: (A) dichloromethane, (B) ethanol.
Yield: 249 mg (0.508 mmol, 96.9%), yellow oil. TLC: R.sub.f=0.242
(ethylacetate/n-heptane, 2:1). LC/MS (ES-API): m/z=491.24
[M+H].sup.+; calculated: 491.19; t.sub.R (.lamda.=220 nm): 1.19 min
(LC/MS-Method 1).
MPLC Gradient
TABLE-US-00027 [0317] start % B end % B duration [min] 0.0 0.0 1.9
0.0 10.1 13.1
.sup.1H-NMR (400 MHz, DMSO-d.sub.6): .delta.=6.84 (m, 2H, ArH),
6.73 (dd, J=1.4 Hz, 1H, ArH), 6.28 (dd, J=1.0 Hz, 1H, CH), 5.97 (s,
2H, O--CH.sub.2--O), 5.08 (d, J=4.2 Hz, 1H, CH), 5.04 (m, 2H,
2.times.CH), 3.31 (m, 4H, 2.times.NCH.sub.2), 2.94 (q, J=14.4 Hz,
2H, CH.sub.2), 2.38 (m, 4H, 2.times.NCH.sub.2), 2.08 (s, 3H,
CH.sub.3), 2.07 (s, 3H, CH.sub.3), 2.03 (s, 3H, CH.sub.3) ppm.
EXAMPLE 48
[(2S,3R,4S)-2,3-Diacetoxy-6-[[4-(4-chlorophenyl)piperazin-1-yl]methyl]-3,4-
-dihydro-2H-pyran-4-yl]acetate
##STR00064##
[0319] Example 48 was synthesized from example 45, step 1 and
1-(4-chlorophenyl)piperazine following the reductive amidation
procedure described in synthesis method D.
[0320] Purification: MPLC CombiFlash Rf (Teledyne ISCO); column:
RediSep Silica 12 g; flow rate: 30 mL/min; wavelength for
detection: 220 nm; eluent: (A) dichloromethane, (B) ethanol.
Yield: 131 mg (0.281 mmol, 53.5%), yellow oil. TLC: R.sub.f=0.636
(ethylacetate/n-heptane, 2:1). LC/MS (ES-API): m/z=467.16
[M+H].sup.+; calculated: 467.91; t.sub.R (.lamda.=220 nm): 1.40 min
(LC/MS-Method 1).
MPLC Gradient
TABLE-US-00028 [0321] start % B end % B duration [min] 0.0 0.0 2.0
0.0 10.1 23.0
.sup.1H-NMR (400 MHz, DMSO-d.sub.6): .delta.=7.21 (d, J=9.1 Hz, 2H,
ArH), 6.93 (d, J=9.1 Hz, 2H, ArH), 6.20 (dd, J=1.1 Hz, 1H, CH),
5.12 (d, J=4.4 Hz, 1H, CH), 5.04 (m, 2H, 2.times.CH), 3.12 (m, 4H,
2.times.NCH.sub.2), 3.02 (q, J=14.1 Hz, 2H, CH.sub.2), 2.51 (m, 4H,
2.times.NCH.sub.2), 2.07 (s, 3H, CH.sub.3), 2.06 (s, 3H, CH.sub.3),
2.04 (s, 3H, CH.sub.3) ppm.
EXAMPLE 49
[(2S,3R,4S)-2,3-Diacetoxy-6-[[4-(2-chlorophenyl)piperazin-1-yl]methyl]-3,4-
-dihydro-2H-pyran-4-yl]acetate
##STR00065##
[0323] Example 49 was synthesized from example 45, step 1 and
1-(2-chlorophenyl)piperazine following the reductive amidation
procedure described in synthesis method D.
[0324] Purification: MPLC CombiFlash Rf (Teledyne ISCO); column:
RediSep Silica 12 g; flow rate: 30 mL/min; wavelength for
detection: 254 nm; eluent: (A) n-heptane, (B) ethylacetate.
Yield: 144 mg (0.308 mmol, 58.9%), yellow oil. TLC: R.sub.f=0.621
(ethylacetate/n-heptane, 2:1). LC/MS (ES-API): m/z=467.16
[M+H].sup.+; calculated: 467.91; t.sub.R (.lamda.=220 nm): 1.38 min
(LC/MS-Method 1).
MPLC Gradient
TABLE-US-00029 [0325] start % B end % B duration [min] 0.0 0.0 1.6
0.0 70.2 23.0
.sup.1H-NMR (400 MHz, DMSO-d.sub.6): .delta.=7.39 (d, J=7.9 Hz, 1H,
ArH), 7.28 (t, J=7.7 Hz, 1H, ArH), 7.17 (d, J=8.1 Hz, 1H, ArH),
7.03 (t, J=7.6 Hz, 1H, ArH), 6.21 (dd, J=1.2 Hz, 1H, CH), 5.13 (d,
J=4.1 Hz, 1H, CH), 5.04 (m, 2H, 2.times.CH), 3.07 (q, J=14.4 Hz,
2H, CH.sub.2), 2.98 (m, 4H, 2.times.NCH.sub.2), 2.59 (m, 4H,
2.times.NCH.sub.2), 2.08 (s, 3H, CH.sub.3), 2.07 (s, 3H, CH.sub.3),
2.03 (s, 3H, CH.sub.3) ppm.
EXAMPLE 50
[(2S,3R,4S)-2,3-Diacetoxy-6-[[4-(4-methoxyphenyl)piperazin-1-yl]methyl]-3,-
4-dihydro-2H-pyran-4-yl]acetate
##STR00066##
[0327] Example 50 was synthesized from example 45, step 1 and
1-(4-methoxyphenyl)piperazine following the reductive amidation
procedure described in synthesis method D.
[0328] Purification: MPLC CombiFlash Rf (Teledyne ISCO); column:
RediSep Silica 12 g; flow rate: 30 mL/min; wavelength for
detection: 254 nm; eluent: (A) n-heptane, (B) ethylacetate.
Yield: 151 mg (0.326 mmol, 62.3%), yellow oil. TLC: R.sub.f=0.530
(ethylacetate/n-heptane, 2:1). LC/MS (ES-API): m/z=463.19
[M+H].sup.+; calculated: 463.20; t.sub.R (.lamda.=220 nm): 1.25 min
(LC/MS-Method 1).
MPLC Gradient
TABLE-US-00030 [0329] start % B end % B duration [min] 0.0 0.0 1.3
0.0 100.0 13.7 100.0 100.0 3.6
.sup.1H-NMR (400 MHz, DMSO-d.sub.6): .delta.=6.88 (d, J=9.1 Hz, 2H,
ArH), 6.80 (d, J=9.1 Hz, 2H, ArH), 6.20 (dd, J=1.0 Hz, 1H, CH),
5.12 (d, J=4.1 Hz, 1H, CH), 5.04 (m, 2H, 2.times.CH), 3.69 (s, 3H,
OCH.sub.3), 3.00 (m, 6H, CH.sub.2+NCH.sub.2), 2.52 (m, 4H,
NCH.sub.2), 2.08 (s, 3H, CH.sub.3), 2.07 (s, 3H, CH.sub.3), 2.04
(s, 3H, CH.sub.3) ppm.
EXAMPLE 51
[(2S,3R,4S)-2,3-Diacetoxy-6-[[4-(3-methoxyphenyl)piperazin-1-yl]methyl]-3,-
4-dihydro-2H-pyran-4-yl]acetate
##STR00067##
[0331] Example 51 was synthesized from example 45, step 1 and
1-(3-methoxyphenyl)piperazine following the reductive amidation
procedure described in synthesis method D.
[0332] Purification: MPLC CombiFlash Rf (Teledyne ISCO); column:
RediSep Silica 12 g; flow rate: 30 mL/min; wavelength for
detection: 254 nm; eluent: (A) n-heptane, (B) ethylacetate.
Yield: 135 mg (0.292 mmol, 55.7%), yellow oil. TLC: R.sub.f=0.591
(ethylacetate/n-heptane, 2:1). LC/MS (ES-API): m/z=463.24
[M+H].sup.+; calculated: 463.20; t.sub.R (.lamda.=220 nm): 1.28 min
(LC/MS-Method 1).
MPLC Gradient
TABLE-US-00031 [0333] start % B end % B duration [min] 0.0 0.0 1.3
0.0 100.0 13.7 100.0 100.0 3.6
.sup.1H-NMR (400 MHz, DMSO-d.sub.6): .delta. =7.09 (t, J=8.2 Hz,
1H, ArH), 6.51 (d, J=8.2 Hz, 1H, ArH), 6.43 (s, 1H, ArH), 6.35 (dd,
J=1.1 Hz, 1H, CH), 5.12 (d, J=4.3 Hz, 1H, CH), 5.04 (m, 1H, CH),
3.71 (s, 3H, OCH.sub.3), 3.11 (m, 4H, 2.times.NCH.sub.2), 3.01 (q,
J=14.3 Hz, 2H, CH.sub.2), 2.52 (m, 4H, 2.times.NCH.sub.2), 2.08 (s,
3H, CH.sub.3), 2.07 (s, 3H, CH.sub.3), 2.04 (s, 3H, CH.sub.3)
ppm.
EXAMPLE 52
[(2S,3R,4S)-2,3-Diacetoxy-6-[[4-(2-methoxyphenyl)piperazin-1-yl]methyl]-3,-
4-dihydro-2H-pyran-4-yl]acetate
##STR00068##
[0335] Example 52 was synthesized from example 45, step 1 and
1-(2-methoxyphenyl)piperazine following the reductive amidation
procedure described in synthesis method D.
[0336] Purification: MPLC CombiFlash Rf (Teledyne ISCO); column:
RediSep Silica 12 g; flow rate: 30 mL/min; wavelength for
detection: 254 nm; eluent: (A) n-heptane, (B) ethylacetate.
Yield: 167 mg (0.361 mmol, 68.9%), yellow oil. TLC: R.sub.f=0.533
(ethylacetate/n-heptane, 2:1). LC/MS (ES-API): m/z=463.24
[M+H].sup.+; calculated: 463.20; t.sub.R (.lamda.=220 nm): 1.26 min
(LC/MS-Method 1).
MPLC Gradient
TABLE-US-00032 [0337] start % B end % B duration [min] 0.0 0.0 1.3
0.0 100.0 13.7 100.0 100.0 3.6
[0338] .sup.1H-NMR (400 MHz, DMSO-d.sub.6): .delta.=6.90 (m, 4H,
ArH), 6.21 (dd, J=1.0 Hz, 1H, CH), 5.12 (d, J=4.4 Hz, 1H, CH), 5.03
(m, 1H, CH), 3.68 (s, 3H, OCH.sub.3), 3.04 (q, J=14.3 Hz, 2H,
CH.sub.2), 2.96 (m, 4H, 2.times.NCH.sub.2), 2.54 (m, 4H,
2.times.NCH.sub.2), 2.10 (s, 3H, CH.sub.3), 2.09 (s, 3H, CH.sub.3),
2.05 (s, 3H, CH.sub.3) ppm.
EXAMPLE 53
[(2S,3R,4S)-2,3-Diacetoxy-6-[[4-(4-bromophenyl)piperazin-1-yl]methyl]-3,4--
dihydro-2H-pyran-4-yl]acetate
##STR00069##
[0340] Example 53 was synthesized from example 45, step 1 and
1-(4-bromophenyl)piperazine following the reductive amidation
procedure described in synthesis method D.
[0341] Purification: MPLC CombiFlash Rf (Teledyne ISCO); column:
RediSep Silica 12 g; flow rate: 30 mL/min; wavelength for
detection: 254 nm; eluent: (A) n-heptane, (B) ethylacetate.
Yield: 248 mg (0.485 mmol, 92.5%), yellow oil. TLC: R.sub.f=0.644
(ethylacetate/n-heptane, 2:1). LC/MS (ES-API): m/z=511.14
[M+H].sup.+; calculated: 511.10; t.sub.R (.lamda.=220 nm): 1.42 min
(LC/MS-Method 1).
MPLC Gradient
TABLE-US-00033 [0342] start % B end % B duration [min] 0.0 0.0 1.3
0.0 100.0 13.7 100.0 100.0 3.6
.sup.1H-NMR (400 MHz, DMSO-d.sub.6): .delta.=7.33 (d, J=9.0 Hz, 2H,
ArH), 6.88 (d, J=9.0 Hz, 2H, ArH), 6.20 (dd, J=1.2 Hz, 1H, CH),
5.12 (d, J=4.3 Hz, 1H, CH), 5.04 (m, 2H, 2.times.CH), 3.12 (m, 4H,
2.times.NCH.sub.2), 3.03 (q, J=14.2 Hz, 2H, CH.sub.2), 2.52 (m, 4H,
2.times.NCH.sub.2), 2.09 (s, 3H, CH.sub.3), 2.08 (s, 3H, CH.sub.3),
2.05 (s, 3H, CH.sub.3) ppm.
[0343] Example 54
5-(Dimethylamino)-N-[[1-[(3R,4R,5S,6R)-2,4,5-trihydroxy-6-(hydroxymethyl)t-
etrahydropyran-3-yl]triazol-4-yl]methyl]naphthalene-1-sulfonamide
##STR00070##
[0344] Step 1:
5-(dimethylamino)-N-prop-2-ynyl-naphthalene-1-sulfonamide
##STR00071##
[0346] To a solution of 405 mg (1.5 mmol) dansylchloride in 4 mL
dimethylformamide were added 91 mg (1.65 mmol) propargylamine and
530 .mu.l (3 mmol, 2 eq.) N,N-diisopropylethylamine. The reaction
mixture was stirred at 100.degree. C. in the microwave for 45
minutes. The reaction mixture was filtered and evaporated.
[0347] Purification: MPLC CombiFlash Rf (Teledyne ISCO); column:
RediSep Silica 24 g Gold; flow rate: 40 mL/min; wavelength for
detection: 254 nm; eluent: (A) dichloromethane, (B) ethanol.
MPLC Gradient
TABLE-US-00034 [0348] start % B end % B duration [min] 0.0 0.0 2.0
0.0 50.0 33.0
Yield: 92 mg (0.319 mmol, 21.5%), yellow oil. TLC: R.sub.f=0.510
(dichloromethane/ethanol, 19:1). LC/MS (ES-API): m/z=289.13
[M+H].sup.+; calculated: 289.09; t.sub.R (.lamda.=220 nm): 1.62 min
(LC/MS-Method 1). .sup.1H-NMR (400 MHz, DMSO-d.sub.6): .delta.=8.48
(d, J=8.6 Hz, 1H, ArH), 8.40 (s, 1H, SO.sub.2NH), 8.25 (d, J=8.6
Hz, 1H, ArH), 8.13 (dd, J=1.1 Hz, 1H, ArH), 7.60 (m, 2H, ArH), 7.26
(d, J=7.4 Hz, 1H, ArH), 3.71 (s, 2H, NCH.sub.2), 2.91 (s, 1H, CH),
2.84 (s, 6H, 2.times.NCH.sub.3) ppm.
Method E
[0349] Copper-Catalyzed Azide-Alkyne-Cycloaddition (CuAAC) with
Azidodeoxyglucose
[0350] To a solution of 146 .mu.mol azidodeoxyglucose
(1-azido-1-deoxyglucose, 2-azido-2-deoxyglucose, or
6-azido-6-deoxyglucose) in 0.5 mL water 1.1 eq. alkyne were added.
If needed dimethylformamide was added until the reaction mixture
was a clear solution. A mixture of 0.1 eq. CuSO.sub.4*5 H.sub.2O
(0.1 M in water), 0.25 eq. sodium ascorbate (1 M in water), and 0.4
eq. THPTA (0.5 M in water) was added. The reaction mixture was
stirred at room temperature for 2-6 hours. The reaction was
controlled by TLC and LC/MS. The reaction mixture was evaporated
and purified by HPLC. Alpha/beta isomers were not separated, NMR
data belong to only one isomer.
[0351] Purification: Agilent 1200 preparative HPLC; column: Agilent
Prep-C.sub.18 column (10 .mu.m, 21.5.times.150 mm); flow rate: 40
mL/min; wavelength for detection: 220 nm; 254 nm; 324 nm; eluent:
(A) water, (B) acetonitrile.
HPLC-Gradient
TABLE-US-00035 [0352] start % B end % B duration [min] 3.0 3.0 5.0
3.0 90.0 7.5 90.0 90.0 2.5 90.0 10.0 0.5 10.0 10.0 2.0
Step 2:
5-(dimethylamino)-N-[[1-[(3R,4R,5S,6R)-2,4,5-trihydroxy-6-(hydroxy-
methyl)-tetrahydropyran-3-yl]triazol-4-yl]methyl]naphthalene-1-sulfonamide
[0353] Example 54 was synthesized from 2-azido-2-deoxyglucose and
example 45, step 1 following the CuAAC procedure described in
synthesis method E.
Yield: 33 mg (67 .mu.mol, 46.7%), yellow oil. TLC: R.sub.f=0.032
(dichloromethane/ethanol, 4:1). LC/MS (ES-API): m/z=494.19
[M+H].sup.+; calculated: 494.16; t.sub.R1 (.lamda.=220 nm): 1.17
min; t.sub.R2 (.lamda.=220 nm): 1.20 min (LC/MS-Method 1).
.sup.1H-NMR (400 MHz, DMSO-d.sub.6): .delta.=8.47 (d, J=8.6 Hz, 2H,
ArH), 8.40 (s, 1H, SNH), 8.13 (d, J=7.3 Hz, 1H, ArH), 7.84 (s, 1H,
NCH), 7.61 (m, 2H, ArH), 7.27 (d, J=7.4 Hz, 1H, ArH), 6.95 (d,
J=6.4Hz, 1H, OH), 5.17 (d, J=6.2 Hz, 1H, OH), 5.09 (d, J=6.2 Hz,
1H, OH), 4.89 (dd, J=6.4 Hz, 1H, CH), 4.61 (dd, J=6.4 Hz, 1H, OH),
4.07 (s, 2H, NCH.sub.2), 4.01 (m, 1H, CH), 3.85 (m, 1H, CH), 3.74
(m, 1H, CH), 3.53 (m, 2H, CH.sub.2), 3.22 (m, 1H, CH.sub.2), 2.84
(s, 6H, 2.times.NCH.sub.3) ppm.
EXAMPLE 55
(3R,4R,5S,6R)-6-(Hydroxymethyl)-3-(4-phenyltriazol-1-yl)tetrahydropyran-2,-
4,5-triol
##STR00072##
[0355] Example 55 was synthesized from 2-azido-2-deoxyglucose and
ethynylbenzene following the CuAAC procedure described in synthesis
method E.
Yield: 15 mg (49 .mu.mol, 33.4%), white solid. TLC: R.sub.f=0.333
(dichloromethane/ethanol, 4:1). LC/MS (ES-API): m/z=308.10
[M+H].sup.+; calculated: 308.12; t.sub.R1 (.lamda.=220 nm): 0.81
min; t.sub.R2 (.lamda.=220 nm): 0.85 min (LC/MS-Method 1).
.sup.1H-NMR (400 MHz, DMSO-d.sub.6): .delta.=8.61 (s, 1H, NCH),
7.86 (dd, J=1.3 Hz, 2H, ArH), 7.45 (m, 2H, ArH), 7.40 (m, 1H, ArH),
7.10 (d, J=6.2 Hz, 1H, OH), 5.48 (d, J=6.2 Hz, 1H, OH), 5.25 (d,
J=6.6 Hz, 1H, OH), 5.25 (t, J=4.2 Hz, 1H, CH), 4.65 (t, J=6.1 Hz,
1H, OH), 4.65 (dd, J=10.8 Hz, J=3.1 Hz, 1H, CH), 4.08 (m, 1H, CH),
3.96 (m, 1H, OH), 3.76 (m, 1H, CH.sub.2), 3.52 (m, 1H, CH.sub.2),
3.36 (m, 1H, CH), 3.29 (m, 1H, CH) ppm. .sup.13C-NMR (150 MHz,
DMSO-d.sub.6): .delta.=145.70 (s, C), 131.00 (s, C), 128.90 (s,
CH), 127.64 (s, CH), 125.05 (s, CH), 120.64 (s, CH), 90.80 (s, CH),
72.42 (s, CH), 70.93 (s, CH), 69.72 (s, CH), 65.24 (s, CH), 60.86
(s, CH.sub.2) ppm.
EXAMPLE 56
(3R,4R,5S,6R)-6-(Hydroxymethyl)-3-[4-(p-tolyl)triazol-1-yl]tetrahydropyran-
-2,4,5-triol
##STR00073##
[0357] Example 56 was synthesized from 2-azido-2-deoxyglucose and
1-ethynyl-4-methylbenzene following the CuAAC procedure described
in synthesis method E.
Yield: 34 mg (106 .mu.mol, 72.4%), white solid. TLC: R.sub.f=0.349
(dichloromethane/ethanol, 4:1). LC/MS (ES-API): m/z=322.19
[M+H].sup.+; calculated: 322.13; t.sub.R1 (.lamda.=220 nm): 1.07
min; t.sub.R2 (.lamda.=220 nm): 1.11 min (LC/MS-Method 1).
.sup.1H-NMR (400 MHz, DMSO-d.sub.6): .delta.=8.47 (s, 1H, NCH),
7.76 (d, J=8.1 Hz, 2H, ArH), 7.24 (d, J=8.1 Hz, 2H, ArH), 6.93 (d,
J=4.4 Hz, 1H, OH), 5.29 (d, J=5.4 Hz, 1H, OH), 5.20 (d, J=5.4 Hz,
1H, OH), 5.00 (dd, J=8.0 Hz, 1H, CH), 4.56 (dd, J=3.2 Hz, 1H, OH),
.delta.=4.09 (m, 1H, CH), 3.96 (m, 1H, CH), 3.76 (m, 1H, CH.sub.2),
3.53 (m, 1H, CH.sub.2), 3.36 (m, 1H, CH), 3.28 (m, 1H, CH), 2.35
(s, 3H, CH.sub.3) ppm.
Example 57
(3R,4R,5S,6R)-6-(Hydroxymethyl)-3-[4-(3-phenylpropyl)triazol-1-yl]tetrahyd-
ropyran-2,4,5-triol
##STR00074##
[0359] Example 57 was synthesized from 2-azido-2-deoxyglucose and
pent-4-yn-1-ylbenzene following the CuAAC procedure described in
synthesis method E.
Yield: 40 mg (114 .mu.mol, 78.3%), white solid. TLC: R.sub.f=0.406
(dichloromethane/ethanol, 4:1). LC/MS (ES-API): m/z=350.19
[M+H].sup.+; calculated: 350.16; t.sub.R1 (.lamda.=220 nm): 1.24
min; t.sub.R2 (.lamda.=220 nm): 1.26 min (LC/MS-Method 1).
.sup.1H-NMR (400 MHz, DMSO-d.sub.6): .delta.=8.78 (s, 1H, NCH),
7.75 (m, 5H, ArH), 6.81 (d, J=5.0 Hz, 1H, OH), 5.22 (d, J=5.7 Hz,
1H, OH), 5.12 (d, J=5.7 Hz, 1H, OH), 4.92 (dd, J=6.7 Hz, 1H, CH),
4.52 (dd, J=5.5 Hz, 1H, OH), 4.09 (m, 1H, CH), 3.95 (m, 1H, CH),
3.76 (m, 1H, CH.sub.2), 3.53 (m, 1H, CH.sub.2), 3.36 (m, 1H, CH),
3.28 (m, 1H, CH), 2.61 (m, 4H, CH.sub.2), 1.90 (m, 2H, CH.sub.2)
ppm.
EXAMPLE 58
Methyl
4-[1-[(3R,4R,5S,6R)-2,4,5-trihydroxy-6-(hydroxymethyl)tetrahydropyr-
an-3-yl]triazol-4-yl]benzoate
##STR00075##
[0361] Example 58 was synthesized from 2-azido-2-deoxyglucose and
4-ethynylmethylbenzoate following the CuAAC procedure described in
synthesis method E.
Yield: 31 mg (85 .mu.mol, 58.0%), white solid. TLC: R.sub.f=0.429
(dichloromethane/ethanol, 4:1). LC/MS (ES-API): m/z=366.12
[M+H].sup.+; calculated: 366.12; t.sub.R1 (.lamda.=220 nm): 0.97
min; t.sub.R2 (.lamda.=220 nm): 1.00 min (LC/MS-Method 1).
.sup.1H-NMR (400 MHz, DMSO-d.sub.6): .delta.=8.80 (s, 1H, NCH),
8.05 (d, J=8.2 Hz, 2H, ArH), 8.00 (d, J=8.2 Hz, 2H, ArH), 7.09 (d,
J=6.4 Hz, 1H, OH), 5.49 (d, J=6.3 Hz, 1H, OH), 5.25 (d, J=5.7 Hz,
1H, OH), 5.00 (dd, J=8.0 Hz, 1H, CH), 4.65 (dd, J=5.2 Hz, 1H, OH),
4.09 (m, 1H, CH), 3.95 (m, 1H, CH), 3.87 (s, 3H, OCH.sub.3), 3.76
(m, 1H, CH.sub.2), 3.53 (m, 1H, CH.sub.2), 3.36 (m, 1H, CH), 3.28
(m, 1H, CH) ppm.
EXAMPLE 59
(3R,4R,5S,6R)-3-[4-[[Benzyl(methyl)amino]methyl]triazol-1-yl]-6-(hydroxyme-
thyl)tetrahydropyran-2,4,5-triol
##STR00076##
[0363] Example 59 was synthesized from 2-azido-2-deoxyglucose and
N-benzyl-N-methylprop-2-yn-1-amine following the CuAAC procedure
described in synthesis method E.
Yield: 32 mg (88 .mu.mol, 60.1%), white solid. TLC: R.sub.f=0.064
(dichloromethane/ethanol, 4:1). LC/MS (ES-API): m/z=365.20
[M+H].sup.+; calculated: 365.17; t.sub.R (ELSD): 0.34 min
(LC/MS-Method 1). .sup.1H-NMR (400 MHz, DMSO-d.sub.6): .delta.=8.00
(s, 1H, NCH), 7.71 (m, 5H, ArH), 6.82 (d, J=6.0 Hz, 1H, OH), 5.25
(d, J=5.8 Hz, 1H, OH), 5.14 (d, J=5.8 Hz, 1H, OH), 4.93 (dd, J=5.7
Hz, 1H, CH), 4.49 (dd, J=5.2 Hz, 1H, OH), 4.08 (m, 1H, CH), 3.95
(m, 1H, CH), 3.76 (m, 2H, CH.sub.2), 3.62 (s, 3H, NCH.sub.3), 3.36
(m, 1H, CH), 3.28 (m, 1H, CH), 2.13 (m, 4H, 2.times.NCH.sub.2)
ppm.
EXAMPLE 60
(3R,4R,5S,6R)-6-(Hydroxymethyl)-3-[4-(6-methoxy-2-naphthyl)triazol-1-yl]te-
trahydropyran-2,4,5-triol
##STR00077##
[0365] Example 60 was synthesized from 2-azido-2-deoxyglucose and
2-ethynyl-6-methoxynaphthalene following the CuAAC procedure
described in synthesis method E.
Yield: 28 mg (72 .mu.mol, 49.4%), white solid. TLC: R.sub.f=0.461
(dichloromethane/ethanol, 4:1). LC/MS (ES-API): m/z=388.16
[M+H].sup.+; calculated: 388.14; t.sub.R1 (.lamda.=220 nm): 1.27
min; t.sub.R2 (.lamda.=220 nm): 1.29 min (LC/MS-Method 1).
.sup.1H-NMR (400 MHz, DMSO-d.sub.6): .delta.=8.64 (s, 1H, NCH),
7.88 (m, 4H, ArH), 7.34 (s, 1H, ArH), 7.07 (d, J=6.5 Hz, 1H, OH),
5.47 (d, J=6.4 Hz, 1H, OH), 5.23 (d, J=6.4 Hz, 1H, OH), 5.00 (dd,
J=6.6 Hz, 1H, CH), 4.49 (dd, J=5.2 Hz, 1H, OH), 4.11 (m, 1H, CH),
3.98 (m, 1H, CH), 3.89 (s, 3H, OCH.sub.3), 3.77 (m, 1H, CH.sub.2),
3.53 (m, 1H, CH.sub.2), 3.46 (m, 1H, CH), 3.38 (m, 1H, CH) ppm.
EXAMPLE 61
(3R,4R,5S,6R)-3-[4-[4-Chloro-6-methyl-2-(p-tolyl)pyrimidin-5-yl]triazol-1--
yl]-6-(hydroxymethyl)tetrahydropyran-2,4,5-triol
##STR00078##
[0367] Example 61 was synthesized from 2-azido-2-deoxyglucose and
4-chloro-5-ethynyl-6-methyl-2-(p-tolyl)pyrimidine following the
CuAAC procedure described in synthesis method E.
Yield: 51 mg (110 .mu.mol, 75.5%), white solid. TLC: R.sub.f=0.556
(dichloromethane/ethanol, 4:1). LC/MS (ES-API): m/z=462.22
[M+H].sup.+; calculated: 462.15; t.sub.R1 (.lamda.=220 nm): 1.51
min; t.sub.R2 (.lamda.=220 nm): 1.52 min (LC/MS-Method 1).
[0368] .sup.1H-NMR (400 MHz, DMSO-d.sub.6): .delta.=8.19 (d, J=8.2
Hz, 2H, ArH), 7.89 (s, 1H, NCH), 7.71 (d, J=8.2 Hz, 2H, ArH), 6.93
(d, J=6.6 Hz, 1H, OH), 5.21 (d, J=6.1 Hz, 1H, OH), 5.14 (d, J=6.1
Hz, 1H, OH), 4.91 (dd, J=6.7 Hz, 1H, CH), 4.50 (dd, J=5.5 Hz, 1H,
OH), 4.21 (s, 3H, CH.sub.3), 4.05 (m, 1H, CH), 3.97 (m, 1H, CH),
3.85 (m, 2H, CH.sub.2), 3.71 (m, 1H, CH), 3.50 (m, 1H, CH), 2.62
(s, 3H, CH.sub.3) ppm.
EXAMPLE 62
Ethyl-2-diethoxyphosphoryl-3-[1-[(3R,4R,5S,6R)-2,4,5-trihydroxy-6-(hydroxy-
methyl)-tetrahydropyran-3-yl]priazol-4-yl]propanoate
##STR00079##
[0370] Example 62 was synthesized from 2-azido-2-deoxyglucose and
ethyl 2-diethoxyphosphorylpent-4-ynoate following the CuAAC
procedure described in synthesis method E.
Yield: 30 mg (64 .mu.mol, 43.9%), white solid. TLC: R.sub.f=0.389
(dichloromethane/ethanol, 4:1). LC/MS (ES-API): m/z=468.17
[M+H].sup.+; calculated: 468.17; t.sub.R1 (.lamda.=220 nm): 0.99
min; t.sub.R2 (.lamda.=220 nm): 1.01 min (LC/MS-Method 1).
.sup.1H-NMR (400 MHz, DMSO-d.sub.6): .delta.=7.89 (s, 1H, NCH),
6.90 (d, J=6.6 Hz, 1H, OH), 5.17 (d, J=6.0 Hz, 1H, OH), 5.10 (d,
J=6.0 Hz, 1H, OH), 4.86 (dd, J=6.5 Hz, 1H, CH), 4.61 (dd, J=5.5 Hz,
1H, OH), 4.01 (m, 1H, CH), 3.88 (m, 1H, CH), 3.61 (m, 1H, CH), 3.50
(m, 1H, CH.sub.2), 3.22 (m, 1H, CH.sub.2), 3.01 (m, 1H, CH), 1.29
(m, 9H, CH.sub.3), 1.14 (m, 6H, CH.sub.2) ppm.
EXAMPLE 63
(3R,4R,5S,6R)-6-(Hydroxymethyl)-3-[4-[[(4-nitro-2,1,3-benzoxadiazol-7-yl)a-
mino]methyl]triazol-1-yl]tetrahydropyran-2,4,5-triol
##STR00080##
[0371] Step 1:
4-nitro-N-prop-2-ynyl-2,1,3-benzoxadiazol-7-amine
##STR00081##
[0373] To a solution of 450 mg (2.23 mmol)
4-chloro-7-nitro-2,1,3-benzoxadiazole in 4 mL dry dimethylformamide
were added 135 mg (2.46 mmol) propargylamine and 395 .mu.l (2.23
mmol) N,N-diisopropylethylamine. The reaction mixture was stirred
at 100.degree. C. in the microwave for 45 minutes and
evaporated.
[0374] Purification: MPLC CombiFlash Rf (Teledyne ISCO); column:
RediSep Silica 40 g; flow rate: 40 mL/min; wavelength for
detection: 254 nm; eluent: (A) n-heptane, (B) ethylacetate.
MPLC Gradient
TABLE-US-00036 [0375] start % B end % B duration [min] 0.0 0.0 1.6
0.0 27.7 16.6 27.7 79.8 19.9 79.8 0.0 0.0 0.0 0.0 2.9
Yield: 98 mg (0.449 mmol, 20.1%), orange solid. TLC: R.sub.f=0.664
(dichloromethane/ethanol, 19:1). LC/MS (ES-API): m/z=219.01
[M+H].sup.+; calculated: 219.04; t.sub.R (.lamda.=220 nm): 1.32 min
(LC/MS-Method 1). .sup.1H-NMR (400 MHz, DMSO-d.sub.6): .delta.=9.62
(s, 1H, NH), 8.62 (d, J=8.7 Hz, 1H, ArH), 6.48 (d, J=8.7 Hz, 1H,
ArH), 4.34 (s, 2H, NCH.sub.2), 3.37 (s, 1H, CH) ppm.
Step 2:
(3R,4R,5S,6R)-6-(hydroxymethyl)-3-[4-[[(4-nitro-2,1,3-benzoxadiazo-
l-7-yl)amino]methyl]triazol-1-yl]tetrahydropyran-2,4,5-triol
[0376] Example 63 was synthesized from 2-azido-2-deoxyglucose and
example 63, step 1 following the CuAAC procedure described in
synthesis method E.
Yield: 43 mg (101 .mu.mol, 69.5%), orange solid. TLC: R.sub.f=0.294
(dichloromethane/ethanol, 4:1). LC/MS (ES-API): m/z=424.10
[M+H].sup.+; calculated: 424.16; t.sub.R1 (.lamda.=220 nm): 0.77
min; t.sub.R2 (.lamda.=220 nm): 0.81 min (LC/MS-Method 1).
.sup.1H-NMR (400 MHz, DMSO-d.sub.6): .delta.=9.83 (s, 1H, NH) 8.62
(d, J=8.7 Hz, 1H, ArH), 8.04 (s, 1H, NCH), 6.93 (d, J=6.4Hz, 1H,
OH), 6.51 (d, J=8.7 Hz, 1H, ArH), 5.21 (d, J=6.2 Hz, 1H, OH), 5.14
(d, J=6.2 Hz, 1H, OH), 4.92 (dd, J=6.4 Hz, 1H, CH), 4.67 (s, 2H,
NCH.sub.2), 4.61 (dd, J=6.4 Hz, 1H, OH), 4.06 (m, 1H, CH), 3.90 (m,
1H, CH), 3.64 (m, 1H, CH), 3.52 (m, 2H, CH.sub.2), 3.22 (m, 1H,
CH.sub.2), 3.01 (m, 1H, CH) ppm.
EXAMPLE 64
(3R,4R,5S,6R)-3-[4-(Cyclohexylmethyl)triazol-1-yl]-6-(hydroxymethyl)tetrah-
ydropyran-2,4,5-triol
##STR00082##
[0378] Example 64 was synthesized from 2-azido-2-deoxyglucose and
prop-2-yn-1-ylcyclohexane following the CuAAC procedure described
in synthesis method E.
Yield: 23 mg (70 .mu.mol, 48.0%), white solid. TLC: R.sub.f=0.437
(dichloromethane/ethanol, 4:1). LC/MS (ES-API): m/z=328.24
[M+H].sup.+; calculated: 328.18; t.sub.R1 (.lamda.=220 nm): 1.23
min; t.sub.R2 (.lamda.=220 nm): 1.26 min (LC/MS-Method 1).
.sup.1H-NMR (400 MHz, DMSO-d.sub.6): .delta.=7.67 (s, 1H, NCH),
6.78 (d, J=6.5 Hz, 1H, OH), 5.19 (d, J=6.3 Hz, 1H, OH), 5.14 (d,
J=5.7 Hz, 1H, OH), 4.90 (dd, J=8.0 Hz, 1H, CH), 4.52 (dd, J=6.2 Hz,
1H, OH), 4.41 (dd, J=4.2 Hz, 1H, OH), 4.07 (m, 1H, CH), 3.76 (m,
2H, CH.sub.2), 3.56 (m, 2H, 2.times.CH), 3.20 (m, 1H, CH), 1.60 (m,
6H, CH.sub.2), 1.55 (m, 1H, CH), 1.21 (m, 4H, CH.sub.2), 0.93 (m,
2H, CH.sub.2) ppm.
EXAMPLE 65
(3R,4R,5S,6R)-6-(Hydroxymethyl)-3-(4-pentyltriazol-1-yl)tetrahydropyran-2,-
4,5-triol
##STR00083##
[0380] Example 65 was synthesized from 2-azido-2-deoxyglucose and
hept-1-yne following the CuAAC procedure described in synthesis
method E.
Yield: 19 mg (63 .mu.mol, 43.1%), white solid. TLC: R.sub.f=0.405
(dichloromethane/ethanol, 4:1). LC/MS (ES-API): m/z=302.22
[M+H].sup.+; calculated: 302.16; t.sub.R1 (.lamda.=220 nm): 1.08
min; t.sub.R2 (.lamda.=220 nm): 1.12 min (LC/MS-Method 1).
.sup.1H-NMR (400 MHz, DMSO-d.sub.6): .delta. =7.73 (s, 1H, NCH),
6.81 (d, J=4.5 Hz, 1H, OH), 5.21 (d, J=5.7 Hz, 1H, OH), 5.16 (d,
J=5.6 Hz, 1H, OH), 4.91 (dd, J=6.7 Hz, 1H, CH), 4.52 (dd, J=5.9 Hz,
1H, OH), 4.39 (dd, J=3.2 Hz, 1H, OH), 4.04 (m, 1H, CH), 3.71 (m,
2H, CH.sub.2), 3.52 (m, 2H, 2.times.CH), 3.20 (m, 1H, CH), 2.59 (m,
2H, CH.sub.2), 1.59 (m, 2H, CH.sub.2), 1.31 (m, 4H, CH.sub.2), 0.93
(m, 3H, CH.sub.3) ppm.
EXAMPLE 66
(3R,4R,5S,6R)-3-[4-(3-Chloropropyl)triazol-1-yl]-6-(hydroxymethyl)tetrahyd-
ropyran-2,4,5-triol
##STR00084##
[0382] Example 66 was synthesized from 2-azido-2-deoxyglucose and
5-chloro-pent-1-yne following the CuAAC procedure described in
synthesis method E.
Yield: 36 mg (117 .mu.mol, 80.0%), white solid. TLC: R.sub.f=0.286
(dichloromethane/ethanol, 4:1). LC/MS (ES-API): m/z=308.21
[M+H].sup.+; calculated: 308.09; t.sub.R1 (.lamda.=220 nm): 0.72
min; t.sub.R2 (.lamda.=220 nm): 0.74 min (LC/MS-Method 1).
.sup.1H-NMR (400 MHz, DMSO-d6): .delta.=7.75 (s, 1H, NCH), 6.32 (d,
J=4.4 Hz, 1H, OH), 5.24 (d, J=5.6 Hz, 1H, OH), 4.91 (dd, J=4.1 Hz,
1H, CH), 4.79 (d, J=5.0 Hz, 1H, OH), 4.63 (dd, J=5.9 Hz, 1H, OH),
4.32 (dd, J=8.2 Hz, 1H, OH), 3.92 (m, 1H, CH), 3.68 (t, J=6.5 Hz,
2H, CH.sub.2), 3.43 (m, 3H, 3.times.CH), 3.10 (m, 1H, CH.sub.2),
2.91 (m, 1H, CH.sub.2), 2.00 (m, 4H, 2.times.CH.sub.2) ppm.
EXAMPLE 67
(3R,4R,5S,6R)-6-(Hydroxymethyl)-3-[4-[7-(4-nitro-2,1,3-benzoxadiazol-7-yl)-
heptyl]triazol-1-yl]tetrahydropyran-2,4,5-triol
##STR00085##
[0383] Step 1: 4-nitro-N-oct-7-ynyl-2,1,3-benzoxadiazol-7-amine
##STR00086##
[0385] To a solution of 300 mg (1.49 mmol)
4-chloro-7-nitrobenzofurazane in 4 mL dry dimethylformamide was
added 216 mg (1.64 mmol) 7-octyn-1-amine and 527 .mu.l (2.98 mmol)
N,N-diisopropylethylamine. The reaction mixture was stirred at room
temperature for 2 days and evaporated.
[0386] Purification: MPLC CombiFlash Rf (Teledyne ISCO); column:
RediSep Silica 40 g; flow rate: 40 mL/min; wavelength for
detection: 254 nm; eluent: (A) n-heptane, (B) ethylacetate.
MPLC Gradient
TABLE-US-00037 [0387] start % B end % B duration [min] 0.0 0.0 1.6
0.0 27.7 16.6 27.7 79.8 19.9 79.8 0.0 0.0 0.0 0.0 2.9
Yield: 96 mg (0.333 mmol, 22.4%), orange solid. TLC: R.sub.f=0.689
(dichloromethane/ethanol, 19:1). LC/MS (ES-API): m/z=289.10
[M+H].sup.+; calculated: 289.30; t.sub.R (.lamda.=220 nm): 1.25 min
(LC/MS-Method 1). .sup.1H-NMR (400 MHz, DMSO-d.sub.6): .delta.=9.53
(s, 1H, NH), 8.51 (d, J=8.8 Hz, 2H, ArH), 6.41 (d, J=8.9 Hz, 1H,
OH), 5.23 (d, J=5.7 Hz, 1H, OH), 4.87 (dd, J=4.2 Hz, 1H, CH), 4.56
(d, J=6.7 Hz, 1H, OH), 4.63 (dd, J=5.9 Hz, 1H, OH), 4.32 (dd, J=8.2
Hz, 1H, OH), 3.71 (m, 2H, 2.times.CH), 3.45 (m, 3H, 3.times.CH),
3.31 (m, 1H, CH.sub.2), 3.20 (m, 1H, CH.sub.2), 2.59 (m, 2H,
CH.sub.2), 1.65 (m, 6H, 3.times.CH.sub.2), 1.36 (m, 4H,
2.times.CH.sub.2) ppm.
Step 2:
(3R,4R,5S,6R)-6-(hydroxymethyl)-3-[4-[7-(4-nitro-2,1,3-benzoxadiaz-
ol-7-yl)heptyl]triazol-1-yl]tetrahydropyran-2,4,5-triol
[0388] Example 67 was synthesized from 2-azido-2-deoxyglucose and
example 67, step 1 following the CuAAC procedure described in
synthesis method E.
Yield: 32 mg (64 .mu.mol, 44.3%), orange solid. TLC: R.sub.f=0.833
(dichloromethane/ethanol, 4:1). LC/MS (ES-API): m/z=494.24
[M+H].sup.+; calculated: 494.19; t.sub.R1 (.lamda.=220 nm): 1.33
min; t.sub.R2 (.lamda.=220 nm): 1.35 min (LC/MS-Method 1).
.sup.1H-NMR (400 MHz, DMSO-d.sub.6): .delta.=9.53 (s, 1H, NH), 8.51
(d, J=8.8 Hz, 2H, ArH), 8.00 (s, 1H, NCH), 6.41 (d, J=8.9 Hz, 1H,
OH), 5.23 (d, J=5.7 Hz, 1H, OH), 4.87 (dd, J=4.2 Hz, 1H, CH), 4.56
(d, J=6.7 Hz, 1H, OH), 4.63 (dd, J=5.9 Hz, 1H, OH), 4.32 (dd, J=8.2
Hz, 1H, OH), 3.71 (m, 2H, 2.times.CH), 3.45 (m, 3H, 3.times.CH),
3.31 (m, 1H, CH.sub.2), 3.20 (m, 1H, CH.sub.2), 2.59 (m, 2H,
CH.sub.2), 1.65 (m, 6H, 3.times.CH.sub.2), 1.36 (m, 4H,
2.times.CH.sub.2) ppm.
EXAMPLE 68
5-(Dimethylamino)-N-[[1-[[(2R,3S,4S,5R)-3,4,5,6-tetrahydroxytetrahydropyra-
n-2-yl]methyl]triazol-4-yl]methyl]naphthalene-1-sulfonamide
##STR00087##
[0390] Example 68 was synthesized from 6-azido-6-deoxyglucose and
example 54, step 1 following the CuAAC procedure described in
synthesis method E.
Yield: 18 mg (36.47 .mu.mol, 49.9%), white-yellow solid. TLC:
R.sub.f=0.045 (dichloromethane/ethanol, 4:1). LC/MS (ES-API):
m/z=494.25 [M+H].sup.+; calculated: 494.16; t.sub.R1 (.lamda.=220
nm): 1.18 min; t.sub.R2 (.lamda.=220 nm): 1.20 min (LC/MS-Method
1). .sup.1H-NMR (400 MHz, DMSO-d.sub.6): .delta.=8.46 (d, J=8.4 Hz,
2H, ArH), 8.13 (d, J=7.0 Hz, 1H, ArH), 7.84 (s, 1H, NCH), 7.67 (s,
1H, SNH), 7.57 (m, 2H, ArH), 7.25 (d, J=7.2 Hz, 1H, ArH), 6.31 (d,
J=6.4Hz, 1H, OH), 5.23 (d, J=6.3 Hz, 1H, OH), 4.92 (d, J=6.2 Hz,
1H, OH), 4.85 (dd, J=6.4 Hz, 1H, CH), 4.58 (d, J=6.0 Hz, 1H, OH),
.delta.=4.21 (m, 1H, CH.sub.2), 4.07 (s, 2H, NCH.sub.2), 3.89 (m,
1H, CH.sub.2), 3.47 (m, 1H, CH), 3.14 (m, 1H, CH), 2.96 (m, 1H,
CH), 2.81 (s, 6H, 2.times.NCH.sub.3) ppm.
EXAMPLE 69
(3R,4S,5S,6R)-6-[(4-Phenyltriazol-1-yl)methyl]tetrahydropyran-2,3,4,5-tetr-
ol
##STR00088##
[0392] Example 69 was synthesized from 6-azido-6-deoxyglucose and
ethynylbenzene following the CuAAC procedure described in synthesis
method E.
Yield: 36 mg (117 .mu.mol, 80.1%), white solid. TLC: R.sub.f=0.242
(dichloromethane/ethanol, 4:1). LC/MS (ES-API): m/z=308.16
[M+H].sup.+; calculated: 308.12; t.sub.R1 (.lamda.=220 nm): 0.90
min; t.sub.R2 (.lamda.=220 nm): 0.97 min (LC/MS-Method 1).
.sup.1H-NMR (400 MHz, DMSO-d.sub.6): .delta.=8.49 (s, 1H, NCH),
7.86 (dd, J=1.3 Hz, 2H, ArH), 7.46 (m, 2H, ArH), 7.42 (m, 1H, ArH),
6.36 (d, J=6.0 Hz, 1H, OH), 5.31 (d, J=5.8 Hz, 1H, OH), 4.96 (t,
J=5.7 Hz, 1H, CH), 4.90 (d, J=4.2 Hz, 1H, OH), 4.65 (d, J=6.1 Hz,
1H, OH), 4.41 (m, 2H, CH.sub.2), 3.99 (m, 1H, CH), 3.49 (m, 1H,
CH), 3.22 (m, 1H, CH), 3.00 (m, 1H, CH) ppm. .sup.13C-NMR (150 MHz,
DMSO-d.sub.6): .delta.=146.03 (s, C), 130.84 (s, C), 128.85 (s,
CH), 127.75 (s, CH), 125.09 (s, CH), 122.25 (s, CH), 92.35 (s, CH),
72.69 (s, CH), 72.07 (s, CH), 71.89 (s, CH), 69.97 (s, CH), 61.26
(s, CH.sub.2) ppm.
EXAMPLE 70
(3R,4S,5S,6R)-6-[[4-(p-Tolyl)triazol-1-yl]methyl]tetrahydropyran-2,3,4,5-t-
etrol
##STR00089##
[0394] Example 70 was synthesized from 6-azido-6-deoxyglucose and
1-ethynyl-4-methyl-benzene following the CuAAC procedure described
in synthesis method E.
Yield: 32 mg (100 .mu.mol, 68.1%), white solid. TLC: R.sub.f=0.250
(dichloromethane/ethanol, 4:1). LC/MS (ES-API): m/z=322.19
[M+H].sup.+; calculated: 322.13; t.sub.R1 (.lamda.=220 nm): 1.12
min; t.sub.R2 (.lamda.=220 nm): 1.16 min (LC/MS-Method 1).
.sup.1H-NMR (400 MHz, DMSO-d.sub.6): .delta.=8.49 (s, 1H, NCH),
7.72 (d, J=8.0 Hz, 2H, ArH), 7.21 (d, J=8.0 Hz, 2H, ArH), 6.43 (d,
J=4.4 Hz, 1H, OH), 5.30 (d, J=5.4 Hz, 1H, OH), 4.94 (d, J=5.4 Hz,
1H, OH), 4.90 (dd, J=4.2 Hz, 1H, CH), 4.58 (d, J=5.3 Hz, 1H, OH),
4.40 (m, 1H, CH.sub.2), 4.00 (m, 1H, CH.sub.2), 3.49 (m, 1H, CH),
3.17 (m, 1H, CH), 3.06 (m, 1H, CH), 2.94 (m, 1H, CH), 2.36 (s, 3H,
CH.sub.3) ppm.
EXAMPLE 71
(3R,4S,5S,6R)-6-[[4-(3-Phenylpropyl)triazol-1-yl]methyl]tetrahydropyran-2,-
3,4,5-tetrol
##STR00090##
[0396] Example 71 was synthesized from 6-azido-6-deoxyglucose and
pent-4-yn-1-ylbenzene following the CuAAC procedure described in
synthesis method E.
Yield: 43 mg (123 .mu.mol, 84.2%), white solid. TLC: R.sub.f=0.273
(dichloromethane/ethanol, 4:1). LC/MS (ES-API): m/z=350.18
[M+H].sup.+; calculated: 350.16; t.sub.R1 (.lamda.=220 nm): 1.29
min; t.sub.R2 (.lamda.=220 nm): 1.35 min (LC/MS-Method 1).
.sup.1H-NMR (400 MHz, DMSO-d.sub.6): .delta.=7.72 (s, 1H, NCH),
7.25 (m, 2H, ArH), 7.20 (m, 3H, ArH), 6.31 (d, J=5.0 Hz, 1H, OH),
5.23 (d, J=5.6 Hz, 1H, OH), 4.89 (d, J=6.5 Hz, 1H, CH), 4.80 (d,
J=5.0 Hz, 1H, OH), 4.55 (dd, J=5.5 Hz, 1H, OH), 4.31 (m, 1H,
CH.sub.2), 3.93 (m, 1H, CH.sub.2), 3.49 (m, 2H, 2.times.CH), 3.11
(m, 1H, CH), 2.97 (m, 1H, CH), 2.63 (m, 4H, CH.sub.2), 1.90 (m, 2H,
CH.sub.2) ppm.
EXAMPLE 72
Methyl-4-[1-[[(2R,3S,4S,5R)-3,4,5,6-tetrahydroxytetrahydropyran-2-yl]methy-
l]triazol-4-yl]-benzoate
##STR00091##
[0398] Example 72 was synthesized from 6-azido-6-deoxyglucose and
4-ethynylmethylbenzoate following the CuAAC procedure described in
synthesis method E.
Yield: 34 mg (93 .mu.mol, 63.6%), white solid. TLC: R.sub.f=0.258
(dichloromethane/ethanol, 4:1). LC/MS (ES-API): m/z=366.23
[M+H].sup.+; calculated: 366.12; t.sub.R1 (.lamda.=220 nm): 1.05
min; t.sub.R2 (.lamda.=220 nm): 1.08 min (LC/MS-Method 1).
.sup.1H-NMR (400 MHz, DMSO-d.sub.6): .delta.=8.59 (s, 1H, NCH),
8.01 (m, 4H, ArH), 6.37 (d, J=5.0 Hz, 1H, OH), 5.31 (d, J=5.6 Hz,
1H, OH), 4.94 (d, J=4.8 Hz, 1H, OH), 4.89 (dd, J=4.3 Hz, 1H, CH),
4.61 (d, J=5.1 Hz, 1H, OH), 4.44 (m, 1H, CH.sub.2), 4.02 (m, 1H,
CH.sub.2), 3.90 (s, 3H, OCH.sub.3), 3.51 (m, 1H, CH), 3.19 (m, 1H,
CH), 3.08 (m, 1H, CH), 2.91 (m, 1H, CH) ppm.
EXAMPLE 73
(3R,4S,5S,6R)-6-[[4-[[Benzyl(methyl)amino]methyl]triazol-1-yl]methyl]tetra-
hydropyran-2,3,4,5-tetrol
##STR00092##
[0400] Example 73 was synthesized from 6-azido-6-deoxyglucose and
N-benzyl-N-methylprop-2-yn-1-amine following the CuAAC procedure
described in synthesis method E.
Yield: 36 mg (99 .mu.mol, 67.6%), white solid. TLC: R.sub.f=0.053
(dichloromethane/ethanol, 4:1). LC/MS (ES-API): m/z=365.20
[M+H].sup.+; calculated: 365.18; t.sub.R (ELSD): 0.37 min
(LC/MS-Method 1). .sup.1H-NMR (400 MHz, DMSO-d.sub.6): .delta.=7.85
(s, 1H, NCH), 7.31 (m, 5H, ArH), 6.33 (d, J=6.1 Hz, 1H, OH), 5.24
(d, J=5.8 Hz, 1H, OH), 4.90 (d, J=5.6 Hz, 1H, OH), 4.87 (dd, J=6.0
Hz, 1H, CH), 4.58 (d, J=5.0 Hz, 1H, OH), 4.41 (m, 1H, CH.sub.2),
3.97 (m, 1H, CH.sub.2), 3.62 (s, 3H, NCH.sub.3), 3.50 (m, 2H,
2.times.CH), 3.15 (m, 1H, CH), 2.96 (m, 1H, CH), 2.10 (m, 4H,
2.times.NCH.sub.2) ppm.
EXAMPLE 74
(3R,4S,5S,6R)-6-[[4-(6-Methoxy-2-naphthyl)triazol-1-yl]methyl]tetrahydropy-
ran-2,3,4,5-tetrol
##STR00093##
[0402] Example 74 was synthesized from 6-azido-6-deoxyglucose and
2-ethynyl-6-methoxynaphthalene following the CuAAC procedure
described in synthesis method E.
Yield: 43 mg (111 .mu.mol, 75.9%), white solid. TLC: R.sub.f=0.288
(dichloromethane/ethanol, 4:1). LC/MS (ES-API): m/z=388.20
[M+H].sup.+; calculated: 388.14; t.sub.R1 (.lamda.=220 nm): 1.30
min; t.sub.R2 (.lamda.=220 nm): 1.33 min (LC/MS-Method 1).
.sup.1H-NMR (400 MHz, DMSO-d.sub.6): .delta.=8.51 (s, 1H, NCH),
7.86 (m, 4H, ArH), 7.34 (s, 2H, ArH), 6.39 (d, J=5.8 Hz, 1H, OH),
5.41 (d, J=6.1 Hz, 1H, OH), 4.94 (d, J=6.0 Hz, 1H, OH), 4.90 (dd,
J=6.3 Hz, 1H, CH), 4.58 (d, J=5.2 Hz, 1H, OH), 4.42 (m, 1H,
CH.sub.2), 4.01 (m, 1H, CH.sub.2), 3.90 (s, 3H, OCH.sub.3), 3.51
(m, 1H, CH), 3.19 (m, 1H, CH), 3.06 (m, 1H, CH), 2.97 (m, 1H, CH)
ppm.
EXAMPLE 75
(3R,4S,5S,6R)-6-[[4-[[(4-Nitro-2,1,3-benzoxadiazol-7-yl)amino]methyl]triaz-
ol-1-yl]methyl]tetrahydropyran-2,3,4,5-tetrol
##STR00094##
[0404] Example 75 was synthesized from 6-azido-6-deoxyglucose and
example 63, step 1 following the CuAAC procedure described in
synthesis method E.
Yield: 56 mg (132 .mu.mol, 90.5%), orange solid. TLC: R.sub.f=0.121
(dichloromethane/ethanol, 4:1). LC/MS (ES-API): m/z=424.16
[M+H].sup.+; calculated: 424.11; t.sub.R1 (.lamda.=220 nm): 0.83
min; t.sub.R2 (.lamda.=220 nm): 0.87 min (LC/MS-Method 1).
[0405] .sup.1H-NMR (400 MHz, DMSO-d.sub.6): .delta.=9.85 (s, 1H,
NH) 8.53 (d, J=8.4 Hz, 1H, ArH), 8.01 (s, 1H, NCH), 6.51 (d, J=8.4
Hz, 1H, ArH), 6.33 (d, J=6.0 Hz, 1H, OH), 5.24 (d, J=6.1 Hz, 1H,
OH), 4.94 (d, J=6.2 Hz, 1H, OH), 4.86 (dd, J=5.8 Hz, 1H, CH), 4.65
(s, 2H, NCH.sub.2), 4.57 (dd, J=6.0 Hz, 1H, OH), 4.37 (m, 1H,
CH.sub.2), 3.91 (m, 1H, CH.sub.2), 3.49 (m, 1H, CH), 3.14 (m, 1H,
CH), 2.95 (m, 1H, CH) ppm.
EXAMPLE 76
(3R,4S,5S,6R)-6-[[4-(Cyclohexylmethyl)triazol-1-yl]methyl]tetrahydropyran--
2,3,4,5-tetrol
##STR00095##
[0407] Example 76 was synthesized from 6-azido-6-deoxyglucose and
prop-2-yn-1-ylcyclohexane following the CuAAC procedure described
in synthesis method E.
Yield: 33 mg (101 .mu.mol, 68.9%), white-yellow solid. TLC:
R.sub.f=0.220 (dichloromethane/ethanol, 4:1). LC/MS (ES-API):
m/z=328.24 [M+H].sup.+; calculated: 328.18; t.sub.R1 (.lamda.=220
nm): 1.23 min; t.sub.R2 (.lamda.=220 nm): 1.29 min (LC/MS-Method
1). .sup.1H-NMR (400 MHz, DMSO-d.sub.6): .delta.=7.68 (s, 1H, NCH),
6.33 (d, J=5.0 Hz, 1H, OH), 5.22 (d, J=5.5 Hz, 1H, OH), 4.87 (dd,
J=4.3 Hz, 1H, CH), 4.78 (d, J=5.0 Hz, 1H, OH), 4.59 (dd, J=2.3 Hz,
1H, OH), 4.55 (d, J=6.7 Hz, 1H, OH), 4.36 (m, 1H, CH.sub.2), 3.92
(m, 1H, CH.sub.2), 3.44 (m, 1H, CH), 3.12 (m, 1H, CH), 2.90 (m, 1H,
CH), 2.49 (m, 2H, CH.sub.2), 1.64 (m, 6H, 3.times.CH.sub.2),
1.53(m, 1H, CH), 1.17 (m, 4H, 2.times.CH.sub.2), 0.95 (m, 2H,
CH.sub.2) ppm.
EXAMPLE 77
(3R,4S,5S,6R)-6-[(4-Pentyltriazol-1-yl)methyl]tetrahydropyran-2,3,4,5-tetr-
ol
##STR00096##
[0409] Example 77 was synthesized from 6-azido-6-deoxyglucose and
hept-1-yne following the CuAAC procedure described in synthesis
method E.
Yield: 7 mg (23 .mu.mol, 15.9%), white-yellow solid. TLC:
R.sub.f=0.235 (dichloromethane/ethanol, 4:1). LC/MS (ES-API):
m/z=302.25 [M+H].sup.+; calculated: 302.16; t.sub.R1 (.lamda.=220
nm): 1.12 min; t.sub.R2 (.lamda.=220 nm): 1.17 min (LC/MS-Method
1). .sup.1H-NMR (400 MHz, DMSO-d.sub.6): =7.70 (s, 1H, NCH), 6.33
(d, J=4.8 Hz, 1H, OH), 5.23 (d, J=5.7 Hz, 1H, OH), 4.87 (dd, J=4.0
Hz, 1H, CH), 4.78 (d, J=4.9 Hz, 1H, OH), 4.60 (dd, J=2.3 Hz, 1H,
OH), 4.55 (d, J=6.6 Hz, 1H, OH), 4.31 (m, 1H, CH.sub.2), 3.92 (m,
1H, CH.sub.2), 3.52 (m, 1H, CH), 3.12 (m, 1H, CH), 2.94 (m, 1H,
CH), 2.58 (t, J=7.5 Hz, 2H, CH.sub.2), 1.58 (m, 2H, CH.sub.2), 1.30
(m, 4H, CH.sub.2), 0.87 (m, 3H, CH.sub.3) ppm.
EXAMPLE 78
(3R,4S,5S,6R)-6-[[4-(3-Chloropropyl)triazol-1-yl]methyl]tetrahydropyran-2,-
3,4,5-tetrol
##STR00097##
[0411] Example 78 was synthesized from 6-azido-6-deoxyglucose and
5-chloro-pent-1-yne following the CuAAC procedure described in
synthesis method E.
Yield: 24 mg (78 .mu.mol, 53.3%), white-yellow solid. TLC:
R.sub.f=0.182 (dichloromethane/ethanol, 4:1). LC/MS (ES-API):
m/z=308.17 [M+H].sup.+; calculated: 308.09; t.sub.R1 (.lamda.=220
nm): 0.70 min; t.sub.R2 (.lamda.=220 nm): 0.72 min (LC/MS-Method
1). .sup.1H-NMR (400 MHz, DMSO-d.sub.6): .delta.=7.78 (s, 1H, NCH),
6.33 (d, J=5.0 Hz, 1H, OH), 5.24 (d, J=5.7 Hz, 1H, OH), 4.89 (dd,
J=4.9 Hz, 1H, CH), 4.79 (d, J=5.3 Hz, 1H, OH), 4.61 (dd, J=2.2 Hz,
1H, OH), 4.55 (d, J=6.6 Hz, 1H, OH), 4.34 (m, 1H, CH.sub.2), 3.93
(m, 1H, CH.sub.2), 3.68 (t, J=6.6 Hz, 2H, CH.sub.2), 3.44 (m, 2H,
2.times.CH), 3.15 (m, 1H, CH), 2.91 (m, 1H, CH), 2.07 (m, 4H,
2.times.CH.sub.2) ppm.
EXAMPLE 79
(3R,4S,5S,6R)-6-[[4-[6-[(4-Nitro-2,1,3-benzoxadiazol-7-yl)amino]hexyl]tria-
zol-1-yl]methyl]tetrahydropyran-2,3,4,5-tetrol
##STR00098##
[0413] Example 79 was synthesized from 6-azido-6-deoxyglucose and
example 67, step 1 following the CuAAC procedure described in
synthesis method E.
Yield: 20 mg (40.53 .mu.mol, 55.4%), orange oil. TLC: R.sub.f=0.222
(dichloromethane/ethanol, 4:1). LC/MS (ES-API): m/z=494.23
[M+H].sup.+; calculated: 494.19; t.sub.R1 (.lamda.=220 nm): 1.33
min; t.sub.R2 (.lamda.=220 nm): 1.35 min (LC/MS-Method 1).
.sup.1H-NMR (400 MHz, DMSO-d.sub.6): .delta.=9.50 (s, 1H, NH), 8.49
(d, J=8.8 Hz, 2H, ArH), 769 (s, 1H, NCH), 6.41 (d, J=5.9 Hz, 1H,
OH), 5.23 (d, J=5.7 Hz, 1H, OH), 4.91 (d, J=6.7 Hz, 1H, OH), 4.87
(dd, J=4.0 Hz, 1H, CH), 4.63 (dd, J=5.9 Hz, 1H, OH), 4.56 (d, J=6.2
Hz, 1H, OH), 4.30 (m, 1H, CH.sub.2), 3.91 (m, 1H, CH.sub.2), 3.49
(m, 2H, 2.times.CH), 3.17 (m, 1H, CH), 2.94 (m, 1H, CH), 2.58 (m,
2H, CH.sub.2), 1.65 (m, 4H, 2.times.CH.sub.2), 1.40 (m, 6H,
3.times.CH.sub.2) ppm.
EXAMPLE 80
5-(Dimethylamino)-N-[[1-[(2R,3R,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethy-
l)tetrahydropyran-2-yl]triazol-4-yl]methyl]naphthalene-1-sulfonamide
##STR00099##
[0415] Example 80 was synthesized from 1-azido-1-deoxyglucose and
example 54, step 1 following the CuAAC procedure described in
synthesis method E.
Yield: 46 mg (94 .mu.mol, 63.7%), white-yellow solid. TLC:
R.sub.f=0.129 (dichloromethane/ethanol, 4:1). LC/MS (ES-API):
m/z=494.26 [M+H].sup.+; calculated: 494.16; t.sub.R (.lamda.=220
nm): 1.20 min (LC/MS-Method 1). .sup.1H-NMR (400 MHz,
DMSO-d.sub.6): .delta.=8.48 (d, J=8.4 Hz, 1H, ArH), 8.32 (d, J=8.6
Hz, 1H, ArH), 8.14 (dd, J=1.1 Hz, 1H, ArH), 8.02 (s, 1H, NCH), 7.62
(m, 2H, ArH), 7.27 (d, J=7.6 Hz, 1H, ArH), 5.49 (d, J=9.2 Hz, 1H,
OH), 5.31 (d, J=6.2 Hz, 1H, OH), 5.23 (d, J=4.9 Hz, 1H, OH), 5.13
(d, J=5.4 Hz, 1H, CH), 4.62 (dd, J=5.4 Hz, 1H, OH), 4.08 (d, J=5.1
Hz, 1H, NCH.sub.2), 3.68 (m, 2H, CH.sub.2), 3.43 (m, 2H,
2.times.CH), 3.19 (m, 1H, CH), 2.84 (s, 6H, 2.times.NCH.sub.3)
ppm.
EXAMPLE 81
(2R,3S,4S,5R,6R)-2-(Hydroxymethyl)-6-(4-phenyltriazol-1-yl)tetrahydropyran-
-3,4,5-triol
##STR00100##
[0417] Example 81 was synthesized from 1-azido-1-deoxyglucose and
ethynylbenzene following the CuAAC procedure described in synthesis
method E.
Yield: 40 mg (130 .mu.mol, 89.0%), white solid. TLC: R.sub.f=0.295
(dichloromethane/ethanol, 4:1). LC/MS (ES-API): m/z=308.14
[M+H].sup.+; calculated: 308.12; t.sub.R (.lamda.=220 nm): 0.92 min
(LC/MS-Method 1). .sup.1H-NMR (400 MHz, DMSO-d.sub.6): .delta.=8.81
(s, 1H, NCH), 7.88 (d, J=8.0 Hz, 2H, ArH), 7.46 (t, J=7.5 Hz, 2H,
ArH), 7.34 (d, J=7.5 Hz, 1H, ArH), 5.57 (d, J=9.2 Hz, 1H, CH), 5.42
(d, J=5.8 Hz, 1H, OH), 5.31 (d, J=4.9 Hz, 1H, OH), 5.16 (d, J=5.5
Hz, 1H, OH), 4.62 (dd, J=5.6 Hz, 1H, OH), 3.81 (m, 1H, CH.sub.2),
3.73 (m, 1H, CH.sub.2), 3.49 (m, 2H, 2.times.CH), 3.17 (m, 1H, CH)
ppm. .sup.13C-NMR (150 MHz, DMSO-d.sub.6): .delta.=146.31 (s, C),
130.62 (s, C), 128.91 (s, CH), 127.92 (s, CH), 125.15 (s, CH),
120.47 (s, CH), 87.68 (s, CH), 79.95 (s, CH), 76.85 (s, CH), 72.19
(s, CH), 69.60 (s, CH), 60.76 (s, CH.sub.2) ppm.
EXAMPLE 82
(2R,3S,4S,5R,6R)-2-(Hydroxymethyl)-6-[4-(p-tolyl)triazol-1-yl]petrahydropy-
ran-3,4,5-triol
##STR00101##
[0419] Example 82 was synthesized from 1-azido-1-deoxyglucose and
1-ethynyl-4-methylbenzene following the CuAAC procedure described
in synthesis method E.
Yield: 39 mg (121 .mu.mol, 83.0%), white solid. TLC: R.sub.f=0.326
(dichloromethane/ethanol, 4:1). LC/MS (ES-API): m/z=322.20
[M+H].sup.+; calculated: 322.13; t.sub.R (.lamda.=220 nm): 1.14 min
(LC/MS-Method 1). .sup.1H-NMR (400 MHz, DMSO-d.sub.6): .delta.=8.76
(s, 1H, NCH), 7.77 (d, J=8.3 Hz, 2H, ArH), 7.27 (d, J=8.3 Hz, 2H,
ArH), 5.55 (d, J=9.1 Hz, 1H, OH), 5.41 (d, J=5.9 Hz, 1H, OH), 5.30
(d, J=5.1 Hz, 1H, OH), 5.15 (d, J=5.5 Hz, 1H, CH), 4.61 (dd, J=5.5
Hz, 1H, OH), 3.80 (m, 1H, CH.sub.2), 3.72 (m, 1H, CH.sub.2), 3.45
(m, 2H, 2.times.CH), 3.26 (m, 1H, CH), 2.34 (s, 3H, CH.sub.3)
ppm.
EXAMPLE 83
(2R,3S,4S,5R,6R)-2-(Hydroxymethyl)-6-[4-(3-phenylpropyl)triazol-1-yl]tetra-
hydropyran-3,4,5-triol
##STR00102##
[0421] Example 83 was synthesized from 1-azido-1-deoxyglucose and
pent-4yn-1ylbenzene following the CuAAC procedure described in
synthesis method E.
Yield: 50 mg (143 .mu.mol, 97.9%), white solid. TLC: R.sub.f=0.356
(dichloromethane/ethanol, 4:1). LC/MS (ES-API): m/z=350.21
[M+H].sup.+; calculated: 350.16; t.sub.R (.lamda.=220 nm): 1.27 min
(LC/MS-Method 1).
[0422] .sup.1H-NMR (400 MHz, DMSO-d.sub.6): .delta.=8.07 (s, 1H,
NCH), 7.25 (m, 5H, ArH), 5.46 (d, J=9.2 Hz, 1H, OH), 5.31 (d, J=5.9
Hz, 1H, OH), 5.24 (d, J=5.0 Hz, 1H, OH), 5.12 (d, J=5.5 Hz, 1H,
CH), 4.59 (dd, J=5.0 Hz, 1H, OH), 3.72 (m, 2H, CH.sub.2), 3.42 (m,
2H, 2.times.CH), 3.21 (m, 1H, CH), 2.63 (m, 4H, 2.times.CH.sub.2),
1.90 (m, 2H, CH.sub.2) ppm.
EXAMPLE 84
Methyl-4-[1-[(2R,3R,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-
pyran-2-yl]triazol-4-yl]benzoate
##STR00103##
[0424] Example 84 was synthesized from 1-azido-1-deoxyglucose and
4-ethynylmethylbenzoate following the CuAAC procedure described in
synthesis method E.
Yield: 44 mg (120 .mu.mol, 82.4%), white solid. TLC: R.sub.f=0.062
(dichloromethane/ethanol, 4:1). LC/MS (ES-API): m/z=366.12
[M+H].sup.+; calculated: 366.12; t.sub.R1 (.lamda.=220 nm): 0.97
min (LC/MS-Method 1). .sup.1H-NMR (400 MHz, DMSO-d.sub.6):
.delta.=9.00 (s, 1H, NCH), 8.05 (s, 4H, ArH), 5.60 (d, J=9.3 Hz,
1H, OH), 5.44 (d, J=5.9 Hz, 1H, OH), 5.32 (d, J=4.8 Hz, 1H, OH),
5.17 (d, J=5.5 Hz, 1H, CH), 4.62 (dd, J=5.8 Hz, 1H, OH), 3.87 (s,
3H, OCH.sub.3), 3.87 (m, 1H, CH.sub.2), 3.80 (m, 1H, CH.sub.2),
3.49 (m, 1H, CH), 3.43 (m, 1H, CH), 3.27 (m, 1H, CH) ppm.
EXAMPLE 85
(2R,3R,4S,5S,6R)-2-[4-[[Benzyl(methyl)amino]methyl]triazol-1-yl]-6-(hydrox-
ymethyl)tetrahydropyran-3,4,5-triol
##STR00104##
[0426] Example 85 was synthesized from 1-azido-1-deoxyglucose and
N-benzyl-N-methylprop-2-yn-1-amine following the CuAAC procedure
described in synthesis method E.
Yield: 51 mg (140 .mu.mol, 95.7%), white solid. TLC: R.sub.f=0.091
(dichloromethane/ethanol, 4:1). LC/MS (ES-API): m/z=365.20
[M+H].sup.+; calculated: 365.17; t.sub.R (ELSD): 0.34 min
(LC/MS-Method 1). .sup.1H-NMR (400 MHz, DMSO-d.sub.6): .delta.=8.19
(s, 1H, NCH), 7.33 (m, 4H, ArH), 7.25 (m, 1H, ArH), 5.50 (d, J=9.4
Hz, 1H, OH), 5.34 (d, J=6.1 Hz, 1H, OH), 5.24 (d, J=5.0 Hz, 1H,
OH), 5.12 (d, J=5.7 Hz, 1H, CH), 4.60 (dd, J=5.6 Hz, 1H, OH), 3.77
(m, 1H, CH.sub.2), 3.70 (m, 1H, CH.sub.2), 3.62 (s, 2H, NCH.sub.2),
3.52 (s, 2H, NCH.sub.2), 3.43 (m, 1H, CH), 3.38 (m, 1H, CH), 3.24
(m, 1H, CH) 2.11 (s, 3H, NCH.sub.3) ppm.
EXAMPLE 86
(2R,3S,4S,5R,6R)-2-(Hydroxymethyl)-6-[4-(6-methoxy-2-naphthyl)triazol-1-yl-
]tetrahydropyran-3,4,5-triol
##STR00105##
[0428] Example 86 was synthesized from 1-azido-1-deoxyglucose and
2-ethynyl-6-methoxynaphthalene following the CuAAC procedure
described in synthesis method E.
Yield: 42 mg (108 .mu.mol, 74.1%), white solid. TLC: R.sub.f=0.318
(dichloromethane/ethanol, 4:1). LC/MS (ES-API): m/z=388.10
[M+H].sup.+; calculated: 388.14; t.sub.R (.lamda.=220 nm): 0.95 min
(LC/MS-Method 1).
[0429] .sup.1H-NMR (400 MHz, DMSO-d.sub.6): .delta.=8.51 (s, 1H,
NCH), 7.86 (m, 4H, ArH), 7.34 (s, 2H, ArH), 6.39 (d, J=5.8 Hz, 1H,
OH), 5.41 (d, J=6.1 Hz, 1H, OH), 4.94 (d, J=6.0 Hz, 1H, OH), 4.90
(dd, J=6.3 Hz, 1H, CH), 4.58 (d, J=5.2 Hz, 1H, OH), 4.42 (m, 1H,
CH.sub.2), 4.01 (m, 1H, CH.sub.2), 3.90 (s, 3H, OCH.sub.3), 3.51
(m, 1H, CH), 3.19 (m, 1H, CH), 3.06 (m, 1H, CH), 2.97 (m, 1H, CH)
ppm.
EXAMPLE 87
Ethyl-2-diethoxyphosphoryl-3-[1-[(2R,3R,4S,5S,6R)-3,4,5-trihydroxy-6-(hydr-
oxymethyl)tetrahydro-pyran-2-yl]triazol-4-yl]propanoate
##STR00106##
[0431] Example 87 was synthesized from 1-azido-1-deoxyglucose and
2-(diethoxyphosphoryl)ethylpent-4-ynoate following the CuAAC
procedure described in synthesis method E.
Yield: 48 mg (103 .mu.mol, 70.2%), white solid. TLC: R.sub.f=0.258
(dichloromethane/ethanol, 4:1). LC/MS (ES-API): m/z=468.26
[M+H].sup.+; calculated: 468.17; t.sub.R (.lamda.=220 nm): 1.04 min
(LC/MS-Method 1). .sup.1H-NMR (400 MHz, DMSO-d.sub.6): .delta.=8.05
(s, 1H, NCH), 5.46 (d, J=9.1 Hz, 1H, OH), 5.30 (d, J=6.3 Hz, 1H,
OH), 5.24 (d, J=4.7 Hz, 1H, OH), 5.12 (d, J=5.5 Hz, 1H, CH), 4.59
(dd, J=5.3 Hz, 1H, OH), 4.08 (m, 2H, CH.sub.2), 3.70 (m, 2H,
CH.sub.2), 3.43 (m, 4H, 4.times.CH), 3.00 (m, 1H, CH), 1.25 (m, 6H,
3.times.CH.sub.2), 1.13 (s, 9H, 3.times.CH.sub.3) ppm.
EXAMPLE 88
(2R,3S,4S,5R,6R)-2-(Hydroxymethyl)-6-[4-[[(4-nitro-2,1,3-benzoxadiazol-7-y-
l)amino]methy]triazol-1-yl]petrahydropyran-3,4,5-triol
##STR00107##
[0433] Example 88 was synthesized from 1-azido-1-deoxyglucose and
example 63, step 1 following the CuAAC procedure described in
synthesis method E.
Yield: 58 mg (138 .mu.mol, 93.7%). TLC: R.sub.f=0.152
(dichloromethane/ethanol, 4:1). LC/MS (ES-API): m/z=424.13
[M+H].sup.+; calculated: 424.16; t.sub.R1 (.lamda.=220 nm): 0.84
min (LC/MS-Method 1). .sup.1H-NMR (400 MHz, DMSO-d.sub.6):
.delta.=9.87 (s, 1H, NH), 8.51 (d, J=8.9 Hz, 1H, ArH), 8.35 (s, 1H,
NCH), 6.55 (d, J=8.9 Hz, 1H, ArH), 5.50 (d, J=9.3 Hz, 1H, OH), 5.32
(d, J=6.0 Hz, 1H, OH), 5.25 (d, J=4.9 Hz, 1H, OH), 5.12 (d, J=5.5
Hz, 1H, CH), 4.80 (s, 2H, NCH.sub.2), 4.60 (dd, J=5.5 Hz, 1H, OH),
3.70 (m, 2H, CH.sub.2), 3.43 (m, 2H, 2.times.CH), 3.19 (m, 1H, CH)
ppm.
EXAMPLE 89
(2R,3R,4S,5S,6R)-2-[4-(Cyclohexylmethyl)triazol-1-yl]-6-(hydroxymethyl)tet-
rahydropyran-3,4,5-triol
##STR00108##
[0435] Example 89 was synthesized from 1-azido-1-deoxyglucose and
prop-2-yn-1-ylcyclohexane following the CuAAC procedure described
in synthesis method E.
Yield: 35 mg (107 .mu.mol, 73.1%), white solid. TLC: R.sub.f=0.258
(dichloromethane/ethanol, 4:1). LC/MS (ES-API): m/z=328.26
[M+H].sup.+; calculated: 328.18; t.sub.R (.lamda.=220 nm): 1.27 min
(LC/MS-Method 1). .sup.1H-NMR (400 MHz, DMSO-d.sub.6): .delta.=7.99
(s, 1H, NCH), 5.45 (d, J=9.3 Hz, 1H, OH), 5.30 (d, J=6.0 Hz, 1H,
OH), 5.23 (d, J=5.0 Hz, 1H, OH), 5.11 (d, J=5.3 Hz, 1H, CH), 4.59
(dd, J=5.5 Hz, 1H, OH), 3.71 (m, 2H, CH.sub.2), 3.42 (m, 2H,
2.times.CH), 3.22 (m, 2H, 2.times.CH), 1.66 (m, 6H,
3.times.CH.sub.2), 1.54 (m, 1H, CH), 1.17 (m, 4H,
2.times.CH.sub.2), 0.95 (m, 2H, CH.sub.2) ppm.
EXAMPLE 90
(2R,3R,4S,5S,6R)-2[4-(3-Chloropropyl)triazol-1-yl]-6-(hydroxymethyl)tetrah-
ydropyran-3,4,5-triol
##STR00109##
[0437] Example 90 was synthesized from 1-azido-1-deoxyglucose and
5-chloro-pent-1-yne following the CuAAC procedure described in
synthesis method E.
Yield: 17 mg (55 .mu.mol, 37.8%), white solid. TLC: R.sub.f=0.288
(dichloromethane/ethanol, 4:1). LC/MS (ES-API): m/z=308.12
[M+H].sup.+; calculated: 308.09; t.sub.R1 (.lamda.=220 nm): 0.62
min (LC/MS-Method 1). .sup.1H-NMR (400 MHz, DMSO-d.sub.6):
.delta.=8.09 (s, 1H, NCH), 5.46 (d, J=9.1 Hz, 1H, OH), 5.31 (d,
J=6.0 Hz, 1H, OH), 5.25 (d, J=4.7 Hz, 1H, OH), 5.12 (d, J=5.8 Hz,
1H, CH), 4.60 (dd, J=5.2 Hz, 1H, OH), 3.74 (m, 2H, CH.sub.2), 3.70
(m, 2H, CH.sub.2), 3.39 (m, 2H, 2.times.CH), 3.22 (m, 2H,
2.times.CH), 2.77 (m, 2H, CH.sub.2), 2.06 (m, 2H, CH.sub.2)
ppm.
EXAMPLE 91
[6-(Diethylamino)-9-[2-[[1-[(2R,3R,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxyme-
thyl)tetrahydropyran-2-yl]triazol-4-yl]methylcarbamoyl]phenyl]xanthen-3-yl-
idene]diethyl-ammonium chloride
##STR00110##
[0438] Step 1:
[6-(diethylamino)-9-[2-(prop-2-ynylcarbamoyl)phenyl]xanthen-3-ylidene]-di-
ethyl-ammonium chloride
##STR00111##
[0440] To a solution of 500 mg (244 .mu.mol) Rhodamin B in 5 mL
dimethylformamide were added 78 mg (1.39 mmol; 1.4 eq.)
propargylamine, 554 mg (1.98 mmol; 2 eq.) 2-bromo-1-ethylpyridinium
tetrafluoroborate, and 348 .mu.l (1.98 mmol; 2 eq.)
N,N-diisopropylethylamine. The reaction mixture was stirred at room
temperature for 4 hours and evaporated.
[0441] Purification: MPLC CombiFlash Rf (Teledyne ISCO); column:
RediSep Silica 24 g Gold; flow rate: 40 mL/min; wavelength for
detection: 254 nm; eluent: (A) dichloromethane, (B) ethanol.
MPLC Gradient
TABLE-US-00038 [0442] start % B end % B duration [min] 0.0 0.0 5.0
0.0 100.0 45.0
Yield: 223 mg (0.432 mmol, 43.6%), light pink solid. TLC:
R.sub.f=0.622 (dichloromethane/ethanol, 19:1). LC/MS (ES-API):
m/z=480.28 [M+H].sup.+; calculated: 480.27; t.sub.R (.lamda.=220
nm): 2.08 min (LC/MS-Method 1). .sup.1H-NMR (400 MHz,
DMSO-d.sub.6): .delta.=7.80 (m, 1H, ArH), 7.51 (m, 1H, ArH), 7.03
(m, 1H, ArH), 6.35 (m, 6H, ArH), 3.79 (d, J=6.9 Hz, 2H, NCH.sub.2),
3.31 (m, 8H, H), 2.65 (m, 8H, CH.sub.2), 1.08 (t, J=6.9 Hz, 12H,
CH.sub.3) ppm.
Step 2:
[6-(diethylamino)-9-[2-[[1-[(2R,3R,4S,5S,6R)-3,4,5-trihydroxy-6-(h-
ydroxymethyl)tetrahydropyran-2-yl]triazol-4-yl]methylcarbamoyl]phenyl]xant-
hen-3-ylidene]-diethyl-ammonium chloride
[0443] Example 91 was synthesized from 1-azido-1-deoxyglucose and
example 91, step 1 following the CuAAC procedure described in
synthesis method E.
Yield: 33 mg (67 .mu.mol, 46.7%), white-yellow solid. TLC:
R.sub.f=0.485 (dichloromethane/ethanol, 4:1). LC/MS (ES-API):
m/z=686.38 [M+H].sup.+; calculated: 686.33; t.sub.R (.lamda.=220
nm): 1.67 min (LC/MS-Method 1). .sup.1H-NMR (400 MHz,
DMSO-d.sub.6): .delta.=8.09 (s, 1H, NCH), 7.82 (m, 1H, ArH), 7.51
(m, 1H, ArH), 7.08 (m, 1H, ArH), 6.33 (m, 6H, ArH), 5.40 (d, J=9.1
Hz, 1H, OH), 5.24 (d, J=6.0 Hz, 1H, OH), 5.19 (d, J=4.7 Hz, 1H,
OH), 5.08 (d, J=5.6 Hz, 1H, CH), 4.63 (dd, J=5.0 Hz, 1H, OH), 3.82
(d, J=6.9 Hz, 2H, NCH.sub.2), 3.77 (m, 2H, CH.sub.2), 3.69 (m, 2H,
CH.sub.2), 3.42 (m, 2H, 2.times.CH), 3.29 (m, 8H, H), 3.18 (m, 2H,
2.times.CH), 2.73 (m, 2H, CH.sub.2), 2.60 (m, 8H, CH.sub.2), 2.03
(m, 2H, CH.sub.2), 1.05 (t, J=6.9 Hz, 12H, CH.sub.3) ppm.
EXAMPLE 92
(2R,3S,4S,5R,6R)-2-(Hydroxymethyl)-6-[4-[6-[(4-nitro-2,1,3-benzoxadiazol-7-
-yl)amino]hexyl]triazol-1-yl]tetrahydropyran-3,4,5-triol
##STR00112##
[0445] Example 92 was synthesized from 1-azido-1-deoxyglucose and
example 67, step 1 following the CuAAC procedure described in
synthesis method E.
Yield: 24 mg (48 .mu.mol, 66.6%), orange solid. TLC: R.sub.f=0.310
(dichloromethane/ethanol, 4:1). LC/MS (ES-API): m/z=494.25
[M+H].sup.+; calculated: 494.19; t.sub.R (.lamda.=220 nm): 1.34 min
(LC/MS-Method 1). .sup.1H-NMR (400 MHz, DMSO-d.sub.6): 67 =8.48 (d,
J=8.6 Hz, 1H, ArH), 8.00 (s, 1H, NCH), 8.14 (d, J=8.6 Hz, 1H, ArH),
5.45 (d, J=9.3 Hz, 1H, OH), 5.29 (d, J=6.1 Hz, 1H, OH), 5.24 (d,
J=4.9 Hz, 1H, OH), 5.11 (d, J=5.6 Hz, 1H, CH), 4.58 (dd, J=5.5 Hz,
1H, OH), 3.93 (s, 1H, NCH.sub.2), 3.72 (m, 2H, CH.sub.2), 3.41 (m,
3H, 3.times.CH), 3.20 (m, 1H, CH), 1.66 (m, 6H, 3.times.CH.sub.2),
1.41 (m, 4H, 2.times.CH.sub.2) ppm.
EXAMPLE 93
[(2S,3R,4S,6S)-2,3-Diacetoxy-6-(4-phenylpiperazine-1-carbonyl)tetrahydropy-
ran-4-yl]acetate
##STR00113##
[0447] A suspension of 7 mg (0.1 eq.) Pd/C (10%) in 3 mL dry
methanol (argon atmosphere) was overlayed with hydrogen and stirred
at room temperature for 30 minutes. A solution of 30 mg example 41,
step 1 in 2 mL dry methanol was added. The reaction mixture was
stirred at room temperature for 3 hours and filtered over
celite.
[0448] Purification: MPLC CombiFlash Rf (Teledyne ISCO); column:
RediSep Silica 4 g; flow rate: 12 mL/min; wavelength for detection:
220 nm; eluent: (A) dichloromethane, (B) ethanol.
MPLC Gradient
TABLE-US-00039 [0449] start % B end % B duration [min] 0 0 4.0 0
5.0 3.0 5.0 5.0 10.0
Yield: 19 mg (42.37 .mu.mol, 63.0%), orange oil. LC/MS (ES-API):
m/z=449.10 [M+H].sup.+; calculated: 449.18; t.sub.R (.lamda.=220
nm): 0.79 min (LC/MS-Method 1). .sup.1H-NMR (400 MHz, CDCl.sub.3):
.delta.=7.28 (m, 2H, ArH), 6.92 (m, 3H, ArH), 5.55 (d, J=7.9 Hz,
1H, CH), 5.13 (d, J=5.2 Hz, 1H, CH), 5.08 (d, J=7.7 Hz, 1H, CH),
4.36 (dd, J=1.7 Hz, J=11.6 Hz 1H, CH), 3.99 (m, 1H, NCH.sub.2),
3.82 (m, 1H, NCH.sub.2), 3.54 (m, 2H, NCH.sub.2), 3.28 (m, 2H,
NCH.sub.2), 3.03 (m, 2H, NCH.sub.2), 2.32 (ddd, J=2.4 Hz, 1H,
CH.sub.2), 2.17 (pseudo-q, J=11.6 Hz, 1H, CH.sub.2), 2.11 (s, 3H,
CH.sub.3), 2.06 (s, 6H, 2.times.CH.sub.3) ppm.
EXAMPLE 94
(4-Phenylpiperazin-1-yl)-[(2S,4S,5R)-4,5,6-tri
hydroxytetrahydropyran-2-yl]methanone
##STR00114##
[0451] To a solution of 10 mg example 93 in 1 mL methanol 4 .mu.l
of a 5.4 M aqueous sodium methoxide solution were added. The
reaction mixture was agitated at room temperature for 10 minutes
and controlled by TLC and LC/MS. The reaction mixture was quenched
and evaporated
Yield: 4 mg (12.41 .mu.mol, 55.6%), orange oil. LC/MS (ES-API):
m/z=323.05 [M+H].sup.+; calculated: 323.15; t.sub.R (ELSD): 0.11
min (LC/MS-Method 1). .sup.1H-NMR (400 MHz, MeOD): .delta. =7.14
(t, J=8.0 Hz, 2H, ArH), 6.88 (d, J=8.0 Hz, 2H, ArH), 6.76 (t, J=7.0
Hz, 1H, ArH), 5.12 (d, J=3.6 Hz, 1H, OH), 4.78 (dd, J=2.0 Hz, 1H,
OH), 4.42 (d, J=7.6 Hz, 1H, OH), 4.33 (dd, J=2.0 Hz, 1H, OH),
3.6-4.0 (m, 4H, 4.times.CH), 3.4-3.0 (m, 8H, 4.times.NCH.sub.2),
1.94 (dd, J=4.7 Hz, 1H, CH.sub.2), 1.91 (qi, J=12.2 Hz, 1H,
CH.sub.2) ppm.
EXAMPLE 95
1-[[(3aR,5R,5aS,8aS,8bR)-2,2,7,7-Tetramethyl-5,5a,8a,8b-tetrahydro-3aH-di[-
1,3]dioxolo[4,5-a:4',5'-c]pyran-5-yl]methyl]-4-(4-benzyloxyphenyl)piperazi-
ne
##STR00115##
[0453] To a solution of
(3aR,5S,5aR,8aS,8bR)-2,2,7,7-tetramethyl-5,5a,8a,8b-tetrahydro-3aH-di[1,3-
]dioxolo[4,5-a:4',5'-c]pyran-5-carbaldehyde (300 mg, 1.16 mmol, 1
eq.), 1-(4-(benzyloxy)phenyl)piperazine (374mg, 1.39 mmol, 1.2
eq.), and acetic acid (132 .mu.l, 2.32 mmol, 2 eq.) in methanol was
added sodium boronhydride (147 mg, 2.32 mmol, 2 eq.). The reaction
mixture was agitated overnight at room temperature and evaporated.
The residue was separated between ethylacetate and a saturated
solution of NaHCO.sub.3. The aqueous phase was extracted twice with
ethylacetate. The organic phase was dried with MgSO.sub.4,
evaporated in vacuo, and purified by flash chromatography on silica
gel (100% heptane till 100% ethylacetate in 40 minutes).
Yield: 394 mg (771 .mu.mol,66%), oil. LC/MS (ES-API): m/z=511.2
[M+H].sup.+; calculated: 511.6 ; t.sub.R (.lamda.=220 nm): 0.77 min
(LC/MS-Method 2). .sup.1H-NMR (400 MHz, DMSO-d.sub.6):
.delta.=7.44-7.28 (m, 5H), 6.86 (s, 4H), 5.45 (d, 1H, J=5.1 Hz),
5.01 (s, 2H), 4.57 (dd, 1H, J=7.9, 2.2 Hz), 4.31 (dd, 1H, J=5.1,
2.34 Hz), 4.21 (dd, 1H, J=7.9, 1.6 Hz), 2.98 (t, 4H, J=4.9 Hz),
2.65-2.57 (m, 3H), 2.42 (dd, 1H, J=12.7, 7.0 Hz), 1.45 (m, 3H),
1.35 (m, 1H), 1.28 (m, 1H) ppm.
EXAMPLE 96
(3R,4S,5R,6R)-6-[[4-(4-Benzyloxyphenyl)piperazin-1-yl]methyl]tetrahydropyr-
an-2,3,4,5-tetrol
##STR00116##
[0454] Method F
[0455] A solution of isopropylidene compounds (1 eq.) in
trifluoroacetic acid/water (4/1, v/v) was agitated at room
temperature for 2 hours, evaporated under vacuo, and
lyophilised.
[0456] Example 96 was obtained from example 95 following the
isopropylidene deprotection described in synthesis method F.
Yield: 21 mg (38.4 .mu.mol, quant.). LC/MS (ES-API): m/z=431.1
[M+H].sup.+; calculated: 431.2 ; t.sub.R (.lamda.=220 nm): 0.620
min (LC/MS-Method 2). .sup.1H-NMR (400 MHz, MeOD): .delta.=7.33 (s,
1H), 7.31 (s, 1H), 7.26 (t, 2H, J=7.1 Hz), 7.22-7.17 (m, 1H), 6.87
(q, 4H, J=6.87 Hz), 5.13 (d, 0.9 H), 4.93 (s, 3H), 4.43 (d, 0.5 H,
J=6.6 Hz), 4.37 (d, 0.5 H, J=9.6 Hz), 3.95 (d, 0.7 H, J=8.4 Hz),
3.76 (s, 1H), 3.74-3.66 (m, 2H), 3.56-3.38 (m, 4H), 3.31 (s, 1H),
3.28 (s, 1H), 3.26 (s, 1H), 3.22 (s, 2H) ppm. Mixture of
diastereoisomers.
EXAMPLE 97
4-[4-[[(3aR,5R,5aS,8aS,8bR)-2,2,7,7-Tetramethyl-5,5a,8a,8b-tetrahydro-3aH--
di[1,3]dioxolo[4,5-a:4',5'-c]pyran-5-yl]methyl]piperazin-1-yl]phenol
##STR00117##
[0458] To a solution of example 95 (395 mg, 77 .mu.mol, 1 eq.) in
methanol (3 mL) under argon was added Pd/C (8.3 mg, 77 .mu.mol, 0.1
eq.). The reaction mixture was purged with a flux of hydrogen. The
reaction mixture was agitated at room temperature for 1 hour. The
reaction mixture was filtrated on celite, rince with methanol, and
evaporated in vacuo.
Yield: 366 mg (870 .mu.mol, quant.). LC/MS (ES-API): m/z=421.1
[M+H]+; calculated: 421.5 ; t.sub.R (.lamda.=220 nm): 0.60 min
(LC/MS-Method 2). .sup.1H-NMR (400 MHz, DMSO-d.sub.6): .delta.=6.72
(d, 2H, J=8.7 Hz), 6.62 (d, 2H, J=8.7 Hz), 5.42 (d, 1H, J=5.1 Hz),
4.53 (dd, 1H, J=7.8, 2.3 Hz), 4.26-4.18 (m, 2H), 3.93-3.86 (m, 1H),
2.94 (t, 4H, J=5.0 Hz), 2.69-2.49 (m, 4H), 1.50 (s, 3H), 1.35 (s,
3H), 1.28 (s, 6H) ppm.
EXAMPLE 98
(2S,3R,4S,5R,6R)-6-[4-(4-Hydroxyphenyl)piperazin-1-yl]methylpetrahydropyra-
n-2,3,4,5-tetrol
##STR00118##
[0460] Example 98 was obtained from example 97 following the
isopropylidene deprotection described in synthesis method F.
Yield: 31 mg (55 .mu.mol, 93%), oil. LC/MS (ES-API): m/z=341.2
[M+H].sup.+; calculated: 341.2 ; t.sub.R=0.07 min (LC/MS-Method 2).
.sup.1H-NMR (400 MHz, MeOD): =6.8 (d, 2H, J=9.0 Hz), 6.64 (d, 2H,
J=9.0 Hz), 5.12 (d, 1H, J=3.6 Hz), 4.40 (dd, 1 H, J=5.8, 1.3 Hz),
4.35 (dd, 1H, J=10.0, 2.5 Hz), 3.95-3.90 (m, 1H), 3.77-3.69 (m,
4H), 3.56-3.46 (m, 4H), 3.43-3.40 (m, 2H), 3.31-3.23 (m, 3H) ppm.
Mixture of diastereoisomers.
EXAMPLE 99
1-[[(3aR,5R,5aS,8aS,8bR)-2,2,7,7-Tetramethyl-5,5a,8a,8b-tetrahydro-3aH-di[-
1,3]dioxolo[4,5-a:4',5'-c]pyran-5-yl]methyl]-4-[4-[2-[2-(2-prop-2-ynoxyeth-
oxy)ethoxy]ethoxy]phenyl]piperazine
##STR00119##
[0461] Step 1: 2-[2-(2-prop-2-ynoxyethoxy)ethoxy]ethyl 4-methyl
benzenesulfonate
##STR00120##
[0463] To a solution of
2-(2-(2-(prop-2-yn-1-yloxy)ethoxy)ethoxy)ethanol (50 mg, 266
.mu.mol, 1 eq.) in pyridine (2 mL) were added
N,N,N',N'-tetramethyl-1,6-diaminohexane (5.8 .mu.l, 27 .mu.mol, 0.1
eq.) and 4-methylbenzene-1-sulfonyl chloride (76 mg, 398 .mu.mol,
1.5 eq.). The reaction mixture was agitated at room temperature for
2 hours. The reaction mixture was evaporated in vacuo.
[0464] Purification: flash chromatography on silica gel (100%
heptane till 100% ethylacetate in 30 minutes).
Yield: 62 mg (181 .mu.mol, 68%). LC/MS (ES-API): m/z=343.1
[M+H].sup.+; calculated: 343.4 ; t.sub.R (.lamda.=220 nm): 0.804
min (LC/MS-Method 2). .sup.1H-NMR (400 MHz, DMSO-d.sub.6):
.delta.=7.78 (d, 2H, J=8.5 Hz), 7.48 (d, 2H, J=8.5 Hz), 4.15-4.08
(m, 4H), 3.61-3.47 (m, 10H), 3.41 (t, 1H, J=2.5 Hz), 2.42 (s, 3H)
ppm.
Step 2:
1-[[(3aR,5R,5aS,8aS,8bR)-2,2,7,7-Tetramethyl-5,5a,8a,8b-tetrahydro-
-3aH-di[1,3]dioxolo[4,5-a:4',5'-c]pyran-5-yl]methyl]-4-[4-[2-[2-(2-prop-2--
ynoxyethoxy)ethoxy]ethoxy]phenyl]piperazine
[0465] A solution of example 99, step 1 (41 mg, 120 .mu.mol, 1.2
eq.), example 97 (42 mg, 100 .mu.mol, 1 eq.), and Cs.sub.2CO.sub.3
(130 mg, 400 .mu.mol, 4 eq.) in dimethylformamide (1 mL) under
argon was irradiated in the microwave for 10 minutes at 70.degree.
C. and 40 minutes at 80.degree. C. The crude mixture was diluted
with ethylacetate. The organic phase was washed with a saturated
solution of NaHCO.sub.3, water, dried with MgSO.sub.4, and
evaporated in vacuo.
[0466] Purification:flash chromatography on silica gel
(dichloromethane till dichloromethane/methanol 9/1 in 30
minutes).
Yield: 16 mg (27 .mu.mol, 27%). LC/MS (ES-API): m/z=591.3
[M+H].sup.+; calculated: 591.3 ; t.sub.R (.lamda.=220 nm): 0.70 min
(LC/MS-Method 2). .sup.1H-NMR (400 MHz, DMSO-d.sub.6):
.delta.=6.89-6.81 (m, 4H), 5.57 (d, 1H, J=5.1 Hz), 4.60 (dd, 1H,
J=7.8, 2.3 Hz), 4.31 (dd, 1H, J=5.1, 2.3 Hz), 4.25-4.19 (m, 4H),
4.07 (t, 2H, J=5.1 Hz), 4.02-3.96 (m, 1H), 3.82 (t, 2H, J=5.1 Hz),
3.75-3.66 (m, 10H), 3.11 (t, 4 H, J=4.5 Hz), 2.80-2.58 (m, 6H),
2.43-2.40 (m, 1H), 2.08 (s, 1H), 1.73 (s, 1H), 1.54 (s, 3H), 1.46
(s, 3H), 1.34 (s, 3H), 1.33 (s, 3H) ppm.
EXAMPLE 100
(3R,4S,5R,6R)-6-[[4-[4-[2-[2-(2-Prop-2-ynoxyethoxy)ethoxy]ethoxy]phenyl]pi-
perazin-1-yl]methyl]tetrahydropyran-2,3,4,5-tetrol
##STR00121##
[0468] Example 100 was obtained from example 99 following the
isopropylidene deprotection described in synthesis method F.
Yield: 18 mg (28 .mu.mol, quant). LC/MS (ES-API): m/z=511.2
[M+H].sup.+; calculated: 511.3 ; t.sub.R (.lamda.=220 nm): 0.552
min (LC/MS-Method 2).
[0469] .sup.1H-NMR (400 MHz, MeOD): .delta.=6. 99 (d, 2H, J=8.9
Hz), 6.91 (d, 2H, J=8.78 Hz), 5.23 (d, 0.6H, J=3.5 Hz), 4.52 (d,
0.5 H, J=6.0 Hz), 4.47 (d, 0.6H, J=10.0 Hz), 4.23-4.16 (m, 3.2H),
4.11-4.02 (m, 2H), 3.88-3.75 (m, 5H), 3.73-3.59 (m, 15H), 3.57-3.49
(m, 1.4H), 3.08 (s1, 2H), 2.84 (s, 1.2H) ppm. Mixture of
diastereoisomers.
EXAMPLE 101
1-[[(3aR,5R,5aS,8aS,8bR)-2,2,7,7-Tetramethyl-5,5a,8a,8b-tetrahydro-3aH-di[-
1,3]dioxolo[4,5-a:4',5'-c]pyran-5-yl]methyl]-4-(4-chlorophenyl)piperazine
##STR00122##
[0471] To a solution of
(3aR,5S,5aR,8aS,8bR)-2,2,7,7-tetramethyl-5,5a,8a,8b-tetrahydro-3aH-di[1,3-
]dioxolo[4,5-a:4',5'-c]pyran-5-carbaldehyde (50 mg, 194 .mu.mol, 1
eq.), 1-(4-chlorophenyl)piperazine (114 mg, 581 .mu.mol, 3 eq.),
and acetic acid (22 .mu.l, 387 .mu.mol, 2 eq.) in methanol was
added sodium cyanoboronhydride (25 mg, 387 .mu.mol, 2 eq.). The
reaction mixture was agitated overnight at room temperature and
evaporated in vacuo. The crude mixture was separated between
ethylacetate and a saturated solution of NaHCO3. The aqueous phase
was extracted twice with ethylacetate. The organic phase was dried
with MgSO.sub.4 and evaporated.
[0472] Purification: flash chromatography on silica gel (100%
heptane till 100% ethylacetate in 40 minutes).
Yield: 63 mg (144 .mu.mol, 74%), oil. LC/MS (ES-API): m/z=439.2
[M+H].sup.+; calculated: 439.9; t.sub.R (.lamda.=220 nm): 0.713 min
(LC/MS-Method 2). .sup.1H-NMR (400 MHz, DMSO-d.sub.6): .delta.=7.21
(d, 2H, J=8.9 Hz), 6.93 (d, 2H, J=8.9 Hz), 5,45 (d, 1H, J=5.1 Hz),
4.58 (dd, 1H, J=7.9, 2.4 Hz), 4.32 (dd, 1H, J=5.2, 2.3 Hz), 4.21
(dd, 1H, J=7.9, 1.7 Hz), 3.90-3.85 (m, 1H), 3.10 (t, 4H, J=4.9 Hz),
2.65-2.57 (m, 4H), 2.43 (dd, 2H, J=13.0, 7.1 Hz), 1.45 (s, 3H),
1.35 (s, 3H), 1.28 (s, 6H) ppm.
EXAMPLE 102
(3R,4S,5R,6R)-6-[[4-(4-Chlorophenyl)piperazin-1-yl]methyl]tetrahydropyran--
2,3,4,5-tetrol
##STR00123##
[0474] Example 102 was obtained from example 101 following the
isopropylidene deprotection described in synthesis method F.
Yiels: 78 mg (133 .mu.mol, 95%). LC/MS (ES-API): m/z=359.1
[M+H].sup.+; calculated: 359.1 ; t.sub.R (.lamda.=220 nm): 0.493
min (LC/MS-Method 2). .sup.1H-NMR (400 MHz, MeOD): .delta.=7.27 (d,
2H, J=8.5 Hz), 7.01 (d, 2H, J=8.5 Hz). 5.24 (d, 0.5 H, J=3.0 Hz),
4.55 (d, 0.5 H, J=6.7 Hz), 4.48 (d, 0.5 H, J=9.8 Hz), 4.07 (d, 0.5
H, J=9.2 Hz), 3.90-3.76 (m, 2H), 3.66-3.30 (m, 9H) ppm. Mixture of
diastereoisomers.
EXAMPLE 103
N-[2-[2-[2-[[(3aR,5R,5aS,8aS,8bR)-2,2,7,7-Tetramethyl-5,5a,8a,8b-tetrahydr-
o-3aH-di[1,3]dioxolo[4,5-a:4',5'-c]pyran
-5-yl]methylamino]ethoxy]ethoxy]ethyl]pent-4-ynamide
##STR00124##
[0475] Step 1: tert-butyl
N-[2-[2-[2-(pent-4-ynoylamino)ethoxy]ethoxy]ethyl]carbamate
##STR00125##
[0477] To a solution of pent-4-ynoic acid (43.5 mg, 442 .mu.mol,
1.1 eq.) and tert-butyl (2-(2-(2-aminoethoxy)ethoxy)ethyl)carbamate
(100 mg, 402 .mu.mol, 1 eq.) in dimethylformamide (3 mL) was added
N,N-diisopropylethylamine (77 .mu.l, 443 .mu.mol, 1.1 eq.), HOBt
(67.8 mg, 443 .mu.mol, 1.1 eq.) and
N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide (84.9 mg, 443
.mu.mol, 1.1 eq.). The reaction mixture was agitated overnight at
room temperature. The crude mixture was diluted with ethylacetate,
washed with 0.5 N aqueous HCl, a saturated solution of NaHCO.sub.3,
and brine, dried with MgSO.sub.4, and concentrated in vacuo.
[0478] Purification: flash chromatography on silica gel (100%
heptane till 100% ethylacetate in 20 minutes, 100% ethylacetate for
10 minutes)
Yield: 99 mg (301 .mu.mol, 75%). LC/MS (ES-API): m/z=351.2
[M+Na].sup.+; calculated: 351.4; t.sub.R: 0.675 min (LC/MS-Method
2). .sup.1H-NMR (400 MHz, DMSO-d.sub.6): .delta.=3.49 (s, 4H),
3.41-3.34 (m, 4H), 3.19 (q, 2H, J=5.8 Hz), 3.05 (q, 2H, J=5.8 Hz),
2.89 (s, 2H), 2.73 (s, 2H), 2.37-2.31 (m, 2H), 2.28-2.24 (m, 2H),
1.90 (s, 1H), 1.37 (s, 9H) ppm.
Step 2: N-[2-[2-(2-aminoethoxy)ethoxy]ethyl]pent-4-ynamide
##STR00126##
[0480] A solution of example 103, step 1 (98 mg, 298 .mu.mol, 1
eq.) in trifluoroacetic acid/dichloromethane (1/1, v/v, 1 mL) was
agitated at room temperature for 30 minutes. The reaction mixture
was evaporated under vacuo and lyophilised.
Yield: 108 mg (315 .mu.mol, quant). LC/MS (ES-API): m/z=229.2
[M+H].sup.+; calculated: 229.1; t.sub.R: 0.123 min (LC/MS-Method
2).
.sup.1H-NMR (400 MHz, MeOD): .delta.=3.72 (t, 2H, J=4.9 Hz), 3.67
(s, 4H), 3.58 (t, 2H, J=5.5 Hz), 3.40 (t, 2H, J=5.5 Hz), 3.15 (t,
2H, J=4.8 Hz), 2.51-2.45 (m, 2H), 4.44-2.39 (m, 2H), 2.28 (s, 1H)
ppm.
Step 3:
N-[2-[2-[2-[[(3aR,5R,5aS,8aS,8bR)-2,2,7,7-tetramethyl-5,5a,8a,8b-t-
etrahydro-3aH-di[1,3]dioxolo[4,5-a:4',5'-c]pyran-5-yl]methylamino]ethoxy]e-
thoxy]ethyl]pent-4-ynamide
[0481] A solution of
(3aR,5S,5aR,8aS,8bR)-2,2,7,7-tetramethyl-5,5a,8a,8b-tetrahydro-3aH-di[1,3-
]dioxolo[4,5-a:4',5'-c]pyran-5-carbaldehyde (41.5 mg, 160 .mu.mol,
1 eq.), example 103, step 2 (55 mg, 160 .mu.mol, 1 eq.), and
triethylamine (44.8 .mu.l, 321 .mu.mol, 2 eq.) in methanol (3 mL)
containing 4 .ANG. molecular sieves was agitated overnight at room
temperature. Sodium boronhydride (12 mg, 321 .mu.mol, 2 eq.) was
added to the reaction mixture. The reaction mixture was agitated at
room temperature for 2 hours. A 1 N aqueous solution of sodium
hydride was added. The reaction mixture was extracted with ether
(three times). The organic phase was dried with MgSO.sub.4 and
evaporated in vacuo.
[0482] Purification: flash chromatography on silica gel
(dichloromethane till dichloromethane/methanol 9/1 in 30
minutes).
Yield: 21.4 mg (45.5 .mu.mol, 28%), oil. LC/MS (ES-API): m/z=471.2
[M+H].sup.+; calculated: 471.5; t.sub.R (.lamda.=220 nm): 0.615 min
(LC/MS-Method 2). .sup.1H-NMR (400 MHz, MeOD): .delta.=6.70 (t, 1H,
J=5.2 Hz), 6.21 (s, 1H), 5.72 (d, 1H, J=7.9 Hz), 5.31 (t, 1H, J=9.3
Hz), 5.20 (t, 1H, J=9.3 Hz), 5.11 (t, 1H, J=8.5 Hz), 4.07 (d, 1H,
J=9.8 Hz), 3.62 (s, 5H), 3.59-3.53 (m, 5H), 3.52-3.46 (m, 4H),
3.40-3.31 (m, 1H) ppm.
EXAMPLE 104
N-[2-[2-[2-[[(3R,4S,5R)-3,4,5,6-Tetrahydroxytetrahydropyran-2-yl]methylami-
no]ethoxy]ethoxy]ethyl]pent-4-ynamide
##STR00127##
[0484] Example 104 was obtained from example 103 following the
isopropylidene deprotection described in synthesis method F.
Yield: 17.3 mg (44 .mu.mol, quant). LC/MS (ES-API): m/z=391.1
[M/5+H].sup.+; calculated: 391.4; t.sub.R (.lamda.=220 nm): 0.07
min (LC/MS-Method 2).
EXAMPLE 105
[(2S,3R,4S,5S)-2,3,5-Triacetoxy-6-[2-[2-[2-(pent-4-ynoylamino)ethoxy]ethox-
y]ethylcarbamoyl]tetrahydropyran-4-yl]acetate
##STR00128##
[0486] To a solution of
(2R,3S,4S,5R)-2,3,4,5-tetraacetoxycyclohexanecarboxylic acid (101
mg, 280 .mu.mol, 1.2 eq.) in dimethylformamide (3 mL) was added
HATU (106.7 mg, 280 .mu.mol, 1.2 eq.) and 4 .ANG. molecular sieves.
After 5 minutes example 103, step 2 (80 mg, 233 .mu.mol, 1 eq.) was
added. The reaction mixture was agitated at room temperature for 72
hours. The reaction mixture was diluted with ethylacetate. The
organic phase was washed with 1N aqueous HCl, saturated
NaHCO.sub.3, and brine, dried with MgSO.sub.4, filtered, and
evaporated in vacuo.
[0487] Purification: flash chromatography on silica gel
(dichloromethane till dichloromethane/methanol 9/1 in 30
minutes)
Yield: 74.45 mg (130 .mu.mol, 56%), oil. LC/MS (ES-API): m/z=513.2
[M-OAc].sup.+; calculated: 513.6; t.sub.R (.lamda.=220 nm): 0.652
min (LC/MS-Method 2). .sup.1H-NMR (400 MHz, DMSO-d.sub.6):
.delta.=8.15 (t, 1H, J=5.6 Hz), 7.93 (t, 1H, J=5.6 Hz), 5.96 (d,
1H, J=8.3 Hz), 5.43 (t, 1H, J=9.8 Hz), 5.08 (t, 1H, J=9.8 hz), 4.98
(dd, 1H, J=9.8, 8.4 Hz), 4.30 (s, 1H, J=9.8 Hz), 3.49 (s, 4H), 3.38
(q, 4H, J=6.0 Hz), 3.24-3.13 (m, 4H), 2.73 (t. 1H, J=2.6 Hz),
2.37-2.31 (m, 2H), 2.30-2.24 (m, 2H), 2.07 (s, 3H), 2.00 (s, 3H),
1.95 (s, 3H), 1.93 (s, 3H) ppm.
EXAMPLE 106
(3S,4S,5R)-3,4,5,6-Tetrahydroxy-N-[2-[2-[2-(pent-4-ynoylamino)ethoxy]ethox-
y]ethyl]tetrahydropyran-2-carboxamide
##STR00129##
[0488] Method G
[0489] To a solution of the tetraacetate derivative (1 eq.) in
methanol/water/THF (5/4/1, v/v/v) at 0.degree. C. was added a
solution of an aqueous 1 N lithium hydroxyde solution (1 eq.). The
reaction mixture was agitated 15 minutes at 0.degree. C., quenched
with a 1 N aqueous solution of HCl, evaporated, and
lyophilised.
[0490] Example 106 was obtained from example 105 using acetyl
deprotection method G.
Yield: 62.2 mg (153 .mu.mol, quant). LC/MS (ES-API): m/z=405.2
[M+H].sup.+; calculated: 404.4; t.sub.R (.lamda.=324 nm): 0.087 min
(LC/MS-Method 2). .sup.1H-NMR (400 MHz, DMSO-d.sub.6): .delta.=6.77
(d, 0.5H, J=6.7 Hz), 6.47 (d, 0.5H, J=4.6 Hz), 4.98-4.92 (m, 2H),
4.90 (d, 0.5H, J=4.3 Hz), 4.77 (d, 0.5H, J=4.9 Hz), 4.57 (d, 0.5H,
J=6.3 Hz), 4.32 (t, 0.5 H, J=7.4 Hz), 3.95 (t, 0.5H, J=10 Hz), 3.50
(d, 4H, J=2.7 Hz), 3.49-3.37 (m, 4H), 3.26-3.09 (m, 6H), 2.98-2.91
(m, 0.5 H), 2.74 (t, 1H, J=2.6 Hz), 2.37-2.32 (m, 2H), 2.99-2.45
(m, 2H) ppm. Mixture of diastereoismers.
EXAMPLE 107
[0491]
N-[[(3aR,5aS,8aS,8bR)-2,2,7,7-Tetramethyl-5,5a,8a,8b-tetrahydro-3aH-
-di[1,3]dioxolo[4,5-a:4',5'-c]pyran-5-yl]methyl]oct-7-yn-1-amine
##STR00130##
[0492] A solution of
(3aR,5S,5aR,8aS,8bR)-2,2,7,7-tetramethyl-5,5a,8a,8b-tetrahydro-3aH-di[1,3-
]dioxolo[4,5-a:4',5'-c]pyran-5-carbaldehyde (50 mg, 174 .mu.mol, 1
eq.), oct-7-yn-1-amine hydrochloride (31 mg, 192 .mu.mol, 1.1 eq.),
and triethylamine (27 .mu.l, 192 .mu.mol, 1.1 eq.) in methanol (1
mL) was agitated at room temperature for 3 hours. The reaction
mixture was cooled to 0.degree. C. and sodium boronhydride (13 mg,
348 .mu.mol, 2 eq.) was added. The reaction mixture was agitated at
0.degree. C. for one hour. A 1 N aqueous solution of sodium
hydroxide was added. The aqueous phase was extracted with ether
(three times), dried over MgSO.sub.4, filtered, and evaporated in
vacuo.
[0493] Purification: flash chromatography on silica gel
(dichloromethane till dichloromethane/methanol 9/1 in 30 min).
Yield: 29 mg (79 .mu.mol, 45%), oil. LC/MS (ES-API): m/z=368.4
[M+H].sup.+; calculated: 368.5; t.sub.R: 0.684 min (LC/MS-Method
2). .sup.1H-NMR (400 MHz, DMSO-d.sub.6): .delta.=5.43 (d, 1H, J=5.2
Hz), 4.55 (dd, 1H, J=7.7, 2.3 Hz), 4.44-4.39 (m, 0.3 H), 4.30 (dd,
1H, J=5.1, 2.3 Hz), 4.20 (dd, 1H, J=7,7, 1.7 Hz), 3.74 (td, 1H,
J=6.5, 1.5 Hz), 3.40-3.37 (m, 0.7 H), 2.72 (t, 0.6 H, J=2.8 Hz),
2.68-2.65 (m, 0.3H), 2.63-2.58 (m, 0.9 H), 1.7 (t, 0.8 H, J=2.5
Hz), 1.48-1.22 (m, 20.6 H) ppm.
EXAMPLE 108
(3R,4S,5R)-6-[(Oct-7-ynylamino)methyl]tetrahydropyran-2,3,4,5-tetrol
##STR00131##
[0495] Example 108 was obtained from example 107 following the
isopropylidene deprotection described in synthesis method F.
Yield: 32 mg (80 .mu.mol, quant). LC/MS (ES-API): m/z=288.2
[M+H].sup.+; calculated: 288.2; t.sub.R=0.648 min (LC/MS-Method 3).
.sup.1H-NMR (400 MHz, DMSO-d.sub.6): .delta.=6.71 (d, 0.5H, J=7.5
Hz), 6.38 (d, 0.5H, J=4.6 Hz), 5.00 (t, 0.5H, J=4.0 Hz), 4.91-4.79
(m, 2H), 4.71 (s, 0.5H), 4.51 (s, 0.5H), 4.29 (t, 0.5H, J=6.8 Hz),
4.12-4.07 (m, 0.5H), 3.70-3.65 (m, 1H), 3.63-3.51 (m, 1.5H),
3.19-3.00 (m, 2H), 2.95-2.83 (m, 2H), 2.75 (t, 0.5H, J=2.6 Hz),
2.19-2.07 (m, 2H), 1.72 (t, 1H, J=2.6 Hz), 1.64-1.52 (m, 2H),
1.48-1.52 (m, 6H) ppm. Mixture of diastereoisomers.
EXAMPLE 109
[(2S,3R,4S,5S)-2,3,5-Triacetoxy-6-(oct-7-ynylcarbamoyl)tetrahydropyran-4-y-
l]acetate
##STR00132##
[0497] To a solution of
(2R,3S,4S,5R)-2,3,4,5-tetraacetoxycyclohexanecarboxylic acid (20
mg, 55 .mu.mol, 1 eq.) in dimethylformamide (2 mL) with 4 .ANG.
molecular sieves was added HATU (29.4 mg, 77 .mu.mol, 1.4 eq.)
followed by oct-7-yn-1-amine hydrochloride (26.8 mg, 166 .mu.mol, 3
eq.) in 0.5 mL dimethylformamide. The reaction mixture was agitated
overnight at room temperature and evaporated in vacuo.
[0498] Purification: flash chromatography on silica gel, (heptane
till ethyl acetate in 20 min, 100% ethyl acetate for 20 min).
Yield: 25.5 mg (54 .mu.mol, 98%), white solid. LC/MS (ES-API):
m/z=492.1 [M+H].sup.+; calculated: 492.5; t.sub.R=0.851 min
(LC/MS-Method 2). .sup.1H-NMR (400 MHz, CDCl.sub.3): .delta.=6.29
(t, 1H, J=4.9 Hz), 5.76 (d, 1H, J=8.0 Hz), 5.30 (t, 1H, J=9.4 Hz),
5.19 (t, 1H J=9.4Hz), 5.11 (td, 1H, J=8.0, 1.5 Hz), 4.05 (d, 1H,
J=9.4 Hz), 3.30-3.13 (m, 2H), 2.28 (td, 1H, J=7.0, 2.6 Hz), 2.14
(s, 3H), 2.07 (s, 3H), 2.04 (s, 3H), 2.03 (s, 3H), 1.95 (t, 1H,
J=2.6 Hz), 1.78 (t, 1H, J=2.3 Hz) , 1.58-1.22 (m, 8H) ppm.
EXAMPLE 110
(3S,4S,5R)-3,4,5,6-Tetrahydroxy-N-oct-7-ynyl-tetrahydropyran-2-carboxamide
##STR00133##
[0500] Example 110 was obtained from example 109 using acetyl
deprotection method G.
Yield: 17 mg (49 .mu.mol, 91%), orange oil. LC/MS (ES-API):
m/z=302.1 [M+H].sup.+; calculated: 302.1; t.sub.R (.lamda.=220 nm):
0.831 min (LC/MS-Method 3). .sup.1H-NMR (400 MHz, MeOD):
.delta.=5,19 (s, 0.5H), 4.55 (d, 0.5H, J=7.5 Hz), 4.17 (d, 0.7H,
J=9.9 Hz), 3.71 (t, 1H, J=9.0 H), 3.58 (s, 0.3H), 3.51-3.39 (m,
1.7H), 3.27-3.18 (m, 3H), 2.18 (s, 3H), 2.12 (s, 1.1H), 1.92 (s,
0.9H), 1.74 (s, 1.4H), 1.59-1.29 (m, 6H) ppm. Mixture of
diastereoisomers.
EXAMPLE 111
N-[6-[1-(4-(Human
Insulin-B29Lys-amino)-4-oxo-butyl)triazol-4-yl]hexyl]-(3S,4S,5R)-3,4,5,6--
tetrahydroxy-tetrahydropyran-2-carboxamide
##STR00134##
[0501] Step 1: 4-azido-butan-(human Insulin-B29Lys)-amide
##STR00135##
[0503] To a solution of human insulin (300 mg, 51 .mu.mol, 1 eq.)
in dimethylformamide/water (1/2, v/v, 9 mL) was added triethylamine
(144 .mu.l, 1.03 mmol, 20 eq.) to get to pH=10. The reaction
mixture was cooled to 0.degree. C. A solution of
2,5-dioxopyrrolidin-1-yl 4-azidobutanoate (12.9 mg, 56 .mu.mol, 1.1
eq.) in dimethylformamide (1 mL) was added dropwise to the reaction
mixture at 0.degree. C. over 10 minutes. The reaction mixture was
agitated 2 hours, quenched with 1 N aqueous HCl till pH=3, and
lyophilized.
[0504] Purification: AEKTA avant 25 (GE Healthcare), HPLC; column:
Kinetex Prep-C18 column (5 .mu.m, 250.times.21.1 mm, Phenomenex,
volume 87 mL); wavelength for detection: 280 nm; eluent: (A)
water+0.5% acetic acid, (B) 60/40 acetonitrile/water+0.5% acetic
acid.
HPLC Gradient:
TABLE-US-00040 [0505] Flow rate Flow rate Column start % B end % B
[cm/h] [mL/min] volume 0 0 80 4.7 2.0 0 100 105 6.2 14.0 100 100
105 6.2 2.0
Yield: 146.5 mg (24.75 .mu.mol, 48%), white powder. LC/MS (ES-API):
m/z=1184.6 [M/5+H].sup.+; calculated: 1184.7; t.sub.R (.lamda.=215
nm): 4.30 min (LC/MS-Method 4).
Step 2: N-[6-[1-(4-(human
Insulin-B29Lys-amino)-4-oxo-butyl)triazol-4-yl]hexyl]-3,4,5,6-tetrahydrox-
y-tetrahydropyran-2-carboxamide
Method H
[0506] To a solution of the alkynes (1.2 eq.) and example 111, step
1 (1 eq.) in dimethylformamide and water was added a mixture of the
click reagents premixed in this order: CuSO.sub.4*5H.sub.2O (0.5
eq.), THPTA (0.8 eq.), and sodium ascorbate (1 eq.). The reaction
mixture was agitated at room temperature for 2 hours. The reaction
mixture was lyophilised. Purification was done on reverse phase
chromatography.
[0507] Example 111, step 2 was obtained from example 111, step 1
and example 110 following the click chemistry procedure described
in synthesis method H.
[0508] Purification and HPLC gradient like in example 111, step
1.
Yield: 2.9 mg (0.46 .mu.mol, 18%), white powder. LC/MS (ES-API):
m/z=1244.9 [M/5+H].sup.+; calculated: 1245.0; t.sub.R (.lamda.=215
nm): 3.95 min (LC/MS-Method 4).
EXAMPLE 112
4-[4-[3-Oxo-3-[1-[2-[2-[[(3R,4S,5R)-3,4,5,6-tetrahydroxytetrahydropyran-2--
yl]methylamino]ethoxy]ethoxy]ethylamino]propyl]triazol-1-yl]butan-(human
Insulin-B29Lys)-amide
##STR00136##
[0510] Example 112 was obtained from example 111, step 1 and
example 114 following the click chemistry procedure described in
synthesis method H.
[0511] Purification and HPLC gradient like in example 111, step
1.
Yield: 1.37 mg (0.22 .mu.mol, 11%), white powder. LC/MS (ES-API):
m/z=1262.7 [M/5+H].sup.+; calculated: 1262.8; t.sub.R (.lamda.=215
nm): 3.75 min (LC/MS-Method 4).
EXAMPLE 113
4-[4-[6-[[(3R,4S,5R)-3,4,5,6-Tetrahydroxytetrahydropyran-2-yl]methylamino]-
hexyl]triazol-1-yl]butan-(human Insulin-B29Lys)-amide
##STR00137##
[0513] Example 113 was obtained from example 111, step 1 and
example 108 following the click chemistry procedure described in
synthesis method H.
[0514] Purification and HPLC gradient like in example 111, step
1.
Yield: 4.36 mg (0.70 .mu.mol, 35%), white powder. LC/MS (ES-API):
m/z=1242.0 [M/5+H].sup.+; calculated: 1242.2; t.sub.R (.lamda.=215
nm): 3.77 min (LC/MS-Method 4).
EXAMPLE 114
(3S,4S,5R)-N-[2-[2-[2-[3-[1-(4-(Humannsulin-B29Lys)-4-oxo-butyl)triazol-4--
yl]propanoylamino]ethoxy]ethoxy]ethyl]-3,4,5,6-tetrahydroxy-tetrahydropyra-
n-2-carboxamide
##STR00138##
[0516] Example 114 was obtained from example 111, step 1 and
example 106 following the click chemistry procedure described in
synthesis method H.
[0517] Purification and HPLC gradient like in example 111, step
1.
Yield: 3.74 mg (0.59 .mu.mol, 29%), white powder. LC/MS (ES-API):
m/z=1265.4[M/5+H].sup.+; calculated: 1265.6; t.sub.R (.lamda.=215
nm): 3.87 min (LC/MS-Method 4).
Example 115
4-[4-[2-[2-[2-[4-[4-[[(3R,4S,5R)-3,4,5,6-Tetrahydroxytetrahydropyran-2-yl]-
methyl]piperazin-1-yl]phenoxy]ethoxy]ethoxy]ethoxymethyl]triazol-1-yl]buta-
n-(human Insulin-B29Lys)-amide
##STR00139##
[0519] Example 115 was obtained from example 111, step 1 and
example 100 following the click chemistry procedure described in
synthesis method H.
[0520] Purification and HPLC gradient like in example 111, step
1.
Yield: 15.2 mg (2.36 .mu.mol, 28%), white powder. LC/MS (ES-API):
m/z=1072.3 [M/6+H].sup.+; calculated: 1072.5; t.sub.R (.lamda.=215
nm): 3.81 min.
EXAMPLE 116
Methy-6-O-p-toluolsulfonyl-.beta.-D-glucopyranoside
##STR00140##
[0522] To a solution of methyl-.beta.-D-glucopyranoside (15 g, 77.3
mmol) in pyridine (50 mL) a solution of toluenesulfonylchloride
(19.1 g, 100.5 mmol) in CH.sub.2Cl.sub.2 (50 mL) was added dropwise
at 0.degree. C. The mixture was then left for 16 h at 8.degree. C.
Methanol (200 mL) was added to the reaction and the solvens
evaporated under reduced pressure. The residue was purified by
column chromatography on silica gel (10:1 CH.sub.2Cl.sub.2/MeOH) to
give the product as a white solid
Yield: 13.1g (49%).
[0523] LC-Mass Method: Mobile phase: A=2.5 mM TFA/H2O, B=2.5 mM
TFA/MeCN; Gradient: B=10%-95% in 1 min; Flow rate: 1.5 mL/min;
Column: Xbridge-C18, 30.times.4.6 mm, 2.5 um.). LC purity: 92% (214
nm); Mass: find peak 370.8 (M+Na)+ at 1.60 min. .sup.1H NMR (400
MHz, DMSO-d6) .delta. 7.79 (d, J=8.0 Hz, 2H), 7.50 (d, J=8.0Hz,
2H), 5.23 (d, J=5.6 Hz 1H), 5.13 (d, J=5.2 Hz, 1H), 5.00 (d, J=5.2
Hz, 1H), 4.20 (dd, J=10.4 Hz, 2.0 Hz, 1H), 4.06-4.02 (m, 2H),
3.36-3.34 (m, 1H), 3.30 (s, 3H), 3.12-3.07 (m, 1H), 3.01-2.95 (m,
1H), 2.93-2.87 (m, 1H), 2.43 (s, 3H).
EXAMPLE 117
Methyl-6-O-(3-phenoxyphenylcarbonyl)-.beta.-D-glucopyranoside
##STR00141##
[0525] Sodium hydride (17,22 mg, 430,58 .mu.mol) was added to a
solution of 3-phenoxybenzoic acid (92.24 mg, 430.58 .mu.mol) in DMF
(4 mL) at 0.degree. C. under argon atmosphere. The reaction mixture
was stirred for about 30 min and
methyl-6-O-p-toluensulfonyl-.beta.-D-glucopyranoside 116 (100 mg,
287.05 .mu.mol) was added and the reaction stirred for 16 h at
80.degree. C. The solvens was evaporated under reduced pressure in
vacuo and the residue extracted with CH.sub.2Cl.sub.2/H.sub.2O
(3.times.). The organic layer was dried, the solvens evaporated and
the product purified by HPLC.
Yield: 53 mg (47%)
[0526] LC/MS (ES-API): m/z=435.20 [M-H+formic acid]-; calculated:
435.16, tR (.lamda.=220 nm): 1.6 min (LC/MS-Method 2) .sup.1H NMR
(400.23 MHz, DMSO-d6) d ppm 7.72 (d, J=7.48 Hz, 1 H), 7.56
(t,J=7.95, 7.95 Hz, 1H),7.44 (m, 3H), 7.33 (ddd, J=8.19, 2.57, 0.86
Hz, 1H), 7.21 (t, J=7.18, 7.18 Hz, 1H), 7.08 (d, J=7.84 Hz, 2H),
5.15 (br s, 1H), 4.55 (dd, J=11.68, 2.02 Hz, 1H), 4.26 (dd,
J=11.74, 6.48 Hz, 1H), 4.08 (d, J=7.82 Hz, 1H), 3.46 (br s, 2H),
3.43 (u), 3.29 (s, 5H), 3.17 (m, 3H), 2.97 (m, 1H).
EXAMPLE 118
Methyl-6-O-(4-phenoxyphenylcarbonyl)-.beta.-D-glucopyranoside
##STR00142##
[0528]
Methyl-6-O-(4-phenoxyphenylcarbonyl)-.beta.-D-glucopyranoside was
synthesized as described for example 117 from
Methyl-6-O-p-toluolsulfonyl-.beta.-D-glucopyranoside (116)
(100 mg, 287.05 .mu.mol) and 4-phenoxybenzoic acid (92.24 mg,
430.58 .mu.mol).
Yield: 49 mg (43.7%)
[0529] LC/MS (ES-API): m/z=435.22 [M-H+formic acid]-; calculated:
435.16, tR (.lamda.=220 nm): 1.6 min (LC/MS-Method 2) .sup.1H NMR
(400.23 MHz, DMSO-d6) d ppm 7.97 (m(para), 2 H), 7.46 (t, J=7.56,
7.56 Hz, 2 H), 7.25 (m, 1H), 7.13 (d, J=7.83 Hz, 2H), 7.08
(m(para), 2H), 4.54 (dd, J=11.80, 1.90 Hz, 1H), 4.29 (dd, J=11.80,
6.17 Hz, 1H), 4.11 (d, J=7.82 Hz, 1H), 3.47 (m, 4H), 3.20 (m, 6H),
2.99 (m, 2H), 2.50 (u), 2.33 (m, 1H)
EXAMPLE 119
Methyl-6-O-(2-Methyl-4-phenoxyphenylcarbonyl)-.beta.-D-glucopyranoside
##STR00143##
[0531]
Methyl-6-O-(2-Methyl-4-phenoxyphenylcarbonyl)-.beta.-D-glucopyranos-
ide was synthesized as described for example 117 from
Methyl-6-O-p-toluolsulfonyl-.beta.-D-glucopyranoside (116; 100 mg,
287.05 .mu.mol) and 2-Methyl-4-phenoxybenzoic acid (98.28 mg,
430.58 .mu.mol)
Yield: 46 mg (39.6%)
[0532] LC/MS (ES-API): m/z=499.17 [M-H+formic acid].sup.-;
calculated: 449.18, tR (.lamda.=220 nm): 1.63 min (LC/MS-Method
2)
EXAMPLE 120
Methyl-6-O-(3-chloro-3'-methoxy-4-phenoxyphenylcarbonyl)-.beta.-D-glucopyr-
anoside
##STR00144##
[0534]
Methyl-6-O-(3-chloro-3'-methoxy-4-phenoxyphenylcarbonyl)-.beta.-D-g-
lucopyranoside was synthesized as described for example 117 from
Methyl-6-O-p-toluolsulfonyl-.beta.-D-glucopyranoside (116; 100 mg,
287.05 .mu.mol) and 3-Chloro-3'-methoxy-4-phenoxybenzoic acid (120
mg, 430.58 .mu.mol).
Yield: 43 mg (32.9%)
[0535] LC/MS (ES-API): m/z=499.17 [M-H+formic acid].sup.-;
calculated: 499.13, tR (.lamda.=220 nm): 1.64 min (LC/MS-Method
2)
EXAMPLE 121
Methyl-6-O-(3-methoxy-4-phenoxyphenylcarbonyl)-.beta.-D-glucopyranoside
##STR00145##
[0537]
Methyl-6-O-(3-methoxy-4-phenoxyphenylcarbonyl)-.beta.-D-glucopyrano-
side was synthesized as described for example 117 from
Methyl-6-O-p-toluolsulfonyl-.beta.-D-glucopyranoside (116; 100 mg,
287.05 .mu.mol) and 3-Methoxy-4-phenoxybenzoic acid (105.17 mg,
430.58 mmol).
Yield: 58 mg (48.1%)
[0538] LC/MS (ES-API): m/z=465.14 [M-H+formic acid].sup.-;
calculated: 465.17, tR (.lamda.=220 nm): 1.53 min (LC/MS-Method 2)
.sup.1H NMR (400.23 MHz, DMSO-d6) d ppm 7.64 (d, J=1.96 Hz, 1 H),
7.59 (dd, J=8.31, 1.96 Hz, 1H), 7.37 (t, J=7.64, 7.64 Hz, 2H), 7.13
(t, J=7.13, 7.13 Hz, 1H), 7.04 (d, J=8.44 Hz, 1H), 6.96 (d, J=7.84
Hz, 2H), 5.24 (d, J=5.01 Hz, 1H), 5.11 (d, J=4.89 Hz, 1H), 5.05 (d,
J=4.03 Hz, 1H), 4.59 (dd, J=11.74, 1.96 Hz, 1H), 4.29 (dd, J=11.74,
6.48 Hz, 1H), 4.12 (d, J=7.70 Hz, 1H), 3.83 (s, 3H), 3.47 (u), 3.20
(m, 3H), 3.00 (m, 1H).
EXAMPLE 122
Methyl-6-O-(1-benzyl-3-bromo-2-oxo-1,2-dihydropyridinyl-4-carbonyl)-.beta.-
-D-glucopyranoside
##STR00146##
[0540]
Methyl-6-O-(1-benzyl-3-bromo-2-oxo-1,2-dihydropyridinyl-4-carbonyl)-
-.beta.-D-glucopyranoside was synthesized as described for example
117 from Methyl-6-O-p-toluolsulfonyl-.beta.-D-glucopyranoside (116;
100 mg, 287.05 .mu.mol) and
1-benzyl-3-bromo-2-oxo-1,2-dihydropyridine-4-carboxylate (400 mg,
649.07 .mu.mol).
Yield: 8 mg
[0541] LC/MS (ES-API): m/z=484.09 [M+H].sup.+; calculated: 484.06,
tR (.lamda.=220 nm): 1.33 min (LC/MS-Method 2)
EXAMPLE 123
Methyl-2-O-(3-methoxy-4-phenoxyphenylcarbonyl)-.alpha.-D-glucopyranoside
##STR00147##
[0543]
Methyl-2-O-(3-methoxy-4-phenoxyphenylcarbonyl)-.alpha.-D-glucopyran-
oside was synthesized according to a method described by Muramatsu
and Takemoto from Methyl-.alpha.-D-glucopyranoside and
3-Methoxy-4-phenoxybenzoic acid.
[0544] 3-Methoxy-4-phenoxybenzoic acid (182.27 mg, 669.47 .mu.mol)
is suspended in CH.sub.2Cl.sub.2 (3 mL) under Argon atmosphere.
1-Chloro-N,N,2-trimethylpropenylamine (88.57 .mu.l, 669.47 .mu.mol)
is added and the reaction mixture stirred for 20 min at 20.degree.
C. to yield the corresponding carboxylic acid chloride.
[0545] A solution of Methyl-.alpha.-D-glucopyranoside (97.05 mg,
257.49 .mu.mol) and Dibutyltinndichloride (16.47 mg, 51.50 .mu.mol)
in THF (3 mL) is stirred for 15 min. Tetrabutylammonium iodide
(97.05 mg, 257.49 .mu.mol) and Diisopropylethylammine (DIPEA;
113.85 .mu.l, 669.47 .mu.mol) are added. Subsequently the solution
of 3-Methoxy-4-phenoxybenzoic acid chloride in CH.sub.2Cl.sub.2 is
added, the reaction mixture stirred for 2 h and left for 18 h at
20.degree. C.
[0546] The reaction is quenched with NH4Cl-solution and the product
extracted with EtOAc (3.times.5 mL). The product is finally
purified by HPLC and freece dried.
Yield: 55 mg (25.4%)
[0547] LC/MS (ES-API): m/z=465.0 [M-H+formic acid].sup.-[M+H
t.sub.R (.lamda.=220 nm): 1.73 min (LC/MS-Method 2) .sup.1H NMR
(400.23 MHz, DMSO-d6) d ppm 7.66 (m, 1H), 7.62 (d, J=1.83 Hz, 1H),
7.37 (m, 2H), 7.13 (m, 1H), 7.06 (d, J=8.46 Hz, 1H), 6.95 (d,
J=7.82 Hz, 2H), 4.88 (d, J=3.67 Hz, 1H), 4.64 (dd, J=9.96, 3.61 Hz,
1H), 3.84 (s, 3H), 3.78 (m, 1H), 3.69 (br d, J=10.15 Hz, 1H), 3.52
(br dd, J=11.80, 5.56 Hz, 2H), 3.44 (u), 3.28 (s, 5H), 3.25 (m,
1H), 2.50 (u), 2.33 (s, 1H)
EXAMPLE 124
Methyl-2-O-(3-chloro-3'-methoxy-4-phenoxyphenylcarbonyl)-.alpha.-D-glucopy-
ranoside
##STR00148##
[0549]
Methyl-2-O-(3-chloro-3'-methoxy-4-phenoxyphenylcarbonyl)-.alpha.-D--
glucopyranoside was synthesized as described for example 123 from
Methyl-.alpha.-D-glucopyranoside (100 mg, 514.98 .mu.mol) and
3-Chloro-3'-methoxy-4-phenoxybenzoic acid (124.24 mg, 669.47
.mu.mol).
Yield: 124 mg (50%)
[0550] LC/MS (ES-API): m/z=499.0 [M-H+formic acid].sup.-t.sub.R
(.lamda.=220 nm): 1.94 min (LC/MS-Method 2) .sup.1H NMR (400.23
MHz, DMSO-d6) d ppm 7.80 (d, J=1.83 Hz, 1H), 7.67 (d, J=1.71 Hz,
1H), 7.31 (t, J=8.01, 8.01 Hz, 2H), 7.05 (t, J=7.40, 7.40 Hz, 1H),
6.82 (d, J=7.95 Hz, 2H), 5.42 (br s, 1H), 5.22 (br s, 1H), 4.91 (d,
J=3.55 Hz, 1H), 4.63 (m, 1H), 3.82 (s, 3H), 3.79 (m, 1H), 3.69 (br
d, J=10.27 Hz, 1H), 3.53 (dd, J=11.80, 5.56 Hz, 1H), 3.44 (u), 3.29
(s, 5H), 3.27 (m, 1H), 3.20 (br s, 1H), 2.33 (s, 1H)
Synthesis of Allyl-6-O-toluenesulfonyl-.beta.-D-glucopyranoside
##STR00149##
[0552] Allyl-6-O-toluenesulfonyl-.beta.-D-glucopyranoside was
synthesized according to a published procedure [R. Brisco et al,.
Carb. Res. 348 (2012), 27-32] starting from commercially available
allyl-.beta.-D-glucopyranoside.
[0553] To a solution of allyl-.beta.-D-glucopyranoside (1 g, 4.54
mmol) in pyridine (30 mL) was added p-toluensulfonylchloride ((1.47
g, 7.72 mmol) at 0.degree. C. The reaction mixture was stirred for
30 min and then stored at 0.degree. C. for 16 h. The reaction was
controlled by TLC (9:1, CH.sub.2Cl.sub.2/MeOH) proving consumption
of starting material. The reaction mixture was quenched with MeOH
and the solvents were removed under reduced pressure. The Product
was purified by flash chromatography (EtOAc/MeOH, 9:1).
Yield: 850 mg (51%)
EXAMPLE 125
[0554]
Allyl-6-O-(3-phenoxyphenylcarbonyl)-.beta.-D-glucopyranoside
##STR00150##
[0555] Allyl-6-O-(3-phenoxyphenylcarbonyl)-.beta.-D-glucopyranoside
was synthesized from
allyl-6-O-toluenesulfonyl-.beta.-D-glucopyranoside (200 mg, 534.18
.mu.mol) and 3-phenoxyphenylcarboxylic acid (171.65 mg, 801.27
.mu.mol) as described for example 117.
Yield: 113 mg (50.8%)
[0556] LC/MS (ES-API): m/z=461.25 [M-H+formic acid].sup.-;
calculated: 461.18 t.sub.R (.lamda.=220 nm): 1.69 min (LC/MS-Method
2) .sup.1H NMR (400.23 MHz, DMSO-d6) d ppm 7.73 (d, J=7.44 Hz, 1
H), 7.57 (t, J=7.95, 7.95 Hz, 1H), 7.48 (s, 1H), 7.43 (t, J=7.27,
7.27 Hz, 2H), 7.33 (ddd, J=8.19, 2.57, 0.98 Hz, 1H), 7.20 (m, 1H),
7.07 (d, J=7.78 Hz, 2H), 5.85 (m, 1 H), 5.23 (m, 2H), 5.09 (m, 3H),
4.54 (dd, J=11.74, 1.96 Hz, 1H), 4.28 (dd, J=11.74, 6.60 Hz, 1H),
4.16 (m, 2H), 3.98 (m, 1H), 3.44 (u), 3.17 (m, 2H), 3.01 (td,
J=8.34, 8.34, 4.95 Hz, 1H).
EXAMPLE 126
Allyl-6-O-(4-phenoxyphenylcarbonyl)-.beta.-D-glucopyranoside
##STR00151##
[0558] Allyl-6-O-(4-phenoxyphenylcarbonyl)-.beta.-D-glucopyranoside
was synthesized from
allyl-6-O-toluenesulfonyl-.beta.-D-glucopyranoside (200 mg, 534.18
.mu.mol) and 3-phenoxyphenylcarboxylic acid (171.65 mg, 801.27
.mu.mol) as described for example 117.
Yield: 116 mg (52.1%)
[0559] LC/MS (ES-API): m/z=461.20 [M-H+formic acid].sup.-;
calculated: 461.18 t.sub.R (.lamda.=220 nm): 1.70 min (LC/MS-Method
2) .sup.1H NMR (400.23 MHz, DMSO-d6) d ppm 7.98 (m(para), 2 H),
7.46 (t, J=7.26, 7.26 Hz, 2H), 7.25 (t, J=7.47, 7.47 Hz, 1H), 7.13
(d, J=7.84 Hz, 2H), 7.08 (m(para), 2 H), 5.87 (m, 1H), 5.26 (m,
2H), 5.09 (br dd, J=10.39, 1.83 Hz, 2H), 4.53 (dd, J=11.74, 1.96
Hz, 1H), 4.22 (m, 3H), 4.02 (m, 1H), 3.46 (u), 3.20 (m, 4 H), 3.03
(m, 1H).
Synthesis of Trimethylsilyethoxy-6-O-tosyl-.beta.-D-glucopyranoside
LCMS Conditions
LCMS-Condition 01: Method:-- LCMS_X-Select (Formic Acid)
[0560] Column: X-Select CSH C18 (4.6*50) mm 2.5u, Mobile Phase:
A.0.1% Formic acid in water B. 0.1% Formic acid in Acetonitrile Inj
Volume; 5.0 .mu.L, Flow Rate: 1.0 mL/minute, Gradient program: 2% B
to 98% B in 2.8 minute, Hold till 4.8 min, At 5.0 min B conc is 2%
up to 7.0 min.
ELSD Conditions:
ELSD-Condition 01: Method:-LCMS_X-Bridge (NH.sub.3)
[0561] Column :X-Bridge C18 (4.6*50)mm 3.5 .mu.; Mobile Phase: A.
0.05% NH3 in water. B: 0.05% NH3 in Acetonitrile Inj Volume; 0.2
.mu.L, Flow Rate: 1.200 mL/minute; Gradient program: 2% B to 100% B
in 3.5 minute, Hold till 4.5 min, At 4.7 min B conc is 2% up to 6.0
min.
Step-1: Synthesis of
(2R,3R,4S,5R,6R)-6-(acetoxymethyl)tetrahydro-2H-pyran-2,3,4,5-tetrayl
tetraacetate (2)
##STR00152##
[0563] To (2S,
3R,4S,5S,6R)-6-(hydroxymethyl)tetrahydro-2H-pyran-2,3,4,5-tetraol 1
(50 g, 277.7 mmol) in pyridine (500 mL) at 0.degree. C. was added
DMAP (339 mg, 2.777 mmol) and acetic anhydride (500 mL). The
reaction mixture was further stirred at room temperature for 20 h.
After completion of the reaction, the pyridine was evaporated under
reduced pressure and the residue was diluted with water and
extracted with CH.sub.2Cl.sub.2 (thrice). The combined organic
layer was dried over anhydrous Na.sub.2SO.sub.4, filtered and
concentrated under reduced pressure. The crude compound was
purified by trituration with n-hexane to afford 100 g (92% yield)
of compound 2 as off white solid.
ELSD-Condition-1: [M+H].sup.+=408.00; R.sub.t=2.88 min .sup.1H NMR
(400 MHz, CDCl.sub.3) .delta.: 6.33 (d, J=3.91 Hz, 1H), 5.47 (t,
J=10.03 Hz, 1H), 5.07-5.17 (m, 2H), 4.24-4.29 (m, 1H), 4.08-4.15
(m, 2H), 2.18 (s, 3H), 2.09 (s, 3H), 2.04 (s, 3H), 2.03 (s, 3H),
2.02 (s, 3H).
Step-2: Synthesis of
(2R,3R,4S,5R,6R)-2-(acetoxymethyl)-6-bromotetrahydro-2H-pyran-3,4,5-triyl
triacetate (3)
##STR00153##
[0565] To
(2R,3R,4S,5R,6R)-6-(acetoxymethyl)tetrahydro-2H-pyran-2,3,4,5-te-
trayl tetraacetate 1 (25 g, 64.04 mmol) in CH.sub.2Cl.sub.2 (500
mL) at 0.degree. C. was added 33% solution of HBr in acetic acid
(250 mL) and stirred for 4 h. After completion of the reaction, the
reaction mixture was poured over ice and the organic layer was
separated and washed with water (400 mL) followed by saturated
NaHCO.sub.3 (200 mL.times.2). The combined organic layer was dried
over anhydrous Na.sub.2SO.sub.4, filtered and concentrated under
reduced pressure. The crude compound was purified by silica gel
column chromatography eluting with 0-50% ethyl acetate in n-hexane
to afford 24 g (89% yield) of compound 3 as off white solid.
.sup.1H NMR (400 MHz, CDCl.sub.3): 6.61 (d, J=3.91 Hz, 1 H), 5.56
(t, J=9.54 Hz, 1 H), 5.17 (t, J=9.78 Hz, 1H), 4.84 (dd, J=3.91,
10.27 Hz, 1H), 4.28-4.36 (m, 2H), 4.10-4.16 (m, 1H), 2.10 (s, 3H),
2.10 (s, 3H), 2.06 (s, 3H), 2.04 (s, 3H).
Step-3: Synthesis of
(2R,3R,4S,5R,6R)-2-(acetoxymethyl)-6-(2-(trimethylsilyl)ethoxy)tetrahydro-
-2H-pyran-3,4,5-triyl triacetate (4)
##STR00154##
[0567] To a stirred solution of HgO (12.1 g, 55.93 mmol),
HgBr.sub.2 (700 mg catalytic), CaSO.sub.4 (15.2 g, 111.86 mmol) and
2-(trimethylsilyl)ethan-1-ol (9.9 g, 83.90 mmol) in CHCl.sub.3 (184
mL) was added
(2R,3R,4S,5R,6R)-2-(acetoxymethyl)-6-bromotetrahydro-2H-pyran-3-
,4,5-triyltriacetate 3 (23 g, 55.93 mmol) and stirred at room
temperature for 1 h. After completion of the reaction, the reaction
mixture was filtered through a pad of Celite and washed with
CH.sub.2Cl.sub.2 and washed with saturated NaHCO.sub.3 solution.
The combined organic layer was dried over anhydrous
Na.sub.2SO.sub.4, filtered and concentrated under reduced pressure.
The crude compound was purified by silica gel column chromatography
eluting with 0-30% ethyl acetate in n-hexane to afford 20 g (83%
yield) of compound 4 as colorless oil.
.sup.1H NMR (400 MHz, CDCl.sub.3): 5.19 (dd, J=8.07, 9.54 Hz, 1 H),
5.08 (dd, J=8.07, 9.54 Hz, 1H), 4.97 (dd, J=8.07, 9.54 Hz, 1H),
4.51 (d, J=8.31 Hz, 1H), 4.23-4.29 (m, 1H), 4.09-4.16 (m, 2H), 3.97
(dt, J=5.87, 10.03 Hz, 1H), 3.66-3.76 (m, 2 H), 3.52-3.60 (m, 1H),
2.08 (s, 2H), 2.03-2.04 (m, 3H), 2.02 (s, 3 H), 2.00 (s, 3H), 1.25
(t, J=7.09 Hz, 1H), 0.85-0.98 (m, 3H), 0.02 (s, 3H), 0.00 (s,
6H).
Step-4: Synthesis of
(2R,3S,4S,5R,6R)-2-(hydroxymethyl)-6-(2-(trimethylsilyl)ethoxy)tetrahydro-
-2H-pyran-3,4,5-triol (5)
##STR00155##
[0569] To
(2R,3R,4S,5R,6R)-2-(acetoxymethyl)-6-(2-(trimethylsilyl)ethoxy)t-
etrahydro-2H-pyran-3,4,5-triyltriacetate 4 (10 g, 22.32 mmol) in
methanol (100 mL) was added solution of sodium methoxide (100 mg
sodium in 7.5 mL methanol) and stirred at room temperature for 12
h. After completion of the reaction, the pyridine was evaporated
under reduced pressure and the residue was stirred in diethyl
ether. The solid precipitated out was filtered and dried to afford
4.2 g (67% yield) of 5 as off white solid.
.sup.1H NMR (400 MHz, D.sub.2O) .delta.: 4.34 (d, J=7.83 Hz, 1H),
3.87-3.96 (m, 1H), 3.78 (d, J=11.74 Hz, 1H), 3.55-3.68 (m, 2H),
3.22-3.38 (m, 4H), 3.11 (t, J=8.56 Hz, 1H), 0.80-1.00 (m, 2H),
-0.10 (s, 9H).
Step-5: Synthesis of
((2R,3S,4S,5R,6R)-3,4,5-trihydroxy-6-(2-(trimethylsilyl)ethoxy)tetrahydro-
-2H-pyran -2-yl)methyl 4-methyl benzenesulfonate (2016-00144)
##STR00156##
[0571] To
(2R,3S,4S,5R,6R)-2-(hydroxymethyl)-6-(2-(trimethylsilyl)ethoxy)t-
etrahydro-2H-pyran-3,4,5-triol 5 (9.5 g, 33.92 mmol) in pyridine
(95 mL) was added DMAP (413 mg, 3.392 mmol) and tosyl chloride (7.1
g, 37.32 mmol) stirred at room temperature for 12 h. After
completion of the reaction, the pyridine was evaporated under
reduced pressure and the residue was diluted with water and
extracted with ethyl acetate. The combined organic layer was dried
over anhydrous Na.sub.2SO.sub.4, filtered and concentrated under
reduced pressure. The crude compound was purified by silica gel
column chromatography eluting with 0-10% methanol in
CH.sub.2Cl.sub.2 to afford 10.2 g (69% yield) of 2016-00144 as off
white solid.
LCMS-Condition-1: [M+18].sup.+=452.15; R.sub.t=1.78 min .sup.1H NMR
(400 MHz, DMSO-d.sub.6) .delta.: 7.76 (d, J=8.31 Hz, 2H), 7.47 (d,
J=8.31 Hz, 2H), 5.16 (d, J=5.38 Hz, 1H), 4.99 (dd, J=4.89, 9.29 Hz,
2H), 4.21 (d, J=10.27 Hz, 1H), 4.12 (d, J=7.83 Hz, 1H), 4.02 (dd,
J=6.60, 10.03 Hz, 1H), 3.69-3.79 (m, 1H), 3.43-3.52 (m, 1H), 3.35
(br. s, 1H), 3.05-3.13 (m, 1H), 2.95-3.03 (m, 1H), 2.84-2.92 (m,
1H), 2.42 (s, 3H), 0.82-0.95 (m, 2H), 0.00 (s, 9H).
EXAMPLE 127
Trimethylsilyethyl-6-O-(2-methyl-3-phenoxyphenylcarbonyl)-.beta.-D-glucopy-
ranoside
##STR00157##
[0573]
Trimethylsilyethyl-6-O-(2-methyl-3-phenoxyphenylcarbonyl)-.beta.-D--
glucopyranoside was syntzhesized as described for example 117 from
trimethylsilyethoxy-6-O-tosyl-.beta.-D-glucopyranoside (100 mg,
230.11 .mu.mol) and 2-methyl-3-phenoxybenzoic acid (78.78 mg
(345.16 .mu.mol).
Yield: 63 mg (55.8%)
[0574] LC/MS (ES-API): m/z=535.23 (M-H+formic acid].sup.-;
calculated: 535.23; tR (.lamda.=220 nm): 1.94 min (LC/MS-Method 2)
.sup.1H NMR (400.23 MHz, DMSO-d6) d ppm 7.95 (d, J=8.68 Hz, 1 H),
7.52 (t, J=7.23, 7.23 Hz, 2H), 7.31 (t, J=7.14, 7.14 Hz, 1H), 7.17
(d, J=7.82 Hz, 2H), 7.00 (d, J=2.32 Hz, 1H), 6.89 (dd, J=8.68, 2.45
Hz, 1H), 5.28 (br s, 1H), 5.07 (br s, 2H), 4.60 (dd, J=11.68, 2.02
Hz, 1H), 4.33 (dd, J=11.68, 7.15 Hz, 1H), 4.27 (d, J=7.82 Hz, 1H),
3.84 (ddd, J=11.06, 9.78, 6.05 Hz, 1H), 3.55 (m, 2H), 3.24 (quin,
J=8.71, 8.71, 8.71, 8.71 Hz, 2H), 3.05 (t, J=8.19, 8.19 Hz, 1H),
2.58 (m, 10H), 2.50 (u), 0.95 (m, 2H).
EXAMPLE 128
Trimethylsilyethyl-6-O-(3-chloro-3'-methoxy-4-phenoxyphenylcarbonyl)-.beta-
.-D-glucopyranoside
##STR00158##
[0576]
trimethylsilyethyl-6-O-(3-chloro-3'-methoxy-4-phenoxyphenylcarbonyl-
)-.beta.-D-glucopyranoside was synthesized as described for example
117 from trimethylsilyethoxy-6-O-tosyl-.beta.-D-glucopyranoside
(100 mg, 230.11 .mu.mol) and 2-methyl-3-phenoxybenzoic acid (78.78
mg, 345.16 .mu.mol).
Yield: 64 mg (51.4%)
[0577] LC/MS (ES-API): m/z=585.15 [M-H+formic acid].sup.-;
calculated: 585.19 t.sub.R (.lamda.=220 nm): 1.92 min (LC/MS-Method
2) .sup.1H NMR (400.23 MHz, DMSO-d6) d ppm 7.79 (d, J=1.83 Hz, 1H),
7.72 (d, J=1.83 Hz, 1H), 7.39 (t, J=7.24, 7.24 Hz, 2H), 7.13 (t,
J=7.34, 7.34 Hz, 1H), 6.88 (d, J=7.82 Hz, 2H), 5.34 (br s, 1H),
5.10 (br s, 1H), 4.66 (dd, J=11.62, 1.96 Hz, 1H), 4.45 (dd,
J=11.68, 7.15 Hz, 1H), 4.30 (d, J=7.82 Hz, 1H), 3.89 (s, 3H), 3.84
(m, 1 H), 3.60 (m, 2H), 3.56 (s, 1H), 3.26 (m, 2H), 3.07 (br t,
J=8.13, 8.13 Hz, 1H), 2.58 (dt, J=3.61, 1.74, 1.74 Hz, 16H), 2.50
(u), 0.95 (m, 2H), 0.08 (s, 1H)
EXAMPLE 129
Trimethylsilyethyl-6-O-(3-methoxy-4-phenoxyphenylcarbonyl)-.beta.-D-glucop-
yranoside
##STR00159##
[0579]
Trimethylsilyethyl-6-O-(3-methoxy-4-phenoxyphenylcarbonyl)-.beta.-D-
-glucopyranoside was synthesized as described for example 117 from
trimethylsilyethoxy-6-O-toluenesulfonyl-.beta.-D-glucopyranoside
(100 mg, 230.11 .mu.mol) and 2-methyl-3-phenoxybenzoic acid (84.31
mg, 345.16 .mu.mol).
Yield: 73 mg (52.6%)
[0580] LC/MS (ES-API): m/z=551.16 [M-H+formic acid].sup.-;
calculated: 551.23 tR (.lamda.=220 nm): 1.84 min (LC/MS-Method
2)
EXAMPLE 130
6-O-(3-phenoxyphenylcarbonyl)-D-glucopyranose
##STR00160##
[0582] To a solution of
allyl-6-O-(3-phenoxyphenylcarbonyl)-.beta.-D-glucopyranoside
(Example 125; 110 mg, 264.15 .mu.mol) in MeOH (2 mL) Pd
(II)chloride (9.37 mg, 52.83 .mu.mol) was added. After 3 h at
25.degree. C. the reaction was controlled by LC/MS. A product with
the desired mass could be detected. MeOH was added (4 mL) and the
product was purified by HPLC.
Yield: 51 mg (53%, mixture of anomers) LC/MS (ES-API): m/z=375.14
[M-H+formic acid].sup.-; calculated: 375.12 tR (.lamda.=220 nm):
1.45/1.43 min (Mixtuure of anomers, LC/MS-Method 2) .sup.1H NMR
(400.23 MHz, DMSO-d6) d ppm 7.72 (m,1 H), 7.48 (m, 4 H), 7.31(ddd,
J=8.10, 2.54, 0.86Hz, 1H), 7.21 (m, 1H),7.08 (m, 2H), 4.91
(d,J=3.55 Hz, 1H), 4.52 (dd,J=11.74, 1.83 Hz, 1H),4.47 (dd,
J=11.68, 1.90 Hz, 1H), 4.31 (m, 2H), 3.88 (m, 1H), 3.48 (br s, 4H),
3.16 (m, 4H).
EXAMPLE 131
6-O-(4-Phenoxyphenylcarbonyl)-D-glucopyranose
##STR00161##
[0584] 6-O-(4-phenoxyphenylcarbonyl)-D-glucopyranose was
syntzhesized as described for example 129 from
Allyl-6-O-(4-phenoxyphenylcarbonyl)-.beta.-D-glucopyranoside
(Example 126; 110 mg, 264.15 mg) as described for Example 129.
Yield: 46 mg (46.3%, mixture of anomers) LC/MS (ES-API): m/z=375.09
[M-H].sup.-; calculated: 375.12 tR (.lamda.=220 nm): 1.46/1.44 min
(Mixture of anomers, LC/MS-Method 2)
EXAMPLE 132
6-O-(2-Methyl-4-phenoxyphenylcarbonyl)-D-glucopyranose
##STR00162##
[0586] To a solution of
Trimethylsilyethyl-6-O-(2-methyl-3-phenoxyphenylcarbonyl)-.beta.-D-glucop-
yranoside (Example 127, 60 mg, 122.29 .mu.mol) in CH.sub.2Cl.sub.2
(1. 8 mL) TFA (200 .mu.l, 2.60 mmol) was added under Argon
atmosphere wurde. After 5 h LC/MS analysis showed consumption of
starting material and one new peak could be detected. The reaction
mixture was diluted with Water and freeze dried. The product was
purified by HPLC.
Yield: 42 mg (88%mixture of anomers) LC/MS (ES-API): m/z=389.16
[M-H].sup.-; calculated: 389.13 tR (.lamda.=220 nm): 1.53/1.51 min
(Mixture of anomers, LC/MS-Method 2)
EXAMPLE 133
6-O-(3-Chloro-3'methyloxy-4-phenoxyphenylcarbonyl)-D-glucopyranose
##STR00163##
[0588]
6-O-(3-Chloro-3'methyloxy-4-phenoxyphenylcarbonyl)-D-glucopyranose
was synthesized from
trimethylsilyethyl-6-O-(3-chloro-3'-methoxy-4-phenoxyphenylcarbonyl)-.bet-
a.-D-glucopyranoside (128; 60 mg, 110.89 .mu.mol) as described for
example 131
Yield: 42 mg (85.9%, mixture of anomers) LC/MS (ES-API):
m/z=485.1/487.0 [M-H+formic acid].sup.-; calculated: 485.12 tR
(.lamda.=220 nm): 1.55/1.53 min (Mixture of anomers, LC/MS-Method
2)
EXAMPLE 134
6-O-(4-Hydroxy-2-methyl-phenylcarbonyl)-D-glucopyranose
##STR00164##
[0590]
6-O-(3-Chloro-3'methyloxy-4-phenoxyphenylcarbonyl)-D-glucopyranose
was synthesized from
trimethylsilyethyl-6-O-(4-hydroxy-2-methylphenyl)carbonyl-.beta.-D-glucop-
yranoside (21 mg, 50.66 .mu.mol) as described for example 131
Yield: 16 mg (quantitative, mixture of anomers) LC/MS (ES-API):
m/z=313.06 [M-H].sup.-; calculated: 313.10 tR (.lamda.=220 nm):
0.72/0.64 min (Mixture of anomers, LC/MS-Method 2) .sup.1H NMR
(400.23 MHz, DMSO-d6) d ppm 10.11 (d, J=4.77 Hz, 1 H), 7.75 (d,
J=7.58 Hz, 1H), 6.67 (m, 2H), 6.32 (br d, J=3.55 Hz, 1H), 5.14 (br
s, 1H), 5.08 (br s, 1H), 4.92 (br s, 1H), 4.74 (br s, 1H), 4.50 (br
s, 1H), 4.43 (ddd, J=17.76, 11.71, 1.71 Hz, 1H), 4.33 (br s, 1H),
4.20 (m, 1H), 3.87 (m, 1H), 3.48 (u), 3.16 (m, 3H), 2.92 (br t,
J=8.19, 8.19 Hz, 1H).
EXAMPLE 135
6-O-(3-Methyloxy-4-phenoxyphenylcarbonyl)-D-glucopyranose
##STR00165##
[0592] 6-O-(3-Methyloxy-4-phenoxyphenylcarbonyl)-D-glucopyranose
was synthesized from
trimethylsilyethyl-6-O-(3-chloro-3'-methoxy-4-phenoxyphenylcarbonyl)-.bet-
a.-D-glucopyranoside (128; 60 mg, 110.89 .mu.mol) as described for
example 131
Yield: 42 mg (85.9%, mixture of anomers) LC/MS (ES-API): m/z=405.09
[M-H].sup.-; calculated: 405.13 tR (.lamda.=220 nm): 1.43/1.41 min
(Mixture of anomers, LC/MS-Method 2) .sup.1H NMR (400.23 MHz,
DMSO-d6) d ppm 7.64 (m, 1H), 7.58 (d, J=8.60 Hz, 1H), 7.37 (t,
J=7.64, 7.64 Hz, 2H), 7.13 (t, J=7.04, 7.04 Hz, 1H), 7.06 (d,
J=8.58 Hz, 1H), 6.95 (d, J=8.44 Hz, 2H), 4.93 (d, J=3.42 Hz, 1H),
4.51 (m, 1H), 4.32 (m, 1H), 3.91 (br dd, J=9.90, 3.91 Hz, 1H), 3.84
(s, 3H), 3.49 (br s, 2H), 3.46 (br d, J=9.17 Hz, 3H), 3.18 (m, 5H),
2.93 (br t, J=8.19, 8.19 Hz, 1H), 2.50 (u), 2.33 (br s, 1H)
EXAMPLE 136
1,2:3,4-Diisopropyliden-6-O-(3-Methoxy-4-phenoxy)-phenylcarbonyl-D-glucopy-
ranose
##STR00166##
[0593] Lit: Angew. Chem. Int. Ed. 2008, 47, 8264 -8267
[0594] A solution of 3-Methoxy-4-phenoxybenzoic acid (93,84 mg,
384, 19 .mu.mol) in CH2Cl2 (1,5 ml) was stirred under argon
atmosphere at 0.degree. C. Consecutively a solution of
1,2:3,4-Di-O-isopropyliden-D-galactose (100 mg, 384.19 .mu.mol) in
DMF (1 mL), DMAP (9.39 mg, 76.84 .mu.mol) and finally DCC (79.27
mg, 384.19 .mu.mol) where added and the reaction was kept at
0.degree. C. for 10 min. The reaction mixture was left at
25.degree. C. for 16 h. The desired mass could be detected by
LC/MS. The reaction mixture was diluted with MeOH (2 mL) filtrated
and the product finally purified by HPLC,
Yield: 154 mg (82.4%)
[0595] LC/MS (ES-API): m/z=487.20 [M-H].sup.-; calculated: 487.19
tR (.lamda.=220 nm): 2.72 min (LC/MS-Method 2) .sup.1H NMR (400.23
MHz, DMSO-d6) d ppm 7.63 (d, J=1.71 Hz, 1H), 7.57 (dd, J=8.38, 0.90
Hz, 1H), 7.38 (t, J=7.95, 7.95 Hz, 2H), 7.14 (t, J=7.16, 7.16 Hz,
1H), 7.00 (m, 3H), 0.48 (d, J=5.01 Hz, 1H), 4.65 (dd, J=7.95, 2.32
Hz, 1H), 4.36 (m, 4H), 4.12 (m, 1H), 3.83 (s, 3H), 3.46 (u), 2.50
(u), 1.44 (s, 3H), 1.39 (s, 3H), 1.31 (s, 3H), 1.28 (s, 3H)
EXAMPLE 137
6-O-(3-Methoxy-4-phenoxyphenylcarbonyl)-D-glucopyranose
##STR00167##
[0597]
1,2:3,4-Diisopropyliden-6-O-(3-methoxy-4-phenoxyphenylcarbonyl)-D-g-
lucopyranose (154 mg, 316.54 pmol) was dissolved in acetonitril.
Under argon atmosphere HCl (2.37 ml, 4.75 mmol) was added and the
Reaction mixture was stirred at 40.degree. C. for 16 h.
[0598] Water was added and the product was freeze dried. The
product was finally purified by HPLC.
Yield: 51 mg (39.6%) (Mixture of Anomers)
[0599] LC/MS (ES-API): m/z=389.1 [M+H-H.sub.2O]+; calculated:
389.11 tR (.lamda.=220 nm): 1.58/1.60 min (LC/MS-Method 2)
EXAMPLE 138
6-O-(3-Chloro-3'Methoxy-4-phenoxyphenylcarbonyl)-D-glucopyranose
##STR00168##
[0601]
6-O-(3-Chloro-3'Methoxy-4-phenoxyphenylcarbonyl)-D-glucopyranose
was synthesized as described for example 136 from
1,2:3,4-Diisopropyliden-6-O-(3-chloro-3'methoxy-4-phenoxy)phenylcarbonyl--
D-glucopyranose (150 mg, 287.93 .mu.mol).
Yield: 93 mg (73. 3%).
[0602] LC/MS (ES-API): m/z=423.1/425.1 [M+H--H.sub.2)].sup.+;
calculated: 423.07 tR (.lamda.=220 nm): 1.76/1.78 min (LC/MS-Method
2)
EXAMPLE 139
Step 1: Amide Coupling
4-(2,4-Dichlorophenyl)piperazin-1yl-(1,2:3,4-diisopropyliden)-D-galacturon-
ic acid amide
##STR00169##
[0604] A solution of 1,2:3,4-Diisopropyliden-D-galacturonic acid
(150 mg, 546.91 .mu.mol) in DMF (5 ml) under argon atmosphere was
treated with HATU (291.14 mg, 765.67 .mu.mol) and the reaction
mixture stirred for 5 min. 1-(2,4-dichlorophenyl)piperazine (176.96
mg, 765.67 .mu.mol) was added and the reaction mixture was stirred
at 25.degree. C. The reaction was monitored by LCMS. After 24 h
starting material could be detected, DIPEA (2,2 equivalents) was
added and the reaction mixture left for another 24 h at 25.degree.
C. CH2Cl2 (1.times.10 ml) and water were added and the product
extracted. The organic layer was dried, the solvens evaporated and
the product purified by HPLC.
Yield: 163 mg (61.2%)
EXAMPLE 140
Step 2: Deprotection
4-(2,4-Dichlorophenyl)piperazin-1-yl-D-galacturonic acid amide
##STR00170##
[0606] A suspension of
4-(2,4-Dichlorophenyl)piperazin-1yl-(1,2:3,4-Diisopropyliden)-D-galacturo-
nic acid amide (163 mg, 334.45 .mu.mol) was treated with aqueous
HCl (2.51 ml, 5.02 mmol) under argon atmosphere and left for 16 h
at 25.degree. C. The reaction was monitored by LC/MS, starting
material could still be detected. The reaction mixture was
dissolved in MeOH and again treated with HCl (2.5 mL) and left at
25.degree. C. for 16 h. Methanol was evaporated, the residue
diluted with water and freeze dried. The product was purified by
HPLC.
Yield: 46 mg (28. 3%)
[0607] LC/MS (ES-API): m/z=407.1/409.0/411. [M+H]+; calculated:
407.24 tR (.lamda.=220 nm): 1.46/1.48 min (LC/MS-Method 2)
EXAMPLE 140
Step 1: Preparation of Substituted Amine
(R)-2-((benzyloxy)methyl)-4-chloro-piperazine
##STR00171##
[0608] Lit.: ACS Med. Chem. Lett., 2015, 6 (10), pp 1041-1046
[0609] A solution of tert-butyl
(R)-2-((benzyloxy)methyl)piperazine-1-carboxylate (333.98 mg, 1.09
mmol), 1-bromo-4-chlorobenzene (227.46 mg, 1.19 mmol) and BINAP
(40.72 mg, 65.40 .mu.mol) in toluene (5 ml) was treated with
Pd(II)acetate(9.79 mg, 43.60 .mu.mol) under argon atmosphere. KoTBu
(183.47 mg, 1.64 mmol) was added and the reaction mixture was
irradiated in the microwave for 30 min at 130.degree. C.
[0610] EtOAc and H.sub.2O where added, the reaction mixture
filtered, and the filtrate washed with brine. The solvens was
evaporated and the residue purified by HPLC.
Yield; 142 mg (41%)
Step 2: Reductive amination
6-Desoxy-6-((R)-2-((benzyloxy)methyl)-4-chlorophenyl)-piperazin-1-yl-(1,2:-
3,4-diisopropyliden)-D-galactopyranose
##STR00172##
[0612] To a solution of
(3aR,5S,5aR,8aS,8bR)-2,2,7,7-tetramethyltetrahydro-5H-bis([1,3]dioxolo)[4-
,5-b:4',5'-d]pyran-5-carbaldehyde (125.54 mg, 486.07 .mu.mol) in
methanol (2 ml),
(R)-3-((benzyloxy)methyl)-1-(4-chlorophenyl)piperazine (140 mg,
441.88 .mu.mol), acetic acid (50.59 .mu.l, 883.76 .mu.mol) and
sodiumcyanoborhydride (58.46 mg, 883.76 .mu.mol) are added. The
reaction mixture was left for 16 h at 25.degree. C. LCMS showed
consumption of starting material. The reaction mixture was
evaporated and the product purified by HPLC.
Yield: 105 mg (40%)
Step 3: Deprotection
Methyl-6-desoxy-6-((R)-2-((benzyloxy)methyl)-4-chlorophenyl)-piperazin-1-y-
l-D-galactopyranoside
##STR00173##
[0614] A suspension of 6-desoxy-
6-(4-chlorophenyl)piperazino-(1,2:3,4-diisopropyliden)-D-galactopyranose
(103 mg, 172,95 .mu.mol) in MeOH (2 mL) is treated with mit HCl
(1.73 ml, 3.46 mmol) and stirred for 2 h and left for 16 h at
25.degree. C.
Yield: 13 mg (12.4%)
[0615] LC/MS (ES-API): m/z=479.3/481.3. [M+H]+; calculated: 478.20
tR (.lamda.=220 nm): 1.37 (LC/MS-Method 2) 1H NMR (400.23 MHz,
DMSO-d6) d ppm 9.62 (br s, 1H), 7.35 (m, 8 H), 7.02 (br d, J=8.80
Hz, 2H), 5.05 (br s, 1H), 4.93 (br s, 1H), 4.58 (m, 3H), 4.38 (br
d, J=5.50 Hz, 1H), 4.28 (br d, J=10.03 Hz, 1H), 3.95 (br s, 1H),
3.89 (br d, J=10.64 Hz, 2H), 3.77 (m, 4H), 3.64 (br d, J=6.85 Hz,
1H), 3.57 (m, 4H), 3.17 (br s, 1H), 3.01 (m, 2H), 2.50 (u) 1H NMR
(250.13 MHz, DMSO-d6) d ppm 7.29 (m, 6H), 6.92 (m, 2H), 5.02 (m,
1H), 4.55 (s, 2H), 3.80 (m, 4H), 3.63 (m, 1H), 3.52 (br s, 2H),
3.18 (m, 7H), 2.93 (br s, 97H), 2.75 (br s, 4H), 2.50 (u).
SYNTHESIS OF EXAMPLE 141
Step 1: Reductive Amination
6-Desoxy-
6-(3-hydroxyphenyl)-piperazin-1-yl-(1,2:3,4-diisopropyliden)-D-g-
alactopyranose
##STR00174##
[0617]
(3aR,5S,5aR,8aS,8bR)-2,2,7,7-tetramethyltetrahydro-5H-bis([1,3]diox-
olo)[4,5-b:4',5'-d]pyran-5-carbaldehyde (239.09 mg, 925.74 .mu.mol)
was dissolved in MeOH (3 ml). 3-(piperazin-1-yl)phenol (150 mg,
841.59 .mu.mol), acetic acid, (96.36 .mu.l, 1.68 mmol) and NaCNBH3
(111.34 mg, 1.68 mmol) where added and the reaction mixture stirred
for 3 h and left for 16 h at 25.degree. C. The reaction was
monitored by LCMS. Starting material was consumed. The solvens was
evaoporated, the resuidue taken up in EtOAc/H2O and the product
extracted with EtOAc and finally purified by HPLC.
Yield: 280 mg (72.8%)
Step 2: Activation of Spacer
Undec-10-yn-1-yl 4-methylbenzenesulfonate
##STR00175##
[0618] Lit.: Tetrahedron 56 (2000) p1233-1245
[0619] Undec-10-yn-1-ol (343.17 .mu.l, 2.97 mmol) was dissolved in
CH.sub.2Cl.sub.2 (10 ml) under argon atmosphere.
4-Methylbenzenesulfonyl chloride (810.01 mg, 4.25 mmol) was added
and the reaction mixture cooled to 0.degree. C. Pyridine (355.67
.mu.l, 4.40 mmol) was added dropwise over 5 min and the reaction
mixture was stirred for 3 h at 0.degree. C. The ice bath was
removed and the solution was left for 16 h at 25.degree. C. The
reaction was monitored by LCMS. 1 N HCl was added and the product
extracted with CH.sub.2Cl.sub.2. The solvens was removed in vacuo
and the product purified by HPLC
Yield: 1.57 g (29.3%)
Step 3: Ether Coupling
6-Desoxy-
6-(3-undecenyloxyphenyl)-4-piperazin-1-yl-(1,2:3,4-diisopropylid-
en)-D-galactopyranose
##STR00176##
[0621] A mixture of 6-desoxy-
6-(3-hydroxyphenyl)-piperazin-1-yl-(1,2:3,4-diisopropyliden)-D-galactopyr-
anose Hydrochlorid (275 mg, 601.80 .mu.mol) and undec-10-yn-1-yl
4-methylbenzenesulfonate (445.78 mg, 842.52 .mu.mol) was treated
with Cesiumcarbonat (980.39 mg, 3.01 mmol). DMF (2mL) was added and
the reaction mixture was iiradiated in the microwave at 80.degree.
C. for 1 h. LCMS showed the expected mass. The product was
extracted with EtOAc/H.sub.2O, the organic layer washed with
aqueous NaCL solution (10%) and dried. The solvens was evaporated
in vacuo and the product purified by HPLC.
Yield: 105 mg (28.7%)
Step 3: Deprotection
EXAMPLE 141
6-Desoxy-
6-(3-undecenyloxypheny)-4-piperazin-1-yl-D-galactopyranose
##STR00177##
[0623] 6-Desoxy-
6-(3-undecenyloxyphenyl)-4-piperazin-1-yl-(1,2:3,4-diisopropyliden)-D-gal-
actopyranose (101 mg, 166,33 pmol) was treated with 2 n HCl (1.66
ml, 3.33 mmol) and then heated for 2 h at 80.degree. C. LCMS showed
consumption of starting material, the reaction mixture was diluted
with H.sub.2O and freeze dried.
Yield; 18 mg (20.5%)
SYNTHESIS OF EXAMPLE 142
6-Desoxy-
6-(1-(4-(2-(2-(2-(prop-2-yn-1-yloxy)ethoxy)ethoxy)ethoxy)phenyl)-
piperazin-1yl-D-galactopyranose
##STR00178##
[0625] The synthesis of example 142 was following the procedure
described for example 141 starting from
2-(2-(2-(prop-2-yn-1-yloxy)ethoxy)ethoxy)ethan-1-ol (500 mg, 2.66
mmol) and 4-methylbenzenesulfonyl chloride (759.63 mg, 3.98 mmol)
to yield the activated spacer
(2-(2-(2-(prop-2-yn-1-yloxy)ethoxy)ethoxy)ethyl
4-methyl-benzenesulfonate, yield: 420 mg, 46.2%).
[0626] In parallel 6-Desoxy-
6-(4-hydroxyphenyl)-piperazin-1-yl-(1,2:3,4-diisopropyliden)-D-galactopyr-
anose (269 mg, 639.71 pmol) was prepared and then coupled with
(2-(2-(2-(prop-2-yn-1-yloxy)ethoxy)ethoxy)ethyl
4-methyl-benzenesulfonate (262.85 mg, 767.65 .mu.mol) to yield.
6-desoxy-
6-(1-(4-(2-(2-(2-(prop-2-yn-1-yloxy)ethoxy)ethoxy)ethoxy)phenyl)piperazin-
-1yl-(1,2:3,4-diisopropyliden)-D-galactopyranose (180 mg, 47.6%),
which was then deprotected with aqueous HCl.
Yield: 130 mg (69.5%)
[0627] LC/MS (ES-API): m/z=511.38 [M+H]+; calculated: 510.25 tR
(.lamda.=220 nm): 0.98 (LC/MS-Method 2)
EXAMPLE 143
Biological Assays
1. HepG2 Assay:
Procedure
[0628] For measurement of .sup.14C 2-deoxy-D-glucose transport into
HepG2 cells, cells were seeded in collagen treated 96-well plates
(Cytostar-T Plates Perkin Elmer, 40.000 cells/200p/well) in medium
complete (MEM W/GLUT-I, EARLES (Gibco #41090)/NEAA/PS/10% FCS) and
grown for 48 hours. After starvation for 2 hours with MEM
W/GLUT-I,EARLES (Gibco #41090)/NEAA/no serum 100 .mu.l/well and
washing twice with 200 .mu.L KRB buffer, cells were stimulated in a
dose dependent manner by adding 20 .mu.L of test compound dilution
0-750 .mu.M (7.5 times higher concentration than final) or 20 .mu.L
of 188 .mu.M Cytochalasin B solution as negative control, to 80
.mu.L KRB buffer and incubated for 30 minutes. After compound
stimulation, the transport of .sup.14C 2-deoxy-D-glucose was
started by adding of 50 .mu.L .sup.14C 2-deoxy-D-glucose solution
(250 .mu.M 2-deoxy-D-glucose cold and 15 .mu.M .sup.14C
2-deoxy-D-glucose 0.13 .mu.Ci/well) for 20 minutes. Transport was
stopped by adding 50 .mu.L/well 40 .mu.M Cytochalasin B solution.
Plates were measured in a 96-well Wallac Microbeta device. The cpm
(counts per minute) values were used to determine the % inhibition
values for the test compounds within each experiment, which then
are averaged over the number of experiments performed.
TABLE-US-00041 Table of results: % inhibition of
.sup.14C-2-deoxy-glucose Example transport [100 .mu.M] SD 1 6.32
6.32 .+-. 3.81 2 6.50 6.50 .+-. 4.16 6 7.75 7.75 .+-. 3.28 7 6.99
6.99 .+-. 2.52 8 7.67 7.67 .+-. 5.12 9 5.11 5.11 .+-. 3.56 12 5.43
5.43 .+-. 6.31 14 5.67 5.67 .+-. 5.11 19 6.89 6.89 .+-. 6.28 21
7.53 7.53 .+-. 3.59 22 11.40 11.40 .+-. 3.95 27 13.06 13.06 .+-.
1.96 28 9.95 9.95 .+-. 1.83 29 18.44 18.44 .+-. 3.80 34 20.47 20.47
.+-. 3.07 36 20.63 20.63 .+-. 3.24 37 6.88 6.88 .+-. 1.71 38 12.70
12.70 .+-. 1.51 39 10.99 10.99 .+-. 4.75 40 10.08 10.08 .+-. 7.98
45 29.31 29.31 .+-. 4.46 46 8.76 8.76 .+-. 2.67 47 13.82 13.82 .+-.
5.23 48 28.82 28.82 .+-. 4.82 49 19.91 19.91 .+-. 3.87 50 13.84
13.84 .+-. 1.91 51 17.86 17.86 .+-. 6.82 52 14.55 14.55 .+-. 3.95
53 23.89 23.89 .+-. 5.32 54 10.48 10.48 .+-. 8.16 55 12.39 12.39
.+-. 2.89 56 21.08 21.08 .+-. 2.76 57 24.14 24.14 .+-. 5.94 58
19.98 19.98 .+-. 5.00 59 7.56 7.56 .+-. 4.50 60 14.75 14.75 .+-.
4.72 61 9.42 9.42 .+-. 6.75 63 37.62 37.62 .+-. 4.09 64 8.86 8.86
.+-. 3.67 65 7.79 7.79 .+-. 2.92 66 5.12 5.12 .+-. 6.07 67 25.77
25.77 .+-. 3.88 69 6.14 6.14 .+-. 6.61 75 31.19 31.19 .+-. 6.64 79
25.54 25.54 .+-. 3.49 88 31.37 31.37 .+-. 1.75 92 23.10 23.10
4.60
2. Glucose Displacement Assay (ATP Measurement)
TABLE-US-00042 [0629] Reagent Provider Catalogue n. CellTiter-Glo
.RTM. Luminescence Promega G-7571 Cell Viability Assay A2780 Human
Carcinoma Cell ECACC 93112519 line 96-well LIA plate, white Greiner
Bio-one 655073 RPMI 1640 medium GlutaMAX Thermo Fisher 61870
Scientitic RPMI 1640 medium (no glucose) Thermo Fisher 11879
Scientific Fetal Bovine Serum Pan Biotech P-30-3305 D-(+)-Glucose
solution Sigma Aldrich G-8644 PBS buffer Life Technologies 14190
KRB buffer PAN P05-32500* DMSO Sigma D-2650 Rotenone Sigma R-8875
*Customer Formulation, sterile filtered: 1.7 mM CaCl.sub.2 .times.
2H.sub.2O; 1.2 mM KH.sub.2PO.sub.4; 4.8 mM KCl; 1.2 mM MgSO.sub.4
.times. 7H.sub.2O; 120 mM NaCl; 26 mM NaHCO.sub.3
[0630] 30.000 A2780 Human Carcinoma Cells are seeded per well in a
Greiner 96-well plate. Cells are expanded and cultured in RPMI 1640
medium+GlutaMAX.RTM. with 10% FCS and 11 mM glucose, at 37.degree.
C. with 5% CO2. After 44 h, culture media is changed and washed
once with PBS to starvation media consisting of RPMI 1640 medium
with 1% FCS without glucose for 2 hours. Cells are then washed with
KRB buffer, followed incubation for 20 min at 37.degree. C. of the
treatment mix consisting of: 60 .mu.L KRB buffer/well with 10 .mu.L
of compound or DMSO 10.times.. 10 .mu.l of rotenone is added to the
mix to a final concentration of 0.5 .mu.M. Cell plates are left for
2 min at room temperature. 20 .mu.L of different glucose
concentrations are added to the mix--typically 0.1 to 20 mM
range--. Cells are incubated for another 15 min at 37.degree. C.,
before measuring ATP with the CellTiter-Glo.RTM. Assay, under
manufacturer's guidance, but without the equilibration step at room
temperature for 30 min. In brief, 100 .mu.l of Cell-Titer-Glo.RTM.
Reagent is added to the wells containing already 100 .mu.l of the
previous reaction mix. Plates are mixed for 2 min at 800 rpm,
followed by incubation at room temperature for 10 min to stabilize
the luminescent signal. Luminescence is then recorded with the
Tekan Ultra Evolution reader.
EXAMPLE 140: IC.sub.50 67.9 .mu.M
EXAMPLE 141: IC.sub.50 84.8 .mu.M
EXAMPLE 138: IC.sub.50 8.37 .mu.M (>100 .mu.M@10 mM Gluc)
EXAMPLE 124: IC.sub.50 33.2 .mu.M (>100 .mu.M@10 mM Gluc)
3. Erythrocyte Dialysis Assay
[0631] 6-NBD Glc
(6-(N-(7-Nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-6-Deoxyglucose) is
a small fluorescent glucose derivative, which has been shown to
bind to GluT-1 in astrocytes (L. F. Barros et al., JOURNAL OF
NEUROCHEMISTRY 109 (2009) 94-100). Here 6-NBD-Glc or alternatively
2-NBD-Glc was used in dialysis based competition experiments.
[0632] Freshly isolated heparinized rat blood samples were
immideately diluted (1:5) in PBS buffer to generate a stable stock
solution. This stock solution was further diluted (1:1 or 1:3) for
the following dialysis experiments in Rapid Equilibrium Dialysis
(RED) devices (Thermo Scientific Pierce).
[0633] The aforementioned stock solution was further diluted (1:1
or 1:3 respectively) and added to the dialysis compartiment of a
RED device. In parallel stock solution was diluted (1:1) with
buffer containing 6 NBD Glc (25 .mu.M).
[0634] Dialyses was started by addition of 6 NBD Glc (25 .mu.M) in
PBS Buffer (k.sup.on) or pure PBS buffer (k.sup.off) respectively
into the buffer compartiment. Aliquots (20 .mu.l) were taken after
defined time intervals from the buffer compartiment and
fluorescence was measured in a UV plate reader (Thermovarioskan,
Thermo).
[0635] As a control, free diffusion of 6-NBD-Glc (25 .mu.M) in PBS
buffer vs pure buffer was measured.
Data are Given in the Following Table:
TABLE-US-00043 [0636] Time 1:1 Dilution 1:1 Dilution Diffusion
(min) k.sup.on k.sup.off (Control) 0 55 0 0 30 50.6 3.4 3.7 60 45.1
6.5 7.5 90 33 12 9.2 120 34 16.1 10
Graphical evaluation of the different slopes gave a K.sub.D=0.68
for 6-NBD-Glc. K.sup.on=0.199; K.sup.off=0.136;
K.sub.D=0.136/0.199; K.sub.D=0.68: 68% free.
[0637] FIG. 1: Fluorescence intensity [AU] is plotted vs time
[min].
[0638] Squares: Decreasing fluorescence intensity measured in
buffer compartiment, due to binding to eyrothrocytes.
[0639] Triangles: Increasing fluorescence intensity measured in
buffer compartiment, due to release from erythrocytes.
[0640] K.sub.D was calculated from the slopes: K.sub.D=0.68 for
6-NBD-Glc.
[0641] Glucose dependency was determined following the protocol
described above, comparing the following solutions: 6 NBD-Glc (100
.mu.M), 6 NBD-Glucose (100 .mu.M)+Glucose 20 mM. Data are shown in
the next table
TABLE-US-00044 Time 6-NBD-Glc NBD-Glc [50 .mu.M] + (min) [50 .mu.M]
Glc [10 mM] 0 0 0 15 7.5 7.2 30 10.6 11.9 60 11.1 17 90 14.9 18.5
120 15.8 20.3 180 16 22.5
Free concentration of 6-NBD-Glc is increased in presence of Glc [10
mM].
[0642] FIG. 2: Fluorescence intensity [AU] is plotted vs time
[min].
[0643] Increase of fluorescence in buffer compartiment in presence
(squares) or absence of glucose (rhombus).
[0644] Example 102 was investigated in a competition experiment
following the same protocol using a 6-NBD-Glc solution (100 .mu.M)
in comparison to 6 NBD-Glucose (100 .mu.M)+Example 102 (50 .mu.M).
The results are shown in the following table
TABLE-US-00045 Time Example 102 (min) [25 .mu.M] Control 0 0 0 15
4.9 0.9 30 11.1 2.2 60 12.5 3.3 90 19.3 3.8 120 25.1 8 180 32.1
10.1
[0645] FIG. 3: Fluorescence intensity [AU] is plotted vs time
[min].
[0646] In presence of the Example 102 a clear increase in the slope
(rhombus) is seen in comparison to control (squares) indicating the
competition with 6-NBD-Glc.
* * * * *