U.S. patent application number 14/132898 was filed with the patent office on 2014-04-17 for methods and compositions for treating amyloid-related diseases.
This patent application is currently assigned to BHI LIMITED PARTNERSHIP. The applicant listed for this patent is Francine Gervais, Xianqi Kong, David Migneault, Isabelle Valade, Xinfu Wu. Invention is credited to Francine Gervais, Xianqi Kong, David Migneault, Isabelle Valade, Xinfu Wu.
Application Number | 20140107027 14/132898 |
Document ID | / |
Family ID | 46325164 |
Filed Date | 2014-04-17 |
United States Patent
Application |
20140107027 |
Kind Code |
A1 |
Kong; Xianqi ; et
al. |
April 17, 2014 |
METHODS AND COMPOSITIONS FOR TREATING AMYLOID-RELATED DISEASES
Abstract
Methods, compounds, pharmaceutical compositions and kits are
described for treating or preventing amyloid-related disease.
Inventors: |
Kong; Xianqi;
(Dollard-des-Ormeaux, CA) ; Migneault; David;
(Laval, CA) ; Valade; Isabelle; (Ste Marthe Sur Le
Lac, CA) ; Wu; Xinfu; (Laval, CA) ; Gervais;
Francine; (L'lle Bizard, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Kong; Xianqi
Migneault; David
Valade; Isabelle
Wu; Xinfu
Gervais; Francine |
Dollard-des-Ormeaux
Laval
Ste Marthe Sur Le Lac
Laval
L'lle Bizard |
|
CA
CA
CA
CA
CA |
|
|
Assignee: |
BHI LIMITED PARTNERSHIP
Laval
CA
|
Family ID: |
46325164 |
Appl. No.: |
14/132898 |
Filed: |
December 18, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12613412 |
Nov 5, 2009 |
8642801 |
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14132898 |
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11316694 |
Dec 21, 2005 |
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12613412 |
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10871514 |
Jun 18, 2004 |
7414076 |
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11316694 |
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60638819 |
Dec 22, 2004 |
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60512047 |
Oct 17, 2003 |
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60480906 |
Jun 23, 2003 |
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Current U.S.
Class: |
514/6.9 ;
514/17.7; 514/17.8; 514/21.9; 514/255.03; 514/305; 514/381;
514/419; 514/550; 514/553; 514/562; 514/576; 514/577; 514/578;
530/331; 544/398; 546/133; 548/252; 548/496; 562/100; 562/104;
562/105; 562/43 |
Current CPC
Class: |
C07C 2601/08 20170501;
C07D 209/44 20130101; C07D 295/088 20130101; C07K 5/0812 20130101;
C07C 307/02 20130101; C07D 211/46 20130101; C07C 309/69 20130101;
C07C 311/32 20130101; C07C 2602/08 20170501; C07C 309/15 20130101;
C07C 335/12 20130101; C07D 209/20 20130101; C07C 309/13 20130101;
C07C 381/02 20130101; C07C 2601/18 20170501; C07C 2602/42 20170501;
C07C 309/14 20130101; C07D 403/04 20130101; A61P 9/00 20180101;
C07C 309/23 20130101; C07C 309/46 20130101; C07C 2601/02 20170501;
C07D 209/14 20130101; C07D 211/70 20130101; C07D 235/28 20130101;
C07C 309/19 20130101; C07D 209/48 20130101; C07D 217/10 20130101;
C07D 401/04 20130101; C07D 317/50 20130101; C07D 453/02 20130101;
C07C 2601/04 20170501; C07D 257/04 20130101; C07D 209/08 20130101;
C07C 2601/10 20170501; C07D 217/04 20130101; C07C 2601/14 20170501;
C07F 9/2458 20130101; C07C 2602/10 20170501; C07C 2603/74 20170501;
C07D 471/04 20130101; C07C 311/46 20130101; A61P 25/28 20180101;
C07C 323/25 20130101; C07C 335/32 20130101; C07D 211/64 20130101;
C07D 403/06 20130101; C07F 9/1651 20130101; A61P 17/00 20180101;
C07C 323/58 20130101; C07D 209/18 20130101; C07D 295/084
20130101 |
Class at
Publication: |
514/6.9 ;
562/100; 514/553; 562/104; 514/578; 562/43; 514/576; 514/562;
514/577; 562/105; 514/550; 548/496; 514/419; 548/252; 514/381;
546/133; 514/305; 544/398; 514/255.03; 530/331; 514/21.9; 514/17.8;
514/17.7 |
International
Class: |
C07C 309/14 20060101
C07C309/14; C07K 5/087 20060101 C07K005/087; C07D 453/02 20060101
C07D453/02; C07D 295/088 20060101 C07D295/088; C07D 209/18 20060101
C07D209/18; C07D 257/04 20060101 C07D257/04 |
Claims
1-232. (canceled)
233. A compound of Formula I or Formula II, or a pharmaceutically
acceptable salt, ester or prodrug thereof: ##STR00685## wherein in
Formula I: R.sup.1 is a substituted or unsubstituted cycloalkyl,
heterocyclic, aryl, arylcycloalkyl, bicyclic or tricyclic ring, a
bicyclic or tricyclic fused ring group, or a substituted or
unsubstituted C.sub.2-C.sub.10 alkyl group; R.sup.2 is hydrogen or
alkyl; Y is SO.sub.3.sup.-X.sup.+; X.sup.+ is hydrogen or a
cationic group; and each of L.sup.1 and L.sup.2 is independently a
substituted or unsubstituted C.sub.1-C.sub.5 alkyl group or absent,
provided that when R.sup.1 is alkyl, L.sup.1 is absent; provided
that when R.sup.2 is benzyl, L.sup.1 is methylene, L.sup.2 is
--(CH.sub.2).sub.3--, Y is SO.sub.3.sup.-X.sup.+, then R.sup.1 is
not phenyl; provided that when R.sup.2 is hydrogen, L.sup.2 is
--(CH.sub.2).sub.3--, L.sup.1 is methylene, Y is
SO.sub.3.sup.-X.sup.+, then R.sup.1 is not 1,3-benzodioxol-5-yl or
3,4-methoxybenzyl; and provided that when R.sup.2 is hydrogen,
L.sup.2 is --(CH.sub.2).sub.3--, L.sup.1 is absent, Y is
SO.sub.3.sup.-X.sup.+, then R.sup.1 is not t-butyl, isobutyl,
pentyl, n-heptyl, n-octyl, n-nonyl, cyclohexyl, isopropyl, isoamyl,
1-hydroxy-2-propyl, 3,5-dimethyl-1-adamantyl, 1-hydroxy-2-pentyl,
3-methyl butyric acid, -4-methyl-pentanoic acid methyl ester, or
2,2-diphenyl-ethyl; and wherein in Formula II: R.sup.1 is a
substituted or unsubstituted cyclic, bicyclic, tricyclic, or
benzoheterocyclic group or a substituted or unsubstituted
C.sub.2-C.sub.10 alkyl group; R.sup.2 is hydrogen, alkyl,
mercaptoalkyl, alkenyl, alkynyl, cycloalkyl, aryl, arylalkyl,
thiazolyl, triazolyl, imidazolyl, benzothiazolyl, benzoimidazolyl,
or linked to R.sup.1 to form a heterocycle; Y is
SO.sub.3.sup.-X.sup.+, OSO.sub.3.sup.-X.sup.+, or
SSO.sub.3.sup.-X.sup.+; X.sup.+ is hydrogen, a cationic group, or
an ester forming moiety; m is 0; n is 1, 2, 3, or 4; L is
substituted or unsubstituted C.sub.1-C.sub.3 alkyl group or absent,
provided that when R.sup.1 is alkyl, L is absent.
234. The compound of claim 233, wherein R.sup.2 is hydrogen.
235. The compound of claim 233, wherein R.sup.1 is straight chain
alkyl.
236. The compound of claim 233, wherein R.sup.1 is t-butyl.
237. The compound of claim 233, wherein R.sup.1 is carbocyclic.
238. The compound of claim 233, wherein R.sup.1 is C.sub.7-C.sub.10
bicycloalkyl.
239. The compound of claim 233, wherein said bicyclic fused ring
group is indolyl.
240. The compound of claim 233, wherein L.sup.1 is CH.sub.2CH.sub.2
or absent.
241. The compound of claim 233, wherein said compound is:
##STR00686## ##STR00687## ##STR00688## ##STR00689## ##STR00690##
##STR00691## ##STR00692## ##STR00693## ##STR00694## ##STR00695##
##STR00696## ##STR00697## ##STR00698## ##STR00699## ##STR00700##
##STR00701## ##STR00702## ##STR00703## ##STR00704## ##STR00705##
##STR00706## ##STR00707## ##STR00708## ##STR00709## ##STR00710##
##STR00711## ##STR00712## ##STR00713## ##STR00714## ##STR00715##
##STR00716## ##STR00717## ##STR00718## ##STR00719## ##STR00720##
##STR00721## ##STR00722## ##STR00723## ##STR00724## ##STR00725##
##STR00726## ##STR00727## ##STR00728## ##STR00729## ##STR00730##
##STR00731## ##STR00732## ##STR00733## or a pharmaceutically
acceptable salt, ester, or prodrug thereof.
242. The compound of claim 233, wherein said compound is a compound
of Formula III: ##STR00734## wherein: A is nitrogen or oxygen;
R.sup.11 is hydrogen, salt-forming cation, ester forming group, or
--(CH.sub.2).sub.X-Q; Q is hydrogen, thiazolyl, triazolyl,
imidazolyl, benzothiazolyl, or benzoimidazolyl; x is 0, 1, 2, 3, or
4; n is 1, 2, 3, or 4; R.sup.3, R.sup.3a, R.sup.4, R.sup.4a,
R.sup.5, R.sup.5a, R.sup.6, R.sup.6a, R.sup.7 and R.sup.7a are each
independently hydrogen, alkyl, mercaptoalkyl, alkenyl, alkynyl,
cycloalkyl, aryl, alkylcarbonyl, arylcarbonyl, alkoxycarbonyl,
cyano, halogen, amino, tetrazolyl, or two R groups on adjacent ring
atoms taken together with the ring atoms form a double bond,
provided that one of R.sup.3, R.sup.3a, R.sup.5, R.sup.5a, R.sup.6,
and R.sup.6a is a moiety of Formula IIIa: ##STR00735## wherein: m
is 0, 1, 2, 3, or 4; R.sup.A, R.sup.B, R.sup.C, R.sup.D, and
R.sup.E are independently selected from a group of hydrogen,
halogen, hydroxyl, alkyl, alkoxyl, halogenated alkyl,
mercaptoalkyl, alkenyl, alkynyl, cycloalkyl, aryl, cyano,
thiazolyl, triazolyl, imidazolyl, tetrazolyl, benzothiazolyl, and
benzoimidazolyl; and pharmaceutically acceptable salts, esters, and
prodrugs thereof, provided that said compound is not
3-(4-phenyl-1,2,3,6-tetrahydro-1-pyridyl)-1-propanesulfonic acid,
and provided that when R.sup.3a, R.sup.4, R.sup.4a, R.sup.5,
R.sup.5a, R.sup.6, R.sup.6a, R.sup.7 and R.sup.7a are hydrogen, and
R.sup.3 is not a moiety of Formula IIIa.
243. The compound of claim 242, wherein said compound is:
##STR00736## ##STR00737## or a pharmaceutically acceptable salt,
ester, or prodrug thereof.
244. The compound of claim 233, wherein said compound is a compound
of Formula IV: ##STR00738## wherein: A is nitrogen or oxygen;
R.sup.11 is hydrogen, salt-forming cation, ester forming group, or
--(CH.sub.2).sub.x-Q; Q is hydrogen, thiazolyl, triazolyl,
imidazolyl, benzothiazolyl, or benzoimidazolyl; x is 0, 1, 2, 3, or
4; n is 1, 2, 3, or 4; R.sup.4, R.sup.4a, R.sup.5, R.sup.5a,
R.sup.6, R.sup.6a, R.sup.7, and R.sup.7a are each independently
hydrogen, alkyl, mercaptoalkyl, alkenyl, alkynyl, cycloalkyl, aryl,
alkylcarbonyl, arylcarbonyl, alkoxycarbonyl, cyano, halogen, amino,
tetrazolyl, R.sup.4 and R.sup.5 taken together, with the ring atoms
they are attached to, form a double bond, or R.sup.6 and R.sup.7
taken together, with the ring atoms they are attached to, form a
double bond; m is 0, 1, 2, 3, or 4; R.sup.8, R.sup.9, R.sup.10,
R.sup.11, and R.sup.12 are independently selected from a group of
hydrogen, halogen, hydroxyl, alkyl, alkoxyl, halogenated alkyl,
mercaptoalkyl, alkenyl, alkynyl, cycloalkyl, aryl, cyano,
thiazolyl, triazolyl, imidazolyl, tetrazolyl, benzothiazolyl, and
benzoimidazolyl; or pharmaceutically acceptable salts, esters, and
prodrugs thereof.
245. The compound of claim 244, wherein said compound is:
##STR00739## or pharmaceutically acceptable salts, esters, or
prodrugs thereof.
246. A compound of the Formula VI: ##STR00740## wherein: n is 1, 2,
3, 4, 5, 6, 7, 8, 9, or 10; A is oxygen or nitrogen; R.sup.11 is
hydrogen, salt-forming cation, ester forming group,
--(CH.sub.2).sub.x-Q, or when A is nitrogen, A and R.sup.11 taken
together may be the residue of a natural or unnatural amino acid or
a salt or ester thereof; Q is hydrogen, thiazolyl, triazolyl,
imidazolyl, benzothiazolyl, or benzoimidazolyl; x is 0, 1, 2, 3, or
4; R.sup.19 is hydrogen, alkyl or aryl; Y.sup.1 is oxygen, sulfur,
or nitrogen; Y.sup.2 is carbon, nitrogen, or oxygen; R.sup.20 is
hydrogen, alkyl, amino, mercaptoalkyl, alkenyl, alkynyl,
cycloalkyl, aryl, arylalkyl, thiazolyl, triazolyl, tetrazolyl,
imidazolyl, benzothiazolyl, or benzoimidazolyl; R.sup.21 is
hydrogen, alkyl, mercaptoalkyl, alkenyl, alkynyl, cycloalkyl, aryl,
arylalkyl, thiazolyl, triazolyl, tetrazolyl, imidazolyl,
benzothiazolyl, benzoimidazolyl, or absent if Y.sup.2 is oxygen;
R.sup.22 is hydrogen, alkyl, mercaptoalkyl, alkenyl, alkynyl,
cycloalkyl, aryl, arylalkyl, thiazolyl, triazolyl, tetrazolyl,
imidazolyl, benzothiazolyl, benzoimidazolyl; or R.sup.22 is
hydrogen, hydroxyl, alkoxy or aryloxy if Y.sup.1 is nitrogen; or
R.sup.22 is absent if Y.sup.1 is oxygen or sulfur; or R.sup.22 and
R.sup.21 may be linked to form a cyclic moiety if Y.sup.1 is
nitrogen; R.sup.23 is hydrogen, alkyl, amino, mercaptoalkyl,
alkenyl, alkynyl, cycloalkyl, aryl, arylalkyl, thiazolyl,
triazolyl, tetrazolyl, imidazolyl, benzothiazolyl, or
benzoimidazolyl, or absent if Y.sup.2 is nitrogen or oxygen; or
pharmaceutically acceptable salts, esters, or prodrugs thereof,
provided that when n is 3, Y.sup.1 is oxygen, Y.sup.2 is oxygen,
R.sup.21 is benzyl, A is oxygen, R.sup.19 is not hydrogen; and
provided that when n is 3, Y.sup.1 is oxygen, Y.sup.2 is carbon,
each of R.sup.20, R.sup.21, and R.sup.23 is methyl, R.sup.19 is not
hydrogen, and provided that R.sup.21 and R.sup.22 are not linked to
form an aryl ring.
247. The compound of claim 246, wherein said compound is selected
from the group consisting of: ##STR00741## ##STR00742## and
pharmaceutically acceptable salts, esters, and prodrugs
thereof.
248. The compound of claim 233, wherein said compound is a compound
of Formula VII: ##STR00743## wherein: n is 2, 3, or 4; A is oxygen
or nitrogen; R.sup.11 is hydrogen, salt-forming cation, ester
forming group, --(CH.sub.2).sub.x-Q, or when A is nitrogen, A and
R.sup.11 taken together may be the residue of a natural or
unnatural amino acid or a salt or ester thereof; Q is hydrogen,
thiazolyl, triazolyl, imidazolyl, benzothiazolyl, or
benzoimidazolyl; x is 0, 1, 2, 3, or 4; G is a direct bond or
oxygen, nitrogen, or sulfur; z is 0, 1, 2, 3, 4, or 5; m is 0 or 1;
R.sup.24 is selected from a group consisting of hydrogen, alkyl,
mercaptoalkyl, alkenyl, alkynyl, cycloalkyl, aryl, arylalkyl,
thiazolyl, triazolyl, imidazolyl, benzothiazolyl, and
benzoimidazolyl; each R.sup.25 is independently selected from
hydrogen, halogen, cyano, hydroxyl, alkoxy, thiol, amino, nitro,
alkyl, aryl, carbocyclic, or heterocyclic; and pharmaceutically
acceptable salts, esters, and prodrugs thereof.
249. The compound of claim 248, wherein said compound is selected
from the group consisting of: ##STR00744## ##STR00745## and
pharmaceutically acceptable salts, esters, and prodrugs
thereof.
250. A compound of the formula: ##STR00746## ##STR00747## and
pharmaceutically acceptable salts and esters thereof.
251. A method of treating or preventing an amyloid-related disease
in a subject comprising administering to a subject in need thereof
a compound of claim 233 in an amount effective to treat or prevent
an amyloid related disease.
252. The method according to claim 251, wherein said
amyloid-related disease is Alzheimer's disease, cerebral amyloid
angiopathy, inclusion body myositis, macular degeneration, MCI, or
Down's syndrome.
253. The method according to claim 251, wherein amyloid fibril
formation or deposition, neurodegeneration, microglial inflammatory
response, or cellular toxicity is reduced or inhibited upon
administration of said compound.
254. The method according to claim 251, wherein said
amyloid-related disease is diabetes, AA amyloidosis, AL
amyloidosis, or hemodialysis related amyloidosis
(.beta..sub.2M).
255. The method of claim 251, wherein said subject has Alzheimer's
disease, Mild Cognitive Impairment, or cerebral amyloid angiopathy,
and stabilization of cognitive function, prevention of a further
decrease in cognitive function, or prevention, slowing, or stopping
of disease progression occurs in said patient upon
administration.
256. A method for inhibiting amyloid deposition in a subject
comprising administering to a subject an effective amount of a
therapeutic compound of claim 233 or a pharmaceutically acceptable
salt thereof.
257. A pharmaceutical composition comprising a compound according
to claim 233 together with a pharmaceutically acceptable
carrier.
258. The method of claim 251, wherein said amyloid-related disease
is familial amyloid polyneuropathy (FAP), senile systemic
amyloidosis, Tenosynovium, familial amyloidosis, Ostertag-type,
non-neuropathic amyloidosis, cranial neuropathy, hereditary
cerebral hemorrhage, familial dementia, chronic dialysis, familial
Creutzfeldt-Jakob disease; Gerstmann-Straussler-Scheinker syndrome,
hereditary spongiform encephalopathies, prion diseases, familial
Mediterranean fever, Muckle-Well's syndrome, nephropathy, deafness,
urticaria, limb pain, cardiomyopathy, cutaneous deposits, multiple
myeloma, benign monoclonal gammopathy, maccoglobulinaemia, myeloma
associated amyloidosis, medullary carcinomas of the thyroid,
isolated atrial amyloid, or diabetes.
259. The method of claim 256, wherein the amyloid is amyloid-.beta.
protein, IAPP protein, AA amyloid protein, AL amyloid protein,
amyloid .lamda., amyloid .kappa., amyloid .kappa.IV, amyloid
.gamma., or amyloid .gamma.1.
Description
RELATED APPLICATIONS
[0001] This application claims priority to U.S. Provisional Patent
Application 60/638,819, filed on Dec. 22, 2004. This application is
also a continuation-in-part of U.S. patent application Ser. No.
10/871,514, filed Jun. 18, 2004, which claims priority to U.S.
patent application Ser. No. 10/871,365 filed Jun. 18, 2004, U.S.
Provisional Patent Application No. 60/512,047, filed Oct. 17, 2003,
and U.S. Provisional Patent Application No. 60/480,906, filed Jun.
23, 2003, all entitled Methods and Compositions for Treating
Amyloid-Related Diseases. This application is also related to U.S.
Provisional Application Ser. No. 60/638,636, filed Dec. 22,
2004.
[0002] This application is also related to U.S. Provisional Patent
Application No. 60/512,017, filed Oct. 17, 2003, U.S. Provisional
Patent Application No. 60/480,918, filed Jun. 23, 2003, and U.S.
patent application Ser. No. 10/871,613, filed Jun. 18, 2004, all
entitled Methods for Treating Protein Aggregation Disorders.
[0003] This application is related to U.S. Provisional Patent
Application No. 60/512,116, filed Oct. 17, 2003, U.S. Provisional
Patent Application No. 60/480,984, filed Jun. 23, 2003, and U.S.
application Ser. No. 10/871,549, filed Jun. 18, 2004, all entitled
Pharmaceutical Formulations of Amyloid-Inhibiting Compounds.
[0004] This application is related to U.S. Provisional Patent
Application No. 60/436,379, filed Dec. 24, 2002, U.S. Provisional
Patent Application No. 60/482,214, filed Jun. 23, 2003, entitled
Combination Therapy for the Treatment of Alzheimer's Disease, U.S.
patent application Ser. No. 10/746,138, filed Dec. 24, 2003,
International Patent Application No. PCT/CA2003/002011, filed Dec.
24, 2003, and U.S. patent application Ser. No. 10/871,537, filed
Jun. 18, 2004, entitled Therapeutic Formulations for the Treatment
of Beta-Amyloid Related Diseases.
[0005] This application is related to U.S. Provisional Patent
Application No. 60/512,135, filed Oct. 17, 2003, U.S. Provisional
Patent Application No. 60/482,058, filed Jun. 23, 2003, both
entitled Synthetic Process for Preparing Compounds for Treating
Amyloidosis, and U.S. patent application Ser. No. 10/871,543, filed
Jun. 18, 2004, entitled Improved Pharmaceutical Drug. Candidates
and Method for Preparation Thereof.
[0006] This application is related to U.S. Provisional Patent
Application Ser. No. 60/512,018, filed on Oct. 17, 2003 and U.S.
Provisional Patent Application Ser. No. 60/480,928, filed on Jun.
23, 2003, and U.S. application Ser. No. 10/871,512, filed Jun. 18,
2004, all entitled Methods and Compositions for Treating Amyloid-
and Epileptogenesis-Associated Diseases.
[0007] This application is also related to Method for Treating
Amyloidosis, U.S. patent application Ser. No. 08/463,548, now U.S.
Pat. No. 5,972,328.
[0008] The entire contents of each of these patent applications and
patents are hereby expressly incorporated herein by reference
including without limitation the specification, claims, and
abstract, as well as any figures, tables, or drawings thereof.
BACKGROUND
[0009] Amyloidosis refers to a pathological condition characterized
by the presence of amyloid fibrils. Amyloid is a generic term
referring to a group of diverse but specific protein deposits
(intracellular or extracellular) which are seen in a number of
different diseases. Though diverse in their occurrence, all amyloid
deposits have common morphologic properties, stain with specific
dyes (e.g., Congo red), and have a characteristic red-green
birefringent appearance in polarized light after staining. They
also share common ultrastructural features and common X-ray
diffraction and infrared spectra.
[0010] Amyloid-related diseases can either be restricted to one
organ or spread to several organs. The first instance is referred
to as "localized amyloidosis" while the second is referred to as
"systemic amyloidosis."
[0011] Some amyloid diseases can be idiopathic, but most of these
diseases appear as a complication of a previously existing
disorder. For example, primary amyloidosis (AL amyloid) can appear
without any other pathology or can follow plasma cell dyscrasia or
multiple myeloma.
[0012] Secondary amyloidosis is usually seen associated with
chronic infection (such as tuberculosis) or chronic inflammation
(such as rheumatoid arthritis). A familial form of secondary
amyloidosis is also seen in other types of familial amyloidosis,
e.g., Familial Mediterranean Fever (FMF). This familial type of
amyloidosis is genetically inherited and is found in specific
population groups. In both primary and secondary amyloidosis,
deposits are found in several organs and are thus considered
systemic amyloid diseases.
[0013] "Localized amyloidoses" are those that tend to involve a
single organ system. Different amyloids are also characterized by
the type of protein present in the deposit. For example,
neurodegenerative diseases such as scrapie, bovine spongiform
encephalitis, Creutzfeldt-Jakob disease, and the like are
characterized by the appearance and accumulation of a
protease-resistant form of a prion protein (referred to as AScr or
PrP-27) in the central nervous system. Similarly, Alzheimer's
disease, another neurodegenerative disorder, is characterized by
neuritic plaques and neurofibrillary tangles. In this case, the
amyloid plaques found in the parenchyma and the blood vessel is
formed by the deposition of fibrillar A.beta. amyloid protein.
Other diseases such as adult-onset diabetes (type II diabetes) are
characterized by the localized accumulation of amyloid fibrils in
the pancreas.
[0014] Once these amyloids have formed, there is no known, widely
accepted therapy or treatment which significantly dissolves amyloid
deposits in situ, prevents further amyloid deposition or prevents
the initiation of amyloid deposition.
[0015] Each amyloidogenic protein has the ability to undergo a
conformational change and to organize into .beta.-sheets and form
insoluble fibrils which may be deposited extracellularly or
intracellularly. Each amyloidogenic protein, although different in
amino acid sequence, has the same property of forming fibrils and
binding to other elements such as proteoglycan, amyloid P and
complement component. Moreover, each amyloidogenic protein has
amino acid sequences which, although different, show similarities
such as regions with the ability to bind to the glycosaminoglycan
(GAG) portion of proteoglycan (referred to as the GAG binding site)
as well as other regions which promote .beta.-sheet formation.
Proteoglycans are macromolecules of various sizes and structures
that are distributed almost everywhere in the body. They can be
found in the intracellular compartment, on the surface of cells,
and as part of the extracellular matrix. The basic structure of all
proteoglycans is comprised of a core protein and at least one, but
frequently more, polysaccharide chains (GAGs) attached to the core
protein. Many different GAGs have been discovered including
chondroitin sulfate, dermatan sulfate, keratan sulfate, heparin,
and hyaluronan.
[0016] In specific cases, amyloid fibrils, once deposited, can
become toxic to the surrounding cells. For example, the A.beta.
fibrils organized as senile plaques have been shown to be
associated with dead neuronal cells, dystrophic neurites,
astrocytosis, and microgliosis in patients with Alzheimer's
disease. When tested in vitro, oligomeric (soluble) as well as
fibrillar A.beta. peptide was shown to be capable of triggering an
activation process of microglia (brain macrophages), which would
explain the presence of microgliosis and brain inflammation found
in the brain of patients with Alzheimer's disease. Both oligomeric
and fibrillar A.beta. peptide can also induce neuronal cell death
in vitro. See, e.g., M P Lambert, et al., Proc. Natl. Acad. Sci.
USA 95, 6448-53 (1998).
[0017] In another type of amyloidosis seen in patients with type II
diabetes, the amyloidogenic protein IAPP, when organized in
oligomeric forms or in fibrils, has been shown to induce
.beta.-islet cell toxicity in vitro. Hence, appearance of IAPP
fibrils in the pancreas of type II diabetic patients contributes to
the loss of the .beta. islet cells (Langerhans) and organ
dysfunction which can lead to insulinemia.
[0018] Another type of amyloidosis is related to .beta..sub.2
microglobulin and is found in long-term hemodialysis patients.
Patients undergoing long term hemodialysis will develop
.beta..sub.2-microglobulin fibrils in the carpal tunnel and in the
collagen rich tissues in several joints. This causes severe pains,
joint stiffness and swelling.
[0019] Amyloidosis is also characteristic of Alzheimer's disease.
Alzheimer's disease is a devastating disease of the brain that
results in progressive memory loss leading to dementia, physical
disability, and death over a relatively long period of time. With
the aging populations in developed countries, the number of
Alzheimer's patients is reaching epidemic proportions.
[0020] People suffering from Alzheimer's disease develop a
progressive dementia in adulthood, accompanied by three main
structural changes in the brain: diffuse loss of neurons in
multiple parts of the brain; accumulation of intracellular protein
deposits termed neurofibrillary tangles; and accumulation of
extracellular protein deposits termed amyloid or senile plaques,
surrounded by misshapen nerve terminals (dystrophic neurites) and
activated microglia (microgliosis and astrocytosis). A main
constituent of these amyloid plaques is the amyloid-.beta. peptide
(A.beta.), a 39-43 amino-acid protein that is produced through
cleavage of the .beta.-amyloid precursor protein (APP). Extensive
research has been conducted on the relevance of A.beta. deposits in
Alzheimer's disease, see, e.g., Selkoe, Trends in Cell Biology 8,
447-453 (1998). A.beta. naturally arises from the metabolic
processing of the amyloid precursor protein ("APP") in the
endoplasmic reticulum ("ER"), the Golgi apparatus, or the
endosomal-lysosomal pathway, and most is normally secreted as a 40
("A.beta.1-40") or 42 ("A.beta.1-42") amino acid peptide (Selkoe,
Annu. Rev. Cell Biol. 10, 373-403 (1994)). A role for A.beta. as a
primary cause for Alzheimer's disease is supported by the presence
of extracellular A.beta. deposits in senile plaques of Alzheimer's
disease, the increased production of A.beta. in cells harboring
mutant Alzheimer's disease associated genes, e.g., amyloid
precursor protein, presenilin I and presenilin II; and the toxicity
of extracellular soluble (oligomeric) or fibrillar A.beta. to cells
in culture. See, e.g., Gervais, Eur. Biopharm. Review, 40-42
(Autumn 2001); May, DDT 6, 459-62 (2001). Although symptomatic
treatments exist for Alzheimer's disease, this disease cannot be
prevented or cured at this time.
[0021] Alzheimer's disease is characterized by diffuse and neuritic
plaques, cerebral angiopathy, and neurofibrillary tangles. Plaque
and blood vessel amyloid is believed to be formed by the deposition
of insoluble A.beta. amyloid protein, which may be described as
diffuse or fibrillary. Both soluble oligomeric A.beta. and
fibrillar A.beta. are also believed to be neurotoxic and
inflammatory.
[0022] Another type of amyloidosis is cerebral amyloid angiopathy
(CAA). CAA is the specific deposition of amyloid-.beta. fibrils in
the walls of leptomingeal and cortical arteries, arterioles and
veins. It is commonly associated with Alzheimer's disease, Down's
syndrome and normal aging, as well as with a variety of familial
conditions related to stroke or dementia (see Frangione et al.,
Amyloid: J. Protein Folding Disord. 8, Suppl. 1, 36-42 (2001)).
[0023] Presently available therapies for treatment of
.beta.-amyloid diseases are almost entirely symptomatic, providing
only temporary or partial clinical benefit. Although some
pharmaceutical agents have been described that offer partial
symptomatic relief, no comprehensive pharmacological therapy is
currently available for the prevention or treatment of for example,
Alzheimer's disease.
SUMMARY OF THE INVENTION
[0024] The present invention relates to the use of certain
compounds in the treatment of amyloid-related diseases. In
particular, the invention relates to a method of treating or
preventing an amyloid-related disease in a subject comprising
administering to the subject a therapeutic amount of a compound of
the invention. The invention also pertains to each of the novel
compounds of the invention as described herein. Among the compounds
for use in the invention are those according to the following
Formulae, such that, when administered, amyloid fibril formation,
organ specific dysfunction (e.g., neurodegeneration), or cellular
toxicity is reduced or inhibited.
[0025] In one embodiment, the invention pertains, at least in part
to compounds of Formula I:
##STR00001##
wherein:
[0026] R.sup.1 is a substituted or unsubstituted cycloalkyl,
heterocyclic, aryl, arylcycloalkyl, bicyclic or tricyclic ring, a
bicyclic or tricyclic fused ring group, or a substituted or
unsubstituted C.sub.2-C.sub.10 alkyl group;
[0027] R.sup.2 is selected from a group consisting of hydrogen,
alkyl, mercaptoalkyl, alkenyl, alkynyl, cycloalkyl, aryl,
arylalkyl, thiazolyl, triazolyl, imidazolyl, benzothiazolyl, and
benzoimidazolyl;
[0028] Y is SO.sub.3.sup.-X.sup.+, OSO.sub.3.sup.-X.sup.+, or
SSO.sub.3X.sup.+;
[0029] X.sup.+ is hydrogen, a cationic group, or an ester-forming
group (i.e., as in a prodrug, which are described elsewhere
herein); and each of L.sup.1 and L.sup.2 is independently a
substituted or unsubstituted C.sub.1-C.sub.5 alkyl group or absent,
or a pharmaceutically acceptable salt thereof, provided that when
R.sup.1 is alkyl, L.sup.1 is absent.
[0030] In another embodiment, the invention pertains, at least in
part to compounds of Formula II:
##STR00002##
wherein:
[0031] R.sup.1 is a substituted or unsubstituted cyclic, bicyclic,
tricyclic, or benzoheterocyclic group or a substituted or
unsubstituted C.sub.2-C.sub.10 alkyl group;
[0032] R.sup.2 is hydrogen, alkyl, mercaptoalkyl, alkenyl, alkynyl,
cycloalkyl, aryl, arylalkyl, thiazolyl, triazolyl, imidazolyl,
benzothiazolyl, benzoimidazolyl, or linked to R.sup.1 to form a
heterocycle;
[0033] Y is SO.sub.3.sup.-X.sup.+, OSO.sub.3.sup.-X.sup.+, or
SSO.sub.3X.sup.+;
[0034] X.sup.+ is hydrogen, a cationic group, or an ester forming
moiety;
[0035] m is 0 or 1;
[0036] n is 1, 2, 3, or 4;
[0037] L is substituted or unsubstituted C.sub.1-C.sub.3 alkyl
group or absent,
or a pharmaceutically acceptable salt thereof, provided that when
R.sup.1 is alkyl, L is absent.
[0038] In yet another embodiment, the invention pertains, at least
in part to compounds of Formula III:
##STR00003##
wherein:
[0039] A is nitrogen or oxygen;
[0040] R.sup.11 is hydrogen, salt-forming cation, ester forming
group, --(CH.sub.2).sub.x-Q, or when A is nitrogen, A and R.sup.11
taken together may be the residue of a natural or unnatural amino
acid or a salt or, ester thereof;
[0041] Q is hydrogen, thiazolyl, triazolyl, imidazolyl,
benzothiazolyl, or benzoimidazolyl;
[0042] x is 0, 1, 2, 3, or 4;
[0043] n is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10;
[0044] R.sup.3, R.sup.3a, R.sup.4, R.sup.4a, R.sup.5, R.sup.5a,
R.sup.6, R.sup.6a, R.sup.7 and R.sup.7a are each independently
hydrogen, alkyl, mercaptoalkyl, alkenyl, alkynyl, cycloalkyl, aryl,
alkylcarbonyl, arylcarbonyl, alkoxycarbonyl, cyano, halogen, amino,
tetrazolyl, or two R groups on adjacent ring atoms taken together
with the ring atoms form a double bond, provided that one of
R.sup.3, R.sup.3a, R.sup.4, R.sup.4a, R.sup.5, R.sup.5a, R.sup.6,
R.sup.6a, R.sup.7 and R.sup.7a is a moiety of Formula IIIa:
##STR00004##
wherein:
[0045] m is 0, 1, 2, 3, or 4;
[0046] R.sup.A, R.sup.B, R.sup.C, R.sup.D, and R.sup.E are
independently selected from a group of hydrogen, halogen, hydroxyl,
alkyl, alkoxyl, halogenated alkyl, mercaptoalkyl, alkenyl, alkynyl,
cycloalkyl, aryl, cyano, thiazolyl, triazolyl, imidazolyl,
tetrazolyl, benzothiazolyl, and benzoimidazolyl; and
pharmaceutically acceptable salts and esters thereof, provided that
said compound is not
3-(4-phenyl-1,2,3,6-tetrahydro-1-pyridyl)-1-propanesulfonic
acid.
[0047] In yet another embodiment, the invention pertains at least
in part to compounds of Formula IV:
##STR00005##
wherein:
[0048] A is nitrogen or oxygen;
[0049] R.sup.11 is hydrogen, salt-forming cation, ester forming
group, --(CH.sub.2).sub.x-Q, or when A is nitrogen, A and R.sup.11
taken together may be the residue of a natural or unnatural amino
acid or a salt or ester thereof;
[0050] Q is hydrogen, thiazolyl, triazolyl, imidazolyl,
benzothiazolyl, or benzoimidazolyl;
[0051] x is 0, 1, 2, 3, or 4;
[0052] n is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10;
[0053] R.sup.4, R.sup.4a, R.sup.5, R.sup.5a, R.sup.6, R.sup.6a,
R.sup.7, and R.sup.7a are each independently hydrogen, alkyl,
mercaptoalkyl, alkenyl, alkynyl, cycloalkyl, aryl, alkylcarbonyl,
arylcarbonyl, alkoxycarbonyl, cyano, halogen, amino, tetrazolyl,
R.sup.4 and R.sup.5 taken together, with the ring atoms they are
attached to, form a double bond, or R.sup.6 and R.sup.7 taken
together, with the ring atoms they are attached to, form a double
bond;
[0054] m is 0, 1, 2, 3, or 4;
[0055] R.sup.8, R.sup.9, R.sup.10, R.sup.11, and R.sup.12 are
independently selected from a group of hydrogen, halogen, hydroxyl,
alkyl, alkoxyl, halogenated alkyl, mercaptoalkyl, alkenyl, alkynyl,
cycloalkyl, aryl, cyano, thiazolyl, triazolyl, imidazolyl,
tetrazolyl, benzothiazolyl, and benzoimidazolyl, and
pharmaceutically acceptable salts and esters thereof.
[0056] In another embodiment, the invention includes compounds of
Formula V:
##STR00006##
wherein:
[0057] A is nitrogen or oxygen;
[0058] R.sup.11 is hydrogen, salt-forming cation, ester forming
group, --(CH.sub.2).sub.x-Q, or when A is nitrogen, A and R.sup.11
taken together may be the residue of a natural or unnatural amino
acid or a salt or ester thereof;
[0059] Q is hydrogen, thiazolyl, triazolyl, imidazolyl,
benzothiazolyl, or benzoimidazolyl;
[0060] x is 0, 1, 2, 3, or 4;
[0061] n is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10;
[0062] aa is a natural or unnatural amino acid residue;
[0063] m is 0, 1, 2, or 3;
[0064] R.sup.14 is hydrogen or protecting group;
[0065] R.sup.15 is hydrogen, alkyl or aryl, and pharmaceutically
acceptable salts and prodrugs thereof.
[0066] In another embodiment, the invention includes compounds of
the Formula VI:
##STR00007##
wherein:
[0067] n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10;
[0068] A is oxygen or nitrogen;
[0069] R.sup.11 is hydrogen, salt-forming cation, ester forming
group, --(CH.sub.2).sub.n-Q, or when A is nitrogen, A and R.sup.11
taken together may be the residue of a natural or unnatural amino
acid or a salt or ester thereof;
[0070] Q is hydrogen, thiazolyl, triazolyl, imidazolyl,
benzothiazolyl, or benzoimidazolyl;
[0071] x is 0, 1, 2, 3, or 4;
[0072] R.sup.19 is hydrogen, alkyl or aryl;
[0073] Y.sup.1 is oxygen, sulfur, or nitrogen;
[0074] Y.sup.2 is carbon, nitrogen, or oxygen;
[0075] R.sup.20 is hydrogen, alkyl, amino, mercaptoalkyl, alkenyl,
alkynyl, cycloalkyl, aryl, arylalkyl, thiazolyl, triazolyl,
tetrazolyl, imidazolyl, benzothiazolyl, or benzoimidazolyl;
[0076] R.sup.21 is hydrogen, alkyl, mercaptoalkyl, alkenyl,
alkynyl, cycloalkyl, aryl, arylalkyl, thiazolyl, triazolyl,
tetrazolyl, imidazolyl, benzothiazolyl, benzoimidazolyl, or absent
if Y.sup.2 is oxygen;
[0077] R.sup.22 is hydrogen, alkyl, mercaptoalkyl, alkenyl,
alkynyl, cycloalkyl, aryl, arylalkyl, thiazolyl, triazolyl,
tetrazolyl, imidazolyl, benzothiazolyl, benzoimidazolyl; or
R.sup.22 is hydrogen, hydroxyl, alkoxy or aryloxy if Y.sup.1 is
nitrogen; or R.sup.22 is absent if Y.sup.1 is oxygen or sulfur; or
R.sup.22 and R.sup.21 may be linked to form a cyclic moiety if
Y.sup.1 is nitrogen;
[0078] R.sup.23 is hydrogen, alkyl, amino, mercaptoalkyl, alkenyl,
alkynyl, cycloalkyl, aryl, arylalkyl, thiazolyl, triazolyl,
tetrazolyl, imidazolyl, benzothiazolyl, or benzoimidazolyl, or
absent if Y.sup.2 is nitrogen or oxygen;
[0079] or pharmaceutically acceptable salts thereof.
[0080] In another embodiment, the invention includes compounds of
Formula VII:
##STR00008##
wherein:
[0081] n is 2, 3, or 4;
[0082] A is oxygen or nitrogen;
[0083] R.sup.11 is hydrogen, salt-forming cation, ester forming
group, --(CH.sub.2).sub.x-Q, or when A is nitrogen, A and R.sup.11
taken together may be the residue of a natural or unnatural amino
acid or a salt or ester thereof;
[0084] Q is hydrogen, thiazolyl, triazolyl, imidazolyl,
benzothiazolyl, or benzoimidazolyl;
[0085] x is 0, 1, 2, 3, or 4;
[0086] G is a direct bond or oxygen, nitrogen, or sulfur;
[0087] z is 0, 1, 2, 3, 4, or 5;
[0088] m is 0 or 1;
[0089] R.sup.24 is selected from a group consisting of hydrogen,
alkyl, mercaptoalkyl, alkenyl, alkynyl, aroyl, alkylcarbonyl,
aminoalkylcarbonyl, cycloalkyl, aryl, arylalkyl, thiazolyl,
triazolyl, imidazolyl, benzothiazolyl, and benzoimidazolyl;
[0090] each R.sup.25 is independently selected from hydrogen,
halogen, cyano, hydroxyl, alkoxy, thiol, amino, nitro, alkyl, aryl,
carbocyclic, or heterocyclic, and pharmaceutically acceptable salts
thereof.
[0091] In one embodiment, the compounds disclosed herein prevent or
inhibit amyloid protein assembly into insoluble fibrils which, in
vivo, are deposited in various organs, or it favors clearance of
pre-formed deposits or slows deposition in patients already having
deposits. In another embodiment, the compound may also prevent the
amyloid protein, in its soluble, oligomeric form or in its
fibrillar form, from binding or adhering to a cell surface and
causing cell damage or toxicity. In yet another embodiment, the
compound may block amyloid-induced cellular toxicity or macrophage
activation. In another embodiment, the compound may block
amyloid-induced neurotoxicity or microglial activation. In another
embodiment, the compound protects cells from amyloid induced
cytotoxicity of B-islet cells. In another embodiment, the compound
may enhance clearance from a specific organ, e.g., the brain or it
decreases concentration of the amyloid protein in such a way that
amyloid fibril formation is prevented in the targeted organ.
[0092] The compounds of the invention may be administered
therapeutically or prophylactically to treat diseases associated
with amyloid fibril formation, aggregation or deposition. The
compounds of the invention may act to ameliorate the course of an
amyloid related disease using any of the following mechanisms (this
list is meant to be illustrative and not limiting): slowing the
rate of amyloid fibril formation or deposition; lessening the
degree of amyloid deposition; inhibiting, reducing, or preventing
amyloid fibril formation; inhibiting neurodegeneration or cellular
toxicity induced by amyloid; inhibiting amyloid induced
inflammation; enhancing the clearance of amyloid; or favoring the
degradation of amyloid protein prior to its organization in
fibrils.
[0093] The compounds of the invention may be administered
therapeutically or prophylactically to treat diseases associated
with amyloid-.beta. fibril formation, aggregation or deposition.
The compounds of the invention may act to ameliorate the course of
an amyloid-.beta. related disease using any of the following
mechanisms (this list is meant to be illustrative and not
limiting): slowing the rate of amyloid-.beta. fibril formation or
deposition; lessening the degree of amyloid-.beta. deposition;
inhibiting, reducing, or preventing amyloid-.beta. fibril
formation; inhibiting neurodegeneration or cellular toxicity
induced by amyloid-.beta.; inhibiting amyloid-.beta. induced
inflammation; enhancing the clearance of amyloid-.beta. from the
brain; or favoring the degradation of amyloid-.beta. protein prior
to its organization in fibrils.
[0094] Therapeutic compounds of the invention may be effective in
controlling amyloid-.beta. deposition either following their entry
into the brain (following penetration of the blood brain barrier)
or from the periphery. When acting from the periphery, a compound
may alter the equilibrium of A.beta. between the brain and the
plasma so as to favor the exit of A.beta. from the brain. It may
also increase the catabolism of neuronal A.beta. and change the
rate of exit from the brain. An increase in the exit of A.beta.
from the brain would result in a decrease in A.beta. brain and
cerebral spinal fluid (CSF) concentration and therefore favor a
decrease in A.beta. deposition. Alternatively, compounds that
penetrate the brain could control deposition by acting directly on
brain A.beta. e.g., by maintaining it in a non-fibrillar form,
favoring its clearance from the brain, or by slowing down APP
processing. These compounds could also prevent A.beta. in the brain
from interacting with the cell surface and therefore prevent
neurotoxicity, neurodegeneration or inflammation. They may also
decrease A.beta. production by activated microglia. The compounds
may also increase degradation by macrophages or neuronal cells.
[0095] In one embodiment, the method is used to treat Alzheimer's
disease (e.g., sporadic, familial, or early A.beta.). The method
can also be used prophylactically or therapeutically to treat other
clinical occurrences of amyloid-.beta. deposition, such as in
Down's syndrome individuals and in patients with cerebral amyloid
angiopathy ("CAA") or hereditary cerebral hemorrhage.
[0096] In another embodiment, the method is used to treat mild
cognitive impairment. Mild Cognitive Impairment ("MCI") is a
condition characterized by a state of mild but measurable
impairment in thinking skills, which is not necessarily associated
with the presence of dementia. MCI frequently, but not necessarily,
precedes Alzheimer's disease.
[0097] Additionally, abnormal accumulation of APP and of
amyloid-.beta. protein in muscle fibers has been implicated in the
pathology of sporadic inclusion body myositis (IBM) (Askanas, et
al., Proc. Natl. Acad. Sci. USA 93, 1314-1319 (1996); Askanas, et
al., Current Opinion in Rheumatology 7, 486-496 (1995)).
Accordingly, the compounds of the invention can be used
prophylactically or therapeutically in the treatment of disorders
in which amyloid-beta protein is abnormally deposited at
non-neurological locations, such as treatment of IBM by delivery of
the compounds to muscle fibers.
[0098] Additionally, it has been shown that A.beta. is associated
with abnormal extracellular deposits, known as drusen, that
accumulate along the basal surface of the retinal pigmented
epithelium in individuals with age-related macular degeneration
(AMD). AMD is a cause of irreversible vision loss in older
individuals. It is believed that A.beta. deposition could be an
important component of the local inflammatory events that
contribute to atrophy of the retinal pigmented epithelium, drusen
biogenesis, and the pathogenesis of AMD (Johnson, et al., Proc.
Natl. Acad. Sci. USA 99(18), 11830-5 (2002)).
[0099] The present invention therefore relates to the use of
compounds of Formulae I, II, III, IV, V, VI, VII, or otherwise
described herein in the prevention or treatment of amyloid-related
diseases, including, inter alia, Alzheimer's disease, cerebral
amyloid angiopathy, mild cognitive impairment, inclusion body
myositis, Down's syndrome, macular degeneration, as well as other
types of amyloidosis like IAPP-related amyloidosis (e.g.,
diabetes), primary (AL) amyloidosis, secondary (AA) amyloidosis and
.beta..sub.2 microglobulin-related (dialysis-related)
amyloidosis.
[0100] In Type II diabetes related amyloidosis (IAPP), the
amyloidogenic protein IAPP induces .beta.-islet cell toxicity when
organized in oligomeric forms or in fibrils. Hence, appearance of
IAPP fibrils in the pancreas of type II diabetic patients
contributes to the loss of the .beta. islet cells (Langerhans) and
organ dysfunction which leads to insulinemia.
[0101] Primary amyloidosis (AL amyloid) is usually found associated
with plasma cell dyscrasia and multiple myeloma. It can also be
found as an idiopathic disease.
[0102] Secondary (AA) amyloidosis is usually seen associated with
chronic infection (such as tuberculosis) or chronic inflammation
(such as rheumatoid arthritis). A familial form of secondary
amyloidosis is also seen in Familial Mediterranean Fever (FMF).
[0103] .beta..sub.2 microglobulin-related (dialysis-related)
amyloidosis is found in long-term hemodialysis patients. Patients
undergoing long term hemodialysis will develop
.beta..sub.2-microglobulin fibrils in the carpal tunnel and in the
collagen rich tissues in several joints.
[0104] This causes severe pains, joint stiffness and swelling.
These deposits are due to the inability to maintain low levels of
.beta..sub.2M in plasma of dialyzed patients. Increased plasma
concentrations of .beta..sub.2M protein will induce structural
changes and may lead to the deposition of modified (.beta..sub.2M
as insoluble fibrils in the joints.
DETAILED DESCRIPTION OF THE INVENTION
[0105] The present invention relates to the use of compounds of
Formulae I, II, III, IV, V, VI, VII, or compounds otherwise
described herein in the treatment of amyloid-related diseases. For
convenience, some definitions of terms referred to herein are set
forth below.
Amyloid-Related Diseases
AA (Reactive) Amyloidosis
[0106] Generally, AA amyloidosis is a manifestation of a number of
diseases that provoke a sustained acute phase response. Such
diseases include chronic inflammatory disorders, chronic local or
systemic microbial infections, and malignant neoplasms. The most
common form of reactive or secondary (AA) amyloidosis is seen as
the result of long-standing inflammatory conditions. For example,
patients with Rheumatoid Arthritis or Familial Mediterranean Fever
(which is a genetic disease) can develop AA amyloidosis. The terms
"AA amyloidosis" and "secondary (AA) amyloidosis" are used
interchangeably.
[0107] AA fibrils are generally composed of 8,000 Dalton fragments
(AA peptide or protein) formed by proteolytic cleavage of serum
amyloid A protein (ApoSAA), a circulating apolipoprotein which is
mainly synthesized in hepatocytes in response to such cytokines as
IL-1, IL-6 and TNF. Once secreted, ApoSAA is complexed with HDL.
Deposition of AA fibrils can be widespread in the body, with a
preference for parenchymal organs. The kidneys are usually a
deposition site, and the liver and the spleen may also be affected.
Deposition is also seen in the heart, gastrointestinal tract, and
the skin.
[0108] Underlying diseases which can lead to the development of AA
amyloidosis include, but are not limited to inflammatory diseases,
such as rheumatoid arthritis, juvenile chronic arthritis,
ankylosing spondylitis, psoriasis, psoriatic arthropathy, Reiter's
syndrome, Adult Still's disease, Behcet's syndrome, and Crohn's
disease. AA deposits are also produced as a result of chronic
microbial infections, such as leprosy, tuberculosis,
bronchiectasis, decubitus ulcers, chronic pyelonephritis,
osteomyelitis, and Whipple's disease. Certain malignant neoplasms
can also result in AA fibril amyloid deposits. These include such
conditions as Hodgkin's lymphoma, renal carcinoma, carcinomas of
gut, lung and urogenital tract, basal cell carcinoma, and hairy
cell leukemia. Other underlying conditions that may be associated
with AA amyloidosis are Castleman's disease and Schnitzler's
syndrome.
AL Amyloidoses (Primary Amyloidosis)
[0109] AL amyloid deposition is generally associated with almost
any dyscrasia of the B lymphocyte lineage, ranging from malignancy
of plasma cells (multiple myeloma) to benign monoclonal gammopathy.
At times, the presence of amyloid deposits may be a primary
indicator of the underlying dyscrasia. AL amyloidosis is also
described in detail in Current Drug Targets, 2004, 5 159-171.
[0110] Fibrils of AL amyloid deposits are composed of monoclonal
immunoglobulin light chains or fragments thereof. More
specifically, the fragments are derived from the N-terminal region
of the light chain (kappa or lambda) and contain all or part of the
variable (V.sub.L) domain thereof. Deposits generally occur in the
mesenchymal tissues, causing peripheral and autonomic neuropathy,
carpal tunnel syndrome, macroglossia, restrictive cardiomyopathy,
arthropathy of large joints, immune dyscrasias, myelomas, as well
as occult dyscrasias. However, it should be noted that almost any
tissue, particularly visceral organs such as the kidney, liver,
spleen and heart, may be involved.
Hereditary Systemic Amyloidoses
[0111] There are many forms of hereditary systemic amyloidoses.
Although they are relatively rare conditions, adult onset of
symptoms and their inheritance patterns (usually autosomal
dominant) lead to persistence of such disorders in the general
population. Generally, the syndromes are attributable to point
mutations in the precursor protein leading to production of variant
amyloidogenic peptides or proteins. Table 1 summarizes the fibril
composition of exemplary forms of these disorders.
TABLE-US-00001 TABLE 1 Fibril Composition of Exemplary
Amyloid-Related Diseases Genetic Fibril Peptide/Protein Variant
Clinical Syndrome ATTR protein from Transthyretin Met30, many
Familial amyloid polyneuropathy (FAP), and fragments others (Mainly
peripheral nerves) ATTR protein from Transthyretin Thr45, Ala60,
Cardiac involvement predominant without and fragments Ser84,
Met111, neuropathy, familial amyloid polyneuropathy, Ile122 senile
systemic amyloidosis, Tenosynovium N-terminal fragment of Arg26
Familial amyloid polyneuropathy (FAP), Apolipoprotein A1 (apoAI)
(mainly peripheral nerves) N-terminal fragment of Arg26, Arg50,
Ostertag-type, non-neuropathic (predominantly Apoliproprotein A1
(AapoAI) Arg 60, others visceral involvement) AapoAII from
Apolipoprotein AII Familial amyloidosis Lysozyme (Alys) Thr56,
His67 Ostertag-type, non-neuropathic (predominantly visceral
involvement) Fibrogen alpha chain fragment Leu554, Val Cranial
neuropathy with lattic corneal 526 dystrophy Gelsolin fragment
(Agel) Asn187, Cranial neuropathy with lattice corneal Tyr187
dystrophy Cystatin C fragment (ACys) Glu68 Hereditary cerebral
hemorrhage (cerebral amyloid angiopathy) - Icelandic type
.beta.-amyloid protein (A.beta.) derived from Gln693 Hereditary
cerebral hemorrhage (cerebral Amyloid Precursor Protein (APP)
amyloid angiopathy) - Dutch type .beta.-amyloid protein (A.beta.)
derived from Ile717, Phe717, Familial Alzheimer's Disease Amyloid
Precursor Protein (APP) Gly717 .beta.-amyloid protein (A.beta.)
derived from Gln 618 Alzheimer's disease, Down's syndrome, Amyloid
Precursor Protein (APP), hereditary cerebral hemorrhage with e.g.,
bPP 695 amyloidosis, Dutch type .beta.-amyloid protein (A.beta.)
derived from Asn670, Familial Dementia - probably Alzheimer's
Amyloid Precursor Protein (APP) Leu671 Disease Prion Protein (PrP,
APrP.sup.SC) derived Leu102, Familial Creutzfeldt-Jakob disease;
from Prp precursor protein (51-91 Val167,
Gerstmann-Straussler-Scheinker syndrome insert) Asn178, (hereditary
spongiform encephalopathies, prion Lys200 diseases) AA derived from
Serum amyloid A Familial Mediterranean fever, predominant protein
(ApoSAA) renal involvement (autosomal recessive) AA derived from
Serum amyloid A Muckle-Well's syndrome, nephropathy, protein
(ApoSAA) deafness, urticaria, limb pain Unknown Cardiomyopathy with
persistent atrial standstill Unknown Cutaneous deposits (bullous,
papular, pustulodermal) AH amyloid protein, derived from A.gamma.I
Myeloma associated amyloidosis immunoglobulin heavy chain (gamma I)
ACal amyloid protein from (Pro) calcitonin Medullary carcinomas of
the thyroid (pro)calcitonin AANF amyloid protein from atrial
Isolated atrial amyloid natriuretic factor Apro from Prolactin
Prolactinomas Abri/ADan from ABri peptide British and Danish
familial Dementia Data derived from Tan SY, Pepys MB. Amyloidosis.
Histopathology, 25(5), 403-414 (November 1994), WHO/IUIS
Nomenclature Subcommittee, Nomenclature of Amyloid and Amyloidosis.
Bulletin of the World Health Organisation 1993; 71: 10508; and
Merlini et al., Clin Chem Lab Med 2001; 39(11): 1065-75.
[0112] The data provided in Table 1 are exemplary and are not
intended to limit the scope of the invention. For example, more
than 40 separate point mutations in the transthyretin gene have
been described, all of which give rise to clinically similar forms
of familial amyloid polyneuropathy.
[0113] In general, any hereditary amyloid disorder can also occur
sporadically, and both hereditary and sporadic forms of a disease
present with the same characteristics with regard to amyloid. For
example, the most prevalent form of secondary AA amyloidosis occurs
sporadically, e.g. as a result of ongoing inflammation, and is not
associated with Familial Mediterranean Fever. Thus general
discussion relating to hereditary amyloid disorders below can also
be applied to sporadic amyloidoses.
[0114] Transthyretin (TTR) is a 14 kiloDalton protein that is also
sometimes referred to as prealbumin. It is produced by the liver
and choroid plexus, and it functions in transporting thyroid
hormones and vitamin A. At least 50 variant forms of the protein,
each characterized by a single amino acid change, are responsible
for various forms of familial amyloid polyneuropathy. For example,
substitution of proline for leucine at position 55 results in a
particularly progressive form of neuropathy; substitution of
methionine for leucine at position 111 resulted in a severe
cardiopathy in Danish patients.
[0115] Amyloid deposits isolated from heart tissue of patients with
systemic amyloidosis have revealed that the deposits are composed
of a heterogeneous mixture of TTR and fragments thereof,
collectively referred to as ATTR, the full length sequences of
which have been characterized. ATTR fibril components can be
extracted from such plaques and their structure and sequence
determined according to the methods known in the art (e.g.,
Gustaysson, A., et al., Laboratory Invest. 73: 703-708, 1995;
Kametani, F., et al., Biochem. Biophys. Res. Commun. 125: 622-628,
1984; Pras, M., et al., PNAS 80: 539-42, 1983).
[0116] Persons having point mutations in the molecule
apolipoprotein Al (e.g., Gly.fwdarw.Arg26; Trp.fwdarw.Arg50;
Leu.fwdarw.Arg60) exhibit a form of amyloidosis ("Ostertag type")
characterized by deposits of the protein apolipoprotein AI or
fragments thereof (AApoAI). These patients have low levels of high
density lipoprotein (HDL) and present with a peripheral neuropathy
or renal failure.
[0117] A mutation in the alpha chain of the enzyme lysozyme (e.g.,
Ile.fwdarw.Thr56 or Asp.fwdarw.His57) is the basis of another form
of Ostertag-type non-neuropathic hereditary amyloid reported in
English families. Here, fibrils of the mutant lysozyme protein
(Alys) are deposited, and patients generally exhibit impaired renal
function. This protein, unlike most of the fibril-forming proteins
described herein, is usually present in whole (unfragmented) form
(Benson, M. D., et al. CIBA Fdn. Symp. 199: 104-131, 1996).
[0118] Immunoglobulin light chains tend to form aggregates in
various morphologies, including fibrillar (e.g., AL amyloidosis and
AH amyloidosis), granular (e.g., light chain deposition disease
(LCDD), heavy chain deposition disease (HCDD), and light-heavy
chain deposition disease (LHCDD)), crystalline (e.g., Acquired
Farconi's Syndome), and microtubular (e.g., Cryoglobulinemia). AL
and AH amyloidosis is indicated by the formation of insoluble
fibrils of immunoglobulin light chains and heavy chain,
respectively, and/or their fragments. In AL fibrils, lambda
(.lamda.) chains such as .lamda. VI chains (.lamda.6 chains), are
found in greater concentrations than kappa (.kappa.) chains.
.lamda.III chains are also slightly elevated. Merlini et CLIN CHEM
LAB MED 39(11):1065-75 (2001). Heavy chain amyloidosis (AH) is
generally characterized by aggregates of gamma chain amyloid
proteins of the IgG 1 subclass. Eulitz et al., PROC NATL ACAD SCI
USA 87:6542-46 (1990).
[0119] Comparison of amyloidogenic to non-amyloidogenic light
chains has revealed that the former can include replacements or
substitutions that appear to destabilize the folding of the protein
and promote aggregation. AL and LCDD have been distinguished from
other amyloid diseases due to their relatively small population
monoclonal light chains, which are manufactured by neoplastic
expansion of an antibody-producing B cell. AL aggregates typically
are well-ordered fibrils of lambda chains. LCDD aggregates are
relatively amorphous aggregations of both kappa and lambda chains,
with a majority being kappa, in some cases .kappa.IV. Bellotti et
al., JOURNAL OF STRUCTURAL BIOLOGY 13:280-89 (2000). Comparison of
amyloidogenic and non-amyloidogenic heavy chains in patients having
AH amyloidosis has revealed missing and/or altered components.
Eulitz et al., PROC NATL ACAD SCI USA 87:6542-46 (1990) (pathogenic
heavy chain characterized by significantly lower molecular mass
than non-amyloidogenic heavy chains); and Solomon et al. AM J HEMAT
45(2) 171-6 (1994) (amyloidogenic heavy chain characterized as
consisting solely of the VH-D portion of the non-amyloidogenic
heavy chain).
[0120] Accordingly, potential methods of detecting and monitoring
treatment of subjects having or at risk of having AL, LCDD, AH, and
the like, include but are not limited to immunoassaying plasma or
urine for the presence or depressed deposition of amyloidogenic
light or heavy chains, e.g., amyloid .lamda., amyloid .kappa.,
amyloid .kappa.IV, amyloid .gamma., or amyloid .gamma.1.
Brain Amyloidosis
[0121] The most frequent type of amyloid in the brain is composed
primarily of A.beta. peptide fibrils, resulting in dementia
associated with sporadic (non-hereditary) Alzheimer's disease. In
fact, the incidence of sporadic Alzheimer's disease greatly exceeds
forms shown to be hereditary. Nevertheless, fibril peptides forming
plaques are very similar in both types. Brain amyloidosis includes
those diseases, conditions, pathologies, and other abnormalities of
the structure or function of the brain, including components
thereof, in which the causative agent is amyloid. The area of the
brain affected in an amyloid-related disease may be the stroma
including the vasculature or the parenchyma including functional or
anatomical regions, or neurons themselves. A subject need not have
received a definitive diagnosis of a specifically recognized
amyloid-related disease. The term "amyloid related disease"
includes brain amyloidosis.
[0122] Amyloid-.beta. peptide ("A.beta.") is a 39-43 amino acid
peptide derived by proteolysis from a large protein known as Beta
Amyloid Precursor Protein (".beta.APP"). Mutations in PAPP result
in familial forms of Alzheimer's disease, Down's syndrome, cerebral
amyloid angiopathy, and senile dementia, characterized by cerebral
deposition of plaques composed of A.beta. fibrils and other
components, which are described in further detail below. Known
mutations in APP associated with Alzheimer's disease occur
proximate to the cleavage sites of .beta. or .gamma.-secretase, or
within A.beta.. For example, position 717 is proximate to the site
of gamma-secretase cleavage of APP in its processing to A.beta.,
and positions 670/671 are proximate to the site of .beta.-secretase
cleavage. Mutations at any of these residues may result in
Alzheimer's disease, presumably by causing an increase in the
amount of the 42/43 amino acid form of A.beta. generated from APP.
The familial form of Alzheimer's disease represents only 10% of the
subject population. Most occurrences of Alzheimer's disease are
sporadic cases where APP and A.beta. do not possess any mutation.
The structure and sequence of A.beta. peptides of various lengths
are well known in the art. Such peptides can be made according to
methods known in the art, or extracted from the brain according to
known methods (e.g., Glenner and Wong, Biochem. Biophys. Res. Comm.
129, 885-90 (1984); Glenner and Wong, Biochem. Biophys. Res. Comm.
122, 1131-35 (1984)). In addition, various forms of the peptides
are commercially available. APP is expressed and constitutively
catabolized in most cells. The dominant catabolic pathway appears
to be cleavage of APP within the A.beta. sequence by an enzyme
provisionally termed .alpha.-secretase, leading to release of a
soluble ectodomain fragment known as APPsa. This cleavage precludes
the formation of A.beta. peptide. In contrast to this
non-amyloidogenic pathway, APP can also be cleaved by enzymes known
as .beta.- and .gamma.-secretase at the N- and C-termini of the
A.beta., respectively, followed by release of A.beta. into the
extracellular space. To date, BACE has been identified as
.beta.-secretase (Vasser, et al., Science 286:735-741, 1999) and
presenilins have been implicated in .gamma.-secretase activity (De
Strooper, et al., Nature 391, 387-90 (1998)). The 39-43 amino acid
A.beta. peptide is produced by sequential proteolytic cleavage of
the amyloid precursor protein (APP) by the .beta. and .gamma.
secretases enzyme. Although A.beta.40 is the predominant form
produced, 5-7% of total A.beta. exists as A.beta.42 (Cappai et al.,
Int. J. Biochem. Cell Biol. 31. 885-89 (1999)).
[0123] The length of the A.beta. peptide appears to dramatically
alter its biochemical/biophysical properties. Specifically, the
additional two amino acids at the C-terminus of A.beta.42 are very
hydrophobic, presumably increasing the propensity of A.beta.42 to
aggregate. For example, Jarrett, et al. demonstrated that A.beta.42
aggregates very rapidly in vitro compared to A.beta.40, suggesting
that the longer forms of A.beta. may be the important pathological
proteins that are involved in the initial seeding of the neuritic
plaques in Alzheimer's disease (Jarrett, et al., Biochemistry 32,
4693-97 (1993); Jarrett, et al., Ann. N.Y. Acad. Sci. 695, 144-48
(1993)). This hypothesis has been further substantiated by the
recent analysis of the contributions of specific forms of A.beta.
in cases of genetic familial forms of Alzheimer's disease ("FAD").
For example, the "London" mutant form of APP (APPV717I) linked to
FAD selectively increases the production of A.beta. 42/43 forms
versus A.beta. 40 (Suzuki, et al., Science 264, 1336-40 (1994))
while the "Swedish" mutant form of APP (APPK670N/M671L) increases
levels of both A.beta.40 and A.beta.42/43 (Citron, et al., Nature
360, 672-674 (1992); Cai, et al., Science 259, 514-16, (1993)).
Also, it has been observed that FAD-linked mutations in the
Presenilin-1 ("PS1") or Presenilin-2 ("PS2") genes will lead to a
selective increase in A.beta.42/43 production but not A.beta.40
(Borchelt, et al., Neuron 17, 1005-13 (1996)). This finding was
corroborated in transgenic mouse models expressing PS mutants that
demonstrate a selective increase in brain A.beta.42 (Borchelt, op
cit.; Duff, et al., Neurodegeneration 5(4), 293-98 (1996)). Thus
the leading hypothesis regarding the etiology of Alzheimer's
disease is that an increase in A.beta.42 brain concentration due to
an increased production and release of A.beta.42 or a decrease in
clearance (degradation or brain clearance) is a causative event in
the disease pathology.
[0124] Multiple mutation sites in either A.beta. or the APP gene
have been identified and are clinically associated with either
dementia or cerebral hemorrhage. Exemplary CAA disorders include,
but are not limited to, hereditary cerebral hemorrhage with
amyloidosis of Icelandic type (HCHWA-I); the Dutch variant of HCHWA
(HCHWA-D; a mutation in A.beta.); the Flemish mutation of A.beta.;
the Arctic mutation of A.beta.; the Italian mutation of A.beta.;
the Iowa mutation of A.beta.; familial British dementia; and
familial Danish dementia. CAA may also be sporadic.
[0125] As used herein, the terms ".beta. amyloid,"
"amyloid-.beta.," and the like refer to amyloid .beta. proteins or
peptides, amyloid .beta. precursor proteins or peptides,
intermediates, and modifications and fragments thereof, unless
otherwise specifically indicated. In particular, "A.beta." refers
to any peptide produced by proteolytic processing of the APP gene
product, especially peptides which are associated with amyloid
pathologies, including A.beta.1-39, A.beta.1-40, A.beta.1-41,
A.beta.1-42, and A.beta.1-43. For convenience of nomenclature,
"A.beta.1-42" may be referred to herein as "A.beta. (1-42)" or
simply as "A.beta.42" or "A.beta.42" (and likewise for any other
amyloid peptides discussed herein). As used herein, the terms
".beta. amyloid," "amyloid-.beta.," and "A.beta." are
synonymous.
[0126] Unless otherwise specified, the term "amyloid" refers to
amyloidogenic proteins, peptides, or fragments thereof which can be
soluble (e.g., monomeric or oligomeric) or insoluble (e.g., having
fibrillary structure or in amyloid plaque). See, e.g., M P Lambert,
et al., Proc. Nat'l Acad. Sci. USA 95, 6448-53 (1998).
"Amyloidosis" or "amyloid disease" or "amyloid-related disease"
refers to a pathological condition characterized by the presence of
amyloid fibers. "Amyloid" is a generic term referring to a group of
diverse but specific protein deposits (intracellular or
extracellular) which are seen in a number of different diseases.
Though diverse in their occurrence, all amyloid deposits hive
common morphologic properties, stain with specific dyes (e.g.,
Congo red), and have a characteristic red-green birefringent
appearance in polarized light after staining. They also share
common ultrastructural features and common X-ray diffraction and
infrared spectra.
[0127] Gelsolin is a calcium binding protein that binds to
fragments and actin filaments. Mutations at position 187 (e.g.,
Asp.fwdarw.Asn; Asp.fwdarw.Tyr) of the protein result in a form of
hereditary systemic amyloidosis, usually found in patients from
Finland, as well as persons of Dutch or Japanese origin. In
afflicted individuals, fibrils formed from gelsolin fragments
(Agel), usually consist of amino acids 173-243 (68 kDa
carboxyterminal fragment) and are deposited in blood vessels and
basement membranes, resulting in corneal dystrophy and cranial
neuropathy which progresses to peripheral neuropathy, dystrophic
skin changes and deposition in other organs. (Kangas, H., et al.
Human Mol. Genet. 5(9): 1237-1243, 1996).
[0128] Other mutated proteins, such as mutant alpha chain of
fibrinogen (AfibA) and mutant cystatin C (Acys) also form fibrils
and produce characteristic hereditary disorders. AfibA fibrils form
deposits characteristic of a normeuropathic hereditary amyloid with
renal disease; Acys deposits are characteristic of a hereditary
cerebral amyloid angiopathy reported in Iceland (Isselbacher,
Harrison's Principles of Internal Medicine, McGraw-Hill, San
Francisco, 1995; Benson, et al.). In at least some cases, patients
with cerebral amyloid angiopathy (CAA) have been shown to have
amyloid fibrils containing a non-mutant form of cystatin C in
conjunction with amyloid beta protein (Nagai, A., et al. Molec.
Chem. Neuropathol. 33: 63-78, 1998).
[0129] Certain forms of prion disease are now considered to be
heritable, accounting for up to 15% of cases, which were previously
thought to be predominantly infectious in nature. (Baldwin, et al.,
in Research Advances in Alzheimer's Disease and Related Disorders,
John Wiley and Sons, New York, 1995). In hereditary and sporadic
prion disorders, patients develop plaques composed of abnormal
isoforms of the normal prion protein (PrP.sup.Sc).
[0130] A predominant mutant isoform, PrP.sup.Sc, also referred to
as AScr, differs from the normal cellular protein in its resistance
to protease degradation, insolubility after detergent extraction,
deposition in secondary lysosomes, post-translational synthesis,
and high .beta.-pleated sheet content. Genetic linkage has been
established for at least five mutations resulting in
Creutzfeldt-Jacob disease (CJD), Gerstmann-Straussler-Scheinker
syndrome (GSS), and fatal familial insomnia (FFI). (Baldwin, supra)
Methods for extracting fibril peptides from scrapie fibrils,
determining sequences and making such peptides are known in the art
(e.g., Beekes, M., et al. J. Gen. Virol. 76: 2567-76, 1995).
[0131] For example, one form of GSS has been linked to a PrP
mutation at codon 102, while telencephalic GSS segregates with a
mutation at codon 117. Mutations at codons 198 and 217 result in a
form of GSS in which neuritic plaques characteristic of Alzheimer's
disease contain PrP instead of A.beta. peptide. Certain forms of
familial CJD have been associated with mutations at codons 200 and
210; mutations at codons 129 and 178 have been found in both
familial CJD and FFI. (Baldwin, supra).
Cerebral Amyloidosis
[0132] Local deposition of amyloid is common in the brain,
particularly in elderly individuals. The most frequent type of
amyloid in the brain is composed primarily of A.beta. peptide
fibrils, resulting in dementia or sporadic (non-hereditary)
Alzheimer's disease. The most common occurrences of cerebral
amyloidosis are sporadic and not familial. For example, the
incidence of sporadic Alzheimer's disease and sporadic CAA greatly
exceeds the incidence of familial A.beta. and CAA. Moreover,
sporadic and familial forms of the disease cannot be distinguished
from each other (they differ only in the presence or absence of an
inherited genetic mutation); for example, the clinical symptoms and
the amyloid plaques formed in both sporadic and familial A.beta.
are very similar, if not identical.
[0133] Cerebral amyloid angiopathy (CAA) refers to the specific
deposition of amyloid fibrils in the walls of leptomingeal and
cortical arteries, arterioles and veins. It is commonly associated
with Alzheimer's disease, Down's syndrome and normal aging, as well
as with a variety of familial conditions related to stroke or
dementia (see Frangione et al., Amyloid: J. Protein Folding Disord.
8, Suppl. 1, 36-42 (2001)). CAA can occur sporadically or be
hereditary.
Senile Systemic Amyloidosis
[0134] Amyloid deposition, either systemic or focal, increases with
age. For example, fibrils of wild type transthyretin (TTR) are
commonly found in the heart tissue of elderly individuals. These
may be asymptomatic, clinically silent, or may result in heart
failure. Asymptomatic fibrillar focal deposits may also occur in
the brain (A.beta.), corpora amylacea of the prostate .beta..sub.2
microglobulin), joints and seminal vesicles.
Dialysis-Related Amyloidosis (DRA)
[0135] Plaques composed of .beta..sub.2 microglobulin
.beta..sub.2M) fibrils commonly develop in patients receiving long
term hemodialysis or peritoneal dialysis. .beta..sub.2
microglobulin is a 11.8 kiloDalton polypeptide and is the light
chain of Class I MHC antigens, which are present on all nucleated
cells. Under normal circumstances, .beta..sub.2M is usually
distributed in the extracellular space unless there is an impaired
renal function, in which case .beta..sub.2M is transported into
tissues where it polymerizes to form amyloid fibrils. Failure of
clearance such as in the case of impaired renal function, leads to
deposition in the carpal tunnel and other sites (primarily in
collagen-rich tissues of the joints). Unlike other fibril proteins,
.beta..sub.2M molecules are not produced by cleavage of a longer
precursor protein and are generally present in unfragmented form in
the fibrils. (Benson, supra). Retention and accumulation of this
amyloid precursor has been shown to be the main pathogenic process
underlying DRA. DRA is characterized by peripheral joint
osteoarthropathy (e.g., joint stiffness, pain, swelling, etc.).
Isoforms of .beta..sub.2M, glycated .beta..sub.2M, or polymers of
.beta..sub.2M in tissue are the most amyloidogenic form (as opposed
to native .beta..sub.2M). Unlike other types of amyloidosis,
.beta..sub.2M is confined largely to osteoarticular sites. Visceral
depositions are rare. Occasionally, these deposits may involve
blood vessels and other important anatomic sites.
[0136] Despite improved dialysis methods for removal of
.beta..sub.2M, the majority of patients have plasmatic
.beta..sub.2M concentrations that remain dramatically higher than
normal. These elevated .beta..sub.2M concentrations generally lead
to Diabetes-Related Amyloidosis (DRA) and cormorbidities that
contribute to mortality.
Islet Amyloid Polypeptide and Diabetes
[0137] Islet hyalinosis (amyloid deposition) was first described
over a century ago as the presence of fibrous protein aggregates in
the pancreas of patients with severe hyperglycemia (Opie, E L., J
Exp. Med. 5: 397-428, 1901). Today, islet amyloid, composed
predominantly of islet amyloid polypeptide (IAPP), or amylin, is a
characteristic histopathological marker in over 90% of all cases of
Type II diabetes (also known as Non-Insulin Dependent Diabetes, or
NIDDM). These fibrillar accumulations result from the aggregation
of the islet amyloid polypeptide (IAPP) or amylin, which is a 37
amino acid peptide, derived from a larger precursor peptide, called
pro-IAPP.
[0138] IAPP is co-secreted with insulin in response to .beta.-cell
secretagogues. This pathological feature is not associated with
insulin-dependent (Type I) diabetes and is a unifying
characteristic for the heterogeneous clinical phenotypes diagnosed
as NIDDM (Type II diabetes).
[0139] Longitudinal studies in cats and immunocytochemical
investigations in monkeys have shown that a progressive increase in
islet amyloid is associated with a dramatic decrease in the
population of insulin-secreting .beta.-cells and increased severity
of the disease. More recently, transgenic studies have strengthened
the relationship between IAPP plaque formation and .beta.-cell
apoptosis and dysfunction, indicating that amyloid deposition is a
principal factor in increasing severity of Type II diabetes.
[0140] IAPP has also been shown to induce .beta.-islet cell
toxicity in vitro, indicating that appearance of IAPP fibrils in
the pancreas of Type II or Type I diabetic patients (post-islet
transplantation) could contribute to the loss of the .beta.-cell
islets (Langerhans) and organ dysfunction. In patients with Type II
diabetes, the accumulation of pancreatic IAPP leads to formation of
oligomeric IAPP, leading to a buildup of IAPP-amyloid as insoluble
fibrous deposits which eventually destroys the insulin-producing
.beta. cells of the islet, resulting in 13 cell depletion and
failure (Westermark, P., Grimelius, L., Acta Path. Microbiol.
Scand., sect. A. 81: 291-300, 1973; de Koning, E J P., et al.,
Diabetologia 36: 378-384, 1993; and Lorenzo, A., et al., Nature
368: 756-760, 1994). Accumulation of IAPP as fibrous deposits can
also have an impact on the ratio of pro-IAPP to IAPP normally found
in plasma by increasing this ratio due to the trapping of IAPP in
deposits. Reduction of 13 cell mass can be manifested by
hyperglycemia and insulinemia. This .beta.-cell mass loss can lead
to a need for insulin therapy.
[0141] Diseases caused by the death or malfunctioning of a
particular type or types of cells can be treated by transplanting
into the patient healthy cells of the relevant type of cell. This
approach has been used for Type I diabetes patients. Often
pancreatic islet cells from a donor are cultured in vitro prior to
transplantation, to allow them to recover after the isolation
procedure or to reduce their immunogenicity. However, in many
instances islet cell transplantation is unsuccessful, due to death
of the transplanted cells. One reason for this poor success rate is
IAPP, which organizes into toxic oligomers. Toxic effects may
result from intracellular and extracellular accumulation of fibril
oligomers. The IAPP oligomers can form fibrils and become toxic to
the cells in vitro. In addition, IAPP fibrils are likely to
continue to grow after the cells are transplanted and cause death
or dysfunction of the cells. This may occur even when the cells are
from a healthy donor and the patient receiving the transplant does
not have a disease that is characterized by the presence of
fibrils. For example, compounds of the present invention may also
be used in preparing tissues or cells for transplantation according
to the methods described in International Patent Application (PCT)
number WO 01/003680.
[0142] The compounds of the invention may also stabilize the ratio
of the concentrations of Pro-IAPP/IAPP, pro-Insulin/Insulin and
C-peptide levels. In addition, as biological markers of efficacy,
the results of the different tests, such as the arginine-insulin
secretion test, the glucose tolerance test, insulin tolerance and
sensitivity tests, could all be used as markers of reduced
.beta.-cell mass and/or accumulation of amyloid deposits. Such
class of drugs could be used together with other drugs targeting
insulin resistance, hepatic glucose production, and insulin
secretion. Such compounds might prevent insulin therapy by
preserving 13-cell function and be applicable to preserving islet
transplants.
Hormone-Derived Amyloidoses
[0143] Endocrine organs may harbor amyloid deposits, particularly
in aged individuals. Hormone-secreting tumors may also contain
hormone-derived amyloid plaques, the fibrils of which are made up
of polypeptide hormones such as calcitonin (medullary carcinoma of
the thyroid), and atrial natriuretic peptide (isolated atrial
amyloidosis). Sequences and structures of these proteins are well
known in the art.
Miscellaneous Amyloidoses
[0144] There are a variety of other forms of amyloid disease that
are normally manifest as localized deposits of amyloid. In general,
these diseases are probably the result of the localized production
or lack of catabolism of specific fibril precursors or a
predisposition of a particular tissue (such as the joint) for
fibril deposition. Examples of such idiopathic deposition include
nodular AL amyloid, cutaneous amyloid, endocrine amyloid, and
tumor-related amyloid. Other amyloid related diseases include those
described in Table 1, such as familial amyloid polyneuropathy
(FAP), senile systemic amyloidosis, Tenosynovium, familial
amyloidosis, Ostertag-type, non-neuropathic amyloidosis, cranial
neuropathy, hereditary cerebral hemorrhage, familial dementia,
chronic dialysis, familial Creutzfeldt-Jakob disease;
Gerstmann-Straussler-Scheinker syndrome, hereditary spongiform
encephalopathies, prion diseases, familial Mediterranean fever,
Muckle-Well's syndrome, nephropathy, deafness, urticaria, limb
pain, cardiomyopathy, cutaneous deposits, multiple myeloma, benign
monoclonal gammopathy, maccoglobulinaemia, myeloma associated
amyloidosis, medullary carcinomas of the thyroid, isolated atrial
amyloid, and diabetes.
[0145] The compounds of the invention may be administered
therapeutically or prophylactically to treat diseases associated
with amyloid fibril formation, aggregation or deposition,
regardless of the clinical setting. The compounds of the invention
may act to ameliorate the course of an amyloid related disease
using any of the following mechanisms, such as, for example but not
limited to: slowing the rate of amyloid fibril formation or
deposition; lessening the degree of amyloid deposition; inhibiting,
reducing, or preventing amyloid fibril formation; inhibiting
amyloid induced inflammation; enhancing the clearance of amyloid
from, for example, the brain; or protecting cells from amyloid
induced (oligomers or fibrillar) toxicity.
[0146] In an embodiment, the compounds of the invention may be
administered therapeutically or prophylactically to treat diseases
associated with amyloid-.beta. fibril formation, aggregation or
deposition. The compounds of the invention may act to ameliorate
the course of an amyloid-.beta. related disease using any of the
following mechanisms (this list is meant to be illustrative and not
limiting): slowing the rate of amyloid-.beta. fibril formation or
deposition; lessening the degree of amyloid-.beta. deposition;
inhibiting, reducing, or preventing amyloid-.beta. fibril
formation; inhibiting neurodegeneration or cellular toxicity
induced by amyloid-.beta.; inhibiting amyloid-.beta. induced
inflammation; enhancing the clearance of amyloid-.beta. from the
brain; or favoring greater catabolism of A.beta..
[0147] Compounds of the invention may be effective in controlling
amyloid-.beta. deposition either following their entry into the
brain (following penetration of the blood brain barrier) or from
the periphery. When acting from the periphery, a compound may alter
the equilibrium of .beta. between the brain and the plasma so as to
favor the exit of .beta. from the brain. An increase in the exit of
.beta. from the brain would result in a decrease in A.beta. brain
concentration and therefore favor a decrease in A.beta. deposition.
In addition, compounds that penetrate the brain may control
deposition by acting directly on brain A.beta., e.g., by
maintaining it in a non-fibrillar form or favoring its clearance
from the brain. The compounds may slow down APP processing; may
increase degradation of A.beta. fibrils by macrophages or by
neuronal cells; or may decrease A.beta. production by activated
microglia. These compounds could also prevent A.beta. in the brain
from interacting with the cell surface and therefore prevent
neurotoxicity, neurodegeneration, or inflammation.
[0148] In a preferred embodiment, the method is used to treat
Alzheimer's disease (e.g., sporadic or familial AD). The method can
also be used prophylactically or therapeutically to treat other
clinical occurrences of amyloid-.beta. deposition, such as in
Down's syndrome individuals and in patients with cerebral amyloid
angiopathy ("CAA"), hereditary cerebral hemorrhage, or early
Alzheimer's disease.
[0149] In another embodiment, the method is used to treat mild
cognitive impairment. Mild Cognitive Impairment ("MCI") is a
condition characterized by a state of mild but measurable
impairment in thinking skills, which is not necessarily associated
with the presence of dementia. MCI frequently, but not necessarily,
precedes Alzheimer's disease.
[0150] Additionally, abnormal accumulation of APP and of
amyloid-.beta. protein in muscle fibers has been implicated in the
pathology of sporadic inclusion body myositis (IBM) (Askanas, V.,
et al. (1996) Proc. Natl. Acad. Sci. USA 93: 1314-1319; Askanas, V.
et al. (1995) Current Opinion in Rheumatology 7: 486-496).
Accordingly, the compounds of the invention can be used
prophylactically or therapeutically in the treatment of disorders
in which amyloid-beta protein is abnormally deposited at
non-neurological locations, such as treatment of IBM by delivery of
the compounds to muscle fibers.
[0151] Additionally, it has been shown that A.beta. is associated
with abnormal extracellular deposits, known as drusen, that
accumulate along the basal surface of the retinal pigmented
epithelium in individuals with age-related macular degeneration
(ARMD). ARMD is a cause of irreversible vision loss in older
individuals. It is believed that A.beta. deposition could be an
important component of the local inflammatory events that
contribute to atrophy of the retinal pigmented epithelium, drusen
biogenesis, and the pathogenesis of ARMD (Johnson, et al., Proc.
Natl. Acad. Sci. USA 99(18), 11830-5 (2002)).
[0152] In another embodiment, the invention also relates to a
method of treating or preventing an amyloid-related disease in a
subject (preferably a human) comprising administering to the
subject a therapeutic amount of a compound according to the
following Formulae or otherwise described herein, such that amyloid
fibril formation or deposition, neurodegeneration, or cellular
toxicity is reduced or inhibited. In another embodiment, the
invention relates to a method of treating or preventing an
amyloid-related disease in a subject (preferably a human)
comprising administering to the subject a therapeutic amount of a
compound according to the following Formulae or otherwise described
herein, such that cognitive function is improved or stabilized or
further deterioration in cognitive function is prevented, slowed,
or stopped in patients with brain amyloidosis, e.g., Alzheimer's
disease, Down's syndrome or cerebral amyloid angiopathy. These
compounds can also improve quality of daily living in these
subjects.
[0153] The therapeutic compounds of the invention may treat
amyloidosis related to type II diabetes by, for example,
stabilizing glycemia, preventing or reducing the loss of .beta.
cell mass, reducing or preventing hyperglycemia due to loss of
.beta. cell mass, and modulating (e.g., increasing or stabilizing)
insulin production. The compounds of the invention may also
stabilize the ratio of the concentrations of pro-IAPP/IAPP.
[0154] The therapeutic compounds of the invention may treat AA
(secondary) amyloidosis and/or AL (primary) amyloidosis, by
stabilizing renal function, decreasing proteinuria, increasing
creatinine clearance (e.g., by at least 50% or greater or by at
least 100% or greater), by leading to remission of chronic diarrhea
or weight gain (e.g., 10% or greater), or by reducing serum
creatinine. Visceral amyloid content as determined, e.g., by SAP
scintigraphy may also be reduced.
Compounds of the Invention
[0155] The present invention relates, at least in part, to the use
of certain chemical compounds (and pharmaceutical formulations
thereof) in the prevention or treatment of amyloid-related
diseases, including, inter alia, Alzheimer's disease, cerebral
amyloid angiopathy, inclusion body myositis, Down's syndrome,
diabetes related amyloidosis, hemodialysis-related amyloidosis
(.beta..sub.2M), primary amyloidosis (e.g., .lamda. or .kappa.
chain-related), familial amyloid polyneuropathy (FAP), senile
systemic amyloidosis, familial amyloidosis, Ostertag-type
non-neuropathic amyloidosis, cranial neuropathy, hereditary
cerebral hemorrhage, familial dementia, chronic dialysis, familial
Creutzfeldt-Jakob disease, Gerstmann-Straussler-Scheinker syndrome,
hereditary spongiform encephalopathies, prion diseases, familial
Mediterranean fever, Muckle-Well's syndrome, nephropathy, deafness,
urticaria, limb pain, cardiomyopathy, cutaneous deposits, multiple
myeloma, benign monoclonal gammopathy, maccoglobulinaemia, myeloma
associated amyloidosis, medullary carcinomas of the thyroid, and
isolated atrial amyloid.
[0156] The chemical structures herein are drawn according to the
conventional standards known in the art. Thus, where an atom, such
as a carbon atom, as drawn appears to have an unsatisfied valency,
then that valency is assumed to be satisfied by a hydrogen atom
even though that hydrogen atom is not necessarily explicitly drawn.
The structures of some of the compounds of this invention include
stereogenic carbon atoms. It is to be understood that isomers
arising from such asymmetry (e.g., all enantiomers and
diastereomers) are included within the scope of this invention
unless indicated otherwise. That is, unless otherwise stipulated,
any chiral carbon center may be of either (R)- or
(S)-stereochemistry. Such isomers can be obtained in substantially
pure form by classical separation techniques and by
stereochemically-controlled synthesis. Furthermore, alkenes can
include either the E- or Z-geometry, where appropriate. In
addition, the compounds of the present invention may exist in
unsolvated as well as solvated forms with acceptable solvents such
as water, THF, ethanol, and the like. In general, the solvated
forms are considered equivalent to the unsolvated forms for the
purposes of the present invention.
[0157] A "small molecule" refers to a compound that is not itself
the product of gene transcription or translation (e.g., protein,
RNA, or DNA) and preferably has a low molecular weight, e.g., less
than about 2500 amu.
[0158] In general, the term "nucleophile" is art-recognized to mean
a chemical group having a reactive pair of electrons that reacts
with a compound by displacing a leaving group (commonly another
nucleophile), such as commonly occur in aliphatic chemistry as
unimolecular (known as "S.sub.N1") or bimolecular ("S.sub.N2")
reactions. Examples of nucleophiles include uncharged compounds
such as amines, mercaptans, and alcohols, and charged groups such
as alkoxides, thiolates, carbanions, and a variety of organic and
inorganic anions. Illustrative anionic nucleophiles include, inter
alia, simple anions such as azide, cyanide, thiocyanate, acetate,
formate, or chloroformate, and bisulfite. Organometallic reagents
such as organocuprates, organozincs, organolithiums, Grignard
reagents, enolates, and acetylides, will under appropriate reaction
conditions, be suitable nucleophiles.
[0159] Similarly, an "electrophile" means an atom, molecule, or ion
able to accept an electron pair, particularly a pair of electrons
from a nucleophile, such as typically occurs during an
electrophilic substitution reaction. In an electrophilic
substitution reaction, an electrophile binds to a substrate with
the expulsion of another electrophile, e.g., the substitution of a
proton by another electrophile such as a nitronium ion on an
aromatic substrate (e.g., benzene). Electrophiles include cyclic
compounds such as epoxides, aziridines, episulfides, cyclic
sulfates, carbonates, lactones, and lactams; and non-cyclic
electrophiles include sulfates, sulfonates (e.g., tosylates),
chlorides, bromides, and iodides. Generally, an electrophile may be
a saturated carbon atom (e.g., a methylene group) bonded to a
leaving group; however, an electrophile may also be an unsaturated
group, such as an aldehyde, ketone, ester, or conjugated
(.alpha.,.beta.-unsaturated) analog thereof, which upon reaction
with a nucleophile forms an adduct.
[0160] The term "leaving group" generally refers to a group that is
readily displaced and substituted by a nucleophile (e.g., an amine,
a thiol, an alcohol, or cyanide). Such leaving groups are well
known and include carboxylates, N-hydroxysuccinimide ("NHS"),
N-hydroxybenzotriazole, a halogen (fluorine, chlorine, bromine, or
iodine), alkoxides, and thioalkoxides. A variety of sulfur-based
leaving groups are routinely used in synthetic chemistry, including
alkane sulfonyloxy groups (e.g., C.sub.1-C.sub.4 alkane such as
methane sulfonyloxy, ethane sulfonyloxy, propane sulfonyloxy, and
butane sulfonyloxy groups) and the halogenated analogs (e.g.,
halogeno(C.sub.1-C.sub.4 alkane) sulfonyloxy groups, such as
trifluoromethane sulfonyloxy (i.e., triflate),
2,2,2-trichloroethane sulfonyloxy, 3,3,3-tribromopropane
sulfonyloxy, and 4,4,4-trifluorobutane sulfonyloxy groups), as well
as arylsulfonyloxy groups (e.g., C.sub.6-C.sub.10 aryl optionally
substituted with 1 to 3 C.sub.1-C.sub.4 alkyl groups, such as
benzene sulfonyloxy, .alpha.-naphthylsulfonyloxy,
.beta.-naphthylsulfonyloxy, p-toluenesulfonyloxy (i.e., tosylates),
4-tert-butylbenzene sulfonyloxy, mesitylene sulfonyloxy, and
6-ethyl-.alpha.-naphthylsulfonyloxy groups).
[0161] "Activated esters" may be represented by the formula --COL,
where L is a leaving group, typical examples of which include
N-hydroxysulfosuccinimidyl and N-hydroxysuccinimidyl groups;
aryloxy groups substituted with electron-withdrawing groups (e.g.,
p-nitro, pentafluoro, pentachloro, p-cyano, or p-trifluoromethyl);
and carboxylic acids activated by a carbodiimide to form an
anhydride or mixed anhydride, e.g., --OCOR.sup.a or
--OCNR.sup.aNHR.sup.b, where R.sup.a and R.sup.b are independently
C.sub.1-C.sub.6 alkyl, C.sub.5-C.sub.8 alkyl (e.g., cyclohexyl),
C.sub.1-C.sub.6 perfluoroalkyl, or C.sub.1-C.sub.6 alkoxy groups.
An activated ester may be formed in situ or may be an isolable
reagent. Sulfosuccinimidyl esters, pentafluorothiophenol esters,
and sulfotetrafluorophenol are preferred activated esters. However,
the ester leaving group may be, for example, substituted or
unsubstituted C.sub.1-C.sub.6 alkyl (such as methyl, ethyl, propyl,
isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, or
hexyl), or substituted or unsubstituted C.sub.6-C.sub.14 aryl or
heterocyclic groups, such as 2-fluoroethyl, 2-chloroethyl,
2-bromoethyl, 2,2-dibromoethyl, 2,2,2-trichloroethyl,
3-fluoropropyl, 4-chlorobutyl, methoxymethyl,
1,1-dimethyl-1-methoxymethyl, ethoxymethyl, N-propoxymethyl,
isopropoxymethyl, N-butoxymethyl, tert-butoxymethyl, 1-ethoxyethyl,
1-methyl-1-methoxyethyl, 1-(isopropoxy)ethyl,
3-methoxypropyl-4-methoxybutyl, fluoromethoxymethyl,
2,2,2-trichloroethoxymethyl, bis(2-chloroethoxy)methyl,
3-fluoropropoxymethyl, 4-chlorobutoxyethyl, dibromomethoxyethyl,
2-chloroethoxypropyl, fluoromethoxybutyl, 2-methoxyethoxymethyl,
ethoxymethoxyethyl, methoxyethoxypropyl, methoxyethoxybutyl,
benzyl, phenethyl, 3-phenylpropyl, 4-phenylbutyl,
.alpha.-naphthylmethyl, .beta.-naphthylmethyl, diphenylmethyl,
triphenylmethyl, .alpha.-naphthyldipheylmethyl, 9-anthiylmethyl,
4-methylbenzyl, 2,4,6-trimethylbenzyl, 3,4,5-trimethylbenzyl,
4-methoxybenzyl, 4-methoxyphenyldiphenylmethyl, 2-nitrobenzyl,
4-nitrobenzyl, 4-chlorobenzyl, 4-bromobenzyl, 4-cyanobenzyl,
4-cyanobenzyldiphenylmethyl, or bis(2-nitrophenyl)methyl
groups.
[0162] The term "electron-withdrawing group" is art-recognized and
describes the ability of a substituent to attract valence electrons
(e.g., pi-electrons) from neighboring atoms, e.g., the substituent
is more electronegative than neighboring atoms, or it draws
electrons to itself more than a hydrogen atom would at the same
position. The Hammett sigma value (.sigma.) is an accepted measure
of a group's electron-donating and withdrawing ability, especially
the sigma para value (.sigma..sub.p). See, e.g., "Advanced Organic
Chemistry" by J. March, 5.sup.th Ed., John Wiley & Sons, Inc.,
New York, pp. 368-75 (2001). The Hammett constant values are
generally negative for electron-donating groups
(.sigma..sub.p=-0.66 for NH.sub.2) and positive for
electron-withdrawing groups (.sigma..sub.p=0.78 for a nitro group),
.sigma..sub.p indicating para substitution. Exemplary
electron-withdrawing groups include nitro, acyl(ketone),
formyl(aldehyde), sulfonyl, trifluoromethyl, halogeno (e.g., chloro
and fluoro), and cyano groups, among others. Conversely, an
"electron-donating group" designates a substituent that contributes
electrons more than hydrogen would if it occupied the same position
in the molecule. Examples include amino (including alkylamino and
dialkylamino), aryl, alkoxy (including aralkoxy), aryloxy, mercapto
and alkylthio, and hydroxyl groups, among others.
[0163] As used herein, "alkyl" groups include saturated
hydrocarbons having one or more carbon atoms, including
straight-chain alkyl groups (e.g., methyl, ethyl, propyl, butyl,
pentyl, hexyl, heptyl, octyl, nonyl, decyl, etc.), cyclic alkyl
groups (or "cycloalkyl" or "alicyclic" or "carbocyclic" groups)
(e.g., cyclopropyl, cyclopentyl, cyclohexyl, cycloheptyl,
cyclooctyl, etc.), branched-chain alkyl groups (isopropyl,
tert-butyl, sec-butyl, isobutyl, etc.), and alkyl-substituted alkyl
groups (e.g., alkyl-substituted cycloalkyl groups and
cycloalkyl-substituted alkyl groups). The term "aliphatic group"
includes organic moieties characterized by straight or
branched-chains, typically having between 1 and 22 carbon atoms. In
complex structures, the chains may be branched, bridged, or
cross-linked. Aliphatic groups include alkyl groups, alkenyl
groups, and alkynyl groups.
[0164] In certain embodiments, a straight-chain or branched-chain
alkyl group may have 30 or fewer carbon atoms in its backbone,
e.g., C.sub.1-C.sub.30 for straight-chain or C.sub.3-C.sub.30 for
branched-chain. In certain embodiments, a straight-chain or
branched-chain alkyl group may have 20 or fewer carbon atoms in its
backbone, e.g., C.sub.1-C.sub.20 for straight-chain or
C.sub.3-C.sub.20 for branched-chain, and more preferably 18 or
fewer. Likewise, preferred cycloalkyl groups have from 4-10 carbon
atoms in their ring structure, and more preferably have 4-7 carbon
atoms in the ring structure. The term "lower alkyl" refers to alkyl
groups having from 1 to 6 carbons in the chain, and to cycloalkyl
groups having from 3 to 6 carbons in the ring structure.
[0165] Unless the number of carbons is otherwise specified, "lower"
as in "lower aliphatic," "lower alkyl," "lower alkenyl," etc. as
used herein means that the moiety has at least one and less than
about 8 carbon atoms. In certain embodiments, a straight-chain or
branched-chain lower alkyl group has 6 or fewer carbon atoms in its
backbone (e.g., C.sub.1-C.sub.6 for straight-chain, C.sub.3-C.sub.6
for branched-chain), and more preferably 4 or fewer. Likewise,
preferred cycloalkyl groups have from 3-8 carbon atoms in their
ring structure, and more preferably have 5 or 6 carbons in the ring
structure. The term "C.sub.1-C.sub.6" as in .sup.13C.sub.1-C.sub.6
alkyl" means alkyl groups containing 1 to 6 carbon atoms.
[0166] Moreover, unless otherwise specified the term alkyl includes
both "unsubstituted alkyls" and "substituted alkyls," the latter of
which refers to alkyl groups having substituents replacing one or
more hydrogens on one or more carbons of the hydrocarbon backbone.
Such substituents may include, for example, alkenyl, alkynyl,
halogeno, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy,
alkoxycarbonyloxy, aryloxy, aryloxycarbonyloxy, carboxylate,
alkylcarbonyl, arylcarbonyl, alkoxycarbonyl, aminocarbonyl,
alkylaminocarbonyl, dialkylaminocarbonyl, alkylthiocarbonyl,
alkoxyl, phosphate, phosphonato, phosphinato, cyano, amino
(including alkylamino, dialkylamino, arylamino, diarylamino, and
alkylarylamino), acylamino (including alkylcarbonylamino,
arylcarbonylamino, carbamoyl and ureido), imino, sulfhydryl,
alkylthio, arylthio, thiocarboxylate, sulfates, alkylsulfinyl,
sulfonato, sulfamoyl, sulfonamido, nitro, trifluoromethyl, cyano,
azido, heterocyclic, alkylaryl, or aromatic (including
heteroaromatic) groups.
[0167] An "arylalkyl" group is an alkyl group substituted with an
aryl group (e.g., phenylmethyl (i.e., benzyl)). An "alkylaryl"
moiety is an aryl group substituted with an alkyl group (e.g.,
p-methylphenyl p-tolyl)). The term "n-alkyl" means a straight-chain
(i.e., unbranched) unsubstituted alkyl group. An "alkylene" group
is a divalent analog of the corresponding alkyl group. The terms
"alkenyl" and "alkynyl" refer to unsaturated aliphatic groups
analogous to alkyls, but which contain at least one double or
triple carbon-carbon bond respectively. Suitable alkenyl and
alkynyl groups include groups having 2 to about 12 carbon atoms,
preferably from 2 to about 6 carbon atoms.
[0168] The term "aromatic group" or "aryl group" includes
unsaturated and aromatic cyclic hydrocarbons as well as unsaturated
and aromatic heterocycles containing one or more rings. Aryl groups
may also be fused or bridged with alicyclic or heterocyclic rings
that are not aromatic so as to form a polycycle (e.g., tetralin).
An "arylene" group is a divalent analog of an aryl group. Aryl
groups can also be fused or bridged with alicyclic or heterocyclic
rings which are not aromatic so as to form a polycycle (e.g.,
tetralin).
[0169] The term "heterocyclic group" includes closed ring
structures analogous to carbocyclic groups in which one or more of
the carbon atoms in the ring is an element other than carbon, for
example, nitrogen, sulfur, or oxygen. Heterocyclic groups may be
saturated or unsaturated. Additionally, heterocyclic groups (such
as pyrrolyl, pyridyl, isoquinolyl, quinolyl, purinyl, and furyl)
may have aromatic character, in which case they may be referred to
as "heteroaryl" or "heteroaromatic" groups.
[0170] Unless otherwise stipulated, aryl and heterocyclic
(including heteroaryl) groups may also be substituted at one or
more constituent atoms. Examples of heteroaromatic and
heteroalicyclic groups may have 1 to 3 separate or fused rings with
3 to about 8 members per ring and one or more N, O, or S
heteroatoms. In general, the term "heteroatom" includes atoms of
any element other than carbon or hydrogen, preferred examples of
which include nitrogen, oxygen, sulfur, and phosphorus.
Heterocyclic groups may be saturated or unsaturated or
aromatic.
[0171] Examples of heterocycles include, but are not limited to,
acridinyl; azocinyl; benzimidazolyl; benzofuranyl;
benzothiofuranyl; benzothiophenyl; benzoxazolyl; benzthiazolyl;
benztriazolyl; benztetrazolyl; benzisoxazolyl; benzisothiazolyl;
benzimidazolinyl; carbazolyl; 4aH-carbazolyl; carbolinyl;
chromanyl; chromenyl; cinnolinyl; decahydroquinolinyl; 2H,
6H-1,5,2-dithiazinyl; dihydrofuro[2,3-b]tetrahydrofuran; furanyl;
furazanyl; imidazolidinyl; imidazolinyl; imidazolyl; 1H-indazolyl;
indolenyl; indolinyl; indolizinyl; indolyl; 3H-indolyl;
isobenzofuranyl; isochromanyl; isoindazolyl; isoindolinyl;
isoindolyl; isoquinolinyl; isothiazolyl; isoxazolyl;
methylenedioxyphenyl; morpholinyl; naphthyridinyl;
octahydroisoquinolinyl; oxadiazolyl; 1,2,3-oxadiazolyl;
1,2,4-oxadiazolyl; 1,2,5-oxadiazolyl; 1,3,4-oxadiazolyl;
oxazolidinyl; oxazolyl; oxazolidinyl; pyrimidinyl; phenanthridinyl;
phenanthrolinyl; phenazinyl; phenothiazinyl; phenoxathiinyl;
phenoxazinyl; phthalazinyl; piperazinyl; piperidinyl; piperidonyl;
4-piperidonyl; piperonyl; pteridinyl; purinyl; pyranyl; pyrazinyl;
pyrazolidinyl; pyrazolinyl; pyrazolyl; pyridazinyl; pyridooxazole;
pyridoimidazole; pyridothiazole; pyridinyl; pyridyl; pyrimidinyl;
pyrrolidinyl; pyrrolinyl; 2H-pyrrolyl; pyrrolyl; quinazolinyl;
quinolinyl; 4H-quinolizinyl; quinoxalinyl; quinuclidinyl;
tetrahydrofuranyl; tetrahydroisoquinolinyl; tetrahydroquinolinyl;
tetrazolyl; 6H-1,2,5-thiadiazinyl; 1,2,3-thiadiazolyl;
1,2,4-thiadiazolyl; 1,2,5-thiadiazolyl; 1,3,4-thiadiazolyl;
thianthrenyl; thiazolyl; thienyl; thienothiazolyl; thienooxazolyl;
thienoimidazolyl; thiophenyl; triazinyl; 1,2,3-triazolyl;
1,2,4-triazolyl; 1,2,5-triazolyl; 1,3,4-triazolyl; and xanthenyl.
Preferred heterocycles include, but are not limited to, pyridinyl;
furanyl; thienyl; pyrrolyl; pyrazolyl; pyrrolidinyl; imidazolyl;
indolyl; benzimidazolyl; 1H-indazolyl; oxazolidinyl;
benzotriazolyl; benzisoxazolyl; oxindolyl; benzoxazolinyl; and
isatinoyl groups. Also included are fused ring and Spiro compounds
containing, for example, the above heterocycles.
[0172] A common hydrocarbon aryl group is a phenyl group having one
ring. Two-ring hydrocarbon aryl groups include naphthyl, indenyl,
benzocyclooctenyl, benzocycloheptenyl, pentalenyl, and azulenyl
groups, as well as the partially hydrogenated analogs thereof such
as indanyl and tetrahydronaphthyl. Exemplary three-ring hydrocarbon
aryl groups include acephthylenyl, fluorenyl, phenalenyl,
phenanthrenyl, and anthracenyl groups.
[0173] Aryl groups also include heteromonocyclic aryl groups, i.e.,
single-ring heteroaryl groups, such as thienyl, furyl, pyranyl,
pyrrolyl, imidazolyl, pyrazolyl, pyridinyl, pyrazinyl, pyrimidinyl,
and pyridazinyl groups; and oxidized analogs thereof such as
pyridonyl, oxazolonyl, pyrazolonyl, isoxazolonyl, and thiazolonyl
groups. The corresponding hydrogenated (i.e., non-aromatic)
heteromonocylic groups include pyrrolidinyl, pyrrolinyl,
imidazolidinyl, imidazolinyl, pyrazolidinyl, pyrazolinyl, piperidyl
and piperidino, piperazinyl, and morpholino and morpholinyl
groups.
[0174] Aryl groups also include fused two-ring heteroaryls such as
indolyl, isoindolyl, indolizinyl, indazolyl, quinolinyl,
isoquinolinyl, phthalazinyl, quinoxalinyl, quinazolinyl,
cinnolinyl, chromenyl, isochromenyl, benzothienyl, benzimidazolyl,
benzothiazolyl, purinyl, quinolizinyl, isoquinolonyl, quinolonyl,
naphthyridinyl, and pteridinyl groups, as well as the partially
hydrogenated analogs such as chromanyl, isochromanyl, indolinyl,
isoindolinyl, and tetrahydroindolyl groups. Aryl groups also
include fused three-ring groups such as phenoxathiinyl, carbazolyl,
phenanthridinyl, acridinyl, perimidinyl, phenanthrolinyl,
phenazinyl, phenothiazinyl, phenoxazinyl, and dibenzofuranyl
groups.
[0175] Some typical aryl groups include substituted or
unsubstituted 5- and 6-membered single-ring groups. In another
aspect, each Ar group may be selected from the group consisting of
substituted or unsubstituted phenyl, pyrrolyl, furyl, thienyl,
thiazolyl, isothiaozolyl, imidazolyl, triazolyl, tetrazolyl,
pyrazolyl, oxazolyl, isooxazolyl, pyridinyl, pyrazinyl,
pyridazinyl, and pyrimidinyl groups. Further examples include
substituted or unsubstituted phenyl, 1-naphthyl, 2-naphthyl,
biphenyl, 1-pyrrolyl, 2-pyrrolyl, 3-pyrrolyl, 3-pyrazolyl,
2-imidazolyl, 4-imidazolyl, pyrazinyl, 2-oxazolyl, 4-oxazolyl,
5-oxazolyl, 3-isoxazolyl, 4-isoxazolyl, 5-isoxazolyl, 2-thiazolyl,
4-thiazolyl, 5-thiazolyl, 2-furyl, 3-furyl, 2-thienyl, 3-thienyl,
2-pyridyl, 3-pyridyl, 4-pyridyl, 2-pyrimidyl, 4-pyrimidyl,
5-benzothiazolyl, purinyl, 2-benzimidazolyl, 5-indolyl,
1-isoquinolyl, 5-isoquinolyl, 2-quinoxalinyl, 5-quinoxalinyl,
3-quinolyl, and 6-quinolyl groups.
[0176] The term "amine" or "amino," as used herein, refers to an
unsubstituted or substituted moiety of the formula
--NR.sup.aR.sup.b, in which R.sup.a and R.sup.b are each
independently hydrogen, alkyl, aryl, or heterocyclyl, or R.sup.a
and R.sup.b, taken together with the nitrogen atom to which they
are attached, form a cyclic moiety having from 3 to 8 atoms in the
ring. Thus, the term amino includes cyclic amino moieties such as
piperidinyl or pyrrolidinyl groups, unless otherwise stated. Thus,
the term "alkylamino" as used herein means an alkyl group having an
amino group attached thereto. Suitable alkylamino groups include
groups having 1 to about 12 carbon atoms, preferably from 1 to
about 6 carbon atoms. The term amino includes compounds or moieties
in which a nitrogen atom is covalently bonded to at least one
carbon or heteroatom. The term "dialkylamino" includes groups
wherein the nitrogen atom is bound to at least two alkyl groups.
The term "arylamino" and "diarylamino" include groups wherein the
nitrogen is bound to at least one or two aryl groups, respectively.
The term "alkylarylamino" refers to an amino group which is bound
to at least one alkyl group and at least one aryl group. The term
"alkaminoalkyl" refers to an alkyl, alkenyl, or alkynyl group
substituted with an alkylamino group. The term "amide" or
"aminocarbonyl" includes compounds or moieties which contain a
nitrogen atom which is bound to the carbon of a carbonyl or a
thiocarbonyl group.
[0177] The term "alkylthio" refers to an alkyl group, having a
sulfhydryl group attached thereto. Suitable alkylthio groups
include groups having 1 to about 12 carbon atoms, preferably from 1
to about 6 carbon atoms.
[0178] The term "alkylcarboxyl" as used herein means an alkyl group
having a carboxyl group attached thereto.
[0179] The term "alkoxy" as used herein means an alkyl group having
an oxygen atom attached thereto. Representative alkoxy groups
include groups having 1 to about 12 carbon atoms, preferably 1 to
about 6 carbon atoms, e.g., methoxy, ethoxy, propoxy, tert-butoxy
and the like. Examples of alkoxy groups include methoxy, ethoxy,
isopropyloxy, propoxy, butoxy, and pentoxy groups. The alkoxy
groups can be substituted with groups such as alkenyl, alkynyl,
halogen, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy,
alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl,
arylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl,
dialkylaminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate,
phosphonato, phosphinato, cyano, amino (including alkyl amino,
dialkylamino, arylamino, diarylamino, and alkylarylamino),
acylamino (including alkylcarbonylamino, arylcarbonylamino,
carbamoyl and ureido), imino, sulfhydryl, alkylthio, arylthio,
thiocarboxylate, sulfates, alkylsulfinyl, sulfonato, sulfamoyl,
sulfonamido, nitro, trifluoromethyl, cyano, azido, heterocyclyl,
alkylaryl, or an aromatic or heteroaromatic moieties. Examples of
halogen substituted alkoxy groups include, but are not limited to,
fluoromethoxy, difluoromethoxy, trifluoromethoxy, chloromethoxy,
dichloromethoxy, trichloromethoxy, etc., as well as perhalogenated
alkyloxy groups.
[0180] The term "acylamino" includes moieties wherein an amino
moiety is bonded to an acyl group. For example, the acylamino group
includes alkylcarbonylamino, arylcarbonylamino, carbamoyl and
ureido groups.
[0181] The terms "alkoxyalkyl", "alkylaminoalkyl" and
"thioalkoxyalkyl" include alkyl groups, as described above, which
further include oxygen, nitrogen or sulfur atoms replacing one or
more carbons of the hydrocarbon backbone.
[0182] The term "carbonyl" or "carboxy" includes compounds and
moieties which contain a carbon connected with a double bond to an
oxygen atom. Examples of moieties which contain a carbonyl include
aldehydes, ketones, carboxylic acids, amides, esters, anhydrides,
etc.
[0183] The term "ether" or "ethereal" includes compounds or
moieties which contain an oxygen bonded to two carbon atoms. For
example, an ether or ethereal group includes "alkoxyalkyl" which
refers to an alkyl, alkenyl, or alkynyl group substituted with an
alkoxy group.
[0184] A "sulfonate" group is a --SO.sub.3H or
--SO.sub.3.sup.-X.sup.+ group bonded to a carbon atom, where
X.sup.+ is a cationic counter ion group. Similarly, a "sulfonic
acid" compound has a --SO.sub.3H or --SO.sub.3.sup.-X.sup.+ group
bonded to a carbon atom, where X+ is a cationic group. A "sulfate"
as used herein is a --OSO.sub.3H or --OSO.sub.3.sup.-X.sup.+ group
bonded to a carbon atom, and a "sulfuric acid" compound has a
--SO.sub.3H or --OSO.sub.3.sup.-X.sup.+ group bonded to a carbon
atom, where X.sup.+ is a cationic group. According to the
invention, a suitable cationic group may be a hydrogen atom. In
certain cases, the cationic group may actually be another group on
the therapeutic compound that is positively charged at
physiological pH, for example an amino group.
[0185] A "counter ion" is required to maintain electroneutrality.
Examples of anionic counter ions include halide, triflate, sulfate,
nitrate, hydroxide, carbonate, bicarbonate, acetate, phosphate,
oxalate, cyanide, alkylcarboxylate, N-hydroxysuccinimide,
N-hydroxybenzotriazole, alkoxide, thioalkoxide, alkane sulfonyloxy,
halogenated alkane sulfonyloxy, arylsulfonyloxy, bisulfate,
oxalate, valerate, oleate, palmitate, stearate, laurate, borate,
benzoate, lactate, citrate, maleate, fumarate, succinate, tartrate,
naphthylate mesylate, glucoheptonate, or lactobionate. Compounds
containing a cationic group covalently bonded to an anionic group
may be referred to as an "internal salt."
[0186] The term "nitro" means --NO.sub.2; the term "halogen" or
"halogeno" or "halo" designates --F, --Cl, --Br or --I; the term
"thiol," "thio," or "mercapto" means SH; and the term "hydroxyl" or
"hydroxy" means --OH.
[0187] The term "acyl" refers to a carbonyl group that is attached
through its carbon atom to a hydrogen (i.e., a formyl), an
aliphatic group (e.g., acetyl), an aromatic group (e.g., benzoyl),
and the like. The term "substituted acyl" includes acyl groups
where one or more of the hydrogen atoms on one or more carbon atoms
are replaced by, for example, an alkyl group, alkynyl group,
halogen, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy,
alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl,
arylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl,
dialkylaminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate,
phosphonato, phosphinato, cyano, amino (including alkyl amino,
dialkylamino, arylamino, diarylamino, and alkylarylamino),
acylamino (including alkylcarbonylamino, arylcarbonylamino,
carbamoyl and ureido), imino, sulfhydryl, alkylthio, arylthio,
thiocarboxylate, sulfates, alkylsulfinyl, sulfonato, sulfamoyl,
sulfonamido, nitro, trifluoromethyl, cyano, azido, heterocyclyl,
alkylaryl, or an aromatic or heteroaromatic moiety.
[0188] Unless otherwise specified, the chemical moieties of the
compounds of the invention, including those groups discussed above,
may be "substituted or unsubstituted." In some embodiments, the
term "substituted" means that the moiety has substituents placed on
the moiety other than hydrogen (i.e., in most cases, replacing a
hydrogen), which allow the molecule to perform its intended
function. Examples of substituents include moieties selected from
straight or branched alkyl (preferably C.sub.1-C.sub.5), cycloalkyl
(preferably C.sub.3-C.sub.8), alkoxy (preferably C.sub.1-C.sub.6),
thioalkyl (preferably C.sub.1-C.sub.6), alkenyl (preferably
C.sub.2-C.sub.6), alkynyl (preferably C.sub.2-C.sub.6),
heterocyclic, carbocyclic, aryl (e.g., phenyl), aryloxy (e.g.,
phenoxy), aralkyl (e.g., benzyl), aryloxyalkyl (e.g.,
phenyloxyalkyl), arylacetamidoyl, alkylaryl, heteroaralkyl,
alkylcarbonyl and arylcarbonyl or other such acyl group,
heteroarylcarbonyl, and heteroaryl groups, as well as
(CR'R'').sub.0-3NR'R'' (e.g., --NH.sub.2), (CR'R'').sub.0-3CN
(e.g., --CN), --NO.sub.2, halogen (e.g., --F, --Cl, --Br, or --I),
(CR'R'').sub.0-3C(halogen).sub.3 (e.g., --CF.sub.3),
(CR'R'').sub.0-3CH(halogen).sub.2,
(CR'R'').sub.0-3CH.sub.2(halogen), (CR'R'').sub.0-3CONR'R'',
(CR'R'').sub.0-3(CNH)NR'R'', (CR'R'').sub.0-3S(O).sub.1-2NR'R'',
(CR'R'').sub.0-3CHO, (CR'R'').sub.0-3O(CR'R'').sub.0-3H,
(CR'R'').sub.0-3S(O).sub.0-3R' (e.g., --SO.sub.3H),
(CR'R'').sub.0-3O(CR'R'').sub.0-3H (e.g., --CH.sub.2OCH.sub.3 and
--OCH.sub.3), (CR'R'').sub.0-3S(CR'R'').sub.0-3H (e.g., --SH and
--SCH.sub.3), (CR'R'').sub.0-3OH (e.g., --OH),
(CR'R'').sub.0-3COR', (CR'R'').sub.0-3 (substituted or
unsubstituted phenyl), (CR'R'').sub.0-3(C.sub.3-C.sub.8
cycloalkyl), (CR'R'').sub.0-3CO.sub.2R' (e.g., --CO.sub.2H), and
(CR'R'').sub.0-3OR' groups, wherein R' and R'' are each
independently hydrogen, a C.sub.1-C.sub.5 alkyl, C.sub.2-C.sub.5
alkenyl, C.sub.2-C.sub.5 alkynyl, or aryl group; or the side chain
of any naturally occurring amino acid.
[0189] In another embodiment, a substituent may be selected from
straight or branched alkyl (preferably C.sub.1-C.sub.5), cycloalkyl
(preferably C.sub.3-C.sub.8), alkoxy (preferably C.sub.1-C.sub.6),
thioalkyl (preferably C.sub.1-C.sub.6), alkenyl (preferably
C.sub.2-C.sub.6), alkynyl (preferably C.sub.2-C.sub.6),
heterocyclic, carbocyclic, aryl (e.g., phenyl), aryloxy (e.g.,
phenoxy), aralkyl (e.g., benzyl), aryloxyalkyl (e.g.,
phenyloxyalkyl), arylacetamidoyl, alkylaryl, heteroaralkyl,
alkylcarbonyl and arylcarbonyl or other such acyl group,
heteroarylcarbonyl, or heteroaryl group, (CR'R'').sub.0-10NR'R''
(e.g., --NH.sub.2), (CR'R'').sub.0-10CN (e.g., --CN), NO.sub.2,
halogen (e.g., F, Cl, Br, or I), (CR'R'').sub.0-10C(halogen).sub.3
(e.g., --CF.sub.3), (CR'R'').sub.0-10CH(halogen).sub.2,
(CR'R'').sub.0-10CH.sub.2(halogen), (CR'R'').sub.0-10CONR'R'',
(CR'R'').sub.0-10(CNH)NR'R'', (CR'R'').sub.0-10S(O).sub.1-2NR'R'',
(CR'R'').sub.0-10CHO, (CR'R'').sub.0-10O(CR'R'').sub.0-10H,
(CR'R'').sub.0-10S(O).sub.0-3R' (e.g., --SO.sub.3H),
(CR'R'').sub.0-10O(CR'R'').sub.0-10H (e.g., --CH.sub.2OCH.sub.3 and
--OCH.sub.3), (CR'R'').sub.0-10S(CR'R'').sub.0-3H (e.g., --SH and
--SCH.sub.3), (CR'R'').sub.0-10OH (e.g., --OH),
(CR'R'').sub.0-10COR', (CR'R'').sub.0-10 (substituted or
unsubstituted phenyl), (CR'R'').sub.0-10(C.sub.3-C.sub.8
cycloalkyl), (CR'R'').sub.0-10CO.sub.2R' (e.g., --CO.sub.2H), or
(CR'R'').sub.0-10OR' group, or the side chain of any naturally
occurring amino acid; wherein R' and R'' are each independently
hydrogen, a C.sub.1-C.sub.5 alkyl, C.sub.2-C.sub.5 alkenyl,
C.sub.2-C.sub.5 alkynyl, or aryl group, or R' and R'' taken
together are a benzylidene group or a
--(CH.sub.2).sub.2--O--(CH.sub.2).sub.2-- group.
[0190] It will be understood that "substitution" or "substituted
with" includes the implicit proviso that such substitution is in
accordance with the permitted valence of the substituted atom and
the substituent, and that the substitution results in a stable
compound, e.g., which does not spontaneously undergo transformation
such as by rearrangement, cyclization, elimination, etc. As used
herein, the term "substituted" is meant to include all permissible
substituents of organic compounds. In a broad aspect, the
permissible substituents include acyclic and cyclic, branched and
unbranched, carbocyclic and heterocyclic, aromatic and nonaromatic
substituents of organic compounds. The permissible substituents can
be one or more.
[0191] In some embodiments, a "substituent" may be selected from
the group consisting of, for example, halogeno, trifluoromethyl,
nitro, cyano, C.sub.1-C.sub.6 alkyl, C.sub.2-C.sub.6 alkenyl,
C.sub.2-C.sub.6 alkynyl, C.sub.1-C.sub.6 alkylcarbonyloxy,
arylcarbonyloxy, C.sub.1-C.sub.6 alkoxycarbonyloxy,
aryloxycarbonyloxy, C.sub.1-C.sub.6 alkylcarbonyl, C.sub.1-C.sub.6
alkoxycarbonyl, C.sub.1-C.sub.6 alkoxy, C.sub.1-C.sub.6 alkylthio,
arylthio, heterocyclyl, aralkyl, and aryl (including heteroaryl)
groups.
[0192] In one embodiment, the invention pertains to compounds of
Formula I:
##STR00009##
wherein:
[0193] R.sup.1 is a substituted or unsubstituted cycloalkyl,
heterocyclic, aryl, arylcycloalkyl, bicyclic or tricyclic ring, a
bicyclic or tricyclic fused ring group, or a substituted or
unsubstituted C.sub.2-C.sub.10 alkyl group;
[0194] R.sup.2 is selected from a group consisting of hydrogen,
alkyl, mercaptoalkyl, alkenyl, alkynyl, cycloalkyl, aryl,
arylalkyl, thiazolyl, triazolyl, imidazolyl, benzothiazolyl, and
benzoimidazolyl;
[0195] Y is SO.sub.3.sup.-X.sup.+, OSO.sub.3.sup.-X.sup.+, or
SSO.sub.3.sup.-X.sup.+;
[0196] X.sup.+ is hydrogen, a cationic group, or ester-forming
group; and
[0197] each of L.sup.1 and L.sup.2 is independently a substituted
or unsubstituted C.sub.1-C.sub.5 alkyl group or absent, or a
pharmaceutically acceptable salt thereof, provided that when
R.sub.1 is alkyl, L.sup.1 is absent.
[0198] In a further embodiment, the invention pertains to compounds
of Formula II:
##STR00010##
wherein:
[0199] R.sup.1 is a substituted or unsubstituted cyclic, bicyclic,
tricyclic, or benzoheterocyclic group or a substituted or
unsubstituted C.sub.2-C.sub.10 alkyl group;
[0200] R.sup.2 is hydrogen, alkyl, mercaptoalkyl, alkenyl, alkynyl,
cycloalkyl, aryl, arylalkyl, thiazolyl, triazolyl, imidazolyl,
benzothiazolyl, benzoimidazolyl, or linked to R.sup.1 to form a
heterocycle;
[0201] Y is SO.sub.3.sup.-X.sup.+, OSO.sub.3.sup.-X.sup.+, or
SSO.sub.3.sup.-X.sup.+;
[0202] X.sup.+ is hydrogen, a cationic group, or an ester forming
moiety;
[0203] m is 0 or 1;
[0204] n is 1, 2, 3, or 4;
[0205] L is substituted or unsubstituted C.sub.1-C.sub.3 alkyl
group or absent, or a pharmaceutically acceptable salt thereof,
provided that when R.sup.1 is alkyl, L is absent.
[0206] In a further embodiment, R.sup.2 is hydrogen. In another
further embodiment, R.sup.1 is straight chain alkyl, for example,
ethyl, n-pentyl, n-heptyl, or n-octyl. In another embodiment,
R.sup.1 is t-butyl. In yet another alternate embodiment, R.sup.1 is
C.sub.7-C.sub.10 bicycloalkyl or tricycloalkyl, such as, for
example, tricyclo[3.3.1.0.sup.3,7]decyl (or adamantyl),
bicyclo[2.1.2]heptyl, or indolyl. In another alternate embodiment,
R.sup.1 is tetrahydronaphthyl.
[0207] In one embodiment, L.sup.2 is --(CH.sub.2).sub.3--. In
another further embodiment, L.sup.2 is --(CH.sub.2).sub.4-- or
--(CH.sub.2).sub.5--. In yet another further embodiment, L.sub.2 is
--(CH.sub.2).sub.2--. In yet another further embodiment, L.sup.2 is
substituted alkyl, e.g., --CH.sub.2--(CHOH)--CH.sub.2--.
[0208] In another embodiment, L.sup.1 is CH.sub.2CH.sub.2 or
absent.
[0209] In a further embodiment, R.sup.1 is branched alkyl, e.g.,
t-butyl. In another embodiment, R.sup.1 is adamanyl. In another
embodiment, R.sup.1 is cyclic alkyl, e.g., cyclopropyl, cyclohexyl,
cycloheptyl, cyclo-octyl, etc. The cycloalkyl moieties may be
substituted further, e.g., with additional alkyl groups or other
groups which allow the molecule to perform its intended function.
In another embodiment, R.sup.1 is alkyl substituted with a
propargyl moiety (e.g., HC.ident.C--). In another embodiment,
R.sup.1 is cyclohexyl substituted with one or more methyl or
propargyl groups.
[0210] In other embodiments, L.sup.1 is a C.sub.1-C.sub.2 alkyl
linker group (e.g., --CH(CH.sub.3)-- or --(CH.sub.2).sub.2--. In a
further embodiment, R.sup.1 is phenyl. In certain embodiments,
R.sup.1 is substituted with a methoxy group. In other embodiments,
L.sup.1 is C.sub.3, e.g., --(CH.sub.2).sub.3-- or
C(CH.sub.3).sub.2--. In certain embodiments, L.sup.1 is
substituted, e.g., with an alkoxy, carboxylate (--COOH), benzyl,
amido (--C.dbd.O--NH--), or ester (C.dbd.O--C--O) group. In certain
embodiment, the ester group is a methyl, ethyl, propyl, butyl,
cyclohexyl, or benzyl ester. In other embodiments, the ester group
may be propenyl. In other embodiments, L.sup.1 is substituted with
a carboxylate group. In a further embodiment, R.sup.1 is
substituted with a substituted amido group, wherein the amido group
is substituted with an alkyl, e.g., methyl, ethyl, propyl, butyl,
pentyl, or hexyl group. In another embodiment, the alkyl R.sup.1
group is a substituted with a --C.dbd.O--NH--OH, C.dbd.O--NH.sub.2,
or an amido group. In certain embodiments, the amido group is
substituted with an alkyl (e.g., methyl, ethyl, propyl, butyl,
pentyl, hexyl, cyclohexyl, etc.), a benzyl or an aryl group. In
another embodiment, the amido group is substituted with a
--CH(CH.sub.2).sub.2 group. R.sup.1 itself may be substituted with
a phenyl or may be branched or straight chain alkyl. In certain
embodiments, R.sup.1 may also be substituted with a thioether
moiety. Examples of thioethers include S-Me, S-Et, etc. In certain
embodiments, the alkyl R.sup.1 moiety is substituted with both an
aryl or a thioether moiety and an amido moiety. In other
embodiments, the alkyl R.sup.1 moiety may be substituted with both
a thioether and a carboxylate moiety. In other embodiments, alkyl
R.sup.1 groups are substituted with hydroxyl. R.sup.1 groups, e.g.,
alkyl R.sup.1 groups, may also be substituted with both thioether
and hydroxyl groups. In other embodiments, R.sup.1 groups, e.g.,
alkyl R.sup.1 groups are substituted with cyano groups. Examples of
R.sup.1 groups including --CN moieties include
--C(CH.sub.3).sub.2CN, cyclohexyl substituted with one or more
cyano groups, etc.
[0211] In other embodiments, alkyl R.sup.1 groups are substituted
with aryl groups. The aryl groups may be substituted phenyl, for
example. The substituted phenyl may be substituted with one or more
substituents such as hydroxy, cyano and alkoxy. In other
embodiments, alkyl R.sup.1 groups are substituted with tetrazolyl
or substituted or unsubstituted benzyl.
[0212] In a further embodiment, L.sup.1 is
--C(CH.sub.3).sub.2--(CH.sub.2)--. In another embodiment, L.sup.1
is --(C(CH.sub.3).sub.2--CHOH--. In yet another embodiment, L.sup.1
is --(C(CH.sub.3).sub.2CH(OMe)-. In another embodiment, R.sup.1 is
substituted or unsubstituted phenyl. In a further embodiment,
R.sup.1 is para-substituted phenyl. Examples of substitutuents
include but are not limited to fluorine, chlorine, bromine, iodine,
methyl, t-butyl, alkoxy, methoxy, etc. In other embodiment, R.sup.1
is substituted at the meta position. Examples of substituents
include methoxy, chloro, methyl, t-butyl, fluoro, alkyl, alkoxy,
iodo, trifluoroalkyl, methoxy, etc. In another embodiment, R.sup.1
is phenyl substituted in the ortho position, with similar
substituents. In another embodiment, L.sup.1 comprises a cycloalkyl
moiety, e.g., cyclopentyl. In another embodiment, L.sup.1 comprises
an alkyenyl group and, optionally, a substituted aryl group, with
substituents similar to those described about.
[0213] In certain embodiments, R.sup.1 is cyclopropyl or
cyclohexyl. In certain embodiments, the cyclopropyl or cyclohexyl
group is substituted with an ether group or an alkyl group. In
certain further embodiments, the ether group is a benzyl ether
group.
[0214] In another embodiment, wherein R.sup.1 is alkyl, it is
substituted with groups such as phenyl, or hydroxy.
[0215] In other embodiments, the compound of the invention is
selected from the group consisting of
##STR00011## ##STR00012## ##STR00013## ##STR00014## ##STR00015##
##STR00016## ##STR00017## ##STR00018## ##STR00019## ##STR00020##
##STR00021## ##STR00022## ##STR00023## ##STR00024## ##STR00025##
##STR00026## ##STR00027## ##STR00028## ##STR00029## ##STR00030##
##STR00031## ##STR00032## ##STR00033## ##STR00034## ##STR00035##
##STR00036## ##STR00037## ##STR00038## ##STR00039## ##STR00040##
##STR00041## ##STR00042## ##STR00043## ##STR00044## ##STR00045##
##STR00046## ##STR00047## ##STR00048## ##STR00049## ##STR00050##
##STR00051## ##STR00052## ##STR00053## ##STR00054## ##STR00055##
##STR00056## ##STR00057## ##STR00058## ##STR00059##
and pharmaceutically acceptable salts, esters, and prodrugs
thereof.
[0216] In another embodiment, the invention pertains to compounds
of Formula III:
##STR00060##
wherein:
[0217] A is nitrogen or oxygen;
[0218] R.sup.11 is hydrogen, salt-forming cation, ester forming
group, --(CH.sub.2).sub.x-Q, or when A is nitrogen, A and R.sup.11
taken together may be the residue of a natural or unnatural amino
acid or a salt or ester thereof;
[0219] Q is hydrogen, thiazolyl, triazolyl, imidazolyl,
benzothiazolyl, or benzoimidazolyl;
[0220] x is 0, 1, 2, 3, or 4;
[0221] n is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10;
[0222] R.sup.3, R.sup.3a, R.sup.4, R.sup.4a, R.sup.5, R.sup.5a,
R.sup.6, R.sup.6a, R.sup.7 and R.sup.7a are each independently
hydrogen, alkyl, mercaptoalkyl, alkenyl, alkynyl, cycloalkyl, aryl,
alkylcarbonyl, arylcarbonyl, alkoxycarbonyl, cyano, halogen, amino,
tetrazolyl, or two R groups on adjacent ring atoms taken together
with the ring atoms form a double bond, provided that one of
R.sup.3, R.sup.3a, R.sup.4, R.sup.5, R.sup.5a, R.sup.6, R.sup.6a,
R.sup.7 and R.sup.7a is a moiety of the Formula IIIa:
##STR00061##
wherein:
[0223] m is 0, 1, 2, 3, or 4;
[0224] R.sup.A, R.sup.B, R.sup.C, R.sup.D, and R.sup.E are
independently selected from a group of hydrogen, halogen, hydroxyl,
alkyl, alkoxyl, halogenated alkyl, mercaptoalkyl, alkenyl, alkynyl,
cycloalkyl, aryl, cyano, thiazolyl, triazolyl, imidazolyl,
tetrazolyl, benzothiazolyl, and benzoimidazolyl; and
pharmaceutically acceptable salts and esters thereof, provided that
said compound is not
3-(4-phenyl-1,2,3,6-tetrahydro-1-pyridyl)-1-propanesulfonic
acid.
[0225] In a further embodiment, n is 2, 3 or 4.
[0226] In another embodiment, R.sup.11 is a salt-forming cation.
Examples of salt forming cations include pharmaceutically
acceptable salts described herein as well as lithium, sodium,
potassium, magnesium, calcium, barium, zinc, iron, and ammonium. In
another embodiment, R.sup.11 is an ester-forming group. An
ester-forming group includes groups which when bound form an ester.
Examples of such groups include substituted or unsubstituted alkyl,
aryl, alkenyl, alkynyl, or cycloalkyl. In another embodiment, A is
oxygen.
[0227] In another embodiment, R.sup.3 and R.sup.4 are taken
together with the carbon atoms to which they are attached to form a
double bond. In another embodiment, R.sup.A, R.sup.B, R.sup.C,
R.sup.D, and R.sup.E are each hydrogen. R.sup.A, R.sup.B, R.sup.D,
and R.sup.E are each hydrogen and R.sup.C is a halogen, such as
fluorine, chlorine, iodine, or bromine.
[0228] In another embodiment, R.sup.3 or R.sup.5a is a moiety of
Formula IIIa.
[0229] In another embodiment, R.sup.4, R.sup.5, R.sup.6, and
R.sup.7 are each hydrogen. In another further embodiment, R.sup.4a,
R.sup.5a, R.sup.6a, and R.sup.7a are each hydrogen.
[0230] In another, R.sup.3a is hydroxyl, cyano, acyl, or
hydroxyl.
[0231] In another further embodiment, R.sup.11 and A taken together
are a natural or unnatural amino acid residue or a pharmaceutically
acceptable salt or ester thereof. Examples of amino acid residues
include esters and salts of phenylalanine and leucine.
[0232] In another embodiment, m is 0, 1, or 3.
[0233] Examples of compounds of Formula III include, but are not
limited to:
##STR00062##
and pharmaceutically acceptable salts, esters, and prodrugs
thereof.
[0234] In another embodiment, the invention pertains to compounds
of Formula IV:
##STR00063##
wherein:
[0235] A is nitrogen or oxygen;
[0236] R.sup.11 is hydrogen, salt-forming cation, ester forming
group, --(CH.sub.2).sub.x-Q, or when A is nitrogen, A and R.sup.11
taken together may be the residue of a natural or unnatural amino
acid or a salt or ester thereof;
[0237] Q is hydrogen, thiazolyl, triazolyl, imidazolyl,
benzothiazolyl, or benzoimidazolyl;
[0238] x is 0, 1, 2, 3, or 4;
[0239] n is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10;
[0240] R.sup.4, R.sup.4a, R.sup.5, R.sup.5a, R.sup.6, R.sup.6a,
R.sup.7, and R.sup.7a are each independently hydrogen, alkyl,
mercaptoalkyl, alkenyl, alkynyl, cycloalkyl, aryl, alkylcarbonyl,
arylcarbonyl, alkoxycarbonyl, cyano, halogen, amino, tetrazolyl,
R.sup.4 and R.sup.5 taken together, with the ring atoms they are
attached to, form a double bond, or R.sup.6 and R.sup.7 taken
together, with the ring atoms they are attached to, form a double
bond;
[0241] m is 0, 1, 2, 3, or 4;
[0242] R.sup.8, R.sup.9, R.sup.10, R.sup.11, and R.sup.12 are
independently selected from a group of hydrogen, halogen, hydroxyl,
alkyl, alkoxyl, halogenated alkyl, mercaptoalkyl, alkenyl, alkynyl,
cycloalkyl, aryl, cyano, thiazolyl, triazolyl, imidazolyl,
tetrazolyl, benzothiazolyl, and benzoimidazolyl, and
pharmaceutically acceptable salts and esters thereof.
[0243] In another embodiment, R.sup.11 is a salt-forming cation.
Examples of salt forming cations include pharmaceutically
acceptable salts described herein as well as lithium, sodium,
potassium, magnesium, calcium, barium, zinc, iron, and ammonium. In
another embodiment, R.sup.11 is an ester-forming group. An
ester-forming group includes groups which when bound form an ester.
Examples of such groups include substituted or unsubstituted alkyl,
aryl, alkenyl, alkynyl, or cycloalkyl. In another embodiment, A is
oxygen.
[0244] In another embodiment, m is 0 or 1. In another further
embodiment, n is 2, 3, or 4. In another further embodiment,
R.sup.4, R.sup.5, R.sup.6 and R.sup.7 are each hydrogen. R.sup.4a,
R.sup.5a, R.sup.6a, and R.sup.7a also may be hydrogen. Examples of
R.sup.8, R.sup.9, R.sup.10, R.sup.11, and R.sup.12 include
hydrogen. In another embodiment R.sup.8, R.sup.9, R.sup.11,
R.sup.12 are each hydrogen, and R.sup.10 is a halogen, (e.g.,
fluorine, chlorine, bromine, or iodine), nitro, or alkyl (e.g.,
methyl; ethyl, butyl).
In another embodiment, A-R.sup.11 may be the residue of an amino
acid, e.g., a phenylalanine residue. In another embodiment,
R.sup.9, R.sup.11 and R.sup.12 are each hydrogen, and R.sup.8 is
not hydrogen, e.g., halogen, e.g., fluorine, bromine, chlorine, or
iodine.
[0245] In another embodiment the compound is:
##STR00064##
[0246] and pharmaceutically acceptable salts, esters, and prodrugs
thereof.
[0247] In another embodiment, the invention pertains to compounds
of Formula V:
##STR00065##
wherein:
[0248] A is nitrogen or oxygen;
[0249] R.sup.11 is hydrogen, salt-forming cation, ester forming
group, --(CH.sub.2), -Q, or when A is nitrogen, A and R.sup.11
taken together may be the residue of a natural or unnatural amino
acid or a salt or ester thereof;
[0250] Q is hydrogen, thiazolyl, triazolyl, imidazolyl,
benzothiazolyl, or benzoimidazolyl;
[0251] x is 0, 1, 2, 3, or 4;
[0252] n is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10;
[0253] aa is a natural or unnatural amino acid residue;
[0254] m is 0, 1, 2, or 3;
[0255] R.sup.14 is hydrogen or protecting group;
[0256] R.sup.15 is hydrogen, alkyl or aryl, and pharmaceutically
acceptable salts, esters and prodrugs thereof.
[0257] In another embodiment, R.sup.11 is a salt-forming cation.
Examples of salt forming cations include pharmaceutically
acceptable salts described herein as well as lithium, sodium,
potassium, magnesium, calcium, barium, zinc, iron, and ammonium. In
another embodiment, R.sup.11 is an ester-forming group. An
ester-forming group includes groups which when bound form an ester.
Examples of such groups include substituted or unsubstituted alkyl,
aryl, alkenyl, alkynyl, or cycloalkyl. In another embodiment, A is
oxygen.
[0258] In an embodiment, n is 2, 3 or 4. In certain embodiments, m
is 0. In certain embodiments, A-R.sup.11 is a residue of a natural
amino acid, or a salt or ester thereof. Examples of amino acid
residues, include, but are not limited to, leucine or phenylalanine
residues, and pharmaceutically acceptable salts and esters thereof.
Examples of possible esters include methyl, ethyl, and t-butyl.
[0259] In another embodiment, m is 1. Examples of aa include
natural and unnatural amino acid residues such as phenylalanine,
glycine, and leucine.
[0260] In another embodiment, (aa).sub.m is a residue of phe-phe,
or an ester thereof.
[0261] In certain embodiments, R.sup.15 is hydrogen or substituted
alkyl, e.g., arylalkyl.
[0262] The term "unnatural amino acid" refers to any derivative of
a natural amino acid including D forms, and .alpha.- and
.beta.-amino acid derivatives. It is noted that certain amino
acids, e.g., hydroxyproline, that are classified as a non-natural
amino acid herein, may be found in nature within a certain organism
or a particular protein. Amino acids with many different protecting
groups appropriate for immediate use in the solid phase synthesis
of peptides are commercially available. In addition to the twenty
most common naturally occurring amino acids, the following examples
of non-natural amino acids and amino acid derivatives may be used
according to the invention (common abbreviations in parentheses):
.beta.-alanine (.beta.-ALA), .gamma.-aminobutyric acid (GABA),
2-aminobutyric acid (2-Abu), .alpha.,.beta.-dehydro-2-aminobutyric
acid (8-AU), 1-aminocyclopropane-1-carboxylic acid (ACPC),
aminoisobutyric acid (Aib), 2-amino-thiazoline-4-carboxylic acid,
5-aminovaleric acid (5-Ava), 6-aminohexanoic acid (6-Ahx),
8-aminooctanoic acid (8-Aoc), 11-aminoundecanoic acid (11-Aun),
12-aminododecanoic acid (12-Ado), 2-aminobenzoic acid (2-Abz),
3-aminobenzoic acid (3-Abz), 4-aminobenzoic acid (4-Abz),
4-amino-3-hydroxy-6-methylheptanoic acid (Statine, Sta),
aminooxyacetic acid (Aoa), 2-aminotetraline-2-carboxylic acid
(ATC), 4-amino-5-cyclohexyl-3-hydroxypentanoic acid (ACHPA),
para-aminophenylalanine (4-NH.sub.2-Phe), biphenylalanine (Bip),
para-bromophenylalanine (4-Br-Phe), ortho-chlorophenylalanine]
(2-Cl-Phe), meta-chlorophenylalanine (3-Cl-Phe),
para-chlorophenylalanine (4-Cl -Phe), meta-chlorotyrosine
(3-Cl-Tyr), para-benzoylphenylalanine (Bpa), tert-butylglycine
(TLG), cyclohexylalanine (Cha), cyclohexylglycine (Chg),
2,3-diaminopropionic acid (Dpr), 2,4-diaminobutyric acid (Dbu),
3,4-dichlorophenylalanine (3,4-C.sub.12-Phe),
3,4-difluororphenylalanine (3,4-F.sub.2-Phe), 3,5-diiodotyrosine
(3,5-I.sub.2-Tyr), ortho-fluorophenylalanine (2-F-Phe),
meta-fluorophenylalanine (3-F-Phe), para-fluorophenylalanine
(4-F-Phe), meta-fluorotyrosine (3-F-Tyr), homoserine (Hse),
homophenylalanine (Hfe), homotyrosine (Htyr), 5-hydroxytryptophan
(5-OH-Trp), hydroxyproline (Hyp), para-iodophenylalanine (4-I-Phe),
3-iodotyrosine (34-Tyr), indoline-2-carboxylic acid (Idc),
isonipecotic acid (Inp), meta-methyltyrosine (3-Me-Tyr),
1-naphthylalanine (1-NaI), 2-naphthylalanine (2-NaI),
para-nitrophenylalanine (4-NO.sub.2-Phe), 3-nitrotyrosine
(3-NO.sub.2-Tyr), norleucine (Nle), norvaline (Nva), ornithine
(Orn), ortho-phosphotyrosine (H.sub.2PO.sub.3-Tyr),
octahydroindole-2-carboxylic acid (Oic), penicillamine (Pen),
pentafluorophenylalanine (F.sub.5-Phe), phenylglycine (Phg),
pipecolic acid (Pip), propargylglycine (Pra), pyroglutamic acid
(PGLU), sarcosine (Sar), tetrahydroisoquinoline-3-carboxylic acid
(Tic), thienylalanine, and thiazolidine-4-carboxylic acid
(thioproline, Th). Additionally, N-alkylated amino acids may be
used, as well as amino acids having amine-containing side chains
(such as Lys and Orn) in which the amine has been acylated or
alkylated.
[0263] Examples of compounds of the invention include, but are not
limited to:
##STR00066## ##STR00067##
and pharmaceutically acceptable salts, esters, and prodrugs
thereof.
[0264] In another embodiment, the invention pertains, at least in
part, to compounds of Formula VI:
##STR00068##
wherein:
[0265] n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10;
[0266] A is oxygen or nitrogen;
[0267] R.sup.11 is hydrogen, salt-forming cation, ester forming
group, --(CH.sub.2).sub.x-Q, or when A is nitrogen, A and R.sup.11
taken together may be the residue of a natural or unnatural amino
acid or a salt or ester thereof;
[0268] Q is hydrogen, thiazolyl, triazolyl, imidazolyl,
benzothiazolyl, or benzoimidazolyl;
[0269] x is 0, 1, 2, 3, or 4;
[0270] R.sup.19 is hydrogen, alkyl or aryl;
[0271] Y.sup.1 is oxygen, sulfur, or nitrogen;
[0272] Y.sup.2 is carbon, nitrogen, or oxygen;
[0273] R.sup.20 is hydrogen, alkyl, amino, mercaptoalkyl, alkenyl,
alkynyl, cycloalkyl, aryl, arylalkyl, thiazolyl, triazolyl,
tetrazolyl, imidazolyl, benzothiazolyl, or benzoimidazolyl;
[0274] R.sup.21 is hydrogen, alkyl, mercaptoalkyl, alkenyl,
alkynyl, cycloalkyl, aryl, arylalkyl, thiazolyl, triazolyl,
tetrazolyl, imidazolyl, benzothiazolyl, benzoimidazolyl, or absent
if Y.sup.2 is oxygen;
[0275] R.sup.22 is hydrogen, alkyl, mercaptoalkyl, alkenyl,
alkynyl, cycloalkyl, aryl, arylalkyl, thiazolyl, triazolyl,
tetrazolyl, imidazolyl, benzothiazolyl, benzoimidazolyl; or
R.sup.22 is hydrogen, hydroxyl, alkoxy or aryloxy if Y.sup.1 is
nitrogen; or R.sup.22 is absent if Y.sup.1 is oxygen or sulfur; or
R.sup.22 and R.sup.21 may be linked to form a cyclic moiety if
Y.sup.1 is nitrogen;
[0276] R.sup.23 is hydrogen, alkyl, amino, mercaptoalkyl, alkenyl,
alkynyl, cycloalkyl, aryl, arylalkyl, thiazolyl, triazolyl,
tetrazolyl, imidazolyl, benzothiazolyl, or benzoimidazolyl, or
absent if Y.sup.2 is nitrogen or oxygen;
[0277] or pharmaceutically acceptable salts thereof.
[0278] In another embodiment, R.sup.11 is a salt-forming cation.
Examples of salt forming cations include pharmaceutically
acceptable salts described herein as well as lithium, sodium,
potassium, magnesium, calcium, barium, zinc, iron, and ammonium. In
a further embodiment, the salt is a sodium salt. In a further,
embodiment, A is oxygen.
[0279] In another embodiment, Y.sup.1 is oxygen or sulfur, and
R.sup.22 is absent.
[0280] In another embodiment, Y.sup.2 is oxygen and R.sup.21 is
absent. Examples of R.sup.20 include benzyl, aryl (e.g., phenyl),
alkyl, cycloalkyl (e.g., adamantyl), etc. In other embodiment,
Y.sup.2 is nitrogen and R.sup.21 is hydrogen. In other embodiment,
R.sup.21 is benzyl. In another further embodiment, R.sup.20 and
R.sup.21 are linked to form a pyridyl ring. In another embodiment,
Y.sup.1 is sulfur.
[0281] Examples of compounds of the invention, include
##STR00069## ##STR00070##
and pharmaceutically acceptable salts, esters, and prodrugs
thereof.
[0282] In another embodiment, the invention pertains to compounds
of Formula VII:
##STR00071##
wherein:
[0283] n is 2, 3, or 4;
[0284] A is oxygen or nitrogen;
[0285] R.sup.11 is hydrogen, salt-forming cation, ester forming
group, --(CH.sub.2), ---Q, or when A is nitrogen, A and R.sup.11
taken together may be the residue of a natural or unnatural amino
acid or a salt or ester thereof;
[0286] Q is hydrogen, thiazolyl, triazolyl, imidazolyl,
benzothiazolyl, or benzoimidazolyl;
[0287] x is 0, 1, 2, 3, or 4;
[0288] G is a direct bond or oxygen, nitrogen, or sulfur;
[0289] z is 0, 1, 2, 3, 4, or 5;
[0290] m is 0 or 1;
[0291] R.sup.24 is selected from a group consisting of hydrogen,
alkyl, mercaptoalkyl, alkenyl, alkynyl, aroyl, alkylcarbonyl,
aminoalkylcarbonyl, cycloalkyl, aryl, arylalkyl, thiazolyl,
triazolyl, imidazolyl, benzothiazolyl, and benzoimidazolyl;
[0292] each R.sup.25 is independently selected from hydrogen,
halogen, cyano, hydroxyl, alkoxy, thiol, amino, nitro, alkyl, aryl,
carbocyclic, or heterocyclic, and pharmaceutically acceptable
salts, esters, and prodrugs thereof.
[0293] In one embodiment, R.sup.11 is hydrogen. In another, A is
oxygen. For example, n may be 3 and m may be 1. In other
embodiments, R.sup.24 is hydrogen or benzyl.
[0294] In certain embodiments, z is 0, 2, or 3. In others, R.sup.25
is hydroxyl or alkoxy, e.g., methoxy, ethoxy, etc. In certain
embodiments, two or more R.sup.25 substituents can be linked to
form a fused ring (e.g., to form a methylendioxyphenyl moiety).
[0295] Examples of compounds of the invention include:
##STR00072##
and pharmaceutically acceptable salts, esters, and prodrugs
thereof.
[0296] Other compounds of the invention include
##STR00073##
and pharmaceutically acceptable salts, esters, and prodrugs
thereof.
[0297] The invention pertains to both salt forms and acid/base
forms of the compounds of the invention. For example, the invention
pertains not only to the particular salt forms of compounds shown
herein as salts, but also the invention includes other
pharmaceutically acceptable salts, and the acid and/or base form of
the compound. The invention also pertains to salt forms of
compounds shown herein.
[0298] Compounds of the invention are also shown in Table 2
below.
TABLE-US-00002 TABLE 2 Structure/ ID Name of Compound B
##STR00074## C ##STR00075## D ##STR00076## E ##STR00077## F
##STR00078## G ##STR00079## H ##STR00080## I ##STR00081## J
##STR00082## K ##STR00083## L ##STR00084## M ##STR00085## N
##STR00086## P ##STR00087## Q ##STR00088## R ##STR00089## S
##STR00090## X ##STR00091## Y ##STR00092## Z ##STR00093## AA
##STR00094## AB ##STR00095## AC ##STR00096## AD ##STR00097## AE
##STR00098## AF ##STR00099## AG ##STR00100## AH ##STR00101## AI
##STR00102## AJ ##STR00103## AK ##STR00104## AL ##STR00105## AM
##STR00106## AU ##STR00107## AV ##STR00108## AW ##STR00109## AX
##STR00110## AY ##STR00111## AZ ##STR00112## BA ##STR00113## BB
##STR00114## BC ##STR00115## BW ##STR00116## BX ##STR00117## BY
##STR00118## BZ ##STR00119## CC ##STR00120## CD ##STR00121## CE
##STR00122## CG ##STR00123## CH ##STR00124## CI ##STR00125## CJ
##STR00126## CK ##STR00127## CL ##STR00128## CM ##STR00129## CN
##STR00130## CO ##STR00131## CV ##STR00132## CY ##STR00133## DC
##STR00134## DD ##STR00135## DE ##STR00136## DG ##STR00137## DH
##STR00138## DI ##STR00139## DJ ##STR00140## DK ##STR00141## DL
##STR00142## DM ##STR00143## DN ##STR00144## DO ##STR00145## DP
##STR00146## DQ ##STR00147## DR ##STR00148## DS ##STR00149## DT
##STR00150## DU ##STR00151## DV ##STR00152## DW ##STR00153## DX
##STR00154## DY ##STR00155## DZ ##STR00156## EA ##STR00157## EB
##STR00158## EC ##STR00159## ED ##STR00160## EE ##STR00161## EF
##STR00162## EG ##STR00163## EH ##STR00164## EI ##STR00165## EJ
##STR00166## EK ##STR00167## EL ##STR00168## EN ##STR00169## EO
##STR00170## EP ##STR00171## EQ ##STR00172## ER ##STR00173## ES
##STR00174## ET ##STR00175## EV ##STR00176## EW ##STR00177## EY
##STR00178## EZ ##STR00179## FA ##STR00180## FH ##STR00181## FL
##STR00182## FM ##STR00183## FN ##STR00184## FO ##STR00185## FP
##STR00186## FQ ##STR00187## FR ##STR00188## FS ##STR00189## FT
##STR00190## FU ##STR00191## FV ##STR00192## FW ##STR00193## FX
##STR00194## FY ##STR00195## FZ ##STR00196##
GA ##STR00197## GB ##STR00198## GC ##STR00199## GD ##STR00200## GE
##STR00201## GF ##STR00202## GH ##STR00203## GI ##STR00204## GJ
##STR00205## GK ##STR00206## GL ##STR00207## GM ##STR00208## GN
##STR00209## GO ##STR00210## GP ##STR00211## GQ ##STR00212## GR
##STR00213## GS ##STR00214## GT ##STR00215## GU ##STR00216## GZ
##STR00217## HA ##STR00218## HB ##STR00219## HC ##STR00220## HD
##STR00221## HE ##STR00222## HF ##STR00223## HG ##STR00224## HI
##STR00225## HJ ##STR00226## HK ##STR00227## HL ##STR00228## HM
##STR00229## HN ##STR00230## HO ##STR00231## HP ##STR00232## HQ
##STR00233## HR ##STR00234## HS ##STR00235## HT ##STR00236## HU
##STR00237## HV ##STR00238## HW ##STR00239## HX ##STR00240## HY
##STR00241## HZ ##STR00242## IA ##STR00243## IB ##STR00244## IC
##STR00245## ID ##STR00246## IE ##STR00247## IF ##STR00248## IG
##STR00249## IH ##STR00250## II ##STR00251## IJ ##STR00252## IK
##STR00253## IL ##STR00254## IM ##STR00255## IN ##STR00256## IO
##STR00257## IP ##STR00258## IR ##STR00259## IS ##STR00260## IT
##STR00261## IU ##STR00262## IV ##STR00263## IW ##STR00264## IX
##STR00265## IY ##STR00266## IZ ##STR00267## JA ##STR00268## JB
##STR00269## JC ##STR00270## JD ##STR00271## JE ##STR00272## JF
##STR00273## JG ##STR00274## JH ##STR00275## JI ##STR00276## JJ
##STR00277## JK ##STR00278## JL ##STR00279## JM ##STR00280## JN
##STR00281## JO ##STR00282## JP ##STR00283## JQ ##STR00284## JR
##STR00285## JS ##STR00286## JT ##STR00287## JU ##STR00288## JV
##STR00289## JW ##STR00290## JX ##STR00291## JY ##STR00292## JZ
##STR00293## KA ##STR00294## KB ##STR00295## KH ##STR00296## KI
##STR00297## KJ ##STR00298## KK ##STR00299## KL ##STR00300## KM
##STR00301## KN ##STR00302## KP ##STR00303## KQ ##STR00304## KR
##STR00305## KS ##STR00306## KT ##STR00307## KV ##STR00308## KW
##STR00309## KX ##STR00310## KY ##STR00311## LA ##STR00312## LC
##STR00313## LD ##STR00314## LE ##STR00315## LF ##STR00316## LG
##STR00317## LH ##STR00318## LI ##STR00319## LJ ##STR00320## LK
##STR00321##
LL ##STR00322## LM ##STR00323## LN ##STR00324## LO ##STR00325## LP
##STR00326## LQ ##STR00327## NE ##STR00328## NG ##STR00329## NH
##STR00330## NI ##STR00331## NJ ##STR00332## NK ##STR00333## NL
##STR00334## ##STR00335##
[0299] It should be noted that in the above table and throughout
the application when an atom is shown without hydrogens, but
hydrogens are required or chemically necessary to form a stable
compound, hydrogens should be inferred to be part of the
compound.
[0300] In one embodiment, the invention does not pertain to the
compounds described in WO 00/64420 and WO 96/28187. In this
embodiment, the invention does not pertain to methods of using the
compounds described in WO 00/64420 and WO 96/28187 for the
treatment of diseases or disorders described therein. In a further
embodiment, the invention pertains to methods of using the
compounds described in WO 00/64420 and WO 96/281.87 for methods
described in this application, which are not described in WO
00/64420 and WO 96/28187. Both of WO 00/64420 and WO 96/28187 are
incorporated by reference herein in their entirety.
[0301] In another embodiment, the invention pertains to methods of
the invention which use and pharmaceutical compositions comprising,
the compounds of Table 2A. In another embodiment, the compounds of
the invention do not include the compounds of Table 2A.
TABLE-US-00003 TABLE 2A ##STR00336##
BzNHCH.sub.2CH.sub.2CH.sub.2SO.sub.3Na ##STR00337## ##STR00338##
##STR00339## ##STR00340## ##STR00341## ##STR00342## ##STR00343##
##STR00344## ##STR00345## ##STR00346## ##STR00347## ##STR00348##
AcNHCH.sub.2CH.sub.2CH.sub.2SO.sub.3Na ##STR00349## ##STR00350##
##STR00351## ##STR00352## ##STR00353## ##STR00354## ##STR00355##
##STR00356## ##STR00357## ##STR00358## ##STR00359## ##STR00360##
##STR00361## ##STR00362## ##STR00363## ##STR00364## ##STR00365##
##STR00366## ##STR00367## ##STR00368## ##STR00369## ##STR00370##
##STR00371## ##STR00372## ##STR00373## ##STR00374## ##STR00375##
##STR00376## ##STR00377## ##STR00378## ##STR00379##
CH.sub.3(CH.sub.2).sub.8NH(CH.sub.2).sub.3SO.sub.3H ##STR00380##
##STR00381## CH.sub.3(CH.sub.2).sub.9NH(CH.sub.2).sub.3SO.sub.3H
##STR00382## CH.sub.3(CH.sub.2).sub.11NH(CH.sub.2).sub.3SO.sub.3H
CH.sub.3(CH.sub.2).sub.10NH(CH.sub.2).sub.3SO.sub.3H
CH.sub.3(CH.sub.2).sub.12NH(CH).sub.3SO.sub.3H ##STR00383##
##STR00384## ##STR00385##
HOCH.sub.2CH.sub.2CH.sub.2NHCH.sub.2CH.sub.2CH.sub.2SO.sub.3]
CH.sub.3(CH.sub.2).sub.13NH(CH.sub.2).sub.3SO.sub.3H
CH.sub.3(CH.sub.2).sub.15NH(CH.sub.2).sub.3SO.sub.3H ##STR00386##
CH.sub.3(CH.sub.2).sub.17NHCH.sub.2CH.sub.2CH.sub.2SO.sub.3H
##STR00387##
CH.sub.3(CH.sub.2).sub.13N.sup.+(CH.sub.3).sub.2[(CH.sub.2).sub.3SO.sub.3.-
sup.-] ##STR00388## ##STR00389## ##STR00390## ##STR00391##
##STR00392## ##STR00393## ##STR00394## ##STR00395## ##STR00396##
##STR00397## ##STR00398## ##STR00399## ##STR00400## ##STR00401##
##STR00402## ##STR00403## ##STR00404## ##STR00405## ##STR00406##
##STR00407## ##STR00408## ##STR00409## ##STR00410## ##STR00411##
##STR00412## ##STR00413## ##STR00414## ##STR00415## ##STR00416##
##STR00417## ##STR00418## ##STR00419##
NaO.sub.3SOCH.sub.2(CH.sub.2).sub.3CH.sub.2OSO.sub.3Na ##STR00420##
##STR00421## ##STR00422## ##STR00423##
HOCH.sub.2CH.sub.2CH.sub.2CH.sub.2SO.sub.3Na
(NaO.sub.3SCH.sub.2CH.sub.2CH.sub.2CH.sub.2).sub.2O ##STR00424##
##STR00425## ##STR00426## ##STR00427## ##STR00428## ##STR00429##
##STR00430## ##STR00431## ##STR00432## ##STR00433## ##STR00434##
CH.sub.3C(CH.sub.2OSO.sub.3Na).sub.3
NH.sub.2CH.sub.2CH.sub.2CH.sub.2SO.sub.3Na
HC(CH.sub.2OSO.sub.3Na).sub.3 NH.sub.2C(CH.sub.2OSO.sub.3Na).sub.3
NH.sub.2CH.sub.2CH.sub.2OSO.sub.3H
NaO.sub.3SNHCH.sub.2CH.sub.2OSO.sub.3Na ##STR00435## ##STR00436##
##STR00437## NaO.sub.3SNHCH.sub.2CH.sub.2CH.sub.2OSO.sub.3Na
HN(CH.sub.2CH.sub.2OSO.sub.3Na).sub.2
H.sub.2NCH.sub.2CH.sub.2CH.sub.2OSO.sub.3Na
NaO.sub.3SN(CH.sub.2CH.sub.2OSO.sub.3Na).sub.2
H.sub.2NCH.sub.2CH.sub.2SO.sub.3H
NaO.sub.3SOCH.sub.2CH.sub.2CH.sub.2SO.sub.3Na ##STR00438##
##STR00439##
##STR00440## ##STR00441## ##STR00442## ##STR00443## ##STR00444##
##STR00445## ##STR00446## ##STR00447## ##STR00448##
CH.sub.3CH.sub.2CH.sub.2CH.sub.2SO.sub.3Na
CH.sub.3(CH.sub.2).sub.8CH.sub.2SO.sub.3Na ##STR00449##
##STR00450## ##STR00451## ##STR00452## ##STR00453## ##STR00454##
##STR00455## ##STR00456## ##STR00457## ##STR00458## ##STR00459##
##STR00460## ##STR00461## ##STR00462## ##STR00463## ##STR00464##
##STR00465## ##STR00466## ##STR00467## ##STR00468## ##STR00469##
##STR00470## ##STR00471## ##STR00472## ##STR00473## ##STR00474##
##STR00475## ##STR00476## ##STR00477## ##STR00478## ##STR00479##
##STR00480## ##STR00481## ##STR00482## ##STR00483## ##STR00484##
##STR00485## ##STR00486## ##STR00487## ##STR00488## ##STR00489##
##STR00490## ##STR00491## ##STR00492## ##STR00493## ##STR00494##
##STR00495## ##STR00496## ##STR00497##
CH.sub.3CH.sub.2CH.sub.2SO.sub.3Na CH.sub.3CH.sub.2SO.sub.3Na
CH.sub.3CH.sub.2CH.sub.2CH.sub.2CH.sub.2SO.sub.3Na
[0302] It should be understood that the use of any of the compounds
described herein or in the applications identified in "The Related
Applications" Section is within the scope of the present invention
and is intended to be encompassed by the present invention and each
of the applications are expressly incorporated herein at least for
these purposes, and are furthermore expressly incorporated for all
other purposes.
Subjects and Patient Populations
[0303] The term "subject" includes living organisms in which
amyloidosis can occur, or which are susceptible to amyloid
diseases, e.g., Alzheimer's disease, Down's syndrome, CAA,
dialysis-related (.beta..sub.2M) amyloidosis, secondary (AA)
amyloidosis, primary (AL) amyloidosis, hereditary amyloidosis,
diabetes, etc. Examples of subjects include humans, chickens,
ducks, peking ducks, geese, monkeys, deer, cows, rabbits, sheep,
goats, dogs, cats, mice, rats, and transgenic species thereof.
Administration of the compositions of the present invention to a
subject to be treated can be carried out using known procedures, at
dosages and for periods of time effective to modulate amyloid
aggregation or amyloid-induced toxicity in the subject as further
described herein. An effective amount of the therapeutic compound
necessary to achieve a therapeutic effect may vary according to
factors such as the amount of amyloid already deposited at the
clinical site in the subject, the age, sex, and weight of the
subject, and the ability of the therapeutic compound to modulate
amyloid aggregation in the subject. Dosage regimens can be adjusted
to provide the optimum therapeutic response. For example, several
divided doses may be administered daily or the dose may be
proportionally reduced as indicated by the exigencies of the
therapeutic situation.
[0304] In certain embodiments of the invention, the subject is in
need of treatment by the methods of the invention, and is selected
for treatment based on this need. A subject in need of treatment is
art-recognized, and includes subjects that have been identified as
having a disease or disorder related to amyloid-deposition or
amyloidosis, has a symptom of such a disease or disorder, or is at
risk of such a disease or disorder, and would be expected, based on
diagnosis, e.g., medical diagnosis, to benefit from treatment
(e.g., curing, healing, preventing, alleviating, relieving,
altering, remedying, ameliorating, improving, or affecting the
disease or disorder, the symptom of the disease or disorder, or the
risk of the disease or disorder).
[0305] In an exemplary aspect of the invention, the subject is a
human. For example, the subject may be a human over 30 years old,
human over 40 years old, a human over 50 years old, a human over 60
years old, a human over 70 years old, a human over 80 years old, a
human over 85 years old, a human over 90 years old, or a human over
95 years old. The subject may be a female human, including a
postmenopausal female human, who may be on hormone (estrogen)
replacement therapy. The subject may also be a male human. In
another embodiment, the subject is under 40 years old.
[0306] A subject may be a human at risk for Alzheimer's disease,
e.g., being over the age of 40 or having a predisposition for
Alzheimer's disease. Alzheimer's disease predisposing factors
identified or proposed in the scientific literature include, among
others, a genotype predisposing a subject to Alzheimer's disease;
environmental factors predisposing a subject to Alzheimer's
disease; past history of infection by viral and bacterial agents
predisposing a subject to Alzheimer's disease; and vascular factors
predisposing a subject to Alzheimer's disease. A subject may also
have one or more risk factors for cardiovascular disease (e.g.,
atherosclerosis of the coronary arteries, angina pectoris, and
myocardial infarction) or cerebrovascular disease (e.g.,
atherosclerosis of the intracranial or extracranial arteries,
stroke, syncope, and transient ischemic attacks), such as
hypercholesterolemia, hypertension, diabetes, cigarette smoking,
familial or previous history of coronary artery disease,
cerebrovascular disease, and cardiovascular disease.
Hypercholesterolemia typically is defined as a serum total
cholesterol concentration of greater than about 5.2 mmol/L (about
200 mg/dL).
[0307] Several genotypes are believed to predispose a subject to
Alzheimer's disease. These include the genotypes such as
presenilin-1, presenilin-2, and amyloid precursor protein (APP)
missense mutations associated with familial Alzheimer's disease,
and .alpha.-2-macroglobulin and LRP-1 genotypes, which are thought
to increase the risk of acquiring sporadic (late-onset) Alzheimer's
disease. E. van Uden, et al., J. Neurosci. 22(21), 9298-304 (2002);
J. J. Goto, et al., J. Mol. Neurosci. 19(1-2), 37-41 (2002).
Another genetic risk factor for the development of Alzheimer's
disease are variants of ApoE, the gene that encodes apolipoprotein
E (particularly the apoE4 genotype), a constituent of the
low-density lipoprotein particle. W J Strittmatter, et al., Annu.
Rev. Neurosci. 19, 53-77 (1996). The molecular mechanisms by which
the various ApoE alleles alter the likelihood of developing
Alzheimer's disease are unknown, however the role of ApoE in
cholesterol metabolism is consistent with the growing body of
evidence linking cholesterol metabolism to Alzheimer's disease. For
example, chronic use of cholesterol-lowering drugs such as statins
has recently been associated with a lower incidence of Alzheimer's
disease, and cholesterol-lowering drugs have been shown to reduce
pathology in APP transgenic mice. These and other studies suggest
that cholesterol may affect APP processing. ApoE4 has been
suggested to alter A.beta. trafficking (in and out of the brain),
and favor retention of A.beta. in the brain. ApoE4 has also been
suggested to favor APP processing toward A.beta. formation.
Environmental factors have been proposed as predisposing a subject
to Alzheimer's disease, including exposure to aluminum, although
the epidemiological evidence is ambiguous. In addition, prior
infection by certain viral or bacterial agents may predispose a
subject to Alzheimer's disease, including the herpes simplex virus
and chlamydia pneumoniae. Finally, other predisposing factors for
Alzheimer's disease can include risk factors for cardiovascular or
cerebrovascular disease, including cigarette smoking, hypertension
and diabetes. "At risk for Alzheimer's disease" also encompasses
any other predisposing factors not listed above or as yet
identified and includes an increased risk for Alzheimer's disease
caused by head injury, medications, diet, or lifestyle.
[0308] The methods of the present invention can be used for one or
more of the following: to prevent Alzheimer's disease, to treat
Alzheimer's disease, or ameliorate symptoms of Alzheimer's disease,
or to regulate production of or levels of amyloid .beta. (A.beta.)
peptides. In an embodiment, the human carries one or more mutations
in the genes that encode) .beta.-amyloid precursor protein,
presenilin-1 or presenilin-2. In another embodiment, the human
carries the Apolipoprotein E4 gene. In another embodiment, the
human has a family history of Alzheimer's Disease or a dementia
illness. In another embodiment, the human has trisomy 21 (Down's
Syndrome). In another embodiment, the subject has a normal or low
serum total blood cholesterol level. In another embodiment, the
serum total blood cholesterol level is less than about 200 mg/dL,
or less than about 180, and it can range from about 150 to about
200 mg/dL. In another embodiment, the total LDL cholesterol level
is less than about 100 mg/dL, or less than about 90 mg/dL and can
range from about 30 to about 100 mg/dL. Methods of measuring serum
total blood cholesterol and total LDL cholesterol are well known to
those skilled in the art and for example include those disclosed in
WO 99/38498 at p. 11, incorporated by reference herein. Methods of
determining levels of other sterols in serum are disclosed in H.
Gylling, et al., "Serum Sterols During Stanol Ester Feeding in a
Mildly Hypercholesterolemic Population", J. Lipid Res. 40: 593-600
(1999).
[0309] In another embodiment, the subject has an elevated serum
total blood cholesterol level. In another embodiment, the serum
total cholesterol level is at least about 200 mg/dL, or at least
about 220 mg/dL and can range from about 200 to about 1000 mg/dL.
In another embodiment, the subject has an elevated total LDL
cholesterol level. In another embodiment, the total LDL cholesterol
level is greater than about 100 mg/dL, or even greater than about
110 mg/dL and can range from about 100 to about 1000 mg/dL.
[0310] In another embodiment, the human is at least about 40 years
of age. In another embodiment, the human is at least about 60 years
of age. In another embodiment, the human is at least about 70 years
of age. In another embodiment, the human is at least about 80 years
of age. In another embodiment, the human is at least about 85 years
of age. In one embodiment, the human is between about 60 and about
100 years of age.
[0311] In still a further embodiment, the subject is shown to be at
risk by a diagnostic brain imaging technique, for example, one that
measures brain activity, plaque deposition, or brain atrophy.
[0312] In still a further embodiment, the subject is shown to be at
risk by a cognitive test such as Clinical Dementia Rating ("CDR"),
Alzheimer's Disease Assessment Scale-Cognition ("ADAS-Cog"),
Disability Assessment for Dementia ("DAD") or Mini-Mental State
Examination ("MMSE"). The subject may exhibit a below average score
on a cognitive test, as compared to a historical control of similar
age and educational background. The subject may also exhibit a
reduction in score as compared to previous scores of the subject on
the same or similar cognition tests.
[0313] In determining the CDR, a subject is typically assessed and
rated in each of six cognitive and behavioural categories: memory,
orientation, judgement and problem solving, community affairs, home
and hobbies, and personal care. The assessment may include
historical information provided by the subject, or preferably, a
corroborator who knows the subject well. The subject is assessed
and rated in each of these areas and the overall rating, (0, 0.5,
1.0, 2.0 or 3.0) determined. A rating of 0 is considered normal. A
rating of 1.0 is considered to correspond to mild dementia. A
subject with a CDR of 0.5 is characterized by mild consistent
forgetfulness, partial recollection of events and "benign"
forgetfulness. In one embodiment the subject is assessed with a
rating on the CDR of above 0, of above about 0.5, of above about
1.0, of above about 1.5, of above about 2.0, of above about 2.5, or
at about 3.0.
[0314] Another test is the Mini-Mental State Examination (MMSE), as
described by Folstein "Mini-mental state. A practical method for
grading the cognitive state of patients for the clinician." J.
Psychiatr. Res. 12:189-198, 1975. The MMSE evaluates the presence
of global intellectual deterioration. See also Folstein
"Differential diagnosis of dementia. The clinical process."
Psychiatr Clin North Am. 20:45-57, 1997. The MMSE is a means to
evaluate the onset of dementia and the presence of global
intellectual deterioration, as seen in Alzheimer's disease and
multi-infart dementia. The MMSE is scored from 1 to 30. The MMSE
does not evaluate basic cognitive potential, as, for example, the
so-called IQ test. Instead, it tests intellectual skills. A person
of "normal" intellectual capabilities will score a "30" on the MMSE
objective test (however, a person with a MMSE score of 30 could
also score well below "normal" on an IQ test). See, e.g., Kaufer,
J. Neuropsychiatry Clin. Neurosci. 10:55-63, 1998; Becke, Alzheimer
Dis Assoc Disord. 12:54-57, 1998; Ellis, Arch. Neurol. 55:360-365,
1998; Magni, Int. Psychogeriatr. 8:127-134, 1996; Monsch, Acta
Neurol. Scand. 92:145-150, 1995. In one embodiment, the subject
scores below 30 at least once on the MMSE. In another embodiment,
the subject scores below about 28, below about 26, below about 24,
below about 22, below about 20, below about 18, below about 16,
below about 14, below about 12, below about 10, below about 8,
below about 6, below about 4, below about 2, or below about 1.
[0315] The Disability Assessment for Dementia ("DAD") scale has
been developed to measure a patient's ability to perform the
activities of daily living (Gelinas I et al. Development of a
Functional Measure for Persons with Alzheimer's Disease: The
Disability Assessment for Dementia. Am. J. Occupational Therapy.
1999; 53: 471-481). Activities of daily living may be assessed
according to self care (i.e., dressing and personal hygiene) and
instrumental activities (e.g., housework, cooking, and using
household devices). The objectives of the DAD scale include
quantitatively measuring functional abilities in activities of
daily living in individuals with cognitive impairments and to help
delineate areas of cognitive deficits that may impair performance
in activities of daily living. The DAD is administered through an
interview with the caregiver. It measures actual performance in
activities of daily living of the individual as observed over a 2
week period prior to the interview. The scale assesses the
following domains of activities: hygiene, dressing, telephoning,
continence, eating, meal preparation, outing activities, finance
and correspondence, medication use, leisure and housework. A total
score is obtained by adding the rating for each question and
converting this total score out of 100. Higher scores represent
less disability in ADL while lower scores indicate more
dysfunction. In one embodiment, the subject scores below 100 at
least once on the DAD. In another embodiment, the subject scores
below about 95, below about 90, below about 85, below about 80,
below about 75, below about 70, below about 65, below about 60,
below about 55, below about 50, below about 45, below about 40,
below about 30, below about 20, or below about 10.
[0316] Another means to evaluate cognition, particularly
Alzheimer's disease, is the Alzheimer's Disease Assessment Scale
(ADAS-Cog), or a variation termed the Standardized Alzheimer's
Disease Assessment Scale (SADAS). It is commonly used as an
efficacy measure in clinical drug trials of Alzheimer's disease and
related disorders characterized by cognitive decline. SADAS and
ADAS-Cog were not designed to diagnose Alzheimer's disease; they
are useful in characterizing symptoms of dementia and are a
relatively sensitive indicator of dementia progression. (See, e.g.,
Doraiswamy, Neurology 48:1511-1517, 1997; and Standish, J. Am.
Geriatr. Soc. 44:712-716, 1996.) Annual deterioration in untreated
Alzheimer's disease patients is approximately 8 points per year
(See, eg., Raskind, M Prim. Care Companion J Clin Psychiatry 2000
August; 2(4):134-138).
[0317] The ADAS-cog is designed to measure, with the use of
questionnaires, the progression and the severity of cognitive
decline as seen in AD on a 70-point scale. The ADAS-cog scale
quantifies the number of wrong answers. Consequently, a high score
on the scale indicates a more severe case of cognitive decline. In
one embodiment, a subject exhibits a score of greater than 0,
greater than about 5, greater than about 10, greater than about 15,
greater than about 20, greater than about 25, greater than about
30, greater than about 35, greater than about 40, greater than
about 45, greater than about 50, greater than about 55, greater
than about 60, greater than about 65, greater than about 68, or
about 70.
[0318] In another embodiment, the subject exhibits no symptoms of
Alzheimer's Disease. In another embodiment, the subject is a human
who is at least 40 years of age and exhibits no symptoms of
Alzheimer's Disease. In another embodiment, the subject is a human
who is at least 40 years of age and exhibits one or more symptoms
of Alzheimer's Disease.
[0319] In another embodiment, the subject has Mild Cognitive
Impairment. In a further embodiment, the subject has a CDR rating
of about 0.5. In another embodiment, the subject has early
Alzheimer's disease. In another embodiment, the subject has
cerebral amyloid angiopathy.
[0320] By using the methods of the present invention, the levels of
amyloid .beta. peptides in a subject's plasma or cerebrospinal
fluid (CSF) can be reduced from levels prior to treatment from
about 10 to about 100 percent, or even about 50 to about 100
percent.
[0321] In an alternative embodiment, the subject can have an
elevated level of amyloid A.beta..sub.40 and A.beta..sub.42 peptide
in the blood and CSF prior to treatment, according to the present
methods, of greater than about 10 pg/mL, or greater than about 20
pg/mL, or greater than about 35 pg/mL, or even greater than about
40 pg/mL. In another embodiment, the elevated level of amyloid
A.beta..sub.42 peptide can range from about 30 pg/mL to about 200
pg/mL, or even to about 500 pg/mL. One skilled in the art would
understand that as Alzheimer's disease progresses, the measurable
levels of amyloid .beta. peptide in the CSF may decrease from
elevated levels present before onset of the disease. This effect is
attributed to increased deposition, i.e., trapping of A.beta.
peptide in the brain instead of normal clearance from the brain
into the CSF.
[0322] In an alternative embodiment, the subject can have an
elevated level of amyloid A.beta..sub.42 peptide in the blood and
CSF prior to treatment, according to the present methods, of
greater than about 5 pg A.beta..sub.42/mL or greater than about 50
pg A.beta..sub.40/mL, or greater than about 400 pg/mL. In another
embodiment, the elevated level of amyloid A.beta..sub.40 peptide
can range from about 200 pg/mL to about 800 pg/mL, to even about
1000 pg/mL.
[0323] In another embodiment, the subject can have an elevated
level of amyloid A.beta..sub.42 peptide in the CSF prior to
treatment, according to the present methods, of greater than about
pg/mL, or greater than about 10 pg/mL, or greater than about 200
pg/mL, or greater than about 500 pg/mL. In another embodiment, the
level of amyloid .beta. peptide can range from about 10 pg/mL to
about 1,000 pg/mL, or even about 100 pg/mL to about 1,000 pg/mL
[0324] In another embodiment, the subject can have an elevated
level of amyloid A.beta..sub.40 peptide in the CSF prior to
treatment according to the present methods of greater than about
pg/mL, or greater than about 50 pg/mL, or even greater than about
100 pg/mL. In another embodiment, the level of amyloid .beta.
peptide can range from about 10 pg/mL to about 1,000 pg/mL.
[0325] The amount of amyloid peptide in the brain, CSF, blood, or
plasma of a subject can be evaluated by enzyme-linked immunosorbent
assay ("ELISA") or quantitative immunoblotting test methods or by
quantitative SELDI-TOF which are well known to those skilled in the
art, such as is disclosed by Zhang, et al., J. Biol. Chem. 274,
8966-72 (1999) and Zhang, et al., Biochemistry 40, 5049-55 (2001).
See also, A. K. Vehmas, et al., DNA Cell Biol. 20(11), 713-21
(2001), P. Lewczuk, et al., Rapid Commun. Mass Spectrom. 17(12),
1291-96 (2003); B. M. Austen, et al., J. Peptide Sci. 6, 459-69
(2000); and H. Davies, et al., BioTechniques 27, 1258-62 (1999).
These tests are performed on samples of the brain or blood which
have been prepared in a manner well known to one skilled in the
art. Another example of a useful method for measuring levels of
amyloid .beta. peptides is by Europium immunoassay (EIA). See,
e.g., WO 99/38498 at p. 11.
[0326] The methods of the invention may be applied as a therapy for
a subject having Alzheimer's disease or a dementia, or the methods
of the invention may be applied as a prophylaxis against
Alzheimer's disease or dementia for subject with such a
predisposition, as in a subject, e.g., with a genomic mutation in
the APP gene, the ApoE gene, or a presenilin gene. The subject may
have (or may be predisposed to developing or may be suspected of
having) vascular dementia, or senile dementia, Mild Cognitive
Impairment, or early Alzheimer's disease. In addition to
Alzheimer's disease, the subject may have another amyloid-related
disease such as cerebral amyloid angiopathy, or the subject may
have amyloid deposits, especially amyloid-.beta. amyloid deposits
in the brain.
Treatment of Amyloid-Related Diseases
[0327] The present invention pertains to methods of using the
compounds and pharmaceutical compositions thereof in the treatment
and prevention of amyloid-related diseases. The pharmaceutical
compositions of the invention may be administered therapeutically
or prophylactically to treat diseases associated with amyloid
(e.g., AL amyloid protein (.lamda. or .kappa.-chain related, e.g.,
amyloid .lamda., amyloid .kappa., amyloid .kappa.IV, amyloid
.lamda.VI, amyloid .gamma., amyloid .gamma.1), A.beta., IAPP,
.beta..sub.2M, AA, or AH amyloid protein) fibril formation,
aggregation or deposition.
[0328] The pharmaceutical compositions of the invention may act to
ameliorate the course of an amyloid-related disease using any of
the following mechanisms (this list is meant to be illustrative and
not limiting): slowing the rate of amyloid fibril formation or
deposition; lessening the degree of amyloid deposition; inhibiting,
reducing, or preventing amyloid fibril formation; inhibiting
neurodegeneration or cellular toxicity induced by amyloid;
inhibiting amyloid induced inflammation; enhancing the clearance of
amyloid from the brain; enhancing degradation of A.beta. in the
brain; or favoring clearance of amyloid protein prior to its
organization in fibrils.
[0329] "Modulation" of amyloid deposition includes both inhibition,
as defined above, and enhancement of amyloid deposition or fibril
formation. The term "modulating" is intended, therefore, to
encompass prevention or stopping of amyloid formation or
accumulation, inhibition or slowing down of further amyloid
formation or accumulation in a subject with ongoing amyloidosis,
e.g., already having amyloid deposition, and reducing or reversing
of amyloid formation or accumulation in a subject with ongoing
amyloidosis; and enhancing amyloid deposition, e.g., increasing the
rate or amount of amyloid deposition in vivo or in vitro.
Amyloid-enhancing compounds may be useful in animal models of
amyloidosis, for example, to make possible the development of
amyloid deposits in animals in a shorter period of time or to
increase amyloid deposits over a selected period of time.
Amyloid-enhancing compounds may be useful in screening assays for
compounds which inhibit amyloidosis in vivo, for example, in animal
models, cellular assays and in vitro assays for amyloidosis. Such
compounds may be used, for example, to provide faster or more
sensitive assays for compounds. Modulation of amyloid deposition is
determined relative to an untreated subject or relative to the
treated subject prior to treatment.
[0330] "Inhibition" of amyloid deposition includes preventing or
stopping of amyloid formation, e.g., fibrillogenesis, clearance of
amyloid, e.g., soluble A.beta. from brain, inhibiting or slowing
down of further amyloid deposition in a subject with amyloidosis,
e.g., already having amyloid deposits, and reducing or reversing
amyloid fibrillogenesis or deposits in a subject with ongoing
amyloidosis. Inhibition of amyloid deposition is determined
relative to an untreated subject, or relative to the treated
subject prior to treatment, or, e.g., determined by clinically
measurable improvement, e.g., or in the case of a subject with
brain amyloidosis, e.g., an Alzheimer's or cerebral amyloid
angiopathy subject, stabilization of cognitive function or
prevention of a further decrease in cognitive function (i.e.,
preventing, slowing, or stopping disease progression), or
improvement of parameters such as the concentration of A.beta. or
tau in the CSF.
[0331] As used herein, "treatment" of a subject includes the
application or administration of a composition of the invention to
a subject, or application or administration of a composition of the
invention to a cell or tissue from a subject, who has an
amyloid-related disease or condition, has a symptom of such a
disease or condition, or is at risk of (or susceptible to) such a
disease or condition, with the purpose of curing, healing,
alleviating, relieving, altering, remedying, ameliorating,
improving, or affecting the disease or condition, the symptom of
the disease or condition, or the risk of (or susceptibility to) the
disease or condition. The term "treating" refers to any indicia of
success in the treatment or amelioration of an injury, pathology or
condition, including any objective or subjective parameter such as
abatement; remission; diminishing of symptoms or making the injury,
pathology or condition more tolerable to the subject; slowing in
the rate of degeneration or decline; making the final point of
degeneration less debilitating; improving a subject's physical or
mental well-being; or, in some situations, preventing the onset of
dementia. The treatment or amelioration of symptoms can be based on
objective or subjective parameters; including the results of a
physical examination, a psychiatric evaluation, or a cognition test
such as CDR, MMSE, DAD, ADAS-Cog, or another test known in the art.
For example, the methods of the invention successfully treat a
subject's dementia by slowing the rate of or lessening the extent
of cognitive decline.
[0332] In one embodiment, the term "treating" includes maintaining
a subject's CDR rating at its base line rating or at 0. In another
embodiment, the term treating includes decreasing a subject's CDR
rating by about 0.25 or more, about 0.5 or more, about 1.0 or more,
about 1.5 or more, about 2.0 or more, about 2.5 or more, or about
3.0 or more. In another embodiment, the term "treating" also
includes reducing the rate of the increase of a subject's CDR
rating as compared to historical controls. In another embodiment,
the term includes reducing the rate of increase of a subject's CDR
rating by about 5% or more, about 10% or more, about 20% or more,
about 25% or more, about 30% or more, about 40% or more, about 50%
or more, about 60% or more, about 70% or more, about 80% or more,
about 90% or more, or about 100%, of the increase of the historical
or untreated controls.
[0333] In another embodiment, the term "treating" also includes
maintaining a subject's score on the MMSE. The term "treating"
includes increasing a subject's MMSE score by about 1, about 2,
about 3, about 4, about 5, about 7.5, about 10, about 12.5, about
15, about 17.5, about 20, or about 25 points. The term also
includes reducing the rate of the decrease of a subject's MMSE
score as compared to historical controls. In another embodiment,
the term includes reducing the rate of decrease of a subject's MMSE
score by about 5% or less, about 10% or less, about 20% or less,
about 25% or less, about 30% or less, about 40% or less, about 50%
or less, about 60% or less, about 70% or less, about 80% or less,
about 90% or less or about 100% or less, of the decrease of the
historical or untreated controls.
[0334] In another embodiment, the term "treating" also includes
maintaining a subject's score on the DAD. The term "treating"
includes increasing a subject's DAD score by about 1, about 5,
about 10, about 15, about 20, about 30, about 35, about 40, about
50, about 60, about 70, or about 80 points. The term also includes
reducing the rate of the decrease of a subject's DAD score as
compared to historical controls. In another embodiment, the term
includes reducing the rate of decrease of a subject's DAD score by
about 5% or less, about 10% or less, about 20% or less, about 25%
or less, about 30% or less, about 40% or less, about 50% or less,
about 60% or less, about 70% or less, about 80% or less, about 90%
or less or about 100% or less, of the decrease of the historical or
untreated controls.
[0335] In yet another embodiment, the term "treating" includes
maintaining a subject's score on the ADAS-Cog. The term "treating"
includes decreasing a subject's ADAS-Cog score by about 1 point or
greater, by about 2 points or greater, by about 3 points or
greater, by about 4 points or greater, by about 5 points or
greater, by about 7.5 points or greater, by about 10 points or
greater, by about 12.5 points or greater, by about 15 points or
greater, by about 17.5 points or greater, by about 20 points or
greater, or by about 25 points or greater. The term also includes
reducing the rate of the increase of a subject's ADAS-Cog score as
compared to historical controls. In another embodiment, the term
includes reducing the rate of increase of a subject's ADAS-Cog
score by about 5% or more, about 10% or more, about 20% or more,
about 25% or more, about 30% or more, about 40% or more, about 50%
or more, about 60% or more, about 70% or more, about 80% or more,
about 90% or more or about 100% of the increase of the historical
or untreated controls.
[0336] In another embodiment, the term "treating" e.g., for AA or
AL amyloidosis, includes an increase in serum creatinine, e.g., an
increase of creatinine clearance of 10% or greater, 20% or greater,
50% or greater, 80% or greater, 90% or greater, 100% or greater,
150% or greater, 200% or greater. The term "treating" also may
induce remission of nephrotic syndrome (NS). It may also include
remission of chronic diarrhea and/or a gain in body weight, e.g.,
by 10% or greater, 15% or greater, or 20% or greater.
[0337] Without wishing to be bound by theory, in some aspects the
pharmaceutical compositions of the invention contain a compound
that prevents or inhibits amyloid fibril formation, either in the
brain or other organ of interest (acting locally) or throughout the
entire body (acting systemically). Pharmaceutical compositions of
the invention may be effective in controlling amyloid deposition
either following their entry into the brain (following penetration
of the blood brain barrier) or from the periphery. When acting from
the periphery, a compound of a pharmaceutical composition may alter
the equilibrium of amyloidogenic peptide between the brain and the
plasma so as to favor the exit of amyloidogenic peptide from the
brain. It may also favor clearance (or catabolism) of the amyloid
protein (soluble), and then prevent amyloid fibril formation and
deposition due to a reduction of the amyloid protein pool in a
specific organ, e.g., liver, spleen, pancreas, kidney, joints,
brain, etc. An increase in the exit of amyloidogenic peptide from
the brain would result in a decrease in amyloidogenic peptide brain
concentration and therefore favor a decrease in amyloidogenic
peptide deposition. In particular, an agent may lower the levels of
amyloid .beta. peptides, e.g., both A.beta.40 and A.beta.42 in the
CSF and the plasma, or the agent may lower the levels of amyloid
.beta. peptides, e.g., A.beta.40 and A.beta.42 in the CSF and
increase it in the plasma. Alternatively, compounds that penetrate
the brain could control deposition by acting directly on brain
amyloidogenic peptide e.g., by maintaining it in a non-fibrillar
form or favoring its clearance from the brain, by increasing its
degradation in the brain, or protecting brain cells from the
detrimental effect of amyloidogenic peptide. An agent can also
cause a decrease of the concentration of the amyloid protein (i.e.,
in a specific organ so that the critical concentration needed to
trigger amyloid fibril formation or deposition is not reached).
Furthermore, the compounds described herein may inhibit or reduce
an interaction between amyloid and a cell surface constituent, for
example, a glycosaminoglycan or proteoglycan constituent of a
basement membrane, whereby inhibiting or reducing this interaction
produces the observed neuroprotective and cell-protective effects.
For example, the compound may also prevent an amyloid peptide from
binding or adhering to a cell surface, a process which is known to
cause cell damage or toxicity. Similarly, the compound may block
amyloid-induced cellular toxicity or microglial activation or
amyloid-induced neurotoxicity, or inhibit amyloid induced
inflammation. The compound may also reduce the rate or amount of
amyloid aggregation, fibril formation, or deposition, or the
compound lessens the degree of amyloid deposition. The foregoing
mechanisms of action should not be construed as limiting the scope
of the invention inasmuch as the invention may be practiced without
such information.
[0338] The A.beta. peptide has been shown by several groups to be
highly toxic to neurons. Amyloid plaques are directly associated
with reactive gliosis, dystrophic neurites and apoptotic cells,
suggesting that plaques induce neurodegenerative changes.
Neurotoxicity may eventually disrupt or even kill neurons. In
vitro, A.beta. has been shown to induce apoptosis in many different
neuronal cell types, such as rat PC-12 cells, primary rat
hippocampal and cortical cultures, and the predifferentiated human
neurotype SH-SY5Y cell line (Dickson D W (2004) J Clin Invest
114:23-7; Canu et al. (2003) Cerebellum 2:270-278; Li et al. (1996)
Brain Research 738:196-204). Numerous reports have shown that
A.beta. fibrils can induce neurodegeneration, and it has been shown
that neuronal cells exposed in vitro to A.beta. can become
apoptotic (Morgan et al. (2004) Prog. Neurobiol. 74:323-349;
Stefani et al. (2003) J. Mol. Med. 81:678-99; La Ferla et al.
(1997) J. Clin. Invest. 100(2):310-320). In Alzheimer's disease, a
progressive neuronal cell loss accompanies the deposition of
A.beta. amyloid fibrils in senile plaques.
[0339] In yet another aspect, the invention pertains to a method
for inhibiting A.beta.-induced neuronal cell death by administering
an effective amount of a compound of the present invention.
[0340] Another aspect of the invention pertains to a method for
providing neuroprotection to a subject having an A.beta.-amyloid
related disease, e.g. Alzheimer's disease, that includes
administering an effective amount of a compound of the present
invention to the subject, such that neuroprotection is
provided.
[0341] In another aspect, methods for inhibiting A.beta.-induced
neuronal cell death are provided that include administration of an
effective amount of a compound of the present invention to a
subject such that neuronal cell death is inhibited.
[0342] In another aspect, methods for treating a disease state
characterized by A.beta.-induced neuronal cell death in a subject
are provided, e.g., by administering an effective amount of a
compound of the present invention. Non-limiting examples of such
disease states include Alzheimer's disease and A.beta.-amyloid
related diseases.
[0343] The term "neuroprotection" includes protection of neuronal
cells of a subject from A.beta.-induced cell death, e.g., cell
death induced directly or indirectly by an A.beta. peptide.
A.beta.-induced cell death may result in initiation of processes
such as, for example: the destabilization of the cytoskeleton; DNA
fragmentation; the activation of hydrolytic enzymes, such as
phospholipase A2; activation of caspases, calcium-activated
proteases and/or calcium-activated endonucleases; inflammation
mediated by macrophages; calcium influx into a cell; membrane
potential changes in a cell; the disruption of cell junctions
leading to decreased or absent cell-cell communication; and the
activation of expression of genes involved in cell death, e.g.,
immediate-early genes.
[0344] The term "amyloid-.beta. disease" (or "amyloid-.beta.
related disease," which terms as used herein are synonymous) may be
used for mild cognitive impairment; vascular dementia; early
Alzheimer's disease; Alzheimer's disease, including sporadic
(non-hereditary) Alzheimer's disease and familial (hereditary)
Alzheimer's disease; age-related cognitive decline; cerebral
amyloid angiopathy ("CAA"); hereditary cerebral hemorrhage; senile
dementia; Down's syndrome; inclusion body myositis ("IBM"); or
age-related macular degeneration ("ARMD"). According to certain
aspects of the invention, amyloid-.beta. is a peptide having 39-43
amino-acids, or amyloid-.beta. is an amyloidogenic peptide produced
from PAPP.
[0345] Mild cognitive impairment ("MCI") is a condition
characterized by a state of mild but measurable impairment in
thinking skills, which is not necessarily associated with the
presence of dementia. MCI frequently, but not necessarily, precedes
Alzheimer's disease. It is a diagnosis that has most often been
associated with mild memory problems, but it can also be
characterized by mild impairments in other thinking skills, such as
language or planning skills. However, in general, an individual
with MCI will have more significant memory lapses than would be
expected for someone of their age or educational background. As the
condition progresses, a physician may change the diagnosis to
"Mild-to-Moderate Cognitive Impairment," as is well understood in
this art.
[0346] Cerebral amyloid angiopathy ("CAA") refers to the specific
deposition of amyloid fibrils in the walls of leptomingeal and
cortical arteries, arterioles and in capillaries and veins. It is
commonly associated with Alzheimer's disease, Down's syndrome and
normal aging, as well as with a variety of familial conditions
related to stroke or dementia (see Frangione, et al., Amyloid: J.
Protein Folding Disord. 8, Suppl. 1, 36-42 (2001)). CAA can occur
sporadically or be hereditary. Multiple mutation sites in either
A.beta. or the APP gene have been identified and are clinically
associated with either dementia or cerebral hemorrhage. Exemplary
CAA disorders include, but are not limited to, hereditary cerebral
hemorrhage with amyloidosis of Icelandic type (HCHWA-I); the Dutch
variant of HCHWA (HCHWA-D; a mutation in A.beta.); the Flemish
mutation of A.beta.; the Arctic mutation of A.beta.; the Italian
mutation of A.beta.; the Iowa mutation of A.beta.; familial British
dementia; and familial Danish dementia. Cerebral amyloid angiopathy
is known to be associated with cerebral hemorrhage (or hemorrhagic
stroke).
[0347] Additionally, abnormal accumulation of APP and of
amyloid-.beta. protein in muscle fibers has been implicated in the
pathology of sporadic inclusion body myositis ("IBM") (Askanas, et
al., Proc. Natl. Acad. Sci. USA 93, 1314-19 (1996); Askanas, et
al., Current Opinion in Rheumatology 7, 486-96 (1995)).
Accordingly, the compounds of the invention can be used
prophylactically or therapeutically in the treatment of disorders
in which amyloid-.beta. protein is abnormally deposited at
non-neurological locations, such as treatment of IBM by delivery of
the compounds to muscle fibers.
[0348] Additionally, it has been shown that A.beta. is associated
with abnormal extracellular deposits, known as drusen, that
accumulate along the basal surface of the retinal pigmented
epithelium in individuals with age-related macular degeneration
(ARMD). ARMD is a cause of irreversible vision loss in older
individuals. It is believed that A.beta. deposition could be an
important component of the local inflammatory events that
contribute to atrophy of the retinal pigmented epithelium, drusen
biogenesis, and the pathogenesis of ARMD (Johnson, et al., Proc.
Natl. Acad. Sci. USA 99(18), 11830-5 (2002)). Therefore, the
invention also relates to the treatment or prevention of
age-related macular degeneration.
[0349] Also, the invention relates to a method for preventing or
inhibiting amyloid deposition in a subject. For example, such a
method comprises administering to a subject a therapeutically
effective amount of a compound capable of reducing the
concentration of amyloid (e.g., AL amyloid protein (.lamda. or
.kappa.-chain related, e.g., amyloid .lamda., amyloid .kappa.,
amyloid .kappa.IV, amyloid .lamda.VI, amyloid .gamma., amyloid
.gamma.1), A.beta., IAPP, .beta..sub.2M, AA, AH amyloid protein, or
other amyloids), such that amyloid accumulation or deposition is
prevented or inhibited.
[0350] In another aspect, the invention relates to a method for
preventing, reducing, or inhibiting amyloid deposition in a
subject. For example, such a method comprises administering to a
subject a therapeutically effective amount of a compound capable of
inhibiting amyloid (e.g., AL amyloid protein (.lamda. or
.kappa.-chain related, e.g., amyloid .lamda., amyloid .kappa.,
amyloid .kappa.IV, amyloid .lamda.VI, amyloid amyloid-.gamma.1),
A.beta., IAPP, .beta..sub.2M, AA, AH amyloid protein, or other
amyloids), such that amyloid deposition is prevented, reduced, or
inhibited.
[0351] The invention also relates to a method for modulating, e.g.,
minimizing, amyloid-associated damage to cells, comprising the step
of administering a compound capable of reducing the concentration
of amyloid (e.g., AL amyloid protein (.lamda. or .kappa.-chain
related, e.g., amyloid .lamda., amyloid .kappa., amyloid .kappa.IV,
amyloid .lamda.VI, amyloid .gamma., amyloid-.gamma.1), A.beta.,
IAPP, .beta..sub.2M, AA, AH amyloid protein, or another amyloid),
such that said amyloid-associated damage to cells is modulated. In
certain aspects of the invention, the methods for modulating
amyloid-associated damage to cells comprise a step of administering
a compound capable of reducing the concentration of amyloid or
reducing the interaction of an amyloid with a cell surface.
[0352] The invention also includes a method for directly or
indirectly preventing cell death in a subject, the method
comprising administering to a subject a therapeutically effective
amount of a compound capable of preventing amyloid (e.g., AL
amyloid protein (.lamda. or .kappa.-chain related, e.g., amyloid
.lamda., amyloid .kappa., amyloid .kappa.IV, amyloid .lamda.VI,
amyloid-.gamma., amyloid .gamma.1), A.beta., IAPP, .beta..sub.2M,
AA, AH amyloid protein, or other amyloid) mediated events that
lead, directly or indirectly, to cell death.
[0353] In an embodiment, the method is used to treat Alzheimer's
disease (e.g. sporadic or familial AD). The method can also be used
prophylactically or therapeutically to treat other clinical
occurrences of amyloid-.beta. deposition, such as in Down's
syndrome individuals and in patients with cerebral amyloid
angiopathy ("CAA") or hereditary cerebral hemorrhage.
[0354] The compounds of the invention may be used prophylactically
or therapeutically in the treatment of disorders in which
amyloid-beta peptide is abnormally deposited at non-neurological
locations, such as treatment of IBM by delivery of the compounds to
muscle fibers, or treatment of macular degeneration by delivery of
the compound(s) of the invention to the basal surface of the
retinal pigmented epithelium.
[0355] The present invention also provides a method for modulating
amyloid-associated damage to cells, comprising the step of
administering a compound capable of reducing the concentration of
A.beta., or capable of mimimizing the interaction of A.beta.
(soluble oligomeric or fibrillary) with the cell surface, such that
said amyloid-associated damage to cells is modulated. In certain
aspects of the invention, the methods for modulating
amyloid-associated damage to cells comprise a step of administering
a compound capable of reducing the concentration of A.beta. or
reducing the interaction of A.beta. with a cell surface.
[0356] In accordance with the present invention, there is further
provided a method for preventing cell death in a subject, said
method comprising administering to a subject a therapeutically
effective amount of a compound capable of preventing
A.beta.-mediated events that lead, directly or indirectly, to cell
death.
[0357] The present invention also provides a method for modulating
amyloid-associated damage to cells, comprising the step of
administering a compound capable of reducing the concentration of
IAPP, or capable of mimimizing the interaction of IAPP (soluble
oligomeric or fibrillary) with the cell surface, such that said
amyloid-associated damage to cells is modulated. In certain aspects
of the invention, the methods for modulating amyloid-associated
damage to cells comprise a step of administering a compound capable
of reducing the concentration of IAPP or reducing the interaction
of IAPP with a cell surface.
[0358] In accordance with the present invention, there is further
provided a method for preventing cell death in a subject, said
method comprising administering to a subject a therapeutically
effective amount of a compound capable of preventing IAPP-mediated
events that lead, directly or indirectly, to cell death.
[0359] This invention also provides methods and compositions which
are useful in the treatment of amyloidosis. The methods of the
invention involve administering to a subject a therapeutic compound
which inhibits amyloid deposition. Accordingly, the compositions
and methods of the invention are useful for inhibiting amyloidosis
in disorders in which amyloid deposition occurs. The methods of the
invention can be used therapeutically to treat amyloidosis or can
be used prophylactically in a subject susceptible to (hereditary)
amyloidosis or identified as being at risk to develop amyloidosis,
e.g., hereditary, or identified as being at risk to develop
amyloidosis. In certain embodiments, the invention includes a
method of inhibiting an interaction between an amyloidogenic
protein and a constituent of basement membrane to inhibit amyloid
deposition. The constituent of basement membrane is a glycoprotein
or proteoglycan, preferably heparan sulfate proteoglycan. A
therapeutic compound used in this method may interfere with binding
of a basement membrane constituent to a target binding site on an
amyloidogenic protein, thereby inhibiting amyloid deposition.
[0360] In some aspects, the methods of the invention involve
administering to a subject a therapeutic compound which inhibits
amyloid deposition. "Inhibition of amyloid deposition," includes
the prevention of amyloid formation, inhibition of further amyloid
deposition in a subject with ongoing amyloidosis and reduction of
amyloid deposits in a subject with ongoing amyloidosis. Inhibition
of amyloid deposition is determined relative to an untreated
subject or relative to the treated subject prior to treatment. In
an embodiment, amyloid deposition is inhibited by inhibiting an
interaction between an amyloidogenic protein and a constituent of
basement membrane. "Basement membrane" refers to an extracellular
matrix comprising glycoproteins and proteoglycans, including
laminin, collagen type IV, fibronectin, perlecan, agrin, dermatan
sulfate, and heparan sulfate proteoglycan (HSPG). In one
embodiment, amyloid deposition is inhibited by interfering with an
interaction between an amyloidogenic protein and a sulfated
glycosaminoglycan such as HSPG, dermatan sulfate, perlecan or agrin
sulfate. Sulfated glycosaminoglycans are known to be present in all
types of amyloids (see Snow, et al. Lab. Invest. 56, 120-23 (1987))
and amyloid deposition and HSPG deposition occur coincidentally in
animal models of amyloidosis (see Snow, et al. Lab. Invest. 56,
665-75 (1987) and Gervais, F. et al. Curr. Med. Chem., 3, 361-370
(2003)). Consensus binding site motifs for HSPG in amyloidogenic
proteins have been described (see, e.g., Cardin and Weintraub
Arteriosclerosis 9, 21-32 (1989)).
[0361] The ability of a compound to prevent or block the formation
or deposition of amyloid may reside in its ability to bind to
non-fibrillar, soluble amyloid protein and to maintain its
solubility.
[0362] The ability of a therapeutic compound of the invention to
inhibit an interaction between an amyloidogenic protein and a
glycoprotein or proteoglycan constituent of a basement membrane can
be assessed by an in vitro binding assay, such as that described in
U.S. Pat. No. 5,164,295, the contents of which are hereby
incorporated by reference. Alternatively, the ability of a compound
to bind to an amyloidogenic protein or to inhibit the binding of a
basement membrane constituent (e.g. HSPG) to an amyloidogenic
protein (e.g. A.beta.) can be measured using a mass spectrometry
assay where soluble protein, e.g. A.beta., IAPP, .beta..sub.2M is
incubated with the compound. A compound which binds to, e.g.
A.beta., will induce a change in the mass spectrum of the protein.
Exemplary protocols for a mass spectrometry assay employing A.beta.
and IAPP can be found in the Examples, the results of which are
provided in Table 3. The protocol can readily be modified to adjust
the sensitivity of the data, e.g., by adjusting the amount of
protein and/or compound employed. Thus, e.g., binding might be
detected for test compounds noted as not having detectable binding
employing less sensitive test protocols.
[0363] Alternative methods for screening compounds exist and can
readily be employed by a skilled practitioner to provide an
indication of the ability of test compounds to bind to, e.g.,
fibrillar A.beta.. One such screening assay is an ultraviolet
absorption assay. In an exemplary protocol, a test compound (20
.mu.M) is incubated with 50 .mu.M A.beta.(1-40) fibers for 1 hour
at 37.degree. C. in Tris buffered saline (20 mM Tris, 150 mM NaCl,
pH 7.4 containing 0.01 sodium azide). Following incubation, the
solution is centrifuged for 20 minutes at 21,000 g to sediment the
A.beta.(1-40) fibers along with any bound test compound. The amount
of test compound remaining in the supernatant can then be
determined by reading the absorbance. The fraction of test compound
bound can then be calculated by comparing the amount remaining in
the supernatants of incubations with A.beta. to the amount
remaining in control incubations which do not contain A.beta.
fibers. Thioflavin T and Congo Red, both of which are known to bind
to A.beta. fibers, may be included in each assay run as positive
controls. Before assaying, test compounds can be diluted to 40
.mu.M, which would be twice the concentration in the final test,
and then scanned using the Hewlett Packard 8453 UV/VIS
spectrophotometer to determine if the absorbance is sufficient for
detection.
[0364] In another embodiment, the invention pertains to a method
for improving cognition in a subject suffering from an
amyloid-related disease. The method includes administering an
effective amount of a therapeutic compound of the invention, such
that the subject's cognition is improved. The subject's cognition
can be tested using methods known in the art such as the Clinical
Dementia Rating ("CDR"), Mini-Mental State Examination ("MMSE"),
Disability Assessment for Dementia ("DAD"), and/or the Alzheimer's
Disease Assessment Scale-Cognition ("ADAS-Cog").
[0365] In another embodiment, the invention pertains to a method
for treating a subject for an amyloid-related disease. The method
includes administering a cognitive test to a subject prior to
administration of a compound of the invention, administering an
effective amount of a compound of the invention to the subject, and
administering a cognitive test to the subject subsequent to
administration of the compound, such that the subject is treated
for the amyloid-related disease, wherein the subject's score on
said cognitive test is improved.
[0366] "Improvement," "improved" or "improving" in cognition is
present within the context of the present invention if there is a
statistically significant difference in the direction of normality
between the performance of subjects treated using the methods of
the invention as compared to members of a placebo group, historical
control, or between subsequent tests given to the same subject.
[0367] In one embodiment, a subject's CDR is maintained at 0. In
another embodiment, a subject's CDR is decreased (e.g., improved)
by about 0.25 or more, about 0.5 or more, about 1.0 or more, about
1.5 or more, about 2.0 or more, about 2.5 or more, or about 3.0 or
more. In another embodiment, the rate of increase of a subject's
CDR rating is reduced by about 5% or more, about 10% or more, about
20% or more, about 25% or more, about 30% or more, about 40% or
more, about 50% or more, about 60% or more, about 70% or more,
about 80% or more, about 90% or more, or about 100% or more of the
increase of the historical or untreated controls.
[0368] In one embodiment, a subject's score on the MMSE is
maintained. Alternatively, the subject's score on the MMSE may be
increased by about 1, about 2, about 3, about 4, about 5, about
7.5, about 10, about 12.5, about 15, about 17.5, about 20, or about
25 points. In another alternative, the rate of the decrease of a
subject's MMSE score as compared to historical controls is reduced.
For example, the rate of the decrease of a subject's MMSE score may
be reduced by about 5% or more, about 10% or more, about 20% or
more, about 25% or more, about 30% or more, about 40% or more,
about 50% or more, about 60% or more, about 70% or more, about 80%
or more, about 90% or more, or about 100% or more of the decrease
of the historical or untreated controls.
[0369] In one embodiment, a subject's score on the DAD is
maintained. Alternatively, the subject's score on the DAD may be
increased by about 1, about 2, about 3, about 4, about 5, about
7.5, about 10, about 15, about 20, about 30, about 40, or about 50
or more points. In another alternative, the rate of the decrease of
a subject's DAD score as compared to historical controls is
reduced. For example, the rate of the decrease of a subject's DAD
score may be reduced by about 5% or more, about 10% or more, about
20% or more, about 25% or more, about 30% or more, about 40% or
more, about 50% or more, about 60% or more, about 70% or more,
about 80% or more, about 90% or more, or about 100% or more of the
decrease of the historical or untreated controls.
[0370] In one embodiment, the invention pertains to a method for
treating, slowing or stopping an amyloid-related disease associated
with cognitive impairment, by administering to a subject an
effective amount of a therapeutic compound of the invention,
wherein the annual deterioration of the subject's cognition as
measured by ADAS-Cog is less than 8 points per year, less the 6
points per year, less than 5 points per year, less than 4 points
per year, or less than 3 points per year. In a further embodiment,
the invention pertains to a method for treating, slowing or
stopping an amyloid-related disease associated with cognition by
administering an effective amount of a therapeutic compound of the
invention such that the subject's cognition as measured by ADAS-Cog
remains constant over a year. "Constant" includes fluctuations of
no more than 2 points. Remaining constant includes fluctuations of
two points or less in either direction. In a further embodiment,
the subject's cognition improves by 2 points or greater per year, 3
points or greater per year, 4 point or greater per year, 5 points
or greater per year, 6 points or greater per year, 7 points or
greater per year, 8 points or greater per year, etc. as measured by
the ADAS-Cog. In another alternative, the rate of the increase of a
subject's ADAS-Cog score as compared to historical controls is
reduced. For example, the rate of the increase of a subject's
ADAS-Cog score may be reduced by about 5% or more, about 10% or
more, about 20% or more, about 25% or more, about 30% or more,
about 40% or more, about 50% or more, about 60% or more, about 70%
or more, about 80% or more, about 90% or more or about 100% of the
increase of the historical or untreated controls.
[0371] In another embodiment, the ratio of A.beta.42:A.beta.40 in
the CSF or plasma of a subject decreases by about 15% or more,
about 20% or more, about 25% or more, about 30% or more, about 35%
or more, about 40% or more, about 45% or more, or about 50% or
more. In another embodiment, the levels of A.beta. in the subject's
cerebrospinal fluid decrease by about 15% or more, about 25% or
more, about 35% or more, about 45% or more, about 55% or more,
about 75% or more, or about 90% or more.
[0372] It is to be understood that wherever values and ranges are
provided herein, e.g., in ages of subject populations, dosages, and
blood levels, all values and ranges encompassed by these values and
ranges, are meant to be encompassed within the scope of the present
invention. Moreover, all values in these values and ranges may also
be the upper or lower limits of a range.
[0373] Furthermore, the invention pertains to any novel chemical
compound described herein. That is, the invention relates to novel
compounds, and novel methods of their use as described herein,
which are within the scope of the Formulae disclosed herein, and
which are not disclosed in the cited Patents and Patent
Applications.
Synthesis of Compounds of the Invention
[0374] In general, the compounds of the present invention may be
prepared by the methods illustrated in the general reaction schemes
as, for example, described below, or by modifications thereof,
using readily available starting materials, reagents and
conventional synthesis procedures. In these reactions, it is also
possible to make use of variants which are in themselves known, but
are not mentioned here. Functional and structural equivalents of
the compounds described herein and which have the same general
properties, wherein one or more simple variations of substituents
are made which do not adversely affect the essential nature or the
utility of the compound are also included.
[0375] The compounds of the present invention may be readily
prepared in accordance with the synthesis schemes and protocols
described herein, as illustrated in the specific procedures
provided. However, those skilled in the art will recognize that
other synthetic pathways for forming the compounds of this
invention may be used, and that the following is provided merely by
way of example, and is not limiting to the present invention. See,
e.g., "Comprehensive Organic Transformations" by R. Larock, VCH
Publishers (1989). It will be further recognized that various
protecting and deprotecting strategies will be employed that are
standard in the art (See, e.g., "Protective Groups in Organic
Synthesis" by Greene and Wuts). Those skilled in the relevant arts
will recognize that the selection of any particular protecting
group (e.g., amine and carboxyl protecting groups) will depend on
the stability of the protected moiety with regards to the
subsequent reaction conditions and will understand the appropriate
selections.
[0376] Further illustrating the knowledge of those skilled in the
art is the following sampling of the extensive chemical literature:
"Chemistry of the Amino Acids" by J. P. Greenstein and M. Winitz,
John Wiley & Sons, Inc., New York (1961); "Comprehensive
Organic Transformations" by R. Larock, VCH Publishers (1989); T. D.
Ocain, et al., J. Med. Chem. 31, 2193-99 (1988); E. M. Gordon, et
al., J. Med. Chem. 31, 2199-10 (1988); "Practice of Peptide
Synthesis" by M. Bodansky and A. Bodanszky, Springer-Verlag, New
York (1984); "Protective Groups in Organic Synthesis" by T. Greene
and P. Wuts (1991); "Asymmetric Synthesis: Construction of Chiral
Molecules Using Amino Acids" by G. M. Coppola and H. F. Schuster,
John Wiley & Sons, Inc., New York (1987); "The Chemical
Synthesis of Peptides" by J. Jones, Oxford University Press, New
York (1991); and "Introduction of Peptide Chemistry" by P. D.
Bailey, John Wiley & Sons, Inc., New York (1992).
[0377] The synthesis of compounds of the invention is carried out
in a solvent. Suitable solvents are liquids at ambient room
temperature and pressure or remain in the liquid state under the
temperature and pressure conditions used in the reaction. Useful
solvents are not particularly restricted provided that they do not
interfere with the reaction itself (that is, they preferably are
inert solvents), and they dissolve a certain amount of the
reactants. Depending on the circumstances, solvents may be
distilled or degassed. Solvents may be, for example, aliphatic
hydrocarbons (e.g., hexanes, heptanes, ligroin, petroleum ether,
cyclohexane, or methylcyclohexane) and halogenated hydrocarbons
(e.g., methylenechloride, chloroform, carbontetrachloride,
dichloroethane, chlorobenzene, or dichlororbenzene); aromatic
hydrocarbons (e.g., benzene, toluene, tetrahydronaphthalene,
ethylbenzene, or xylene); ethers (e.g., diglyme, methyl-tert-butyl
ether, methyl-tert-amyl ether, ethyl-tert-butyl ether,
diethylether, diisopropylether, tetrahydrofuran or
methyltetrahydrofurans, dioxane, dimethoxyethane, or
diethyleneglycol dimethylether); nitriles (e.g., acetonitrile);
ketones (e.g., acetone); esters (e.g., methyl acetate or ethyl
acetate); and mixtures thereof.
[0378] In general, after completion of the reaction, the product is
isolated from the reaction mixture according to standard
techniques. For example, the solvent is removed by evaporation or
filtration if the product is solid, optionally under reduced
pressure. After the completion of the reaction, water may be added
to the residue to make the aqueous layer acidic or basic and the
precipitated compound filtered, although care should be exercised
when handling water-sensitive compounds. Similarly, water may be
added to the reaction mixture with a hydrophobic solvent to extract
the target compound. The organic layer may be washed with water,
dried over anhydrous magnesium sulphate or sodium sulphate, and the
solvent is evaporated to obtain the target compound. The target
compound thus obtained may be purified, if necessary, e.g., by
recrystallization, reprecipitation, chromatography, or by
converting it to a salt by addition of an acid or base.
[0379] The compounds of the invention may be supplied in a solution
with an appropriate solvent or in a solvent-free form (e.g.,
lyophilized). In another aspect of the invention, the compounds and
buffers necessary for carrying out the methods of the invention may
be packaged as a kit, optionally including a container. The kit may
be commercially used for treating or preventing amyloid-related
disease according to the methods described herein and may include
instructions for use in a method of the invention. Additional kit
components may include acids, bases, buffering agents, inorganic
salts, solvents, antioxidants, preservatives, or metal chelators.
The additional kit components are present as pure compositions, or
as aqueous or organic solutions that incorporate one or more
additional kit components. Any or all of the kit components
optionally further comprise buffers.
[0380] The term "container" includes any receptacle for holding the
therapeutic compound. For example, in one embodiment, the container
is the packaging that contains the compound. In other embodiments,
the container is not the packaging that contains the compound,
i.e., the container is a receptacle, such as a box or vial that
contains the packaged compound or unpackaged compound and the
instructions for use of the compound. Moreover, packaging
techniques are well known in the art. It should be understood that
the instructions for use of the therapeutic compound may be
contained on the packaging containing the therapeutic compound, and
as such the instructions form an increased functional relationship
to the packaged product.
Pharmaceutical Preparations
[0381] In another embodiment, the present invention relates to
pharmaceutical compositions comprising agents according to any of
the Formulae herein for the treatment of an amyloid-related
disease, as well as methods of manufacturing such pharmaceutical
compositions.
[0382] In general, the agents of the present invention may be
prepared by the methods illustrated in the general reaction schemes
as, for example, in the patents and patent applications referred to
herein, or by modifications thereof, using readily available
starting materials, reagents and conventional synthesis procedures.
In these reactions, it is also possible to make use of variants
which are in themselves known, but are not mentioned here.
Functional and structural equivalents of the agents described
herein and which have the same general properties, wherein one or
more simple variations of substituents are made which do not
adversely affect the essential nature or the utility of the agent
are also included.
[0383] The agents of the invention may be supplied in a solution
with an appropriate solvent or in a solvent-free form (e.g.,
lyophilized). In another aspect of the invention, the agents and
buffers necessary for carrying out the methods of the invention may
be packaged as a kit. The kit may be commercially used according to
the methods described herein and may include instructions for use
in a method of the invention. Additional kit components may include
acids, bases, buffering agents, inorganic salts, solvents,
antioxidants, preservatives, or metal chelators. The additional kit
components are present as pure compositions, or as aqueous or
organic solutions that incorporate one or more additional kit
components. Any or all of the kit components optionally further
comprise buffers.
[0384] The therapeutic agent may also be administered parenterally,
intraperitoneally, intraspinally, or intracerebrally. Dispersions
can be prepared in glycerol, liquid polyethylene glycols, and
mixtures thereof and in oils. Under ordinary conditions of storage
and use, these preparations may contain a preservative to prevent
the growth of microorganisms.
[0385] To administer the therapeutic agent by other than parenteral
administration, it may be necessary to coat the agent with, or
co-administer the agent with, a material to prevent its
inactivation. For example, the therapeutic agent may be
administered to a subject in an appropriate carrier, for example,
liposomes, or a diluent. Pharmaceutically acceptable diluents
include saline and aqueous buffer solutions. Liposomes include
water-in-oil-in-water CGF emulsions as well as conventional
liposomes (Strejan et al., J. Neuroimmunol. 7, 27 (1984)).
[0386] Pharmaceutical compositions suitable for injectable use
include sterile aqueous solutions (where water soluble) or
dispersions and sterile powders for the extemporaneous preparation
of sterile injectable solutions or dispersion. In all cases, the
composition must be sterile and must be fluid to the extent that
easy syringability exists. It must be stable under the conditions
of manufacture and storage and must be preserved against the
contaminating action of microorganisms such as bacteria and
fungi.
[0387] Suitable pharmaceutically acceptable vehicles include,
without limitation, any non-immunogenic pharmaceutical adjuvants
suitable for oral, parenteral, nasal, mucosal, transdermal,
intravascular (IV), intraarterial (IA), intramuscular (IM), and
subcutaneous (SC) administration routes, such as phosphate buffer
saline (PBS).
[0388] The vehicle can be a solvent or dispersion medium
containing, for example, water, ethanol, polyol (for example,
glycerol, propylene glycol, and liquid polyethylene glycol, and the
like), suitable mixtures thereof, and vegetable oils. The proper
fluidity can be maintained, for example, by the use of a coating
such as lecithin, by the maintenance of the required particle size
in the case of dispersion and by the use of surfactants. Prevention
of the action of microorganisms can be achieved by various
antibacterial and antifungal agents, for example, parabens,
chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In
many cases, isotonic agents are included, for example, sugars,
sodium chloride, or polyalcohols such as mannitol and sorbitol, in
the composition. Prolonged absorption of the injectable
compositions can be brought about by including in the composition
an agent which delays absorption, for example, aluminum
monostearate or gelatin.
[0389] Sterile injectable solutions can be prepared by
incorporating the therapeutic agent in the required amount in an
appropriate solvent with one or a combination of ingredients
enumerated above, as required, followed by filtered sterilization.
Generally, dispersions are prepared by incorporating the
therapeutic agent into a sterile vehicle which contains a basic
dispersion medium and the required other ingredients from those
enumerated above. In the case of sterile powders for the
preparation of sterile injectable solutions, the methods of
preparation are vacuum drying and freeze-drying which yields a
powder of the active ingredient (i.e., the therapeutic agent) plus
any additional desired ingredient from a previously
sterile-filtered solution thereof.
[0390] The therapeutic agent can be orally administered, for
example, with an inert diluent or an assimilable edible carrier.
The therapeutic agent and other ingredients may also be enclosed in
a hard or soft shell gelatin capsule, compressed into tablets, or
incorporated directly into the subject's diet. For oral therapeutic
administration, the therapeutic agent may be incorporated with
excipients and used in the form of ingestible tablets, buccal
tablets, troches, capsules, elixirs, suspensions, syrups, wafers,
and the like. The percentage of the therapeutic agent in the
compositions and preparations may, of course, be varied. The amount
of the therapeutic agent in such therapeutically useful
compositions is such that a suitable dosage will be obtained.
[0391] It is especially advantageous to formulate parenteral
compositions in dosage unit form for ease of administration and
uniformity of dosage. Dosage unit form as used herein refers to
physically discrete units suited as unitary dosages for the
subjects to be treated; each unit containing a predetermined
quantity of therapeutic agent calculated to produce the desired
therapeutic effect in association with the required pharmaceutical
vehicle. The specification for the dosage unit forms of the
invention are dictated by and directly dependent on (a) the unique
characteristics of the therapeutic agent and the particular
therapeutic effect to be achieved, and (b) the limitations inherent
in the art of compounding such a therapeutic agent for the
treatment of amyloid deposition in subjects.
[0392] The present invention therefore includes pharmaceutical
formulations comprising the agents of the Formulae described
herein, including pharmaceutically acceptable salts thereof, in
pharmaceutically acceptable vehicles for aerosol, oral and
parenteral administration. Also, the present invention includes
such agents, or salts thereof, which have been lyophilized and
which may be reconstituted to form pharmaceutically acceptable
formulations for administration, as by intravenous, intramuscular,
or subcutaneous injection. Administration may also be intradermal
or transdermal.
[0393] In accordance with the present invention, an agent of the
Formulae described herein, and pharmaceutically acceptable salts
thereof, may be administered orally or through inhalation as a
solid, or may be administered intramuscularly or intravenously as a
solution, suspension or emulsion. Alternatively, the agents or
salts may also be administered by inhalation, intravenously or
intramuscularly as a liposomal suspension.
[0394] Pharmaceutical formulations are also provided which are
suitable for administration as an aerosol, by inhalation. These
formulations comprise a solution or suspension of the desired agent
of any Formula herein, or a salt thereof, or a plurality of solid
particles of the agent or salt. The desired formulation may be
placed in a small chamber and nebulized. Nebulization may be
accomplished by compressed air or by ultrasonic energy to form a
plurality of liquid droplets or solid particles comprising the
agents or salts. The liquid droplets or solid particles should have
a particle size in the range of about 0.5 to about 5 microns. The
solid particles can be obtained by processing the solid agent of
any Formula described herein, or a salt thereof, in any appropriate
manner known in the art, such as by micronization. The size of the
solid particles or droplets will be, for example, from about 1 to
about 2 microns. In this respect, commercial nebulizers are
available to achieve this purpose.
[0395] A pharmaceutical formulation suitable for administration as
an aerosol may be in the form of a liquid, the formulation will
comprise a water-soluble agent of any Formula described herein, or
a salt thereof, in a carrier which comprises water. A surfactant
may be present which lowers the surface tension of the formulation
sufficiently to result in the formation of droplets within the
desired size range when subjected to nebulization.
[0396] Peroral compositions also include liquid solutions,
emulsions, suspensions, and the like. The pharmaceutically
acceptable vehicles suitable for preparation of such compositions
are well known in the art. Typical components of carriers for
syrups, elixirs, emulsions and suspensions include ethanol,
glycerol, propylene glycol, polyethylene glycol, liquid sucrose,
sorbitol and water. For a suspension, typical suspending agents
include methyl cellulose, sodium carboxymethyl cellulose,
tragacanth, and sodium alginate; typical wetting agents include
lecithin and polysorbate 80; and typical preservatives include
methyl paraben and sodium benzoate. Peroral liquid compositions may
also contain one or more components such as sweeteners, flavoring
agents and colorants disclosed above.
[0397] Pharmaceutical compositions may also be coated by
conventional methods, typically with pH or time-dependent coatings,
such that the subject agent is released in the gastrointestinal
tract in the vicinity of the desired topical application, or at
various times to extend the desired action. Such dosage forms
typically include, but are not limited to, one or more of cellulose
acetate phthalate, polyvinylacetate phthalate, hydroxypropyl methyl
cellulose phthalate, ethyl cellulose, waxes, and shellac.
[0398] Other compositions useful for attaining systemic delivery of
the subject agents include sublingual, buccal and nasal dosage
forms. Such compositions typically comprise one or more of soluble
filler substances such as sucrose, sorbitol and mannitol; and
binders such as acacia, microcrystalline cellulose, carboxymethyl
cellulose and hydroxypropyl methyl cellulose. Glidants, lubricants,
sweeteners, colorants, antioxidants and flavoring agents disclosed
above may also be included.
[0399] The compositions of this invention can also be administered
topically to a subject, e.g., by the direct laying on or spreading
of the composition on the epidermal or epithelial tissue of the
subject, or transdermally via a "patch". Such compositions include,
for example, lotions, creams, solutions, gels and solids. These
topical compositions may comprise an effective amount, usually at
least about 0.1%, or even from about 1% to about 5%, of an agent of
the invention. Suitable carriers for topical administration
typically remain in place on the skin as a continuous film, and
resist being removed by perspiration or immersion in water.
Generally, the carrier is organic in nature and capable of having
dispersed or dissolved therein the therapeutic agent. The carrier
may include pharmaceutically acceptable emollients, emulsifiers,
thickening agents, solvents and the like.
[0400] In one embodiment, active agents are administered at a
therapeutically effective dosage sufficient to inhibit amyloid
deposition in a subject. A "therapeutically effective" dosage
inhibits amyloid deposition by, for example, at least about 20%, or
by at least about 40%, or even by at least about 60%, or by at
least about 80% relative to untreated subjects. In the case of an
Alzheimer's subject, a "therapeutically effective" dosage
stabilizes cognitive function or prevents a further decrease in
cognitive function (i.e., preventing, slowing, or stopping disease
progression). The present invention accordingly provides
therapeutic drugs. By "therapeutic" or "drug" is meant an agent
having a beneficial ameliorative or prophylactic effect on a
specific disease or condition in a living human or non-human
animal.
[0401] In the case of AA or AL amyloidosis, the agent may improve
or stabilize specific organ function. As an example, renal function
may be stabilized or improved by 10% or greater, 20% or greater,
30% or greater, 40% or greater, 50% or greater, 60% or greater, 70%
or greater, 80% or greater, or by greater than 90%.
[0402] In the case of IAPP, the agent may maintain or increase
.beta.-islet cell function, as determined by insulin concentration
or the Pro-IAPP/IAPP ratio. In a further embodiment, the
Pro-IAPP/IAPP ratio is increased by about 10% or greater, about 20%
or greater, about 30% or greater, about 40% or greater, or by about
50%. In a further embodiment, the ratio is increased up to 50%. In
addition, a therapeutically effective amount of the agent may be
effective to improve glycemia or insulin levels.
[0403] In another embodiment, the active agents are administered at
a therapeutically effective dosage sufficient to treat AA
(secondary) amyloidosis and/or AL (primary) amyloidosis, by
stabilizing renal function, decreasing proteinuria, increasing
creatinine clearance (e.g., by at least 50% or greater or by at
least 100% or greater), remission of chronic diarrhea, or by weight
gain (e.g., 10% or greater). In addition, the agents may be
administered at a therapeutically effective dosage sufficient to
improve nephrotic syndrome.
[0404] Furthermore, active agents may be administered at a
therapeutically effective dosage sufficient to decrease deposition
in a subject of amyloid protein, e.g., A.beta.40 or A.beta.42. A
therapeutically effective dosage decreases amyloid deposition by,
for example, at least about 15%, or by at least about 40%, or even
by at least 60%, or at least by about 80% relative to untreated
subjects.
[0405] In another embodiment, active agents are administered at a
therapeutically effective dosage sufficient to increase or enhance
amyloid protein, e.g., A.beta.40 or A.beta.42, in the blood, CSF,
or plasma of a subject. A therapeutically effective dosage
increases the concentration by, for example, at least about 15%, or
by at least about 40%, or even by at least 60%, or at least by
about 80% relative to untreated subjects.
[0406] In yet another embodiment, active agents are administered at
a therapeutically effective dosage sufficient to maintain a
subject's CDR rating at its base line rating or at 0. In another
embodiment, the active agents are administered at a therapeutically
effective dosage sufficient to decrease a subject's CDR rating by
about 0.25 or more, about 0.5 or more, about 1.0 or more, about 1.5
or more, about 2.0 or more, about 2.5 or more, or about 3.0 or
more. In another embodiment, the active agents are administered at
a therapeutically effective dosage sufficient to reduce the rate of
the increase of a subject's CDR rating as compared to historical or
untreated controls. In another embodiment, the therapeutically
effective dosage is sufficient to reduce the rate of increase of a
subject's CDR rating (relative to untreated subjects) by about 5%
or greater, about 10% or greater, about 20% or greater, about 25%
or greater, about 30% or greater, about 40% or greater, about 50%
or greater, about 60% or greater, about 70% or greater, about 80%
or greater, about 90% or greater or about 100% or greater.
[0407] In yet another embodiment, active agents are administered at
a therapeutically effective dosage sufficient to maintain a
subject's score on the MMSE. In another embodiment, the active
agents are administered at a therapeutically effective dosage
sufficient to increase a subject's MMSE score by about 1, about 2,
about 3, about 4, about 5, about 7.5, about 10, about 12.5, about
15, about 17.5, about 20, or about 25 points. In another
embodiment, the active agents are administered at a therapeutically
effective dosage sufficient to reduce the rate of the decrease of a
subject's MMSE score as compared to historical controls. In another
embodiment, the therapeutically effective dosage is sufficient to
reduce the rate of decrease of a subject's MMSE score may be about
5% or less, about 10% or less, about 20% or less, about 25% or
less, about 30% or less, about 40% or less, about 50% or less,
about 60% or less, about 70% or less, about 80% or less, about 90%
or less or about 100% or less, of the decrease of the historical or
untreated controls.
[0408] Another means to evaluate cognition, particularly
Alzheimer's disease, is the Alzheimer's Disease Assessment Scale
(ADAS-Cog), or a variation termed the Standardized Alzheimer's
Disease Assessment Scale (SADAS). It is commonly used as an
efficacy measure in clinical drug trials of Alzheimer's disease and
related disorders characterized by cognitive decline. SADAS and
ADAS-Cog were not designed to diagnose Alzheimer's disease; they
are useful in characterizing symptoms of dementia and are a
relatively sensitive indicator of dementia progression. (See, e.g.,
Doraiswamy, Neurology 48:1511-1517, 1997; and Standish, J. Am.
Geriatr. Soc. 44:712-716, 1996.) Annual deterioration in untreated
Alzheimer's disease patients is approximately 8 points per year
(See, eg., Raskind, M Prim. Care Companion J Clin Psychiatry 2000
August; 2(4):134-138).
[0409] In yet another embodiment, active agents are administered at
a therapeutically effective dosage sufficient to maintain a
subject's score on the DAD. In another embodiment, the active
agents are administered at a therapeutically effective dosage
sufficient to increase a subject's DAD score by about 1, about 2,
about 3, about 4, about 5, about 10, about 15, about 20, about 25,
about 30, about 40, or about 50 points or greater. In another
embodiment, the active agents are administered at a therapeutically
effective dosage sufficient to reduce the rate of the decrease of a
subject's DAD score as compared to historical controls. In another
embodiment, the therapeutically effective dosage is sufficient to
reduce the rate of decrease of a subject's DAD score by about 5% or
less, about 10% or less, about 20% or less, about 25% or less,
about 30% or less, about 40% or less, about 50% or less, about 60%
or less, about 70% or less, about 80% or less, about 90% or less or
about 100% or less, of the decrease of the historical or untreated
controls.
[0410] In yet another embodiment, active agents are administered at
a therapeutically effective dosage sufficient to maintain a
subject's score on the ADAS-Cog. In another embodiment, the active
agents are administered at a therapeutically effective dosage
sufficient to decrease a subject's ADAS-Cog score by about 2 points
or greater, by about 3 points or greater, by about 4 points or
greater, by about 5 points or greater, by about 7.5 points or
greater, by about 10 points or greater, by about 12.5 points or
greater, by about 15 points or greater, by about 17.5 points or
greater, by about 20 points or greater, or by about 25 points or
greater. In another embodiment, the active agents are administered
at a therapeutically effective dosage sufficient to reduce the rate
of the increase of a subject's ADAS-Cog scores as compared to
historical or untreated controls. In another embodiment, the
therapeutically effective dosage is sufficient to reduce the rate
of increase of a subject's ADAS-Cog scores (relative to untreated
subjects) by about 5% or greater, about 10% or greater, about 20%
or greater, about 25% or greater, about 30% or greater, about 40%
or greater, about 50% or greater, about 60% or greater, about 70%
or greater, about 80% or greater, about 90% or greater or about
100% or greater.
[0411] In another embodiment, active agents are administered at a
therapeutically effective dosage sufficient to decrease the ratio
of A.beta.42:A.beta.40 in the CSF or plasma of a subject by about
15% or more, about 20% or more, about 25% or more, about 30% or
more, about 35% or more, about 40% or more, about 45% or more, or
about 50% or more.
[0412] In another embodiment, active agents are administered at a
therapeutically effective dosage sufficient to lower levels of
A.beta. in the CSF or plasma of a subject by about 15% or more,
about 25% or more, about 35% or more, about 45% or more, about 55%
or more, about 75% or more, or about 95% or more.
[0413] Toxicity and therapeutic efficacy of such agents can be
determined by standard pharmaceutical procedures in cell cultures
or experimental animals, e.g., for determining the LD50 (the dose
lethal to 50% of the population) and the ED50 (the dose
therapeutically effective in 50% of the population). The dose ratio
between toxic and therapeutic effects is the therapeutic index and
can be expressed as the ratio LD50/ED50, and usually a larger
therapeutic index is more efficacious. While agents that exhibit
toxic side effects may be used, care should be taken to design a
delivery system that targets such agents to the site of affected
tissue in order to minimize potential damage to unaffected cells
and, thereby, reduce side effects.
[0414] It is understood that appropriate doses depend upon a number
of factors within the ken of the ordinarily skilled physician,
veterinarian, or researcher. The dose(s) of the small molecule will
vary, for example, depending upon the identity, size, and condition
of the subject or sample being treated, further depending upon the
route by which the composition is to be administered, if
applicable, and the effect which the practitioner desires the small
molecule to have upon the subject. Exemplary doses include
milligram or microgram amounts of the small molecule per kilogram
of subject or sample weight (e.g., about 1 microgram per kilogram
to about 500 milligrams per kilogram, about 100 micrograms per
kilogram to about 5 milligrams per kilogram, or about 1 microgram
per kilogram to about 50 micrograms per kilogram). It is
furthermore understood that appropriate doses depend upon the
potency. Such appropriate doses may be determined using the assays
described herein. When one or more of these compounds is to be
administered to an animal (e.g., a human), a physician,
veterinarian, or researcher may, for example, prescribe a
relatively low dose at first, subsequently increasing the dose
until an appropriate response is obtained. In addition, it is
understood that the specific dose level for any particular animal
subject will depend upon a variety of factors including the
activity of the specific agent employed, the age, body weight,
general health, gender, and diet of the subject, the time of
administration, the route of administration, the rate of excretion,
and any drug combination.
[0415] The ability of an agent to inhibit amyloid deposition can be
evaluated in an animal model system that may be predictive of
efficacy in inhibiting amyloid deposition in human diseases, such
as a transgenic mouse expressing human APP or other relevant animal
models where A.beta. deposition is seen or for example in an animal
model of AA amyloidosis. Likewise, the ability of an agent to
prevent or reduce cognitive impairment in a model system may be
indicative of efficacy in humans. Alternatively, the ability of an
agent can be evaluated by examining the ability of the agent to
inhibit amyloid fibril formation in vitro, e.g., using a
fibrillogenesis assay such as that described herein, including a
ThT, CD, or EM assay. Also the binding of an agent to amyloid
fibrils may be measured using a MS assay as described herein. The
ability of the agent to protect cells from amyloid induced toxicity
is determined in vitro using biochemical assays to determine
percent cell death induced by amyloid protein. The ability of an
agent to modulate renal function may also be evaluated in an
appropriate animal model system.
[0416] The therapeutic agent of the invention may be also be
administered ex vivo to inhibit amyloid deposition or treat certain
amyloid-related diseases, such as NM amyloidosis and other
amyloidoses related to dialysis. Ex vivo administration of the
therapeutic agents of the invention can be accomplished by
contacting a body fluid (e.g., blood, plasma, etc.) with a
therapeutic compound of the invention such that the therapeutic
compound is capable of performing its intended function and
administering the body fluid to the subject. The therapeutic
compound of the invention may perform its function ex vivo (e.g.,
dialysis filter), in vivo (e.g., administered with the body fluid),
or both. For example, a therapeutic compound of the invention may
be used to reduce plasma .beta..sub.2M levels and/or maintain
.beta..sub.2M in its soluble form ex vivo, in vivo, or both.
Prodrugs
[0417] The present invention is also related to prodrugs of the
agents of the Formulae disclosed herein. Prodrugs are agents which
are converted in vivo to active forms (see, e.g., R. B. Silverman,
1992, "The Organic Chemistry of Drug Design and Drug Action,"
Academic Press, Chp. 8). Prodrugs can be used to alter the
biodistribution (e.g., to allow agents which would not typically
enter the reactive site of the protease) or the pharmacokinetics
for a particular agent. For example, a carboxylic acid group, can
be esterified, e.g., with a methyl group or an ethyl group to yield
an ester. When the ester is administered to a subject, the ester is
cleaved, enzymatically or non-enzymatically, reductively,
oxidatively, or hydrolytically, to reveal the anionic group. An
anionic group can be esterified with moieties (e.g., acyloxymethyl
esters) which are cleaved to reveal an intermediate agent which
subsequently decomposes to yield the active agent. The prodrug
moieties may be metabolized in vivo by esterases or by other
mechanisms to carboxylic acids.
[0418] Examples of prodrugs and their uses are well known in the
art (see, e.g., Berge, et al., "Pharmaceutical Salts", J. Pharm.
Sci. 66, 1-19 (1977)). The prodrugs can be prepared in situ during
the final isolation and purification of the agents, or by
separately reacting the purified agent in its free acid form with a
suitable derivatizing agent. Carboxylic acids can be converted into
esters via treatment with an alcohol in the presence of a
catalyst.
[0419] Examples of cleavable carboxylic acid prodrug moieties
include substituted and unsubstituted, branched or unbranched lower
alkyl ester moieties, (e.g., ethyl esters, propyl esters, butyl
esters, pentyl esters, cyclopentyl esters, hexyl esters, cyclohexyl
esters), lower alkenyl esters, dilower alkyl-amino lower-alkyl
esters (e.g., dimethylaminoethyl ester), acylamino lower alkyl
esters, acyloxy lower alkyl esters (e.g., pivaloyloxymethyl ester),
aryl esters (phenyl ester), aryl-lower alkyl esters (e.g., benzyl
ester), substituted (e.g., with methyl, halo, or methoxy
substituents) aryl and aryl-lower alkyl esters, amides, lower-alkyl
amides, dilower alkyl amides, and hydroxy amides.
Pharmaceutically Acceptable Salts
[0420] Certain embodiments of the present agents can contain a
basic functional group, such as amino or alkylamino, and are, thus,
capable of forming pharmaceutically acceptable salts with
pharmaceutically acceptable acids. The term "pharmaceutically
acceptable salts" in this respect, refers to the relatively
non-toxic, inorganic and organic acid addition salts of agents of
the present invention. These salts can be prepared in situ during
the final isolation and purification of the agents of the
invention, or by separately reacting a purified agent of the
invention in its free base form with a suitable organic or
inorganic acid, and isolating the salt thus formed.
[0421] Representative salts include the hydrohalide (including
hydrobromide and hydrochloride), sulfate, bisulfate, phosphate,
nitrate, acetate, valerate, oleate, palmitate, stearate, laurate,
benzoate, lactate, phosphate, tosylate, citrate, maleate, fumarate,
succinate, tartrate, napthylate, mesylate, glucoheptonate,
lactobionate, 2-hydroxyethanesulfonate, and laurylsulphonate salts
and the like. See, e.g., Berge et al., "Pharmaceutical Salts", J.
Pharm. Sci. 66, 1-19 (1977).
[0422] In other cases, the agents of the present invention may
contain one or more acidic functional groups and, thus, are capable
of forming pharmaceutically acceptable salts with pharmaceutically
acceptable bases. The term "pharmaceutically acceptable salts" in
these instances refers to the relatively non-toxic, inorganic and
organic base addition salts of agents of the present invention.
[0423] These salts can likewise be prepared in situ during the
final isolation and purification of the agents, or by separately
reacting the purified agent in its free acid form with a suitable
base, such as the hydroxide, carbonate or bicarbonate of a
pharmaceutically acceptable metal cation, with ammonia, or with a
pharmaceutically acceptable organic primary, secondary or tertiary
amine. Representative alkali or alkaline earth salts include the
lithium, sodium, potassium, calcium, magnesium, and aluminum salts
and the like. Representative organic amines useful for the
formation of base addition salts include ethylamine, diethylamine,
ethylenediamine, ethanolamine, diethanolamine, piperazine and the
like.
[0424] "Pharmaceutically acceptable salts" also includes, for
example, derivatives of agents modified by making acid or base
salts thereof, as described further below and elsewhere in the
present application. Examples of pharmaceutically acceptable salts
include mineral or organic acid salts of basic residues such as
amines; and alkali or organic salts of acidic residues such as
carboxylic acids. Pharmaceutically acceptable salts include the
conventional non-toxic salts or the quaternary ammonium salts of
the parent agent formed, for example, from non-toxic inorganic or
organic acids. Such conventional non-toxic salts include those
derived from inorganic acids such as hydrochloric, hydrobromic,
sulfuric, sulfamic, phosphoric, and nitric acid; and the salts
prepared from organic acids such as acetic, propionic, succinic,
glycolic, stearic, lactic, malic, tartaric, citric, ascorbic,
palmoic, maleic, hydroxymaleic, phenylacetic, glutamic, benzoic,
salicylic, sulfanilic, 2-acetoxybenzoic, fumaric, toluenesulfonic,
methanesulfonic, ethane disulfonic, oxalic, and isethionic acid.
Pharmaceutically acceptable salts may be synthesized from the
parent agent which contains a basic or acidic moiety by
conventional chemical methods. Generally, such salts may be
prepared by reacting the free acid or base forms of these agents
with a stoichiometric amount of the appropriate base or acid in
water or in an organic solvent, or in a mixture of the two.
[0425] All acid, salt, base, and other ionic and non-ionic forms of
the compounds described are included as compounds of the invention.
For example, if a compound is shown as an acid herein, the salt
forms of the compound are also included. Likewise, if a compound is
shown as a salt, the acid and/or basic forms are also included.
[0426] Those skilled in the art will recognize, or be able to
ascertain using no more than routine experimentation, numerous
equivalents to the specific procedures, embodiments, claims, and
examples described herein. Such equivalents are considered to be
within the scope of this invention and covered by the claims
appended hereto. The contents of all references, issued patents,
and published patent applications cited throughout this application
are hereby incorporated by reference. The invention is further
illustrated by the following examples, which should not be
construed as further limiting.
EXAMPLES
Binding and Antifibrillogenic Assays
[0427] The test compounds were synthesized and screened by mass
spectrometry ("MS") assays, except for selected compounds which
were purchased from a commercial source. The MS assay gives data on
the ability of compounds to bind to proteins, in this example, to
.beta.-amyloid and LAPP.
[0428] In the MS assay for A.beta.40, samples were prepared as
aqueous solutions (adding 20% ethanol if necessary to solubilize in
water), 200 .mu.M of a test compound and 20 .mu.M of solubilized
A.beta.40, or 400 .mu.M of a test compound and 40 .mu.M of
solubilized A.beta.40. The pH value of each sample was adjusted to
7.4 (.+-.0.2) by addition of 0.1% aqueous sodium hydroxide. The
solutions were then analyzed by electrospray ionization mass
spectrometry using a Waters ZQ 4000 mass spectrometer. Samples were
introduced by direct infusion at a flow-rate of 25 .mu.L/min
within. 2 hours after sample preparation. The source temperature
was kept at 70.degree. C. and the cone voltage was 20 V for all the
analysis. Data were processed using Masslynx 3.5 software. The MS
assay gives data on the ability of compounds to bind to soluble
A.beta., whereas the ThT, EM and CD assays give data on inhibition
of fibrillogenesis. The results from the assay for binding to
A.beta. are summarized in Tables 3A-3D. In Tables 3A-3D, a blank
box means that a value was not determined for that compound in that
assay.
[0429] The assay for IAPP was conducted under the same conditions
except that 200 .mu.M or 100 .mu.M of test compound and 20 .mu.M of
solubilized IAPP were employed. The key below describes the codes
used in Table 3A to quantify the binding based on the intensity of
the absorption.
TABLE-US-00004 Key to Table 3A A.beta.1-40 Code 400 .mu.M 200 .mu.M
Strong Binding +++ 90-100% 60-100% Moderate Binding ++ 70-89%
30-69% Weak Binding + 45-59% 20-44% Little/no detectable - 20-39%
20-39% binding IAPP 200 .mu.M 100 .mu.M (20% EtOH) (20% EtOH)
Strong Binding +++ >75% >50% Moderate Binding ++ 40-75%
30-50% Weak Binding + 20-40% 15-30% Little/no detectable - 0-20%
0-15% binding
TABLE-US-00005 TABLE 3A Relative Binding Affinities of Compounds of
the Invention MS binding ID IAPP A.beta.1-40 B ++ + C + D + E +++ +
F +++ + G ++ + H +++ + I +++ ++ J +++ + K ++ - L ++ + M ++ - N + -
P ++ - Q - - AC ++ + AD + AE + AF +++ + AG ++ ++ AH ++ + AK +++ ++
AL +++ ++ AM ++ + AW ++ ++ AX + ++ AY ++ +++ AZ +++ ++ BA ++ ++ BB
+++ +++ BC - + BV + +++ BW ++ +++ BX ++ +++ BY ++ +++ BZ +++ CC -
++ CD +++ ++ CE + +++ CG ++ +++ CH +++ +++ CI + +++ CJ ++ +++ CK
+++ +++ CM + CV + +++ CY +++ +++ DC ++ +++ DD +++ DE + - DF +++ -
DG - + DH + ++ DI - - DJ +++ - DK ++ +++ DL - + DM + ++ DN + ++ DO
++ +++ DP - + DQ - + DR + + DS + + DT + + DU +++ +++ DV +++ +++ DW
+++ ++ DX +++ +++ DY ++ +++ DZ +++ +++ EA + ++ EB +++ +++ EC +++ ED
++ +++ EE ++ +++ EF - + EG +++ +++ EH +++ +++ EI - - EJ + ++ EK +++
+++ EL + ++ EN + + EO - + EP ++ + EQ - + ER ++ ES +++ +++ ET +++ EV
+++ EW +++ EY +++ +++ EZ +++ +++ FA ++ +++ FH - - FO - - FP +++ ++
FQ ++ + FR +++ ++ FS +++ +++ FT - - FU ++ ++ FV - - FW + - FX +++
+++ FY +++ +++ FZ ++ + GA + - GB +++ + GC - - GD +++ ++ GE ++ GF +
GH ++ + GI ++ + GJ ++ ++ GK + + GL ++ ++ GM ++ - GN - - GO - - GP +
- GQ - - GR - - GS ++ + GT + - GU ++ + GZ + - HA - - HB - - HC ++ +
HD - - HE + - HF ++ - HG + + HI ++ HJ ++ - HK - - NG ++ NH + NI +
NJ +++ NK + NL +++ Compounds in Table 3B were tested under the
following conditions: 30 .mu.M A.beta. peptide, 150 .mu.M compound;
60-100% binding = (+++), 40-59% binding = (++), 20-39% binding =
(+), <20% = (-), blank = not determined.
TABLE-US-00006 TABLE 3B Relative Binding Affinities of Compounds of
the Invention MS binding ID IAPP A.beta.1-40 AI +++ FN ++ IY +++ ++
JH +++ ++ JI +++ +++ JP +++ JQ +++ JR +++ ++ JV ++ KB +++ KH ++ KJ
+++ KM +++ KN ++ KS +++ KT ++ LK ++ LN +++ Compounds in Table 3C
were tested under the following conditions: 20 .mu.M A.beta.
peptide, 100 .mu.M compound; 75-100% binding = (+++), 40-74%
binding = (++), 20-39% binding = (+), <20% = (-), blank = not
determined.
TABLE-US-00007 TABLE 3C Relative Binding Affinities of Compounds of
the Invention MS binding ID IAPP A.beta.1-40 IW +++ ++ IX +++ ++ IZ
+++ - JA ++ + JB + + JC + JD ++ + JE ++ - JF ++ + JG +++ JJ +++ ++
JK + - JL ++ ++ JM + - JN + ++ JO ++ + JS + JT + JU ++ JW ++ JX ++
JY +++ JZ +++ KA ++ KI + KK +++ KL ++ KQ + LD ++ LE ++ LF + LG ++
LH + LI +++ LJ +++ LL ++ LM +++ LQ +++ Compounds in Table 3D were
tested under the following conditions: 40 .mu.M A.beta. peptide,
400 .mu.M compound; 75-100% binding = (+++), 40-74% binding = (++),
20-39% binding = (+), <20% = (-), blank = not determined.
TABLE-US-00008 TABLE 3D Relative Binding Affinities of Compounds of
the Invention MS binding ID IAPP A.beta.1-40 S ++ - HL + ++ HM - +
HN - HP - HQ + HR - ++ HS - HT + ++ HU + +++ HV + ++ HW - HX - HY -
HZ - IA + +++ IB + IF - IG ++ +++ IH + II - IJ + IK + IL + IM - IN
+ IP + IR + IS + IT + IU - KV + KW + KX + KY + LA - LC - LP +++
Effects of Short Term Treatment in Adult Transgenic CRNDS Mice
Overexpressing .beta.APP
[0430] Transgenic mice, TgCRND8, expressing the human amyloid
precursor protein (hAPP) develop a pathology resembling Alzheimer's
disease. In particular, high levels of A.beta.40 and A.beta.42 have
been documented in the plasma and the brain of these animals at 8-9
weeks of age, followed by early accumulation of amyloid plaques
similar to the senile plaques observed in A.beta. patients. These
animals also display progressive cognitive deficits that parallel
the appearance of degenerative changes. See, e.g., (Chishti, et
al., J. Biol. Chem. 276, 21562-70 (2001).
[0431] The short term therapeutic effect of 19 compounds of the
invention was studied. These compounds were administered over a 14
or 28 day period at the end of which the levels of A.beta. peptides
in the plasma and brain of TgCRND8 animals were determined.
Methods
[0432] Male and female transgenic mice from the 3.sup.th and
4.sup.th B6C3F1 generations were used in this example and given
daily subcutaneous or oral administrations of one of a series of
compounds for 14 or 28 days. The following abbreviations are used
to designate these animals from the 3.sup.rd and 4th generation
backcross in the present protocol: TgCRND8-2.B6C3F1(N.sub.3);
TgCRND8-2.B6C3F1(N.sub.4).
[0433] Baseline animals (Group 1) consisted of naive TgCRND8-2.
B6C3F1(N.sub.3) at 11.+-.1 weeks of age. These mice were used to
determine the A.beta. levels in the plasma and brain of naive
transgenic animals at the initiation of treatment.
[0434] Starting at 11 weeks of age (.+-.1 week) animals received
daily administration of their respective treatment for a period of
14 or 28 days (groups 2-21), at a dose of 250 mg/kg at 10 ml/kg or
of vehicle only (water; group 2) or 1% methyl cellulose only (group
21). The route of administration was subcutaneous for water-soluble
compounds and oral for compounds solubilized in methylcellulose 1%
(MC 1%). At the end of the treatment periods, plasma and perfused
brains were collected for quantification of A.beta. levels.
TABLE-US-00009 TABLE 4 Test System Species: Mouse Strain:
TgCRND8-2.B6C3F1(N.sub.3) & (N.sub.4) Genotype: hAPP+/- Gender:
Male and Female Age at Day 1: 11 .+-. 1 weeks Body Weight at 10 to
30 g Day 1: Number of Baseline: 8 Animals/Group at Day 1: Vehicle
and 12-15 Treated: Suppliers: TgCRND8-2 founders were obtained from
the Centre for Research in Neurodegenerative Diseases, University
of Toronto. The inbred B6C3F1 were obtained from Charles River
(Quebec, Canada).
[0435] The mice used in this study were derived from a breeding
colony at Institut Armand Frappier, and were well-acclimated to the
animal facility environment prior to initiation of the study.
Animals were assigned, according to age and gender, into the
following experimental groups:
TABLE-US-00010 TABLE 5 Groups of Mice Daily Dose Duration of
Treatment Group No. Treatment (mg/kg) (days) 1 Baseline NA NA 2
Water NA 14 & 28 4 BY 250 14 & 28 6 CV 250 14 & 28 12
CY NA 14 & 28 15 BW 250 14 & 28 16 BZ 250 14 & 28 18 BX
250 14 & 28 20 DC 250 14 & 28 21 Methylcellulose 1% 100 14
& 28 22 DD 250 14 & 28 23 DH 250 14 & 28 24 DM 250 14
& 28 25 DX 250 14 & 28 26 DY 250 14 & 28 27 DZ 250 14
& 28 28 ED 250 14 & 28 29 EG 250 14 & 28
Animal Health Monitoring
[0436] All animals were examined daily for signs of ill health when
handled in the morning for their daily treatment and twice a day
for mortality checks (once daily during weekends and holidays).
Detailed examinations were performed on the treatment initiation,
weekly during the study, and once before terminal procedures. More
frequent observations were undertaken when considered appropriate.
Death and all individual clinical signs were individually recorded.
Individual body weights were recorded at randomization, once weekly
during the study, and once before terminal procedures.
Sample Collection
[0437] At 11.+-.1 weeks of age for the Baseline group, and at the
end of the treatment period (14 or 28 days) for Groups 2 to 21, at
24 hours after the last compound administration animals were
sacrificed and samples collected. An approximate blood volume of
500 .mu.l was collected from the orbital sinus and kept on ice
until centrifugation at 4.degree. C. at a minimum speed of 3,000
rpm for 10 minutes. Plasma samples were immediately frozen and
stored at -80.degree. C. pending analysis. The brains were removed,
frozen, and stored at -80.degree. C. awaiting analysis.
Measurements of A.beta. Levels
[0438] Brains were weighted frozen and homogenized with 4 volumes
of ice cold 50 mM Tris-Cl pH 8.0 buffer with protease inhibitor
cocktail (4 mL of buffer for 1 g of wet brain). Samples were spun
at 15000 g for 20 minutes and the supernatants were transferred to
fresh tubes. One hundred fifty (150) .mu.l from each supernatant
were mixed with 250 .mu.l of 8M guanidine-HCL/50 mM Tris-HCL pH 8.0
(ratio of 0.6 vol supernatant:1 vol 8M guanidium/Tris-HCL 50 mM
pH8.0) and 400 .mu.L 5 M guanidium/Tris-HCL 50 mM pH8.0 were added.
The tubes were vortexed for 30 seconds and frozen at -80.degree. C.
In parallel, pellets were treated with 7 volumes of 5 M
guanidine-HCL/50 mM Tris-HCL pH 8.0 (7 mL of guanidine for 1 g of
wet brain), vortexed for 30 seconds and frozen at -80.degree. C.
Samples were thawed at room temperature, sonicated at 80.degree. C.
for 15 minutes and frozen again. This cycle was repeated 3 times to
ensure homogeneity and samples were returned to -80.degree. C.
pending analysis.
[0439] A.beta. levels were evaluated in plasma and brain samples by
ELISA using Human A.beta.40 and A.beta.42 Fluorometric ELISA kits
from Biosource (Cat. No. 89-344 and 89-348) according to
manufacturer's recommended procedures. In short, samples were
thawed at room temperature, sonicated for 5 minutes at 80.degree.
C. (sonication for brain homogenates; no sonication for plasma
samples) and kept on ice. A.beta. peptides were captured using 100
.mu.l of the diluted samples to the plate and incubated without
shaking at 4.degree. C. overnight. The samples were aspirated and
the wells were rinsed 4 times with wash buffer obtained from the
Biosource ELISA kit. The anti-A.beta.40 or anti-A.beta.42 rabbit
polyclonal antiserum (specific for the A.beta.40 or A342 peptide)
was added (100 .mu.l) and the plate was incubated at room
temperature for 2 hours with shaking. The wells were aspirated and
washed 4 times before adding 100 .mu.l of the alkaline phosphatase
labeled anti-rabbit antibody and incubating at room temperature for
2 hours with shaking. The plates were then rinsed 5 times and the
fluorescent substrate (100 .mu.l) was added to the plate. The plate
was incubated for 35 minutes at room temperature and the plate was
read using a titer plate reader at an excitation wavelength of 460
nm and emission at 560 nm.
[0440] Compounds were scored based on their ability to modulate
levels of A.beta. peptides in the plasma and the cerebral
soluble/insoluble levels in the brain. Levels of A.beta. observed
in the plasma and brain of treated animals were normalized using
values from vehicle-treated (water) or methylcellulose-treated
control groups and ranked according to the strength of the
pharmacological effect. Results are shown in Tables 3 to 11.
Increases in the levels of A.beta. peptides are indicated using "+"
symbols, while decreases in the levels of A.beta. peptides are
indicated using "-" symbols. The strongest effects are recorded as
"+++" or "---" while the weakest are shown as "+" or "-".
[0441] Specifically, increases in the levels of A.beta. (relative
to untreated control) of 20 to 39% are scored as "+"; increases of
40 to 69% are scored as "++"; and increases of 70% or higher are
scored as "+++". Decreases in the levels of A.beta. of 5 to 19% are
scored as "-"; decreases of 20 to 38% are scored as "--"; and
decreases of 39% or more are scored as "---".
[0442] The data are presented in Tables 6-11. Treatment with these
compounds after 14 and/or 28 days resulted in a significant change
in the cerebral levels of A.beta.40 and/or A.beta.42 (Tables 8-11).
Furthermore, treatment with these compounds, for instance, Compound
BX (3-(t-butyl)amino-1-propanesulfonic acid), resulted after 14 and
28 days (Tables 6-7) in a significant increase in the levels of
A.beta. peptides in the plasma. This suggests that some of these
compounds may act by a peripheral sink effect, sequestering A.beta.
peptides in the plasma and thereby facilitating their clearance
from the CNS as previously suggested for treatment by passive
immunization using anti-A.beta. monoclonal antibody m266 (DeMattos
et al., Science 295(5563):2264-7).
[0443] The tables show levels of A.beta. peptides in the plasma and
brain of TgCRND8 mice treated for 14 and 28 days with compounds of
the invention.
[0444] Tables 6A and 6C show the data from Day 14 and Day 28 for
the Plasma Vehicle group, respectively. Tables 6B and 7 show the
data for the Plasma Methylcellulose group on Days 14 and 28,
respectively. Tables 8 and 10 show the data on Days 14 and 28 for
the Brain homogenate vehicle group, respectively. Tables 9 and 11
show the data for brain homogenate for the Methylcellulose group on
Days 14 and 28, respectively.
TABLE-US-00011 TABLE 6A Plasma Vehicle Group, Day 14 Treatment
A.beta.40 Change A.beta.42 Change BY + + CV + ++ DC +\+ ++ BX +++
++
TABLE-US-00012 TABLE 6B Plasma Methylcellulose Group, Day 14
Treatment A.beta.40 Change A.beta.42 Change BZ + BW CY
TABLE-US-00013 TABLE 6C Plasma Vehicle Group, Day 28 Treatment
A.beta.40 Change A.beta.42 Change BY CV ++ ++ DC ++ BX +++
TABLE-US-00014 TABLE 7 Plasma Methylcellulose Group, Day 28
Treatment A.beta.40 Change A.beta.42 Change BZ ++ ++ BW + CY +
TABLE-US-00015 TABLE 8 Brain Homogenate Vehicle Group, Day 14
A.beta.40 Change A.beta.42 Change Treatment Soluble Insoluble
Soluble Insoluble BY +++ +++ +++ CV** - DC -- + -- BX - --- -- ---
DD - DX -- - DY -- -- DZ -- EG - -- -- -- DH -- --- --- DM + - - ED
- + **The effect of this compound in the brain has only been tested
on its ability to modulate the total levels of A.beta.40 and
A.beta.42 peptides rather than measuring soluble and insoluble
levels independently.
TABLE-US-00016 TABLE 9 Brain Homogenate Methylcellulose Group, Day
14 A.beta.40 Change A.beta.42 Change Treatment Soluble Insoluble
Soluble Insoluble BZ --- -- BW --- -- --- CY - +++ ++
TABLE-US-00017 TABLE 10 Brain Homogenate Vehicle Group, Day 28
A.beta.40 Change A.beta.42 Change Treatment Soluble Insoluble
Soluble Insoluble BY + +++ +++ CV** ++ +++ DC - + ++ +++ BX --- ---
-- - DD - DX -- - -- DY --- - -- DZ - -- -- -- EG -- -- DH - - DM -
-- -- -- ED - -- - - **The effect of this compound in the brain has
only been tested on its ability to modulate the total levels of
A.beta.40 and 42 peptides rather than measuring soluble and
insoluble levels independently.
TABLE-US-00018 TABLE 11 Brain Homogenate Methylcellulose Group, Day
28 A.beta.40 Change A.beta.42 Change Treatment Soluble Insoluble
Soluble Insoluble BZ -- -- -- BW - - - -- CY ++ +++ +++ +
Effects of Long Term Treatment in Adult Transgenic CRND8 Mice
Overexpressing .beta.APP
[0445] Transgenic mice, TgCRND8, as those used in the short term
treatment, overexpress a human APP gene with the Swedish and
Indiana mutations leading to the production of high levels of the
amyloid peptides and to the development of an early-onset,
aggressive development of brain amyloidosis. The high levels of
A.beta. peptides and the relative overabundance of A.beta..sub.42
compared to A.beta..sub.40 are believed to be associated with the
severe and early degenerative pathology observed. The pattern of
amyloid deposition, presence of dystrophic neuritis, and cognitive
deficit has been well documented in this transgenic mouse line. The
levels of A.beta. peptides in the brain of these mice increase
dramatically as the animals age. While the total amyloid peptide
levels increase from .about.1.6.times.10.sup.5 pg/g of brain to
.about.3.8.times.10.sup.6 between 9 and 17 weeks of age.
[0446] While the early deposition of amyloid in this model allows
the rapid testing of compounds in a relatively short time frame,
the aggressivity of this model and the high levels of A.beta.
peptides renders therapeutic assessment in the longer term a more
difficult task.
[0447] The long-term therapeutic effects of compounds of the
present invention on cerebral amyloid deposition and .beta.-amyloid
(A.beta.) levels in the plasma and in the brains of transgenic
mice, TgCRND8, expressing the human amyloid precursor protein
(hAPP) was studied. These compounds were administered over an 8 or
16 week period at the end of which the levels of A.beta. peptides
in the plasma and brain of TgCRND8 animals were determined. The
goal of this study was to evaluate the efficacy of the compounds at
modulating the progression of the amyloidogenic process in the
brain and in the plasma of a transgenic mouse model of Alzheimer's
disease (AD)
Methods
[0448] The mice used in the study consisted of animals bearing one
copy of the hAPP gene (+/-) from the 2.sup.nd and 3.sup.rd
generation progenies (N.sub.2 and N.sub.3) derived from backcrosses
from TgCRND8.FVB(N.sub.2)AJ(N.sub.3) with B6AF1/J hybrid
animals.
N.sub.1=TgCRND8.FVB(N.sub.2)AJ(N.sub.3).times.B6AF1/J
N.sub.2=TgCRND8.FVB(N.sub.2)AJ(N.sub.3).B6AF1/J(N.sub.1).times.B6AF1/J
N.sub.3=TgCRND8.FVB(N.sub.2)AJ(N.sub.3).B6AF1/J(N.sub.2).times.B6AF1/J
[0449] The following abbreviations are used to designate these
animals in the present study: TgCRND8.B6AF1/J(N.sub.2);
TgCRND8.B6AF1/J(N.sub.3). Male and female transgenic mice were
given daily subcutaneous (compound BX) or oral (compounds BW and
BZ) administrations of the appropriate compounds for 8 or 16
weeks.
[0450] Baseline animals consisted of 9.+-.1 week old naive
TgCRND8.B6AF1/J animals from the 2.sup.nd and 3.sup.rd generations.
These mice were used to determine the extent of cerebral amyloid
deposits and A.beta. levels in the plasma and brain of naive
transgenic animals at the initiation of treatment.
[0451] Starting at 9 weeks of age (.+-.1 week) animals received
daily administration of their respective treatment for a period of
8 or 16 weeks, at a dose of 30 or 100 mg/kg at 10 ml/kg. The route
of administration was subcutaneous for water-soluble compounds
(Compound BX) and oral for compounds solubilized in methylcellulose
1% (MC 1%) (Compounds BW and BZ). At the end of the treatment
periods, plasma and perfused brains were collected for
quantification of A.beta. levels.
[0452] Animal health was monitored, samples were collected and
A.beta. levels were measured as described above in the short term
treatment study. Compounds were scored based on their ability to
modulate levels of A.beta. peptides in the plasma and the cerebral
soluble/insoluble levels in the brain. Levels of A.beta. observed
in the plasma and brain of treated animals were compared to that of
vehicle-treated (water) or methylcellulose-treated control groups
and ranked according to the strength of the pharmacological effect.
Results are shown in Table 12. Increases in the levels of A.beta.
peptides are indicated using "+" symbols, while decreases in the
levels of A.beta. peptides are indicated using "-" symbols. The
strongest effects are recorded as "+++" or "---" while the weakest
are shown as "+" or "-".
[0453] Specifically, increases in the levels of A.beta. (relative
to vehicle treated control) of 5 to 14% are scored as "+";
increases of 15 to 29% are scored as "++"; and increases of 30% or
higher are scored as "+++". Decreases in the levels of A.beta. of 5
to 14% are scored as "-"; decreases of 15 to 29% are scored as
"--"; and decreases of 30% or more are scored as "---".
Additionally, changes of 4% or less in either direction are scored
as "0".
[0454] Table 12 shows levels of A.beta. peptides in the plasma and
brain of TgCRND8 mice treated for 8 and 16 weeks with compounds of
the invention. Treatment with these compounds after 8 and/or 16
weeks in many cases resulted in a change in the levels of
A.beta..sub.40 and/or A.beta..sub.42 in the plasma and/or brain.
For example, administration of compound BX generally resulted in a
dramatic decrease in the amount of A.beta. in the brain at both 8
and 16 weeks. Compound BW also resulted in a dramatic decrease in
brain and plasma levels of A.beta. at 8 weeks and plasma levels at
16 weeks.
[0455] For the ThioS studies, the plaques in the brains of the mice
were quantified as follows. Mice were transcardially perfused with
saline solution. Brains were dissected out and separated in 2
hemispheres. The left hemisphere was immersed in freshly-prepared
4% paraformaldehyde for 3 hrs at 4.degree. C., then transferred
into 30% sucrose at 4.degree. C. When cryoprotection was achieved
(24-48 hours), brains were frozen in isopentane at -45.degree. C.
and subsequently stored at -80.degree. C. until sectioning. Coronal
40 .mu.m-thick sections were performed, and stained with thioflavin
S (1% solution in water) for 8 min. After differentiation of the
thioflavin S staining, sections were counterstained with
hematoxylin for 5 minutes. Two sets of pictures were captured
simultaneously. A first set of pictures was captured under
brightfield illumination to obtain morphological details of the
section; a second set of pictures was captured under a green,
specific, fluorescent filter (fluorescein filter, Ex 495 nm, Em 525
nm). Image analysis to quantify the number of plaques and the area
occupied by these plaques was performed using Image Pro Plus
software (Media Cybernetics, MD, USA).
[0456] The data from the histological ThioS studies is summarized
in Table 13. Increases in the areas and numbers of plaques are
indicated using "+" symbols, while decreases in the areas and
numbers of the plaques are indicated using "-" symbols. Preliminary
histochemical experiments using ThioS staining of brain sections
indicated that both the number of plaques and the area occupied by
the plaques were decreased in mice treated with 30 mg/kg of
compound BX.
[0457] Specifically, increases in the areas and numbers of plaques
(relative to vehicle treated control) of 10 to 19.99% are scored as
"+". Decreases in the areas and numbers of plaques of 10 to 19.99%
are scored as "-". Additionally, changes of 9.99% or less in either
direction are scored as "0".
TABLE-US-00019 TABLE 12 Effects of Compounds BX, BW and BZ on
levels of A.beta. in plasma and brain Brain Dose Timepoint Plasma
Abeta40 Abeta42 Compound (mg/kg) (weeks) Abeta40 Abeta42 soluble
insoluble soluble insoluble BX 30 8 wks + + --- --- --- -- BX 100 8
wks ++ +++ + ++ + 0 BX 30 16 wks - - -- --- 0 - BX 100 16 wks - 0 -
--- 0 -- BW 30 8 wks --- --- - -- -- 0 BW 100 8 wks - - -- --- --
--- BW 30 16 wks -- -- + ++ - + BW 100 16 wks - - ++ + + ++ BZ 30 8
wks 0 0 0 -- 0 --- BZ 100 8 wks ++ +++ ++ 0 0 - BZ 30 16 wks 0 + 0
+ + 0 BZ 100 16 wks ++ ++ -- 0 - +
TABLE-US-00020 TABLE 13 Histological effects of compounds BW and BX
on numbers of plaques and areas occupied by plaques ThioS Sites
Analyzed Change Change Dose Timepoint Surface Area in Plaque in
Plaque Compound (mg/kg) (weeks) (.mu.m.sup.2) Number Area BX 30 16
wks 7,773,230 - - BX 100 16 wks 7,803,230 0 + BW 30 16 wks
7,563,737 0 0 BW 100 16 wks 7,812,844 - 0
Evaluation of Compound S Binding to NAC Peptide by Mass
Spectrometry
[0458] Recent findings have demonstrated that a high percentage of
Alzheimer Disease (AD) patients also form Lewy bodies, most
abundantly in the amygdala (Hamilton. 2000. Brain Pathol, 10:378;
Mukaetova-Ladinska, et al. 2000. J Neuropathol Exp Neurol 59:408).
Interestingly, the highly hydrophobic non-amyloid component (NAC)
region of .alpha.-synuclein has also been described as the second
most abundant component of amyloid plaques in the brain of AD
patients, after. Alpha-synuclein has been shown to form fibrils in
vitro. Furthermore it binds to A.beta. and promotes its aggregation
(Yoshimoto, et al. 1995. Proc Natl Acad Sci USA 92:9141). It was in
fact originally identified as the precursor of the non-amyloid beta
(A.beta.) component (NAD) of AD plaques (Ueda, et al. 1993. Proc
Natl Acad Sci USA 90:11282; Iwai. 2000. Biochem Biophys Acta
1502:95; Masliah, et al. 1996. Am J Pathol 148:201). NAC is a 35
amino acid long peptide with highly hydrophobic stretches which can
self-aggregate and form fibrils in vitro. Moreover, these fibrils
can efficiently seed the formation of A.beta. fibrils in vitro
(Han, et al. 1995. Chem Biol. 2: 163-169; Iwai, et al. 1995.
Biochemistry 34:10139). It is in fact through its NAC domain that
alpha-synuclein retains its fibrillogenic properties. Modulating
the properties of NAC or targeting NAC with the compounds of the
invention could therefore be a valid therapeutic avenue to inhibit
the formation of protein aggregates and inclusions associated with
alpha-synucleopathies, as well as the formation of aggregates
between the beta-amyloid peptide and NAC of alpha-synuclein.
[0459] The ability of the compounds of the present invention to
bind to NAC peptide in aqueous solution was evaluated. The binding
ability correlates to the intensities of the peptide-compound
complex peaks observed by the Electrospray Mass Spectrum. Millipore
distilled deionized water was used to prepare all aqueous
solutions. For pH determination a Beckman .PHI.36 pH meter fitted
with a Corning Semi-Micro Combination pH Electrode was
employed.
[0460] NAC (MW 3260.6 Da) at 20 .mu.M was first analyzed at pH 7.40
and the usual sodium clusters was observed at +2, +3 and +4 at m/z
1335.5, 1116.7 and 843.4 respectively. The optimal cone voltage was
determined to be 20V.
[0461] Mass Spectrometry
[0462] Mass spectrometric analysis was performed using a Waters ZQ
4000 mass spectrometer equipped with a Waters 2795 sample manager.
MassLynx 4.0 (earlier by MassLynx 3.5) was used for data processing
and analysis. Test compounds were mixed with disaggregated peptides
in aqueous media (6.6% EtOH) at a 5:1 ratio (20 .mu.M NAC:100 .mu.M
of test compound or 40 .mu.M NAC:200 .mu.M of test compound). The
pH of the mixture was adjusted to 7.4 (.+-.0.2) using 0.1% NaOH
(3-5 .mu.L). Periodically, NAC peptide solution at 20 .mu.M or 40
.mu.M was also prepared in the same fashion and run as control. The
spectra were obtained by introducing the solutions to the
electrospray source by direct infusion using a syringe pump at a
flow rate of 25 .mu.l/min, and scanning from 100 to 2100 Da in the
positive mode. The scan time was 0.9 sec per scan with an
inter-scan delay of 0.1 sec and the run time was 5 min for each
sample. All the mass spectra were sum of 300 scans. The desolvation
and source temperature was 70.degree. C. and the cone and capillary
voltage were maintained at 20 V and 3.2 kV respectively.
[0463] The total area under the peaks for the bound NAC-compound
complex divided by total area under the peaks for unbound NAC was
determined for each compound tested. The results are summarized in
Table 14 below.
TABLE-US-00021 TABLE 14 NAC Peptide Binding Data Structure Binding
Strength* NaO.sub.3SCH.sub.2(CH.sub.2).sub.2CH.sub.2SO.sub.3Na -
NaO.sub.3SOCH.sub.2CH.sub.2CH.sub.2OSO.sub.3Na -
NH.sub.2CH.sub.2CH.sub.2OSO.sub.3H -
H.sub.2NCH.sub.2CH.sub.2CH.sub.2OSO.sub.3Na ++
H.sub.2NCH.sub.2CH.sub.2SO.sub.3H + ##STR00498## ++ ##STR00499## ++
##STR00500## + ##STR00501## +++ ##STR00502## *+++ = Strong; when
the total binding is 120% and higher ++ = Moderate; when the total
binding is between 120% and 70% + = Weak; when the total binding is
between 70% and 30% - = None; when the total binding is between 30%
and 0%
[0464] The present invention also relates to novel compounds and
the synthesis thereof. Accordingly, the following examples are
presented to illustrate how some of those compounds may be
prepared.
Synthesis of Compounds of the Invention
Preparation of 3-isopropylamino-1-propanesulfonic acid (Compound
CG)
##STR00503##
[0466] Isopropylamine (2.5 mL, 29 mmol) was added to a solution of
1,3-propane sultone (3.05 g, 25 mmol) in a mixture of
dichloromethane/ether (40 mL, 1:1). The mixture was heated at
reflux for 3 hours. The reaction mixture was cooled to room
temperature and hexane (10 mL) was added. The solid material was
collected by filtration, rinsed with ether (10 mL), and dried in
vacuo. Compound CG was obtained as a fine white powder (2.98 g,
65.6% yield). m.p. 240-43.degree. C. .sup.1H NMR (500 MHz,
D.sub.2O) .delta. 1.06 (d, J=6.3 Hz, 6H), 1.86 (qt, J=7.6 Hz, 2H),
2.76 (t, J=7.6 Hz, 2H), 3.14 (t, J=7.8 Hz, 2H), 3.13-3.21 (m, J=6.6
Hz, 1H). .sup.13C NMR (125 MHz, D.sub.2O) .delta. 18.2, 21.5, 25.8,
43.4, 47.9, 50.8.
Preparation of 3-cyclopropylamino-1-propanesulfonic acid (Compound
CI)
##STR00504##
[0468] Cyclopropylamine (3.7 mL, 52 mmol) was added to a solution
of 1,3-propane sultone (6.9 g, 55.3 mmol) in THF (60 mL). The
mixture was heated with an oil-bath at 42.degree. C. for 2 hours.
Stirring was difficult and part of the solid formed a crust above
the stirred mixture. The mixture was heated at reflux for 1 hour,
cooled to room temperature. The solid material was collected by
filtration, dried in a vacuum oven for 2 hours at 60.degree. C.
(4.95 g). The solid was recrystallized in methanol/water (35 mL/5
mL, v/v). The mixture was cooled in a fridge then the solid was
collected by filtration, rinsed with methanol (15 mL), air-dried
for 15 minutes, and further dried in a vacuum oven at 60.degree. C.
overnight. Compound CI was obtained as long, fine, white needles
(3.74 g, 40% yield). m.p. 234-236.degree. C. .sup.1H NMR (500 MHz,
D.sub.2O) .delta. 0.62-0.71 (m, 4H), 1.92 (qt, J=7.6 Hz, 2H),
2.51-2.55 (m, J=3.7 Hz, 1H), 2.78 (d, 7.3 Hz, 2H), 3.09 (t, J=7.8
Hz, 2H). .sup.13C NMR (125 MHz, D.sub.2O) .delta. 3.0, 21.2, 30.0,
46.8, 47.9. FT-IR (KBr) .nu..sub.max 3041, 1570, 1465, 1039
Preparation of 3-cyclopentylamino-1-propanesulfonic acid (Compound
CJ)
##STR00505##
[0470] Cyclopentylamine (3.95 mL, 40 mmol) was added to a solution
of 1,3-propane sultone (5.5 g, 45 mmol) in THF (80 mL). The mixture
was heated at reflux with an oil-bath for 4 hours. Stirring was
difficult, some acetone and ethanol were added to restore stirring.
The mixture was cooled to room temperature. The solid was collected
by filtration, dried in a vacuum oven for 1 hour at 60.degree. C.
(5.47 g). The solid material was dissolved in methanol/water (35
mL/2.5 mL, v/v) at reflux. The mixture was cooled slowly to room
temperature overnight, and further cooled in a fridge. The product
was collected by filtration, rinsed with methanol (15 mL),
air-dried for 15 minutes, and further dried in a vacuum oven at
60.degree. C. overnight. The product, Compound CJ, was obtained as
long fine white needles (4.79 g). A second crop was obtained from
the combined crude and first recrystallization mother liquor (0.84
g). Both crops were pure and were combined, total 5.63 g, 68%
yield. m.p. 280-82.degree. C. .sup.1H NMR (500 MHz, D.sub.2O)
.delta. 1.34-1.43 (m, 4H), 1.46-1.54 (m, 2H), 1.82-1.90 (m, 4H),
2.76 (7, J=7.6 Hz, 2H), 2.95 (t, J=7.8 Hz, 2H), 3.35 (qt, J=7.2 Hz,
1H). .sup.13C NMR (125 MHz, D.sub.2O) .delta. 21.5, 23.6, 29.3,
45.1, 47.9, 59.5. FT-IR (KBr) .nu..sub.max 3558, 3501, 2972, 1647,
1587, 1466
Preparation of 3-cycloheptylamino-1-propanesulfonic acid (Compound
CK)
##STR00506##
[0472] Cycloheptylamine (3.9 mL, 30 mmol) was added to a solution
of 1,3-propane sultone (4.1 g, 33 mmol) in THF (65 mL). The mixture
was heated at reflux for 5 hours with a heating mantle. The mixture
was cooled to room temperature and the solid was collected by
filtration, and then dried in a vacuum oven for 1 hour at
60.degree. C. (6.21 g). The solid material was dissolved in
methanol/water (30 mL/3 mL, v/v). The solution was cooled slowly to
room temperature, and further cooled with an ice-bath. The solid
product was collected by filtration, rinsed with methanol,
air-dried for 15 minutes, and further dried in a vacuum oven at
60.degree. C. Compound CK was obtained as small white flakes (5.08
g, 72% yield). m.p. 341-43.degree. C. .sup.1H NMR (500 MHz,
D.sub.2O) .delta. 1.21-1.42 (m, 8H), 1.45-1.51 (m, 2H), 1.79-1.89
(m, 4H), 2.76 (7, J=7.3 Hz, 2H), 2.96 (t, J=7.8 Hz, 2H), 3.35 (m,
J=4.6 Hz, 1H). .sup.13C NMR (125 MHz, D.sub.2O) .delta. 21.6, 23.3,
27.3, 30.5, 43.6, 48.0, 59.6. FT-IR (KBr) .nu..sub.max 2924, 1615,
1464, 1243.
Preparation of 3-benzyloxycarbonylamino-2-hydroxy-1-propanesulfonic
acid, sodium salt (Compound AC)
##STR00507##
[0474] 3-Amino-2-hydroxy-1-propanesulfonic acid (15.51 g, 100 mmol)
was dissolved in water (150 mL) with the help of 1 equivalent of
NaOH (4.08 g). A solution of CBZ-OSuc (27.4 g, 110 mmol) in MeCN
(300 mL) was added. After stirring for 4 hours at room temperature,
the solvent was evaporated under reduced pressure. The wet cake
(one equivalent in weight of water) was then suspended in acetone
(400 mL) and heated under reflux for 20 minutes. The mixture was
cooled to room temperature and the solid was collected by
filtration, washed with acetone and dried overnight in then vacuum
oven at 40.degree. C. Compound AC was obtained as a white fine
solid (28.85 g, 87.6 mmol, 88%). The .sup.1H and .sup.13C NMR were
consistent with the structure.
Preparation of 4-benzyloxycarbonylamino-1-butanesulfonic acid,
sodium salt (Compound AD)
##STR00508##
[0476] 4-Amino-1-butanesulfonic acid sodium salt (0.516 g, 2.95
mmol. was dissolved in water for a final concentration of 0.5 M
(slightly yellow solution). A solution of CBZ-OSuc in CH.sub.3CN (2
M, 1.55 mL, 3.1 mmol, 1.05 eq.) was added. The reagent
precipitated. A mixture of 1,4-dioxane and ethanol was added until
almost all of the solid was dissolved. After 3.75 h, the solvent
was evaporated under reduced pressure. The solid was dried in vacuo
over the weekends. The solid was then suspended in acetone and
heated under reflux for 30 minutes. The mixture was cooled to room
temperature and the solid was collected by filtration, washed with
acetone and dried overnight in vacuo. Compound A.beta. was obtained
as a white fine solid (0.7610 g, 2.32 mmol, 78%). The .sup.1H NMR
was consistent with the structure.
Preparation of 3-benzyloxycarbonylamino-1-propanesulfonic acid
sodium salt (Compound AE)
##STR00509##
[0478] 3-Amino-1-propanesulfonic acid sodium salt (1.09 g, 6.76
mmol) was dissolved in water for a final concentration of 0.5 M. A
solution of CBZ-OSuc in CH.sub.3CN (2 M, 3.55 mL, 7.1 mmol, 1.05
eq.) was added. The reagent precipitated. A mixture of 1,4-dioxane
and ethanol was added until almost all the solid was dissolved.
After 3.75 hours, the solvent was evaporated under reduced
pressure. The solid was dried in vacuo over the weekends. The solid
was then suspended in acetone and heated under reflux for 30
minutes. The mixture was cooled to room temperature and the solid
was collected by filtration, washed with acetone and dried
overnight in vacuo. Compound AE was obtained as a white fine solid
(1.58 g, 5.06 mmol, 75%). The .sup.1H NMR was consistent with the
structure. 90% pure (10% mol of homotaurine).
Preparation of L-(N-Boc)-Phe-L-Phe-homotaurine isobutyl ester
##STR00510##
[0479] Component 1: L-(N-Boc)-Phe-L-Phe
##STR00511##
[0481] A solution of (Boc).sub.2O (800 mg, 3.5 mmol) in 1,4-dioxane
(5 mL) was added to a cold (0.degree. C.) solution of di-L-Phe-Phe
(1 g, 3.20 mmol) in 1,4-dioxane (6 mL) and 1N NaOH (3.3 mL). The
mixture was stirred at 0-5.degree. C. for 2 hours. Another portion
of (BOC).sub.2O (100 mg) was added and the mixture was stirred for
an additional 60 minutes at 0-5.degree. C. then at room temperature
for 30 minutes. The mixture was then evaporated to dryness. The
solid was taken in a mixture of water/EtOAc and pH was adjusted to
2 with 2N HCl. The aqueous layer was extracted 3 times with EtOAc.
The combined organic layers were dried with brine and the solvent
was evaporated. Some solid was insoluble in a mixture of
EtOAc/CHCl.sub.3: it was removed by filtration. The desired
N-Boc-L-Phe-L-Phe 1 was obtained as a white foamy solid (913.7 mg,
2.215 mmol, 71% yield).
Component 2: 3-amino1-propanesulfonic acid isobutyl ester
##STR00512##
[0482] Step 1: 3-azido-1-propanesulfonic acid, sodium salt (5)
[0483] A solution of 1,3-propane sultone 4 (6.12 g, 49.1 mmol) in
acetone (30 mL) was added to a mixture of sodium azide (3.22 g,
49.1 mmol) in water/acetone (70 mL, 20 to 50). The clear solution
was stirred at room temperature. The reaction was completed within
1 hour. The solvent was removed by evaporation under reduced
pressure. The solid obtained was rinsed with hot ether (50 mL) and
then with ether at room temperature (150 ml). The solid was then
dried in the vacuum oven at 40.degree. C. for overnight. The title
compound 5 was obtained as a white solid (8.69 g, 46.4 mmol, 95%
yield).
Step 2: 3-azido-1-propanesulfonyl chloride
[0484] 3-Azidopropanesulfonic acid, sodium salt 5 (1.87 g, 10.0
mmol) was suspended in dry benzene (20 mL), and PCl.sub.5 (2.3 g,
10.5 mmol) was added to the suspension. The mixture was stirred at
room temperature for 30 minutes, then at gentle reflux for about 1
hour. The benzene and P(O)C13 were removed by evaporation under
reduced pressure. Benzene was added to the crude mixture and the
solvent was removed again under reduced pressure. The residue was
dried in vacuo. The dried residue was dissolved in dichloromethane
(anhydrous, 15 mL) and cooled at -10.degree. C. using an
ice/acetone bath.
Step 3: 3-azido-1-propanesulfonic acid, isobutyl ester
[0485] A solution of isobutanol (1.00 mL, 10.8 mmol) and
2,6-lutidine (1.3 mL, 11.2 mmol) in dichloromethane (10 mL) was
added slowly to the cold sulfonyl chloride solution. The mixture
was stirred at -10.degree. C. for 5 minutes then at room
temperature for 2 hours. The reaction was quenched with water and
dichloromethane was added to extract the product. The organic layer
was washed once with water, aqueous saturated NaHCO.sub.3, brine,
and then dried over magnesium sulfate. The solvent was removed by
evaporation under reduced pressure and the residue was dried in
vacuo. The remaining 2,6-lutidine hydrochloride was removed by
washing the residue with ether. The resulting oil (1.78 g) was then
applied on flash column (silica gel, EtOAc in Hexanes from 15% to
20%) to give afford the desired ester as an oil (790 mg, 35%).
Step 4: 3-amino-1-propanesulfonic acid isobutyl ester
[0486] A solution of isobutyl 3-azidopropanesulfonate (1.13 g, 5.11
mmol) in isobutanol (10 mL) was added under H.sub.2, via a cannula,
to a suspension of Pd/C (10%, 200 mg) in isobutanol (4 mL) which
had been saturated with H.sub.2. The mixture was then stirred under
H.sub.2 (40 psi) at room temperature overnight. The solid was then
removed by filtration. The filtrate was evaporated to dryness. And
the residue was dried in vacuo. The title compound 2, homotaurine
isobutyl ester, was obtained as brown oil (808.7 mg, 81%).
Reaction of Component 1 with Component 2
[0487] HOBT (340 mg, 2.215 mmol) was added to a cold (0-5.degree.
C.) solution of N-Boc-L-Phe-L-Phe 1 (913.7 mg, 2.215 mmol) and
homotaurine isobutyl ester 2 (423 mg, 2.21 mmol) in dichloromethane
(anhydrous, 30 mL). After 5 minutes, a solution of
1-cyclohexyl-3-(2-morpholinoethyl)carbodiimide
metho-p-toluenesulfonate (982 mg, 2.215 mmol) in dichloromethane
(10 mL) was added dropwise. The solution was stirred overnight at
room temperature. The mixture was diluted with dichloromethane (50
mL) and the organic layer was washed sequentially with 1N
NaHSO.sub.4, aqueous saturated NaHCO.sub.3, and brine, and dried
over sodium sulfate. The solvent was removed by evaporation under
reduced pressure. Three compounds in the mixture were shown on TLC.
Since the impurities were less soluble in methanol, repeated
treatment with methanol followed by filtration removed most of the
impurities. Column chromatography on silica gel (2% MeOH in
CHCl.sub.3) afforded the tripeptide 3 as amber glassy solid (156.1
mg, 12%).
Preparation of L-Phe-L-Phe-homotaurine isobutyl ester
##STR00513##
[0489] Concentrated HCl (0.7 mL) was added to a cold (0.degree. C.)
solution of N-Boc-L-Phe-L-Phe-homotaurine isobutyl ester (202 mg,
0.343 mmol) in methanol. The mixture was stirred at room
temperature for 2 hours and then left standing in the refrigerator
overnight. The solvent was removed under reduced pressure and the
residual solid was dried in vacuo to afford the
L-Phe-L-Phe-homotaurine isobutyl ester as a white solid (171.8 mg,
95%).
Preparation of L-Phe-L-Phe-homotaurine (Compound X)
##STR00514##
[0491] A solution of N--BOC-L-phenylalanine-N-hydroxusuccinimide
ester (400 mg, 1.1 mmol) in a mixture of ethanol (6 mL) and
1,4-dioxane (4 mL) was added to a solution of L-Phe-Homotaurine
(273 mg, 1.0 mmol) in 1N NaOH (1.05 mL), water (3 mL) and ethanol
(4 mL). The mixture was stirred at room temperature overnight. The
solvent was removed under reduced pressure and the solid (601.9 mg)
was suspended in a mixture of acetone (8 mL) and isopropanol (0.2
mL) and stirred overnight at room temperature. The mixture was
heated under refluxed for 30 minutes and then was cooled to room
temperature. The white solid was collected by filtration, washed
with ether, then dried in the vacuum oven for 45 minutes. The
resulting solid (423.1 mg) was dissolved in a mixture of
water/tert-butanol (7:3, 5 mL) and treated with Amberlite IR-120
plus (washed, 15 g dry weight) for 2 minutes at room temperature.
The resin was removed by filtration and washed 3 times with the
mixed solvents of water and tert-butanol (10 mL). Concentrated HCl
(4 mL) was added. The solvents were removed under reduced pressure,
and the resulting solid was dried in vacuum. The compound was
purified by recrystallization from a mixed solvent of THF and MeOH.
The resulting solid was heated under reflux in methanol (about 3
mL) to remove the yellowish color. The solid was dried in vacuo.
Compound X was obtained as white solid (84.4 mg, 20%). The .sup.1H
and .sup.13C NMR were consistent with the structure.
Preparation of N-(3-aminopropane-1-sulfonyl)-phenylalanine, ethyl
ester (Compound CL)
##STR00515##
[0493] The 3-chloropropane-1-sulfonyl chloride (10 mmol, 1.21 mL)
was added dropwise to a cold (-10.degree. C.) solution of
L-phenylalanine ethyl ester (10 mmol, 2.3 g) and 4-methylmorpholine
(20 mmol, 2.2 mL) in dichloromethane (30 mL). The mixture was
stirred for 30 minutes at -10.degree. C. and for 2 hours at room
temperature. The mixture was diluted with dichloromethane (40 mL)
and washed twice with water, once with brine, dried over sodium
sulfate. Evaporation of the solvent under reduced pressure gave
rise to almost pure 3-chloropropyl sulfonamide as a yellow-oil in
quantitative yield. The .sup.1H and .sup.13C NMR were consistent
with the structure.
[0494] A mixture of the 3-chloropropyl sulfonamide (10 mmol),
sodium azide (20 mmol) and a catalytic amount of Bu.sub.4NI in DMF
(40 ml) was heated at 60.degree. C. for 24 hours. The mixture was
diluted with ethyl acetate, washed with water three times, and once
with brine, dried over sodium sulfate. Evaporation of the solvent
gave rise to the azide as a brown-oil (3.0489 g, 8.96 mmol, 90%).
The .sup.1H and .sup.13C NMR were consistent with the
structure.
[0495] The azide (2.70 g, 7.94 mmol) was stirred under H.sub.2 (40
psi) with 10% Pd/C (348 mg) in ethanol (16 mL) at room temperature
overnight. The solid was removed by filtration over Celite. The
filtrate was treated with TMSCl/EtOH in attempt to obtain a
crystalline hydrochloride salt of the product. The solvent was
evaporated to give a thick residue (2.2 g, 6.27 mmol, 79%) that
crystallized under none of the conditions tried. The crude
hydrochloric acid salt was dissolved in dichloromethane and washed
once with saturated sodium bicarbonate. The organic layer was
recovered and dried over sodium sulfate. The solvent was removed by
evaporation under reduced pressure. The residue was dissolved in
methanol and treated with activated charcoal. The solid was removed
by filtration over Celite and the filtrate was evaporated to
dryness. The residue was dried in vacuo to afford a tan oil (1.3969
g, 56% from the azide). The .sup.1H and .sup.13C NMR were
consistent with the structure of Compound CL.
Preparation of N-Boc-L-Phe-(3-aminopropane-1-sulfonyl)-L-Phe, ethyl
ester (Compound Y)
##STR00516##
[0497] A solution of N-t-BOC-L-Phe N-hydroxysuccinimide ester (2.80
mmol, 1.01 g) in dichloromethane (12 mL) was added to a cold
(0.degree. C.) solution of 3-aminopropane-1-sulfonyl-L-Phe-OEt
(2.66 mmol, 839 mg) in dichloromethane (10 mL). The mixture was
stirred overnight at room temperature. The mixture was diluted with
dichloromethane, and washed with 2N HCl, aqueous saturated
NaHCO.sub.3, and brine. The organic layer was dried over magnesium
sulfate and the solvent was removed under reduced pressure. The
residue was applied on flash column chromatography on silica gel
(2% MeOH in CHCl.sub.3). A portion of the pure desired material
(600 mg) was isolated. The remaining product was mixed with 40% of
the succinimide. Some 3-aminopropanol (70 .mu.L) was added to the
mixture that was dissolved in dichloromethane (8 mL) and cooled to
0.degree. C. The mixture was stirred for 1 hour at room
temperature. The mixture was diluted with dichloromethane, and
washed with 2N HCl, aqueous saturated NaHCO.sub.3, brine. The
organic layer was dried over magnesium sulfate and the solvent was
removed under reduced pressure. The product was purified by flash
column chromatography on silica gel (2% MeOH in CHCl.sub.3).
Another portion of the pure desired material (432.4 mg) was
isolated along with the adduct of the succinimide and
3-aminopropanol (220.6 mg, 0.684 mmol). Compound Y was obtained as
a white crystalline foamy solid (1030 mg, 1.83 mmol, 65%). The
.sup.1H and .sup.13C NMR were consistent with the structure.
Preparation of L-Phe-(3-aminopropane-1-sulfonyl)-L-Phe, ethyl ester
(Compound Z)
##STR00517##
[0499] Concentrated HCl (0.8 mL) was added to a cold (0.degree. C.)
solution of the N-Boc-L-Phe-(3-Aminopropane-1-Sulfonyl)-L-Phe,
Ethyl Ester (197 mg, 0.350 mmol) in ethanol (8 mL). The mixture was
cooled using an ice/water bath and stirred for 45 min and for 3
hours at room temperature. And the mixture was kept in freezer
(-20.degree. C.) over the weekend. The solvent was removed under
reduced pressure. The residual ethanol was removed by coevaporation
3 times with chloroform under reduced pressure. The residue was
dried in vacuo to give an off-white crystalline solid (175.3 mg) in
quantitative yield. The .sup.1H and .sup.13C NMR were consistent
with the structure of Compound Z.
Preparation of L-(N-Boc)-Phe-(3-aminopropane-1-sulfonyl)-L-Phe,
sodium salt (Compound AA)
##STR00518##
[0501] One equivalent of 1N NaOH (202 .mu.L) was added to a
solution of L-(N-Boc)-Phe-(3-aminopropane-1-sulfonyl)-L-Phe, ethyl
ester (110 mg, 0.197 mmol) in methanol (2 mL). The mixture was
stirred overnight at room temperature. A white suspension was
observed after overnight stirring. MeOH (1 mL); water (1 mL) and 1N
NaOH (10 pit) were added. The mixture was stirred in a warm water
bath for about 2 hours. The solvent methanol was removed under
reduced pressure. The wet residue was then freeze-dried to give
compound AA as a white powder in quantitative yield (110.2 mg). The
.sup.1H and .sup.13C NMR were consistent with the structure.
Preparation of L-Phe-(3-aminopropane-1-sulfonyl)-L-Phe, methyl
ester (Compound AB)
##STR00519##
[0503] The hydrolysis was done by the conventional LiOH/MeOH
method. The product was purified by recrystallization from EtOAc
and Hexanes.
[0504] The L-(N-Boc)-Phe-(3-aminopropane-1-sulfonyl)-L-Phe-OH (203
mg, 0.382 mmol) was dissolved in methanol (4 mL) and the solution
was cooled to 0.degree. C. Concentrated HCl (0.35 mL) was added and
the mixture was stirred for 2 hours at 0.degree. C. and for 2.5 h
at room temperature. The volatile solvents were removed under
reduced pressure. The aqueous residue was freeze-dried to give the
product as a white solid (171.4 mg). The NMR and MS showed to
product to be a mixture of the free acid and the methyl ester. The
MS showed also a strong association of the peptide. The dimer was
the major species on the MS. The solid was dissolved into methanol
and treated with HCl overnight at room temperature. The solvent was
evaporated and the residue was dried in vacuo. The product was
obtained as a white foamy solid (180.4 mg, 97%). The .sup.1H and
.sup.13C NMR were consistent with the structure of compound AB.
Preparation of 4-iodo-N-(3-Sulfopropyl)-L-phenylalanine amide
(Compound CO)
##STR00520##
[0506] Thionyl chloride (8.2 mL, 112.5 mmol) was added to a cold
MeOH (60 mL, in an ice-bath). The ice bath was removed and
4-iodo-L-phenylalanine (6.55 g, 22.4 mmol) was added to the
mixture. The solution was stirred at reflux for 2 h. The solvent
was removed under reduced pressure. The residual solid was
dissolved in MeOH (40 mL) and the solution was poured into
Et.sub.2O (300 mL). The solid was collected by filtration, washed
with Et.sub.2O (2.times.50 mL) and dried in vacuo.
[0507] The solid (1.96 g, 5.8 mmol) was dissolved in a minimum
amount of water. To the solution was added aqueous NH.sub.4OH
(28-30%, 15 mL). The reaction mixture was stirred at room
temperature over weekend. The solvent was removed under reduced
pressure and EtOAc (15 mL) was added. The mixture was heated under
reflux. The hot solution was filtered. The filtrate was cooled to
room temperature and was stored in the fridge. The solid was
collected by filtration, washed with EtOAc, to give
4-iodophenylalanine amide.
[0508] The amide (1.3 g, 4.4 mmol) was dissolved in 15 mL of
2-butanone with a few drops of DMF before 1,3-propane sultone (560
mg, 4.9 mmol) was added. The reaction mixture was stirred at reflux
for 2 hours. The mixture was cooled to room temperature. The solid
was collected by filtration, washed with acetone (2.times.20 mL)
and dried in vacuo. The solid was suspended in MeOH (25 mL) and a
small amount of water (1 mL). The suspension was stirred at reflux.
The solid material was collected by filtration while the mixture
was still hot. The solid was washed with hot MeOH (2.times.10 mL).
Compound CO was obtained as a white solid (320 mg).
Preparation of
3-[4-(4-fluorophenyl)-1,2,3,6-tetrahydropyridin-1-yl]-1-propanesulfonic
acid (Compound F)
##STR00521##
[0510] The 4-(4-fluorophenyl)-1,2,3,6-tetrahydropyridine
hydrochloride (2.58 g, 14.5 mmol) was treated with 1N NaOH (20 mL).
The aqueous mixture was extracted with CH.sub.2Cl.sub.2 (20 mL).
The organic layer was separated and dried over MgSO.sub.4. The
solvents were removed by evaporation under reduced pressure.
[0511] To a solution of
4-(4-fluorophenyl)-1,2,3,6-tetrahydropyridine (1.96 g, 13.7 mmol)
in acetone (30 mL) was added 1,3-propane sultone (1.74 g, 14.5
mmol). The mixture was stirred at reflux overnight. Only a small
amount of compound precipitated. The resulting suspension was
cooled to room temperature with stirring and a larger amount of
solid precipitated. The suspension was heated with the addition of
a small amount of MeOH until complete dissolution of the solid. The
resulting solution was stirred at reflux for a few minutes and was
cooled to room temperature with stirring. The solid was collected
by filtration, washed with MeOH and dried in vacuo. This allowed
the isolation of compound F, 1.33 g (32%).
Preparation of
3-[4-(4-bromophenyl)-4-hydroxypiperidin-1-yl]-1-propanesulfonic
acid (Compound G)
##STR00522##
[0513] To a solution of 4-(4-bromophenyl)-4-piperidinol (2.51 g,
9.8 mmol) in MeOH (25 mL) was added 1,3-propane sultone (1.28 g,
10.7 mmol). The mixture was stirred at reflux for 2 h. Only a small
amount of compound precipitated. The resulting suspension was
cooled to room temperature with stirring and a solution of 50%
MeOH/Acetone was added to precipitate a maximum of compound. The
solid was collected by filtration, washed with 50% MeOH/Acetone
(2.times.25 mL) and dried in vacuo. This allowed the isolation of
compound G, 2.11 g (57%).
Preparation of
3-[4-(4-chlorophenyl)-4-hydroxypiperidin-1-yl]-1-propanesulfonic
acid (Compound H)
##STR00523##
[0515] To a solution of 4-(4-chlorophenyl)-4-piperidinol (2.5 g,
11.8 mmol) in acetone (25 mL) was added 1,3-propane sultone (1.56
g, 13.0 mmol). The mixture was stirred at reflux for 2 h. The
reaction mixture was cooled to room temperature. The solid was
collected by filtration, washed with acetone (2.times.20 mL) and
dried in vacuo. This allowed the isolation of compound H, 2.83 g
(72%).
Preparation of
3-(4-acetyl-4-phenylpiperidin-1-yl)-1-propanesulfonic acid
(Compound I)
##STR00524##
[0517] 4-Acetyl-4-phenylpiperidine hydrochloride (3.32 g, 12.5
mmol) was treated with 1N NaOH (20 mL). The aqueous mixture was
extracted with CH.sub.2Cl.sub.2 (20 mL). The organic layer was
separated, dried over Na.sub.2SO.sub.4, filtered, and the solvent
was removed under reduced pressure.
[0518] To a solution of 4-acetylphenylpiperidine (1.83 g, 9.0 mmol)
in acetone (22 mL) was added 1,3-propane sultone (1.20 g, 10.0
mmol). The mixture was stirred at reflux for 2 hours. The reaction
mixture was cooled to room temperature. The solid was collected by
filtration, washed with acetone (2.times.20 mL) and dried in vacuo.
This allowed the isolation of compound I, 2.65 g (90%).
Preparation of
3-[4-(4-chlorophenyl)-1,2,3,6-tetrahydropyridin-1-yl]-1-propanesulfonic
acid (Compound J)
##STR00525##
[0520] The 4-(4-chlorophenyl)-1,2,3,6-tetrahydropyridine
hydrochloride (2.52 g, 10.9 mmol) was treated with 1N NaOH (20 mL);
and aqueous mixture was extracted with CH.sub.2Cl.sub.2 (20 mL).
The organic layer was separated, dried over Na.sub.2SO.sub.4, and
filtered. The solvent was removed under reduced pressure.
[0521] To a solution of
4-(4-chlorophenyl)-1,2,3,6-tetrahydropyridine (2.07 g, 10.7 mmol)
in acetone (25 mL) was added 1,3-propane sultone (1.41 g, 11.8
mmol). The mixture was stirred at reflux for 2 h. The reaction
mixture was cooled to room temperature. The solid was collected by
filtration, washed with acetone (2.times.20 mL) and dried in vacuo.
The product was suspended in 50% MeOH/acetone (75 mL). The
suspension was stirred at reflux for 5 minutes before 25 mL of cold
acetone was added. The solid material was filtered and washed with
acetone (2.times.25 mL). This allowed the isolation of compound J,
1.48 g (44%).
Preparation of 3-(4-phenylpiperazin-1-yl)-1-propanesulfonic acid
(Compound K)
##STR00526##
[0523] To a solution of 1-phenylpiperazine (2.0 g, 1.9 mL, 12.3
mmol) in acetone (20 mL) was added 1,3-propane sultone (1.53 g,
12.9 mmol). The mixture was stirred at reflux for 2 hours. The
reaction mixture was cooled to room temperature. The solid was
collected by filtration, washed with acetone (2.times.25 mL) and
dried in vacuo. This allowed the isolation of compound K, 3.04 g
(87%).
Preparation of
3-[4-(4-chlorophenyl)piperazin-1-yl]-1-propanesulfonic acid
(Compound L)
##STR00527##
[0525] The 1-(4-chlorophenyl)piperazine dihydrochloride (2.5 g, 9.3
mmol) was treated with 1N NaOH (40 mL); and the aqueous mixture was
extracted with CH.sub.2Cl.sub.2 (40 mL). The organic layer was
separated, dried over Na.sub.2SO.sub.4, filtered and solvent was
removed under reduced pressure.
[0526] To a solution of 1-(4-chlorophenyl)piperazine (1.62 g, 8.2
mmol) in acetone (20 mL) was added 1,3-propane sultone (1.06 g, 8.6
mmol). The mixture was stirred at reflux for 2 h. The reaction
mixture was cooled to room temperature. The solid was collected by
filtration, washed with acetone (2.times.25 mL) and dried in vacuo.
This allowed the isolation of compound L, 2.11 g (81%).
Preparation of
3-[4-(2-fluorophenyl)piperazin-1-yl]-1-propanesulfonic acid
(Compound
##STR00528##
[0528] To a solution of 1-(2-fluorophenyl)piperazine (2.5 g, 2.2
mL, 13.9 mmol) in acetone (25 mL) was added 1,3-propane sultone
(1.73 g, 14.6 mmol). The mixture was stirred at reflux for 2 hours.
The reaction mixture was cooled to room temperature. The solid was
collected by filtration, washed with acetone (2.times.25 mL) and
dried in vacuo. This allowed the isolation of compound M, 3:56 g
(85%).
Preparation of
3-[4-(4-nitrophenyl)piperazin-1-yl]-1-propanesulfonic acid
(Compound N)
##STR00529##
[0530] To a solution of 1-(4-nitrophenyl)piperazine (2.58 g, 12.1
mmol) in acetone (25 mL) was added 1,3-propane sultone (1.06 g, 8.6
mmol). The mixture was stirred at reflux for 2 h. The reaction was
cooled to room temperature. The solid was collected by filtration,
washed with acetone (2.times.25 mL) and dried in vacuo. This
allowed the isolation of compound N, 2.85 g (71%).
Preparation of
3-[4-(4-fluorophenyl)piperazin-1-yl]-1-propanesulfonic acid
(Compound P)
##STR00530##
[0532] To a solution of 1-(4-fluorophenyl)piperazine (2.0 g, 11.1
mmol) in acetone (20 mL) was added 1,3-propane sultone (1.46 g,
11.7 mmol). The mixture was stirred at reflux for 2 hours. The
reaction mixture was cooled to room temperature. The solid was
collected by filtration, washed with acetone (2.times.25 mL) and
dried in vacuo. This allowed the isolation of compound P, 2.62 g
(78%).
Preparation of 3-(4-phenyl-1,2,3,6-tetrahydropyridin-1-yl)propanoic
acid (Compound Q)
##STR00531##
[0534] The 4-phenyl-1,2,3,6-tetrahydropyridine hydrochloride (1.5
g, 7:8 mmol) suspended in 16 mL of CH.sub.2Cl.sub.2. To this
suspension was added triethylamine (2.1 mL, 15.3 mmol) followed by
methyl 3-bromopropionate (1:0 mL, 9.2 mmol). The reaction was
stirred at room temperature for 4 h and at reflux for 2 h. The
reaction mixture was washed with water, 1N HCl(2.times.20 mL), 1N
NaOH (2.times.20 mL) and Brine (1.times.20 mL). The organic layer
was separated, dried over Na.sub.2SO.sub.4, filtered; and solvent
was removed under reduced pressure.
[0535] To the crude product was added 2N NaOH (15 mL). The mixture
was stirred at reflux for 1 h. The reaction mixture was washed with
CH.sub.2Cl.sub.2 (3.times.20 mL) and neutralized with concentrated
HCl. The aqueous solution was concentrated to dryness under reduced
pressure, to give a solid residue. Sodium chloride in the residue
was removed in the following way (three repeats): dissolving the
residue in a minimum amount of water, treating the aqueous solution
with acetone, removing the resultant solid material by filtration,
and concentrate the filtrate to dryness under reduced pressure.
This allowed the isolation of compound Q (159.4 mg).
Preparation of 3-dibenzylamino-1-propanesulfonic acid (Compound
AV)
##STR00532##
[0537] To a solution of dibenzylamine (9.8 mL, 50.8 mmol) in
toluene (50 mL) was added 1,3-propane sultone (6.50 g, 53.3 mmol).
The mixture was stirred at reflux for 3 h. A sticky paste was
formed at the bottom of the flask. The reaction mixture was cooled
to room temperature. The top layer was decanted; and the paste was
partially dissolved in EtOAc with heating. The mixture was poured
in a 10% EtOAc/Hexanes (200 mL). The mixture was heated and the
paste was spread on the walls of the conical flask. The solvent was
removed. This process was repeated twice. MeOH (75 mL) was added to
the paste. The mixture was heated until a white solid appeared. The
solid was collected by filtration, washed with cold MeOH, and dried
in vacuo, affording compound AV, 7.03 g (43%).
Preparation of 3-(4-cyano-4-phenylpiperidin-1-yl)-1-propanesulfonic
acid (Compound E)
##STR00533##
[0539] The 4-cyano-4-phenylpiperidine hydrochloride (2.0 g, 9.0
mmol) was treated with 1N NaOH (20 mL) and the aqueous phase was
extracted with CH.sub.2Cl.sub.2 (20 mL). The organic layer was
separated, dried over MgSO.sub.4, filtered and solvent was removed
under reduced pressure.
[0540] To a solution of piperidine (1.43 g, 7.7 mmol) in acetone
(20 mL) was added 1,3-propane sultone (1.02 g, 8.5 mmol). The
mixture was stirred at reflux for 2 h. The resulting suspension was
cooled to room temperature. The solid was collected by filtration,
washed with acetone and dried in vacuo. The solid was
recrystallized from MeOH (and traces of water) to afford compound
E, 800 mg (34%).
Preparation of 3-(4-phenyl-1,2,3,6-tetrahydropyridin-1-yl)butanoic
acid hydrochloride (Compound R)
##STR00534##
[0542] The 4-phenyl-1,2,3,6-tetrahydropyridine hydrochloride (2.01
g, 10.2 mmol) was treated with 1N NaOH (20 mL) and the aqueous
phase was extracted with CH.sub.2Cl.sub.2 (20 mL). The organic
layer was separated, dried over MgSO.sub.4, filtered and solvent
was removed under reduced pressure.
[0543] The resulting 4-phenyl-1,2,3,6-tetrahydropyridine (1.55 g,
9.7 mmol) was dissolved in 20 mL of 2-butanone. To this solution
was added potassium carbonate (2.02 g, 14.6 mmol). The mixture was
stirred 30 minutes at room temperature; ethyl 4-bromobutyrate (1.46
mL, 10.1 mmol) was added. The reaction mixture was stirred at
reflux for 5 hours. After cooling to room temperature, inorganic
salts were filtered. The solvent was evaporated under reduced
pressure. The residue was dissolved in CH.sub.2Cl.sub.2 (30 mL).
The organic phase was washed with water (2.times.30 mL), 2N HCl
(2.times.30 mL) and Brine (2.times.30 mL). The organic layer was
dried with Na.sub.2SO.sub.4, filtered, evaporated under reduced
pressure and dried in vacuo. This allowed the isolation of 1.55 g
(58%) of the desired ester.
[0544] The ester (5.7 mmol) was dissolved in 6N HCl (40 mL). The
reaction mixture was stirred at room temperature for 5 hours and at
reflux for 1 hour before it was cooled to room temperature. The
reaction mixture was extracted with CH.sub.2Cl.sub.2 (3.times.30
mL). The aqueous phase was evaporated under reduced pressure. The
residue was dissolved in water (20 mL); and the aqueous solution
was concentrated to dryness under reduced pressure. The resultant
material was further dried in vacuo, affording compound R, 973 mg
(61%).
Preparation of 3-piperonylamino-1-propanesulfonic acid (Compound
AW)
##STR00535##
[0546] To a solution of piperonylamine (2.5 mL, 19.8 mmol) in
acetone (30 mL) was added 1,3-propane sultone (2.52 g, 20.8 mmol).
The mixture stirred at reflux for 2 hours. The reaction mixture was
cooled to room temperature. The solid material was collected by
filtration, washed with acetone (2.times.25 mL) and dried in vacuo.
The product was suspended in 90% Acetone/MeOH (75 mL). The
suspension was stirred at reflux for 30 sec., the solid was
collected by filtration and dried in vacuo. This allowed the
isolation of compound AW, 2.56 g (45%).
Preparation of 3-(3,4,5-trimethoxybenzyl)amino-1-propanesulfonic
acid (Compound AY)
##STR00536##
[0548] To a solution of 3,4,5-trimethoxybenzylamine (2.2 mL, 12.7
mmol) in 2-butanone (20 mL) was added 1,3-propane sultone (1.66 g,
13.3 mmol). The mixture was stirred at reflux for 2 hours. The
reaction mixture was cooled to room temperature. The solid was
collected by filtration, washed with acetone (2.times.25 mL) and
dried in vacuo. The product was suspended in 90% Acetone/MeOH (75
mL). The suspension was stirred at reflux for 30 sec., the solid
was collected by filtration, and dried in vacuo; affording compound
AY, 2.61 g (64%).
Preparation of 3-(2,3-dimethoxybenzyl)amino-1-propanesulfonic acid
(Compound AZ)
##STR00537##
[0550] To a solution of 2,3-dimethoxybenzylamine (2.2 mL, 15.0
mmol) in 2-butanone (20 mL) was added 1,3-propane sultone (1.97 g,
15.8 mmol). The mixture was stirred at reflux for 2 hours. The
reaction mixture was cooled to room temperature. The solid was
collected by filtration, washed with acetone (2.times.25 mL) and
dried in vacuo. The crude product was suspended in 90% Acetone/MeOH
(75 mL). The suspension was stirred at reflux for 30 seconds, the
solid was collected by filtration, and dried in vacuo; affording
compound AZ, 1.95 g (45%).
Preparation of 3-(N-benzhydrylcarbamyl)amino-1-propanesulfonic acid
(Compound AF)
##STR00538##
[0552] The 3-amino-1-propanesulfonic acid (1.0 g, 7.2 mmol) was
dissolved in 3N NaOH (370 mg, 9.4 mmol in 3 mL of water). After the
solution was cooled to 0.degree. C., diphenylmethyl isocyanate (1.4
mL, 7.2 mmol) was added. The reaction mixture was allowed to warm
up to room temperature, stirred for 8 h (r.t.), and followed by
addition of 3N NaOH (3 mL). The reaction mixture was stirred for 18
h. The pH of the reaction mixture was brought to 3 with 5N HCl. The
solvent was evaporated under reduced pressure. EtOH (15 mL) was
added and the mixture was stirred at reflux for 30 sec. The hot
mixture was filtered. The filtrate was evaporated to dryness. This
process was repeated 2 more times. The final product was dried in
vacuo, affording compound AF, 837 mg (34%).
Preparation of 3-(3,5-dimethoxybenzyl)amino-1-propanesulfonic acid
(Compound BA)
##STR00539##
[0554] To a solution of 3,5-dimethoxybenzylamine (2.5 g, 15.0 mmol)
in 2-butanone (22 mL) was added 1,3-propane sultone (1.95 g, 15.8
mmol). The mixture was stirred at reflux for 2 hours. The reaction
mixture was cooled to room temperature. The solid was collected by
filtration, washed with acetone (2.times.25 mL), and dried in
vacuo. This allowed the isolation of compound BA, 2.89 g (67%).
Preparation of 3-(2,4-dimethoxybenzyl)amino-1-propanesulfonic acid
(Compound BB)
##STR00540##
[0556] The 2,4-dimethoxybenzylamine hydrochloride (2.51 g, 12.3
mmol) was treated with 1N NaOH (20 mL) and the aqueous phase was
extracted with CH.sub.2Cl.sub.2 (20 mL). The organic layer was
separated, dried over MgSO.sub.4, and filtered. Solvent was removed
under reduced pressure to give the amine (free base form).
[0557] To a solution of 2,4-dimethoxybenzylamine (1.71 g, 10.3
mmol) in 2-butanone (15 mL) was added 1,3-propane sultone (1.31 g,
10.7 mmol). The mixture was stirred at reflux for 2.5 h. The
reaction mixture was cooled to room temperature. The supernatant
was decanted; and the paste was washed with acetone (2.times.30
mL), and dissolved in MeOH with heating. Acetone addition to the
methanolic solution caused precipitation. The solid material was
collected by filtration, washed with acetone (2.times.25 mL), and
dried in vacuo; affording compound BB, 1.14 g (38%).
Preparation of 3-(phenylacetamido)-1-propanesulfonic acid, sodium
salt (Compound AG)
##STR00541##
[0559] 3-amino-1-propanesulfonic acid (1.0 g, 7.2 mmol) was
dissolved in solution of 3M NaOH (7.2 mL). The mixture was cooled
to 0.degree. C. before phenylacetyl chloride (1.4 mL, 10.8 mmol)
was added. The reaction mixture was allowed to warm up to room
temperature and it was stirred for 22 h. The solvent was evaporated
under reduced pressure. The residue was suspended in 50%
EtOH/Acetone. The mixture was stirred at reflux for 30 sec. The
solid material was collected by filtration and dried in vacuo. The
product was recrystallized from 95% EtOH/H.sub.2O and dried in
vacuo. This allowed the isolation of compound AG, 880 mg (44%).
Preparation of 3-(N-benzylcarbamyl)amino-1-propanesulfonic acid,
sodium salt (Compound AH)
##STR00542##
[0561] 3-amino-1-propanesulfonic acid (1.06 g, 7.7 mmol) was
dissolved in 1.5N NaOH (5.3 mL). To this solution was added benzyl
isocyanate (927 .mu.L, 7.7 mmol). The reaction mixture was stirred
at 70.degree. C. for 30 min, followed by addition to the mixture
one-equivalent of benzyl isocyanate (927 .mu.L, 7.7 mmol). The
reaction mixture was stirred for 1 hour. The solvent was evaporated
under reduced pressure. The residue was suspended in hot acetone.
The solid material was collected by filtration, washed with hot
acetone, and dried in vacuo; affording compound AH, 2.07 g
(92%).
Preparation of 3-(N-n-dodecylcarbamyl)amino-1-propanesulfonic acid,
sodium salt (Compound AJ)
##STR00543##
[0563] 3-amino-1-propanesulfonic acid (1.06 g, 7.7 mmol) was
dissolved in 1.5N NaOH (5.3 mL). To this solution was added
n-dodecyl isocyanate (1.7 mL, 7.7 mmol). The reaction mixture was
stirred at 70.degree. C. for 30 min followed by addition to the
mixture one-equivalent of n-dodecyl isocyanate (1.7 mL, 7.7 mmol).
The reaction mixture was stirred for 1 h. The solvent was removed
under reduced pressure. The residual material was suspended in hot
acetone. The solid material was collected by filtration, washed
with hot acetone, and dried in vacuo; affording compound AJ, 2.47 g
(86%).
Preparation of 3-(N-1-adamantylcarbamyl)amino-1-propanesulfonic
acid, sodium salt (Compound AK)
##STR00544##
[0565] 3-amino-1-propanesulfonic acid (1.06 g, 7.7 mmol) was
dissolved in 1.5N NaOH (5.3 mL). To this solution was added
1-adamantyl isocyanate (1.36 g 7.7 mmol) in hot EtOH (5 mL). The
reaction mixture was stirred at 70.degree. C. for 30 min followed
by addition (to the mixture) of one-equivalent of 1-adamantyl
isocyanate (1.37, 7.7 mmol) in hot EtOH (5 mL). The reaction
mixture was stirred for 1 h. The solvent was removed under reduced
pressure. The residue was suspended in hot acetone. The solid
material was collected by filtration, washed with hot acetone, and
dried in vacuo. The solid was recrystallized from EtOH, affording
compound AK, 519.4 mg (20%).
Preparation of
3-[2-(4-isobutylphenyl)propanoyl]amino-1-propanesulfonic acid,
sodium salt (Compound AL)
##STR00545##
[0567] Thionyl chloride (1.6 mL, 21.1 mmol) was added to ibuprofen
(1.02 g, 4.9 mmol). The reaction mixture was heated to reflux for 4
h. The solvent was evaporated, dried in vacuo, giving corresponding
acid chloride.
[0568] 3-amino-1-propanesulfonic acid (308 mg, 2.2 mmol) was
dissolved in 1.5N NaOH (3 mL). To this solution was added dropwise
the acid chloride (500.8 mg, 4.4 mmol, prepared above). The
reaction mixture was stirred at 70.degree. C. overnight. The
solvent was removed under reduced pressure. The residue was
suspended in acetone. The suspension was stirred at reflux for 30
seconds. The solid material was removed by filtration. The filtrate
was evaporated to dryness under reduced pressure. The residual
material was subjected to separation by flash chromatography (80%
CH.sub.2Cl.sub.2/MeOH). This allowed the isolation of compound AL,
237 mg (14%).
Preparation of 3-[(benzylamino)thiocarbonyl]amino-1-propanesulfonic
acid, sodium salt (Compound AM)
##STR00546##
[0570] 3-amino-1-propanesulfonic acid (1.07 g, 7.7 mmol) was
dissolved in 1.5N NaOH (5.3 mL). To this solution was added benzyl
isothiocyanate (1.02 mL, 7.7 mmol). The reaction mixture was
stirred at 70.degree. C. for 0.5 h; a second-equivalent of benzyl
isocyanate (1.02 mL, 7.7 mmol) was added. The reaction mixture was
stirred for 1 h. The solvent was evaporated under reduced pressure.
The residue was suspended in hot acetone. The solid material was
collected by filtration, washed with hot acetone, and dried in
vacuo. The residual material was recrystallized from MeOH (traces
of water), affording compound AM, 1.00 g (42%).
Preparation of 3-(3,4-dihydroxybenzyl)amino-1-propanesulfonic acid
(Compound S)
##STR00547##
[0572] To a solution of 3,4-dimethoxybenzylamine (2.2 mL, 15.0
mmol) in 2-butanone (20 mL) was added 1,3-propane sultone (1.98 g,
15.8 mmol). The mixture was stirred at reflux for 2 hours. The
reaction mixture was cooled to room temperature. The solid was
collected by filtration, washed with acetone (2.times.25 mL) and
dried in vacuo. The crude product was suspended in 75% Acetone/MeOH
(75 mL). The suspension was stirred at reflux for 30 sec.; the
solid material was collected by filtration, washed with acetone
(2.times.25 mL), and dried in vacuo. The solid (1.94 g, 6.7 mmol)
was dissolved hydrobromic acid (48%, 27 mL). The solution was
stirred at 100.degree. C. for 4 h. The solvent was removed under
reduced pressure. The residue was dissolved in water (20 mL). The
aqueous phase was washed with CH.sub.2Cl.sub.2 (3.times.20 mL), and
evaporated under educed pressure. The solid residue was suspended
in hot MeOH (75 mL). The suspension was stirred at reflux for 30
sec.; the solid was collected by filtration, washed with 50%
MeOH/acetone, and dried in vacuo. This allowed the isolation of
compound S, 1.22 g (70%).
Preparation of 4-(3-phenylpropyl)-1-sulfopropylpyridinium
hydroxide, inner salt (Compound C)
##STR00548##
[0574] To a solution of 4-(3-phenylpropyl)pyridine-(14.5 mL, 76
mmol) in 2-butanone (150 mL) was added 1,3-propane sultone (10.0 g,
83.6 mmol). The mixture was stirred at reflux for 1.5 h. Once the
reaction mixture was cooled to room temperature, the precipitate
was collected by filtration and washed with acetone. The solid
material was recrystallized from EtOH (traces of Et.sub.2O),
affording compound C, 15.8 g (66%).
Preparation of
4-(3-phenylpropyl)-1-sulfopropyl-1,2,3,6-tetrahydropyridine
(Compound D)
##STR00549##
[0576] 4-(3-phenylpropyl)-1-sulfopropylpyridine (10.7 g, 33.5 mmol)
was dissolved in 60 mL of MeOH. The solution was cooled to
0.degree. C. before sodium borohydride (2.55 g, 67.0 mmol) was
added portionwise. The reaction mixture was stirred for 0.5 hour at
room temperature. Water (10 mL) and concentrated HCl (5 mL) were
successively added to the mixture. The inorganic material was
removed by filtration. The filtrate was concentrated to dryness
under reduced pressure and dried in vacuo. The resultant viscous
residue was dissolved in MeOH (60 mL). The solution was stirred
with Amberlite IR-120 ion exchange resin (8.3 g) for 15 min. The
resin was removed by filtration and washed with MeOH. The filtrate
and washing was combined and concentrated to dryness under reduced
pressure. The residual material was recrystallized from water,
affording compound D (8.05 g, 75%) as white crystals.
Preparation of 3-ethylamino-1-propanesulfonic acid (Compound
CV)
##STR00550##
[0578] Tetrahydrofuran (THF, 800 mL) was placed a 3 neck 2-L flask
(equipped with a condenser) and cooled to 5.degree. C. with an
ice-bath. To the cold THF was added aqueous ethylamine (70 wt. %
solution in water, 85 mL, 1.07 mol), followed by addition of a cold
solution of 1,3-propane sultone (25.08 g, 201 mmol) in THF (100 mL)
over 24-min period. The mixture was stirred, while cooled with an
ice-bath, for 1 h. The ice-bath was removed and the mixture was
stirred at room temperature overnight. It was then heated under
reflux for 1 h to distill off the ethylamine. The hot mixture was
biphasic. Upon cooling, a solid crystallized at the bottom of the
flask. Ether (400 mL) was added and the mixture was cooled to
-20.degree. C. The supernatant was decanted. Methanol (about 120
mL) was added to the residue. The mixture was heated to reflux; and
complete dissolution of the solid material was achieved. After the
solution was cooled to room temperature, precipitates formed. The
mixture was cooled in an ice-bath; and the solid material was
collected by filtration, rinsed with cold methanol and dried in
vacuo (20.66 g, pure by NMR analysis). The solid material was
recrystallized from methanol (100 mL). After the mixture was cooled
using an ice-bath, the solid was collected by filtration, rinsed
with cold methanol, and dried in a vacuum oven at 40.degree. C.
Compound CV was obtained as white fine needles (19.12 g, 57%). The
.sup.1H and .sup.13C NMR were consistent with the structure.
Preparation of 3-(1-adamantyl)amino-1-propanesulfonic acid
(Compound BW)
##STR00551##
[0580] The 1-adamantanamine hydrochloride (80 g, 0.426 mol) was
treated with NaOH (10%, 400 mL) in water. The aqueous mixture was
extracted with dichloromethane (1.times.400 mL, 2.times.100 mL).
The combined organic layers were washed with brine (50 mL) and
dried over sodium sulfate (10 g). Solvent was removed under reduced
pressure. The resulting white waxy solid was co-evaporated with
acetonitrile (50 mL). The wet solid was suspended in acetonitrile
(200 mL). The suspension mixture was added dropwise over 20 min to
a solution of 1,3-propane sultone (53 g, 0.426 mol) in acetonitrile
(300 mL) and THF (200 mL). The thick mixture was stirred for 2
hours under reflux with a mechanical stirrer. The suspension was
then cooled to 13.degree. C. The solid was collected by
suction-filtration, rinsed with acetonitrile (2.times.100 mL) and
ether (1.times.100 mL), air-dried for 30 min, and further dried in
vacuo at 60.degree. C. overnight (104.17 g for crop 1). Another
crop was collected from the filtrate and dried in vacuo in the same
manner (3.39 g for crop 2). Both crops gave identical proton NMR
spectrum. The two batches were combined for further
purification.
[0581] The solid was suspended in methanol (720 mL) and the mixture
was heated to reflux. Water (490 mL) was added dropwise over 45
min, while maintaining reflux. After complete dissolution of the
solid, the solution was kept under reflux for 30 minute. The
mixture was left in the power-off heating mantle and allowed to
cool slowly. After 90 min, the temperature reached 40.degree. C.
The heating mantle was replaced by a thermostated water bath. The
mixture was cooled to 5.degree. C. and stirred overnight at this
temperature. The white flaky solid was collected by filtration,
rinsed with cold (0.degree. C.) methanol (2.times.125 mL),
air-dried for 60 minutes, and then dried in the vacuum oven at
60.degree. C. overnight. Compound BW was obtained as a white flaky
solid (white plates, 88.48 g, 0.76% yield for the first crop). The
.sup.1H NMR and MS were consistent with the structure. A second
crop (8.62 g) of compound BW was obtained from the mother liquid.
The .sup.1H NMR was identical to that of the first crop. This made
the reaction a total yield of 83% from the hydrochloride.
Preparation of 3-(2-norbornyl)amino-1-propanesulfonic acid
(Compound BY)
##STR00552##
[0583] To a solution of 2-aminonorbornane (7.3 g, 65.7 mmol) in
2-butanone (50 mL) was added dropwise a solution of 1,3-propane
sulfone (8.1 g, 65.7 mmol) in 2-butanone (10 mL). The mixture was
stirred at 60.degree. C. for 1 h. The suspension was cooled to room
temperature. The solid material was collected by filtration and
washed with ethanol (2.times.20 mL). The crude material was
recrystallized from 95% EtOH to afford compound BY as a white
crystalline solid (8.2 g, 53% yield).
Preparation of 3-(2-adamantyl)amino-1-propanesulfonic acid
(Compound BZ)
##STR00553##
[0585] The 2-aminoadamantane hydrochloride (2.times.5 g) was
treated with NaOH in water. The aqueous mixture was extracted with
dichloromethane. The organic layer was dried over magnesium
sulfate. The solvent was removed under reduced pressure. The
resulting white solid was dried 30 minutes at room temperature
under vacuum. A solution of 1,3-propane sultone (7.4 g, 60 mmol) in
THF was added to a solution of the free amine (7.98 g, 52 mmol) in
THF (70 mL, total). The mixture was heated under reflux for 4 h,
cooled into an ice bath. The solid was collected by filtration,
air-dried for 15 min., and further dried in vacuo (11.2 g).
Recrystallization was done with methanol/water (60 mL/35 mL). After
cooled in a fridge, the solid was collected by filtration, rinsed
with methanol, dried in the vacuum oven at 60.degree. C. overnight.
A white crystalline sandy solid (small plates, 10.45 g, 74% yield)
was obtained. The and .sup.13C NMR were consistent with the
structure of Compound BZ.
Preparation of 3-(3,4-dimethoxybenzyl)amino-1-propanesulfonic acid
(Compound S)
##STR00554##
[0587] To a solution of 3,4-dimethoxybenzylamine (2.2 mL, 15.0
mmol) in acetone (20 mL) was added 1,3-propane sultone (1.97 g,
15.8 mmol). The mixture was stirred at reflux for 2 hours. The
reaction mixture was cooled to room temperature. The solid material
was collected by filtration, washed with acetone (2.times.25 mL)
and dried in vacuo. The crude product was suspended in 90%
acetone/MeOH (75 mL). The suspension was stirred at reflux for 30
sec., the solid material was collected by filtration, and dried in
vacuo. Compound S (1.84 g, 43%) was isolated as a white solid.
.sup.1H NMR (D.sub.2O, 500 MHz) .delta. ppm 6.96 (m, 3H), 4.06 (s,
2H), 3.74 (s, 6H), 3.07 (t, 1H, J=7.8 Hz), 2.86 (t, 1H, J=7.8 Hz),
2.01 (m, 2H). .sup.13C NMR (D.sub.2O, 125 MHz) .delta. ppm 149.23,
148.50, 123.63, 123.37, 55.90, 55.86, 50.96, 48.06, 45.62, 21.39.
ES-MS 290 (M+1).
Preparation of 3-(2,2-diphenylethyl)amino-1-propanesulfonic acid
(Compound ET)
##STR00555##
[0589] To a solution of 1,2-diphenylethylamine (2.49 g, 12.7 mmol)
in 2-butanone (15 mL) was added 1,3-propane sultone (1.67 g, 13.3
mmol). The mixture was stirred at reflux for 2 hours. The reaction
mixture was cooled to room temperature. The solid material was
collected by filtration, washed with acetone (2.times.25 mL), and
dried in vacuo, to give compound ET: 3.02 g (74%). .sup.1H NMR
(DMSO, 500 MHz) .delta. ppm 8.58 (s (broad), 1H), 7.34 (m, 8H),
7.23 (t, 2H, J=7.3 Hz), 4.32 (t, 1H, J=7.8 Hz), 3.68 (d, 2H, J=7.3
Hz), 3.08 (t, 2H, J=6.1 Hz), 2.57 (t, 2H, J=7.3 Hz), 1.92 (m, 2H).
.sup.13C NMR (DMSO, 125 MHz) .delta. ppm 141.63, 129.46, 128.47,
127.76, 50.85, 49.91, 48.53, 22.07. ES-MS 318 (M-1).
Preparation of 4-(tert-butylamino)-2-butanesulfonic acid (Compound
ES)
##STR00556##
[0591] To a solution of tert-butylamine (1.0 mL, 9.5 mmol) in
tetrahydrofuran (15 mL) was added 2,4-butane sultone (1.33 g, 10.0
mmol). The mixture was stirred at reflux for 2 hours. The reaction
mixture was cooled to room temperature. The solid product was
collected by filtration, washed with THF (2.times.20 mL), and dried
in vacuo; affording compound ES: .sup.1H NMR (DMSO, 500 MHz)
.delta. ppm 2.97 (t, 2H, J=6.6 Hz), 2.62 (m, 1H), 1.95 (m, 0.5H),
1.750 (m, 0.5H), 1.22 (s, 9H), 1.12 (d, 3H, J=6.8 Hz). .sup.13C NMR
(DMSO, 125 MHz) .delta. ppm 56.17, 53.15, 29.87, 25.87, 17.05.
ES-MS 207 (M-1).
Preparation of 4-(tert-butylamino)-1-butanesulfonic acid (Compound
ER)
##STR00557##
[0593] To a solution of tert-butylamine (1.0 mL, 9.5 mmol) in
tetrahydrofuran (4 mL) was added 1,4-butane sultone (1.36 g, 10.0
mmol) at room temperature. The solution was stirred at reflux for 2
hours. The reaction mixture was cooled to room temperature. The
solid product was collected by filtration, washed with acetone
(2.times.20 mL), and dried in vacuo; affording compound ER 690 mg;
(34%); .sup.1H NMR (D.sub.2O, 500 MHz) .delta. ppm 2.92 (t, 2H,
J=7.1 Hz), 2.82 (t, 2H, J=7.1 Hz), 1.68 (m, 4H), 1.22 (s, 9H).
.sup.13C NMR (D.sub.2O, 125 MHz) .delta. ppm 57.07, 50.30, 40.95,
25.28, 24.96, 21.62. ES-MS 210 (M-1).
Preparation of 3-(3-pentyl)amino-1-propanesulfonic acid (Compound
DD)
##STR00558##
[0595] To a solution of 1-ethylpropylamine (10.0 g, 115 mmol) in
tetrahydrofuran (80 mL) was added a solution of 1,3-propane sultone
(13.7 g, 110 mmol) in 20 mL of THF. The solution was stirred at
reflux for 2 hours. The reaction mixture was cooled to room
temperature. The solid product was collected by filtration, washed
with acetone (2.times.50 mL), and dried in vacuo., to afford
compound DD (18.1, 80%): .sup.1H NMR (D.sub.2O, 500 MHz) .delta.
ppm 3.08 (t, 2H, J=7.3 Hz), 3.01 (m, 1H), 2.87 (t, 2H, J=7.3 Hz)
2.00 (m, 2H), 1.59 (m, 4H), 0.82 (t, 6H, J=7.3 Hz). .sup.13C NMR
(D.sub.2O, 125 MHz) .delta. ppm 60.87, 48.14, 43.68, 21.81, 21.60,
8.25. ES-MS 208 (M-1).
Preparation of 3-(tert-amyl)amino-1-propanesulfonic acid (Compound
DG)
##STR00559##
[0597] To a solution of tert-amylamine (2.0 g, 23.3 mmol) in
tetrahydrofuran (15 mL) was added 1,3-propane sultone (2.76 g, 22.2
mmol). The solution was stirred at reflux for 2 hours. The reaction
mixture was cooled to room temperature. The solid product was
collected by filtration, washed with acetone (2.times.25 mL), and
dried in vacuo, to afford compound DG (3.3 g, 73%): .sup.1H NMR
(D.sub.2O, 500 MHz) .delta. ppm 3.04 (t, 2H, J=7.8 Hz), 2.89 (t,
2H, J=7.8 Hz), 2.87 (t, 2H, J=7.3 Hz), 1.97 (m, 2H), 1.55 (m, 2H),
1.18 (s, 6H), 0.82 (t, 6H, J=7.3 Hz). .sup.13C NMR (D.sub.2O, 125
MHz) .delta. ppm 60.42, 48.17, 39.88, 30.81, 22.23, 21.98, 7.25
ES-MS 208 (M-1).
Preparation of
3-(1,1-dimethyl-2-hydroxyethyl)amino-1-propanesulfonic acid
(Compound DH)
##STR00560##
[0599] To a solution of 2-amino-2-methyl-1-propanol (2.0 g, 21.4
mmol) in tetrahydrofuran (15 mL) was added 1,3-propane sultone
(2.66 g, 21.4 mmol). The mixture was stirred at reflux for 2 hours.
The reaction mixture was cooled to room temperature. The crude
product was collected by filtration, washed with acetone
(2.times.25 mL). The solid was suspended in EtOH (50 mL). The
suspension was stirred at reflux for 5 minutes. The solid was
collected by filtration and dried in a vacuum oven (50.degree. C.),
affording compound DH (2.5 g, 58%): .sup.1H NMR (D.sub.2O, 500 MHz)
.delta. ppm 3.48 (s, 2H), 3.04 (t, 2H, J=7.8 Hz), 2.90 (t, 2H,
J=7.3 Hz), 2.00 (m, 2H), 1.18 (s, 6H). .sup.13C NMR (D.sub.2O, 125
MHz) .delta. ppm 64.88, 60.27, 48.19, 40.10, 21.92, 20.02. ES-MS
210 (M-1).
Preparation of 3-(1-carboxy-1-methylethylamino)-1-propanesulfonic
acid (Compound DI)
##STR00561##
[0601] To a cold (5.degree. C.) mixture of 2-aminoisobutyric acid
(2.0 g, 19.4 mmol), NaOH (776 mg, 19.4 mmol) in 1,4-dioxane (10 mL)
and water (4 mL) was added via syringe pump (over a 4-hour period),
a solution 1,3-propane sultone (2.02 g, 16.2 mmol) in 1,4-dioxane
(total: 4 mL). The solution was stirred at room temperature for 2
hours before it was allowed to warm up to room temperature. The
reaction mixture was stirred under these conditions overnight. The
solvent was evaporated under reduced pressure. The resultant solid
material was recrystallized from 5% water/EtOH. The resulting solid
was dissolved in water; and the aqueous solution was passed through
an ion exchange column (Dowex 50WX 8, 100 g, solvent: water). The
solvent was evaporated under reduced pressure. The product was
lyophilized to afford Compound DI (880 mg, 28%). .sup.1H NMR
(D.sub.2O, 500 MHz) .delta. ppm 3.00 (t, 2H, J=7.6 Hz), 2.91 (t,
2H, J=7.3 Hz), 2.01 (m, 2H), 1.34 (s, 6H). .sup.13C NMR (D.sub.2O,
125 MHz) .delta. ppm 176.93, 63.89, 48.13, 42.04, 22.15, 21.86.
ES-MS 224 (M-1).
Preparation of
3-[(1R,2S)-2-methylcyclohexyl]amino-1-propanesulfonic acid
(Compound DJ)
##STR00562##
[0603] To a solution of 2-methylcyclohexylamine (98% cis and trans
isomers, 10.0 g, 88.3 mmol) in tetrahydrofuran (60 mL) was slowly
added a solution of 1,3-propane sultone (10.5 g, 84.1 mmol) in THF
(20 mL). The mixture was stirred at reflux for 2 hours. The
reaction mixture was cooled to room temperature. The solid material
was collected by filtration, washed with acetone (2.times.50 mL).
The solid was dissolved in 50% EtOH/water (200 mL); and solution
was treated with Dowex 50WX8 resin (15 g). The suspension stirred
at room temperature for 15 min. The resin was removed by
filtration. The filtrate was concentrated to half of the original
volume on a rotary evaporator. The solid product slowly
crystallized. The product was collected by filtration, washed with
acetone (2.times.50 mL) and dried in a vacuum oven (50.degree. C.),
to afford compound DJ (10.4 g, 53%): NMR (D.sub.2O, 500 MHz)
.delta. ppm 3.15 (m, 1H), 3.03 (m, 1H), 2.88 (t, 2H, J=7.3 Hz),
2.76 (m, 1H), 1.98 (m, 3H), 1.68 (m, 2H), 1.51 (m, 2H), 1.18 (m,
3H), 1.01 (m, 1H), 0.92 (m, 3H). .sup.13C NMR (D.sub.2O, 125 MHz)
.delta. ppm 62.97, 48.16, 42.72, 34.77, 33.50, 27.57, 24.51, 24.23,
21.51, 17.80. ES-MS 234 (M-1).
Preparation of 3-(2,3-dimethylcyclohexyl)amino-1-propanesulfonic
acid (Compound DK)
##STR00563##
[0605] To a solution of 2,3-dimethylcyclohexylamine (10.0 g, 79.0
mmol) in tetrahydrofuran (60 mL) was slowly added a solution of
1,3-propane sultone (9.3 g, 75.0 mmol) in THF (20 mL). The solution
was stirred at reflux for 2 hours. The reaction mixture was cooled
to room temperature. The solid was collected by filtration, washed
with THF (50 mL) and acetone (50 mL). The solid was dissolved in
25% EtOH/water (150 mL), and treated with Dowex 50WX8 resin (15 g).
The suspension stirred at room temperature for 5 minutes. The resin
was removed by filtration. The filtrate was concentrated to dryness
under reduced pressure; and the solid residue was suspended in
acetone (100 mL). The solid material was collected by filtration,
and dried in vacuo; affording compound DK (7.4 g, 43%). .sup.1H NMR
(D.sub.2O, 500 MHz) .delta. ppm 3.29 (m, 0.5H), 3.09 (m, 2H), 2.88
(t, 2H, J=7.3 Hz), 2.80 (m, 0.5H), 1.99 (m, 3H), 1.40 (m, 7H), 0.81
(m, 6H). .sup.13C NMR (D.sub.2O, 125 MHz) .delta. ppm 62.85, 61.56,
59.88, 58.22, 48.26, 48.15, 44.29, 43.84, 43.59, 42.65, 42.01,
41.03, 37.34, 36.12, 34.73, 34.45, 33.88, 33.64, 29.39, 28.00,
26.50, 24.25, 24.02, 23.64, 22.42, 21.55, 21.45, 21.35, 19.38,
19.13, 18.82, 18.40, 14.38, 13.39, 4.59. ES-MS 248 (M-1).
Preparation of 3-neopentylamino-1-propanesulfonic acid (Compound
DL)
##STR00564##
[0607] To a solution of neopentylamine (8.5 g, 98 mmol) in
tetrahydrofuran (75 mL) was slowly added a solution of 1,3-propane
sultone (11.5 g, 93 mmol) in THF (20 mL). The solution was stirred
at reflux for 2 hours. The reaction mixture was cooled to room
temperature. The solid was collected by filtration, washed with
acetone (2.times.50 mL). The solid was suspended in EtOH (150 mL).
The suspension was stirred at reflux for 15 minutes. The solid
product was collected by filtration, washed with acetone
(2.times.50 mL), and dried in vacuo, to afford compound DL (13.3 g,
69%). .sup.1H NMR (D.sub.2O, 500 MHz) .delta. ppm 3.09 (t, 2H,
J=7.3 Hz), 2.89 (t, 2H, J=7.3 Hz), 2.78 (s, 2H), 2.04 (m, 2H),
0.901 (s, 9H). .sup.13C NMR (D.sub.2O, 125 MHz) .delta. ppm 59.44,
48.31, 47.83, 29.91, 26.42, 21.06. ES-MS 208 (M-1).
Preparation of 3-cumylamino-1-propanesulfonic acid (Compound
DM)
##STR00565##
[0609] To a solution of cumylamine (10.5 g, 78 mmol) in
tetrahydrofuran (75 mL) was slowly added a solution of 1,3-propane
sultone (9.2 g, 74 mmol) in THF (20 mL). The mixture was stirred at
reflux for 4 h. The reaction mixture was cooled to room
temperature. The solid was collected by filtration, and washed with
THF (2.times.35 mL). The solid was suspended in EtOH (80 mL). The
suspension was stirred at reflux for 15 minutes. The solid product
was collected by filtration, washed with EtOH (35 mL) and acetone
(35 mL). The resulting solid was dried in vacuo, affording compound
DM (5.6 g, 30%). .sup.1H NMR (D.sub.2O, 500 MHz) .delta. ppm 7.41
(m, 5H), 2.74 (m, 4H), 1.88 (m, 2H), 1.66 (s, 6H). .sup.13C NMR
(D.sub.2O, 125 MHz) .delta. ppm 138.36, 129.44, 129.41, 126.52,
61.56, 48.04, 41.21, 24.80, 21.81. ES-MS 256 (M-1).
Preparation of
3-[(1R)-1-(4-methylphenyl)ethyl]amino-1-propanesulfonic acid
(Compound FN)
##STR00566##
[0611] To a solution of (R)-(+)-1-(4-methoxyphenyl)ethylamine (5.83
g, 38.6 mmol) in tetrahydrofuran (25 mL) was slowly added
1,3-propanesultone (4.56 g, 36.8 mmol). The solution was stirred at
reflux for 4 hours. The reaction mixture was cooled to room
temperature. The solid was collected by filtration, washed with THF
(25 mL) and acetone (25 mL). The solid was suspended in EtOH (200
mL). The suspension was stirred at reflux for 15 min. The solid was
collected by filtration, washed with cold EtOH (50 mL), and dried
in a vacuum oven (50.degree. C.), to afford compound FN (5.6 g,
56%). .sup.1H NMR (D.sub.2O, 500 MHz) .delta. ppm 7.26 (d, 2H,
J=8.3 Hz), 6.90 (d, 2H, J=8.3 Hz), 4.22 (m, 1H), 3.68 (s, 3H), 2.92
(m, 1H), 2.76 (m, 3H), 1.90 (m, 2H), 1.49 (d, 3H, J=6.8 Hz).
.sup.13C NMR (D.sub.2O, 125 MHz) .delta. ppm 159.87, 129.34,
128.18, 114.85, 57.99, 55.58, 48.05, 44.21, 21.45, 18.21. ES-MS 272
(M-1).
Preparation of 3-[(1R)-1-indanamino]-1-propanesulfonic acid
(Compound DO)
##STR00567##
[0613] To a solution of (R)-(-)-1-aminoindan (1.0 g, 7.5 mmol) in
tetrahydrofuran (10 mL) was slowly added 1,3-propane sultone (890
mg, 7.1 mmol). The mixture was stirred at reflux for 2 hours. The
reaction mixture was cooled to room temperature. The solid material
was collected by filtration, and washed with THF (20 mL) and
acetone (20 mL). The solid was suspended in 80% acetone/EtOH (40
mL). The suspension was stirred at reflux for 30 sec. The solid
product was collected by filtration, washed with acetone
(2.times.20 mL), and dried in vacuo, to afford compound DO (1.1 g,
61%). .sup.1H NMR (D.sub.2O, 500 MHz) .delta. ppm 7.41 (d, 1H,
J=7.3 Hz), 7.30 (m, 2H), 7.24 (m, 1H), 4.72 (m, 1H), 3.14 (t, 2H,
J=7.8 Hz), 3.02 (m, 1H), 2.88 (m, 3H), 2.43 (m, 1H), 2.12 (m, 1H),
2.01 (m, 2H). .sup.13C NMR (D.sub.2O, 125 MHz) .delta. ppm 145.38,
136.39, 130.31, 127.20, 125.72, 125.54, 62.98, 48.10, 43.93, 29.84,
28.62, 21.64. [.alpha.].sub.D=-1.3.degree. (c=0.00515 in water).
ES-MS 254 (M-1).
Preparation of 3-(N-tert-butylcarbamyl)amino-1-propanesulfonic
acid, sodium salt (Sodium Salt of Compound DP)
##STR00568##
[0615] 3-amino-1-propanesulfonic acid (2.0 g, 14.3 mmol) was
dissolved in 1.6M NaOH (10 mL). To this solution was added
tert-butyl isocyanate (1.1 g, 14.3 mmol). The reaction mixture was
stirred at 70.degree. C. for 1 h, followed by addition of one
equivalent of tert-butyl isocyanate (1.1 g, 14.3 mmol). The
reaction mixture was stirred for 1 h. The solvent was evaporated
under reduced pressure. The residue was suspended in EtOH (30 mL).
The solid product was collected by filtration, washed with EtOH (20
mL) and acetone (20 mL). The resulting solid was dried in vacuo, to
afford the sodium salt of compound DP (2.1 g, 66%). .sup.1H NMR
(D.sub.2O, 500 MHz) .delta. ppm 3.03 (t, 2H, J=6.6 Hz), 2.76 (t,
2H, J=7.6 Hz), 1.72 (m, 2H), 1.12 (s, 9H). .sup.13C NMR (D.sub.2O,
125 MHz) .delta. ppm 160.35, 50.26, 48.74, 38.31, 28.86, 25.24.
ES-MS 280 (M+Na).
Preparation of 3-(1,2-dimethyl-1-propyl)amino-1-propanesulfonic
acid (Compound DQ)
##STR00569##
[0617] To a solution of 1,2-dimethylpropylamine (10.0 g, 115 mmol)
in tetrahydrofuran (80 mL) was slowly added a solution of
1,3-propane sultone (13.7 g, 110 mmol) in THF (20 mL). The solution
was stirred at reflux for 2 hours. The reaction mixture was cooled
to room temperature. The solid product was collected by filtration,
washed with THF (50 mL) and EtOH (50 mL). The resulting solid was
dried in vacuo, to afford compound DQ (17.5 g, 76%). .sup.1H NMR
(D.sub.2O, 500 MHz) .delta. ppm 3.07 (m, 3H), 2.88 (t, 2H, J=7.3
Hz), 1.97 (m, 3H), 1.10 (d, 3H, J=6.8 Hz), 0.85 (d, 3H, J=6.8 Hz),
0.81 (d, 3H, J=6.8 Hz). .sup.13C NMR (D.sub.2O, 125 MHz) .delta.
ppm 59.79, 48.19, 44.18, 29.72, 21.51, 18.44, 15.02, 10.71. ES-MS
232 (M+Na).
Preparation of 3-(4-methylcyclohexyl)amino-1-propanesulfonic acid
(Compound DR)
##STR00570##
[0619] To a solution of 4-methylcyclohexylamine (97% cis and trans
isomers, 11.0 g, 97.4 mmol) in tetrahydrofuran (70 mL) was slowly
added a solution of 1,3-propane sultone (11.5 g, 92.8 mmol) in THF
(20 mL). The mixture was stirred at reflux for 2 hours. The
reaction mixture was cooled to room temperature. The solid material
was collected by filtration, washed with THF (50 mL) and acetone
(50 mL). The solid was suspended EtOH. The suspension was stirred
at room temperature for 5 minutes. The solid product was collected
by filtration, washed with EtOH (50 mL), and dried in a vacuum oven
(50.degree. C.), to afford compound DR (16.1 g, 75%). .sup.1H NMR
(D.sub.2O, 500 MHz) .delta. ppm 3.08 (m, 2.5H), 2.93 (m, 0.5H),
2.87 (m, 2H), 1.99 (m, 4H), 1.64 (m, 3H), 1.47 (m, 1H), 1.24 (m,
2H), 0.88 (m, 1H), 0.78 (m, 3H). .sup.13C NMR (D.sub.2O, 125 MHz)
.delta. ppm 57.35, 56.52, 48.16, 18.05, 43.73, 43.30, 32.45, 31.13,
28.92, 28.69, 27.77, 24.55, 21.65, 21.53, 21.20, 18.38. ES-MS 236
(M+1).
Preparation of 3-(2-methyl-1-butyl)amino-1-propanesulfonic acid
(Compound DS)
##STR00571##
[0621] To a solution of (+/-)-2-methylbutylamine (10 g, 115 mmol)
in tetrahydrofuran (80 mL) was slowly added a solution of
1,3-propane sultone (13.5 g, 109 mmol) in THF (20 mL). The mixture
was stirred at reflux for 2 hours. The reaction mixture was cooled
to room temperature. The solid material was collected by
filtration, washed with acetone (2.times.30 mL). The solid was
suspended 95% Acetone/EtOH (200 mL). The suspension was stirred at
room temperature for 5 minutes. The solid product was collected by
filtration, and dried in a vacuum oven (50.degree. C.), to afford
compound DS (17.6 g, 78%). .sup.1H NMR (D.sub.2O, 500 MHz) .delta.
ppm 3.07 (t, 2H, J=7.8 Hz), 2.93 (m, 3H), 2.76 (m, 1H), 2.01 (m,
2H), 1.67 (m, 1H), 1.30 (m, 1H), 1.09 (m, 1H), 0.81 (d, 3H, J=6.8
Hz), 0.77 (t, 3H, J=6.8 Hz). .sup.13C NMR (D.sub.2O, 125 MHz)
.delta. ppm 53.48, 48.13, 46.92, 31.96, 26.38, 21.29, 16.11, 10.19.
ES-MS 210 (M+1).
Preparation of 3-pivaloylamino-1-propanesulfonic acid (Compound
DT)
##STR00572##
[0623] 3-amino-1-propanesulfonic acid (2.0 g, 14.4 mmol) was
dissolved a NaOH (1.2 g, 30.2 mmol) solution in a mixture of
1,4-dioxane (5 mL) and water (15 mL). The mixture was cooled to
0.degree. C. before pivaloyl chloride (2.8 mL, 21.6 mmol) in
1,4-dioxane (5 mL) was added dropwise. The reaction mixture was
allowed to warm up to room temperature and it was stirred at
65.degree. C. for 4 h. The solvent was evaporated under reduced
pressure. The resulting solid was dissolved in water (30 mL), and
treated with Dowex 50WX8 resin. The suspension was stirred for 5
minutes and the resin was removed by filtration. The filtrate was
evaporated under reduced pressure. The residual material was
suspended in 20% EtOH/Acetone. The mixture was stirred at reflux
for 30 seconds. The solid product was collected by filtration, and
dried in vacuo, to afford compound DT (1.3 g, 41%). NMR (D.sub.2O,
500 MHz) .delta. ppm 3.16 (t, 2H, J=6.8 Hz), 2.75 (t, 2H, J=7.8
Hz), 1.78 (m, 2H), 1.1 (s, 9H). .sup.13C NMR (D.sub.2O, 125 MHz)
.delta. ppm 182.75, 48.70, 38.57, 38.18, 26.65, 24.27. ES-MS 222
(M-1).
Preparation of
3-(3,3,5-trimethylcyclohexyl)amino]-1-propanesulfonic acid
(Compound ED)
##STR00573##
[0625] To a solution of 3,3,5-trimethylcyclohexylamine (5.0 g, 35.4
mmol) in tetrahydrofuran (35 mL) was slowly added a solution of
1,3-propane sultone (4.17 g, 33.7 mmol) in THF. The mixture was
stirred at reflux for 2 hours. The reaction mixture was cooled to
room temperature. The solid material was collected by filtration,
washed with acetone (2.times.25 mL). The solid was suspended 90%
Acetone/EtOH (100 mL) The suspension was stirred at room
temperature for 5 min. The solid product was collected by
filtration, and dried in a vacuum oven (50.degree. C.), affording
compound ED (5.9 g, 67%). .sup.1H NMR (D.sub.2O, 500 MHz) .delta.
ppm 3.20 (m, 1H), 3.08 (m, 2H), 2.87 (t, 2H, J=6.8 Hz), 1.96 (m,
3H), 1.60 (m, 2H), 1.29 (m, 1H), 1.01 (m, 1H), 0.84 (s, 3H), 0.73
(m, 8H). .sup.13C NMR (D.sub.2O, 125 MHz) .delta. ppm 54.98, 48.06,
46.51, 43.28, 40.96, 37.02, 32.07, 31.23, 26.68, 24.28, 21.67,
21.52. ES-MS 262 (M-1).
Preparation of 3-(2-indanamino)-1-propanesulfonic acid (Compound
EE)
##STR00574##
[0627] To a solution of 2-aminoindan (2.50 g, 18.8 mmol) in
tetrahydrofuran (25 mL) was slowly added 1,3-propane sultone (2.24
g, 17.9 mmol). The mixture was stirred at reflux for 2.5 h. The
reaction mixture was cooled to room temperature. The solid material
was collected by filtration, washed with acetone (2.times.25 mL).
The crude product was suspended in 90% Acetone/EtOH (100 mL). The
suspension was stirred at room temperature for 5 minutes. The solid
product was collected by filtration, and dried in a vacuum oven
(50.degree. C.), to afford compound EE (3.1 g, 67%). .sup.1H NMR
(DMSO, 500 MHz) .delta. ppm 7.25 (m, 2H), 7.20 (m, 2H), 4.00 (m,
1H), 3.30 (m, 2H), 3.13 (m, 2H), 3.02 (m, 2H), 2.64 (m, 2H), 1.96
(m, 2H). .sup.13C NMR (DMSO, 125 MHz) .delta. ppm 130.98, 127.80,
125.25, 57.70, 49.24, 45.87, 36.32, 22.66. ES-MS 254 (M-1).
Preparation of 3-(4-biphenylamino)-1-propanesulfonic acid (Compound
EF)
##STR00575##
[0629] To a solution of 4-aminobiphenyl (3.0 g, 17.8 mmol) in
tetrahydrofuran (25 mL) was slowly added 1,3-propane sultone (2.11
g, 16.9 mmol). The solution was stirred at reflux for 3 h. The
reaction mixture was cooled to room temperature. The solid material
was collected by filtration, washed with acetone (2.times.25 mL).
The crude product was dissolved in a hot solution of 80%
MeOH/H.sub.2O (120 mL). To this warm solution was added Dowex 50WX8
ion exchange resin (10 g). The hot suspension was stirred for 5
minutes and the resin was removed by filtration. The filtrate was
concentrated to dryness under reduced pressure. The residual solid
was dried in a vacuum oven (50.degree. C.), affording compound EF
(283 mg, 6%). .sup.1H NMR (DMSO, 500 MHz) .delta. ppm 7.77 (d, 2H,
J=7.8 Hz), 7.66 (d, 2H, J=7.8 Hz), 7.46 (m, 3H), 7.37 (m, 1H), 3.44
(m, 2H), 2.68 (m, 2H), 1.98 (m, 2H). .sup.13C NMR (DMSO, 125 MHz)
.delta. ppm 139.74, 129.69, 128.74, 128.33, 127.31, 122.23, 49.70,
22.93. ES-MS 290 (M-1).
Preparation of
3-[(1R,2S)-2-hydroxy-1-(methoxymethyl)-2-phenylethyl]amino-1-propanesulfo-
nic acid (Compound EG)
##STR00576##
[0631] To a solution of
(1S,2S)-2-amino-3-methoxy-1-phenyl-1-propanol (1.0 g, 5.5 mmol) in
tetrahydrofuran (10 mL) was slowly added 1,3-propane sultone (662
mg, 5.3 mmol). The mixture was stirred at reflux for 2.5 h. The
reaction mixture was cooled to room temperature. The solid material
was collected by filtration, washed with acetone (2.times.25 mL).
The crude product was suspended 80% Acetone/EtOH. The suspension
was stirred at reflux for 30 seconds. The solid product was
collected by filtration and dried in a vacuum oven (50.degree. C.),
to afford compound EG (1.0 g, 63%). .sup.1H NMR (D.sub.2O, 500 MHz)
.delta. ppm 7.32 (m, 5H), 4.77 (d, 1H, J=9.8 Hz), 3.53 (m, 1H),
3.37 (m, 1H), 3.26 (m, 1H), 3.17 (m, 6H), 2.91 (t, 2H, J=7.3 Hz),
2.07 (m, 2H). .sup.13C NMR (D.sub.2O, 125 MHz) .delta. ppm 139.09,
129.33, 129.27, 127.11, 70.85, 66.29, 62.62, 58.76, 48.14, 44.24,
21.47. [.alpha.].sub.D=+42.6.degree. (c=0.00091 in water), ES-MS
302 (M-1).
Preparation of
3-[(1R,2R,3R,5S)-1,2,6,6-tetramethylbicyclo[3.1.1]hept-3-yl]amino-1-propa-
nesulfonic acid (Compound EH)
##STR00577##
[0633] To a solution of (1R,2R,3R,5S)-(-)-isopinocampheylamine (2.0
g, 13.0 mmol) in tetrahydrofuran (20 mL) was slowly added
1,3-propanesultone (1.56 g, 12.5 mmol). The mixture was stirred at
reflux for 2 hours. The reaction mixture was cooled to room
temperature. The solid product was collected by filtration, washed
with acetone (2.times.25 mL), and dried in a vacuum oven
(50.degree. C.), to afford compound EH (2.7 g, 80%). .sup.1H NMR
(DMSO, 500 MHz) .delta. ppm 3.32 (m, 2H), 3.09 (d, 2H), 2.67 (m,
2H), 2.30 (m, 2H), 1.96 (m, 4H), 1.75 (m, 2H), 1.18 (s, 3H), 1.11
(m, 4H), 0.90 (s, 3H). .sup.13C NMR (DMSO, 125 MHz) .delta. ppm
55.97, 50.11, 47.55, 45.86, 41.15, 40.91, 38.94, 32.81, 31.61,
27.96, 23.85, 22.59, 21.21, [.alpha.].sub.D=-17.2.degree.
(c=0.00083 in water), ES-MS 274 (M-1).
Preparation of 3-(2-methoxy-1-methylethyl)amino-1-propanesulfonic
acid (Compound EI)
##STR00578##
[0635] To a solution of 2-amino-1-methoxypropane (5.0 g, 17.8 mmol)
in tetrahydrofuran (25 mL) was slowly added 1,3-propane sultone
(2.12 g, 17.0 mmol). The mixture was stirred at reflux for 4 h. The
reaction mixture was cooled to room temperature. The solid product
was collected by filtration, washed with acetone (2.times.25 mL),
and dried in a vacuum oven (50.degree. C.), to afford compound EI
(3.1 g, 86%). NMR (D.sub.2O, 500 MHz) .delta. ppm 3.53 (m, 1H),
3.41 (m, 2H), 3.27 (s, 3H), 3.11 (m, 2H), 2.89 (t, 2H, J=7.3 Hz),
2.00 (m, 2H), 1.18 (d, 3H, J=5.9 Hz). .sup.13C NMR (D.sub.2O, 125
MHz) .delta. ppm 71.73, 58.80, 53.79, 48.08, 43.55, 21.52, 12.79.
ES-MS 210 (M-1).
Preparation of
3-[(1R)-2-benzyl-1-hydroxyethyl]amino-1-propanesulfonic acid
(Compound EJ)
##STR00579##
[0637] To a solution of (R)-(+)-2-amino-3-phenyl-1-propanol (1.0 g,
6.6 mmol) in tetrahydrofuran (10 mL) was slowly added 1,3-propane
sultone (785 mg, 6.3 mmol). The mixture was stirred at reflux for 2
hours. The reaction mixture was cooled to room temperature. The
solid material was collected by filtration, washed with acetone
(2.times.25 mL). The crude product was suspended 80% acetone/EtOH
(100 mL). The suspension was stirred at reflux for 30 seconds. The
solid product was collected by filtration, washed with acetone
(2.times.25 mL), and dried in a vacuum oven (50.degree. C.), to
afford compound EJ (890 mg, 52%). .sup.1H NMR (DMSO, 500 MHz)
.delta. ppm 8.58 (s (broad), 1H), 7.26 (m, 5H), 5.30 (s (broad),
1H), 3.53 (m, 1H), 3.31 (m, 2H, 3.14 (t, 2H), 2.98 (m, 1H), 2.80
(m, 1H), 2.62 (t, 2H), 1.98 (m, 2H). .sup.13C NMR (DMSO, 125 MHz)
.delta. ppm 137.31, 130.00, 129.26, 127.49, 60.16, 57.78, 49.90,
45.34, 33.78, 22.49. [.alpha.].sub.D=+9.7.degree. (c=0.00118 in
water). ES-MS 272 (M-1).
Preparation of
3-[(1S)-2-benzyl-1-hydroxyethyl]amino-1-propanesulfonic acid
(Compound EK)
##STR00580##
[0639] To a solution of (S)-(-)-2-amino-3-phenyl-1-propanol (2.0 g,
13.2 mmol) in tetrahydrofuran (20 mL) was slowly added 1,3-propane
sultone (1.57, 12.6 mmol). The mixture was stirred at reflux for 2
hours. The reaction mixture was cooled to room temperature. The
solid material was collected by filtration, washed with acetone
(2.times.25 mL). The crude product was suspended 80% Acetone/EtOH.
The suspension was stirred at reflux for 30 seconds. The solid
product was collected by filtration, and dried in a vacuum oven
(50.degree. C.), to afford compound EK (1.9 g, 56%). .sup.1H NMR
(DMSO, 500 MHz) .delta. ppm 8.63 (s (broad), 1H), 7.27 (m, 5H),
5.31 (s (broad), 1H), 3.53 (m, 1H), 3.25 (m, 2H), 3.15 (t, 2H),
2.98 (m, 1H), 2.80 (m, 1H), 2.61 (t, 2H), 1.99 (m, 2H). .sup.13C
NMR (DMSO, 125 MHz) .delta. ppm 137.31, 130.01, 129.27, 127.49,
60.18, 57.77, 49.88, 45.32, 33.78, 22.49.
[.alpha.].sub.o=-7.5.degree. (c=0.00118, H.sub.2O). ES-MS 272
(M-1).
Preparation of 3-(N-methyl-N-tert-butylamino)-1-propanesulfonic
acid (Compound EN)
##STR00581##
[0641] To a solution of N-methyl-tert-butylamine (2.0 g, 22.9 mmol)
in acetone (25 mL) was slowly added 1,3-propane sultone (2.72 g,
21.8 mmol). The mixture was stirred at reflux for 3 h. The reaction
mixture was cooled to room temperature. The solid material was
collected by filtration, washed with acetone (2.times.25 mL). The
crude product was suspended in 80% Acetone/EtOH. The suspension was
stirred at reflux for 30 seconds. The solid product was collected
by filtration, and dried in a vacuum oven (50.degree. C.), to
afford compound EN (2.9 g, 65%). NMR (DMSO, 500 MHz) .delta. ppm
9.40 (s (broad), 1H), 3.45 (m, 1H), 2.85 (m, 1H), 2.59 (m, 5H),
1.99 (m, 2H), 1.28 (s, 9H). .sup.13C NMR (DMSO, 125 MHz) .delta.
ppm 63.23, 51.12, 49.73, 34.79, 25.50, 21.72. ES-MS 208 (M-1).
Preparation of 3-[(1R,2S)-2-hydroxyindan-1-amino]-1-propanesulfonic
acid (Compound EO)
##STR00582##
[0643] To a solution of (1R,2S)-1-amino-2-indanol (2.37 g, 15.9
mmol) in tetrahydrofuran (25 mL) was slowly added 1,3-propane
sultone (1.89 g, 15.1 mmol). The mixture was stirred at reflux for
2 hours. The reaction mixture was cooled to room temperature. The
solid material was collected by filtration, and washed with acetone
(2.times.25 mL). The crude product was suspended in 80%
acetone/ethanol (75 mL). The suspension was stirred at reflux for
30 seconds. The solid product was collected by filtration, and
dried in a vacuum oven (50.degree. C.), to afford the compound EO
(2.7 g, 65%). .sup.1H NMR (D.sub.2O, 500 MHz) .delta. ppm 7.36 (d,
1H, J=7.4 Hz), 7.24 (m, 3H), 4.70 (q, 1H, J=5.5 Hz), 4.53 (d, 1H,
J=5.4 Hz), 3.23 (t, 2H, J=7.8 Hz), 3.10 (m, 1H), 2.89 (m, 3H), (m,
1H), 2.08 (m, 2H). .sup.13C NMR (D.sub.2O, 125 MHz) .delta. ppm
141.60, 134.30, 130.53, 127.61, 126.10, 125.69, 70.42, 64.08,
48.25, 44.73, 38.33, 21.50. [.alpha.].sub.D=+3.0.degree. (c=0.0018,
water) ES-MS 272 (M+1).
Preparation of
3-[(1S)-1-(hydroxymethyl)-2-methylpropyl]amino-1-propanesulfonic
acid (Compound EP)
##STR00583##
[0645] To a solution of (S)-(-)-2-amino-3-methyl-1-butanol (2.50 g,
24.2 mmol) in tetrahydrofuran (35 mL) was slowly added 1,3-propane
sultone (2.89 g, 23.0 mmol). The mixture was stirred at reflux for
3 h. The reaction mixture was cooled to room temperature. The solid
material was collected by filtration, washed with acetone
(2.times.25 mL). The crude product was suspended in 80%
acetone/ethanol (75 mL). The suspension was stirred at reflux for
30 seconds. The solid product was collected by filtration, and
dried in a vacuum oven (50.degree. C.), to afford compound EP (2.9
g, 56%). .sup.1H NMR (D.sub.2O, 500 MHz) .delta. ppm 3.78 (dd, 1H),
3.62 (dd, 1H), 3.13 (m, 2H), 2.90 (m, 1H), 2.88 (t, 3H), 1.90 (m,
3H), 0.90 (d, 3H), 0.84 (d, 3H). .sup.13C NMR (D.sub.2O, 125 MHz)
.delta. ppm 64.74, 57.24, 48.20, 44.44, 26.98, 21.49, 18.53, 17.00.
[.alpha.].sub.D=+4.1.degree. (c=0.0017 in water), ES-MS 224
(M-1).
Preparation of
3-[(1S)-1-carbamoyl-2-methylpropyl]amino-1-propanesulfonic acid
(Compound EQ)
##STR00584##
[0647] L-valinamide hydrochloride (2.50 g, 16.4 mmol) was treated
with a saturated solution of K.sub.2CO.sub.3 (75 mL). The mixture
was extracted with EtOAc (3.times.75 mL). The organic extracts were
combined, dried over Na.sub.2SO.sub.4. The solid material was
removed by filtration, and the filtrate was concentrated to dryness
under reduced pressure. The residual material was dried in
vacuo.
[0648] To a solution of L-valinamide (1.57 g, 13.5 mmol) in
tetrahydrofuran (20 mL) was slowly added 1,3-propane sultone (1.61
g, 12.9 mmol). The mixture was stirred at reflux for 2 hours. The
reaction mixture was cooled to room temperature. The solid material
was collected by filtration, washed with acetone (2.times.25 mL).
The crude product was dissolved in water (60 mL) and treated with
ion exchange resin Dowex Marathon C (strongly acidic, 15 g). The
mixture was stirred for 15 minutes. The resin was removed by
filtration. The filtrate was poured into EtOH (250 mL). The solid
product, after the completion of the precipitation, was collected
by filtration, and dried in vacuo, to afford compound EQ (1.6 g,
51%). .sup.1H NMR (D.sub.2O, 500 MHz) .delta. ppm 3.63 (d, 1H),
3.05 (m, 2H), 2.85 (m, 2H), 2.05 (m, 4H), 0.93 (d, 3H), 0.88 (d,
3H). .sup.13C NMR (D.sub.2O, 125 MHz) .delta. ppm 170.24, 65.92,
48.16, 46.31, 29.59, 21.31, 18.02, 17.07.
[.alpha.].sub.D=+10.5.degree. (c=0.0027 in water), ES-MS 237
(M-1).
Preparation of 3-isobutylamino-1-propanesulfonic acid (Compound
CE)
##STR00585##
[0650] Isobutylamine (2.4 mL, 24 mmol) was added to a solution of
1,3-propane sultone (3.04 g, 24.5 mmol) in 2-butanone (20 mL). The
mixture was heated to reflux. After about 10 minutes, the mixture
had turned into a lump. It was cooled to room temperature. Acetone
was added and the lump was crushed. The solid was collected by
filtration and dried in-vacuo (2.2 g). The white solid was
suspended in ethanol (10 mL) and the mixture brought to reflux. A
significant amount of the solid dissolved. Water was added slowly
until a clear pink solution was obtained. The solution was left at
room temperature overnight. The flask was placed in a fridge for 2
hours. The solid product was collected by filtration, rinsed with
ethanol (5 mL), washed with ether (10 mL), and dried in-vacuo.
Compound CE was obtained as long, fine white needles (1.87 g, 40%
yield). m.p. 255-57.degree. C. .sup.1H NMR (500 MHz, D.sub.2O)
.delta. 0.88 (d, J=6.8 Hz, 6H), 1.85-1.93 (m, J=6.8 Hz, 1H), 2.03
(qt, J=7.6 Hz, 2H), 2.80 (d, J=7.3 Hz, 2H), 2.90 (t, J=7.3 Hz, 2H),
3.08 (t, J=8.1 Hz, 2H). .sup.13C NMR (125 MHz, D.sub.2O) .delta.
19.2, 21.2, 25.8, 46.9, 48.1, 54.9 ES-MS 196 (M+1). FT-IR (KBr)
.nu..sub.max 3566, 2973, 2021, 1719.
Preparation of 3-isoamylamino-1-propanesulfonic acid (Compound
CH)
##STR00586##
[0652] Isoamylamine (4 mL, 34.5 mmol) was added to a solution of
1,3-propane sultone (4.7 g, 38 mmol) in 2-butanone (70 mL). The
mixture was warmed to reflux. After 30 minutes, the mixture was too
thick to stir. Acetone (15 mL) was added. The reflux was maintained
for a total of 4 hours. The suspension was cooled at room
temperature. The white solid was collected by filtration, rinsed
with acetone (10 mL), then with ether (10 mL). Compound CH was
obtained as a very light, fluffy white solid (4.48 g, 62% yield).
m.p. 220.degree. C.: decomposed. .sup.1H NMR (500 MHz, D.sub.2O)
.delta. 0.65 (d, J=6.3 Hz, 6H), 1.29 (q, J=7.7 Hz, 2H), 1.35-1.42
(m, J=6.6 Hz, 1H), 1.86 (qt, J=7.6 Hz, 2H), 2.74 (t, J=7.3 Hz, 2H),
2.81 (t, 8.1 Hz, 2H), 2.93 (t, J=7.8 Hz, 2H). .sup.13C NMR (125
MHz, D.sub.2O) .delta.21.3, 21.4, 25.2, 34.2, 46.1, 46.2, 47.9
Preparation of 2-(tert-butyl)amino-1-ethanesulfonic Acid (Compound
DU)
##STR00587##
[0654] A solution of 2-bromoethanesulfonic acid, sodium salt (4.2
g, 20 mmol) in water (total 12 mL) was added over 6 hours to a
42.degree. C. solution of t-butylamine (10 mL, 94 mmol) in a
mixture of water (10 mL) and 1,4-dioxane (10 mL). The mixture was
stirred at 42.degree. for 18 hours. The mixture was then heated to
60.degree. C. for 24 h. By proton NMR, 30% of elimination product
(vinylsulfonic acid) was observed. The mixture was concentrated to
dryness and treated with ethanol at refluxing temperature. The
solid material was collected (crop 1). The mother liquor was
concentrated to dryness and the solid was again treated with
ethanol at refluxing temperature, and the solid material was
collected (crop 2). Both crops of the solid material were dissolved
in water, and the resultant aqueous solutions passed in sequence
through a Dowex 50 W X 8 ion-exchange column (100 g resin). The
fractions containing the title compound were collected and
concentrated to dryness. The solid material obtained was
recrystallized from a mixture of ethanol (20 mL) and water (2 mL).
The crystals were collected by filtration, dried in a vacuum oven
at 60.degree. C. for 18 hours. Compound DU was obtained as fine
white needles (860 mg, 24% yield). .sup.1H NMR (500 MHz, D.sub.2O)
.delta. 1.16 (s, 9H), 3.02 (t, J=6.8 Hz, 2H), 3.19 (t, J=6.8 Hz,
2H). .sup.13C NMR (125 MHz, D.sub.2O) .delta. 24.8, 37.3, 47.0,
57.8. ES-MS 182 (M+1)
Preparation of 3-(cyclohexanemethyl)amino-1-propanesulfonic acid
(Compound DV)
##STR00588##
[0656] A mixture of cyclohexanemethylamine (11.12 mL, 0.085 mol)
and 1,3-propane sultone (11.00 g, 0.090 mol) in acetonitrile (120
mL) was heated at reflux for 2 hours. The mixture was cooled to
room temperature. The solid was collected by filtration, air-dried
for 20 minutes (19 g). The solid was suspended in methanol (100 mL)
and the suspension was heat to reflux. Water (4 mL) was added
dropwise until a clear solution was obtained at refluxing
temperature. The mixture was then cooled to 5.degree. C. with
stirring. The solid was collected by suction filtration, air-dried
for 45 minutes, and further dried in a vacuum oven at 60.degree. C.
for over 3 days. Compound DV was obtained as white flakes, (16.23
g, 81% yield.). .sup.1H NMR (500 MHz, D.sub.2O) .delta. 0.82 (br q,
J=11 Hz, 2H), 0.91-1.09 (m, 3H), 1.43-1.53 (m, 6H), 1.93 (qt, J=7.3
Hz, 2H), 2.71 (d, J=6.3 Hz, 2H), 2.80 (t, J=7.3 Hz, 2H), 2.71 (t,
J=7.8 Hz, 2H). .sup.13C NMR (125 MHz, D.sub.2O) .delta. 21.2, 25.0,
25.5, 29.9, 34.7, 46.8, 48.1, 53.7. ES-MS 236 (M+1)
Preparation of 3-(1,1-diethylpropargyl)amino-1-propanesulfonic acid
(Compound DW)
##STR00589##
[0658] A mixture of 1,1-diethylpropargylamine (5 g, 45 mmol) and
1,3-propane sultone (6.05 g, 49.5 mmol) in THF (25 mL) was heated
at reflux for 5 hours. The mixture was cooled to room temperature.
The solid was collected by filtration, rinsed with diisopropylether
(2.times.10 mL) then dried overnight in the vacuum oven (7.16 g).
The solid was suspended in ethanol (30 mL) and the suspension was
heated at reflux for 1 hour. The mixture was then cooled to room
temperature and the solid was collected by suction filtration,
air-dried for 5 m, and further dried in a vacuum oven at 60.degree.
C. overnight (5.86 g). There was still a significant amount of
ethanol present. The solid was further dried in the vacuum oven for
40 hours. Compound DW was obtained as a fine white solid, (5.66 g,
81% yield). NMR (500 MHz, D.sub.2O) .delta. 0.92 (t, J=7.6 Hz, 2H),
1.71 (q, J=7.3 Hz, 3H), 1.93 (qt, J=7.3 Hz, 2H), 2.81 (t, J=7.3 Hz,
2H), 2.94 (s, 1H), 3.13 (t, J=7.6 Hz, 2H). .sup.13C NMR (125 MHz,
D.sub.2O) .delta. 7.2, 21.6, 27.8, 41.4, 48.1, 62.2, 78.6, 78.9.
ES-MS 234 (M+1)
Preparation of 3-(1-ethynylcyclohexyl)amino-1-propanesulfonic acid
(Compound DX)
##STR00590##
[0660] A mixture of 1-ethynylcyclohexylamine (6 g, 48.7 mmol) and
1,3-propane sultone (6.55 g, 53.6 mmol) in THF (35 mL) was heated
at reflux for 2 hours (thick paste). The mixture was cooled to room
temperature. The solid was collected by filtration, rinsed with THF
(3.times.5 mL), air-dried 15 minutes (7.3 g). The solid was
suspended in ethanol (30 mL) and the suspension was heated at
reflux for 1 hour. The mixture was then cooled to room temperature
and the solid was collected by suction filtration, rinsed with
ethanol (2.times.5 mL), air-dried for 10 min, and further dried in
a vacuum oven at 60.degree. C. overnight (crop1, 7.10 g). The
combined mother liquors were stirred overnight at room temperature.
There was a lot of solid. The solid was collected by filtration,
rinsed with acetone (3.times.5 mL), air-dried for 30 minutes, and
then suspended in ethanol (12 mL). The suspension was heated at
reflux for 1 hour. The mixture was then cooled to room temperature
and the solid was collected by suction filtration, rinsed with
ethanol (2.times.5 mL), air-dried for 2 minutes, and further dried
in a vacuum oven at 60.degree. C. overnight (crop 2: 1.85 g).
Compound DX was obtained as a fine white solid (two crops in total
8.95 g, 75% yield). .sup.1H NMR (500 MHz, D.sub.2O) .delta.
0.95-1.05 (m, 1H), 1.38-1.54 (m, 5H), 1.60-1.64 (m, 2H), 1.94 (qt,
J=7.8 Hz, 2H), 2.85 (t, J=7.3 Hz, 2H), 3.01 (s, 1H), 3.22 (t, J=7.8
Hz, 2H). .sup.13C NMR (125 MHz, D.sub.2O) .delta. 21.8, 22.3, 24.2,
34.4, 40.9, 48.0, 59.2, 78.6, 79.3. ES-MS 243.0 (M-1).
Preparation of (3-(2-hydroxy-2-phenyl)amino-1-propanesulfonic acid
(Compound DY)
##STR00591##
[0662] A mixture of (.+-.)-2-Amino-1-phenylethanol (9.9 g, 72 mmol)
and 1,3-propane sultone (9.3 g, 76 mmol) in acetonitrile (70 mL)
and ethanol (2 mL) was heated at reflux for 1.5 hours. The mixture
was cooled to room temperature. The solid was collected by
filtration, rinsed with acetonitrile (2.times.25 mL) air-dried for
20 minutes (21.3 g). The solid was suspended in methanol (110 mL)
and the suspension was heated to reflux. Water (4 mL) was added
dropwise until a clear solution was obtained. The mixture was then
cooled to room temperature. The solid was collected by suction
filtration, air-dried for 30 minutes, and further dried in a vacuum
oven at 60.degree. C. for 40 hours (crop 1, 4.47 g). The combined
mother liquor was stored at -20.degree. C. for 40 hours. A second
crop of the solid was collected by filtration, rinsed with acetone
(2.times.15 mL), air-dried (1 hour), and further dried in a vacuum
oven at 60.degree. C. for 24 hours. Compound DY was obtained in two
crops (total 7.82 g, 42% yield). .sup.1H NMR (500 MHz, D.sub.2O) 8,
2.03-2.06 (m, 2H), 2.90 (t, J=7.3 Hz, 2H), 3.16 (t, J=7.3 Hz, 2H),
3.20-3.24 (m, 2H), 4.92-4.95 (m, 1H), 7.30-7.37 (m, 5H). .sup.13C
NMR (125 MHz, D.sub.2O) .delta. 21.3, 46.6, 78.1, 53.2, 69.0,
126.1, 129.0, 129.2, 139.6. ES-MS 260 (M+1).
Preparation of
3-[(S)-1-(4-methoxyphenyl)ethyl]-amino-1-propanesulfonic acid
(Compound DZ)
##STR00592##
[0664] A mixture of (S)-(-)-(4-methoxyphenyl)ethylamine (1.83 g,
12.1 mmol) and 1,3-propane sultone (1.6 g, 13 mmol) in acetonitrile
(25 mL) was heated at reflux for 2.5 hours. The mixture was cooled
to room temperature. The solid was collected by filtration, rinsed
with acetonitrile (2.times.5 mL) air-dried for 15 minutes (3.07 g).
The solid was suspended in ethanol (15 mL) and the suspension was
heated at reflux for 1 hour. The mixture was then cooled to room
temperature. The solid was collected by suction filtration, rinsed
with ethanol (2.times.10 mL), air-dried for 15 minutes, and further
dried in a vacuum oven at 60.degree. C. for 18 hours. Compound DZ
was obtained as a white solid (2.95 g, 10.8 mmol, 89% yield).
.sup.1H NMR (500 MHz, D.sub.2O) .delta. 1.52 (d, J=6.8 Hz, 3H),
1.88-1.98 (m, 2H), 2.76-2.79 (m, 3H), 2.80-2.98 (m, 1H), 3.71 (s,
3H), 4.25 (qt, J=6.7 Hz, 2H), 6.93 (d, J=8.3 Hz, 2H), 7.29 (d,
J=8.3 Hz, 2H). .sup.13C NMR (125 MHz, D.sub.2O) .delta. 18.3, 21.5,
44.2, 48.1, 55.6, 58.0, 114.9, 128.2, 129.4, 159.9. ES-MS 274.
(M+1). [.alpha.].sub.D=-28.8.degree. (c=0.0038 in water)
Preparation of 3-(4-bromophenethyl)amino-1-propanesulfonic acid
(Compound EA)
##STR00593##
[0666] A mixture of 4-Bromophenethylamine (4 g, 20 mmol) and
1,3-propane sultone (2.56 g, 21 mmol) in acetonitrile (30 mL) was
heated at reflux for 2.5 hours. The mixture was cooled to room
temperature. The solid was collected by filtration, rinsed with
acetonitrile (2.times.5 mL) air-dried for 15 minutes (9.57 g), and
further dried for 15 minutes in vacuo (8.02 g). The solid was
suspended in ethanol (40 mL) and the suspension was heated at
reflux for 1 hour. The mixture was then cooled to room temperature.
The solid was collected by suction filtration, rinsed with ethanol
(2.times.5 mL), air-dried for 15 minutes, and further dried in a
vacuum oven at 60.degree. C. for 18 hours. Compound EA was obtained
as a white solid (6.04 g, 18.8 mmol, 94% yield). .sup.1H NMR (500
MHz, DMSO) .delta. 1.95 (t, J=6.3 Hz, 3H), 2.63 (t, J=6.1 Hz, 2H),
2.88 (t, J=7.6 Hz, 2H), 3.09 (t, J=6.3 Hz, 2H), 3.15 (t, J=7.8 Hz,
2H), 7.25 (2, J=7.8 Hz, 2H), 7.53 (2, J=7.8 Hz, 2H), 8.63 (br s,
2H). .sup.13C NMR (125 MHz, DMSO) .delta. 21.8, 31.0, 46.7, 47.2,
48.8, 119.9, 131.0, 131.4, 136.5. ES-MS 324 (M+1).
Preparation of 3-[(S)-1-indanamino]-1-propanesulfonic acid
(Compound EB)
##STR00594##
[0668] A mixture of (S)-(-)-1-aminoindan (0.92 g, 6.9 mmol) and
1,3-propane sultone (0.93 g, 7.6 mmol) in acetonitrile (15 mL) was
heated at reflux for 2.5 hours. The mixture was cooled to room
temperature. The solid was collected by filtration, rinsed with
acetonitrile (2.times.4 mL), air-dried for 15 minutes. The solid
was suspended in ethanol (12 mL) and the suspension was heated at
reflux for 1 hour. The mixture was then cooled to room temperature.
The solid was collected by suction filtration, rinsed with ethanol
(2.times.4 mL), air-dried for 15 minutes, and further dried in a
vacuum oven at 60.degree. C. over the weekend. Compound EB was
obtained as a light pink solid (1.54 g, 87% yield). .sup.1H NMR
(500 MHz, D.sub.2O) .delta. 2.00 (qt, J=7.3 Hz, 2H), 2.09-2.13 (m,
1H), 2.40-2.45 (m, 1H), 2.84-2.87 (m, 3H), 2.98-3.04 (m. 1H), 3.12
(t, J=7.8 Hz, 2H), 4.67-4.70 (m, 1H), 7.22 (m, 2H), 7.29-7.32 (m,
2H), 7.40 (d, J=7.8 Hz, 1H). .sup.13C NMR (125 MHz, D.sub.2O)
.delta. 21.6, 28.6, 29.8, 43.9, 48.1, 63.0, 125.5, 125.7, 127.2,
130.3, 136.3, 145.4. ES-MS 256 (M+1). [.alpha.].sub.D=-1.0.degree.
(c=0.003095 in water).
Preparation of 3-cyclobutylamino-1-propanesulfonic acid (Compound
EC)
##STR00595##
[0670] A mixture of cyclobutylamine (1.11 g, 15.6 mmol) and
1,3-propane sultone (2 g, 17 mmol) in acetonitrile (18 mL) was
heated at reflux. The mixture turned to a lump within 15 minutes.
THF (10 mL) was added. The reflux was maintained for 1 hour. The
mixture was cooled to room temperature. The solid was collected by
filtration, rinsed with acetonitrile (2.times.4 mL), air-dried for
60 minutes (2.41 g). The solid was suspended in methanol (20 mL)
and the suspension was heated at reflux until all the solid
material was dissolved. The mixture was then cooled to room
temperature. The solid thus formed was collected by suction
filtration, rinsed with methanol (2.times.4 mL), air-dried for 20
minutes, and further dried in a vacuum oven at 40.degree. C. for 18
hours. Compound EC was obtained as a white solid (1.81 g, 60%
yield). .sup.1H NMR (500 MHz, D.sub.2O) .delta. 1.70-1.77 (m, 2H),
1.94-2.03 (m, 4H), 2.18 (br s, 2H), 2.85 (t, J=6.8 Hz, 1H), 2.95
(t, J=7.3 Hz, 1H), 3.63-3.66 (m, 1H). .sup.13C NMR (125 MHz,
D.sub.2O) .delta. 14.5, 21.5, 26.1, 43.6, 48.0, 51.8. ES-MS 194
(M+1).
Preparation of 3-(4-mexiletino)-1-propanesulfonic acid (Compound
EV)
##STR00596##
[0672] Mexiletine hydrochloride (2.45 g, 11.3 mmol) was freed with
1N NaOH (50 mL), extracted with ethyl acetate (2.times.50 mL). The
combined extract was dried over sodium sulfate. The solvent was
evaporated. A solution of 1,3-propane sultone (1.46 g, 11.9 mmol)
in THF (35 mL) was added to the free amine. The mixture was heated
at reflux for 4 hours. The mixture was cooled to room temperature;
and the resultant solid material was collected by filtration,
rinsed with THF (5 mL). The solid was dried overnight at 40.degree.
C. The filtrated dried in air overnight to afford a brownish solid
(1.45 g) it was less pure than the first crop thus discarded.
Compound EV was obtained as a white solid (1.19 g, 56% (99% crude)
yield). .sup.1H NMR (500 MHz, DMSO) .delta. 1.39 (d, J=6.3 Hz, 3H),
2.02 (t, J=5.9 Hz, 2H), 2.26 (s, 6H), 2.67 (t, J=5.9 Hz, 2H), 3.21
(br d, J=19.5 Hz, 2H), 3.61 (br s, 1H), 3.83-3.91 (m, 2H), 6.95 (t,
J=7.3 Hz, 1H), 7.04 (m, 2H), 8.91 (br s, 2H). .sup.13C NMR (125
MHz, DMSO) .delta. 13.3, 16.0, 21.8, 44.6, 49.2, 52.9, 70.8, 124.3,
128.9, 130.4, 154.2. ES-MS 302 (M+1).
Preparation of 3-(1-benzyl-2-methoxyethyl))amino-1-propanesulfonic
acid (Compound EW)
##STR00597##
[0674] S-(+)-2-Amino-1-methoxy-3-phenylpropane hydrochloride (2.06
g, 10.0 mmol) was freed with saturated potassium carbonate (20 mL).
The aqueous mixture was extracted with ethyl acetate (3.times.15
mL); and the combined extract was dried over sodium sulfate. The
solvent was evaporated. A solution of 1,3-propane sultone (1.29 g,
10.5 mmol) in THF (15 mL) was added to the free amine. The mixture
was heated at reflux for 4 hours. The mixture was cooled to room
temperature and stirred for 1 hour. The solid was collected by
filtration, rinsed with acetone (5 mL). The solid was dried
overnight at 40.degree. C. Compound EW was obtained as a white
solid (2.48 g, 83% yield). .sup.1H NMR (500 MHz, DMSO) .delta. 1.99
(m, 2H), 2.65 (t, J=6.1 Hz, 2H), 2.80 (t, J=12.0 Hz, 1H), 3.05 (m,
2H), 3.17 (m, 3H), 3.22 (dd, J=3.4 Hz, 10.7 Hz, 2H), 3.33 (m, 1H),
3.43 (d, J=10.7 Hz, 2H), 3.50 (m, 1H), 7.27 (m, 3H), 7.35 (t, J=7.1
Hz, 2H), 8.86 (br d, 1H). .sup.13C NMR (125 MHz, DMSO) .delta.
21.8, 33.4, 45.0, 49.2, 57.8, 58.5, 68.1, 126.9, 128.7, 129.2,
136.3. ES-MS 310 (M+1). [.alpha.].sub.D=-0.8.degree. (c=0.0025 in
water).
Preparation of
341-(N-hydroxycarbamoyl)-2-phenylethyl)amino-1-propanesulfonic acid
(Compound FO)
##STR00598##
[0676] A solution of hydroxylamine 50% in water (wt./wt, 7 mL) was
added to a solution of L-N-(3-sulfopropyl)phenylalanine ethyl ester
(1.00 g, 3.17 mmol in water (5 mL). The mixture was stirred at room
temperature for 24 hours. The mixture was concentrated to dryness.
The resulting solid was dissolved in a mixture of hot methanol (10
mL) and water; and the mixture was stored at 5.degree. C. for 3
days. Only a very small amount of solid had formed. Upon addition
of acetone (3 mL), a large amount of solid formed. The solid was
collected by suction-filtration, rinsed with acetone (2.times.5 mL)
then dried overnight at 40.degree. C. in a vacuum oven. Compound FO
was obtained as a white solid (700 mg, 73%). NMR (500 MHz,
D.sub.2O) .delta. 2.03-2.07 (m, 2H), 2.88-2.90 (M, 2H), 2.99-3.03
(M, 1H), 3.07-3.12 (M, 1H), 3.18-3.23 (m, 1H), 3.82-3.85 (m, 1H),
7.15 (d, J=6.8 Hz, 2H), 7.26-7.31 (m, 3H). .sup.13C (125 MHz,
D.sub.2O) .delta. 19.3, 33.8, 43.2, 45.8, 57.9, 126.0, 127.1,
127.3, 131.4, 162.2. ES-MS 303 (M+Na). [.alpha.].sub.D=40.degree.
(c=0.001983 in water).
Preparation of
3-{[(1S)-1-benzyl-2-(benzyloxy)-2-oxoethyl]amino}propane-1-sulfonic
acid (Compound EL)
##STR00599##
[0678] L-Phenylalanine benzylester hydrochloride (2.0 g, 6.9 mmol)
was treated with a saturated aqueous solution of K.sub.2CO.sub.3
(50 mL) and EtOAc (3.times.50 mL) was added. The organic extracts
were separated, combined, dried with Na.sub.2SO.sub.4, filtered,
evaporated under reduced pressure and dried in vacuo.
[0679] To a solution of L-Phenylalanine benzylester (1.8 g, 6.8
mmol) in 1,4-dioxane (10 mL) was added 1,3-propanesultone (708 mg,
6.5 mmol). The solution was stirred at reflux. After 1 hour, 20 mL
of 1,4-dioxane was added to allow for good stirring. The reaction
was stirred at reflux for an additional 1 hour. It was cooled to
room temperature. The solid was collected by filtration, washed
with acetone (2.times.25 mL). The product was suspended 80%
Acetone/EtOH. The suspension was stirred at reflux for 30 seconds.
The solid was filtered and dried in the vacuum oven (50.degree. C.)
affording the title compound (1.14 g, 46%). .sup.1H NMR (DMSO, 500
MHz) .delta. ppm 7.27 (m, 6H), 7.12 (m, 4H), 5.05 (dd, 2H, J=12.3
Hz), 4.49 (m, 1H), 3.29 (m, 1H), 3.00 (m, 1H), 2.98 (m, 1H), 2.61
(t, 2H, J=6.5 Hz), 1.97 (m, 2H). .sup.13C (DMSO, 125 MHz) .delta.
ppm 168.88, 135.21, 134.90, 130.00, 129.31, 129.07, 128.01, 67.98,
60.50, 49.77, 46.72, 35.87, 22.43. [.alpha.].sub.D=+ 4.8.degree.
(c=0.00073 in water), ES-MS 378 (M+1).
Preparation of
3-{[(1S)-1-(methoxycarbonyl)-2-methylpropyl]amino}-1-propanesulfonic
acid (Compound FT)
##STR00600##
[0681] L-valine methylester hydrochloride (5.0 g, 29.8 mmol) was
treated with a saturated solution of K.sub.2CO.sub.3 (75 mL) and
EtOAc (3.times.75 mL) was added. The organic extracts were
separated, combined, dried with Na.sub.2SO.sub.4, filtered,
evaporated under reduced pressure and dried in vacuo.
[0682] To a solution of L-valine methylester in THF (25 mL) was
slowly added 1,3-propanesultone (2.49 g, 19.9 mmol). The solution
was stirred at reflux for 2 hours. The reaction was cooled to room
temperature. The solid was collected by filtration, washed with
acetone (2.times.30 mL). It was dried in the vacuum oven
(50.degree. C.) affording the title compound (2.52 g, 50%). .sup.1H
NMR (D.sub.2O, 500 MHz) .delta. ppm 3.92 (m, 1H), 3.75 (s, 3H),
3.13 (t, 2H, J=6.8 Hz), 2.88 (t, 2H, J=6.8 Hz), 2.24 (m, 1H), 2.06
(m, 2H)), 0.96 (d, 3H, J=6.8 Hz), 0.88 (d, 3H, J=6.8 Hz). .sup.13C
(D.sub.2O, 125 MHz) .delta. ppm 169.35, 65.85, 55.61, 48.14, 45.59,
29.48, 21.32, 18.25, 16.57. [.alpha.].sub.D=+9.6.degree. (c=0.0014
in water), ES-MS 254 (M+1).
Preparation of 3-{[(1R)-1-cyclohexylethyl]amino}-1-propanesulfonic
acid (Compound FU)
##STR00601##
[0684] To a solution of (R)-(-)-cyclohexylethylamine (2.5 g, 19.7
mmol) in tetrahydrofuran (25 mL) was slowly added
1,3-propanesultone (2.33 g, 18.7 mmol). The solution was stirred at
reflux for 2 hours. The reaction was cooled to room temperature.
The solid was collected by filtration, washed with acetone
(2.times.25 mL) and dried in vacuo, affording the title compound
(3.47 g, 74%). .sup.1H NMR (D.sub.2O, 500 MHz) .delta. ppm 3.09 (m,
3H), 2.88 (t, 2H, J=7.3 Hz), 2.00 (m, 2H), 1.58 (m, 6H), 1.13 (m,
5H), 1.03 (m, 3H). .sup.13C (D.sub.2O, 125 MHz) .delta. ppm 58.37,
48.17, 44.00, 39.84, 29.00, 26.01, 25.82, 25.73, 25.47, 21.51,
11.79. [.alpha.].sub.D=+4.5.degree. (c=0.0022 in water), ES-MS 250
(M-1).
Preparation of 3-{[(1S)-1-cyclohexylethyl]amino}-1-propanesulfonic
acid (Compound FW)
##STR00602##
[0686] To a solution of (S)-(+)-cyclohexylethylamine (5.0 g, 39.3
mmol) in tetrahydrofuran (50 mL) was slowly added
1,3-propanesultone (4.66 g, 37.4 mmol). The solution was stirred at
reflux for 2 hours. The reaction was cooled to room temperature.
The solid was collected by filtration and washed with acetone
(2.times.25 mL). The solid was suspended in 80% acetone/EtOH (200
mL). The suspension was stirred at reflux for 30 seconds before the
solid was filtered and dried in vacuo, affording the title compound
(6.13 g, 66%). .sup.1H NMR (D.sub.2O, 500 MHz) .delta. ppm 3.09 (m,
3H), 2.88 (t, 2H, J=7.3 Hz), 2.00 (m, 2H), 1.55 (m, 6H), 1.13 (m,
5H), 1.03 (m, 3H). .sup.13C (D.sub.2O, 125 MHz) .delta. ppm 59.37,
48.17, 44.00, 39.84, 29.00, 26.01, 25.82, 25.73, 25.47, 21.51,
11.78. [.alpha.].sub.D=-2.8.degree. (c=0.0014 in water), ES-MS 250
(M-1).
Preparation of 3-[(4-tert-butylcyclohexyl)amino]-1-propanesulfonic
acid (Compound FX)
##STR00603##
[0688] To a solution of 4-tert-butylcyclohexylethylamine (mixture
of cis and trans isomers, 2.5 g, 16.1 mmol) in tetrahydrofuran (30
mL) was slowly added 1,3-propanesultone (1.84 g, 15.3 mmol). The
solution was stirred at reflux for 2 hours. The reaction was cooled
to room temperature. The solid was collected by filtration and
washed with acetone (2.times.35 mL). The solid was suspended in 80%
acetone/EtOH (200 mL). The suspension was stirred at reflux for 30
seconds before the solid was filtered and dried in vacuo, affording
the title compound (3.07 g, 72%). .sup.1H NMR (DMSO, 500 MHz)
.delta. ppm 3.21 (m, 0.5H), 3.04 (m, 2H), 2.89 (m, 1H), 2.67 (m,
0.5H), 1.97 (m, 4H), 1.77 (m, 1H), 1.52 (m, 1H), 1.19 (m, 2H), 0.96
(m, 2H), 0.81 (s, 9H). .sup.13C (DMSO, 125 MHz) .delta. ppm 56.47,
53.62, 50.44, 49.77, 47.56, 47.04, 46.28, 44.49, 32.98, 32.74,
29.61, 28.10, 28.03, 25.57, 22.68, 22.48, 21.01. ES-MS 276
(M-1).
Preparation of
3-{[(1S,2S)-2-(benzyloxy)cyclopentyl]amino}-1-propanesulfonic acid
(Compound FY)
##STR00604##
[0690] To a solution of (1S,25)-2-benzyloxycyclopentylamine (1.0 g,
5.2 mmol) in tetrahydrofuran (12 mL) was slowly added
1,3-propanesultone (601 mg, 5.0 mmol). The solution was stirred at
reflux for 2 hours. The reaction was cooled to room temperature.
The solid was collected by filtration, washed with acetone
(2.times.25 mL) and dried in vacuo, affording the title compound
(1.36 g, 87%). .sup.1H NMR (D.sub.2O, 500 MHz) .delta. ppm 7.32 (m,
5H), 4.53 (d, 1H, J=11.2 Hz), 4.41 (d, 1H, J=11.2 Hz), 4.01 (m,
1H), 3.36 (m, 1H), 3.00 (t, 2H, J=7.8 Hz), 2.80 (t, 2H, J=7.8 Hz),
2.00 (m, 4H), 1.64 (m, 3H), 1.49 (m, 1H). .sup.13C NMR (D.sub.2O,
125 MHz) .delta. ppm 136.99, 129.06, 129.01, 128.77, 81.78, 71.81,
63.88, 48.01, 45.33, 29.91, 27.43, 21.60, 20.93.
[.alpha.].sub.D=+31.1.degree. (c=0.0064 in water). ES-MS 314
(M+1).
Preparation of
3-{[(1R,2R)-2-(benzyloxy)cyclopentyl]amino}-1-propanesulfonic acid
(Compound FZ)
##STR00605##
[0692] To a solution of (1R,2R)-2-benzyloxycyclopentylamine (1.0 g,
5.2 mmol) in tetrahydrofuran (12 mL) was slowly added
1,3-propanesultone (601 mg, 5.0 mmol). The solution was stirred at
reflux for 2 hours. The reaction was cooled to room temperature.
The solid was collected by filtration and washed with acetone
(2.times.15 mL). The product was suspended in EtOH and the solvent
was evaporated (to remove THF residue). The solid was dried in
vacuo, affording the title compound (717 mg, 46%). NMR (D.sub.2O,
500 MHz) S ppm 7.32 (m, 5H), 4.53 (d, 1H, J=11.2 Hz), 4.42 (d, 1H,
J=11.2 Hz), 4.02 (m, 1H), 3.36 (m, 1H), 3.01 (t, 2H, J=7.8 Hz),
2.81 (t, 2H, J=7.8 Hz), 2.01 (m, 4H), 1.65 (m, 3H), 1.49 (m, 1H).
.sup.13C NMR (D.sub.2O, 125 MHz) .delta. ppm 137.00, 129.07,
129.01, 128.77, 81.78, 71.81, 63.89, 48.02, 45.34, 29.93, 27.43,
21.61, 20.94. [.alpha.].sub.D=-38.8.degree. (c=0.00122 in water).
ES-MS 314 (M+1).
Preparation of
3-{[(1S)-1-benzyl-2-(cyclohexylamino)-2-oxoethyl]amino}-1-propanesulfonic
acid (Compound GA)
##STR00606##
[0694] To a solution of L-Phenylalanine cyclohexylamide (2.5 g,
10.1 mmol) in tetrahydrofuran (25 mL) was added 1,3-propanesultone
(1.17 g, 9.7 mmol). The solution was stirred at reflux for 2 hours.
It was cooled to room temperature. The solid was collected by
filtration, washed with acetone (2.times.25 mL) and dried in the
vacuum oven (50.degree. C.), affording the title compound (1.39 g,
39%). .sup.1H NMR (D.sub.2O, 500 MHz) .delta. ppm 7.21 (m, 3H),
7.08 (m, 2H), 4.42 (m, 0.5H), 3.83 (m, 1H), 3.29 (m, 1H), 3.15 (m,
1H), 3.02 (m, 2H), 2.86 (m, 3H), 2.49 (m, 0.5H), 2.01 (m, 2H), 1.54
(m, 1H), 1.45 (m, 1H), 1.33 (m, 2H), 1.02 (m, 4H), 0.55 (m, 1H).
.sup.13C (D.sub.2O, 125 MHz) .delta. ppm 133.71, 129.54, 129.18,
128.03, 62.08, 49.25, 47.97, 45.41, 36.29, 31.73, 31.46, 24.86,
24.28, 24.20, 21.39. [.alpha.].sub.D=+36.4.degree. (c=0.0019 in
water), ES-MS 369 (M+1).
Preparation of
3-{[(1S,2S)-2-(benzyloxy)cyclopentyl]amino}-1-propanesulfonic acid
(Compound GB)
##STR00607##
[0696] To a solution of (1S,2S)-2-benzyloxycyclohexylamine (1.0 g,
5.2 mmol) in tetrahydrofuran (12 mL) was slowly added
1,3-propanesultone (601 mg, 5.0 mmol). The solution was stirred at
reflux for 2 hours. The reaction was cooled to room temperature.
The solid was collected by filtration, washed with acetone
(2.times.20 mL) and dried in the vacuum oven (50.degree. C.),
affording the title compound (1.15 g, 75%). NMR (D.sub.2O, 500 MHz)
.delta. ppm 7.34 (m, 5H), 4.62 (d, 1H, J=11.2 Hz), 4.42 (d, 1H,
J=11.2 Hz), 3.40 (m, 1H), 2.97 (m, 2H), 2.90 (m, 1H), 2.76 (t, 2H,
J=6.5 Hz), 2.26 (m, 1H), 1.92 (m, 3H), 1.66 (m, 2H), 1.18 (m, 4H).
.sup.13C NMR (D.sub.2O, 125 MHz) .delta. ppm 137.22, 129.34,
129.11, 128.84, 76.74, 70.26, 60.84, 48.02, 42.74, 29.49, 26.43,
23.57, 23.02, 21.53. [.alpha.].sub.D=+74.8.degree. (c=0.00207 in
water). ES-MS 326 (M-1).
Preparation of
3-{[(1R,2R)-2-(benzyloxy)cyclopentyl]amino}-1-propanesulfonic acid
(Compound GD)
##STR00608##
[0698] To a solution of (1R,2R)-2-benzyloxycyclohexylamine (1.0 g,
5.2 mmol) in tetrahydrofuran (12 mL) was slowly added
1,3-propanesultone (601 mg, 5.0 mmol). The solution was stirred at
reflux for 2 hours. The reaction was cooled to room temperature.
The solid was collected by filtration and washed with acetone
(2.times.35 mL). The solid was suspended in 80% acetone/EtOH (200
mL). The suspension was stirred at reflux for 30 seconds before the
solid was filtered and dried in the vacuum oven (50.degree. C.),
affording the title compound (844 mg. 55%). .sup.1H NMR (D.sub.2O,
500 MHz) .delta. ppm 7.32 (m, 5H), 4.60 (d, 1H, J=11.2 Hz), 4.40
(d, 1H, J=11.2 Hz), 3.39 (m, 1H), 2.94 (m, 2H), 2.85 (m, 1H), 2.74
(t, 2H, J=6.5 Hz), 2.24 (m, 1H), 1.90 (m, 3H), 1.64 (m, 2H), 1.14
(m, 4H). .sup.13C NMR (D.sub.2O, 125 MHz) 8 ppm 137.35, 129.28,
129.19, 128.90, 76.90, 70.35, 60.97, 48.12, 42.90, 29.59, 26.53,
23.64, 23.09, 21.63. [.alpha.].sub.D=-68.9.degree. (c=0.0026 in
water). ES-MS 326 (M-1).
Preparation of
3-({(1S)-1-[(benzyloxy)carbonyl]-2-methylpropyl}amino)-1-propanesulfonic
acid (Compound GE)
##STR00609##
[0700] L-valine benzylester p-tosylate (2.5 g, 6.6 mmol) was
treated with a saturated solution of K.sub.2CO.sub.3 (50 mL) and
EtOAc (3.times.50 mL). The organic extracts were separated,
combined, dried with Na.sub.2SO.sub.4, filtered, evaporated under
reduced pressure and dried in vacuo.
[0701] To a solution of L-valine benzylester in MeOH (12 mL) was
slowly added 1,3-propanesultone (604 mg, 5.0 mmol). The solution
was stirred at reflux for 2 hours. The reaction was cooled to room
temperature. The solid was collected by filtration, washed with
cold MeOH and acetone (2.times.25 mL). It was dried in the vacuum
oven (50.degree. C.), affording the title compound (649 mg, 39%).
.sup.1H NMR (D.sub.2O, 500 MHz) .delta. ppm 7.47 (m, 5H), 5.41 (d,
1H, J=11.7 Hz), 5.31 (d, 3H, J=11.7 Hz), 4.04 (m, 1H), 3.19 (m,
2H), 2.95 (t, 2H, J=6.8 Hz), 2.35 (m, 1H), 2.14 (m, 2H), 1.04 (d,
3H, J=6.3 Hz), 0.95 (d, 3H, J=6.3 Hz). .sup.13C (D.sub.2O, 125 MHz)
.delta. ppm 168.75, 134.81, 129.38, 129.31, 129.17, 69.00, 65.96,
48.24, 46.71, 29.67, 21.40, 18.39, 16.65.
[.alpha.].sub.D=-7.2.degree. (c=0.0015 in water), ES-MS 330
(M+1).
Preparation of
3-{[(1S)-2-ethoxy-1-methyl-2-oxoethyl]amino}-1-propanesulfonic acid
(Compound GF)
##STR00610##
[0703] L-alanine ethyl ester hydrochloride (2.5 g, 16.3 mmol) was
treated with a saturated solution of K.sub.2CO.sub.3 (50 mL) and
EtOAc (3.times.50 mL). The organic extracts were separated,
combined, dried with Na.sub.2SO.sub.4, filtered, evaporated under
reduced pressure and dried in vacuo.
[0704] To a solution of L-alanine ethyl ester (1.67 g, 14.3 mmol)
in tetrahydrofuran (25 mL) was slowly added 1,3-propanesultone
(1.42 g, 11.9 mmol). The solution was stirred at reflux for 2
hours. The reaction was cooled to room temperature. The solid was
collected by filtration, washed with acetone (2.times.25 mL) and
dried in vacuo, affording the title compound (1.19 g, 42%). .sup.1H
NMR (D.sub.2O, 500 MHz) .delta. ppm 4.16 (m, 2H), 4.01 (m, 1H),
3.12 (m, 2H), 2.87 (t, 2H, J=7.3 Hz), 2.01 (m, 2H), 1.43 (d, 3H,
J=7.3 Hz), 1.14 (t, 3H, J=6.8 Hz). .sup.13C (D.sub.2O, 125 MHz)
.delta. ppm 170.22, 63.84, 55.69, 47.94, 44.73, 21.51, 14.05,
13.25. [.alpha.].sub.D=-2.4.degree. (c=0.0022 in water), ES-MS 240
(M+1).
Preparation of (2S)-3-methyl-2-[(3-sulfopropyl)amino]butanoic acid
(Compound GC)
##STR00611##
[0706] L-valine methylester hydrochloride (5.0 g, 29.8 mmol) was
treated with a saturated solution of K.sub.2CO.sub.3 (75 mL) and
EtOAc (3.times.75 mL). The organic extracts were separated,
combined, dried with Na.sub.2SO.sub.4, filtered, evaporated under
reduced pressure and dried in vacuo.
[0707] To a solution of L-valine methylester in tetrahydrofuran (25
mL) was slowly added 1,3-propanesultone (2.49 g, 19.9 mmol). The
solution was stirred at reflux for 2.5 hours. The reaction was
cooled to room temperature. The solid was collected by filtration,
washed with acetone (2.times.30 mL) and dried in vacuo affording
the desired ester.
[0708] The ester (860 mg, 3.4 mmol) was dissolved in 2M NaOH (1.20
g of NaOH and 15 mL of water). The reaction was stirred at room
temperature overnight. Dowex Marathon C ion exchange resin
(strongly acidic) was added to the solution. The suspension was
stirred for 15 minutes before the resin was removed by filtration.
The filtrate was evaporated under reduced pressure and dried in
vacuo, affording the title compound (645 mg, 79%). .sup.1H NMR
(D.sub.2O, 500 MHz) .delta. ppm 3.66 (m, 1H), 3.09 (t, 2H, J=6.3
Hz), 2.86 (t, 2H, J=7.3 Hz), 2.17 (m, 1H), 2.07 (m, 2H)), 0.93 (d,
3H, J=6.8 Hz), 0.87 (d, 3H, J=6.8 Hz). .sup.13C (D.sub.2O, 125 MHz)
.delta. ppm 171.24, 66.83, 48.28, 46.77, 29.33, 21.44, 18.30,
16.98. [.alpha.].sub.D=-16.5.degree. (c=0.0020 in water), ES-MS 238
(M-1).
Preparation of
3-{[(1S)-1-(methoxycarbonyl)-3-methylbutyl]amino}-1-propanesulfonic
acid (Compound GH)
##STR00612##
[0710] L-Leucine methylester hydrochloride (5.0 g, 27.5 mmol) was
treated with a saturated solution of K.sub.2CO.sub.3 (50 mL) and
EtOAc (3.times.50 mL) was added. The organic extracts were
separated, combined, dried with Na.sub.2SO.sub.4, filtered,
evaporated under reduced pressure and dried in vacuo.
[0711] To a solution of L-valine methylester (3.74 g, 25.6 mmol) in
tetrahydrofuran (35 mL) was slowly added 1,3-propanesultone (2.04
g, 17.2 mmol). The solution was stirred at reflux for 3 hours. The
reaction was cooled to room temperature. The solid was collected by
filtration. Dowex Marathon C ion exchange resin (strongly acidic)
was added to the solution. The suspension was stirred for 15
minutes before the resin was removed by filtration. The filtrate
was evaporated under reduced pressure. The solid was suspended in
acetone (50 mL), filtered and dried in vacuo, affording the title
compound (1.80 g, 39%). .sup.1H NMR (D.sub.2O, 500 MHz) .delta. ppm
3.99 (m, 1H), 3.72 (s, 3H), 3.12 (m, 2H), 2.87 (t, 2H, J=7.3 Hz),
2.02 (m, 2H), 1.74 (m, 1H), 1.60 (m, 2H), 0.81 (d, 3H, J=5.4 Hz),
0.87 (d, 3H, J=6.8 Hz). .sup.13C (D.sub.2O, 125 MHz) .delta. ppm
170.60, 58.91, 53.81, 48.08, 45.50, 38.17, 24.44, 22.15, 21.59,
20.93. [.alpha.].sub.D=+13.8.degree. (c=0.0016 in water), ES-MS 268
(M+1).
Preparation of
3-({(1S)-1-[(tert-butylamino)carbonyl]-2-methylpropyl}amino)-1-propanesul-
fonic acid (Compound GI)
##STR00613##
[0713] L-valine tert-butylamide hydrochloride (2.5 g, 12.0 mmol)
was treated with a saturated solution of K.sub.2CO.sub.3 (50 mL)
and EtOAc (3.times.50 mL) was added. The organic extracts were
separated, combined, dried with Na.sub.2SO.sub.4, filtered,
evaporated under reduced pressure and dried in vacuo.
[0714] To a solution of L-valine tert-butylamide (1.87 g, 11.0
mmol) in 1,4-dioxane (20 mL) was added 1,3-propanesultone (1.07 g,
9.0 mmol). The solution was stirred at reflux for 5 hours. The
reaction was cooled to room temperature. The solid was collected by
filtration, washed with acetone (2.times.30 mL) and dried in vacuo,
affording the title compound (801 mg, 25%). .sup.1H NMR (D.sub.2O,
500 MHz) .delta. ppm 3.43 (m, 1H), 3.00 (m, 2H), 2.85 (m, 2H), 2.03
(m, 3H), 1.21 (m, 9H), 0.92 (d, 3H, J=6.3 Hz), 0.85 (d, 3H, J=6.8
Hz). .sup.13C (D.sub.2O, 125 MHz) ppm 166.47, 66.55, 52.56, 48.23,
46.11, 29.91, 27.81, 21.29, 18.30, 17.44.
[.alpha.].sub.D=-11.6.degree. (c=0.0023 in water), ES-MS 293
(M-1).
Preparation of
3-{[(1S)-1-(hydroxymethyl)-3-methylbutyl]amino}-1-propanesulfonic
acid (Compound GJ)
##STR00614##
[0716] To a solution of L-(+)-leucinol (5.0 g, 42.8 mmol) in THF
(65 mL) was slowly added 1,3-propanesultone (4.85 g, 40.7 mmol in
THF (10 mL)). The solution was stirred at reflux for 2 hours. The
reaction was cooled to room temperature. The solid was collected by
filtration and washed with acetone (2.times.50 mL). The solid was
dissolved in 50% water/EtOH (400 mL). Dowex Marathon C ion exchange
resin (strongly acidic) was added to the solution. The suspension
was stirred for 15 minutes before the resin was removed by
filtration. The filtrate was evaporated under reduced pressure. The
solid was suspended-in acetone (150 mL), filtered and dried in the
vacuum oven (50.degree. C.), affording the title compound (6.11 g,
63%). .sup.1H NMR (D.sub.2O, 500 MHz) .delta. ppm 3.77 (m, 1H),
3.59 (m, 1H), 3.23 (m, 1H), 3.13 (m, 2H), 2.90 (m, 2H), 2.02 (m,
2H), 1.53 (m, 2H), 1.35 (m, 1H), 0.81 (d, 3H, J=16.1 Hz). .sup.13C
(D.sub.2O, 125 MHz) .delta. ppm 58.72, 58.00, 48.17, 43.54, 35.96,
24.34, 22.53, 21.62, 20.89. [.alpha.].sub.D+16.6.degree. (c=0.0022
in water), ES-MS 240 (M+1).
Preparation of
3-{[(1S)-1-(hydroxymethyl)-2-methylbutyl]amino}-1-propanesulfonic
acid (Compound GK)
##STR00615##
[0718] To a solution of L-(+)-isoleucinol (2.0 g, 17.1 mmol) in THF
(30 mL) was slowly added 1,3-propanesultone (1.94 g, 16.3 mmol).
The solution was stirred at reflux for 2 hours. The reaction was
cooled to room temperature. The solid was collected by filtration
and washed with acetone (2.times.20 mL). The solid was dissolved in
70% water/EtOH (240 mL). Dowex Marathon C ion exchange resin
(strongly acidic, 15 g) was added to the solution. The suspension
was stirred for 15 minutes before the resin was removed by
filtration. The filtrate was evaporated under reduced pressure. The
solid was suspended in acetone (60 mL), filtered and dried in the
vacuum oven (50.degree. C.), affording the title compound (1.70 g,
44%). .sup.1H NMR
[0719] (D.sub.2O, 500 MHz) .delta. ppm 3.78 (d, 1H, J=13.1 Hz),
3.64 (m, 1H), 3.14 (m, 3H), 2.03 (m, 2H), 1.75 (m, 1H), 1.32 (m,
1H), 1.17 (m, 1H), 0.79 (m, 6H). .sup.13C (D.sub.2O, 125 MHz)
.delta. ppm 63.42, 57.38, 48.27, 44.77, 33.64, 25.91, 21.52, 13.31,
10.94. [.alpha.].sub.D=+20.4.degree. (c=0.00212 in water), ES-MS
240 (M+1).
Preparation of
3-{[(1R)-1-(hydroxymethyl)-3-methylbutyl]amino}-1-propanesulfonic
acid (Compound GL)
##STR00616##
[0721] To a solution of D-(-)-leucinol (2.0 g, 17.1 mmol) in THF
(30 mL) was slowly added 1,3-propanesultone (1.94 g, 16.3 mmol).
The solution was stirred at reflux for 2 hours. The reaction was
cooled to room temperature. The solid was collected by filtration
and washed with acetone (2.times.20 mL). The solid was dissolved in
50% water/EtOH (240 mL). Dowex Marathon C ion exchange resin
(strongly acidic) was added to the solution. The suspension was
stirred for 15 minutes before the resin was removed by filtration.
The filtrate was evaporated under reduced pressure. The solid was
suspended in acetone (50 mL), filtered and dried in the vacuum oven
(50.degree. C.), affording the title compound (2.55 g, (65%).
.sup.1H NMR (D.sub.2O, 500 MHz) .delta. ppm 3.74 (d, 1H, J=12.7
Hz), 3.56 (d, 1H, J=12.7 Hz), 3.20 (m, 1H), 3.10 (t, 2H, J=7.3 Hz),
2.87 (t, 2H, J=7.3 Hz), 2.00 (m, 2H), 1.49 (m, 2H), 1.31 (m, 1H),
0.80 (d, 3H, J=6.3 Hz), 0.76 (d, 3H, J=6.3 Hz). .sup.13C (D.sub.2O,
125 MHz) .delta. ppm 58.74, 58.01, 48.19, 43.56, 35.98, 24.34,
22.54, 21.63, 20.90. [.alpha.].sub.D=-16.3.degree. (c=0.0019 in
water), ES-MS 238 (M-1).
Preparation of
3-{[(1S)-2-amino-2-oxo-1-phenylethyl]amino}-1-propanesulfonic acid
(Compound GN)
##STR00617##
[0723] L-Phenylglycine amide hydrochloride (1.0 g, 6.7 mmol) was
treated with a solution of K.sub.2CO.sub.3 (20 mL). The resultant
mixture was extracted with EtOAc (3.times.20 mL). The organic
extracts were separated, combined, dried over Na.sub.2SO.sub.4. The
solid material was removed by filtration, and the filtrate was
concentrated to dryness under reduced pressure.
[0724] To a solution of L-Phenylglycine amide (670 mg, 5.9 mmol) in
tetrahydrofuran (10 mL) and 1,4-dioxane (4 mL) was added
1,3-propanesultone (674 mg, 5.6 mmol). The solution stirred at
reflux for 2 hours. The reaction was cooled to room temperature.
The solid was collected by filtration and washed with acetone
(2.times.20 mL). The solid was dissolved in 50% water/EtOH mL).
Dowex Marathon C ion exchange resin (strongly acidic) was added to
the solution. The suspension was stirred for 15 minutes before the
resin was removed by filtration. The filtrate was evaporated under
reduced pressure. The solid was suspended in acetone (50 mL),
filtered and dried in the vacuum oven (50.degree. C.), affording
the title compound (743 mg, 50%). NMR (D.sub.2O, 500 MHz) .delta.
ppm 7.38 (m, 5H), 4.92, (5, 1H), 3.01 (m, 1H), 2.91 (m, 1H), 2.78
(t, 2H, J=7.3 Hz), 2.0 (m, 2H). .sup.13C (D.sub.2O, 125 MHz)
.delta. ppm 170.15, 130.95, 130.24, 129.94, 128.74, 63.40, 47.99,
44.92, 21.27. [.alpha.].sub.D=-124.degree. (c=0.0041 in water),
ES-MS 271 (M-1).
Preparation of
3-{[(1S)-2-tert-butoxy-1-methyl-2-oxoethyl]amino}-1-propanesulfonic
acid (Compound GO)
##STR00618##
[0726] L-Alanine tert-butylester hydrochloride (2.61 g, 14.4 mmol)
was treated with a solution of K.sub.2CO.sub.3 (75 mL). The
resultant mixture was extracted with EtOAc (3.times.75 mL). The
organic extracts were separated, combined, dried over
Na.sub.2SO.sub.4. The solid material was removed by filtration, and
the filtrate was concentrated to dryness under reduced
pressure.
[0727] To a solution of L-Alanine tert-butylester (1.53 g, 10.5
mmol) in tetrahydrofuran (20 mL) was added 1,3-propanesultone (1.16
g, 9.6 mmol). The solution stirred at reflux for 2 hours. The
reaction was cooled to room temperature. The solid was collected by
filtration and washed with acetone (2.times.20 mL). The solid was
dissolved in water (80 mL). Dowex Marathon C ion exchange resin
(strongly acidic) was added to the solution. The suspension was
stirred for 15 minutes before the resin was removed by filtration.
The filtrate was evaporated under reduced pressure. The solid was
suspended in acetone (80 mL), filtered and dried in the vacuum oven
(50.degree. C.), affording the title compound (1.37 g, 54%).
.sup.1H NMR (D.sub.2O, 500 MHz) .delta. ppm 3.88 (m, 1H), 3.09 (m,
2H), 2.86 (t, 2H, J=7.3 Hz), 2.00 (m, 2H), 1.39 (d, 3H, J=7.3 Hz),
1.35 (s, 9H). .sup.13C (D.sub.2O, 125 MHz) .delta. ppm 169.13,
86.12, 56.24, 47.94, 44.71, 27.11, 21.52, 14.17.
[.alpha.].sub.D=-1.1.degree. (c=0.0027 in water), ES-MS 266
(M-1).
Preparation of
3-{[(1S)-2-amino-2-oxo-1-phenylethyl]amino}-1-propanesulfonic acid
(Compound GP)
##STR00619##
[0729] D-Phenylglycine amide hydrochloride (1.0 g, 6.7 mmol) was
treated with a solution of K.sub.2CO.sub.3 (20 mL). The resultant
mixture was extracted with EtOAc (3.times.20 mL). The organic
extracts were separated, combined, dried over Na.sub.2SO.sub.4. The
solid material was removed by filtration, and the filtrate was
concentrated to dryness under reduced pressure.
[0730] To a solution of D-Phenylglycine amide (850 mg, 7.5 mmol) in
tetrahydrofuran (10 mL) and 1,4-dioxane (4 mL) was added
1,3-propanesultone (818 mg, 6.8 mmol). The solution stirred at
reflux for 2 hours. The reaction was cooled to room temperature.
The solid was collected by filtration and washed with acetone
(2.times.20 mL). The solid was dissolved in 50% water/EtOH mL).
Dowex Marathon C ion exchange resin (strongly acidic) was added to
the solution. The suspension was stirred for 15 minutes before the
resin was removed by filtration. The filtrate was evaporated under
reduced pressure. The solid was suspended in acetone (50 mL),
filtered and dried in the vacuum oven (50.degree. C.), affording
the title compound (720 mg, 34%). .sup.1H NMR (D.sub.2O, 500 MHz)
.delta. ppm 7.38 (m, 5H), 4.92, (s, 1H), 3.00 (m, 1H), 2.90 (m,
1H), 2.78 (m, 2H), 1.97 (m, 2H). .sup.13C (D.sub.2O, 125 MHz)
.delta. ppm 170.14, 130.95, 130.24, 129.94, 128.74, 63.40, 47.99,
44.92, 21.27. [.alpha.].sub.D=+106.degree. (c=0.0016 in water),
ES-MS 273 (M+1).
Preparation of (2S)-2-[(3-sulfopropyl)amino]propanoic acid
(Compound GQ)
##STR00620##
[0732] L-alanine methylester hydrochloride (5.0 g, 35.8 mmol) was
treated with a saturated solution of K.sub.2CO.sub.3 (75 mL). The
mixture was extracted with EtOAc (3.times.75 mL). The organic
extracts were separated, combined, dried with Na.sub.2SO.sub.4,
filtered, evaporated under reduced pressure and dried in vacuo.
[0733] To a solution of L-alanine methylester (2.37 g, 23.3 mmol)
in tetrahydrofuran (35 mL) was added 1,3-propanesultone (2.41 g,
20.0 mmol). The solution was stirred at reflux for 2 hours. The
reaction was cooled to room temperature. The solid was collected by
filtration, washed with acetone (2.times.30 mL) and dried in
vacuo.
[0734] The ester (2.21 g, 9.8 mmol) was dissolved in 2M NaOH (2.40
g of NaOH and 30 mL of water). The reaction was stirred at room
temperature overnight. Dowex Marathon C ion exchange resin
(strongly acidic) was added to the solution. The suspension was
stirred for 15 minutes before the resin was removed by filtration.
The filtrate was evaporated under reduced pressure and lyophilized,
affording the title compound (1.81 g, 87%). .sup.1H NMR (D.sub.2O,
500 MHz) .delta. ppm 3.76 (m, 1H), 3.07 (m, 2H), 2.85 (t, 2H, J=7.3
Hz), 1.99 (m, 2H), 1.38 (d, 3H, J=7.3 Hz), 0.87 (d, 3H, J=6.8 Hz).
.sup.13C (D.sub.2O, 125 MHz) .delta. ppm 173.31, 56.66, 47.97,
44.76, 21.56, 14.51. [.alpha.].sub.D=+3.5.degree. (c=0.0023 in
water), ES-MS 210 (M-1).
Preparation of (2S)-3-phenyl-2-[3-sulfopropyl)amino]propanoic acid
(Compound GR)
##STR00621##
[0736] The N-(3-sulfo-propyl)-phenylalanine ethyl ester
(DM-258-069, 860 mg, 2.7 mmol) was dissolved in 2N NaOH (1.2 g of
NaOH and 15 mL of water). The reaction was stirred at room
temperature overnight. Dowex Marathon C ion exchange resin
(strongly acidic) was added to the solution. The suspension was
stirred for 15 minutes before the resin was removed by filtration.
The filtrate was evaporated under reduced pressure and lyophilized,
affording the title compound (654 mg, 84%). .sup.1H NMR (D.sub.2O,
500 MHz) .delta. ppm 7.20 (m, 5H), 3.96 (t, 1H, J=6.3 Hz), 3.11 (m,
4H), 2.80 (t, 2H, J=7.3 Hz), 1.95 (m, 2H). .sup.13C (D.sub.2O, 125
MHz) .delta. ppm 171.46, 134.03, 129.50, 129.28, 128.10, 62.02,
47.97, 45.64, 35.23, 21.39. [.alpha.].sub.D=+14.9.degree. (c=0.0013
in water), ES-MS 286 (M-1).
Preparation of
3-{[(1S)-1-isopropyl-2-oxopent-4-enyl]amino}-1-propanesulfonic acid
(Compound GS)
##STR00622##
[0738] L-Valine allylester p-tosylate (3.0 g, 9.1 mmol) was treated
with a saturated solution of K.sub.2CO.sub.3 (30 mL). The mixture
was extracted with EtOAc (3.times.30 mL). The organic extracts were
separated, combined, dried with Na.sub.2SO.sub.4, filtered and
evaporated under reduced pressure.
[0739] To a solution of L-valine allylester (1.30 g, 8.3 mmol) in
tetrahydrofuran (6 mL), 1,4-dioxane (6 mL) and MeOH (0.5 mL) was
added 1,3-propanesultone (910 mg, 7.5 mmol). The solution was
stirred at reflux for 2 hours. The reaction was cooled to room
temperature. The solvent was evaporated under reduced pressure. The
sticky paste was suspended in 20% acetone/ether. The solid was
filtered and dissolved EtOH (75 mL). Dowex Marathon C ion exchange
resin (strongly acidic) was added to the solution. The suspension
was stirred for 15 minutes before the resin was removed by
filtration. The filtrate was evaporated to dryness under reduced
pressure, affording the title compound (605 mg, 26%). .sup.1H NMR
(D.sub.2O, 500 MHz) .delta. ppm 5.84 (m, 1H), 5.27 (d, 1H, J=17.1
Hz), 5.19 (m, 1H, J=10.3 Hz), 3.91 (d, 1H, 3.9 Hz), 3.10 (t, 2H,
J=7.3 Hz), 2.85 (t, 2H, J=7.3 Hz), 2.22 (m, 1H), 2.03 (m, 2H), 0.93
(d, 3H, J=6.8 Hz), 0.85 (d, 3H, J=6.8 Hz). .sup.13C (D.sub.2O, 125
MHz) .delta. ppm 168.55, 130.90, 120.31, 67.58, 65.82, 48.09,
46.57, 29.53, 21.29, 18.25, 16.57. [.alpha.].sub.D=+5.0.degree.
(c=0.0011 in water), ES-MS 278 (M-1).
Preparation of
3-{[(1S)-1-(aminocarbonyl)-3-methylbutyl]amino}-1-propanesulfonic
acid (Compound GT)
##STR00623##
[0741] L-Leucinamide hydrochloride (5.0 g, 30.0 mmol) was treated
with a saturated solution of K.sub.2CO.sub.3 (100 mL). The mixture
was extracted with EtOAc (3.times.100 mL). The organic extracts
were separated, combined, dried with Na.sub.2SO.sub.4, filtered and
evaporated under reduced pressure.
[0742] To a solution of L-Leucinamide (3.20 g, 24.5 mmol) in
tetrahydrofuran (35 mL) was added 1,3-propanesultone (2.82 g, 23.3
mmol). The solution was stirred at reflux for 2 hours. The reaction
mixture was cooled to room temperature. The solid was filtered and
washed with acetone (2.times.25 mL). The solid was dissolved in 50%
EtOH/water (200 mL). Dowex Marathon C ion exchange resin (strongly
acidic, 25 g) was added to the solution. The suspension was stirred
for 15 minutes before the resin was removed by filtration. The
filtrate was evaporated to dryness under reduced pressure. The
solid was suspended in acetone (75 mL), and it was then filtered
and dried in vacuo, affording the title compound (3.13 g, 53%).
.sup.1H NMR (D.sub.2O, 500 MHz) .delta. ppm 3.79 (m, 1H), 3.04 (m,
2H), 2.85 (m, 2H), 2.02 (m, 2H), 1.65 (m, 1H), 1.54 (m, 2H), 0.80
(m, 6H). .sup.13C (D.sub.2O, 125 MHz) .delta. ppm 171.46, 59.42,
48.04, 45.46, 39.04, 24.27, 22.24, 21.49, 21.17.
[.alpha.].sub.D=+13.5.degree. (c=0.0026 in water), ES-MS 251
(M-1).
Preparation of
3-{[(1S)-1-(benzyloxycarbonyl)-3-methylbutyl]amino}-1-propanesulfonic
acid (Compound GU)
##STR00624##
[0744] L-Leucine benzylester p-tosylate (5.0 g, 12.7 mmol) was
treated with a saturated solution of K.sub.2CO.sub.3 (100 mL). The
mixture was extracted with EtOAc (3.times.100 mL). The organic
extracts were separated, combined, dried with Na.sub.2SO.sub.4,
filtered and evaporated under reduced pressure.
[0745] To a solution of L-Leucine benzylester (2.81 g, 12.7 mmol)
in tetrahydrofuran (6 mL), 1,4-dioxane (6 mL) and MeOH (6 mL) was
added 1,3-propane sultone (1.40 g, 11.5 mmol). The solution was
stirred at reflux for 2.5 hours. The reaction mixture was cooled to
room temperature. The solid was filtered and washed with acetone
(2.times.20 mL). The filtrate was evaporated under reduced
pressure. The residue was dissolved in acetone (20 mL). The product
was precipitated with Et.sub.2O (200 mL). The solid material was
filtered. Both solids were combined and dissolved in 50% EtOH/water
(200 mL). Dowex Marathon C ion exchange resin (strongly acidic) was
added to the solution. The suspension was stirred for 15 minutes
before the resin was removed by filtration. The filtrate was
evaporated under reduced pressure and lyophilized, affording the
title compound (1.87 g, 47%). .sup.1H NMR (DMSO, 500 MHz) .delta.
ppm 9.34 (s (broad), 1H), 7.39 (m, 5H), 5.25 (s, 2H), 4.10 (m, 1H),
3.09 (m, 2H), 2.60 (m, 2H), 1.95 (m, 2H), 1.64 (m, 3H), 0.86 (m,
6H). .sup.13C (DMSO, 125 MHz) .delta. ppm 168.90, 134.91, 128.53,
128.50, 128.41, 67.38, 57.37, 49.17, 45.79, 38.06, 24.07, 22.79,
21.78, 21.33. [.alpha.].sub.D=+1.8.degree. (c=0.0017 in water),
ES-MS 344 (M+1).
Preparation of
3-{[(1S)-1-(methyloxycarbonyl)-3-methylbutyl]amino}-1-propanesulfonic
acid (Compound GZ)
##STR00625##
[0747] L-Isoleucine methylester hydrochloride (5.0 g, 27.5 mmol)
was treated with a saturated solution of K.sub.2CO.sub.3 (100 mL).
The mixture was extracted with EtOAc (3.times.100 mL).
[0748] The organic layers were separated, combined, dried with
Na.sub.2SO.sub.4, filtered and evaporated under reduced
pressure:
[0749] To a solution of L-Isoleucine methlylester (3.43 g, 23.6
mmol) in acetone (30 mL) was added 1,3-propane sultone (2.62 g,
21.5 mmol). The solution was stirred at reflux for 2 h. The
reaction mixture was cooled to room temperature. The solid was
filtered and washed with acetone (2.times.20 mL). The filtrate was
evaporated under reduced pressure. The residue was suspended in
acetone (50 mL). The solid was filtered. The solid materials were
combined and dissolved in water (100 mL). Dowex Marathon C ion
exchange resin (strongly acidic) was added to the solution. The
suspension was stirred for 15 minutes before the resin was removed
by filtration. The filtrate was evaporated under reduced pressure.
The solid product was suspended in acetone (100 mL), filtered and
dried in vacuo, affording the title compound (3.23 g, 56%). .sup.1H
NMR (D.sub.2O, 500 MHz) .delta. ppm 4.00 (m, 1H), 3.74 (s, 3H),
3.14 (t, 2H, J=7.8 Hz), 2.89 (t, 2H, J=7.3 Hz), 2.05 (m, 2H), 1.97
(m, 1H), 1.41 (m. 1H), 1.23 (m, 1H), 0.83 (m, 6H). .sup.13C
(D.sub.2O, 125 MHz) .delta. ppm 169.29, 64.51, 53.55, 48.14, 46.52,
36.07, 25.92, 21.34, 13.76, 11.09. [.alpha.].sub.D=+22.6.degree.
(c=0.0023 in water), ES-MS 266 (M-1).
Preparation of
3-{[(1S)-1-(oxycarbonyl)-3-methylbutyl]amino}-1-propanesulfonic
acid (Compound HA)
##STR00626##
[0751] L-Isoleucine methylester hydrochloride (5.0 g, 27.5 mmol)
was treated with a saturated solution of K.sub.2CO.sub.3 (100 mL).
The mixture was extracted with EtOAc (3.times.100 mL). The organic
layers were separated, combined, dried with Na.sub.2SO.sub.4,
filtered and evaporated under reduced pressure.
[0752] To a solution of L-Isoleucine methlylester (3.43 g, 23.6
mmol) in acetone (30 mL) was added 1,3-propane sultone (2.62 g,
21.5 mmol). The solution was stirred at reflux for 2 hours. The
reaction mixture was cooled to room temperature. The solid was
filtered and washed with acetone (2.times.20 mL). The filtrate was
evaporated under reduced pressure. The residue was suspended in
acetone (50 mL). The solid was filtered. The solid materials were
combined and dissolved in water (100 mL). Dowex Marathon C ion
exchange resin (strongly acidic) was added to the solution. The
suspension was stirred for 15 minutes before the resin was removed
by filtration. The filtrate was evaporated under reduced pressure.
The solid product was suspended in acetone (100 mL), filtered and
dried in vacuo (3.23 g, 56%).
[0753] The solid (1.0 g, 3.7 mmol) was dissolved in 2M NaOH (30
mL). The reaction mixture was stirred at room temperature
overnight. Dowex Marathon C ion exchange resin (strongly acidic, 15
g) was added to the solution. The suspension was stirred for 15
minutes before the resin was removed by filtration. The filtrate
was coevaporated with EtOH and lyophilized, affording the title
compound (740 mg, 83%). .sup.1H NMR (D.sub.2O, 500 MHz) .delta. ppm
4.00 (m, 1H), 3.59 (m, 3H), 3.07 (t, 2H, J=7.3 Hz), 2.86 (m, 2H),
2.02 (m, 2H), 1.84 (m, 1H), 1.39 (m. 1H), 1.19 (m, 1H), 0.81 (m,
6H). .sup.13C (D.sub.2O, 125 MHz) .delta. ppm 169.29, 64.51, 53.55,
48.14, 46.52, 36.07, 25.92, 21.34, 13.76, 11.09.
[.alpha.].sub.D=+30.4.degree. (c=0.0031 in water), ES-MS 252
(M-1).
Preparation of
3-{[(1S)-1-carbamoyl-2-phenylethyl]amino}-1-propanesulfonic acid
(Compound HB)
##STR00627##
[0755] L-Phenylalaninamide hydrochloride (5.0 g, 24.9 mmol) was
treated with a saturated solution of K.sub.2CO.sub.3 (75 mL). The
mixture was extracted with EtOAc (3.times.75 mL). The organic
layers were separated, combined, dried with Na.sub.2SO.sub.4,
filtered and evaporated under reduced pressure.
[0756] To a solution of L-Phenylalaninamide (3.93 g, 23.9 mmol) in
acetonitrile (25 mL) was added 1,3-propane sultone (2.70 g, 21.8
mmol). The solution was stirred at reflux for 2 hours. The reaction
mixture was cooled to room temperature. The solid was filtered and
washed with acetonitrile (2.times.25 mL). The solid product was
suspended in EtOH (100 mL). The suspension was stirred at reflux
for 1 hour. The solid material was filtered and dried in vacuo,
affording the title compound (5.18 g, 83%). .sup.1H NMR (D.sub.2O,
500 MHz) .delta. ppm 7.24 (m, 3H), 7.16 (m, 2H), 4.02 (m, 1H), 3.15
(m, 1H), 3.01 (m, 3H), 2.83 (m, 2H), 2.02 (m, 2H), 1.98 (m, 2H).
.sup.13C (D.sub.2O, 125 MHz) .delta. ppm 170.48, 133.72, 129.58,
129.21, 128.11, 61.55, 47.95, 45.52, 36.21, 21.44.
[.alpha.].sub.D=+23.1.degree. (c=0.0021 in water), ES-MS 285
(M-1).
Preparation of
3-{[(1R)-1-(methoxycarbonyl)-3-methylbutyl]amino}-1-propanesulfonic
acid (Compound HC)
##STR00628##
[0758] D-Leucine methylester hydrochloride (2.63 g, 14.5 mmol) was
treated with a saturated solution of K.sub.2CO.sub.3 (50 mL). The
aqueous mixture was extracted with EtOAc (3.times.50 mL). The
organic layers were separated, combined, dried with
Na.sub.2SO.sub.4, filtered and evaporated under reduced
pressure.
[0759] To a solution of D-Leucine methylester (1.58 g, 10.9 mmol)
in acetonitrile (35 mL) was added 1,3-propanesultone (1.21 g, 9.9
mmol). The solution was stirred at reflux for 2 hours. The reaction
mixture was cooled to room temperature. The solid material was
collected by filtration, recrystallized from EtOH and dried in
vacuo, affording the title compound (1.59 g, 60%). .sup.1H NMR
(D.sub.2O, 500 MHz) .delta. ppm 3.98 (m, 1H), 3.70 (s, 3H), 3.11
(m, 2H), 2.85 (m, 2H), 2.00 (m, 2H), 1.72 (m, 1H), 1.60 (m, 2H),
0.81 (m, 6H). .sup.13C (D.sub.2O, 125 MHz) .delta. ppm 170.51,
58.78, 53.69, 47.97, 45.36, 38.09, 24.34, 22.07, 21.51, 20.82.
[.alpha.].sub.D=+13.1.degree. (c=0.0019 in water), ES-MS 266
(M-1).
Preparation of
3-{[(1R)-1-(aminocarbonyl)-2-methylpropyl]amino}-1-propanesulfonic
acid (Compound HD)
##STR00629##
[0761] D-Valinamide hydrochloride (2.49 g, 14.7 mmol) was treated
with a solution of K.sub.2CO.sub.3 (50 mL). The organic mixture was
extracted with EtOAc (3.times.50 mL). The organic extracts were
separated, combined, dried with Na.sub.2SO.sub.4, filtered,
evaporated under reduced pressure and dried in vacuo.
[0762] To a solution of D-valinamide (1.76 g, 14.7 mmol) in
acetonitrile (30 mL) was slowly added 1,3-propanesultone (1.75 g,
14.4 mmol). The solution was stirred at reflux for 2 hours. The
reaction mixture was cooled to room temperature. The solid was
collected by filtration, washed with acetonitrile (2.times.25 mL).
The solid product was dissolved in water (75 mL). Dowex Marathon C
resin (strongly acidic) was added to the solution. The suspension
was stirred for 15 minutes before the resin was removed by
filtration. The filtrate was evaporated under reduced pressure. The
solid material was suspended in acetone (50 mL), filtered and dried
in vacuo, affording the title compound (1.57 g, 51%). .sup.1H NMR
(D.sub.2O, 500 MHz) .delta. ppm 3.80 (m, 1H), 3.19 (m, 2H), 3.00
(m, 2H), 2.25 (m, 1H), 2.16 (m, 2H), 1.08 (d, 3H, J=6.8 Hz), 1.02
(d, 3H, J=6.8 Hz). .sup.13C (D.sub.2O, 125 MHz) .delta. ppm 169.94,
65.86, 48.10, 46.27, 29.54, 21.23, 17.99, 17.02.
[.alpha.].sub.D=-12.4.degree. (c=0.0037 in water), ES-MS 237
(M-1).
Preparation of
3-{[(1R)-1-carbamoyl-2-phenylethyl]amino}-1-propanesulfonic acid
(Compound HE)
##STR00630##
[0764] D-Phenylalaninamide hydrochloride (2.53 g, 12.6 mmol) was
treated with a saturated solution of K.sub.2CO.sub.3 (50 mL). The
mixture was extracted with EtOAc (3.times.50 mL). The organic
layers were separated, combined, dried with Na.sub.2SO.sub.4,
filtered and evaporated under reduced pressure.
[0765] To a solution of D-Phenylalaninamide (1.83 g, 11.1 mmol) in
acetonitrile (20 mL) was added 1,3-propane sultone (1.29 g, 10.6
mmol). The solution was stirred at reflux for 2.5 hours. The
reaction mixture was cooled to room temperature. The solid was
filtered and washed with acetonitrile (2.times.20 mL). The solid
product was suspended in EtOH (75 mL). The suspension was stirred
at reflux for 1 hours. The solid material was filtered, washed with
acetone (1.times.25 mL) and dried in vacuo, affording the title
compound (2.62 g, 89%). .sup.1H NMR (D.sub.2O, 500 MHz) .delta. ppm
7.28 (m, 3H), 7.19 (m, 2H), 4.05 (m, 1H), 3.19 (dd, 1H, J=5.3 Hz,
14.2 Hz), 3.04 (m, 3H), 2.86 (t, 2H, J=5.8 Hz), 2.03 (m, 2H).
.sup.13C (D.sub.2O, 125 MHz) .delta. ppm 170.39, 133.73, 129.62,
129.26, 128.15, 61.57, 47.99, 45.57, 36.21, 21.45.
[.alpha.].sub.D=-20.7.degree. (c=0.0038 in water), ES-MS 285
(M-1).
Preparation of
3-({(1S)-1-[(benzyloxy)carbonyl]-2-methylbutyl}amino)-1-propanesulfonic
acid (Compound HF)
##STR00631##
[0767] L-Isoleucine benzylester p-tosylate (2.50 g, 6.4 mmol) was
treated with a saturated solution of K.sub.2CO.sub.3 (30 mL). The
mixture was extracted with EtOAc (3.times.30 mL). The organic
extracts were separated, combined, dried with Na.sub.2SO.sub.4,
filtered and evaporated under reduced pressure.
[0768] To a solution of L-isoleucine benzylester (1.41 g, 6.4 mmol)
in acetonitrile (12 mL) was added 1,3-propane sultone (706 mg, 5.8
mmol). The solution was stirred at reflux for 2 hours. The reaction
mixture was cooled to room temperature. The solid was filtered and
washed with acetone (2.times.20 mL). The solid material was
dissolved in 50% EtOH/water (50 mL). Dowex Marathon C ion exchange
resin (strongly acidic, 10 g) was added to the solution. The
suspension was stirred for 15 minutes before the resin was removed
by filtration. The filtrate was evaporated under reduced pressure
and lyophilized affording the title compound (778 mg, 39%). .sup.1H
NMR (D.sub.2O, 500 MHz) .delta. ppm 7.49 (m, 5H), 5.42 (d, 1H,
J=11.7 Hz), 5.31 (d, 1H, J=11.7 Hz), 3.24 (m, 2H), 2.98 (m, 2H),
2.13 (m, 3H), 1.46 (m, 1H), 1.34 (m, 1H), 0.93 (m, 6H). .sup.13C
(D.sub.2O, 125 MHz) .delta. ppm 168.53, 134.69, 129.28, 129.22,
129.06, 68.86, 64.45, 48.12, 46.56, 36.21, 25.97, 21.31, 13.73,
11.05. [.alpha.].sub.D=-1.5.degree. (c=0.0031 in water), ES-MS 342
(M-1).
Preparation of
3-{[(1R)-1-(aminocarbonyl)-3-methylbutyl]amino}-1-propanesulfonic
acid (Compound HG)
##STR00632##
[0770] D-Leucinamide hydrochloride (1.0 g, 6.0 mmol) was treated
with a saturated solution of K.sub.2CO.sub.3 (30 mL). The aqueous
mixture was extracted with EtOAc (3.times.30 mL). The organic
extracts were separated, combined, dried with Na.sub.2SO.sub.4,
filtered and evaporated under reduced pressure.
[0771] To a solution of D-Leucinamide (6.0 mmol) in acetonitrile
(35 mL) was added 1,3-propanesultone (666 mg, 5.5 mmol). The
solution was stirred at reflux for 2 hours. The reaction mixture
was cooled to room temperature. The solid was filtered and washed
with MeCN (2.times.20 mL). The solid was suspended in EtOH (50 mL).
The suspension was stirred at reflux for 1 hour. The mixture was
cooled to room temperature. The solid material was filtered, washed
with acetone (1.times.20 mL) and dried in a vacuum oven (50.degree.
C.), affording the title compound (1.03 g, 74%): .sup.1H NMR
(D.sub.2O, 500 MHz) .delta. ppm 3.81 (m, 1H), 3.07 (m, 2H), 2.85
(t, 2H, J=7.3 Hz), 2.03 (m, 2H), 1.68 (t, 1H, J=7.8 Hz), 1.58 (m,
2H), 0.83 (m, 6H). .sup.13C (D.sub.2O, 125 MHz) .delta. ppm 171.45,
59.39, 48.01, 45.41, 39.02, 24.24, 22.21, 21.47, 21.13.
[.alpha.].sub.D=-13.7.degree. (c=0.0019 in water), ES-MS 251
(M-1).
3-[(1-methylcyclopentyl)amino]-1-propanesulfonic acid (Compound
FQ)
##STR00633##
[0773] For the Ritter reaction, the flask was closed with a septum
and connected to a 20% NaOH scrubber. Sodium cyanide (powdered, 5.5
g, 112 mmol) was added to acetic acid (30 mL) in one portion. The
mixture was stirred for 10 minutes at room temperature. A solution
of sulfuric acid (16 mL) in acetic acid (15 mL) was added dropwise
over a 20 minute period. Then, a solution of
1-methyl-1-cyclopentanol (10 g, 99.8 mmol) in acetic acid (5 mL)
was added dropwise over a 5 minute period. The mixture was stirred
at room temperature for 22 hours then poured over ice (approx. 100
g). The pH of the solution was adjusted to 9 with the addition of
50% NaOH (about 135 g). The layers were separated and the aqueous
layer was extracted with ether (1.times.40 mL). The combined
organic layers were washed with saturated sodium carbonate
(1.times.10 mL) then dried over sodium sulfate. The ether was
evaporated under reduced pressure to afford light brown oil (12.04
g, 94.7 mmol, 95%). The oil showed to be a mixture of cis and trans
formamide but what otherwise pure enough to be used as such.
.sup.1H NMR (500 MHz, CDCl.sub.3) .delta. [1.40 and 1.45 (s, 3H)],
1.68-1.76 (m, 7H), 1.97-1.98 (m, 1H), [5.42 and 6.24 (br s, 1H)],
[8.05 (s) and 8.24 (d, J=12.2 Hz) for 1H]; .sup.13C NMR (125 MHz,
CDCl.sub.3) .delta. 23.1, 23.6, 25.5, 28.2, 39.5, 40.7, 60.7, 61.3,
160.7, 163.9
[0774] A solution of NaOH (25%, 80 mL) was added to the crude
1-methyl-1-cyclopentylformamide (12.00 g, 94.7 mmol). The mixture
was heated to reflux for 2.5 hours then cooled at room temperature.
Some sodium chloride (20 g) was added to facilitate the phase
separation. The layers were separated and the aqueous layer was
extracted with toluene (1.times.15 mL). The combined organic layers
were agitated. The addition of isopropyl ether (2.5 mL, chloroform
(1 g) and cyclohexane (6.5 g) did not improved the separation of
the solution. The combined organic layers were washed with brine
(1.times.10 mL) then dried over sodium sulfate and filtered. The
filtrate was used as such in the next step. .sup.1H NMR (500 MHz,
CDCl.sub.3) .delta. 1.22 (s, 3H), 1.47-1.75 (4 m, 9H): .sup.13C NMR
(125 MHz, CDCl.sub.3) .delta. 24.1, 29.6, 42.2, 58.4
[0775] A solution of 1,3-propanesultone (9.4 g, 75 mmol) in
2-butanone (35 mL) was added dropwise to a the crude solution of
1-methyl-1-cyclopentylamine (mixture of solvent from previous
step).
[0776] The mixture was heated to reflux for 20 hours then was
cooled to room temperature. The solid was collected by
suction-filtration and rinsed with acetone (2.times.10 mL). The
solid was dried overnight at 45.degree. C. in the vacuum oven. The
title compound was obtained as a fine white solid (16.26 g, 73.47
mmol, 74% overall yield). .sup.1H NMR (500 MHz, D.sub.2O) .delta.
1.31 (s, 3H), 1.591.6-1-85 (m, 8H), 2.03-2.06 (m, 2H), 2.96 (t,
J=6.8 Hz, 2H), 3.15 (t, J=7.6 Hz, 2H); .sup.13C NRM (125 MHz,
D.sub.2O) .delta. 22.1, 22.5, 23.6, 36.5, 41.4, 48.1, 66.8.1; ES-MS
220 (M-H)
3-[(1-methylcyclohexyl)amino]-1-propanesulfonic acid (Compound
FR)
##STR00634##
[0778] For the Ritter reaction, the flask was closed with a septum
and connected to a 20% NaOH scrubber. Potassium cyanide (powdered,
3.3 g, 50 mmol) was added in portions to acetic acid (13 mL). The
mixture was stirred for 10 minutes at room temperature. A solution
of sulfuric acid (7 mL) in acetic acid (7 mL) was added drop-wise
over a 10 minute period. Then, a solution of
1-methyl-1-cyclohexanol (5 g, 43.8 mmol) in acetic acid (4 mL) was
added dropwise over a 5 minute period. The mixture was stirred at
room temperature for 22 hours then poured over ice (approx. 50 g).
The pH of the solution was adjusted to 9 with the addition of 50%
NaOH (about 70 g). The layers were separated and the aqueous layer
was extracted with ether (2.times.20 mL). The combined organic
layers were washed with saturated sodium carbonate (1.times.10 mL)
then dried over sodium sulfate. The ether was evaporated under
reduced pressure to afford a clear yellow oil (6.56 g,
quantitative). The oil showed to be a mixture of cis and trans
formamide but what otherwise pure enough to be used as such.
.sup.1H NMR (500 MHz, CDCl.sub.3) .delta. 1.33-1.53 (3m, 11H), 1.67
(br s, 1H), 1.99 (m, 1H), [5.16 and 6.09 (br s, 1H)], [8.11 (s) and
8.25 (d, J=12.2 Hz) for 1H]; .sup.13C NMR (125 MHz, CDCl.sub.3)
23.1, 23.6, 25.5, 28.2, 39.5, 40.7, 60.7, 61.3, 160.7, 163.9
[0779] A solution of NaOH (20%, 40 mL) was added to the crude
1-methyl-1-cyclohexylformamide (43.8 mmol). The mixture was heated
to reflux for 3 hours then cooled at room temperature. Some sodium
chloride (7.5 g) was added to facilitate the phase separation. The
layers were separated and the aqueous layer was extracted with MTBK
(1.times.10 mL). The combined organic layers were washed with brine
(1.times.5 mL) the dried over sodium sulfate and filtered. The
filtrate was used as such in the next step. .sup.1H NMR (500 MHz,
CDCl.sub.3) .delta. 1.08 (s, 3H), 1.33-1.51 (m, 10H); .sup.13C NMR
(125 MHz, CDCl.sub.3) .delta. 22.8, 25.8, 29.6, 40.8, 48.6
[0780] A solution of 1,3-propanesultone (5.00 g, 40 mmol) in
toluene (10 mL) was added dropwise to a the crude solution of
1-methyl-1-cyclohexylamine in MTBK (total volume 30 mL). The
mixture was heated to reflux for 18 hours then cooled to room
temperature. The solid was collected by suction filtration, rinsed
with acetone (2.times.8 mL). The solid was dried overnight at
45.degree. C. in the vacuum oven. The title compound was obtained
as a fine white solid (9.22 g). However, the proton NMR and the
ES-MS were not clean. The solid was suspended in methanol (45 mL)
and the suspension was warmed to reflux. Water (12 mL) was added
dropwise until a clear yellow solution was obtained. The mixture
was slowly cool to room temperature with stirring. The solid was
collected by suction-filtration, rinsed with methanol (2.times.5
mL). Another crop was collected from the filtrate. Both crops were
dried overnight at 45.degree. C. in the vacuum oven. The title
compound was obtained as a fine white solid (6.82 g, 29.0 mmol, 66%
overall yield). Both crops were identical and were mixed for
submitting the compound. .sup.1H NMR (500 MHz, D.sub.2O) .delta.
1.04-1.11 (m, 1H), 1.19 (s, 3H), 1.31 (q, J=12.2 Hz, 2H), 1.40 (qt,
J=12.2 Hz, 2H), 1.46-1.62 (m, 2H), 1.63 (br d, J=11.7 Hz, 2H), 1.94
(q, J=7.3 Hz, 2H), 2.86 (t, J=7.1 Hz, 2H), 3.03 (t, J=7.6 Hz, 2H);
.sup.13C NMR (125 MHz, D.sub.2O) .delta. 19.1, 21.5, 22.0, 24.6,
34.1, 39.1, 48.2, 60.2; ES-MS 236 (M+H).
3[(1-methylcycloheptyl)amino]-1-propanesulfonic acid (Compound
FS)
##STR00635##
[0782] For the Ritter reaction, the flask was closed with a septum
and connected to a 20% NaOH scrubber. Potassium cyanide (powdered,
2.8 g, 43 mmol) was added in portions to acetic acid (10 mL). The
mixture was stirred for 10 minutes at room temperature. A solution
of sulfuric acid (7 mL) in acetic acid (7 mL) was added drop-wise
over a 20 minute period. Then, the 1-methyl-1-cycloheptanol (5 g,
39.0 mmol) was added drop-wise over 5 minutes. The mixture was
stirred at room temperature for 22 h then cooled to 0.degree. C.
with a ice/water bath. The pH of the solution was adjusted to 9
with the addition of 50% NaOH (about 70 g). The layers were
separated and the aqueous layer was extracted with ether
(1.times.20 mL). The combined organic layers were washed with
saturated sodium carbonate (1.times.5 mL) then dried over sodium
sulfate. The ether was evaporated under reduced pressure to afford
a clear yellow oil (5.71 g, 94%). The oil showed to be a mixture of
cis and trans formamide but what otherwise pure enough to be used
as such. .sup.1H NMR (500 MHz, CDCl.sub.3) .delta. 1.34 (s, 1.5H),
1.43 (s, 1.5H), 1.49-1.60 (m, 8H), 1.96-2.00 (m, 1H), [5.28 and
5.95 (br s, 1H)], [8.06 (s) and 8.28 (d, J=12.2 Hz) for 1H];
.sup.13C NMR (125 MHz, CDCl.sub.3) .delta. 22.2, 22.4, 27.7, 29.3,
29.4, 30.7, 40.5, 42.5, 56.0, 57.4, 160.5, 163.3
[0783] A solution of NaOH (25%, 40 mL) was added to the crude
1-methyl-1-cycloheptylformamide (5.7 g). The mixture was heated to
reflux for 3 hours then cooled at room temperature. Some sodium
chloride (7.5 g) was added to facilitate the phase separation. The
layers were separated and the aqueous layer was extracted with MTBK
(1.times.10 mL). The combined organic layers were washed with brine
(1.times.5 mL) the dried over sodium sulfate and filtered. The
filtrate was used as such in the next step. .sup.1H NMR (500 MHz,
CD.sub.3OD) .delta. 1.10 (s, 3H), 1.40-1.48 (m, 2H), 1.5-1.65 (m,
10H); .sup.13C NMR (125 MHz, CD.sub.3OD) .delta. 24.0, 31.2, 31.4,
44.4, 53.6
[0784] A solution of 1,3-propanesultone (4.3 g, 35 mmol) in toluene
(10 mL) was added dropwise to a the crude solution of
1-methyl-1-cycloheptylamine in MTBK (total volume 30 mL). The
mixture was heated to reflux for 18 hours then was cooled to room
temperature. The solid was collected by suction filtration, rinsed
with acetone (2.times.5 mL). The solid was dried overnight at
45.degree. C. in the vacuum oven. The title compound was obtained
as a fine white solid (7.77 g, 31.2 mmol, 80% overall yield).
.sup.1H NMR (500 MHz, D.sub.2O) .delta. 1.27 (s, 3H), 1.40-1.60 (m,
8H), 1.71-1.81 (m, 4H), 2.00-2.06 (m, 2H), 2.95 (t, J=6.3 Hz, 2H),
3.13 (t, J=7.1 Hz, 2H); .sup.13C NMR (125 MHz, D.sub.2O) .delta.
22.0, 22.1, 23.3, 29.5, 37.1, 40.0, 48.3, 64.0; ES-MS 250 (M+H)
Preparation of
3-{[(1R)-1-(benzyloxycarbonyl)-3-methylbutyl]amino}-1-propanesulfonic
acid (Compound HI)
##STR00636##
[0786] D-Leucine benzylester p-tosylate (2.5 g, 6.3 mmol) was
treated with an aqueous solution of K.sub.2CO.sub.3 (30 mL). The
mixture was extracted with EtOAc (3.times.30 mL). The organic
extracts were separated, combined, dried with Na.sub.2SO.sub.4,
filtered, evaporated under reduced pressure and dried in vacuo.
[0787] To a solution of D-Leucine benzylester (6.3 mmol) in
acetonitrile (9 mL) and MeOH (3 mL) was added 1,3-propane sultone
(691 mg, 5.7 mmol). The solution was stirred at reflux for 2.5
hours. The reaction mixture was cooled to room temperature. The
solid material was filtered and washed with aconitrile (2.times.20
mL). The solid was dissolved in 20% water/EtOH (75 mL). Dowex
Marathon C ion exchange resin (strongly acidic) was added to the
solution. The suspension was stirred for 15 minutes before the
resin was removed by filtration. The filtrate was evaporated under
reduced pressure and dried in vacuo, affording the title compound
(960 mg, 49%). .sup.1H NMR (D.sub.2O, 500 MHz) .delta. ppm 7.52 (m,
5H), 5.41 (d, 1H, J=12.2 Hz), 5.35 (d, 1H, J=12.2 Hz), 4.16 (m,
1H), 3.22 (m, 2H), 2.97 (t, 2H, J=6.8 Hz), 2.16 (m, 2H), 1.88 (m,
1H), 1.79 (m, 1H), 1.76 (m, 1H), 0.94 (d, 6H, J=3.9 Hz). .sup.13C
(DMSO, 125 MHz) .delta. ppm 169.60, 135.62, 129.24, 129.21, 129.11,
68.08, 58.09, 49.87, 46.48, 24.77, 23.50, 22.50, 22.04.
[.alpha.].sub.D=-2.1.degree. (c=0.00095 in water), ES-MS 344
(M+1).
Preparation of
3-[(5-hydroxy-1,5-dimethylhexyl)amino]-1-propanesulfonic acid
(Compound HJ)
##STR00637##
[0789] To a solution of 6-amino-2-methyl-2-heptanol (2.5 g, 17.2
mmol) in acetonitrile (22 mL) was added 1,3-propane sultone (2.0 g,
16.4 mmol). The solution was stirred at reflux for 2 hours. The
reaction mixture was cooled to room temperature. The solid material
was filtered and washed with acetonitrile (2.times.20 mL). The
solid was dissolved in 20% MeOH (75 mL). Dowex Marathon C ion
exchange resin (strongly acidic) was added to the solution. The
suspension was stirred for 15 minutes before the resin was removed
by filtration. The filtrate was evaporated under reduced pressure.
The solid was suspended in acetone (150 mL), and then the solid
material was filtered and dried in vacuo, affording the title
compound (3.08 g, 70%). .sup.1H NMR (D.sub.2O, 500 MHz) .delta. ppm
3.19 (m, 1H), 3.08 (m, 2H), 2.88 (t, 2H, J=7.3 Hz), 1.99 (m, 2H),
1.60 (m, 2H), 1.36 (m, 4H), 1.18 (d, 3H, J=6.8 Hz), 1.07 (s, 6H).
.sup.13C (D.sub.2O, 125 MHz) .delta. ppm 71.63, 54.73, 48.08,
43.46, 42.27, 32.97, 27.78, 27.73, 21.64, 19.67, 15.43. ES-MS 268
(M+1).
Preparation of
3-{[(1R)-2-methoxy-1-methyl-2-oxoethyl]amino}-1-propanesulfonic
acid (Compound HK)
##STR00638##
[0791] D-Alanine methylester hydrochloride (3.0 g, 21.5 mmol) was
treated with a aqueous solution of K.sub.2CO.sub.3 (50 mL). The
mixture was extracted with EtOAc (3.times.50 mL). The organic
extracts were separated, combined, dried with Na.sub.2SO.sub.4,
filtered and evaporated under reduced pressure.
[0792] To a solution of D-Alanine methylester (1.33 g, 12.9 mmol)
in acetonitrile (15 mL was added 1,3-propane sultone (1.42 g, 11.7
mmol). The solution was stirred at reflux for 2 hours. The reaction
mixture was cooled to room temperature. The solid material was
filtered and washed with acetonitrile (2.times.15 mL). The solid
was dissolved in water (30 mL). Dowex Marathon C ion exchange resin
(strongly acidic) was added to the solution. The suspension was
stirred for 15 minutes before the resin was removed by filtration.
The filtrate was evaporated under reduced pressure and dried in
vacuo, affording the title compound (1.52 g, 42%). NMR (D.sub.2O,
500 MHz) .delta. ppm 4.07 (m, 1H), 3.72 (s, 3H), 3.14 (m, 2H), 2.89
(t, 2H, J=7.3 Hz), 2.03 (m, 2H), 1.46 (dd, 3H, J=1.95 Hz, 7.3 Hz).
.sup.13C (DMSO, 125 MHz) .delta. ppm 170.74, 55.62, 53.82, 47.96,
44.76, 21.53, 14.03. [.alpha.].sub.D=+1.4.degree. (c=0.0088 in
water), ES-MS 224 (M-1).
Preparation of
4-(1,2,3,4-tetrahydro-1-naphthylamino)-2-butanesulfonic acid
(Compound JF)
##STR00639##
[0794] To a solution of 1,2,3,4-tetrahydro-1-naphthylamine (2.01 g,
13.6 mmol) in 2-butanone (15 mL) was added 2,4-butane sultone (1.95
g, 14.3 mmol). The solution was stirred at for 2 hours. The
reaction was cooled to room temperature. The solid was collected by
filtration, washed with acetone (2.times.25 mL) and dried in vacuo.
.sup.1H NMR (DMSO, 500 MHz) .delta. ppm 8.56 (s (broad), 1H), 7.49
(dd, 1H, J=8.0 Hz, 11.9 Hz), 7.29 (m, 1H), 7.26 (m, 1H), 7.19 (d,
1H, J=8.0 Hz), 4.40 (d, 1H, J=13.7 Hz), 3.14 (m, 2H), 2.75 (m, 3H),
1.96 (m, 5H), 1.40 (m, 1H), 1.23 (m, 3H). .sup.13C (DMSO, 125 MHz)
.delta. ppm 138.81, 131.81, 130.40, 130.28, 130.16, 129.41, 126.76,
126.73, 54.97, 54.58, 54.08, 44.18, 43.23, 29.50, 28.84, 24.84,
24.76, 18.23, 18.20, 17.79, 17.01. ES-MS 284 (M+1).
Preparation of 4-(octylamino)-2-butanesulfonic acid (Compound
JG)
##STR00640##
[0796] To a solution of octylamine (2.00 g, 15.5 mmol) in
2-butanone (17 mL) was added 2,4-butane sultone (2.21 g, 16.2
mmol). The solution was stirred at reflux for 2 hours. The reaction
was cooled to room temperature. The solid was collected by
filtration, washed with acetone (2.times.25 mL) and dried in vacuo.
It was suspended in a solution of 25% EtOH/acetone (50 mL). The
suspension was stirred for 5 minutes. The solid was collected by
filtration, washed with acetone (2.times.25 mL) and dried in vacuo.
NMR (DMSO, 500 MHz) .delta. ppm 8.45 (s (broad), 1H), 3.01 (m, 1H),
2.84 (m, 2H), 2.58 (m, 1H), 1.92 (m, 1H), 1.75 (m, 1H), 1.51 (m,
2H), 1.10 (d, 1H, J=6.8 Hz), 0.85 (t, 3H, J=6.8 Hz). .sup.13C
(DMSO, 125 MHz) .delta. ppm 53.05, 47.27, 46.15, 31.83, 29.42,
29.13, 26.51, 26.27, 22.75, 17.19, 14.64. ES-MS 266 (M+1).
Preparation of 4-(adamantyl)amino-2-butanesulfonic acid (Compound
JH)
##STR00641##
[0798] 1-adamantaneamine hydrochloride (2.51 g, 13.3 mmol) was
treated with 1N NaOH (20 mL) and CH.sub.2Cl.sub.2 (3.times.20 mL).
The organic extracts were combined, dried with Na.sub.2SO.sub.4,
filtered, evaporated under reduced pressure and dried in vacuo.
[0799] To a solution of 2-adamantanamine (1.99 g, 13.1 mmol) in
acetonitrile (15 mL) was added 2,4-butane sultone (1.87 g, 13.8
mmol). The solution was stirred at reflux for 2 hours. The reaction
was cooled to room temperature. The solid was collected by
filtration, washed with acetonitrile (3.times.25 mL) and dried in
vacuo. NMR (DMSO, 500 MHz) .delta. ppm 8.53 (s (broad), 1H), 3.33
(m, 2H), 2.61 (m, 1H), 2.10 (s, 3H), 1.93 (m, 1H), 1.77 (m, 7H),
1.61 (m, 6H), 1.12 (d, 1H, J=6.8 Hz). .sup.13C (DMSO, 125 MHz)
.delta. ppm 56.20, 53.34, 35.85, 29.75, 29.04, 17.206. ES-MS 288
(M+1).
Preparation of 4-(2-adamantyl)amino-2-butanesulfonic acid (Compound
JI)
##STR00642##
[0801] The 2-adamantanamine hydrochloride (2.50 g, 13.3 mmol) was
treated with 1N NaOH (2 0 mL) and CH.sub.2Cl.sub.2 (3.times.20 mL).
The organic extracts were combined, dried with Na.sub.2SO.sub.4,
filtered, evaporated under reduced pressure and dried in vacuo.
[0802] To a solution of 1-adamantanamine (1.99 g, 13.1 mmol) in
acetonitrile (15 mL) was added 2,4-butane sultone (1.87 g, 13.8
mmol). The solution was stirred at reflux for 2 hours. The reaction
was cooled to room temperature. The solid was collected by
filtration, washed with acetonitrile (2.times.25 mL) and dried in
vacuo. .sup.1H NMR (DMSO, 500 MHz) .delta. ppm 3.20 (m, 1H), 3.05
(m, 2H), 2.67 (m, 1H), 2.07 (m, 2H), 2.00 (m, 1H), 1.95 (m, 4H),
1.82 (m, 4H), 1.69 (m, 4H), 1.55 (m, 4H), 1.12 (d, 1H, J=8.0 Hz).
.sup.13C (DMSO, 125 MHz) .delta. ppm 62.27, 53.91, 44.33, 37.30,
36.82, 36.77, 30.30, 30.20, 29.57, 29.50, 28.95, 17.12, 26.85,
17.44. ES-MS 288 (M+1).
Preparation of 4-(bicyclo[2.2.1]hept-2-ylamino)-2-butanesulfonic
acid (Compound JJ)
##STR00643##
[0804] To a solution of exo-2-aminonorbornane (1.0 g, 9.0 mmol) in
tetrahydrofuran (THF, 10 mL) was added 2,4-butane sultone (1.28 g,
9.3 mmol). The solution was stirred at reflux for 3 hours. The
reaction was cooled to room temperature. The solid was collected by
filtration, washed with THF (2.times.20 mL) and dried in vacuo.
.sup.1H NMR (DMSO, 500 MHz) .delta. ppm 8.43 (s (broad), 1H), 2.96
(m, 3H), 2.62 (m, 1H), 2.38 (m, 1H), 2.28, (m, 1H), 1.91 (m, 1H),
1.82 (m, 1H), 1.61 (m, 1H), 1.54 (m, 2H), 1.42 (m, 2H), 1.12 (m,
6H). .sup.13C (DMSO, 125 MHz) .delta. ppm 60.92, 60.79, 53.61,
53.21, 44.55, 44.36, 39.80, 39.55, 36.27, 36.15, 36.11, 35.98,
35.19, 35.13, 29.62, 29.43, 28.07, 26.88, 17.56, 14.11. ES-MS 248
(M+1).
Preparation of
4-(azoniabicyclo[2.2.2]oct-2-ylamino)-2-butanesulfonate (Compound
JK)
##STR00644##
[0806] Quinuclidine hydrochloride (2.50 g, 16.9 mmol) was treated
with 1N NaOH (20 mL) and CH.sub.2Cl.sub.2 (4.times.20 mL). The
organic extracts were combined, dried with Na.sub.2SO.sub.4,
filtered, evaporated under reduced pressure and dried in vacuo.
[0807] To a solution of quinuclidine (900 g, 8.2 mmol) in
tetrahydrofuran (THF, 18 mL) and MeOH (0.5 mL) was added 2,4-butane
sultone (1.16 g, 8.6 mmol). The solution was stirred at reflux
overnight. The reaction was cooled to room temperature. The solid
was collected by filtration, washed with THF (2.times.25 mL) and
dried in vacuo. NMR (DMSO, 500 MHz) .delta. ppm 3.40 (m, 7H), 3.20
(td, 1H, J=3.9 Hz, 12.7 Hz), 2.38 (m, 1H), 2.01 (m, 2H), 1.83, (m,
6H), 1.70 (m, 1H), 1.10 (d, 3H, J=12.7 Hz). .sup.13C (DMSO, 125
MHz) .delta. ppm 62.63, 54.23, 52.18, 25.67, 24.07, 19.78, 17.04.
ES-MS 208 (M+1).
Preparation of 4-[(dl)-1-hydroxy-2-pentyl]amino-2-butane sulfonic
acid (Compound JL)
##STR00645##
[0809] To a solution of DL-2-aminopentanol (1.0 g, 9.7 mmol) in
tetrahydrofuran (11 mL) was added 2,4-butane sultone (1.45 g, 10.2
mmol). The solution was stirred at reflux for 4 hours. The reaction
was cooled to room temperature. The supernatant was removed and the
solid was dried in vacuo. The product was suspended in 2-propanol
(100 mL) and the mixture was stirred for 5 minutes. The white solid
was filtered, washed with 2-propanol and dried in vacuo. NMR
(D.sub.2O, 500 MHz) .delta. ppm 3.74 (d, 1H, J=12.7 Hz,), 3.59 (dd,
1H, 1=5.4 Hz, 12.9 Hz), 3.13 (m, 3H), 2.89 (m, 1H), 2.07 (m, 1H),
1.82, (m, 1H), 1.52 (m, 2H), 1.28 (m, 2H), 1.18 (d, 3H, J=6.8 Hz).
.sup.13C (D.sub.2O, 125 MHz) .delta. ppm 59.43, 58.80, 53.47,
42.88, 29.30, 28.43, 18.40, 14.99, 13.23. ES-MS 240 (M+1).
Preparation of 4-(nonylamino)-2-butanesulfonic acid (Compound
JN)
##STR00646##
[0811] To a solution of nonylamine (2.00 g, 14.0 mmol) in
tetrahydrofuran (THF, 15 mL) was added 2,4-butane sultone (2.08 g,
14.7 mmol). The solution stirred at reflux for 5 hours. The
reaction was cooled to room temperature. The solid was collected by
filtration, washed with THF (2.times.25 mL) and dried in vacuo.
[0812] The product (1.10 g, 3.9 mmol) was dissolved with heating in
a solution of EtOH (20 mL), water (600 uL) and NaOH (163 mg, 4.1
mmol). After a few minutes a white solid precipitated. The solid
was collected by filtration, washed with acetone (2.times.25 mL)
and dried in vacuo. .sup.1H NMR (D.sub.2O, 500 MHz) .delta. ppm
2.70 (m, 1H), 2.52 (m, 2H), 2.37 (m, 1H), 1.95 (m, 1H), 1.46 (m,
1H), 1.34 (m, 1H), 1.14 (m, 17H), 0.70 (t, 3H, J=6.8 Hz). .sup.13C
(D.sub.2O, 125 MHz) .delta. ppm 54.31, 52.90, 50.44, 31.31, 28.80,
28.74, 28.57, 27.31, 26.95, 22.20, 14.60, 13.57. ES-MS 302
(M+1).
Preparation of 4-(dimethylamino)-2-butanesulfonic acid (Compound
JO)
##STR00647##
[0814] 2,4-butanesultone (1.27 g, 8.9 mmol) was added to an
ice-chilled solution of dimethylamine (40% w/w in water). The
solution was stirred at 0.degree. C. for 4 hours. The solvent was
evaporated in vacuo until complete dryness. The solid was washed
with acetone (50 mL), collected by filtration and dried in vacuo.
.sup.1H NMR (D.sub.2O, 500 MHz) .delta. ppm 3.18 (t, 2H, J=8.1 Hz),
2.85 (m, 1H), 2.76 (s, 6H), 2.09 (m, 1H), 1.81 (m, 1H), 1.17 (d,
3H, J=7.3 Hz). .sup.13C (D.sub.2O, 125 MHz) .delta. ppm 55.65,
53.05, 42.88, 26.72, 14.81. ES-MS 182 a (M+1).
Preparation of 4-(benzylamino)-2-butanesulfonic acid, sodium salt
(Compound JP)
##STR00648##
[0816] To a solution of benzylamine (1.50 g, 14.0 mmol) in
tetrahydrofuran (THF, 18 mL) was added 2,4-butane sultone (1.98 g,
14.6 mmol). The solution was stirred at reflux for 2 hours. The
reaction was cooled to room temperature. The solid was collected by
filtration, washed with THF (2.times.25 mL) and dried in vacuo.
[0817] The product (2.55 g, 10.5 mmol) was dissolved with heating
in a solution of EtOH (25 mL), water (1.6 mL) and NaOH (440 mg,
11.0 mmol). Diethyl ether (150 mL) was added to the filtrate. The
solid was filtered and dried in vacuo. Yield: 27%. .sup.1H NMR
(DMSO, 500 MHz) .delta. ppm 7.29 (m, 4H), 7.20 (m, 1H), 3.67 (m,
2H), 2.56 (m, 1H), 2.45 (m, 2H), 1.98 (m, 1H), 1.36 (m, 1H), 1.04
(d, 3H, J=6.8 Hz). .sup.13C (DMSO, 125 MHz) .delta. ppm 141.49,
128.73, 128.61, 127.16, 53.49, 52.88, 47.31, 32.67, 16.53. ES-MS
266 (M+1).
Preparation of 4-(ethylamino)-2-butanesulfonic acid, sodium salt
(Compound JQ)
##STR00649##
[0819] A solution of 2,4-butanesultone (1.33 g, 9.3 mmol) in
tetrahydrofuran (THF, 3.0 mL) was added via syringe pump over a 2 h
period to ethylamine (70% w/w in water, 12.0 mL, 186.0 mmol) at
5.degree. C. The solution was stirred at 5.degree. C. for an
additional 2 hours. The solvent was coevaporated with EtOH
(3.times.25 mL). The solid was suspended in acetone (25 mL). The
suspension was stirred for 5 minutes the solid was filtered, washed
with acetone (2.times.25 mL) and dried in vacuo. Yield: 70%.
.sup.1H NMR (D.sub.2O, 500 MHz) .delta. ppm 3.06 (t, 2H, J=8.1 Hz),
2.97 (m, 2H), 2.87 (m, 2H), 2.06 (m, 1H), 1.77 (m, 1H), 1.18 (d,
3H, J=7.3 Hz), 1.14 (t, 3H, J=7.3 Hz). .sup.13C (D.sub.2O, 125 MHz)
.delta. ppm 53.16, 44.91, 43.03, 28.20, 14.76, 10.66. ES-MS 182
(M+1).
Preparation of 4-(tert-butylamino)-1-butanesulfonic acid (Compound
LD)
##STR00650##
[0821] To a solution of tert-butylamine (1.0 mL, 9.5 mmol) in
tetrahydrofuran (4 mL) was added 1,4-butane sultone (1.36 g, 10.0
mmol) at room temperature. The solution was stirred at reflux for 2
hours. The reaction was cooled to room temperature. The solid was
collected by filtration, washed with acetone (2.times.20 mL) and
dried in vacuo. Yield: 690 mg (34%). .sup.1H NMR (D.sub.2O, 500
MHz) .delta. ppm 2.92 (t, 2H, J=7.1 Hz), 2.82 (t, 2H, J=7.1 Hz),
1.68 (m, 4H), 1.22 (s, 9H). .sup.13C (D.sub.2O, 125 MHz) .delta.
ppm 57.07, 50.30, 40.95, 25.28, 24.96, 21.62. ES-MS 210 (M-1).
Preparation of 4-amino-2-butanesulfonic acid (Compound JR)
##STR00651##
[0823] A solution of 2,4-butanesultone (1.0 g, 7 mmol) in
tetrahydrofuran (THF, 4.0 mL) was added via syringe pump over a 4 h
period to ammonium hydroxide (28-30% NH.sub.3, 43 mL, 350 mmol) at
5.degree. C. The solution was stirred at 5.degree. C. for an
additional 30 minutes. The solvent was coevaporated with EtOH
(3.times.25 mL). The solid was dried in vacuo. Yield: 94%. .sup.1H
NMR (D.sub.2O, 500 MHz) .delta. ppm 3.05 (m, 2H), 2.90 (m, 1H),
2.05 (m, 1H), 2.06 (m, 1H), 1.77 (m, 1H), 1.18 (d, 3H, J=6.8 Hz),
1.14 (t, 3H, J=7.3 Hz). .sup.13C (DMSO, 125 MHz) .delta. ppm 52.75,
38.20, 30.87, 17.27. ES-MS 154 (M+1).
Preparation of 4-piperidin-1-yl-2-butanesulfonic acid (Compound
JS)
##STR00652##
[0825] To a solution of piperidine (1.50 g, 17.6 mmol) in
tetrahydrofuran (THF, 20 mL) was added 2,4-butanesultone (2.50 g,
18.5 mmol). The solution was stirred at reflux for 3 hours. The
reaction was cooled to room temperature. The solid was collected by
filtration, washed with THF (2.times.20 mL) and dried in vacuo.
[0826] The product (3.53 g, 15.9 mmol) was dissolved with heating
in a solution of EtOH (30 mL), water (1.3 mL) and NaOH (670 mg,
16.7 mmol). The solution was poured in a large excess of Et.sub.2O
(500 mL). The solid was filtered, washed with Et.sub.2O (1.times.25
mL) and acetone (1.times.20 mL) and dried in vacuo. Yield: 64%.
.sup.1H NMR (D.sub.2O, 500 MHz) .delta. ppm 2.72 (m, 1H), 2.33 (m,
6H), 1.97 (m, 1H), 1.48 (m, 1H), 1.43 (m, 4H), 1.31 (m, 1H), 1.13
(d, 3H, J=6.8 Hz). .sup.13C (D.sub.2O, 125 MHz) .delta. ppm 55.78,
54.42, 53.61, 27.73, 24.92, 23.59, 14.61. ES-MS 244 (M+1).
Preparation of 4-(ethylamino)-1-butanesulfonic acid (Compound
LE)
##STR00653##
[0828] A solution of 1,4-butanesultone (2.66 g, 18.6 mmol) in
tetrahydrofuran (total volume: 4 mL) was added via syringe pump
over a 4 hour period to ethylamine (70% w/w in water, 24 mL, 372
mmol) at 5.degree. C. The solution was stirred at 5.degree. C. for
an additional 3 hours before it was warm up to room temperature.
The reaction was stirred in these conditions overnight. The solvent
was coevaporated with EtOH (1.times.25 mL). The solid was suspended
in 50% acetone/EtOH (50 mL). The suspension was stirred for 5
minutes, the solid was filtered and dried in vacuo. Yield: 75%.
.sup.1H NMR (D.sub.2O, 500 MHz) .delta. ppm 2.95 (m, 4H), 2.82 (m,
2H), 1.68 (m, 4H), 1.13 (t, 3H, J=7.3 Hz), 1.14 (t, 3H, J=7.3 Hz).
.sup.13C (D.sub.2O, 125 MHz) .delta. ppm 50.27, 46.68, 42.99,
24.66, 21.48, 10.64. ES-MS 182 (M+1).
Preparation of
4-(azoniabicyclo[2.2.2]oct-2-ylamino)-1-butanesulfonate (Compound
LF)
##STR00654##
[0830] To a solution of quinuclidine (1.5 g, 13.5 mmol) in
tetrahydrofuran (THF, 15 mL) was added 1,4-butanesultone (2.0 g,
14.4 mmol) at room temperature. The solution was stirred at reflux
for 2 hours. The reaction was cooled to room temperature. The solid
was collected by filtration, washed with THF (2.times.25 mL) and
dried in vacuo. NMR (D.sub.2O, 500 MHz) .delta. ppm 3.26 (m, 6H),
3.02 (m, 2H), 2.82 (t, 2H, J=17.3 Hz), 2.04 (m, 1H), 1.84, (m, 6H),
1.75 (m, 2H), 1.64 (m, 2H). .sup.13C (D.sub.2O, 125 MHz) .delta.
ppm 63.68, 54.81, 50.14, 23.51, 21.45, 20.58, 19.19. ES-MS 248
(M+1).
Preparation of 3-(dimethylamino)-2-hydroxy-1-propane sulfonic acid,
sodium salt (Compound JT)
##STR00655##
[0832] A solution of 3-chloro-2-hydroxy-1-propanesulfonic acid,
sodium salt (10 g, 48.3 mmol) in water (40 mL total volume) was
added via a syringe pump over 4 hours to a cold (2.8-3.1.degree.
C.) solution of dimethylamine (40% wt in water, 300 mL) with
stirring. The mixture was slowly warmed to room temperature
overnight. The mixture was then coevaporated with absolute ethanol
(20 mL) and concentrated to dryness. The solid was dried overnight
at 60.degree. C. in the vacuum oven. The solid was suspended in
ethanol (40 mL) stirred at reflux for 2 hours. The suspension was
cooled to 5.degree. C. and the solid was collected by
suction-filtration, aspirator-dried 5 minutes, then dried for the
weekend at 60.degree. C. in the vacuum oven (wet cake: 13.74 g).
The desired material was obtained as an off-white solid (11.65 g,
quantitative).
Preparation of 4-Dimethylamino-1-butanesulfonic acid (Compound
LH)
##STR00656##
[0834] A solution of 1,4-butane sultone (7.5 mL, 73.6 mmol) in
1,4-dioxane (total volume: 10 mL) was added over 4 hours via a
syringe pump to a cold (4.3.degree. C.) solution of dimethylamine
(40% wt in water, 275 mL). The mixture was stirred for 3 hours at
4.degree. C. after the end of the addition, then overnight at room
temperature. The mixture was concentrated to dryness. The solid was
suspended in absolute ethanol (50 mL) and the mixture was heated to
reflux for 90 minutes. The suspension was cooled to 5.degree. C.
and the solid was collected by suction-filtration, rinsed with
ethanol (2.times.10 mL). The solid was dried for 18 h at 60.degree.
C. in the vacuum oven. The desired material was obtained as a fine
white powder 13.21 g, 72.9 mmol, 99% yield. The .sup.1H and
.sup.13C NMR and MS were consistent with the structure.
Preparation of 3-(ethylamino)-2-hydroxy-1-propanesulfonic acid
(Compound JU)
##STR00657##
[0836] A solution of 3-chloro-2-hydroxy-1-propanesulfonic acid
sodium salt (10 g, 50.9 mmol) in water (total volume: 40 mL) was
added over 5 hours, via a syringe pump, to a cold (4.7.degree. C.)
solution of ethyl amine in water. The mixture was stirred for an
additional 2 hours at 4.7.degree. C. then for 18 hours at room
temperature. NMR: quantitative yield. The mixture was concentrated.
A solid could not be obtained: the sodium salt was too hygroscopic.
The solution was treated with Amberlite IR-120 Plus, acid form,
ion-exchange resin to give the free acid. It was still too
hygroscopic to be obtained as a solid form. Submitted as a
solution: d=1.314 g/mL, 62.5% w/w of the free acid in water. The
.sup.1H and .sup.13C NMR and MS were consistent with the
structure.
Preparation of 3-(tert-butylamino-2-hydroxy-1-propanesulfonic acid
(Compound JV)
##STR00658##
[0838] A solution of 3-chloro-2-hydroxy-1-propanesulfonic acid,
sodium salt (15 g, 25 mmol) in water (12 mL total volume) was added
over 5 minutes to a mixture of tert-butylamine (12.5 mL), water (6
mL) and methanol (3 mL). The mixture was heated at 35.degree. C.
for 1 h, 40.degree. C. for 1 h, 45.degree. C. for 1.5 hours. The
mixture was the concentrated to a thick oil. The crude reaction
mixture was passed over a column of Dowex 50.times.8 (125 g). The
fractions containing the product were concentrated to dryness. The
solid was dried overnight at 60.degree. C. in the vacuum oven. The
solid was recrystallized in a mixture of methanol (25 mL) and water
(7 mL). The mixture was cooled slowly to room temperature, then to
5.degree. C. The solid was collected by suction-filtration, rinsed
with ethanol (1.times.10 mL). The solid was then dried for 18 hours
at 60.degree. C. in the vacuum oven. The desired material was
obtained as an off-white solid (3.11 g, 14.7 mmol, 59%). The
.sup.1H and .sup.13C NMR and MS were consistent with the
structure.
Preparation of 1-(N-octylamino)-2-hydroxy-1-propanesulfonic acid
(Compound JW)
##STR00659##
[0840] A solution of 3-chloro-2-hydroxy-1-propanesulfonic acid,
sodium salt (4 g, 20 mmol) in water (17.5 mL total volume) was
added over 2 hours to a mixture of octylamine (8 mL), water (20 mL)
and 1,4-dioxane (11 mL) at 70-75.degree. C. The mixture was stirred
at this temperature for another 2 hours after the end of the
addition. The 1,4-dioxane was removed under reduced pressure and
the mixture was diluted with water (10 mL). The mixture was
extracted with 40% ethyl acetate/hexane (3.times.40 mL). The
aqueous layer was concentrated then the mixture was passed over a
column of Dowex 50.times.8 (125 g). The fractions containing the
pure product were concentrated to a thick oil then freeze-dried.
The desired material was obtained as a white fluffy solid (150 mg,
0.56 mmol, 3%). The .sup.1H and .sup.13C NMR and MS were consistent
with the structure.
Preparation of 1-(3-sulfo-2-hydroxypropyl)quinuclidinium, inner
salt (Compound JX)
##STR00660##
[0842] A solution of 3-chloro-2-hydroxy-1-propanesulfonic acid,
sodium salt (2 g, 10 mmol) in water (12 mL total volume) was added
over 1 hour to a mixture of quinuclidine (1.63 g, 4.7 mmol), water
(10 mL) and 1,4-dioxane (10 mL) at 80.degree. C. The mixture was
stirred at this temperature for another 2 hours after the end of
the addition. The reaction mixture was concentrated then the
mixture was passed over a column of Dowex 50.times.8 (125 g). The
fractions containing the pure product were concentrated to a white
solid. The solid was dried for 18 hours at 60.degree. C. in the
vacuum oven. The desired material was obtained as a white solid
(1.92 g, 7.7 mmol, 77%). The .sup.1H and .sup.13C NMR and MS were
consistent with the structure.
Preparation of 1-(N-benzylamino)-2-hydroxy-1-propanesulfonic acid,
sodium salt (Compound JY)
##STR00661##
[0844] A solution of 3-chloro-2-hydroxy-1-propanesulfonic acid,
sodium salt (4 g, 20 mmol) in water (12.5 mL total volume) was
added over 2 hours to a mixture of benzylamine (418 g, 40 mmol),
water (10 mL) and 1,4-dioxane (5 mL) at 80.degree. C. The mixture
was stirred at this temperature for another 2.5 hours after the end
of the addition. The reaction mixture was extracted with chloroform
(2.times.40 mL). It was then concentrated to dryness. The crude
solid was recrystallized in a mixture of ethanol (30 mL) and water
(4 mL). The mixture was left to cool to room temperature for the
night. The solid was collected by suction-filtration, rinsed with
ethanol (10 mL) and dried in the vacuum oven at 60.degree. C. The
desired material was obtained as a white solid (2.67 g, 10 mmol,
50%). The .sup.1H and .sup.13C NMR and MS were consistent with the
structure.
Preparation of
2-hydroxy-3-(1,2,3,4-tetrahydronaphthalen-1-ylamino)propane-1-sulfonic
acid (Compound JZ)
##STR00662##
[0846] A solution of 3-chloro-2-hydroxy-1-propanesulfonic acid,
sodium salt (2 g, 10 mmol) in water (9.75 mL total volume) was
added over 8 hours to a mixture of 1,2,3,4-tetrahydro-1-nahtylamine
(2 g, 13.6 mmol), water (10 mL) and 1,4-dioxane (4 mL) at
40.degree. C. The mixture was stirred at this temperature for
another 18 hours after the end of the addition. The reaction was
not completed. The mixture was heated for 2 hours at reflux. The
mixture was diluted with water (10 mL) and 50% w/w NaOH (0.25 mL)
was added. The reaction mixture was extracted with chloroform
(2.times.25 mL). It was then concentrated to a thick oil. The
solution was applied on a Dowex 50 W 8 column (100 g). The
fractions containing the product were concentrated, treated with
activated charcoal (no effect) and freeze-dried. The desired
material was obtained as a glassy solid (0.85 g, 3 mmol, 30%). The
.sup.1H and .sup.13C NMR and MS were consistent with the
structure.
Preparation of 2-hydroxy-3-piperidin-1-ylpropane-1-sulfonic acid
(Compound KA)
##STR00663##
[0848] A solution of 3-chloro-2-hydroxy-1-propanesulfonic acid,
sodium salt (4 g, 20 mmol) in water (13.35 mL total volume) was
added over 5 hours to a solution of piperidine (8 mL g, 80 mmol),
in water (15 mL) at 70.degree. C. The mixture was stirred at
80.degree. C. for 2 hours. The reaction was completed. The mixture
was stirred at room temperature for the night. The mixture was
diluted with water (10 mL) and was extracted with chloroform
(3.times.30 mL). It was then concentrated to a thick oil. The
solution was applied on a Dowex 50 W 8 column (100 g). The
fractions containing the product were concentrated to dryness then
recrystallized in a mixture of ethanol (30 mL) and water (2.1 mL).
The mixture was cooled slowly at room temperature. The solid was
collected by suction filtration, rinsed with ethanol (2.times.5 mL)
air dried 5 minutes, then 18 hours at 60.degree. C. in the vacuum
oven. The desired material was obtained as a fine white solid (3.06
g, 13.7 mmol, 68%). The .sup.1H and .sup.13C NMR and MS were
consistent with the structure.
Preparation of 4-(adamantyl)amino-1-butanesulfonic acid (Compound
LI)
##STR00664##
[0850] 1-adamantaneamine hydrochloride (2.67 g, 13.3 mmol) was
treated with 1N NaOH (20 mL) and CH.sub.2Cl.sub.2 (3.times.20 mL).
The biphasic solution was shaken. The organic extracts were
combined, dried with Na.sub.2SO.sub.4, filtered, evaporated under
reduced pressure and dried in vacuo.
[0851] To a solution of 2-adamantanamine (1.87 g, 12.4 mmol) in
tetrahydrofuran (THF, 15 mL) was added 1,4-butane sultone (1.76 g,
13.0 mmol). The solution was stirred at reflux overnight. The
reaction was cooled to room temperature. The solid was collected by
filtration, washed with THF (1.times.15 mL) and dried in vacuo. A
suspension of the solid in EtOH (25 mL) was stirred at reflux for 1
hour. The warm mixture was filtered. The solid was dried in vacuo.
.sup.1H NMR (D.sub.2O, 500 MHz) .delta. ppm 2.92 (m, 2H), 2.82 (m,
1H), 2.05 (s, 3H), 1.75 (s, 6H), 1.63 (m, 6H), 1.52 (m, 3H).
.sup.13C (D.sub.2O, 125 MHz) .delta. ppm 57.62, 50.30, 39.03,
38.14, 35.09, 28.98, 25.25, 21.63. ES-MS 288 (M+1).
Preparation of 4-(octylamino)-1-butanesulfonic acid (Compound
LJ)
##STR00665##
[0853] To a solution of octylamine (2.20 g, 17.0 mmol) in
tetrahydrofuran (11 mL) was added 1,4-butane sultone (2.30 g, 16.2
mmol). The solution was heated to reflux for 5 hours. The reaction
was cooled to room temperature. The product formed a gel. A few
drops of EtOH were added to dissolve the product. The solution was
poured in a large excess of acetone (25 mL). After 5 minutes, a
white solid precipitated. The solid was collected by filtration and
dried in vacuo. The product was dissolved in EtOH and Dowex
50.times.8 resin (pre-washed, 6 g) was added to the solution. The
suspension was stirred for 15 minutes and the resin was filtered.
The filtrate was evaporated under educed pressure and the product
was dried in vacuo. Yield: 31%. .sup.1H NMR (DMSO, 500 MHz) .delta.
ppm 8.24 (s (broad), 1H), 2.85 (m, 4H), 2.45 (m, 2H), 1.64 (m, 4H),
1.61 (m, 2H), 1.25 (m, 10H), (m, 2H), 0.85 (t, 3H, J=6.8 Hz).
.sup.13C (DMSO, 125 MHz) .delta. ppm 51.22, 47.54, 47.38, 31.82,
29.14, 26.59, 26.13, 25.51, 22.95, 22.75, 14.64. ES-MS 266
(M+1).
Preparation of 4-(cyclohexylamino)-2-butanesulfonic acid (Compound
KM)
##STR00666##
[0855] To a solution of cyclohexylamine (1.50 g, 15.1 mmol) in
tetrahydrofuran (15 mL) was added 2,4-butane sultone (2.04 g, 14.4
mmol). The solution stirred at reflux for 2 hours. The reaction was
cooled to room temperature. The solid was collected by filtration
and dried in vacuo. Yield: 59%. .sup.1H NMR (DMSO, 500 MHz) .delta.
ppm 8.50 (s (broad), 1H), 3.02 (m, 2H), 2.93 (m, 1H), 2.60 (m, 1H),
1.93 (m, 3H), 1.75 (m, 3H), 1.57 (m, 1H), 1.21 (m, 4H), 1.11 (m,
4H). .sup.13C (DMSO, 125 MHz) .delta. ppm 56.23, 53.20, 43.09,
29.54, 29.41, 29.39, 25.40, 24.42, 17.23. ES-MS 234 (M-1).
Preparation of 4-[(dl)-1-hydroxy-2-pentyl]amino-1-butanesulfonic
acid (Compound LL)
##STR00667##
[0857] To a solution of DL-2-aminopentanol (1.0 g, 9.7 mmol) in
tetrahydrofuran (6 mL) was added 1,4-butane sultone (1.31 g, 9.2
mmol) at room temperature. The solution was stirred at reflux for 5
hours. The reaction was cooled to room temperature. The supernatant
was removed and the solid was dried in vacuo. The white solid was
filtered, washed with acetone (2.times.25 mL) and dried in vacuo.
Yield: 45%. .sup.1H NMR (DMSO, 500 MHz) .delta. ppm 8.20 (s
(broad), 1H), 5.23 (m, 1H), 3.66 (m, 1H), 3.49 (m, 1H), 3.02 (m,
1H), 2.91 (m, 2H), 2.46 (t, 2H, J=7.3 Hz), 1.65, (m, 4H), 1.54 (m,
2H), 1.38 (m, 2H), 0.88 (t, 3H, J=7.3 Hz). .sup.13C (DMSO, 125 MHz)
.delta. ppm 58.83, 58.54, 51.23, 44.77, 29.91, 25.56, 23.06, 18.95,
14.48. ES-MS 238 (M-1).
Preparation of 3-[(3,4-dimethoxybenzyl)amino]-1-butanesulfonic acid
(Compound LM)
##STR00668##
[0859] To a solution of veratrylamine (1.50 g, 9.0 mmol) in
1,4-dioxane (8 mL) was added 1,4-butane sultone (1.21 g, 8.5 mmol)
at room temperature. The mixture was then heated at reflux for 2
hours. The reaction was cooled to room temperature. The solid was
collected by filtration, washed with acetone (2.times.25 mL) and
dried on pump. Yield: 18%. .sup.1H NMR (D.sub.2O, 500 MHz) .delta.
6.96 (m, 3H), 4.04 (s, 2H), 3.74 (m, 6H), 2.95 (t, 2H, J=6.8 Hz),
2.80 (t, 2H, 7.3 Hz), 1.68, (m, 4H). .sup.13C (D.sub.2O, 125 MHz)
.delta. ppm 149.19, 148.50, 123.82, 123.36, 113.25, 112.17, 55.91,
50.88, 50.24, 46.41, 24.55, 21.50. ES-MS 302 (M-1).
Preparation of 4-(adamantan-1-ylamino)-2-hydroxy-1-propanesulfonic
acid (Compound KB)
##STR00669##
[0861] 1-adamantaneamine hydrochloride (2.67 g, 14.2 mmol) was
treated with 1N NaOH (20 mL) and CH.sub.2Cl.sub.2 (3.times.20 mL).
The organic extracts were combined, dried with Na.sub.2SO.sub.4,
filtered, evaporated under reduced pressure and dried in vacuo.
[0862] To an 80.degree. C. solution of 1-adamantanamine (2.15 g,
14.2 mmol) in 1,4-dioxane (10 mL) and water (5 mL) was added via
syringe pump (1 h addition) a solution of
3-chloro-2-hydroxy-propanesulfonic acid, sodium salt (1.93 g, 9.7
mmol) in 1,4-dioxane (0.5 mL) and water (10 mL). The solution was
stirred at reflux overnight. The reaction was cooled to room
temperature. The solvent was evaporated under reduced pressure. The
solid was suspended in 25% acetone/EtOH. The mixture was heated to
reflux for 1 minute. The solid was collected by filtration. The
pure product crystallized in the filtrate. The product was
filtered, washed with EtOH (2.times.10 mL), dissolved in water and
lyophilized. Yield: 15%. .sup.1H NMR (D.sub.2O, 500 MHz) .delta.
ppm 4.18 (m, 1H), 3.22 (m, 1H), 3.01 (m, 2H), 2.94 (m, 1H), 2.06
(s, 3H), 1.77 (m, 7H), 1.61 (d, 3H), 1.53 (m, 3H). .sup.13C
(D.sub.2O, 125 MHz) .delta. ppm 64.23, 57.99, 55.05, 44.10, 38.10,
35.07, 29.03. ES-MS 288 (M-Na (23)).
Preparation of 4-(2-adamantyl)amino-1-butanesulfonic acid (Compound
LN)
##STR00670##
[0864] 2-adamantanamine hydrochloride (2.50 g, 13.3 mmol) was
treated with 1N NaOH (20 mL) and CH.sub.2Cl.sub.2 (3.times.20 mL).
The organic extracts were combined, dried with Na.sub.2SO.sub.4,
filtered, evaporated under reduced pressure and dried in vacuo.
[0865] To a solution of 2-adamantanamine (1.06 g, 7.0 mmol) in
1,4-dioxane (6 mL) was added 1,4-butane sultone (955 mg, 6.7 mmol).
The solution was stirred at reflux for 5 hours. The reaction was
cooled to room temperature. The solid was collected by filtration.
It was suspended in EtOH (25 mL) and the mixture was heated to
reflux for 1 minute before the solid was filtered. It was washed
with EtOH (1.times.15 mL) and dried in vacuo. Yield: 55%. .sup.1H
NMR (D.sub.2O, 500 MHz) .delta. ppm 3.29 (m, 1H), 2.97 (m, 2H),
2.83 (m, 2H), 2.02 (m, 2H), 1.83 (m, 2H), 1.68 (m, 14H). .sup.13C
NMR (D2O, 125 MHz) .delta. ppm 63.07, 50.25, 45.03, 36.55, 36.31,
29.85, 29.05, 26.68, 26.41, 24.47, 21.61. ES-MS 286 (M-1).
Preparation of 3-(2-adamantylamino)-2-hydroxy-1-propanesulfonic
acid (Compound KJ)
##STR00671##
[0867] To an 80.degree. C. solution of 2-adamantanamine
hydrochloride (2.50 g, 13.3 mmol) and sodium hydroxide (586 mg,
14.6 mmol) in 1,4-dioxane (7 mL) and water (7 mL) was added via
syringe pump (1 hour addition) a solution of
3-chloro-2-hydroxy-propane sulfonic acid, sodium salt (1.76 g, 8.9
mmol) in 1,4-dioxane (1 mL) and water (9 mL). The solution was
stirred at 80.degree. C. for an additional 4 hours. The reaction
was cooled to room temperature. The solvent was evaporated under
reduced pressure. The solid was suspended in EtOH (25 mL). The
mixture was heated to reflux for 1 minute. The solid was removed by
filtration. The pure product crystallized in the filtrate. The
product was filtered, washed with EtOH (1.times.10 mL) and dried in
vacuo. Yield: 30%. .sup.1H NMR (D.sub.2O, 500 MHz) .delta. ppm 4.30
(m, 1H), 3.35 (m, 2H), 3.03 (m, 3H), 2.07 (m, 2H), 1.84 (m, 2H),
1.75 (m, 4H), 1.65 (d, 6H). .sup.13C (D.sub.2O, 125 MHz) .delta.
ppm 63.31, 55.03, 49.51, 36.51, 36.33, 36.26, 29.78, 29.23, 28.81,
26.63, 26.38. ES-MS 289 (M+1).
Preparation of
3-(bicyclo[2.2.1]hept-2-ylamino)-2-hydroxy-1-propanesulfonic acid
(Compound KI)
##STR00672##
[0869] To an 80.degree. C. solution of exo-2-aminonorbornane (910
mg, 8.2 mmol) and sodium hydroxide (242 mg, 6.1 mmol) in
1,4-dioxane (4 mL) and water (4 mL) was added via syringe pump (1
hour addition) a solution of 3-chloro-2-hydroxy-propane sulfonic
acid, sodium salt (1.09 g, 5.5 mmol) in 1,4-dioxane (0.5 mL) and
water (5.5 mL). The solution was stirred at 80.degree. C. for an
additional 5 hours. The reaction was cooled to room temperature.
The solvent was evaporated under reduced pressure. The solid was
suspended in EtOH (25 mL). The mixture was heated to reflux for 1
minute. The solid was recovered by filtration and it was passed
through an ion exchange column (Dowex 50.times.8, 100 g, solvent:
water). The product was recrystallized in EtOH/water (99/1). Yield:
17%. NMR (D.sub.2O, 500 MHz) .delta. ppm 4.25 (m, 1H), 3.25 (m,
2H), 3.01 (m, 4H), 2.39 (m, 1H), 2.27 (m, 1H), 1.69 (m, 1H), 1.51
(m, 1H), 1.38 (m, 3H), 1.19 (m, 1H), 1.07 (m, 2H). .sup.13C
(D.sub.2O, 125 MHz) .delta. ppm 63.66, 63.50, 62.21, 61.98, 54.94,
50.11, 50.04, 39.26, 39.21, 36.02, 35.97, 35.91, 35.80, 34.70,
34.61, 27.11, 27.08, 26.50, 26.45. ES-MS 250 (M-1).
Preparation of 4-[(3-methylbutyl)amino]-2-butanesulfonic acid
(Compound KH)
##STR00673##
[0871] To a hot solution of isoamylamine (2.0 g, 22.9 mmol) in
tetrahydrofuran (THF, 11 mL) was added via syringe pump (2 hour
addition) a solution of 2,4-butane sultone (3.1 g, 21.8 mmol in THF
(total of 5 mL)). The solution was stirred at reflux for an
additional 2 hours. The reaction was cooled to room temperature.
The solid was recovered by filtration and it was washed with THF
(25 mL) and acetone (25 mL). The solid was dissolved in water (20
mL) and Dowex 50.times.8 (10 g) was suspended in the solution. The
mixture was stirred for 15 minutes and the resin was filtered. The
solvent was evaporated under reduced pressure. Yield: 28%. .sup.1H
NMR (H.sub.2O, 500 MHz) .delta. ppm 3.07 (t, 2H, J=7.8 Hz), 2.92
(t, 2H, J=7.8 Hz), 2.87 (m, 1H), 2.06 (m, 1H), 1.77 (m, 1H), 1.51
(m, 1H), 1.42 (m, 2H), 1.18 (d, 3H, J=6.8 Hz), 0.78 (d, 3H, J=6:3
Hz). .sup.13C(H.sub.2O, 125 MHz) .delta. ppm 53.21, 46.32, 45.37,
34.36, 28.16, 25.35, 21.51, 14.79. ES-MS 224 (M+1).
Preparation of 2-hydroxy-3-[(3-methylbutyl)amino]-1-propane
sulfonic acid (Compound KK)
##STR00674##
[0873] To a 80.degree. C. solution of isoamylamine (2.0 g, 22.9
mmol) in 1,4-dioxane (9 mL) and water (3 mL) was added via syringe
pump (1 h addition) a solution of 3-chloro-2-hydroxy-propane
sulfonic acid, sodium salt (3.04 g, 15.3 mmol) in 1,4-dioxane (9.5
mL) and water (0.5 mL). The solution was stirred overnight at
80.degree. C. The solvent was evaporated. The product was passed
through an ion exchange column (Dowex 50.times.8, 100 g, solvent:
water). It was recrystallized in absolute EtOH and lyophilized.
Yield: 27%. .sup.1H NMR (H.sub.2O, 500 MHz) .delta. ppm 4.24 (m,
1H), 3.22 (m, 1H), 3.02 (m, 5H), 1.49 (m, 3H), 0.79 (d, 3H,
J=6.3
[0874] Hz). .sup.13C(H.sub.2O, 125 MHz) .delta. ppm 63.54, 54.89,
51.53, 46.48, 34.12, 25.46, 21.56, 21.46. ES-MS 226 (M+1).
Preparation of 3-[(dl)-1-Hydroxy-2-pentyl]amino-1-propane sulfonic
acid (Compound KL)
##STR00675##
[0876] To a 80.degree. C. solution of DL-2-amino-1-pentanol (1.0 g,
9.7 mmol) in 1,4-dioxane (5 mL) and water (3 mL) was added via
syringe pump (1 hour addition) a solution of
3-chloro-2-hydroxy-propane sulfonic acid, sodium salt (1.84 g, 9.2
mmol) in 1,4-dioxane (6 mL) and water (0.5 mL). The solution was
stirred overnight at 80.degree. C. The solvent was evaporated. The
product was passed through an ion exchange column (Dowex
50.times.8, 100 g, solvent: water). The product was dissolved. It
was recrystallized in absolute EtOH and lyophilized. Yield: 27%.
.sup.1H NMR (H.sub.2O, 500 MHz) .delta. ppm 4.26 (m, 1H), 3.77 (m,
1H), 3.32 (m, 1H), 3.24 (m, 1H), 3.03 (m, 3H), 1.54 (m, 2H), 1.29
(m, 2H), 0.81 (t, 3H, J=7.3 Hz). .sup.13C(H.sub.2O, 125 MHz)
.delta. ppm 63.69, 63.60, 59.49, 59.38, 58.81, 58.36, 54.98, 48.68,
48.27, 29.32, 28.85, 18.40, 18.38, 13.12. ES-MS 242 (M+1).
Preparation of 4-(1H-benzimidazol-2-ylthio)-2-butanesulfonic acid
(Compound NE)
##STR00676##
[0878] To a hot solution of 2-mercaptobenzimidazole (2.0 g, 13.3
mmol) in 1,4-dioxane (12 mL) and water (3 mL) was added via syringe
pump (1 hour addition) a solution of 2,4-butane sultone (1.80 g,
12.7 mmol in 1,4-dioxane (total of 3 mL)). The solution was stirred
at reflux for an additional 3 hours. The solid was collected by
filtration. It was washed with acetone (2.times.20 mL) and dried in
vacuo. Yield: 86%. .sup.1H NMR (DMSO, 500 MHz) .delta. ppm 7.64 (m,
2H), 7.44 (m, 2H), 3.63 (t, 2H, J=7.3 Hz), 2.68 (m, 1H), 2.10 (m,
1H), 1.90 (m, 1H), 1.15 (d, 3H, J=6.8 Hz). .sup.13C (DMSO, 125 MHz)
.delta. ppm 152.64, 133.32, 125.66, 113.67, 53, 10, 39.72, 33.48,
30.14, 16.85. ES-MS 287 (M+1).
Preparation of 4-(cyclohexylamino)-1-butanesulfonic acid (Compound
LK)
##STR00677##
[0880] To a solution of cyclohexylamine (2.0 g, 20.2 mmol) in
1,4-dioxane (13 mL) was added 1,4-butane sultone (2.61 g, 19.2
mmol). The solution was heated to reflux for 2 hours. The reaction
was cooled to room temperature. The solid was collected by
filtration, washed with acetone (2.times.20 mL) and dried in vacuo.
Yield: 52%. .sup.1H NMR (D.sub.2O, 500 MHz) .delta. ppm 2.95 (m,
3H), 2.81 (m, 2H), 1.92 (m, 2H), 1.67 (m, 6H), 1.52 (m, 1H), 1.18
(m, 4H), 1.02 (m, 1H). .sup.13C (D2O, 125 MHz) .delta. ppm 57.32,
50.31, 44.01, 29.02, 24.84, 24.68, 24.07, 24.55. ES-MS 236
(M+1).
Preparation of 3-[(1-ethyl-1-methylpropyl)amino]-1-propanesulfonic
acid (Compound FP)
##STR00678##
[0882] The flask was closed with a septum and connected to a 20%
NaOH scrubber for the Ritter Reaction. Potassium cyanide (3.25 g,
50 mmol) was added to acetic acid (13 mL) and the mixture was
stirred for 10 min at room temperature. A solution of sulfuric acid
(7 mL) in acetic acid (6 mL) was added and the resulting suspension
was stirred 10 minutes at room temperature. The 3-methyl-3-pentanol
(5 g, 48.9 mmol) was added drop-wise over a 5 minute period. The
mixture was stirred at room temperature for 4 hours, at which time
some chunks of potassium cyanide were still visible.
[0883] Another portion of potassium cyanide (0.6 g, powdered) was
added and the mixture was stirred for 18 hours at room temperature.
The mixture was purged with nitrogen for 1 h then poured over ice
(approx. 50 g). The pH of the solution was adjusted to 9 with the
addition of 20% NaOH (use 50% next time to reduce the volume). The
layers were separated and the aqueous layer was extracted with
ether (1.times.20 mL). The combined organic layers were washed with
saturated potassium carbonate (1.times.5 mL) then dried over
magnesium sulfate. The ether was evaporated under reduced pressure
to afford a yellow oil (4.11 g, 31.8 mmol, 64%). The oil showed to
be a mixture of cis and trans formamide but what otherwise pure
enough to be used as such. .sup.1H NMR (500 MHz, DMSO-d6) .delta.
0.75-0.80 (m, 6H), 1.11-1.12 (m, 3H), 1.40-1.54 (m, 2H), 1.66-1.73
(m, 2H), 7.35-7.45 (br s and br d, 1H), [7.88 (s) and 8.08 (d,
J=11.7 Hz) for 1H); .sup.13C NMR (125 MHz, DMSO-d6) .delta. 7.7,
7.9, 23.4, 24.2, 30.6, 33.8, 55.6, 160.3, 163.3
[0884] The 1-ethyl-1-methyl-propylformamide (4.00 g, 31.1 mmol) was
added to 20% NaOH (40 mL). The mixture was heated to reflux for 4
hours then was left overnight at room temperature. Toluene (10 mL)
was added and the layers were separated. The organic layer was
dried over sodium sulfate then filtered. The final volume of the
filtrate was about 30 mL. It was used as such in the next step.
[0885] A solution of 1,3-propanesultone (2.5 g, 20 mmol) in
2-butanone (10 mL) was added to a solution of
3-methyl-3-ethyl-3-propylamine in toluene (total volume: 30 mL).
The mixture was heated to reflux for 5 hours then was cooled to
room temperature. The solid was collected by suction-filtration and
rinsed with acetone (2.times.5 mL). The solid was dried overnight
at 45.degree. C. in the vacuum oven. The title compound was
obtained as a fine white solid (3.63 g, 16.3 mmol, 33% overall
yield). .sup.1H NMR (500 MHz, DMSO-d6) .delta. 0.79 (t, J=7.3 Hz,
6H), 1.15 (s, 3H), 1.53-1.59 (m, 4H), 1.97-2.00 (m, 2H), 2.89 (t,
J=7.1 Hz, 2H), 3.03 (t, J=7.6 Hz, 2H); .sup.13C NMR (125 MHz,
DMSO-d6) .delta. 6.8, 20.2, 21.9, 27.7, 39.7, 48.2, 63.3 ES-MS 224;
(M+H)
Preparation of
3-({2-hydroxy-1,1-dimethyl-2-(3-methoxyphenyl)ethyl]amino)-1-propanesulfo-
nic acid (Compound NG)
##STR00679##
[0887] To a cooled solution of sodium methoxide (0.5 M in MeOH, 25
mL1) was added via syringe over a 10 minutes period 2-nitropropane
(5.0 g, 56 mmol). The reaction mixture was stirred at room
temperature for 30 minutes and recooled before m-anisaldehyde (6.8
mL, 56 mmol) was added. The reaction mixture was stirred at room
temperature overnight. The mixture was neutralized with Amberlite
1R-120 (strongly acidic). The resin was removed by filtration and
washed with MeOH (2.times.20 mL). The filtrate was evaporated. The
resulting oil was purified by flash chromatography: 98%
Hexanes/EtOAc to 90% Hexanes/EtOAc, affording the desired nitro
compound (5.70 g, 45%).
[0888] To a solution of the nitro compound (5.70 g, 25.3 mmol)) in
MeOH (25 mL) was added 6M HCl (25 mL). After cooling to 5.degree.
C., zinc powder (8.2 g, 125 mmol) was added. The suspension was
stirred at 0-5.degree. C. and at room temperature overnight. The
mixture was filtered on a celite pad. The filter cake was washed
with MeOH (2.times.20 mL). The combined filtrates were evaporated
under reduced pressure. The residue was dissolved in EtOAc (40 mL).
The mixture was extracted with 5% NaOH (1.times.40 mL). The aqueous
phase was extracted with EtOAc (2.times.40 mL). The combined
organic extracts were dried with Na.sub.2SO.sub.4, filtered,
evaporated and dried in vacuo to afford the corresponding amine.
The amine (2.15 g, 44%) was used without further purification.
[0889] To a solution of amine (2.15 g, 11.0 mmol) in Pinacolone (6
mL) and toluene (6 mL) was added 1,3-propane sultone (1.28 g, 10.5
mmol). The solution was stirred at reflux overnight. The reaction
mixture was cooled to room temperature. The solid material was
collected by filtration, was washed with acetone (2.times.20 mL).
The solid was suspended in EtOH (30 mL). The suspension was stirred
at reflux for 1 hour. The mixture was cooled to room temperature.
The white solid was filtered, washed with acetone (2.times.15 mL)
and dried in a vacuum oven at 50.degree. C., affording the title
compound, 2.26 g (66%). .sup.1H NMR (DMSO, 500 MHz) .delta. ppm
8.45 (s (broad), 1H), 7.26 (t, 1H, J=7.9 Hz), 6.89 (m, 3H), 6.30
(d, 1H, J=3.2 Hz), 4.69 (d, 1H, J=3.8 Hz), 3.74 (s, 3H), 3.10 (m,
2H), 2.62 (t, 2H, J=6.7 Hz), 2.00 (m, 2H), 1.07 (m, 6H). .sup.13C
(DMSO, 125 MHz) .delta. ppm 159.24, 142.09, 129.45, 120.80, 114.30,
113.76, 74.16, 62.48, 55.92, 50.10, 41.57, 23.30, 21.18, 19.37.
ES-MS 316 (M-1).
Preparation of
3-{[1-(4-methylbenzyl)cyclohexyl]amino}-1-propane-1-sulfonic acid
(Compound NH)
##STR00680##
[0891] NaOMe (0.5M, 40 mL) was added to nitrocyclohexane (2.58 g,
20 mmol) and the solution was stirred for 30 minutes then
concentrated to afford a white solid. To this solid was added
4-methylbenzylpyrridinium (6.6 g, 13 mmol) and DMSO (20 mL). The
mixture was heated at 100.degree. C. for 15 hours then cooled to rt
and diluted with HCl (IM) and EtOAc. After separation of the two
phases, the organic layer was washed twice with HCl (1M) then
concentrated to obtain an oily crude. Methanol was added to
precipitate the pyridinium byproduct which was filtered off, and
the filtrate was concentrated and purified by column using
Hex:EtOAc 90:10 to obtain the desired nitro (still contaminated
with the pyridinium salt). 2 g, 66% yield.
[0892] To a stirred solution of the nitro (2.0 g, 8.58 mmol) in
methanol (20 mL) was added a spatula of Raney-Ni in water. The
suspension was hydrogenated under atmospheric pressure of hydrogen
for 15 hours (TLC indicates complete consumption of the starting
material) then filtered on celite and concentrated under reduced
pressure. The crude was purified by column using
CH.sub.2Cl.sub.2:MeOH 80:10 to afford 1.2 g of the corresponding
amine.
[0893] To a stirred solution of the amine (800 mg, 3.93 mmol) in
THF (8 mL) was added 1,3-propane sultone (480 mg, 3.93 mmol). The
reaction mixture was stirred at reflux for 15 hours then cooled to
room temperature. The solid was collected by filtration and was
washed with THF. The solid was suspended in EtOH (10 mL) and
stirred at reflux for 1 hour. The suspension was then cooled to
room temperature. The solid was collected by filtration, washed
with ethanol and dried under high vacuum to afford the title
compound, 1.1 g (86%). .sup.1H NMR (500 MHz, DMSO-d.sub.6) .delta.
1.18-1.78 (m, 10H), 2.00 (m, 2H), 2.29 (s, 3H), 2.65 (m, 2H), 2.92
(s, 2H), 3.12 (m, 2H), 7.10-7.16 (m, 2H), 8.39 (bs, 2H). .sup.13NMR
(125 MHz, DMSO-d.sub.6) .delta. 20.74, 21.35, 22.80, 25.05, 31.60,
41.04, 50.24, 61.63, 129.71, 131.38, 132.44, 136.80. ES-MS 324
(M-1).
Preparation of
3-{[2-(4-methoxyphenyl)-1,1-dimethylethyl]amino}-1-propanesulfonic
acid (Compound NI)
##STR00681##
[0895] To a stirred solution of the phenol (233 mg, 1 mmol) in
DMF/THF (2.5 mL/2.5 mL) was added MeI (93 uL, 1.5 mmol) followed by
K.sub.2CO.sub.3 (276 mg, 2 mmol). The suspension was heated at
reflux for 15 hours then diluted with HCl (1M) and with EtOAc. The
organic layer was washed with HCl(1M) then concentrated under high
vacuum. The crude was purified by column using Hex:EtOAc 90:10 to
obtain 215 mg of the methoxy (87% yield).
[0896] To a stirred solution of the nitro (300 mg, 1.2 mmol) in
methanol (5 mL) was added a small spatula of Raney-Ni in water. The
suspension was hydrogenated under atmospheric pressure of hydrogen
for 3 hours (TLC indicates complete consumption of the starting
material) then filtered on prewashed celite and concentrated under
reduced pressure. The crude amine was used as such in the next
step.
[0897] To the crude amine (240 mg, 1.34 mmol) in solution in THF (3
mL) was added 1,3-sultone (181 mg, 1.48 mmol) and the mixture was
heated at reflux of THF for 12 hours. The suspension of was cooled
down and filtered. The solid was dried to afford 270 mg of the
homotaurin as a white solid (67% yield). .sup.1H NMR (500 MHz,
D.sub.2O) .delta. 1.11 (s, 6H), 2.00 (m, 2H), 2.67 (m, 2H), 2.80
(m, 2H), 3.12 (m, 2H), 3.74 (s, 3H), 6.90 (m, 2H), 7.14 (m, 2H),
8.61 (bs, 2H). ES-MS 272 (M-1). ES-MS 300 (M-1).
Preparation of
3-{[2-hydroxy-1,1-dimethyl-2-(4-methylphenyl)ethyl]amino}-1-propanesulfon-
ic acid (Compound NJ)
##STR00682##
[0899] To a solution of 2-nitropropane (3.0 g, 34 mmol),
p-tolualdehyde (4.0 mL, 34 mmol) and Tetrahydrofuran (30 mL) was
added Amberlyst A-21 (7 g). The reaction mixture was stirred at
room temperature for 40 hours. The resin was removed by filtration
and washed with THF (2.times.20 mL). The filtrate was evaporated.
The resulting oil was purified by flash chromatography: 98%
Hexanes/EtOAc to 90% Hexanes/EtOAc, affording the desired nitro
compound (820 mg, 12%).
[0900] A suspension of Pd/C and the nitro compound (820 mg, 3.9
mmol) in EtOAc (10 mL) was stirred under H.sub.2 (1 atm) overnight.
The mixture was filtered on a celite pad. The celite was washed
with EtOAc (2.times.15 mL). The combined filtrates were evaporated
under reduced pressure to afford the corresponding amine. The amine
(470 mg, 67%) was used without further purification.
[0901] To a solution of amine (470 mg, 2.6 mmol) in pinacolone (5
mL) and Toluene (5 mL) was added 1,3-propane sultone (310 mg, 2.5
mmol). The solution was stirred at reflux for 4 hours. The reaction
mixture was cooled to room temperature. The solid was collected by
filtration, was washed with acetone (2.times.10 mL) and dried in
vacuo, affording the title compound, 196 mg (26%). NMR (DMSO, 500
MHz) .delta. ppm 8.46 (s (broad), 1H), 7.24 (d, 2H, J=7.8 Hz), 7.16
(d, 2H, J=8.3 Hz), 6.23 (d, 1H, J=3.9 Hz), 4.68 (d, 1H, J=3.9 Hz),
3.11 (m, 2H), 2.63 (t, 2H, J=6.8 Hz), 2.29 (s, 3H), 2.00 (m, 2H),
1.04 (s, 6H). .sup.13C (DMSO, 125 MHz) .delta. ppm 137.69, 137.56,
129.03, 128.45, 74.07, 62.38, 49.91, 41.35, 22.99, 21.39, 20.81,
18.76. ES-MS 300 (M-1).
Preparation of
3-{[1,1-dimethyl-2-(4-methylphenyl)ethyl]amino}-1-propanesulfonic
acid (Compound NK)
##STR00683##
[0903] NaOMe (0.5M, 20 mL) was added to 2-nitropropane (890 mg, 10
mmol) and the solution was stirred for 30 minutes then concentrated
to afford a white solid. To this solid was added
4-methylbenzylpyrridinium (3.3 g, 15 mmol) and DMSO (15 mL). The
mixture was heated at 100.degree. C. for 15 hours then cooled to
room temperature and diluted with HCl (1M) and EtOAc. After
separation of the two phases, the organic layer was washed twice
with HCl (1M) then concentrated to obtain an oily crude product.
Methanol was added to precipitate the pyridinium byproduct which
was filtered off, and the filtrate was concentrated and purified by
column using Hex:EtOAc 90:10 to obtain the desired nitro but still
contaminated with the pyridinium salt. 1.32 g, 66% yield.
[0904] To a stirred solution of the nitro (700 mg, 3.62 mmol) in
methanol (10 mL) was added a small spatula of Raney-Ni in water.
The suspension was hydrogenated under atmospheric pressure of
hydrogen for 15 hours (TLC indicates complete consumption of the
starting material) then filtered on celite and concentrated under
reduced pressure. The crude amine was used as such in the next
step.
[0905] To a stirred solution of the amine (550 mg, 3.39 mmol) in
THF (8 mL) was added 1,3-propane sultone (414 mg, 3.39 mmol). The
reaction mixture was stirred at reflux for 6 hours then cooled to
room temperature. The solid was collected by filtration and was
washed with THF. The solid was suspended in EtOH (5 mL) and stirred
at reflux for 1 hour. The suspension was then cooled to room
temperature. The solid was collected by filtration, washed with
ethanol and dried under high vacuum to afford the title compound,
210 mg (22%). .sup.1H NMR (500 MHz, DMSO-d.sub.6) .delta. 1.13 (s,
6H), 2.00 (m, 2H), 2.66 (dd, J=7.0 & 7.0 Hz, 2H), 2.75 (s, 2H),
3.10 (dd, J=7.0 & 7.0 Hz, 2H), 6.72 (d, J=8.3 Hz, 2H), 7.00 (d,
J=8.3 Hz, 2H), 8.60 (bs, 2H), 9.36 (s, 1H). .sup.13NMR (125 MHz,
DMSO-d.sub.6) .delta. 23.1, 41.2, 43.2, 49.8, 59.4, 115.7, 125.8,
132.3, 157.1. ES-MS 284 (M-1).
Preparation of
3-[2-(4-fluorophenyl)-2-hydroxy-1,1-dimethylethyl)amino]-1-propanesulfoni-
c acid (Compound NL)
##STR00684##
[0907] 14 g of washed Amberlyst A21 ion exchange resin were placed
in a round bottom flask to which was added nitropropane (14 mL, 120
mmol) and 4F-fluorobenzaldehyde (7.45 g, 60 mmol). The reaction
mixture was stirred overnight then diluted with Et.sub.2O and
filtered. The filtrate was concentrated under rotavap vaccuo then
pump vaccuo by heating at 120.degree. C. to remove the excess of
aldehyde. The crude was purified by column using Hex:EA 90:10 to
afford 3.3 g (25%) of the Henry-aldol product as a colorless
solid.
[0908] To a solution of the nitro compound (5 g, 23.5 mmol) in MeOH
(100 mL) was added 6M HCl (25 mL). After cooling to 5.degree. C.,
zinc powder (7.6 g, 117 mmol) was added. The suspension was stirred
at room temperature for 3 hours then filtered on a celite pad. The
filter cake was washed with MeOH (2.times.20 mL). The combined
filtrates were evaporated under reduced pressure. The residue was
dissolved in EtOAc (40 mL), then K.sub.2CO.sub.3 (1M) was added
until basic pH. The organic phase was dried with Na.sub.2SO.sub.4,
filtered, evaporated and dried in vacuo to afford 3.5 g (83% yield)
of the corresponding amine. The amine was used without further
purification.
[0909] To a stirred solution of the amine (3.3 g, 18.0 mmol) in THF
(20 mL) was added 1,3-propane sultone (2.2 g, 18.0 mmol). The
reaction mixture was stirred at reflux for 15 hours then cooled to
room temperature. The solid was collected by filtration, washed
with ethanol and with Et.sub.2O then dried under high vacuum to
afford the title compound, 4.25 g (77% yield). .sup.1H NMR (500
MHz, DMSO-d.sub.6) .delta. 1.13 (s, 6H), 2.00 (m, 2H), 2.66 (dd,
J=7.0 & 7.0 Hz, 2H), 2.75 (s, 2H), 3.10 (dd, J=7.0 & 7.0
Hz, 2H), 6.72 (d, J=8.3 Hz, 2H), 7.00 (d, J=8.3 Hz, 2H), 8.60 (bs,
2H), 9.36 (s, 1H). .sup.13NMR (125 MHz, DMSO-d.sub.6) .delta. 23.1,
41.2, 43.2, 49.8, 59.4, 115.7, 125.8, 132.3, 157.1. .sup.19F (282
MHz, DMSO-d.sub.6) .delta. -115.15 (m, 1F). ES-MS 304 (M-1).
* * * * *