U.S. patent application number 17/311064 was filed with the patent office on 2022-02-10 for quaternary ammonium salts as inhibitors of trimethylamine production.
The applicant listed for this patent is Senda Biosciences, Inc.. Invention is credited to Kehinde AJAYI, Gabriel BILLINGS, Elijah BOGART, Timothy BRIGGS, Devin Forest Reed DOUD, Dinara Shashanka GUNASEKERA, Danny LAFRANCE, Jenny LIU, Ana MARTINEZ-DEL CAMPO, Kathleen NUDEL, Spencer Cory PECK, John PROUDFOOT, Cheri ROSS, Steven TAYLOR, Yohannes TEFFERA, Koji YASUDA.
Application Number | 20220041641 17/311064 |
Document ID | / |
Family ID | |
Filed Date | 2022-02-10 |
United States Patent
Application |
20220041641 |
Kind Code |
A1 |
NUDEL; Kathleen ; et
al. |
February 10, 2022 |
QUATERNARY AMMONIUM SALTS AS INHIBITORS OF TRIMETHYLAMINE
PRODUCTION
Abstract
Provided are compounds that can inhibit pathogenic, bacterial
metabolite production and conjugates of the same. Also provided are
pharmaceutical compositions comprising the same and methods of
using the same.
Inventors: |
NUDEL; Kathleen; (Jamaica
Plain, MA) ; LIU; Jenny; (Cambridge, MA) ;
BRIGGS; Timothy; (Waltham, MA) ; GUNASEKERA; Dinara
Shashanka; (Cambridge, MA) ; MARTINEZ-DEL CAMPO;
Ana; (Somerville, MA) ; BOGART; Elijah;
(Cambridge, MA) ; TAYLOR; Steven; (Winchester,
MA) ; PROUDFOOT; John; (Newtown, CT) ; ROSS;
Cheri; (Winthrop, MA) ; TEFFERA; Yohannes;
(Topsfield, MA) ; YASUDA; Koji; (Boston, MA)
; DOUD; Devin Forest Reed; (Arlington, MA) ;
BILLINGS; Gabriel; (Somerville, MA) ; PECK; Spencer
Cory; (Watertown, MA) ; LAFRANCE; Danny;
(Natick, MA) ; AJAYI; Kehinde; (Medford,
MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Senda Biosciences, Inc. |
Cambridge |
MA |
US |
|
|
Appl. No.: |
17/311064 |
Filed: |
December 6, 2019 |
PCT Filed: |
December 6, 2019 |
PCT NO: |
PCT/US2019/065045 |
371 Date: |
June 4, 2021 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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62776459 |
Dec 6, 2018 |
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International
Class: |
C07H 15/12 20060101
C07H015/12; C07D 493/04 20060101 C07D493/04; C07C 215/40 20060101
C07C215/40; C07D 207/08 20060101 C07D207/08; C07C 215/44 20060101
C07C215/44; C07D 205/04 20060101 C07D205/04; C07D 231/04 20060101
C07D231/04; C07C 317/28 20060101 C07C317/28; C07D 295/15 20060101
C07D295/15; C07C 229/12 20060101 C07C229/12; C07F 9/38 20060101
C07F009/38; C07D 453/02 20060101 C07D453/02; C07D 257/04 20060101
C07D257/04; C07D 303/36 20060101 C07D303/36; C07D 305/06 20060101
C07D305/06; C07D 265/32 20060101 C07D265/32; C07D 265/30 20060101
C07D265/30; C07D 271/06 20060101 C07D271/06; C07F 5/02 20060101
C07F005/02; C07C 311/05 20060101 C07C311/05; C07D 263/32 20060101
C07D263/32; C07F 9/40 20060101 C07F009/40; C07C 211/63 20060101
C07C211/63; C12Q 1/68 20060101 C12Q001/68 |
Claims
1. A compound consisting of a cation and a pharmaceutically
acceptable counterion, wherein the cation is chosen from:
##STR00375## ##STR00376## ##STR00377## ##STR00378## ##STR00379##
##STR00380## ##STR00381## ##STR00382## ##STR00383## ##STR00384##
##STR00385## ##STR00386## ##STR00387## ##STR00388## ##STR00389##
##STR00390## ##STR00391## ##STR00392## ##STR00393## ##STR00394##
##STR00395## ##STR00396## ##STR00397##
2. The compound according to claim 1, wherein the cation is chosen
from: ##STR00398## ##STR00399## ##STR00400##
3. A compound consisting of a cation and a pharmaceutically
acceptable counterion, wherein the cation is chosen from:
##STR00401## ##STR00402## ##STR00403## ##STR00404## ##STR00405##
##STR00406## ##STR00407## ##STR00408## ##STR00409## ##STR00410##
##STR00411##
4. A compound consisting of a cation and a pharmaceutically
acceptable counterion, wherein the cation is a structure of formula
(I): ##STR00412## wherein R.sup.1 is C.sub.2-6 alkyl substituted
with --O-(acylated sugar) or isosorbide, wherein said C.sub.2-6
alkyl is further optionally substituted with oxo and/or methene;
C.sub.4 cycloalkyl optionally substituted with hydroxyl, ethynyl,
or --O-(acylated sugar); or C.sub.3 cycloalkyl substituted with
C.sub.1-6 alkyl, hydroxyl, ethynyl, or --O-(acylated sugar)
C.sub.3-4 cycloalkyl C.sub.1-2 alkyl; R.sup.2 is C.sub.2-6 alkyl
optionally substituted with one or two hydroxyl, oxo, and
--O-(acylated sugar); or R.sup.1 and R.sup.2, together with the
nitrogen atom to which both are attached, combine to form a 4- or
5-membered heterocyclic ring optionally substituted with ethynyl or
--(CH.sub.2).sub.n--OR.sup.5 or an acylated sugar, wherein n is 0
or 1, R.sup.5 is hydrogen or an acylated sugar; R.sup.3 is
C.sub.1-6 alkyl optionally substituted with a halogen or hydroxyl;
and R.sup.4 is C.sub.1-6 alkyl or propargyl.
5. The compound of claim 4, wherein R.sup.1 is C.sub.2-6 alkyl
substituted with --O-(acylated sugar) and is optionally further
substituted with oxo.
6. The compound of claim 4 or 5, wherein R.sup.2 is methyl.
7. The compound of any one of claims 4, 5, and 6, wherein R.sup.3
is C.sub.1-6 alkyl.
8. The compound of any one of claims 4-7, wherein R.sup.4 is
propargyl.
9. The compound of claim 4, wherein R.sup.1 is C.sub.2-6 alkyl
substituted with --O-(acylated sugar) and is optionally further
substituted with oxo; R.sup.2 is methyl; R.sup.3 is C.sub.1-6
alkyl; and R.sup.4 is propargyl.
10. The compound of claim 4, wherein R.sup.1 is C.sub.2-6 alkyl
substituted with isosorbide and is optionally further substituted
with oxo and/or methene; R.sup.2 is methyl; R.sup.3 is C.sub.1-6
alkyl; and R.sup.4 is propargyl.
11. The compound of claim 4, wherein R.sup.1 is chosen from C.sub.2
alkyl substituted with --O-(acylated sugar) and optionally further
substituted with oxo and C.sub.4 alkyl substituted with
--O-(acylated sugar) and optionally further substituted with oxo;
wherein the acylated sugar is chosen from groups of formula (A),
groups of formula (B), and groups of formula (C): ##STR00413##
wherein each R.sup.A is independently H or fatty acid acyl; and
R.sup.B is H, --CH.sub.3, --CH.sub.2--OR.sup.A, --OCH.sub.3,
--COOCH.sub.3, or --COOH; R.sup.2 is methyl; R.sup.3 is methyl; and
R.sup.4 is propargyl.
12. The compound of claim 4, wherein R.sup.1 is chosen from C.sub.2
alkyl substituted with isosorbide and optionally further
substituted with oxo and C.sub.3 alkyl substituted with isosorbide
and optionally further substituted with oxo and methene; wherein
isosorbide is chosen from groups of formula (C): ##STR00414##
R.sup.2 is methyl; R.sup.3 is methyl; and R.sup.4 is propargyl.
13. The compound of claim 4, wherein R.sup.1 is C.sub.3-4
cycloalkyl C.sub.1-2 alkyl; R.sup.2 is C.sub.2-6 alkyl optionally
substituted with one or two hydroxyl, oxo, and --O-(acylated
sugar); or R.sup.1 and R.sup.2, together with the nitrogen atom to
which both are attached, combine to form a 4- or 5-membered
heterocyclic ring optionally substituted with ethynyl or
--(CH.sub.2).sub.n--OR.sup.s or an acylated sugar, wherein n is 0
or 1, R.sup.s is hydrogen or an acylated sugar; R.sup.3 is
C.sub.1-6 alkyl optionally substituted with a halogen or hydroxyl;
and R.sup.4 is C.sub.1-6 alkyl or propargyl.
14. The compound of claim 13, wherein R.sup.1 is C.sub.3-4
cycloalkyl C.sub.1 alkyl.
15. The compound of claim 13 or 14, wherein R.sup.2 is C.sub.2
alkyl optionally substituted with one or two hydroxyl groups.
16. The compound of claim 13, wherein R.sup.1 and R.sup.2, together
with the nitrogen atom to which both are attached, combine to form
a 5-membered heterocyclic ring optionally substituted with ethynyl,
--OH, or --CH.sub.2OH.
17. The compound of any one of claims 13-16, wherein R.sup.3 is
C.sub.1 alkyl optionally substituted with a halogen.
18. The compound of any one of claims 13-17, wherein R.sup.4 is
C.sub.1 alkyl or propargyl.
19. A compound consisting of a cation and a pharmaceutically
acceptable counterion, wherein the cation is a structure of formula
(II): ##STR00415## wherein R.sup.1 is C.sub.1-6 alkyl optionally
substituted with one or more oxo, hydroxyl, halogen, cyano,
--COOMe, amino, methene, ethenyl, ethynyl, hydroxyphenyl, C.sub.3-4
cycloalkyl, --OCH.sub.2CH.sub.2OH, --HNC(O)OCMe.sub.3, --SMe,
--OMe, --HNS(O).sub.2Me, --SO.sub.3H, B(OH).sub.2, PO.sub.3H.sub.2,
PO.sub.2H.sub.2, --P(O)(OCH.sub.2CH.sub.3).sub.2,
--P(O)(OH)(OCH.sub.2CH.sub.3), heteroaryl ring; phenyl; benzyl;
C.sub.3-4 heterocyclyl optionally substituted with C.sub.1 alkyl
C.sub.4 heterocyclylalkyl C.sub.1 alkyl; or C.sub.4 cycloalkyl;
R.sup.2 is C.sub.1-6 alkyl optionally substituted with one or two
hydroxyl, oxo, methene, cyano, ethynyl, --HNC(O)H, or
--C.ident.C--CH.sub.2OH; or --HN(CH.sub.2).sub.3C(O)OH; or R.sup.1
and R.sup.2, together with the nitrogen atom to which both are
attached and optionally one or more additional heteroatoms, combine
to form a 4-8 membered mono- or bi-cyclic heterocycle optionally
substituted with ethynyl, trifluoromethyl, --CH.sub.2Ph, --OH, or
--(CH.sub.2)OH; R.sup.3 is methyl or propargyl; and R.sup.4 is
methyl or propargyl, with the proviso that (1) when two of R.sup.1,
R.sup.2, R.sup.3, and R.sup.4 are methyl and one of the other two
of R.sup.1, R.sup.2, R.sup.3, and R.sup.4 is propargyl, then the
other of R.sup.1, R.sup.2, R.sup.3, and R.sup.4 is not methyl, a
monohalomethyl, --CH.sub.2CH.sub.2OH, --COOH,
--(CH.sub.2).sub.4C(O)OH, or --CH.sub.2C(CH.sub.2)CN; and (2) when
two of R.sup.1, R.sup.2, R.sup.3, and R.sup.4 are methyl and one of
the other two of R.sup.1, R.sup.2, R.sup.3, and R.sup.4 is
--CH.sub.2CH.sub.2OH or --CH.sub.2C(O)OH, then the other of
R.sup.1, R.sup.2, R.sup.3, and R.sup.4 is not a monohalomethyl.
20. A compound consisting of a cation and a pharmaceutically
acceptable counterion, wherein the cation is a structure of formula
(III): ##STR00416## wherein R.sup.1 is C.sub.1-6 alkyl optionally
substituted with one or more oxo, hydroxyl, halogen, cyano,
--COOMe, --COOH, methene, ethenyl, ethynyl, C.sub.3-4 cycloalkyl,
--OCH.sub.2CH.sub.2OH, --SMe, --OMe, --HNS(O).sub.2Me,
--P(O)(OCH.sub.2CH.sub.3).sub.2, --P(O)(OH)(OCH.sub.2CH.sub.3), or
5-membered heteroaryl ring; R.sup.2 is C.sub.1-6 alkyl optionally
substituted with one or two hydroxyl, oxo, methene, cyano, ethynyl,
--HNC(O)H, or --C.ident.C--CH.sub.2OH; or
--HN(CH.sub.2).sub.3C(O)OH; or R.sup.1 and R.sup.2, together with
the nitrogen atom to which both are attached and optionally one or
more additional heteroatoms, combine to form a 4-8 membered mono-
or bi-cyclic heterocycle optionally substituted with ethynyl, --OH,
or --(CH.sub.2)OH; R.sup.3 is C.sub.1-6 alkyl optionally
substituted with a halogen, hydroxyl, or ethynyl; and R.sup.4 is
C.sub.1-6 alkyl or propargyl; with the proviso that (1) when two of
R.sup.1, R.sup.2, R.sup.3, and R.sup.4 are methyl and one of the
other two of R.sup.1, R.sup.2, R.sup.3, and R.sup.4 is propargyl,
then the other of R.sup.1, R.sup.2, R.sup.3, and R.sup.4 is not
methyl, a monohalomethyl, --CH.sub.2CH.sub.2OH, --COOH,
--(CH.sub.2).sub.4C(O)OH, or --CH.sub.2C(CH.sub.2)CN; and (2) when
two of R.sup.1, R.sup.2, R.sup.3, and R.sup.4 are methyl and one of
the other two of R.sup.1, R.sup.2, R.sup.3, and R.sup.4 is
--CH.sub.2CH.sub.2OH or --CH.sub.2C(O)OH, then the other of
R.sup.1, R.sup.2, R.sup.3, and R.sup.4 is not a monohalomethyl.
21. A pharmaceutical composition comprising a pharmaceutically
acceptable excipient and the compound of any one of claims 1 to
20.
22. A method of modulating a trimethylaminuria marker in a subject
in need thereof, the method comprising administering to the subject
in need thereof a therapeutically effective amount of the compound
of any one of claims 1 to 20 or the pharmaceutical composition of
claim 21.
23. The method of claim 22, wherein the trimethylaminuria marker is
the trimethylamine and/or trimethylamine oxide levels in the
subject's blood, plasma, serum, or urine.
24. A method of treating or preventing a disease associated with
elevated levels of trimethylamine (TMA), a decreased rate of
conversion of TMA to trimethylamine oxide (TMAO), or a high ratio
of TMA to TMAO in a subject in need thereof, the method comprising
administering a therapeutically effective amount of the compound of
any one of claims 1 to 20 or the pharmaceutical composition of
claim 21 to the subject.
25. The method of claim 24, further comprising detecting the
presence of one or more genetic variants of the FMO3 gene of the
subject in need before the administering step.
26. The method of claim 24, wherein the disease associated with
elevated levels of trimethylamine (TMA) or trimethylamine N-oxide
(TMAO), a decreased rate of conversion of TMA to TMAO, or a high
ratio of TMA to TMAO is a cardiovascular disease, reduced or
impaired kidney function, kidney disease, chronic kidney disease,
end-stage renal disease, or diabetes mellitus.
27. A method of inhibiting a CutC choline lyase-mediated conversion
of choline to trimethylamine, the method comprising contacting the
compound of any one of claims 1 to 20 with the CutC choline
lyase.
28. A method of inhibiting a CntA carnitine monooxygenase-mediated
conversion of carnitine to trimethylamine, the method comprising
contacting the compound of any one of claims 1 to 20 with the CntA
carnitine monooxygenase.
29. A method of treating a subject in need of treatment for
trimethylaminuria comprising contacting bacteria in vivo with a
therapeutically effective amount of the compound of any one of
claims 1 to 20 or the pharmaceutical composition of claim 21 to the
subject.
30. The method of claim 29, wherein the bacteria are localized in
the colon of the subject.
31. A method of identifying a subject suffering from
trimethylaminuria, or predicting a predisposition for developing
trimethylaminura in a subject, comprising: (i) analyzing a sample
from the patient to detect the presence of at least one FMO3
genetic variant in the patient, and (ii) identifying a subject
suffering from trimethylaminuria or predicting a predisposition for
developing trimethylaminura in a subject.
32. The method of claim 31, further comprising (iii) administering
a therapeutically effective amount of the compound of any one of
claims 1 to 20 or the pharmaceutical composition of claim 21.
33. The method of claim 31 or 32, wherein the FMO3 genetic variant
is chosen from g.-2092 to 10145del, g.94G>A+A29A2:A30,
g.110T>C, g.11145A>G, g.11148G>T, g.11166G>A,
g.11177A>G, g.11185delA, g.11192G>T, g.11239T>C,
g.15036A>G, g.15123T>A, g.15137G>T, g.15153C>T,
g.15526_15527delTG, g.15531T>A, g.15533T>C, g.15539C>A,
g.18177G>A, g.18225G>C, g.21429G>T, g.21460G>T,
g.21680G>T, g.21684G>A, g.21702delG, g.23580delG,
g.24486G>A, g.24592C>T, g.24608G>A, g.24658C>T, and
g.24682C>T.
Description
[0001] This application claims priority to U.S. provisional
application No. 62/776,459, filed Dec. 6, 2018, the disclosure of
which is incorporated herein by reference in its entirety.
[0002] Disclosed herein are novel compounds, pharmaceutical
compositions comprising the same, and methods of using the
same.
[0003] Mammalian microbiota can engage in a bidirectional
communication with the mammalian host system. In some instances,
mammalian microbiota may be responsible for producing enzymes that
can mediate formation of pathologic metabolites. These metabolites,
in sufficient quantities, can compromise the host's health and
often lead to debilitating diseases.
[0004] For example, choline-trimethylamine lyase (CutC) from
Clostridium sporogenes and from Proteus mirabilis can mediate
production of trimethylamine (TMA) from choline. Trimethylamine can
be also oxidized in vivo (e.g., by Flavin monooxygenase 3 (FMO3))
to produce a trimethylamine N-oxide (TMAO). Trimethylaminuria can
be characterized by elevated TMA and/or TMAO levels in a subject, a
decreased rate of conversion of TMA to TMAO in a subject, or a high
ratio of TMA to TMAO in a subject. Trimethylaminuria often leads
to, e.g., cardiovascular disease, reduced or impaired kidney
function, kidney disease, chronic kidney disease, end-stage renal
disease, or diabetes mellitus.
[0005] There is a need for therapeutic strategies that address
disease etiology. In particular, there is a need for therapeutic
strategies that target enzymatic production of the pathogenic,
bacterial metabolites.
[0006] The present disclosure provides novel compounds which may
act as inhibitors of enzymatic production of pathogenic, bacterial
metabolites, pharmaceutical compositions comprising at least one of
such compounds, and methods of using the same, such as, for
example, in methods for modulating an autoimmunity marker in a
subject, and in methods of treating an autoimmunity disorder in a
subject. In some embodiments, the disclosure provides compounds for
the inhibition of choline-trimethylamine lyase (CutC). In some
embodiments, the disclosure provides compounds for the inhibition
of carnitine monooxygenase (CntA). In some embodiments, the
disclosure provides compounds for the inhibition of betaine
reductase. In some embodiments, compounds of the present disclosure
consist of a cation and a pharmaceutically acceptable counterion.
In some embodiments, the cation includes at least one glycoside or
acylated sugar, and thus the cation is a conjugate which can be
advantageous for targeted delivery to a tissue (e.g., a small
intestine) having the highest abundance of CutC from Clostridium
sporogenes and/or Proteus mirabilis. CutC may be found and targeted
in a host of other bacterial species, including, but not limited
to, Proteus hauseri, Klebsiella oxytoca, Escherichia coli,
Escherichia fergusonii, Paenibacillus thiaminolyticus,
Paenibacillus alvei, Paenibacillus uliginis, Streptococcus
dysgalactiae, Streptococcus intermedius, Streptococcus suis,
Streptococcus castoreus, Streptococcus merionis, Streptococcus
ovis, Collinsella tanakaei, Anaerococcus hydrogenalis, Anaerococcus
tetradius, Anaerococcus obesiensis, Anaerococcus vaginalis,
Eubacterium sp., Lachnospiraceae bacterium, Hungatella hathewayi,
Clostridium phytofermentans, Clostridium saccharolyticum,
Clostridium indolis, Clostridium methoxybenzovorans, Clostridium
citroniae, Clostridium asparagiforme, Clostridium chauvoei,
Clostridium sartagoforme, Clostridium tetani, Clostridium
botulinum, Desulfovibrio desulfuricans, and Desulfovibrio
alaskensis. Among these species, strains exhibiting a high
abundance of CutC include, but are not limited to, Proteus hauseri
ZMd44, Proteus mirabilis ATCC29906, Klebsiella oxytoca-,
Escherichia coli MS 69-1, Escherichia fergusonii ECD227,
Paenibacillus thiaminolyticus, Paenibacillus alvei TS-15,
Paenibacillus uliginis N3/975, Streptococcus dysgalactiae subsp.
equisimilis ATCC 12394, Streptococcus intermedius ATCC 27335,
Streptococcus suis YS10-2, Streptococcus castoreus DSM 17536,
Streptococcus merionis, Streptococcus ovis, Collinsella tanakaei
YIT 12063, Anaerococcus hydrogenalis DSM 7454, Anaerococcus
tetradius ATCC 35098, Anaerococcus obesiensis, Anaerococcus
vaginalis ATCC 51170, Eubacterium sp. 68-3-10, Eubacterium sp.
AB3007, Lachnospiraceae bacterium, Hungatella hathewayi,
Clostridium phytofermentans ISDg, Clostridium saccharolyticum WM1,
Clostridium indolis DSM 755, Clostridium methoxybenzovorans SR3,
Clostridium citroniae WAL-17108, Clostridium asparagiforme,
Clostridium chauvoei JF4335, Clostridium sartagoforme MU1,
Clostridium tetani 12124569, Clostridium botulinum F 230613,
Clostridium sporogenes ATCC15579, Desulfovibrio desulfuricans
subsp. desulfuricans ATCC 27774, and Desulfovibrio alaskensis
G20.
[0007] In some embodiments, disclosed are compounds consisting of a
cation and a pharmaceutically acceptable counterion, wherein the
cation is a structure of formula (I):
##STR00001##
wherein
[0008] R.sup.1 is [0009] C.sub.2-6 alkyl substituted with
--O-(acylated sugar) or isosorbide, wherein said C.sub.2-6 alkyl is
further optionally substituted with oxo and/or methene; [0010]
C.sub.4 cycloalkyl optionally substituted with hydroxyl, ethynyl,
or --O-(acylated sugar); or [0011] C.sub.3 cycloalkyl substituted
with C.sub.1-6 alkyl, hydroxyl, ethynyl, or --O-(acylated sugar)
[0012] C.sub.3-4 cycloalkyl C.sub.1-2 alkyl;
[0013] R.sup.2 is C.sub.2-6 alkyl optionally substituted with one
or two hydroxyl, oxo, and --O-(acylated sugar);
[0014] or R.sup.1 and R.sup.2, together with the nitrogen atom to
which both are attached, combine to form a 4- or 5-membered
heterocyclic ring optionally substituted with ethynyl or
--(CH.sub.2).sub.n--OR.sup.s or an acylated sugar, wherein n is 0
or 1, R.sup.s is hydrogen or an acylated sugar;
[0015] R.sup.3 is C.sub.1-6 alkyl optionally substituted with a
halogen or hydroxyl; and
[0016] R.sup.4 is C.sub.1-6 alkyl or propargyl.
[0017] In some embodiments, provided herein are compounds
consisting of a cation and a pharmaceutically acceptable
counterion, wherein the cation is a structure of formula (II):
##STR00002##
wherein
[0018] R.sup.1 is [0019] C.sub.1-6 alkyl optionally substituted
with one or more oxo, hydroxyl, halogen, cyano, --COOMe, amino,
methene, ethenyl, ethynyl, hydroxyphenyl, C.sub.3-4 cycloalkyl,
--OCH.sub.2CH.sub.2OH, --HNC(O)OCMe.sub.3, --SMe, --OMe,
--HNS(O).sub.2Me, --SO.sub.3H, B(OH).sub.2, PO.sub.3H.sub.2,
PO.sub.2H.sub.2, --P(O)(OCH.sub.2CH.sub.3).sub.2,
--P(O)(OH)(OCH.sub.2CH.sub.3), heteroaryl ring; [0020] phenyl;
[0021] benzyl; [0022] C.sub.3-4 heterocyclyl optionally substituted
with C.sub.1 alkyl [0023] C.sub.4 heterocycloalkyl C.sub.1 alkyl;
or [0024] C.sub.4 cycloalkyl;
[0025] R.sup.2 is [0026] C.sub.1-6 alkyl optionally substituted
with one or two hydroxyl, oxo, methene, cyano, ethynyl, --HNC(O)H,
or --C.ident.C--CH.sub.2OH; or [0027]
--HN(CH.sub.2).sub.3C(O)OH;
[0028] or R.sup.1 and R.sup.2, together with the nitrogen atom to
which both are attached and optionally one or more additional
heteroatoms, combine to form a 4-8 membered mono- or bi-cyclic
heterocycle optionally substituted with ethynyl, trifluoromethyl,
--CH.sub.2Ph, --OH, or --(CH.sub.2)OH;
[0029] R.sup.3 is methyl or propargyl; and
[0030] R.sup.4 is methyl or propargyl,
[0031] with the proviso that
[0032] (1) when two of R.sup.1, R.sup.2, R.sup.3, and R.sup.4 are
methyl and one of the other two of R.sup.1, R.sup.2, R.sup.3, and
R.sup.4 is propargyl, then the other of R.sup.1, R.sup.2, R.sup.3,
and R.sup.4 is not methyl, a monohalomethyl, --CH.sub.2CH.sub.2OH,
--COOH, --(CH.sub.2).sub.4C(O)OH, or --CH.sub.2C(CH.sub.2)CN;
and
[0033] (2) when two of R.sup.1, R.sup.2, R.sup.3, and R.sup.4 are
methyl and one of the other two of R.sup.1, R.sup.2, R.sup.3, and
R.sup.4 is --CH.sub.2CH.sub.2OH or --CH.sub.2C(O)OH, then the other
of R.sup.1, R.sup.2, R.sup.3, and R.sup.4 is not a
monohalomethyl.
[0034] In some embodiments, provided herein are compounds
consisting of a cation and a pharmaceutically acceptable
counterion, wherein the cation is a structure of formula (III):
##STR00003##
wherein
[0035] R.sup.1 is [0036] C.sub.1-6 alkyl optionally substituted
with one or more oxo, hydroxyl, halogen, cyano, --COOMe, --COOH,
methene, ethenyl, ethynyl, C.sub.3-4 cycloalkyl,
--OCH.sub.2CH.sub.2OH, --SMe, --OMe, --HNS(O).sub.2Me,
--P(O)(OCH.sub.2CH.sub.3).sub.2, --P(O)(OH)(OCH.sub.2CH.sub.3), or
5-membered heteroaryl ring;
[0037] R.sup.2 is [0038] C.sub.1-6 alkyl optionally substituted
with one or two hydroxyl, oxo, methene, cyano, ethynyl, --HNC(O)H,
or --C.ident.C--CH.sub.2OH; or [0039]
--HN(CH.sub.2).sub.3C(O)OH;
[0040] or R.sup.1 and R.sup.2, together with the nitrogen atom to
which both are attached and optionally one or more additional
heteroatoms, combine to form a 4-8 membered mono- or bi-cyclic
heterocycle optionally substituted with ethynyl, --OH, or
--(CH.sub.2)OH;
[0041] R.sup.3 is C.sub.1-6 alkyl optionally substituted with a
halogen, hydroxyl, or ethynyl; and
[0042] R.sup.4 is C.sub.1-6 alkyl or propargyl,
with the proviso that
[0043] (1) when two of R.sup.1, R.sup.2, R.sup.3, and R.sup.4 are
methyl and one of the other two of R.sup.1, R.sup.2, R.sup.3, and
R.sup.4 is propargyl, then the other of R.sup.1, R.sup.2, R.sup.3,
and R.sup.4 is not methyl, a monohalomethyl, --CH.sub.2CH.sub.2OH,
--COOH, --(CH.sub.2).sub.4C(O)OH, or --CH.sub.2C(CH.sub.2)CN;
and
[0044] (2) when two of R.sup.1, R.sup.2, R.sup.3, and R.sup.4 are
methyl and one of the other two of R.sup.1, R.sup.2, R.sup.3, and
R.sup.4 is --CH.sub.2CH.sub.2OH or --CH.sub.2C(O)OH, then the other
of R.sup.1, R.sup.2, R.sup.3, and R.sup.4 is not a
monohalomethyl.
[0045] In some embodiments, the disclosure provides a
pharmaceutical composition comprising a pharmaceutically acceptable
excipient and at least one of the compounds described above.
[0046] In some embodiments, provided herein is a method of
modulating a trimethylaminuria marker in a subject in need thereof,
the method comprising administering to the subject in need thereof
a therapeutically effective amount of at least one of the compounds
or pharmaceutical compositions disclosed herein. In some
embodiments, the trimethylaminuria marker is the trimethylamine
and/or trimethylamine oxide level in the subject's blood, plasma,
serum, or urine.
[0047] In some embodiments, provided herein is a method of treating
or preventing a disease associated with elevated levels of
trimethylamine (TMA) or trimethylamine N-oxide (TMAO), a decreased
rate of conversion of TMA to TMAO, or a high ratio of TMA to TMAO
in a subject in need thereof, the method comprising administering a
therapeutically effective amount of at least one of the compounds
or pharmaceutical compositions disclosed herein to the subject. In
some embodiments, the method further comprises detecting the
presence of one or more genetic variants of the FMO3 gene of the
subject in need before the administering step. In some embodiments,
the disease associated with elevated levels of TMA or TMAO, a
decreased rate of conversion of TMA to TMAO, or a high ratio of TMA
to TMAO is a cardiovascular disease, reduced or impaired kidney
function, kidney disease, chronic kidney disease, end-stage renal
disease, or diabetes mellitus. In some embodiments, the
cardiovascular disease is angina, arrhythmia, atherosclerosis,
cardiomyopathy, congestive heart failure, coronary artery disease,
carotid artery disease, endocarditis, coronary thrombosis,
myocardial infarction, high blood pressure/hypertension,
hypercholesterolemia, hyperlipidemia, mitral valve prolapse,
peripheral artery disease, or stroke.
[0048] In some embodiments, provided herein is a method of
inhibiting a CutC choline lyase-mediated conversion of choline to
trimethylamine, the method comprising contacting at least one of
the compounds or pharmaceutical compositions disclosed herein with
the CutC choline lyase.
[0049] In some embodiments, provided herein is a method of
inhibiting a CntA carnitine monooxygenase-mediated conversion of
carnitine to trimethylamine, the method comprising contacting at
least one of the compounds or pharmaceutical compositions disclosed
herein with the CntA carnitine monooxygenase.
[0050] In some embodiments, provided herein is a method of
inhibiting a betaine reductase-mediated conversion of betaine or
gamma-butyrobetaine to trimethylamine, the method comprising
contacting at least one of the compounds or pharmaceutical
compositions disclosed herein with the betaine reductase.
[0051] In some embodiments, provided herein is a method of treating
a subject in need of treatment for trimethylaminuria comprising
contacting bacteria in vivo with a therapeutically effective amount
of at least one of the compounds or pharmaceutical compositions
disclosed herein to the subject. In some embodiments, the bacteria
are localized in the colon of the subject.
[0052] In some embodiments, the present disclosure provides a
method of modulating a trimethylamine marker in a subject in need
thereof, the method consisting of administering to the subject in
need thereof a therapeutically effective amount of at least one
compound of the present disclosure or a pharmaceutical composition
of the present disclosure.
[0053] In some embodiments, the trimethylaminuria marker is the
trimethylamine and/or trimethylamine oxide levels in the subject's
blood, plasma, serum, or urine.
[0054] In some embodiments, the present disclosure provides a
method of treating or preventing a disease associated with
trimethylaminuria in a subject in need thereof, the method
comprising administering a therapeutically effective amount of at
least one pharmaceutically acceptable salt of at least one compound
of the present disclosure or a pharmaceutical composition of the
present disclosure to the subject.
[0055] In some embodiments, provided herein is a method of
identifying a subject suffering from trimethylaminuria, or
predicting a predisposition for developing trimethylaminura in a
subject, comprising: (i) analyzing a sample from the patient to
detect the presence of at least one FMO3 genetic variant in the
patient, and (ii) identifying identifying a subject suffering from
trimethylaminuria or predicting a predisposition for developing
trimethylaminura in a subject. In some embodiments, the method
further comprises (iii) administering a therapeutically effective
amount of at least one of the compounds or pharmaceutical
compositions disclosed herein to the subject.
[0056] In some embodiments, the FMO3 genetic variant is identified
based on public databases reporting pathogenic variants associated
with TMAU. In some embodiments, the FMO3 genetic variant is
identified based on predicted loss of function of the FMO3 gene
product, such as variants introducing frameshifts or premature stop
codons into the coding sequence of FMO3. In some embodiments, the
variant is identified based on predicted structural modification to
the exonic sequence of FMO3. In some embodiments, the FMO3 genetic
variant is chosen from g.-2092 to 10145del, g.94G>A+A29A2:A30,
g.110T>C, g.11145A>G, g.11148G>T, g.11166G>A,
g.11177A>G, g.11185delA, g.11192G>T, g.11239T>C,
g.15036A>G, g.15123T>A, g.15137G>T, g.15153C>T,
g.15526_15527delTG, g.15531T>A, g.15533T>C, g.15539C>A,
g.18177G>A, g.18225G>C, g.21429G>T, g.21460G>T,
g.21680G>T, g.21684G>A, g.21702delG, g.23580delG,
g.24486G>A, g.24592C>T, g.24608G>A, g.24658C>T, and
g.24682C>T.
[0057] As used herein, the term "acyl," as used herein, represents
a chemical substituent of formula --C(O)--R, where R is alkyl,
alkenyl, aryl, arylalkyl, cycloalkyl, heterocyclyl, heterocyclyl
alkyl, heteroaryl, or heteroaryl alkyl. An optionally substituted
acyl is an acyl that is optionally substituted as described herein
for each group R. Non-limiting examples of acyl include fatty acid
acyls (e.g., short chain fatty acid acyls (e.g., acetyl, propionyl,
or butyryl)).
[0058] The term "acylated sugar," as used herein, refers to a
carbohydrate, sugar acid, or sugar alcohol having one or more
hydroxyls substituted with an acyl (e.g., a fatty acid acyl). In
some embodiments, the carbohydrate is a monosaccharide. In some
embodiments, the carbohydrate is a disaccharide. In some
embodiments, the fatty acid acyl is a short chain fatty acid acyl
(e.g., propionyl or butyryl). An acylated sugar can be a compound
or a monovalent group. When an acylated sugar is a monovalent
group, the group includes one and only one valency for attaching to
another molecular fragment. When an acylated sugar is covalently
bonded to a carbon atom of another molecular fragment, the valency
is on an oxygen atom of the acylated sugar. When an acylated sugar
is covalently bonded to an oxygen atom of another molecular
fragment, the valency is on the anomeric carbon atom of the
acylated sugar. Non-limiting examples of a monosaccharide include
arabinose, xylose, fructose, galactose, glucose, glucosinolate,
ribose, tagatose, fucose, and rhamnose. Non-limiting examples of a
disaccharide include lactose, sucrose, melibiose, gentiobiose,
kojibiose, cellobiose, maltose, trehalose and chitobiose.
Non-limiting examples of a sugar acid include xylonic acid,
gluconic acid, glucuronic acid, galacturonic acid, tartaric acid,
saccharic acid, or mucic acid. Non-limiting examples of sugar
alcohols are glycerol, erythritol, theritol, arabitol, xylitol,
tibitol, mannitol, sorbitol, galactitol, fucitol, iditol, or
inositol.
[0059] The term "acyloxy," as used herein, represents a chemical
substituent of formula --OR, where R is acyl. An optionally
substituted acyloxy is an acyloxy that is optionally substituted as
described herein for acyl.
[0060] The term "alcohol oxygen atom," as used herein, refers to a
divalent oxygen atom, where at least one valency of the oxygen atom
is bonded to an sp.sup.3-hybridized carbon atom.
[0061] The term "alkanoyl," as used herein, represents a chemical
substituent of formula --C(O)--R, where R is alkyl. An optionally
substituted alkanoyl is an alkanoyl that is optionally substituted
as described herein for alkyl.
[0062] The term "alkoxy," as used herein, represents a chemical
substituent of formula --OR, where R is a C.sub.1-6 alkyl group,
unless otherwise specified. An optionally substituted alkoxy is an
alkoxy group that is optionally substituted as defined herein for
alkyl.
[0063] The term "alkenyl," as used herein, represents acyclic
monovalent straight or branched chain hydrocarbon groups containing
one, two, or three carbon-carbon double bonds. Alkenyl, when
unsubstituted, has from 2 to 12 carbon atoms (e.g., 1 to 8
carbons), unless specified otherwise. Non-limiting examples of the
alkenyl groups include ethenyl, prop-1-enyl, prop-2-enyl,
1-methylethenyl, but-1-enyl, but-2-enyl, but-3-enyl,
1-methylprop-1-enyl, 2-methylprop-1-enyl, and 1-methylprop-2-enyl.
Alkenyl groups may be optionally substituted as defined herein for
alkyl.
[0064] The term "alkyl," as used herein, refers to an acyclic,
straight or branched, saturated hydrocarbon group, which, when
unsubstituted, has from 1 to 12 carbons (e.g., 1 to 6 carbons),
unless otherwise specified. Alkyl groups are exemplified by methyl;
ethyl; n- and iso-propyl; n-, sec-, iso- and tert-butyl; neopentyl,
and the like, and may be optionally substituted, valency
permitting, with one, two, three, or, in the case of alkyl groups
of two carbons or more, four or more substituents independently
selected from the group consisting of: alkoxy; acyloxy;
alkylsulfinyl; alkylsulfonyl; amino; aryl; aryloxy; azido;
cycloalkyl; cycloalkoxy; halo; heterocyclyl; heteroaryl;
heterocyclylalkyl; heteroarylalkyl; heterocyclyloxy; heteroaryloxy;
hydroxy; nitro; thioalkyl; thioalkenyl; thioaryl; thiol; cyano; oxo
(.dbd.O); thio (.dbd.S); and imino (.dbd.NR'), where R' is H,
alkyl, aryl, or heterocyclyl. Each of the substituents may itself
be unsubstituted or, valency permitting, substituted with
unsubstituted substituent(s) defined herein for each respective
group.
[0065] The term "alkylene," as used herein, refers to a divalent,
straight or branched, saturated hydrocarbon, in which two valencies
replace two hydrogen atoms. Alkyl, when unsubstituted, has from 2
to 12 carbon atoms (e.g., 2 to 6 carbons), unless specified
otherwise. Non-limiting examples of the alkylene group include
methylene, ethane-1,2-diyl, ethane-1,1-diyl, propane-1,3-diyl,
propane-1,2-diyl, propane-1,1-diyl, propane-2,2-diyl,
butane-1,4-diyl, butane-1,3-diyl, butane-1,2-diyl, butane-1,1-diyl,
and butane-2,2-diyl, butane-2,3-diyl. An optionally substituted
alkylene is an alkylene that is optionally substituted as described
herein for alkyl.
[0066] The term "alkylsulfinyl," as used herein, represents a group
of formula --S(O)-(alkyl). An optionally substituted alkylsulfinyl
is an alkylsulfinyl that is optionally substituted as described
herein for alkyl.
[0067] The term "alkylsulfonyl," as used herein, represents a group
of formula --S(O).sub.2-(alkyl). An optionally substituted
alkylsulfonyl is an alkylsulfonyl that is optionally substituted as
described herein for alkyl.
[0068] The term "alkynyl," as used herein, represents an acyclic,
monovalent, straight or branched chain hydrocarbon groups
containing one, two, or three carbon-carbon triple bonds. Alkynyl,
when unsubstituted, has from 2 to 12 carbon atoms (e.g., 2 to 6
carbons), unless specified otherwise. Non-limiting examples of the
alkynyl groups include ethynyl, prop-1-ynyl, prop-2-ynyl,
but-1-ynyl, but-2-ynyl, but-3-ynyl, and 1-methylprop-2-ynyl. An
optionally substituted alkynyl is an alkynyl that is optionally
substituted as defined herein for alkyl.
[0069] The term "aryl," as used herein, represents a mono-,
bicyclic, or multicyclic carbocyclic ring system having one or two
aromatic rings. Aryl group may include from 6 to 10 carbon atoms.
All atoms within an unsubstituted carbocyclic aryl group are carbon
atoms. Non-limiting examples of carbocyclic aryl groups include
phenyl, naphthyl, 1,2-dihydronaphthyl, 1,2,3,4-tetrahydronaphthyl,
fluorenyl, indanyl, indenyl, etc. The aryl group may be
unsubstituted or substituted with one, two, three, four, or five
substituents independently selected from the group consisting of:
alkyl; alkenyl; alkoxy; acyloxy; amino; aryl; aryloxy; azido;
cycloalkyl; cycloalkoxy; halo; heterocyclyl; heteroaryl;
heterocyclylalkyl; heteroarylalkyl; heterocyclyloxy; heteroaryloxy;
hydroxy; nitro; thioalkyl; thioalkenyl; thioaryl; thiol; and cyano.
Each of the substituents may itself be unsubstituted or substituted
with unsubstituted substituent(s) defined herein for each
respective group.
[0070] The term "aryl alkyl," as used herein, represents an alkyl
group substituted with an aryl group. An optionally substituted
aryl alkyl is an aryl alkyl, in which aryl and alkyl portions may
be optionally substituted as the individual groups as described
herein.
[0071] The term "aryloxy," as used herein, represents a group --OR,
where R is aryl. Aryloxy may be an optionally substituted aryloxy.
An optionally substituted aryloxy is aryloxy that is optionally
substituted as described herein for aryl.
[0072] The term "carbamate linker," as used herein, refers to a
group R.sup.1--(CO)--R.sup.2, where R.sup.1 is a bond to an alcohol
or phenolic oxygen atom, and R.sup.2 is a bond to a nitrogen
atom.
[0073] The term "carbohydrate," as used herein, refers to a
monosaccharide, disaccharide, or an oligosaccharide or an analog of
the following structure:
##STR00004##
where R.sup.B is H, optionally substituted C.sub.1-6 alkyl, or
--CH.sub.2--OH.
[0074] The term "carbohydrate" may refer to a compound or to a
monovalent or multivalent chemical substituent. When the term
"carbohydrate" refers to a chemical substituent, the valence(s)
reside on the anomeric carbon atom and/or alcohol oxygen atoms. An
optionally substituted carbohydrate is a carbohydrate, in which at
least one hydroxyl is substituted with an acyl (e.g., a fatty acid
acyl).
[0075] The term "carbonate linker," as used herein, refers to a
group R.sup.1--C(O)--R.sup.2 where R.sup.1 is a bond to a first
alcohol or phenolic oxygen atom, and R.sup.2 is a bond to a second
alcohol or phenolic oxygen atom.
[0076] The term "carbonyl," as used herein, refers to a divalent
group --C(O)--.
[0077] The term "carboxylate," as used herein, represents group
--COOH or a salt thereof.
[0078] The term "cycloalkoxy," as used herein, represents a group
--OR, where R is cycloalkyl. An optionally substituted cycloalkoxy
is cycloalkoxy that is optionally substituted as described herein
for cycloalkyl.
[0079] The term "ester bond," as used herein, refers to a covalent
bond between an alcohol or phenolic oxygen atom and a carbonyl
group that is further bonded to a carbon atom.
[0080] The term "fatty acid," as used herein, refers to a
short-chain fatty acid, a medium chain fatty acid, a long chain
fatty acid, a very long chain fatty acid, or an unsaturated
analogue thereof, or a phenyl-substituted analogue thereof. Short
chain fatty acids contain from 1 to 6 carbon atoms, medium chain
fatty acids contain from 7 to 13 carbon atoms, and long-chain fatty
acids contain from 14 to 22 carbon atoms. A fatty acid may be
saturated or unsaturated. An unsaturated fatty acid includes 1, 2,
3, 4, 5, or 6 carbon-carbon double bonds. In some embodiments, the
carbon-carbon double bonds in unsaturated fatty acids have Z
stereochemistry. In some embodiments, the carbon-carbon double
bonds in unsaturated fatty acids have E stereochemistry.
[0081] The term "fatty acid acyl," as used herein, refers to a
fatty acid, in which the hydroxyl group is replaced with a valency.
In some embodiments, a fatty acid acyl is a short chain fatty acid
acyl.
[0082] The term "fatty acid acyloxy," as used herein, refers to
group --OR, where R is a fatty acid acyl.
[0083] The term "glycoside," as used herein, refers to a monovalent
group that is a monosaccharide, disaccharide, or sugar acid having
a valency on an anomeric carbon. Non-limiting examples of
monosaccharides include arabinose, xylose, fructose, galactose,
glucose, ribose, tagatose, fucose, and rhamnose. Non-limiting
examples of disaccharides include lactose, sucrose, melibiose,
gentiobiose, kojibiose, cellobiose, maltose, trehalose and
chitobiose. Non-limiting examples of sugar acids include xylonic
acid, gluconic acid, glucuronic acid, galacturonic acid, tartaric
acid, saccharic acid, or mucic acid.
[0084] The term "glycosidic bond," as used herein, refers to a
covalent bond between an oxygen atom and an anomeric carbon atom in
a monosaccharide, disaccharide, or sugar acid having an anomeric
carbon atom.
[0085] The term "halogen," as used herein, represents a halogen
selected from bromine, chlorine, iodine, and fluorine.
[0086] The term "heteroaryl," as used herein, represents a
monocyclic 5-, 6-, 7-, or 8-membered ring system, or a fused or
bridging bicyclic, tricyclic, or tetracyclic ring system; the ring
system contains one, two, three, or four heteroatoms independently
selected from the group consisting of nitrogen, oxygen, and sulfur;
and at least one of the rings is an aromatic ring. Non-limiting
examples of heteroaryl groups include benzimidazolyl, benzofuryl,
benzothiazolyl, benzothienyl, benzoxazolyl, furyl, imidazolyl,
indolyl, isoindazolyl, isoquinolinyl, isothiazolyl, isothiazolyl,
isoxazolyl, oxadiazolyl, oxazolyl, purinyl, pyrrolyl, pyridinyl,
pyrazinyl, pyrimidinyl, qunazolinyl, quinolinyl, thiadiazolyl
(e.g., 1,3,4-thiadiazole), thiazolyl, thienyl, triazolyl,
tetrazolyl, dihydroindolyl, tetrahydroquinolyl,
tetrahydroisoquinolyl, etc. The term bicyclic, tricyclic, and
tetracyclic heteroaryls include at least one ring having at least
one heteroatom as described above and at least one aromatic ring.
For example, a ring having at least one heteroatom may be fused to
one, two, or three carbocyclic rings, e.g., an aryl ring, a
cyclohexane ring, a cyclohexene ring, a cyclopentane ring, a
cyclopentene ring, or another monocyclic heterocyclic ring.
Examples of fused heteroaryls include
1,2,3,5,8,8a-hexahydroindolizine; 2,3-dihydrobenzofuran;
2,3-dihydroindole; and 2,3-dihydrobenzothiophene. Heteroaryl may be
optionally substituted with one, two, three, four, or five
substituents independently selected from the group consisting of:
alkyl; alkenyl; alkoxy; acyloxy; aryloxy; alkylsulfinyl;
alkylsulfonyl; amino; arylalkoxy; cycloalkyl; cycloalkoxy; halogen;
heterocyclyl; heterocyclyl alkyl; heteroaryl; heteroaryl alkyl;
heterocyclyloxy; heteroaryloxy; hydroxyl; nitro; thioalkyl;
thioalkenyl; thioaryl; thiol; cyano; .dbd.O; --NR.sub.2, where each
R is independently hydrogen, alkyl, acyl, aryl, arylalkyl,
cycloalkyl, heterocyclyl, or heteroaryl; --COOR.sup.A, where
R.sup.A is hydrogen, alkyl, aryl, arylalkyl, cycloalkyl,
heterocyclyl, or heteroaryl; and --CON(R.sup.B).sub.2, where each
R.sup.B is independently hydrogen, alkyl, aryl, arylalkyl,
cycloalkyl, heterocyclyl, or heteroaryl. Each of the substituents
may itself be unsubstituted or substituted with unsubstituted
substituent(s) defined herein for each respective group.
[0087] The term "heteroaryloxy," as used herein, refers to a
structure --OR, in which R is heteroaryl. Heteroaryloxy can be
optionally substituted as defined for heteroaryl.
[0088] The term "heterocyclyl," as used herein, represents a
monocyclic, bicyclic, tricyclic, or tetracyclic non-aromatic ring
system having fused or bridging 4-, 5-, 6-, 7-, or 8-membered
rings, unless otherwise specified, the ring system containing one,
two, three, or four heteroatoms independently selected from the
group consisting of nitrogen, oxygen, and sulfur. Non-aromatic
5-membered heterocyclyl has zero or one double bonds, non-aromatic
6- and 7-membered heterocyclyl groups have zero to two double
bonds, and non-aromatic 8-membered heterocyclyl groups have zero to
two double bonds and/or zero or one carbon-carbon triple bond.
Heterocyclyl groups have a carbon count of 1 to 16 carbon atoms
unless otherwise specified. Certain heterocyclyl groups may have a
carbon count up to 9 carbon atoms. Non-aromatic heterocyclyl groups
include pyrrolinyl, pyrrolidinyl, pyrazolinyl, pyrazolidinyl,
imidazolinyl, imidazolidinyl, piperidinyl, homopiperidinyl,
piperazinyl, pyridazinyl, oxazolidinyl, isoxazolidiniyl,
morpholinyl, thiomorpholinyl, thiazolidinyl, isothiazolidinyl,
thiazolidinyl, tetrahydrofuranyl, dihydrofuranyl,
tetrahydrothienyl, dihydrothienyl, pyranyl, dihydropyranyl,
dithiazolyl, etc. The term "heterocyclyl" also represents a
heterocyclic compound having a bridged multicyclic structure in
which one or more carbons and/or heteroatoms bridges two
non-adjacent members of a monocyclic ring, e.g., quinuclidine,
tropanes, or diaza-bicyclo[2.2.2]octane. The term "heterocyclyl"
includes bicyclic, tricyclic, and tetracyclic groups in which any
of the above heterocyclic rings is fused to one, two, or three
carbocyclic rings, e.g., a cyclohexane ring, a cyclohexene ring, a
cyclopentane ring, a cyclopentene ring, or another heterocyclic
ring. Examples of fused heterocyclyls include
1,2,3,5,8,8a-hexahydroindolizine; 2,3-dihydrobenzofuran;
2,3-dihydroindole; and 2,3-dihydrobenzothiophene. The heterocyclyl
group may be unsubstituted or substituted with one, two, three,
four or five substituents independently selected from the group
consisting of: alkyl; alkenyl; alkoxy; acyloxy; alkylsulfinyl;
alkylsulfonyl; aryloxy; amino; arylalkoxy; cycloalkyl; cycloalkoxy;
halogen; heterocyclyl; heterocyclyl alkyl; heteroaryl; heteroaryl
alkyl; heterocyclyloxy; heteroaryloxy; hydroxyl; nitro; thioalkyl;
thioalkenyl; thioaryl; thiol; cyano; .dbd.O; .dbd.S; --NR.sub.2,
where each R is independently hydrogen, alkyl, acyl, aryl,
arylalkyl, cycloalkyl, heterocyclyl, or heteroaryl; --COOR.sup.A,
where R.sup.A is hydrogen, alkyl, aryl, arylalkyl, cycloalkyl,
heterocyclyl, or heteroaryl; and --CON(R.sup.B).sub.2, where each
R.sup.B is independently hydrogen, alkyl, aryl, arylalkyl,
cycloalkyl, heterocyclyl, or heteroaryl.
[0089] The term "heterocyclyl alkyl," as used herein, represents an
alkyl group substituted with a heterocyclyl group. The heterocyclyl
and alkyl portions of an optionally substituted heterocyclyl alkyl
are optionally substituted as the described for heterocyclyl and
alkyl, respectively.
[0090] The term "heterocyclyloxy," as used herein, refers to a
structure --OR, in which R is heterocyclyl. Heterocyclyloxy can be
optionally substituted as described for heterocyclyl.
[0091] The terms "hydroxyl" and "hydroxy," as used interchangeably
herein, represent --OH. A hydroxyl substituted with an acyl is an
acyloxy. A protected hydroxyl is a hydroxyl, in which the hydrogen
atom is replaced with an O-protecting group.
[0092] The term "hydroxyalkyl," as used herein, refers to a
C.sub.1-6 alkyl group that is substituted with one or more
hydroxyls, provided that each carbon atom in the hydroxyalkyl is
attached either to no more than one hydroxyl. Non-limiting examples
of hydroxyalkyls include hydroxymethyl, 2-hydroxyethyl, and
1-hydroxyethyl.
[0093] The term "methene," as used herein, represents a double
bonded carbon atom (e.g., the structure of methene may be shown as
.dbd.CH.sub.2).
[0094] The term "modulating," as used herein, refers to an
observable change in the level of a marker in a subject, as
measured using techniques and methods known in the art for the
measurement of the marker. Modulating the marker level in a subject
may result in a change of at least 1% relative to prior to
administration (e.g., at least 5%, 10%, 15%, 20%, 25%, 30%, 35%0,
40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or at
least 98% or more relative to prior to administration; e.g., up to
100% relative to prior to administration). In some embodiments,
modulating is increasing the level of a marker in a subject.
Increasing the marker level in a subject may result in an increase
of at least 1% relative to prior to administration (e.g., at least
5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%,
70%, 75%, 80%, 85%, 90%, 95% or at least 98% or more relative to
prior to administration; e.g., up to 100% relative to prior to
administration). In other embodiments, modulating is decreasing the
level of a marker in a subject. Decreasing the marker level in a
subject may result in a decrease of at least 1% relative to prior
to administration (e.g., at least 5%, 10%, 15%, 20%, 25%, 30%, 35%,
40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or at
least 98% or more relative to prior to administration; e.g., up to
100% relative to prior to administration). In embodiments in which
a parameter is increased or decreased (or reduced) in a subject
following a step of administering a composition described herein,
the increase or decrease may take place and/or be detectable within
a range of time following the administration (e.g., within six
hours, 24 hours, 3 days, a week or longer), and may take place
and/or be detectable after one or more administrations (e.g., after
2, 3, 4, 5, 6, 7, 8, 9, 10 or more administrations, e.g., as part
of a dosing regimen for the subject).
[0095] The term "oxo," as used herein, represents a divalent oxygen
atom (e.g., the structure of oxo may be shown as .dbd.O).
[0096] The term "pharmaceutically acceptable salt," as used herein,
represents those salts which are, within the scope of sound medical
judgment, suitable for use in contact with the tissues of humans
and animals without undue toxicity, irritation, allergic response
and the like and are commensurate with a reasonable benefit/risk
ratio. Principles for preparing pharmaceutically acceptable salts
are well known in the art. For example, pharmaceutically acceptable
salts are described in Berge et al., J. Pharmaceutical Sciences
66:1-19, 1977 and in Pharmaceutical Salts: Properties, Selection,
and Use, (Eds. P. H. Stahl and C. G. Wermuth), Wiley-VCH, 2008. The
salts can be prepared in situ during the final isolation and
purification of the compounds described herein, by reacting the
free base group with a suitable electrophile, by use of an
alkylating agent or via exchange resin, or by exchanging in
solution. Representative counterions useful for pharmaceutically
acceptable salts include acetate, adipate, alginate, ascorbate,
aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate,
camphorate, camphorsulfonate, citrate, cyclopentanepropionate,
digluconate, dodecylsulfate, ethanesulfonate, fumarate,
glucoheptonate, glycerophosphate, hemisulfate, heptonate,
hexanoate, bromide, chloride, iodide, 2-hydroxy-ethanesulfonate,
lactobionate, lactate, laurate, lauryl sulfate, malate, maleate,
malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate,
nitrate, oleate, oxalate, palmitate, pamoate, pectinate,
persulfate, 3-phenylpropionate, phosphate, picrate, pivalate,
propionate, stearate, succinate, sulfate, tartrate, thiocyanate,
toluenesulfonate, undecanoate, valerate salts, and the like.
[0097] The term "phenolic oxygen atom," as used herein, refers to a
divalent oxygen atom within the structure of a compound, where at
least one valency of the phenolic oxygen atom is bonded to an
sp.sup.2-hybridized carbon atom within an aromatic ring.
[0098] The term "physiological conditions," as used herein, refers
to the conditions prevalent in vivo. For example, incubation in
simulated gastric fluid (SGF) or simulated intestinal fluid (SIF)
at physiologically relevant temperatures (e.g., about 36-37.degree.
C.) may be used to simulate physiological conditions representative
of a stomach or upper intestine, respectively. Colon conditions may
be simulated using a slurry of a healthy human fecal matter under
anaerobic conditions.
[0099] The term "prevent," as used herein in reference to the
medical effect of a compound of the disclosure on a subject, refers
to minimizing or partially or completely inhibiting the development
of the associated disease, disorder, or condition. Non-limiting
examples of the disease, disorder, or condition are those described
herein.
[0100] The term "protecting group," as used herein, represents a
group intended to protect a hydroxy, an amino, or a carbonyl from
participating in one or more undesirable reactions during chemical
synthesis. The term "O-protecting group," as used herein,
represents a group intended to protect a hydroxy or carbonyl group
from participating in one or more undesirable reactions during
chemical synthesis. The term "N-protecting group," as used herein,
represents a group intended to protect a nitrogen containing (e.g.,
an amino or hydrazine) group from participating in one or more
undesirable reactions during chemical synthesis. Commonly used O-
and N-protecting groups are disclosed in Greene, "Protective Groups
in Organic Synthesis," 3.sup.rd Edition (John Wiley & Sons, New
York, 1999), which is incorporated herein by reference. Exemplary
O- and N-protecting groups include alkanoyl, aryloyl, or carbamyl
groups such as formyl, acetyl, propionyl, pivaloyl, t-butylacetyl,
2-chloroacetyl, 2-bromoacetyl, trifluoroacetyl, trichloroacetyl,
phthalyl, o-nitrophenoxyacetyl, .alpha.-chlorobutyryl, benzoyl,
4-chlorobenzoyl, 4-bromobenzoyl, t-butyldimethylsilyl,
tri-iso-propylsilyloxymethyl, 4,4'-dimethoxytrityl, isobutyryl,
phenoxyacetyl, 4-isopropylpehenoxyacetyl, dimethylformamidino, and
4-nitrobenzoyl.
[0101] Exemplary O-protecting groups for protecting carbonyl
containing groups include, but are not limited to: acetals,
acylals, 1,3-dithianes, 1,3-dioxanes, 1,3-dioxolanes, and
1,3-dithiolanes.
[0102] Other O-protecting groups include, but are not limited to:
substituted alkyl, aryl, and aryl-alkyl ethers (e.g., trityl;
methylthiomethyl; methoxymethyl; benzyloxymethyl; siloxymethyl;
2,2,2,-trichloroethoxymethyl; tetrahydropyranyl; tetrahydrofuranyl;
ethoxyethyl; 1-[2-(trimethylsilyl)ethoxy]ethyl;
2-trimethylsilylethyl; t-butyl ether; p-chlorophenyl,
p-methoxyphenyl, p-nitrophenyl, benzyl, p-methoxybenzyl, and
nitrobenzyl); silyl ethers (e.g., trimethylsilyl; triethylsilyl;
triisopropylsilyl; dimethylisopropylsilyl; t-butyldimethylsilyl;
t-butyldiphenylsilyl; tribenzylsilyl; triphenylsilyl; and
diphenymethylsilyl); carbonates (e.g., methyl, methoxymethyl,
9-fluorenylmethyl; ethyl; 2,2,2-trichloroethyl;
2-(trimethylsilyl)ethyl; vinyl, allyl, nitrophenyl; benzyl;
methoxybenzyl; 3,4-dimethoxybenzyl; and nitrobenzyl).
[0103] Other N-protecting groups include, but are not limited to,
chiral auxiliaries such as protected or unprotected D, L or D,
L-amino acids such as alanine, leucine, phenylalanine, and the
like; sulfonyl-containing groups such as benzenesulfonyl,
p-toluenesulfonyl, and the like; carbamate forming groups such as
benzyloxycarbonyl, p-chlorobenzyloxycarbonyl,
p-methoxybenzyloxycarbonyl, p-nitrobenzyloxycarbonyl,
2-nitrobenzyloxycarbonyl, p-bromobenzyloxycarbonyl,
3,4-dimethoxybenzyloxycarbonyl, 3,5-dimethoxybenzyl oxycarbonyl,
2,4-dimethoxybenzyloxycarbonyl, 4-methoxybenzyloxycarbonyl,
2-nitro-4,5-dimethoxybenzyloxycarbonyl,
3,4,5-trimethoxybenzyloxycarbonyl,
1-(p-biphenylyl)-1-methylethoxycarbonyl,
.alpha.,.alpha.-dimethyl-3,5-dimethoxybenzyloxycarbonyl,
benzhydryloxy carbonyl, t-butyloxycarbonyl,
diisopropylmethoxycarbonyl, isopropyloxycarbonyl, ethoxycarbonyl,
methoxycarbonyl, allyloxycarbonyl, 2,2,2,-trichloroethoxycarbonyl,
phenoxycarbonyl, 4-nitrophenoxy carbonyl,
fluorenyl-9-methoxycarbonyl, cyclopentyloxycarbonyl,
adamantyloxycarbonyl, cyclohexyloxycarbonyl, phenylthiocarbonyl,
and the like, aryl-alkyl groups such as benzyl, triphenylmethyl,
benzyloxymethyl, and the like and silyl groups such as
trimethylsilyl, and the like. Useful N-protecting groups are
formyl, acetyl, benzoyl, pivaloyl, t-butylacetyl, alanyl,
phenylsulfonyl, benzyl, t-butyloxycarbonyl (Boc), and
benzyloxycarbonyl (Cbz).
[0104] The term "subject," as used herein, represents a human or
non-human animal (e.g., a mammal) that is suffering from, or is at
risk of, disease, disorder, or condition, as determined by a
qualified professional (e.g., a doctor or a nurse practitioner)
with or without known in the art laboratory test(s) of sample(s)
from the subject. Non-limiting examples of diseases, disorders, and
conditions include those described herein.
[0105] The term "sugar acid," as used herein, refers to a
monosaccharide, in the linear form of which one or both terminal
positions are oxidized to a carboxylic acid. There are at least
four classes of sugar acids: aldonic acid, ulosonic acid, uronic
acid, and aldaric acid. Any of the four sugar acid classes may be
used in conjugates disclosed herein. Non-limiting examples of sugar
acids include xylonic acid, gluconic acid, glucuronic acid,
galacturonic acid, tartaric acid, saccharic acid, or mucic
acid.
[0106] The term "sugar acid acyl," as used herein, refers to a
monovalent group that is a sugar acid having a carboxylate, in
which --OH is replaced with a valency.
[0107] The term "thioalkenyl," as used herein, represents a group
--SR, where R is alkenyl. An optionally substituted thioalkenyl is
thioalkenyl that is optionally substituted as described herein for
alkenyl.
[0108] The term "thioalkyl," as used herein, represents a group
--SR, where R is alkyl. An optionally substituted thioalkyl is
thioalkyl that is optionally substituted as described herein for
alkyl.
[0109] The term "thioaryl," as used herein, represents a group
--SR, where R is aryl. An optionally substituted thioaryl is
thioaryl that is optionally substituted as described herein for
aryl.
[0110] "Treatment" and "treating," as used herein, refer to the
medical management of a subject with the intent to improve,
ameliorate, stabilize, or cure a disease, disorder, or condition.
This term includes active treatment (treatment directed to improve
the disease, disorder, or condition); causal treatment (treatment
directed to the cause of the associated disease, disorder, or
condition); palliative treatment (treatment designed for the relief
of symptoms of the disease, disorder, or condition); and supportive
treatment (treatment employed to supplement another therapy).
[0111] The compounds described herein, unless otherwise noted,
encompass isotopically enriched compounds (e.g., deuterated
compounds), tautomers, and all stereoisomers and conformers (e.g.
enantiomers, diastereomers, E/Z isomers, atropisomers, etc.), as
well as racemates thereof and mixtures of different proportions of
enantiomers or diastereomers, or mixtures of any of the foregoing
forms as well as salts (e.g., pharmaceutically acceptable
salts).
[0112] Other features and advantages of the disclosure will be
apparent from the disclosure.
[0113] As mentioned above, provided herein are compounds,
pharmaceutical compositions, and methods of their use. The
compounds of the disclosure may target the production of pathogenic
metabolites by certain bacteria in a subject, thereby reducing the
pathogenic metabolite levels in the subject.
[0114] The compounds of the disclosure may be a conjugate of the
disclosure, e.g., those compounds including a glycoside or an
acylated sugar. Upon administration of the conjugate of the
disclosure, the conjugate may be cleaved in vivo to remove the
glycoside or an acylated sugar from the compound and to release the
corresponding unconjugated compound of the disclosure. Conjugates
of the disclosure may be advantageous in therapeutic applications
benefitting from a particular tissue-targeted delivery of an
unconjugated compound of the disclosure.
[0115] Compounds of the disclosure that include at least one
glycoside or at least one acylated sugar are conjugates. Compounds
having a fatty acid acyl (e.g., a short chain fatty acid acyl)
attached through an ester bond are also conjugates.
[0116] Acylated sugars that may be used in the conjugates disclosed
herein include an acyl (e.g., a fatty acid acyl) and a core
selected from the group consisting of a carbohydrate (e.g., a
monosaccharide or disaccharide), sugar acid, and sugar alcohol. For
example, an acylated sugar may be a monovalent group of the
following formula:
##STR00005##
where
[0117] L is a bond to a pharmaceutically active agent, a compound
of formula (I), a compound of formula (II), a compound of formula
(III), a carbonate linker, or a carbamate linker;
[0118] group A is a core selected from the group consisting of
carbohydrate (e.g., a monosaccharide or dissaccharide), sugar acid,
and sugar alcohol;
[0119] each R is independently an acyl bonded to an oxygen atom in
group A; and
[0120] m is an integer from 0 to the total number of available
hydroxyl groups in group A (e.g., 1, 2, 3, 4, or 5).
[0121] In some embodiments, L may be attached to a carbon atom in
group A (e.g., an anomeric carbon atom or a carbonyl carbon atom).
In some embodiments, L may be attached to an oxygen atom in group A
(e.g., an alcoholic oxygen atom, a phenolic oxygen atom, or a
carboxylate oxygen atom).
[0122] In some embodiments, at least one R is a fatty acid
acyl.
[0123] In some embodiments, the fatty acid(s) are short chain fatty
acid acyls. In some embodiments, the short chain fatty acid acyl is
a C.sub.3-6 short chain fatty acid acyl (e.g., propionyl or
butyryl).
[0124] In some embodiments, the acylated sugar is peracylated,
i.e., all of the available hydroxyls in the acylated sugar are
substituted with an acyl.
[0125] A monosaccharide may be, e.g., arabinose, xylose, fructose,
galactose, glucose, ribose, tagatose, fucose, or rhamnose. In some
embodiments, the monosaccharide is L-arabinose, D-xylose, fructose,
galactose, D-glucose, D-ribose, D-tagatose, L-fucose, or L-rhamnose
(e.g., the monosaccharide is D-xylose). A disaccharide may be,
e.g., lactose, sucrose, melibiose, gentiobiose, kojibiose,
cellobiose, maltose, trehalose and chitobiose. A sugar acid may be,
e.g., aldonic acid, ulosonic acid, uronic acid, or aldaric acid. A
sugar acid may be, e.g., xylonic acid, gluconic acid, glucuronic
acid, galacturonic acid, tartaric acid, saccharic acid, or mucic
acid. A sugar alcohol may be, e.g., glycerol, erythritol, threitol,
arabitol, xylitol, tibitol, mannitol, sorbitol, galactitol,
fucitol, iditol, or inositol.
[0126] An acylated sugar may be covalently linked to a
pharmaceutically active agent through a carbon-oxygen bond that is
cleavable in vivo, a carbonate linker, or a carbamate linker. The
carbon-oxygen bond may be, e.g., a glycosidic bond or ester bond.
Acylated sugars having a monosaccharide, disaccharide, or a sugar
acid as a core may be covalently linked to a pharmaceutically
active agent through a carbon-oxygen bond that is cleavable in vivo
(e.g., a glycosidic bond or ester bond), a carbonate linker, or a
carbamate linker. In the sugar acid core, one or both carboxylates
may be present as O-protected versions (e.g., as alkyl esters
(e.g., methyl or ethyl esters)). Acylated sugars having a sugar
alcohol as a core may be covalently linked to a pharmaceutically
active agent through a carbon-oxygen bond that is cleavable in vivo
(e.g., an ester bond), a carbonate linker, or a carbamate
linker.
[0127] Non-limiting examples of acylated sugars are:
##STR00006##
[0128] where
[0129] R is H, --CH.sub.3, or --CH.sub.2OR.sup.FA;
[0130] each R.sup.FA is independently H or a fatty acid acyl (e.g.,
a short chain fatty acid acyl);
[0131] each R.sup.A is independently H or fatty acid acyl; and
[0132] R.sup.B is H, --CH.sub.3, --CH.sub.2--OR.sup.A, --OCH.sub.3,
--COOCH.sub.3, or --COOH.
[0133] In some embodiments, the disclosure provides a compound of a
cation and a pharmaceutically acceptable counterion, where the
cation is chosen from the following cations:
##STR00007## ##STR00008## ##STR00009## ##STR00010## ##STR00011##
##STR00012## ##STR00013## ##STR00014## ##STR00015## ##STR00016##
##STR00017## ##STR00018## ##STR00019## ##STR00020## ##STR00021##
##STR00022## ##STR00023## ##STR00024## ##STR00025## ##STR00026##
##STR00027## ##STR00028## ##STR00029## ##STR00030##
##STR00031##
[0134] In some embodiments, the disclosure provides a compound of a
cation and a pharmaceutically acceptable counterion, where the
cation is chosen from the following cations:
##STR00032## ##STR00033## ##STR00034## ##STR00035##
[0135] In some embodiments, provided here are compounds consisting
of a cation and a pharmaceutically acceptable counterion, wherein
the cation is chosen from the following cations:
##STR00036## ##STR00037## ##STR00038## ##STR00039## ##STR00040##
##STR00041## ##STR00042## ##STR00043## ##STR00044## ##STR00045##
##STR00046## ##STR00047## ##STR00048## ##STR00049##
[0136] The disclosure provides a compound consisting of a cation
and a pharmaceutically acceptable counterion, where the cation is a
structure of formula (I):
##STR00050##
wherein
[0137] R.sup.1 is [0138] C.sub.2-6 alkyl substituted with
--O-(acylated sugar) or isosorbide, wherein said C.sub.2-6 alkyl is
further optionally substituted with oxo and/or methene; [0139]
C.sub.4 cycloalkyl optionally substituted with hydroxyl, ethynyl,
or --O-(acylated sugar); or [0140] C.sub.3 cycloalkyl substituted
with C.sub.1-6 alkyl, hydroxyl, ethynyl, or --O-(acylated sugar)
[0141] C.sub.3-4 cycloalkyl C.sub.1-2 alkyl;
[0142] R.sup.2 is C.sub.2-6 alkyl optionally substituted with one
or two hydroxyl, oxo, and --O-(acylated sugar);
[0143] or R.sup.1 and R.sup.2, together with the nitrogen atom to
which both are attached, combine to form a 4- or 5-membered
heterocyclic ring optionally substituted with ethynyl or
--(CH.sub.2).sub.n--OR.sup.s or an acylated sugar, wherein n is 0
or 1, R.sup.s is hydrogen or an acylated sugar;
[0144] R.sup.3 is C.sub.1-6 alkyl optionally substituted with a
halogen or hydroxyl; and
[0145] R.sup.4 is C.sub.1-6 alkyl or propargyl.
[0146] In some embodiments, the cation comprises at least one
acylated sugar. In some embodiments, the acylated sugar is chosen
from groups of formula (A), groups of formula (B), and groups of
formula (C):
##STR00051##
wherein
[0147] each R.sup.A is independently H or fatty acid acyl; and
[0148] R.sup.B is H, --C.sub.1-6alkyl,
--C.sub.1-6alkylene-OR.sup.A, --OC.sub.1-6alkyl,
--COOC.sub.1-6alkyl, or --COOH.
[0149] In some embodiments, acylated sugar is a group of formula
(A).
##STR00052##
where
[0150] each R.sup.A is independently H or fatty acid acyl; and
[0151] R.sup.B is H, --CH.sub.3, --CH.sub.2--OR.sup.A, --OCH.sub.3,
--COOCH.sub.3, or --COOH.
[0152] In some embodiments, acylated sugar is a group of formula
(B):
##STR00053##
where
[0153] each R.sup.A is independently H or fatty acid acyl; and
[0154] R.sup.B is H, --CH.sub.3, --CH.sub.2--OR.sup.A, --OCH.sub.3,
--COOCH.sub.3, or --COOH.
[0155] In some embodiments, acylated sugar is a group of formula
(C):
##STR00054##
where
[0156] each R.sup.A is independently H or fatty acid acyl; and
[0157] R.sup.B is H, --CH.sub.3, --CH.sub.2--OR.sup.A, --OCH.sub.3,
--COOCH.sub.3, or --COOH.
[0158] In some embodiments, in groups of formula (A), (B), and (C),
each R.sup.A is independently chosen from fatty acid acyls. In some
embodiments, each R.sup.A is independently chosen from short chain
fatty acid acyls.
[0159] In some embodiments, the acylated sugar is chosen from
groups of formula (A).
[0160] In some embodiments, at least one R.sup.A is H. In some
embodiments, at least one R.sup.A is --COCH.sub.3. In some
embodiments, at least one R.sup.A is --COCH.sub.2CH.sub.3. In some
embodiments, at least one R.sup.A is
--COCH.sub.2CH.sub.2CH.sub.3.
[0161] In some embodiments, at least three R.sup.A are H. In some
embodiments, at least three R.sup.A are --COCH.sub.3. In some
embodiments, at least three R.sup.A are --COCH.sub.2CH.sub.3. In
some embodiments at least three R.sup.A are
--COCH.sub.2CH.sub.2CH.sub.3.
[0162] In some embodiments, R.sup.B is H. In some embodiments,
R.sup.B is --CH.sub.3. In some embodiments, R.sup.B is
--CH.sub.2OH. In some embodiments, R.sup.B is
--CH.sub.2--OCOCH.sub.3. In some embodiments, R.sup.B is
--CH.sub.2--OCOCH.sub.2CH.sub.3. In some embodiments, R.sup.B is
--COOH. In some embodiments, R.sup.B is --COOCH.sub.3.
[0163] In some embodiments, R.sup.B is H, --CH.sub.3,
--CH.sub.2--OR.sup.A, --OCH.sub.3, --COOCH.sub.3, or --COOH. In
some embodiments, R.sup.B is H, --C.sub.1-6alkyl, or
--C.sub.1-6alkylene-OR.sup.A wherein R.sup.A is H or fatty acid
acyl.
[0164] In some embodiments, R.sup.B is H, --CH.sub.3, or
--CH.sub.2--OR.sup.A, wherein R.sup.A is H or fatty acid acyl. In
some embodiments, R.sup.B is --OC.sub.1-6alkyl,
--COOC.sub.1-6alkyl, or --COOH. In some embodiments, R.sup.B is
--OCH.sub.3, --COOCH.sub.3, or --COOH.
[0165] In some embodiments, the acylated sugar is a group of the
following formula:
##STR00055##
[0166] In some embodiments, the acylated sugar is a group of the
following formula:
##STR00056##
[0167] In some embodiments, R.sup.1 is C.sub.2-6 alkyl substituted
with --O-(acylated sugar), wherein said C.sub.2-6 alkyl is further
optionally substituted with oxo. In some embodiments, R.sup.1 is
C.sub.2-6 alkyl substituted with oxo and --O-(acylated sugar). In
some embodiments, R.sup.1 is C.sub.3-4 cycloalkyl substituted with
hydroxyl, ethynyl, or --O-(acylated sugar). In some embodiments,
R.sup.1 is C.sub.3-4 cycloalkyl C.sub.1-2 alkyl. In some
embodiments, R.sup.2 is methyl. In some embodiments, R.sup.2 is
C.sub.2-6 alkyl, wherein the C.sub.2-6 alkyl is substituted with
one or two substituents independently selected from the group
consisting of hydroxyl, oxo, and --O-(acylated sugar). In some
embodiments, R.sup.1 and R.sup.2, together with the nitrogen atom
to which both are attached, combine to form a 4- or 5-membered
heterocyclic ring optionally substituted with ethynyl or
--(CH.sub.2).sub.n--OR.sup.A, wherein n is 0 or 1, and R.sup.A is
hydrogen or an acylated sugar. In some embodiments, R.sup.S is
hydrogen. In another embodiment, R.sup.3 is C.sub.1-6 alkyl
substituted with a halogen or hydroxyl. In some embodiments,
R.sup.4 is propargyl. In some embodiments, R.sup.4 is C.sub.1-6
alkyl.
[0168] In some embodiments, R.sup.1 is C.sub.2-6 alkyl substituted
with --O-(acylated sugar) and is optionally further substituted
with oxo; R.sup.2 is methyl; R.sup.3 is C.sub.1-6 alkyl; and
R.sup.4 is propargyl. In some embodiments, R.sup.1 is C.sub.2-6
alkyl substituted with isosorbide and is optionally further
substituted with oxo and/or methene; R.sup.2 is methyl; R.sup.3 is
C.sub.1-6 alkyl; and R.sup.4 is propargyl. In some embodiments,
R.sup.1 is C.sub.2-6 alkyl substituted with --O-(acylated sugar)
and is optionally further substituted with oxo. In some
embodiments, R.sup.1 is C.sub.2-6 alkyl substituted with
--O-(acylated sugar) and further substituted with oxo.
[0169] In some embodiments, R.sup.1 is chosen from C.sub.2 alkyl
substituted with --O-(acylated sugar) and optionally further
substituted with oxo and C.sub.4 alkyl substituted with
--O-(acylated sugar) and optionally further substituted with
oxo.
[0170] In some embodiments, R.sup.1 is chosen from C.sub.2 alkyl
substituted with --O-(acylated sugar) and optionally further
substituted with oxo. In some embodiments, R.sup.1 is chosen from
C.sub.2 alkyl substituted with --O-(acylated sugar) and further
substituted with oxo. In some embodiments, R.sup.1 is chosen from
C.sub.4 alkyl substituted with --O-(acylated sugar) and optionally
further substituted with oxo. In some embodiments, R.sup.1 is
chosen from C.sub.4 alkyl substituted with --O-(acylated sugar) and
further substituted with oxo.
[0171] In some embodiments, R.sup.1 is C.sub.2-6 alkyl substituted
with isosorbide and is optionally further substituted with oxo
and/or methene. In some embodiments, isosorbide is chosen from
groups of formula (C):
##STR00057##
[0172] In some embodiments, R.sup.1 is C.sub.2 alkyl substituted
with isosorbide. In some embodiments, R.sup.1 is C.sub.2-6 alkyl
substituted with isosorbide and further substituted with oxo and/or
methene. In some embodiments, R.sup.1 is C.sub.2-6 alkyl
substituted with isosorbide and further substituted with oxo. In
some embodiments, R.sup.1 is C.sub.2 alkyl substituted with
isosorbide and further substituted with oxo. In some embodiments,
R.sup.1 is C.sub.2-6 alkyl substituted with isosorbide and further
substituted with methene. In some embodiments, R.sup.1 is C.sub.2-6
alkyl substituted with isosorbide and further substituted with oxo
and methene. In some embodiments, R.sup.1 is
##STR00058##
In some embodiments, R.sup.1 is
##STR00059##
[0173] In some embodiments, R.sup.1 is C.sub.3-4 cycloalkyl
C.sub.1-2 alkyl. In some embodiments, R.sup.1 is C.sub.3-4
cycloalkyl C.sub.1 alkyl. In some embodiments, R.sup.1 is
cyclopropylmethyl. In some embodiments, R.sup.1 is
cyclobutylmethyl.
[0174] In some embodiments, R.sup.2 is C.sub.2-6 alkyl optionally
substituted with one or two hydroxyl, oxo, and --O-(acylated
sugar). In some embodiments, R.sup.2 is methyl.
[0175] In some embodiments, R.sup.1 and R.sup.2, together with the
nitrogen atom to which both are attached, combine to form a 4- or
5-membered heterocyclic ring optionally substituted with ethynyl or
--(CH.sub.2).sub.n--OR.sup.s or an acylated sugar, wherein n is 0
or 1, R.sup.s is hydrogen or an acylated sugar.
[0176] In some embodiments, R.sup.3 is C.sub.1-6 alkyl optionally
substituted with a halogen or hydroxyl. In some embodiments,
R.sup.3 is C.sub.1-6 alkyl. In some embodiments, R.sup.3 is
C.sub.1-6 alkyl optionally substituted with a halogen or hydroxyl.
In some embodiments, R.sup.3 is C.sub.1-6 alkyl optionally
substituted with a halogen and/or hydroxyl. In some embodiments,
R.sup.3 is C.sub.1-6 alkyl optionally substituted with a halogen or
hydroxyl. In some embodiments, R.sup.3 is C.sub.1-6 alkyl
substituted with a halogen. In some embodiments, R.sup.3 is
C.sub.1-6 alkyl substituted with a hydroxyl.
[0177] In some embodiments, R.sup.3 is C.sub.1 alkyl optionally
substituted with a halogen. In some embodiments, R.sup.3 is C.sub.1
alkyl substituted with a halogen.
[0178] In some embodiments, R.sup.4 is C.sub.1-6 alkyl or
propargyl. In some embodiments, R.sup.4 is C.sub.1 alkyl or
propargyl. In some embodiments, R.sup.4 is propargyl.
[0179] In some embodiments, R.sup.1 is C.sub.2-6 alkyl substituted
with --O-(acylated sugar) and is optionally further substituted
with oxo; R.sup.2 is methyl; R.sup.3 is C.sub.1-6 alkyl; and
R.sup.4 is propargyl.
[0180] In some embodiments, R.sup.1 is C.sub.2-6 alkyl substituted
with isosorbide and is optionally further substituted with oxo
and/or methene; R.sup.2 is methyl; R.sup.3 is C.sub.1-6 alkyl; and
R.sup.4 is propargyl.
[0181] In some embodiments, R.sup.1 is chosen from C.sub.2 alkyl
substituted with --O-(acylated sugar) and optionally further
substituted with oxo and C.sub.4 alkyl substituted with
--O-(acylated sugar) and optionally further substituted with oxo;
wherein the acylated sugar is chosen from groups of formula (A),
groups of formula (B), and groups of formula (C):
##STR00060##
[0182] wherein [0183] each R.sup.A is independently H or fatty acid
acyl; and [0184] R.sup.B is H, --CH.sub.3, --CH.sub.2--OR.sup.A,
--OCH.sub.3, --COOCH.sub.3, or --COOH.
[0185] R.sup.2 is methyl;
[0186] R.sup.3 is methyl; and
[0187] R.sup.4 is propargyl.
[0188] In some embodiments, R.sup.1 is chosen from C.sub.2 alkyl
substituted with isosorbide and optionally further substituted with
oxo and C.sub.3 alkyl substituted with isosorbide and optionally
further substituted with oxo and methene;
[0189] wherein isosorbide is chosen from groups of formula (C):
##STR00061##
[0190] R.sup.2 is methyl;
[0191] R.sup.3 is methyl; and
[0192] R.sup.4 is propargyl.
[0193] In some embodiments, R.sup.1 is C.sub.3-4 cycloalkyl
C.sub.1-2 alkyl; R.sup.2 is C.sub.2-6 alkyl optionally substituted
with one or two hydroxyl, oxo, and --O-(acylated sugar); or R.sup.1
and R.sup.2, together with the nitrogen atom to which both are
attached, combine to form a 4- or 5-membered heterocyclic ring
optionally substituted with ethynyl or --(CH.sub.2).sub.n--OR.sup.s
or an acylated sugar, wherein n is 0 or 1, R.sup.s is hydrogen or
an acylated sugar; R.sup.3 is C.sub.1-6 alkyl optionally
substituted with a halogen or hydroxyl; and R.sup.4 is C.sub.1-6
alkyl or propargyl. In some embodiments, R.sup.1 is C.sub.3-4
cycloalkyl C.sub.1 alkyl. In some embodiments, R.sup.2 is C.sub.2
alkyl optionally substituted with one or two hydroxyl groups. In
some embodiments, R.sup.1 and R.sup.2 together with the nitrogen
atom to which both are attached, combine to form a 5-membered
heterocyclic ring optionally substituted with ethynyl, --OH, or
--CH.sub.2OH. In some embodiments, R.sup.3 is C.sub.1 alkyl
optionally substituted with a halogen. In some embodiments, R.sup.4
is C.sub.1 alkyl or propargyl.
[0194] In some embodiments, provided herein are a method of
treating a subject in need thereof, the method comprising
administering to the subject in need thereof a therapeutically
effective amount of a compound of the invention, a pharmaceutical
composition of the invention, or the compound of formula (I-a) or
(I-b), where the compound of formula (I-a) is:
##STR00062##
where
[0195] R.sup.1 is H or C.sub.1-3 alkyl optionally substituted with
hydroxyl;
[0196] R.sup.2 is H or C.sub.1-3 alkyl optionally substituted with
hydroxyl;
[0197] R.sup.3 is absent or alkyl;
[0198] Z.sup.q is absent or a pharmaceutically acceptable
counterion present in the stoichiometric ratio appropriate for the
compound of formula (I-a) to have no net charge,
[0199] when R.sup.3 is absent, Z.sup.q is absent; and
[0200] where the compound of formula (I-b) is:
##STR00063##
where
[0201] n is 2, 3, or 4;
[0202] X.sup.1 is C, S, or P;
[0203] R.sup.1 is propargyl or C.sub.1-6 alkyl;
[0204] R.sup.2 is absent, hydroxyl, or oxo;
[0205] Z.sup.q is a pharmaceutically acceptable counterion present
in the stoichiometric ratio appropriate for the compound of formula
(I-b) to have no net charge.
[0206] In some embodiments, provided herein are compounds
consisting of a cation and a pharmaceutically acceptable
counterion, wherein the cation is a structure of formula (II):
##STR00064##
wherein
[0207] R.sup.1 is [0208] C.sub.1-6 alkyl optionally substituted
with one or more oxo, hydroxyl, halogen, cyano, --COOMe, amino,
methene, ethenyl, ethynyl, hydroxyphenyl, C.sub.3-4 cycloalkyl,
--OCH.sub.2CH.sub.2OH, --HNC(O)OCMe.sub.3, --SMe, --OMe,
--HNS(O).sub.2Me, --SO.sub.3H, B(OH).sub.2, PO.sub.3H.sub.2,
PO.sub.2H.sub.2, --P(O)(OCH.sub.2CH.sub.3).sub.2,
--P(O)(OH)(OCH.sub.2CH.sub.3), heteroaryl ring; [0209] phenyl;
[0210] benzyl; [0211] C.sub.3-4 heterocyclyl optionally substituted
with C.sub.1 alkyl [0212] C.sub.4 heterocyclylalkyl C.sub.1 alkyl;
or [0213] C.sub.4 cycloalkyl;
[0214] R.sup.2 is [0215] C.sub.1-6 alkyl optionally substituted
with one or two hydroxyl, oxo, methene, cyano, ethynyl, --HNC(O)H,
or --C.ident.C--CH.sub.2OH; or [0216]
--HN(CH.sub.2).sub.3C(O)OH;
[0217] or R.sup.1 and R.sup.2, together with the nitrogen atom to
which both are attached and optionally one or more additional
heteroatoms, combine to form a 4-8 membered mono- or bi-cyclic
heterocycle optionally substituted with ethynyl, trifluoromethyl,
--CH.sub.2Ph, --OH, or --(CH.sub.2)OH;
[0218] R.sup.3 is methyl or propargyl; and
[0219] R.sup.4 is methyl or propargyl,
[0220] with the proviso that
[0221] (1) when two of R.sup.1, R.sup.2, R.sup.3, and R.sup.4 are
methyl and one of the other two of R.sup.1, R.sup.2, R.sup.3, and
R.sup.4 is propargyl, then the other of R.sup.1, R.sup.2, R.sup.3,
and R.sup.4 is not methyl, a monohalomethyl, --CH.sub.2CH.sub.2OH,
--COOH, --(CH.sub.2).sub.4C(O)OH, or --CH.sub.2C(CH.sub.2)CN;
and
[0222] (2) when two of R.sup.1, R.sup.2, R.sup.3, and R.sup.4 are
methyl and one of the other two of R.sup.1, R.sup.2, R.sup.3, and
R.sup.4 is --CH.sub.2CH.sub.2OH or --CH.sub.2C(O)OH, then the other
of R.sup.1, R.sup.2, R.sup.3, and R.sup.4 is not a
monohalomethyl.
[0223] In some embodiments, provided herein are compounds
consisting of a cation and a pharmaceutically acceptable
counterion, wherein the cation is a structure of formula (III):
##STR00065##
wherein
[0224] R.sup.1 is [0225] C.sub.1-6 alkyl optionally substituted
with one or more oxo, hydroxyl, halogen, cyano, --COOMe, --COOH,
methene, ethenyl, ethynyl, C.sub.3-4cycloalkyl,
--OCH.sub.2CH.sub.2OH, --SMe, --OMe, --HNS(O).sub.2Me, --P(O)
(OCH.sub.2CH.sub.3).sub.2, --P(O)(OH)(OCH.sub.2CH.sub.3), or
5-membered heteroaryl ring;
[0226] R.sup.2 is [0227] C.sub.1-6 alkyl optionally substituted
with one or two hydroxyl, oxo, methene, cyano, ethynyl, --HNC(O)H,
or --C.ident.C--CH.sub.2OH; or [0228]
--HN(CH.sub.2).sub.3C(O)OH;
[0229] or R.sup.1 and R.sup.2, together with the nitrogen atom to
which both are attached and optionally one or more additional
heteroatoms, combine to form a 4-8 membered mono- or bi-cyclic
heterocycle optionally substituted with ethynyl, --OH, or
--(CH.sub.2)OH;
[0230] R.sup.3 is C.sub.1-6 alkyl optionally substituted with a
halogen, hydroxyl, or ethynyl; and
[0231] R.sup.4 is C.sub.1-6 alkyl or propargyl,
with the proviso that
[0232] (1) when two of R.sup.1, R.sup.2, R.sup.3, and R.sup.4 are
methyl and one of the other two of R.sup.1, R.sup.2, R.sup.3, and
R.sup.4 is propargyl, then the other of R.sup.1, R.sup.2, R.sup.3,
and R.sup.4 is not methyl, a monohalomethyl, --CH.sub.2CH.sub.2OH,
--COOH, --(CH.sub.2).sub.4C(O)OH, or --CH.sub.2C(CH.sub.2)CN;
and
[0233] (2) when two of R.sup.1, R.sup.2, R.sup.3, and R.sup.4 are
methyl and one of the other two of R.sup.1, R.sup.2, R.sup.3, and
R.sup.4 is --CH.sub.2CH.sub.2OH or --CH.sub.2C(O)OH, then the other
of R.sup.1, R.sup.2, R.sup.3, and R.sup.4 is not a
monohalomethyl.
[0234] In some embodiments, in cations of formula (II) or (III),
R.sup.2, R.sup.3, and R.sup.4 are methyl.
[0235] Production of trimethylamine by CutC may be inhibited using
the compounds described above. Accordingly, a method of inhibiting
a CutC choline lyase-mediated conversion of choline to
trimethylamine includes contacting the compound described above
with the CutC choline lyase.
[0236] Inhibition of CutC may lead to the modulation of
trimethylaminuria markers. Accordingly, a method of modulating a
trimethylamine marker in a subject in need thereof includes
administering to the subject in need thereof a therapeutically
effective amount of the compound described above (e.g., as a
pharmaceutical composition). The trimethylaminuria marker can be,
e.g., trimethylamine and/or trimethylamine oxide levels in the
subject's blood, plasma, serum, or urine. The amount of the
trimethylaminuria marker can be reduced in accordance with the
methods of the disclosure.
[0237] Trimethylaminuria and the associated cardiovascular
disorders can be treated using the compounds described above.
Accordingly, a method of treating a subject in need thereof
includes administering to the subject in need thereof a
therapeutically effective amount of the compound described above.
For example, a method of treating or preventing a disease
associated with trimethylaminuria in a subject in need thereof
includes administering a therapeutically effective amount of the
compound described above (e.g., as a pharmaceutical composition).
The disease associated with trimethylaminuria may be, e.g., a
cardiovascular disease, reduced or impaired kidney function, kidney
disease, chronic kidney disease, end-stage renal disease, or
diabetes mellitus. The cardiovascular disease may be, e.g., angina,
arrhythmia, atherosclerosis, cardiomyopathy, congestive heart
failure, coronary artery disease, carotid artery disease,
endocarditis, coronary thrombosis, myocardial infarction, high
blood pressure/hypertension, hypercholesterolemia, hyperlipidemia,
mitral valve prolapse, peripheral artery disease, or stroke.
[0238] In some embodiments, provided herein is a method of
modulating a trimethylaminuria marker in a subject in need thereof,
the method comprising administering to the subject in need thereof
a therapeutically effective amount of at least one of the compounds
or pharmaceutical compositions disclosed herein. In some
embodiments, the trimethylaminuria marker is the trimethylamine
and/or trimethylamine oxide level in the subject's blood, plasma,
serum, or urine.
[0239] In some embodiments, provided herein is a method of treating
or preventing a disease associated with elevated levels of
trimethylamine (TMA) or trimethylamine N-oxide (TMAO), a decreased
rate of conversion of TMA to TMAO, or a high ratio of TMA to TMAO
in a subject in need thereof, the method comprising administering a
therapeutically effective amount of at least one of the compounds
or pharmaceutical compositions disclosed herein to the subject. In
some embodiments, the method further comprises detecting the
presence of one or more genetic variants of the FMO3 gene of the
subject in need before the administering step. In some embodiments,
the disease associated with elevated levels of TMA or TMAO, a
decreased rate of conversion of TMA to TMAO, or a high ratio of TMA
to TMAO is a cardiovascular disease, reduced or impaired kidney
function, kidney disease, chronic kidney disease, end-stage renal
disease, or diabetes mellitus. In some embodiments, the
cardiovascular disease is angina, arrhythmia, atherosclerosis,
cardiomyopathy, congestive heart failure, coronary artery disease,
carotid artery disease, endocarditis, coronary thrombosis,
myocardial infarction, high blood pressure/hypertension,
hypercholesterolemia, hyperlipidemia, mitral valve prolapse,
peripheral artery disease, or stroke.
[0240] In some embodiments, provided herein is a method of
inhibiting a CutC choline lyase-mediated conversion of choline to
trimethylamine, the method comprising contacting at least one of
the compounds or pharmaceutical compositions disclosed herein with
the CutC choline lyase.
[0241] In some embodiments, provided herein is a method of
inhibiting a CntA carnitine monooxygenase-mediated conversion of
carnitine to trimethylamine, the method comprising contacting at
least one of the compounds or pharmaceutical compositions disclosed
herein with the CntA carnitine monooxygenase.
[0242] In some embodiments, provided herein is a method of
inhibiting a betaine reductase-mediated conversion of betaine or
gamma-butyrobetaine to trimethylamine, the method comprising
contacting at least one of the compounds or pharmaceutical
compositions disclosed herein with the betaine reductase.
[0243] In some embodiments, provided herein is a method of treating
a subject in need of treatment for trimethylaminuria comprising
contacting bacteria in vivo with a therapeutically effective amount
of at least one of the compounds or pharmaceutical compositions
disclosed herein to the subject. In some embodiments, the bacteria
are localized in the colon of the subject.
[0244] In some embodiments, the present disclosure provides a
method of modulating a trimethylamine marker in a subject in need
thereof, the method consisting of administering to the subject in
need thereof a therapeutically effective amount of at least one
compound of the present disclosure or a pharmaceutical composition
of the present disclosure.
[0245] In some embodiments, the trimethylaminuria marker is the
trimethylamine and/or trimethylamine oxide levels in the subject's
blood, plasma, serum, or urine.
[0246] In some embodiments, the present disclosure provides a
method of treating or preventing a disease associated with
trimethylaminuria in a subject in need thereof, the method
comprising administering a therapeutically effective amount of at
least one pharmaceutically acceptable salt of at least one compound
of the present disclosure or a pharmaceutical composition of the
present disclosure to the subject.
[0247] The compounds disclosed herein may be formulated into
pharmaceutical compositions for administration to human subjects in
a biologically compatible form suitable for administration in vivo.
Pharmaceutical compositions typically include a compound as
described herein and a physiologically acceptable excipient (e.g.,
a pharmaceutically acceptable excipient).
[0248] The compound described herein can also be used in the form
of the free acid/base, in the form of salts, zwitterions, or as
solvates. All forms are within the scope of the disclosure. The
compounds, salts, zwitterions, solvates, or pharmaceutical
compositions thereof, may be administered to a subject in a variety
of forms depending on the selected route of administration, as will
be understood by those skilled in the art. The compounds described
herein may be administered, for example, by oral, parenteral,
buccal, sublingual, nasal, rectal, patch, pump, or transdermal
administration, and the pharmaceutical compositions formulated
accordingly. Parenteral administration includes intravenous,
intraperitoneal, subcutaneous, intramuscular, transepithelial,
nasal, intrapulmonary, intrathecal, rectal, and topical modes of
administration. Parenteral administration may be by continuous
infusion over a selected period of time.
[0249] For human use, a compound disclosed herein can be
administered alone or in admixture with a pharmaceutical carrier
selected regarding the intended route of administration and
standard pharmaceutical practice. Pharmaceutical compositions for
use in accordance with the present disclosure thus can be
formulated in a conventional manner using one or more
physiologically acceptable carriers having excipients and
auxiliaries that facilitate processing of compounds disclosed
herein into preparations which can be used pharmaceutically.
[0250] This disclosure also includes pharmaceutical compositions
which can contain one or more physiologically acceptable carriers.
In making the pharmaceutical compositions of the disclosure, the
active ingredient is typically mixed with an excipient, diluted by
an excipient or enclosed within such a carrier in the form of, for
example, a capsule, sachet, paper, or other container. When the
excipient serves as a diluent, it can be a solid, semisolid, or
liquid material (e.g., normal saline), which acts as a vehicle,
carrier or medium for the active ingredient. Thus, the compositions
can be in the form of tablets, powders, lozenges, sachets, cachets,
elixirs, suspensions, emulsions, solutions, syrups, and soft and
hard gelatin capsules. As is known in the art, the type of diluent
can vary depending upon the intended route of administration. The
resulting compositions can include additional agents, e.g.,
preservatives.
[0251] The excipient or carrier is selected on the basis of the
mode and route of administration. Suitable pharmaceutical carriers,
as well as pharmaceutical necessities for use in pharmaceutical
formulations, are described in Remington: The Science and Practice
of Pharmacy, 21st Ed., Gennaro, Ed., Lippencott Williams &
Wilkins (2005), a well-known reference text in this field, and in
the USP/NF (United States Pharmacopeia and the National Formulary).
Examples of suitable excipients are lactose, dextrose, sucrose,
sorbitol, mannitol, starches, gum acacia, calcium phosphate,
alginates, tragacanth, gelatin, calcium silicate, microcrystalline
cellulose, polyvinylpyrrolidone, cellulose, water, syrup, and
methyl cellulose. The formulations can additionally include
lubricating agents, e.g., talc, magnesium stearate, and mineral
oil; wetting agents; emulsifying and suspending agents; preserving
agents, e.g., methyl- and propylhydroxy-benzoates; sweetening
agents; and flavoring agents. Other exemplary excipients are
described in Handbook of Pharmaceutical Excipients, 6th Edition,
Rowe et al., Eds., Pharmaceutical Press (2009).
[0252] These pharmaceutical compositions can be manufactured in a
conventional manner, e.g., by conventional mixing, dissolving,
granulating, dragee-making, levigating, emulsifying, encapsulating,
entrapping, or lyophilizing processes. Methods well known in the
art for making formulations are found, for example, in Remington:
The Science and Practice of Pharmacy, 21st Ed., Gennaro, Ed.,
Lippencott Williams & Wilkins (2005), and Encyclopedia of
Pharmaceutical Technology, eds. J. Swarbrick and J. C. Boylan,
1988-1999, Marcel Dekker, New York. Proper formulation is dependent
upon the route of administration chosen. The formulation and
preparation of such compositions is well-known to those skilled in
the art of pharmaceutical formulation. In preparing a formulation,
the compounds can be milled to provide the appropriate particle
size prior to combining with the other ingredients. If the compound
is substantially insoluble, it can be milled to a particle size of
less than 200 mesh. If the compound is substantially water soluble,
the particle size can be adjusted by milling to provide a
substantially uniform distribution in the formulation, e.g., about
40 mesh.
[0253] The dosage of the compound used in the methods described
herein, or pharmaceutically acceptable salts or prodrugs thereof,
or pharmaceutical compositions thereof, can vary depending on many
factors, e.g., the pharmacodynamic properties of the compound; the
mode of administration; the age, health, and weight of the
recipient; the nature and extent of the symptoms; the frequency of
the treatment, and the type of concurrent treatment, if any; and
the clearance rate of the compound in the subject to be treated.
One of skill in the art can determine the appropriate dosage based
on the above factors. The compounds used in the methods described
herein may be administered initially in a suitable dosage that may
be adjusted as required, depending on the clinical response. In
general, a suitable daily dose of a compound disclosed herein will
be that amount of the compound that is the lowest dose effective to
produce a therapeutic effect. Such an effective dose will generally
depend upon the factors described above.
[0254] A compound disclosed herein may be administered to the
subject in a single dose or in multiple doses. When multiple doses
are administered, the doses may be separated from one another by,
for example, 1-24 hours, 1-7 days, or 1-4 weeks. The compound may
be administered according to a schedule, or the compound may be
administered without a predetermined schedule. It is to be
understood that, for any particular subject, specific dosage
regimes should be adjusted over time according to the individual
need and the professional judgment of the person administering or
supervising the administration of the compositions.
[0255] The compounds may be provided in a dosage form. In some
embodiments, the unit dosage form may be an oral unit dosage form
(e.g., a tablet, capsule, suspension, liquid solution, powder,
crystals, lozenge, sachet, cachet, elixir, syrup, and the like) or
a food product serving (e.g., the active agents may be included as
food additives or dietary ingredients). In certain embodiments, the
dosage form is designed for administration of at least one compound
disclosed herein, where the total amount of an administered
compound is from 0.1 g to 10 g (e.g., 0.5 g to 9 g, 0.5 g to 8 g,
0.5 g to 7 g, 0.5 g to 6 g, 0.5 g to 5 g, 0.5 g to 1 g, 0.5 g to
1.5 g, 0.5 g to 2 g, 0.5 g to 2.5 g, 1 g to 1.5 g, 1 g to 2 g, 1 g
to 2.5 g, 1.5 g to 2 g, 1.5 g to 2.5 g, or 2 g to 2.5 g). In other
embodiments, the compound is consumed at a rate of 0.1 g to 10 g
per day (e.g., 0.5 g to 9 g, 0.5 g to 8 g, 0.5 g to 7 g, 0.5 g to 6
g, 0.5 g to 5 g, 0.5 g to 1 g per day, 0.5 g to 1.5 g per day, 0.5
g to 2 g per day, 0.5 g to 2.5 g per day, 1 g to 1.5 g per day, 1 g
to 2 g per day, 1 g to 2.5 g per day, 1.5 g to 2 g per day, 1.5 g
to 2.5 g per day, or 2 g to 2.5 g per day) or more. The attending
physician ultimately will decide the appropriate amount and dosage
regimen, an effective amount of the compound disclosed herein may
be, for example, a total daily dosage of, e.g., between 0.5 g and 5
g (e.g., 0.5 to 2.5 g) of any of the compound described herein.
Alternatively, the dosage amount can be calculated using the body
weight of the subject. When daily dosages exceed 5 g/day, the
dosage of the compound may be divided across two or three daily
administration events.
[0256] In the methods of the disclosure, the time period during
which multiple doses of a compound disclosed herein are
administered to a subject can vary. For example, in some
embodiments doses of the compounds are administered to a subject
over a time period that is 1-7 days; 1-12 weeks; or 1-3 months. In
other embodiments, the compounds are administered to the subject
over a time period that is, for example, 4-11 months or 1-30 years.
In yet other embodiments, the compounds disclosed herein are
administered to a subject at the onset of symptoms. In any of these
embodiments, the amount of the compound that is administered may
vary during the time period of administration. When a compound is
administered daily, administration may occur, for example, 1, 2, 3,
or 4 times per day.
[0257] A compound described herein may be administered to a subject
with a pharmaceutically acceptable diluent, carrier, or excipient,
in unit dosage form. Conventional pharmaceutical practice may be
employed to provide suitable formulations or compositions to
administer the compound to subjects suffering from a disorder.
Administration may begin before the subject is symptomatic.
[0258] Exemplary routes of administration of the compounds
disclosed herein or pharmaceutical compositions thereof, used in
the present disclosure include oral, sublingual, buccal,
transdermal, intradermal, intramuscular, parenteral, intravenous,
intra-arterial, intracranial, subcutaneous, intraorbital,
intraventricular, intraspinal, intraperitoneal, intranasal,
inhalation, and topical administration. The compounds desirably are
administered with a physiologically acceptable carrier (e.g., a
pharmaceutically acceptable carrier). Pharmaceutical formulations
of the compounds described herein formulated for treatment of the
disorders described herein are also part of the present disclosure.
In some embodiments, the compounds disclosed herein are
administered to a subject orally. In some embodiments, the
compounds disclosed herein are administered to a subject
topically.
[0259] The pharmaceutical compositions contemplated by the
disclosure include those formulated for oral administration ("oral
dosage forms"). Oral dosage forms can be, for example, in the form
of tablets, capsules, a liquid solution or suspension, a powder, or
liquid or solid crystals, which contain the active ingredient(s) in
a mixture with physiologically acceptable excipients (e.g.,
pharmaceutically acceptable excipients). These excipients may be,
for example, inert diluents or fillers (e.g., sucrose, sorbitol,
sugar, mannitol, microcrystalline cellulose, starches including
potato starch, calcium carbonate, sodium chloride, lactose, calcium
phosphate, calcium sulfate, or sodium phosphate); granulating and
disintegrating agents (e.g., cellulose derivatives including
microcrystalline cellulose, starches including potato starch,
croscarmellose sodium, alginates, or alginic acid); binding agents
(e.g., sucrose, glucose, sorbitol, acacia, alginic acid, sodium
alginate, gelatin, starch, pregelatinized starch, microcrystalline
cellulose, magnesium aluminum silicate, carboxymethylcellulose
sodium, methylcellulose, hydroxypropyl methylcellulose,
ethylcellulose, polyvinylpyrrolidone, or polyethylene glycol); and
lubricating agents, glidants, and antiadhesives (e.g., magnesium
stearate, zinc stearate, stearic acid, silicas, hydrogenated
vegetable oils, or talc). Other physiologically acceptable
excipients (e.g., pharmaceutically acceptable excipients) can be
colorants, flavoring agents, plasticizers, humectants, buffering
agents, and the like.
[0260] Formulations for oral administration may also be presented
as chewable tablets, as hard gelatin capsules where the active
ingredient is mixed with an inert solid diluent (e.g., potato
starch, lactose, microcrystalline cellulose, calcium carbonate,
calcium phosphate or kaolin), or as soft gelatin capsules where the
active ingredient is mixed with water or an oil medium, for
example, peanut oil, liquid paraffin, or olive oil. Powders,
granulates, and pellets may be prepared using the ingredients
mentioned above under tablets and capsules in a conventional manner
using, e.g., a mixer, a fluid bed apparatus or a spray drying
equipment.
[0261] Controlled release compositions for oral use may be
constructed to release the active drug by controlling the
dissolution and/or the diffusion of the active drug substance. Any
of a number of strategies can be pursued in order to obtain
controlled release and the targeted plasma concentration versus
time profile. In one example, controlled release is obtained by
appropriate selection of various formulation parameters and
ingredients, including, e.g., various types of controlled release
compositions and coatings. Examples include single or multiple unit
tablet or capsule compositions, oil solutions, suspensions,
emulsions, microcapsules, microspheres, nanoparticles, patches, and
liposomes. In certain embodiments, compositions include
biodegradable, pH, and/or temperature-sensitive polymer
coatings.
[0262] Dissolution or diffusion-controlled release can be achieved
by appropriate coating of a tablet, capsule, pellet, or granulate
formulation of compounds, or by incorporating the compound into an
appropriate matrix. A controlled release coating may include one or
more of the coating substances mentioned above and/or, e.g.,
shellac, beeswax, glycowax, castor wax, carnauba wax, stearyl
alcohol, glyceryl monostearate, glyceryl distearate, glycerol
palmitostearate, ethylcellulose, acrylic resins, dl-polylactic
acid, cellulose acetate butyrate, polyvinyl chloride, polyvinyl
acetate, vinyl pyrrolidone, polyethylene, polymethacrylate,
methylmethacrylate, 2-hydroxymethacrylate, methacrylate hydrogels,
1,3 butylene glycol, ethylene glycol methacrylate, and/or
polyethylene glycols. In a controlled release matrix formulation,
the matrix material may also include, e.g., hydrated
methylcellulose, carnauba wax and stearyl alcohol, carbopol 934,
silicone, glyceryl tristearate, methyl acrylate-methyl
methacrylate, polyvinyl chloride, polyethylene, and/or halogenated
fluorocarbon.
[0263] The liquid forms in which the compounds and compositions of
the present disclosure can be incorporated for administration
orally include aqueous solutions, suitably flavored syrups, aqueous
or oil suspensions, and flavored emulsions with edible oils, e.g.,
cottonseed oil, sesame oil, coconut oil, or peanut oil, as well as
elixirs and similar pharmaceutical vehicles.
[0264] Dosages for buccal or sublingual administration typically
are 0.1 to 500 mg per single dose as required. In practice, the
physician determines the actual dosing regimen which is most
suitable for an individual subject, and the dosage varies with the
age, weight, and response of the particular subject. The above
dosages are exemplary of the average case, but individual instances
exist where higher or lower dosages are merited, and such are
within the scope of this disclosure.
[0265] For buccal administration, the compositions may take the
form of tablets, lozenges, etc. formulated in a conventional
manner. Liquid drug formulations suitable for use with nebulizers
and liquid spray devices and electrohydrodynamic (EHD) aerosol
devices will typically include a compound disclosed herein with a
pharmaceutically acceptable carrier. In some embodiments, the
pharmaceutically acceptable carrier is a liquid, e.g., alcohol,
water, polyethylene glycol, or a perfluorocarbon. Optionally,
another material may be added to alter the aerosol properties of
the solution or suspension of compounds disclosed herein.
Desirably, this material is liquid, e.g., an alcohol, glycol,
polyglycol, or a fatty acid. Other methods of formulating liquid
drug solutions or suspension suitable for use in aerosol devices
are known to those of skill in the art (see, e.g., U.S. Pat. Nos.
5,112,598 and 5,556,611, each of which is herein incorporated by
reference).
[0266] The compounds may also be formulated for nasal
administration. Compositions for nasal administration also may
conveniently be formulated as aerosols, drops, gels, and powders.
The formulations may be provided in a single or multidose form. In
the case of a dropper or pipette, dosing may be achieved by the
subject administering an appropriate, predetermined volume of the
solution or suspension. In the case of a spray, this may be
achieved, for example, by means of a metering atomizing spray
pump.
[0267] The compounds may further be formulated for aerosol
administration, particularly to the respiratory tract by inhalation
and including intranasal administration. The compounds for nasal or
inhalation administration will generally have a small particle size
for example on the order of five (5) microns or less. Such a
particle size may be obtained by means known in the art, for
example by micronization. The active ingredient is provided in a
pressurized pack with a suitable propellant, e.g., a
chlorofluorocarbon (CFC), for example, dichlorodifluoromethane,
trichlorofluoromethane, or dichlorotetrafluoroethane, or carbon
dioxide, or other suitable gas. The aerosol may conveniently also
contain a surfactant, e.g., lecithin. The dose of drug may be
controlled by a metered valve. Alternatively, the active
ingredients may be provided in a form of a dry powder, e.g., a
powder mix of the compound in a suitable powder base, e.g.,
lactose, starch, and starch derivatives, e.g., hydroxypropylmethyl
cellulose, and polyvinylpyrrolidine (PVP). The powder carrier will
form a gel in the nasal cavity. The powder composition may be
presented in unit dose form for example in capsules or cartridges
of e.g., gelatin or blister packs from which the powder may be
administered by means of an inhaler.
[0268] Aerosol formulations typically include a solution or fine
suspension of the active substance in a physiologically acceptable
aqueous or non-aqueous solvent and are usually presented in single
or multidose quantities in sterile form in a sealed container,
which can take the form of a cartridge or refill for use with an
atomizing device. Alternatively, the sealed container may be a
unitary dispensing device, e.g., a single dose nasal inhaler or an
aerosol dispenser fitted with a metering valve which is intended
for disposal after use. Where the dosage form comprises an aerosol
dispenser, it will contain a propellant, which can be a compressed
gas, e.g., compressed air or an organic propellant, e.g.,
fluorochlorohydrocarbon. The aerosol dosage forms can also take the
form of a pump-atomizer.
[0269] The compounds described herein for use in the methods of the
disclosure can be administered in a pharmaceutically acceptable
parenteral (e.g., intravenous or intramuscular) formulation as
described herein. The pharmaceutical formulation may also be
administered parenterally (intravenous, intramuscular, subcutaneous
or the like) in dosage forms or formulations containing
conventional, non-toxic pharmaceutically acceptable carriers and
adjuvants. In particular, formulations suitable for parenteral
administration include aqueous and non-aqueous sterile injection
solutions which may contain anti-oxidants, buffers, bacteriostats,
and solutes which render the formulation isotonic with the blood of
the intended recipient; and aqueous and non-aqueous sterile
suspensions which may include suspending agents and thickening
agents. For example, to prepare such a composition, the compounds
disclosed herein may be dissolved or suspended in a parenterally
acceptable liquid vehicle. Among acceptable vehicles and solvents
that may be employed are water, water adjusted to a suitable pH by
addition of an appropriate amount of hydrochloric acid, sodium
hydroxide or a suitable buffer, 1,3-butanediol, Ringer's solution
and isotonic sodium chloride solution. The aqueous formulation may
also contain one or more preservatives, for example, methyl, ethyl
or n-propyl p-hydroxybenzoate. Additional information regarding
parenteral formulations can be found, for example, in the United
States Pharmacopeia-National Formulary (USP-NF), herein
incorporated by reference.
[0270] The parenteral formulation can be any of the five general
types of preparations identified by the USP-NF as suitable for
parenteral administration: [0271] (1) "Drug Injection:" a liquid
preparation that is a drug substance (e.g., a compound disclosed
herein or a solution thereof); [0272] (2) "Drug for Injection:" the
drug substance (e.g., a compound disclosed herein) as a dry solid
that will be combined with the appropriate sterile vehicle for
parenteral administration as a drug injection; [0273] (3) "Drug
Injectable Emulsion:" a liquid preparation of the drug substance
(e.g., a compound disclosed herein) that is dissolved or dispersed
in a suitable emulsion medium; [0274] (4) "Drug Injectable
Suspension:" a liquid preparation of the drug substance (e.g., a
compound disclosed herein) suspended in a suitable liquid medium;
and [0275] (5) "Drug for Injectable Suspension:" the drug substance
(e.g., a compound disclosed herein) as a dry solid that will be
combined with the appropriate sterile vehicle for parenteral
administration as a drug injectable suspension.
[0276] Exemplary formulations for parenteral administration include
solutions of the compounds prepared in water suitably mixed with a
surfactant, e.g., hydroxypropylcellulose. Dispersions can also be
prepared in glycerol, liquid polyethylene glycols, DMSO and
mixtures thereof with or without alcohol, and in oils. Under
ordinary conditions of storage and use, these preparations may
contain a preservative to prevent the growth of microorganisms.
Conventional procedures and ingredients for the selection and
preparation of suitable formulations are described, for example, in
Remington: The Science and Practice of Pharmacy, 21st Ed., Gennaro,
Ed., Lippencott Williams & Wilkins (2005) and in The United
States Pharmacopeia: The National Formulary (USP 36 NF31),
published in 2013.
[0277] Formulations for parenteral administration may, for example,
contain excipients, sterile water, or saline, polyalkylene glycols,
e.g., polyethylene glycol, oils of vegetable origin, or
hydrogenated napthalenes. Biocompatible, biodegradable lactide
polymer, lactide/glycolide copolymer, or
polyoxyethylene-polyoxypropylene copolymers may be used to control
the release of the compounds or biologically active agents within
the compounds. Other potentially useful parenteral delivery systems
for compounds include ethylene-vinyl acetate copolymer particles,
osmotic pumps, implantable infusion systems, and liposomes.
Formulations for inhalation may contain excipients, for example,
lactose, or may be aqueous solutions containing, for example,
polyoxyethylene-9-lauryl ether, glycocholate and deoxycholate, or
may be oily solutions for administration in the form of nasal
drops, or as a gel.
[0278] The parenteral formulation can be formulated for prompt
release or for sustained/extended release of the compound.
Exemplary formulations for parenteral release of the compound
include: aqueous solutions, powders for reconstitution, cosolvent
solutions, oil/water emulsions, suspensions, oil-based solutions,
liposomes, microspheres, and polymeric gels.
[0279] Non-limiting embodiments of the present disclosure include
the following:
1. A compound consisting of a cation and a pharmaceutically
acceptable counterion, wherein the cation is chosen from:
##STR00066## ##STR00067## ##STR00068## ##STR00069## ##STR00070##
##STR00071## ##STR00072## ##STR00073## ##STR00074## ##STR00075##
##STR00076## ##STR00077## ##STR00078## ##STR00079## ##STR00080##
##STR00081## ##STR00082## ##STR00083## ##STR00084## ##STR00085##
##STR00086## ##STR00087## ##STR00088## ##STR00089##
##STR00090##
2. The compound according to embodiment 1, wherein the cation is
chosen from:
##STR00091## ##STR00092## ##STR00093##
3. A compound consisting of a cation and a pharmaceutically
acceptable counterion, wherein the cation is chosen from:
##STR00094## ##STR00095## ##STR00096## ##STR00097## ##STR00098##
##STR00099## ##STR00100## ##STR00101## ##STR00102## ##STR00103##
##STR00104##
4. A compound consisting of a cation and a pharmaceutically
acceptable counterion, wherein the cation is a structure of formula
(I):
##STR00105##
wherein
[0280] R.sup.1 is [0281] C.sub.2-6 alkyl substituted with
--O-(acylated sugar) or isosorbide, wherein said C.sub.2-6 alkyl is
further optionally substituted with oxo and/or methene; [0282]
C.sub.4 cycloalkyl optionally substituted with hydroxyl, ethynyl,
or --O-(acylated sugar); or [0283] C.sub.3 cycloalkyl substituted
with C.sub.1-6 alkyl, hydroxyl, ethynyl, or --O-(acylated sugar)
[0284] C.sub.3-4 cycloalkyl C.sub.1-2 alkyl;
[0285] R.sup.2 is C.sub.2-6 alkyl optionally substituted with one
or two hydroxyl, oxo, and --O-(acylated sugar);
[0286] or R.sup.1 and R.sup.2, together with the nitrogen atom to
which both are attached, combine to form a 4- or 5-membered
heterocyclic ring optionally substituted with ethynyl or
--(CH.sub.2).sub.n--OR.sup.s or an acylated sugar, wherein n is 0
or 1, R.sup.s is hydrogen or an acylated sugar;
[0287] R.sup.3 is C.sub.1-6 alkyl optionally substituted with a
halogen or hydroxyl; and
[0288] R.sup.4 is C.sub.1-6 alkyl or propargyl.
5. The compound of embodiment 4, wherein R.sup.1 is C.sub.2-6 alkyl
substituted with --O-(acylated sugar) and is optionally further
substituted with oxo. 6. The compound of embodiment 4 or 5, wherein
R.sup.2 is methyl. 7. The compound of any one of embodiments 4, 5,
and 6, wherein R.sup.3 is C.sub.1-6 alkyl. 8. The compound of any
one of embodiments 4-7, wherein R.sup.4 is propargyl. 9. The
compound of embodiment 4, wherein
[0289] R.sup.1 is C.sub.2-6 alkyl substituted with --O-(acylated
sugar) and is optionally further substituted with oxo;
[0290] R.sup.2 is methyl;
[0291] R.sup.3 is C.sub.1-6 alkyl; and
[0292] R.sup.4 is propargyl.
10. The compound of embodiment 4, wherein
[0293] R.sup.1 is C.sub.2-6 alkyl substituted with isosorbide and
is optionally further substituted with oxo and/or methene;
[0294] R.sup.2 is methyl;
[0295] R.sup.3 is C.sub.1-6 alkyl; and
[0296] R.sup.4 is propargyl.
11. The compound of embodiment 4, wherein
[0297] R.sup.1 is chosen from C.sub.2 alkyl substituted with
--O-(acylated sugar) and optionally further substituted with oxo
and C.sub.4 alkyl substituted with --O-(acylated sugar) and
optionally further substituted with oxo;
[0298] wherein the acylated sugar is chosen from groups of formula
(A), groups of formula (B), and groups of formula (C):
##STR00106##
[0299] wherein [0300] each R.sup.A is independently H or fatty acid
acyl, provided at least one R.sup.A is a fatty acid acyl; and
[0301] R.sup.B is H, --CH.sub.3, --CH.sub.2--OR.sup.A, --OCH.sub.3,
--COOCH.sub.3, or --COOH;
[0302] R.sup.2 is methyl;
[0303] R.sup.3 is methyl; and
[0304] R.sup.4 is propargyl.
12. The compound of embodiment 4, wherein
[0305] R.sup.1 is chosen from C.sub.2 alkyl substituted with
isosorbide and optionally further substituted with oxo and C.sub.3
alkyl substituted with isosorbide and optionally further
substituted with oxo and methene;
[0306] wherein isosorbide is chosen from groups of formula (C):
##STR00107##
[0307] R.sup.2 is methyl;
[0308] R.sup.3 is methyl; and
[0309] R.sup.4 is propargyl.
13. The compound of embodiment 4, wherein
[0310] R.sup.1 is C.sub.3-4 cycloalkyl C.sub.1-2 alkyl;
[0311] R.sup.2 is C.sub.2-6 alkyl optionally substituted with one
or two hydroxyl, oxo, and --O-(acylated sugar);
[0312] or R.sup.1 and R.sup.2, together with the nitrogen atom to
which both are attached, combine to form a 4- or 5-membered
heterocyclic ring optionally substituted with ethynyl or
--(CH.sub.2).sub.n--OR.sup.s or an acylated sugar, wherein n is 0
or 1, R.sup.s is hydrogen or an acylated sugar;
[0313] R.sup.3 is C.sub.1-6 alkyl optionally substituted with a
halogen or hydroxyl; and
[0314] R.sup.4 is C.sub.1-6 alkyl or propargyl.
14. The compound of embodiment 13, wherein R.sup.1 is C.sub.3-4
cycloalkyl C.sub.1 alkyl. 15. The compound of embodiment 13 or 14,
wherein R.sup.2 is C.sub.2 alkyl optionally substituted with one or
two hydroxyl groups. 16. The compound of embodiment 13, wherein
R.sup.1 and R.sup.2, together with the nitrogen atom to which both
are attached, combine to form a 5-membered heterocyclic ring
optionally substituted with ethynyl, --OH, or --CH.sub.2OH. 17. The
compound of any one of embodiments 13-16, wherein R.sup.3 is
C.sub.1 alkyl optionally substituted with a halogen. 18. The
compound of any one of embodiments 13-17, wherein R.sup.4 is
C.sub.1 alkyl or propargyl. 19. A compound consisting of a cation
and a pharmaceutically acceptable counterion, wherein the cation is
a structure of formula (II):
##STR00108##
wherein
[0315] R.sup.1 is [0316] C.sub.1-6 alkyl optionally substituted
with one or more oxo, hydroxyl, halogen, cyano, --COOMe, amino,
methene, ethenyl, ethynyl, hydroxyphenyl, C.sub.3-4 cycloalkyl,
--OCH.sub.2CH.sub.2OH, --HNC(O)OCMe.sub.3, --SMe, --OMe,
--HNS(O).sub.2Me, --SO.sub.3H, B(OH).sub.2, PO.sub.3H.sub.2,
PO.sub.2H.sub.2, --P(O)(OCH.sub.2CH.sub.3).sub.2,
--P(O)(OH)(OCH.sub.2CH.sub.3), heteroaryl ring; [0317] phenyl;
[0318] benzyl; [0319] C.sub.3-4 heterocyclyl optionally substituted
with C.sub.1 alkyl [0320] C.sub.4 heterocycloalkyl C.sub.1 alkyl;
or [0321] C.sub.4 cycloalkyl;
[0322] R.sup.2 is [0323] C.sub.1-6 alkyl optionally substituted
with one or two hydroxyl, oxo, methene, cyano, ethynyl, --HNC(O)H,
or --C.ident.C--CH.sub.2OH; or [0324]
--HN(CH.sub.2).sub.3C(O)OH;
[0325] or R.sup.1 and R.sup.2, together with the nitrogen atom to
which both are attached and optionally one or more additional
heteroatoms, combine to form a 4-8 membered mono- or bi-cyclic
heterocycle optionally substituted with ethynyl, trifluoromethyl,
--CH.sub.2Ph, --OH, or --(CH.sub.2)OH;
[0326] R.sup.3 is methyl or propargyl; and
[0327] R.sup.4 is methyl or propargyl,
[0328] with the proviso that
[0329] (1) when two of R.sup.1, R.sup.2, R.sup.3, and R.sup.4 are
methyl and one of the other two of R.sup.1, R.sup.2, R.sup.3, and
R.sup.4 is propargyl, then the other of R.sup.1, R.sup.2, R.sup.3,
and R.sup.4 is not methyl, a monohalomethyl, --CH.sub.2CH.sub.2OH,
--COOH, --(CH.sub.2).sub.4C(O)OH, or --CH.sub.2C(CH.sub.2)CN;
and
[0330] (2) when two of R.sup.1, R.sup.2, R.sup.3, and R.sup.4 are
methyl and one of the other two of R.sup.1, R.sup.2, R.sup.3, and
R.sup.4 is --CH.sub.2CH.sub.2OH or --CH.sub.2C(O)OH, then the other
of R.sup.1, R.sup.2, R.sup.3, and R.sup.4 is not a
monohalomethyl.
20. A compound consisting of a cation and a pharmaceutically
acceptable counterion, wherein the cation is a structure of formula
(III):
##STR00109##
wherein
[0331] R.sup.1 is [0332] C.sub.1-6 alkyl optionally substituted
with one or more oxo, hydroxyl, halogen, cyano, --COOMe, --COOH,
methene, ethenyl, ethynyl, C.sub.3-4 cycloalkyl,
--OCH.sub.2CH.sub.2OH, --SMe, --OMe, --HNS(O).sub.2Me,
--P(O)(OCH.sub.2CH.sub.3).sub.2, --P(O)(OH)(OCH.sub.2CH.sub.3), or
5-membered heteroaryl ring;
[0333] R.sup.2 is [0334] C.sub.1-6 alkyl optionally substituted
with one or two hydroxyl, oxo, methene, cyano, ethynyl, --HNC(O)H,
or --C.ident.C--CH.sub.2OH; or [0335]
--HN(CH.sub.2).sub.3C(O)OH;
[0336] or R.sup.1 and R.sup.2, together with the nitrogen atom to
which both are attached and optionally one or more additional
heteroatoms, combine to form a 4-8 membered mono- or bi-cyclic
heterocycle optionally substituted with ethynyl, --OH, or
--(CH.sub.2)OH;
[0337] R.sup.3 is C.sub.1-6 alkyl optionally substituted with a
halogen, hydroxyl, or ethynyl; and
[0338] R.sup.4 is C.sub.1-6 alkyl or propargyl;
[0339] with the proviso that
[0340] (1) when two of R.sup.1, R.sup.2, R.sup.3, and R.sup.4 are
methyl and one of the other two of R.sup.1, R.sup.2, R.sup.3, and
R.sup.4 is propargyl, then the other of R.sup.1, R.sup.2, R.sup.3,
and R.sup.4 is not methyl, a monohalomethyl, --CH.sub.2CH.sub.2OH,
--COOH, --(CH.sub.2).sub.4C(O)OH, or --CH.sub.2C(CH.sub.2)CN;
and
[0341] (2) when two of R.sup.1, R.sup.2, R.sup.3, and R.sup.4 are
methyl and one of the other two of R.sup.1, R.sup.2, R.sup.3, and
R.sup.4 is --CH.sub.2CH.sub.2OH or --CH.sub.2C(O)OH, then the other
of R.sup.1, R.sup.2, R.sup.3, and R.sup.4 is not a
monohalomethyl.
21. A pharmaceutical composition comprising a pharmaceutically
acceptable excipient and the compound of any one of embodiments 1
to 20. 22. A method of modulating a trimethylaminuria marker in a
subject in need thereof, the method comprising administering to the
subject in need thereof a therapeutically effective amount of the
compound of any one of embodiments 1 to 20 or the pharmaceutical
composition of embodiment 21. 23. The method of embodiment 22,
wherein the trimethylaminuria marker is the trimethylamine and/or
trimethylamine oxide levels in the subject's blood, plasma, serum,
or urine. 24. A method of treating or preventing a disease
associated with elevated levels of trimethylamine (TMA) or
trimethylamine N-oxide (TMAO), a decreased rate of conversion of
TMA to TMAO, or a high ratio of TMA to TMAO in a subject in need
thereof, the method comprising administering a therapeutically
effective amount of the compound of any one of embodiments 1 to 20
or the pharmaceutical composition of embodiment 21 to the subject.
25. The method of embodiment 24, further comprising detecting the
presence of one or more genetic variants of the FMO3 gene of the
subject in need before the administering step. 26. The method of
embodiment 24, wherein the disease associated with elevated levels
of trimethylamine (TMA) or trimethylamine N-oxide (TMAO), a
decreased rate of conversion of TMA to TMAO, or a high ratio of TMA
to TMAO is a cardiovascular disease, reduced or impaired kidney
function, kidney disease, chronic kidney disease, end-stage renal
disease, or diabetes mellitus. 27. A method of inhibiting a CutC
choline lyase-mediated conversion of choline to trimethylamine, the
method comprising contacting the compound of any one of embodiments
1 to 20 with the CutC choline lyase. 28. A method of inhibiting a
CntA carnitine monooxygenase-mediated conversion of carnitine to
trimethylamine, the method comprising contacting the compound of
any one of embodiments 1 to 20 with the CntA carnitine
monooxygenase. 29. A method of treating a subject in need of
treatment for trimethylaminuria comprising contacting bacteria in
vivo with a therapeutically effective amount of the compound of any
one of embodiments 1 to 20 or the pharmaceutical composition of
embodiment 21 to the subject. 30. The method of embodiment 29,
wherein the bacteria are localized in the colon of the subject.
[0342] The following examples are meant to illustrate the
disclosure. They are not meant to limit the disclosure in any
way.
EXAMPLES
Example 1: Preparation of Exemplary Compounds
[0343] In the following examples, the title compounds may exist as
a trifluoroacetate salt after prep-HPLC but one of ordinary skill
in the art would understand that the salt may be changed by
conventional methods of salt formation.
##STR00110##
Compound 1:
N,N-dimethyl-N-(2-(((2R,3R,4S,5R,6R)-3,4,5-tris(butyryloxy)-6-((butyrylox-
y)methyl)tetrahydro-2H-pyran-2-yl)oxy)ethyl)prop-2-yn-1-aminium
iodide
[0344] Step 1:
[0345] A mixture of
(3R,4S,5S,6R)-6-(hydroxymethyl)tetrahydropyran-2,3,4,5-tetrol (20
g, 111.01 mmol, 1 eq) and butanoyl butanoate (96.59 g, 610.58 mmol,
99.89 mL, 5.5 eq) in Pyridine (200 mL) was degassed and purged with
N2 for 3 times, and then the mixture was stirred at 20.degree. C.
for 12 h. TLC indicated the starting material was consumed
completely and one new spot formed. The reaction mixture was
filtered and concentrated under reduced pressure to give a residue.
The residue was purified by column chromatography (SiO.sub.2,
Petroleum ether/Ethyl acetate=1 to 20:1).
[(2R,3R,4S,5R)-3,4,5,6-tetra(butanoyloxy)tetrahydropyran-2-yl]methyl
butanoate (100 g, crude) was obtained as colorless oil.
[0346] Step 2:
[0347] To a mixture of
[(2R,3R,4S,5R)-3,4,5,6-tetra(butanoyloxy)tetrahydropyran-2-yl]methyl
butanoate (10 g, 18.85 mmol, 1 eq) in THF (100 mL) was added
MeNH.sub.2 aq. solution (2.4 g, 30.91 mmol, 40% purity, 1.64 eq) in
one portion at 20.degree. C. under N2. The mixture was stirred at
20.degree. C. for 12 h. TLC indicated
[(2R,3R,4S,5R)-3,4,5,6-tetra(butanoyloxy)tetrahydropyran-2-yl]methyl
butanoate was consumed completely and one new spot formed. The
reaction mixture was filtered and concentrated under reduced
pressure to give a residue. The residue was purified by column
chromatography (SiO.sub.2, Petroleum ether/Ethyl acetate=1/0 to
20:1). Compound
[(2R,3R,4S,5R)-3,4,5-tri(butanoyloxy)-6-hydroxy-tetrahydropyran-2-yl]meth-
yl butanoate (5 g, 10.86 mmol, 57.6% yield) was obtained as
colorless oil.
[0348] Step 3:
[0349] To a mixture of
[(2R,3R,4S,5R)-3,4,5-tri(butanoyloxy)-6-hydroxy-tetrahydropyran-2-yl]meth-
yl butanoate (2 g, 4.34 mmol, 1 eq) and DBU (330.58 mg, 2.17 mmol,
327.31 uL, 0.5 eq) in DCM (20 mL) was added
2,2,2-trichloroacetonitrile (6.27 g, 43.43 mmol, 4.35 mL, 10 eq) in
one portion at 20.degree. C. The mixture was stirred at 20.degree.
C. for 12 h. TLC showed
[(2R,3R,4S,5R)-3,4,5-tri(butanoyloxy)-6-hydroxy-tetrahydropyran-2-yl]meth-
yl butanoate was consumed completely and one new spot formed. The
reaction mixture was filtered and concentrated under reduced
pressure to give a residue. The residue was purified by column
chromatography (SiO.sub.2, Petroleum ether/Ethyl acetate=30/1 to
20:1). Compound
[(2R,3R,4S,5R)-3,4,5-tri(butanoyloxy)-6-(2,2,2-trichloroethanimidoyl)oxy--
tetrahydropyran-2-yl]methyl butanoate (1.8 g, 2.98 mmol, 68.5%
yield) was obtained as colorless oil.
[0350] Step 4:
[0351] To a solution of
[(2R,3R,4S,5R)-3,4,5-tri(butanoyloxy)-6-(2,2,2-trichloroethanimidoyl)
oxy-tetrahydropyran-2-yl]methyl butanoate (1 g, 1.65 mmol, 1 eq)
and 2-bromoethanol (247.90 mg, 1.98 mmol, 140.85 uL, 1.2 eq) in DCM
(10 mL) was added BF.sub.3.Et.sub.2O (1.17 g, 8.27 mmol, 1.02 mL, 5
eq). The mixture was stirred at 0.degree. C. for 3 h and then
warmed to 20.degree. C. stirred for 12 h. TLC indicated
[(2R,3R,4S,5R)-3,4,5-tri(butanoyloxy)-6-(2,2,2-trichloroethanimidoyl)oxy--
tetrahydropyran-2-yl]methyl butanoate was consumed completely. The
reaction mixture was filtered and concentrated under reduced
pressure to give a residue. The residue was purified by column
chromatography (SiO.sub.2, Petroleum ether/Ethyl acetate=30/1 to
20:1). Compound
[(2R,3R,4S,5R)-6-(2-bromoethoxy)-3,4,5-tri(butanoyloxy)tetrahydropyran-2--
yl]methyl butanoate (0.8 g, 1.41 mmol, 85.3% yield) was obtained as
colorless oil.
[0352] Step 5:
[0353] A mixture of
[(2R,3R,4S,5R)-6-(2-bromoethoxy)-3,4,5-tri(butanoyloxy)tetrahydropyran-2--
yl] methyl butanoate (0.1 g, 176.22 umol, 1 eq),
N-methylprop-2-yn-1-amine (36.53 mg, 528.67 umol, 44.02 uL, 3 eq)
in THF (3 mL) was degassed and purged with N.sub.2 for 3 times, and
then the mixture was stirred at 20.degree. C. for 2 hr under
N.sub.2 atmosphere, then warmed to 70.degree. C. and stirred for 12
h. TLC indicated
[(2R,3R,4S,5R)-6-(2-bromoethoxy)-3,4,5-tri(butanoyloxy)tetrahydropyran-2--
yl]methyl butanoate was consumed completely and one new spot
formed. The reaction mixture was filtered and concentrated under
reduced pressure to give a residue. The residue was purified by
prep-TLC (SiO.sub.2, Petroleum ether/Ethyl acetate=2:1). Compound
[(2R,3R,4S,5R)-3,4,5-tri(butanoyloxy)-6-[2-[methyl(prop-2-ynyl)amino]etho-
xy]tetrahydropyran-2-yl]methyl butanoate (0.03 g, 54 umol, 30.6%
yield) was obtained as colorless oil.
[0354] Step 6:
[0355] A mixture of
[(2R,3R,4S,5R)-3,4,5-tri(butanoyloxy)-6-[2-[methyl(prop-2-ynyl)amino]etho-
xy] tetrahydropyran-2-yl]methyl butanoate (0.06 g, 107.98 umol, 1
eq), MeI (30.65 mg, 215.96 umol, 13.44 uL, 2 eq) in THF (3 mL) was
degassed and purged with N.sub.2 for 3 times, and then the mixture
was stirred at 20.degree. C. for 2 hr under N.sub.2 atmosphere.
LC-MS showed
[(2R,3R,4S,5R)-3,4,5-tri(butanoyloxy)-6-[2-[methyl(prop-2-ynyl)amino]etho-
xy]tetrahydropyran-2-yl]methyl butanoate was consumed. The reaction
mixture was filtered and concentrated under reduced pressure to
give a residue. The residue was purified by prep-HPLC [water (0.1%
TFA)-ACN]. The title compound (0.007 g, 12.1 umol, 11% yield) was
obtained as colorless oil. LCMS (M.sup.+): 570.3 .sup.1H NMR (400
MHz, DMSO-d.sub.6) .delta. 5.33 (t, J=9.5 Hz, 1H), 5.04-4.81 (m,
3H), 4.37 (d, J=2.5 Hz, 2H), 4.22-4.00 (m, 6H), 3.62 (t, J=4.9 Hz,
2H), 3.09 (s, 6H), 2.33-2.11 (m, 8H), 1.61-1.38 (m, 8H), 0.93-0.78
(m, 12H
##STR00111##
Compound 2 and Compound 51:
N,N-dimethyl-N-(2-(((3R,4S,5R)-3,4,5-tris(butyryloxy)tetrahydro-2H-pyran--
2-yl)oxy)ethyl)prop-2-yn-1-aminium Iodide
[0356] This compound may be synthesized according to the
experimental procedure described for Compound 1.
##STR00112##
Compound 3:
N,N-dimethyl-N-(2-(((3R,4R,5R)-3,4,5-tris(butyryloxy)tetrahydro-2H-pyran--
2-yl)oxy)ethyl)prop-2-yn-1-aminium Iodide
[0357] This compound may be synthesized according to the
experimental procedure described for Compound 1.
##STR00113##
Compound 4 and Compound 53 and Compound 84:
N,N-dimethyl-N-(2-(((3R,4S,5S)-3,4,5-tris(butyryloxy)tetrahydro-2H-pyran--
2-yl)oxy)ethyl)prop-2-yn-1-aminium iodide
[0358] This compound may be synthesized according to the
experimental procedure described for Compound 1.
##STR00114##
Compound 5:
N,N-dimethyl-N-(2-(((3R,4R,5S,6S)-3,4,5-tris(butyryloxy)-6-methyltetrahyd-
ro-2H-pyran-2-yl)oxy)ethyl)prop-2-yn-1-aminium Iodide
[0359] This compound may be synthesized according to the
experimental procedure described for Compound 1.
##STR00115##
Compound 6:
N,N-dimethyl-N-(2-(((3S,4R,5R,6S)-3,4,5-tris(butyryloxy)-6-methyltetrahyd-
ro-2H-pyran-2-yl)oxy)ethyl)prop-2-yn-1-aminium Iodide
[0360] This compound may be synthesized according to the
experimental procedure described for Compound 1.
##STR00116##
Compound 7:
N,N-dimethyl-N-(2-(((2R,3R,4S,5R,6R)-3,4,5-triacetoxy-6-(acetoxymethyl)te-
trahydro-2H-pyran-2-yl)oxy)ethyl)prop-2-yn-1-aminium Iodide
[0361] Step 1:
[0362] Compound .beta.-D-Glucose pentaacetate (20 g, 51.2 mmol, 1
eq) was dissolved in DCM (100 mL) and cooled to 0.degree. C. under
N.sub.2, followed by addition of 2-bromoethanol (5.5 mL, 77.6 mmol,
1.5 eq) and boron trifluoride diethyl etherate (32 mL, 259 mmol, 5
eq). The ice bath was removed and the reaction was stirred at room
temperature overnight. Silica was added to the reaction mixture and
solution was concentrated to dryness to dry-load onto silica before
purification by column chromatography (gradient: 0-100% EtOAc in
hexanes, product eluted around 40-50% EtOAc in hexanes). Fractions
containing product were concentrated and recrystallized by
dissolution in ethyl acetate, followed by slow addition of hexanes
until crystals started to form, at which point it was placed in the
fridge for further crystallization. The white crystals were
filtered and washed with hexanes to yield
1-(2-bromoethoxy)-2,3,4,6-tetra-O-acetyl-beta-D-glucopyranose (8.9
g, 19.5 mmol, 38%).
[0363] Step 2:
[0364] Compound
1-(2-bromoethoxy)-2,3,4,6-tetra-O-acetyl-beta-D-glucopyranose (1.7
g, 3.7 mmol, 1 eq) was stirred with sodium iodide (0.614 g, 4.1
mmol, 1.1 eq) for a few minutes, followed by addition of
3-dimethylamino-1-propyne (1.2 mL, 11.1 mmol, 3 eq). The reaction
mixture was stirred overnight at 40.degree. C. filtered, then
cooled to 4.degree. C. in the fridge for recrystallization. The
crystals were washed with cold acetone to yield the title compound
as a white crystalline solid (300 mg, 0.51 mmol, 14%, iodide
salt-confirmed by ion chromatography). The remaining filtrate was
concentrated and washed with cold acetone and ethyl acetate to
yield the title compound as an amorphous powder (1.2 g, 2.05 mmol,
55%, iodide salt-confirmed by ion chromatography). LCMS (M+): 458.1
.sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. 5.26 (t, J=9.5 Hz, 1H),
4.98-4.77 (m, 3H), 4.36 (d, J=2.5 Hz, 2H), 4.24-3.97 (m, 6H),
3.67-3.54 (m, 2H), 3.08 (s, 6H), 2.05-1.89 (m, 12H).
##STR00117##
Compound 8:
N,N-dimethyl-N-(2-(((3R,4S,5R)-3,4,5-triacetoxytetrahydro-2H-pyran-2-yl)o-
xy)ethyl)prop-2-yn-1-aminium iodideiodideiodideiodideiodide
[0365] Step 1:
[0366] To a solution of (3R,4S,5R)-tetrahydropyran-2,3,4,5-tetrol
(20 g, 133.22 mmol, 1 eq) in Pyridine (200 mL) was added acetyl
acetate (108.80 g, 1.07 mol, 99.82 mL, 8 eq) at 15.degree. C. The
mixture was stirred at 15.degree. C. for 12 hr. TLC indicated new
spots formed. The reaction mixture was concentrated under reduced
pressure to give yellow oil. The oil was diluted with ethyl acetate
100 mL and washed with H.sub.2O (100 mL*3). The combined organic
layers were washed with brine 100 mL, dried over anhydrous sodium
sulfate, filtered and the filtrate was concentrated under reduced
pressure to give yellow oil. The residue was purified by column
chromatography (SiO.sub.2, Petroleum ether/Ethyl acetate=1:0).
Compound [(3R,4S,5R)-4,5,6-triacetoxytetrahydropyran-3-yl] acetate
(42 g, crude) was obtained as a yellow oil.
[0367] Step 2:
[0368] To a solution of
[(3R,4S,5R)-4,5,6-triacetoxytetrahydropyran-3-yl] acetate (21 g,
65.98 mmol, 1 eq) in THF (120 mL) was added aq. MeNH.sub.2 (7.51 g,
72.58 mmol, 30% purity, 1.1 eq) at 15.degree. C. The mixture was
stirred at 15.degree. C. for 12 hr. TLC indicated new spots formed.
The reaction mixture was concentrated under reduced pressure to
give a residue. The residue was purified by column chromatography
(SiO.sub.2, Petroleum ether/Ethyl acetate=1:1 to 0:1). Compound
[(3R,4S,5R)-4,5-diacetoxy-6-hydroxy-tetrahydropyran-3-yl] acetate
(9 g, 32.58 mmol, 49.38% yield) was obtained as a yellow solid.
[0369] Step 3:
[0370] To a solution of
[(3R,4S,5R)-4,5-diacetoxy-6-hydroxy-tetrahydropyran-3-yl]acetate (5
g, 18.10 mmol, 1 eq) in DCM (20 mL) was added DBU (1.38 g, 9.05
mmol, 1.36 mL, 0.5 eq) and 2,2,2-trichloroacetonitrile (26.13 g,
181.00 mmol, 18.15 mL, 10 eq) at 15.degree. C. The mixture was
stirred at 15.degree. C. for 1 hr. TLC indicated a new spot formed.
The reaction mixture was concentrated under reduced pressure to
give a residue. The residue was purified by column chromatography
(SiO.sub.2, Petroleum ether/Ethyl acetate=1:1). Compound
[(3R,4S,5R)-4,5-diacetoxy-6-(2,2,2-trichloroethanimidoyl)oxy-tetrahydropy-
ran-3-yl] acetate (6 g, 14.26 mmol, 78.81% yield) was obtained as a
yellow oil.
[0371] Step 4:
[0372] To a solution of
[(3R,4S,5R)-4,5-diacetoxy-6-(2,2,2-trichloroethanimidoyl)oxy-tetrahydropy-
ran-3-yl] acetate (3 g, 7.13 mmol, 1 eq) and 2-bromoethanol (891.27
mg, 7.13 mmol, 506.41 uL, 1 eq) in DCM (30 mL) was added
BF.sub.3.Et.sub.2O (5.06 g, 35.66 mmol, 4.40 mL, 5 eq) at 0.degree.
C. The mixture was stirred at 0.degree. C. for 2 hr. TLC indicated
new spots formed. The reaction mixture was quenched by addition
H.sub.2O (30 mL) at 0.degree. C., and extracted with ethyl acetate
(50 mL*3). The combined organic layers were washed with brine 30
mL, dried over anhydrous sodium sulfate, filtered and the filtrate
was concentrated under reduced pressure to give a residue. The
residue was purified by column chromatography (SiO.sub.2, Petroleum
ether/Ethyl acetate=10:1 to 1:1). Compound
[(3R,4S,5R)-4,5-diacetoxy-6-(2-bromoethoxy)tetrahydropyran-3-yl]
acetate (1.3 g, 3.39 mmol, 47.57% yield) was obtained as a white
solid.
[0373] Step 5:
[0374] To a solution of
[(3R,4S,5R)-4,5-diacetoxy-6-(2-bromoethoxy)tetrahydropyran-3-yl]
acetate (1.3 g, 3.39 mmol, 1 eq) in acetone (20 mL) was added
N,N-dimethylprop-2-yn-1-amine (564.06 mg, 6.79 mmol, 719.46 uL, 2
eq) and NaI (1.02 g, 6.79 mmol, 2 eq) at 15.degree. C. The mixture
was stirred at 80.degree. C. for 4 hr. LCMS showed the desired
compound was detected. TLC indicated 50% of
[(3R,4S,5R)-4,5-diacetoxy-6-(2-bromoethoxy)tetrahydropyran-3-yl]
acetate (1.3 g, 3.39 mmol, 1 eq) remained. The mixture was stirred
at 80.degree. C. for 8 hr. The reaction mixture was concentrated
under reduced pressure to give a residue. The residue was purified
by column chromatography (SiO.sub.2, Petroleum ether/Ethyl
acetate=1:1 to Ethyl acetate:Methanol=1:1) The title compound (530
mg, 1.03 mmol, 30.30% yield) was obtained as a white solid. LCMS:
(M+H.sup.+): 386.1 .sup.1H NMR (400 MHz, Deuterium Oxide) .delta.
5.10 (t, J=7.6 Hz, 1H), 4.93-4.79 (m, 2H), 4.71 (d, J=6.1 Hz, 1H),
4.20-4.14 (m, 3H), 4.06 (dd, J=12.3, 4.7 Hz, 1H), 3.98-3.90 (m,
1H), 3.70-3.59 (m, 2H), 3.50 (dd, J=12.3, 7.9 Hz, 1H), 3.19-3.13
(m, 1H), 3.11 (s, 6H), 2.02-1.92 (m, 9H).
##STR00118##
Compound 9:
N,N-dimethyl-N-(2-(((3R,4R,5R)-3,4,5-triacetoxytetrahydro-2H-pyran-2-yl)o-
xy)ethyl)prop-2-yn-1-aminium bromide
[0375] This compound may be synthesized according to the
experimental procedure described for Compound 7.
##STR00119##
Compound 10 and Compound 56:
N,N-dimethyl-N-(2-(((3R,4S,5S)-3,4,5-triacetoxytetrahydro-2H-pyran-2-yl)o-
xy)ethyl)prop-2-yn-1-aminium Iodide
[0376] Step 1:
[0377] To a mixture of (2R,3S,4S)-2,3,4,5-tetrahydroxypentanal (10
g, 66.61 mmol, 1 eq) in pyridine (100 mL) was added Ac.sub.2O
(40.80 g, 399.65 mmol, 37.43 mL, 6 eq) in one portion at 25.degree.
C. under N.sub.2. The mixture was stirred at 25.degree. C. for 12
hours. TLC indicated one new spot formed. The reaction mixture was
concentrated under reduced pressure to remove pyridine. The residue
was purified by column chromatography (SiO.sub.2, Petroleum
ether/Ethyl acetate=30/1 to 0/1). Compound
[(3S,4S,5R,6S)-4,5,6-triacetoxytetrahydropyran-3-yl] acetate (20 g,
crude) was obtained as yellow oil.
[0378] Step 2:
[0379] To a mixture of
[(3S,4S,5R,6S)-4,5,6-triacetoxytetrahydropyran-3-yl] acetate (10 g,
31.42 mmol, 1 eq) in DCM (100 mL) was added 2-bromoethanol (5.89 g,
47.13 mmol, 3.35 mL, 1.5 eq) and BF.sub.3.Et.sub.2O (22.30 g,
157.10 mmol, 19.39 mL, 5 eq) in one portion at 0.degree. C. under
N.sub.2. The mixture was heated to 25.degree. C. and stirred for 12
hours. TLC indicated
[(3S,4S,5R,6S)-4,5,6-triacetoxytetrahydropyran-3-yl] acetate was
consumed and one new spot formed. The reaction mixture was quenched
by addition of H.sub.2O (50 mL), and then diluted with H.sub.2O
(100 mL) and extracted with DCM (100 mL*2). The combined organic
layers were dried over Na.sub.2SO.sub.4, filtered and the filtrate
was concentrated under reduced pressure to give a residue. The
residue was purified by column chromatography (SiO.sub.2, Petroleum
ether/Ethyl acetate=20/1 to 0/1). Then the residue was purified by
prep-HPLC [water (0.10% TFA)-ACN]. .sup.1HNMR indicated the
compound
[(3S,4S,5R,6S)-4,5-diacetoxy-6-(2-bromoethoxy)tetrahydropyran-3-yl]
acetate (1.2 g, 3.13 mmol, 9.97% yield) was obtained as yellow oil
and
compound[(3S,4S,5R,6R)-4,5-diacetoxy-6-(2-bromoethoxy)tetrahydropyran-3-y-
l] acetate (2.3 g, 6.00 mmol, 19.10% yield) was obtained as yellow
oil.
[0380] Step 3:
[0381] To a mixture of
[(3S,4S,5R,6S)-4,5-diacetoxy-6-(2-bromoethoxy)tetrahydropyran-3-yl]
acetate (1 g, 2.61 mmol, 1 eq) in acetone (10 mL) was added NaI
(430.29 mg, 2.87 mmol, 1.1 eq) in one portion at 25.degree. C.
under N.sub.2. Then N,N-dimethylprop-2-yn-1-amine (1.08 g, 13.05
mmol, 1.38 mL, 5 eq) was added. The mixture was heated to
90.degree. C. and stirred for 2 hours. TLC indicated
[(3S,4S,5R,6S)-4,5-diacetoxy-6-(2-bromoethoxy)tetrahydropyran-3-yl]
acetate was consumed and one new spot formed. The reaction mixture
was filtered and the filtrate was concentrated under reduced
pressure to give a residue. The residue was purified by column
chromatography (SiO.sub.2, Petroleum ether/Ethyl acetate=10/1 Ethyl
acetate:Methanol=3/1).dimethyl-prop-2-ynyl-[2-[(2R,3R,4S,5S)-3,4,5-triace-
toxytetrahydropyran-2-yl]oxyethyl]ammonium (318 mg, 617.64 umol,
23.67% yield, 99.7% purity, I) was obtained as white solid. LCMS:
(M+): 386.2 1H NMR (400 MHz, Chloroform-d) .delta. 5.26-5.20 (m,
1H), 5.08 (dd, J=10.0, 7.4 Hz, 1H), 4.99 (dd, J=10.0, 3.5 Hz, 1H),
4.75 (t, J=3.0 Hz, 2H), 4.48 (d, J=7.4 Hz, 1H), 4.37-4.26 (m, 1H),
4.17-4.06 (m, 2H), 4.09-4.00 (m, 1H), 3.95 (dd, J=13.3, 2.5 Hz,
1H), 3.66 (dd, J=13.4, 1.5 Hz, 1H), 3.46 (s, 6H), 2.82 (t, J=2.5
Hz, 1H), 2.10 (s, 3H), 2.04 (s, 3H), 1.95 (s, 3H)))).).
##STR00120##
Compound 11:
N,N-dimethyl-N-(2-(((3R,4R,5S,6S)-3,4,5-triacetoxy-6-methyltetrahydro-2H--
pyran-2-yl)oxy)ethyl)prop-2-yn-1-aminium bromide
[0382] This compound may be synthesized according to the
experimental procedure described for Compound 7.
##STR00121##
Compound 12:
N,N-dimethyl-N-(2-(((3S,4R,5R,6S)-3,4,5-triacetoxy-6-methyltetrahydro-2H--
pyran-2-yl)oxy)ethyl)prop-2-yn-1-aminium bromide
[0383] This compound may be synthesized according to the
experimental procedure described for Compound 7.
##STR00122##
Compound 13:
N,N-dimethyl-N-(2-oxo-2-(((3R,4S,5R,6R)-3,4,5-tris(butyryloxy)-6-((butyry-
loxy)methyl)tetrahydro-2H-pyran-2-yl)oxy)ethyl)prop-2-yn-1-aminium
chloride
[0384] Step 1:
[0385] To a mixture of
[(2R,3R,4S,5R)-3,4,5-tri(butanoyloxy)-6-hydroxy-tetrahydropyran-2-yl]meth-
yl butanoate (1 g, 2.17 mmol, 1 eq) and 2-chloroacetyl chloride
(735.76 mg, 6.51 mmol, 518.14 uL, 3 eq) in DCM (10 mL) was added
pyridine (858.82 mg, 10.86 mmol, 876.35 uL, 5 eq) in one portion at
20.degree. C. The mixture was stirred at 20.degree. C. for 12 h.
TLC showed
[(2R,3R,4S,5R)-3,4,5-tri(butanoyloxy)-6-hydroxy-tetrahydropyran-2-yl]meth-
yl butanoate was consumed completely and one new spot formed. The
reaction mixture was filtered and concentrated under reduced
pressure to give a residue. The residue was purified by column
chromatography (SiO.sub.2, Petroleum ether/Ethyl acetate=1/0 to
20:1). Compound
[(2R,3R,4S,5R)-3,4,5-tri(butanoyloxy)-6-(2-chloroacetyl)oxy-tetrahydropyr-
an-2-yl]methyl butanoate (0.8 g, crude) was obtained as colorless
oil.
[0386] Step 2:
[0387] A mixture of
[(2R,3R,4S,5R)-3,4,5-tri(butanoyloxy)-6-(2-chloroacetyl)oxy-tetrahydropyr-
an-2-yl] methyl butanoate (0.2 g, 372.44 umol, 1 eq) and
N,N-dimethylprop-2-yn-1-amine (92.89 mg, 1.12 mmol, 118.48 uL, 3
eq) in acetone (3 mL) was stirred at 90.degree. C. for 0.5 h. LCMS
showed
[(2R,3R,4S,5R)-3,4,5-tri(butanoyloxy)-6-(2-chloroacetyl)oxy-tetrahydropyr-
an-2-yl]methyl butanoate was consumed completely. The reaction
mixture was filtered and concentrated under reduced pressure to
give a residue. The residue was purified by prep-HPLC [water (0.1%
TFA)-ACN]. The title compound (0.049 g, 67 umol, 18% yield) was
obtained as colorless oil. LCMS (M.sup.+): 584.3 .sup.1H NMR (400
MHz, DMSO-d.sub.6) .delta. 6.30 (d, J=3.4 Hz, 1H), 5.46-5.35 (m,
1H), 5.25-5.11 (m, 2H), 4.80-4.63 (m, 2H), 4.61-4.46 (m, 2H),
4.30-3.98 (m, 4H), 3.30-3.16 (m, 6H), 2.37-2.10 (m, 8H), 1.63-1.41
(m, 8H), 0.95-0.75 (m, 12H).
##STR00123##
Compound 14 and Compound 52:
N,N-dimethyl-N-(2-oxo-2-(((3R,4S,5R)-3,4,5-tris(butyryloxy)tetrahydro-2H--
pyran-2-yl)oxy)ethyl)prop-2-yn-1-aminium chloride
[0388] This compound may be synthesized according to the
experimental procedure described for Compound 13.
##STR00124##
Compound 15:
N,N-dimethyl-N-(2-oxo-2-(((3R,4R,5R)-3,4,5-tris(butyryloxy)tetrahydro-2H--
pyran-2-yl)oxy)ethyl)prop-2-yn-1-aminium chloride
[0389] This compound may be synthesized according to the
experimental procedure described for Compound 13.
##STR00125##
Compound 16 and Compound 54:
N,N-dimethyl-N-(2-oxo-2-(((3R,4S,5S)-3,4,5-tris(butyryloxy)tetrahydro-2H--
pyran-2-yl)oxy)ethyl)prop-2-yn-1-aminium chloride
[0390] This compound may be synthesized according to the
experimental procedure described for Compound 13.
##STR00126##
Compound 17 and Compound 60:
N,N-dimethyl-N-(2-oxo-2-(((3R,4R,5S,6S)-3,4,5-tris(butyryloxy)-6-methylte-
trahydro-2H-pyran-2-yl)oxy)ethyl)prop-2-yn-1-aminium chloride
[0391] This compound may be synthesized according to the
experimental procedure described for Compound 13.
##STR00127##
Compound 18:
N,N-dimethyl-N-(2-oxo-2-(((3S,4R,5R,6S)-3,4,5-tris(butyryloxy)-6-methylte-
trahydro-2H-pyran-2-yl)oxy)ethyl)prop-2-yn-1-aminium chloride
[0392] This compound may be synthesized according to the
experimental procedure described for Compound 13.
##STR00128##
Compound 19:
N,N-dimethyl-N-(2-oxo-2-(((2R,3R,4S,5R,6R)-3,4,5-triacetoxy-6-(acetoxymet-
hyl)tetrahydro-2H-pyran-2-yl)oxy)ethyl)prop-2-yn-1-aminium
trifluoroacetate
[0393] Step 1
[0394] Compound 2,3,4,6-tetra-O-acetyl-D-glucopyranose (Carbosynth,
2 g, 5.7 mmol, 1 eq) was dissolved in DCM (10 mL), followed by
addition of 2-chloroacetyl chloride (1.4 mL, 17.6 mmol, 3 eq), DMAP
(0.6 g, 4.9 mmol, 0.9 eq), and DIPEA (3 mL, 17.2 mmol, 3 eq). The
reaction mixture was stirred at room temperature overnight,
followed by filtration and multiple purifications by column
chromatography (0-100% EtOAc in hexanes) to yield
1-(2-chloroacetoxy)-2,3,4,6-tetra-O-acetyl-D-glucopyranose (0.8 g,
1.9 mmol, mixture of anomers, 33% yield).
[0395] Step 2
[0396] Compound
1-(2-chloroacetoxy)-2,3,4,6-tetra-O-acetyl-D-glucopyranose (0.6 g,
1.4 mmol, 1 eq) was dissolved in acetone, followed by addition of
sodium iodide (0.6 g, 4.0 mmol, 3 eq). The reaction was stirred for
a few minutes, and dimethylamino-1-propyne (0.5 mL, 4.6 mmol, 3.3
eq) was added. The reaction was stirred at room temperature
overnight, then filtered. The filtrate was concentrated, diluted
with DMSO and water, then purified by reverse phase C.sub.18 column
chromatography (0.1% TFA in 95% water/5% MeCN to 100% MeCN).
Fractions containing product were lyophilized to yield an off-white
powder (230 mg, 0.39 mmol, 28% yield, TFA salt). LCMS (M+): 472.4
.sup.1H NMR (400 MHz, DMSO-d6) .delta. 6.27 (d, J=3.6 Hz, 1H), 5.35
(t, J=9.9 Hz, 1H), 5.17-5.08 (m, 2H), 4.76-4.62 (m, 2H), 4.53 (d,
J=2.5 Hz, 2H), 4.30-3.97 (m, 4H), 3.26 (s, 6H), 2.08-1.91 (m,
12H).
##STR00129##
Compound 20 and Compound 55:
N,N-dimethyl-N-(2-oxo-2-(((3R,4S,5R)-3,4,5-triacetoxytetrahydro-2H-pyran--
2-yl)oxy)ethyl)prop-2-yn-1-aminium chloride
[0397] This compound may be synthesized according to the
experimental procedure described for Compound 19.
##STR00130##
Compound 21:
N,N-dimethyl-N-(2-oxo-2-(((3R,4R,5R)-3,4,5-triacetoxytetrahydro-2H-pyran--
2-yl)oxy)ethyl)prop-2-yn-1-aminium chloride
[0398] This compound may be synthesized according to the
experimental procedure described for Compound 19.
##STR00131##
Compound 22 and Compound 57:
N,N-dimethyl-N-(2-oxo-2-(((3R,4S,5S)-3,4,5-triacetoxytetrahydro-2H-pyran--
2-yl)oxy)ethyl)prop-2-yn-1-aminium chloride
[0399] This compound may be synthesized according to the
experimental procedure described for Compound 19.
##STR00132##
Compound 23:
N,N-dimethyl-N-(2-oxo-2-(((3R,4R,5S,6S)-3,4,5-triacetoxy-6-methyltetrahyd-
ro-2H-pyran-2-yl)oxy)ethyl)prop-2-yn-1-aminium chloride
[0400] This compound may be synthesized according to the
experimental procedure described for Compound 19.
##STR00133##
Compound 24:
N,N-dimethyl-N-(2-oxo-2-(((3S,4R,5R,6S)-3,4,5-triacetoxy-6-methyltetrahyd-
ro-2H-pyran-2-yl)oxy)ethyl)prop-2-yn-1-aminium chloride
[0401] This compound may be synthesized according to the
experimental procedure described for Compound 19.
##STR00134##
Compound 25:
N,N-dimethyl-N-(prop-2-yn-1-yl)-4-(((3R,4S,5R,6R)-3,4,5-tris(butyryloxy)--
6-((butyryloxy)methyl)tetrahydro-2H-pyran-2-yl)oxy)butan-1-aminium
bromide
[0402] Step 1:
[0403] To a solution of
[(2R,3R,4S,5R)-3,4,5-tri(butanoyloxy)-6-(2,2,2-trichloroethanimidoyl)oxy--
tetrahydropyran-2-yl]methyl butanoate (1.5 g, 2.48 mmol, 1 eq) and
4-bromobutan-1-ol (455.33 mg, 2.98 mmol, 1.2 eq) in DCM (15 mL) was
added BF.sub.3.Et.sub.2O (1.76 g, 12.40 mmol, 1.53 mL, 5 eq). The
mixture was stirred at 0.degree. C. for 3 h and then warmed to
20.degree. C. and stirred for 12 h. LC-MS
showed[(2R,3R,4S,5R)-3,4,5-tri(butanoyloxy)-6-(2,2,2-trichloroethanimidoy-
l)oxy-tetrahydropyran-2-yl]methyl butanoate was consumed
completely. The reaction mixture was filtered and concentrated
under reduced pressure to give a residue. The residue was purified
by column chromatography (SiO.sub.2, Petroleum ether/Ethyl
acetate=20/1 to 3:1). Desired compound
[(2R,3R,4S,5R)-6-(4-bromobutoxy)-3,4,5-tri(butanoyloxy)tetrahydropyran-2--
yl]methyl butanoate (1 g, crude) was obtained as colorless oil.
[0404] Step 2:
[0405] To a mixture of
[(2R,3R,4S,5R)-6-(4-bromobutoxy)-3,4,5-tri(butanoyloxy)tetrahydropyran-2--
yl]methyl butanoate (0.2 g, 335.84 umol, 1 eq) and
N,N-dimethylprop-2-yn-1-amine (83.76 mg, 1.01 mmol, 106.83 uL, 3
eq) in Acetone (3 mL) stirred at 90.degree. C. for 2 h under
N.sub.2. LCMS showed
[(2R,3R,4S,5R)-6-(4-bromobutoxy)-3,4,5-tri(butanoyloxy)tetrahydrop-
yran-2-yl]methyl butanoate was consumed completely. The reaction
mixture was filtered and concentrated under reduced pressure to
give a residue. The residue was purified by prep-HPLC [water (0.10%
TFA)-ACN]. The title compound (0.019 g, 28 .mu.mol, 8.3% yield,
mixture of anomers, Br) was obtained as colorless oil. LCMS
(M.sup.-): 598.4 .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta.
5.41-5.27 (m, 1H), 5.11-4.77 (m, 3H), 4.35 (dd, J=10.3, 2.6 Hz,
2H), 4.23-3.97 (m, 4H), 3.79-3.65 (m, 1H), 3.59-3.44 (m, 1H),
3.11-3.04 (m, 6H), 2.34-2.11 (m, 8H), 1.80-1.39 (m, 12H), 0.96-0.78
(m, 12H).
##STR00135##
Compound 26:
N,N-dimethyl-N-(prop-2-yn-1-yl)-4-(((3R,4S,5R)-3,4,5-tris(butyryloxy)tetr-
ahydro-2H-pyran-2-yl)oxy)butan-1-aminium bromide
[0406] Step 1
[0407] To a solution of (3R,4S,5R)-tetrahydropyran-2,3,4,5-tetrol
(10 g, 66.61 mmol, 1 eq) in Pyridine (100 mL) was added butanoyl
butanoate (63.22 g, 399.66 mmol, 65.38 mL, 6 eq). The mixture was
stirred at 25.degree. C. for 10 h. TLC showed the starting reactant
was consumed. The mixture was concentrated. The crude product
[(3R,4S,5R)-4,5,6-tri(butanoyloxy)tetrahydropyran-3-yl] butanoate
(64.89 g, crude) was obtained as yellow oil.
[0408] Step 2
[0409] To a solution of
[(3R,4S,5R)-4,5,6-tri(butanoyloxy)tetrahydropyran-3-yl]butanoate
(10 g, 23.23 mmol, 1 eq) in THF (100 mL) was added MeNH.sub.2 aq.
solution (4.33 g, 41.81 mmol, 30% purity, 1.8 eq) in H.sub.2O. The
mixture was stirred at 25.degree. C. for 10 h. TLC showed the
starting reactant was consumed. The mixture reaction was
concentrated. The residue was purified by flash silica gel
chromatography (PE:EA=5:1).
[(3R,4S,5R)-4,5-di(butanoyloxy)-6-hydroxy-tetrahydropyran-3-yl]
butanoate (2.65 g, 6.61 mmol, 28.4% yield, 90% purity) was obtained
as yellow oil.
[0410] Step 3
[0411] To a solution of
[(3R,4S,5R)-4,5-di(butanoyloxy)-6-hydroxy-tetrahydropyran-3-yl]
butanoate (500 mg, 1.39 mmol, 1 eq) in DCM (10 mL) was added
2,2,2-trichloroacetonitrile (2.00 g, 13.87 mmol, 1.39 mL, 10 eq)
and DBU (105.61 mg, 693.68 umol, 104.56 uL, 0.5 eq). The mixture
was stirred at 25.degree. C. for 10 h. TLC showed the starting
reactant was consumed. The mixture was concentrated. The residue
was purified by flash silica gel chromatography (PE:EA=5:1).
Compound
[(3R,4S,5R)-4,5-di(butanoyloxy)-6-(2,2,2-trichloroethanimidoyl)oxy-tetrah-
ydropyran-3-yl] butanoate (0.5 g, 792.42 umol, 57.1% yield, 80%
purity) was obtained as a white solid.
[0412] Step 4
[0413] To a solution of
[(3R,4S,5R)-4,5-di(butanoyloxy)-6-(2,2,2-trichloroethanimidoyl)oxy-tetrah-
y-dropyran-3-yl] butanoate (400 mg, 792.42 umol, 1 eq) and
4-bromobutan-1-ol (181.88 mg, 1.19 mmol, 1.5 eq) in DCM (40 mL) was
added BF.sub.3.Et.sub.2O (562.34 mg, 3.96 mmol, 489 uL, 5 eq) under
dark at 0.degree. C. and stirred for 3 h. Then the mixture was
stirred at 25.degree. C. for 12 h. TLC showed the starting reactant
was consumed. The mixture was concentrated. The residue was
purified by silica gel chromatography (PE:EA=5:1).
[(3R,4S,5R)-6-(4-bromobutoxy)-4,5-di(butanoyloxy)
tetrahydropyran-3-yl] butanoate (160 mg, crude) was obtained as
yellow oil.
[0414] Step 5
[0415] A solution of
[(3R,4S,5R)-6-(4-bromobutoxy)-4,5-di(butanoyloxy)tetrahydropyran-3-yl]
butanoate (643 mg, 1.30 mmol, 1 eq) and
N,N-dimethylprop-2-yn-1-amine (323.70 mg, 3.89 mmol, 412.88 uL, 3
eq) in acetone (10 mL) was stirred at 90.degree. C. for 3 h. LCMS
showed the product formed. The mixture was concentrated. The
residue was purified by prep-HPLC (column: Xbridge 150*30 mm*10 um;
mobile phase: [water (0.1% TFA)-ACN]; B %: 25%-55%, 10 min). The
title compound (10 mg, 16.9 umol, 1.3% yield, mixture of anomers,
Br) was obtained as yellow oil. LCMS (M.sup.+): 498.3 .sup.1H NMR
(400 MHz, Chloroform-d) .delta. 5.45-5.12 (m, 1H), 4.95-4.74 (m,
3H), 4.46-4.32 (m, 2H), 3.80-3.67 (m, 2H), 3.58-3.35 (m, 4H),
3.31-3.18 (m, 6H), 2.91-2.79 (m, 1H), 2.32-2.10 (m, 6H), 1.87-1.45
(m, 10H), 0.93-0.79 (m, 9H).
##STR00136##
Compound 27 and Compound 67:
N,N-dimethyl-N-(prop-2-yn-1-yl)-4-(((3R,4R,5R)-3,4,5-tris(butyryloxy)tetr-
ahydro-2H-pyran-2-yl)oxy)butan-1-aminium bromide
[0416] This compound may be synthesized according to the
experimental procedure described for Compound 26.
##STR00137##
Compound 28 and Compound 59:
N,N-dimethyl-N-(prop-2-yn-1-yl)-4-(((2S,3R,4S,5S)-3,4,5-tris(butyryloxy)t-
etrahydro-2H-pyran-2-yl)oxy)butan-1-aminium bromide
[0417] Step 1:
[0418] To a solution of (3R,4S,5S)-tetrahydropyran-2,3,4,5-tetrol
(5 g, 33.30 mmol, 1 eq) in pyridine (50 mL) was added butanoyl
butanoate (31.61 g, 199.83 mmol, 32.69 mL, 6 eq). The mixture was
stirred at 25.degree. C. for 12 h. TLC showed the starting reactant
was consumed. The mixture was concentrated.
[(3S,4S,5R)-4,5,6-tri(butanoyloxy)tetrahydropyran-3-yl] butanoate
(15 g, crude) was obtained as yellow oil.
[0419] Step 2
[0420] To a solution of
[(3S,4S,5R)-4,5,6-tri(butanoyloxy)tetrahydropyran-3-yl]butanoate
(15 g, 34.84 mmol, 1 eq) in THF (50 mL) was added MeNH.sub.2 aq.
solution (5.41 g, 52.27 mmol, 30% purity, 1.5 eq) and stirred at
25.degree. C. for 12 h. TLC showed the starting reactant was
consumed. The mixture reaction was concentrated. The residue was
purified by column chromatography (SiO.sub.2, Petroleum ether/Ethyl
acetate=10/1 to 5:1).
[(3S,4S,5R)-4,5-di(butanoyloxy)-6-hydroxy-tetrahydropyran-3-yl]
butanoate (4 g, 8.88 mmol, 25.5% yield, 80% purity) was obtained as
yellow oil.
[0421] Step 3
[0422] To a solution of
[(3S,4S,5R)-4,5-di(butanoyloxy)-6-hydroxy-tetrahydropyran-3-yl]
butanoate (2 g, 5.55 mmol, 1 eq) and 2,2,2-trichloroacetonitrile
(8.01 g, 55.49 mmol, 5.56 mL, 10 eq) in DCM (20 mL) was added DBU
(422.42 mg, 2.77 mmol, 418.24 uL, 0.5 eq) at 25.degree. C. and
stirred for 12 h. TLC showed the starting reactant was consumed.
The mixture was concentrated. The residue was purified by column
chromatography (SiO.sub.2, Petroleum ether/Ethyl acetate=10/1 to
3:1).
[(3S,4S,5R)-4,5-di(butanoyloxy)-6-(2,2,2-trichloroethanimidoyl)oxy-tetrah-
ydropyran-3-yl] butanoate (1.5 g, 2.67 mmol, 48.2% yield, 90%
purity) was obtained as yellow oil.
[0423] Step 4
[0424]
[(3S,4S,5R)-4,5-di(butanoyloxy)-6-(2,2,2-trichloroethanimidoyl)oxy--
tetrahydropyran-3-yl]butanoate (1.5 g, 2.97 mmol, 1 eq) and
4-bromobutan-1-ol (682.05 mg, 4.46 mmol, 1.5 eq) was dissolved with
DCM (20 mL) and stirred at 0.degree. C. for 3 h in dark.
BF.sub.3.Et.sub.2O (2.11 g, 14.86 mmol, 1.83 mL, 5 eq) was added
into the mixture at 0.degree. C. and stirred for 12 h at 25.degree.
C. TLC showed the starting reactant was consumed. The mixture was
concentrated. The residue was purified by flash silica gel
chromatography (SiO.sub.2, Petroleum ether/Ethyl acetate=5/1).
Compound
[(3S,4S,5R)-6-(4-bromobutoxy)-4,5-di(butanoyloxy)tetrahydropyran-3-yl]
butanoate (1.6 g, crude) was obtained as brown oil.
[0425] Step 5
[0426] To a solution of
[(3S,4S,5R)-6-(4-bromobutoxy)-4,5-di(butanoyloxy)tetrahydropyran-3-yl]
butanoate (1.6 g, 3.23 mmol, 1 eq) in acetone (10 mL) was added
N,N-dimethylprop-2-yn-1-amine (805.47 mg, 9.69 mmol, 1.03 mL, 3 eq)
and stirred at 90.degree. C. for 2 h. LCMS showed the product
formed. The mixture was concentrated. The residue was purified by
prep-HPLC (column: Luna C18 100*30 5u; mobile phase: [water (0.1%
TFA)-ACN]; B %: 25%-50%, 12 min). The title compound (42 mg, 69
.mu.mol, 2.1% yield, Br) as a yellow oil was obtained. The anomer
(Compound 29) was also isolated. LCMS (M.sup.+): 498.3 .sup.1H NMR
(400 MHz, DMSO-d.sub.6) .delta. 5.30-5.24 (m, 1H), 5.22 (dd, J=9.9,
3.6 Hz, 1H), 5.06-4.97 (m, 2H), 4.33 (d, J=2.6 Hz, 2H), 4.04 (t,
J=2.4 Hz, 1H), 3.92 (dd, J=13.4, 1.6 Hz, 1H), 3.66-3.57 (m, 2H),
3.50-3.28 (m, 3H), 3.05 (s, 6H), 2.37-2.29 (m, 2H), 2.25 (td,
J=7.2, 2.3 Hz, 2H), 2.14 (td, J=7.2, 1.8 Hz, 2H), 1.78-1.66 (m,
2H), 1.62-1.39 (m, 8H), 0.94-0.77 (m, 9H).
##STR00138##
Compound 29 and Compound 66:
N,N-dimethyl-N-(prop-2-yn-1-yl)-4-(((2R,3R,4S,5S)-3,4,5-tris(butyryloxy)t-
etrahydro-2H-pyran-2-yl)oxy)butan-1-aminium bromide
[0427] This compound was synthesized according to the experimental
procedure described for Compound 28. The title compound (21 mg,
34.5 .mu.mol, 1.1% yield, Br) was isolated as a yellow oil. LCMS
(M.sup.+): 498.2 .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta.
5.20-5.14 (m, 1H), 5.11 (dd, J=10.1, 3.6 Hz, 1H), 4.95 (dd, J=10.1,
7.7 Hz, 1H), 4.57 (d, J=7.7 Hz, 1H), 4.30 (d, J=2.6 Hz, 2H),
4.06-4.00 (m, 1H), 3.85-3.76 (m, 2H), 3.76-3.66 (m, 1H), 3.56-3.42
(m, 1H), 3.03 (s, 6H), 2.31 (t, J=7.2 Hz, 2H), 2.24 (td, J=7.2, 3.5
Hz, 2H), 2.17-2.09 (m, 2H), 1.73-1.62 (m, 2H), 1.60-1.38 (m, 8H),
0.95-0.76 (m, 9H).
##STR00139##
Compound 30:
N,N-dimethyl-N-(prop-2-yn-1-yl)-4-(((2R,3R,4R,5S,6S)-3,4,5-tris(butyrylox-
y)-6-methyltetrahydro-2H-pyran-2-yl)oxy)butan-1-aminium bromide
[0428] Step 1:
[0429] To a solution of
(3R,4R,5R,6S)-6-methyltetrahydro-2H-pyran-2,3,4,5-tetraol (10 g,
60.92 mmol, 1 eq) in Pyridine (100 mL) was added butanoyl butanoate
(57.82 g, 365.51 mmol, 59.79 mL, 6 eq) at 25.degree. C. The mixture
was stirred at 25.degree. C. for 12 h. TLC indicated new spots
formed. The reaction mixture was concentrated under reduced
pressure to give a residue. The reaction mixture was washed with
saturated sodium bicarbonate solution 300 mL (100 mL*3) and
extracted with ethyl acetate 100 mL. The organic layer were washed
with brine 50 mL, dried over anhydrous sodium sulfate, filtered and
concentrated under reduced pressure to give
(3R,4R,5S,6S)-6-methyltetrahydro-2H-pyran-2,3,4,5-tetrayl
tetrabutyrate (34 g, crude) as yellow oil.
[0430] Step 2:
[0431] To a solution of
(3R,4R,5S,6S)-6-methyltetrahydro-2H-pyran-2,3,4,5-tetrayl
tetrabutyrate (34 g, 76.49 mmol, 1 eq) in THF (20 mL) was added
MeNH.sub.2 aq. solution (10.69 g, 137.68 mmol, 40% purity, 1.8 eq)
at 25.degree. C. The mixture was stirred at 25.degree. C. for 12
hr. TLC indicated new spots formed. The reaction mixture was
concentrated under reduced pressure to give a residue. The residue
was purified by column chromatography (SiO.sub.2, Petroleum
ether/Ethyl acetate=10/1 to 1:1) to give
(3R,4R,5S,6S)-2-hydroxy-6-methyltetrahydro-2H-pyran-3,4,5-triyl
tributyrate (18 g, 48.1 mmol, 62.9% yield) as yellow oil.
[0432] Step 3:
[0433] To a solution of
(3R,4R,5S,6S)-2-hydroxy-6-methyltetrahydro-2H-pyran-3,4,5-triyl
tributyrate (5 g, 13.35 mmol, 1 eq) in DCM (50 mL) was added DBU
(1.02 g, 6.68 mmol, 1.01 mL, 0.5 eq) and
2,2,2-trichloroacetonitrile (19.28 g, 133.54 mmol, 13.39 mL, 10 eq)
at 25.degree. C. The mixture was stirred at 25.degree. C. for 2 h.
TLC indicated new spots formed. The reaction mixture was
concentrated under reduced pressure to give a residue. The residue
was purified by column chromatography (SiO.sub.2, Petroleum
ether/Ethyl acetate=10/1 to 1:1) to give
(2S,3S,4R,5R)-2-methyl-6-(2,2,2-trichloro-1-iminoethoxy)tetrahydro-2H-pyr-
an-3,4,5-triyl tributyrate (4 g, 7.7 mmol, 57.7% yield) as yellow
oil.
[0434] Step 4:
[0435] To a solution of 4-bromobutan-1-ol (766.84 mg, 5.01 mmol, 1
eq) and
(2S,3S,4R,5R)-2-methyl-6-(2,2,2-trichloro-1-iminoethoxy)tetrahydro-2H-pyr-
an-3,4,5-triyl tributyrate (2.6 g, 5.01 mmol, 1 eq) in DCM (30 mL)
was added BF.sub.3.Et.sub.2O (3.56 g, 25.06 mmol, 3.09 mL, 5 eq) at
0.degree. C. The mixture was stirred at 0.degree. C. for 3 hr. The
mixture was stirred at 25.degree. C. for 12 hr. TLC indicated new
spots formed. The reaction mixture was concentrated under reduced
pressure to give a residue. The residue was purified by column
chromatography (SiO.sub.2, Petroleum ether/Ethyl acetate=20:1) to
give
(3R,4R,5S,6S)-2-(4-bromobutoxy)-6-methyltetrahydro-2H-pyran-3,4,5-triyl
tributyrate (600 mg, 1.2 mmol, 23.5% yield) as yellow oil.
[0436] Step 5:
[0437] To a solution of
(3R,4R,5S,6S)-2-(4-bromobutoxy)-6-methyltetrahydro-2H-pyran-3,4,5-triyl
tributyrate (600 mg, 1.18 mmol, 1 eq) in Acetone (5 mL) was added
N,N-dimethylprop-2-yn-1-amine (293.73 mg, 3.53 mmol, 374.66 uL, 3
eq) at 25.degree. C. The mixture was stirred at 90.degree. C. for 2
h. LCMS showed the desired compound was detected. The reaction
mixture was concentrated under reduced pressure to give a residue.
The residue was purified by prep-HPLC (column: Luna C18 100*30 5u;
mobile phase: [water (0.1% TFA)-ACN]; B %: 25%-55%, 12 min) to give
the title compound (150 mg, 253 .mu.mol, 21.5% yield) as a yellow
oil. LCMS (M.sup.+): 512.3 .sup.1H NMR (400 MHz, DMSO-d.sub.6)
.delta. 5.16-5.06 (m, 2H), 4.91 (t, J=9.9 Hz, 1H), 4.78 (d, J=1.6
Hz, 1H), 4.34 (d, J=2.5 Hz, 2H), 4.00 (t, J=2.5 Hz, 1H), 3.87-3.76
(m, 1H), 3.65 (dt, J=9.9, 6.1 Hz, 1H), 3.50-3.40 (m, 1H), 3.40-3.31
(m, 2H), 3.06 (s, 6H), 2.38-2.08 (m, 6H), 1.81-1.68 (m, 2H),
1.62-1.39 (m, 8H), 1.10 (d, J=6.2 Hz, 3H), 0.90 (t, J=7.4 Hz, 3H),
0.87-0.75 (m, 6H).
##STR00140##
Compound 31 and Compound 58:
N,N-dimethyl-N-(prop-2-yn-1-yl)-4-(((3S,4R,5R,6S)-3,4,5-tris(butyryloxy)--
6-methyltetrahydro-2H-pyran-2-yl)oxy)butan-1-aminium bromide
[0438] This compound may be synthesized according to the
experimental procedure described for Compound 30.
##STR00141##
Compound 32:
N,N-dimethyl-N-(prop-2-yn-1-yl)-4-(((3R,4S,5R,6R)-3,4,5-triacetoxy-6-(ace-
toxymethyl)tetrahydro-2H-pyran-2-yl)oxy)butan-1-aminium bromide
[0439] Step 1:
[0440] To a mixture of
[(2R,3R,4S,5R)-3,4,5-triacetoxy-6-(2,2,2-trichloroethanimidoyl)oxy-tetrah-
ydropyran-2-yl]methyl acetate (1 g, 2.03 mmol, 1 eq) and
4-bromobutan-1-ol (372.69 mg, 2.44 mmol, 1.2 eq) in DCM (10 mL) was
added BF.sub.3.Et.sub.2O (1.44 g, 10.15 mmol, 1.25 mL, 5 eq) in one
portion at 0.degree. C. under N.sub.2. The mixture was stirred at
0.degree. C. for 3 h and then heated to 20.degree. C. and stirred
for 12 h. TLC indicated the starting material was consumed
completely. The reaction mixture was filtered and concentrated
under reduced pressure to give a residue. The residue was purified
by column chromatography (SiO.sub.2, Petroleum ether/Ethyl
acetate=5/1 to 2:1).
[(2R,3R,4S,5R)-3,4,5-triacetoxy-6-(4-bromobutoxy)tetrahydropyran-2-yl]met-
hyl acetate (1 g, crude) was obtained as colorless oil.
[0441] Step 2:
[0442] To a mixture of
[(2R,3R,4S,5R)-3,4,5-triacetoxy-6-(4-bromobutoxy)tetrahydropyran-2-yl]met-
hyl acetate (0.2 g, 413.82 umol, 1 eq) and
N,N-dimethylprop-2-yn-1-amine (103.20 mg, 1.24 mmol, 131.64 uL, 3
eq) in acetone (3 mL) was stirred at 90.degree. C. for 2 h. LC-MS
showed
[(2R,3R,4S,5R)-3,4,5-triacetoxy-6-(4-bromobutoxy)tetrahydropyran-2-yl]met-
hyl acetate was consumed completely. The reaction mixture was
filtered and concentrated under reduced pressure to give a residue.
The residue was purified by prep-HPLC [water (0.1% TFA)-ACN]. The
title compound (0.006 g, 10.5 .mu.mol, 2.5% yield, mixture of
anomers) was obtained as colorless oil. LCMS (M.sup.+): 486.2
.sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. 5.44-5.19 (m, 1H),
5.15-4.65 (m, 3H), 4.39-4.32 (m, 2H), 4.22-3.94 (m, 4H), 3.75-3.65
(m, 1H), 3.59-3.35 (m, 3H), 3.13-3.04 (m, 6H), 2.15-1.89 (m, 12H),
1.79-1.74 (m, 2H), 1.66-1.58 (m, 2H).
##STR00142##
Compound 33:
N,N-dimethyl-N-(prop-2-yn-1-yl)-4-(((3R,4S,5R)-3,4,5-triacetoxytetrahydro-
-2H-pyran-2-yl)oxy)butan-1-aminium bromide
[0443] Step 1
[0444] To a solution of (3R,4S,5R)-tetrahydropyran-2,3,4,5-tetrol
(10 g, 66.61 mmol, 1 eq) in Pyridine (100 mL) was added acetyl
acetate (40.80 g, 399.65 mmol, 37.43 mL, 6 eq). The mixture was
stirred at 25.degree. C. for 10 h. TLC showed the starting reactant
was consumed. The mixture was concentrated. Compound
[(3R,4S,5R)-4,5,6-triacetoxytetrahydropyran-3-yl] acetate (10.6 g,
crude) was obtained as a white solid.
[0445] Step 2
[0446] To a solution of
[(3R,4S,5R)-4,5,6-triacetoxytetrahydropyran-3-yl] acetate (5 g,
15.71 mmol, 1 eq) in THF (50 mL) was added MeNH.sub.2 aq. solution
(2.93 g, 28.28 mmol, 30% purity, 1.8 eq). The mixture was stirred
at 25.degree. C. for 10 h. TLC showed the starting reactant was
consumed. The mixture reaction was concentrated. The residue was
purified by flash silica gel chromatography (PE:EA=2:1).
[(3R,4S,5R)-4,5-diacetoxy-6-hydroxy-tetrahydropyran-3-yl] acetate
(1.53 g, 4.98 mmol, 31.7% yield, 90% purity) was obtained as a
yellow solid.
[0447] Step 3
[0448] To a solution of
[(3R,4S,5R)-4,5-diacetoxy-6-hydroxy-tetrahydropyran-3-yl]acetate (1
g, 3.62 mmol, 1 eq) in DCM (20 mL) was added
2,2,2-trichloroacetonitrile (5.23 g, 36.20 mmol, 3.63 mL, 10 eq)
and DBU (275.55 mg, 1.81 mmol, 272.82 uL, 0.5 eq). The mixture was
stirred at 25.degree. C. for 10 h. TLC showed the starting reactant
was consumed. The mixture was concentrated. The residue was
purified by flash silica gel chromatography (PE:EA=5:1). Compound
[(3R,4S,5R)-4,5-diacetoxy-6-(2,2,2-trichloroethanimidoyl)oxy-tetrahydropy-
ran-3-yl] acetate (1.09 g, 2.07 mmol, 57.3% yield, 80% purity) was
obtained as a white solid.
[0449] Step 4
[0450] To a solution of
[(3R,4S,5R)-4,5-diacetoxy-6-(2,2,2-trichloroethanimidoyl)oxy-tetrahydropy-
ran-3-yl] acetate (1.09 g, 2.59 mmol, 1 eq) and 4-bromobutan-1-ol
(594.79 mg, 3.89 mmol, 1.5 eq) in DCM (10 mL) was added
BF.sub.3.Et.sub.2O (1.84 g, 12.96 mmol, 1.60 mL, 5 eq) in dark and
stirred at 0.degree. C. for 3 h. Then the mixture was stirred at
25.degree. C. for 12 h. TLC showed the starting reactant was
consumed. The mixture was concentrated and purified by flash silica
gel chromatography (PE:EA=5:1).
[(3R,4S,5R)-4,5-diacetoxy-6-(4-bromobutoxy)tetrahydropyran-3-yl]
acetate (429 mg, 835 umol, 32.2% yield, 80% purity) was obtained as
yellow oil.
[0451] Step 5
[0452] The solution of
[(3R,4S,5R)-4,5-diacetoxy-6-(4-bromobutoxy)tetrahydropyran-3-yl]
acetate (429 mg, 1.04 mmol, 1 eq) and N,N-dimethylprop-2-yn-1-amine
(260.16 mg, 3.13 mmol, 331.84 uL, 3 eq) in acetone (10 mL) was
stirred at 90.degree. C. for 3 h. LCMS showed the product formed.
The mixture was concentrated. The residue was purified by prep-HPLC
(column: Xbridge 150*30 mm*10 um; mobile phase: [water (0.1%
TFA)-ACN]; B %: 25%-55%, 10 min). The title compound (10 mg, 23.2
.mu.mol, 2.2% yield, mixture) was obtained as a yellow oil. LCMS
(M.sup.+) 414.2 .sup.1H NMR (400 MHz, Chloroform-d) .delta. 5.5-5.1
(m, 1H), 5.0-4.8 (m, 3H), 4.6-4.4 (m, 2H), 3.9-3.7 (m, 2H), 3.7-3.4
(m, 4H), 3.4-3.2 (m, 6H), 3.0-2.6 (m, 1H), 2.4-2.0 (m, 9H), 2.0-1.6
(m, 4H).
##STR00143##
Compound 34:
N,N-dimethyl-N-(prop-2-yn-1-yl)-4-(((3R,4R,5R)-3,4,5-triacetoxytetrahydro-
-2H-pyran-2-yl)oxy)butan-1-aminium bromide
[0453] This compound may be synthesized according to the
experimental procedure described for Compound 33.
##STR00144##
Compound 35 and compound 62:
N,N-dimethyl-N-(prop-2-yn-1-yl)-4-(((2S,3R,4S,5S)-3,4,5-triacetoxytetrahy-
dro-2H-pyran-2-yl)oxy)butan-1-aminium bromide
[0454] Step 1
[0455] To a solution of (3R,4S,5S)-tetrahydropyran-2,3,4,5-tetrol
(3 g, 19.98 mmol, 1 eq) in Pyridine (50 mL) was added acetyl
acetate (12.24 g, 119.90 mmol, 11.23 mL, 6 eq) and then the mixture
was stirred at 25.degree. C. for 12 h. TLC showed the starting
reactant was consumed. To the mixture was added H.sub.2O (250 mL)
at 0.degree. C., extracted with EtOAc (300 mL) and the organic
layer was concentrated. The residue was purified by column
chromatography (SiO.sub.2, Petroleum ether/Ethyl acetate=10/1 to
4:1). [(3S,4S,5R)-4,5,6-triacetoxytetrahydropyran-3-yl] acetate (5
g, crude) was obtained as yellow solid.
[0456] Step 2
[0457] To a solution of
[(3S,4S,5R)-4,5,6-triacetoxytetrahydropyran-3-yl] acetate (5 g,
15.71 mmol, 1 eq) in THF (50 mL) was added MeNH.sub.2 aq. solution
(2.44 g, 23.56 mmol, 30% purity, 1.5 eq) and stirred at 25.degree.
C. for 12 h. TLC showed the starting reactant was consumed. The
mixture reaction was concentrated. The residue was purified by
column chromatography (SiO.sub.2, Petroleum ether/Ethyl
acetate=10/1 to 5:1).
[(3S,4S,5R)-4,5-diacetoxy-6-hydroxy-tetrahydropyran-3-yl] acetate
(2 g, 6.5 mmol, 41.5% yield, 90% purity) was obtained as yellow
solid.
[0458] Step 3
[0459] To a solution of
[(3S,4S,5R)-4,5-diacetoxy-6-hydroxy-tetrahydropyran-3-yl]acetate (2
g, 7.24 mmol, 1 eq) and 2,2,2-trichloroacetonitrile (10.45 g, 72.40
mmol, 7.26 mL, 10 eq) in DCM (10 mL) was added DBU (551.11 mg, 3.62
mmol, 545.65 uL, 0.5 eq) at 25.degree. C. and stirred for 12 h. TLC
showed the starting reactant was consumed. The mixture was combined
work-up with another batch in Et16408-556. The combined mixture was
concentrated. The residue was purified by column chromatography
(SiO.sub.2, Petroleum ether/Ethyl acetate=20/1 to 3:1).
[(3S,4S,5R)-4,5-diacetoxy-6-(2,2,2-trichloroethanimidoyl)oxy-tetrahydropy-
ran-3-yl] acetate (1.5 g in total) was obtained as white solid.
[0460] Step 4
[0461]
[(3S,4S,5R)-4,5-diacetoxy-6-(2,2,2-trichloroethanimidoyl)oxy-tetrah-
ydropyran-3-yl] acetate (500 mg, 1.19 mmol, 1 eq) and
4-bromobutan-1-ol (236.46 mg, 1.55 mmol, 1.3 eq) was dissolved with
DCM (5 mL) and stirred at 0.degree. C. for 3 h in dark.
BF.sub.3.Et.sub.2O (843.55 mg, 5.94 mmol, 733.52 uL, 5 eq) was
added into the mixture at 0.degree. C. and stirred for 12 h at
25.degree. C. TLC showed the starting reactant was consumed. The
mixture was concentrated. The residue was purified by flash silica
gel chromatography (SiO.sub.2, Petroleum ether/Ethyl acetate=5/1).
[(3S,4S,5R)-4,5-diacetoxy-6-(4-bromobutoxy)tetrahydropyran-3-yl]
acetate (500 mg, crude) was obtained as brown oil.
[0462] Step 5
[0463] To a solution of
[(3S,4S,5R)-4,5-diacetoxy-6-(4-bromobutoxy)tetrahydropyran-3-yl]
acetate (500 mg, 1.22 mmol, 1 eq) in acetone (5 mL) was added
N,N-dimethylprop-2-yn-1-amine (303.22 mg, 3.65 mmol, 386.76 uL, 3
eq) and stirred at 90.degree. C. for 2 h. LCMS showed the product
formed. The reaction mixture was concentrated. The residue was
purified by prep-HPLC (column: Luna C18 100*30 5u; mobile phase:
[water (0.1% TFA)-ACN]; B %: 1%-30%, 12 min). The title compound
(120 mg, 290 umol, 23.8% yield) was obtained as colorless oil. LCMS
(M.sup.+): 414.2 .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta.
5.30-5.25 (m, 1H), 5.22 (dd, J=9.9, 3.6 Hz, 1H), 5.06-4.97 (m, 2H),
4.36 (d, J=2.6 Hz, 2H), 4.07 (t, J=2.5 Hz, 1H), 3.93 (dd, J=13.4,
1.5 Hz, 1H), 3.71-3.61 (m, 2H), 3.51-3.34 (m, 3H), 3.08 (s, 6H),
2.13-2.01 (m, 6H), 1.96 (s, 3H), 1.82-1.72 (m, 2H), 1.61 (q, J=6.7
Hz, 2H).
##STR00145##
Compound 36:
N,N-dimethyl-N-(prop-2-yn-1-yl)-4-(((3R,4R,5S,6S)-3,4,5-triacetoxy-6-meth-
yltetrahydro-2H-pyran-2-yl)oxy)butan-1-aminium bromide
[0464] Step 1
[0465] To a solution of
(3R,4R,5R,6S)-6-methyltetrahydropyran-2,3,4,5-tetrol (10 g, 60.92
mmol, 1 eq) in Pyridine (100 mL) was added acetyl acetate (37.31 g,
365.51 mmol, 34.23 mL, 6 eq) at 25.degree. C. The mixture was
stirred at 25.degree. C. for 12 hr. TLC indicated new spots formed.
The reaction mixture was concentrated under reduced pressure to
give a residue. The residue was purified by column chromatography
(SiO.sub.2, Petroleum ether/Ethyl acetate=10:1 to 1:1) to give
[(2S,3S,4R,5R)-4,5,6-triacetoxy-2-methyl-tetrahydropyran-3-yl]
acetate (16 g, 48 mmol, 79% yield) as a colorless oil.
[0466] Step 2
[0467] To a solution of
[(2S,3S,4R,5R)-4,5,6-triacetoxy-2-methyl-tetrahydropyran-3-yl]
acetate (16 g, 48.15 mmol, 1 eq) in THF (160 mL) was added
MeNH.sub.2 aq. (6.73 g, 86.67 mmol, 40% purity, 1.8 eq) at
25.degree. C. The mixture was stirred at 25.degree. C. for 12 hr.
TLC indicated new spots formed. The reaction mixture was
concentrated under reduced pressure to give a residue. The residue
was purified by column chromatography (SiO.sub.2, Petroleum
ether/Ethyl acetate=10/1 to 0:1) to give
[(2S,3S,4R,5R)-4,5-diacetoxy-6-hydroxy-2-methyl-tetrahydropyran-3-yl]
acetate (8 g, 28 mmol, 57% yield) as a white solid.
[0468] Step 3
[0469] To a solution of
[(2S,3S,4R,5R)-4,5-diacetoxy-6-hydroxy-2-methyl-tetrahydropyran-3-yl]
acetate (3 g, 10.34 mmol, 1 eq) in DCM (30 mL) was added DBU
(786.71 mg, 5.17 mmol, 778.92 uL, 0.5 eq) and
2,2,2-trichloroacetonitrile (14.92 g, 103.35 mmol, 10.36 mL, 10 eq)
at 25.degree. C. The mixture was stirred at 25.degree. C. for 2 hr.
TLC indicated new spots formed. The reaction mixture was
concentrated under reduced pressure to give a residue. The residue
was purified by column chromatography (SiO.sub.2, Petroleum
ether/Ethyl acetate=10:1 to 9:1) to give
[(2S,3S,4R,5R)-4,5-diacetoxy-2-methyl-6-(2,2,2-trichloroethanimidoyl)oxy--
tetrahydropyran-3-yl] acetate (3 g, 6.9 mmol, 66.8% yield) as a
yellow oil.
[0470] Step 4
[0471] To a solution of
[(2S,3S,4R,5R)-4,5-diacetoxy-2-methyl-6-(2,2,2-trichloroethanimidoyl)oxy--
tetrahydropyran-3-yl] acetate (1.5 g, 3.45 mmol, 1 eq) and
4-bromobutan-1-ol (686.49 mg, 4.49 mmol, 1.3 eq) in DCM (15 mL) was
added BF.sub.3.Et.sub.2O (2.45 g, 17.26 mmol, 2.13 mL, 5 eq) at
0.degree. C. The mixture was stirred at 0.degree. C. for 3 hr. The
mixture was stirred at 25.degree. C. for 12 hr. TLC indicated new
spots formed. The reaction mixture was concentrated under reduced
pressure to give a residue. The residue was purified by column
chromatography (SiO.sub.2, Petroleum ether/Ethyl acetate=10:1 to
1:1) to give
[(2S,3S,4R,5R)-4,5-diacetoxy-6-(4-bromobutoxy)-2-methyl-tetrahydropyran-3-
-yl]acetate (300 mg, 705 umol, 20% yield) as a yellow oil.
[0472] Step 5
[0473] To a solution of
[(2S,3S,4R,5R)-4,5-diacetoxy-6-(4-bromobutoxy)-2-methyl-tetrahydropyran-3-
-yl] acetate (250 mg, 587.9 umol, 1 eq) in acetone (5 mL) was added
N,N-dimethylprop-2-yn-1-amine (146.61 mg, 1.76 mmol, 187.00 uL, 3
eq) at 25.degree. C. The mixture was stirred at 90.degree. C. for 4
hr. LCMS showed the desired compound was detected. The reaction
mixture was concentrated under reduced pressure to give a residue.
The residue was purified by prep-HPLC (column: Luna C18 100*30 5u;
mobile phase: [water (water (0.1% TFA)-ACN]; B %: 10%-35%, 12 min)
to give the title compound (47 mg, 90.6 umol, 15.4% yield, mixture
of anomers) as a yellow solid. LCMS (M.sup.+): 428.2 .sup.1H NMR
(400 MHz, DMSO-d.sub.6) .delta. 5.12-5.03 (m, 2H), 4.87 (t, J=9.8
Hz, 1H), 4.79 (d, J=1.6 Hz, 1H), 4.34 (d, J=2.6 Hz, 2H), 4.01 (t,
J=2.5 Hz, 1H), 3.86-3.75 (m, 1H), 3.70-3.60 (m, 1H), 3.50-3.36 (m,
1H), 3.39-3.31 (m, 2H), 3.06 (s, 6H), 2.09 (s, 3H), 2.02 (s, 3H),
1.92 (s, 3H), 1.79-1.68 (m, 2H), 1.64-1.46 (m, 2H), 1.11 (d, J=6.2
Hz, 3H).
##STR00146##
Compound 37 and Compound 37:
N,N-dimethyl-N-(prop-2-yn-1-yl)-4-(((3S,4R,5R,6S)-3,4,5-triacetoxy-6-meth-
yltetrahydro-2H-pyran-2-yl)oxy)butan-1-aminium bromide
[0474] This compound may be synthesized according to the
experimental procedure described for Compound 36.
##STR00147##
Compound 38:
N,N-dimethyl-4-oxo-N-(prop-2-yn-1-yl)-4-(((3R,4S,5R,6R)-3,4,5-tris(butyry-
loxy)-6-((butyryloxy)methyl)tetrahydro-2H-pyran-2-yl)oxy)butan-1-aminium
bromide
[0475] Step 1:
[0476] To a mixture of
[(2R,3R,4S,5R)-3,4,5-tri(butanoyloxy)-6-hydroxy-tetrahydropyran-2-yl]meth-
yl butanoate (1 g, 2.17 mmol, 1 eq) in DCM (10 mL) was added
4-bromobutanoyl chloride (1.21 g, 6.51 mmol, 755.05 uL, 3 eq) and
Pyridine (858.82 mg, 10.86 mmol, 876.35 uL, 5 eq) in one portion at
20.degree. C. under N.sub.2. The mixture was stirred at 20.degree.
C. for 12 h. TLC showed
indicated[(2R,3R,4S,5R)-3,4,5-tri(butanoyloxy)-6-hydroxy-tetrahydropyran--
2-yl]methyl butanoate was consumed completely. The reaction mixture
was filtered and concentrated under reduced pressure to give a
residue. The residue was purified by column chromatography
(SiO.sub.2, Petroleum ether/Ethyl acetate=10/1 to 5:1). Compound
[(3R,4S,5R,6R)-3,4,5-tri(butanoyloxy)-6-(butanoyloxymethyl)tetrahydropyra-
n-2-yl]4-bromobutanoate (1.5 g, crude) was obtained as colorless
oil.
[0477] Step 2:
[0478] To a mixture of
[(3R,4S,5R,6R)-3,4,5-tri(butanoyloxy)-6-(butanoyloxymethyl)tetrahydropyra-
n-2-yl] 4-bromobutanoate (0.2 g, 328.14 umol, 1 eq) and
N,N-dimethylprop-2-yn-1-amine (81.84 mg, 984.41 umol, 104.38 uL, 3
eq) in Acetone (3 mL) was stirred at 90.degree. C. for 2 h. LCMS
showed
[(3R,4S,5R,6R)-3,4,5-tri(butanoyloxy)-6-(butanoyloxymethyl)tetrahydropyra-
n-2-yl]4-bromobutanoate (0.2 g, 328.14 umol, 1 eq) was consumed
completely. The reaction mixture was filtered and concentrated
under reduced pressure to give a residue. The residue was purified
by prep-HPLC [water (0.1% TFA)-ACN]. The title compound (0.028 g,
40.4 .mu.mol, 12.3% yield, mixture of anomers, Br) was obtained as
a colorless oil. LCMS (M.sup.+): 612.3 .sup.1H NMR (400 MHz,
DMSO-d.sub.6) .delta. 6.28-5.95 (m, 1H), 5.56-5.34 (m, 1H),
5.20-4.94 (m, 2H), 4.44-4.35 (m, 2H), 4.30-4.12 (m, 2H), 4.10-3.98
(m, 2H), 3.45-3.30 (m, 2H), 3.14-3.06 (m, 6H), 2.78-2.40 (m, 2H),
2.34-2.13 (m, 8H), 2.06-1.88 (m, 2H), 1.61-1.40 (m, 8H), 0.93-0.79
(m, 12H).
##STR00148##
Compound 39:
N,N-dimethyl-4-oxo-N-(prop-2-yn-1-yl)-4-(((3R,4S,5R)-3,4,5-tris(butyrylox-
y)tetrahydro-2H-pyran-2-yl)oxy)butan-1-aminium bromide
[0479] This compound may be synthesized according to the
experimental procedure described for Compound 38.
##STR00149##
Compound 40 and Compound 68:
N,N-dimethyl-4-oxo-N-(prop-2-yn-1-yl)-4-(((3R,4R,5R)-3,4,5-tris(butyrylox-
y)tetrahydro-2H-pyran-2-yl)oxy)butan-1-aminium bromide
[0480] This compound may be synthesized according to the
experimental procedure described for Compound 38.
##STR00150##
Compound 41A:
N,N-dimethyl-4-oxo-N-(prop-2-yn-1-yl)-4-(((2R,3R,4S,5S)-3,4,5-tris(butyry-
loxy)tetrahydro-2H-pyran-2-yl)oxy)butan-1-aminium bromide
[0481] Step 1
[0482]
[(3S,4S,5R)-4,5-di(butanoyloxy)-6-hydroxy-tetrahydropyran-3-yl]
butanoate (500 mg, 1.39 mmol, 1 eq) and 4-bromobutanoyl chloride
(771.84 mg, 4.16 mmol, 482.40 uL, 3 eq) was dissolved with DCM (5
mL). Then Pyridine (548.70 mg, 6.94 mmol, 559.90 uL, 5 eq) was
added into the mixture and stirred for 12 h at 25.degree. C. TLC
showed the starting reactant was consumed. The reaction mixture was
concentrated.
[(3R,4S,5S)-3,4,5-tri(butanoyloxy)tetrahydropyran-2-yl]
4-bromobutanoate (0.5 g, crude) was obtained as colorless oil.
[0483] Step 2
[0484] To a solution of
[(3R,4S,5S)-3,4,5-tri(butanoyloxy)tetrahydropyran-2-yl]
4-bromobutanoate (500 mg, 981.58 umol, 1 eq) in Acetone (5 mL) was
added N,N-dimethylprop-2-yn-1-amine (244.80 mg, 2.94 mmol, 312.24
uL, 3 eq) and stirred at 90.degree. C. for 2 h. LCMS showed the
product formed. The mixture was concentrated. The residue was
purified by prep-HPLC (column: Luna C18 100*30 Su; mobile phase:
[water (0.1% TFA)-ACN]; B %: 25%-50%, 12 min). The title compound
(67 mg, 107 umol, 11% yield, Br) as a yellow oil was obtained. The
anomer (Compound 42) was also isolated. LCMS (M.sup.+): 512.3
.sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. 6.23 (d, J=3.6 Hz, 1H),
5.41-5.35 (m, 1H), 5.32 (dd, J=10.7, 3.4 Hz, 1H), 5.18 (dd, J=10.7,
3.6 Hz, 1H), 4.39 (d, J=2.6 Hz, 2H), 4.17-4.06 (m, 2H), 3.80 (dd,
J=13.4, 2.0 Hz, 1H), 3.41-3.34 (m, 2H), 3.10 (s, 6H), 2.70-2.58 (m,
2H), 2.43-2.34 (m, 2H), 2.34-2.14 (m, 4H), 2.05-1.90 (m, 2H),
1.66-1.44 (m, 6H), 0.97-0.81 (m, 9H).
##STR00151##
Compound 41B and Compound 65:
N,N-dimethyl-4-oxo-N-(prop-2-yn-1-yl)-4-(((2S,3R,4S,5S)-3,4,5-tris(butyry-
loxy)tetrahydro-2H-pyran-2-yl)oxy)butan-1-aminium bromide
[0485] This compound was synthesized according to the experimental
procedure described for Compound 41. The title compound (30 mg,
48.1 .mu.mol, 4.9% yield, Br) was obtained as a yellow oil. LCMS
(M.sup.+): 512.3 .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. 5.81
(d, J=7.8 Hz, 1H), 5.40-5.28 (m, 1H), 5.27-5.08 (m, 2H), 4.43-4.35
(m, 2H), 4.15-3.84 (m, 4H), 3.40-3.31 (m, 2H), 3.09 (s, 6H),
2.49-2.14 (m, 8H), 1.97-1.90 (m, 2H), 1.66-1.42 (m, 6H), 0.93 (t,
J=7.4 Hz, 3H), 0.92-0.80 (m, 6H).
##STR00152##
Compound 42:
N,N-dimethyl-4-oxo-N-(prop-2-yn-1-yl)-4-(((3R,4R,5S,6S)-3,4,5-tris(butyry-
loxy)-6-methyltetrahydro-2H-pyran-2-yl)oxy)butan-1-aminium
bromide
[0486] This compound may be synthesized according to the
experimental procedure described for Compound 38.
##STR00153##
Compound 43:
N,N-dimethyl-4-oxo-N-(prop-2-yn-1-yl)-4-(((2S,3S,4R,5R,6S)-3,4,5-tris(but-
yryloxy)-6-methyltetrahydro-2H-pyran-2-yl)oxy)butan-1-aminium
bromide
[0487] Step 1:
[0488] (3S,4R,5S,6S)-6-methyltetrahydro-2H-pyran-2,3,4,5-tetraol (5
g, 30.46 mmol, 1 eq) was dissolved in Pyridine (50 mL) and butanoyl
butanoate (28.91 g, 182.75 mmol, 29.90 mL, 6 eq) was added. The
mixture was stirred at 25.degree. C. for 12 h. TLC indicated the
starting material was consumed completely. The reaction mixture was
concentrated under reduced pressure to give a residue.
(3S,4R,5R,6S)-6-methyltetrahydro-2H-pyran-2,3,4,5-tetrayl
tetrabutyrate (12.8 g, crude) was obtained as yellow oil.
[0489] Step 2:
[0490] To a solution of
(3S,4R,5R,6S)-6-methyltetrahydro-2H-pyran-2,3,4,5-tetrayl
tetrabutyrate (12.5 g, 28.12 mmol, 1 eq) in THF (100 mL) was added
MeNH.sub.2 aq. solution (5.24 g, 50.62 mmol, 30% purity, 1.8 eq).
The mixture was stirred at 25.degree. C. for 12 h. TLC indicated
the starting material was consumed completely. The reaction mixture
was concentrated under reduced pressure to give a residue as
colorless oil. The residue was purified by column chromatography
(SiO.sub.2, Petroleum ether/Ethyl acetate=10/1 to 3:1).
(3S,4R,5R,6S)-2-hydroxy-6-methyltetrahydro-2H-pyran-3,4,5-triyl
tributyrate (4.0 g, crude) was obtained as a colorless oil.
[0491] Step 3:
[0492] To a solution of
(3S,4R,5R,6S)-2-hydroxy-6-methyltetrahydro-2H-pyran-3,4,5-triyl
tributyrate (1 g, 2.67 mmol, 1 eq) in DCM (10 mL) was added
4-bromobutanoyl chloride (1.49 g, 8.01 mmol, 928.66 uL, 3 eq) and
Pyridine (1.06 g, 13.35 mmol, 1.08 mL, 5 eq). The mixture was
stirred at 25.degree. C. for 12 h. TLC indicated the reaction was
completed. The reaction mixture was concentrated under reduced
pressure to give a residue as yellow oil. The residue was purified
by column chromatography (SiO.sub.2, Petroleum ether/Ethyl
acetate=15:1 to 5:1).
(3S,4R,5R,6S)-2-((4-bromobutanoyl)oxy)-6-methyltetrahydro-2H-pyran-3,4,5--
triyl tributyrate (600 mg, crude) was obtained as a yellow oil.
[0493] Step 4:
[0494] To a solution of
(3S,4R,5R,6S)-2-((4-bromobutanoyl)oxy)-6-methyltetrahydro-2H-pyran-3,4,5--
triyl tributyrate (0.3 g, 573.16 umol, 1 eq) in acetone (10 mL) was
added N,N-dimethylprop-2-yn-1-amine (142.94 mg, 1.72 mmol, 182.33
uL, 3 eq). The mixture was stirred at 90.degree. C. for 2 h. LCMS
showed the desired compound was detected. The reaction mixture was
concentrated under reduced pressure to give a residue as brown
liquid. The residue was purified by prep-HPLC (column: Xbridge
150*30 mm*10 um; mobile phase: [water (0.1% TFA)-ACN]) to give the
crude product (0.12 g, 227.86 umol, 39.75% yield) as a brown
liquid. Then the crude product was purified twice by prep-HPLC
(column: Luna C18 100*30 5u; mobile phase: [water (0.1% TFA)-ACN])
to afford the title compound (9.5 mg, 16 .mu.mol, 2.7% yield) and
the anomer (Compound 45) as yellow oils. LCMS (M.sup.+): 526.3
.sup.1H NMR (400 MHz, Chloroform-d) .delta. 5.60 (d, J=8.3 Hz, 1H),
5.28-5.18 (m, 2H), 5.05 (dd, J=10.4, 3.5 Hz, 1H), 4.43 (s, 2H),
3.90 (q, J=6.4 Hz, 1H), 3.57-3.53 (m, 2H), 3.25 (s, 6H), 2.81 (s,
1H), 2.52-2.46 (m, 2H), 2.36 (td, J=7.3, 2.2 Hz, 2H), 2.23-2.09 (m,
4H), 2.02 (s, 2H), 1.71-1.59 (m, 2H), 1.57-1.44 (m, 4H), 1.15 (d,
J=6.4 Hz, 3H), 0.93 (t, J=7.4 Hz, 3H), 0.84 (t, J=7.4 Hz, 6H).
##STR00154##
Compound 44:
N,N-dimethyl-4-oxo-N-(prop-2-yn-1-yl)-4-(((2R,3S,4R,5R,6S)-3,4,5-tris(but-
yryloxy)-6-methyltetrahydro-2H-pyran-2-yl)oxy)butan-1-aminium
bromide
[0495] This compound was synthesized according to the experimental
procedure described for Compound 44. The title compound (19 mg, 31
.mu.mol, 5.5% yield) was isolated as a yellow oil. LCMS (M.sup.+):
526.3 .sup.1H NMR (400 MHz, Chloroform-d) .delta. 6.38 (s, 1H),
5.41-5.35 (m, 3H), 4.54-4.50 (m, 2H), 4.33-4.26 (m, 1H), 3.75-3.71
(m, 2H), 3.35 (s, 6H), 2.89 (s, 1H), 2.67-2.62 (m, 2H), 2.44 (t,
J=7.3 Hz, 2H), 2.21 (dd, J=24.3, 16.5 Hz, 6H), 1.80-1.66 (m, 2H),
1.68-1.57 (m, 4H), 1.18 (d, J=6.1 Hz, 3H), 1.02 (t, J=7.4 Hz, 3H),
0.93 (t, J=7.3 Hz, 6H).
##STR00155##
Compound 45:
N,N-dimethyl-4-oxo-N-(prop-2-yn-1-yl)-4-(((3R,4S,5R,6R)-3,4,5-triacetoxy--
6-(acetoxymethyl)tetrahydro-2H-pyran-2-yl)oxy)butan-1-aminium
bromide
[0496] Step 1:
[0497] To a mixture of
[(2R,3R,4S,5R)-3,4,5-triacetoxy-6-hydroxy-tetrahydropyran-2-yl]methyl
acetate (2 g, 5.74 mmol, 1 eq) and Pyridine (2.27 g, 28.71 mmol,
2.32 mL, 5 eq) in DCM (10 mL) was added 4-bromobutanoyl chloride
(3.19 g, 17.23 mmol, 2.00 mL, 3 eq) in one portion at 25.degree. C.
under N.sub.2. The mixture was stirred at 25.degree. C. for 12
hours. TLC indicated SM 1 was consumed completely and one new spot
formed. The reaction mixture was filtered and concentrated under
reduced pressure to give a residue. The residue was purified by
column chromatography (SiO.sub.2, Petroleum ether/Ethyl acetate=5/1
to 2:1). Desired compound
[(3R,4S,5R,6R)-3,4,5-triacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]
4-bromobutanoate (1.5 g, crude) was obtained as colorless oil.
[0498] Step 2:
[0499] To a mixture of
[(3R,4S,5R,6R)-3,4,5-triacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]
4-bromobutanoate (0.2 g, 402.18 umol, 1 eq) and
N,N-dimethylprop-2-yn-1-amine (100.30 mg, 1.21 mmol, 127.94 uL, 3
eq) in Acetone (3 mL) was stirred at 90.degree. C. for 2 hours.
LC-MS showed the reaction was completed. The reaction mixture was
filtered and concentrated under reduced pressure to give a residue.
The residue was purified by prep-HPLC [water (0.1% TFA)-ACN]. The
title compound (0.039 g, 67.2 umol, 16.7% yield, mixture of
anomers, Br) was obtained as colorless oil. LCMS (M.sup.+): 500.2
.sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. 6.30-5.92 (m, 1H),
5.52-5.21 (m, 1H), 5.17-4.90 (m, 2H), 4.43-4.36 (m, 2H), 4.28-4.10
(m, 2H), 4.09-3.97 (m, 2H), 3.45-3.32 (m, 2H), 3.14-3.07 (m, 6H),
2.75-2.43 (m, 2H), 2.08-1.89 (m, 14H).
##STR00156##
Compound 46 and Compound 53:
N,N-dimethyl-4-oxo-N-(prop-2-yn-1-yl)-4-(((3R,4S,5R)-3,4,5-triacetoxytetr-
ahydro-2H-pyran-2-yl)oxy)butan-1-aminium bromide
[0500] This compound may be synthesized according to the
experimental procedure described for Compound 45.
##STR00157##
Compound 47:
N,N-dimethyl-4-oxo-N-(prop-2-yn-1-yl)-4-(((3R,4R,5R)-3,4,5-triacetoxytetr-
ahydro-2H-pyran-2-yl)oxy)butan-1-aminium bromide
[0501] This compound may be synthesized according to the
experimental procedure described for Compound 45.
##STR00158##
Compound 48 and Compound 64:
N,N-dimethyl-4-oxo-N-(prop-2-yn-1-yl)-4-(((3R,4S,5S)-3,4,5-triacetoxytetr-
ahydro-2H-pyran-2-yl)oxy)butan-1-aminium bromide
[0502] Step 1
[0503] [(3S,4S,5R)-4,5-diacetoxy-6-hydroxy-tetrahydropyran-3-yl]
acetate (200 mg, 724.01 umol, 1 eq) and 4-bromobutanoyl chloride
(402.80 mg, 2.17 mmol, 251.75 uL, 3 eq) was dissolved with DCM (2
mL). Then Pyridine (286.35 mg, 3.62 mmol, 292.19 uL, 5 eq) was
added into the mixture and stirred for 12 h at 25.degree. C. TLC
showed the starting reactant was consumed. The reaction mixture was
concentrated. The residue was purified by column chromatography
(SiO.sub.2, Petroleum ether/Ethyl acetate=10/1).
[(3R,4S,5S)-3,4,5-triacetoxytetrahydropyran-2-yl] 4-bromobutanoate
(200 mg, 400 umol, 55% yield, 85% purity) was obtained as a yellow
oil.
[0504] Step 2
[0505] To a solution of
[(3R,4S,5S)-3,4,5-triacetoxytetrahydropyran-2-yl] 4-bromobutanoate
(200 mg, 470.34 umol, 1 eq) in acetone (5 mL) was added
N,N-dimethylprop-2-yn-1-amine (117.30 mg, 1.41 mmol, 149.62 uL, 3
eq) and stirred at 90.degree. C. for 2 h. LCMS showed the product
formed and the starting reactant was consumed. The mixture reaction
was concentrated. The residue was purified by prep-HPLC (column:
Luna C18 100*30 5u; mobile phase: [water (0.1% TFA)-ACN]; B %:
1%-30%, 12 min). The title compound (30 mg, 63.0 .mu.mol, 13.4%
yield, mixture of anomers) was obtained as a colorless oil. LCMS
(M.sup.+): 428.1 .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta.
6.26-5.75 (m, 1H), 5.37-5.23 (m, 2H), 5.22-5.04 (m, 1H), 4.42-4.35
(m, 2H), 4.16-3.77 (m, 3H), 3.43-3.30 (m, 2H), 3.12-3.06 (m, 6H),
2.66-2.57 (m, 2H), 2.19-1.94 (m, 11H).
##STR00159##
Compound 49:
N,N-dimethyl-4-oxo-N-(prop-2-yn-1-yl)-4-(((3R,4R,5S,6S)-3,4,5-triacetoxy--
6-methyltetrahydro-2H-pyran-2-yl)oxy)butan-1-aminium bromide
[0506] Step 1
[0507] To a solution of
[(2S,3S,4R,5R)-4,5-diacetoxy-6-hydroxy-2-methyl-tetrahydropyran-3-yl]
acetate (1 g, 3.45 mmol, 1 eq) in DCM (10 mL) was added pyridine
(1.36 g, 17.23 mmol, 1.39 mL, 5 eq) and 4-bromobutanoyl chloride
(1.92 g, 10.34 mmol, 1.20 mL, 3 eq) at 25.degree. C. The mixture
was stirred at 25.degree. C. for 12 hr. TLC indicated new spots
formed. The reaction mixture was concentrated under reduced
pressure to give a residue. The residue was purified by column
chromatography (SiO.sub.2, Petroleum ether/Ethyl acetate=15:1 to
5:1) to give
[(3R,4R,5S,6S)-3,4,5-triacetoxy-6-methyl-tetrahydropyran-2-yl]
4-bromobutanoate (1.2 g, 2.73 mmol, 79.3% yield) as a yellow
oil.
[0508] Step 2
[0509] To a solution of
[(3R,4R,5S,6S)-3,4,5-triacetoxy-6-methyl-tetrahydropyran-2-yl]
4-bromobutanoate (200 mg, 455.32 umol, 1 eq) in acetone (5 mL) was
added N,N-dimethylprop-2-yn-1-amine (113.55 mg, 1.37 mmol, 144.84
uL, 3 eq) at 25.degree. C. The mixture was stirred at 90.degree. C.
for 4 hr. LCMS showed the desired compound was detected. The
reaction mixture was concentrated under reduced pressure to give a
residue. The residue was purified by prep-HPLC (column: Luna C18
100*30 5u; mobile phase: [water (0.1% TFA)-ACN]; B %: 5%-35%, 12
min) to afford the title compound (71 mg, 132 .mu.mol, 30.0% yield,
mixture of anomers) as a yellow solid. LCMS (M.sup.+): 442.2
.sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. 5.97-5.74 (m, 1H),
5.18-4.71 (m, 3H), 4.39-4.32 (m, 2H), 4.07-3.88 (m, 2H), 3.40-3.29
(m, 2H), 3.10-3.03 (m, 6H), 2.67-2.50 (m, 2H), 2.19-1.86 (m, 11H),
1.13-0.98 (m, 3H).
##STR00160##
Compound 50:
N,N-dimethyl-4-oxo-N-(prop-2-yn-1-yl)-4-(((3S,4R,5R,6S)-3,4,5-triacetoxy--
6-methyltetrahydro-2H-pyran-2-yl)oxy)butan-1-aminium bromide
[0510] Step 1
[0511] (3S,4R,5S,6S)-6-methyltetrahydropyran-2,3,4,5-tetrol (5 g,
30.46 mmol, 1 eq) was dissolved with Pyridine (50 mL) and acetyl
acetate (18.66 g, 182.76 mmol, 17.12 mL, 6 eq) was added to the
mixture. The mixture was stirred at 25.degree. C. for 12 h. TLC
showed the reaction was completed. The mixture was concentrated.
[(2S,3R,4R,5S)-4,5,6-triacetoxy-2-methyl-tetrahydropyran-3-yl]
acetate (10 g, crude) was obtained as yellow oil.
[0512] Step 2
[0513] To a solution of
[(2S,3R,4R,5S)-4,5,6-triacetoxy-2-methyl-tetrahydropyran-3-yl]
acetate (10 g, 30.09 mmol, 1 eq) in THF (50 mL) was added
MeNH.sub.2 aq. solution (5.61 g, 54.17 mmol, 30% purity, 1.8 eq)
and stirred at 25.degree. C. for 12 h. TLC showed the starting
reactant consumed. The mixture reaction was concentrated. The
residue was purified by column chromatography (SiO.sub.2, Petroleum
ether/Ethyl acetate=10/1 to 5:1).
[(2S,3R,4R,5S)-4,5-diacetoxy-6-hydroxy-2-methyl-tetrahydropyran-3-yl]
acetate (6 g, 18.60 mmol, 61.8% yield, 90% purity) was obtained as
yellow oil.
[0514] Step 3
[0515] To a solution of
[(2S,3R,4R,5S)-4,5-diacetoxy-6-hydroxy-2-methyl-tetrahydropyran-3-yl]
acetate (1 g, 3.45 mmol, 1 eq) and 4-bromobutanoyl chloride (1.66
g, 8.96 mmol, 1.04 mL, 2.6 eq) in DCM (20 mL) was added Pyridine
(1.36 g, 17.23 mmol, 1.39 mL, 5 eq). The mixture was stirred at
25.degree. C. for 12 h. TLC showed the starting reactant consumed.
The mixture was concentrated. The residue was purified by flash
silica gel chromatography (PE:EA=5:1).
[(3S,4R,5R,6S)-3,4,5-triacetoxy-6-methyl-tetrahydropyran-2-yl]
4-bromobutanoate (1.93 g, crude) was obtained as yellow oil.
[0516] Step 4
[0517] To a solution of
[(3S,4R,5R,6S)-3,4,5-triacetoxy-6-methyl-tetrahydropyran-2-yl]
4-bromobutanoate (1.83 g, 4.17 mmol, 1 eq) in acetone (20 mL) was
added N,N-dimethylprop-2-yn-1-amine (1.04 g, 12.50 mmol, 1.33 mL, 3
eq). The mixture was stirred at 90.degree. C. for 12 h. LCMS showed
the starting reactant was consumed. The mixture was concentrated.
The residue was purified by prep-HPLC (column: Xbridge 150*30 mm*10
um; mobile phase: [water (0.1% TFA)-ACN]; B %: 1%-60%, 10 min). The
title compound (100.9 mg, 183.5 .mu.mol, 4.4% yield, mixture of
anomers, Br) was obtained as a colorless oil. LCMS (M.sup.+): 442.2
.sup.1H NMR (400 MHz, Chloroform-d) .delta. 6.4-6.3 (m, 1H),
5.4-5.3 (m, 3H), 4.6-4.4 (m, 2H), 4.3-4.2 (m, 1H), 3.8-3.6 (m, 2H),
3.34 (s, 6H), 2.9 (s, 1H), 2.7-2.5 (m, 2H), 2.2-2.0 (m, 11H), 1.16
(d, 3H).
##STR00161##
Compound 51:
dimethyl(prop-2-yn-1-yl)(2-{[(2R,3R,4S,5R)-3,4,5-tris(butanoyloxy)oxan-2--
yl]oxy}ethyl)azanium
[0518] This compound may be synthesized according to the
experimental procedure described for Compound 1.
##STR00162##
Compound 69:
N,N'-((((3R,3aR,6S,6aR)-hexahydrofuro[3,2-b]furan-3,6-diyl)bis(oxy))bis(2-
-oxoethane-2,1-diyl))bis(N,N-dimethylprop-2-yn-1-aminium)
bistrifluoroacetate
[0519] Step 1:
[0520] To a solution of
(3R,3aR,6S,6aR)-2,3,3a,5,6,6a-hexahydrofuro[3,2-b]furan-3,6-diol
(500 mg, 3.42 mmol, 1 eq) in CHCl3 (5 mL) was added pyridine (1.62
g, 20.53 mmol, 1.66 mL, 6 eq) and (2-chloroacetyl) 2-chloroacetate
(2.34 g, 13.69 mmol, 4 eq) at 0.degree. C. The mixture was stirred
at 15.degree. C. for 12 h. TLC indicated new spots formed. The
reaction mixture was quenched by addition aq. HCl (1M, 10 mL) and
extracted with ethyl acetate (20 mL*3). The combined organic layers
were washed with brine (20 mL), dried over anhydrous sodium
sulfate, filtered and concentrated under reduced pressure to give a
residue. The residue was purified by column chromatography
(SiO.sub.2, Petroleum ether/Ethyl acetate=20/1 to 5:1). Compound
[(3R,3aR,6S,6aR)-6-(2-chloroacetyl)oxy-2,3,3a,5,6,6a-hexahydrofu-
ro[3,2-b]furan-3-yl] 2-chloroacetate (750 mg, 2.51 mmol, 73.29%
yield) was obtained as a yellow oil.
[0521] Step 2:
[0522] To a solution of
[(3R,3aR,6S,6aR)-6-(2-chloroacetyl)oxy-2,3,3a,5,6,6a-hexahydrofuro[3,2-b]
furan-3-yl]2-chloroacetate (550 mg, 1.84 mmol, 1 eq) in ACN (3 mL)
was added N,N-dimethylprop-2-yn-1-amine (336.30 mg, 4.05 mmol,
428.95 uL, 2.2 eq) at 15.degree. C. The mixture was stirred at
80.degree. C. for 4 h. The reaction mixture was concentrated under
reduced pressure to give a residue. The residue was purified by
prep-HPLC (column: Xbridge 150*30 mm*10 um; mobile phase: [water
(0.1% TFA)-ACN]; B %:1%-20%, 10 min). The title compound (190 mg,
305.22 umol, 16.60% yield, 2CF.sub.3COO--)--) was obtained as a
colorless oil. LCMS: (M+/2): 197.1 .sup.1H NMR (400 MHz,
Methanol-d4) .delta. 5.39-5.33 (m, 2H), 4.93 (t, J=5.4 Hz, 1H),
4.62-4.45 (m, 8H), 4.16-3.88 (m, 4H), 3.64 (t, J=2.5 Hz, 2H), 3.38
(d, J=2.8 Hz, 12H).
##STR00163##
Compound 70:
N-(2-(((3S,3aR,6R,6aR)-6-hydroxyhexahydrofuro[3,2-b]furan-3-yl)oxy)-2-oxo-
ethyl)-N,N-dimethylprop-2-yn-1-aminium trifluoroacetate
[0523] Step 1:
[0524] To a solution of
(3R,3aR,6S,6aR)-2,3,3a,5,6,6a-hexahydrofuro[3,2-b]furan-3,6-diol
(300 mg, 2.05 mmol, 1 eq) in ACN (8 mL) was added K.sub.2CO.sub.3
(283.71 mg, 2.05 mmol, 1 eq) and 2-chloroacetyl chloride (231.85
mg, 2.05 mmol, 163.27 uL, 1 eq) at 0.degree. C. Then the mixture
was stirred at 15.degree. C. for 2 h under N.sub.2. TLC indicated
one major new spot was detected. Compound
[(3S,3aR,6R,6aR)-3-hydroxy-2,3,3a,5,6,6a-hexahydrofuro[3,2-b]furan-6-yl]
2-chloroacetate was obtained as liquid (.about.2.05 mmol, 8 mL in
ACN). The crude product was used into the next step without further
purification.
[0525] Step 2:
[0526] To a solution of
[(3S,3aR,6R,6aR)-3-hydroxy-2,3,3a,5,6,6a-hexahydrofuro[3,2-b]furan-6-yl]
2-chloroacetate (2.05 mmol, 8 mL in ACN, 1 eq) was added
N,N-dimethylprop-2-yn-1-amine (170.65 mg, 2.05 mmol, 217.67 uL, 1
eq). The mixture was stirred at 70.degree. C. for 10 h under
N.sub.2. LCMS showed expected mass was detected. The reaction
mixture was filtered and concentrated under reduced pressure to
give a residue. The residue was purified by prep-HPLC (column:
Xbridge 150*30 mm*10 um; mobile phase: [water (0.1% TFA)-ACN]; B %:
1%-10%, 10 min) to give
N-(2-(((3S,3aR,6R,6aR)-6-hydroxyhexahydrofuro[3,2-b]furan-3-yl)oxy)-2-oxo-
ethyl)-N,N-dimethylprop-2-yn-1-aminium trifluoroacetate (34 mg,
107.86 umol, 5.25% yield, CF.sub.3COO--) as colorless oil. LCMS:
(M+) 270.1 .sup.1H NMR (400 MHz, Methanol-d4) .delta. 5.33 (t,
J=2.3 Hz, 1H), 4.59 (t, J=2.7 Hz, 4H), 4.53 (s, 2H), 4.41-4.22 (m,
1H), 4.08 (d, J=2.3 Hz, 2H), 3.89 (dd, J=8.9, 6.3 Hz, 1H),
3.68-3.62 (m, 1H), 3.55 (dd, J=8.9, 7.0 Hz, 1H), 3.41 (s, 6H).
##STR00164##
Compound 71:
N,N'-((((3R,3aR,6S,6aR)-hexahydrofuro[3,2-b]furan-3,6-diyl)bis(oxy))bis(e-
thane-2,1-diyl))bis(N,N-dimethylprop-2-yn-1-aminium)
bistrifluoroacetate
[0527] Step 1:
[0528] To a solution of
(3R,3aR,6S,6aR)-2,3,3a,5,6,6a-hexahydrofuro[3,2-b]furan-3,6-diol (4
g, 27.37 mmol, 1 eq) in DMF (40 mL) was added NaH (3.28 g, 82.11
mmol, 60% purity, 3 eq) at 0.degree. C. for 0.5 h. Then to the
mixture was added 2-bromoethoxy-tert-butyl-dimethyl-silane (19.64
g, 82.11 mmol, 3 eq). The mixture was stirred at 15.degree. C. for
11.5 h. TLC showed the starting reactant was consumed and three new
spots formed. The reaction mixture was quenched by addition of
H.sub.2O (30 mL) at 0.degree. C., and extracted with EtOAc (30
mL*3). Then the organic layer was dried over Na.sub.2SO.sub.4 and
concentrated under reduced pressure. The residue was purified by
column chromatography (SiO.sub.2, Petroleum ether/Ethyl
acetate=50:1 to 20:1). Compound
2-[[(3R,3aR,6S,6aR)-3-[2-[tert-butyl(dimethyl)silyl]oxyethoxy]-2,3,3a,5,6-
,6a-hexahydrofuro[3,2-b]furan-6-yl]oxy]ethoxy-tert-butyl-dimethyl-silane
(3.35 g, 7.24 mmol, 26.45% yield) was obtained as yellow oil.
[0529] Step 2:
[0530] To a solution of
2-[[(3R,3aR,6S,6aR)-3-[2-[tert-butyl(dimethyl)silyl]oxyethoxy]-2,3,3a,5,6-
,6a-hexahydrofuro[3,2-b]furan-6-yl]oxy]ethoxy-tert-butyl-dimethyl-silane
(2.3 g, 4.97 mmol, 1 eq) in THF (20 mL) was added
pyridine.hydrofluoride (2.81 g, 19.88 mmol, 2.56 mL, 70%, 4 eq).
The mixture was stirred at 15.degree. C. for 12 h. TLC showed the
starting reactant was consumed and one new spot formed. The mixture
was concentrated under reduced pressure. The residue was purified
by column chromatography (SiO.sub.2, Petroleum Ether:Ethyl
Aacetate=0:1 to EA:EtOH=20:1). Compound
2-[[(3R,3aR,6S,6aR)-6-(2-hydroxyethoxy)-2,3,3a,5,6,6a-hexahydrofuro[3,2-b-
]furan-3-yl]oxy]ethanol (800 mg, 3.42 mmol, 68.72% yield) was
obtained as yellow oil.
[0531] Step 3:
[0532] To a solution of
2-[[(3R,3aR,6S,6aR)-6-(2-hydroxyethoxy)-2,3,3a,5,6,6a-hexahydrofuro[3,2-b-
] furan-3-yl]oxy]ethanol (1 g, 4.27 mmol, 1 eq) in THF (10 mL) was
added imidazole (1.16 g, 17.08 mmol, 4 eq) and PPh3 (4.48 g, 17.08
mmol, 4 eq). Then to the mixture was added iodine (4.33 g, 17.08
mmol, 3.44 mL, 4 eq). The mixture was stirred at 15.degree. C. for
2 h. LCMS showed the starting reactant was consumed. The reaction
mixture was quenched by addition 15% aq. Na.sub.2S2O.sub.4 (10 mL)
at 0.degree. C., and extracted with EtOAc (10 mL*3). Then the
organic layer was dried over Na.sub.2SO.sub.4 and concentrated
reduced pressure. The residue was purified by column chromatography
(SiO.sub.2, Petroleum ether/Ethyl acetate=50/1 to 20:1). Compound
(3R,3aR,6S,6aR)-3,6-bis(2-iodoethoxy)-2,3,3a,5,6,6a-hexahydrofuro[3,2-b]f-
uran (1.2 g, 2.64 mmol, 61.91% yield) was obtained as yellow
oil.
[0533] Step 4:
[0534] To a solution of N,N-dimethylprop-2-yn-1-amine (1.10 g,
13.21 mmol, 1.40 mL, 6 eq) in acetone (10 mL) was added
(3R,3aR,6S,6aR)-3,6-bis(2-iodoethoxy)-2,3,3a,5,6,6a-hexahydrofuro[3,2-b]
furan (1 g, 2.20 mmol, 1 eq). The mixture was stirred at 90.degree.
C. for 4 h. LCMS showed the starting reactant was consumed. The
mixture was concentrated under reduced pressure. The residue was
purified by prep-HPLC (column: Xbridge 150*30 mm*10 um; mobile
phase: [water (0.1% TFA)-ACN]; B %: 1%-60%, 10 min).
N,N'-((((3R,3aR,6S,6aR)-hexahydrofuro[3,2-b]furan-3,6-diyl)bis(oxy))bis(e-
thane-2,1-diyl))bis(N,N-dimethylprop-2-yn-1-aminium)
bistrifluoroacetate (470 mg, 793.22 umol, 36.02% yield, 100%
purity, 2CF3CO2-) was obtained as colorless oil. LCMS:
(M.sup.2+/2): 183.2 .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta.
4.63 (t, J=4.6 Hz, 1H), 4.51 (d, J=4.5 Hz, 1H), 4.39 (dd, J=11.9,
2.5 Hz, 4H), 4.13-4.01 (m, 4H), 4.01-3.83 (m, 5H), 3.81 (dd, J=8.9,
6.5 Hz, 1H), 3.72 (dd, J=10.3, 3.6 Hz, 1H), 3.68-3.51 (m, 4H), 3.45
(dd, J=8.9, 7.1 Hz, 1H), 3.14-3.07 (m, 12H).
##STR00165##
Compound 72: 343:
N-(2-(((3R,3aR,6S,6aR)-6-hydroxyhexahydrofuro[3,2-b]furan-3-yl)oxy)ethyl)-
-N,N-dimethylprop-2-yn-1-aminium trifluoroacetate
[0535] Step 1:
[0536] To a solution of
(3R,3aR,6S,6aR)-6-benzyloxy-2,3,3a,5,6,6a-hexahydrofuro[3,2-b]furan-3-ol
(1.9 g, 8.04 mmol, 1 eq) in DMF (30 mL) was added NaH (482.47 mg,
12.06 mmol, 60% purity, 1.5 eq) at 0.degree. C. and stirred for 0.5
h. Then 2-bromoethoxy-tert-butyl-dimethyl-silane (2.89 g, 12.06
mmol, 1.5 eq) was added to the mixture and stirred at 15.degree. C.
for 9.5 h. TLC indicated three new spots formed. The reaction
mixture was quenched by addition of H.sub.2O (40 mL) at 0.degree.
C., and then diluted with EtOAc (40 mL) and extracted with EtOAc
(40 mL*3). The combined organic layers were dried over
Na.sub.2SO.sub.4, filtered and the filtrate was concentrated under
reduced pressure to give a residue. The residue was purified by
column chromatography (SiO.sub.2, Petroleum ether/Ethyl
acetate=20/1 to 0:1) to give
2-[[(3R,3aR,6S,6aR)-6-benzyloxy-2,3,3a,5,6,6a-hexahydrofuro[3,2-b]
furan-3-yl]oxy]ethoxy-tert-butyl-dimethyl-silane (1.9 g, 4.82 mmol,
59.88% yield) as yellow oil.
[0537] Step 2:
[0538] To a solution of
2-[[(3R,3aR,6S,6aR)-6-benzyloxy-2,3,3a,5,6,6a-hexahydrofuro[3,2-b]
furan-3-yl]oxy]ethoxy-tert-butyl-dimethyl-silane (1.9 g, 4.82 mmol,
1 eq) in THF (20 mL) was added pyridine; hydrofluoride (2.05 g,
14.45 mmol, 1.86 mL, 70% purity, 3 eq). The mixture was stirred at
15.degree. C. for 10 hr under N.sub.2. TLC indicated one new spot
formed. The reaction mixture was filtered and the filtrate was
concentrated under reduced pressure to give a residue. The residue
was purified by column chromatography (SiO.sub.2, Petroleum
ether/Ethyl acetate=10/1 to 0/1) to give
2-[[(3R,3aR,6S,6aR)-6-benzyloxy-2,3,3a,5,6,6a-hexahydrofuro[3,2-b]fu-
ran-3-yl]oxy]ethanol (1.1 g, 3.92 mmol, 81.49% yield) as yellow
oil.
[0539] Step 3:
[0540] To a solution of
2-[[(3R,3aR,6S,6aR)-6-benzyloxy-2,3,3a,5,6,6a-hexahydrofuro[3,2-b]
furan-3-yl]oxy]ethanol (1.1 g, 3.92 mmol, 1 eq) in THF (50 mL) was
added PPh3 (2.32 g, 8.83 mmol, 2.25 eq) and imidazole (801.44 mg,
11.77 mmol, 3 eq). Then 12 (2.49 g, 9.81 mmol, 1.98 mL, 2.5 eq) was
added to the mixture and stirred at 15.degree. C. for 2 hr. TLC
indicated one new spot was detected. The reaction mixture was
filtered and the filtrate was concentrated under reduced pressure
to give a residue. The residue was purified by column
chromatography (SiO.sub.2, Petroleum ether/Ethyl acetate=20/1 to
0/1) to give
(3R,3aR,6S,6aR)-6-benzyloxy-3-(2-iodoethoxy)-2,3,3a,5,6,6a-hexahydrofuro[-
3,2-b]furan (1.5 g, 3.84 mmol, 97.96% yield) as yellow oil.
[0541] Step 4:
[0542] A mixture of
(3R,3aR,6S,6aR)-6-benzyloxy-3-(2-iodoethoxy)-2,3,3a,5,6,6a-hexahydrofuro
[3,2-b]furan (1 g, 2.56 mmol, 1 eq) in THF (10 mL) was dropped to
dimethylamine (2 M, 10.00 mL, 7.80 eq, THF) and degassed and purged
with N.sub.2 3 times, and then the mixture was stirred at
15.degree. C. for 10 hr under N.sub.2 atmosphere. TLC indicated one
new spot was detected. LCMS showed desired compound was detected.
The reaction mixture was quenched by addition of H.sub.2O (10 mL)
at 15.degree. C., and then diluted with EtOAc (10 mL) and extracted
with EtOAc (10 mL*3). The combined organic layers were dried over
Na.sub.2SO.sub.4, filtered and the filtrate was concentrated under
reduced pressure to give
2-[[(3R,3aR,6S,6aR)-6-benzyloxy-2,3,3a,5,6,6a-hexahydrofuro[3,2-b]furan-3-
-yl]oxy]-N,N-dimethyl-ethanamine (0.8 g, crude) as yellow oil.
LCMS: (M+H+) 308.1
[0543] Step 5:
[0544] To a solution of
2-[[(3R,3aR,6S,6aR)-6-benzyloxy-2,3,3a,5,6,6a-hexahydrofuro[3,2-b]furan-3-
-yl]oxy]-N,N-dimethyl-ethanamine (0.9 g, 2.93 mmol, 1 eq) in EtOH
(100 mL) was added Pd(OH)2/C (0.9 g, 20% purity). The mixture was
stirred at 80.degree. C. for 12 hr under H2 (50 Psi). TLC indicated
one major new spot was detected. The reaction mixture was filtered
and the filtrate was concentrated under reduced pressure to give a
residue. The residue was purified by column chromatography
(SiO.sub.2, Petroleum ether/Ethyl acetate=5/1 to 0/1) to give
(3S,3aR,6R,6aR)-6-[2-(dimethylamino)ethoxy]-2,3,3a,5,6,6a-hexahydrofuro
[3,2-b]furan-3-ol (0.5 g, 2.30 mmol, 78.60% yield) as yellow
oil.
[0545] Step 6:
[0546] To a solution of
(3S,3aR,6R,6aR)-6-[2-(dimethylamino)ethoxy]-2,3,3a,5,6,6a-hexahydrofuro
[3,2-b]furan-3-ol (500 mg, 2.30 mmol, 1 eq) in ACN (10 mL) was
added 3-bromoprop-1-yne (301.15 mg, 2.53 mmol, 218.22 uL, 1.1 eq).
The mixture was stirred at 15.degree. C. for 5 hr under N.sub.2.
LCMS showed desired compound was detected. The reaction mixture was
filtered and the filtrate was concentrated under reduced pressure
to give a residue. The residue was purified by prep-HPLC (column:
Waters Xbridge Prep OBD C18 100*19 mm*5 um; mobile phase: [water
(0.1% TFA)-ACN]; B %: 1%-10%, 10 min) to
N-(2-(((3R,3aR,6S,6aR)-6-hydroxyhexahydrofuro[3,2-b]furan-3-yl)oxy)ethyl)-
-N,N-dimethylprop-2-yn-1-aminium trifluoroacetate (290 mg, 785.20
umol, 34.12% yield, CF.sub.3COO--) as colorless oil. LCMS: (M+)
256.1 1H NMR (400 MHz, Methanol-d4) .delta. 4.80 (t, J=4.4 Hz, 1H),
4.48 (d, J=2.6 Hz, 2H), 4.44 (dd, J=4.2, 1.2 Hz, 1H), 4.27-4.21 (m,
1H), 4.23-4.13 (m, 2H), 3.99-3.93 (m, 2H), 3.96-3.86 (m, 2H), 3.79
(ddd, J=14.2, 8.8, 2.4 Hz, 1H), 3.67 (ddd, J=14.2, 5.0, 2.2 Hz,
1H), 3.62-3.53 (m, 2H), 3.30 (d, J=1.8 Hz 6H).
##STR00166##
Compound 73:
N-(2-(((3R,3aR,6S,6aR)-6-hydroxyhexahydrofuro[3,2-b]furan-3-yl)oxy)-2-oxo-
ethyl)-N,N-dimethylprop-2-yn-1-aminium bromide
[0547] Step 1:
[0548] To a solution of
(3R,3aR,6S,6aR)-6-benzyloxy-2,3,3a,5,6,6a-hexahydrofuro[3,2-b]furan-3-ol
(500 mg, 2.12 mmol, 1 eq) in DCM (5 mL) was added TEA (428.29 mg,
4.23 mmol, 589.13 uL, 2 eq). Then 2-chloroacetyl chloride (262.92
mg, 2.33 mmol, 185.16 uL, 1.1 eq) was dropped to the mixture at
0.degree. C. and stirred at 15.degree. C. for 10 hr. TLC indicated
(3R,3aR,6S,6aR)-6-benzyloxy-2,3,3a,5,6,6a-hexahydrofuro[3,2-b]furan-3-ol
was consumed completely and one new spot formed. The reaction
mixture was quenched by addition of H.sub.2O (5 mL) at 15.degree.
C., and then diluted with EtOAc (5 mL) and extracted with EtOAc (5
mL*3). The combined organic layers were dried over
Na.sub.2SO.sub.4, filtered and the filtrate was concentrated under
reduced pressure to give a residue. The residue was purified by
column chromatography (SiO.sub.2, Petroleum ether/Ethyl acetate=0/1
to 1/1) to give
[(3S,3aR,6R,6aR)-3-benzyloxy-2,3,3a,5,6,6a-hexahydrofuro[3,2-b]furan-6-yl-
] 2-chloroacetate (230 mg, 735.42 umol, 34.75% yield) as yellow
oil.
[0549] Step 2:
[0550] A mixture of
[(3S,3aR,6R,6aR)-3-benzyloxy-2,3,3a,5,6,6a-hexahydrofuro[3,2-b]furan-6-yl-
] 2-chloroacetate (230 mg, 735.42 umol, 1 eq) in THF (5 mL) was
dropped to dimethylamine (2 mL, 2M, in THF) and degassed and purged
with N.sub.2 3 times. And then the mixture was stirred at
15.degree. C. for 10 hr under N.sub.2 atmosphere. TLC indicated
[(3S,3aR,6R,6aR)-3-benzyloxy-2,3,3a,5,6,6a-hexahydrofuro[3,2-b]furan-6-yl-
] 2-chloroacetate was consumed completely and one new spot formed.
LCMS showed desired mass was detected. The reaction mixture was
quenched by addition of H.sub.2O (5 mL) at 15.degree. C., and then
diluted with EtOAc (5 mL) and extracted with EtOAc (5 mL*3). The
combined organic layers were dried over Na.sub.2SO.sub.4, filtered
and the filtrate was concentrated under reduced pressure to give a
residue. The residue was purified by prep-TLC (SiO.sub.2,
DCM:MeOH=10:1) to give
[(3S,3aR,6R,6aR)-3-benzyloxy-2,3,3a,5,6,6a-hexahydrofuro[3,2-b]furan-6-yl-
] 2-(dimethylamino)acetate (180 mg, 503.03 umol, 68.40% yield, HCl)
as yellow oil. LCMS: (M+H+) 322.1
[0551] Step 3:
[0552] A mixture of
[(3S,3aR,6R,6aR)-3-benzyloxy-2,3,3a,5,6,6a-hexahydrofuro[3,2-b]furan-6-yl-
] 2-(dimethylamino)acetate (180 mg, 503.03 umol, 1 eq, HCl) and
Pd(OH).sub.2/C (0.18 g, 20% purity) in EtOH (20 mL) was degassed
and purged with H.sub.2 3 times. And then the mixture was stirred
at 60.degree. C. for 10 hr under H.sub.2 (15 Psi) atmosphere. LCMS
showed desired compound was detected. The reaction mixture was
filtered and the filtrate was concentrated under reduced pressure
to give a residue. The residue was purified by prep-TLC (SiO.sub.2,
DCM:MeOH=10:1, 5% NH.sub.3H.sub.2O) to give compound
[(3S,3aR,6R,6aR)-3-hydroxy-2,3,3a,5,6,6a-hexahydrofuro[3,2-b]furan-6-yl]
2-(dimethylamino)acetate (100 mg, 432.44 umol, 85.97% yield) as
colorless oil. LCMS: (M+H+) 232.1
[0553] Step 4:
[0554] To a solution of
[(3S,3aR,6R,6aR)-3-hydroxy-2,3,3a,5,6,6a-hexahydrofuro[3,2-b]furan-6-yl]
2-(dimethylamino)acetate (100 mg, 432.44 umol, 1 eq) in ACN (5 mL)
was added 3-bromoprop-1-yne (56.59 mg, 475.69 umol, 41.01 uL, 1.1
eq). The mixture was stirred at 15.degree. C. for 10 hr. LCMS
showed desired mass was detected. The reaction mixture was filtered
and the filter cake was concentrated under reduced pressure to give
N-(2-(((3R,3aR,6S,6aR)-6-hydroxyhexahydrofuro[3,2-b]furan-3-yl)oxy)-2-oxo-
ethyl)-N,N-dimethylprop-2-yn-1-aminium bromide (60 mg, 131.92 umol,
30.51% yield, 77% purity, Br--) as a white solid. LCMS: (M+) 270.1
.sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. 5.26-5.16 (m, 2H), 4.75
(t, J=5.3 Hz, 1H), 4.57-4.40 (m, 4H), 4.21 (d, J=4.8 Hz, 1H),
4.17-4.11 (m, 1H), 4.08 (t, J=3.5 Hz, 1H), 3.77 (d, J=4.2 Hz, 2H),
3.71 (d, J=9.4 Hz, 1H), 3.64 (dd, J=9.4, 3.2 Hz, 1H), 3.24 (s,
6H).
##STR00167##
Compound 74:
N-(2-(((3S,3aR,6R,6aR)-6-hydroxyhexahydrofuro[3,2-b]furan-3-yl)oxy)ethyl)-
-N,N-dimethylprop-2-yn-1-aminium trifluoroacetate
[0555] Step 1:
[0556] To a solution of
(3S,3aR,6R,6aR)-6-benzyloxy-2,3,3a,5,6,6a-hexahydrofuro[3,2-b]furan-3-ol
(7 g, 29.63 mmol, 1 eq) in DMF (70 mL) was added NaH (1.78 g, 44.44
mmol, 60% purity, 1.5 eq) at 0.degree. C. and stirred for 0.5 h.
Then 2-bromoethoxy-tert-butyl-dimethyl-silane (10.63 g, 44.44 mmol,
1.5 eq) was added to the mixture and stirred at 15.degree. C. for
11.5 h. TLC indicated
(3S,3aR,6R,6aR)-6-benzyloxy-2,3,3a,5,6,6a-hexahydrofuro[3,2-b]f-
uran-3-ol was consumed completely and two new spots formed. The
reaction mixture was quenched by addition of H.sub.2O (80 mL) at
0.degree. C., diluted with EtOAc (80 mL) and extracted with EtOAc
(100 mL*4). The combined organic layers were dried over
Na.sub.2SO.sub.4, filtered and the filtrate was concentrated under
reduced pressure to give a residue. The residue was purified by
column chromatography (SiO.sub.2, Petroleum ether/Ethyl acetate=0/1
to 2/1) to give
2-[[(3S,3aR,6R,6aR)-6-benzyloxy-2,3,3a,5,6,6a-hexahydrofuro[3,2-b]furan-3-
-yl]oxy]ethoxy-tert-butyl-dimethyl-silane (4.2 g, 10.64 mmol,
35.93% yield) as yellow oil.
[0557] Step 2:
[0558] To a solution of
2-[[(3S,3aR,6R,6aR)-6-benzyloxy-2,3,3a,5,6,6a-hexahydrofuro[3,2-b]
furan-3-yl]oxy]ethoxy-tert-butyl-dimethyl-silane (4.2 g, 10.64
mmol, 1 eq) in THF (40 mL) was added HF.Py (4.52 g, 31.93 mmol,
4.11 mL, 70%, 3 eq). The mixture was stirred at 15.degree. C. for
12 hr under N.sub.2. LCMS showed desired mass was detected. TLC
indicated
2-[[(3S,3aR,6R,6aR)-6-benzyloxy-2,3,3a,5,6,6a-hexahydrofuro[3,2-b]furan-3-
-yl]oxy]ethoxy-tert-butyl-dimethyl-silane was consumed completely
and one new spot formed. The reaction mixture was filtered and the
filtrate was concentrated under reduced pressure to give a residue.
The residue was purified by column chromatography (SiO.sub.2, Ethyl
acetate/Methanol=1/0 to 0/1) to give
2-[[(3S,3aR,6R,6aR)-6-benzyloxy-2,3,3a,5,6,6a-hexahydrofuro[3,2-b]furan-3-
-yl]oxy]ethanol (1.67 g, 5.96 mmol, 55.97% yield) as yellow oil.
LCMS: (M+H+) 281.1
[0559] Step 3:
[0560] To a solution of
2-[[(3S,3aR,6R,6aR)-6-benzyloxy-2,3,3a,5,6,6a-hexahydrofuro[3,2-b]furan-3-
-yl] oxy]ethanol (1.67 g, 5.96 mmol, 1 eq) in THF (17 mL) was added
PPh3 (3.52 g, 13.40 mmol, 2.25 eq) and imidazole (1.22 g, 17.87
mmol, 3 eq). Then 12 (3.78 g, 14.89 mmol, 3.00 mL, 2.5 eq) was
added to the mixture and stirred at 15.degree. C. for 12 hr. TLC
indicated two new spots formed. The reaction mixture was filtered
and the filtrate was concentrated under reduced pressure to give a
residue. The residue was purified by column chromatography
(SiO.sub.2, Petroleum ether/Ethyl acetate=50/1 to 2/1) to give
(3S,3aR,6R,6aR)-6-benzyloxy-3-(2-iodoethoxy)-2,3,3a,5,6,6a-hexahydrofuro[-
3,2-b]furan (2 g, 5.13 mmol, 86.03% yield) as yellow oil.
[0561] Step 4:
[0562] A mixture of
(3S,3aR,6R,6aR)-6-benzyloxy-3-(2-iodoethoxy)-2,3,3a,5,6,6a-hexahydrofuro
[3,2-b]furan (2 g, 5.13 mmol, 1 eq) in THF (20 mL) was dropped to
dimethylamine (20 mL, 2 M, in THF) and degassed and purged with
N.sub.2 3 times, and then the mixture was stirred at 15.degree. C.
for 10 hr under N.sub.2 atmosphere. TLC indicated one new spot
formed. LCMS showed desired compound was detected. The reaction
mixture was quenched by addition of H.sub.2O (30 mL) at 15.degree.
C., and then diluted with EtOAc (30 mL) and extracted with EtOAc
(30 mL*4). The combined organic layers were dried over
Na.sub.2SO.sub.4, filtered, and the filtrate was concentrated under
reduced pressure to give a residue. The residue was purified by
column chromatography (SiO.sub.2, Ethyl acetate/Methanol=1/0 to
0/1) to give
2-[[(3S,3aR,6R,6aR)-6-benzyloxy-2,3,3a,5,6,6a-hexahydrofuro[3,2-b]furan-3-
-yl]oxy]-N,N-dimethyl-ethanamine (1.9 g, 4.36 mmol, 85.16% yield,
HI) as yellow oil. LCMS: (M+H+) 308.1
[0563] Step 5:
[0564] To a solution of
2-[[(3S,3aR,6R,6aR)-6-benzyloxy-2,3,3a,5,6,6a-hexahydrofuro[3,2-b]
furan-3-yl]oxy]-N,N-dimethyl-ethanamine (1.9 g, 4.36 mmol, 1 eq,
HI) in EtOH (40 mL) was added Pd(OH).sub.2/C (1.4 g, 20% purity).
The mixture was stirred at 80.degree. C. for 10 hr under H.sub.2
(15 Psi). TLC indicated one new spot formed. The reaction mixture
was filtered and the filtrate was concentrated under reduced
pressure to give
(3R,3aR,6S,6aR)-6-[2-(dimethylamino)ethoxy]-2,3,3a,5,6,6a-hexahydrofuro[3-
,2-b]furan-3-ol as yellow oil without further purification.
[0565] Step 6:
[0566] To a solution of
(3R,3aR,6S,6aR)-6-[2-(dimethylamino)ethoxy]-2,3,3a,5,6,6a-hexahydrofuro
[3,2-b]furan-3-ol (0.88 g, 4.05 mmol, 1 eq) in ACN (10 mL) was
added 3-bromoprop-1-yne (530.02 mg, 4.46 mmol, 384.07 uL, 1.1 eq).
The mixture was stirred at 15.degree. C. for 10 hr. LCMS showed
desired mass was detected. The reaction mixture was concentrated
under reduced pressure to give a residue. The residue was purified
by prep-HPLC (column: Waters Xbridge BEH C18 100*30 mm*10 um;
mobile phase: [water (0.1% TFA)-ACN]; B %: 1%-10%, 10 min) to give
N-(2-(((3S,3aR,6R,6aR)-6-hydroxyhexahydrofuro[3,2-b]furan-3-yl)oxy)ethyl)-
-N,N-dimethylprop-2-yn-1-aminium trifluoroacetate (671 mg, 1.82
mmol, 44.85% yield, 100.0% purity, CF3CO2-) as yellow oil. LCMS:
(M+) 256.1 1H NMR (400 MHz, Methanol-d4) .delta. 4.56-4.46 (m, 2H),
4.38 (d, J=2.6 Hz, 2H), 4.31-4.22 (m, 1H), 4.15-3.96 (m, 4H),
3.93-3.80 (m, 2H), 3.75-3.65 (m, 2H), 3.56 (t, J=2.5 Hz, 1H), 3.48
(dd, J=8.8, 7.2 Hz, 1H), 3.23 (s, 6H).
##STR00168##
Compound 75:
(3-{[(3S,3aR,6R,6aR)-6-hydroxy-hexahydrofuro[3,2-oxopropyl)dimethyl(prop--
2-yn-1-yl)azanium
[0567] This compound may be synthesized according to the
experimental procedure described for Compound 344.
##STR00169##
Compound 76:
(3-{[(3R,3aR,6S,6aR)-6-hydroxy-hexahydrofuro[3,2-b]furan-3-yl]oxy}-2-meth-
ylidene-3-oxopropyl)dimethyl(prop-2-yn-1-yl)azanium
[0568] This compound may be synthesized according to the
experimental procedure described for Compound 344.
##STR00170##
Compound 102:
N,N-dimethyl-N-(2-oxo-2-(((2S,3R,4S,5R,6R)-2,3,5-triacetoxy-6-(acetoxymet-
hyl)tetrahydro-2H-pyran-4-yl)oxy)ethyl)prop-2-yn-1-aminium
trifluoroacetate
[0569] Step 1:
[0570] Tetrahydrofuran (5 mL) was added to 60% NaH (0.2 g, 5.0
mmol, 1.3 eq) in mineral oil under nitrogen, followed by addition
of TBAI (71 mg, 0.22 mmol, 0.05 eq), then cooled to 0.degree. C.
Compound 1,2:5,6-Di-O-isopropylidene-.alpha.-D-glucofuranose (Sigma
Aldrich, CAS: 582-52-5, 1 g, 3.8 mmol, 1 eq) in THF (3 mL) was
added dropwise, followed by dropwise addition of benzyl bromide
(0.55 mL, 4.6 mmol, 1.2 eq). The ice bath was removed and the
reaction was warmed to 55.degree. C., and stirred overnight. The
reaction was quenched with methanol (1 mL), then filtered and
loaded onto silica. Purification by column chromatography (100%
hexane to 100% ethyl acetate) yielded
3-benzyl-1,2:5,6-Di-O-isopropylidene-.alpha.-D-glucofuranose as a
viscous oil (877 mg, 2.5 mmol 65% yield).
[0571] Step 2:
[0572] Compound
3-benzyl-1,2:5,6-Di-O-isopropylidene-.alpha.-D-glucofuranose (877
mg, 2.5 mmol, 1 eq) was dissolved in TFA (1 mL) followed by
addition of water (1 mL), then stirred overnight. The reaction
solution was concentrated by rotary evaporation, and the solids
were precipitated out in ethyl acetate and washed with diethyl
ether. Solids were dried in vacuo to 3-benzyl-beta-D-glucopyranose
as a white solid (400 mg, 1.48 mmol, 59% yield).
[0573] Step 3:
[0574] Compound 3-benzyl-beta-D-glucopyranose (400 mg, 1.48 mmol, 1
eq) was stirred in acetic anhydride (1.6 mL, 16.9 mmol, 11.4 eq)
and pyridine (1.6 mL, 19.9 mmol, 13.4 eq) overnight at room
temperature. The reaction was concentrated by rotary evaporation
and purified by column chromatography (100% hexanes to 100% ethyl
acetate) to yield
3-benzyl-1,2,4,6-tetra-O-acetyl-beta-D-glucopyranose as a waxy
solid (436 mg, 0.99 mmol, 67% yield).
[0575] Step 4:
[0576] Compound
3-benzyl-1,2,4,6-tetra-O-acetyl-beta-D-glucopyranose (500 mg, 1.14
mmol, 1 eq) was dissolved in methanol (approximately 10 mL), and
flowed through an H-cube hydrogenation flow system with a 10% Pd/C
catalyst cartridge at 0.4 mL/min and hydrogen pressure at 30 bar.
Reaction was continuously fed through the H-cube until completed as
monitored by LCMS. If necessary, the cartridge was replaced. The
solution was concentrated by rotary evaporation and dried in vacuo
to yield 1,2,4,6-tetra-O-acetyl-beta-D-glucopyranose (170 mg, 0.49
mmol, 43% yield).
[0577] Step 5:
[0578] Compound
3-benzyl-1,2,4,6-tetra-O-acetyl-beta-D-glucopyranose (170 mg, 0.49
mmol, 1 eq) was dissolved in DCM (2 mL), followed by addition of
chloroacetyl chloride (0.11 mL, 1.38 mmol, 2.8 eq) and pyridine
(0.12 mL, 1.49 mmol, 3 eq). The reaction was stirred overnight,
filtered, concentrated, and purified by column chromatography (100%
hexanes to 100% ethyl acetate) to yield
3-(2-chloroacetoxy)-1,2,4,6-tetra-O-acetyl-beta-D-glucopyranose as
a yellow viscous oil (0.19 g, 0.45 mmol, 91% yield).
[0579] Step 6:
[0580] Compound
3-(2-chloroacetoxy)-1,2,4,6-tetra-O-acetyl-beta-D-glucopyranose
(0.19 g, 0.45 mmol, 1 eq) was dissolved in acetone (4 mL), followed
by addition of sodium iodide (0.1 g, 0.68 mmol, 1.5 eq). The
reaction was stirred for a few minutes, and
3-dimethylamino-1-propyne (0.144 mL, 1.3 mmol, 3 eq) was added. The
reaction was stirred at room temperature overnight, then filtered.
The filtrate was concentrated, diluted with DMSO and water, and
injected onto reverse phase C18 column chromatography (0.1% TFA in
95% water/5% MeCN to 100% MeCN). Fractions containing product were
lyophilized to yield an off-white powder (113 mg, 0.19 mmol, 43%
yield). LCMS (M+): 472.1 .sup.1H NMR (400 MHz, DMSO-d.sub.6)
.delta. 6.00 (d, J=8.2 Hz, 1H), 5.64 (t, J=9.5 Hz, 1H), 5.11-4.98
(m, 2H), 4.58-4.44 (m, 4H), 4.29-4.10 (m, 3H), 4.00 (dd, J=12.5,
2.3 Hz, 1H), 3.21 (s, 6H), 2.12-1.93 (m, 12H).
##STR00171##
Compound 77:
(fluoromethyl)(methyl)(prop-2-yn-1-yl)(2-{[(2R,3R,4S,5R,6R)-3,4,5-tris(ac-
etyloxy)-6-[(acetyloxy)methyl]oxan-2-yl]oxy}ethyl)azanium
[0581] Step 1:
[0582] To a mixture of
[(2R,3R,4S,5R,6S)-3,4,5,6-tetraacetoxytetrahydropyran-2-yl]methyl
acetate (20 g, 51.24 mmol, 1 eq) in DCM (300 mL) was added
2-bromoethanol (9.60 g, 76.86 mmol, 5.46 mL, 1.5 eq) and
BF.sub.3.Et.sub.2O (36.36 g, 256.19 mmol, 31.62 mL, 5 eq) in one
portion at 0.degree. C. under N.sub.2. The mixture stirred at
25.degree. C. for 12 hours. TLC indicated the starting material was
consumed and one new spot formed. The reaction mixture was
concentrated under reduced pressure to give a residue. The residue
was purified by column chromatography (SiO.sub.2, Petroleum
ether/Ethyl acetate=20/1 to 0/1). Compound
[(2R,3R,4S,5R,6R)-3,4,5-triacetoxy-6-(2-bromoethoxy)tetrahydropyran-2-yl]-
methyl acetate (10 g, 21.97 mmol, 42.87% yield) was obtained as
white solid.
[0583] Step 2:
[0584] To a mixture of
[(2R,3R,4S,5R,6R)-3,4,5-triacetoxy-6-(2-bromoethoxy)tetrahydropyran-2-yl]-
methyl acetate (2 g, 4.39 mmol, 1 eq) and N-methylprop-2-yn-1-amine
(1.67 g, 24.16 mmol, 2.01 mL, 5.5 eq) in THF (5 mL) was added
Na.sub.2CO.sub.3 (931.26 mg, 8.79 mmol, 2 eq) and NaI (658.51 mg,
4.39 mmol, 1 eq) in one portion at 25.degree. C. under at
25.degree. C. under N.sub.2. Then mixture was heated to 70.degree.
C. and stirred for 12 hours. TLC indicated the stating material was
consumed and one new spot formed. LCMS showed the expected mass was
detected. The reaction mixture was filtered and the filtrate was
concentrated under reduced pressure to give a residue. The residue
was purified by column chromatography (SiO.sub.2, Petroleum
ether/Ethyl acetate=20/1 to 0/1).
Compound[(2R,3R,4S,5R,6R)-3,4,5-triacetoxy-6-[2-[methyl(prop-2-ynyl)amino-
]ethoxy]tetrahydropyran-2-yl]methyl acetate (1.6 g, crude) was
obtained as a yellow oil. LCMS: (M+H+): 444.3@ 0.125 min
[0585] Step 3:
[0586] To a mixture of
[(2R,3R,4S,5R,6R)-3,4,5-triacetoxy-6-[2-[methyl(prop-2-ynyl)amino]ethoxy]-
tetrahydropyran-2-yl]methyl acetate (1 g, 2.26 mmol, 1 eq) in
acetone (10 mL) was added fluoro(iodo)methane (1.80 g, 11.28 mmol,
5 eq) in one portion at 25.degree. C. under N.sub.2. The mixture
was heated to 90.degree. C. and stirred for 2 hours. TLC indicated
the starting material was consumed and one new spot formed. LCMS
showed one main peak with expected mass was detected. The reaction
mixture was concentrated under reduced pressure to give a residue.
The residue was purified by column chromatography (SiO.sub.2,
Petroleum ether/Ethyl acetate=10/1 to Ethyl acetate/Methanol=3/1).
Then the residue was purified by prep-HPLC [water (0.1% TFA)-ACN].
The title compound (266 mg, 441.46 umol, 19.58% yield, 98% purity,
TFA) was obtained as colorless oil. LCMS: (M+): 476.2 .sup.1H NMR
(400 MHz, Chloroform-d) .delta. 5.72 (d, J=48.1 Hz, 2H), 5.24 (t,
J=9.5 Hz, 1H), 5.08 (t, J=9.4 Hz, 1H), 4.99 (t, J=8.8 Hz, 1H),
4.65-4.58 (m, 3H), 4.40 (s, 1H), 4.32-4.18 (m, 5H), 3.82-3.73 (m,
1H), 3.42 (s, 3H), 2.91 (s, 1H), 2.07 (dd, J=21.9, 12.1 Hz,
12H).
##STR00172##
Compound 78:
N-(chloromethyl)-N,N-dimethyl-2-(((2S,3S,4R,5S,6S)-3,4,5-triacetoxy-6-(ac-
etoxymethyl)tetrahydro-2H-pyran-2-yl)oxy)ethan-1-aminium Iodide
[0587] Step 1:
[0588] To a solution of
(2S,3R,4S,5R,6R)-6-(acetoxymethyl)tetrahydro-2H-pyran-2,3,4,5-tetrayl
tetraacetate (60.0 g, 1 Eq, 154 mmol) in anhydrous DCM (300 mL) at
0.degree. C. under nitrogen, 2-bromoethan-1-ol (28.8 g, 16.3 mL,
1.5 Eq, 231 mmol) was added followed by BF.sub.3OEt.sub.2 (109 g,
97.4 mL, 5 Eq, 769 mmol). The reaction was stirred at room
temperature for 18 hrs. Reaction was quenched slowly with 300 mL of
H.sub.2O in an ice bath and 50 mL of saturated sodium bicarbonate
was added. The layers were separated. The water layer was washed
2.times. with 200 mL DCM. Organic layers were combined,
concentrated and residue was purified by normal phase flash
chromatography (0-40% ethyl acetate in hexanes). Filtrate was
concentrated and recrystallized by dissolving in minimal ethyl
acetate and adding hexanes until crystals began forming, then
solution was cooled to 4.degree. C. overnight. Crystals were
collected and washed with cold 1:2 ethyl acetate/hexanes followed
by cold hexanes.
(2R,3R,4S,5R,6R)-2-(acetoxymethyl)-6-(2-bromoethoxy)tetrahydro-2H-pyran-3-
,4,5-triyl triacetate (39.41 g, 56.3%) was obtained as a white
crystalline solid.
[0589] Step 2:
[0590] To a stirred solution of
(2S,3S,4R,5S,6S)-2-(acetoxymethyl)-6-(2-bromoethoxy)tetrahydro-2H-pyran-3-
,4,5-triyl triacetate (3.60 g, 1 Eq, 7.90 mmol) in 16 mL anhydrous
THF, 2 M dimethylamine in THF (16.0 mL, 4.0 Eq, 32.0 mmol) was
added followed by sodium iodide (1.2 g, 1 Eq, 7.9 mmol). Reaction
was stirred under nitrogen at room temperature for 18 hours,
filtered, and concentrated. Residue was purified by normal phase
flash chromatography (0-10% methanol in DCM) to give
(2S,3S,4R,5S,6S)-2-(acetoxymethyl)-6-(2-(dimethylamino)ethoxy)tetrahydro--
2H-pyran-3,4,5-triyl triacetate, Iodide (1.6 g, 37%) as a yellow
oil that crystallized upon standing.
[0591] Step 3:
[0592] To a stirred solution of
(2S,3S,4R,5S,6S)-2-(acetoxymethyl)-6-(2-(dimethylamino)ethoxy)tetrahydro--
2H-pyran-3,4,5-triyl triacetate, iodide (1.15 g, 1 Eq, 2.10 mmol)
in 10 mL anhydrous DCM at room temperature, chloroiodomethane (766
.mu.L, 5 Eq, 10.5 mmol) was added followed by DIEA (0.40 mL, 1.1
Eq, 2.32 mmol). Vial was sealed and flushed with nitrogen. Reaction
was heated to 40.degree. C. and stirred for 2.5 hours. Reaction was
allowed to come to room temperature, concentrated by rotary
evaporation, and purified by normal phase HPLC (0-10% methanol in
DCM) to give
N-(chloromethyl)-N,N-dimethyl-2-(((2S,3S,4R,5S,6S)-3,4,5-triacetoxy-6-(ac-
etoxymethyl)tetrahydro-2H-pyran-2-yl)oxy)ethan-1-aminium, Iodide
(250 mg, 19.9%) as a slightly yellow solid. LCMS (M.sup.+): 468.3.
.sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. 5.33 (s, 2H), 5.28 (t,
1H), 4.99-4.81 (m, 3H), 4.19 (dd, 1H), 4.16-4.01 (m, 4H), 3.72-3.67
(m, 2H), 3.15 (s, 6H), 2.03 (s, 3H), 2.02 (s, 3H), 1.99 (s, 3H),
1.94 (s, 3H).
##STR00173##
Compound 79:
N-(fluoromethyl)-N,N-dimethyl-2-(((2S,3S,4R,5S,6S)-3,4,5-triacetoxy-6-(ac-
etoxymethyl)tetrahydro-2H-pyran-2-yl)oxy)ethan-1-aminium Iodide
[0593] Step 1:
[0594] To a solution of
(2S,3R,4S,5R,6R)-6-(acetoxymethyl)tetrahydro-2H-pyran-2,3,4,5-tetrayl
tetraacetate (60.0 g, 1 Eq, 154 mmol) in anhydrous DCM (300 mL) at
0.degree. C. under nitrogen, 2-bromoethan-1-ol (28.8 g, 16.3 mL,
1.5 Eq, 231 mmol) was added followed by BF.sub.3OEt.sub.2 (109 g,
97.4 mL, 5 Eq, 769 mmol). The reaction was stirred at room
temperature for 18 hrs. Reaction was quenched slowly with 300 mL of
H.sub.2O in an ice bath and 50 mL of saturated sodium bicarbonate
was added. The layers were separated. The water layer was washed
2.times. with 200 mL DCM. Organic layers were combined,
concentrated and residue was purified by normal phase flash
chromatography (0-40% ethyl acetate in hexanes). Filtrate was
concentrated and recrystallized by dissolving in minimal ethyl
acetate and adding hexanes until crystals began forming, then
solution was cooled to 4.degree. C. overnight. Crystals were
collected and washed with cold 1:2 ethyl acetate/hexanes followed
by cold hexanes. Yielded
(2R,3R,4S,5R,6R)-2-(acetoxymethyl)-6-(2-bromoethoxy)tetrahydro-2H-pyran-3-
,4,5-triyl triacetate (39.41 g, 56.3%) as a white crystalline
solid.
[0595] Step 2:
[0596] To a stirred solution of
(2S,3S,4R,5S,6S)-2-(acetoxymethyl)-6-(2-bromoethoxy)tetrahydro-2H-pyran-3-
,4,5-triyl triacetate (2.00 g, 1 Eq, 4.39 mmol) in 8.8 mL anhydrous
THF, dimethylamine (792 mg, 8.8 mL, 4.0 Eq, 17.6 mmol) was added
followed by sodium iodide (658 mg, 1 Eq, 4.39 mmol). Reaction was
stirred under nitrogen at room temperature for 18 hours, filtered,
and concentrated. Residue was purified by normal phase flash
chromatography (0-10% methanol in DCM) to give
(2S,3S,4R,5S,6S)-2-(acetoxymethyl)-6-(2-(dimethylamino)ethoxy)tetrahydro--
2H-pyran-3,4,5-triyl triacetate, Iodide (1.0 g, 42%) as a yellow
oil that crystallized upon standing.
[0597] Step 3:
[0598] To a stirred solution of
(2S,3S,4R,5S,6S)-2-(acetoxymethyl)-6-(2-(dimethylamino)ethoxy)tetrahydro--
2H-pyran-3,4,5-triyl triacetate, Iodide (517 mg, 1 Eq, 946 .mu.mol)
in 15 mL anhydrous dichloromethane at room temperature,
fluoroiodomethane (757 mg, 320 .mu.L, 5 Eq, 4.73 mmol) was added
followed by DIEA (135 mg, 0.18 mL, 1.1 Eq, 1.04 mmol). Reaction
vessel was flushed with nitrogen, then reaction was stirred for 2
hours at 40.degree. C. Reaction was then allowed to come to room
temperature, concentrated, and purified by normal phase flash HPLC
0-10% MeOH in DCM to give
N-(fluoromethyl)-N,N-dimethyl-2-(((2S,3S,4R,5S,6S)-3,4,5-triacetoxy-6-(ac-
etoxymethyl)tetrahydro-2H-pyran-2-yl)oxy)ethan-1-aminium, Iodide
(231 mg, 42.1%) as a light yellow solid. LC-MS (M.sup.+): 452.3.
.sup.1H NMR (400 MHz, Chloroform-d) .delta. 5.75 (s, 1H), 5.63 (s,
1H), 5.22 (t, 1H), 5.06 (t, 1H), 4.95 (dd, 1H), 4.75-4.68 (m, 1H),
4.49-4.37 (m, 1H), 4.33-4.14 (m, 5H), 3.85 (ddd, 1H), 3.51 (t, 6H),
2.12 (s, 3H), 2.09 (d, 3H), 2.04 (d, 3H), 2.00 (s, 3H).
##STR00174##
Compound 80:
N,N-dimethyl-N-(2-{[(3R,4S,5R,6R)-3,4,5-tris(acetyloxy)-6-[(acetyloxy)met-
hyl]oxan-2-yl]oxy}ethyl)cyclopropanaminium Iodide
[0599] Step 1:
[0600] To a mixture of
[(2R,3R,4S,5R,6S)-3,4,5,6-tetraacetoxytetrahydropyran-2-yl]methyl
acetate (20 g, 51.24 mmol, 1 eq) in DCM (300 mL) was added
2-bromoethanol (9.60 g, 76.86 mmol, 5.46 mL, 1.5 eq) and
BF.sub.3.Et.sub.2O (36.36 g, 256.19 mmol, 31.62 mL, 5 eq) in one
portion at 0.degree. C. under N.sub.2. The mixture stirred at
25.degree. C. for 12 hours. TLC indicated the starting material was
consumed and one new spot formed. The reaction mixture was
concentrated under reduced pressure to give a residue. The residue
was purified by column chromatography (SiO.sub.2, Petroleum
ether/Ethyl acetate=20/1 to 0/1). Compound
[(2R,3R,4S,5R,6R)-3,4,5-triacetoxy-6-(2-bromoethoxy)tetrahydropyran-2-yl]-
methyl acetate (10 g, 21.97 mmol, 42.87% yield) was obtained as
white solid.
[0601] Step 2:
[0602] To a mixture of
[(2R,3R,4S,5R,6R)-3,4,5-triacetoxy-6-(2-bromoethoxy)tetrahydropyran-2-yl]-
methyl acetate (2 g, 4.39 mmol, 1 eq) in THF (20 mL) was added NaI
(658.51 mg, 4.39 mmol, 1 eq) and Na.sub.2CO.sub.3 (931.26 mg, 8.79
mmol, 2 eq) in one portion at 25.degree. C. under N.sub.2. Then
N-methylcyclopropanamine (2.60 g, 24.16 mmol, 5.5 eq, HCl) was
added. The mixture was heated to 70.degree. C. and stirred for 12
hours. TLC indicated
[(2R,3R,4S,5R,6R)-3,4,5-triacetoxy-6-(2-bromoethoxy)tetrahydropyran-2-yl]-
methyl acetate was consumed and one new spot formed. The reaction
mixture was filtered and the filtrate was concentrated under
reduced pressure to give a residue. The residue was purified by
column chromatography (SiO.sub.2, Petroleum ether/Ethyl
acetate=20/1 to 0/1).
Compound[(2R,3R,4S,5R)-3,4,5-triacetoxy-6-[2-[cyclopropyl(methyl)amino]et-
hoxy]tetrahydropyran-2-yl]methyl acetate (1.4 g, 3.14 mmol, 71.54%
yield) was obtained as yellow oil. LCMS: (M+H+): 446.3
[0603] Step 3:
[0604] To a mixture of
[(2R,3R,4S,5R)-3,4,5-triacetoxy-6-[2-[cyclopropyl(methyl)amino]ethoxy]tet-
rahydropyran-2-yl]methyl acetate (1.4 g, 3.14 mmol, 1 eq) in
Acetone (15 mL) was added MeI (2.23 g, 15.71 mmol, 978.26 uL, 5 eq)
in one portion at 25.degree. C. under N.sub.2. Then mixture was
heated to 90.degree. C. and stirred for 2 hours. TLC indicated the
starting material was consumed and one new spot formed. The
reaction mixture was concentrated under reduced pressure to give a
residue. The residue was purified by column chromatography
(SiO.sub.2, Petroleum ether/Ethyl acetate=10/1 to Ethyl
acetate:Methanol=3/1). Compound
N,N-dimethyl-N-(2-{[(3R,4S,5R,6R)-3,4,5-tris(acetyloxy)-6-[(acetyloxy)met-
hyl]oxan-2-yl]oxy}ethyl)cyclopropanaminium iodide (1 g, 1.70 mmol,
54.17% yield, 100% purity, I) was obtained as yellow solid. LCMS:
(M+): 460.2 .sup.1H NMR (400 MHz, Chloroform-d) .delta. 5.21 (t,
J=9.6 Hz, 1H), 5.04 (t, J=9.7 Hz, 1H), 4.94 (dd, J=9.7, 8.0 Hz,
1H), 4.67 (d, J=8.0 Hz, 1H), 4.51-4.42 (m, 1H), 4.36-4.06 (m, 6H),
3.83 (dt, J=10.2, 3.5 Hz, 1H), 3.70-3.60 (m, 1H), 3.20 (d, J=9.6
Hz, 6H), 2.16-1.97 (m, 12H), 1.29-1.21 (m, 2H), 1.09-0.96 (m,
2H).
##STR00175##
Compound 81:
dimethyl(prop-2-yn-1-yl)(2-{[(2R,3R,4S,5R,6R)-3,4,5-tris(acetyloxy)-6-({[-
(2S,3R,4S,5S,6R)-3,4,5-tris(acetyloxy)-6-[(acetyloxy)methyl]oxan-2-yl]oxy}-
methyl)oxan-2-yl]oxy}ethyl)azanium Iodide
[0605] Step 1:
[0606] Preparation of solution 1: Ac.sub.2O (64.31 g, 629.94 mmol,
59 mL, 21.56 eq) and NaOAc (4.21 g, 51.32 mmol, 1.76 eq) was
stirred at reflux under N.sub.2.
(3R,4S,5S,6R)-6-[[(2S,3R,4S,5R,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetr-
ahydropyran-2-yl]oxymethyl]tetrahydropyran-2,3,4,5-tetrol (10 g,
29.21 mmol, 1 eq) in toluene (150 mL) was stirred at 100.degree. C.
Then the mixture was added to solution 1 and stirred at 100.degree.
C. for 1 hour. TLC indicated one new spot formed. The reaction
mixture was poured into ice water (400 mL), extracted with ethyl
acetate (200 mL*3). The combined organic layers were washed with
NaHCO.sub.3 (200 mL*2), dried over Na.sub.2SO.sub.4, filtered and
the filtrate was concentrated under reduced pressure to give a
residue. The residue was purified by column chromatography
(SiO.sub.2, Petroleum ether/Ethyl acetate=20/1 to 0/1). Compound
[(2R,3S,4S,5R,6S)-3,4,5-triacetoxy-6-[[(2R,3R,4S,5R,6S)-3,4,5,6--
tetraacetoxytetrahydropyran-2-yl]methoxy]tetrahydropyran-2-yl]methyl
acetate (5.8 g, 8.55 mmol, 29.26% yield) was obtained as white
solid.
[0607] Step 2:
[0608] To a mixture of
[(2R,3S,4S,5R,6S)-3,4,5-triacetoxy-6-[[(2R,3R,4S,5R,6S)-3,4,5,6-tetraacet-
oxytetrahydropyran-2-yl]methoxy]tetrahydropyran-2-yl]methyl acetate
(5.8 g, 8.55 mmol, 1 eq) in DCM (50 mL) was added 2-bromoethanol
(1.60 g, 12.82 mmol, 910.31 uL, 1.5 eq) and BF.sub.3.Et.sub.2O
(6.07 g, 42.74 mmol, 5.27 mL, 5 eq) in one portion at 0.degree. C.
under N.sub.2. Then the mixture was heated to 25.degree. C. and
stirred for 12 hours. TLC indicated
[(2R,3S,4S,5R,6S)-3,4,5-triacetoxy-6-[[(2R,3R,4S,5R,6S)-3,4,5,6-
-tetraacetoxytetrahydropyran-2-yl]methoxy]tetrahydropyran-2-yl]methyl
acetate was consumed and one new spot formed. The reaction mixture
was quenched by addition of H.sub.2O (50 mL), diluted with H.sub.2O
(50 mL) and extracted with ethyl acetate (50 mL*3). The combined
organic layers were dried over Na.sub.2SO.sub.4, filtered and the
filtrate was concentrated under reduced pressure to give a residue.
The residue was purified by column chromatography (SiO.sub.2,
Petroleum ether/Ethyl acetate=20/1 to 0/1).
[(2R,3S,4S,5R,6S)-3,4,5-triacetoxy-6-[[(2R,3R,4S,5R)-3,4,5-triacetoxy-6-(-
2-bromoethoxy)tetrahydropyran-2-yl]methoxy]tetrahydropyran-2-yl]methyl
acetate (2 g, crude) was obtained as yellow oil.
[0609] Step 3:
[0610] To a mixture of
[(2R,3S,4S,5R,6S)-3,4,5-triacetoxy-6-[[(2R,3R,4S,5R)-3,4,5-triacetoxy-6-(-
2-bromoethoxy)tetrahydropyran-2-yl]methoxy]tetrahydropyran-2-yl]methyl
acetate (2.00 g, 2.69 mmol, 1 eq) in Acetone (20 mL) was added NaI
(443.53 mg, 2.96 mmol, 1.1 eq) and N,N-dimethylprop-2-yn-1-amine
(1.12 g, 13.45 mmol, 1.43 mL, 5 eq) in one portion at 25.degree. C.
under N.sub.2. Then the mixture was heated to 80.degree. C. and
stirred for 10 hours. TLC indicated
[(2R,3S,4S,5R,6S)-3,4,5-triacetoxy-6-[[(2R,3R,4S,5R)-3,4,5-triacetoxy-6-(-
2-bromoethoxy)tetrahydropyran-2-yl]methoxy]tetrahydropyran-2-yl]methyl
acetate was consumed and one new spot formed. LCMS showed desired
m/z was detected. The reaction mixture was filtered and the
filtrate was concentrated under reduced pressure to give a residue.
The residue was purified by prep-HPLC [water (0.1% TFA)-ACN]. The
title compound (136 mg, 155.67 umol, 5.6% yield, 100% purity, I--)
was obtained as white solid. LCMS: (M+): 746.3 .sup.1H NMR (400
MHz, Methanol-d4) .delta. 5.49-5.43 (m, 1H), 5.37-5.27 (m, 3H),
5.24-5.02 (m, 3H), 4.95 (dd, J=9.7, 8.0 Hz, 1H), 4.79 (d, J=8.0 Hz,
1H), 4.51-4.39 (m, 2H), 4.38-4.26 (m, 2H), 4.24-4.16 (m, 1H),
4.14-4.05 (m, 2H), 3.95-3.87 (m, 1H), 3.84-3.72 (m, 3H), 3.65-3.58
(m, 1H), 3.28 (s, 6H), 2.20-1.95 (m, 21H).
##STR00176##
Compound 82:
dimethyl(prop-2-yn-1-yl)(2-{[(2R,3R,4S,5S,6S)-3,4,5-tris(acetyloxy)-6-car-
boxyoxan-2-yl]oxy}ethyl)azanium trifluoroacetate
[0611] Step 1:
[0612] To a solution of
(2S,3S,4S,5R)-3,4,5,6-tetrahydroxytetrahydropyran-2-carboxylic acid
(10 g, 51.51 mmol, 1 eq) in DMF (100 mL) was added DBU (8.63 g,
56.66 mmol, 8.54 mL, 1.1 eq), stirred for 15 min at 25.degree. C.,
and then 3-bromoprop-1-ene (7.48 g, 61.81 mmol, 1.2 eq) was added
to the mixture at 0.degree. C. The mixture was stirred for 10 h at
25.degree. C. under N.sub.2. TLC showed the starting reactant was
consumed and one new spot formed. The mixture was concentrated. The
residue was purified by column chromatography (SiO.sub.2, Petroleum
ether/Ethyl acetate=30/1 to 0/1). Allyl
(2S,3S,4S,5R)-3,4,5,6-tetrahydroxytetrahydropyran-2-carboxylate (10
g, 38.43 mmol, 74.60% yield, 90% purity) was obtained as a white
solid.
[0613] Step 2:
[0614] To a solution of allyl
(2S,3S,4S,5R)-3,4,5,6-tetrahydroxytetrahydropyran-2-carboxylate (10
g, 42.70 mmol, 1 eq) in Pyridine (100 mL) was added Ac.sub.2O
(43.59 g, 426.98 mmol, 39.99 mL, 10 eq). The mixture was stirred at
25.degree. C. for 12 h. TLC showed the starting reactant was
consumed completely and one new spot formed. The reaction was clean
according to TLC. The residue was purified by column chromatography
(SiO.sub.2, Petroleum ether/Ethyl acetate=20/1 to 2/1). Allyl
(2S,3S,4S,5R)-3,4,5,6-tetraacetoxytetrahydropyran-2-carboxylate (13
g, 29.08 mmol, 68.10% yield, 90% purity) was obtained as a yellow
solid.
[0615] Step 3:
[0616] To a mixture of allyl
(2S,3S,4S,5R)-3,4,5,6-tetraacetoxytetrahydropyran-2-carboxylate (5
g, 12.43 mmol, 1 eq), 2-bromoethanol (4.66 g, 37.28 mmol, 2.65 mL,
3 eq) in DCM (50 mL) at 0.degree. C. was added BF.sub.3.Et.sub.2O
(17.64 g, 124.27 mmol, 15.34 mL, 10 eq), purged with N.sub.2 3
times, and then the mixture was stirred at 25.degree. C. for 12 h
under N.sub.2 atmosphere. To the mixture was added H.sub.2O (30
mL), the mixture was extracted with EtOAc (10 mL*3), the organic
layer was dried over Na.sub.2SO.sub.4, filtered, and the filtrate
was concentrated. The residue was purified by prep-HPLC [water
(0.10% TFA)-ACN]. Allyl
(2S,3S,4S,5R)-3,4,5-triacetoxy-6-(2-bromoethoxy)tetrahydropyran-2-carboxy-
late (660 mg, 1.27 mmol, 10.23% yield, 90% purity) was obtained as
yellow oil.
[0617] Step 4:
[0618] To a solution of allyl
(2S,3S,4S,5R)-3,4,5-triacetoxy-6-(2-bromoethoxy)tetrahydropyran-2-carboxy-
late (280 mg, 599.24 umol, 1 eq) in ACN (10 mL) was added pyridine
(56.00 mg, 787.39 umol, 65.73 uL, 1.31 eq) and Pd(PPh3).sub.4
(84.00 mg, 72.69 umol, 1.21 eq) at 0.degree. C. The mixture was
stirred at 0.degree. C. for 1 h. TLC showed the starting reactant
was consumed. The reaction mixture was concentrated. The residue
was purified by prep-HPLC [water (0.1% TFA)-ACN]. Compound
(2S,3S,4S,5R)-3,4,5-triacetoxy-6-(2-bromoethoxy)tetrahydropyran-2-carboxy-
lic acid (156 mg, 346.91 umol, 28.95% yield, 95% purity) was
obtained as a yellow solid. LCMS: (M+H+): 427.0.
[0619] Step 5:
[0620] To a solution of
(2S,3S,4S,5R)-3,4,5-triacetoxy-6-(2-bromoethoxy)tetrahydropyran-2-carboxy-
lic acid (67 mg, 156.84 umol, 1 eq) in acetone (5 mL) was added
N,N-dimethylprop-2-yn-1-amine (65.19 mg, 784.18 umol, 83.15 uL, 5
eq). The mixture was stirred at 80.degree. C. for 12 h. LCMS showed
the reactant was consumed completely. The reaction mixture was
concentrated. The residue was purified by prep-HPLC [water (0.1%
TFA)-ACN]. The title compound (6 mg, 12.84 umol, 8.19% yield, 90%
purity) was obtained as a yellow solid. LCMS: (M): 430.2 .sup.1H
NMR (400 MHz, Deuterium Oxide) .delta. 5.35 (t, J=9.6 Hz, 1H),
5.13-4.97 (m, 3H), 4.31-4.15 (m, 3H), 4.12-4.04 (m, 1H), 3.98-3.88
(m, 1H), 3.84-3.74 (m, 1H), 3.58 (dd, J=14.5, 4.2 Hz, 1H),
3.20-3.11 (m, 7H), 1.98-1.87 (m, 9H).
##STR00177##
Compound 83:
(2-{[(2R,3R,4R,5S,6R)-4,5-bis(acetyloxy)-6-[(acetyloxy)methyl]-3-acetamid-
ooxan-2-yl]oxy}ethyl)dimethyl(prop-2-yn-1-yl)azanium
[0621] This compound may be synthesized according to the
experimental procedure described for Compound 7.
##STR00178##
Compound 85:
dimethyl(prop-2-yn-1-yl)(2-{[(2R,3R,4S,5S,6S)-3,4,5-tris(acetyloxy)-6-(me-
thoxycarbonyl)oxan-2-yl]oxy}ethyl)azanium bromide
[0622] Step 1:
[0623] To a solution of methyl
(2S,3S,4S,5R,6S)-3,4,5,6-tetraacetoxytetrahydropyran-2-carboxylate
(5 g, 13.29 mmol, 1 eq) and 2-bromoethanol (2.49 g, 19.93 mmol,
1.42 mL, 1.5 eq) in DCM (50 mL) was added dropwise
BF.sub.3.Et.sub.2O (9.43 g, 66.43 mmol, 8.20 mL, 5 eq) at 0.degree.
C., stirred at 0.degree. C. for 1 h, then stirred at 25.degree. C.
for 11 h. TLC showed the starting reactant was consumed. The
mixture was poured into H.sub.2O (50 mL) at 0.degree. C., and
extracted with DCM (20 mL*3). The combined organic layer was dried
over Na.sub.2SO.sub.4, filtered, and the filtrate concentrated. The
residue was purified by column chromatography (SiO.sub.2, Petroleum
ether/Ethyl acetate=30/1 to 3/1). Methyl
(2S,3S,4S,5R,6R)-3,4,5-triacetoxy-6-(2-bromoethoxy)tetrahydropyran-2-carb-
oxylate (3 g, 5.44 mmol, 40.94% yield, 80% purity) was obtained as
colorless oil.
[0624] Step 2:
[0625] To a solution of methyl
(2S,3S,4S,5R,6R)-3,4,5-triacetoxy-6-(2-bromoethoxy)tetrahydropyran-2-carb-
oxylate (260 mg, 589.27 umol, 1 eq) in acetone (10 mL) was added
N,N-dimethylprop-2-yn-1-amine (244.94 mg, 2.95 mmol, 312.42 uL, 5
eq), stirred at 90.degree. C. for 2 h. LCMS showed the starting
reactant was consumed. The mixture was concentrated. The residue
was purified by prep-HPLC (column: Phenomenex Luna C18 100*30 mm*5
um; mobile phase: [water (0.1% TFA)-ACN]; B %: 1%-28%, 12 min). The
title compound (50 mg, 82.01 umol, 13.92% yield, 86% purity, Br--)
was obtained as white solid.
[0626] LCMS: (M+): 444.1 .sup.1H NMR (400 MHz, Deuterium Oxide)
.delta. 5.5 (m, 1H), 5.2-5.1 (m, 4H), 4.5 (m, 1H), 4.4-4.3 (m, 2H),
4.2 (m, 1H), 4.1 (m, 1H), 3.9 (m, 1H), 3.7 (m, 3H), 3.3-3.2 (m,
7H), 2.1-2.0 (m, 9H).
##STR00179##
Compound 86:
N-(fluoromethyl)-N-methyl-N-(2-{[(2R,3R,4S,5R,6R)-3,4,5-tris(acetyloxy)-6-
-[(acetyloxy)methyl]oxan-2-yl]oxy}ethyl)cyclopropanaminium
Iodide
[0627] Step 1:
[0628] To a mixture of
[(2R,3R,4S,5R,6R)-3,4,5-triacetoxy-6-(2-bromoethoxy)tetrahydropyran-2-yl]-
methyl acetate (2 g, 4.39 mmol, 1 eq) in THF (20 mL) was added NaI
(658.51 mg, 4.39 mmol, 1 eq), Na.sub.2CO.sub.3 (931.26 mg, 8.79
mmol, 2 eq) and N-methylcyclopropanamine (2.60 g, 24.16 mmol, 5.5
eq, HCl) in one portion at 25.degree. C. under N.sub.2. The mixture
was heated to 70.degree. C. and stirred for 12 hours. TLC indicated
[(2R,3R,4S,5R,6R)-3,4,5-triacetoxy-6-(2-bromoethoxy)tetrahydropyran-2-yl]-
methyl acetate was consumed and one new spot formed. LCMS showed
one main peak with expected mass was detected. The reaction mixture
was filtered and the filtrate was concentrated under reduced
pressure to give a residue. The residue was purified by column
chromatography (SiO.sub.2, Petroleum ether/Ethyl acetate=50/1 to
0/1). 1.2 g crude product was obtained. 200 mg of the crude product
was further purified by prep-HPLC [water (0.10% TFA)-ACN] to get 82
mg of product (99% purity, TFA) as white solid. The crude compound
[(2R,3R,4S,5R,6R)-3,4,5-triacetoxy-6-[2-[cyclopropyl(methyl)amino]ethoxy]-
tetrahydropyran-2-yl]methyl acetate (1 g, 40.28% yield, crude) as
white solid was used in the next step directly. LCMS: (M+H)+:
446.2
[0629] Step 2:
[0630] To a mixture of
[(2R,3R,4S,5R,6R)-3,4,5-triacetoxy-6-[2-[cyclopropyl(methyl)amino]ethoxy]-
tetrahydropyran-2-yl]methyl acetate (1.78 g, 2.20 mmol, 1 eq) in
acetone (20 mL) was added fluoro(iodo)methane (1.76 g, 11.00 mmol,
5 eq) in one portion at 25.degree. C. under N.sub.2. The mixture
was heated to 90.degree. C. and stirred for 2 hours. TLC indicated
[(2R,3R,4S,5R,6R)-3,4,5-triacetoxy-6-[2-[cyclopropyl(methyl)amino]ethoxy]-
tetrahydropyran-2-yl]methyl acetate was consumed and one new spot
formed. LCMS showed one main peak with expected mass was detected.
The reaction mixture was concentrated under reduced pressure to
give a residue. The residue was purified by column chromatography
(SiO.sub.2, Ethyl acetate:Methanol=1/0 to 3/1). The title compound
(1.11 g, 58% yield 99% purity, I--) was obtained as yellow solid.
LCMS: (M+): 478.2 .sup.1H NMR (400 MHz, Methanol-d4) .delta. 5.58
(s, 1H), 5.46 (s, 1H), 5.32 (t, J=9.5 Hz, 1H), 5.08 (t, J=9.8 Hz,
1H), 4.98 (dd, J=9.7, 8.0 Hz, 1H), 4.86-4.81 (m, 1H), 4.50-4.35 (m,
1H), 4.30 (d, J=3.5 Hz, 2H), 4.27-4.10 (m, 1H), 4.02-3.93 (m, 1H),
3.92-3.82 (m, 2H), 2.97 (dd, J=6.0, 2.2 Hz, 3H), 2.14-2.02 (m, 9H),
2.00 (s, 3H), 1.47-1.30 (m, 2H), 1.12-0.95 (m, 2H).
##STR00180##
Compound 88:
(chloromethyl)dimethyl(2-{[(2R,3R,4S,5R)-3,4,5-tris(acetyloxy)oxan-2-yl]o-
xy}ethyl)azanium Iodide
[0631] Step 1:
[0632] To a mixture of (2R,3R,4S,5R)-tetrahydropyran-2,3,4,5-tetrol
(50 g, 333.04 mmol, 1 eq) in pyridine (300 mL) was added acetyl
acetate (204.00 g, 2.00 mol, 187.16 mL, 6 eq) in one portion at
0.degree. C. under N.sub.2. The mixture was heated to 25.degree. C.
and stirred for 12 hours. TLC indicated one new spot formed. The
reaction was clean according to TLC. The reaction mixture was
concentrated under reduced pressure to give a residue. The residue
was purified by column chromatography (SiO.sub.2, Petroleum
ether/Ethyl acetate=50/1 to 0/1). Compound
[(3R,4S,5R)-4,5,6-triacetoxytetrahydropyran-3-yl]acetate (80 g,
251.35 mmol, 75.47% yield) was obtained as yellow oil.
[0633] Step 2:
[0634] To a mixture of
[(3R,4S,5R)-4,5,6-triacetoxytetrahydropyran-3-yl] acetate (20 g,
62.84 mmol, 1 eq) in DCM (200 mL) was added 2-bromoethanol (11.78
g, 94.26 mmol, 6.69 mL, 1.5 eq) and BF.sub.3.Et.sub.2O (9.10 g,
62.84 mmol, 7.91 mL, 98% purity, 1 eq) in one portion at 0.degree.
C. under N.sub.2. The mixture was heated to 25.degree. C. and
stirred for 12 hours. TLC indicated
[(3R,4S,5R)-4,5,6-triacetoxytetrahydropyran-3-yl] acetate was
consumed and one new spot formed. The reaction mixture was quenched
by addition of H.sub.2O (100 mL), and then diluted with H.sub.2O
(200 mL) and extracted with ethyl acetate (200 mL*3). The combined
organic layers were dried over Na.sub.2SO.sub.4, filtered and the
filtrate was concentrated under reduced pressure to give a residue.
The crude product was purified by re-crystallization from ethyl
acetate:Petroleum ether (40 ml:30 mL) at 0.degree. C. for 12h. The
mixture was filtered and the filter cake was concentrated under
reduced pressure to give a residue. The filtrate was purified by
prep-HPLC [water (0.1%
TFA)-ACN].[(3R,4S,5R,6S)-4,5-diacetoxy-6-(2-bromoethoxy)tetrahydrop-
yran-3-yl] acetate (3.4 g, 8.87 mmol, 14.12% yield) was obtained as
a white solid. Compound
[(3R,4S,5R,6R)-4,5-diacetoxy-6-(2-bromoethoxy)tetrahydropyran-3-yl]
acetate (3.5 g, 9.13 mmol, 14.54% yield) was obtained as yellow
oil.
[0635] Step 3:
[0636] To a mixture of
[(3R,4S,5R,6S)-4,5-diacetoxy-6-(2-bromoethoxy)tetrahydropyran-3-yl]
acetate (3.4 g, 8.87 mmol, 1 eq) in THF (30 mL) was added NaI (1.33
g, 8.87 mmol, 1 eq) and Na.sub.2CO.sub.3 (1.88 g, 17.75 mmol, 2 eq)
in one portion at 25.degree. C. under N.sub.2. N-methylmethanamine
(2 M, 24.40 mL, 5.5 eq) was added. The mixture was heated to
70.degree. C. and stirred for 12 hours. TLC indicated
[(3R,4S,5R,6S)-4,5-diacetoxy-6-(2-bromoethoxy)tetrahydropyran-3-yl]
acetate was consumed incompletely and one new spot formed. LCMS
showed
[(3R,4S,5R,6S)-4,5-diacetoxy-6-(2-bromoethoxy)tetrahydropyran-3-yl]
acetate was consumed and one main peak with expected mass was
detected. The reaction mixture was filtered and concentrated under
reduced pressure to give a residue. The residue was purified by
column chromatography (SiO.sub.2, Ethyl acetate:Methanol=1/0 to
1/1). The 200 mg crude product was purified by prep-HPLC [water
(0.1% TFA)-ACN]. Compound
[(3R,4S,5R,6R)-4,5-diacetoxy-6-[2-(dimethylamino)ethoxy]tetrahydropyran-3-
-yl] acetate (3.1 g, crude) was obtained as yellow oil. Compound
[(3R,4S,5R,6R)-4,5-diacetoxy-6-[2-(dimethylamino)ethoxy]tetrahydropyran-3-
-yl] acetate (14.7 mg, 98% purity) was obtained as white solid.
LCMS: (M+H).sup.+: 348.1
[0637] Step 4:
[0638] To a mixture of
[(3R,4S,5R,6R)-4,5-diacetoxy-6-[2-(dimethylamino)ethoxy]tetrahydropyran-3-
-yl] acetate (3 g, 8.64 mmol, 1 eq) in acetone (30 mL) was added
chloro(iodo)methane (7.62 g, 43.18 mmol, 3.13 mL, 5 eq) in one
portion at 25.degree. C. under N.sub.2. The mixture was heated to
90.degree. C. and stirred for 2 hours. LCMS showed
[(3R,4S,5R,6R)-4,5-diacetoxy-6-[2-(dimethylamino)ethoxy]tetrahydropyran-3-
-yl]acetate was consumed and one main peak with expected mass was
detected. The reaction mixture was filtered and concentrated under
reduced pressure to give a residue. The residue was purified by
prep-HPLC [water (0.1% TFA)-ACN]. Compound
chloromethyl-dimethyl-[2-[(2R,3R,4S,5R)-3,4,5-triacetoxytetrahydropyran-2-
-yl]oxyethyl]ammonium (315 mg, 589.41 umol, 6.82% yield, 98%
purity, I--) was obtained as yellow oil. LCMS: (M+).sup.+:
396.1
[0639] Step 5:
[0640] To a mixture of
[(3R,4S,5R,6R)-4,5-diacetoxy-6-[2-(dimethylamino)ethoxy]tetrahydropyran-3-
-yl] acetate (2.8 g, 8.06 mmol, 1 eq) in DCM (20 mL) was added
chloro(iodo)methane (7.11 g, 40.30 mmol, 2.93 mL, 5 eq) and DIPEA
(1.06 g, 8.06 mmol, 1.43 mL, 98% purity, 1 eq) in one portion at
25.degree. C. under N.sub.2. The mixture was stirred at 40.degree.
C. for 12 hours in a 50 mL sealed tube. TLC indicated
[(3R,4S,5R,6R)-4,5-diacetoxy-6-[2-(dimethylamino)ethoxy]tetrahydropyran-3-
-yl] acetate was consumed incompletely and one new spot formed.
LCMS showed one main peak with expected mass was detected. The
reaction mixture was concentrated under reduced pressure to give a
residue. The residue was purified by column chromatography
(SiO.sub.2, Ethyl acetate:Methanol=1/0 to 2/1). The residue was
then purified by prep-HPLC [water (0.1% TFA)-ACN]. Compound
chloromethyl-dimethyl-[2-[(2R,3R,4S,5R)-3,4,5-triacetoxytetrahydropyran-2-
-yl]oxyethyl]ammonium (617 mg, 1.18 mmol, 14.6% yield, 100% purity,
I--) was obtained as yellow solid. LCMS: (M+).sup.+: 396.1 .sup.1H
NMR (400 MHz, Methanol-d4) .delta. 5.31 (s, 2H), 5.32-5.22 (m, 1H),
5.06-4.91 (m, 2H), 4.73 (d, J=7.4 Hz, 1H), 4.35-4.26 (m, 1H),
4.19-4.02 (m, 2H), 3.87-3.73 (m, 2H), 3.52 (dd, J=11.7, 9.6 Hz,
1H), 3.29 (s, 6H), 2.15-1.99 (m, 9H).
##STR00181##
Compound 89:
(chloromethyl)dimethyl(2-{[(3R,4S,5S)-3,4,5-tris(acetyloxy)oxan-2-yl]oxy}-
ethyl)azanium
[0641] This compound may be synthesized according to the
experimental procedure described for Compound 325.
##STR00182##
Compound 90:
(fluoromethyl)dimethyl(2-{[(2R,3R,4S,5R)-3,4,5-tris(acetyloxy)oxan-2-yl]o-
xy}ethyl)azanium Iodide
[0642] Step 1:
[0643] To a solution of
(2R,3R,4S,5R)-tetrahydropyran-2,3,4,5-tetrol (50 g, 333.04 mmol, 1
eq) in pyridine (500 mL) was added Ac.sub.2O (204.00 g, 2.00 mol,
187.16 mL, 6 eq). The mixture was stirred at 25.degree. C. for 12
hr. TLC indicated (2R,3R,4S,5R)-tetrahydropyran-2,3,4,5-tetrol was
consumed completely and one new spot formed. The reaction mixture
was concentrated under reduced pressure. The residue was purified
by column chromatography (SiO.sub.2, Petroleum ether/Ethyl
acetate=80/1 to 0/1). Compound
[(3R,4S,5R)-4,5,6-triacetoxytetrahydropyran-3-yl] acetate (100 g,
314.19 mmol, 94.34% yield) was obtained as a yellow oil.
[0644] Step 2:
[0645] To a solution of
[(3R,4S,5R)-4,5,6-triacetoxytetrahydropyran-3-yl] acetate (20 g,
62.84 mmol, 1 eq) in DCM (150 mL) was added dropwise 2-bromoethanol
(11.78 g, 94.26 mmol, 6.69 mL, 1.5 eq) and BF.sub.3.Et.sub.2O
(45.50 g, 314.19 mmol, 39.57 mL, 98% purity, 5 eq) at 0.degree. C.
The mixture was warmed to 25.degree. C. and was stirred at
25.degree. C. for 12 hr. TLC indicated
[(3R,4S,5R)-4,5,6-triacetoxytetrahydropyran-3-yl] acetate was
consumed completely and new spots formed. 5 batches were combined
for work-up. The reaction mixture was quenched by addition of
H.sub.2O (500 mL) at 25.degree. C. and extracted with EtOAc (900
mL, 300 mL*3). The combined organic layers were concentrated under
reduced pressure to give a residue. The residue was purified by
column chromatography (SiO.sub.2, Petroleum ether/Ethyl
acetate=20/1 to 0/1). The crude product was purified by
re-crystallization from ethyl acetate:Petroleum ether (40 ml:30 mL)
at 0.degree. C. for 12h. Compound
[(3R,4S,5R,6S)-4,5-diacetoxy-6-(2-bromoethoxy)tetrahydropyran-3-yl]
acetate (3 g, 7.83 mmol, 2.49% yield) was obtained as a white
solid. The filtrate was purified by prep-HPLC [water (0.1%
TFA)-ACN]. Compound
[(3R,4S,5R,6R)-4,5-diacetoxy-6-(2-bromoethoxy)tetrahydropyran-3-yl]
acetate (7 g, 18.27 mmol, 5.81% yield) was obtained as a colorless
oil.
[0646] Step 3:
[0647] To a solution of
[(3R,4S,5R,6S)-4,5-diacetoxy-6-(2-bromoethoxy)tetrahydropyran-3-yl]
acetate (1 g, 2.61 mmol, 1 eq) in THF (10 mL) was added
Me.sub.2NH.THF (2 M, 7.18 mL, 5.50 eq) and DIEA (674.57 mg, 5.22
mmol, 909.12 uL, 2 eq). The mixture was stirred at 70.degree. C.
for 12 hr. LCMS showed
[(3R,4S,5R,6S)-4,5-diacetoxy-6-(2-bromoethoxy)tetrahydropyran-3-yl]
acetate was consumed completely and the expected mass was detected.
The reaction mixture was concentrated under reduced pressure. The
residue was purified by column chromatography (SiO.sub.2, Ethyl
acetate/Methanol=100/1 to 0/1). Compound
[(3R,4S,5R,6R)-4,5-diacetoxy-6-[2-(dimethylamino)ethoxy]tetrahydropyran-3-
-yl] acetate (500 mg, 1.44 mmol, 55.16% yield) was obtained as a
yellow solid.
[0648] Step 4:
[0649] To a solution of
[(3R,4S,5R,6R)-4,5-diacetoxy-6-[2-(dimethylamino)ethoxy]tetrahydropyran-3-
-yl] acetate (500 mg, 1.44 mmol, 1 eq) in DCM (5 mL) was added
fluoro(iodo)methane (1.15 g, 7.20 mmol, 5 eq) and DIEA (186.04 mg,
1.44 mmol, 250.72 uL, 1 eq). The mixture was stirred at 40.degree.
C. for 3 hr. LCMS showed the reaction was completed and one main
peak with expected mass was detected. The mixture was filtered and
the filter cake was concentrated under reduced pressure to give a
residue. The crude product was washed with ethyl acetate (10 mL).
The mixture was filtered and the filter cake was concentrated under
reduced pressure to give a residue. The title compound (397 mg,
782.59 umol, 54.37% yield, 100% purity, I--) was obtained as a
white solid. LCMS: (M+): 380.1 .sup.1H NMR (400 MHz, Deuterium
Oxide) .delta. 5.35 (s, 1H), 5.24 (s, 1H), 5.14 (t, J=7.6 Hz, 1H),
4.96-4.83 (m, 2H), 4.75 (d, J=6.1 Hz, 1H), 4.21 (d, J=13.8 Hz, 1H),
4.10 (dd, J=12.3, 4.7 Hz, 1H), 3.99 (d, J=13.6 Hz, 1H), 3.70-3.63
(m, 2H), 3.54 (dd, J=12.3, 7.9 Hz, 1H), 3.13 (d, J=2.0 Hz, 6H),
2.06-1.97 (m, 9H).
##STR00183##
Compound 91 and Compound 97:
(fluoromethyl)dimethyl(2-{[(3R,4S,5S)-3,4,5-tris(acetyloxy)oxan-2-yl]oxy}-
ethyl)azanium
[0650] This compound may be synthesized according to the
experimental procedure described for Compound 311.
##STR00184##
Compound 92:
(fluoromethyl)(methyl)(prop-2-yn-1-yl)(2-{[(2R,3R,4S,5R)-3,4,5-tris(acety-
loxy)oxan-2-yl]oxy}ethyl)azanium Iodide
[0651] Step 1:
[0652] To a mixture of (2R,3R,4S,5R)-tetrahydropyran-2,3,4,5-tetrol
(50 g, 333.05 mmol, 1 eq) in pyridine (300 mL) was added acetyl
acetate (204.00 g, 2.00 mol, 187.16 mL, 6 eq) in one portion at
0.degree. C. under N.sub.2. The mixture was heated to 25.degree. C.
and stirred for 12 hours. TLC indicated one new spot formed. The
reaction mixture was concentrated under reduced pressure to give a
residue. The residue was purified by column chromatography
(SiO.sub.2, Petroleum ether/Ethyl acetate=50/1 to 0/1). Compound
[(3R,4S,5R)-4,5,6-triacetoxytetrahydropyran-3-yl] acetate (100 g,
314.19 mmol, 94.34% yield) was obtained as yellow oil.
[0653] Step 2:
[0654] To a solution of [of
[(3R,4S,5R)-4,5,6-triacetoxytetrahydropyran-3-yl] acetate (20 g,
62.84 mmol, 1 eq) in DCM (150 mL) was added dropwise 2-bromoethanol
(11.78 g, 94.26 mmol, 6.69 mL, 1.5 eq) and BF.sub.3.Et.sub.2O
(45.50 g, 314.19 mmol, 39.57 mL, 98% purity, 5 eq) at 0.degree. C.
The mixture was warmed to 25.degree. C. and stirred at 25.degree.
C. for 12 hr. TLC indicated
[(3R,4S,5R)-4,5,6-triacetoxytetrahydropyran-3-yl] acetate was
consumed completely and two new spots formed. The reaction mixture
was quenched by addition of H.sub.2O (500 mL) and extracted with
EtOAc (900 mL, 300 mL*3). The combined organic layers were
concentrated under reduced pressure to give a residue. The residue
was purified by column chromatography (SiO.sub.2, Petroleum
ether/Ethyl acetate=20/1 to 0/1). Two batches were obtained
successively. The batch 1 was further purified by recrystallization
from ethyl acetate:Petroleum etherat 0.degree. C. for 12h. Compound
[(3R,4S,5R,6S)-4,5-diacetoxy-6-(2-bromoethoxy)tetrahydropyran-3-yl]
acetate (3 g, 2.5% yield) was obtained as a white solid. The batch
2 was further purified by prep-HPLC [water (0.1% TFA)-ACN].
Compound
[(3R,4S,5R,6R)-4,5-diacetoxy-6-(2-bromoethoxy)tetrahydropyran-3-yl]
acetate (7 g, 5.8% yield) was obtained as a colorless oil.
[0655] Step 3:
[0656] To a solution of
[(3R,4S,5R,6S)-4,5-diacetoxy-6-(2-bromoethoxy)tetrahydropyran-3-yl]
acetate (1.8 g, 4.70 mmol, 1 eq) in THF (20 mL) was added
N-methylprop-2-yn-1-amine (1.79 g, 25.84 mmol, 2.15 mL, 5.5 eq) and
DIPEA (1.21 g, 9.39 mmol, 1.64 mL, 2 eq). The mixture was stirred
at 70.degree. C. for 12 hr. LCMS showed
[(3R,4S,5R,6S)-4,5-diacetoxy-6-(2-bromoethoxy)tetrahydropyran-3-yl]
acetate was consumed completely and one main peak with expected
mass was detected. The reaction mixture was concentrated under
reduced pressure. The crude product (300 mg) was purified by
prep-HPLC [water (0.1% TFA)-ACN]. Compound
[(3R,4S,5R,6R)-4,5-diacetoxy-6-[2-[methyl(prop-2-ynyl)amino]ethoxy]tetrah-
ydropyran-3-yl] acetate (97 mg, 100% purity, TFA) was obtained as
white solid. The other crude
product[(3R,4S,5R,6R)-4,5-diacetoxy-6-[2-[methyl(prop-2-ynyl)amino]ethoxy-
]tetrahydropyran-3-yl] acetate (1.4 g) was used into the next step
without further purification. LCMS: (M+H).sup.+: 372.1
[0657] Step 4:
[0658] To a solution of
[(3R,4S,5R,6R)-4,5-diacetoxy-6-[2-[methyl(prop-2-ynyl)amino]ethoxy]tetrah-
ydropyran-3-yl] acetate (800 mg, 2.15 mmol, 1 eq) in DCM (8 mL) was
added fluoro(iodo)methane (1.72 g, 10.77 mmol, 5 eq) and DIPEA
(278.40 mg, 2.15 mmol, 375.21 uL, 1 eq). The mixture was stirred at
40.degree. C. for 3 hr. LCMS showed
[(3R,4S,5R,6R)-4,5-diacetoxy-6-[2-[methyl(prop-2-ynyl)amino]ethoxy]tetrah-
ydropyran-3-yl] acetate was consumed completely and expected mass
was detected. The reaction mixture was concentrated under reduced
pressure. The residue was purified by prep-HPLC [water (0.10%
TFA)-ACN]. The title compound (234 mg, 20.5% yield, 100% purity,
I--) was obtained as yellow oil. LCMS:(M+): 404.1 .delta. 1H NMR
(400 MHz, Methanol-d4) .delta. 5.65 (s, 1H), 5.54 (s, 1H), 5.26 (t,
J=9.1 Hz, 1H), 5.03-4.91 (m, 2H), 4.73 (d, J=7.3, 0.7 Hz, 1H),
4.57-4.51 (m, 2H), 4.38-4.27 (m, 1H), 4.19-4.05 (m, 2H), 3.93-3.79
(m, 2H), 3.66 (t, J=2.6 Hz, 1H), 3.52 (dd, J=11.8, 9.6 Hz, 1H),
3.30 (d, J=2.1 Hz, 3H), 2.12-2.01 (m, 9H).
##STR00185##
Compound 93:
(fluoromethyl)(methyl)(prop-2-yn-1-yl)(2-{[(3R,4S,5S)-3,4,5-tris(acetylox-
y)oxan-2-yl]oxy}ethyl)azanium
[0659] This compound may be synthesized according to the
experimental procedure described for Compound 309.
##STR00186##
##STR00187##
Compound 94:
(chloromethyl)dimethyl(2-{[(2S,3R,4S,5R)-3,4,5-tris(acetyloxy)oxan-2-yl]o-
xy}ethyl)azanium Iodide
[0660] Step 1:
[0661] To a mixture of (2R,3R,4S,5R)-tetrahydropyran-2,3,4,5-tetrol
(50 g, 333.04 mmol, 1 eq) in pyridine (300 mL) was added acetyl
acetate (204.00 g, 2.00 mol, 187.16 mL, 6 eq) in one portion at
0.degree. C. under N.sub.2. The mixture was heated to 25.degree. C.
and stirred for 12 hours. TLC indicated one new spot. The reaction
was clean according to TLC. The reaction mixture was concentrated
under reduced pressure to give a residue. The residue was purified
by column chromatography (SiO.sub.2, Petroleum ether/Ethyl
acetate=50/1 to 0/1). Compound
[(3R,4S,5R)-4,5,6-triacetoxytetrahydropyran-3-yl] acetate (80 g,
251.35 mmol, 75.47% yield) was obtained as yellow oil.
[0662] Step 2:
[0663] To a mixture of
[(3R,4S,5R)-4,5,6-triacetoxytetrahydropyran-3-yl] acetate (20 g,
62.84 mmol, 1 eq) in DCM (200 mL) was added 2-bromoethanol (11.78
g, 94.26 mmol, 6.69 mL, 1.5 eq) and BF.sub.3.Et.sub.2O (9.10 g,
62.84 mmol, 7.91 mL, 98% purity, 1 eq) in one portion at 0.degree.
C. under N.sub.2. The mixture was heated to 25.degree. C. and
stirred for 12 hours. TLC indicated
[(3R,4S,5R)-4,5,6-triacetoxytetrahydropyran-3-yl] acetate was
consumed and one new spot formed. The reaction mixture was quenched
by addition of H.sub.2O (100 mL), and then diluted with H.sub.2O
(200 mL) and extracted with ethyl acetate (200 mL*3). The combined
organic layers were dried over Na.sub.2SO.sub.4, filtered, and the
filtrate was concentrated under reduced pressure to give a residue.
The crude product was purified by re-crystallization from ethyl
acetate: Petroleum ether (40 ml:30 mL) at 0.degree. C. for 12h. The
mixture was filtered and the filter cake was concentrated under
reduced pressure to give a residue. The filtrate was purified by
prep-HPLC [water (0.1% TFA)-ACN].
[(3R,4S,5R,6S)-4,5-diacetoxy-6-(2-bromoethoxy)tetrahydropyran-3-yl]
acetate (3.4 g, 8.87 mmol, 14.12% yield) was obtained as a white
solid. Compound
[(3R,4S,5R,6R)-4,5-diacetoxy-6-(2-bromoethoxy)tetrahydropyran-3--
yl] acetate (3.5 g, 9.13 mmol, 14.54% yield) was obtained as yellow
oil.
[0664] Step 3:
[0665] To a mixture of
[(3R,4S,5R,6S)-4,5-diacetoxy-6-(2-bromoethoxy)tetrahydropyran-3-yl]
acetate (3.4 g, 8.87 mmol, 1 eq) in THF (30 mL) was added NaI (1.33
g, 8.87 mmol, 1 eq) and Na.sub.2CO.sub.3 (1.88 g, 17.75 mmol, 2 eq)
in one portion at 25.degree. C. under N.sub.2. Then
N-methylmethanamine (2 M, 24.40 mL, 5.5 eq) was added. The mixture
was heated to 70.degree. C. and stirred for 12 hours. TLC indicated
[(3R,4S,5R,6S)-4,5-diacetoxy-6-(2-bromoethoxy)tetrahydropyran-3-yl]
acetate was consumed incompletely and one new spot formed. LCMS
showed
[(3R,4S,5R,6S)-4,5-diacetoxy-6-(2-bromoethoxy)tetrahydropyran-3-yl]
acetate was consumed and one main peak with desired m/z was
detected. The reaction mixture was filtered and concentrated under
reduced pressure to give a residue. The residue was purified by
column chromatography (SiO.sub.2, Ethyl acetate:Methanol=1/0 to
1/1). The 200 mg crude product was purified by prep-HPLC [water
(0.1% TFA)-ACN]. Compound
[(3R,4S,5R,6R)-4,5-diacetoxy-6-[2-(dimethylamino)ethoxy]tetrahydropyran-3-
-yl] acetate (3.1 g, crude) was obtained as yellow oil. Compound
[(3R,4S,5R,6R)-4,5-diacetoxy-6-[2-(dimethylamino)ethoxy]tetrahydropyran-3-
-yl] acetate (14.7 mg, 98% purity) was obtained as white solid.
LCMS: (M+H).sup.+: 348.1
[0666] Step 4:
[0667] To a mixture of
[(3R,4S,5R,6R)-4,5-diacetoxy-6-(2-bromoethoxy)tetrahydropyran-3-yl]
acetate (3.5 g, 9.13 mmol, 1 eq) in THF (30 mL) was added NaI (1.37
g, 9.13 mmol, 1 eq), Na.sub.2CO.sub.3 (1.94 g, 18.27 mmol, 2 eq)
and N-methylmethanamine (2 M, 25.12 mL, 5.5 eq) in one portion at
25.degree. C. under N.sub.2. The mixture was heated to 70.degree.
C. and stirred for 12 hours. LCMS showed
[(3R,4S,5R,6R)-4,5-diacetoxy-6-(2-bromoethoxy)tetrahydropyran-3-yl]
acetate was consumed and one main peak with desired m/z was
detected. The reaction mixture was filtered and concentrated under
reduced pressure to give a residue. The 500 mg crude product was
purified by prep-HPLC [water (0.10% TFA)-ACN]. Compound
[(3R,4S,5R,6S)-4,5-diacetoxy-6-[2-(dimethylamino)ethoxy]tetrahydropyran-3-
-yl] acetate (34 mg, 73.69 umol, 5.12% yield, 100% purity, TFA) was
obtained as colorless oil. Compound
[(3R,4S,5R,6S)-4,5-diacetoxy-6-[2-(dimethylamino)ethoxy]tetrahydropyran-3-
-yl] acetate (4.2 g, crude) was obtained as yellow oil and the
crude product was used into the next step without further
purification. LCMS: (M+H).sup.+: 348.1
[0668] Step 5:
[0669] To a mixture of
[(3R,4S,5R,6S)-4,5-diacetoxy-6-[2-(dimethylamino)ethoxy]tetrahydropyran-3-
-yl] acetate (4.2 g, 12.09 mmol, 1 eq) in acetone (40 mL) was added
chloro(iodo)methane (10.66 g, 60.46 mmol, 4.39 mL, 5 eq) in one
portion at 25.degree. C. under N.sub.2. The mixture was heated to
90.degree. C. and stirred for 2 hours. TLC indicated
[(3R,4S,5R,6S)-4,5-diacetoxy-6-[2-(dimethylamino)ethoxy]tetrahydropyran-3-
-yl] acetate was consumed and one new spot formed. LCMS showed one
main peak with desired m/z. The reaction mixture was concentrated
under reduced pressure to give a residue. The residue was purified
by column chromatography (SiO.sub.2, Ethyl acetate: Methanol=1/0 to
2/1). Then the residue was purified by prep-HPLC [water (0.1%
TFA)-ACN]. The title compound (26 mg, 49.64 umol, 9.85% yield, I--)
was obtained as yellow oil. LCMS: (M.sup.+).sup.+: 396.0 .sup.1H
NMR (400 MHz, Methanol-d4) .delta. 5.45-5.25 (m, 4H), 5.18 (s, 1H),
4.42-4.31 (m, 2H), 4.30-4.15 (m, 2H), 4.07-3.97 (m, 1H), 3.82 (t,
J=4.7 Hz, 2H), 3.32 (s, 6H), 2.16-2.04 (m, 9H).
##STR00188##
Compound 95:
(fluoromethyl)dimethyl(2-{[(3R,4S,5S,6R)-3,4,5-tris(acetyloxy)-6-methylox-
an-2-yl]oxy}ethyl)azanium
[0670] This compound may be synthesized according to the
experimental procedure described for Compound 311.
##STR00189##
Compound 96:
(fluoromethyl)(methyl)(prop-2-yn-1-yl)(2-{[(2S,3R,4S,5R)-3,4,5-tris(acety-
loxy)oxan-2-yl]oxy}ethyl)azanium Iodide
[0671] Step 1:
[0672] To a mixture of (2R,3R,4S,5R)-tetrahydropyran-2,3,4,5-tetrol
(50 g, 333.05 mmol, 1 eq) in pyridine (300 mL) was added acetyl
acetate (204.00 g, 2.00 mol, 187.16 mL, 6 eq) in one portion at
0.degree. C. under N.sub.2. The mixture was heated to 25.degree. C.
and stirred for 12 hours. TLC indicated one new spot formed. The
reaction mixture was concentrated under reduced pressure to give a
residue. The residue was purified by column chromatography
(SiO.sub.2, Petroleum ether/Ethyl acetate=50/1 to 0/1). Compound
[(3R,4S,5R)-4,5,6-triacetoxytetrahydropyran-3-yl] acetate (100 g,
314.19 mmol, 94.34% yield) was obtained as yellow oil.
[0673] Step 2:
[0674] To a solution of [of
[(3R,4S,5R)-4,5,6-triacetoxytetrahydropyran-3-yl] acetate (20 g,
62.84 mmol, 1 eq) in DCM (150 mL) was added dropwise 2-bromoethanol
(11.78 g, 94.26 mmol, 6.69 mL, 1.5 eq) and BF.sub.3.Et.sub.2O
(45.50 g, 314.19 mmol, 39.57 mL, 98% purity, 5 eq) at 0.degree. C.
The mixture was warmed to 25.degree. C. and stirred at 25.degree.
C. for 12 hr. TLC indicated
[(3R,4S,5R)-4,5,6-triacetoxytetrahydropyran-3-yl] acetate was
consumed completely and two new spots formed. The reaction mixture
was quenched by addition of H.sub.2O (500 mL) and extracted with
EtOAc (900 mL, 300 mL*3). The combined organic layers were
concentrated under reduced pressure to give a residue. The residue
was purified by column chromatography (SiO.sub.2, Petroleum
ether/Ethyl acetate=20/1 to 0/1). Two batches were obtained
successively. The batch 1 was further purified by
re-crystallization from ethyl acetate:Petroleum ether at 0.degree.
C. for 12h. Compound
[(3R,4S,5R,6S)-4,5-diacetoxy-6-(2-bromoethoxy)tetrahydropyran-3-yl]
acetate (3 g, 2.5% yield) was obtained as a white solid. The batch
2 was further purified by prep-HPLC [water (0.10% TFA)-ACN]. The
title compound (7 g, 5.8% yield) was obtained as colorless oil.
[0675] Step 3:
[0676] To a solution of
[(3R,4S,5R,6R)-4,5-diacetoxy-6-(2-bromoethoxy)tetrahydropyran-3-yl]
acetate (1.50 g, 3.91 mmol, 1 eq) in THF (15 mL) was added
N-methylprop-2-yn-1-amine (1.49 g, 21.53 mmol, 1.79 mL, 5.5 eq) and
DIPEA (1.01 g, 7.83 mmol, 1.36 mL, 2 eq). The mixture was stirred
at 70.degree. C. for 12 hr. LCMS showed
[(3R,4S,5R,6R)-4,5-diacetoxy-6-(2-bromoethoxy)tetrahydropyran-3-yl]
acetate was consumed completely and one main peak with expected
mass was detected. The reaction mixture was concentrated under
reduced pressure. The crude product (300 mg) was purified by
prep-HPLC [water (0.1% TFA)-ACN].
Compound[(3R,4S,5R,6S)-4,5-diacetoxy-6-[2-[methyl(prop-2-ynyl)amino]ethox-
y]tetrahydropyran-3-yl] acetate (110 mg, 100% purity, TFA) was
obtained as yellow oil. The other crude product
[(3R,4S,5R,6S)-4,5-diacetoxy-6-[2-[methyl(prop-2-ynyl)amino]ethoxy]tetrah-
ydropyran-3-yl] acetate (600 mg, crude) was used in the next step
without further purification. LCMS: (M+H).sup.+: 372.1
[0677] Step 4:
[0678] To a solution of
[(3R,4S,5R,6S)-4,5-diacetoxy-6-[2-[methyl(prop-2-ynyl)amino]ethoxy]tetrah-
ydropyran-3-yl] acetate (300 mg, 807.80 umol, 1 eq) in DCM (3 mL)
was added fluoro(iodo)methane (645.95 mg, 4.04 mmol, 5 eq) and
DIPEA (104.40 mg, 807.80 umol, 140.70 uL, 1 eq). The mixture was
stirred at 40.degree. C. for 3 hr. LCMS showed
[(3R,4S,5R,6S)-4,5-diacetoxy-6-[2-[methyl(prop-2-ynyl)amino]ethoxy]tetrah-
ydropyran-3-yl] acetate was consumed completely and the expected
mass was detected. The reaction mixture was concentrated under
reduced pressure. The residue was purified by prep-HPLC [water
(0.10% TFA)-ACN]. Compound
fluoromethyl-methyl-prop-2-ynyl-[2-[(2R,3R,4S,5R)-3,4,5-triacetoxytetrahy-
dropyran-2-yl]oxyethyl]ammonium (95 mg, 29.1% yield, 100% purity,
I--) was obtained as yellow oil. LCMS: (M+): 404.1 .sup.1H NMR (400
MHz, Methanol-d4) .delta. 5.72 (d, J=2.1 Hz, 1H), 5.61 (d, J=2.1
Hz, 1H), 5.41 (td, J=9.8, 2.5 Hz, 1H), 5.11-5.05 (m, 1H), 5.05-4.94
(m, 2H), 4.61 (dd, J=2.8, 1.4 Hz, 2H), 4.19 (d, J=12.4 Hz, 1H),
4.07-3.91 (m, 2H), 3.91-3.83 (m, 2H), 3.77-3.61 (m, 2H), 3.34 (d,
J=2.1 Hz, 3H), 2.14-1.97 (m, 9H).
Compound 97:
(fluoromethyl)dimethyl(2-{[(2S,3R,4S,5R)-3,4,5-tris(acetyloxy)oxan-2-yl]o-
xy}ethyl)azanium Iodide
[0679] Step 1:
[0680] To a solution of
(2R,3R,4S,5R)-tetrahydropyran-2,3,4,5-tetrol (50 g, 333.04 mmol, 1
eq) in pyridine (500 mL) was added Ac.sub.2O (204.00 g, 2.00 mol,
187.16 mL, 6 eq). The mixture was stirred at 25.degree. C. for 12
hr. TLC indicated (2R,3R,4S,5R)-tetrahydropyran-2,3,4,5-tetrol was
consumed completely and one new spot formed. The reaction mixture
was concentrated under reduced pressure. The residue was purified
by column chromatography (SiO.sub.2, Petroleum ether/Ethyl
acetate=80/1 to 0/1). Compound
[(3R,4S,5R)-4,5,6-triacetoxytetrahydropyran-3-yl] acetate (100 g,
314.19 mmol, 94.34% yield) was obtained as a yellow oil.
[0681] Step 2:
[0682] To a solution of
[(3R,4S,5R)-4,5,6-triacetoxytetrahydropyran-3-yl] acetate (20 g,
62.84 mmol, 1 eq) in DCM (150 mL) was added dropwise 2-bromoethanol
(11.78 g, 94.26 mmol, 6.69 mL, 1.5 eq) and BF.sub.3.Et.sub.2O
(45.50 g, 314.19 mmol, 39.57 mL, 98% purity, 5 eq) at 0.degree. C.
The mixture was warmed to 25.degree. C. and was stirred at
25.degree. C. for 12 hr. TLC indicated
[(3R,4S,5R)-4,5,6-triacetoxytetrahydropyran-3-yl] acetate was
consumed completely and new spots formed. 5 batches were combined
for work-up. The reaction mixture was quenched by addition of
H.sub.2O (500 mL) at 25.degree. C. and extracted with EtOAc (900
mL, 300 mL*3). The combined organic layers were concentrated under
reduced pressure to give a residue. The residue was purified by
column chromatography (SiO.sub.2, Petroleum ether/Ethyl
acetate=20/1 to 0/1). The crude product was purified by
re-crystallization from ethyl acetate:Petroleum ether (40 ml:30 mL)
at 0.degree. C. for 12h. Compound
[(3R,4S,5R,6S)-4,5-diacetoxy-6-(2-bromoethoxy)tetrahydropyran-3-yl]
acetate (3 g, 7.83 mmol, 2.49% yield) was obtained as a white
solid. The filtrate was purified by prep-HPLC [water (0.1%
TFA)-ACN]. Compound
[(3R,4S,5R,6R)-4,5-diacetoxy-6-(2-bromoethoxy)tetrahydropyran-3-yl]
acetate (7 g, 18.27 mmol, 5.81% yield) was obtained as a colorless
oil.
[0683] Step 3:
[0684] To a mixture of
[(3R,4S,5R,6R)-4,5-diacetoxy-6-(2-bromoethoxy)tetrahydropyran-3-yl]
acetate (0.5 g, 1.30 mmol, 1 eq) in THF (5 mL) was added MeNH.sub.2
(810.49 mg, 5.22 mmol, 20% purity, 4 eq) and NaI (195.59 mg, 1.30
mmol, 1 eq) in one portion at 25.degree. C. under N.sub.2. The
mixture was stirred at 25.degree. C. for 12 hours. LCMS showed the
expected mass was detected. The reaction mixture was concentrated
under reduced pressure to give a residue. The residue was purified
by column chromatography (SiO.sub.2, Ethyl acetate/MeOH=0/1 to
10/1). The crude product
[(3R,4S,5R,6S)-4,5-diacetoxy-6-[2-(dimethylamino)ethoxy]tetrahydropyran-3-
-yl] acetate (0.38 g, 1.09 mmol, 83.84% yield) was obtained as
yellow oil, and used into the next step without further
purification.
[0685] Step 4:
[0686] To a mixture of
[(3R,4S,5R,6S)-4,5-diacetoxy-6-[2-(dimethylamino)ethoxy]tetrahydropyran-3-
-yl] acetate (370 mg, 1.07 mmol, 1 eq) in DCM (5 mL) was added
fluoro(iodo)methane (851.76 mg, 5.33 mmol, 5 eq). The mixture was
stirred at 40.degree. C. for 3 hours. LCMS showed desired mass was
detected. The reaction mixture was filtered and concentrated under
reduced pressure to give a residue. The residue was purified by
prep-HPLC [water (0.10% TFA)-ACN]. The title compound (14 mg, 36.07
umol, 3.39% yield, 98% purity) was obtained as colorless oil. (M+):
380.1 .sup.1H NMR (400 MHz, Methanol-d4) .delta. 5.58 (s, 1H), 5.47
(s, 1H), 5.42 (t, J=9.7 Hz, 1H), 5.11 (d, J=3.7 Hz, 1H), 5.07-4.96
(m, 2H), 4.20 (d, J=13.2 Hz, 1H), 4.02 (t, J=11.0 Hz, 1H),
3.94-3.82 (m, 2H), 3.82-3.74 (m, 1H), 3.68 (t, J=10.8 Hz, 1H), 3.31
(d, J=2.1 Hz, 6H), 3.04-2.94 (m, 1H), 2.09-2.01 (m, 9H).
##STR00190##
Compound 98:
(fluoromethyl)dimethyl(2-{[(2R,3R,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxyme-
thyl)oxan-2-yl]oxy}ethyl)azanium Chloride
[0687]
N-(fluoromethyl)-N,N-dimethyl-2-(((3R,4S,5R,6R)-3,4,5-triacetoxy-6--
(acetoxymethyl)tetrahydro-2H-pyran-2-yl)oxy)ethan-1-aminium iodide
(1.00 g, 1 Eq, 1.73 mmol) is dissolved in methanol (3 mL), followed
by addition of 0.5M NaOMe in methanol (9.32 mg, 0.35 mL, 0.1 Eq,
173 .mu.mol). The reaction was stirred at room temperature for a
few hours, then concentrated by rotary evaporation. The sample was
redissolved in MeOH, then purified by column chromatography (C18AQ
flash column, elutes at 100% Water with 0.1% TFA). The pure
fractions were lyophilized and collected. The product was
redissolved in water and stirred with Amberlyst IRA-400 Cl-resin
(300 mg resin/100 mg of TFA salt), then lyophilized to yield the
title compound (0.400 g, 72.5%)). LCMS M+: 284.2. .sup.1H NMR (400
MHz, Deuterium Oxide) .delta. 5.39 (s, 1H), 5.28 (s, 1H), 4.41 (dd,
J=8.0, 0.9 Hz, 1H), 4.35-4.25 (m, 1H), 4.09-3.99 (m, 1H), 3.87-3.79
(m, 1H), 3.74-3.59 (m, 3H), 3.45-3.33 (m, 2H), 3.33-3.27 (m, 1H),
3.31-3.19 (m, 1H), 3.16 (d, J=2.1 Hz, 6H).
##STR00191##
Compound 99:
(chloromethyl)dimethyl(2-{[(2R,3R,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxyme-
thyl)oxan-2-yl]oxy}ethyl)azanium Chloride
[0688]
N,N-dimethyl-N-(2-(((3R,4S,5R,6R)-3,4,5-triacetoxy-6-(acetoxymethyl-
)tetrahydro-2H-pyran-2-yl)oxy)ethyl)cyclopropanaminium iodide (1.00
g, 1 Eq, 1.70 mmol, as synthesized in Compound 310) is dissolved in
methanol (3 mL), followed by addition of 0.5M NaOMe in methanol
(18.4 mg, 0.68 mL, 0.2 Eq, 340 .mu.mol). The reaction was stirred
at room temperature for a few hours, then concentrated by rotary
evaporation. The sample was redissolved in MeOH, then purified by
column chromatography (C18AQ flash column, elutes at 100% Water
with 0.1% TFA). The pure fractions were lyophilized and collected.
The product was redissolved in water and stirred with Amberlyst
IRA-400 Cl-resin (300 mg resin/100 mg of TFA salt), then
lyophilized to yield the title compound (0.200 g, 35.8%). LCMS M+:
300.2. .sup.1H NMR (400 MHz, Deuterium Oxide) .delta. 5.15 (s, 2H),
4.41 (d, J=7.9 Hz, 1H), 4.35-4.25 (m, 1H), 4.09-3.99 (m, 1H), 3.83
(dd, J=12.3, 2.2 Hz, 1H), 3.79-3.69 (m, 2H), 3.63 (dd, J=12.3, 5.8
Hz, 1H), 3.44-3.33 (m, 2H), 3.33-3.17 (m, 2H), 3.20 (s, 6H).
##STR00192##
Compound 100:
N,N-dimethyl-N-(2-{[(2R,3R,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)ox-
an-2-yl]oxy}ethyl)cyclopropanaminium Chloride
[0689]
N,N-dimethyl-N-(2-(((3R,4S,5R,6R)-3,4,5-triacetoxy-6-(acetoxymethyl-
)tetrahydro-2H-pyran-2-yl)oxy)ethyl)cyclopropanaminium iodide (1.00
g, 1 Eq, 1.70 mmol, as synthesized in Compound 312) is dissolved in
methanol (3 mL), followed by addition of 0.5M NaOMe in methanol
(18.4 mg, 0.68 mL, 0.2 Eq, 340 .mu.mol). The reaction was stirred
at room temperature for a few hours, then concentrated by rotary
evaporation. The sample was redissolved in MeOH, then purified by
column chromatography (C18AQ flash column, elutes at 100% Water
with 0.1% TFA). The pure fractions were lyophilized and collected.
The product was redissolved in water and stirred with Amberlyst
IRA-400 Cl-resin (300 mg resin/100 mg of TFA salt), then
lyophilized to yield the title compound (0.200 g, 35.8%). LCMS M+:
292.3. .sup.1H NMR (400 MHz, Deuterium Oxide) .delta. 4.40 (dd,
J=8.0, 1.0 Hz, 1H), 4.37-4.28 (m, 1H), 4.12-4.02 (m, 1H), 3.82 (dd,
J=12.4, 2.1 Hz, 1H), 3.69-3.57 (m, 3H), 3.44-3.33 (m, 2H),
3.33-3.24 (m, 1H), 3.24-3.16 (m, 1H), 3.16-3.08 (m, 1H), 2.93 (d,
J=2.0 Hz, 6H), 1.18-1.09 (m, 2H), 0.91-0.80 (m, 2H).
##STR00193##
Compound 101:
N,N-dimethyl-N-(2-{[(2S,3R,4S,5R,6R)-3,4,5-tris(acetyloxy)-6-[(acetyloxy)-
methyl]oxan-2-yl]oxy}ethyl)cyclopropanaminium
[0690] This compound may be synthesized according to the
experimental procedure described for Compound 312.
##STR00194##
Compound 103:
N,N-dimethyl-N-(prop-2-yn-1-yl)-2-((((2R,3R,4S,5R)-3,4,5-triacetoxy-6-met-
hoxytetrahydro-2H-pyran-2-yl)methoxy)carbonyl)prop-2-en-1-aminium
2,2,2-trifluoroacetate
[0691] Compound
2-carboxy-N,N-dimethyl-N-(prop-2-yn-1-yl)prop-2-en-1-aminium (315
mg, 2 Eq, 1.87 mmol) and EDC (359 mg, 2 Eq, 1.87 mmol) were stirred
in DMF (3 mL). Next
(2R,3R,4S,5R)-2-(hydroxymethyl)-6-methoxytetrahydro-2H-pyran-3,-
4,5-triyl triacetate (300 mg, 1 Eq, 937 .mu.mol) was added to the
solution and the reaction was stirred at room temperature
overnight. The reaction mixture was injected directly onto reverse
phase C18 column chromatography (5% ACN in water with 0.1% TFA to
100% ACN), and product containing fractions were lyophilized to
yield
N,N-dimethyl-N-(prop-2-yn-1-yl)-2-((((2R,3R,4S,5R)-3,4,5-triacetoxy-6-met-
hoxytetrahydro-2H-pyran-2-yl)methoxy)carbonyl)prop-2-en-1-aminium
2,2,2-trifluoroacetate (18 mg, 3.3%). LCMS M+: 470.1. .sup.1H NMR
(400 MHz, DMSO-d6) .delta. 6.90 (s, 1H), 6.52 (s, 1H), 5.31 (t,
J=9.7 Hz, 1H), 5.07 (t, J=9.9 Hz, 1H), 4.96-4.83 (m, 2H), 4.37 (d,
J=2.5 Hz, 2H), 4.33-4.21 (m, 4H), 4.11-4.05 (m, 1H), 4.00 (dt,
J=10.3, 3.3 Hz, 1H), 3.34 (s, 3H), 3.03 (s, 6H), 2.02-1.93 (m,
9H).
##STR00195##
Compound 104:
N,N-dimethyl-N-(prop-2-yn-1-yl)-2-((((2R,3R,4S,5R)-3,4,5,6-tetraacetoxyte-
trahydro-2H-pyran-2-yl)methoxy)carbonyl)prop-2-en-1-aminium2,2,2-trifluoro-
acetate
[0692] 2-carboxy-N,N-dimethyl-N-(prop-2-yn-1-yl)prop-2-en-1-aminium
(193 mg, 2 Eq, 1.15 mmol) and EDC (220 mg, 2 Eq, 1.15 mmol) were
stirred in DMF (3 mL). Next
(3R,4S,5R,6R)-6-(hydroxymethyl)tetrahydro-2H-pyran-2,3,4,5-tetrayltetraac-
etate (200 mg, 1 Eq, 574 mol) was added to the solution and the
reaction was stirred at room temperature overnight. The reaction
mixture was injected directly onto reverse phase C18 column
chromatography (5% ACN in water with 0.1% TFA to 100% ACN; two
column purifications were performed), and product containing
fractions were lyophilized to yield
N,N-dimethyl-N-(prop-2-yn-1-yl)-2-((((2R,3R,4S,5R)-3,4,5,6-tetraacetoxyte-
trahydro-2H-pyran-2-yl)methoxy)carbonyl)prop-2-en-1-aminium2,2,2-trifluoro-
acetate (33 mg, 9.4%). LCMS M+: 498.1. .sup.1HNMR (400 MHz,
DMSO-d6) .delta. 6.88 (s, 1H), 6.51 (s, 1H), 5.94 (d, J=8.3 Hz,
1H), 5.45 (t, J=9.6 Hz, 1H), 5.08 (t, J=9.4 Hz, 1H), 5.00 (dd,
J=9.7, 8.3 Hz, 1H), 4.36 (d, J=2.5 Hz, 2H), 4.32-4.16 (m, 5H), 4.08
(t, J=2.4 Hz, 1H), 3.02 (s, 6H), 2.11-1.90 (m, 12H).
##STR00196##
Compound 105:
N,N-dimethyl-N-(2-(((3R,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetra-
hydro-2H-pyran-2-yl)oxy)ethyl)prop-2-yn-1-aminium
2,2,2-trifluoroacetate
[0693] Step 1:
[0694] To a solution of compound 1 (200 g, 512 mmol, 1.00 eq) in
DCM (1000 mL) was added 4A molecular sieves (100 g). 2-iodoethanol
(88.1 g, 512 mmol, 40.0 mL, 1.00 eq) was added to the suspension
drop-wise at 0.degree. C. and the resulting suspension was stirred
at 0.degree. C. for 30 mins. BF.sub.3.Et.sub.2O (218 g, 1.54 mol,
190 mL, 3.00 eq) was added to the suspension drop-wise at 0.degree.
C. under N.sub.2. After addition, the suspension was stirred at
20.degree. C. for 18 hrs. TLC (Petroleum ether:Ethyl acetate=2:1,
R.sub.f of material=0.4) showed the reaction was completed.
K.sub.2CO.sub.3 (280 g) was added to the suspension in one portion,
and the suspension was stirred for 30 mins. The suspension was
filtered. The filtrate was washed by water (1500 mL.times.2)
followed by brine (1000 mL). The organic layer was dried over
Na.sub.2SO.sub.4, filtered and concentrated under vacuum to give a
crude product. The crude product was purified by column
chromatography (SiO.sub.2, Petroleum ether/Ethyl acetate=100/1 to
5/1) to give a solid. The solid was re-crystallized from
MTBE/Petroleum ether (3:1, 300 mL) to give compound 2 (195 g, 388
mmol, 37.9% yield) as a white solid.
[0695] Step 2:
[0696] To a solution of compound 2 (80 g, 159 mmol, 1.00 eq) in
acetone (500 mL) was added compound 2A (19.9 g, 239 mmol, 25.3 mL,
1.50 eq). After addition, the solution was stirred at 40.degree. C.
for 16 hrs. TLC (Petroleum ether:Ethyl acetate=2:1, Rf of
material=0.5) showed the reaction was completed. The solution was
cooled to 0.degree. C. and solid precipitated out. The solid was
filtered, collected and concentrated under vacuum to give 3 (100 g,
171 mmol, 53.6% yield) as a white solid.
[0697] Step 3:
[0698]
N,N-dimethyl-N-(2-(((3R,4S,5R,6R)-3,4,5-triacetoxy-6-(acetoxymethyl-
)tetrahydro-2H-pyran-2-yl)oxy)ethyl)prop-2-yn-1-aminium iodide
(3.00 g, 1 Eq, 5.12 mmol) is dissolved in methanol (3 mL), followed
by addition of 0.5M NaOMe in methanol (sodium methoxide (277 mg, 10
mL, 1 Eq, 5.12 mmol)). The reaction was stirred at room temperature
for a few hours, then concentrated by rotary evaporation. The
sample was dissolved in water, then purified by column
chromatography (C18AQ flash column, elutes at 100% Water with 0.1%
TFA). The pure fractions were lyophilized to yield product
N,N-dimethyl-N-(2-(((3R,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetra-
hydro-2H-pyran-2-yl)oxy)ethyl)prop-2-yn-1-aminium
2,2,2-trifluoroacetate (590 mg, 28.5%)).
[0699] Step 4:
[0700] Compound
N,N-dimethyl-N-(2-(((2R,3R,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)te-
trahydro-2H-pyran-2-yl)oxy)ethyl)prop-2-yn-1-aminium (0.300 g, 1
Eq, 1.03 mmol) was stirred in acetic-2,2,2-d3 anhydride (1.12 g,
977 .mu.L, 10 Eq, 10.3 mmol) and pyridine (817 mg, 836 .mu.L, 10
Eq, 10.3 mmol). The reaction was diluted in dichloromethane then
purified by column chromatography (100% DCM to 10% MeOH in DCM;
product elutes around 8% MeOH in DCM). Fractions containing product
were dried in vacuo to yield
N,N-dimethyl-N-(2-(((2R,3R,4S,5R,6R)-3,4,5-tris(acetoxy-d3)-6-((acetoxy-d-
3)methyl)tetrahydro-2H-pyran-2-yl)oxy)ethyl)prop-2-yn-1-aminium
2,2,2-trifluoroacetate (75 mg, 12%). LCMS M+: 470.4. .sup.1H NMR
(400 MHz, DMSO-d6) .delta. 5.26 (t, J=9.5 Hz, 1H), 4.99-4.74 (m,
3H), 4.36 (d, J=2.5 Hz, 2H), 4.24-3.94 (m, 6H), 3.61 (t, J=5.0 Hz,
2H), 3.08 (s, 6H).
##STR00197##
Compound 106:
N,N-dimethyl-N-(2-(((3R,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetra-
hydro-2H-pyran-2-yl)oxy)ethyl)prop-2-yn-1-aminium
2,2,2-trifluoroacetate
[0701] Step 1:
[0702] To a solution of compound 1 (200 g, 512 mmol, 1.00 eq) in
DCM (1000 mL) was added 4A molecular sieves (100 g). 2-iodoethanol
(88.1 g, 512 mmol, 40.0 mL, 1.00 eq) was added to the suspension
drop-wise at 0.degree. C. and the resulting suspension was stirred
at 0.degree. C. for 30 mins. BF.sub.3.Et.sub.2O (218 g, 1.54 mol,
190 mL, 3.00 eq) was added to the suspension drop-wise at 0.degree.
C. under N.sub.2. After addition, the suspension was stirred at
20.degree. C. for 18 hrs. TLC (Petroleum ether:Ethyl acetate=2:1,
Rf of material=0.4) showed the reaction was completed.
K.sub.2CO.sub.3 (280 g) was added to the suspension in one portion,
and the suspension was stirred for 30 mins. The suspension was
filtered. The filtrate was washed by water (1500 mL.times.2)
followed by brine (1000 mL). The organic layer was dried over
Na.sub.2SO.sub.4, filtered and concentrated under vacuum to give a
crude product. The crude product was purified by column
chromatography (SiO.sub.2, Petroleum ether/Ethyl acetate=100/1 to
5/1) to give a solid. The solid was re-crystallized from
MTBE/Petroleum ether (3:1, 300 mL) to give compound 2 (195 g, 388
mmol, 37.9% yield) as a white solid.
[0703] Step 2:
[0704] To a solution of compound 2 (80 g, 159 mmol, 1.00 eq) in
acetone (500 mL) was added compound 2A (19.9 g, 239 mmol, 25.3 mL,
1.50 eq). After addition, the solution was stirred at 40.degree. C.
for 16 hrs. TLC (Petroleum ether:Ethyl acetate=2:1, Rf of
material=0.5) showed the reaction was completed. The solution was
cooled to 0.degree. C. and lots of solid precipitated out. The
solid was filtered, collected and concentrated under vacuum to give
3 (100 g, 171 mmol, 53.6% yield) as a white solid.
[0705] Step 3:
[0706]
N,N-dimethyl-N-(2-(((3R,4S,5R,6R)-3,4,5-triacetoxy-6-(acetoxymethyl-
)tetrahydro-2H-pyran-2-yl)oxy)ethyl)prop-2-yn-1-aminium iodide
(3.00 g, 1 Eq, 5.12 mmol) is dissolved in methanol (3 mL), followed
by addition of 0.5M NaOMe in methanol (sodium methoxide (277 mg, 10
mL, 1 Eq, 5.12 mmol)). The reaction was stirred at room temperature
for a few hours, then concentrated by rotary evaporation. The
sample was dissolved in water, then purified by column
chromatography (C18AQ flash column, elutes at 100% Water with 0.1%
TFA). The pure fractions were lyophilized to yield product
N,N-dimethyl-N-(2-(((3R,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetra-
hydro-2H-pyran-2-yl)oxy)ethyl)prop-2-yn-1-aminium
2,2,2-trifluoroacetate (590 mg, 28.5%)). LCMS M+: 290.2. .sup.1H
NMR (400 MHz, Deuterium Oxide) .delta. 4.43 (d, J=7.9 Hz, 1H),
4.36-4.23 (m, 3H), 4.11-4.00 (m, 1H), 3.84 (dd, J=12.4, 2.2 Hz,
1H), 3.73-3.61 (m, 3H), 3.46-3.35 (m, 2H), 3.34-3.18 (m, 3H), 3.18
(s, 6H).
##STR00198##
Compound 107 and Compound 131:
N-(cyclopropylmethyl)-2-hydroxy-N,N-dimethylethan-1-aminium
Bromide
[0707] To a solution of 2-(dimethylamino) ethanol (1 g, 11.22 mmol,
1.13 mL, 1 eq) in THF (10 mL) was added (bromomethyl) cyclopropane
(1.67 g, 12.34 mmol, 1.18 mL, 1.1 eq) at 20.degree. C. The mixture
was stirred at 20.degree. C. for 12 h. The reaction mixture was
filtered to give a residue. The residue was diluted with H.sub.2O
(10 mL) and extracted with ethyl acetate (10 mL). The water layer
was freeze-drying to give N-(cyclopropylmethyl)-2-hydroxy-N,
N-dimethylethanaminium bromide (170 mg, 758.46 umol, 6.76% yield)
as a white solid. MS: M.sup.+=144.1 .sup.1H NMR (400 MHz, D2O)
.delta. 4.0 (m, 2H), 3.5 (m, 2H), 3.2 (d, 2H), 3.1 (s, 6H), 1.2 (m,
1H), 0.8 (m, 2H), 0.4 (m, 2H) ppm.
##STR00199##
Compound 108:
(2S)-2-(hydroxymethyl)-1-methyl-1-(prop-2-yn-1-yl)pyrrolidin-1-ium
bromide
[0708] To a solution of [(2S)-1-methylpyrrolidin-2-yl] methanol
(200 mg, 1.74 mmol, 206.19 uL, 1 eq) in DCM (5 mL) was added
3-bromoprop-1-yne (247.89 mg, 2.08 mmol, 179.63 uL, 1.2 eq). The
mixture was stirred at 25.degree. C. for 5 h. Some solid
precipitate out. The mixture was filtered and the filter cake was
washed with DCM (5 mL*2) and concentrated to dryness. Compound
(2S)-2-(hydroxymethyl)-1-methyl-1-(prop-2-yn-1-yl) pyrrolidin-1-ium
bromide (346 mg, crude) was obtained as a white solid. .sup.1H NMR
(400 MHz, DMSO) .delta. 5.5 (m, 1H), 4.3 (m, 2H), 3.8 (m, 5H), 3.3
(s 6H), 2.0 (m, 4H) ppm.
##STR00200##
Compound 109:
(2S)-1-(fluoromethyl)-2-(hydroxymethyl)-1-methylpyrrolidin-1-ium
iodide
[0709] To a solution of (3S)-1-methylpyrrolidin-3-ol (200 mg, 1.98
mmol, 201.41 uL, 1 eq) in DCM (5 mL) was added fluoro (iodo)
methane (379.48 mg, 2.37 mmol, 1.2 eq). The mixture was stirred at
25.degree. C. for 5 h. MS showed desired compound was detected. The
reaction mixture was concentrated under reduced pressure to remove
solvent. The residue was dissolved in H.sub.2O (1 mL), washed with
EtOAc (2 mL*2) and lyophilized the aqueous phase to give a residue.
Compound
(2S)-1-(fluoromethyl)-2-(hydroxymethyl)-1-methylpyrrolidin-1-ium
iodide (111 mg, crude) was obtained as a white solid. .sup.1H NMR
(400 MHz, DMSO) .delta. 5.6 (m, 3H), 3.8 (m, 4H), 3.5 (m, 1H), 3.1
(s, 3H), 2.0 (m, 4H) ppm
##STR00201##
Compound 110: 2-(hydroxymethyl)-1,1-dimethylpyrrolidin-1-ium
Iodide
[0710] To a solution of (1-methylpyrrolidin-2-yl) methanol (300 mg,
2.60 mmol, 1 eq) and DMAP (159.11 mg, 1.30 mmol, 0.5 eq) in THF (5
mL) was added (Boc).sub.2O (1.42 g, 6.51 mmol, 1.50 mL, 2.5 eq) at
25.degree. C. The mixture was stirred at 25.degree. C. for 12 h.
LCMS showed the desired compound was detected. The reaction mixture
was filtered and the filtrate was concentrated under reduced
pressure to give a residue. The residue was purified by column
chromatography (SiO.sub.2, Petroleum ether/Ethyl acetate=3:1 to
1:1) to give tert-butyl ((1-methylpyrrolidin-2-yl) methyl)
carbonate (190 mg, 882.54 umol, 33.88% yield) as a yellow oil.
[0711] To a solution of tert-butyl ((1-methylpyrrolidin-2-yl)
methyl) carbonate (190 mg, 882.54 umol, 1 eq) in THF (5 mL) was
added MeI (250.53 mg, 1.77 mmol, 109.88 uL, 2 eq) at 25.degree. C.
The mixture was stirred at 25.degree. C. for 5 h. LCMS showed the
desired compound was detected. The reaction mixture was diluted
with H.sub.2O (10 mL) and extracted with ethyl acetate (10 mL). The
water layer was freeze-drying to give
2-(((tert-butoxycarbonyl)oxy)methyl)-1,1-dimethylpyrrolidin-1-ium
iodide (200 mg, 559.87 umol, 63.44% yield) as a yellow solid.
[0712] To a solution of
2-(((tert-butoxycarbonyl)oxy)methyl)-1,1-dimethylpyrrolidin-1-ium
iodide (180 mg, 503.88 umol, 1 eq) in HCl/dioxane (0.5 mL, 4M) and
dioxane (1 mL) was stirred at 25.degree. C. for 2 h. The reaction
mixture was concentrated under reduced pressure to give a residue.
The residue was diluted with H.sub.2O (10 mL) and extracted with
ethyl acetate (10 mL). The water layer was freeze-drying to give
2-(hydroxymethyl)-1,1-dimethylpyrrolidin-1-ium iodide (70 mg,
272.26 umol, 54.03% yield) as a yellow solid. MS: M.sup.+=130.1
.sup.1H NMR (400 MHz, DMSO) .delta. 3.9 (m, 2H), 3.7 (m, 1H), 3.5
(m, 2H), 3.2 (s, 3H), 3.0 (s, 3H), 2.3 m, 1H0, 2.1 (m, 2H), 1.8 (m,
1H) ppm.
##STR00202##
Compound 111: N-(2-hydroxyethyl)-N,N-dimethylcyclopropanaminium
Iodide
[0713] To a solution of N-methylcyclopropanamine (150 mg, 2.11
mmol, 1.68 eq) and (2-bromoethoxy)(tert-butyl) dimethylsilane (300
mg, 1.25 mmol, 1 eq) in ACN (5 mL) was added K.sub.2CO.sub.3
(173.32 mg, 1.25 mmol, 1 eq) at 25.degree. C. The mixture was
stirred at 50.degree. C. for 12 h. TLC indicated new spot formed.
The reaction mixture was filtered and concentrated under reduced
pressure to give a residue. The residue was purified by column
chromatography (SiO.sub.2, Petroleum ether/Ethyl acetate=5:1 to
1:1) to give
N-(2-((tert-butyldimethylsilyl)oxy)ethyl)-N-methylcyclopropanamine
(120 mg, 523.03 umol, 41.71% yield) as colorless oil.
[0714] To a solution of N-(2-((tert-butyldimethylsilyl) oxy)
ethyl)-N-methylcyclopropanamine (70 mg, 305.10 umol, 1 eq) in THF
(2 mL) was added MeI (86.61 mg, 610.20 umol, 37.99 uL, 2 eq) at
25.degree. C. The mixture was stirred at 25.degree. C. for 12 h.
The solid was filtered and the filter cake was concentrated to give
N-(2-((tert-butyldimethylsilyl) oxy) ethyl)-N,
N-dimethylcyclopropanaminium iodide (50 mg, 134.64 umol, 44.13%
yield) as a white solid.
[0715] To a solution of N-(2-((tert-butyldimethylsilyl) oxy)
ethyl)-N, N-dimethylcyclopropanaminium iodide (20 mg, 53.85 umol, 1
eq) in H.sub.2O (2 mL) was added KF (31.29 mg, 538.54 umol, 12.62
uL, 10 eq) at 25.degree. C. The mixture was stirred at 25.degree.
C. for 12 h. The precipitation was separated out by filtration. And
the filtrate was diluted with H.sub.2O (10 mL) and extracted with
ethyl acetate (10 mL). The water layer was concentrated under
reduced pressure to give a residue. The residue was washed by
acetonitrile (2 mL). The acetonitrile layer was filtered and the
filtrate was concentrated under reduced pressure to give
N-(2-hydroxyethyl)-N, N-dimethylcyclopropanaminium iodide (10 mg,
38.89 umol, 72.22% yield) as a white solid. .sup.1H NMR (400 MHz,
D2O) .delta. 4.1 (m, 2H), 3.6 (m, 2H), 3.2 (m, 1H), 3.0 (s, 6H),
1.2 (m, 2H), 0.9 (m, 2H) ppm.
##STR00203##
Compound 112:
(3S)-3-hydroxy-1-methyl-1-(prop-2-yn-1-yl)pyrrolidin-1-ium
bromide
[0716] To a solution of (3S)-1-methylpyrrolidin-3-ol (200 mg, 1.98
mmol, 201.41 uL, 1 eq) in DCM (5 mL) was added 3-bromoprop-1-yne
(282.27 mg, 2.37 mmol, 204.54 uL, 1.2 eq). The mixture was stirred
at 25.degree. C. for 5 h. MS showed desired compound was detected.
The reaction mixture was concentrated under reduced pressure to
remove solvent. The residue was dissolved in H.sub.2O (1 mL),
washed with EtOAc (2 mL*2) and lyophilized the aqueous phase to
give a residue. Compound (3S)-3-hydroxy-1-methyl-1-(prop-2-yn-1-yl)
pyrrolidin-1-ium bromide (311 mg, crude) was obtained as yellow
oil. .sup.1H NMR (400 MHz, DMSO) .delta. 5.6 (m, 1H), 4.4 (m, 3H),
4.0 (m, 1H), 3.6 (m, 4H), 3.1 (m, 3H), 2.4 (m, 1H), 1.9 (m 1H)
ppm.
##STR00204##
Compound 113:
(3R)-3-hydroxy-1-methyl-1-(prop-2-yn-1-yl)pyrrolidin-1-ium
bromide
[0717] To a solution of (3R)-1-methylpyrrolidin-3-ol (200 mg, 1.98
mmol, 217.16 uL, 1 eq) in DCM (5 mL) was added 3-bromoprop-1-yne
(282.27 mg, 2.37 mmol, 204.54 uL, 1.2 eq). The mixture was stirred
at 25.degree. C. for 5 h. MS showed desired compound was detected.
The reaction mixture was concentrated under reduced pressure to
remove solvent. The residue was dissolved in H.sub.2O (1 mL),
washed with EtOAc (2 mL*2) and lyophilized the aqueous phase to
give a residue. Compound (3R)-3-hydroxy-1-methyl-1-(prop-2-yn-1-yl)
pyrrolidin-1-ium bromide (368 mg, crude) was obtained as yellow
oil. .sup.1H NMR (400 MHz, DMSO) .delta. 5.6 (m, 1H), 4.4 (m, 3H),
4.0 (m, 1H), 3.6 (m, 4H), 3.1 (m, 3H), 2.4 (m, 1H), 1.9 (m 1H)
ppm.
##STR00205##
Compound 114:
(3S)-1-(fluoromethyl)-3-hydroxy-1-methylpyrrolidin-1-ium Iodide
[0718] To a solution of (3S)-1-methylpyrrolidin-3-ol (200 mg, 1.98
mmol, 201.41 uL, 1 eq) in DCM (5 mL) was added fluoro (iodo)
methane (379.48 mg, 2.37 mmol, 1.2 eq). The mixture was stirred at
25.degree. C. for 5 h. MS showed desired compound was detected. The
reaction mixture was concentrated under reduced pressure to remove
solvent. The residue was dissolved in H.sub.2O (1 mL), washed with
EtOAc (2 mL*2) and lyophilized the aqueous phase to give a residue.
Compound (3S)-1-(fluoromethyl)-3-hydroxy-1-methylpyrrolidin-1-ium
iodide (111 mg, crude) was obtained as a white solid. .sup.1H NMR
(400 MHz, DMSO) .delta. 5.7 (m, 1H), 5.4 (m, 2H), 4.5 (m, 1H), 3.6
(m, 4H), 3.2 (s, 3H), 2.4 (m, 1H), 1.9 (m, 1H) ppm.
##STR00206##
Compound 115:
(3R)-1-(fluoromethyl)-3-hydroxy-1-methylpyrrolidin-1-ium Iodide
[0719] To a solution of (3R)-1-methylpyrrolidin-3-ol (0.2 g, 1.98
mmol, 217.16 uL, 1 eq) in DCM (5 mL) was added fluoro (iodo)
methane (379.48 mg, 2.37 mmol, 1.2 eq). The mixture was stirred at
25.degree. C. for 5 h. MS showed desired compound was detected. The
reaction mixture was concentrated under reduced pressure to remove
solvent. The residue was dissolved in H.sub.2O (1 mL), washed with
EtOAc (2 mL*2) and lyophilized the aqueous phase to give a residue.
Compound (3R)-1-(fluoromethyl)-3-hydroxy-1-methylpyrrolidin-1-ium
iodide (358 mg, crude) was obtained as yellow oil. .sup.1H NMR (400
MHz, DMSO) .delta. 5.7 (m, 1H), 5.4 (m, 2H), 4.5 (m, 1H), 3.6 (m,
4H), 3.2 (s, 3H), 2.4 (m, 1H), 1.9 (m, 1H) ppm.
##STR00207##
Compound 116: (R)-3-hydroxy-1,1-dimethylpyrrolidin-1-ium Iodide
[0720] To a solution of (3R)-pyrrolidin-3-ol (4 g, 32.37 mmol, 3.81
mL, 1 eq, HCl) in THF (50 mL) was added NaOH (1.29 g, 32.37 mmol, 1
eq) at 25.degree. C. After addition, the mixture was stirred at
this temperature for 20 min. Then paraformaldehyde (1.17 g, 38.84
mmol, 1.2 eq) and HCOOH (4.82 g, 100.34 mmol, 3.1 eq) were added
25.degree. C. The resulting mixture was stirred at 60.degree. C.
for 5 h 40 min. TLC indicated the reaction was completed. The
reaction mixture was cooled to 0.degree. C., quenched by addition
10N NaOH to pH about 10, and extracted with EtOAc (30 mL*3). The
combined organic layers were washed with H.sub.2O (100 mL*1), dried
over Na.sub.2SO.sub.4, filtered and concentrated under reduced
pressure to give a residue. The crude product
(3R)-1-methylpyrrolidin-3-ol (0.7 g, crude) as yellow oil was used
into the next step without further purification.
[0721] To a solution of (3R)-1-methylpyrrolidin-3-ol (0.2 g, 1.98
mmol, 217.16 uL, 1 eq) in DCM (10 mL) was added MeI (561.32 mg,
3.95 mmol, 246.19 uL, 2 eq). The mixture was stirred at 25.degree.
C. for 12 h. Some solid precipitate out and the solid was filtered.
The filter cake was washed with DCM (5 mL*2) and concentrated to
dryness. Compound (R)-3-hydroxy-1,1-dimethylpyrrolidin-1-ium iodide
(333 mg, crude) was obtained as a white solid. .sup.1H NMR (400
MHz, DMSO) .delta. 5.5 (s, 1H), 4.5 (m, 1H), 3.6 (4H), 3.2 (s, 3H),
3.1 (s, 3H), 2.4 (m, 1H), 1.9 (m, 1H) ppm.
##STR00208##
Compound 117: (S)-3-hydroxy-1,1-dimethylpyrrolidin-1-ium Iodide
[0722] To a solution of (3S)-pyrrolidin-3-ol (4 g, HCl salt, 32.37
mmol, 3.70 mL, 1 eq) in THF (50 mL) was added NaOH (1.29 g, 32.37
mmol, 1 eq) at 25.degree. C. After addition, the mixture was
stirred at this temperature for 20 min. Then Paraformaldehyde (1.13
g, 38.84 mmol, 1.2 eq) and HCOOH (4.82 g, 100.34 mmol, 3.1 eq) were
added at 25.degree. C. The resulting mixture was stirred at
60.degree. C. for 5 h 40 min. TLC indicated the reaction was
completed. The reaction mixture was cooled to 0.degree. C. and
quenched by addition 10N NaOH to pH about 10, and extracted with
EtOAc (30 mL*3). The combined organic layers were washed with
H.sub.2O (100 mL*1), dried over Na.sub.2SO.sub.4, filtered and
concentrated under reduced pressure to give a residue. The crude
product (3S)-1-methylpyrrolidin-3-ol (0.7 g, crude) as yellow oil
was used into the next step without further purification.
[0723] To a solution of (3S)-1-methylpyrrolidin-3-ol (0.2 g, 1.98
mmol, 201.41 uL, 1 eq) in DCM (10 mL) was added MeI (561.32 mg,
3.95 mmol, 246.19 uL, 2 eq). The mixture was stirred at 25.degree.
C. for 12 h. Some solid precipitate out and the solid was filtered.
The filter cake was washed with DCM (5 mL*2) and concentrated to
dryness. Compound (S)-3-hydroxy-1,1-dimethylpyrrolidin-1-ium iodide
(272 mg, crude) was obtained as a white solid. .sup.1H NMR (400
MHz, DMSO) .delta. 5.5 (s, 1H), 4.5 (m, 1H), 3.6 (m, 4H), 3.2 (s,
3H), 3.1 (s, 3H), 2.4 (m, 1H), 1.0 (m, 1H) ppm.
##STR00209##
Compound 118:
(1R,2S)-2-hydroxy-N,N,N-trimethylcyclopropan-1-aminium Iodide
[0724] (1R, 2S), 2-dimethylamino cyclopropanol is dissolved in THF
followed by the dropwise addition of methyl iodide. The resulting
precipitate is collected and dried and recrystallized to afford the
title compound.
##STR00210##
Compound 119:
(1S,2R)-2-hydroxy-N,N,N-trimethylcyclopropan-1-aminium Iodide
[0725] (1S, 2R), 2-dimethylamino cyclopropanol is dissolved in THF
followed by the dropwise addition of methyl iodide. The resulting
precipitate is collected and dried and recrystallized to afford the
title compound.
##STR00211##
Compound 120:
(1S,2R)-2-hydroxy-N,N-dimethyl-N-(prop-2-yn-1-yl)cyclopropan-1-aminium
Iodide
[0726] (1S, 2R), 2-dimethylamino cyclopropanol is dissolved in THF
followed by the dropwise addition of propargyl iodide. The
resulting precipitate is collected and dried and recrystallized to
afford the title compound.
##STR00212##
Compound 121:
(1R,2S)-2-hydroxy-N,N-dimethyl-N-(prop-2-yn-1-yl)cyclopropan-1-aminium
Iodide
[0727] (1R, 2S), 2-dimethylamino cyclopropanol is dissolved in THF
followed by the dropwise addition of propargyl iodide. The
resulting precipitate is collected and dried and recrystallized to
afford the title compound.
##STR00213##
Compound 122:
(2R)-2-ethynyl-1-(2-hydroxyethyl)-1-methylazetidin-1-ium Iodide
[0728] (2R) 2-ethynyl azetidine is dissolved in THF water and
2-hydroxyacetaldehyde is added to the mixture. The resulting
mixture is stirred at room temperature and then sodium
cyanoborohydide is added portion wise to the mixture. The mixture
is stirred for 1 hour then quenched by the addition of water ethyl
acetate. The resulting material is taken up in ether, and treated
with dropwise addition of methyl iodide. The resulting precipitate
is collected and dried and recrystallized to afford the title
compound.
##STR00214##
Compound 123: (1S,2R)-2-hydroxy-N,N,N-trimethylcyclobutan-1-aminium
Iodide
[0729] (1S,2R)-2-hydroxy dimethylamino cyclobutanol is dissolved in
THF and then treated with the dropwise addition of methyl iodide.
The resulting precipitate is collected and dried and recrystallized
to afford the title compound.
##STR00215##
Compound 124:
(2S)-2-ethynyl-1-(2-hydroxyethyl)-1-methylazetidin-1-ium Iodide
[0730] (2S) 2-ethynyl azetidine is dissolved in THF water and
2-hydroxyacetaldehyde is added to the mixture. The resulting
mixture is stirred at room temperature and then sodium
cyanoborahydide is added portion wise to the mixture. The mixture
is stirred for 1 hour then quenched by the addition of water ethyl
acetate. The resulting material is taken up in ether, and treated
with dropwise addition of methyl iodide. The resulting precipitate
is collected and dried and recrystallized to afford the title
compound.
##STR00216##
Compound 125: 1-methyl-1-(prop-2-yn-1-yl)pyrazolidin-1-ium
Iodide
[0731] Step 1:
[0732] To a solution of tert-butyl pyrazolidine-1-carboxylate (200
mg, 1.16 mmol, 1 eq) in THF (10 mL) was added K2CO3 (176.54 mg,
1.28 mmol, 1.1 eq) at 15.degree. C. and stirred at 15.degree. C.
for 10 min. Then 3-bromoprop-1-yne (207.22 mg, 1.74 mmol, 150.16
uL, 1.5 eq) was added to the mixture and the mixture was stirred at
15.degree. C. for 12 h. TLC indicated reactant was consumed
completely. The reaction mixture was filtered and the filtrate was
concentrated under reduced pressure to give a residue. The residue
was purified by prep-TLC (SiO.sub.2, PE:EtOAc=1:1). Compound
tert-butyl 2-prop-2-ynylpyrazolidine-1-carboxylate (170 mg, 808.48
umol, 69.62% yield) was obtained as a white solid.
[0733] Step 2:
[0734] To a solution of tert-butyl
2-prop-2-ynylpyrazolidine-1-carboxylate (170 mg, 808.48 umol, 1 eq)
in THF (5 mL) was added MeI (688.52 mg, 4.85 mmol, 301.98 uL, 6
eq). The mixture was stirred at 15.degree. C. for 12 h. MS showed
the desired compound was detected. The reaction mixture was
concentrated under reduced pressure to give a residue. The crude
product tert-butyl
2-methyl-2-prop-2-ynyl-pyrazolidin-2-ium-1-carboxylate (200 mg,
crude, I--) as a yellow solid was used into the next step without
further purification. LCMS: (M+): 225.2
[0735] Step 3:
[0736] To a solution of tert-butyl
2-methyl-2-prop-2-ynyl-pyrazolidin-2-ium-1-carboxylate (200 mg,
887.68 umol, 1 eq) in HCl/dioxane (4 M, 5 mL, 22.53 eq). The
mixture was stirred at 15.degree. C. for 4 h. MS showed the desired
compound was detected. The reaction mixture was concentrated under
reduced pressure to give a residue. The residue was purified by
prep-HPLC ([water (0.1% TFA)-ACN]). Compound
1-methyl-1-(prop-2-yn-1-yl)pyrazolidin-1-ium iodide (26 mg, 103.14
umol, 11.6% yield) was obtained as a colorless oil. LCMS: (M+):
125.1 .sup.1H NMR (400 MHz, Deuterium Oxide) .delta. 4.42-4.28 (m,
2H), 3.91-3.80 (m, 1H), 3.75-3.64 (m, 1H), 3.44-3.23 (m, 5H),
3.16-3.11 (m, 1H), 2.37 (p, J=7.4 Hz, 2H).
##STR00217##
Compound 126:
(1S,3S)-3-hydroxy-N,N-dimethyl-N-(prop-2-yn-1-yl-)cyclobutan-1-aminium
Iodide
[0737] Cis-3-dimethylamine cyclobutanol is taken up in THF and
treated with drop wise addition of propargyl iodide. The resulting
precipitate is collected and dried and recrystallized to afford the
title compound.
##STR00218##
Compound 127: 3-carboxy-N,N,N-trimethylpropan-1-aminium Iodide
[0738] 4-(dimethylamino)butanoic acid is taken up in THF and
treated with drop wise addition of methyl iodide. The resulting
precipitate is collected and dried and recrystallized to afford the
title compound.
##STR00219##
Compound 129:
(1R,2R)-2-(hydroxymethyl)-1-methyl-1-(prop-2-yn-1-yl)pyrrolidin-1-ium
Bromide
[0739] To a solution of [(2R)-1-methylpyrrolidin-2-yl]methanol (200
mg, 1.74 mmol, 1 eq) in THF (10 mL) was added 3-bromoprop-1-yne
(826.30 mg, 6.95 mmol, 598.77 uL, 4 eq). The mixture was stirred at
15.degree. C. for 24 hr. MS showed the desired compound was
detected. The reaction mixture was concentrated under reduced
pressure to give a residue. The residue was purified by prep-HPLC
([water (0.10% TFA)-ACN]). The title compound (60 mg, 389.03 umol,
22.40% yield, Br) was obtained as a colorless oil. LCMS:(M+) 154.1
.sup.1H NMR (400 MHz, Deuterium Oxide) .delta. 4.46-4.23 (m, 1H),
4.15 (d, J=2.5 Hz, 1H), 4.04-3.69 (m, 4H), 3.63-3.39 (m, 1H), 3.24
(s, 1H), 3.14-3.01 (m, 3H), 2.33-2.02 (m, 3H), 1.99-1.77 (m,
1H).
##STR00220##
Compound 132: 4-hydroxy-N,N,N-trimethylbut-2-yn-1-aminium
Chloride
[0740] To a solution of 4-chlorobut-2-yn-1-ol (88.17 mg, 843.45
umol, 1 eq) in THE (5 mL) was added N, N-dimethylmethanamine (3 M,
562.30 uL, 2 eq, in THF). The mixture was stirred at 25.degree. C.
for 12 h. The reaction mixture was diluted with H.sub.2O (10 mL)
and extracted with ethyl acetate (10 mL). The water layer was
freeze-drying to give 4-hydroxy-N, N, N-trimethylbut-2-yn-1-aminium
chloride (55 mg, 336.09 umol, 39.85% yield) as colorless oil. MS:
M.sup.+=128.1 .sup.1H NMR (400 MHz, DMSO) .delta. 4.3 (m, 4H), 3.1
(s, 9H) ppm.
##STR00221##
Compound 134: 2-[(benzyloxy)methyl]-1,1-dimethylpyrrolidin-1-ium
Iodide
[0741] The title compound may be synthesized by reacting Compound
57 with benzyl bromide, followed by purification by
recrystallization.
##STR00222##
Compound 135:
1-(carboxymethyl)-1-(prop-2-yn-1-yl)azetidin-1-ium
[0742] This compound may be synthesized according to the
experimental procedure described for Compound 133.
##STR00223##
Compound 137: 3-ethynyl-1,1-dimethylpyrrolidin-1-ium Iodide
[0743] This compound may be synthesized according to the
experimental procedure described for Compound 262.
##STR00224##
Compound 138: N-(3-hydroxypropyl)-N,N-dimethylprop-2-yn-1-aminium
Trifluoroacetate
[0744] The mixture of N,N-dimethylprop-2-yn-1-amine (500 mg, 6.01
mmol, 637.76 uL, 1 eq) and 3-bromopropan-1-ol (1.00 g, 7.22 mmol,
651.40 uL, 1.2 eq) in EtOH (5 mL) was stirred at 80.degree. C.
under N.sub.2 2 hours. LCMS showed desired compound was detected.
The reaction mixture was concentrated under reduced pressure to
give a residue. Then the residue was purified by prep-HPLC (column:
Xbridge 150*30 mm*10 um; mobile phase: [water (0.10% TFA)-ACN]; B
%: 1%-10%, 10 min) to give
N-(3-hydroxypropyl)-N,N-dimethylprop-2-yn-1-aminium
trifluoroacetate (107 mg, 409.35 umol, 6.81% yield, TFA) as a
colorless oil. LCMS: (M+) 142.2 .sup.1H NMR (400 MHz, Deuterium
Oxide) .delta. 4.08 (d, J=2.6 Hz, 2H), 3.53 (t, J=5.9 Hz, 2H),
3.40-3.32 (m, 2H), 3.07 (t, J=2.5 Hz, 1H), 3.01 (s, 6H), 1.91-1.79
(m, 2H).
##STR00225##
Compound 139:
5-hydroxy-N,N-dimethyl-N-(prop-2-yn-1-yl)pentan-1-aminium
Bromide
[0745] A mixture of N,N-dimethylprop-2-yn-1-amine (500 mg, 6.01
mmol, 637.76 uL, 1 eq) and 5-bromopentan-1-ol (1.05 g, 6.31 mmol,
1.05 eq) in EtOH (5 mL) was degassed and purged with N.sub.2 3
times, and then the mixture was stirred at 80.degree. C. for 10 hr
under N.sub.2 atmosphere. LCMS showed desired mass was detected.
The reaction mixture was concentrated under reduced pressure to
give a residue. The residue was diluted with H.sub.2O 20 mL and
washed with EtOAc (20 mL*3). Then the water layers were lyophilized
to afford 5-hydroxy-N,N-dimethyl-N-(prop-2-yn-1-yl)pentan-1-aminium
bromide (625 mg, 1.98 mmol, 32.90% yield) as a yellow oil. LCMS:
(M+) 170.1 .sup.1H NMR (400 MHz, Deuterium Oxide) .delta. 4.17 (s,
2H), 3.55 (t, J=6.5 Hz, 2H), 3.42-3.33 (m, 2H), 3.19-3.14 (m, 1H),
3.10 (s, 6H), 1.81-1.69 (m, 2H), 1.61-1.49 (m, 2H), 1.42-1.30 (m,
2H).
##STR00226##
Compound 140: (methanesulfinylmethyl)trimethylazanium Chloride
[0746] Step 1:
[0747] To a solution of chloro(methylsulfanyl)methane (200 mg, 2.07
mmol, 173.91 uL, 1 eq) in THF (5 mL) was added
N,N-dimethylmethanamine (1 M, 4.14 mL, 2 eq, in THF) at 15.degree.
C. The mixture was stirred at 70.degree. C. for 12 hr. The
precipitation was found. The reaction mixture was filtered to give
a residue. Compound
methylsulfanyl-[N,N,N-(trimethyl)-azanyl]methane (100 mg, crude)
was obtained as a white solid.
[0748] Step 2:
[0749] A mixture of methylsulfanyl-[N,N,N
(trimethyl)-azanyl]methane (50 mg, 321.15 umol, 1 eq) in
H.sub.2O.sub.2 (2 mL, 30%) and H.sub.2O (2 mL) was stirred at
80.degree. C. for 12 hr. LCMS showed the desired compound was
detected. The reaction mixture was quenched by saturated sodium
sulfite (30 mL), and the aqueous phase was freeze-dried. The
residue was diluted with methanol (50 mL) and filtered. The
filtrate was concentrated under reduced pressure to give a residue.
The residue was purified by prep-HPLC (column: Waters Xbridge BEH
C18 100*30 mm*10 um; mobile phase: [water (0.1% TFA)-ACN]; B %:
1%-20%, 10 min). The residue was purified by prep-HPLC (column:
YMC-Actus Triart Diol-Hilic 100*30 mm Sum; mobile phase: [water
(0.1% TFA)-ACN]; B %: 99%-85%, 12 min). The title compound (5 mg,
28.46 umol, Cl-salt, 24.43% yield, 97.73% purity) was obtained as a
colorless oil. LCMS: (M+): 136.1 1H NMR (400 MHz, Deuterium Oxide)
.delta. 4.62-4.53 (m, 2H), 3.25 (s, 9H), 2.75 (s, 3H).
##STR00227##
Compound 140A: 3-(trimethylammonio)propane-1-sulfonate
[0750] To a solution of oxathiolane 2, 2-dioxide (200 mg, 1.64
mmol, 143.88 uL, 1 eq) in acetone (5 mL) was added
N,N-dimethylmethanamine (322.63 mg, 1.64 mmol, 377.34 uL, 1 eq) at
25.degree. C. The mixture was stirred at 25.degree. C. for 12 h.
The precipitate was separated from the reaction mixture by
filtration and the solid was concentrated under reduced pressure to
dryness. Compound 3-(trimethylammonio) propane-1-sulfonate (157 mg,
0.87 mmol, 53% yield) was obtained as a white solid. LCMS
(M+H.sup.+): 182.0 .sup.1H NMR (400 MHz, Deuterium Oxide) .delta.
3.47-3.43 (m, 2H), 3.11 (s, 9H), 2.95 (t, 2H), 2.24-2.18 (m,
2H).2.95 (t, 2H), 2.24-2.18 (m, 2H).
##STR00228##
Compound 141:
4-hydroxy-N,N-dimethyl-N-(prop-2-yn-1-yl)butan-1-aminium
Trifluoroacetate
[0751] A mixture of N,N-dimethylprop-2-yn-1-amine (500 mg, 6.01
mmol, 637.76 uL, 1 eq) and 4-bromobutan-1-ol (966.35 mg, 6.32 mmol,
1.05 eq) in EtOH (5 mL) was degassed and purged with N.sub.2 3
times. Then the mixture was stirred at 80.degree. C. for 10 hr
under N.sub.2 atmosphere. LCMS showed expected mass was detected.
The reaction mixture was concentrated under reduced pressure to
give a residue. The residue was purified by prep-HPLC (column:
Xbridge 150*30 mm*10 um; mobile phase: [water (0.1% TFA)-ACN]; B %:
1%-5%, 10 min) to give
4-hydroxy-N,N-dimethyl-N-(prop-2-yn-1-yl)butan-1-aminium
trifluoroacetate (150 mg, 529.64 umol, 8.81% yield) as colorless
oil. LCMS: (M+) 156.3 1H NMR (400 MHz, Deuterium Oxide) .delta. 4.1
(d, 2H), 3.6-3.5 (m, 2H), 3.4-3.3 (m, 2H), 3.1-3.0 (m, 1H), 3.0 (s,
6H), 1.8-1.7 (m, 2H), 1.5-1.4 (m, 2H).
##STR00229##
Compound 142: N-(2-carboxyethyl)-N,N-dimethylprop-2-yn-1-aminium
Bromide
[0752] A mixture of N,N-dimethylprop-2-yn-1-amine (500 mg, 6.01
mmol, 637.76 uL, 1 eq) 3-bromopropanoic acid (966.08 mg, 6.32 mmol,
652.76 uL, 1.05 eq) in EtOH (5 mL) was degassed and purged with
N.sub.2 3 times. And then the mixture was stirred at 80.degree. C.
for 10 hr under N.sub.2 atmosphere. LCMS showed desired mass was
detected. The reaction mixture was concentrated under reduced
pressure to give a residue. The residue was purified by prep-HPLC
(column: Xbridge 150*30 mm*10 um; mobile phase: [water (0.04%
NH.sub.3H.sub.2O)-ACN]; B %: 1%-10%, 10 min) to give
N-(2-carboxyethyl)-N,N-dimethylprop-2-yn-1-aminium bromide (95 mg,
345.71 umol, 5.75% yield) as colorless oil. LCMS: (M+) 156.1
.sup.1H NMR (400 MHz, Deuterium Oxide) .delta. 4.2 (d, 2H), 3.8-3.6
(m, 2H), 3.2-3.1 (m, 1H), 3.1 (s, 6H), 2.7-2.6 (m, 2H).
##STR00230##
Compound 143: N,N,N-trimethyl-3-phosphonopropan-1-aminium
Trifluoroacetatetrifluoroacetatetrifluoroacetatetrifluoroacetatetrifluoro-
acetate
[0753] To a solution of 1-bromo-3-diethoxyphosphoryl-propane (500
mg, 1.93 mmol, 370.37 uL, 1 eq) in ACN (5 mL) was added
1-bromo-3-diethoxyphosphoryl-propane (500 mg, 1.93 mmol, 370.37 uL,
1 eq.). The mixture was stirred at 15.degree. C. for 48 h. Then the
reaction mixture was concentrated under reduced pressure to give a
residue. Then HCl aq. (5 mL, 12 M) was added to the residue at
15.degree. C. and the mixture was stirred at 80.degree. C. for 12
h. LC-MS showed the desired compound was detected. The reaction
mixture was concentrated under reduced pressure to give a residue.
The residue was purified by prep-HPLC ([water (0.10% TFA)-ACN]).
The title compound (225 mg, 1.22 mmol, 63.36% yield) was obtained
as a white solid. LCMS: (M+): 182.2 .sup.1H NMR (400 MHz, Deuterium
Oxide) .delta. 3.36-3.26 (m, 2H), 3.06-3.00 (m, 9H), 2.02-1.92 (m,
2H), 1.78-1.65 (m, 2H).
##STR00231##
Compound 144: N-(3-carboxypropyl)-N,N-dimethylprop-2-yn-1-aminium
Iodide
[0754] 4-(dimethylamino)butanoic acid is taken up in THF and
treated with drop wise addition of propargyl iodide. The resulting
precipitate is collected and dried and recrystallized to afford the
title compound.
Compound 147:
3-(hydroxyhydrophosphoryl)-N,N,N-trimethylpropan-1-aminium
Chloridechloridechloridechloridechloride
[0755] Step 1:
[0756] To a solution of 1-[ethoxy(ethoxyphosphonoyl)methoxy]ethane
(1 g, 5.10 mmol, 1 eq) in THF (40 mL) was added slowly NaH (224.26
mg, 5.61 mmol, 60% purity, 1.1 eq) at 0.degree. C. under N.sub.2.
Then the mixture was stirred at 15.degree. C. for 1 h under N.sub.2
atmosphere. Then the mixture was dropwise added to the solution of
1,3-dibromopropane (2.79 g, 13.81 mmol, 1.41 mL, 2.71 eq) in THF
(20 mL) at 15.degree. C. Then the mixture was stirred at 15.degree.
C. for 9 h under N.sub.2 atmosphere. TLC indicated
1-[ethoxy(ethoxyphosphonoyl)methoxy]ethane was consumed completely
and one new spot formed. The reaction mixture was quenched by
addition of H.sub.2O (100 mL) at 0.degree. C. and extracted with
EtOAc (20 mL*3). The combined organic layers were washed with brine
(30 mL). The combined organic layer was dried over
Na.sub.2SO.sub.4, filtered and the filtrate was concentrated under
reduced pressure to give a residue. The residue was purified by
column chromatography (SiO.sub.2, Petroleum ether/Ethyl
acetate=1/1). Compound 1-bromo-3-[diethoxymethyl(ethoxy)phosphoryl]
propane (400 mg, 1.26 mmol, 24.74% yield) was obtained as yellow
oil.
[0757] Step 2:
[0758] A mixture of
1-bromo-3-[diethoxymethyl(ethoxy)phosphoryl]propane (400 mg, 1.26
mmol, 1 eq), trimethylamine (1.49 g, 7.57 mmol, 30% purity, 6 eq.,
in EtOH) in ACN (5 mL) was degassed and purged with N.sub.2 3
times, and then the mixture was stirred at 15.degree. C. for 96 hr
under N.sub.2 atmosphere. TLC indicated 20% of
1-bromo-3-[diethoxymethyl(ethoxy)phos-phoryl]propane (400 mg, 1.26
mmol, 1 eq) remained and one major new spot with larger polarity
was detected. The reaction mixture was concentrated under reduced
pressure to give a residue. The residue was purified by prep-HPLC
(TFA condition; column: Luna C18 100*30 5u; mobile phase: [water
(0.1% TFA)-ACN]; B %: 1%-25%, 12 min). Compound
3-[diethoxymethyl(ethoxy)phosphoryl] propyl-trimethyl-ammonium (150
mg, 366.41 umol, 29.05% yield, CF3CO2-) was obtained as colorless
oil.
[0759] Step 3:
[0760] A mixture of
3-[diethoxymethyl(ethoxy)phosphoryl]propyl-trimethyl-ammonium (150
mg, 366.41 umol, 1 eq, CF.sub.3COO--), in conc. HCl (10 mL) was
degassed and purged with N.sub.2 3 times, and then the mixture was
stirred at 110.degree. C. for 10 hr under N.sub.2 atmosphere. LC-MS
showed 100% desired compound was detected. The reaction mixture was
concentrated under reduced pressure to give a colorless oil.
Compound 3-(hydroxyhydrophosphoryl)-N,N,N-trimethylpropan-1-aminium
chloride (120 mg, quantitative yield, Cl--) was obtained as
colorless oil. LCMS: (M+): 166.1 .sup.1H NMR (400 MHz, Deuterium
Oxide) .delta. 6.95 (dt, J=546.5, 1.8 Hz, 1H), 3.31-3.22 (m, 2H),
2.99 (s, 9H), 1.98-1.83 (m, 2H), 1.71-1.58 (m, 2H).)
##STR00232##
Compound 148: (1s,4s)-3-hydroxy-1-methylquinuclidin-1-ium
[0761] (S)-(+)-3-quinuclidinol (1 equiv) is stirred with methyl
iodide (1.1 equiv) to yield the title compound.
##STR00233##
Compound 149: 4-hydroxy-1-methylquinuclidin-1-ium
[0762] 1-azabicyclo[2.2.2]octan-4-ol (1 equiv) is stirred with
methyl iodide (1.1 equiv) to yield the title compound.
##STR00234##
Compound 151:
(2R)-1-(fluoromethyl)-2-(hydroxymethyl)-1-methylpyrrolidin-1-ium
Iodide
[0763] A mixture of [(2R)-1-methylpyrrolidin-2-yl]methanol (251 mg,
2.18 mmol, 1 eq), fluoro(iodo)methane (348.54 mg, 2.18 mmol, 1 eq)
in THF (5 mL) was degassed and purged with N.sub.2 3 times, and
then the mixture was stirred at 15.degree. C. for 2 hr under
N.sub.2 atmosphere. TLC indicated
[(2R)-1-methylpyrrolidin-2-yl]methanol was consumed completely and
one new spot formed. The reaction mixture was filtered and the
filter cake was concentrated under reduced pressure to give a
residue. Then the crude product was purified by p-HPLC (column:
Phenomenex Luna C18 100*30 mm*5 um; mobile phase: [water (0.1%
TFA)-ACN]; B %: 1%-3%, 4 min) to get the desired product
(2R)-1-(fluoromethyl)-2-(hydroxymethyl)-1-methylpyrrolidin-1-ium
iodide (150 mg, 545.25 umol, 25.02% yield) as a yellow solid. LCMS
(M+): 148.1 .sup.1H NMR (400 MHz, Deuterium Oxide) .delta.
5.47-5.31 (m, 1H), 5.34-5.22 (m, 1H), 3.93-3.60 (m, 4H), 3.54-3.36
(m, 1H), 3.15-2.94 (m, 3H), 2.21-1.71 (m, 4H).
Compound 152: (S)-1-carboxy-N,N,N-trimethylbut-3-yn-1-aminium
Iodide
[0764] To a solution of (2S)-2-aminopent-4-ynoic acid (1 g, 8.84
mmol, 1 eq) in MeOH (10 mL) was added NaOH (800.06 mg, 20.00 mmol,
2.26 eq) in EtOH (2 mL) and MeI (3.76 g, 26.52 mmol, 1.65 mL, 3 eq)
at 15.degree. C. The mixture was stirred at 15.degree. C. for 12
hr. LCMS showed the desired compound was detected. The reaction
mixture was concentrated under reduced pressure to give a residue.
The residue was purified by prep-HPLC (column: Phenomenex Luna C18
100*30 mm*5 um; mobile phase: [water (0.2% FA)-ACN]; B %: 1%-1%, 4
min). The mixture was freeze-dried. The residue was re-purified by
prep-HPLC (column: Phenomenex Luna C18 100*30 mm*5 um; mobile
phase: [water (0.2% FA)-ACN]; B %: 1%-1%, 4 min). The title
compound (48 mg, 158.73 umol, I-salt, 1.80% yield) was obtained as
a white solid. LCMS: (M+): 156.1 .sup.1H NMR (400 MHz, Deuterium
Oxide) .delta. 3.82 (ddd, J=8.9, 5.1, 0.9 Hz, 1H), 3.16 (s, 9H),
2.96-2.77 (m, 2H), 2.49-2.40 (m, 1H).
##STR00235##
Compound 153:
N-(fluoromethyl)-N-(2-hydroxyethyl)-N-methylprop-2-yn-1-aminium
Iodide
[0765] To a solution of 2-[methyl(prop-2-ynyl)amino]ethanol (400
mg, 3.53 mmol, 1 eq) in THF (5 mL) was added fluoro(iodo)methane
(565.33 mg, 3.53 mmol, 1 eq) at 15.degree. C. The mixture was
stirred at 15.degree. C. for 12 hr. TLC indicated
2-[methyl(prop-2-ynyl)amino]ethanol (400 mg, 3.53 mmol, 1 eq) was
consumed completely. The reaction mixture was diluted with H2O (10
mL) and extracted with ethyl acetate 40 mL. The aqueous phase is
freeze-dried. Compound
N-(fluoromethyl)-N-(2-hydroxyethyl)-N-methylprop-2-yn-1-aminium
iodide (247 mg, 882.41 umol, 24.96% yield) was obtained as a white
solid. LCMS (M+): 146.1 .sup.1H NMR (400 MHz, Deuterium Oxide)
.delta. 5.56-5.37 (m, 2H), 4.35 (s, 2H), 3.95 (s, 2H), 3.63-3.56
(m, 2H), 3.19-3.14 (m, 4H).
##STR00236##
Compound 154: 1-(2-hydroxyethyl)-1-(prop-2-yn-1-yl)piperidin-1-ium
Bromide
[0766] To a solution of 2-(1-piperidyl)ethanol (300 mg, 2.32 mmol,
308.32 uL, 1 eq) in THF (5 mL) was added 3-bromoprop-1-yne (303.85
mg, 2.55 mmol, 220.18 uL, 1.1 eq). The mixture was stirred at
15.degree. C. for 12 hr. The precipitation was found. The reaction
mixture was filtered to give a residue. The residue was washed with
ethyl acetate 20 mL. Compound
1-(2-hydroxyethyl)-1-(prop-2-yn-1-yl)piperidin-1-ium bromide (554
mg, 2.18 mmol, 94% yield) was obtained as a white solid. LCMS:
(M+): 168.1 .sup.1H NMR (400 MHz, DMSO-d6) .delta. 5.36 (t, J=4.9
Hz, 1H), 4.53 (d, J=2.5 Hz, 2H), 4.04 (t, J=2.5 Hz, 1H), 3.86 (q,
J=5.1 Hz, 2H), 3.59-3.49 (m, 4H), 3.49-3.38 (m, 2H), 1.83 (p, J=5.9
Hz, 4H), 1.65-1.45 (m, 2H).
##STR00237##
Compound 155: 2-carboxy-N,N,N-trimethylprop-2-en-1-aminium
Bromide
[0767] To a solution of 2-(bromomethyl)prop-2-enoic acid (200 mg,
1.21 mmol, 1 eq) in THF (3 mL) was added N,N-dimethylmethanamine (1
M, 1.21 mL, 1 eq., in THF) at 15.degree. C. The mixture was stirred
at 15.degree. C. for 12 hr. The precipitation was found. The
reaction mixture was filtered to give a residue. The residue was
washed by ethyl acetate 10 mL. Compound
2-carboxy-N,N,N-trimethylprop-2-en-1-aminium bromide (131 mg,
561.19 umol, 46.29% yield) was obtained as a white solid. LCMS:
(M+): 144.1 .sup.1H NMR (400 MHz, Deuterium Oxide) .delta. 6.77 (s,
1H), 6.23 (s, 1H), 4.09 (s, 2H), 2.96 (s, 9H).
##STR00238##
Compound 156:
2-carboxy-N-(2-carboxyallyl)-N,N-dimethylprop-2-en-1-aminium
Bromide
[0768] To a solution of 2-(bromomethyl)prop-2-enoic acid (600 mg,
3.64 mmol, 3 eq) in acetone (5 mL) was added N-methylmethanamine (2
M, 606.12 uL, 1 eq., in THF) at 15.degree. C. The mixture was
stirred at 15.degree. C. for 12 hr, and a precipitate formed. The
reaction mixture was filtered to give a residue. The residue was
washed by acetone 10 mL. Compound
2-carboxy-N-(2-carboxyallyl)-N,N-dimethylprop-2-en-1-aminium
bromide (100 mg, 310.22 umol, 25.59% yield) was obtained as a white
solid. LCMS: (M+): 214.1 .sup.1H NMR (400 MHz, Deuterium Oxide)
.delta. 6.9 (s, 2H), 6.4 (s, 2H), 4.2 (s, 4H), 2.9 (s, 6H).
##STR00239##
Compound 157:
2-hydroxy-N-(2-hydroxyethyl)-N,N-dimethylethan-1-aminium Iodide
[0769] To a solution of 2-(dimethylamino)ethanol (100 mg, 1.12
mmol, 112.61 uL, 1 eq) in THF (5 mL) was added 2-iodoethanol
(578.77 mg, 3.37 mmol, 263.08 uL, 3 eq) at 15.degree. C. The
mixture was stirred at 50.degree. C. for 12 hr. The precipitation
was found. The reaction mixture was filtered to give a residue. The
residue was washed by ethyl acetate 10 mL. Compound
2-hydroxy-N-(2-hydroxyethyl)-N,N-dimethylethan-1-aminium iodide
(230 mg, 872.08 umol, 77.73% yield) was obtained as a white solid.
LCMS: (M+): 134.2 .sup.1H NMR (400 MHz, Deuterium Oxide) .delta.
3.95-3.87 (m, 4H), 3.48-3.40 (m, 4H), 3.07 (s, 6H).
##STR00240##
Compound 158: 1,1-dimethylpyrazolidin-1-ium Iodide
[0770] Step 1
[0771] To a solution of NaH (600.78 mg, 15.02 mmol, 60% purity, 2
eq.) in DMF (20 mL) at 0.degree. C. was added tert-butyl
N-(benzyloxycarbonylamino)carbamate (2 g, 7.51 mmol, 1 eq) at
0.degree. C. and stirred at 0.degree. C. for 20 min. Then
1,3-dibromopropane (1.52 g, 7.51 mmol, 765.80 uL, 1 eq) was added
to the mixture at 0.degree. C. The mixture was stirred at
15.degree. C. for 12 h. TLC indicated reactant was consumed
completely. The reaction mixture was quenched by addition of
H.sub.2O 100 mL at 0.degree. C., and then diluted with H.sub.2O 100
mL and extracted with EtOAc (200 mL*2). The combined organic layers
were washed with saturate brine 300 mL (150 mL*2), dried over,
filtered and the filtrate was concentrated under reduced pressure
to give a residue. The residue was purified by column
chromatography (SiO.sub.2, Petroleum ether/Ethyl acetate=20/1 to
4/1). Compound 02-benzyl 01-tert-butyl
pyrazolidine-1,2-dicarboxylate (1.5 g, 4.90 mmol, 65.19% yield) was
obtained as a colorless oil.
[0772] Step 2
[0773] To a solution of 02-benzyl 01-tert-butyl
pyrazolidine-1,2-dicarboxylate (800 mg, 2.61 mmol, 1 eq) in THF (15
mL) was added Pd/C (0.5 g, 2.61 mmol, 10% purity). The suspension
was degassed and purged with H.sub.2 3 times. The mixture was
stirred under H.sub.2 (15 Psi.) at 15.degree. C. for 3 h. TLC
indicated reactant was consumed completely. The reaction mixture
was filtered and the filtrate was concentrated under reduced
pressure to give a residue. Compound tert-butyl
pyrazolidine-1-carboxylate (400 mg, 2.32 mmol, 88.94% yield) was
obtained as a colorless oil and used into the next step without
further purification.
[0774] Step 3
[0775] To a solution of tert-butyl pyrazolidine-1-carboxylate (200
mg, 1.16 mmol, 1 eq) in THF (10 mL) was added MeI (494.49 mg, 3.48
mmol, 216.88 uL, 3 eq). The mixture was stirred at 15.degree. C.
for 12 h. LC-MS showed the desired compound was detected. The
reaction mixture was concentrated under reduced pressure to give a
residue. Compound tert-butyl
2,2-dimethylpyrazolidin-2-ium-1-carboxylate (150 mg, crude, I--)
was obtained as a yellow solid was used into the next step without
further purification. LCMS: (M+): 201.2.
[0776] Step 4
[0777] A solution of tert-butyl
2,2-dimethylpyrazolidin-2-ium-1-carboxylate (150 mg, 745.21 umol, 1
eq) in HCl/dioxane (4 M, 10 mL, 53.68 eq) was stirred at 15.degree.
C. for 1 h. MS showed the desired mass was detected. The reaction
mixture was concentrated under reduced pressure to give a residue.
The residue was purified by prep-HPLC ([water (0.1% TFA)-ACN]).
Compound 1,1-dimethylpyrazolidin-1-ium iodide (10 mg, 43.85 umol,
5.88% yield, I--) was obtained as a colorless oil. MS: (M+): 101.1.
.sup.1H NMR (400 MHz, Chloroform-d) .delta. 3.77 (t, J=7.7 Hz, 2H),
3.42 (t, J=7.5 Hz, 2H), 3.36 (s, 6H), 2.43 (p, J=7.6 Hz, 2H).
##STR00241##
Compound 159: 1-(carboxymethyl)-1-(prop-2-yn-1-yl)pyrrolidin-1-ium
Bromide
[0778] Step 1:
[0779] A mixture of pyrrolidine (5 g, 70.30 mmol, 5.87 mL, 1 eq),
benzyl 2-bromoacetate (14.49 g, 63.27 mmol, 9.93 mL, 0.9 eq),
Na.sub.2CO.sub.3 (7.45 g, 70.30 mmol, 1 eq) in THF (100 mL) was
degassed and purged with N.sub.2 3 times, and then the mixture was
stirred at 15.degree. C. for 10 hr under N.sub.2 atmosphere. TLC
indicated reactant was consumed completely and one new spot formed.
The reaction was clean according to TLC. The reaction mixture was
filtered and the filtrate was concentrated under reduced pressure
to give a residue. The residue was purified by column
chromatography (SiO.sub.2, Petroleum ether/Ethyl acetate=10/1 to
1:1). Compound benzyl 2-pyrrolidin-1-ylacetate (8 g, 36.48 mmol,
51.89% yield) was obtained as yellow oil.
[0780] Step 2:
[0781] A mixture of benzyl 2-pyrrolidin-1-ylacetate (5 g, 22.80
mmol, 1 eq), Pd/C (2 g, 22.80 mmol, 10% purity) in EtOH (100 mL)
was degassed and purged with H.sub.2 for 3 times, and then the
mixture was stirred at 15.degree. C. for 10 hr under H.sub.2
atmosphere at 15 psi. TLC indicated benzyl 2-pyrrolidin-1-ylacetate
was consumed completely and one new spot formed. The reaction
mixture was filtered and the filtrate was concentrated to give the
crude product. Compound 2-pyrrolidin-1-ylacetic acid (2 g, 15.49
mmol, 67.91% yield) was obtained as a white solid. The crude was
used directly in next step.
[0782] Step 3:
[0783] A mixture of 2-pyrrolidin-1-ylacetic acid (300 mg, 2.32
mmol, 1 eq), 3-bromoprop-1-yne (303.95 mg, 2.56 mmol, 220.25 uL,
1.1 eq) in DMF (5 mL) was stirred at 15.degree. C. for 10 hrs under
N.sub.2. LCMS indicated desired mass was detected. The reaction
mixture was concentrated under reduced pressure to give the crude
product. The residue was purified by prep-HPLC (column: Waters
Xbridge BEH C18 100*30 mm*10 um; mobile phase: [water (0.10%
TFA)-ACN]; B %: 1%). Compound
1-(carboxymethyl)-1-(prop-2-yn-1-yl)pyrrolidin-1-ium bromide (48
mg, 193.03 umol, 8.31% yield) was obtained as a white solid. LCMS:
(M+): 168.1 .sup.1H NMR (400 MHz, Deuterium Oxide) .delta. 4.40 (d,
J=2.6 Hz, 2H), 4.11 (s, 2H), 3.77-3.69 (m, 2H), 3.61-3.54 (m, 2H),
3.03 (d, J=2.6 Hz, 1H), 2.12-2.06 (m, 4H).
##STR00242##
Compound 160: 1-(carboxymethyl)-1-(prop-2-yn-1-yl)piperidin-1-ium
bromide
[0784] Step 1:
[0785] A mixture of piperidine (5 g, 58.72 mmol, 5.80 mL, 1 eq),
benzyl 2-bromoacetate (13.45 g, 58.72 mmol, 9.21 mL, 1 eq), Na2CO3
(6.22 g, 58.72 mmol, 1 eq), in THF (100 mL) was degassed and purged
with N.sub.2 3 times, and then the mixture was stirred at
15.degree. C. for 10 hr under N.sub.2 atmosphere. LC-MS showed
15.64% of desired compound was detected. The reaction mixture was
filtered. Then the filtrate was concentrated under reduced pressure
to give a residue. The residue was purified by column
chromatography (SiO.sub.2, Petroleum ether/Ethyl acetate=1/1).
Compound benzyl 2-(1-piperidyl)acetate (10 g, 42.86 mmol, 72.99%
yield) was obtained as a yellow oil. LCMS: (M+H.sup.+): 234.2
[0786] Step 2:
[0787] A mixture of benzyl 2-(1-piperidyl)acetate (10 g, 42.86
mmol, 1 eq), Pd/C (2 g, 10% purity), in EtOH (100 mL) was degassed
and purged with H2 for 3 times, and then the mixture was stirred at
15.degree. C. for 10 hr under H2 atmosphere at 15 psi. TLC
indicated benzyl 2-(1-piperidyl)acetate (10 g, 42.86 mmol, 1 eq)
was consumed completely and one new spot formed. LC-MS showed
desired compound was detected. The reaction mixture was filtered
and the filtrate was concentrated under reduced pressure to give a
residue. Compound 2-(1-piperidyl)acetic acid (2.5 g, 17.46 mmol,
40.74% yield) was obtained as a white solid. The crude was used
directly for next step. LCMS: (M+H.sup.+): 144.1
[0788] Step 3:
[0789] A mixture of 2-(1-piperidyl)acetic acid (300 mg, 2.10 mmol,
1 eq), 3-bromoprop-1-yne (288.60 mg, 2.30 mmol, 209.13 uL, 1.1 eq),
in DMF (20 mL) was stirred at 15.degree. C. for 10 hr under N.sub.2
atmosphere. LC-MS showed desired mass was detected. The reaction
mixture was concentrated under reduced pressure to give the crude
product. The residue was purified by prep-HPLC (column: Waters
Xbridge BEH C18 100*30 mm*10 um; mobile phase: [water (0.1%
TFA)-ACN]; B %:1%-10%, 10 min). Compound
2-(1-prop-2-ynylpiperidin-1-ium-1-yl)acetic acid (18 mg, 68.52
umol, 3.27% yield, 99.79% purity, Br--)--) was obtained as white
solid. Compound 1-(carboxymethyl)-1-(prop-2-yn-1-yl)piperidin-1-ium
bromide (19 mg, 64.35 umol, 3.07% yield) was obtained as white
solid. LCMS: (M+): 182 .sup.1H NMR (400 MHz, Deuterium Oxide)
.delta. 4.49 (s, 2H), 4.04-4.00 (m, 2H), 3.63-3.55 (m, 2H),
3.51-3.43 (m, 2H), 3.06 (s, 1H), 1.81-1.74 (m, 4H), 1.60-1.50 (m,
2H).
##STR00243##
Compound 161:
(2R)-1-(fluoromethyl)-2-(hydroxymethyl)-1-methylpiperidin-1-ium
Iodide
[0790] To a solution of [(2R)-1-methyl-2-piperidyl]methanol (80 mg,
619.20 umol, 1 eq) in THF (5 mL) was added fluoro(iodo)methane
(148.54 mg, 928.79 umol, 1.5 eq). The mixture was stirred at
15.degree. C. for 12 hr. LCMS showed the desired compound was
detected. The precipitation was found. The reaction mixture was
filtered to give a residue. The residue was washed by acetone 20
mL. The residue was purified by prep-HPLC (column: Waters Xbridge
BEH C18 100*30 mm*10 um; mobile phase: [water (0.1% TFA)-ACN]; B %:
1%-10%, 10 min). Compound
(2R)-1-(fluoromethyl)-2-(hydroxymethyl)-1-methylpiperidin-1-ium
iodide (40 mg, 138.14 umol, 22.31% yield) was obtained as a yellow
oil. LCMS: (M+): 162.2 .sup.1H NMR (400 MHz, Deuterium Oxide)
.delta. 5.56 (d, J=6.1 Hz, 0.5H), 5.51-5.38 (m, 1H), 5.30 (d, J=6.0
Hz, 0.5H), 3.84-3.70 (m, 2H), 3.70-3.58 (m, 1H), 3.49-3.38 (m, 2H),
3.19-2.96 (m, 3H), 1.81-1.73 (m, 5H), 1.57-1.35 (m, 1H).
##STR00244##
Compound 162:
(R)--N-(2-carboxypropyl)-N,N-dimethylprop-2-yn-1-aminium
Chloride
[0791] Step 1:
[0792] To a solution of methyl (2S)-3-bromo-2-methyl-propanoate
(300 mg, 1.66 mmol, 1.3 eq) in ACN (5 mL) was added
N,N-dimethylprop-2-yn-1-amine (105.97 mg, 1.27 mmol, 135.17 uL, 1
eq) at 15.degree. C. The mixture was stirred at 80.degree. C. for
12 hr. LCMS showed the desired compound was detected. The reaction
mixture was concentrated under reduced pressure to give a residue.
The residue was purified by prep-HPLC (column: Waters Xbridge BEH
C18 100*30 mm*10 um; mobile phase: [water (0.1% TFA)-ACN]; B %:
1%-10%, 10 min). Compound
[(2R)-3-methoxy-2-methyl-3-oxo-propyl]-dimethyl-prop-2-ynyl-ammonium
(15 mg, 48.50 umol, 3.80% yield, CF.sub.3COO--) was obtained as a
colorless oil.
[0793] Step 2:
[0794] To a solution of
[(2R)-3-methoxy-2-methyl-3-oxo-propyl]-dimethyl-prop-2-ynyl-ammonium
(10 mg, 33.64 umol, 1 eq, CF.sub.3COO--) in dioxane (2 mL) and aq.
HCl (8 mL, 6 M) was stirred at 50.degree. C. for 4 hr. LCMS showed
the desired compound was detected and 50% of
[(2R)-3-methoxy-2-methyl-3-oxo-propyl]-dimethyl-prop-2-ynyl-ammonium
(10 mg, 33.64 umol, 1 eq, CF.sub.3COO--) remained. The mixture was
stirred at 50.degree. C. for 12 hr. LCMS showed the desired
compound was detected. The reaction mixture was concentrated under
reduced pressure to give a residue. The residue was purified by
prep-HPLC (column: Waters Xbridge BEH C18 100*30 mm*10 um; mobile
phase: [water (0.10% TFA)-ACN]; B %: 1%-10%, 10 min). Compound
(2R)-3-(N,N-dimethyl-prop-2-ynyl-azanyl)-2-methyl-propanoic acid
(Cl-salt) was obtained as a yellow oil. LCMS: (M+): 170.2 .sup.1H
NMR (400 MHz, Deuterium Oxide) .delta. 4.20 (d, J=2.6 Hz, 2H), 3.91
(dd, J=13.8, 8.9 Hz, 1H), 3.36 (dd, J=13.9, 2.2 Hz, 1H), 3.20 (t,
J=2.6 Hz, 1H), 3.10 (s, 6H), 3.03 (ddd, J=9.3, 7.2, 2.1 Hz, 1H),
1.25 (d, J=7.3 Hz, 3H).
##STR00245##
Compound 164: 1-(fluoromethyl)-1-(prop-2-yn-1-yl)piperidin-1-ium
Iodide
[0795] To a solution of 1-prop-2-ynylpiperidine (100 mg, 811.72
umol, 1 eq) in THE (5 mL) was added fluoro(iodo)methane (389.45 mg,
2.44 mmol, 3 eq) at 15.degree. C. The mixture was stirred at
15.degree. C. for 12 hr. The precipitation was found. The reaction
mixture was diluted with H2O6060 mL and extracted with ethyl
acetate 60 mL. The aqueous phase is freeze-dried to give the
residue. The residue was purified by prep-HPLC (column: Waters
Xbridge BEH C18 100*30 mm*10 um; mobile phase: [water (0.1%
TFA)-ACN]; B %: 1%-10%, 10 min). Compound
1-(fluoromethyl)-1-(prop-2-yn-1-yl)piperidin-1-ium iodide (60 mg,
211.92 umol, 41.10% yield) was obtained as a colorless oil. LCMS
(M+): 156.2 .sup.1H NMR (400 MHz, Deuterium Oxide) .delta. 5.51 (d,
J=44.9 Hz, 2H), 4.39 (d, J=1.8 Hz, 2H), 3.61-3.33 (m, 5H), 1.89 (p,
J=6.3 Hz, 4H), 1.77-1.58 (m, 2H).
##STR00246##
Compound 165: N,N-dimethyl-N-(prop-2-yn-1-yl)prop-2-yn-1-aminium
Bromide
[0796] To a solution of N,N-dimethylprop-2-yn-1-amine (100 mg, 1.20
mmol, 127.55 uL, 1 eq) in THF (5 mL) was added 3-bromoprop-1-yne
(143.10 mg, 1.20 mmol, 103.69 uL, 1 eq) at 15.degree. C. The
mixture was stirred at 15.degree. C. for 12 hr. The precipitation
was found. The reaction mixture was filtered to give a residue. The
residue was purified by prep-HPLC (column: Waters Xbridge BEH C18
100*30 mm*10 um; mobile phase: [water (0.1% TFA)-ACN]; B %: 1%-3%,
10 min). Compound
N,N-dimethyl-N-(prop-2-yn-1-yl)prop-2-yn-1-aminium bromide (88 mg,
435.45 umol, 56.77% yield) was obtained as a white solid. LCMS:
(M+): 122.1 .sup.1H NMR (400 MHz, DMSO-d6) .delta. 4.4 (s, 4H), 4.1
(s, 2H), 3.1 (s, 6H).
##STR00247##
Compound 166:
(S)--N-(2-carboxypropyl)-N,N-dimethylprop-2-yn-1-aminium
Chloride
[0797] Step 1:
[0798] To a solution of methyl (2R)-3-bromo-2-methyl-propanoate
(500 mg, 2.76 mmol, 1 eq) in ACN (10 mL) was added
N,N-dimethylprop-2-yn-1-amine (160.73 mg, 1.93 mmol, 205.01 uL, 0.7
eq) at 15.degree. C. The mixture was stirred at 80.degree. C. for
12 hr. TLC indicated most of N,N-dimethylprop-2-yn-1-amine
remained. The mixture was stirred at 80.degree. C. for 24 hr. The
reaction mixture was concentrated under reduced pressure to give a
residue. The residue was purified by prep-HPLC (column: Waters
Xbridge BEH C18 100*30 mm*10 um; mobile phase: [water (0.1%
TFA)-ACN]; B %: 1%-20%, 10 min). Compound methyl
(2S)-3-(N,N-dimethyl-prop-2-ynyl-azanyl)-2-methyl-propanoate (10
mg, 37.86 umol, Br-salt, 3.33% yield) was obtained as a colorless
oil.
[0799] Step 2:
[0800] A solution of methyl
(2S)-3-(N,N-dimethyl-prop-2-ynyl-azanyl)-2-methyl-propanoate (10
mg, 37.86 umol, 1 eq) in dioxane (2 mL) and HCl (8 mL) (6 M, H2O)
was stirred at 50.degree. C. for 16 hr. LCMS showed the desired
compound was detected. The reaction mixture was concentrated under
reduced pressure to give a residue. The residue was purified by
prep-HPLC (column: Waters XbridgeBEH C18 100*30 mm*10 um; mobile
phase: [water (0.1% TFA)-ACN]; B %: 1%-10%, 10 min). The residue
was further purified by prep-HPLC (column: Waters Xbridge BEH C18
100*30 mm*10 um; mobile phase: [water (0.1% TFA)-ACN]; B %: 1%-10%,
10 min) to give the title compound (6 mg, 29.17 umol, Cl-salt,
60.00% yield) as a yellow oil. LCMS: (M+): 170.2 .sup.1H NMR (400
MHz, Deuterium Oxide) .delta. 4.15 (d, J=2.6 Hz, 2H), 3.85 (dd,
J=13.8, 8.9 Hz, 1H), 3.30 (dd, J=13.8, 2.2 Hz, 1H), 3.14 (t, J=2.5
Hz, 1H), 3.04 (s, 6H), 3.01-2.90 (m, 1H), 1.18 (d, J=7.2 Hz,
3H).
##STR00248##
Compound 167: (3-hydroxy-2-methylidenepropyl)trimethylazanium
trifluoroacetate
[0801] Step 1:
[0802] To a solution of 2-methylenepropane-1,3-diol (1 g, 11.35
mmol, 925.93 uL, 1 eq) in DCM (10 mL) was added SOCl2 (2.03 g,
17.03 mmol, 1.24 mL, 1.5 eq) at -78.degree. C. and the mixture was
stirred at 15.degree. C. for 1.5 h. TLC indicated Reactant was
consumed completely. The reaction mixture was concentrated under
reduced pressure to give a residue. The crude product
5-methylene-1,3,2-dioxathiane 2-oxide (1.5 g, crude) as a brown
liquid was used into the next step without further
purification.
[0803] Step 2:
[0804] To a solution of 5-methylene-1,3,2-dioxathiane 2-oxide (600
mg, 4.47 mmol, 1 eq) in THF (10 mL) was added
N,N-dimethylmethanamine (1 M, 4.92 mL, 1.1 eq. in THF) at
15.degree. C. and the mixture was stirred at 70.degree. C. for 12
h. MS showed the desired compound was detected. The reaction
mixture was concentrated under reduced pressure to give a residue.
The residue was purified by prep-HPLC ([water (0.10% TFA)-ACN]) to
give the title compound (21 mg, 85.82 umol, 1.92% yield, 99.4%
purity, CF3CO2-) was obtained as a colorless oil. MS: (M+) 130.1
.sup.1H NMR (400 MHz, Methanol-d4) .delta. 5.84 (s, 1H), 5.62 (s,
1H), 4.23 (s, 2H), 4.04 (s, 2H), 3.17 (s, 9H).
##STR00249##
Compound 168: (cyanomethyl)dimethyl(prop-2-yn-1-yl)azanium
Bromide
[0805] A mixture of N,N-dimethylprop-2-yn-1-amine (500 mg, 6.01
mmol, 637.76 uL, 1 eq), 2-bromoacetonitrile (721.43 mg, 6.01 mmol,
400.80 uL, 1 eq) in THF (5 mL) degassed and purged with N.sub.2 3
times, and then the mixture was stirred at 15.degree. C. for 10 hr
under N.sub.2 atmosphere. LC-MS showed 80.4% desired compound was
detected. The reaction mixture was filtered and the filter cake was
concentrated under reduced pressure to give a residue. Then the
residue was washed with THF (4 mL) to give the title compound (1.0
g, 4.15 mmol, 69.07% yield, 84.37% purity, Br--) as a white solid.
LCMS: (M+) 123.1 1H NMR (400 MHz, DMSO-d.sub.6) .delta. 4.98 (s,
2H), 4.61 (d, J=2.6 Hz, 2H), 4.23 (t, J=2.5 Hz, 1H), 3.28 (s,
6H).
##STR00250##
Compound 169: (cyanomethyl)trimethylazanium Iodide
[0806] A mixture of N,N-dimethylmethanamine (1 M, 8.46 mL, 1 eq),
2-bromoacetonitrile (1.01 g, 8.46 mmol, 563.68 uL, 1 eq) in THF (5
mL) was degassed and purged with N2 for 3 times, and then the
mixture was stirred at 15.degree. C. for 10 hr under N2 atmosphere.
TLC indicated N,N-dimethylmethanamine was consumed completely and
one new spot larger polarity was formed. The reaction mixture was
filtered and the filter cake was concentrated under reduced
pressure to give a residue. Then the residue was washed with THF (4
mL) to give a residue as compound cyanomethyl(trimethyl)azanium
(1.38 g, 7.71 mmol, 91.11% yield, 100% purity, I--) was obtained as
a white solid. LCMS: (M+) 99.1 1H NMR (400 MHz, DMSO-d6) .delta.
4.98 (s, 2H), 3.26 (s, 9H).
##STR00251##
Compound 170:
N-(3-methoxy-2-methylidene-3-oxopropyl)-N,N-dimethylcyclopropanaminium
Iodide
[0807] Step 1:
[0808] To a solution of N-methylcyclopropanamine (300 mg, 4.22
mmol, 1 eq) in THF (10 mL) was added methyl
2-(bromomethyl)prop-2-enoate (755.43 mg, 4.22 mmol, 1 eq) and K2CO3
(874.84 mg, 6.33 mmol, 1.5 eq) at 25.degree. C. The mixture was
stirred at 70.degree. C. for 12 hr. LCMS showed the desired
compound was detected. The reaction mixture was filtered and the
filtrate was concentrated under reduced pressure to give a residue.
The residue was purified by column chromatography (SiO.sub.2,
Petroleum ether/Ethyl acetate=10:1 to 1:1). Compound methyl
2-[[cyclopropyl(methyl)amino]methyl]prop-2-enoate (400 mg, 2.36
mmol, 56.01% yield) was obtained as a colorless liquid.
[0809] Step 2:
[0810] To a solution of methyl
2-[[cyclopropyl(methyl)amino]methyl]prop-2-enoate (100 mg, 590.95
umol, 1 eq) in THF (10 mL) was added MeI (503.27 mg, 3.55 mmol,
220.73 uL, 6 eq) at 25.degree. C. The mixture was stirred at
25.degree. C. for 12 hr. The precipitation was found. The reaction
mixture was filtered to give a residue. The title compound (106 mg,
340.66 umol, I-salt, 57.65% yield, 100% purity) was obtained as a
white solid. LCMS: (M+): 184.1 1H NMR (400 MHz, Deuterium Oxide)
.delta. 6.79 (s, 1H), 6.28 (s, 1H), 4.24 (s, 2H), 3.71 (s, 3H),
3.05-2.98 (m, 1H), 2.81 (s, 6H), 1.07-0.99 (m, 2H), 0.78-0.68 (m,
2H).
##STR00252##
Compound 171:
(2-carboxy-2-methylideneethyl)(fluoromethyl)dimethylazanium
Iodide
[0811] To a solution of 2-[(dimethylamino)methyl]prop-2-enoic acid
(30 mg, 232.28 umol, 1 eq) in ACN (5 mL) was added fluoro added
fluoro(iodo)methane (185.74 mg, 1.16 mmol, 5 eq) at 15.degree. C.
The mixture was stirred at 15.degree. C. for 24 hr. LCMS showed the
LCMS showed the desired compound was detected. The reaction mixture
was concentrated under reduced pressure to give a residue. The
residue was purified by prep-HPLC (column: Waters Xbridge BEH C18
100*30 mm*10 um; mobile phase: [water (0.1% TFA)-ACN]; B %: 1%-10%,
10 min). The title compound (3 mg, 8.05 umol, I-salt, 3.47% yield,
77.59% purity) was obtained as a colorless oil. LCMS: (M+): 162.1
1H NMR (400 MHz, Deuterium Oxide) .delta. 6.76 (s, 1H), 6.31 (s,
1H), 5.78 (s, 1H), 5.65 (s, 1H), 3.92 (s, 2H), 2.75 (s, 6H).
##STR00253##
Compound 172:
N-(fluoromethyl)-N-(2-hydroxyethyl)-N-methylcyclopropanaminium
Iodide
[0812] Step 1:
[0813] To a solution of N-methylcyclopropanamine (1 g, 14.06 mmol,
1.68 eq) and 2-bromoethoxy-tert-butyl-dimethyl-silane (2.00 g, 8.37
mmol, 1 eq) in acetonitrile (20 mL) was added K2CO3 (1.16 g, 8.37
mmol, 1 eq) at 25.degree. C. The mixture was stirred at 50.degree.
C. for 12 hr. TLC indicated new spots formed. The reaction mixture
was filtered and concentrated under reduced pressure to give a
residue. The residue was purified by column chromatography
(SiO.sub.2, Petroleum ether/Ethyl acetate=10:1 to 1:1). Compound
N-[2-[tert-butyl(dimethyl)silyl]oxyethyl]-N-methyl-cyclopropanamine
(3.2 g, 13.95 mmol, 83.32% yield) was obtained as a colorless
oil.
[0814] Step 2:
[0815] To a solution of
N-[2-[tert-butyl(dimethyl)silyl]oxyethyl]-N-methyl-cyclopropanamine
(200 mg, 871.71 umol, 1 eq) in THF (10 mL) was added
fluoro(iodo)methane (418.24 mg, 2.62 mmol, 3 eq) at 25.degree. C.
The mixture was stirred at 25.degree. C. for 12 hr. LCMS showed 50%
of
N-[2-[tert-butyl(dimethyl)silyl]oxyethyl]-N-methyl-cyclopropanamine
remained. Fluoro(iodo)methane (418.24 mg, 2.62 mmol, 3 eq) was
added. The mixture was stirred at 25.degree. C. for 12 hr. The
reaction mixture was concentrated under reduced pressure to give a
residue. Compound
tert-butyl-[2-[N-cyclopropyl-(fluoromethyl)-methyl-azanyl]ethoxy]-dimethy-
l-silane (200 mg, 513.66 umol, 58.93% yield) was obtained as a
white solid.
[0816] Step 3:
[0817] To a solution of
tert-butyl-[2-[N-cyclopropyl-(fluoromethyl)-methyl-azanyl]ethoxy]-dimethy-
l-silane (200 mg, 513.66 umol, 1 eq) in H.sub.2O (10 mL) was added
KF (298.42 mg, 5.14 mmol, 120.33 uL, 10 eq) at 25.degree. C. The
mixture was stirred at 25.degree. C. for 12 hr. LCMS showed the
desired compound was detected. The reaction mixture was extracted
with ethyl acetate (50 mL). The aqueous phase is freeze-dried. The
residue was washed with washed by acetonitrile (100 mL). The
acetonitrile layer was filtered and the filtrate was concentrated
under reduced pressure to give a residue. The solid was diluted
with H.sub.2O (3 mL) and extracted with ethyl acetate (5 mL*4). The
aqueous phase was freeze-dried. The title compound (36 mg, 130.86
umol, I-salt, 85.71% yield) was obtained as a white solid. LCMS:
(M+): 148.1 1H NMR (400 MHz, Deuterium Oxide) .delta. 5.39 (q,
J=5.8 Hz, 1H), 5.28 (q, J=5.8 Hz, 1H), 4.02-3.97 (m, 2H), 3.58-3.51
(m, 2H), 3.16-3.12 (m, 1H), 2.78 (d, J=2.2 Hz, 3H), 1.18-1.14 (m,
2H), 0.96-0.79 (m, 2H).
##STR00254##
Compound 172A: (cyanomethyl)(2-hydroxyethyl)dimethylazanium
Bromide
[0818] A mixture of 2-(dimethylamino)ethanol (500 mg, 5.61 mmol,
563.06 uL, 1 eq) in THF (5 mL) was added slowly 2-bromoacetonitrile
(672.84 mg, 5.61 mmol, 373.80 uL, 1 eq) at 15.degree. C., and then
the mixture was stirred at 15.degree. C. for 10 hr under N2
atmosphere. TLC indicated 2-(dimethylamino) ethanol was consumed
completely and one new spot formed. The reaction was clean
according to TLC. The reaction mixture was filtered and the filter
cake was concentrated under reduced pressure to give a residue. The
crude product was washed with THE (4 mL). The title compound (200
mg, 956.55 umol, 17.05% yield, 100% purity, Br--) was obtained as a
white solid. LCMS: (M+) 129.1 1H NMR (400 MHz, DMSO-d6) .delta.
5.45 (t, J=4.8 Hz, 1H), 4.91 (s, 2H), 3.91-3.82 (m, 2H), 3.63-3.53
(m, 2H), 3.26 (s, 6H).
##STR00255##
Compound 173: benzyl(2-hydroxyethyl)dimethylazanium
[0819] A mixture of 2-(dimethylamino)ethanol (500 mg, 5.61 mmol,
563.06 uL, 1 eq) and bromomethylbenzene (959.40 mg, 5.61 mmol,
666.25 uL, 1 eq) in THF (5 mL) was degassed and purged with N.sub.2
for 3 times. And then the mixture was stirred at 15.degree. C. for
10 hr under N.sub.2 atmosphere. TLC indicated
2-(dimethylamino)ethanol was consumed completely and one new spot
larger polarity formed. The reaction mixture was filtered and the
filter cake was concentrated under reduced pressure to give a
residue. The crude product was triturated with THF (30 mL) at
15.degree. C. for 30 min. Then the reaction mixture was filtered
and the filter cake was concentrated under reduced pressure to give
a residue. The title compound (200 mg, 699.54 umol, 12.47% yield,
91% purity, Br--) was obtained as a white solid. LCMS: (M+) 180.1
1H NMR (400 MHz, Methanol-d4) .delta. 7.64-7.48 (m, 5H), 4.63 (s,
2H), 4.11-4.03 (m, 2H), 3.52-3.45 (m, 2H), 3.12 (s, 6H).
##STR00256##
Compound 174:
[(2R)-3-methoxy-2-methyl-3-oxopropyl]dimethyl(prop-2-yn-1-yl)azanium
[0820] To a solution of methyl (2S)-3-bromo-2-methyl-propanoate
(300 mg, 1.66 mmol, 2 eq) in ACN (10 mL) was added
N,N-dimethylprop-2-yn-1-amine (68.88 mg, 828.60 umol, 87.86 uL, 1
eq) at 15.degree. C. The mixture was stirred at 80.degree. C. for
12 hr. LCMS showed most of N,N-dimethylprop-2-yn-1-amine remained.
Then the mixture Then the mixture was stirred at 80.degree. C. for
24 hr. LCMS showed the desired compound was detected. The reaction
mixture was concentrated under reduced pressure to give a residue.
The residue was purified by prep-HPLC (column: Waters Xbridge BEH
C18 100*30 mm*10 um; mobile phase: [water (0.1% TFA)-ACN]; B %:
1%-20%, 10 min). The title compound (7 mg, 26.50 umol, Br-salt,
3.20% yield, 100% purity) was obtained as a yellow oil. LCMS: (M+):
184.1 1H NMR (400 MHz, Deuterium Oxide) .delta. 4.18 (d, J=2.5 Hz,
2H), 3.94 (dd, J=13.9, 8.7 Hz, 1H), 3.69 (s, 3H), 3.37 (dd, J=13.9,
2.2 Hz, 1H), 3.26-3.14 (m, 1H), 3.17-3.08 (m, 1H), 3.07 (s, 6H),
1.23 (d, J=7.2 Hz, 3H).
##STR00257##
Compound 175:
[(2S)-3-methoxy-2-methyl-3-oxopropyl]dimethyl(prop-2-yn-1-yl)azanium
[0821] To a solution of methyl (2R)-3-bromo-2-methyl-propanoate
(300 mg, 1.66 mmol, 1 eq) in ACN (10 mL) was added
N,N-dimethylprop-2-yn-1-amine (68.88 mg, 828.60 umol, 87.86 uL, 0.5
eq) at 15.degree. C. The mixture was stirred at 80.degree. C. for
24 hr. LCMS showed the desired compound was detected. The reaction
mixture was concentrated under reduced pressure to give a residue.
The residue was purified by prep-HPLC (column: Waters Xbridge BEH
C18 100*30 mm*10 um; mobile phase: [water (0.1% TFA)-ACN]; B %:
1%-20%, 10 min). The title compound (5 mg, 18.93 umol, Br-salt,
1.14% yield, 100% purity) was obtained as a yellow oil. LCMS: (M+):
184.1 1H NMR (400 MHz, Deuterium Oxide) .delta. 4.17 (d, J=2.5 Hz,
2H), 3.93 (ddd, J=13.9, 8.7, 1.3 Hz, 1H), 3.67 (d, J=1.3 Hz, 3H),
3.35 (dd, J=13.9, 2.1 Hz, 1H), 3.18 (t, J=2.5 Hz, 1H), 3.12 (dd,
J=8.9, 7.0 Hz, 1H), 3.05 (s, 6H), 1.22 (dd, J=7.2, 1.3 Hz, 3H).
##STR00258##
Compound 176: trimethyl[(3-methyloxiran-2-yl)methyl]azanium
[0822] Step 1:
[0823] To a solution of mCPBA (14.29 g, 66.26 mmol, 1.2 eq) (80%
purity) in CHCl3 (100 mL) was added (E)-1-chlorobut-2-ene (5 g,
55.22 mmol, 1 eq) at 0.degree. C. The mixture was stirred at
25.degree. C. for 12 h. TLC indicated the reactant was consumed
completely. The reaction mixture was quenched by addition
Na.sub.2SO.sub.3 aqueous solution (500 ml), and then the mixture
was partitioned and the organic phase was separated, dried over
Na.sub.2SO.sub.4, then filtered and the crude solution as 0.55
mol/L of 2-(chloromethyl)-3-methyl-oxirane (crude) in CHCl3 (100
ml) was used in the next step without further purification.
[0824] Step 2:
[0825] A solution of N,N-dimethylmethanamine (1 M, 57.86 mL, 1.23
eq) (THF) and 0.55 mol/L of 2-(chloromethyl)-3-methyl-oxirane (55
mmol, 100 mL, 1 eq) in CHCl.sub.3 (100 ml) was stirred at
25.degree. C. for 12 h. MS showed the desired compound was
detected. The reaction mixture was concentrated under reduced
pressure to give a residue. The residue was purified by prep-HPLC
([water (0.10% TFA)-ACN]). The title compound (5 mg, 19.12 umol,
93.00% yield, 93% purity, TFA-) was obtained as colorless oil.
LCMS: (M+): 130.2. 1H NMR (400 MHz, Methanol-d4) .delta. 3.89 (dd,
J=13.6, 1.8 Hz, 1H), 3.29-3.21 (m, 10H), 3.15 (dd, J=13.6, 9.1 Hz,
1H), 3.00 (qd, J=5.2, 1.9 Hz, 1H), 1.38 (d, J=5.2 Hz, 3H).
##STR00259##
Compound 177:
dimethyl(prop-2-yn-1-yl)[(1H-1,2,3,4-tetrazol-5-yl)methyl]azanium
[0826] A mixture of 5-(chloromethyl)-1H-tetrazole (500 mg, 4.22
mmol, 1 eq) and N,N-dimethylprop-2-yn-1-amine (350.69 mg, 4.22
mmol, 447.31 uL, 1 eq) in THF (5 mL) was degassed and purged with
N.sub.2 3 times, and then the mixture was stirred at 15.degree. C.
for 10 hr under N2 atmosphere. LCMS showed desired compound was
detected. The reaction mixture was filtered and the filtrate was
concentrated under reduced pressure to give a residue. The residue
was purified by prep-HPLC (column: Waters Xbridge BEH C18 100*30
mm*10 um; mobile phase: [water (0.04% NH3H2O)-ACN]; B %: 1%-3%, 10
min). The residue was further purified by SFC (column: DAICEL
CHIRALPAK AD (250 mm*30 mm, 10 um); mobile phase: [0.1% NH3H2O
EtOH]; B %: 20%-20%, 20 min). The title compound (6 mg, 29.75 umol,
60.00% yield, 100% purity) was obtained as a white solid. LCMS:
(M+): 166.1 1H NMR (400 MHz, DMSO-d6) .delta. 4.68 (s, 2H), 4.36
(d, J=2.5 Hz, 2H), 4.04 (t, J=2.4 Hz, 1H), 3.00 (s, 6H).
##STR00260##
Compound 178: (cyclopropylmethyl)trimethylazanium Iodide
[0827] To a solution of cyclopropylmethanamine (1 g, 9.30 mmol, 1
eq, HCl) in DCM (10 mL) was added dropwise MeI (26.39 g, 185.91
mmol, 11.57 mL, 20 eq) at 0.degree. C. After addition, the mixture
was stirred at 25.degree. C. for 12h. TLC indicated reactant 1 was
consumed completely. The reaction mixture was concentrated under
reduced pressure to give a residue. The residue was dissolved in
MeOH (7 mL), then to the solution was added MTBE (70 mL) and
precipitation formed, was filtered and the filter cake was
dissolved in MeOH (7 mL), then MTBE (70 mL) was added and
precipitation formed, was filtered and the filter cake was
concentrated under reduce pressure to give the target. The title
compound (676 mg, 2.78 mmol, 29.86% yield, 99% purity, I--) was
obtained as a white solid. LCMS: (M+): 114.2 1H NMR (400 MHz,
Deuterium Oxide) .delta. 3.00 (d, J=7.3 Hz, 2H), 2.91 (s, 9H),
1.01-0.86 (m, 1H), 0.62-0.51 (m, 2H), 0.24-0.15 (m, 2H).
##STR00261##
Compound 179: (fluoromethyl)dimethyl(prop-2-yn-1-yl)azanium
Iodide
[0828] To a mixture of N,N-dimethylprop-2-yn-1-amine (0.20 g, 2.39
mmol, 1 eq) in THF (5 mL) was added fluoro(iodo)methane (1.15 g,
7.18 mmol, 3 eq). The mixture was stirred at 25.degree. C. for 10
h. The reaction mixture was filtered and the filter cake was dried
under reduced pressure to give a crude product. The crude product
was washed with EtOAc (10 mL) and then dried. The title compound
(0.35 g, 1.44 mmol, 60.19% yield, 100% purity, I--) was obtained as
a white solid. LCMS: (M+): 116.2 1H NMR (400 MHz, Chloroform-d)
.delta. 6.29 (d, J=1.7 Hz, 1H), 5.79 (d, J=1.5 Hz, 1H), 3.79 (s,
3H), 3.39 (d, J=2.4 Hz, 2H), 2.35 (s, 3H), 2.25 (t, J=2.3 Hz,
1H).
##STR00262##
Compound 181:
dimethyl[(3-methyloxiran-2-yl)methyl](prop-2-yn-1-yl)azanium
[0829] Step 1:
[0830] To a solution of m-CPBA (8.58 g, 39.76 mmol, 80% purity, 1.2
eq) in CHCl3 (100 mL) was added (E)-1-chlorobut-2-ene (3 g, 33.13
mmol, 1 eq) at 0.degree. C. The mixture was stirred at 25.degree.
C. for 12 hr. TLC indicated (E)-1-chlorobut-2-ene was consumed
completely. The reaction mixture was filtered to give organic
layers. The combined organic layers were quenched by saturated
sodium sulfite solution (200 mL), and then extracted to give
organic layers. Compound 0.33 mol/L of
2-(chloromethyl)-3-methyl-oxirane (crude) in CHCl.sub.3 (100 mL)
was obtained as a colorless liquid and used into the next step
without further purification.
[0831] Step 2:
[0832] To a solution of 2-(chloromethyl)-3-methyl-oxirane (0.33 M,
100 mL, 1 eq) (CHCl.sub.3) was added N,N-dimethylprop-2-yn-1-amine
(2.74 g, 33.00 mmol, 3.50 mL, 1 eq) at 25.degree. C. The mixture
was stirred at 25.degree. C. for 12 hr. LCMS indicated desired
compound was detected. The reaction mixture was concentrated under
reduced pressure to give a liquid. The liquid was purified liquid
was purified by prep-HPLC (column: Waters Xbridge BEH C18 100*30
mm*10 um; mobile phase: [water (0.1% TFA)-ACN]; B %: 1%-10%, 10
min). Compound
2-[(N,N-dimethyl-prop-2-ynyl-azanyl)methyl]-3-methyl-oxirane (20
mg, 105.44 umol, 0.32% yield) was obtained as a colorless oil. The
oil was further purified by prep-HPLC (column: Waters XbridgeBEH
C.sub.18 100*30 mm*10 um; mobile phase: [water (0.1% TFA)-ACN]; B
%: 1%-5%, 10 min). The title compound (7 mg, 36.90 umol, Cl-salt,
35.00% yield, 100% purity) was obtained as a colorless oil. LCMS:
(M+): 154.2 .sup.1H NMR (400 MHz, Methanol-d4) .delta. 4.49-4.35
(m, 2H), 3.99 (d, J=12.3 Hz, 1H), 3.33-3.18 (m, 9H), 3.07-2.97 (m,
1H), 1.36 (d, J=5.2 Hz, 3H).
##STR00263##
Compound 182: (2-hydroxyethyl)dimethyl(prop-2-en-1-yl)azanium
Bromide
[0833] To a solution of 2-(dimethylamino)ethanol (200 mg, 2.24
mmol, 225.23 uL, 1 eq) in THF (10 mL) was added 3-bromoprop-1-ene
(271.44 mg, 2.24 mmol, 1 eq) at 0.degree. C. The mixture was
stirred at 25.degree. C. for 12 hr. The precipitation was found.
LCMS showed desired compound was detected. The reaction mixture was
diluted with H.sub.2O (60 mL) and extracted with ethyl acetate (60
mL). The aqueous phase is freeze-dried. The residue was washed with
ethyl acetate (50 mL) at 0.degree. C. Compound
(2-hydroxyethyl)dimethyl(prop-2-en-1-yl)azanium (160 mg, 761.50
umol, 33.94% yield, 100% purity) was obtained as a white solid.
LCMS: (M+): 130. 1H NMR (400 MHz, Deuterium Oxide) .delta.
6.00-5.82 (m, 1H), 5.64-5.51 (m, 2H), 3.96-3.83 (m, 4H), 3.37-3.30
(m, 2H), 2.98 (s, 6H).
##STR00264##
Compound 183: (2-aminoethyl)dimethyl(prop-2-yn-1-yl)azanium
[0834] Step 1:
[0835] To a solution of tert-butyl N-(2-bromoethyl)carbamate (200
mg, 892.48 umol, 1 eq) in THF (10 mL) was added
N,N-dimethylprop-2-yn-1-amine (74.19 mg, 892.48 umol, 94.63 uL, 1
eq) at 25.degree. C. The mixture was stirred at 25.degree. C. for 5
hr. LCMS showed LCMS showed most of tert-butyl
N-(2-bromoethyl)carbamate was remained. The mixture was stirred at
70.degree. C. for 12 hr. LCMS showed desired compound was detected.
The reaction mixture was concentrated under reduced pressure to
give a residue. The residue was diluted with H.sub.2O 60 mL and
extracted with ethyl acetate 60 mL. The aqueous phase is
freeze-dried. The residue was purified by prep-HPLC (column: Waters
Xbridge BEH C18 100*30 mm*10 um; mobile phase: [water (0.1%
TFA)-ACN]; B %: 1%-60%, 9 min). Compound tert-butyl
N-[2-(N,N,N-dimethyl-prop-2-ynyl-azanyl)ethyl]carbamate (120 mg,
390.59 umol, Br-salt, 80.00% yield, 100% purity) was obtained as a
colorless soil. LCMS: (M+): 227.2
[0836] Step 2:
[0837] To a solution of tert-butyl
N-[2-(N,N-dimethyl-prop-2-ynyl-azanyl)ethyl]carbamate (50 mg,
162.75 umol, 1 eq) in DCM (3 mL) and TFA (0.3 mL) was stirred at
25.degree. C. for 12 hr. LCMS showed tert-butyl
N-[2-(N,N-dimethyl-prop-2-ynyl-azanyl)ethyl]carbamate was consumed
completely and the desired compound was detected. The reaction
mixture was concentrated under reduced pressure to give a residue.
The title compound (30 mg, 144.85 umol, Br-salt, 89.00% yield, 100%
purity) was obtained as a colorless oil. LCMS: (M+): 127.1 1H NMR
(400 MHz, Deuterium Oxide) .delta. 4.24 (d, J=2.5 Hz, 2H),
3.73-3.64 (m, 2H), 3.47-3.38 (m, 2H), 3.23 (t, J=2.4 Hz, 1H), 3.15
(s, 6H).
##STR00265##
Compound 184: (2-hydroxyethyl)dimethyl[(oxetan-3-yl)methyl]azanium
Bromide
[0838] To a solution of 2-(dimethylamino)ethanol (100 mg, 1.12
mmol, 112.61 uL, 1 eq) in ACN (5 mL) was added
3-(bromomethyl)oxetane (169.41 mg, 1.12 mmol, 1 eq) at 25.degree.
C. The mixture was stirred at 70.degree. C. for 12 hr. The reaction
mixture was diluted with H.sub.2O 60 mL and extracted with ethyl
acetate 60 mL. The aqueous phase is freeze-dried. Compound
2-[N,N-dimethyl-(oxetan-3-ylmethyl)-azanyl]ethanol (70 mg, 290.26
umol, 25.87% yield, Br-salt, 99.576% purity) was obtained as a
white solid. LCMS: (M+): 160.1 1H NMR (400 MHz, Deuterium Oxide)
.delta. 4.81-4.73 (m, 2H), 4.55-4.47 (m, 2H), 3.94-3.86 (m, 2H),
3.76-3.61 (m, 3H), 3.37-3.30 (m, 2H), 2.95 (s, 6H).
##STR00266##
Compound 185:
(2-chloroprop-2-en-1-yl)dimethyl(prop-2-yn-1-yl)azanium
Chloride
[0839] To a mixture of N of N,N-dimethylprop-2-yn-1-amine (200 mg,
2.41 mmol, 255.10 uL, 1 eq) in THF (3 mL) was added
2,3-dichloroprop-1-ene (533.95 mg, 4.81 mmol, 441.28 uL, 2 eq) in
one portion at 25.degree. C. under N.sub.2. Then the mixture was
heated to 66.degree. C. and stirred for 12 hours. LCMS showed one
main peak with desired m/z was detected. The reaction mixture was
diluted with H.sub.2O (55 mL) and extracted with ethyl acetate (9
mL, 3 mL*3). The combined aqueous layer was lyophilized. Compound
2-chloroallyl-dimethyl-prop-2-ynyl-ammonium (266 mg, 1.36 mmol,
56.67% yield, 100% purity, Cl--) was obtained as yellow solid.
LCMS: (M+): 158.1 1H NMR (400 MHz, Deuterium Oxide) .delta. 5.89
(dd, J=20.2, 2.3 Hz, 2H), 4.27-4.20 (m, 4H), 3.19 (t, J=2.6 Hz,
1H), 3.15 (s, 6H).
##STR00267##
Compound 186: (2-fluoroethyl)(2-hydroxyethyl)dimethylazanium
[0840] To a solution of 2-(dimethylamino)ethanol (100 mg, 1.12
mmol, 112.61 uL, 1 eq) in THF (5 mL) was added
1-fluoro-2-iodo-ethane (195.16 mg, 1.12 mmol, 1 eq) at 25.degree.
C. The mixture was stirred at 25.degree. C. for 5 hr. The mixture
was stirred at 70.degree. C. for 12 hr. The precipitate was washed
with tetrahydrofuran (10 mL). The mixture was diluted with H.sub.2O
60 mL and extracted with ethyl acetate 100 mL. The aqueous phase is
freeze-dried. The title compound (83 mg, 315.48 umol, I-salt,
28.12% yield, 100% purity) was obtained as a white solid. LCMS:
(M+): 136.1 1H NMR (400 MHz, Deuterium Oxide) .delta. 4.93-4.86 (m,
1H), 4.82-4.74 (m, 1H), 3.97-3.89 (m, 2H), 3.79-3.73 (m, 1H),
3.72-3.66 (m, 1H), 3.51-3.44 (m, 2H), 3.11 (s, 6H).
##STR00268##
Compound 187:
1-(fluoromethyl)-1-(prop-2-yn-1-yl)pyrrolidin-1-ium
[0841] Step 1:
[0842] To a mixture of pyrrolidine (1 g, 14.06 mmol, 1.17 mL, 1 eq)
and 3-bromoprop-1-yne (1.84 g, 15.47 mmol, 1.33 mL, 1.1 eq) in EtOH
(10 mL) was added NaHCO.sub.3 (2.95 g, 35.15 mmol, 1.37 mL, 2.5
eq). The mixture was stirred at 25.degree. C. for 5 h. LC-MS showed
the desired product was detected. The residue was purified by
prep-HPLC (column: Phenomenex Luna C18 100*30 mm*5 um; mobile
phase: [water (0.04% HCl)-ACN]; B %: 1%-1%, 12 min). The solution
of 1-prop-2-ynylpyrrolidine (after prep-HPLC) was adjusted to pH=8
by NaHCO.sub.3. The mixture was extracted with ethyl acetate (40
mL*2). The combined organic phase dried with anhydrous
Na.sub.2SO.sub.4, filtered, and concentrated in vacuum.
1-prop-2-ynylpyrrolidine (in EtOAc (80 mL) was obtained. LCMS:
(M+): 110.2
[0843] Step 2:
[0844] To the solution of 1-prop-2-ynylpyrrolidine (60 mL, 1 eq) in
EtOAc (60 mL) was added fluoro(iodo)methane (0.8 g, 5.00 mmol, 1
eq) at 25.degree. C. The mixture was stirred at 25.degree. C. for 5
hr. TLC (Dichloromethane:Methanol=10:1, Rf=0.04) indicated reactant
was consumed completely. The mixture was concentrated in reduced
pressure. To the mixture was added H.sub.2O (10 mL). The aqueous
phase was extracted with ethyl acetate (10 mL*2). The aqueous phase
was lyophilized to give a yellow solid.
1-(fluoromethyl)-1-prop-2-ynyl-pyrrolidin-1-ium (70 mg, 254.78
umol, 97.942% purity, I--) was obtained as yellow solid. LCMS:
(M+): 142.1 .sup.1H NMR (400 MHz, Methanol-d4) .delta. 5.55 (d,
J=45.1 Hz, 2H), 4.50 (d, J=1.6 Hz, 2H), 3.82-3.71 (m, 4H), 3.55 (s,
1H), 2.30-2.23 (m, 4H).
##STR00269##
Compound 188: 1,1-bis(prop-2-yn-1-yl)pyrrolidin-1-ium
Bromidebromide
[0845] To pyrrolidine (1 g, 14.06 mmol, 1.17 mL, 1 eq) was added
3-bromoprop-1-yne (836.32 mg, 7.03 mmol, 606.03 uL, 0.5 eq) at
-10.degree. C. under N.sub.2. The mixture was stirred at 25.degree.
C. for 12 h. LCMS showed the desired compound was detected. The
mixture was purified directly without work-up. The residue was
purified by prep-HPLC (column: Phenomenex Luna C18 100*30 mm*5 um;
mobilephase: [water (0.04% HCl)-ACN]; B %: 1%-1%, 12 min). The
title compound (150 mg, 98.62% purity) was obtained as yellow
solid. LCMS: (M+): 148.2. .sup.1H NMR (400 MHz, Deuterium Oxide)
.delta. 4.70-4.60 (m, 4H), 3.72 (t, 4H), 3.20-3.10 (m, 2H),
2.25-2.15 (m, 4H).
##STR00270##
Compound 189:
N-(cyanomethyl)-2-(methoxycarbonyl)-N,N-dimethylprop-2-en-1-aminium
Bromide
[0846] Methyl 2-(bromomethyl)acrylate (0.200 g, 1 Eq, 1.12 mmol)
was dissolved in DCM (3 mL), followed by addition of
2-(dimethylamino)acetonitrile (94.0 mg, 109 .mu.L, 1 Eq, 1.12
mmol). The reaction was stirred at 40.degree. C. overnight. The
solution was concentrated by rotary evaporation until product
started to precipitate out. More DCM was added and material was
triturated in DCM. The solids were further washed in DCM and dried
in vacuo to yield product
N-(cyanomethyl)-2-(methoxycarbonyl)-N,N-dimethylprop-2-en-1-aminiumbromid-
e (0.24 g, 82%) as a white solid. LCMS M+: 183.3 .sup.1H NMR (400
MHz, DMSO-d6) .delta. 6.86 (s, 1H), 6.55 (s, 1H), 4.90 (s 2H), 4.41
(s, 2H), 3.76 (s, 3H), 3.20 (s, 6H).
##STR00271##
Compound 190:
N-((1-(methoxycarbonyl)cyclopropyl)methyl)-N,N-dimethylprop-2-yn-1-aminiu-
m Bromide
[0847] Compound methyl 1-(bromomethyl)cyclopropane-1-carboxylate
(0.100 g, 1 Eq, 518 .mu.mol) was dissolved in DCM (3 mL), followed
by addition of N,N-dimethylprop-2-yn-1-amine (45.2 mg, 58.6 .mu.L,
1.05 Eq, 544 .mu.mol). The reaction was stirred at 40.degree. C.
overnight, after which an immiscible viscous oil appeared. The oil
was washed with DCM multiple times and dried in vacuo to yield
product
N-((1-(methoxycarbonyl)cyclopropyl)methyl)-N,N-dimethylprop-2-yn-1-aminiu-
m bromide (0.025 g, 17%) as a viscous glassy oil. LCMS M+: 196.3
.sup.1H NMR (400 MHz, DMSO-d6) .delta. 4.38 (d, J=2.5 Hz, 2H), 4.06
(t, J=2.5 Hz, 1H), 3.77 (s, 2H), 3.63 (s, 3H), 3.09 (s, 6H), 1.44
(q, J=4.5 Hz, 2H), 1.27 (q, J=4.4 Hz, 2H).
##STR00272##
Compound 191:
[2-(2-hydroxyethoxy)ethyl]dimethyl(prop-2-yn-1-yl)azanium
[0848] To a solution of 2-(2-chloroethoxy)ethanol (299.68 mg, 2.41
mmol, 253.97 uL, 1 eq) in THF (10 mL) was added NaI (1.08 g, 7.22
mmol, 3 eq) at 25.degree. C. The mixture was stirred at 70.degree.
C. for 0.5 hr. N,N-dimethylprop-2-yn-1-amine (200 mg, 2.41 mmol,
255.10 uL, 1 eq) was added and stirred at 70.degree. C. for 12 hr.
The reaction mixture was concentrated under reduced pressure to
give a residue. The residue was purified by prep-HPLC (column:
Waters Xbridge BEH C18 100*30 mm*10 um; mobile phase: [water (0.10%
TFA)-ACN]; B %: 1%-5%, 9 min). The title compound (79 mg, 262.62
umol, I-salt, 10.92% yield, 99.447% purity) was obtained as yellow
oil. LCMS: (M+): 172.1 1H NMR (400 MHz, Deuterium Oxide) .delta.
4.17 (d, J=2.6 Hz, 2H), 3.86-3.79 (m, 2H), 3.61-3.51 (m, 4H),
3.54-3.45 (m, 2H), 3.11-3.06 (m, 7H).
##STR00273##
Compound 192: tert-butyl
N-{2-[dimethyl(prop-2-yn-1-yl)azaniumyl]ethyl}carbamate Bromide
[0849] To a solution of tert-butyl N-(2-bromoethyl)carbamate (200
mg, 892.48 umol, 1 eq) in THF (10 mL) was added
N,N-dimethylprop-2-yn-1-amine (74.19 mg, 892.48 umol, 94.63 uL, 1
eq) at 25.degree. C. The mixture was stirred at 25.degree. C. for 5
hr. LCMS showed most of the tert-butyl N-(2-bromoethyl)carbamate
remained. The mixture was stirred at 70.degree. C. for 12 hr. LCMS
showed desired compound was detected. The reaction mixture was
concentrated under reduced pressure to give a residue. The residue
was diluted with H.sub.2O 60 mL and extracted with ethyl acetate 60
mL. The aqueous phase is freeze-dried. The residue was purified by
prep-HPLC (column: Waters Xbridge BEH C18 100*30 mm*10 um; mobile
phase: [water (0.1% TFA)-ACN]; B %: 1%-60%, 9 min). The title
compound (120 mg, 390.59 umol, Br-salt, 80.00% yield, 100% purity)
was obtained as a colorless oil. LCMS: (M+): 227.2 1H NMR (400 MHz,
Deuterium Oxide) .delta. 4.22 (d, J=2.5 Hz, 2H), 3.51 (s, 4H), 3.23
(s, 1H), 3.16 (s, 6H), 1.37 (s, 9H).
##STR00274##
Compound 193: (difluoromethyl)(2-hydroxyethyl)dimethylazanium
[0850] To a solution of 2-(dimethylamino)ethanol (500 mg, 5.61
mmol, 563.06 uL, 1 eq) in DCM (10 mL) was added MeOH (197.71 mg,
6.17 mmol, 249.70 uL, 1.1 eq) and
1-[[bromo(difluoro)methyl]-ethoxy-phosphoryl]oxyethane (4.49 g,
16.83 mmol, 3 eq) at 25.degree. C. The mixture was stirred at
25.degree. C. for 12 hr. The precipitation was formed. The reaction
mixture was filtered to give a residue. To the residue was added
solvent (ethyl acetate:petroleum ether=1:1, 1 mL). The mixture was
diluted with H.sub.2O (1 mL). The aqueous phase was freeze-dried.
Compound 2-[N-(difluoromethyl)-dimethyl-azanyl]ethanol (39 mg,
177.23 umol, Br-salt, 4.2% yield, 100% purity) was obtained n as a
white solid. LCMS: (M+): 140.1 1H NMR (400 MHz, Deuterium Oxide)
.delta. 7.02 (t, J=58.7 Hz, 1H), 3.96 (t, J=4.8 Hz, 2H), 3.64-3.57
(m, 2H), 3.18 (t, J=1.5 Hz, 6H).
##STR00275##
Compound 194: (cyclobutylmethyl)(2-hydroxyethyl)dimethylazanium
Bromide
[0851] To a mixture of 2-(dimethylamino)ethanol (200 mg, 2.24 mmol,
225.23 uL, 1 eq) in THF (3 mL) was added bromomethylcyclobutane
added bromomethylcyclobutane (668.77 mg, 4.49 mmol, 502.83 uL, 2
eq) in one portion at 25.degree. C. under N.sub.2. Then the mixture
was heated to 66.degree. C. and stirred for 12 hours. LCMS showed
one main peak with desired m/z was detected. The reaction mixture
was diluted with H.sub.2O (5 mL) and extracted with ethyl acetate
(9 mL, 3 mL*3). The aqueous layer was lyophilized. The residue was
purified by prep-HPLC [water (0.10% TFA)-ACN]. The title compound
(76 mg, 319.11 umol, 14.22% yield, 100% purity, Br--) was obtained
as colorless oil. LCMS: (M+): 158.2 1H NMR (400 MHz, Deuterium
Oxide) .delta. 3.90-3.83 (m, 2H), 3.33-3.24 (m, 4H), 2.92 (s, 6H),
2.74 (p, J=7.9 Hz, 1H), 2.06-1.95 (m, 2H), 1.93-1.61 (m, 4H).
##STR00276##
Compound 195:
(2-hydroxyethyl)dimethyl[(methylsulfanyl)methyl]azanium
[0852] To a mixture of 2-(dimethylamino)ethanol (200 mg, 2.24 mmol,
225.23 uL, 1 eq) in THF (3 mL) was added chloro added
chloro(methylsulfanyl)methane (433.40 mg, 4.49 mmol, 376.87 uL, 2
eq) in one portion at 25.degree. C. under N.sub.2. Then the mixture
was heated to 66.degree. C. and stirred for 12 hours. LCMS showed
one main peak with desired m/z was detected. The reaction mixture
was diluted with H.sub.2O (5 mL) and extracted with ethyl acetate
(9 acetate 9 mL, 3 mL*3). The aqueous layer was lyophilized was
lyophilized. The residue was purified by prep-HPLC [water (0.10%
TFA)-ACN]. Compound
2-hydroxyethyl-dimethyl-(methylsulfanylmethyl)azanium (72 mg,
387.69 umol, 17.28% yield, 100% purity, Cl--)--) was obtained as
colorless oil. LCMS: (M+): 150.1 1H NMR (400 MHz, Deuterium Oxide)
.delta. 4.47 (s, 2H), 3.91-3.83 (m, 2H), 3.43-3.36 (m, 2H), 2.99
(s, 6H) 2.23 (s, 3H).
##STR00277##
Compound 196:
dimethyl[(methylsulfanyl)methyl](prop-2-yn-1-yl)azanium
[0853] To a mixture of N,N-dimethylprop-2-yn-1-amine (200 mg, 2.41
mmol, 255.10 uL, 1 eq) in THF (3 mL) was added
chloro(methylsulfanyl)methane (464.71 mg, 4.81 mmol, 404.09 uL, 2
eq) in one portion at 25.degree. C. under N.sub.2. Then the mixture
was heated to 66.degree. C. and stirred for 12 hours. LCMS showed
one main peak with desired m/z was detected. The reaction mixture
was diluted with H.sub.2O (5 mL) and extracted with ethyl acetate
(9 mL, 3 mL*3). The aqueous layer was lyophilized. The residue was
purified by prep-HPLC [water (0.10% TFA)-ACN]. Compound
dimethyl[(methylsulfanyl)methyl](prop-2-yn-1-yl)azanium (93 mg,
507.15 umol, 21.08% yield, 98% purity, TFA) was obtained as
colorless oil. LCMS: (M+): 144.1 1H NMR (400 MHz, Deuterium Oxide)
.delta. 4.59 (s, 2H), 4.23 (d, J=2.6 Hz, 2H), 3.17 (t, J=2.6 Hz,
1H), 3.10 (s, 6H), 2.34 (s, 3H).
##STR00278##
Compound 197: dimethyl(2-oxopropyl)(prop-2-yn-1-yl)azanium
Bromide
[0854] To a mixture of N,N-dimethylprop-2-yn-1-amine (200 mg, 2.41
mmol, 255.10 uL, 1 eq) in THF (3 mL) was added 1-bromopropan-2-one
(659.07 mg, 4.81 mmol, 2 eq) in one portion at 25.degree. C. under
N.sub.2. Then the mixture the mixture was heated to 66.degree. C.
and stirred for 12 hours. LCMS showed one main peak with desired
m/z was detected. The reaction mixture was diluted with H.sub.2O (5
mL) and extracted with ethyl acetate9 mL (3 mL*3). The aqueous
layer was lyophilized. The residue was purified by prep-HPLC [water
(0.10% TFA)-ACN]. The title compound (155 mg, 704.21 umol, 29.27%
yield, 100% purity, Br--)--) was obtained as white solid. LCMS:
(M+): 140.1 1H NMR (400 MHz, Deuterium Oxide) .delta. 4.55 (s, 2H),
4.38 (d, J=2.6 Hz, 2H), 3.24 (s, 6H), 3.20 (t, J=2.6 Hz, 1H), 2.18
(s, 3H).
##STR00279##
Compound 198: (2-chloroethyl)(2-hydroxyethyl)dimethylazanium
Iodide
[0855] To a solution of 2-(dimethylamino)ethanol (200 mg, 2.24
mmol, 225.23 uL, 1 eq) in THF (10 mL) was added
1-chloro-2-iodo-ethane (427.24 mg, 2.24 mmol, 1 eq) at 25.degree.
C. The mixture was stirred at 25.degree. C. for 12 hr. LCMS showed
most of 2-(dimethylamino)ethanol was remained and a little of
desired compound was detected. The mixture was stirred at
70.degree. C. for 6 hr. The precipitation was found. The reaction
mixture was diluted with H.sub.2O (60 mL) and extracted with ethyl
acetate (100 mL). The aqueous phase is freeze-dried. The residue
was purified by prep-HPLC (column: Phenomenex Luna C18 100*30 mm*5
um; mobile phase: [water (0.1% TFA)-ACN]; B %: 1%-1%, 12 min). The
title compound (39 mg, 135.99 umol, I-salt, 9.50% yield, 97.478%
purity) was obtained as a colorless oil. LCMS: (M+): 152.1 1H NMR
(400 MHz, Deuterium Oxide) .delta. 3.95-3.82 (m, 4H), 3.68 (t,
J=6.7 Hz, 2H), 3.47-3.40 (m, 2H), 3.08 (s, 6H).
##STR00280##
Compound 199: N-(2-hydroxyethyl)-N,N-dimethylcyclobutanaminium
[0856] Step 1:
[0857] To a mixture of 2 of 2-(methylamino)ethanol (500 mg, 6.66
mmol, 534.76 uL, 1 eq) in DCE (5 mL) was added HOAc (799.53 mg,
13.31 mmol, 761.46 uL, 2 eq) in one portion at 25.degree. C. under
N.sub.2. The mixture was stirred at 25.degree. C. for 0.5 h, then
cyclobutanone (933.17 mg, 13.31 mmol, 994.85 uL, 2 eq) and
NaBH(OAc).sub.3 (2.82 g, 13.31 mmol, 2 eq) was added. Then the
mixture was stirred at 25.degree. C. for 11.5 hours. LCMS showed
one main peak with expected mass was detected. The reaction mixture
was filtered and concentrated under reduced pressure to give a
residue. Compound 2-[cyclobutyl(methyl)amino]ethanol (1 g, crude)
was obtained as yellow oil. LCMS: (M+): 130.1
[0858] Step 2:
[0859] To a mixture of 2-[cyclobutyl(methyl)amino]ethanol (430 mg,
3.33 mmol, 1 eq) in THF (3 mL) was added MeI (2.36 g, 16.64 mmol,
1.04 mL, 5 eq) in one portion at 25.degree. C. under N.sub.2. The
mixture was stirred at 25.degree. C. for 12 hours. LCMS showed one
main peak with desired m/z was detected. The reaction mixture was
concentrated under reduced pressure to give a residue. The residue
was purified by prep-HPLC [water (0.10% TFA)-ACN] and by prep-HPLC
[water (0.2% FA)-ACN]. Compound
cyclobutyl-(2-hydroxyethyl)-dimethyl-ammonium (38 mg, 140.15 umol,
4.21% yield, I--)--) was obtained as colorless oil. LCMS: (M+):
144.1 1H NMR (400 MHz, Deuterium Oxide) .delta. 3.99 (p, J=8.9 Hz,
1H), 3.90-3.86 (m, 2H), 3.25-3.18 (m, 2H), 2.88 (s, 6H), 2.24 (p,
J=10.3 Hz, 2H), 2.13-2.02 (m, 2H), 1.76-1.51 (m, 2H).
##STR00281##
Compound 200:
(3R)-3-hydroxy-1,1-bis(prop-2-yn-1-yl)pyrrolidin-1-ium
[0860] To a mixture of (3R)-pyrrolidin-3-ol (0.5 g, 5.74 mmol,
476.19 uL, 1 eq) in THF (10 mL) and EtOH (10 mL) was added 3 added
3-bromoprop-1-yne (1.37 g, 11.48 mmol, 989.61 uL, 2 eq) in one
portion at -5.degree. C. under N.sub.2. The mixture was stirred at
25.degree. C. for 5h. LCMS (product: RT=0.142 min) showed the
starting material was consumed completely. The mixture was
concentrated in reduced pressure at 45.degree. C. The residue was
poured into water (20 mL). The aqueous phase was extracted with
ethyl acetate (10 mL*3). The aqueous phase was lyophilized to give
yellow oil. The crude product was purified by prep-HPLC (column:
PhenomenexLuna C18 100*30 mm*5 um; mobile phase: [water (0.1%
TFA)-ACN]; B %: 1%-1%, 12 min). The product was added to ACN (10
mL). The mixture was stirred at 25.degree. C. for 30 min. The
mixture was filtered. The organic phase was concentrated in vacuum.
The title compound (60 mg, 4.4% yield, Br--)--) was obtained as
yellow oil. LCMS: (M+): 164.1 .sup.1H NMR (400 MHz, Methanol-d4)
.delta. 4.67 (s, 1H), 4.61 (d, J=2.5 Hz, 2H), 4.55-4.42 (m, 2H),
4.03-3.91 (m, 1H), 3.91-3.81 (m, 1H), 3.83-3.72 (m, 2H), 3.54-3.47
(m, 2H), 2.59-2.45 (m, 1H), 2.25-2.17 (m, 1H).
##STR00282##
Compound 201: 1,1-bis(prop-2-yn-1-yl)pyrazolidin-1-ium Bromide
[0861] Step 1:
[0862] To a solution of tert-butyl pyrazolidine-1-carboxylate (2 g,
11.61 mmol, 1 eq) in THF (20 mL) was added K.sub.2CO.sub.3 (1.77 g,
12.77 mmol, 1.1 eq) and 3-bromoprop-1-yne (1.66 g, 13.94 mmol, 1.20
mL, 1.2 eq). The mixture was stirred at 25.degree. C. for 12 hr.
LC-MS showed the desired compound was detected. The reaction
mixture was filtered and the filtrate was concentrated under
reduced pressure to give a residue. The residue was purified by
column chromatography (SiO.sub.2, Petroleum ether/Ethyl
acetate=10:1 to 1:1). Compound tert-butyl
2-prop-2-ynylpyrazolidine-1-carboxylate (1.8 g, 8.56 mmol, 73.71%
yield) was obtained as a yellow solid. LCMS: (M-56): 155.1
[0863] Step 2:
[0864] To a solution of tert-butyl
2-prop-2-ynylpyrazolidine-1-carboxylate (50 mg, 237.79 umol, 1 eq)
in THF (5 mL) was added 3-bromoprop-1-yne (113.15 mg, 951.15 umol,
81.99 uL, 4 eq) at 25.degree. C. The mixture was stirred at
25.degree. C. for 12 hr. LC-MS showed the desired compound was
detected. The reaction mixture was concentrated under reduced
pressure to give a residue. The residue was washed with
tetrahydrofuran (20 mL). Compound
tert-butyl-2,2-bis(prop-2-ynyl)-1,2-diazolidine-1-carboxylate (50
mg, 151.87 umol, Br-salt, 63.87% yield) was obtained as a white
solid. LCMS: (M+): 249.2
[0865] Step 3:
[0866] A solution of tert-butyl
2,2-bis(prop-2-ynyl)-1,2-diazolidine-1-carboxylate (50 mg, 151.87
umol, 1 eq) in TFA (0.5 mL) and DCM (5 mL) was stirred at
25.degree. C. for 12 hr. LCMS showed the desired compound was
detected. The reaction mixture was concentrated under reduced
pressure to give a residue. Compound
1,1-bis(prop-2-ynyl)-1,2-diazolidine (22 mg, 96.02 umol, Br-salt,
63.23% yield, 100% purity) was obtained as a yellow oil. LCMS:
(M+H+): 149.1 1H NMR (400 MHz, Deuterium Oxide) .delta. 4.50-4.36
(m, 4H), 3.84 (t, J=7.6 Hz, 2H), 3.31 (t, J=7.0 Hz, 2H), 3.10 (t,
J=2.5 Hz, 2H), 2.36-2.24 (m, 2H).
##STR00283##
Compound 202: diethyl
{2-[dimethyl(prop-2-yn-1-yl)azaniumyl]ethyl}phosphonate Bromide
[0867] A mixture of N,N-dimethylprop-2-yn-1-amine (0.5 g, 6.01
mmol, 637.76 uL, 1 eq) and 1-bromo-2-diethoxyphosphoryl-ethane
(1.77 g, 7.22 mmol, 1.2 eq) in THF (10 mL) was degassed and purged
with N.sub.2 3 times, and then the mixture was stirred at 25 at
25.degree. C. for 10 hr under N.sub.2 atmosphere. LC-MS showed
N,N-dimethylprop-2-yn-1-amine was remained. Desired compound was
detected by LCMS. Then crude product was added water added water
(20 mL), and the aqueous phase is freeze-dried. The title compound
(100 mg, 398.75 umol, 6.63% yield, 99% purity) was obtained as a
white oil. LCMS (M+): 248.1. .sup.1H NMR (400 MHz, Methanol-d4)
.delta. 4.40 (d, J=2.5 Hz, 2H), 4.24-4.11 (m, 4H), 3.75-3.64 (m,
2H), 3.62-3.56 (m, 1H), 3.21 (s, 6H), 2.53-2.39 (m, 2H), 1.36 (t,
J=7.1 Hz, 6H).
##STR00284##
Compound 203:
N-allyl-2-(methoxycarbonyl)-N,N-dimethylprop-2-en-1-aminium
2,2,2-trifluoroacetate
[0868] Methyl 2-(bromomethyl)acrylate (0.200 g, 1 Eq, 1.12 mmol)
was dissolved in DCM (3 mL), followed by addition of
N,N-dimethylprop-2-en-1-amine (95.1 mg, 132 .mu.L, 1 Eq, 1.12
mmol). The reaction was stirred at 40.degree. C. overnight, then
concentrated by rotary evaporation. The reaction mixture was
purified by reverse phase column chromatography on an C18AQ column
(100% water with 0.1% TFA). Fractions containing product were
lyophilized to yield
N-allyl-2-(methoxycarbonyl)-N,N-dimethylprop-2-en-1-aminium
2,2,2-trifluoroacetate (150 mg, 45.2%) as a gel-like solid. LCMS
M+: 184.3. .sup.1H NMR (400 MHz, Deuterium Oxide) .delta. 6.90 (s,
1H), 6.34 (s, 1H), 6.09-5.91 (m, 1H), 5.74-5.55 (m, 2H), 4.16 (s,
2H), 3.86 (d, J=7.4 Hz, 2H), 3.76 (s, 3H), 2.91 (s, 6H).
##STR00285##
Compound 204: dimethyl(prop-2-yn-1-yl)propylazanium Iodide
[0869] N,N-dimethylprop-2-yn-1-amine (500 mg, 6.01 mmol, 637.76 uL,
1 eq) and 1-iodopropane (2.04 g, 12.03 mmol, 1.18 mL, 2 eq) in THF
(2 mL) was stirred at 25.degree. C. for 10 h. LCMS showed the
starting reactant consumed completely. The mixture was filtered and
filter cake was washed by EtOAc (10 mL*3). The filter cake was
concentrated. The title compound (250 mg, 4% yield, I--) was
obtained as a white solid. LCMS: (M+): 126.2 1H NMR (400 MHz,
Deuterium Oxide) .delta. 4.10 (s, 2H), 3.30-3.21 (m, 2H), 3.03 (s,
6H), 1.75-1.60 (m, 2H), 0.85 (t, J=7.3 Hz, 3H).
##STR00286##
Compound 205:
(2S)-1-(fluoromethyl)-2-(hydroxymethyl)-1-(prop-2-yn-1-yl)pyrrolidin-1-iu-
m Iodide
[0870] To a mixture of [of
[(2S)-1-prop-2-ynylpyrrolidin-2-yl]methanol (100 mg, 718.42 umol, 1
eq) in THF (3 mL) was added fluoro added fluoro(iodo)methane
(574.48 mg, 3.59 mmol, 5 eq) in one portion at 25.degree. C. under
N.sub.2. The mixture was stirred at 25.degree. C. for 12 at
25.degree. C. for 12 hours. LCMS showed one main peak with desired
m/z was detected. The reaction mixture was diluted with H.sub.2O (3
mL). The aqueous layer was washed with ethyl acetate (3 mL*4). Then
the aqueous layer was lyophilized. Compound
[(2S)-1-(fluoromethyl)-1-prop-2-ynyl-pyrrolidin-1-ium-2-yl]methanol
(115 mg, 384.46 umol, 53.51% yield, I--) was obtained as white
solid. LCMS: (M+): 172.1 1H NMR (400 MHz, Deuterium Oxide) .delta.
5.65-5.39 (m, 2H), 4.49-4.40 (m, 1H), 4.40-4.30 (m, 1H), 4.20-3.99
(m, 1H), 3.99-3.91 (m, 1H), 3.90-3.64 (m, 3H), 3.12 (t, J=2.6 Hz,
1H), 2.28-2.18 (m, 1H), 2.18-2.03 (m, 2H), 2.06-1.87 (m, 1H).
##STR00287##
Compound 206: ethyldimethyl(prop-2-yn-1-yl)azanium Iodide
[0871] A mixture of N,N-dimethylprop-2-yn-1-amine (500 mg, 6.01
mmol, 637.76 uL, 1 eq) and iodoethane (1.88 g, 12.03 mmol, 962.11
uL, 2 eq) in THF (2 mL) was stirred at 25.degree. C. for 10 h. TLC
showed Reactant was consumed completely. The reaction mixture was
concentrated under reduced pressure to remove THF. The residue was
diluted with water (30 mL) and extracted with EtOAc (10 mL*3). The
water layer was concentrated by lyophilization. The title compound
(200 mg, 794.66 umol, 13.21% yield, 95% purity, I--) was obtained
as a yellow solid. LCMS: (M+): 112.1 1H NMR (400 MHz, Deuterium
Oxide) .delta. 4.09 (s, 2H), 3.39 (q, J=7.3 Hz, 2H), 3.03-2.99 (m,
6H), 1.25 (t, J=7.4 Hz, 3H).
##STR00288##
Compound 207:
dimethyl[(1,3-oxazol-2-yl)methyl](prop-2-yn-1-yl)azanium
Chloride
[0872] A mixture of N,N-dimethylprop-2-yn-1-amine (100 mg, 1.20
mmol, 127.55 uL, 1 eq) and 2-(chloromethyl)oxazole (141.38 mg, 1.20
mmol, 1 eq) in THF (2 mL) was stirred at 25.degree. C. for 10 h.
LCMS showed the starting reactant was consumed completely. The
reaction mixture was concentrated. The residue was dissolved with
H.sub.2O (10 mL) and extracted with EtOAc (10 mL*3), Water phase
was freeze-dried. The title compound (95 mg, crude, Cl--) was
obtained as yellow solid. LCMS: (M+): 165.1 1H NMR (400 MHz,
Deuterium Oxide) .delta. 7.95 (d, J=0.9 Hz, 1H), 7.25 (d, J=0.9 Hz,
1H), 4.74 (s, 2H), 4.22 (d, J=2.5 Hz, 2H), 3.28-3.17 (m, 1H), 3.15
(s, 6H).
##STR00289##
Compound 208: N,N,2-trimethyl-N-(prop-2-yn-1-yl)prop-2-en-1-aminium
2,2,2-trifluoroacetate
[0873] N,N-dimethylprop-2-yn-1-amine (116 mg, 0.150 mL, 1 Eq, 1.39
mmol) and 3-bromo-2-methylprop-1-ene (188 mg, 1 Eq, 1.39 mmol) were
added to a vial with DCM (2 mL), and the reaction was stirred at 40
C overnight. The mixture was concentrated and re-dissolved in water
before purification by reverse phase column chromatography on a
C18AQ column (100% water with 0.1% TFA). Fractions containing
product were lyophilized to yield
N,N,2-trimethyl-N-(prop-2-yn-1-yl)prop-2-en-1-aminium
2,2,2-trifluoroacetate (0.0290 g, 8.29%) as a clear viscous oil.
LCMS M+: 138.2 1H NMR (400 MHz, Deuterium Oxide) .delta. 5.52 (s,
1H), 5.35 (s, 1H), 4.14 (d, J=2.6 Hz, 2H), 3.91 (s, 2H), 3.20 (t,
J=2.6 Hz, 1H), 3.09 (s, 6H), 1.90 (s, 3H).
##STR00290##
Compound 209:
2-fluoro-N,N-dimethyl-N-(prop-2-yn-1-yl)prop-2-en-1-aminium
2,2,2-trifluoroacetate
[0874] N,N-dimethylprop-2-yn-1-amine (116 mg, 0.150 mL, 1 Eq, 1.39
mmol) and 3-bromo-2-fluoroprop-1-ene (194 mg, 1 Eq, 1.39 mmol) were
added to a vial with DCM (2 mL), and the reaction was stirred at
40.degree. C. overnight. The mixture was concentrated and
re-dissolved in water before purification by reverse phase column
chromatography on an C18AQ column (100% water with 0.1% TFA).
Fractions containing product were lyophilized to yield
2-fluoro-N,N-dimethyl-N-(prop-2-yn-1-yl)prop-2-en-1-aminium
2,2,2-trifluoroacetate (262 mg, 73.7%) as a clear viscous oil. LCMS
M+: 142.2. .sup.1H NMR (400 MHz, Deuterium Oxide) .delta. 5.31 (dd,
J=15.8, 4.0 Hz, 1H), 5.12 (dd, J=47.9, 3.9 Hz, 1H), 4.31-4.17 m 4H
3.24 (t, J=2.6 Hz, 1H), 3.17 (s, 6H).
##STR00291##
Compound 210:
{2-[dimethyl(prop-2-yn-1-yl)azaniumyl]ethyl}phosphonic Acid
[0875] A mixture/n of
3-[N-(2-diethoxyphosphorylethyl)-dimethyl-azanyl]prop-1-yne (300
mg, 914.13 umn/ol, 1 eq) in aq. HCl (20 mL, 12 N) was stirred at
100.degree. C. for 15 hr. LC-MS showed the desired compound was
detected. The reaction mixture was concentrated under reduced
pressure to give a residue. The residue was purified by prep-HPLC
(column: Phenomenex Luna C18 100*30 mm*5 um; mobile phase: [water
(0.10% TFA)-ACN]; B %: 1%-1%, 12 min). Compound
2-(N,N-dimethyl-prop-2-ynyl-azanyl)ethylphosphonic acid (44 mg,
161.72 umol, Br-salt, 17.69% yield, 100% purity) was obtained as a
white solid. LCMS: (M+): 192.1. .sup.1H NMR (400 MHz, Deuterium
Oxide) .delta. 4.12 (d, J=2.6 Hz, 2H), 3.54-3.44 (m, 2H), 3.12 (t,
J=2.5 Hz, 1H), 3.04 (s, 6H), 2.05-1.91 (m, 2H).
##STR00292##
Compound 211: N,N-dimethyl-N-(prop-2-yn-1-yl)prop-2-en-1-aminium
2,2,2-trifluoroacetate
[0876] Compound 3-bromoprop-1-yne (200 mg, 0.150 mL, 1 Eq, 1.68
mmol) and N,N-dimethylprop-2-en-1-amine (143 mg, 199 .mu.L, 1 Eq,
1.68 mmol) were added to a vial with DCM (2 mL), and the reaction
was stirred at 40 C overnight. The mixture was concentrated and
re-dissolved in water before purification by reverse phase column
chromatography on a C18AQ column (100% water with 0.1% TFA).
Fractions containing product were lyophilized to yield
N,N-dimethyl-N-(prop-2-yn-1-yl)prop-2-en-1-aminium
2,2,2-trifluoroacetate (260 mg, 65.1%) as a clear viscous oil. LCMS
M+: 124.2. .sup.1H NMR (400 MHz, Deuterium Oxide) .delta. 6.03-5.88
(m, 1H), 5.75-5.61 (m, 2H), 4.12 (d, J=2.4 Hz, 2H), 3.95 (d, J=7.4
Hz, 2H), 3.21-3.14 (m, 1H), 3.07 (s, 6H).
##STR00293##
Compound 212: (2-hydroxyethyl)(methoxymethyl)dimethylazanium
trifluoroacetate
[0877] To a solution of 2-(dimethylamino)ethanol (500 mg, 5.61
mmol, 563.06 uL, 1 eq) in DCM (5 mL) was added MOMCl (677.45 mg,
8.41 mmol, 639.10 uL, 1.5 eq) at 0.degree. C. The mixture was
stirred at 25.degree. C. for 5 h. LCMS showed Reactant was consumed
completely. The mixture was quenched with water (10 mL) then
extracted with EtOAc (10 mL*3). The aqueous phase was concentrated
by lyophilization. The residue was purified by prep-HPLC (column:
Phenomenex Luna C18 100*30 mm*5 um; mobile phase: [water (0.10%
TFA)-ACN]. The title compound (135 mg, 905.39 umol, 16.14% yield,
90% purity, TFA salt) was obtained as yellow oil. LCMS: (M): 134.1
1H NMR (400 MHz, Deuterium Oxide) .delta. 4.50 (s, 2H), 3.91-3.82
(m, 2H), 3.52 (s, 3H), 3.35-3.28 (m, 2H), 2.94 (s, 6H).
##STR00294##
Compound 213: ethyl(fluoromethyl)(2-hydroxyethyl)(methyl)azanium
Iodide
[0878] Step 1:
[0879] To a solution of 2-bromoethanol (1 g, 8.00 mmol, 568.18 uL,
1 eq) in THF (10 mL) was added K.sub.2CO.sub.3 (1.22 g, 8.80 mmol,
1.1 eq) and N-methylethanamine (520.31 mg, 8.80 mmol, 756.27 uL,
1.1 eq). The mixture was stirred at 25.degree. C. for 12 hr. LCMS
showed the desired compound was detected. The reaction mixture was
filtered and the filtrate was concentrated under reduced pressure
to give a residue. The residue was purified by column
chromatography (SiO.sub.2, Petroleum ether/Ethyl acetate=0:1 to
Ethyl acetate:Methanol=1:1). Compound 2-[ethyl(methyl)amino]ethanol
(120 mg, 1.14 mmol, 14.25% yield, 98% purity) was obtained as a
yellow oil. LCMS: (M+H+): 104.1
[0880] Step 2:
[0881] To a solution of 2-[ethyl(methyl)amino]ethanol (100 mg,
969.35 umol, 1 eq) in THF (5 mL) was added fluoro(iodo)methane
(775.13 mg, 4.85 mmol, 5 eq). The mixture was stirred at 25.degree.
C. for 12 hr. LCMS showed the desired compound was detected. The
reaction mixture was concentrated under reduced pressure to give a
residue. The residue was diluted with H.sub.2O (50 mL) and
extracted with ethyl acetate (50 mL*3). The aqueous phase is
freeze-dried. Compound
2-[N-ethyl-N-(fluoromethyl)-methyl-azanyl]ethanol (60 mg, 221.85
umol, 23.53% yield, I-salt, 97.28% purity) was obtained as a yellow
solid. LCMS: (M+): 136.1 1H NMR (400 MHz, Deuterium Oxide) .delta.
5.46 (s, 1H), 5.35 (s, 1H), 4.01-3.95 (m, 2H), 3.61-3.46 (m, 4H),
3.10 (d, J=2.2 Hz, 3H), 1.37-1.22 (m, 3H).
##STR00295##
Compound 214:
(3R)-1-(fluoromethyl)-3-hydroxy-1-(prop-2-yn-1-yl)pyrrolidin-1-ium
[0882] Fluoro(iodo)methane (1.28 g, 7.99 mmol, 5 eq) and
(3R)-1-prop-2-ynylpyrrolidin-3-ol (200 mg, 1.60 mmol, 1 eq) in DCM
(2 mL) was stirred at 25.degree. C. for 12 h. LCMS showed the
starting reactant was consumed completely. The reaction mixture was
concentrated. The residue was dissolved by water (30 mL) and
extracted with ethyl acetate (10 mL*3). Water phase was
concentrated by lyophilization. The residue was purified by
prep-HPLC [water (0.1% TFA)-ACN]. Compound
(3R)-1-(fluoromethyl)-1-prop-2-ynyl-pyrrolidin-1-ium-3-ol (80 mg,
252.55 umol, 15.81% yield, 90% purity, CF3CO2-) was obtained as
yellow oil. LCMS: (M+): 158. .sup.1H NMR (400 MHz, Deuterium Oxide)
.delta. 5.56-5.48 (m, 1H), 5.46-5.37 (m, 1H), 4.30 (d, J=1.2 Hz,
2H), 3.87-3.74 (m, 3H), 3.78-3.61 (m, 3H), 2.45-2.26 (m, 1H),
2.15-2.10 (m, 1H).
Compound 215:
[2-(methanesulfonylcarbamoyl)ethyl]dimethyl(prop-2-yn-1-yl)azanium
[0883] Step 1:
[0884] To a solution of 3-bromopropanoyl chloride (5 g, 29.17 mmol,
2.94 mL, 1 eq) in toluene (50 mL) was added methanesulfonamide
(1.14 g, 11.98 mmol, 4.11e-1 eq). The mixture was stirred at
110.degree. C. for 5 hr. LCMS showed 3-bromopropanoyl chloride was
consumed completely and desired m/z was detected. The reaction
mixture was cooled to 0-5.degree. C. with ice, and the resulting
solid was filtered. The filter cake was washed with cold toluene.
Compound 3-bromo-N-methylsulfonyl-propanamide (2.8 g, crude) was
obtained as white solid and used into the next step without further
purification. LCMS: (M+H)+: 230.1
[0885] Step 2:
[0886] To a solution of 3-bromo-N-methylsulfonyl-propanamide (500
mg, 2.17 mmol, 1.2 eq) in ACN (5 mL) was added
N,N-dimethylprop-2-yn-1-amine (150.55 mg, 1.81 mmol, 192.03 uL, 1
eq). The mixture was stirred at 25 at 25.degree. C. for 5 hr. LCMS
showed N,N-dimethylprop-2-yn-1-amine was consumed completely and
desired m/z was detected. The reaction mixture was concentrated
under reduced pressure. The residue was purified by prep-HPLC
[water (0.1% TFA)-ACN]. The title compound (93 mg, 575.82 umol,
31.80% yield, TFA) was obtained as a white solid. Mass: (M+): 233.2
1H NMR (400 MHz, Methanol-d4) .delta. 4.37 (d, J=2.6 Hz, 2H), 3.80
(t, J=7.3 Hz, 2H), 3.61 (t, J=2.6 Hz, 1H), 3.25 (s, 3H), 3.19 (s,
6H), 2.99-2.90 (m, 2H).
##STR00296##
Compound 216:
[(methanesulfonylcarbamoyl)methyl]dimethyl(prop-2-yn-1-yl)azanium
[0887] Step 1:
[0888] To a solution of methanesulfonamide (2 g, 21.03 mmol, 1 eq)
in EtOAc (30 mL) was added 2-bromoacetyl bromide (4.24 g, 21.01
mmol, 1.83 mL, 1 eq). The mixture was stirred at 65.degree. C. for
5 hr. LCMS showed methanesulfonamide was consumed completely and
desired m/z was detected. The reaction was cooled, and a large
amount of white solid precipitated. The mixture was filtered and
the filter cake was concentrated under reduced pressure to give a
residue. Compound 2-bromo-N-methylsulfonyl-acetamide (1.7 g, crude)
was obtained as white solid and used in the next step without
further purification. LCMS: (M+H)+: 216.0
[0889] Step 2:
[0890] To a solution of N,N-dimethylprop-2-yn-1-amine (160.32 mg,
1.93 mmol, 204.49 uL, 1 eq) in THF (4 mL) was added
2-bromo-N-methylsulfonyl-acetamide (500 mg, 2.31 mmol, 1.2 eq). The
mixture was stirred at 25.degree. C. for 5 hr. LCMS showed
N,N-dimethylprop-2-yn-1-amine was consumed completely and one main
peak with desired m/z was detected. The reaction mixture was
concentrated under reduced pressure and the residue was purified by
prep-HPLC [water (0.1%
TFA)-ACN].[2-(methanesulfonamido)-2-oxo-ethyl]-dimethyl-prop-2-ynyl-
-ammonium (68 mg, 227.28 umol, 11.79% yield, 100% purity, TFA salt)
was obtained as white solid. LCMS: (M+): 219.1 1H NMR (400 MHz,
Methanol-d4) .delta. 4.61 (d, J=2.6 Hz, 2H), 4.31 (s, 2H), 3.64 (t,
J=2.6 Hz, 1H), 3.41 (s, 6H), 3.26 (s, 3H).
##STR00297##
Compound 217:
(2-bromoprop-2-en-1-yl)dimethyl(prop-2-yn-1-yl)azanium
[0891] To a solution of N,N-dimethylprop-2-yn-1-amine (200 mg, 2.41
mmol, 255.10 uL, 1 eq) in THF (4 mL) was added 2 added
2,3-dibromoprop-1-ene (528.94 mg, 2.65 mmol, 258.02 uL, 1.1 eq).
The mixture was stirred at 25 at 25.degree. C. for 5 hr. LCMS
showed N,N-dimethylprop-2-yn-1-amine was consumed completely and
one main peak with desired m/z was detected. The mixture was
filtered and the filter cake was concentrated under reduced
pressure to give a residue. The title compound (90 mg, 318.02 umol,
13.22% yield, 100% purity, Br--) was obtained as a yellow solid.
LCMS: (M+): 202.0 1H NMR (400 MHz, Deuterium Oxide) .delta. 6.38
(d, J=2.5 Hz, 1H), 6.28 (d, J=2.6 Hz, 1H), 4.40 (s, 2H), 4.31 (d,
J=2.1 Hz, 2H), 3.27 (t, J=2.5 Hz, 1H), 3.23 (s, 6H).
##STR00298##
Compound 218:
2-hydroxy-4,4-dimethyl-2-(trifluoromethyl)morpholin-4-ium
trifluoroacetate
[0892] Step 1:
[0893] To a mixture of 2-(dimethylamino)ethanol (1 g, 11.22 mmol,
1.13 mL, 1 eq) in THF (10 mL) was added DMAP (137.06 mg, 1.12 mmol,
0.1 eq) and Boc2O (2.69 g, 12.34 mmol, 2.84 mL, 1.1 eq) in one
portion at 25 at 25.degree. C. under N.sub.2. The mixture was
heated to 50.degree. C. and stirred for 12 hours. TLC indicated
2-(dimethylamino) ethanol was consumed and one new spot formed.
LCMS showed one main peak with desired m/z was detected. The
reaction mixture was concentrated under reduced pressure to give a
residue. The residue was purified by column chromatography
(SiO.sub.2, Petroleum ether/Ethyl acetate=50/1 to 0/1). Desired
compound tert-butyl 2-(dimethylamino)ethyl carbonate (850 mg, 4.49
mmol, 40.03% yield) was obtained as colorless oil.
[0894] Step 2:
[0895] To a mixture of tert-butyl 2-(dimethylamino)ethyl carbonate
(850 mg, 4.49 mmol, 1 eq) in THF (8 mL) was added 3 added
3-bromo-1,1,1-trifluoro-propan-2-one (1.72 g, 8.98 mmol, 932.19 uL,
2 eq) in one portion at 0.degree. C. under N.sub.2. The mixture was
stirred at 25.degree. C. for 10 hours. LCMS showed desired compound
was detected. The reaction mixture was concentrated under reduced
pressure to remove THF. The residue was diluted with H.sub.2O (3
mL). The residue was purified by prep-HPLC [water (0.1% TFA)-ACN].
Compound
2-tert-butoxycarbonyloxyethyl-dimethyl-(3,3,3-trifluoro-2-oxo-propyl)ammo-
nium (50 mg, 166.50 umol, 3.71% yield) was obtained as a white
solid.
[0896] Step 3:
[0897]
2-tert-butoxycarbonyloxyethyl-dimethyl-(3,3,3-trifluoro-2-oxo-propy-
l)ammonium (0.05 g, 166.50 umol, 1 eq) in DCM (3 mL) and TFA (1 mL)
was stirred at 25.degree. C. for 3 hours. LCMS showed LCMS showed
desired compound was detected. The reaction mixture was
concentrated under reduced pressure to give a residue. The residue
was purified by prep-HPLC[water (0.1% TFA)-ACN]. The title compound
(0.029 g, 92.30 umol, 55.43% yield, TFA) was obtained as white
solid.
[0898] MS: (M+): 200.1 1H NMR (400 MHz, Deuterium Oxide) .delta.
4.48-4.36 (m, 1H), 4.10 (d, J=14.1 Hz, 1H), 3.69 (d, J=13.4 Hz,
1H), 3.65-3.51 (m, 3H), 3.41 (s, 3H), 3.23 (s, 3H).
##STR00299##
Compound 219: N-(2-hydroxyethyl)-N,N-dimethyloxetan-3-aminium
trifluoroacetate
[0899] Step 1:
[0900] To a mixture of 2-(methylamino)ethanol (1 g, 13.31 mmol,
1.07 mL, 1 eq) in DCM in DCM (5 mL) was added TBDPSCl (4.03 g,
14.65 mmol, 3.76 mL, 1.1 eq), TEA (1.48 g, 14.65 mmol, 2.04 mL, 1.1
eq) and DMAP (813.26 mg, 6.66 mmol, 0.5 eq) in one portion at
25.degree. C. under N.sub.2, then heated to 40.degree. C. and
stirred for 12 hours. LCMS showed desired compound was detected.
The reaction mixture was concentrated under reduced pressure to
give a residue. The residue was purified by column chromatography
(SiO.sub.2, Petroleum ether/Ethyl acetate=30/1 to 1/1). .sup.1HNMR
indicated the desired compound
2-[tert-butyl(diphenyl)silyl]oxy-N-methyl-ethanamine (1.2 g, crude)
was obtained as colorless oil.
[0901] Step 2:
[0902] To a mixture of
2-[tert-butyl(diphenyl)silyl]oxy-N-methyl-ethanamine (1 g, 3.19
mmol, 1 eq) in DCE (10 mL) was added AcOH added AcOH (1.92 g, 31.90
mmol, 1.82 mL, 10 eq) in one portion at 25.degree. C. under
N.sub.2. The mixture was stirred at 25.degree. C. for 0.5h, then
oxetan-3-one (459.72 mg, 6.38 mmol, 2 eq) and NaBH(OAc).sub.3 (1.35
g, 6.38 mmol, 2 eq) were added, and the mixture stirred for 11.5
hours. LC-MS showed the desired compound was detected. The reaction
mixture was concentrated under reduced pressure to give a residue.
The residue was purified by column chromatography (SiO.sub.2,
Petroleum ether/Ethyl acetate=20/1 to 0/1). The crude product
N-[2-[tert-butyl(diphenyl)silyl]oxyethyl]-N-methyl-oxetan-3-amine
(0.72 g, crude) as white solid.
[0903] Step 3:
[0904] To a mixture of
N-[2-[tert-butyl(diphenyl)silyl]oxyethyl]-N-methyl-oxetan-3-amine
(0.72 g, 1.95 mmol, 1 eq) in THF (5 mL) was added MeI (1.66 g,
11.69 mmol, 727.70 uL, 6 eq) in one portion at 25.degree. C. under
N.sub.2. The mixture was stirred at 70.degree. C. for 12 hours.
LCMS showed the desired compound was detected. The reaction mixture
was concentrated under reduced pressure to give a residue. The
residue was purified by column chromatography (SiO.sub.2, Petroleum
ether/Ethyl acetate=20/1 to 1/1). The crude product
2-[tert-butyl(diphenyl)silyl]oxyethyl-dimethyl-(oxetan-3-yl)ammonium
iodide (0.67 g, crude, I) was obtained as yellow solid.
[0905] Step 4:
[0906] To a mixture of
2-[tert-butyl(diphenyl)silyl]oxyethyl-dimethyl-(oxetan-3-yl)ammonium
(300 mg, 586.50 umol, 1 eq, I) in THF (3 mL) was added pyridine
hydrofluoride (174.38 mg, 1.76 mmol, 158.52 uL, 3 eq) in one
portion at 5.degree. C. under N.sub.2. The mixture was stirred at
25.degree. C. for 12 hours. LCMS showed desired mass was detected.
The reaction mixture was concentrated under reduced pressure to
give a residue. The residue was purified by prep-HPLC [water (0.10%
TFA)-ACN]. The title compound (38 mg, 146.02 umol, 24.90% yield,
TFA) as obtained as white gum. LCMS: (M+): 146.1 .sup.1H NMR (400
MHz, Deuterium Oxide) .delta. 4.93-4.85 (m, 2H), 4.89-4.70 (m, 3H),
3.86 (dq, J=5.2, 2.6 Hz, 2H), 3.42-3.35 (m, 2H), 3.12 (s, 6H).
##STR00300##
Compound 220: 3-(hydroxymethyl)-4,4-dimethylmorpholin-4-ium
[0907] Step 1:
[0908] To a mixture of 2-(methylamino)ethanol (1 g, 13.31 mmol,
1.07 mL, 1 eq) in DCM (5 mL) was added TBDPSCI added TBDPSCl (4.03
g, 14.65 mmol, 3.76 mL, 1.1 eq), TEA (1.48 g, 14.65 mmol, 2.04 mL,
1.1 eq) and DMAP (813.26 mg, 6.66 mmol, 0.5 eq) in one portion at
25.degree. C. under N2, then heated to 40.degree. C. and stirred
for 12 hours. LCMS showed LCMS showed desired compound was
detected. The reaction mixture was concentrated under reduced
pressure to give a residue. The residue was purified by column
chromatography (SiO.sub.2, Petroleum ether/Ethyl acetate=30/1 to
1/1). 1HNMR indicated the desired compound
2-[tert-butyl(diphenyl)silyl]oxy-N-methyl-ethanamine (1.2 g, crude)
was obtained as colorless oil.
[0909] Step 2:
[0910] To a mixture of
2-[tert-butyl(diphenyl)silyl]oxy-N-methyl-ethanamine (1 g, 3.19
mmol, 1 eq) in DCE (10 mL) was added AcOH added AcOH (1.92 g, 31.90
mmol, 1.82 mL, 10 eq) in one portion at 25.degree. C. under
N.sub.2. The mixture was stirred at 25.degree. C. for 0.5h, then
oxetan-3-one (459.72 mg, 6.38 mmol, 2 eq) and NaBH(OAc).sub.3 (1.35
g, 6.38 mmol, 2 eq) were added, and the mixture stirred for 11.5
hours. LC-MS showed the desired compound was detected. The reaction
mixture was concentrated under reduced pressure to give a residue.
The residue was purified by column chromatography (SiO.sub.2,
Petroleum ether/Ethyl acetate=20/1 to 0/1). The crude product
N-[2-[tert-butyl(diphenyl)silyl]oxyethyl]-N-methyl-oxetan-3-amine
(0.72 g, crude) as white solid.
[0911] Step 3:
[0912] To a mixture of
N-[2-[tert-butyl(diphenyl)silyl]oxyethyl]-N-methyl-oxetan-3-amine
(0.72 g, 1.95 mmol, 1 eq) in THF (5 mL) was added MeI added MeI
(1.66 g, 11.69 mmol, 727.70 uL, 6 eq) in one portion at 25.degree.
C. under N.sub.2. The mixture was stirred at 70.degree. C. for 12
hours. LCMS showed the desired compound was detected. The reaction
mixture was concentrated under reduced pressure to give a residue.
The residue was purified by column chromatography (SiO.sub.2,
Petroleum ether/Ethyl acetate=20/1 to 1/1). The crude product
2-[tert-butyl(diphenyl)silyl]oxyethyl-dimethyl-(oxetan-3-yl)ammonium
(0.67 g, crude, I) was obtained as yellow solid.
[0913] Step 4:
[0914]
2-[tert-butyl(diphenyl)silyl]oxyethyl-dimethyl-(oxetan-3-yl)ammoniu-
m (0.2 g, 520.01 umol, 1 eq) and KF (151.06 mg, 2.60 mmol, 60.91
uL, 5 eq) in THF (5 mL) was stirred at 25 at 25.degree. C. for 12
hours. LC-MS showed desired mass was detected. The reaction mixture
was concentrated under reduced pressure to give a residue. The
residue was purified by prep-HPLC [water (0.1% TFA)-ACN]. The title
compound (54 mg, 207.51 umol, 39.90% yield, TFA) was obtained as
colorless oil. MS: (M+): 146.1 1H NMR (400 MHz, Deuterium Oxide)
.delta. 3.99-3.77 (m, 6H), 3.57-3.43 (m, 2H), 3.42-3.33 (m, 1H),
3.19 (s, 3H), 3.08 (s, 3H).
##STR00301##
Compound 221: dimethyl(prop-2-yn-1-yl)(2-sulfoethyl)azanium
Trifluoroacetate
[0915] To a solution of 2-(methylamino)ethanesulfonic acid (100 mg,
718.53 umol, 1 eq) in H.sub.2O (3 mL) and EtOH (3 mL) was added
NaCO.sub.3 (178.91 mg, 2.16 mmol, 3 eq) and 3-bromoprop-1-yne
(85.48 mg, 718.53 umol, 61.94 uL, 1 eq). The mixture was stirred at
25.degree. C. for 12 hr. The mixture was added MeI (305.96 mg, 2.16
mmol, 134.19 uL, 3 eq) at 25.degree. C. The mixture was stirred at
25.degree. C. for 12 hr. LCMS showed desired compound was detected.
The reaction mixture was concentrated under reduced pressure to
give a residue. The residue was purified by prep-HPLC [water (0.1%
TFA)-ACN]. Compound 2-[dimethyl(prop-2-ynyl)ammonio]ethanesulfonate
(72 mg, 376.48 umol, 52.4% yield) was obtained as white solid.
LCMS: (M+H)+: 192.1. .sup.1H NMR (400 MHz, Deuterium Oxide) .delta.
4.27-4.22 (m, 2H), 3.81-3.72 (m, 2H), 3.43-3.34 (m, 2H), 3.27-3.21
(m, 1H), 3.17 (s, 6H).
##STR00302##
Compound 222: {3-[dimethyl(prop-2-yn-1-yl)azaniumyl]propyl}boronic
Acid
[0916] Step 1:
[0917] To 3-bromoprop-1-ene (500 mg, 4.13 mmol, 1 eq) was added
1,3,2-benzodioxaborole (495.61 mg, 4.13 mmol, 517.34 uL, 1 eq). The
mixture was stirred at 100.degree. C. for 4 hr. LCMS showed
3-bromoprop-1-ene was consumed completely and desired mass was
detected. The reaction mixture was concentrated under reduced
pressure. Compound 2-(3-bromopropyl)-1,3,2-benzodioxaborole
(.about.1 g, crude) as a yellow oil was obtained and the crude
product was used in the next step without further purification.
[0918] Step 2:
[0919] To a solution of 2-(3-bromopropyl)-1,3,2-benzodioxaborole (1
g, 4.15 mmol, 1 eq) in THF (3 mL) was added N added
N,N-dimethylprop-2-yn-1-amine (345.10 mg, 4.15 mmol, 440.18 uL, 1
eq). The mixture was stirred at 25.degree. C. for 12 hr. LCMS
showed 2-(3-bromopropyl)-1,3,2-benzodioxaborole was consumed
completely and desired mass was detected. The reaction mixture was
concentrated under reduced pressure. The residue was purified by
prep-HPLC [water (0.10% TFA)-ACN]. The title compound (40 mg,
139.41 umol, 3.36% yield, 99% purity, TFA) was obtained as white
solid. LCMS: (M+): 170.0 1H NMR (400 MHz, Deuterium Oxide) .delta.
4.14 (d, J=2.6 Hz, 2H), 3.35-3.27 (m, 2H), 3.16 (t, J=2.5 Hz, 1H),
3.08 (s, 6H), 1.84-1.71 (m, 2H), 0.74 (t, J=8.0 Hz, 2H).
##STR00303##
Compound 223: methylbis(prop-2-yn-1-yl)(2-sulfoethyl)azanium
[0920] To a solution of 2-(methylamino)ethanesulfonic acid (100 mg,
718.53 umol, 1 eq) in DMF (5 mL) was added NaH (57.48 mg, 1.44
mmol, 60% purity, 2 eq) at 0.degree. C. The mixture was stirred at
0.degree. C. for 0.5 hr. Then 3-bromoprop-1-yne (94.02 mg, 790.38
umol, 68.13 uL, 1.1 eq) in DMF (1 mL) was added slowly via syringe
at 0.degree. C. The mixture was stirred at 25.degree. C. for 12 hr.
LC-MS showed the desired compound was detected. The reaction
mixture was quenched by addition of H.sub.2O (1 mL) at 0.degree. C.
and concentrated under reduced pressure to give a residue. The
residue was purified by prep-HPLC [water (0.1% TFA)-ACN]. The title
compound (13 mg, 43.89 umol, 6.11% yield, 100% purity) was obtained
as a white solid. LCMS: (M+): 216.0 .sup.1H NMR (400 MHz, Deuterium
Oxide) .delta. 4.38 (d, J=2.5 Hz, 4H), 3.92-3.84 (m, 2H), 3.45-3.37
(m, 2H), 3.29 (t, J=2.5 Hz, 2H), 3.24 (s, 3H).
##STR00304##
Compound 227:
{2-[dimethyl(prop-2-yn-1-yl)azaniumyl]ethyl}phosphinic Acid
[0921] This compound may be synthesized according to the
experimental procedure described for Compound 226.
##STR00305##
Compound 228: (fluoromethyl)(2-methoxyethyl)dimethylazanium
Iodide
[0922] 2-methoxy-N,N-dimethyl-ethanamine (50 mg, 484.67 umol, 1 eq)
and fluoro(iodo)methane (232.54 mg, 1.45 mmol, 3 eq) in THF (3 mL)
was stirred at 25 was stirred at 25.degree. C. for 12 hours. The
reaction mixture was filtered and the filter cake was concentrated
under reduced pressure to give a residue. The title compound (71
mg, 269.87 umol, 55.68% yield, I) was obtained as white solid. MS:
(M+): 136.2 1H NMR (400 MHz, Deuterium Oxide) .delta. 5.40 (s, 1H),
5.29 (s, 1H), 3.90-3.81 (m, 2H), 3.67-3.60 (m, 2H), 3.35 (s, 3H),
3.17 (s, 6H).
##STR00306##
Compound 229:
(fluoromethyl)dimethyl[2-(methylsulfanyl)ethyl]azanium
[0923] Step 1:
[0924] To a mixture of 1-chloro-2-methylsulfanyl-ethane (500 mg,
4.52 mmol, 446.43 uL, 1 eq) in THF (5 mL) was added NaI (1.36 g,
9.04 mmol, 2 eq) and N-methylmethanamine (2 M, 6.78 mL, 3 eq) in
one portion at 25.degree. C. under N.sub.2. The mixture was heated
to 50.degree. C. and stirred for 12 hours. LCMS showed one main
peak with desired mass was detected. The crude product
N,N-dimethyl-2-methylsulfanyl-ethanamine (500 mg, crude) was
obtained as colorless oil and used into the next step without
further purification. LCMS: (M+H+): 120.1
[0925] Step 2:
[0926] To a mixture of N,N-dimethyl-2-methylsulfanyl-ethanamine
(200 mg, 1.68 mmol, 1 eq) in THF (5 mL) was added NaI added NaI
(502.88 mg, 3.35 mmol, 2 eq) and fluoro(iodo)methane (804.82 mg,
5.03 mmol, 3 eq) in one portion at 25.degree. C. under N.sub.2. The
mixture was stirred at 25.degree. C. for 12 hours. LCMS showed
N,N-dimethyl-2-methylsulfanyl-ethanamine was consumed incompletely
and one new peak with desired mass was detected. The reaction
mixture was concentrated under reduced pressure to give a residue.
The residue was purified by prep-HPLC [water (0.10% TFA)-ACN]. The
title compound (62 mg, 222.10 umol, 13.24% yield, I--)--) was
obtained as yellow oil. LCMS: (M+): 152.1 1H NMR (400 MHz,
Deuterium Oxide) .delta. 5.27 (s, 1H), 5.16 (s, 1H), 3.54-3.45 (m,
2H), 3.02 (d, J=2.0 Hz, 6H), 2.83-2.70 (m, 2H), 1.99 (d, J=1.0 Hz,
3H).
##STR00307##
Compound 230:
[(2E)-2-ethylidene-3-methoxy-3-oxopropyl]dimethyl(prop-2-yn-1-yl)azanium
[0927] This compound may be synthesized according to the
experimental procedure described for Compound 52.
##STR00308##
Compound 231:
(4-methoxy-3-methylidene-4-oxobutyl)dimethyl(prop-2-yn-1-yl)azanium
[0928] This compound may be synthesized according to the
experimental procedure described for Compound 52.
##STR00309##
Compound 236:
dimethyl[2-(methylcarbamoyl)-2-methylideneethyl](prop-2-yn-1-yl)azanium
[0929] This compound may be synthesized by reacting Compound 88
with methylamine, followed by concentration in vacuo and
purification by recrystallization.
##STR00310##
Compound 237:
[(1E)-3-methoxy-3-oxoprop-1-en-1-yl]dimethyl(prop-2-yn-1-yl)azanium
[0930] This compound may be synthesized according to the
experimental procedure described for Compound 52.
##STR00311##
Compound 238:
[(1Z)-3-methoxy-3-oxoprop-1-en-1-yl]dimethyl(prop-2-yn-1-yl)azanium
[0931] This compound may be synthesized according to the
experimental procedure described for Compound 52.
##STR00312##
Compound 240:
dimethyl[2-(1,3-oxazol-2-yl)ethyl](prop-2-yn-1-yl)azanium
[0932] This compound may be synthesized according to the
experimental procedure described for Compound 239.
##STR00313##
Compound 241:
dimethyl(prop-2-yn-1-yl)[2-(1H-1,2,4-triazol-5-yl)ethyl]azanium
[0933] This compound may be synthesized according to the
experimental procedure described for Compound 239.
##STR00314##
Compound 242:
dimethyl(prop-2-yn-1-yl)[(1H-1,2,4-triazol-5-yl)methyl]azanium
[0934] This compound may be synthesized according to the
experimental procedure described for Compound 239.
##STR00315##
Compound 243:
dimethyl[2-(1,2,4-oxadiazol-5-yl)ethyl](prop-2-yn-1-yl)azanium
[0935] This compound may be synthesized according to the
experimental procedure described for Compound 239.
##STR00316##
Compound 246:
{3-[dimethyl(prop-2-yn-1-yl)azaniumyl]prop-1-en-2-yl}phosphonic
Acid
[0936] This compound may be synthesized according to the
experimental procedure described for Compound 245, followed by
hydrolysis of the phosphate esters with NaOH in water and
purification by recrystallization.
##STR00317##
Compound 248:
(2,2-dimethoxyethyl)(2-hydroxyethyl)dimethylazanium
[0937] This compound may be synthesized according to the
experimental procedure described for Compound 247.
##STR00318##
Compound 249:
(2-hydroxyethyl)dimethyl(3,3,3-trifluoro-2-oxopropyl)azanium
[0938] This compound may be synthesized according to the
experimental procedure described for Compound 247.
##STR00319##
Compound 250:
(2-hydroxyethyl)dimethyl[2-(methylsulfanyl)-2-oxoethyl]azanium
[0939] This compound may be synthesized according to the
experimental procedure described for Compound 247.
##STR00320##
Compound 253:
(2-hydroxyethyl)dimethyl(4-methylpenta-2,3-dien-1-yl)azanium
[0940] This compound may be synthesized according to the
experimental procedure described for Compound 60.
##STR00321##
Compound 254:
(2-hydroxyethyl)dimethyl(3-methylbuta-1,2-dien-1-yl)azanium
[0941] This compound may be synthesized according to the
experimental procedure described for Compound 60.
##STR00322##
Compound 255:
N-(2-hydroxyethyl)-N,N,2-trimethylcyclopropan-1-aminium
[0942] Step 1:
[0943] To a mixture of 2-methylcyclopropanamine (0.2 g, 1.86 mmol,
1 eq, HCl) in HCO2H (3 mL) was added HCHO (1.51 g, 18.59 mmol, 1.38
mL, 37% purity, 10 eq) in one portion at 25.degree. C. under
N.sub.2. The mixture was stirred at 90.degree. C. for 12 hours.
LCMS showed desired m/z was detected. The reaction mixture was
concentrated under reduced pressure to remove HCHO and HCO2H. The
residue was diluted with H.sub.2O (3 mL). The residue was purified
by prep-HPLC [water (0.10% TFA)-ACN]. Compound
N,2-trimethylcyclopropanamine (0.176 g, 825.53 umol, 44.41% yield,
TFA) was obtained as colorless oil.
[0944] Step 2:
[0945] To a mixture of N,N,2-trimethylcyclopropanamine (0.1 g, 1.01
mmol, 1 eq) and 2-bromoethanol (252.01 mg, 2.02 mmol, 143.19 uL, 2
eq) in THF (3 mL) was added Na.sub.2CO.sub.3 (213.74 mg, 2.02 mmol,
2 eq) and NaI (151.14 mg, 1.01 mmol, 1 eq) in one portion at
25.degree. C. under N.sub.2. The mixture was stirred at 70.degree.
C. for 12 hours. LCMS showed desired mass was detected. The
reaction mixture was concentrated under reduced pressure to give a
residue. The residue was purified by prep-HPLC [water (0.1%
TFA)-ACN]). The title compound (72 mg, 265.55 umol, 26.34% yield,
I) was obtained as colorless oil. MS: (M+): 144.2. .sup.1H NMR (400
MHz, Deuterium Oxide) .delta. 4.07-3.99 (m, 2H), 3.58-3.45 (m, 2H),
2.95 (d, J=6.4 Hz, 6H), 2.88-2.81 (m, 1H), 1.59-1.51 (m, 1H),
1.34-1.24 (m, 1H), 1.05 (d, J=6.1 Hz, 3H), 0.72 (q, J=7.3 Hz,
1H).
##STR00323##
Compound 256:
2-fluoro-N-(2-hydroxyethyl)-N,N-dimethylcyclopropan-1-aminium
[0946] This compound may be synthesized according to the
experimental procedure described for Compound 255.
##STR00324##
Compound 257:
2,2-difluoro-N-(2-hydroxyethyl)-N,N-dimethylcyclopropan-1-aminium
[0947] This compound may be synthesized according to the
experimental procedure described for Compound 255.
##STR00325##
Compound 258:
N-(2-hydroxyethyl)-N,N,2,2-tetramethylcyclopropan-1-aminium
[0948] This compound may be synthesized according to the
experimental procedure described for Compound 255.
##STR00326##
Compound 259:
1-fluoro-N-(2-hydroxyethyl)-N,N-dimethylcyclopropan-1-aminium
[0949] This compound may be synthesized according to the
experimental procedure described for Compound 255.
##STR00327##
Compound 260:
1-methyl-1-(prop-2-yn-1-yl)-4,5-dihydro-1H-pyrazol-1-ium
[0950] This compound may be synthesized according to the
experimental procedure described for Compound 208.
##STR00328##
Compound 261: 1-methyl-1-(prop-2-yn-1-yl)imidazolidin-1-ium
[0951] This compound may be synthesized according to the
experimental procedure described for Compound 208 using
boc-protected imidazolidine, followed by deprotection to yield the
title compound.
##STR00329##
Compound 264: 3-bromo-1,1-dimethylpyrrolidin-1-ium Bromide
[0952] This compound may be synthesized according to the
experimental procedure described for Compound 262.
##STR00330##
Compound 265: 1,1-dimethyl-2-(prop-2-yn-1-yl)pyrazolidin-1-ium
[0953] The title compound may be synthesized by reaction Compound
127 with propargyl bromide, followed by purification by
recrystallization.
##STR00331##
Compound 266:
(carbamothioylmethyl)(2-hydroxyethyl)dimethylazanium
[0954] This compound may be synthesized according to the
experimental procedure described for Compound 247.
##STR00332##
Compound 267: 2-hydroxy-N,N,N-trimethylcyclopropan-1-aminium
[0955] This compound may be synthesized according to the
experimental procedure described for Compound 184.
##STR00333##
Compound 271:
(2-iodoprop-2-en-1-yl)dimethyl(prop-2-yn-1-yl)azanium
[0956] This compound may be synthesized according to the
experimental procedure described for Compound 270.
##STR00334##
Compound 272:
[2-(fluoromethyl)prop-2-en-1-yl]dimethyl(prop-2-yn-1-yl)azanium
[0957] This compound may be synthesized according to the
experimental procedure described for Compound 270.
##STR00335##
Compound 273:
(2-chloroprop-2-en-1-yl)(2-hydroxyethyl)dimethylazanium
[0958] This compound may be synthesized according to the
experimental procedure described for Compound 270.
##STR00336##
Compound 274:
N-cyclopropyl-N-(2-hydroxyethyl)-N-methylcyclopropanaminium
[0959] This compound may be synthesized according to the
experimental procedure described for Compound 58.
##STR00337##
Compound 275: N,N-diethyl-N-(2-hydroxyethyl)cyclopropanaminium
[0960] This compound may be synthesized according to the
experimental procedure described for Compound 58.
##STR00338##
Compound 276: (2-carboxyethyl)(fluoromethyl)dimethylazanium
[0961] This compound may be synthesized according to the
experimental procedure described for Compound 111.
##STR00339##
Compound 277: (2-carboxyethyl)(chloromethyl)dimethylazanium
[0962] This compound may be synthesized according to the
experimental procedure described for Compound 111.
##STR00340##
Compound 278: 1-(fluoromethyl)-1-methylpyrazolidin-1-ium
[0963] This compound may be synthesized according to the
experimental procedure described for Compound 74.
##STR00341##
Compound 279:
(4R)-4-hydroxy-1-methyl-1-(prop-2-yn-1-yl)pyrazolidin-1-ium
[0964] This compound may be synthesized according to the
experimental procedure described for Compound 74, followed by SFC
purification of stereoisomers.
##STR00342##
Compound 280:
(4S)-4-hydroxy-1-methyl-1-(prop-2-yn-1-yl)pyrazolidin-1-ium
[0965] This compound may be synthesized according to the
experimental procedure described for Compound 74, followed by SFC
purification of stereoisomers.
##STR00343##
Compound 284: {2-[dimethyl(prop-2-yn-1-yl)azaniumyl]ethyl}boronic
Acid
[0966] This compound may be synthesized according to the
experimental procedure described for Compound 283.
##STR00344##
Compound 285: [2-(trimethylazaniumyl)ethyl]boronic Acid
[0967] This compound may be synthesized according to the
experimental procedure described for
##STR00345##
Compound 289:
(fluoromethyl)[(2R)-3-methoxy-2-methyl-3-oxopropyl]dimethylazanium
[0968] Step 1:
[0969] To a solution of (2R)-3-amino-2-methyl-propanoic acid (300
mg, 2.91 mmol, 1 eq) in HCOOH (5 mL) was added formaldehyde (2.36
g, 29.09 mmol, 2.17 mL, 37% purity, 10 eq). The mixture was stirred
at 90.degree. C. for 12 hr. LCMS showed the starting reactant was
consumed and the product had the desired mass. The mixture was
concentrated. The residue was purified by prep-HPLC [water (0.04%
HCl)-ACN]. Compound (2R)-3-(dimethylamino)-2-methyl-propanoic acid
(300 mg, 2.29 mmol, 78.61% yield) was obtained as a white
solid.
[0970] Step 2:
[0971] To a solution of (2R)-3-(dimethylamino)-2-methyl-propanoic
acid (250.00 mg, 1.91 mmol, 1 eq) in MeOH (5 mL) was added SOCl2
(453.49 mg, 3.81 mmol, 276.52 uL, 2 eq) at 0.degree. C. The mixture
was stirred at 15.degree. C. for 12 hr. LCMS showed the starting
reactant was consumed and have the desired mass. The mixture was
concentrated. Compound methyl
(2R)-3-(dimethylamino)-2-methyl-propanoate (313 mg, crude) was
obtained as a white solid and used in the next step without further
purification.
[0972] Step 3:
[0973] To a solution of methyl
(2R)-3-(dimethylamino)-2-methyl-propanoate (100 mg, 688.71 umol, 1
eq) in THF (5 mL) was added Na.sub.2CO.sub.3 (73.00 mg, 688.71
umol, 1 eq) and fluoro(iodo)methane (330.43 mg, 2.07 mmol, 3 eq).
The mixture was stirred at 15.degree. C. for 10 h and stirred at
50.degree. C. for 2 h. LCMS showed the starting reactant was
consumed and the product had the desired mass. The mixture was
filtered and the filtrate was concentrated. The residue was
purified by prep-HPLC [water (0.04% HCl)-ACN]. Compound
fluoromethyl-[(2R)-3-methoxy-2-methyl-3-oxo-propyl]-dimethyl-ammonium
(19 mg, 62.27 umol, 9.04% yield, I--)--) was obtained as a white
solid. MS: (M+): 178.1 .sup.1H NMR (400 MHz, Deuterium Oxide)
.delta. 5.30 (q, J=6.0 Hz, 1H), 5.18 (q, J=6.0 Hz, 1H), 3.92 (dd,
J=14.0, 8.9 Hz, 1H), 3.65 (s, 3H), 3.37-3.29 (m, 1H), 3.17-3.10 (m,
1H), 3.04 (dd, J=7.2, 2.1 Hz, 6H), 1.19 (d, J=7.2 Hz, 3H).
##STR00346##
Compound 290:
(fluoromethyl)[(2S)-3-methoxy-2-methyl-3-oxopropyl]dimethylazanium
[0974] Step 1:
[0975] To a mixture of (2S)-3-amino-2-methyl-propanoic acid (0.3 g,
2.91 mmol, 1 eq) in HCOOH (5 mL) was added aq. HCHO (2.36 g, 29.09
mmol, 2.17 mL, 37% purity, 10 eq) in one portion at 25.degree. C.
under N.sub.2. The mixture was stirred at 90.degree. C. for 12
hours. LCMS showed the desired mass was detected. The reaction
mixture was filtered and concentrated under reduced pressure to
give a residue. The residue was purified by prep-HPLC [water (0.1%
TFA)-ACN]; B %:1%-5%, 12 min). Compound
(2S)-3-(dimethylamino)-2-methyl-propanoic acid (0.2 g, 815.68 umol,
28.04% yield, TFA) was obtained as colorless oil.
[0976] Step 2:
[0977] To a mixture of (2S)-3-(dimethylamino)-2-methyl-propanoic
acid (0.2 g, 1.13 mmol, 1 eq) in MeOH (5 mL) was added SOCl.sub.2
(268.56 mg, 2.26 mmol, 163.76 uL, 2 eq) in one portion at
25.degree. C. under N.sub.2. The mixture was stirred at 25.degree.
C. for 12 hours. LCMS showed desired mass was detected. The
reaction mixture was concentrated under reduced pressure to give a
residue. No further purification was performed. Compound methyl
(2S)-3-(dimethylamino)-2-methyl-propanoate (150 mg, 1.03 mmol,
91.53% yield) was obtained as a white solid.
[0978] Step 3:
[0979] To a mixture of methyl
(2S)-3-(dimethylamino)-2-methyl-propanoate (50 mg, 276.78 umol, 1
eq, C.sub.1) and fluoro(iodo)methane (132.79 mg, 830.33 umol, 3 eq)
in THF (5 mL) was added Na2CO3 added Na2CO3 (88.01 mg, 830.33 umol,
3 eq) in one portion at 25.degree. C. under N.sub.2. The mixture
was stirred at 25.degree. C. for 12 hours. LCMS showed desired mass
was detected. The reaction mixture was filtered and concentrated
under remove THF, then the residue was diluted with H.sub.2O (3
ml), washed by EtOAc 9 ml (3 ml*3). The water phase was
lyophilized. The title compound (32 mg, 179.55 umol, 64.87% yield)
was obtained as white solid. MS: (M+): 178.1 1H NMR (400 MHz,
Deuterium Oxide) .delta. 5.28 (q, J=6.0 Hz, 1H), 5.17 (q, J=6.0 Hz,
1H), 3.91 (dd, J=14.0, 9.0 Hz, 1H), 3.64 (s, 3H), 3.36-3.28 (m,
1H), 3.16-3.08 (m, 1H), 3.03 (dd, J=7.4, 2.1 Hz, 6H), 1.18 (d,
J=7.3 Hz, 3H).
##STR00347##
Compound 291:
(fluoromethyl)[(2R)-2-hydroxypropyl]dimethylazanium
[0980] Step 1:
[0981] To a solution of methyl (2R)-2-hydroxypropanoate (10 g,
96.06 mmol, 9.17 mL, 1 eq) and imidazole (13.08 g, 192.12 mmol, 2.0
eq) in DCM (100 mL) was added tert added
tert-butyl-chloro-dimethyl-silane (28.96 g, 192.12 mmol, 23.54 mL,
2 eq) at 0.degree. C. The mixture was stirred at 25.degree. C. for
12 hr. TLC indicated methyl (2R)-2-hydroxypropanoate was consumed
completely and many new spots formed. The reaction mixture was
diluted with H.sub.2O (150 mL) and extracted with ethyl acetate 450
mL (150 mL*3). The combined organic layers were dried over
anhydrous sodium sulfate, filtered, and concentrated under reduced
pressure to give a residue. The residue was purified by column
chromatography (SiO.sub.2, Petroleum ether/Ethyl acetate=1:0).
Compound methyl (2R)-2-[tert butyl(dimethyl)silyl]oxypropanoate (15
g, 68.69 mmol, 71.51% yield) was obtained as a colorless oil.
[0982] Step 2:
[0983] To a solution of methyl
(2R)-2-[tert-butyl(dimethyl)silyl]oxypropanoate (5 g, 22.90 mmol, 1
eq) in THF (100 mL) was added DIBAL added DIBAL-H (1 M, 45.79 mL, 2
eq, in Tol.) at 0.degree. C. The mixture was stirred at 25 at
25.degree. C. for 12 hr. TLC indicated new spots formed. The
reaction mixture was quenched by addition of H.sub.2O (100 mL) at
0.degree. C., and then diluted with ethyl acetate 50 mL. The
mixture was filtered. The filtrate was extracted with ethyl acetate
300 mL (100 mL*3). The combined organic layers were dried over
anhydrous sodium sulfate, filtered, and the filtrate was
concentrated under reduced pressure to give a residue. The residue
was purified by column chromatography (SiO.sub.2, Petroleum
ether/Ethyl acetate=1:0 to 10:1). Compound
(2R)-2-[tert-butyl(dimethyl)silyl]oxypropan-1-ol (6 g, 36.77 mmol,
80.30% yield) was obtained as a colorless oil without further
purification.
[0984] Step 3:
[0985] To a solution of
(2R)-2-[tert-butyl(dimethyl)silyl]oxypropan-1-ol (6 g, 31.52 mmol,
1 eq) in DCM (60 mL) was added imidazole (10.73 g, 157.60 mmol, 5
eq), PPh3 (20.67 g, 78.80 mmol, 2.5 eq) and 12 (24.00 g, 94.56
mmol, 19.05 mL, 3 eq) at 25.degree. C. The mixture was stirred at
25.degree. C. for 12 hr. TLC indicated
(2R)-2-[tert-butyl(dimethyl)silyl]oxypropan-1-ol was consumed
completely. The reaction mixture was concentrated under reduced
pressure to give a residue. The residue was purified by column
chromatography (SiO.sub.2, Petroleum ether/Ethyl acetate=1:0).
Compound tert-butyl-[(1R)-2-iodo-1-methyl-ethoxy]-dimethyl-silane
(6 g, 19.98 mmol, 63.40% yield) was obtained as a colorless
oil.
[0986] Step 4:
[0987] To a solution of
tert-butyl-[(1R)-2-iodo-1-methyl-ethoxy]-dimethyl-silane (4 g,
13.32 mmol, 1 eq) in THF (50 mL) was added N-methylmethanamine (2
M, 33.31 mL, 5 eq)(in THF) at 25.degree. C. The mixture was stirred
at 25.degree. C. for 12 hr. LCMS showed the desired compound was
detected. TLC indicated most of
tert-butyl-[(1R)-2-iodo-1-methyl-ethoxy]-dimethyl-silane remained.
The mixture was stirred at 70.degree. C. for 12 hr. The reaction
mixture was purified by column chromatography (SiO.sub.2, Petroleum
ether/Ethyl acetate=1:0 to 0:1). Compound
(2R)-2-[tert-butyl(dimethyl)silyl]oxy-N,N-dimethyl-propan-1-amine
(3 g, 8.69 mmol, 65.21% yield, HI) was obtained as yellow oil.
[0988] Step 5:
[0989] To a solution of
(2R)-2-[tert-butyl(dimethyl)silyl]oxy-N,N-dimethyl-propan-1-amine
(3 g, 8.69 mmol, 1 eq, HI) in THF (30 mL) was added
fluoro(iodo)methane (6.95 g, 43.44 mmol, 5 eq) at 25.degree. C. The
mixture was stirred at 25.degree. C. for 12 hr. TLC indicated
(2R)-2-[tert-butyl(dimethyl)silyl]oxy-N,N-dimethyl-propan-1-amine
was remained. The mixture was added another batch of
fluoro(iodo)methane (4.17 g, 26.06 mmol, 3 eq) at 25.degree. C. The
mixture was stirred at 50.degree. C. for 3 hr. TLC indicated
(2R)-2-[tert-butyl(dimethyl)silyl]oxy-N,N-dimethyl-propan-1-amine
was consumed completely. The reaction mixture was concentrated
under reduced pressure to give a residue. The residue was diluted
with H.sub.2O 100 mL and extracted with ethyl acetate 150 mL (50
mL*3). The aqueous phase was freeze-dried.
[0990] Compound
tert-butyl-[(1R)-2-[N-(fluoromethyl)-dimethyl-azanyl]-1-methyl-ethoxy]-di-
methyl-silane (900 mg, 2.39 mmol, 27.45% yield) was obtained as a
white solid without further purification.
[0991] Step 6:
[0992] To a solution of
tert-butyl-[(1R)-2-[N-(fluoromethyl)-dimethyl-azanyl]-1-methyl-ethoxy]-di-
methyl-silane (500 mg, 1.33 mmol, 1 eq) in H.sub.2O (20 mL) was
added KF (1.54 g, 26.50 mmol, 620.80 uL, 20 eq) at 25.degree. C.
The mixture was stirred at 50.degree. C. for 12 hr. LC-MS showed
the desired compound was detected. The reaction mixture was
concentrated under reduced pressure to give a residue. The residue
was diluted with H.sub.2O 5 mL and extracted with ethyl acetate 15
mL (5 mL*3). The residue was purified by prep-HPLC [water (0.1%
TFA)-ACN]. Compound
fluoromethyl-[(2R)-2-hydroxypropyl]-dimethyl-ammonium (98 mg,
372.49 umol, 28.11% yield, I--) was obtained as yellow oil. MS:
(M+): 136.1 .sup.1H NMR (400 MHz, Deuterium Oxide) .delta.
5.48-5.37 (m, 1H), 5.37-5.26 (m, 1H), 4.37 (q, J=6.4 Hz, 1H),
3.42-3.36 (m, 2H), 3.20-3.14 (m, 6H), 1.18 (d, J=6.4 Hz, 3H).
##STR00348##
Compound 292:
(fluoromethyl)[(2S)-2-hydroxypropyl]dimethylazanium
[0993] This compound may be synthesized according to the
experimental procedure described for Compound 169.
##STR00349##
Compound 294: trimethyl(2-methylpent-4-yn-2-yl)azanium
[0994] This compound may be synthesized according to the
experimental procedure described for Compound 174.
##STR00350##
Compound 295:
(fluoromethyl)dimethyl[(pyrrolidin-2-yl)methyl]azanium Iodide
[0995] Step 1:
[0996] To a mixture of pyrrolidin-2-ylmethanol (5 g, 49.43 mmol, 1
eq) and ethyl 2,2,2-trifluoroacetate (10.53 g, 74.15 mmol, 10.23
mL, 1.5 eq) in MeOH (50 mL) was added TEA added TEA (5.50 g, 54.38
mmol, 7.57 mL, 1.1 eq) in one portion at 25.degree. C. under
N.sub.2. The mixture as heated to 70.degree. C. and stirred for 12
hours. TLC indicated pyrrolidin-2-ylmethanol was consumed and one
new spot formed. The reaction mixture was concentrated under
reduced pressure to give a residue. The residue was purified by
column chromatography (SiO.sub.2, Petroleum ether/Ethyl
acetate=50/1 to 0/1). Compound
2,2,2-trifluoro-1-[2-(hydroxymethyl)pyrrolidin-1-yl]ethanone (5 g,
25.36 mmol, 51.30% yield) was obtained as yellow oil. LCMS: (M+H+):
198.0.
[0997] Step 2:
[0998] To a mixture of PPh3 (6.39 g, 24.35 mmol, 2.4 eq) in DCM (15
mL) was added imidazole (3.45 g, 50.72 mmol, 5 eq) and 12 (6.44 g,
25.36 mmol, 2.5 eq) in one portion at 25.degree. C. under N.sub.2.
The mixture was stirred at 25.degree. C. for 6 min, then
2,2,2-trifluoro-1-[2-(hydroxymethyl)pyrrolidin-1-yl]ethanone (2 g,
10.14 mmol, 1 eq) was added. The mixture was stirred at 25.degree.
C. for 11.9 hours. TLC indicated
2,2,2-trifluoro-1-[2-(hydroxymethyl)pyrrolidin-1-yl]ethanone was
consumed and many new spots formed. The reaction mixture was
concentrated under reduced pressure to give a residue. The residue
was purified by column chromatography (SiO.sub.2, Petroleum
ether/Ethyl acetate=100/1 to 5/1). Compound
2,2,2-trifluoro-1-[2-(iodomethyl)pyrrolidin-1-yl]ethanone (2.7 g,
crude) was obtained as colorless oil.
[0999] Step 3:
[1000] To a mixture of
2,2,2-trifluoro-1-[2-(iodomethyl)pyrrolidin-1-yl]ethanone (1 g,
3.26 mmol, 1 eq) in THF (5 mL) was added Na.sub.2CO.sub.3 (690.37
mg, 6.51 mmol, 2 eq) and N-methylmethanamine (2 M, 4.89 mL, 3 eq)
in one portion at 25.degree. C. under N.sub.2. The mixture was
stirred at 25.degree. C. for 12 hours. TLC indicated
2,2,2-trifluoro-1-[2-(iodomethyl)pyrrolidin-1-yl]ethanone was
consumed and one new spot formed. The reaction mixture was
concentrated under reduced pressure to give a residue. The residue
was purified by column chromatography (SiO.sub.2, Petroleum
ether/Ethyl acetate=80/1 to 0/1). Compound
1-[2-[(dimethylamino)methyl]pyrrolidin-1-yl]-2,2,2-trifluoro-eth-
anone (150 mg, crude) was obtained as yellow oil. LCMS: (M+H+):
225.1
[1001] Step 4:
[1002] To a mixture of
1-[2-[(dimethylamino)methyl]pyrrolidin-1-yl]-2,2,2-trifluoro-ethanone
(100 mg, 445.98 umol, 1 eq) in THF (3 mL) was added NaI (133.70 mg,
891.97 umol, 2 eq), Na.sub.2CO.sub.3 (94.54 mg, 891.97 umol, 2 eq)
and fluoro(iodo)methane (213.98 mg, 1.34 mmol, 3 eq) in one portion
at 25.degree. C. under N.sub.2. The mixture was stirred at
25.degree. C. for 12 hours. LCMS showed desired compound was
detected. The reaction mixture was concentrated under reduced
pressure to give a residue. The residue was purified by prep-HPLC
[water (0.1% TFA)-ACN]. The title compound (88 mg, 305.40 umol,
68.48% yield, I) was obtained as colorless oil. LCMS: (M+H+): 161.2
1H NMR (400 MHz, Deuterium Oxide) .delta. 5.42 (s, 1H), 5.31 (s,
1H), 4.12-4.03 (m, 1H), 3.96-3.81 (m, 2H), 3.31 (dd, J=8.5, 6.4 Hz,
2H), 3.17 (s, 6H), 2.40-2.30 (m, 1H), 2.05-2.00 (m, 1H), 1.98-1.84
(m, 1H), 1.84-1.69 (m, 1H).
##STR00351##
Compound 296:
(fluoromethyl)dimethyl{[1-(2,2,2-trifluoroacetyl)pyrrolidin-2-yl]methyl}a-
zanium
[1003] This compound may be synthesized by reacting Compound 295
with trifluoracetic anhydride, followed by purification by
recrystallization.
##STR00352##
Compound 297:
(fluoromethyl)(3-methoxy-2-methyl-3-oxopropyl)dimethylazanium
Iodide
[1004] Step 1:
[1005] To a mixture of 3-amino-2-methyl-propanoic acid (300 mg,
2.91 mmol, 1 eq) in HCOOH (5 mL) was added HCHO (2.36 g, 29.09
mmol, 2.17 mL, 37% purity, 10 eq) in one portion at 25.degree. C.
under N.sub.2. The mixture was stirred at 90.degree. C. for 12
hours. LCMS showed desired mass was detected. The reaction mixture
was concentrated under reduced pressure to give a residue. The
residue was purified by prep-HPLC [water (0.10% TFA)-ACN]. Compound
3-(dimethylamino)-2-methyl-propanoic acid (100 mg, 762.36 umol,
26.20% yield) was obtained as colorless oil.
[1006] Step 2:
[1007] To a mixture of 3-(dimethylamino)-2-methyl-propanoic acid
(50 mg, 381.18 umol, 1 eq) in MeOH (3 mL) was added SOCl.sub.2
(90.70 mg, 762.35 umol, 55.30 uL, 2 eq) in one portion at
25.degree. C. under N.sub.2. The mixture was stirred at 25.degree.
C. for 12 hours. LCMS showed desired mass was detected. The
reaction mixture was concentrated under reduced pressure to give a
residue. No further purification was performed. The crude product
methyl 3-(dimethylamino)-2-methyl-propanoate (50 mg, crude) was
obtained as colorless oil, and used in the next step without
further purification.
[1008] Step 3:
[1009] To a mixture of methyl 3-(dimethylamino)-2-methyl-propanoate
(50 mg, 344.35 umol, 1 eq) in THF (3 mL) was added Na.sub.2CO.sub.3
(73.00 mg, 688.71 umol, 2 eq) in one portion at 25.degree. C. under
N.sub.2, then fluoro(iodo)methane (165.22 mg, 1.03 mmol, 3 eq) was
added. The mixture was stirred at 25.degree. C. for 12 hours. LCMS
showed desired compound was detected. The reaction mixture was
filtered and concentrated under reduced pressure to remove THF,
then 5 ml H.sub.2O was added, extracted with EtOAc (15 ml, 5 ml*3).
The water phase was lyophilized. The title compound (39.4 mg,
221.07 umol, 64.20% yield) was obtained as white solid. LCMS:
(M+H+): 178.1 .sup.1H NMR (400 MHz, Deuterium Oxide) .delta. 5.4
(m, 1H), 5.3 (m, 1H), 4.1-4.0 (m, 1H), 3.8 (s, 3H), 3.4 (m, 1H),
3.2 (m, 1H), 3.2-3.1 (m, 6H), 1.3 (m, 3H).
##STR00353##
Compound 298:
[2-(benzyloxy)ethyl](chloromethyl)methyl(prop-2-yn-1-yl)azanium
[1010] Step 1:
[1011] To a mixture of 2-benzyloxyacetaldehyde (500 mg, 3.33 mmol,
467.29 uL, 1 eq) and N-methylprop-2-yn-1-amine (230.08 mg, 3.33
mmol, 277.21 uL, 1 eq) in THF (3 mL) was added AcOH (10.00 mg,
166.47 umol, 9.52 uL, 0.05 eq) in one portion at 0.degree. C. under
N.sub.2. The mixture was stirred at 0.degree. C. for 30 min, then
NaBH(OAc).sub.3 (1.41 g, 6.66 mmol, 2 eq) was added heated to
25.degree. C. and stirred for 11.5 hours. LC-MS showed desired mass
was detected. The reaction mixture was diluted with H.sub.2O (3 mL)
and extracted with EtOAc 15 mL (5 mL*3), filtered and concentrated
under reduced pressure to give a residue. The residue was purified
by column chromatography (SiO.sub.2, Petroleum ether/Ethyl
acetate=20/1 to 1/1). compound
N-(2-benzyloxyethyl)-N-methyl-prop-2-yn-1-amine (600 mg, 2.95 mmol,
88.65% yield) was obtained as yellow oil.
[1012] Step 2:
[1013] To a mixture of
N-(2-benzyloxyethyl)-N-methyl-prop-2-yn-1-amine (20 mg, 98.39 umol,
1 eq) in acetone (0.5 mL) was added chloro(iodo)methane (86.77 mg,
491.93 umol, 35.71 uL, 5 eq). The mixture was stirred at 25.degree.
C. for 12 hours. LCMS showed desired mass was detected. The
reaction mixture was concentrated under reduced pressure to give a
residue. The residue was purified by prep-HPLC (column: Waters
Xbridge BEH C18 100*30 mm*10 um; mobile phase: [water (0.04%
HCl)-ACN]; B %: 5%-35%, 12 min). The title compound (18 mg, 47.41
umol, 48.19% yield, I--) was obtained as colorless oil. LCMS: (M+):
252.1 .sup.1H NMR (400 MHz, Deuterium Oxide) .delta. 7.45-7.36 (m,
5H), 5.30 (s, 2H), 4.60 (s, 2H), 4.44 (d, J=2.5 Hz, 2H), 4.03-3.97
(m, 2H), 3.87-3.80 (m, 2H), 3.27 (s, 4H).
##STR00354##
Compound 299:
[2-(benzyloxy)ethyl](fluoromethyl)methyl(prop-2-yn-1-yl)azanium
[1014] Step 1:
[1015] To a mixture of 2-benzyloxyacetaldehyde (500 mg, 3.33 mmol,
467.29 uL, 1 eq) and N-methylprop-2-yn-1-amine (230.08 mg, 3.33
mmol, 277.21 uL, 1 eq) in THF (3 mL) was added AcOH (10.00 mg,
166.47 umol, 9.52 uL, 0.05 eq) in one portion at 0.degree. C. under
N.sub.2. The mixture was stirred at 0.degree. C. for 30 min, then
NaBH(OAc).sub.3 (1.41 g, 6.66 mmol, 2 eq) was added heated to
25.degree. C. and stirred for 11.5 hours. LC-MS showed desired mass
was detected. The reaction mixture was diluted with H.sub.2O (3 mL)
and extracted with EtOAc 15 mL (5 mL*3), filtered and concentrated
under reduced pressure to give a residue. The residue was purified
by column chromatography (SiO.sub.2, Petroleum ether/Ethyl
acetate=20/1 to 1/1). compound
N-(2-benzyloxyethyl)-N-methyl-prop-2-yn-1-amine (600 mg, 2.95 mmol,
88.65% yield) was obtained as yellow oil.
[1016] Step 2:
[1017] To a solution of
N-(2-benzyloxyethyl)-N-methyl-prop-2-yn-1-amine (100 mg, 491.93
umol, 1 eq) in THF (5 mL) was added fluoro(iodo)methane (236.02 mg,
1.48 mmol, 3 eq) at 25.degree. C. The mixture was stirred at
25.degree. C. for 12 hr. LCMS showed the desired compound was
detected. The reaction mixture was concentrated under reduced
pressure to give a residue. The residue was diluted with H.sub.2O 5
mL and extracted with ethyl acetate 15 mL (5 mL*3). The residue was
purified by prep-HPLC [water (0.04% HCl)-ACN]. Desired compound
2-benzyloxyethyl-(fluoromethyl)-methyl-prop-2-ynyl-ammonium (58 mg,
159.69 umol, 32.46% yield, I--) was obtained as colorless oil.
LCMS: (M+): 236.1 1H NMR (400 MHz, Methanol-d4) .delta. 7.41-7.27
(m, 5H), 5.70-5.62 (m, 1H), 5.59-5.51 (m, 1H), 4.60 (s, 2H),
4.57-4.51 (m, 2H), 3.99-3.94 (m, 2H), 3.86-3.79 (m, 2H), 3.62 (t,
J=2.6 Hz, 1H), 3.28 (d, J=2.1 Hz, 3H).
##STR00355##
Compound 300:
(fluoromethyl)(methyl)(prop-2-yn-1-yl)(2-{[(2S)-3,3,3-trifluoro-2-methoxy-
-2-phenylpropanoyl]oxy}ethyl)azanium
[1018] Step 1:
[1019] A mixture of
(2S)-3,3,3-trifluoro-2-methoxy-2-phenyl-propanoic acid (0.6 g, 2.56
mmol, 461.54 uL, 1 eq), 2-[methyl(prop-2-ynyl)amino]ethanol (318.65
mg, 2.82 mmol, 1.1 eq), DCC (581.02 mg, 2.82 mmol, 569.62 uL, 1.1
eq), DMAP (156.38 mg, 1.28 mmol, 0.5 eq) in DCM (15 mL) was
degassed and purged with N.sub.2 for 3 times, and then the mixture
was stirred at 0.degree. C. for 0.5 hr. LCMS showed desired mass
was detected. The reaction mixture was filtered and concentrated
under reduced pressure to give a residue. The residue was purified
by column chromatography (SiO.sub.2, Petroleum ether/Ethyl
acetate=90/1 to 30/1). Compound 2-[methyl(prop-2-ynyl)amino]ethyl
(2S)-3,3,3-trifluoro-2-methoxy-2-phenyl-propanoate (100 mg, 303.66
umol, 11.86% yield) was obtained as colorless oil.
[1020] Step 2:
[1021] 2-[methyl(prop-2-ynyl)amino]ethyl
(2S)-3,3,3-trifluoro-2-methoxy-2-phenyl-propanoate (200 mg, 607.32
umol, 1 eq) in fluoro(iodo)methane (1.94 g, 12.15 mmol, 20 eq) was
stirred at 25.degree. C. for 12 hours. LC-MS showed desired mass
was detected. The reaction mixture was concentrated under reduced
pressure to give a residue. The residue was purified by
prep-HPLC
[water (0.04% HCl)-ACN]. The title compound (47 mg, 96.07 umol,
15.82% yield, I--) was obtained as white solid. MS (M+): 362.0 1H
NMR (400 MHz, Deuterium Oxide) .delta. 7.58-7.47 (m, 5H), 5.46-5.36
(m, 1H), 5.34-5.25 (m, 1H), 4.84 (s, 2H), 4.31-4.17 (m, 2H), 3.95
(s, 2H), 3.47 (s, 3H), 3.27-3.22 (m, 1H), 3.08 (s, 3H).
##STR00356##
Compound 301:
1-(fluoromethyl)-1-(2-hydroxyethyl)pyrrolidin-1-ium
[1022] To a mixture of 2-pyrrolidin-1-ylethanol (100 mg, 868.26
umol, 101.52 uL, 1 eq) in THF (3 mL) was added fluoro added
fluoro(iodo)methane (416.58 mg, 2.60 mmol, 3 eq) in one portion at
25.degree. C. under N.sub.2. The mixture was stirred at 25.degree.
C. for 12 hours. LCMS showed desired mass was detected. The
reaction mixture was concentrated under reduced pressure to remove
THF, and diluted with H.sub.2O 3 mL and extracted with EtOAc (15
mL, 5 mL*3). The water phase was lyophilized. The title compound
(90 mg, 327.15 umol, 37.68% yield, I--) was obtained as yellow
solid. MS (M+): 148.0 1H NMR (400 MHz, Deuterium Oxide) .delta.
5.47 (s, 1H), 5.35 (s, 1H), 4.03-3.96 (m, 2H), 3.78-3.68 (m, 2H),
3.70-3.59 (m, 4H), 2.24-2.05 (m, 4H).
##STR00357##
Compound 303:
N,N-dimethyl-N-(2-oxo-2-(((2S,3R,4S,5R,6R)-3,4,5-triacetoxy-6-(acetoxymet-
hyl)tetrahydro-2H-pyran-2-yl)oxy)ethyl)prop-2-yn-1-aminium
Trifluoroacetate
[1023] Step 1
[1024] Compound 2,3,4,6-tetra-O-acetyl-D-glucopyranose (Carbosynth,
2 g, 5.7 mmol, 1 eq) was dissolved in DCM (10 mL), and cooled to
0.degree. C. under N.sub.2. Then 2-chloroacetyl chloride (1.4 mL,
17.6 mmol, 3 eq) and pyridine (1.4 mL, 17.4 mmol, 3 eq) in DCM (10
mL) were added over ice, the reaction was stirred overnight. The
reaction mixture was filtered and purified by column chromatography
(0-100% EtOAc in hexanes) to yield
1-(2-chloroacetoxy)-2,3,4,6-tetra-O-acetyl-D-glucopyranose (1.18 g,
2.8 mmol, 48% yield).
[1025] Step 2
[1026] Compound
1-(2-chloroacetoxy)-2,3,4,6-tetra-O-acetyl-D-glucopyranose (1.18 g,
2.8 mmol, 1 eq) was dissolved in acetone, followed by addition of
sodium iodide (0.625 g, 4.2 mmol, 1.5 eq). The reaction was stirred
for a few minutes, followed by addition of dimethylamino-1-propyne
(0.76 mL, 7.1 mmol, 2.5 eq). The reaction was stirred at room
temperature overnight, then filtered. The filtrate was
concentrated, diluted with DMSO and water, then purified by reverse
phase C18 column chromatography (0.1% TFA in 95% water/5% MeCN to
100% MeCN). Fractions containing product were lyophilized to yield
an off-white powder (420 mg, 0.72 mmol, 26% yield). LCMS (M+):
472.4 .sup.1H NMR (400 MHz, DMSO-d6) .delta. 6.13 (d, J=8.2 Hz,
1H), 5.48 (t, J=9.6 Hz, 1H), 5.08-4.94 (m, 2H), 4.75-4.46 (m, 4H),
4.35-3.95 (m, 4H), 3.28-3.15 (m, 6H), 2.07-1.86 (m, 12H).
##STR00358##
Compound 304:
N,N-dimethyl-N-(2-oxo-2-(((2R,3R,4S,5R)-3,4,5,6-tetraacetoxytetrahydro-2H-
-pyran-2-yl)methoxy)ethyl)prop-2-yn-1-aminium Trifluoroacetate
[1027] Step 1:
[1028] Compound 1,2,3,4-tetra-O-acetyl-beta-D-glucopyranose (Alfa
Aesar, CAS: 13100-46-4, 0.5 g, 1.44 mmol, 1 eq) was dissolved in
DCM (5 mL) and cooled to 0.degree. C. Chloroacetyl chloride (0.3425
mL, 4.3 mmol, 3 eq) was added, followed by dropwise addition of
pyridine (0.35 mL, 4.3 mmol, 3 eq). The reaction was directly
loaded onto silica, and purified by column chromatography (100%
hexanes to 100% ethyl acetate) to yield
6-(2-chloroacetoxy)-1,2,3,4-tetra-O-acetyl-beta-D-glucopyranose as
an off-white to yellow solid (0.5 g, 1.18 mmol, 82% yield).
[1029] Step 2:
[1030] Compound
6-(2-chloroacetoxy)-1,2,3,4-tetra-O-acetyl-beta-D-glucopyranose
(0.5 g, 1.18 mmol, 1 eq) was dissolved in acetone, followed by
addition of sodium iodide (0.35 g, 2.3 mmol, 2 eq). The reaction
was stirred for a few minutes, followed by addition of
3-dimethylamino-1-propyne (0.25 mL, 2.3 mmol, 2 eq) was added. The
reaction was stirred at room temperature overnight, then filtered.
The filtrate was concentrated, diluted with DMSO and water and
purified by reverse phase C18 column chromatography (0.1% TFA in
water 95%/MeCN 5% to 100% MeCN). Fractions containing product was
lyophilized to yield an off-white powder (476 mg, 0.81 mmol, 69%
yield). LCMS (M+): 472.1 1H NMR (400 MHz, DMSO-d6) .delta. 5.98 (d,
J=8.3 Hz, 1H), 5.47 (t, J=9.6 Hz, 1H), 5.10-4.84 (m, 2H), 4.50 (t,
J=2.2 Hz, 4H), 4.41-4.17 (m, 3H), 4.16-4.07 (m, 1H), 3.23 (d,
J=145.8 Hz, 6H), 2.15-1.88 (m, 12H).
Compound 306:
dimethyl(prop-2-yn-1-yl)(2-{[(2S,3R,4S,5S)-3,4,5-tris(acetyloxy)oxan-2-yl-
]oxy}ethyl)azanium Iodide
[1031] Step 1:
[1032] To a mixture of (2R,3S,4S)-2,3,4,5-tetrahydroxypentanal (10
g, 66.61 mmol, 1 eq) in pyridine (100 mL) was added Ac.sub.2O
(40.80 g, 399.65 mmol, 37.43 mL, 6 eq) in one portion at 25.degree.
C. under N.sub.2. The mixture was stirred at 25.degree. C. for 12
hours. TLC indicated one new spot formed. The reaction mixture was
concentrated under reduced pressure to remove pyridine. The residue
was purified by column chromatography (SiO.sub.2, Petroleum
ether/Ethyl acetate=30/1 to 0/1). Compound
[(3S,4S,5R,6S)-4,5,6-triacetoxytetrahydropyran-3-yl] acetate (20 g,
crude) was obtained as yellow oil.
[1033] Step 2:
[1034] To a mixture of
[(3S,4S,5R,6S)-4,5,6-triacetoxytetrahydropyran-3-yl] acetate (10 g,
31.42 mmol, 1 eq) in DCM (100 mL) was added 2-bromoethanol (5.89 g,
47.13 mmol, 3.35 mL, 1.5 eq) and BF.sub.3.Et.sub.2O (22.30 g,
157.10 mmol, 19.39 mL, 5 eq) in one portion at 0.degree. C. under
N.sub.2. The mixture was heated to 25.degree. C. and stirred for 12
hours. TLC indicated
[(3S,4S,5R,6S)-4,5,6-triacetoxytetrahydropyran-3-yl] acetate was
consumed and one new spot formed. The reaction mixture was quenched
by addition of H.sub.2O (50 mL), and then diluted with H.sub.2O
(100 mL) and extracted with DCM (100 mL*2). The combined organic
layers were dried over Na.sub.2SO.sub.4, filtered and the filtrate
was concentrated under reduced pressure to give a residue. The
residue was purified by column chromatography (SiO.sub.2, Petroleum
ether/Ethyl acetate=20/1 to 0/1). Then the residue was purified by
prep-HPLC [water (0.1% TFA)-ACN]. .sup.1HNMR indicated compound
[(3S,4S,5R,6S)-4,5-diacetoxy-6-(2-bromoethoxy)tetrahydropyran-3-yl]
acetate (1.2 g, 3.13 mmol, 9.97% yield) was obtained as yellow oil
and
compound[(3S,4S,5R,6R)-4,5-diacetoxy-6-(2-bromoethoxy)tetrahydropyran-3-y-
l] acetate (2.3 g, 6.00 mmol, 19.10% yield) was obtained as yellow
oil.
[1035] Step 3:
[1036] To a mixture of
[(3S,4S,5R,6R)-4,5-diacetoxy-6-(2-bromoethoxy)tetrahydropyran-3-yl]
acetate (1.00 g, 2.61 mmol, 1 eq) in acetone (10 mL) was added NaI
(430.29 mg, 2.87 mmol, 1.1 eq) in one portion at 25.degree. C.
under N.sub.2. Then N,N-dimethylprop-2-yn-1-amine (1.08 g, 13.05
mmol, 1.38 mL, 5 eq) was added. The mixture was heated to
90.degree. C. and stirred for 2 hours. TLC indicated
[(3S,4S,5R,6R)-4,5-diacetoxy-6-(2-bromoethoxy)tetrahydropyran-3-yl]
acetate was consumed and one new spot formed. The reaction mixture
was filtered and the filtrate was concentrated under reduced
pressure to give a residue. The residue was purified by column
chromatography (SiO.sub.2, Petroleum ether/Ethyl acetate=10/1 to
Ethyl acetate:Methanol=3/1). The title compound (109 mg, 210.22
umol, 8.06% yield, 99% purity, I) was obtained as white solid.
LCMS: (M+): 386.1 1H NMR (400 MHz, Methanol-d4) .delta. 5.21 (s,
1H), 5.12-5.06 (m, 2H), 4.48 (d, J=2.5 Hz, 2H), 4.43 (dd, J=11.6,
3.9 Hz, 1H), 4.38 (q, J=4.4 Hz, 1H), 4.29-4.16 (m, 2H), 4.06-3.96
(m, 1H), 3.86-3.74 (m, 2H), 3.61 (t, J=2.5 Hz, 1H), 3.31 (s, 6H),
2.20-1.95 (m, 9H).
##STR00359##
Compound 308:
dimethyl(prop-2-yn-1-yl)(2-{[(3R,4R,5R)-3,4,5-tris(acetyloxy)oxan-2-yl]ox-
y}ethyl)azanium
[1037] Step 1:
[1038] To a mixture of (2R,3R,4R)-2,3,4,5-tetrahydroxypentanal (10
g, 66.61 mmol, 1 eq) in pyridine (100 mL) was added Ac.sub.2O
(40.80 g, 399.65 mmol, 37.43 mL, 6 eq) in one portion at 25.degree.
C. under N.sub.2. The mixture was stirred at 25.degree. C. for 12
hours. TLC indicated one new spot formed. The reaction mixture was
concentrated under reduced pressure to remove the pyridine. The
residue was purified by column chromatography (SiO.sub.2, Petroleum
ether/Ethyl acetate=30/1 to 0/1). Compound
[(3R,4R,5R)-4,5,6-triacetoxytetrahydropyran-3-yl] acetate (20 g,
crude) was obtained as white solid.
[1039] Step 2:
[1040] To a mixture of
[(3R,4R,5R)-4,5,6-triacetoxytetrahydropyran-3-yl] acetate (10 g,
31.42 mmol, 1 eq) in DCM (50 mL) was added 2-bromoethanol (5.89 g,
47.13 mmol, 3.35 mL, 1.5 eq) in one portion at 0.degree. C. under
N.sub.2. BF.sub.3.Et.sub.2O was added (22.30 g, 157.10 mmol, 19.39
mL, 5 eq) and stirred at 0.degree. C. for 6 minutes, then heated to
25.degree. C. and stirred for 11.9 hours. TLC indicated
[(3R,4R,5R)-4,5,6-triacetoxytetrahydropyran-3-yl] acetate was
consumed and one new spot formed. The reaction mixture was quenched
by addition H.sub.2O (20 mL), then diluted with H.sub.2O (50 mL)
and extracted with DCM (100 mL, 50 mL*2). The combined organic
layers were dried over Na.sub.2SO.sub.4, filtered and the filtrate
was concentrated under reduced pressure to give a residue. The
residue purified by column chromatography (SiO.sub.2, Petroleum
ether/Ethyl acetate=20/1 to 0/1). Then the residue was purified by
prep-HPLC [water (0.1% TFA)-ACN]. Compound
[(3R,4R,5R)-4,5-diacetoxy-6-(2-bromoethoxy)tetrahydropyran-3-yl]
acetate (2.6 g, 6.79 mmol, 21.60% yield) was obtained as a yellow
oil.
[1041] Step 3:
[1042] To a mixture of
[(3R,4R,5R)-4,5-diacetoxy-6-(2-bromoethoxy)tetrahydropyran-3-yl]
acetate (1.5 g, 3.91 mmol, 1 eq) in acetone (15 mL) was added
N,N-dimethylprop-2-yn-1-amine (1.63 g, 19.57 mmol, 2.08 mL, 5 eq)
in one portion at 25.degree. C. under N.sub.2. Then the mixture was
heated to 90.degree. C. and stirred for 2 hours. LCMS showed one
main peak with expected mass was detected. The reaction mixture was
concentrated under reduced pressure to give a residue. Then the
residue was purified by prep-HPLC [water (0.10% TFA)-ACN]. The
title compound (619 mg, 1.33 mmol, 33.91% yield, 100% purity, Br--)
was obtained as colorless oil. LCMS: (M+): 386.2 .sup.1H NMR (400
MHz, Methanol-d4) .delta. 5.49 (t, J=3.3 Hz, 1H), 5.11 (dt, J=6.8,
3.5 Hz, 1H), 5.01 (dd, J=5.2, 3.3 Hz, 1H), 4.96 (d, J=5.1 Hz, 1H),
4.42 (d, J=2.6 Hz, 2H), 4.26 (dt, J=13.5, 4.4 Hz, 1H), 4.13-3.99
(m, 2H), 3.86 (dd, J=12.1, 6.6 Hz, 1H), 3.78 (t, J=4.7 Hz, 2H),
3.59 (t, J=2.5 Hz, 1H), 3.27 (s, 6H), 2.16-1.99 (m, 9H).
##STR00360##
Compound 336:
(2-{[(2R,3R,4R,5S,6R)-4,5-bis(acetyloxy)-6-[(acetyloxy)methyl]-3-acetamid-
ooxan-2-yl]oxy}ethyl)(fluoromethyl)dimethylazanium
[1043] This compound may be synthesized according to the
experimental procedure described for Compound 311.
##STR00361##
Compound 337:
(2-{[(2R,3R,4R,5S,6R)-3-acetamido-4,5-dihydroxy-6-(hydroxymethyl)oxan-2-y-
l]oxy}ethyl)(fluoromethyl)dimethylazanium
[1044] This compound may be synthesized according to the
experimental procedure described for Compound 317.
##STR00362##
Compound 338:
(fluoromethyl)dimethyl(2-{[(2R,3R,4S,5S,6S)-3,4,5-tris(acetyloxy)-6-carbo-
xyoxan-2-yl]oxy}ethyl)azanium
[1045] This compound may be synthesized according to the
experimental procedure described for Compound 321.
##STR00363##
Compound 339:
(2-{[(2R,3R,4S,5S,6S)-6-carboxy-3,4,5-trihydroxyoxan-2-yl]oxy}ethyl)(fluo-
romethyl)dimethylazanium
[1046] This compound may be synthesized according to the
experimental procedure described for Compound 321, followed by
deacetylation following procedure for Compound 317.
##STR00364##
Compound 159: N-(carboxymethyl)-N,N-dimethylprop-2-yn-1-aminium
Bromide
[1047] To the mixture of N,N-dimethylprop-2-yn-1-amine (1 g, 12.03
mmol, 1.28 mL, 1 eq) in Acetone (20 mL) was added dropwise
2-bromoacetic acid (1.82 g, 13.11 mmol, 943.97 uL, 1.09 eq), and
then the mixture was stirred at 15.degree. C. for 20 min. TLC
indicated N,N-dimethylprop-2-yn-1-amine was consumed completely and
one new spot formed. The reaction mixture was filtered and filter
cake was concentrated under reduced pressure to give a residue.
Compound carboxymethyl-dimethyl-prop-2-ynyl-ammonium (700 mg, 3.15
mmol, 26.20% yield, Br--) was obtained as a white solid. .sup.1H
NMR (400 MHz, DMSO) .delta. 4.5 (s, 2H), 4.0 (m, 3H), 3.2 (s, 6H)
ppm.
##STR00365##
[1048] Compound J60:
N-(2-hydroxyethyl)-N,N-dimethylprop-2-yn-1-aminium Iodide
[1049] To a solution of N,N-dimethylprop-2-yn-1-amine (1 g, 12.03
mmol, 1.28 mL, 1 eq) in THF (3 mL) was added dropwise 2-iodoethanol
(2.17 g, 12.63 mmol, 987.35 uL, 1.05 eq) at 0.degree. C. The
mixture was warmed to 15.degree. C. for 5 hr. TLC indicated
N,N-dimethylprop-2-yn-1-amine was consumed completely and one new
spot formed. The reaction was clean according to TLC. The reaction
mixture was filtered and filter cake was concentrated under reduced
pressure to give a residue. Compound
N-(2-hydroxyethyl)-N,N-dimethyl-prop-2-ynyl-ammonium iodide (2 g,
7.84 mmol, 65.17% yield, I--) was obtained as a white solid.
.sup.1H NMR (400 MHz, DMSO) .delta. 5.3 (m, 1H), 4.4 (s, 2H), 4.0
(s, 1H), 3.8 (m, 2H), 3.4 (m, 2H), 3.1 (s, 6H) ppm.
##STR00366##
Compound Z76: 1-(prop-2-yn-1-yl)-1H-imidazole
[1050] Imidazole is reacted with propargyl bromide in THF and
diisopropyl ethyl amine. The resulting compound is purified to
afford the title compound.
##STR00367##
Compound A51:
4-carboxy-N,N-dimethyl-N-(prop-2-yn-1-yl)butan-1-aminium
Bromide
[1051] To a solution of N, N-dimethylprop-2-yn-1-amine (300 mg,
3.61 mmol, 382.65 uL, 1.08 eq) in acetone (5 mL) was added
5-bromopentanoic acid (604.89 mg, 3.34 mmol, 1 eq) at 15.degree. C.
The mixture was stirred at 60.degree. C. for 16 h. The reaction
mixture was concentrated under reduced pressure. The residue was
diluted with H.sub.2O (10 mL) and extracted with ethyl acetate (10
mL*2). The combined water layer was freeze-dried to get
4-carboxy-N, N-dimethyl-N-(prop-2-yn-1-yl) butan-1-aminium bromide
(89 mg, 336.92 umol, 10.08% yield) as a yellow solid. .sup.1H NMR
(400 MHz, D2O) .delta. 4.22 brS, 2H), 3.4 (m, 2H), 3.1 s (1H), 3.05
(brs, 6H), 2.4 (t, 2H), 1.8 (m, 2H), 1.6 m, 2H).
##STR00368##
[1052] Compound B52:
2-carboxy-N,N-dimethyl-N-(prop-2-yn-1-yl)prop-2-en-1-aminium
Bromide
[1053] To a solution of N,N-dimethylprop-2-yn-1-amine (200 mg, 2.41
mmol, 255.10 uL, 1 eq) in ACN (10 mL) was added dropwise
2-(bromomethyl)prop-2-enoic acid (417.49 mg, 2.53 mmol, 1.05 eq) at
15.degree. C. After addition the mixture was stirred at 15.degree.
C. for 12 h. The white precipitate was formed and TLC showed the
starting reactant was consumed. The mixture reaction was filtered
to obtain white solid. Compound
2-carboxy-N,N-dimethyl-N-(prop-2-yn-1-yl)prop-2-en-1-aminium
bromide (103 mg, 551.09 umol, 22.87% yield) was obtained as a white
solid. LCMS: (M+): 168.1 1H NMR (400 MHz, Deuterium Oxide) .delta.
6.79 (s, 1H), 6.30 (s, 1H), 4.19 (s, 2H), 4.10 (d, J=2.6 Hz, 2H),
3.14 (t, J=2.6 Hz, 1H), 2.99 (s, 6H).
##STR00369##
Compound AA 77: (1-(prop-2-yn-1-yl)-1H-imidazol-2-yl)methanol
[1054] 1-(prop-2-yn-1-yl)-1H-imidazole is dissolved in THF and
cooled to -78.degree. C. n-Butyl lithium is added followed by
paraformaldehyde. The resulting mixture is stirred at -78 then
quenched with water followed by ethyl acetate. The material was
purified to afford the title compound.
##STR00370##
Compound AB78: 3-methyl-1-(prop-2-yn-1-yl)-1H-imidazol-3-ium
Iodide
[1055] Imidazole is reacted with propargyl bromide in THF and
diisopropyl ethyl amine. The resulting compound is purified to
afford 1-(prop-2-yn-1-yl)-1H-imidazole. This material is taken up
in diethyl ether and treated with dropwise addition of methyl
iodide. The resulting precipitate is collected and dried and
recrystallized to afford the title compound.
##STR00371##
Compound AC79: (1-(prop-2-yn-1-yl)-1H-imidazol-5-yl)methanol
[1056] (1H-imidazol-4-yl)methanol is treated with propargyl bromide
and diisopropyl ethyl amine in THF overnight. The resulting
material is purified to afford the title compound.
##STR00372##
Compound AE81:
N-(3-(hydroxyhydrophosphoryl)propyl)-N,N-dimethylprop-2-yn-1-aminium
[1057] (3-(dimethylamino)propyl)phosphinic acid is taken up in THF
and treated with drop wise addition of propargyl iodide. The
resulting precipitate is collected and dried and recrystallized to
afford the title compound.
##STR00373##
Compound AG83:
N,N-dimethyl-N-(3-phosphonopropyl)prop-2-yn-1-aminium Iodide
[1058] (3-(dimethylamino)propyl)phosphonic acid is taken up in THF
and treated with drop wise addition of propargyl iodide. The
resulting precipitate is collected and dried and recrystallized to
afford the title compound.
##STR00374##
Compound AI85: N,N-dimethyl-N-(3-sulfopropyl)prop-2-yn-1-aminium
Iodide
[1059] 3-(dimethylamino)propane-1-sulfonic acid is taken up in THF
and treated with drop wise addition of propargyl iodide. The
resulting precipitate is collected and dried and recrystallized to
afford the title compound.
Example 2: Cell-Based Assay for TMA Production from a Native
Producer
[1060] Trimethylamine lyase (CutC) from Clostridium sporogenes ATCC
15579 (protein EDU36695.1) and Proteus mirabilis strain HI4320
(protein WP_012368484.1) was identified as an enzyme that converts
choline into trimethylamine. In each bacterium, CutC was modified
into its activated form by a cognate activating enzyme, CutD
(protein EDU36696.1 for C. sporogenes; protein WP_004249185.1 for
P. mirabilis). We have also identified additional enzymes that
convert carnitine into trimethylamine. Trimethylamine may
subsequently be converted by human enzymes such as flavin
monooxygenase 3 to trimethylamine N-oxide, which might be
associated with negative health consequences in humans.
[1061] These examples of bacterial enzymes producing metabolites
negatively associated with human health are not intended to be
limiting.
[1062] A compound's ability to inhibit CutC from Proteus mirabilis
and C. sporogenes from converting choline into trimethylamine was
tested in cells as described. A BHI blood plate was inoculated with
a frozen stock of Proteus mirabilis or Clostridium sporogenes under
anaerobic conditions. A single colony was isolated from the plate
and inoculated into 5 mL BHI supplemented with 1 mM choline under
anaerobic conditions. The liquid cultures were grown overnight at
37.degree. C.
[1063] To prepare each sample, 300 .mu.L of the saturated culture
was then inoculated into a 30 mL dilution blank under anaerobic
conditions. 1 mM (trimethyl-d.sub.9)-choline and a sufficient
amount of a compound to obtain a final concentration of 10 .mu.M
were added to the inoculated dilution blank. Optionally, the
IC.sub.50 of an inhibitor may instead be determined by adding an
inhibitor across a range of concentrations, for example, 0, 0.001,
0.01, 0.1, 1, and 10 .mu.M. The mixture was incubated at 37.degree.
C. for 5 h. Chilled 85% acetonitrile, 15% methanol and 0.2% formic
acid was added to the reaction and the mixture was centrifuged.
After centrifugation, the samples were analyzed by LCMS to
determine the amount of d.sub.9-TMA produced. Results are shown in
Table 1.
TABLE-US-00001 TABLE 1 IC.sub.50 Standard Number Whole Deviation of
assay Com- cell C. Average IC.sub.50 for IC.sub.50 in runs for
pound sporogene Whole cell P. P. P. No. s (uM)* mirabilis (uM)
mirabilis mirabilis 127 10.00 5.01 7.05 2 B52 0.01 0.03 0.04 3 Z76
10.00 4.04 5.26 3 128 10.00 10.00 N/A 1 159 0.76 3.51 5.63 3 159
10.00 0.28 0.37 2 129 10.00 10.00 0.00 2 J60 0.00 0.18 0.13 7 130
10.00 3.47 1.71 2 A51 0.93 1.10 1.39 2 107 10.00 10.00 0.00 2 132
10.00 10.00 N/A 1 133 0.44 0.66 N/A 1 134 10.00 10.00 N/A 1 110
10.00 10.00 N/A 1 111 -- 0.14 0.12 2 137 10.00 10.00 N/A 1 117
10.00 10.00 N/A 1 116 10.00 10.00 N/A 1 109 0.42 0.62 0.58 6 108
2.46 2.19 N/A 1 113 1.25 0.70 N/A 1 114 1.63 2.08 N/A 1 115 9.85
2.07 N/A 1 116 10.00 10.00 N/A 1 138 5.35 1.05 0.28 3 112 0.28 0.55
0.30 2 140 10.00 10.00 N/A 1 141 10.00 5.25 4.20 3 111 0.22 0.27
N/A 1 143 10.00 10.00 0.00 2 144 0.70 0.58 N/A 1 145 1.38 10.00
0.00 2 146 1.50 10.00 N/A 1 147 10.00 0.00 2 148 10.00 N/A 1 149
10.00 N/A 1 Prodrug 7 3.16 N/A 1 Prodrug 19 1.730 N/A 1 Prodrug 8
10.00 N/A 1 Prodrug 69 10.00 N/A 1 Prodrug 73 10.00 N/A 1 Prodrug
70 10.00 N/A 1 Prodrug 71 10.00 N/A 1 Prodrug 72 10.00 N/A 1
Prodrug 73 10.00 N/A 1 Prodrug 74 10.00 N/A 1 Prodrug 106 10.00 N/A
1 Prodrug 78 10.00 N/A 1 Prodrug 78 10.00 N/A 1 Prodrug 77 10.00
N/A 1 Prodrug 79 10.00 N/A 1 Prodrug 79 10.00 N/A 1 Prodrug 80
10.00 N/A 1 Prodrug 86 10.00 N/A 1 Prodrug 98 0.00 N/A 1 Prodrug 99
0.00 N/A 1 151 0.89 1.16 2 152 10.00 N/A 1 153 0.02 0.01 2 159
10.00 N/A 1 160 10.00 N/A 1 161 10.00 N/A 1 162 0.29 0.23 2 125
0.19 0.02 2 164 10.00 N/A 1 165 2.13 N/A 1 J60 0.09 0.02 2 B52 0.04
N/A 1 166 1.26 N/A 1 167 10.00 N/A 1 168 0.87 N/A 1 169 10.00 N/A 1
171 10.00 N/A 1 172 10.00 N/A 1 173 10.00 N/A 1 174 0.17 N/A 1 175
0.17 N/A 1 154 10.00 N/A 1 155 2.58 N/A 1 156 10.00 N/A 1 157 10.00
N/A 1 176 10.00 N/A 1 177 7.57 N/A 1 178 10.00 N/A 1 179 0.11 N/A 1
180 >10 N/A 1 181 >10 N/A 1 182 3.150 N/A 1 183 >10 N/A 1
184 >10 N/A 1 185 0.235 N/A 1 186 >10 N/A 1 187 0.763 N/A 1
188 >10 N/A 1 189 >10 N/A 1 190 1.280 N/A 1 191 2.165 N/A 1
192 >10 N/A 1 193 1.375 N/A 1 194 >10 N/A 1 195 >10 N/A 1
196 7.780 N/A 1 197 4.970 N/A 1 198 >10 N/A 1 199 >10 N/A 1
200 2.740 N/A 1 201 >10 N/A 1 202 0.242 N/A 1 203 1.720 N/A 1
204 0.660 N/A 1 205 8.300 N/A 1 206 0.069 N/A 1 207 0.310 N/A 1 208
>10 N/A 1 209 6.800 N/A 1 210 >10 N/A 1 211 0.211 N/A 1 212
1.990 N/A 1 213 0.180 N/A 1 214 3.870 N/A 1 215 0.409 N/A 1 216
>10 N/A 1 217 0.771 N/A 1 218 >10 N/A 1 219 >10 N/A 1 220
>10 N/A 1 221 >10 N/A 1 222 >10 N/A 1 223 2.280 N/A 1 224
0.223 0.102 2 225 0.629 0.095 2 226 0.244 0.007 2 227 >10 N/A 1
228 >10 N/A 2 229 >100.000 N/A 1 IC.sub.50 values were given
the value "10" when a best fit ICso line could not calculated or
when the % TMA remaining at the highest concentration of inhibitor
(10 .mu.M) was higher than 15% * C. sporogenes was run once
Example 3: Cell-Based Assay with Bacterial Enzymes
[1064] A vector may be designed to express an enzyme, such as those
identified in Example 2, in its wild-type form or a catalytically
inactive version. The vector may be transformed into a heterologous
host, for example an E. coli expression strain. Alternatively, a
strain natively encoding one of the enzymes identified in Example 2
may be procured.
[1065] The heterologous host (containing the wild-type enzyme, a
catalytically inert version, or the empty vector) or the native
strain may be grown to a desirable level.
[1066] The native substrate or an appropriately-chosen substrate
analog may be added to the bacterial growth or uninoculated medium.
Optionally, an inhibitor might be added to prevent conversion of
substrate to product. The mixture may be incubated for a designated
period of time.
[1067] After incubation, the mixture may be rendered compatible
with LCMS analysis via addition of organic solvent or
lyophilization followed by dissolution in organic solvent. The
amount of residual starting material and of the product may be
quantified by LCMS analysis.
[1068] For example, the following cell-based assay for TMA
production from a heterologous host is a representative example of
the above-described assays. E. coli strains capable of
co-expressing full-length WT CutC and CutD, the catalytically inert
G821A CutC and CutD, or empty pET28 can be produced by transforming
the aforementioned plasmids into E. coli BL21(DE3) by heat shocking
the cells. Transformants may be selected by growing on LB plates
supplemented with kanamycin (50 .mu.g/mL).
[1069] A single colony may be isolated from the plate and
inoculated into 10 mL LB supplemented with kanamycin (50 .mu.g/mL).
The liquid cultures may be grown overnight at 37.degree. C. with
rocking on a nutator.
[1070] 500 .mu.L of the saturated overnight culture may be
inoculated into 30 mL LB-Kan50 under anaerobic conditions. The
cultures may be grown at 37.degree. C. until the optical density at
600 nm reached 0.6 A.U. Then, IPTG may be added to a final
concentration of 0.5 mM.
[1071] To 1 mL of induced culture or a media blank may be added 1
mM (trimethyl-d.sub.9)-choline. A compound to be tested for
inhibition of CutC may also be added at this time at a fixed final
concentration of, for example, 10 .mu.M. Optionally, the IC.sub.50
of an inhibitor may instead be determined by adding an inhibitor
across a range of concentrations, for example, 0, 0.001, 0.01, 0.1,
1, and 10 .mu.M. The mixture may be incubated at 37.degree. C. for
8 h. Chilled 85% acetonitrile, 15% methanol and 0.2% formic acid
may be added to the reaction. After centrifugation, the samples may
be analyzed by LCMS, for example, to determine the amount of
d.sub.9-TMA that has been produced from the d.sub.9-choline.
[1072] Another representative example of the above-described assays
involves assaying active and inactive versions of the proteins
listed above and in Example 2 for their ability to react with their
native substrate or an appropriately chosen substrate analog in a
cell-based assay. In each instance, this incubation may be carried
out in the presence of an inhibitor. LCMS may be used to quantify
the amount of product that is produced from the native substrate or
the isotope-labeled substrate.
Example 4: Determination of Inhibitor Effect on Bacterial
Growth
[1073] To verify that the inhibition of d.sub.9-TMA (such as
assayed by a method disclosed in Example 3 above) is due to
inhibition of enzymatic activity and not cell death, the lag phase
of the growth curve from either P. mirabilis or C. sporogenes was
measured.
[1074] A single colony was isolated from the plate and inoculated
into 5 mL BHI supplemented with 1 mM choline under anaerobic
conditions. The liquid cultures were grown overnight at 37.degree.
C.
[1075] 300 .mu.L of the saturated overnight culture was inoculated
into 30 mL BHI under anaerobic conditions supplemented in the
presence of 10 .mu.M of compound to be tested. The growth of the
bacteria was monitored with a spectrophotometer at an Optical
Density of 600 nm for 24 hours. Results are shown in Table 2.
TABLE-US-00002 TABLE 2 Avg Lag Time Avg Lag Time (% compared (%
compared to control) [C. Number of SD of C. to control) [P. Number
of SD of P. sporogenes, C.sporogenes sporogenes mirabilis,
P.mirabilis mirabilis Cmpd No. Conc: 10 .mu.M] Replicates Lag Time
Conc: 10 .mu.M] Replicates Lag Time B52 102.55 2 17.04 100.40 2
10.32 Z76 100.20 1 N/A 92.30 1 N/A J60 98.30 2 2.26 101.93 3 7.29
H58 N/A 0 N/A 106.10 1 N/A 109 92.70 1 N/A 99.40 2 4.38 138 102.60
1 N/A 82.80 1 N/A 112 97.20 1 N/A 97.20 1 N/A 145 107.70 1 N/A
107.10 1 N/A 153 N/A 0 N/A 98.20 1 N/A X74 N/A 0 N/A 98.40 1 N/A 78
93.8 109.4 79 83.5 103.3 80 95.9 95.3 228 108.3 112.2 317 83 111.1
318 83.1 103 229 96.9 112.4
Example 5: Inhibition of Enzymatic Activity in Fecal Matter
[1076] Inhibition of enzymatic activity in fecal matter was
determined by adding a substrate (d9-choline, 1 mM final
concentration) and a compound in a range of concentrations in order
to determine an IC.sub.50. After incubation for a designated 16
hours at 37.degree. C. in anaerobic conditions, samples were be
prepared for LCMS analysis, and the amount of d9-TMA produced was
determined using LCMS analysis as in the Examples above. Results
are shown in Table 3.
TABLE-US-00003 TABLE 3 Compound IC.sub.50 Number of Runs 78 0.39 6
79 1.17 6 80 0.38 5 317 0.43 3
Example 6: Acute Mouse Model
[1077] The following acute mouse model may be used to determine
inhibition of the CutC/D enzyme and thus the inhibition of the
formation of trimethylamine (TMA) from choline.
[1078] On day 1, male C57BL/6 mice were given a chemically defined
diet (Tekland Global Rodent Diet 2018) containing 1.0% choline
(g/g) for the duration of the experiment. Concurrently on day 1,
mice were orally gavaged with 200 .mu.L of compounds of interest
(formulated in water). Mice were orally gavaged with the compounds
and vehicle control again on day 2 and day 3 at a fixed time.
Twenty hours after the last gavage, mice were sacrificed, and
plasma was collected and prepared for detection of
trimethylamine-N-oxide (TMAO) via liquid chromatography with
on-line tandem mass spectrometry (LC-MS/MS). Food consumption and
mouse weight were measured each day.
[1079] Plasma samples were prepared for LCMS as follows: An aliquot
of 20 .mu.L plasma sample was protein precipitated with 200 .mu.L
internal standard solution (100 ng/mL Labetalol & 100 ng/mL
Tolbutamide & 100 ng/mL Diclofenac in acetonitrile, the mixture
was vortex-mixed and centrifuged at 4000 rpm for 15 min, 4.degree.
C. An aliquot of 100 .mu.L supernatant was transferred to the
sample plate for LCMS injection. 1 .mu.L of sample was injected
onto an LC column for TMAO LCMS analysis.
[1080] Concentrations of TMAO were determined with a calibration
curve ranging 0.05-100 .mu.M for d9-TMAO in control plasma. Results
are shown in Tables 3a-3e.
TABLE-US-00004 TABLE 4a Mean % change plasma from vehicle TMAO
control + Diet + Compound (.mu.M) choline diet Normal Diet +
Vehicle Control 4.56 -86.75 Choline Diet + Vehicle Control 34.39
0.00 Choline diet + Compound I59 (100 mg/kg) 10.85 -68.44 Choline
diet + Compound 19 (333 mg/kg) 5.40 -84.29 Choline diet + Compound
J60 (10 mg/kg) 31.06 -28.46 Choline diet + Compound J60 (33 mg/kg)
17.20 -60.39 Choline diet + Compound J60 (100 mg/kg) 5.52 -87.29
Choline diet + Compound 7 (358 mg/kg) 6.90 -79.95
TABLE-US-00005 TABLE 4b Mean % change plasma from vehicle TMAO
control + Diet + Compound (.mu.M) choline diet normal diet +
vehicle 2.55 -89.74 choline diet + vehicle 24.9 0.00 choline diet +
Compound J60 (10 mg/kg) 24.7 -0.69 choline diet + Compound B52 (10
mg/kg) 30.0 20.76 choline diet + Compound 141 (10 mg/kg) 2.92
-88.23 choline diet + Compound J60 (10 mg/kg) 27.4 10.16
TABLE-US-00006 TABLE 4c Mean % change plasma from vehicle TMAO
control + Diet + Compound (.mu.M) choline diet normal diet +vehicle
2.17 -89.66 choline diet + vehicle 21.0 0.00 choline diet +
Compound J60 (100 mg/kg) 5.41 -74.22 choline diet + Compound 7
(35.8 mg/kg) 20.4 -2.97 choline diet + Compound 7 (107 mg/kg) 15.8
-24.83 choline diet + Compound 7 (358 mg/kg) 6.41 -69.46
TABLE-US-00007 TABLE 4d Mean % change plasma from vehicle TMAO
control + Diet + Compound (.mu.M) choline diet normal diet +
vehicle 2.49 -87 choline diet + vehicle 18.8 0 choline diet +
Compound J60 (100 mg/kg) 6.00 -68 choline diet + Compound 58 (10
mg/kg) 10.1 -46 choline diet + Compound 310 (33.8 mg/kg) 2.3 -88
choline diet + Compound 312 (35.4 mg/kg) 11.69 -38 choline diet +
Compound 309 (32.6 mg/kg) 1.9 -90 choline diet + Compound 311 (37.0
mg/kg) 1.8 -90 choline diet + Compound 8 (100 mg/kg) 12.5 -33
choline diet + Compound 10 (100 mg/kg) 14.9 -21 choline diet +
Compound 9 (100 mg/kg) 33.91 80
TABLE-US-00008 TABLE 4e Mean % change plasma from vehicle TMAO
control + Diet + Compound (.mu.M) choline diet normal diet +
vehicle 1.90 -93 choline diet + vehicle 26.4 0 choline diet +
Compound J60 (100 mg/kg) 3.81 -86 choline diet + Compound 185
(123.7 mg/kg) 22.2 -16
TABLE-US-00009 TABLE 4f Mean % change plasma from vehicle TMAO
control + Diet + Compound (.mu.M) choline diet normal diet +
vehicle 3.91 -88 choline diet + vehicle 32.3 0 choline diet +
Compound J60 (100 mg/kg) 6.20 -81 choline diet + Compound 98 (36.6
mg/kg) 10.1 -69 choline diet + Compound 98 (11.0 mg/kg) 34.3 6
choline diet + Compound 98 (3.70 mg/kg) 29.7 -8 choline diet +
Compound 309 (37.2 mg/kg) 5.45 -83 choline diet + Compound 309
(11.0 mg/kg) 15.4 -52 choline diet + Compound 309 (3.70 mg/kg) 35.2
9 choline diet + Compound 311 (35.3 mg/kg) 11.23 -65 choline diet +
Compound 311 (10.6 mg/kg) 17.4 -46 choline diet + Compound 311(3.5
mg/kg) 20.22 -37
TABLE-US-00010 TABLE 4g Mean % change plasma from vehicle TMAO
control + Diet + Compound (.mu.M) choline diet normal diet +
vehicle 3.04 -89 choline diet + vehicle 27.8 0 choline diet +
Compound J60 (100 mg/kg) 13.1 -53 choline diet + Compound 125 (97.7
mg/kg) 32.8 18 choline diet + Compound 125 (9.8 mg/kg) 26.2 -6
choline diet + Compound 86 (112 mg/kg) 4.72 -83 choline diet +
Compound 86 (37.3 mg/kg) 18.6 -33 choline diet + Compound 109 (116
mg/kg) 2.71 -90 choline diet + Compound 151 (116 mg/kg) 29.4 6
TABLE-US-00011 TABLE 4h Mean % change plasma from vehicle TMAO
control + Diet + Compound (.mu.M) choline diet normal diet +
vehicle 3.07 -93 choline diet + vehicle 44.4 0 choline diet +
Compound J60 (100 mg/kg) 8.72 -80 choline diet + Compound 315
(226.5 mg/kg) 8.96 -80 choline diet + Compound 315 (67.9 mg/kg)
26.6 -40
[1081] The data in Tables 4a-4h show that compounds of the present
disclosure can inhibit the production of trimethyl amine and thus
can be useful in treating diseases in which CutC and the production
of trimethylamine are implications (e.g. treating
trimethylaminuria).
Example 7: Compound Efficacy in a Preclinical Chronic Kidney
Disease Mouse Model
[1082] The following preclinical model has been used to determine
the effect of TMA/TMAO on chronic kidney disease (CKD) (Tang et al
2015; Circulation Research, 116(3): 448-455.). C57BL/6 mice may be
fed a chemically defined diet with or without 1.0% choline (g/g)
for 16 weeks. Earlier time points of compound intervention may
define the potential to prevent the progression of kidney disease.
Later time points of intervention may determine the ability of the
compounds to treat an established kidney disease phenotype. The
weight and food consumption of each mouse will be monitored at
regular intervals throughout the study.
[1083] All mice may be sacrificed after 16 weeks of defined diet
and compound intervention. As described in the acute mouse model,
plasma may be collected and prepared for detection of TMA and TMAO
via LCMS. To assess the effect of compounds on renal function,
protein in urine may be quantified and blood urea nitrogen (BUN)
levels may be measured in the blood. To assess the effect of
compounds on kidney injury, kidneys may be collected, and levels of
fibrosis may be quantified via histological examination as well as
expression of markers of kidney damage and inflammation.
Additionally, aortas will be collected and examined for the
presence of vascular injury via protein markers of vascular
damage.
[1084] For histological examination, kidneys may be fixed and
embedded. Fibrosis may be quantified via collagen deposition using
the Mason trichome staining procedure (Tang et al 2015, supra, and
Sun et al 2017; Biochem. Biophys. Res. Commun. 493(2):
964-970).
[1085] Expression of kidney damage markers, such as pSMAD3, kidney
injury molecule (KIM)-1, TNF.alpha., TL-1.beta., neutrophil
gelatinase-associated lipocalin (NGAL), plasma cystatin C, urine
albumin, and NOX-4, may be determined via Western blot analysis of
homogenized kidneys. Expression of vascular damage markers, such as
ICAM, VCAM, TNF.alpha., and IL-1.beta., may be determined via the
MesoScale Discovery instrument.
Example 8: Release of Active Agents from Conjugates
[1086] Sprague-Dawley rats (three rats per compound) were treated
with a single intravenous (IV) 1 mg/kg dose or orally (PO) a single
10 mg/kg dose. Plasma concentrations of the conjugates were
determined by LCMS at 5 minutes, 15 minutes, 30 minutes, 1 hour, 2
hours, 4 hours, 8 and 24 hours post compound administration. For
all conjugates, PO doses displayed substantially lower plasma
exposure compared to IV administered rats and therefor all
conjugates have a low bioavailability (% F) as shown in Table 5
below. In addition, the plasma exposure and bioavailability of the
active agent of was lower when dosed as a conjugate instead of the
active agent alone.
TABLE-US-00012 TABLE 5 Compound Compound IV C.sub.max PO C.sub.max
Dosed Measured (nmol/L) (nmol/L) % F 78 78 8310 130 0.99 78 Active
6 70 1.91 agent of 78 Active Active 17200 970 4.20 agent of 78
agent of 78 79 79 13600 260 2.16 79 Active 1 50 1.60 agent of 79
Active Active 41700 2360 5.99 agent of 79 agent of 79 98 98 23600
306 0.72 Active Active 134 142 3.78 agent of 98 agent of 98 80 80
7680 100 0.68 80 Active 0 20 1.5 agent of 80 Active Active 36900
1000 2.16 agent of 80 agent of 80 7 7 19700 16.2 1.1
Example 9. Targeted Delivery of Active Agents by Non-Systemic
Conjugates
[1087] To confirm that the active agent component of the conjugate
is released in the gastrointestinal tract, a pharmacokinetic study
was conducted in male C57B3L/6 mice examining conjugate and active
agent of concentrations in both the colon contents and plasma after
oral dose of conjugate. Conjugate and active agent of were
quantified by LCMS at 0 (pre-dose), 0.25, 0.5, 1, 2, 4, 6, 8, 12,
and 24 hours post-dose of compound. The results are shown in Table
6 below. The active agents of the conjugate was at least
100,000-fold more concentrated in the colon versus the plasma,
suggesting non-systemic, gut restrictive delivery of the active
agent by the conjugate.
TABLE-US-00013 TABLE 6 Colon Plasma Compound Compound C.sub.max
C.sub.max Dosed Measured (.mu.mol/L) (.mu.mol/L) 78 78 0.07 Not
detected 78 Active agent 7077 0.09 of 78 79 79 Not 0.005 detected
79 Active agent 4081 0.16 of 79 98 98 21.83 1.3 98 Active agent
5522 0.40 of 98 80 80 0.13 Not detected 80 Active agent 4759 Not of
80 detected
Example 10: In Vitro Stability Studies of Conjugates
Assay 1. Stability of Conjugates in Simulated Gastric Fluid
(SGF)
[1088] This assay was used to assess the stability of a conjugate
in a stomach.
[1089] Medium was prepared by dissolving 2 g of sodium chloride in
0.6 L in ultrapure water (MilliQ.RTM., Millipore Sigma, Darmstadt,
Germany). The pH was adjusted to 1.6 with 1N hydrochloric acid, and
the volume was then adjusted to 1 L with purified water.
[1090] 60 mg FaSSIF powder (Biorelevant.TM., London, UK) were
dissolved in 500 mL buffer (above). Pepsin was added (0.1 mg/mL)
(Millipore Sigma, Darmstadt, Germany), and the solution was
stirred. The resulting SGF media were used fresh for each
experiment.
[1091] Test compounds were dissolved in DMSO stock to 1 mM. An
aliquot of the DMSO stock solution was removed and diluted in the
SGF Media in 15 mL falcon tubes to generate a total compound
concentration of 1 .mu.M. A 1 mL aliquot was immediately removed
and diluted once with 1 volume of acetonitrile for T0 timepoint.
The mixture was sealed and mixed at 37.degree. C. in an incubator.
Aliquots (1 mL) were removed at regular intervals and immediately
quenched by the addition of 1 volume of acetonitrile. The resulting
samples were analyzed by LCMS to determine degradation rates in
SGF. The results for percent of compound remaining at 1 hour are
shown in Table 7.
Assay 2. Stability of Conjugates in Simulated Intestinal Fluid
(SIF).
[1092] This assay was used to assess the stability of a conjugate
in a small intestine.
[1093] Phosphate buffer was prepared by dissolving 0.42 g of sodium
hydroxide pellets and 3.95 g of monobasic sodium phosphate
monohydrate and 6.19 g of sodium chloride in ultrapure water
(MilliQ.RTM., Millipore Sigma, Darmstadt, Germany). The pH was
adjusted to 6.7 using aq. HCl and aq. NaOH, as necessary, and the
solution was diluted with ultrapure water to produce 1 L of the pH
6.7 buffer.
[1094] 112 mg FaSSIF powder (Biorelevant.TM., London, UK) was
dissolved in 50 mL of the pH 6.7 buffer. 2 to 3 mL of the resulting
solution were then added to 500 mg pancreatin (Millipore Sigma,
Darmstadt, Germany). The resulting mixture was agitated by finger
tapping the vessel containing the mixture until milky suspension
formed. At this time, the remainder of the 50 mL FaSSiF/pH 6.7
buffer solution was added. The resulting suspension was flipped
upside down 10 times to produce SIF, which was used fresh.
[1095] Test compounds were dissolved in DMSO stock to 1 mM. An
aliquot of the DMSO stock solution was removed and diluted in the
SIF media in 15 mL falcon tubes to produce a mixture with a tested
compound concentration of 1 .mu.M. A 1 mL aliquot was immediately
removed and diluted once with 1 volume of acetonitrile for T0
timepoint. The mixture was sealed and agitated at 37.degree. C. in
an incubator. Aliquots (1 mL) were removed at regular intervals and
immediately quenched by the addition of 1 volume of acetonitrile.
The resulting samples were analyzed by LCMS to determine
degradation rates. The results for percent of compound remaining at
4 hours are shown in Table 7.
Assay 3. In Vitro Colonic Material Stability Assay.
[1096] This assay was used to assess the stability of a conjugate
in a large intestine.
[1097] All experiments were performed in an anaerobic chamber
containing 90% nitrogen, 5% hydrogen and 5% carbon dioxide. Colonic
material was resuspended as a slurry (15% w/v final concentration)
in pre-reduced, anaerobically sterilized dilution blanks (Anaerobe
Systems AS-908). The colonic material was then inoculated into 96
well plates containing YCFAC media (Anaerobe Systems AS-680, 6.7
.mu.L slurry into 1 mL total media). A compound was added to each
individual well to reach a final analyte concentration of 1 .mu.M,
and the material was mixed by pipetting. Each sample was removed
after set timepoints (0, 120, 240, 480, 1440, 2880 minutes after
initiation of the assay), quenched with acetonitrile containing
internal standard, and analyzed by LCMS. Results are shown in Table
7.
TABLE-US-00014 TABLE 7 Assay 3- Colonic Assay 1- Assay 2- Material
FaSSGF FaSSIF Stability Stability: Stability: Assay % % Remaining %
Remaining Remaining Compound at 1 hr at 4 hr at 24 hr 1 84.79 30.79
80 7 85.06 121.01 56 7 100.35 93.47 51 19 101.36 11.06 2.5 19 85.52
1.52 0.18 303 60.49 0.03 0.16 304 114.28 13.44 0.94 70 98.36 28.06
47 305 92.73 0.25 0.04 72 102.71 -- 110 73 107.49 62.73 44 74 107.3
121.56 99 8 105.93 113.28 17 315 109.96 120.35 68 310 97.99 115.11
55 309 97.94 94.72 16 311 104.43 97.27 33 80 145.32 99.34 25 86 --
-- 56 317 96.24 82.34
Example 11: Caco-2 Permeability Assay
[1098] Preparation of Caco-2 Cells: 50 .mu.L and 25 mL of cell
culture medium were added to each well of the Transwell insert and
reservoir, respectively. The HTS transwell plates were incubated at
37.degree. C., 5% CO.sub.2 for 1 hour before cell seeding. Caco-2
cells were diluted to 6.86.times.105 cells/mL with culture medium
and 50 .mu.L of cell suspension were dispensed into the filter well
of the 96-well HTS Transwell plate. Cells were cultivated for 14-18
days in a cell culture incubator at 37.degree. C., 5% CO.sub.2, 95%
relative humidity. Cell culture medium was replaced every other
day, beginning no later than 24 hours after initial plating.
[1099] Assessment of Cell Monolayer Integrity: Medium was removed
from the reservoir and each Transwell insert and replaced with
prewarmed fresh culture medium. Transepithelial electrical
resistance (TEER) across the monolayer was measured using Millicell
Epithelial Volt-Ohm measuring system (Millipore, USA). The Plate
was returned to the incubator once the measurement was done. The
TEER value was calculated according to the following equation:
TEER measurement (ohms).times.Area of membrane (cm2)=TEER value
(ohmcm2)
TEER value should be greater than 230 ohmcm2, which indicates the
well-qualified Caco-2 monolayer.
[1100] Preparation of Solutions: 2 mM stock solutions in DMSO of
control compounds were prepared and diluted with HBSS (10 mM HEPES,
pH 7.4) to get 10 .mu.M working solution. 0.2 mM stock solutions of
test compounds in DMSO were prepared and diluted with HBSS (10 mM
HEPES, pH 7.4 with 0.5% BSA) to get 1 .mu.M working solution.
Metoprolol, erythromycin and cimetidine were used as control
compounds.
[1101] Performing the Drug Transport Assay.
[1102] The Caco-2 plate was removed from the incubator. The
monolayer was washed twice with pre-warmed HBSS (10 mM HEPES, pH
7.4). The plate was incubated at 37.degree. C. for 30 minutes. To
determine the rate of drug transport in the apical to basolateral
direction, 125 .mu.L of the working solution was added to the
Transwell insert (apical compartment). A 50 .mu.L sample was
transferred immediately from the apical compartment to 200 .mu.L of
acetonitrile containing IS (100 nM alprazolam, 200 nM Caffeine and
100 nM tolbutamide) in a new 96-well plate as the initial donor
sample (A-B) and it was vortexed at 1000 rpm for 10 minutes. The
wells in the receiver plate (basolateral compartment) were filled
with 235 .mu.L of transport buffer. To determine the rate of drug
transport in the basolateral to apical direction, 285 .mu.L of the
working solution were added to the receiver plate wells
(basolateral compartment). A 50 .mu.L sample was transferred
immediately from the basolateral compartment to 200 .mu.L of
acetonitrile containing IS (100 nM alprazolam, 200 nM Caffeine and
100 nM tolbutamide) in a new 96-well plate as the initial donor
sample (B-A) and it was vortexed at 1000 rpm for 10 minutes. The
Transwell insert (apical compartment) was filled with 75 .mu.L of
transport buffer. The apical to basolateral direction and the
basolateral to apical direction need to be done at the same time.
The plates were incubated at 37.degree. C. for 2 hours. At the end
of the incubation, 50 .mu.L samples from donor sides (apical
compartment for Ap.fwdarw.Bl flux, and basolateral compartment for
B1.fwdarw.Ap) and receiver sides (basolateral compartment for
Ap.fwdarw.Bl flux, and apical compartment for B1.fwdarw.Ap) were
transferred to wells of a new 96-well plate, followed by the
addition of 4 volume of acetonitrile containing IS (100 nM
alprazolam, 200 nM Caffeine and 100 nM tolbutamide). Samples were
vortexed for 10 minutes, 50 .mu.L samples were transferred to wells
of a new 96-well plate, followed by the addition of 50 .mu.L Hepes
and 200 .mu.L IS. All samples were vortexed for 10 minutes, and
then centrifuged at 3,220 g for 40 minutes. An aliquot of 150 .mu.L
of the supernatant was mixed with an appropriate volume of
ultra-pure water before LC-MS/MS analysis.
[1103] Data Analysis
[1104] All calculations were carried out using Microsoft Excel.
Peak areas were determined from extracted ion chromatograms.
Lucifer yellow leakage of monolayer can be calculated using the
following equation:
LY .times. .times. Leakage = ( I acceptor .times. 0.3 I acceptor
.times. 0.3 + I donor .times. 0.1 ) .times. 100 .times. %
##EQU00001##
where I.sub.acceptor is the fluorescence intensity in the acceptor
well (0.3 mL), and I.sub.donor is the fluorescence intensity in the
donor well (0.1 mL) and expressed as % leakage. Lucifer yellow
percentage amount transported values should be less than 1.5%.
However, if the lucifer yellow percentage amount transported value
for a particular transwell is higher than 1.5 but the determined
digoxin P.sub.app in that transwell is qualitatively similar to
that determined in the replicate transwells then, based upon the
scientific judgement of the responsible scientist, the monolayer is
considered acceptable.
[1105] Apparent permeability (P.sub.app) can be calculated for drug
transport assays using the following equation:
P app = dQ / dt A .times. D o ##EQU00002##
where:
[1106] P.sub.app is apparent permeability
(cm/s.times.10.sup.-6);
[1107] dQ/dt is the rate of drug transport (pmol/second);
[1108] A is the surface area of the membrane (cm.sup.2); and
[1109] D.sub.0 is the initial donor concentration (nM;
pmol/cm.sup.3).
[1110] Efflux ratio can be determined using the following
equation:
Efflux .times. .times. Ratio = P app .function. ( B - A ) P app
.function. ( A - B ) ##EQU00003##
where
[1111] P.sub.app (B-A) indicates the apparent permeability
coefficient in basolateral to apical direction, and
[1112] P.sub.app (A-B) indicates the apparent permeability
coefficient in apical to basolateral direction. The results are
shown in Table 8 below.
TABLE-US-00015 TABLE 8 Compound Papp A-B Papp B-A No. (*10.sup.-6
cm/s) (*10.sup.-6 cm/s) 159 0.894 1.43 77 0.218 0.337 79 0.214
0.255 80 0.28 0.313 J60 11.07 6.23 107 26.8 31.1 7 0.1 0.15 111 0.8
0.8 109 0.6 0.5 315 0.3 0.3 125 0.466 0.482 310 0.206 0.23
OTHER EMBODIMENTS
[1113] Various modifications and variations of the described
disclosure will be apparent to those skilled in the art without
departing from the scope and spirit of the disclosure. Although the
disclosure has been described in connection with specific
embodiments, it should be understood that the disclosure as claimed
should not be unduly limited to such specific embodiments. Indeed,
various modifications of the described modes for carrying out the
disclosure that are obvious to those skilled in the art are
intended to be within the scope of the disclosure.
[1114] Other embodiments are in the claims.
* * * * *