U.S. patent application number 13/538676 was filed with the patent office on 2013-02-07 for cationic steroid antimicrobial compositions and methods of use.
The applicant listed for this patent is Donald Y.M. Leung, Paul B. Savage. Invention is credited to Donald Y.M. Leung, Paul B. Savage.
Application Number | 20130034500 13/538676 |
Document ID | / |
Family ID | 38318649 |
Filed Date | 2013-02-07 |
United States Patent
Application |
20130034500 |
Kind Code |
A1 |
Savage; Paul B. ; et
al. |
February 7, 2013 |
Cationic Steroid Antimicrobial Compositions and Methods of Use
Abstract
The invention provides methods for decreasing or inhibiting
herpesviridae (HV) infection or pathogenesis of a cell in vitro, ex
vivo or in vivo, a symptom or pathology associated with a
herpesviridae (HV) infection or pathogenesis in vitro, ex vivo or
in vivo, or an adverse side effect of herpesviridae (HV) infection
or pathogenesis in vitro, ex vivo or in vivo. In one embodiment, a
method of the invention includes treating a subject with an
invention compound (e.g., cationic steroid antimicrobial or
CSA).
Inventors: |
Savage; Paul B.; (Mapleton,
UT) ; Leung; Donald Y.M.; (Denver, CO) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Savage; Paul B.
Leung; Donald Y.M. |
Mapleton
Denver |
UT
CO |
US
US |
|
|
Family ID: |
38318649 |
Appl. No.: |
13/538676 |
Filed: |
June 29, 2012 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12876993 |
Sep 7, 2010 |
8211879 |
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13538676 |
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11669803 |
Jan 31, 2007 |
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12876993 |
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60764129 |
Feb 1, 2006 |
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Current U.S.
Class: |
424/9.2 ;
424/159.1; 435/375; 435/5; 514/170; 514/171; 514/182 |
Current CPC
Class: |
A61P 31/16 20180101;
A61K 31/56 20130101; A61K 31/568 20130101; A61K 31/568 20130101;
A61K 31/57 20130101; A61K 31/575 20130101; G01N 2333/03 20130101;
C12Q 1/18 20130101; A61K 31/56 20130101; A61K 2300/00 20130101;
A61K 31/57 20130101; A61P 31/22 20180101; A61K 31/575 20130101;
A61K 2300/00 20130101; A61K 45/06 20130101; A61K 2300/00 20130101;
A61K 2300/00 20130101 |
Class at
Publication: |
424/9.2 ;
514/182; 514/170; 514/171; 424/159.1; 435/375; 435/5 |
International
Class: |
A61K 31/575 20060101
A61K031/575; C12Q 1/70 20060101 C12Q001/70; A61P 31/22 20060101
A61P031/22; C12N 5/07 20100101 C12N005/07; A61K 39/42 20060101
A61K039/42; A61K 49/00 20060101 A61K049/00 |
Goverment Interests
GOVERNMENT FUNDING
[0002] Work described herein was supported in part by grants
N01-AI-40029, awarded by the National Institutes of Health. The
United States Government may have certain rights in this invention.
Claims
1. A method for providing a subject with protection against a
herpesviridae (HV) infection or pathogenesis, comprising
administering a sufficient amount of cationic steroid antimicrobial
(CSA) to provide the subject with protection against herpesviridae
(HV) infection or pathogenesis.
2. A method for treating a subject in need of treatment for
herpesviridae (HV) infection or pathogenesis, comprising
administering a sufficient amount of cationic steroid antimicrobial
(CSA) to treat the subject for the herpesviridae (HV) infection or
pathogenesis.
3. A method for decreasing susceptibility or inhibiting
herpesviridae (HV) reactivation from latency in a subject,
comprising administering a sufficient amount of cationic steroid
antimicrobial (CSA) to decrease susceptibility or inhibit
herpesviridae (HV) reactivation from latency in the subject.
4. The method of claim 1, wherein the CSA is administered prior to,
concurrently with, or following infection of the subject with HV,
exposure to or contact of the subject with HV, or reactivation of
HV.
5. The method of claim 1, wherein the CSA is administered prior to,
concurrently with, or following development of a symptom or
pathology of acute or chronic HV infection, or reactivation of HV
from latency.
6. The method of claim 1, wherein the CSA is administered to a
biological fluid, an immune cell or tissue, mucosal cell or tissue,
neural cell or tissue, or epithelial cell or tissue.
7. The method of claim 1, wherein the HV is present in a biological
fluid, cell, tissue or organ.
8-9. (canceled)
10. The method of claim 1, wherein the HV is present in an immune
cell tissue or organ, mucosal cell, tissue or organ, neural cell,
tissue or organ, or epithelial cell, tissue or organ.
11-14. (canceled)
15. The method of claim 1, wherein the HV comprises an
alpha-herpesvirus, beta-herpesvirus or gamma-herpesvirus.
16-17. (canceled)
18. The method of any of claim 1, wherein the CSA is selected from
CSA-7, CSA-8, CSA-10, CSA-11, CSA-13, CSA-15, CSA-17, CSA-21,
CSA-25, CSA-26, CSA-31, CSA-46, CSA-54 and CSA-59, as set forth in
FIG. 10.
19-24. (canceled)
25. The method of claim 1, wherein the CSA has a shorter tether
length between the steroid scaffold and the amine groups at
positions C3, C7 and C12, relative to the tether length of CSA-7,
CSA-8, CSA-10, CSA-11, CSA-13, CSA-15, CSA-17, CSA-21, CSA-25,
CSA-26, CSA-31, CSA-46, CSA-54 or CSA-59, as set forth in FIG.
10.
26. The method of claim 1, wherein the CSA comprises a
pharmaceutically acceptable carrier or excipient.
27. The method of claim 1, wherein the CSA comprises a sterile
formulation.
28. The method of claim 1, wherein the CSA comprises a composition
comprising one or more additional CSAs or biologically active
ingredients.
29-46. (canceled)
47. The method of claim 1, further comprising administering to the
subject an additional CSA or treatment.
48-52. (canceled)
53. The method of claim 47, wherein the additional treatment
comprises an antibody that binds to an HV protein.
54. The method of claim 53, wherein the HV protein is selected
from: envelope protein, tegument protein, capsid protein, core
protein and polymerase.
55. The method of claim 54, wherein the envelope protein comprises
glycoprotein gp42, gp350, gpK8.1A, B, C, D, E, H, L (gB, gC, gD,
gE, gH, gL).
56. The method of claim 54, wherein the tegument protein comprises:
UL17, UL36, UL37, UL48, UL49, US11, UL11, UL14, UL16, UL21, UL41,
UL46, UL47, VP13/14, VP16 and VP22.
57-59. (canceled)
60. A method for decreasing or inhibiting herpesviridae (HV)
infection of a cell or herpesviridae (HV) reactivation from
latency, in vitro or in vivo, comprising administering a
composition comprising a sufficient amount of cationic steroid
antimicrobial (CSA) to inhibit herpesviridae (HV) infection of the
cell.
61-65. (canceled)
66. A method for reducing, decreasing, inhibiting, ameliorating or
preventing onset, severity, duration, progression, frequency or
probability of one or more symptoms or pathologies associated with
or caused by herpesviridae (HV) infection or pathogenesis, or
reactivation of herpesviridae (HV) from latency, in a subject,
comprising administering a sufficient amount of CSA-7, CSA-8,
CSA-10, CSA-11, CSA-13, CSA-15, CSA-17, CSA-21, CSA-25, CSA-26,
CSA-31, CSA-46, CSA-54 and CSA-59, as set forth in FIG. 10, to
decrease, inhibit, ameliorate or prevent onset, severity, duration,
progression, frequency or probability of one or more symptoms or
pathologies associated with or caused by herpesviridae (HV)
infection or pathogenesis, or reactivation of herpesviridae (HV)
from latency in the subject.
67. (canceled)
68. A method for identifying a candidate agent for treating a
subject for an HV infection or pathogenesis, or reactivation from
latency, comprising: a) providing a test agent, said test agent
comprising a cationic steroid antimicrobial (CSA); b) contacting
said test agent with HV and ascertaining whether the test agent
inhibits HV infection or pathogenesis, or reactivation from
latency, wherein a test agent identified as inhibiting HV infection
or pathogenesis or reactivation from latency is a candidate agent
for treating a subject for HV infection or pathogenesis.
69. A method for identifying a candidate agent for decreasing
susceptibility or inhibiting HV reactivation from latency,
comprising: a) providing a test agent, said test agent comprising a
cationic steroid antimicrobial (CSA); b) contacting said test agent
with HV and ascertaining whether the test agent decreases
susceptibility or inhibits HV reactivation from latency, wherein a
test agent identified as decreasing susceptibility or inhibiting HV
reactivation from latency is a candidate agent for decreasing
susceptibility or inhibiting HV reactivation from latency.
70. A method for identifying a candidate agent for decreasing,
inhibiting, ameliorating or preventing onset, severity, duration,
progression, frequency or probability of one or more symptoms or
pathologies caused by or associated with HV infection or
pathogenesis or reactivation from latency comprising: a) providing
a test agent, said test agent comprising a cationic steroid
antimicrobial (CSA); b) administering said test agent to a subject
infected with or exposed to HV and ascertaining whether the test
agent decreases, inhibits, ameliorates or prevents onset, severity,
duration, progression, frequency or probability of one or more
symptoms or pathologies associated with or caused by HV infection
or pathogenesis, or reactivation from latency, wherein a test agent
identified is a candidate agent for decreasing, inhibiting,
ameliorating or preventing onset, severity, duration, progression,
frequency or probability of one or more symptoms or pathologies
associated with or caused by HV infection or pathogenesis or
reactivation from latency.
71-72. (canceled)
Description
RELATED APPLICATIONS
[0001] This application claims the benefit of priority of
provisional application Ser. No. 60/764,129, filed Feb. 1, 2006,
which is expressly incorporated herein by reference.
TECHNICAL FIELD
[0003] The invention relates to methods for decreasing or
inhibiting Herpesviridae (HV) infection or pathogenesis of a cell
in vitro, ex vivo or in vivo, a symptom or pathology associated
with a herpesviridae (HV) infection or pathogenesis in vitro, ex
vivo or in vivo, or an adverse side effect of herpesviridae (HV)
infection or pathogenesis in vitro, ex vivo or in vivo. In one
embodiment, a method of the invention includes treating a subject
with an invention compound (e.g., cationic steroid antimicrobial or
CSA).
INTRODUCTION
[0004] Vaccination has remained the best method for preventing
virus spread. The herpes simplex virus (HV) candidate vaccines
tested till now were mostly purified subunit vaccines and/or
recombinant envelope glycoproteins (such as gB and gD). In various
animal studies, protection against acute virus challenge was
demonstrated along with the reduction of the extent of latency,
when established in the immunized host. However, the
immunotherapeutic effect of herpes vaccines seems less
convincing.
SUMMARY
[0005] Cationic steroid antimicrobials (CSAs) were developed as
functional mimics of endogenous peptide antibiotics such as LL-37.
A series of CSAs have been developed and CSAs are highly active
against specific lipid-enveloped viruses including herpesviridae
(HV) (e.g., herpes simplex virus). Antiviral activities of multiple
CSAs have been measured, and active and inactive forms have been
identified.
DESCRIPTION OF THE DRAWINGS
[0006] FIG. 1 is a drawing showing compounds of the invention.
[0007] FIG. 2 is a drawing showing compounds CSA-26 and CSA-46.
[0008] FIG. 3 is a drawing showing compound 134.
[0009] FIG. 4 is a drawing showing compound CSA-10.
[0010] FIG. 5 is a dr awing showing compound 140.
[0011] FIG. 6 is a drawing showing compound CSA-31.
[0012] FIG. 7 is a drawing showing compounds 352-354.
[0013] FIG. 8 is a drawing showing compounds 341-343 and
324-327.
[0014] FIG. 9 is a drawing showing compounds 358.
[0015] FIG. 10 is a drawing showing various compounds of the
invention (CSAs).
[0016] FIG. 11 shows antiviral activity of CSA-8, CSA-13, CSA-31
and CSA-54, as determined by a viral killing assay.
DETAILED DESCRIPTION
[0017] In accordance with the invention, there are provided methods
for decreasing or inhibiting herpesviridae (HV) infection or
pathogenesis of a cell in vitro, ex vivo or in vivo, a symptom or
pathology associated with a herpesviridae (HV) infection or
pathogenesis in vitro, ex vivo or in vivo, or an adverse side
effect of herpesviridae (HV) infection or pathogenesis in vitro, ex
vivo or in vivo. In one embodiment, a method of the invention
includes treating a subject with an invention compound (e.g.,
cationic steroid antimicrobial or CSA), wherein the subject is in
need of treatment with CSA anti-herpesviridae (HV) activity or
function, in order to provide the subject with a beneficial effect
or improvement. In another embodiment, a method of the invention
includes providing a subject with protection against a
herpesviridae (HV) infection or pathogenesis by administering a
composition comprising a sufficient amount of CSA to provide the
subject with protection against a herpesviridae (HV) infection or
pathogenesis. In a further embodiment, a method of the invention
includes treating a subject for herpesviridae (HV) infection or
pathogenesis by administering a composition comprising a sufficient
amount of CSA to treat the subject for the herpesviridae (HV)
infection or pathogenesis. In an additional embodiment, a method of
the invention includes decreasing susceptibility of a subject to a
herpesviridae (HV) infection or pathogenesis by administering a
composition comprising a sufficient amount of CSA to decrease
susceptibility of the subject to a herpesviridae (HV) infection or
pathogenesis. Methods of the invention include administering CSA
prior to, concurrently with, or following contact of the subject
with, exposure of the subject to, infection with or reactivation of
a herpesviridae (HV); and administering CSA prior to, concurrently
with, or following development of a symptom or pathology associated
with or caused by herpesviridae (HV) infection or reactivation. In
various aspects, a compound of the invention (e.g., CSA) is
administered prior to (prophylaxis), concurrently with or following
infection, contact or exposure of the subject to HV, or
reactivation of HV (therapeutic).
[0018] The invention treatment methods therefore include, among
other things, therapeutic and prophylactic methods. Subjects can be
contacted with, administered ex vivo or in vivo delivered a
compound of the invention (e.g., GSA) prior to, concurrently with
or following HV exposure or contact, HV infection, development of a
symptom or pathology associated with or caused by a HV infection or
pathogenesis, or reactivation of HV from latency.
[0019] The term "therapeutic" and grammatical variations thereof
means the subject has a herpesviridae (HV) infection, for example,
the subject exhibits one or more symptoms or pathologies associated
with or caused by an acute or chronic HV infection, reactivation or
pathogenesis as set forth herein or known in the art. The term
"therapeutic" also includes a subject that has been exposed to or
contacted with HV but may not exhibit one or more symptoms or
pathologies associated with or caused by acute or chronic HV
infection, reactivation or pathogenesis, as set forth herein or
known in the art.
[0020] "Prophylaxis" and grammatical variations thereof refer to
contact, administration or in vivo delivery to a subject prior to a
known contact with or exposure to herpesviridae (HV). In situations
where it is not known if a subject has been contacted with or
exposed to HV, contact with, administration or in vivo delivery of
a compound to a subject occurs prior to manifestation or onset of a
symptom associated with or caused by HV infection or pathogenesis.
In such a method, the effect of contact with, administration or in
vivo delivery of a compound of the invention (e.g., CSA) can be to
eliminate, prevent, inhibit, decrease or reduce the probability of
or susceptibility towards developing an HV infection, reactivation
or pathogenesis, or a symptom or pathology associated with or
caused by HV infection, reactivation or pathogenesis.
[0021] As used herein, the term "associated with," when used in
reference to the relationship between a symptom, pathology or
adverse side effect of herpesviridae (HV), means that the symptom,
pathology or side effect is caused by HV infection, reactivation
from latency, or pathogenesis, or is a secondary effect of HV
infection, reactivation from latency, or pathogenesis. A symptom,
pathology or side effect that is present in a subject may therefore
be the direct result of or caused by the herpesviridae (HV)
infection, reactivation or pathogenesis, or may be clue at least in
part to the subject reacting or responding to HV infection,
reactivation, or pathogenesis (e.g., the immunological response).
For example, a symptom or pathology that occurs during a
herpesviridae (HV) infection, reactivation or pathogenesis may be
due in part to an inflammatory response of the subject.
[0022] The invention also provides methods for decreasing or
preventing an adverse side effect caused by vaccination of a
subject with or against a herpesviridae (HV). In one embodiment, a
method includes administering a sufficient amount of CSA to the
subject to decrease or prevent an adverse side effect caused by
vaccination with a herpesviridae (HV). In one aspect, the
herpesviridae (HV) comprises an alpha-, beta- or gamma-herpesvirus
(e.g., herpes simplex virus-1 (HSV-1), herpes simplex virus-2
(HSV-2), varicella zoster virus (VZV/HHV-3), cytomegalovirus (CMV),
Epstein-Barr virus (EBV), human herpes virus-6, -7 or -8 (HHV-6,
HHV-7, or HHV-81 Kaposi's sarcoma herpesvirus/KSHV)).
[0023] Herpesviridae (HV) is typically found in biological fluids,
cells, tissues or organs, in vivo. Accordingly, HV present in any
biological fluid, cell, tissue or organ, is treatable with the
invention compounds and methods, locally, regionally or
systemically. In particular embodiments, HV is present in a
biological fluid (e.g., mucus, saliva, blood, serum, plasma,
cerebrospinal fluid, urine, or placenta); in a tissue or organ
comprising a transplant; in an immune cell, tissue or organ,
mucosal cell, tissue or organ, neural cell, tissue or organ, or
epithelial cell, tissue or organ. In particular aspects, an immune
cell is a T cell or a B cell; a mucosal cell or tissue is mouth,
buccal cavity, labia, nasopharynx, esophagus, trachea, lung,
stomach, small intestine, vagina, rectum, or colon; a neural cell
or tissue is ganglia, motor or sensory neuron; and an epithelial
cell or tissue is nose, fingers, ears, cornea, conjunctiva, skin or
dermis.
[0024] In particular embodiments of the compounds and methods of
the invention, a CSA is selected from: CSA-7, CSA-8, CSA-10,
CSA-11, CSA-13, CSA-15, CSA-17, CSA-21, CSA-25, CSA-26, CSA-31,
CSA-46, CSA-54 and CSA-59, as set forth in FIG. 10. In other
embodiments, a CSA does not have a charged group at position C24 or
a CSA has a hydrophobic moiety at position C24 (e.g., a lipid). In
additional embodiments, a CSA has a charged group at position C7.
In further embodiments, a CSA comprises a multimer (e.g., a dimer,
trimer, tetramer or higher order polymer). In yet additional
embodiments, a CSA has a shorter tether length between the steroid
scaffold and any amine group at positions C3, C7 or C12, relative
to the tether length between the steroid scaffold and any amine
group at positions C3, C7 or C12 of CSA-7, CSA-8, CSA-10, CSA-11,
CSA-13, CSA-15, CSA-17, CSA-21, CSA-25, CSA-26, CSA-31, CSA-46,
CSA-54 or CSA-59, as set forth in FIG. 10.
[0025] Methods of treatment include reducing, decreasing,
inhibiting, ameliorating or preventing onset, severity, duration,
progression, frequency or probability of one or more adverse side
effects associated with herpesviridae (HV) vaccination (e.g., a
live or attenuated pathogenic or non-pathogenic HV, a vaccine
comprising an HV protein, such as glycoprotein D, etc.).
Non-limiting examples of adverse side affects associated with HV
vaccination treatable with a compound of the invention include
fatigue, weakness, headache, fever, stomach ache/nausea, flu-like
symptoms, rash, vomiting, inflammation (cerebral or ocular) and
fainting.
[0026] Methods of the invention, including, for example,
prophylactic and therapeutic treatment methods, as well as methods
for decreasing or preventing an adverse side effect caused by
vaccination with or against herpesvirus, are applicable to HV
generally, more specifically, the members of the family
Herpesviridae. Herpesviridae (HV) includes any strain or isolate or
subtype or a species of HV, or combination of strains or isolates
or subtypes or species of herpesviruses. Particular examples are
infectious or pathogenic viruses. Specific non-limiting examples of
HV the subject of treatment with an invention compound (e.g., CSA)
include, for example, live or attenuated pathogenic and
non-pathogenic HV. Exemplary I-IV include, alpha-, beta- and
gamma-herpesvirus. Particular non-limiting examples of alpha-virus
include herpes simplex virus-1 (HSV-1), herpes simplex virus-2
(HSV-2) and varicella zoster virus (VZV/HHV-3). Particular
non-limiting examples of beta- and gamma-herpesvirus include
cytomegalovirus (CMV), Epstein-Barr virus (EBV), human herpes
virus-6, -7 and -8 (HHV-6, HHV-7, or HHV-8/Kaposi's sarcoma
herpesvirus/KSHV).
[0027] Methods of the invention include methods of treatment that
results in a beneficial effect. Particular non-limiting examples of
beneficial effects include providing a subject with partial or
complete protection against HV infection, reactivation or
pathogenesis, or a symptom caused by a HV infection, reactivation
or pathogenesis (e.g., inhibit or reduce probability or
susceptibility). Particular non-limiting examples of beneficial
effects also include reducing, decreasing, inhibiting, delaying or
preventing HV infection, reactivation or pathogenesis, and
reducing, decreasing, inhibiting, ameliorating or preventing onset,
severity, duration, progression, frequency or probability of one or
more symptoms or pathologies associated with a HV infection,
reactivation or pathogenesis. Additional non-limiting examples of
beneficial effects also include reducing, decreasing, amounts of,
or inhibiting, delaying or preventing increases in HV titer or
viral load, proliferation or replication. Further non-limiting
particular examples of beneficial effects include reducing,
decreasing, inhibiting, delaying, ameliorating or preventing onset,
progression, severity, duration, frequency, probability or
susceptibility of a subject to HV infection, reactivation or
pathogenesis, or accelerating, facilitating or hastening recovery
of a subject from HV infection, reactivation or pathogenesis or one
or more associated symptoms or pathologies.
[0028] Methods of the invention therefore include providing a
beneficial or therapeutic effect to a subject, for example,
reducing, decreasing, inhibiting, delaying, ameliorating or
preventing onset, progression, severity, duration, frequency or
probability of HV infection, reactivation or pathogenesis or one or
more symptoms or pathologies associated with or caused by HV
infection, reactivation or pathogenesis; reducing, decreasing,
inhibiting, delaying or preventing increases in HV titer, viral
load, replication, proliferation, or an amount of a viral protein
of one or more HV strains or isolates or subtypes. Stabilizing the
infection, reactivation, or a symptom or pathology thereof, or
preventing, inhibiting or delaying reactivation, worsening or
progression of infection, reactivation or a symptom or pathology
associated with or caused by HV infection, reactivation or
pathogenesis, or progression of the underlying HV infection, are
also included in various embodiments of the methods of the
invention.
[0029] Invention methods are applicable to providing a subject with
protection against HV infection, reactivation or pathogenesis,
treating a subject for HV infection, reactivation and pathogenesis;
and decreasing susceptibility or inhibiting HV reactivation from
latency in a subject. The invention methods are therefore
applicable to HV infection that is in an active state, latent state
or reactivated state.
[0030] The term "infection," when used in reference to means a
initial or primary infection. An infection may be "infectious" in
the sense that HV infects other sites in the infected host subject,
or contagious to other subjects (cross-infection), or may be
latent, in which case HV does not generally infect other sites or
is contagious to other subjects. In immunocompetent subjects,
initial/primary infection is usually either asymptomatic or causes
mild pathogenesis or symptoms; only a small proportion of subjects
develop more severe clinical illness. Primary infection is
self-limiting in immunocompetent patients. In contrast, primary HV
infection in immunocompromised subjects (e.g., immunosuppressant
treatment, HIV+, newborns/neonates, pregnant, elderly subjects,
etc.), can result in severe symptoms and even be fatal.
[0031] Following a primary or initial HV infection, the virus
establishes "latency," in the host subject which allows the virus
to evade immune clearance and remain in the host subject, and
infection is lifelong. In the latent state HV does not typically
cause illness or symptoms, there is little if any viral replication
and the subject is not infectious or contagious. Latency, also
referred to as "latent infection" may occur in a different cell
type from that of the initial/primary HV infection.
[0032] The term "reactivation," when used in reference to HV, means
activation of HV in the host subject following a period of latency.
Reactivation is associated with increased viral replication and
proliferation in an HV infected host subject, who becomes
infectious and contagious again. Symptoms and pathologies
associated with or caused by HV reactivation may or may not be the
same type, severity, frequency or duration as initial HV infection
and subsequent pathogenesis. For example, VZV/HHV-3 causes
chickenpox (primary infection) and shingles (reactivation).
Reactivation can be milder (e.g., asymptomatic) than an initial HV
infection/pathogenesis, in which case it would not be obvious
whether a host subject is in a latent or reactivated state. In
immunocompetent host subjects reactivation is typically mild,
whereas in immunocompromised host subjects, symptoms associated
with or caused by reactivation can be severe and lead to death.
Thus, clinical manifestations associated with reactivation may be
different from that observed with an initial/primary infection.
Accordingly, a single HV can cause different clinical symptoms or
pathologies. One symptom of HV reactivation is the appearance of
"cold sores" around mucosal areas (e.g., mouth, lips, tongue,
genitalia, etc.). Reactivation occurs periodically and can be
induced by stress, immune suppression, etc.
[0033] Specific examples of symptoms and pathologies associated
with or caused by herpesviridae (HV) infection, reactivation or
pathogenesis, whose onset, progression, severity, frequency,
duration or probability can be reduced, decreased inhibited,
delayed ameliorated or prevented include, for example, lesions,
ulcers, canker sore, cold sore, rash, boils, Gingivostomatitis,
Herpetic whitlow Traumatic herpes (herpes gladiatorum), Eczema
herpeticum, fever, fatigue, headache, sore throat, swollen lymph
nodes, pneumonitis, pneumonia, hepatitis, meningitis, myelitis,
Encephalitis, keratitis, Genital herpes, esophagitis, dysphasia,
hemiparesis, coma, shingles, chicken pox, mononucleosis, chronic or
acute pelvic inflammatory disease (PID), proctitis, colitis, nerve
damage and death. Other symptoms and pathologies of HV infection,
reactivation or pathogenesis, are known in the art and treatment
thereof in accordance with the invention is provided.
[0034] The methods of the invention, including, among other
methods, providing a subject with protection against a
herpesviridae (HV) infection, reactivation or pathogenesis,
treatment of a herpesviridae (HV) infection, reactivation or
pathogenesis, or a symptom or pathology associated with or caused
by herpesviridae (HV) infection, reactivation or pathogenesis, or
decreasing susceptibility of a subject to a herpesviridae (HV)
infection, reactivation or pathogenesis, can therefore result in an
improvement in the subjects' condition. An improvement is therefore
any objective or subjective reduction, decrease, inhibition, delay,
ameliorating or prevention of onset, progression, severity,
duration, frequency or probability of one or more symptoms or
pathologies associated with or caused by HV infection, reactivation
or pathogenesis (e.g., illness), or virus titer, viral load,
replication, proliferation, or an amount of a viral protein. An
improvement would also include reducing, inhibiting or preventing
increases in virus titer, viral load, replication, proliferation,
or an amount of a viral protein of one or more HV strains or
isolates or subtypes or species. An improvement would further
include stabilizing a symptom or pathology associated with or
caused by HV infection, reactivation or pathogenesis, or
inhibiting, decreasing, delaying or preventing a worsening or
progression of the symptom or pathology associated with or caused
by HV infection, reactivation or pathogenesis, or progression of
the underlying HV infection. An improvement can therefore be, for
example, in any of lesions, ulcers, canker sore, cold sore, rash,
boils, Gingivostomatitis, Herpetic whitlow Traumatic herpes (herpes
gladiatorum), Eczema herpeticum, fever, fatigue, headache, sore
throat, swollen lymph nodes, pneumonitis, pneumonia, hepatitis,
meningitis, myelitis, Encephalitis, keratitis, Genital herpes,
esophagitis, dysphasia, hemiparesis, coma, shingles, chicken pox,
mononucleosis, chronic or acute pelvic inflammatory disease (PID),
proctitis, colitis, nerve damage and death to any degree or for any
duration of time (hours, days, weeks, months, years, or cure).
[0035] An improvement would also include reducing or eliminating a
need, dosage amount or frequency of another treatment, such as an
antiviral drug or other agent used for treating a subject having or
at risk of having a herpesviridae (HV) infection, reactivation or
pathogenesis, a symptom or pathology associated with or caused by
herpesviridae (HV) infection, reactivation or pathogenesis, or
decreasing or preventing an adverse side effect caused by
vaccination with or against a herpesviridae (HV). Thus, reducing an
amount of another treatment for HV infection, reactivation or
pathogenesis, a symptom or pathology associated with or caused by
HV, or an adverse side effect caused by vaccination with or against
a HV is considered to provide a benefit and, therefore, is
considered within the invention methods. Non-limiting exemplary HV
treatments that may be eliminated or used at reduced doses or
frequencies of administration include protease inhibitors, reverse
transcriptase inhibitors, virus fusion inhibitors and virus entry
inhibitors. Additional non-limiting exemplary HV and other
treatments include AK602, AMD070, APV, ATV, ATZ, AVX754, AZT,
Abacavir, Acyclovir, Adefovir dipivoxil, Adriamycin, Agenerase,
Aldesleukin, Alovudine, AmBisome, Amdoxovir, Amphocin, Amphotec,
Amphotericin B, Ampligen, Amprenavir, Androderm, Androgel, Aptivus,
Atazanavir, Azithromycin, BMS-488043, Bactrim, Baraclude, Biaxin,
BufferGel, C31G, CD4-IgG2, CPV, CS, Calanolide A, Capravirine,
Carbopol 974P, Carrageenan, Carraguard, Cellulose sulfate,
Cidofovir, Clarithromycin, Combivir, Copegus, Cotrimoxazole,
Crixivan, Cyanovirin-N, Cytovene, DAPD, DLV, DPC 817, DS,
Delavirdine, Depo-Testosterone, Dextran sulfate, Didanosine,
Diflucan, Docosanol, Doxil, Doxorubicin, Dronabinol, EFV,
Efavirenz, Elvucitabine, Emtricitabine, Emtriva, Enfuvirtide,
Entecavir, Epivir, Epoetin alfa, Epogen, Epzicom, Etopophos
(phosphate salt), Etoposide, Etravirine, Famcyclovir, Fluconazole,
Foscarnet, Fortovase, Fosamprenavir, Fungizone, Fuzeon, GSK-873,140
(aplaviroc), GW433908, Gammar-P, Ganciclovir, Growth hormone, Human
growth hormone, HEC, Hepsera, Hivid, Hydroxyethyl cellulose, IDV,
IGIV, Interleukin-2 (IL-2), INH, Immune Globulin, Indinavir,
Interferon alfa-2, Intron A (2b), Invirase, Isoniazid,
Isoprinosine, Itraconazole, KP-1461, Kaletra, L-000870810, LPV/RTV,
Lamivudine, Lexiva, Marinol, Megace, Megestrol, Mycobutin, NFV,
NVP, Naphthalene 2-sulfonate polymer, Nebupent, Nelfinavir,
Neutrexin, Nevirapine, New-Fill, Norvir, Nydrazid, Onxol, PA-457,
PMPA, PRO2000, PRO542, Paclitaxel, Paxene, Pegasys (2a),
Pentamidine, Peptide T, Poly(I)-Poly(C12U), Poly-L-lactic acid,
Polygam SID, Procrit, Proleukin, RCV, RTV, RVT, Racivir, Rebetol,
Rescriptor, Retrovir, Reverset, Reyataz, Ribavirin, Rifabutin,
Rifadin, Rifampin, Rimactane, Ritonavir, Roferon-A (2a), SCH-C,
SCH-D (vicriviroc), SQV, Saquinavir, Savvy, Sculptra, Septra,
Serostim, Somatropin, Sporanox, Stavudine, Sulfarnethoxazole,
Sustanon, Sustiva, T-20, TDF, THC, TMC114, TMC125, TNX-355, Taxol,
Tenofovir, Tenofovir disoproxil fumarate, Testosterone, Tipranavir,
Toposar, Trimethoprim, Trimetrexate, Trizivir, Truvada, UC-781,
UK-427,857 (maraviroc), Ushercell, Valacyclovir, Vaicyte,
Valgancielovir, Valproic acid, VePesid, Vicriviroc, Vidabrine,
Videx, Viracept, Viramune, Virazole, Viread, Vitrasert, ZDV,
Zalcitabine, Zerit, Ziagen, Zidovudine, Zithromax, Zovirax, D4T,
ddC, .beta.-LFddC, P-LFd4C, DDI, f-APV, 3TC and human
erythropoietin (EPO). Further non-limiting exemplary treatments
include cytokines, chemokines, interferons and interleukins. Yet
additional non-limiting exemplary HV treatments include an antibody
that binds to an HV protein, such as an envelope protein (e.g.,
glycoprotein gp42, gp350, gpK8.1A, B, C, D, E, H, L (gB, gC, gD,
gE, gH, gL)), tegument protein (e.g., UL17, UL36, UL37, UL48, UL49,
US11, UL11, UL14, UL16, UL21, UL41, UL46, UL47, VP13/14, VP16,
VP22, etc.), capsid protein (e.g., VP5, VP19c, VP21, VP23, VP24,
VP26, etc.), core protein or polymerase. Still further non-limiting
exemplary HV treatments include vaccination, such as with an
attenuated or live HV.
[0036] A treatment or improvement need not be complete ablation of
any particular infection, reactivation, pathogenesis, symptom,
pathology or adverse side effect, or all of the infection,
reactivation, pathology, symptoms, pathologies or adverse side
effects associated with or caused by HV infection, reactivation or
pathogenesis, or vaccination with or against HV. Rather, treatment
may be any objective or subjective measurable or detectable
anti-virus effect or improvement in a treated subject. Thus,
reducing, inhibiting decreasing, eliminating, delaying, halting or
preventing a progression or worsening of the infection,
reactivation or pathogenesis, a symptom or pathology of the
infection, reactivation or pathogenesis, or an adverse side effect
caused by vaccination is a satisfactory outcome. For example, a
compound of the invention (e.g., CSA) may reduce, inhibit, delay
formation of, or stabilize lesions, ulcers, canker sores, or cold
sores, but not have a measurable effect on rash, boils,
Gingivostomatitis, Herpetic whitlow Traumatic herpes (herpes
gladiatorum), Eczema herpeticum, fever, fatigue, headache, sore
throat, swollen lymph nodes, pneumonitis, pneumonia, hepatitis,
meningitis, myelitis, Encephalitis, keratitis, Genital herpes,
esophagitis, dysphasia, hemiparesis, coma, shingles, chicken pox,
mononucleosis, chronic or acute pelvic inflammatory disease (PID),
proctitis, colitis, nerve damage or death. Another example is where
a compound of the invention reduces fever or fatigue, without a
detectable improvement in one or more other symptoms or
pathologies. Thus, a satisfactory clinical endpoint is achieved
when there is an incremental improvement in the subject's condition
or a partial reduction or a stabilization of a HV infection,
reactivation, pathogenesis or a symptom, pathology or adverse side
effect thereof, or an inhibition or prevention of worsening or
progression of the HV infection, reactivation, pathogenesis,
symptom, pathology or adverse side effect thereof (stabilizing one
or more symptoms or pathologies), over a short or long duration
(hours, days, weeks, months, years, or cure).
[0037] In the methods of the invention in which there is a desired
outcome, for example, a therapeutic or prophylactic method that
provides an objective or subjective improvement in a HV infection,
reactivation or pathogenesis, a symptom or pathology associated
with or caused by HV, or an adverse side effect caused by
vaccination with or against HV or an HV treatment, a compound of
the invention (e.g., CSA) can be administered in a sufficient or
effective amount. As used herein, a "sufficient amount" or
"effective amount" or an "amount sufficient" or an "amount
effective" refers to an amount that provides, in single or multiple
doses, alone or in combination with one or more other compounds,
treatments, agents (e.g., a drug) or therapeutic regimens, a long
term or a short term detectable or measurable improvement or
beneficial effect to a given subject of any degree or for any time
period or duration (e.g., for minutes, hours, days, months, years,
or cured).
[0038] A "sufficient amount" or "effective amount" therefore
includes decreasing, reducing, inhibiting, preventing, or delaying
onset; decreasing, reducing, inhibiting, delaying, or preventing a
progression or worsening of or reducing, relieving, ameliorating,
or alleviating, severity, frequency, duration, susceptibility or
probability of HV infection, reactivation or pathogenesis, one or
more symptoms associated with or caused by HV infection,
reactivation or pathogenesis, or an adverse side effect of
vaccination with or against a HV or an HV treatment. In addition,
hastening a subject's recovery from HV infection, reactivation or
pathogenesis, one or more symptoms associated with or caused by HV
infection, reactivation or pathogenesis, or an adverse side effect
of vaccination with or against a HV or an HV treatment is
considered to be a sufficient or effective amount. Various
beneficial effects and indicia of therapeutic and prophylactic
benefit are as set forth herein and are known to the skilled
artisan.
[0039] A sufficient amount or an effective amount can but need not
be provided in a single administration and can but need not be
administered alone (i.e., without a second drug, agent, treatment
or therapeutic regimen), or in combination with another compound,
agent, treatment or therapeutic regimen. In addition, a sufficient
amount or an effective amount need not be sufficient or effective
if given in single or multiple doses without a second compound,
treatment, agent, or therapeutic regimen, since additional doses,
amounts, frequency or duration of administration above and beyond
such doses, or additional compounds, agents, treatments or
therapeutic regimens may be included in order to be effective or
sufficient in a given subject.
[0040] A sufficient amount or an effective amount need not be
effective in each and every subject, nor a majority of subjects in
a given group or population. Thus, a sufficient amount or an
effective amount means sufficiency or effectiveness in a particular
subject, not a group or the general population. As is typical for
such methods, some subjects will exhibit a greater or less response
to a method of the invention than other subjects.
[0041] Amounts, frequencies or duration also considered sufficient
and effective and are therefore beneficial are those that result in
the elimination or a reduction in amount, frequency or duration of
another compound, agent, treatment or therapeutic regimen. For
example, a compound of the invention is considered as having a
beneficial or therapeutic effect if contact, administration or
delivery in vivo results in the use of a lesser amount, frequency
or duration of another compound, agent, treatment or therapeutic
regimen to treat the infection, pathogenesis, symptom or pathology,
or adverse side effect of vaccination.
[0042] Any compound, agent, treatment (e.g., a biologically active
ingredient) or other therapeutic regimen having a beneficial,
additive, synergistic or complementary activity or effect can be
formulated or used in combination with or in addition to the
invention compounds (e.g., CSAs). In various embodiments, the
compound, agent, treatment or therapeutic regimen is for providing
a subject with protection against HV infection, reactivation or
pathogenesis; treating a subject for HV infection, reactivation or
pathogenesis; decreasing susceptibility of a subject to a HV
infection, reactivation or pathogenesis; or decreasing or
preventing an adverse side effect caused by HV vaccination or an HV
treatment. Thus, compositions of the invention include CSA
combinations with other CSAs, CSA combinations with other agents or
treatments (e.g., biologically active ingredients such as
anti-herpesvirus drugs, such as acyclovir, herpesvirus proteins,
herpesvirus antibodies, herpesvirus vaccines, etc.), and methods of
the invention include contact with, administration in vitro or in
vivo, with another compound (e.g., another CSA or biologically
active ingredient), agent, treatment or therapeutic regimen
appropriate for the condition to be treated. The compound (e.g.,
another CSA or biologically active ingredient), agent, treatment or
therapeutic regimen appropriate may be used in accordance with the
prophylactic and therapeutic treatment methods, as well as methods
for decreasing or preventing an adverse side effect caused by HV
vaccination or HV treatment, as set forth herein, prior to,
concurrently or following contacting or administering a compound of
the invention (e.g., CSA) in vitro or in vivo.
[0043] Examples of such combination compositions and methods
include protease inhibitors, reverse transcriptase inhibitors,
virus fusion inhibitors and virus entry inhibitors. Additional
examples of combination compositions and methods include other
treatments such as AK602, AMD070, APV, ATV, ATZ, AVX754, AZT,
Abacavir, Acyclovir, Adefovir dipivoxil, Adriamycin, Agenerase,
Aldesleukin, Alovudine, AmBisome, Amdoxovir, Amphocin, Amphotec,
Amphotericin B, Ampligen, Amprenavir, Androderm, Androgel, Aptivus,
Atazanavir, Azithromycin, BMS-488043, Bactrim, Baraclude, Biaxin,
BufferGel, C31G, CD4-IgG2, CPV, CS, Calanolide A, Capravirine,
Carbopol 974P, Carrageenan, Carraguard, Cellulose sulfate,
Cidofovir, Clarithromycin, Combivir, Copegus, Cotrimoxazole,
Crixivan, Cyanovirin-N, Cytovene, DAPD, DLV, DPC 817, DS,
Delavirdine, Depo-Testosterone, Dextran sulfate, Didanosine,
Diflucan, Docosanol, Doxil, Doxorubicin, Dronabinol, EFV,
Efavirenz, Elvucitabine, Emtricitabine, Emtriva, Enfuvirtide,
Entecavir, Epivir, Epoetin alfa, Epogen, Epzicom, Etopophos
(phosphate salt), Etoposide, Etravirine, Famcyclovir, Fluconazole,
Foscarnet, Fortovase, Fosamprenavir, Fungizone, Fuzeon, GSK-873,140
(aplaviroc), GW433908, Gammar-P, Ganciclovir, Growth hormone, Human
growth hormone, HEC, Hepsera, Hivid, Hydroxyethyl cellulose, IDV,
IGIV, Interleukin-2 (IL-2), INH, Immune Globulin, Indinavir,
Interferon alfa-2, Intron A (2b), Invirase, Isoniazid,
Isoprinosine, Itraconazole, KP-1461, Kaletra, L-000870810, LPV/RTV,
Lamivudine, Lexiva, Marinol, Megace, Megestrol, Mycobutin, NFV,
NVP, Naphthalene 2-sulfonate polymer, Nebupent, Nelfinavir,
Neutrexin, Nevirapine, New-Fill, Norvir, Nydrazid, Onxol, PA-457,
PMPA, PRO2000, PRO542, Paclitaxel, Panne, Pegasys (2a),
Pentamidine, Peptide T, Poly(I)-Poly(C12U), Poly-L-lactic acid,
Polygam S/D, Procrit, Proleukin, RCV, RTV, RVT, Racivir, Rebetol,
Rescriptor, Retrovir, Reverset, Reyataz, Ribavirin, Rifabutin,
Rifadin, Rifampin, Rimactane, Ritonavir, Roferon-A (2a), SCH-C,
SCH-D (vicriviroc), SQV, Saquinavir, Savvy, Sculptra, Septra,
Serostim, Somatropin, Sporanox, Stavudine, Sulfamethoxazole,
Sustanon, Sustiva, T-20, TDF, THC, TMC114, TMC125, TNX-355, Taxol,
Tenofovir, Tenofovir disoproxil fumarate, Testosterone, Tipranavir,
Toposar, Trimethoprim, Trimetrexate, Trizivir, Truvada, UC-781,
UK-427,857 (maraviroc), Ushercell, Valacyclovir, Valcyte,
Valganciclovir, Valproic acid, VePesid, Vicriviroc, Vidabrine,
Videx, Viracept, Viramune, Virazole, Viread, Vitrasert, ZDV,
Zalcitabine, Zerit, Ziagen, Zidovudine, Zithromax, Zovirax, D4T,
ddC, J3-LFddC, P-LFd4C, DDI, f-APV, 3TC and human erythropoietin
(EPO). Further examples of combination compositions and methods
include cytokines, chemokines, interferons and interleukins.
[0044] Yet additional examples of combination compositions and
methods include an herpesvirus protein or antibodies that bind to
herpesvirus proteins. A pool of HV proteins or HV binding
antibodies (e.g., monoclonal or polyclonal) can be combined with a
compound of the invention or administered separately (prior to,
concurrently with or following) administration of a compound in
accordance with the invention. In particular embodiments, an
additional herpesvirus protein is an envelope protein (e.g.,
glycoprotein gp42, gp350, gpK8.1A, B, C, D, E, H, L (gB, gC, gD,
gE, gH, gL)), tegument protein (e.g., UL17, UL36, UL37, UL48, UL49,
US11, UL11, UL14, UL16, UL21, UL41, UL46, UL47, VP13/14, VP16,
VP22, etc.), capsid protein (e.g., VP5, VP19c, VP21, VP23, VP24,
VP26, etc.), core protein or polymerase.
[0045] Antibodies include proteins that bind to other molecules
(antigens) via heavy and light chain variable domains, V.sub.H and
V.sub.L, respectively. An antibody is any polyclonal or monoclonal
immunoglobulin molecule, or mixture thereof, such as IgM, IgG, IgA,
IgE, IgD, and any subclass thereof, such as IgG.sub.1, IgG.sub.2,
IgG.sub.3, IgG.sub.4, etc. A monoclonal antibody, refers to an
antibody that is based upon, obtained from or derived from a single
clone, including any eukaryotic, prokaryotic, or phage clone. An
antibody also includes a functional (e.g., binding) fragment or
subsequence, such as, for example, Fab, Fab', F(ab').sub.2, Fv, Fd,
scFv and sdFv, unless otherwise expressly stated.
[0046] Antibodies include those specific or selective for binding
to an HV protein or a homolog. That is, binding to proteins other
than the HV protein or a homolog is such that the binding does not
significantly interfere with detection of the HV protein or
homolog, unless such other proteins have a similar or same epitope
the HV protein or homolog that is recognized by the HV antibody.
Selective binding can be distinguished from non-selective binding
using specificity, affinity and other binding assays, competitive
and non-competitive, known in the art.
[0047] Antibodies include "human" forms, which mean that the amino
acid sequence of the antibody is fully human or can or do exist in
a human antibody. An antibody that is non-human may be made fully
human by substituting non-human amino acid residues with amino acid
residues that can or do exist in a human antibody. Amino acid
residues present in human antibodies, CDR region maps and human
antibody consensus residues are known in the art (see, e.g., Kabat,
Sequences of Proteins of Immunological Interest, 4.sup.th Ed. US
Department of Health and Human Services. Public Health Service
(1987); Chothia and Lesk J. Mol. Biol. 186:651 (1987); Padlan Mol.
Immunol. 31:169 (1994); and Padlan Mol. Immunol. 28:489
(1991)).
[0048] Antibodies include "human" forms, which means that the amino
acid sequence of the antibody has non-human amino acid residues
(e.g., mouse, rat, goat, rabbit, etc.) of one or more
complementarity determining regions (CDRs) that specifically bind
to the desired antigen in an acceptor human immunoglobulin
molecule, and one or more human amino acid residues in the Fv
framework region (FR), which are amino acid residues that flank the
CDRs. Antibodies referred to as "primatized" in the art are within
the meaning of "humanized" as used herein, except that the acceptor
human immunoglobulin molecule and framework region amino acid
residues may be any primate amino acid residue (e.g., ape, gibbon,
gorilla, chimpanzees orangutan, macaque), in addition to any human
residue.
[0049] Antibodies include "chimeric" forms, which means that the
amino acid sequence of the antibody contains one or more portions
that are derived from, obtained or isolated from, or based upon two
or more different species. That is, for example, a portion of the
antibody may be human (e.g., a constant region) and another portion
of the antibody may be non-human (e.g., a murine heavy or light
chain variable region). Thus, a chimeric antibody is a molecule in
which different portions of the antibody are of different species
origins. Unlike a humanized antibody, a chimeric antibody can have
the different species sequences in any region of the antibody.
[0050] The term "subject" refers to an animal, typically mammalian
animals, such as but not limited to non-human primates (apes,
gibbons, gorillas, chimpanzees, orangutans, macaques), domestic
animals (dogs and cats), a farm animals (chickens, ducks, horses,
cows, goats, sheep, pigs), experimental animal (mouse, rat, rabbit,
guinea pig) and humans. Subjects include animal models, for
example, a mouse model of herpesvirus infection (e.g., alpha, beta-
or gamma-herpesvirus). Subjects include naturally occurring or
non-naturally occurring mutated or non-human genetically engineered
(e.g., transgenic or knockout) animals. Subjects further include
animals having or at risk of having a chronic or acute HV
infection, reactivation or pathogenesis, symptom or pathology of HV
infection, reactivation or pathogenesis, or adverse side effect
caused by vaccination with or against HV or an HV treatment.
Subjects can be any age. For example, a subject (e.g., human) can
be a newborn, infant, toddler, child, teenager, or adult, e.g., 50
years or older.
[0051] Subjects include those in need of a method of the invention,
e.g., in need of a therapeutic or prophylactic treatment. A subject
is considered to be in need of a method of the invention where a
method is likely to provide some benefit to a subject. Various
benefits provided to a subject are as set forth herein and known in
the art for HV infection, reactivation or pathogenesis, symptoms or
pathologies caused by or associated with HV infection, reactivation
or pathogenesis, and adverse side effects caused by vaccination
with or against a HV or treatment of HV.
[0052] Subjects appropriate for treatment include those having HV
infection, reactivation or pathogenesis or currently or previously
having any symptom or pathology associated with or caused by HV
infection, reactivation or pathogenesis (e.g., diagnosed as HV+),
HV vaccination or an HV treatment, Target subjects therefore
include subjects infected with HV that are infectious or
contagious, subjects infected with HV that is in a latent state,
and subjects in which HV is or has been reactivated from latency.
Thus, subjects that have been exposed to a HV (e.g., subjects that
do produce an antibody against an HV protein) are appropriate
targets. Such subjects may or may not have developed one or more
adverse symptoms or pathologies associated with or caused by HV
infection, reactivation or pathogenesis, regardless of the virus
type, timing or degree of onset, progression, severity, frequency,
duration of any infection, pathogenesis, symptom, pathology or
adverse side effect. A subject may therefore be symptomatic or
asymptomatic for HV infection, reactivation or pathogenesis.
[0053] Subjects appropriate for treatment also include those at
risk of HV infection, reactivation or pathogenesis or at risk of
having or developing a symptom or pathology associated with or
caused by HV infection, reactivation or pathogenesis. Candidate
subjects therefore include subjects that have been exposed to or
contacted with HV, or that are at risk of exposure to or contact
with HV, regardless of the type, timing or extent of exposure or
contact. The invention methods are therefore applicable to a
subject who is at risk of HV infection, reactivation or
pathogenesis, but has not yet been exposed to or contacted with
herpesviridae (HV). Thus, subjects that have not been exposed to a
HV (e.g., subjects that do not produce an antibody against an HV
protein) are appropriate targets. Prophylactic methods are
therefore included. Subjects targeted for prophylaxis can be at
increased risk (probability or susceptibility) of herpesviridae
(HV) infection or pathogenesis, as set forth herein and known in
the art.
[0054] At risk subjects appropriate for treatment include subjects
exposed to other subjects having an HV infection or reactivation
(infectious or contagious), or where the risk of HV infection is
increased due to changes in virus infectivity or cell tropism,
immunological susceptibility (e.g., an immunocompromised subject),
or environmental risk. At risk subjects appropriate for treatment
therefore include human subjects exposed to or at risk of exposure
to other humans that have HV infection or reactivation (infectious
or contagious), or are at risk of a HV infection or reactivation
(infectious or contagious).
[0055] Subjects also appropriate for treatment also include those
vaccinated against or a candidate for vaccination against HV (e.g.,
vaccinated with live or attenuated HV or an HV protein or antibody
that binds to an HV protein). Subjects therefore include vaccinated
subjects that have not or have been exposed to or contacted with
HV, as well as candidate subjects for vaccination that have not or
have been exposed to or contacted with HV, regardless of the type,
timing or extent of exposure or contact. A subject can be
administered a compound of the invention (e.g., CSA) prior to,
concurrently with, or following vaccination (e.g., within 0-2, 2-4,
4-12 or 12-24 hours or days of vaccination).
[0056] Subjects further include immunocompromised subjects due to
an immunological disorder (e.g., autoimmunity) or disease, or an
immune-suppressing treatment (e.g., cyclophosphamide). Subjects
also include those having been exposed to or diagnosed as HV+.
Subjects further include those receiving or candidates for a tissue
or organ transplant.
[0057] Compounds of the invention, including CSAs, can be
incorporated into pharmaceutical compositions or formulations. Such
pharmaceutical compositions/formulations are useful for
administration to a subject, in vivo or ex vivo.
[0058] Pharmaceutical compositions and formulations include
carriers or excipients for administration to a subject. As used
herein the terms "pharmaceutically acceptable" and "physiologically
acceptable" mean a biologically compatible formulation, gaseous,
liquid or solid, or mixture thereof, which is suitable for one or
more routes of administration, in vivo delivery or contact. A
formulation is compatible in that it does not destroy activity of
an active ingredient therein (e.g., a GSA), or induce adverse side
effects that fax outweigh any prophylactic or therapeutic effect or
benefit.
[0059] Such formulations include solvents (aqueous or non-aqueous),
solutions (aqueous or non-aqueous), emulsions (e.g., oil-in-water
or water-in-oil), suspensions, syrups, elixirs, dispersion and
suspension media, coatings, isotonic and absorption promoting or
delaying agents, compatible with pharmaceutical administration or
in vivo contact or delivery. Aqueous and non-aqueous solvents,
solutions and suspensions may include suspending agents and
thickening agents. Such pharmaceutically acceptable carriers
include tablets (coated or uncoated), capsules (hard or soft),
microbeads, powder, granules and crystals. Supplementary active
compounds (e.g., preservatives, antibacterial, antiviral and
antifungal agents) can also be incorporated into the
compositions.
[0060] The formulations may, for convenience, be prepared or
provided as a unit dosage form. Preparation techniques include
bringing into association the active ingredient (e.g., GSA) and a
pharmaceutical carrier(s) or excipient(s). In general, formulations
are prepared by uniformly and intimately associating the active
ingredient with liquid carriers or finely divided solid carriers or
both, and then, if necessary, shaping the product. For example, a
tablet may be made by compression or molding. Compressed tablets
may be prepared by compressing, in a suitable machine, an active
ingredient (e.g., a CSA) in a free-flowing form such as a powder or
granules, optionally mixed with a binder, lubricant, inert diluent,
preservative, surface-active or dispersing agent. Molded tablets
may be produced by molding, in a suitable apparatus, a mixture of
powdered compound (e.g., CSA) moistened with an inert liquid
diluent. The tablets may optionally be coated or scored and may be
formulated so as to provide a slow or controlled release of the
active ingredient therein.
[0061] Cosolvents and adjuvants may be added to the formulation.
Non-limiting examples of cosolvents contain hydroxyl groups or
other polar groups, for example, alcohols, such as isopropyl
alcohol; glycols, such as propylene glycol, polyethyleneglycol,
polypropylene glycol, glycol ether; glycerol; polyoxyethylene
alcohols and polyoxyethylene fatty acid esters. Adjuvants include,
for example, surfactants such as, soya lecithin and oleic acid;
sorbitan esters such as sorbitan trioleate; and
polyvinylpyrrolidone.
[0062] Supplementary active compounds (e.g., preservatives,
antioxidants, antimicrobial agents including biocides and biostats
such as antibacterial, antiviral and antifungal agents) can also be
incorporated into the compositions. Preservatives and other
additives include, for example, antimicrobials, anti-oxidants,
chelating agents and inert gases (e.g., nitrogen). Pharmaceutical
compositions may therefore include preservatives, antimicrobial
agents, anti-oxidants, chelating agents and inert gases.
[0063] Preservatives can be used to inhibit microbial growth or
increase stability of the active ingredient thereby prolonging the
shelf life of the pharmaceutical formulation. Suitable
preservatives are known in the art and include, for example, EDTA,
EGTA, benzalkonium chloride or benzoic acid or benzoates, such as
sodium benzoate. Antioxidants include, for example, ascorbic acid,
vitamin A, vitamin E, tocopherols, and similar vitamins or
provitamins.
[0064] An antimicrobial agent or compound directly or indirectly
inhibits, reduces, delays, halts, eliminates, arrests, suppresses
or prevents contamination by or growth, infectivity, replication,
proliferation, reproduction, of a pathogenic or non-pathogenic
microbial organism. Classes of antimicrobials include,
antibacterial, antiviral, antifungal and antiparasitics.
Antimicrobials include agents and compounds that kill or destroy
(-cidal) or inhibit (-static) contamination by or growth,
infectivity, replication, proliferation, reproduction of the
microbial organism.
[0065] Exemplary antibacterials (antibiotics) include penicillins
(e.g., penicillin G, ampicillin, methicillin, oxacillin, and
amoxicillin), cephalosporins (e.g., cefadroxil, ceforanid,
cefotaxime, and ceftriaxone), tetracyclines (e.g., doxycycline,
chlortetracycline, minocycline, and tetracycline), aminoglycosides
(e.g., amikacin, gentamycin, kanamycin, neomycin, streptomycin,
netilmicin, paromomycin and tobramycin), macrolides (e.g.,
azithromycin, clarithromycin, and erythromycin), fluoroquinolones
(e.g., ciprofloxacin, lomefloxacin, and norfloxacin), and other
antibiotics including chloramphenicol, clindamycin, cycloserine,
isoniazid, rifampin, vancomycin, aztreonam, clavulanic acid,
imipenem, polymyxin, bacitracin, amphotericin and nystatin.
[0066] Particular non-limiting classes of anti-virals include
reverse transcriptase inhibitors; protease inhibitors; thymidine
kinase inhibitors; sugar or glycoprotein synthesis inhibitors;
structural protein synthesis inhibitors; nucleoside analogues; and
viral maturation inhibitors. Specific non-limiting examples of
anti-virals include those set forth above and, nevirapine,
delavirdine, efavirenz, saquinavir, ritonavir, indinavir,
nelfinavir, amprenavir, zidovudine (AZT), stavudine (d4T),
lamivudine (3TC), didanosine (DDE), zalcitabine (ddC), abacavir,
acyclovir, penciclovir, valacyclovir, ganciclovir,
1,-D-ribofuranosyl-1,2,4-triazole-3 carboxamide,
9->2-hydroxy-ethoxy methylguanine, adamantanamine,
5-iodo-2'-deoxyuridine, trifluorothymidine, interferon and adenine
arabinoside.
[0067] Exemplary antifungals include agents such as benzoic acid,
undecylenic alkanolamide, ciclopiroxolamine, polyenes, imidazoles,
allylamine, thicarbamates, amphotericin B, butylparaben,
clindamycin, econaxole, ammolfine, butenafine, naftifine,
terbinafine, ketoconazole, elubiol, econazole, econaxole,
itraconazole, isoconazole, miconazole, sulconazole, clotrimazole,
enilconazole, oxiconazole, tioconazole, terconazole, butoconazole,
thiabendazole, voriconazole, saperconazole, sertaconazole,
fenticonazole, posaconazole, bifonazole, fluconazole, flutrimazole,
nystatin, pimaricin, amphotericin B, flucytosine, natamycin,
tolnaftate, mafenide, dapsone, caspofungin, actofunicone,
griseofulvin, potassium iodide, Gentian Violet, ciclopirox,
ciclopirox olamine, haloprogin, ketoconazole, undecylenate, silver
sulfadiazine, undecylenic acid, undecylenic alkanolamide and
Carbol-Fuchsin.
[0068] Pharmaceutical compositions can optionally be formulated to
be compatible with a particular route of administration. Exemplary
routes of administration include administration to a biological
fluid, an immune cell (e.g., T or B cell) or tissue, mucosal cell
or tissue (e.g., mouth, buccal cavity, labia, nasopharynx,
esophagus, trachea, lung, stomach, small intestine, vagina, rectum,
or colon), neural cell or tissue (e.g., ganglia, motor or sensory
neurons) or epithelial cell or tissue (e.g., nose, fingers, ears,
cornea, conjunctiva, skin or dermis). Thus, pharmaceutical
compositions include carriers (excipients, diluents, vehicles or
filling agents) suitable for administration to any cell, tissue or
organ, in vivo, ex vivo (e.g., tissue or organ transplant) or in
vitro, by various routes and delivery, locally, regionally or
systemically.
[0069] Exemplary routes of administration for contact or in vivo
delivery which a compound of the invention (e.g., CSA) can
optionally be formulated include inhalation, respiration,
intubation, intrapulmonary instillation, oral (buccal, sublingual,
mucosal), intrapulmonary, rectal, vaginal, intrauterine,
intradermal, topical, dermal, parenteral (e.g., subcutaneous,
intramuscular, intravenous, intradermal, intraocular, intratracheal
and epidural), intranasal, intrathecal, intraarticular,
intracavity, transdermal, iontophoretic, ophthalmic, optical (e.g.,
corneal), intraglandular, intraorgan, intralymphatic.
[0070] Formulations suitable for parenteral administration include
aqueous and non-aqueous solutions, suspensions or emulsions of the
compound, which may include suspending agents and thickening
agents, which preparations are typically sterile and can be
isotonic with the blood of the intended recipient. Non-limiting
illustrative examples of aqueous carriers include water, saline
(sodium chloride solution), dextrose (e.g., Ringer's dextrose),
lactated Ringer's, fructose, ethanol, animal, vegetable or
synthetic oils. Examples of non-aqueous solvents are propylene
glycol, polyethylene glycol, vegetable oils such as olive oil, and
injectable organic esters such as ethyl oleate. Intravenous
vehicles include fluid and nutrient replenishers, electrolyte
replenishers (such as those based on Ringer's dextrose). The
formulations may be presented in unit-dose or multi-dose kits, for
example, ampules and vials, and may be stored in a freeze-dried
(lyophilized) condition requiring addition of a sterile liquid
carrier, for example, water for injections, prior to use.
[0071] For transmucosal or transdermal administration (e.g.,
topical contact), penetrants can be included in the pharmaceutical
composition. Penetrants are known in the art, and include, for
example, for transmucosal administration, detergents, bile salts,
and fusidic acid derivatives. For transdermal administration, the
active ingredient can be formulated into aerosols, sprays,
ointments, salves, gels, pastes, lotions, oils or creams as
generally known in the art.
[0072] For topical administration, for example, to skin,
pharmaceutical compositions typically include ointments, creams,
lotions, pastes, gels, sprays, aerosols or oils, Carriers which may
be used include Vaseline, lanolin, polyethylene glycols, alcohols,
transdermal enhancers, and combinations thereof. An exemplary
topical delivery system is a transdermal patch containing an active
ingredient (e.g., CSA).
[0073] For oral administration, pharmaceutical compositions include
capsules, cachets, lozenges, tablets or troches, as powder or
granules. Oral administration formulations also include a solution
or a suspension (e.g., aqueous liquid or a non-aqueous liquid; or
as an oil-in-water liquid emulsion or a water-in-oil emulsion).
[0074] For airway or nasal administration, pharmaceutical
compositions can be formulated in a dry powder for delivery, such
as a fine or a coarse powder having a particle size, for example,
in the range of 20 to 500 microns which is administered in the
manner by inhalation through the airways or nasal passage.
Depending on delivery device efficiency, effective dry powder
dosage levels typically fall in the range of about 10 to about 100
mg. Appropriate formulations, wherein the carrier is a liquid, for
administration, as for example, a nasal spray or as nasal drops,
include aqueous or oily solutions of the active ingredient.
[0075] For airway or nasal administration, aerosol and spray
delivery systems and devices, also referred to as "aerosol
generators" and "spray generators," such as metered dose inhalers
(MDI), nebulizers (ultrasonic, electronic and other nebulizers),
nasal sprayers and dry powder inhalers can be used. MDIs typically
include an actuator, a metering valve, and a container that holds a
suspension or solution, propellant, and surfactant (e.g., oleic
acid, sorbitan trioleate, lecithin). Activation of the actuator
causes a predetermined amount to be dispensed from the container in
the form of an aerosol, which is inhaled by the subject. MDIs
typically use liquid propellant and typically, MDIs create droplets
that are 15 to 30 microns in diameter, optimized to deliver doses
of 1 microgram to 10 mg of a therapeutic. Nebulizers are devices
that turn medication into a fine mist inhalable by a subject
through a face mask that covers the mouth and nose. Nebulizers
provide small droplets and high mass output for delivery to upper
and lower respiratory airways. Typically, nebulizers create
droplets down to about 1 micron in diameter.
[0076] Dry-powder inhalers (DPI) can be used to deliver the
compounds of the invention, either alone or in combination with a
pharmaceutically acceptable carrier. DPIs deliver active ingredient
to airways and lungs while the subject inhales through the device.
DPIs typically do not contain propellants or other ingredients,
only medication, but may optionally include other components. DPIs
are typically breath-activated, but may involve air or gas pressure
to assist delivery.
[0077] For rectal administration, pharmaceutical compositions can
be included as a suppository with a suitable base comprising, for
example, cocoa butter or a salicylate. For vaginal administration,
pharmaceutical compositions can be included as pessaries, tampons,
creams, gels, pastes, foams or spray formulations containing in
addition to the active ingredient (e.g., CSA) a carrier, examples
of appropriate carriers which are known in the art.
[0078] Pharmaceutical formulations and delivery systems appropriate
for the compositions and methods of the invention are known in the
art (see, e.g., Remington: The Science and Practice of Pharmacy
(2003) 20.sup.th ed., Mack Publishing Co., Easton, Pa.; Remington's
Pharmaceutical Sciences (1990) 18.sup.th ed., Mack Publishing Co.,
Easton, Pa.; The Merck Index (1996) 12.sup.th ed., Merck Publishing
Group, Whitehouse, N.J.; Pharmaceutical Principles of Solid Dosage
Forms (1993), Technonic Publishing Co., Inc., Lancaster, Pa.; Ansel
and Stoklosa, Pharmaceutical Calculations (2001) 11.sup.th ed.,
Lippincott Williams & Wilkins, Baltimore, Md.; and Poznansky et
al., Drug Delivery Systems (1980), R. L. Juliano, ed., Oxford,
N.Y., pp. 253-315).
[0079] Compounds of the invention (e.g., CSAs), including
pharmaceutical formulations can be packaged in unit dosage forms
for ease of administration and uniformity of dosage. A "unit dosage
form" as used herein refers to a physically discrete unit suited as
unitary dosages for the subject to be treated; each unit containing
a predetermined quantity of compound optionally in association with
a pharmaceutical carrier (excipient, diluent, vehicle or filling
agent) which, when administered in one or more doses, is calculated
to produce a desired effect (e.g., prophylactic or therapeutic
effect or benefit). Unit dosage forms can contain a daily dose or
unit, daily sub-dose, or an appropriate fraction thereof, of an
administered compound (e.g., CSA). Unit dosage forms also include,
for example, capsules, troches, cachets, lozenges, tablets, ampules
and vials, which may include a composition in a freeze-dried or
lyophilized state; a sterile liquid carrier, for example, can be
added prior to administration or delivery in vivo. Unit dosage
forms additionally include, for example, ampules and vials with
liquid compositions disposed therein. Unit dosage forms further
include compounds for transdermal administration, such as "patches"
that contact with the epidermis of the subject for an extended or
brief period of time. The individual unit dosage forms can be
included in multi-dose kits or containers. Pharmaceutical
formulations can be packaged in single or multiple unit dosage
forms for ease of administration and uniformity of dosage.
[0080] Compounds of the invention (e.g., CSAs) can be administered
in accordance with the methods at any frequency as a single bolus
or multiple dose e.g., one, two, three, four, five, or more times
hourly, daily, weekly, monthly or annually or between about 1 to 10
days, weeks, months, or for as long as appropriate. Exemplary
frequencies are typically from 1-7 times, 1-5 times, 1-3 times,
2-times or once, daily, weekly or monthly. Timing of contact,
administration ex vivo or in vivo delivery can be dictated by the
infection, reactivation, pathogenesis, symptom, pathology or
adverse side effect to be treated. For example, an amount can be
administered to the subject substantially contemporaneously with,
or within about 1-60 minutes or hours of the onset of a symptom or
adverse side effect of HV infection, reactivation, pathogenesis,
vaccination or treatment.
[0081] Doses may vary depending upon whether the treatment is
therapeutic or prophylactic, the onset, progression, severity,
frequency, duration, probability of or susceptibility of the
symptom, the type of virus infection, reactivation or pathogenesis
to which treatment is directed, clinical endpoint desired,
previous, simultaneous or subsequent treatments, general health,
age, gender or race of the subject, bioavailability, potential
adverse systemic, regional or local side effects, the presence of
other disorders or diseases in the subject, and other factors that
will be appreciated by the skilled artisan (e.g., medical or
familial history). Dose amount, frequency or duration may be
increased or reduced, as indicated by the clinical outcome desired,
status of the infection, reactivation, pathology or symptom, or any
adverse side effects of the treatment or therapy. The skilled
artisan will appreciate the factors that may influence the dosage,
frequency and timing required to provide an amount sufficient or
effective for providing a prophylactic or therapeutic effect or
benefit.
[0082] Typically, for therapeutic treatment, a compound of the
invention (e.g., CSA) will be administered as soon as practical,
typically within 0-72 hours after a subject is exposed to or
contacted with HV, or within 0-72 hours after development of one or
more symptoms or pathologies associated with HV infection,
reactivation or pathogenesis (e.g., onset of lesions, ulcers,
canker sores, cold sores, rash, boils, etc.) or a symptom
associated with or caused by HV.
[0083] For prophylactic treatment, a compound of the invention can
be administered immediately or within 0-72 after suspected contact
with, or 0-4 weeks, e.g., 1-3 weeks, prior to anticipated or
possible exposure to or contact or infection with or reactivation
of HV. For prophylactic treatment in connection with
immunization/vaccination of a subject, a compound can be
administered prior to, concurrently with or following
immunization/vaccination of the subject.
[0084] Doses can be based upon current existing treatment protocols
(e.g., acyclovir), empirically determined, determined using animal
disease models or optionally in human clinical studies. For
example, initial study doses can be based upon animal studies, such
as a mouse, which weighs about 30 grams, and the amount of compound
administered to achieve a prophylactic or therapeutic effect or
benefit. The dose can be adjusted according to the mass of a
subject, and will generally be in a range from about 0.1-1 ug/kg,
1-10 ug/kg, 10-25 ug/kg, 25-50 ug/kg, 50-100 ug/kg, 100-500 ug/kg,
500-1,000 ug/kg, 1-5 mg/kg, 5-10 mg/kg, 10-20 mg/kg, 20-50 mg/kg,
50-100 mg/kg, 100-250 mg/kg, 250-500 mg/kg, or more, of subject
body weight, two, three, four, or more times per hour, day, week,
month or annually. Of course, doses can be more or less, as
appropriate, for example, 0.00001 mg/kg of subject body weight to
about 10,000.0 mg/kg of subject body weight, about 0.001 mg/kg, to
about 100 mg/kg, about 0.01 mg/kg, to about 10 mg/kg, or about 0.1
mg/kg, to about 1 mg/kg of subject body weight over a given time
period, e.g., 1, 2, 3, 4, 5 or more hours, days, weeks, months,
years. A subject may be administered in single bolus or in
divided/metered doses, which can be adjusted to be more or less
according to the various consideration set forth herein and known
in the art.
[0085] Dose amount, frequency or duration may be increased or
reduced, as indicated by the status of the HV infection,
reactivation or pathogenesis, associated symptom or pathology, or
any adverse side effect(s) of vaccination, treatment or anti-HV
therapy. For example, once control or a particular endpoint is
achieved, for example, reducing, decreasing, inhibiting,
ameliorating or preventing onset, severity, duration, progression,
frequency or probability of one or more symptoms associated with a
HV infection, reactivation or pathogenesis of one or more symptoms
or pathologies associated with or caused by HV infection,
reactivation or pathogenesis, dose amount, frequency or duration
can be reduced.
[0086] The invention provides kits including compounds of the
invention (e.g., CSA), combination compositions and pharmaceutical
compositions/formulations thereof, packaged into a suitable
packaging material. In one embodiment, a kit includes packaging
material, a cationic steroid antimicrobial (CSA) and instructions.
In various aspects, the instructions are for administering the CSA
to: provide a subject with protection against a herpesviridae (HV)
infection, reactivation or pathogenesis; treat a subject for
herpesviridae (HV) infection, reactivation or pathogenesis;
decrease susceptibility of a subject to a herpesviridae (HV)
infection, reactivation or pathogenesis; decrease, inhibit,
ameliorate or prevent onset, severity, duration, progression,
frequency or probability of one or more symptoms or pathologies
associated with or caused by HV infection, reactivation or
pathogenesis; or decrease or prevent an adverse side effect caused
by vaccination of a subject with a herpesviridae (HV) or a
herpesviridae (HV) treatment.
[0087] The term "packaging material" refers to a physical structure
housing one or more components of the kit. The packaging material
can maintain the components sterilely, and can be made of material
commonly used for such purposes (e.g., paper, corrugated fiber,
glass, plastic, foil, ampules, vials, tubes, etc.). A kit can
contain a plurality of components, e.g., two or more compounds of
the invention alone or in combination with an anti-HV agent or
treatment (e.g., an anti-viral, a herpesvirus protein or an
antibody that binds to a herpesvirus protein, HV vaccine, etc.) or
drug, optionally sterile.
[0088] A kit optionally includes a label or insert including a
description of the components (type, amounts, doses, etc.),
instructions for use in vitro, in vivo, or ex vivo, and any other
components therein. Labels or inserts include "printed matter,"
e.g., paper or cardboard, or separate or affixed to a component, a
kit or packing material (e.g., a box), or attached to an ampule,
tube or vial containing a kit component. Labels or inserts can
additionally include a computer readable medium, such as a disk
(e.g., floppy diskette, hard disk, ZIP disk), optical disk such as
CD- or DVD-ROM/RAM, DVD, MP3, magnetic tape, or an electrical
storage media such as RAM and ROM or hybrids of these such as
magnetic/optical storage media, FLASH media or memory type
cards.
[0089] Labels or inserts can include identifying information of one
or more components therein, dose amounts, clinical pharmacology of
the active ingredient(s) including mechanism of action,
pharmacokinetics and pharmacodynamics. Labels or inserts can
include information identifying manufacturer, lot numbers,
manufacturer location and date, expiration dates.
[0090] Labels or inserts can include information on a condition,
disorder or disease (e.g., virus pathogenesis or infection) for
which a kit component may be used. Labels or inserts can include
instructions for a clinician or subject for using one or more of
the kit components in a method, treatment protocol or
therapeutic/prophylactic regimen, including the methods of the
invention. Instructions can include amounts of compound, frequency
or duration of administration, and instructions for practicing any
of the methods, treatment protocols or prophylactic or therapeutic
regimes described herein. Exemplary instructions include,
instructions for treating HV infection, reactivation or
pathogenesis. Kits of the invention therefore can additionally
include labels or instructions for practicing any of the methods of
the invention described herein including treatment, screening or
other methods. Thus, for example, a kit can include a compound of
the invention (e.g., CSA) that has one or more anti-HV activities
as set forth herein, together with instructions for administering
the compound in a prophylactic or therapeutic treatment method of
the invention, for example to a subject in need of such treatment.
Exemplary instructions include administering the CSA to: provide a
subject with protection against a HV infection, reactivation or
pathogenesis; treat a subject for HV infection, reactivation or
pathogenesis; decrease susceptibility of a subject to a HV
infection, reactivation or pathogenesis; or decrease or prevent an
adverse side effect caused by vaccination of a subject with or
against a HV or an HV treatment.
[0091] Labels or inserts can include information on any effect or
benefit a kit component may provide, such as a prophylactic or
therapeutic effect or benefit. For example, a label or insert could
provide a description of one or more symptoms which can be
improved, i.e., reducing, decreasing, inhibiting, ameliorating or
preventing onset, severity, duration, progression, frequency or
probability of one or more symptoms or pathologies associated with
a HV infection, reactivation or pathogenesis, or one or more
adverse side effects associated with HV vaccination or an HV
treatment. HV symptoms and pathologies are as set forth herein or
known in the art (e.g., lesions, ulcers, canker sore, cold sore,
rash, boils, Gingivostomatitis, Herpetic whitlow Traumatic herpes
(herpes gladiatorum), Eczema herpeticum, fever, fatigue, headache,
sore throat, swollen lymph nodes, pneumonitis, pneumonia,
hepatitis, meningitis, myelitis, Encephalitis, keratitis, Genital
herpes, esophagitis, dysphasia, hemiparesis, coma, shingles,
chicken pox, mononucleosis, chronic or acute pelvic inflammatory
disease (PID), proctitis, colitis, nerve damage, death, etc.),
Adverse side effects associated with HV vaccination are as set
forth herein or known in the art (e.g., fatigue, weakness,
headache, fever, stomach ache/nausea, flu-like symptoms, rash,
vomiting, inflammation (cerebral or ocular), fainting, etc.)
[0092] Labels or inserts can include information on potential
adverse side effects of treatment. Labels or inserts can further
include warnings to the clinician or subject regarding situations
or conditions where a subject should stop or reduce use of a
particular kit component. Adverse side effects could also occur
when the subject has, will be or is currently taking one or more
other medications that may be incompatible with a compound of the
invention, or the subject has, will be or is currently undergoing
another treatment protocol or therapeutic regimen which would be
incompatible with the compound and, therefore, labels or inserts
could include information regarding such side effects or
incompatibilities.
[0093] Invention kits can additionally include a buffering agent,
or a preservative or a stabilizing agent in a pharmaceutical
formulation containing a compound of the invention. Each component
of the kit can be enclosed within an individual container and all
of the various containers can be within a single package. Invention
kits can be designed for cold storage.
[0094] Invention kits can include components, such as devices for
practicing a method of the invention or administering a compound of
the invention (e.g., GSA) to a subject, ex vivo or in vivo. The
device can be a delivery device, such as a syringe, a compressible
(e.g., squeezable) tube or dermal patch for mucosal, skin/dermis or
corneal delivery, or an aerosol delivery device for administration
to lungs or airways.
[0095] Compounds useful in accordance with the invention, are
described herein, both generically and with particularity, and in
U.S. Pat. Nos. 6,350,738; 6,486,148; and 6,767,904, which are
incorporated herein by reference. Compounds include steroid
derivatives, such as cationic steroid antimicrobials (CSA) that
exhibit one or more anti-herpesviridae (HV) activities or
functions. The skilled artisan will recognize the compounds within
the generic formula set forth herein. Additional compounds of the
invention having one or more anti-herpesviridae (HV) activities or
functions are described and can be characterized using the assays
set forth herein and in the art.
[0096] Compounds of formula I, also referred to as cationic steroid
antimicrobials (CSA), comprise:
##STR00001##
wherein: fused rings A, B, C, and D are independently saturated or
fully or partially unsaturated; and each of R.sub.1 through
R.sub.4, R.sub.6, R.sub.7, R.sub.11, R.sub.12, R.sub.15, R.sub.16,
and R.sub.17 is independently selected from the group consisting of
hydrogen, hydroxyl, a substituted or unsubstituted (C1-C10) alkyl,
(C1-C10) hydroxyalkyl, (C1-C10) alkyloxy-(C1-C10) alkyl, (C1-C10)
alkylcarboxy-(C1-C10) alkyl, (C1-C10) alkylamino-(C1-C10) alkyl,
(C1-C10) alkylamino-(C1-C10) alkylamino, (C1-C10)
alkylamino-(C1-C10) alkylamino-(C1-C10) alkylamino, a substituted
or unsubstituted (C1-C10) aminoalkyl, a substituted or
unsubstituted aryl, a substituted or unsubstituted
arylamino-(C1-C10) alkyl, (C1-C10) haloalkyl, C2-C6 alkenyl, C2-C6
alkynyl, oxo, a linking group attached to a second steroid, a
substituted or unsubstituted (C1-C10) aminoalkyloxy, a substituted
or unsubstituted (C1-C10) aminoalkyloxy-(C1-C10) alkyl, a
substituted or unsubstituted (C1-C10) aminoalkylcarboxy, a
substituted or unsubstituted (C1-C10) aminoalkylaminocarbonyl, a
substituted or unsubstituted (C1-C10) aminoalkylcarhoxamido,
H.sub.2N--HC(Q5)-C(O)--O--, H2N--HC(Q5)-C(O)--N(H)--, (C1-C10)
azidoalkyloxy, (C1-C10) cyanoalkyloxy, P.G.-HN--HC(Q5)-C(O)--O--,
(C1-C10) guanidinoalkyl oxy, (C1-C10)
quaternaryammoniumalkylcarboxy, and (C1-C10) guanidinoalkyl
carboxy, where Q5 is a side chain of any amino acid (including the
side chain of glycine, i.e., H), P.G. is an amino protecting group,
and R.sub.5, R.sub.8, R.sub.9, R.sub.10, R.sub.13, and R.sub.14 is
each independently: deleted when one of fused rings A, B, C, or D
is unsaturated so as to complete the valency of the carbon atom at
that site, or selected from the group consisting of hydrogen,
hydroxyl, a substituted or unsubstituted (C1-C10) alkyl, (C1-C10)
hydroxyalkyl, (C1-C10) alkyloxy-(C1-C10) alkyl, a substituted or
unsubstituted (C1-C10) aminoalkyl, a substituted or unsubstituted
aryl, C1-C10 haloalkyl, C2-C6 alkenyl, C2-C6 alkynyl, oxo, a
linking group attached to a second steroid, a substituted or
unsubstituted (C1-C10) aminoalkyloxy, a substituted or
unsubstituted (C1-C10) aminoalkylcarboxy, a substituted or
unsubstituted (C1-C10) aminoalkylaminocarbonyl,
H.sub.2N--HC(Q5)-C(O)--O--, H.sub.2N--HC(Q5)-C(O)--N(H), (C1-C10)
azidoalkyloxy, (C1-C10) cyanoalkyloxy, P.G.-HN--HC(Q5)-C(O)--O--,
(C1-C10) guanidinoalkyloxy, and (C1-C10) guanidinoalkylcarboxy,
where Q5 is a side chain of any amino acid, P.G. is an amino
protecting group, and provided that at least two of R.sub.1 through
R.sub.14 are independently selected from the group consisting of a
substituted or unsubstituted (C1-C10) aminoalkyloxy, (C1-C10)
alkylcarboxy-(C1-C10) alkyl, (C1-C10) alkylamino-(C1-C10)
alkylamino, (C1-C10) alkylamino-(C1-C10) alkylamino-(C1-C10)
alkylamino, a substituted or unsubstituted (C1-C10)
aminoalkylcarboxy, a substituted or unsubstituted
arylamino-(C1-C10) alkyl, a substituted or unsubstituted (C1-C10)
aminoalkyloxy-(C1-C10) alkyl, a substituted or unsubstituted
(C1-C10) aminoalkylaminocarbonyl, (C1-C10) quaternaryammonium
alkylcarboxy, H2N--HC(Q5)-C(O)--O--, H.sub.2N--HC(Q5)-C(O)--N(H)--,
(C1-C10) azidoalkyloxy, (C1-C10) cyanoalkyloxy,
P.G.-HN--HC(Q5)-C(O)--O--, (C1-C10) guanidinoalkyloxy, and (C1-C10)
guanidinoalkylcarboxy; or a pharmaceutically acceptable salt
thereof.
[0097] A "ring" as used herein can be heterocyclic or carbocyclic.
The term "saturated" used herein refers to the fused ring of
formula I having each atom in the fused ring either hydrogenated or
substituted such that the valency of each atom is filled. The term
"unsaturated" used herein refers to the fused ring of formula I
where the valency of each atom of the fused ring may not be filled
with hydrogen or other substituents. For example, adjacent carbon
atoms in the fused ring can be doubly bound to each other.
Unsaturation can also include deleting at least one of the
following pairs and completing the valency of the ring carbon atoms
at these deleted positions with a double bond; such as R.sub.5 and
R.sub.9; R.sub.8 and R.sub.10; and R.sub.13 and R.sub.14.
[0098] The term "unsubstituted" used herein refers to a moiety
having each atom hydrogenated such that the valency of each atom is
filled.
[0099] The term "halo" used herein refers to a halogen atom such as
fluorine, chlorine, bromine, or iodine.
[0100] Examples of amino acid side chains include but are not
limited to H (glycine), methyl (alanine),
--CH.sub.2--(C.dbd.O)--NH.sub.2 (asparagine), --CH.sub.2--SH
(cysteine), and --CH(OH)CH.sub.3 (threonine).
[0101] An alkyl group is a branched or unbranched hydrocarbon that
may be substituted or unsubstituted. Examples of branched alkyl
groups include isopropyl, sec-butyl, isobutyl, tert-butyl,
sec-pentyl, isopentyl, tert-pentyl, isohexyl. Substituted alkyl
groups may have one, two, three or more substituents, which may be
the same or different, each replacing a hydrogen atom. Substituents
are halogen (e.g., F, Cl, Br, and I), hydroxyl, protected hydroxyl,
amino, protected amino, carboxy, protected carboxy, cyano,
methylsulfonylamino, alkoxy, acyloxy, nitro, and lower
haloalkyl.
[0102] The term "substituted" used herein refers to moieties having
one, two, three or more substituents, which may be the same or
different, each replacing a hydrogen atom. Examples of substituents
include but are not limited to halogen (e.g., F, Cl, Br, and I),
hydroxyl, protected hydroxyl, amino, protected amino, carboxy,
protected carboxy, cyano, methylsulfonylamino, alkoxy, alkyl, aryl,
aralkyl, acyloxy, nitro, and lower haloalkyl.
[0103] An aryl group is a C6-20 aromatic ring, wherein the ring is
made of carbon atoms (e.g., C6-C14, C6-10 aryl groups). Examples of
haloalkyl include fluoromethyl, di chloromethyl, trifluoromethyl,
1,1-difluoroethyl, and 2,2-dibromoethyl.
[0104] An aralkyl group is a group containing 6-20 carbon atoms
that has at least one aryl ring and at least one alkyl or alkylene
chain connected to that ring. An example of an aralkyl group is a
benzyl group.
[0105] A linking group is any divalent moiety used to link a
compound of formula to another steroid, e.g., a second compound of
formula I. An example of a linking group is (C1-C10)
alkyloxy-(C1-C10) alkyl.
[0106] Amino-protecting groups are known to those skilled in the
art. In general, the species of protecting group is not critical,
provided that it is stable to the conditions of any subsequent
reaction(s) on other positions of the compound and can be removed
at the appropriate point without adversely affecting the remainder
of the molecule. In addition, a protecting group may be substituted
for another after substantive synthetic transformations are
complete. Clearly, where a compound differs from a compound
disclosed herein only in that one or more protecting groups of the
disclosed compound has been substituted with a different protecting
group, that compound is within the invention. Further examples and
conditions are found in T. W. Greene, Protective Groups in Organic
Chemistry, (1st ed., 1981, 2nd ed., 1991).
[0107] The invention also includes compounds comprising a ring
system of at least 4 fused rings, where each of the rings has from
5-7 atoms. The ring system has two faces, and contains 3 chains
attached to the same face. Each of the chains contains a
nitrogen-containing group that is separated from the ring system by
at least one atom; the nitrogen-containing group is an amino group,
e.g., a primary amino group, or a guanidino group. The compound can
also contain a hydrophobic group, such as a substituted (C3-10)
aminoalkyl group, a (C1-10) alkyloxy (C3-10) alkyl group, or a
(C1-10) alkylamino (C3-10)alkyl group, attached to the steroid
backbone.
[0108] For example, the compound may have the formula V, where each
of the three chains containing nitrogen-containing groups is
independently selected from R.sub.1 through R.sub.4, R.sub.6,
R.sub.7, R.sub.11, R.sub.12, R.sub.15, R.sub.16, R.sub.17, and
R.sub.18, defined below.
##STR00002##
where: each of fused rings A, B, C, and D is independently
saturated, or is fully or partially unsaturated, provided that at
least two of A, B, C, and D are saturated, wherein rings A, B, C,
and D form a ring system; each of m, n, p, and q is independently 0
or 1; each of R.sub.1 through R.sub.4, R.sub.6, R.sub.7, R.sub.11,
R.sub.12, R.sub.15, R.sub.16, R.sub.17, and R.sub.18 is
independently selected from the group consisting of hydrogen,
hydroxyl, a substituted or unsubstituted (C1-C10) alkyl, (C1-C10)
hydroxyalkyl, (C1-C10) alkyloxy-(C1-C10) alkyl,
(C1-C10)alkylcarboxy-(C1-C10 alkyl, (C1-C10) alkylamino-(C1-C10)
alkyl, (C1-C10) alkylamino-(C1-C10) alkylamino, (C1-C10
alkylamino-(C1-C10) alkylamino-(C1-C10) alkylamino, a substituted
or unsubstituted (C1-C10) aminoalkyl, a substituted or
unsubstituted aryl, a substituted or unsubstituted
arylamino-(C1-C10) alkyl, (C1-C10) haloalkyl, C2-C6 alkenyl, C2-C6
alkynyl, oxo, a linking group attached to a second steroid, a
substituted or unsubstituted (C1-C10) aminoalkyloxy, a substituted
or unsubstituted (C1-C10) aminoalkyloxy-(C1-C10) alkyl, a
substituted or unsubstituted (C1-C10) aminoalkylcarboxy, a
substituted or unsubstituted (C1-C10) aminoalkylaminocarbonyl, a
substituted or unsubstituted (C1-C10) aminoalkylcarboxamido,
H.sub.2N--HC(Q5)-C(O)--O--, H2N--HC(Q5)-C(O)--N(H)--, (C1-C10)
azidoalkyloxy, (C1-C10) cyanoalkyloxy, P.G.-HN--HC(Q5)-C(O)--O--,
(C1-C10) guanidinoalkyl oxy, (C1-C10)
quaternaryammoniumalkylcarboxy, and (C1-C10) guanidinoalkyl
carboxy, where Q5 is a side chain of any amino acid (including a
side chain of glycine, i.e., H). P.G. is an amino protecting group:
and each of R.sub.5, R.sub.8, R.sub.9, R.sub.10, R.sub.13, and
R.sub.14 is independently: deleted when one of fused rings A, B, C,
or D is unsaturated so as to complete the valency of the carbon
atom at that site, or selected from the group consisting of
hydrogen, hydroxyl, a substituted or unsubstituted (C1-C10) alkyl,
(C1-C10) hydroxyalkyl, (C1-C10) alkyloxy-(C1-C10) alkyl, a
substituted or unsubstituted (C1-C10) aminoalkyl, a substituted or
unsubstituted aryl, C1-C10 haloalkyl, C2-C6 alkenyl, C2-C6 alkynyl,
oxo, a linking group attached to a second steroid, a substituted or
unsubstituted (C1-C10) aminoalkyloxy, a substituted or
unsubstituted (C1-C10) aminoalkylcarboxy, a substituted or
unsubstituted (C1-C10) aminoalkylaminocarbonyl,
H2N--HC(Q5)-C(O)--O--, H2N--HC(Q5)-C(O)--O--N(H), (C1-C10)
azidoalkyloxy, (C1-C10) cyanoalkyloxy, P. G.-HN--HC(Q5)-C(O)--O--,
(C1-C10) guanidinoalkyloxy, and (C1-C10) guanidinoalkylcarboxy,
where Q5 is a side chain of any amino acid, P.G. is an amino
protecting group, provided that at least three of R.sub.1 through
R.sub.4, R.sub.6, R.sub.7, R.sub.11, R.sub.12, R.sub.15, R.sub.16,
R.sub.17, and R.sub.18 are disposed on the same face of the ring
system and are independently selected from the group consisting of
a substituted or unsubstituted (C1-C10) aminoalkyl, a substituted
or unsubstituted (C1-C10) aminoalkyloxy, (C1-C10)
alkylcarboxy-(C1-C10) alkyl, (C1-C10) alkylamino-(C1-C10)
alkylamino, (C1-C10) alkylamino-(C1-C10) alkylamino-(C1-C10)
alkylamino, a substituted or unsubstituted (C1-C10)
aminoalkylcarboxy, a substituted or unsubstituted
arylamino-(C1-C10) alkyl, a substituted or unsubstituted (C1-C10)
aminoalkyloxy-(C1-C10) aminoalkylaminocarbonyl, a substituted or
unsubstituted (C1-C10) aminoalkylaminocarbonyl, a substituted or
unsubstituted (C1-C5) amino alkylcarboxamido, a (C1-C10)
quaternaryammoniumalkylcarboxy, H2N--HC(Q5)-C(O)--O--,
H2N--HC(Q5)-C(O)--N(H)--, (C1-C10) azidoalkyloxy, (C1-C10)
cyanoalkylox, P.G.-HN--HC(Q5)-C(O)--O--, (C1-C10)
guanidinoalkyloxy, and a (C1-C10) guanidinoalkylcarboxy; or a
pharmaceutically acceptable salt thereof. In various aspects, at
least two, or at least, three, of m, n, p, and q are 1.
[0109] Compounds set forth herein preserve certain stereochemical
and electronic characteristics found in steroids. The term "same
configuration" as used herein refers to substituents on the fused
steroid having the same stereochemical orientation. For example
substituents R.sub.3, R.sub.7 and R.sub.12 are all
.beta.-substituted or .alpha.-substituted.
[0110] Compounds of the invention include but are not limited to
compounds having amine or guanidine groups covalently attached to a
steroid backbone or scaffold at any carbon position, e.g., cholic
acid. In various embodiments, a group is covalently attached at any
one, or more, of positions C3, C7 and C12 of the steroid backbone
or scaffold. In additional embodiments, a group is absent from any
one, or more, of positions C3, C7 and C12 of the steroid backbone
or scaffold.
[0111] Compounds of the invention that include such groups can
include a tether, the tether having variable chain length or size.
As used herein, the terms "tether" or "tethered," when used in
reference to a compound of the invention, refers to the chain of
atoms between the steroid backbone or scaffold and a terminal amino
or guanidine group. In various embodiments, a tether is covalently
attached at any one, or more, of positions C3, C7 and C12. In
additional embodiments, a tether is lacking at any one, or more, of
positions C3, C7 and C12. A tether length may include the
heteroatom (O or N) covalently attached to the steroid
backbone.
[0112] Other ring systems can also be used, e.g., 5-member fused
rings. Compounds with backbones having a combination of 5- and
6-membered rings are also included in the invention. Amine or
guanidine groups can be separated from the backbone by at least
one, two, three, four or more atoms. The backbone can be used to
orient the amine or guanidine groups on one face, or plane, of the
steroid. For example, a scheme showing a compound having primary
amino groups on one face, or plane, of a backbone is shown
below:
##STR00003##
[0113] Methods of synthesizing compounds of formula I are provided,
wherein for example, at least two of R.sub.1 through R.sub.14 are
independently selected from the group consisting of a substituted
or unsubstituted (C1-C10) aminoalkyloxy. In one embodiment, a
method includes the step of contacting a compound of formula
IV,
##STR00004##
where at least two of R.sub.1 through R.sub.14 are hydroxyl, and
the remaining moieties on the fused rings A, B, C, and D are
defined for formula I, with an electrophile to produce an alkyl
ether compound of formula IV, wherein at least two of R.sub.1
through R.sub.14 are (C1-C10)alkyloxy. The alkyl ether compounds
are converted into an amino precursor compound wherein at least two
of R.sub.1 through R.sub.14 are independently selected from the
group consisting of (C1-C10) azidoalkyloxy and (C1-C10)
cyanoalkyloxy and the amino precursor compound is reduced to form a
compound of formula I.
[0114] The electrophiles used in a method include but are not
limited to 2-(2-bromoethyl)-1,3-dioxolane, 2-iodoacetamide,
2-chloroacetamide, N-(2-bromoethyl)phthalimide,
N-(3-bromopropyl)phthalimide, and allybromide. An exemplary
electrophile is allylbromide.
[0115] The invention also includes methods of producing a compound
of formula I where at least two of R.sub.1 through R.sub.14 are
(C1-C10) guanidoalkyloxy. In one embodiment, a method includes
contacting a compound of formula IV, where at least two of R.sub.1
through R.sub.14 are hydroxyl, with an electrophile to produce an
alkyl ether compound of formula IV, where at least two of R.sub.1
through R.sub.14 are (C1-C10)alkyloxy. The allyl ether compound is
converted into an amino precursor compound where at least two of
R.sub.1 through R.sub.14 are independently selected from the group
consisting of (C1-C10) azidoalkyloxy and (C1-C10) cyanoalkyloxy.
The amino precursor compound is reduced to produce an aminoalkyl
ether compound wherein at least two of R.sub.1 through R.sub.14 are
(C1-C10) aminoalkyloxy. The aminoalkyl ether compound is contacted
with a guanidino producing electrophile to form a compound of
formula I.
[0116] The term "guanidino producing electrophile" used herein
refers to an electrophile used to produce a guanidino compound of
formula I. An example of an guanidino producing electrophile is
HSO.sub.3--C(NH)--NH.sub.2.
[0117] The invention also includes methods of producing a compound
of formula I where at least two of R.sub.1 through R.sub.14 are
H2N--HC(Q5)-C(O)--O-- and Q5 is the side chain of any amino acid.
In one embodiment, a method includes the step of contacting a
compound of formula IV, where at least two of R.sub.1 through
R.sub.14 are hydroxyl, with a protected amino acid to produce a
protected amino acid compound of formula IV where at least two of
at least two of R.sub.1 through R.sub.14 are
P.G.-HN--HC(Q5)-C(O)--O-- and Q5 is the side chain of any amino
acid and P.G. is an amino protecting group. The protecting group of
the protected amino acid compound is removed to form a compound of
formula I.
[0118] Exemplary non-limiting synthesis schemes for preparing
compounds of the invention include the following:
##STR00005## ##STR00006## ##STR00007##
##STR00008##
##STR00009##
##STR00010## ##STR00011##
##STR00012##
##STR00013## ##STR00014##
##STR00015##
##STR00016##
##STR00017##
##STR00018##
##STR00019##
##STR00020##
##STR00021##
##STR00022##
##STR00023##
##STR00024##
##STR00025##
[0119] Compounds of the invention and precursors to the compounds
according to the invention are available commercially, e.g., from
Sigma-Aldrich Co., St, Louis; MO; and Research Plus, Inc.,
Manasquan, N.J. Other compounds according to the invention can be
synthesized according to methods disclosed herein, in U.S. Pat.
Nos. 6,350,738; 6,486,148; and 6,767,904, and in the art.
[0120] Methods for identifying a candidate agent for treating a
subject for a HV infection, reactivation or pathogenesis, for
decreasing susceptibility of a subject to a HV infection,
reactivation or pathogenesis, for decreasing, inhibiting,
ameliorating or preventing onset, severity, duration, progression,
frequency or probability of one or more symptoms or pathologies
caused by or associated with HV infection or pathogenesis or
reactivation from latency, and for decreasing or preventing an
adverse side effect caused by vaccination of a subject with or
against a HV or a HV treatment, are provided. In one embodiment, a
method includes providing a test agent comprising a cationic
steroid antimicrobial (CSA); contacting the test agent with HV and
ascertaining whether the test agent inhibits HV infection or
pathogenesis, or reactivation from latency. A test agent identified
as inhibiting HV infection or pathogenesis or reactivation from
latency is a candidate agent for treating a subject for HV
infection, reactivation or pathogenesis. A test agent identified as
inhibiting HV infection, reactivation or pathogenesis is also a
candidate agent for decreasing susceptibility of a subject to a HV
infection, reactivation or pathogenesis. A test agent identified is
further a candidate agent for decreasing. inhibiting, ameliorating
or preventing onset, severity, duration, progression, frequency or
probability of one or more symptoms or pathologies associated with
or caused by HV infection or pathogenesis or reactivation from
latency. A test agent identified is moreover a candidate agent for
decreasing or preventing an adverse side effect caused by or
associated with vaccination of a subject with a HV or a HV
treatment. In various aspects, the subject is a mammal. For
example, a mammal can comprise an animal model for HV infection,
reactivation or pathogenesis.
[0121] Unless otherwise defined, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs. Although
methods and materials similar or equivalent to those described
herein can be used in the practice or study of the present
invention, suitable methods and materials are described herein.
[0122] All of the features disclosed herein may be combined in any
combination. Each feature disclosed in the specification may be
replaced by an alternative feature serving a same, equivalent, or
similar purpose. Thus, unless expressly stated otherwise, disclosed
features (e.g., compound structures) are an example of a genus of
equivalent or similar features.
[0123] All applications, publications, patents and other
references, GenBank citations and ATCC citations cited herein are
incorporated by reference in their entirety. In case of conflict,
the specification, including definitions, will control.
[0124] As used herein, the singular forms "a", "and," and "the"
include plural referents unless the context clearly indicates
otherwise. Thus, for example, reference to "a compound" includes a
plurality of compounds and reference to "an anti-herpesviridae (HV)
effect, activity or function" can include reference to one or more
effects, activities or functions, and so forth.
[0125] As used herein, all numerical values or numerical ranges
include integers within such ranges and fractions of the values or
the integers within ranges unless the context clearly indicates
otherwise. Thus, to illustrate, reference to a range of 90-100%,
includes 91%, 92%, 93%, 94%, 95%, 95%, 97%, etc., as well as 91.1%,
91.2%, 91.3%, 91.4%, 91.5%, etc., 92.1%, 92.2%, 92.3%, 92.4%,
92.5%, etc., and so forth, Reference to a range of 0-72 hrs,
includes 1, 2, 3, 4, 5, 6, 7 hrs, etc., as well as 1, 2, 3, 4, 5,
6, 7 minutes, etc., and so forth. Reference to a range of 0-72 hrs,
includes 1, 2, 3, 4, 5, 6, 7 hrs, etc., as well as 1, 2, 3, 4, 5,
6, 7 minutes, etc., and so forth. Reference to a range of doses,
such as 0.1-1 ug/kg, 1-10 ug/kg, 10-25 ug/kg, 25-50 ug/kg, 50-100
ug/kg, 100-500 ug/kg, 500-1,000 ug/kg, 1-5 mg/kg, 5-10 mg/kg, 10-20
mg/kg, 20-50 mg/kg, 50-100 mg/kg, 100-250 mg/kg, 250-500 mg/kg,
includes 0.11-0.9 ug/kg, 2-9 ug/kg, 11.5-24.5 ug/kg, 26-49 ug/kg,
55-90 ug/kg, 125-400 ug/kg, 750-800 ug/kg, 1.1-4.9 mg/kg, 6-9
mg/kg, 11.5-19.5 mg/kg, 21-49 mg/kg, 55-90 mg/kg, 125-200 mg/kg,
275.5-450.1 mg/kg, etc.
[0126] The invention is generally disclosed herein using
affirmative language to describe the numerous embodiments. The
invention also includes embodiments in which subject matter is
excluded, in full or in part, such as substances or materials,
method steps and conditions, protocols, or procedures. Thus, even
though the invention is generally not expressed herein in terms of
what the invention does not include aspects that are not expressly
excluded in the invention are nevertheless disclosed herein.
[0127] A number of embodiments of the invention have been
described. Nevertheless, one skilled in the art, without departing
from the spirit and scope of the invention, can make various
changes and modifications of the invention to adapt it to various
usages and conditions. For example, salts, esters, ethers and
amides of invnetion compounds disclosed herein are within the scope
of this invention. Accordingly, the following examples are intended
to illustrate but not limit the scope of invention described in the
claims.
EXAMPLES
[0128] CSA compounds and intermediates were characterized using the
following instruments: .sup.1H and .sup.13C NMR spectra were
recorded on a Varian Gemini 2000 (200 MHz), Varian Unity 300 (300
MHz), or Varian VXR 500 (500 MHz) spectrometer and are referenced
to TMS, residual CHCl.sub.3 (.sup.1H) or CDCl.sub.3 (.sup.13C), or
residual CHD.sub.2OD (.sup.1H), or CD.sub.3OD (.sup.13C), IR
spectra were recorded on a Perkin Elmer 1600 FTIR instrument. Mass
spectrometric data were obtained on a JOEL SX 102A spectrometer.
THE solvent was dried over Na/benzophenone and CH.sub.2Cl.sub.2 was
dried over CaH.sub.2 prior to use. Other reagents and solvents were
obtained commercially and were used as received.
Example 1
[0129] This example includes a description of one or more exemplary
synthetic procedures for obtaining Compounds 1-5, 13-20 and
22-27.
[0130] Compound 13: To a 1 L round-bottom flask were added methyl
cholate (30.67 g, 72.7 mmol) in dry THF (600 mL) and LiAlH.sub.4
(4.13 g, 109 mmol). After reflux for 48 hours, saturated aqueous
Na.sub.2SO.sub.4 (100 mL) was introduced slowly, and the resulted
precipitate was filtered out and washed with hot THF and MeOH.
Recrystallization from MeOH gave colorless crystals of 13 (28.0 g,
98% yield). m.p. 236.5-238.degree. C.; IR (KBr) 3375, 2934, 1373,
1081 cm.sup.-1; .sup.1H NMR (CDCl.sub.3/MeOH-d.sub.4, 200 MHz)
.delta. 3.98 (bs, 1H), 3.83 (bs, 1H), 3.60-3.46 (m, 2H), 3.38 (bs,
5H), 2.30-2.10 (m, 2H), 2.05-1.05 (series of multiplets, 22H), 1.03
(bs, 3H), 0.92 (s, 3H), 0.71 (s, 3H); .sup.13C NMR
(CDCl.sub.3/MeOH-d.sub.4, 50 MHz) .delta. 73.89, 72.44, 68.99,
63.51, 48.05, 47.12, 42.49, 40.37, 39.99, 36.62, 36.12, 35.58,
35.40, 32.77, 30.69, 30.04, 29.02, 28.43, 27.27, 23.96, 23.08,
18.00, 13.02; HRFAB-MS (thioglycerol+Na.sup.+ matrix) m/e:
([M+Na].sup.+) 417.2992 (55.3%); calcd. 417.2981.
[0131] Compound 14: To a round-bottom flask were added 13 (28.2 g,
71.7 mmol) in DMF (300 ml), Et.sub.3 N (20 mL, 143.4 mmol), trityl
chloride (25.98 g, 93.2 mmol) and DMAP (0.13 g, 1.07 mmol). The
mixture was stirred at 50.degree. C. under N.sub.2 for 30 hours
followed by the introduction of water (1000 mL) and extraction with
EtOAc (5.times.200 mL). The combined extracts were washed with
water and brine and then dried over MgSO.sub.4. After removal of
solvent in vacuo, the residue was purified using SiO.sub.2
chromatography (CH.sub.2Cl.sub.2, Et.sub.2O and MeOH as eluents) to
give 14 as a pale yellow solid (31.9 g, 70% yield). m.p.
187.degree. C. (decomposition); IR (KBr) 3405, 2935, 1448, 1075
cm.sup.-1; .sup.1H NMR (CDCl.sub.3, 200 MHz) .delta. 7.46-7.42 (m,
6H), 7.32-7.17 (m, 9H), 3.97 (bs, 1H), 3.83 (bs, 1H), 3.50-3.38 (m,
1H), 3.01 (bs, 1H), 2.94 (dd, J=14.2, 12.2 Hz, 2H), 2.64 (bs, 1H),
2.51 (bs, 1H), 2.36-2.10 (m, 2H), 2.00-1.05 (series of multiplets,
22H), 096 (d, J=5.8 Hz, 3H), 0.87 (s, 3H), 0.64 (s, 3H); .sup.13C
NMR (CDCl.sub.3, 50 MHz) .delta. 144.77, 128.93, 127.91, 127.01,
86.43, 73.35, 72.06, 68.66, 64.28, 47.47, 46.53, 41.74, 41.62,
39.64, 35.57, 35.46, 34.91, 34.82, 32.40, 30.55, 28.21, 27.69,
26.80, 26.45, 23.36, 22.59, 17.83, 12.61; HRFAB-MS
(thioglycerol+Na.sup.+ matrix) m/e: ([M+Na].sup.+) 659.4069 (100%);
calcd. 659.4076.
[0132] Compound 15: To a round-bottom flask were added 14 (20.0 g,
31.4 mmol) in dry TI-IF (600 mL) and NaH (60% in mineral oil, 6.3
g, 157.2 mmol). The mixture was refluxed for 30 min under N.sub.2
followed by addition of allyl bromide (27 mL, 314 mmol). After 60
hours of reflux, additional Nail (3 eq.) and allyl bromide (4 eq.)
were added. Following another 50 hours of reflux, water (20 mL) was
introduced slowly followed by addition of 1% HCl until the aqueous
layer became neutral. The mixture was then extracted with ether
(3.times.100 mL) and the combined extracts were washed with water
(100 mL) and brine (2.times.100 mL). The ether solution was dried
over anhydrous Na.sub.2SO.sub.4, and after removal of solvent, the
residue was purified using SiO.sub.2 chromatography (hexanes and
EtOAc/hexanes 1:8 as eluents) to give 15 (22.76 g, 96% yield) as a
pale yellow glass. IR (neat) 2930, 1448, 1087 cm.sup.-1; .sup.1H
NMR (CDCl.sub.3, 200 MHz) .delta. 7.48-7.30 (m, 6H), 7.32-7.14 (m,
9H), 6.04-5.80 (m, 3H), 5.36-5.04 (series of multiplets, 6H),
4.14-3.94 (m, 4H), 3.74 (td, J=13.8, 5.8 Hz, 2H), 3.53 (bs, 1H),
3.20-2.94 (m, 3H), 3.31 (hs, 1H), 2.38-1.90 (m, 4H), 1.90-0.96
(series of multiplets, 20H), 0.90 (d, J=5.4 Hz, 3H), 0.89 (s, 3H),
0.64 (s, 3H); .sup.13C NMR (CDCl.sub.3, 50 MHz) .delta. 144.83,
136.27, 136.08, 128.94, 127.90, 126.98, 116.46, 115.70, 86.42,
80.94, 79.29, 74.98, 69.52, 69.39, 68.86, 64.39, 46.51, 46.42,
42.67, 42.14, 39.92, 35.63, 35.51, 35.13, 32.45, 28.98, 28.09,
27.66, 27.57, 26.72, 23.32, 23.11, 17.92, 12.69; HRFAB-MS
(thioglycerol+Na.sup.+ matrix) m/e: ([M+Na].sup.+) 779.5013
(86.1%); calcd. 779.5015.
[0133] Compound 16: To a three-necked round bottom flask was added
15 (3.34 g, 4.4 mmol) in CH.sub.2Cl.sub.2 (200 mL) and methanol
(100 mL). Through the cold solution (-78.degree. C.) ozone was
bubbled through until a blue color persisted. Excess ozone was
removed with oxygen flow. The mixture was left in a dry ice-acetone
bath for an hour. Methyl sulfide (2.4 mL) was added and 15 minutes
later, the mixture was treated with NaBH.sub.4 (1.21 g, 32 mmol) in
5% aqueous NaOH solution (10 mL)/methanol (10 mL) and allowed to
warm to room temperature. The mixture was washed with brine
(3.times.50 mL), and the combined brine wash was extracted with
CH.sub.2Cl.sub.2 (2.times.50 mL). The organic solution was dried
over MgSO.sub.4. After SiO.sub.2 chromatography (MeOH (5%) in
CH.sub.2 Cl.sub.2), 3.30 g (95% yield) of 16 was isolated as an
oil. IR (neat) 3358, 2934, 1448, 1070 cm.sup.-1; .sup.1H NMR
(CDCl.sub.3, 200 MHz) .delta. 7.50-7.42 (m, 6H), 7.32-7.17 (m, 9H),
3.80-2.96 (series of multiplets, 20H), 2.25-0.96 (series of
multiplets, 24H), 0.89 (bs, 6H), 0.65 (s, 3H); .sup.13C NMR
(CDCl.sub.3, 50 MHz) .delta. 144.73, 128.88, 127.87, 126.96, 86.38,
81.05, 79.75, 76.59, 70.33, 69.66, 69.30, 64.20, 62.25, 62.16,
62.03, 46.77, 46.36, 42.63, 41.77, 39.60, 35.43, 35.23, 35.05,
34.89, 32.42, 28.91, 27.93, 27.56, 27.15, 26.68, 23.35, 22.98,
22.85, 18.15, 12.60; HRFAB-MS (thioglycerol+Na.sup.+ matrix) m/e:
([M+Na].sup.+) 791.4860 (100%), calcd. 791.4863.
[0134] Compound 17: To a round-bottom flask was added 16 (1.17 g,
1.55 mmol) in dry THF (30 mL) under N.sub.2 in ice-bath followed by
9-BBN/THF solution (0.5 M, 10.2 mL, 5.51 mmol). The mixture was
stirred at room temperature for 12 hours. Aqueous NaOH (20%) (2 mL)
and hydrogen peroxide (30%) (2 mL) were added in sequence. The
mixture was refluxed for 1 hour followed by the addition of brine
(60 mL) and extraction with EtOAc (4.times.30 mL). The combined
extracts were dried over anhydrous Na.sub.2 SO.sub.4. The product
(1.01 g, 80% yield) was obtained as a colorless oil after SiO.sub.2
chromatography (5% MeOH in CH.sub.2 Cl.sub.2). IR (neat) 3396,
2936, 1448, 1365, 1089 cm.sup.-1; .sup.1H NMR (CDCl.sub.3, 200 MHz)
.delta. 7.50-7.42 (m, 6H), 7.34-7.16 (m, 9H), 3.90-3.56 (m, 13H),
3.50 (bs, 1H), 3.40-2.96 (series of multiplets, 6H), 2.30-0.94
(series of multiplets, 30H), 0.90 (s, 3H), 0.88 (d, Hz, 3H), 0.64
(s, 3H); .sup.13C NMR (CDCl.sub.3, 50 MHz) .delta. 144.73, 128.88,
127.85, 126.94, 86.36, 80.52, 78.90, 76.36, 66.82, 66.18, 65.77,
64.22, 61.53, 61.41, 61.34, 46.89, 46.04, 42.60, 41.59, 39.60,
35.37, 35.27, 34.88, 32.75, 32, 44, 32.31, 28.82, 27.65, 27.48,
27.13, 26.77, 23.35, 22.74, 22.38, 18.08, 12.48; HRFAB-MS
(thioglycerol+Na.sup.+ matrix) m/e: ([M+Na].sup.+) 833.5331 (100%),
calcd. 833.5332.
[0135] Compound 18: To a round-bottom flask were added 16 (3.30 g,
4.29 mmol) in CH.sub.2Cl.sub.2 (150 mL) and NEt.sub.3 (2.09 mL,
15.01 mmol). The mixture was put in ice-bath under N.sub.2 followed
by addition of mesyl chloride (1.10 mL, 14.16 mmol). After 30
minutes, water (30 mL) and brine (200 mL) were added. The
CH.sub.2Cl.sub.2 layer was washed with brine (2.times.50 mL) and
dried over anhydrous Na.sub.2SO.sub.4. The combined aqueous mixture
was extracted with EtOAc (3.times.100 mL). The combined extracts
were washed with brine and dried over anhydrous Na.sub.2SO.sub.4.
The desired product (3.35 g, 78% yield) was isolated as a pale
yellow oil after SiO.sub.2 chromatography (EtOAc/hexanes 1:1). IR
(neat) 2937, 1448, 1352, 1174, 1120, 924 cm.sup.-1; .sup.1H NMR
(CDCl.sub.3, 200 MHz) .delta. 7.52-7.40 (m, 6H), 7.34-7.20, (m,
9H), 4.42-4.24 (m, 6H), 3.90-3.64 (m, 4H), 3.60-3.30 (m, 4H),
3.24-3.00 (m, 3H), 3.10 (s, 6H), 3.05 (s, 3H), 2.20-1.96 (m, 3H)
1.96-1.60 (m, 8H), 1.60-0.94 (series of multiplets, 13H), 0.91 (bs,
6H), 0.65 (s, 3H); .sup.13C NMR (CDCl.sub.3, 50 MHz) .delta.
114.68, 128.85, 127.85, 126.96, 86.37, 81.37, 79.58, 76.58, 69.95,
69.43, 69.34, 66.52, 66.31, 65.59, 64.11, 46.80, 46.20, 42.65,
41.48, 39.35, 37.82, 37, 48, 35.36, 34.92, 34.73, 32.37, 28.66,
28.01, 27.44, 27.03, 26.72, 23.17, 22.91, 22.72, 18.13, 12.50;
HRFAB-MS (thioglycerol+Na.sup.+ matrix) m/e: ([M+Na].sup.+)
1205.4176 (81.5%), calcd. 1205.4189.
[0136] Compound 19: To a round-bottom flask were added 17 (1.01 g,
1.25 mmol) in CH.sub.2Cl.sub.2 (50 mL) and NEt.sub.3 (0.608 mL,
4.36 mmol). The mixture was put in ice-bath under N.sub.2 followed
by addition of mesyl chloride (0.318 mL, 4.11 mmol). After 30
minutes, water (10 mL) and then brine (80 mL) were added. The
CH.sub.2 Cl.sub.2 layer was washed with brine (2.times.20 mL) and
dried over anhydrous Na.sub.2SO.sub.4. The combined aqueous mixture
was extracted with EtOAc (3.times.40 mL). The combined extracts
were washed with brine and dried over anhydrous Na.sub.2 SO.sub.4.
The desired product (1.07 g, 82%) was isolated as a pale yellowish
oil after SiO.sub.2 chromatography (EtOAc/hexanes 1:1). IR (neat)
2938, 1356, 1176, 1112 cm.sup.-1; .sup.1H NMR (CDCl.sub.3, 300 MHz)
.delta. 7.46-7.43, (m, 6H), 7.32-7.22 (m, 9H), 4.40-4.31 (m, 6H),
3.72-3.64 (m, 2H), 3.55 (dd, J=6.3, 5.8 Hz, 2H), 3.51 (bs, 1H),
3.32-3.14 (m, 3H), 3.14-2.92 (m, 3H), 3.01 (s, 3H), 3.01 (s, 3H),
3.00 (s, 3H), 2.10-1.92 (m, 10H), 1.92-1.58 (m, 8H), 1.56-0.92
(series of multiplets, 12H), 0.90 (s, 3H), 0.89 (d, J=5.4 Hz, 3H),
0.64 (s, 3H); .sup.13C NMR (CDCl.sub.3, 75 MHz) .delta. 144.67,
128.85, 127.85, 126.96, 86.42, 81.06, 79.83, 76.81, 68.12, 68.06,
68.02, 64.26, 64.06, 63.42, 46.76, 46.38, 42.73, 41.87, 39.73, 37,
44, 37.32, 37.29, 35.52, 35.48, 35.32, 35.06, 32.53, 30.55, 30.28,
30.02, 29.15, 27.96, 27.69, 27.61, 26.75, 23.52, 23.02, 18.17,
12.64; HRFAB-MS (thioglycerol+Na.sup.+ matrix) m/e: ([M+Na].sup.+)
1067.4672 (100%), calcd. 1067.4659.
[0137] Compound 20: To a round-bottom flask were added 18 (1.50 g,
1.50 mmol) in dry DMSO (20 mL) and NaN.sub.3 (0.976 g, 15 mmol).
The mixture was heated to 80.degree. C. and stirred under N.sub.2
overnight then diluted with water (100 mL). The resulted aqueous
mixture was extracted with EtOAc (3.times.50 mL), and the combined
extracts washed with brine and dried over anhydrous Na.sub.2
SO.sub.4. The desired product (0.83 g, 66% yield) was isolated as a
clear glass after SiO.sub.2 chromatography (EtOAc/hexanes 1:5). IR
(neat) 2935, 2106, 1448, 1302, 1114 cm.sup.-1; .sup.1H NMR
(CDCl.sub.3, 200 MHz) .delta. 7.50-7.42 (m, 6H), 7.36-7.20 (m, 9H),
3.84-3.70 (m, 2H), 3.65 (t, J=4.9 Hz, 2H), 3.55 (bs, 1H), 3.44-3.08
(m, 10H), 3.02 (t, J=6.4 Hz, 2H), 2.38-0.96 (series of multiplets,
24H), 0.92 (d, J=5.6 Hz, 3H), 0.91 (s, 3H), 0.65 (s, 3H); .sup.13C
NMR (CDCl.sub.3, 50 MHz) .delta. 114.84, 128.97, 127.92, 126.99,
86.42, 81.24, 80.12, 76.59, 67.84, 67.29, 66.66, 64.36, 51.67,
51.44, 51.18, 46.53, 46.23, 42.21, 41.93, 39.73, 35, 66, 35.36,
35.06, 34.78, 32.40, 28.95, 27.76, 27.39, 26.87, 23.45, 22.98,
22.92, 17.98, 12.53; HRFAB-MS (thioglycerol.sup.+ Na.sup.+ matrix)
m/e: ([M+Na].sup.+) 866.5040 (100%), calcd. 866.5057.
[0138] Compound 22: To a round-bottom flask were added 20 (830 mg,
0.984 mmol) in MeOH (30 mL) and CH.sub.2 Cl.sub.2 (30 mL) and
p-toluenesulfonic acid (9.35 mg, 0.0492 mmol). The solution was
stirred at room temperature for 2.5 hours then saturated aqueous
NaHCO.sub.3 (10 mL) was introduced. Brine (30 mL) was added, and
the mixture was extracted with EtOAc (4.times.20 mL). The combined
extracts were dried over anhydrous Na.sub.2 SO.sub.4. The desired
product (0.564 g, 95% yield) was isolated as a pale yellowish oil
after SiO.sub.2 chromatography (EtOAc/hexanes 1:2). IR (neat) 3410,
2934, 2106, 1301, 1112 cm.sup.-1; .sup.1H NMR (CDCl.sub.3, 200 MHz)
.delta. 3.80-3.54 (m, 7H), 3.44-3.20 (m, 10H), 2.35-0.96 (series of
multiplets, 24H), 0.95 (d, J=6.4 Hz, 3H), 0.92 (s, 3H), 0.68 (s,
3H); .sup.13C NMR (CDCl.sub.3, 50 MHz) .delta. 81.10, 80.01, 76.60,
67.75, 67.16, 66.56, 63.63, 51.57, 51.34, 51.06, 46.29, 46.12,
42.12, 41.81, 39.60, 35.55, 35.23, 34.94, 34.66, 31.75, 29.48,
28.81, 27.72, 27.66, 27.29, 23.32, 22.86, 22.80, 17.85, 12.39;
HRFAB-MS (thioglycerol+Na.sup.+ matrix) m/e: ([M+Na].sup.+)
624.3965 (100%), calcd. 624.3962.
[0139] Compound 23: To a round-bottom flask were added 19 (1.07 g,
1.025 mmol) and NaN.sub.3 (0.666 g, 10.25 mmol) followed the
introduction of dry DMSO (15 mL). The mixture was heated up to
80.degree. C. under N.sub.2 overnight. After the addition of
H.sub.2 O (100 mL), the mixture was extracted with EtOAc
(4.times.40 mL) and the combined extracts were washed with brine
(2.times.50 mL) and dried over anhydrous Na.sub.2 SO.sub.4. After
removal of solvent, the residue was dissolved in MeOH (15 mL) and
CH.sub.2 Cl.sub.2 (15 mL) followed by the addition of catalytic
amount of p-toluenesulfonic acid (9.75 mg, 0.051 mmol). The
solution was stirred at room temperature for 2.5 hours before the
addition of saturated NaHCO.sub.3 solution (15 mL). After the
addition of brine (60 mL), the mixture was extracted with EtOAc
(5.times.30 mL). The combined extracts were washed with brine (50
mL) and dried over anhydrous Na.sub.2SO.sub.4. The desired product
(0.617 g, 94% yield for two steps) was obtained as a yellowish oil
after SiO.sub.2 chromatography (EtOAc/hexanes 1:2). IR (neat) 3426,
2928, 2094, 1456, 1263, 1107 cm.sup.-1; .sup.1H NMR (CDCl.sub.3,
300 MHz) .delta. 3.68-3.56 (m, 3H), 3.56-3.34 (series of
multiplets, 10H), 3.28-3.00 (series of multiplets, 4H), 2.20-2.00
(m, 3H), 1.98-1.55 (series of multiplets, 15H), 1.55-0.96 (series
of multiplets, 13H), 0.92 (d, J=6.6 Hz, 3H), 0.89 (s, 3H), 0.66 (s,
3H); .sup.13C NMR (CDCl.sub.3, 75 MHz) .delta. 80.63, 79.79, 76.04,
64.99, 64.45, 64.30, 63.72, 49.01, 48.94, 48.74, 46.49, 46.39,
42.70, 41.98, 39.80, 35.65, 35.42, 35.28, 35.08, 31.99, 29.78,
29.75, 29.70, 29.49, 29.06, 27.87, 27.79. 27.65, 23.53, 23.04,
22.85, 18.05, 12.59; HRFAB-MS (thioglycerol+Na matrix) m/e:
([M+Na].sup.+) 666.4415 (100%), calcd. 666.4431.
[0140] Compound 24: To a round-bottom flask were added 22 (0.564 g,
0.938 mmol) in CH.sub.2Cl.sub.2 (30 mL) and NEt.sub.3 (0.20 mL,
1.40 mmol). The mixture was put in ice-bath under N.sub.2 followed
by addition of mesyl chloride (0.087 mL, 1.13 mmol). After 30
minutes, water (20 mL) and brine (100 mL) were added. The CH.sub.2
Cl.sub.2 layer was washed with brine (2.times.20 mL) and dried over
anhydrous Na.sub.2 SO.sub.4. The combined aqueous mixture was
extracted with EtOAc (3.times.30 mL). The combined extracts were
washed with brine and dried over anhydrous Na.sub.2 SO.sub.4. The
desired product (0.634 g, 99% yield) was isolated as a pale
yellowish oil after SiO.sub.2 chromatography (EtOAc/hexanes 1:2).
JR (neat) 2935, 2106, 1356, 1175, 1113 cm.sup.-1; NMR (CDCl.sub.3,
300 MHz) .delta. 4.20 (t, J=6.8 Hz, 2H), 3.80-3.75 (m, 1H),
3.70-3.64 (m, 3H), 3.55 (bs, 1H), 3.44-3.01 (m, 10H), 3.00 (s, 3H),
2.32-2.17 (m, 3H), 2.06-2.03 (m, 1H), 1.90-0.88 (series of
multiplets, 20H), 0.95 (d, J=6.6 Hz, 3H), 0.91 (s, 3H), 0.68 (s,
3H); .sup.13C NMR (CDCl.sub.3, 75 MHz) .delta. 80.90, 79.86, 76.43,
70.78, 67.64, 66.99, 66.48, 51.50, 51.26, 50.97, 46.05, 45.96,
42.08, 41.71, 39.51, 37.33, 35.15, 34.86, 34.60, 31.34, 28.73,
27.62, 27.59, 27.51, 25.68, 23.22, 22.80, 22.70, 17.62, 12.33;
HRFAB-MS (thioglycerol+Na.sup.+ matrix) m/e: ([M+Na].sup.+)
702.3741 (100%), calcd. 702.3737.
[0141] Compound 25: To a round-bottom flask were added 23 (0.617 g,
0.96 mmol) in CH.sub.2 (30 mL) and NEt.sub.3 (0.20 mL, 1.44 mmol).
The mixture was put in ice-bath under N.sub.2 followed by addition
of mesyl chloride (0.089 mL, 1.15 mmol). After 30 minutes, water
(20 mL) and brine (120 mL) were added. The CH.sub.2 Cl.sub.2 layer
was washed with brine (2.times.20 mL) and dried over anhydrous
Na.sub.2 SO.sub.4. The combined aqueous mixture was extracted with
EtOAc (3.times.30 mL). The combined extracts were washed with brine
and dried over anhydrous Na.sub.2 SO.sub.4. The desired product
(0.676 g, 97% yield) was isolated as a pale yellowish oil after
removal of solvent. IR (neat) 2934, 2094, 1454, 1360, 1174, 1112
cm.sup.-1; .sup.1H NMR (CDCl.sub.3, 300 MHz) .delta. 4.17 (t, J=6.6
Hz, 2H), 3.65-3.28 (series of multiplets, 11H), 3.64-3.00 (series
of multiplets, 4H), 2.97 (s, 3H), 2.18-1.96 (series of multiplets,
16H), 1.54-0.94 (series of multiplets, 11H), 0.89 (d, Hz, 3H), 0.86
(s, 3H), 0.63 (s, 3H); .sup.13C NMR (CDCl.sub.3, 75 MHz) .delta.
80.47, 79.67, 75.92, 70.84, 64.90, 64.37, 64.17, 48.90, 48.86,
48.66, 46.32, 46.26, 42.63, 41.87, 39.70, 37.39, 35.34, 35.28,
35.20, 34.99, 31.61, 29.68, 29.60, 28.96, 27.78, 27.68, 27.57,
25.79, 23.41, 22.95, 22.74, 17.82, 12.50; HRFAB-MS (thioglycerol
matrix) m/e: ([M+H].sup.+) 722.4385 (22.1%), calcd. 722.4387.
[0142] Compound 26: To a 50 mL round-bottom flask was added 24
(0.634 g, 0.936 mmol) and N-benzylmethylamine (2 mL). The mixture
was heated under N.sub.2 at 80.degree. C. overnight. Excess
N-benzylmethylamine was removed under vacuum, and the residue was
subjected to SiO.sub.2 chromatography (EtOAc/hexanes 1:2). The
desired product (0.6236 g, 95% yield) was isolated as a pale yellow
oil. IR (neat) 2935, 2106, 1452, 1302, 1116 cm.sup.-1; .sup.1H NMR
(CDCl.sub.3, 200 MHz) .delta. 7.32-7.24 (m, 5H), 3.80-3.76 (m, 1H),
3.70-3.60 (m, 3H), 3.54 (bs, 1H), 3.47 (s, 2H), 3.42-3.10 (m, 10H),
2.38-2.05 (m, 5H), 2.17 (s, 3H), 2.02-0.88 (series of multiplet,
21H), 0.93 (d, J=7.0 Hz, 3H), 0.91 (s, 3H), 0.66 (s, 3H); .sup.13C
NMR (CDCl.sub.3, 50 MHz) .delta. 139.60, 129.34, 128.38, 127.02,
81.22, 80.10, 76.71, 67.85, 67.29, 66.65, 62.45, 58.38, 51.65,
51.44, 51.16, 46.50, 46.21, 42.40, 42.20, 41.93, 39.72, 35, 80,
35.34, 35.05, 34.76, 33.65, 28.93, 27082, 27.75, 27.38, 24.10,
23.45, 22.98, 22.91, 18.05, 12.50; HRFAB-MS (thioglycerol+Na.sup.4
matrix) m/e: ([M-H].sup.+) 703.4748 (90.2%), calcd. 703.4772;
([M+H].sup.+) 705.4911 (100%), calcd. 705.4928; ([M+Na].sup.+)
727.4767 (1.5%), calcd. 727.4748.
[0143] Compound 27: To a 50 mL round-bottom flask was added 25
(0.676 g, 0.937 mmol) and N-benzylmethylamine (2 mL). The mixture
was heated under N.sub.2 at 80.degree. C. overnight. Excess
N-benzylmethylamine was removed under vacuum and the residue was
subjected to SiO.sub.2 chromatography (EtOAc/hexanes 1:2). The
desired product (0.672 g, 96% yield) was isolated as a pale yellow
oil. IR (neat) 2934, 2096, 1452, 1283, 1107 cm.sup.-1; .sup.1H NMR
(CDCl.sub.3, 300 MHz) .delta. 7.34-7.20 (m, 5H), 3.68-3.37 (series
of multiplets, 13H), 3.28-3.04 (m, 4H), 2.33 (t, Hz, 2H), 2.18 (s,
3H), 2.20-2.00 (m, 3H), 1.96-1.56 (series of multiplets, 14H),
1.54-1.12 (m, 10H), 1.10-0.96 (m, 3H), 0.91 (d, J=8.7 Hz, 3H), 0.89
(s, 3H), 0.65 (s, 3H); .sup.13C NMR (CDCl.sub.3, 75 MHz) .delta.
139.48, 129.23, 128.30, 126.96, 80.66, 79.81, 76.08, 65.00, 64.46,
64.34, 62.50, 58.37, 49.02, 48.95, 48.75, 46.65, 46.40, 42.69,
42.43, 42.00, 39.83, 35.86, 35.45, 35.30, 35.10, 33.83, 29.81,
29.78, 29.72, 29.09, 27.88, 27.81, 27.66, 24.19, 23.57, 23.06,
22.87, 18.15, 12.62; HRFAB-MS (thioglycerol matrix) m/e:
([M+H].sup.+) 747.5406 (77.2%), calcd. 747.5398.
[0144] Compound 1: To a round-bottom flask were added 26 (0.684 g,
0.971 mmol) in dry THF (30 mL) and LiAlH.sub.4 (113.7 mg, 3.0 mmol)
under N.sub.2. The mixture was stirred at room temperature for 12
hours, and then Na.sub.2SO.sub.4.10 H.sub.2O powder (10 g) was
added slowly. After the grey color disappeared, the mixture was
filtered through Celite and washed with dry THF. The product (0.581
g, 95% yield) was obtained as a colorless glass. IR (neat) 3372,
2937, 1558, 1455, 1362, 1102 cm.sup.-1; .sup.1H NMR (CDCl.sub.3,
300 MHz) .delta. 7.34-7.20 (m, 5H), 3.68-3.48 (m, 5H), 3.47 (s,
2H), 3.29 (bs, 1H), 3.22-3.00 (m, 3H), 2.96-2.80 (m, 6H), 2.32 (t,
J=6.8, 5.4 Hz, 2H), 2.17 (s, 3H), 2.20-2.00 (m, 3H), 1.96-0.96
(series of multiplets, 27H), 0.93 (d, J=6.8 Hz, 3H), 0.90, (s, 3H),
0.67 (s, 3H); .sup.13C NMR (CDCl.sub.3, 75 MHz) .delta. 139.50,
129.22, 128.31, 126.96, 80.76, 79.85, 76.10, 70.90, 70.33, 70.24,
62.48, 58.27, 46.55, 46.45, 42.72, 42.58, 42.33, 41.99, 39.77,
35.78, 35.37, 35.01, 33.73, 29.07, 27.95, 27.71, 24.06, 23.46,
22.99, 18.14, 12.55; HRFAB-MS (thioglycerol matrix) m/e:
([M+H].sup.+) 627.5211 (100%), calcd. 627.5213.
[0145] HCl salt of compound 1: Compound 1 was dissolved in a
minimum amount of CH.sub.2 Cl.sub.2 and excess HCl in ether was
added. Solvent and excess HCl were removed in vacuo and a
noncrystalline white powder was obtained. .sup.1H NMR
(methanol-d4/15% (CDCl.sub.3, 300 MHz) .delta. 7.61-7.57 (m, 2H),
7.50-7.48 (m, 3H), 4.84 (bs, 10H), 4.45 (bs, 1H), 4.30 (bs, 1H),
3.96-3.82 (m, 2H), 3.78-3.69 (m, 2H), 3.66 (bs, 1H), 3.59-3.32
(series of multiplets, 4H), 3.28-3.02 (m, 8H), 2.81 (s, 3 PT),
2.36-2.15 (m, 4H), 2.02-1.68 (m, 8H), 1.64-0.90 (series of
multiplets, 12H), 1.01 (d, J=6.35 Hz, 3H), 0.96 (s, 3H), 0.73 (s,
3H); .sup.13C NMR (methanol-d4/15% (CDCl.sub.3, 75 MHz) .delta.
132.31, 131.20, 130.92, 130.40, 83.13, 81.09, 78, 48, 65.54, 64.98,
64.11, 60.87, 57.66, 47.51, 46.91, 43.52, 43.00, 41.38, 41.19,
41.16, 40, 75, 40.30, 36.37, 36.08, 36.00, 35.96, 33.77, 29.68,
29.34, 28.65, 28.37, 24.42, 24.25, 23.33, 21.51, 18.80, 13.04.
[0146] Compound 2: To a round-bottom flask were added 27 (0.82 g,
1.10 mmol) in dry THF (150 mL) and LiAlH.sub.4 (125 mg, 3.30 mmol)
under N.sub.2. The mixture was stirred at room temperature for 12
hours and Na.sub.2 SO.sub.4.10 H.sub.2O powder (10 g) was added
slowly. After the grey color disappeared, the mixture was filtered
through a cotton plug and washed with dry THF. TI-IF was removed in
vacuo and the residue dissolved in CH.sub.2 Cl.sub.2 (50 mL). After
filtration, the desired product was obtained as a colorless glass
(0.73 g, 99% yield). IR (neat) 3362, 2936, 2862, 2786, 1576, 1466,
1363, 1103 cm.sup.-1; .sup.1H NMR (CDCl.sub.3, 300 MHz) .delta.
7.32-7.23 (m, 5H), 3.67-3.63 (m, 1H), 3.60-3.57 (m, 1H), 3.53 (t,
J=6.4 Hz, 2H), 3.47 (s, 2H), 3.46 (bs, 1H), 3.24-3.17 (m, 2H),
3.12-2.99 (m, 2H), 2.83-2.74 (series of multiplets, 6H), 2.30 (t,
J=7.3 Hz, 2H), 2.15 (s, 3H), 2.20-2.00 (m, 3H), 1.95-1.51 (series
of multiplets, 20H), 1.51-1.08, (series of multiplets, 10H),
1.06-0.80 (m, 3H), 0.87 (d, J=8.1 Hz, 3H), 0.86 (s, 3H), 0.61 (s,
3H); .sup.13C NMR (CDCl.sub.3, 75 MHz).
[0147] 139.35, 129.16, 128.22, 126.88, 80.44, 79.29, 75.96, 66.70,
66.52, 66.12, 62.45, 58.26, 46.76, 46.27, 42.69, 42.41, 42.02,
40.68, 40.10, 40.02, 39.82, 35.84, 35.47, 35.30, 35.06, 34.15,
34.09, 34.03, 33.80, 28.96, 27.93, 27.75, 27.71, 24.32, 23.53,
23.03, 22.75, 18.17, 12.58; HRFAH-MS (thioglycerol+Na.sup.+ matrix)
m/e: ([M+Na].sup.+) 691.5504 (38.5%), calcd. 691.5502.
[0148] HCl salt of compound 2: Compound 2 was dissolved in a
minimum amount of CH.sub.2 Cl.sub.2 and excess HCl in ether was
added. Removal of the solvent and excess HCl gave a noncrystalline
white powder. NMR (methanol-d.sub.4/15% (CDCl.sub.3, 300 MHz)
.delta. 7.60-7.59 (m, 2H), 7.50-7.47 (m, 3H), 4.82 (bs, 10H), 4.43
(bs, 1 FT), 4.32 (bs, 1H), 3.85-3.79 (m, 1H), 3.75-3.68 (m, 1H),
3.64 (t, J=5.74 Hz, 2H), 3.57 (bs, 1H), 3.36-3.28 (m, 2H),
3.25-3.00 (series of multiplets, 10H), 2.82 (s, 3H), 2.14-1.68
(series of multiplets, 19H), 1.65-1.15 (series of multiplets, 11H),
0.98 (d, J=6.6 Hz, 3H), 0.95 (s, 3H), 0.72 (s, 3H); .sup.13C NMR
(methanol-d4/15% (CDCl.sub.3, 75 MHz) .delta. 132.21, 131.10,
130.58, 130.28, 81.96, 80.72, 77.60, 66.84, 66.58, 66.12, 61.03,
57.60, 44.16, 42.77, 40.62, 39.57, 39.43, 36.28, 36.03, 35.96,
35.78, 33.65, 29.48, 29.27, 29.11, 29.01, 28.61, 28.56, 28.35,
24.25, 23.56, 23.30, 21.17, 18.64, 12.90.
[0149] Compound 4: A suspension of 1 (79.1 mg, 0.126 mmol) and
aminoiminomethanesulfonic acid (50.15 mg, 0.404 mmol) in methanol
and chloroform was stirred at room temperature for 24 hours, and
the suspension became clear. An ether solution of HCl (1 M, 1 mL)
was added followed by the removal of solvent with N.sub.2 flow. The
residue was dissolved in H.sub.2 O (5 mL) followed by the addition
of 20% aqueous NaOH (0.5 mL). The resulting cloudy mixture was
extracted with CH.sub.2Cl.sub.2 (4.times.5 mL). The combined
extracts were dried over anhydrous Na.sub.2SO.sub.4. Removal of
solvent gave the desired product (90 mg, 95%) as white powder. m.p.
111-112.degree. C. IR (neat) 3316, 2937, 1667, 1650, 1556, 1454,
1348, 1102 cm.sup.-1; .sup.1H NMR (5% methanol-d4/CDCl.sub.3, 300
MHz) .delta. 7.26-7.22 (m, 5H), 4.37 (bs, 3H), 3.71-3.51 (series of
multiplets, 5H), 3.44 (s, 2H), 3.39-3.10 (series of multiplets,
10H), 2.27 (t, J=6.83 Hz, 2H), 2.13 (s, 3H), 2.02-0.94 (series of
multiplets, 33H), 0.85 (d, J=5.62 Hz, 3H), 0.84 (s, 3H), 0.61 (s,
3H); .sup.13C NMR (5% methanol-d4/CDCl.sub.3, 75 MHz) .delta.
158.54, 158.48, 158.43, 138.27, 129.47, 128.32, 127.19, 81.89,
80.30, 77.34, 69.02, 68.46, 67, 21, 62.36, 58.00, 47.36, 46, 18,
43.26, 43.00, 42.73, 42.18, 41.48, 39.32, 35.55, 34.97, 34.89, 34,
67, 33.63, 28.93, 28.28, 27.53, 27.16, 23.96, 23.28, 23.16, 22.77,
18, 36, 12.58; HRFAB-MS (thioglycerol+Na.sup.+ matrix) m/e:
([M+H].sup.+) 753.5858 (100%), calcd. 753.5867.
[0150] HCl salt of compound 4: Compound 4 was dissolved in minimum
amount of CH.sub.2Cl.sub.2 and MeOH followed by addition of excess
HCl in ether. The solvent was removed by N.sub.2 flow, and the
residue was subjected to high vacuum overnight. The desired product
was obtained as noncrystalline white powder. .sup.1H NMR
(methanol-d4/20% (CDCl.sub.3, 300 MHz) .delta. 7.58 (bs, 2H),
7.50-7.48 (m, 3H), 4.76 (bs, 13H), 4.45 (d, J=12.9 Hz, 1H), 4.27
(dd, 1 H, J=12.9, 5.4 Hz), 3.82-3.00 (series of multiplets, 17H),
2.81-2.80 (m, 3H), 2.20-1.02 (series of multiplets, 27H), 0.98 (d,
J=6.59 Hz, 3H), 0.95 (s, 3H), 0.72 (s, 3H); .sup.13C NMR
(methanol-d4/20% CDCl.sub.3, 75 MHz) .delta. 158.88, 158.72,
132.00, 131.96, 130.98, 130.15, 82.51, 81.07, 78.05, 68.50, 68.02,
67.94, 67.10, 60.87, 60.53, 57.38, 47, 16, 46.91, 43.91, 43.11,
43.01, 42.91, 42.55, 40.28, 39.88, 39.95, 35.90, 35.73, 35.64,
33.53, 29.18, 28.35, 27.99, 24.02, 23.30, 21.35, 18.52, 18.44,
13.06.
[0151] Compound 5: A suspension of 2 (113 mg, 0.169 mmol) and
aminoiminomethanesulfonic acid (67.1 mg, 0.541 mmol) in methanol
and chloroform was stirred at room temperature for 24 hours. HCl in
ether (1 M, 1 mL) was added followed by the removal of solvent with
N.sub.2 flow. The residue was subject to high vacuum overnight and
dissolved in H.sub.2O (5 mL) followed by the addition of 20% NaOH
solution (1.0 mL). The resulting mixture was extracted with
CH.sub.2 Cl.sub.2 (5.times.5 mL). The combined extracts were dried
over anhydrous Na.sub.2 SO.sub.4, Removal of solvent gave desired
the product (90 mg, 95% yield) as a white solid. m.p.
102-104.degree. C. IR (neat) 3332, 3155, 2939, 2863, 1667, 1651,
1558, 1456, 1350, 1100 cm.sup.-1; .sup.1H NMR (5%
methanol-d4/CDCl.sub.3, 300 MHz) i 7.35-7.24 (m, 5H), 3.75-3.64 (m,
1H), 3.57 (bs, 5H), 3.50 (s, 2H), 3.53-3.46 (m, 1H), 3.40-3.10
(series of multiplets, 14H), 2.34 (t, J=7.31 Hz, 2H), 2.19 (s, 3H),
2.13-0.96 (series of multiplets, 36H), 0.91 (bs, 6H), 0.66 (s, 3H);
.sup.13C NMR (5% methanol-d4/CDCl.sub.3, 75 MHz) 157.49, 157.31,
157.23, 138.20, 129.52, 128.34, 127.23, 81.17, 79.19, 76.42, 65.63,
65.03, 64.70, 62.36, 58.02, 47.23, 46.24, 42.89, 42.18, 41.45,
39.45, 39.40, 39.30, 38.71, 35.61, 35.55, 35.02, 34.82, 33.69,
29.87, 29.59, 29.42, 28.84, 27.96, 27.56, 23.95, 23.40, 22.82,
22.64, 18.28, 12.54; HRFAB-MS (thioglycerol+Na.sup.+ matrix) m/e:
([M+H].sup.+) 795.6356 (84.3%), calcd. 795.6337.
[0152] HCl salt of compound 5: Compound 5 was dissolved in minimum
amount of CH.sub.2 Cl.sub.2 and MeOH followed by the addition of
excess HCl in ether. The solvent and excess HCl were removed by
N.sub.2 flow and the residue was subject to high vacuum overnight.
The desired product was obtained as noncrystalline white powder.
.sup.1H NMR (methanol-d4/10% CDCl.sub.3, 300 MHz) .delta. 7.62-7.54
(m, 2H), 7.48-7.44 (m, 3H), 4.84 (bs, 16H), 4.46 (d, J=12.7 Hz,
1H), 4.26 (dd, J=12.7, 3.42 Hz, 1H), 3.78-3.56 (series of
multiplets, 5H), 3.38-3.05 (series of multiplets, 13H), 2.80 (d,
3H), 2.19-2.04 (m, 3H), 2.02-1.04 (series of multiplets, 30H), 0.98
(d, J=6.35 Hz, 3H), 0.95 (s, 3H), 0.72 (s, 3H); .sup.13C NMR
(methanol-d4/10% CDCl.sub.3, 75 MHz) .delta. 158.75, 158.67,
132.32, 131.24, 130.83, 130.43, 82.49, 81.02, 77.60, 66.47, 65.93,
61.19, 60.85, 57.69, 47.79, 47.60, 44.29, 43.07, 40.86, 40.42,
40.19, 40.09, 39.76, 36.68, 36.50, 36.15, 35.94, 33.91, 30.75,
30.46, 29.74, 29.33, 28.71, 24.41, 24.03, 23.38, 22.21, 22.16,
18.59, 18.52, 13.09.
[0153] Compound CSA-26 was synthesized according to Scheme 1 and
Example 1 using 7-deoxycholic acid in place of cholic acid and
methyl cholate.
Example 2
[0154] This example includes a description of one or more exemplary
synthetic procedures for obtaining Compounds 3, 28 and 29.
[0155] Compound 28: A suspension of 19 (0.641 g, 0.614 mmol) and
KCN (0.40 g, 6.14 mmol) in anhydrous DMSO (5 mL) was stirred under
N.sub.2 at 80.degree. C. overnight followed by the addition of
H.sub.2 O (50 mL). The aqueous mixture was extracted with EtOAc
(4.times.20 mL). The combined extracts were washed with brine once,
dried over anhydrous Na.sub.2 SO.sub.4 and concentrated in vacuo.
The residue was dissolved in CH.sub.2 Cl.sub.2 (3 mL) and MeOH (3
mL) and catalytic amount of p-toluenesulfonic acid (5.84 mg, 0.03
mmol) was added. The solution was stirred at room temperature for 3
hours before the introduction of saturated NaHCO.sub.3 solution (10
mL). After the addition of brine (60 mL), the mixture was extracted
with EtOAc (4.times.30 mL). The combined extracts were washed with
brine once and dried over anhydrous Na.sub.2 SO.sub.4 and
concentrated. The residue afforded the desired product (0.342 g,
92% yield) as pale yellowish oil after column chromatography
(silica gel, EtOAc/hexanes 2:1). IR (neat) 3479, 2936, 2864, 2249,
1456, 1445, 1366, 1348, 1108 cm.sup.-1H NMR (CDCl.sub.3, 300 MHz)
.delta. 3.76-3.53 (m, 7H), 3.32-3.06 (series of multiplets, 4H),
2.57-2.46 (m, 6H), 2.13-1.00 (series of multiplets, 31H), 0.93 (d,
J=6.35 Hz, 3H), 0.90 (s, 3H), 0.67 (s, 3H); .sup.13C NMR
(CDCl.sub.3, 75 MHz) .delta. 119.91, 119.89, 80.75, 79.65, 76.29,
65.83, 65.37, 65.19, 63.63, 46, 57, 46.44, 42.77, 41.79, 39.71,
35.63, 35.26, 35.02, 32.00, 29.46, 29.03, 27.96, 27.74, 26.64,
26.42, 26.12, 23.56, 22.98, 22.95, 18.24, 14.65. 14.54, 14.30,
12.60; HRFAB-MS (thioglycerol+Na.sup.+ matrix) m/e: ([M+Na].sup.+)
618.4247 (67.8%), calcd. 618.4247.
[0156] Compound 29: To a solution of 28 (0.34 g, 0.57 mmol) in dry
CH.sub.2 Cl.sub.2 (15 mL) under N.sub.2 at 0.degree. C. was added
NEt.sub.3 (119.5 .mu.L, 0.857 mmol) followed by the addition of
mesyl chloride (53.1.mu.L, 0.686 mmol). The mixture was allowed to
stir at 0.degree. C. for 30 minutes before the addition of H.sub.2
O (6 mL). After the introduction of brine (60 mL), the aqueous
mixture was extracted with EtOAc (4.times.20 mL). The combined
extracts were washed with brine once, dried over anhydrous Na.sub.2
SO.sub.4 and concentrated. To the residue was added N-benzylmethyl
amine (0.5 mL) and the mixture was stirred under N.sub.2 at
80.degree. C. overnight. Excess N-benzylmethylamine was removed in
vacuo and the residue was subject to column chromatography (silica
gel, EtOAc/hexanes 2:1 followed by EtOAc) to afford product (0.35
g, 88% yield) as a pale yellow oil. IR (neat) 2940, 2863, 2785,
2249, 1469, 1453, 1366, 1348, 1108 cm.sup.-1; .sup.1H NMR
(CDCl.sub.3, 300 MHz) .delta. 7.34-7.21 (m, 5H), 3.76-3.69 (m, 1H),
3.64-3.50 (m, 4H), 3.48 (s, 2H), 3.31-3.05 (series of multiplets,
4H), 2.52-2.46 (m, 6H), 2.33 (t, J=7.32 H, 2 Hz), 2.18 (s, 3H),
2.13-0.95 (series of multiplets, 30H), 0.91 (d, J=6.80 H, 3 Hz),
0.90 (s, 3H), 0.66 (s, 3H); .sup.13C NMR (CDCl.sub.3, 75 MHz)
.delta. 139.37, 129.17, 128.26, 126.93, 119.96, 119.91, 80.73,
79.59, 76.26, 65.79, 65.35, 65.13, 62.47, 58.25, 46.74, 46.40,
42.72, 42.38, 41.76, 39.68, 35.78, 35.22, 34.98, 33.79, 28.99,
27.92, 27.71, 26.63, 26.38, 26.09, 24.21, 23.54, 22.96, 22.90,
18.28, 14.62, 14.51, 14.26, 12.58; HRFAB-MS (thioglycerol+Na.sup.+
matrix) m/e: ([M+H].sup.+) 699.5226 (100%), calcd. 699.5213.
[0157] Compound 3: A solution of 29 (0.074 g, 0.106 mmol) in
anhydrous THF (10 mL) was added dropwise to a mixture of AlCl.sub.3
(0.1414 g, 1.06 mmol) and LiAlH.sub.4 (0.041 g, 1.06 mmol) in dry
THF (10 mL). The suspension was stirred for 24 hours followed by
the addition of 20% NaOH aqueous solution (2 mL) at ice-bath
temperature. Anhydrous Na.sub.2 SO.sub.4 was added to the aqueous
slurry. The solution was filtered and the precipitate washed twice
with THF. After removal of solvent, the residue was subject to
column chromatography (silica gel, MeOH/CH.sub.2 Cl.sub.2 1:1
followed by MeOH/CH.sub.2 Cl.sub.2/NH.sub.3.H.sub.2 O 4:4:1) to
afford the desired product (0.038 g, 50% yield) as a clear oil. IR
(neat) 3362, 2935, 2863, 2782, 1651, 1574, 1568, 1557, 1471, 1455,
1103 cm.sup.-1; .sup.1H NMR (CDCl.sub.3, 300 MHz) .delta. 7.32-7.22
(m, 5H), 3.60-3.02 (series of broad multiplets, 18H), 2.90-2.70 (m,
5H), 2.33 (t, J=7.20 Hz, 2H), 2.24-2.04 (m, 3H), 2.18 (s, 3H),
1.96-0.96 (series of multiplets, 30H), 0.90 (d, J=7.57 Hz, 3H),
0.89 (s, 3H), 0.64 (s, 3H); .sup.13C NMR (CDCl.sub.3, 75 MHz)
.delta. 139.44, 129.24, 128.31, 126.97, 80.63, 79.65, 75.97, 68.44,
68.00. 67.96, 62.54, 58.40, 46.77, 46.30, 42.73, 42.43, 42.07,
41.92, 41.74, 41.72, 39.81, 35.82, 35.48, 35.07, 33.84, 31.04,
30.30, 30.10, 29.03, 28.11, 27.82, 27.81, 27.74, 27.67, 27.64,
24.31, 23.50, 23.04, 22.93, 18.22, 12.63; HRFAB-MS
(thioglycerol+Na.sup.+ matrix) m/e: ([M+H].sup.+) 711.6139 (100%),
calcd. 711.6152; ([M+Na].sup.+) 733.5974 (46.1%), calcd.
733.5972.
Example 3
[0158] This example includes a description of one or more exemplary
synthestic procedures for obtaining Compounds 6, 7 and 30-33.
[0159] Compound 30: Cholic acid (3.0 g, 7.3 mmol) was dissolved in
CH.sub.2Cl.sub.2 (50 mL) and methanol (5 mL).
Dicyclohexylcarbodiimide (DCC) (1.8 g, 8.8 mmol) was added followed
by N-hydroxysuccinimide (about 100 mg) and benzylmethylamine (1.1
g, 8.8 mmol). The mixture was stirred for 2 hours, then filtered.
The filtrate was concentrated and chromatographed (SiO.sub.2, 3%
MeOH in CH.sub.2Cl.sub.2) to give 3.0 g of a white solid (81%
yield). m.p. 184-186.degree. C.; IR (neat) 3325, 2984, 1678
cm.sup.-1; .sup.1H NMR (CDCl.sub.3, 200 MHz) .delta. 7.21 (m, 5H),
4.51 (m, 2H), 3.87 (m, 1H), 3.74 (m, 2H), 3.36 (m, 2H), 2.84 (s,
3H), 2.48-0.92 (series of multiplets, 28H), 0.80 (s, 3H), 0.58 (d,
J=6.5 Hz, 3H); .sup.13C NMR (CDCl.sub.3, 50 MHz) .delta. 174.30,
173.94, 137.36, 136.63, 128.81, 128.46, 127.85, 127.50, 127.18,
126.28, 72.96, 71.76, 68.35, 53.39, 50.65, 48.77, 46.91, 46.33,
41.44, 39.36, 39.18, 35.76, 35.27, 34.76, 33.87, 31.54, 34.19,
31.07, 30.45, 28.11, 27.63, 26.14, 25.59, 24.92, 23.26, 17.51,
12.41; FAB-MS (thioglycerol+Na.sup.4 matrix) m/e: ([M+H].sup.+) 512
(100%), calcd. 512.
[0160] Compound 31: Compound 30 (2.4 g, 4.7 mmol) was added to a
suspension of LiAlH.sub.4 (0.18 g, 4.7 mmol) in THF (50 mL). The
mixture was refluxed for 24 hours, then cooled to 0.degree. C. An
aqueous solution of Na.sub.2SO.sub.4 was carefully added until the
grey color of the mixture dissipated. The salts were filtered out,
and the filtrate was concentrated in vacuo to yield 2.1 g of a
white solid (88%). The product proved to be of sufficient purity
for further reactions. m.p. 70-73.degree. C.; IR (neat) 3380, 2983,
1502 cm.sup.-1H NMR (CDCl.sub.3, 300 MHz) .delta. 7.23 (m, 5H),
3.98 (bs, 2H), 3.81 (m, 3H), 3.43 (m, 3H), 2.74 (m, 2H), 2.33 (m,
3H), 2.25 (s, 3H), 2.10-0.90 (series of multiplets, 24H), 0.98 (s,
3H), 0.78 (s, 3H); .sup.13C NMR (CDCl.sub.3, 75 MHz) .delta.
135.72, 129.63, 128.21, 128.13, 125.28, 72.91, 71, 63, 62.05,
60.80, 56.79, 47.00, 46.23, 41.44, 40.81, 39.41, 35.42, 35.24,
34.63, 34.02, 33.22, 31.73, 30.17, 29, 33, 29.16, 28.02, 27.49,
26.17, 25.55, 23.10, 22.48, 22.33, 17.54, 12.65; FAB-MS
(thioglycerol matrix) m/e: 498 (100%), calcd. 498.
[0161] Compound 32: Compound 31 (0.36 g, 0.72 mmol) was dissolved
in CH.sub.2Cl.sub.2 (15 mL) and Bocglycine (0.51 g, 2.89 mmol), DCC
(0.67 g, 3.24 mmol) and dimethylaminopyridine (DMAP) (about 100 mg)
were added. The mixture was stirred under N.sub.2 for 4 hours then
filtered. After concentration and chromatography (SiO.sub.2, 5%
MeOH in CH.sub.2Cl.sub.2), the product was obtained as a 0.47 g of
a clear glass (68%). .sup.1H NMR (CDCl.sub.3, 300 MHz) .delta. 7.30
(m, 5H), 5.19 (bs, 1H), 5.09 (bs, 3H), 5.01 (bs, 1H), 4.75 (m, 1H),
4.06-3.89 (m, 6H), 2.33 (m, 2H), 2.19 (s, 3H) 2.05-1.01 (series of
multiplets, 26H), 1.47 (s, 9H), 1.45 (s, 18H), 0.92 (s, 3H), 0.80
(d, J=6.4 Hz, 3H), 0.72 (s, 3H). .sup.13C NMR (CDCl.sub.3, 75 MHz)
.delta. 170.01, 169.86, 169.69, 155.72, 155.55, 139.90, 129.05,
128.17, 126.88, 79.86, 76.53, 75.09, 72.09, 62, 35, 57.88, 47.78,
45.23, 43.12, 42.79, 42.16, 40.81, 37.94, 35.51, 34.69, 34.57,
34.36, 33.30, 31.31, 29.66, 28.80, 28.34, 27.22, 26.76, 25.61,
24.02, 22.83, 22.47, 17.93, 12.19; FAB-MS (thioglycerol matrix)
m/e: ([M+H].sup.+) 970 (100%), calcd. 970.
[0162] Compound 33: Compound 31 (0.39 g, 0.79 mmol) was dissolved
in CH.sub.2Cl.sub.2 (15 mL) and Boc-.beta.-alanine (0.60 g, 3.17
mmol), DCC (0.73 g, 3.56 mmol) and dimethylaminopyridine (DMAP)
(about 100 mg) were added. The mixture was stirred under N.sub.2
for 6 hours then filtered. After concentration and chromatography
(SiO.sub.2, 5% MeOH in CH.sub.2Cl.sub.2), the product was obtained
as a 0.58 g of a clear glass (72%). IR (neat) 3400, 2980, 1705,
1510 cm.sup.-1; .sup.1H NMR (CDCl.sub.3, 300 MHz) .delta. 7.27 (m,
5H), 5.12 (bs, 4H), 4.93 (bs, 1H), 4.71 (m, 1H), 3.40 (m, 12H),
2.59-2.48 (m, 6H), 2.28 (m, 2H), 2.17 (s, 3H), 2.05-1.01 (series of
multiplets, 26H), 1.40 (s, 27H), 0.90 (s, 3H), 0.77 (d, J=6.1 Hz,
3H), 0.70 (s, 3H). .sup.13C NMR (CDCl.sub.3, 75 MHz) .delta.
171.85, 171.50, 171.44, 155.73, 138.62, 129.02, 128.09, 126.87,
79.18, 75.53, 74.00, 70.91, 62.20, 57.67, 47.84, 44.99, 43.28,
41.98, 40.73, 37.67, 36.12, 34.94, 34.65, 34.47, 34.20, 33.29,
31.23, 29.57, 28.74, 28.31, 28.02, 27.86, 27.12, 26.73, 25.46,
24.86, 23.95, 22.77, 22.39, 17.91, 12.14; HRFAB-MS
(thioglycerol+Na.sup.+ matrix) m/e: ([M+H].sup.+) 1011.6619 (100%),
calcd. 1011.6634.
[0163] Compound 6: Compound 32 (0.15 g, 0.15 mmol) was stirred with
excess 4 N HCl in dioxane for 40 minutes. The dioxane and HCl were
removed in vacuo leaving 0.12 g of a clear glass (about 100%).
.sup.1H NMR (CD.sub.3OD, 300 MHz) .delta. 7.62 (bs, 2H), 7.48 (bs,
3H), 5.30 (bs, 1H), 5.11 (bs, 1H), 4.72 (bs (1H), 4.46 (m, 1H),
4.32 (m, 1H) 4.05-3.91 (m, 4H), 3.10 (m, 2H), 2.81 (s, 3H),
2.15-1.13 (series of multiplets, 25H), 1.00 (s, 3H), 0.91 (bs, 3H),
0.82 (s, 3H). .sup.13C NMR (CD.sub.3OD, 125 MHz) .delta. 166.86,
166.50, 131.09, 130.18, 129.17, 128.55, 76.60, 75.43, 72.61, 72.04,
70.40, 66.22, 60.07, 58.00, 57.90, 54.89, 54.76, 46.44, 44.64,
43.39, 42.22, 38.56, 36.78, 34.14, 33.92, 33.84, 31.82, 30.54,
29.67, 28.79, 27.96, 26.79, 26.00, 24.99, 23.14, 22.05, 21.82,
19.91, 17.27, 11.60; HRFAB-MS (thioglycerol+Na.sup.+ matrix) m/e:
([M-4 Cl-3 H].sup.+) 669.4576 (100%), calcd. 669.4591.
[0164] Compound 7: Compound 33 (0.20 g, 0.20 mmol) was stirred with
excess 4 N HCl in dioxane for 40 minutes. The dioxane and HCl were
removed in vacuo leaving 0.12 g of a clear glass (about 100%).
.sup.1H NMR (CD.sub.3OD, 500 MHz) .delta. 7.58 (bs, 2H), 7.49 (bs,
3H), 5.21 (bs, 1H), 5.02 (bs, 1H), 4.64 (m, 1H), 4.44 (m, 1H), 4.28
(m, 1H), 3.30-2.84 (m, 14H), 2.80 (s, 3H), 2.11-1.09 (series of
multiplets, 25H), 0.99 (s, 3H), 0.89 (d, Hz, 3H), 0.80 (s, 3H);
.sup.13C NMR (CD.sub.3 OD, 125 MHz) .delta. 171.92, 171.56, 171.49,
132.44, 131.32, 131.02, 130.51, 78.13, 76.61, 61.45, 57.94, 46.67,
44.80, 42.36, 40.85, 39.33, 37.03, 36.89, 36.12, 36.09, 35.79,
35.63, 33.81, 33.10, 32.92, 32.43, 30.28, 28.43, 28.04, 26.65,
24.02, 22.86, 21.98, 18.70, 12.68; HRFAB-MS (thioglycerol+Na.sup.+
matrix) m/e: ([M-4 Cl-3 H].sup.+) 711.5069 (43%), calcd.
711.5061.
Example 4
[0165] This example includes a description of one or more exemplary
synthestic procedures for obtaining Compounds 8, CSA-7, CSA-8 and
34-40.
[0166] Compound 34: Diisopropyl azodicarboxylate (DIAD) (1.20 mL,
6.08 mmol) was added to triphenylphosphine (1.60 g, 6.08 mmol) in
THF (100 mL) at 0.degree. C. and was stirred for half an hour
during which time the yellow solution became a paste. Compound 14
(2.58 g, 4.06 mmol) and p-nitrobenzoic acid (0.81 g, 4.87 mmol)
were dissolved in THF (50 mL) and added to the paste. The resulted
mixture was stirred at ambient temperature overnight. Water (100
mL) was added and the mixture was made slightly basic by adding
NaHCO.sub.3 solution followed by extraction with EtOAc (3.times.50
mL). The combined extracts were washed with brine once and dried
over anhydrous Na.sub.2 SO.sub.4. The desired product (232 g, 85%
yield) was obtained as white powder after SiO.sub.2 chromatography
(Et.sub.2O/hexanes 1:2). m.p. 207-209.degree. C.; IR (KBr) 3434,
3056, 2940, 2868, 1722, 1608, 1529, 1489, 1448, 1345 cm.sup.-1; NMR
(CDCl.sub.3, 300 MI-1z) .delta. 8.30-8.26 (m, 2H), 8.21-8.16 (m,
2H), 7.46-7.42 (m, 6H), 7.31-7.18 (m, 9H) 5.33 (bs, 1H), 4.02 (bs,
1H), 3.90 (bs, 1H), 3.09-2.97 (m, 2H), 2.68 (td, J=14.95, 2.56 Hz,
1H), 2.29-2.19 (m, 1H), 2.07-1.06 (series of multiplets, 24H), 1.01
(s, 3H), 0.98 (d, J=6.6 Hz, 3H), 0.70 (s, 3H); .sup.13C NMR
(CDCl.sub.3, 75 MHz) .delta. 164.21, 150.56, 144.70, 136.79,
130.77, 128.88, 127.86, 126.98, 123.70, 86.47, 73.24, 73.00, 68.70,
64.22, 47.79, 46.79, 42.15, 39.76, 37.47, 35.52, 35.34, 34.23,
33.79, 32.46, 31.12, 28.74, 27.71, 26.85, 26.30, 25.16, 23.41,
17.98, 12.77; HRFAB-MS (thioglycerol+Na.sup.+ matrix) m/e:
([M+Na].sup.+) 808.4203 (53.8%), calcd. 808.4189. Nitrobenzoate
(2.75 g, 3.5 mmol) was dissolved in CH.sub.2Cl.sub.2 (40 mL) and
MeOH (20 mL) and 20% aqueous NaOH (5 mL) were added. The mixture
was heated up to 60.degree. C. for 24 hours. Water (100 mL) was
introduced and extracted with EtOAc. The combined extracts were
washed with brine and dried over anhydrous Na.sub.2 SO.sub.4. The
desired product (1.89 g, 85% yield) was obtained as white solid
after SiO.sub.2 chromatography (3% MeOH in CH.sub.2 Cl.sub.2 as
eluent). m.p. 105-106.degree. C.; IR (KBr) 3429, 3057, 2936, 1596,
1489, 1447, 1376, 1265, 1034, 704 cm.sup.-1; NMR (CDCl.sub.3, 300
MHz) .delta. 7.46-7.42 (m, 6H), 7.32-7.19 (m, 9H), 4.06 (bs, 1H),
3.99 (bs, 1H), 3.86 (bd, J=2.44 Hz, 1H), 3.09-2.97 (m, 2H), 2.47
(td, J=14.03, 2.44 Hz, 1H), 2.20-2.11 (m, 1H), 2.04-1.04 (series of
multiplets, 25H), 0.97 (d, J=6.59 Hz, 3H), 0.94 (s, 3H), 0.68 (s,
3H); .sup.13C NMR (CDCl.sub.3, 75 MHz) .delta. 144.70, 128.88,
127.86, 126.97, 86.45, 73.31, 68.84, 67.10, 64.23, 47.71, 46.74,
42.10, 39.70, 36.73, 36.73, 36.15, 35.53, 35.45, 34.45, 32.46,
29.93, 28.67, 27.86, 27.71, 26.87, 26.04, 23.43, 23.16, 17.94,
12.75; HRFAB-MS (thioglycerol+Na.sup.+ matrix) m/e: ([M+Na].sup.+)
659.4064 (100%), calcd. 659.4076.
[0167] Compound 35: To a round-bottom flask were added 34 (2.0 g,
3.14 mmol), NaH (60% in mineral oil, 3.8 g, 31.4 mmol) and THF (150
mL). The suspension was refluxed for 2 hours followed by the
addition of allyl bromide (2.72 mL, 31.4 mL). After refluxing for
28 hours, another 10 eq. of Nail and allyl bromide were added.
After 72 hours, another 10 eq. of NaH and allyl bromide were added.
After 115 hours, TLC showed almost no starting material or
intermediates. Water (100 mL) was added to the suspension
carefully, followed by extraction with EtOAc (5.times.50 mL). The
combined extracts were washed with brine and dried over anhydrous
Na.sub.2SO.sub.4. The desired product (1.81 g, 79% yield) was
obtained as a yellowish glass after SiO.sub.2 chromatography (5%
EtOAc/hexanes). IR (neat) 3060, 3020, 2938, 2865, 1645, 1596, 1490,
1448, 1376, 1076, 705 cm.sup.-1; .sup.1H NMR (CDCl.sub.3, 300 MHz)
.delta. 7.46-7.42 (m, 6H), 7.31-7.18 (m, 9H), 6.06-5.85 (m, 3H),
5.35-5.20 (m, 3H), 5.15-5.06 (m, 3H), 4.10-4.00 (m, 2H), 3.93-3.90
(m, 2H), 3.85-3.79 (ddt, J=13.01, 4.88, 1.59 Hz, 1H), 3.73-3.66
(ddt, J=13.01, 5.38, 1.46 Hz, 1H), 3.58 (bs, 1H), 3.54 (bs, 1H),
3.32 (d, J=2.93 Hz, 1H), 3.07-2.96 (m, 2H), 2.36 (td, J=13.67, 2.68
Hz, 1H), 2.24-2.10 (m, 2H), 2.03-1.94 (m, 1H), 1.87-0.86 (series of
multiplets, 20H), 0.91 (s, 3H), 0.90 (d, J=6.83 Hz, 3H), 0.64 (s,
3H); .sup.13C NMR (CDCl.sub.3, 75 MHz) .delta. 144.77, 136.29,
136.21, 136.13, 128.90, 127.86, 126.94, 116.13, 115.51, 115.42,
86.44, 81.11, 75, 65, 73.92, 69.40, 68.81, 64.43, 46.68, 46.54,
42.93, 39.93, 36.98, 35.66, 35.14, 35.14, 32.83, 32.54, 30.48,
28.51, 27.72, 27.64, 26.82, 24.79, 23.65, 23.43, 23.40, 18.07,
12.80; HRFAB-MS (thioglycerol+Na.sup.+ matrix) m/e: ([M+H].sup.+)
757.5185 (12.9%), calcd. 757.5196.
[0168] Compound 36: Ozone was bubbled through a solution of 35
(0.551 g, 0.729 mmol) in CH.sub.2 Cl.sub.2 (40 mL) and MeOH (20 mL)
at -78.degree. C. until the solution turned a deep blue. Excess
ozone was blown off with oxygen. Methylsulfide (1 mL) was added
followed by the addition of NaBH.sub.4 (0.22 g, 5.80 mmol) in 5%
NaOH solution and methanol. The resulted mixture was stirred
overnight at room temperature and washed with brine. The brine was
then extracted with EtOAc (3.times.20 mL). The combined extracts
were dried over Na.sub.2SO.sub.4. The desired product (0.36 g, 65%
yield) was obtained as a colorless glass after SiO.sub.2
chromatography (5% MeOH/CH.sub.2 Cl.sub.2). IR (neat) 3396, 3056,
2927, 1596, 1492, 1462, 1448, 1379, 1347, 1264, 1071 cm.sup.-1;
.sup.1H NMR (CDCl.sub.3, 300 MHz) .delta. 7.46-7.42 (m, 6H),
7.32-7.18 (m, 9H), 3.77-3.57 (series of multiplets, 10H), 3.48-3.44
(m, 2H), 3.36-3.30 (m, 2H), 3.26-3.20 (m, 1H), 3.04-2.99 (m, 2H),
2.37-0.95 (series of multiplets, 27 H), 0.92 (s, 3H), 0.91 (d,
J=6.59 Hz, 3H), 0.67 (s, 3H); .sup.13C NMR (CDCl.sub.3, 75 MHz)
.delta. 144.69, 128.87, 127.84, 126.94, 86.44, 81.05, 76.86, 74.65,
69.91, 69.22, 68.77, 64.24, 62.44, 62.42, 62.26, 46.92, 46.54,
42.87, 39, 73, 36.86, 35.52, 35.13, 32.82, 32.54, 30.36, 28.71,
27.61, 27.44, 26.79, 24.82, 23.51, 23.38, 23.31, 18.28, 12.74;
HRFAB-MS (thioglycerol+Na.sup.+ matrix) ride: ([M+Na].sup.+)
791.4844 (96.4%), calcd. 791.4863.
[0169] Compound 37: NEt.sub.3 (0.23 mL, 1.66 mmol) was added to a
solution of 36 (0.364 g, 0.47 mmol) in dry CH.sub.2 Cl.sub.2 (30
mL) at 0.degree. C. under N.sub.2 followed by the introduction of
mesyl chloride (0.12 mL, 1.56 mmol). The mixture was stirred for 10
minutes and H.sub.2 O (10 mL) added to quench the reaction,
followed by extraction with EtOAc (3.times.30 mL). The combined
extracts were washed with brine and dried over anhydrous Na.sub.2
SO.sub.4. SiO.sub.2 chromatography (EtOAc/hexanes 1:1) gave the
desired product (0.411 g, 86% yield) as white glass. IR (neat)
3058, 3029, 2939, 2868, 1491, 1461, 1448, 1349, 1175, 1109, 1019
cm.sup.-1; .sup.1H NMR (CDCl.sub.3, 300 MHz) .delta. 7.46-7.42 (m,
6H), 7.31-7.19 (m, 9H), 4.35-4.26 (m, 6H), 3.84-3.74 (m, 2H),
3.64-3.56 (m, 4H), 3.49-3.34 (m, 3H), 3.06 (s, 3H), 3.04 (s, 3H),
3.02 (s, 3H), 3.09-2.95 (m, 2H), 2.28 (bt, J=14.89 Hz, 1H),
2.09-0.86 (series of multiplets, 21H), 0.92 (s, 3H), 0.90 (d,
J=6.78 Hz, 3H), 0.66 (s, 3H); .sup.13C NMR (CDCl.sub.3, 75 MHz)
.delta. 144.66, 128.86, 127.86, 126.97, 86.46, 81.28, 77.18, 75.00,
70.14, 69.89, 69.13, 66.49, 65.85, 65.72, 64.22, 47.06, 46.35,
42.77, 39.58, 37.81, 37.64, 37.55, 36.75, 35.48, 35.02, 32.59,
32.52, 30.27, 28.43, 27.56, 27.52, 26, 92, 24.62, 23.34, 23.25,
23.10, 18.24, 12.64; HRFAB-MS (thioglycerol+Na.sup.+ matrix) m/e:
([M+Na].sup.+) 1025.4207 (100%), calcd. 1025.4189.
[0170] Compound 38: The suspension of 37 (0.227 g, 0.227 mmol) and
NaN.sub.3 (0.147 g, 2.27 mmol) in dry DMSO (5 mL) was stirred at
80.degree. C. overnight, diluted with H.sub.2 O (50 mL) and
extracted with EtOAc (3.times.20 mL). The extracts were washed with
brine once and dried over anhydrous Na.sub.2 SO.sub.4. SiO.sub.2
chromatography (EtOAc/hexanes 1:8) afforded the desired product
(0.153 g, 80% yield) as a yellow oil. IR (neat) 2929, 2866, 2105,
1490, 1466, 1448, 1107, 705 cm.sup.-1; .sup.1H NMR (CDCl.sub.3, 300
MHz) .delta. 7.46-7.42 (m, 6H), 7.32-7.19 (m, 9H), 3.80-3.74 (m,
1H), 3.70-3.55 (series of multiplets, 5H), 3.41-3.19 (series of
multiplets, 9H), 3.04-2.98 (m, 2H), 2.41 (td, J=13.1, 2.44 Hz, 1H),
2.29-2.14 (m, 2 H), 2.04-0.86 (series of multiplets, 20H), 0.93 (s,
3H), 0.91 (d, J=6.60 Hz, 3H), 0.66 (s, 3H); .sup.13C NMR
(CDCl.sub.3, 75 MHz) .delta. 144.78, 128.93, 127.87, 126.96, 86.46,
81.30, 77.16, 75.21, 67.99, 67.44, 67.03, 64.41, 51.64, 51.57, 51,
33, 46.71, 46.30, 42.35, 39.75, 36.72, 35.64, 35.20, 32.52, 32.42,
30.17, 28.63, 27.80, 27.22, 26.90, 24.80, 23.55, 23.30, 23.24,
18.23, 12.65; HRFAB-MS (thioglycerol+Na.sup.+ matrix) m/e:
([M+Na].sup.+) 866.5049 (96.9%), calcd. 866.5057.
[0171] Compound 39: p-Toluenesulfonic acid (1.72 mg) was added into
the solution of 38 (0.153 g, 0.18 mmol) in CH.sub.2 Cl.sub.2 (5 mL)
and MeOH (5 mL), and the mixture was stirred for 2.5 hours.
Saturated NaHCO.sub.3 solution (5 mL) was introduced followed by
the introduction of brine (30 mL). The aqueous mixture was
extracted with EtOAc and the combined extracts washed with brine
and dried over Na.sub.2 SO.sub.4. The desired product (0.10 g, 92%
yield) was obtained as a pale yellowish oil after SiO.sub.2
chromatography (EtOAc/hexanes 1:3). IR (neat) 3426, 2926, 2104,
1467, 1441, 1347, 1107 cm.sup.-1; .sup.1H NMR (CDCl.sub.3, 300 MHz)
.delta. 3.81-3.74 (m, 1H), 3.71-3.54 (m, 7H), 3.41-3.19 (m, 9H),
2.41 (td, J=13.61, 2.32 Hz, 1H), 2.30-2.14 (m, 2H), 2.07-1.98 (m,
1H), 1.94-0.95 (series of multiplets, 21H), 0.95 (d, J=6.35 Hz,
3H), 0.93 (s, 3H), 0.69 (s, 3H); .sup.13C NMR (CDCl.sub.3, 75 MHz)
.delta. 81.22, 77.08, 75.13, 67.94, 67.36, 66, 97, 63.76, 51.59,
51.51, 51.26, 46.51, 46.24, 42.31, 39.68, 36.64, 35.58, 35.12,
32.34, 31.92, 30.11, 29.55, 28.54, 27.82, 27.16, 24, 75, 23.47,
23.23, 23.18, 18.15, 12.56; HRFAB-MS (thioglycerol+Na.sup.+ matrix)
m/e: ([M+Na].sup.+) 624.3966 (54.9%), calcd. 624.3962.
[0172] Compound 40: To a solution of 39 (0.10 g, 0.166 mmol) in
CH.sub.2Cl.sub.2 (8 mL) at 0.degree. C. was added NEt.sub.3 (34.8
.mu.L, 0.25 mmol) under N.sub.2 followed by the introduction of
mesyl chloride (15.5.mu.L, 0.199 mmol). The mixture was stirred 15
minutes. Addition of H.sub.2O (3 mL) and brine (20 mL) was followed
by extraction with EtOAc (4.times.10 mL). The combined extracts
were washed with brine once and dried over Na.sub.2 SO.sub.4. After
removal of solvent, the residue was mixed with N-benzylmethylamine
(0.5 mL) and heated to 80.degree. C. under N.sub.2 overnight.
Excess N-benzyl methylamine was removed in vacuo and the residue
was subjected to SiO.sub.2 chromatography (EtOAc/hexanes 1:4) to
give the product (0.109 g, 93% yield) as a yellow oil. IR (neat)
2936, 2784, 2103, 1467, 1442, 1346, 1302, 1106, 1027 cm.sup.-1;
.sup.1H NMR (CDCl.sub.3, 300 MHz) .delta. 7.32-7.23 (m, 5H),
3.81-3.74 (m, 1H), 3.71-3.55 (m, 5H), 3.47 (s, 2H), 3.41-3.19 (m,
9H), 2.46-2.11 (m, 5H), 2.18 (s, 3H), 2.03-0.85 (series of
multiplets, 20H), 0.93 (s, 3H), 0.93 (d, J=6.35 Hz, 3H,), 0.67 (s,
3H); .sup.13C NMR (CDCl.sub.3, 75 MHz) .delta. 139.54, 129.26,
128.32, 126.97, 81.26, 77.12, 75.17, 67.98, 67.42, 67.00, 62.50,
58.41, 51.61, 51.54, 51.29, 46.66, 46.28, 42.46, 42.32, 39.72,
36.68, 35.76, 35.16, 33.75, 32.38, 30.15, 28.59, 27.85, 27.19,
24.77, 24, 15, 23, 53, 23.28, 23.22, 18.28, 12.60; HRFAB-MS
(thioglycerol+Na.sup.+ matrix) m/e: ([M+H].sup.+) 705.4929 (100%),
calcd. 705.4928.
[0173] Compound 8: A suspension of 40 (0.109 g, 0.155 mmol) and
LiAlH.sub.4 (23.5 mg, 0.62 mmol) in THF (20 mL) was stirred under
N.sub.2 overnight. Na.sub.2 SO.sub.4.10H.sub.2 O was carefully
added and stirred until no grey color persisted. Anhydrous
Na.sub.2SO.sub.4 was added and the white precipitate was filtered
out and rinsed with dry THF. After removal of solvent, the residue
was dissolved in minimum CH.sub.2Cl.sub.2 and filtered. The desired
product (0.091 g, 94% yield) was obtained as a colorless oil after
the solvent was removed. IR (neat) 3371, 3290, 3027, 2938, 2862,
2785, 1586, 1493, 1453, 1377, 1347, 1098 cm.sup.-1; .sup.1H NMR
(CDCl.sub.3, 300 MHz) .delta. 7.31-7.21 (m, 5H), 3.65-3.53 (m, 4H),
3.47 (s, 2H), 3.42-3.34 (m, 2H), 3.30 (bs, 1H), 3.26-3.20 (m, 1H),
3.14-3.09 (m, 1H), 2.89-2.81 (m, 6H), 2.39-2.27 (m, 3H), 2.17 (s,
3H), 2.15-0.88 (series of multiplets, 29H), 0.93 (d, J=6.59 Hz,
3H), 0.92 (s, 3H), 0.67 (s, 3H); .sup.13C NMR (CDCl.sub.3, 75 MHz)
.delta. 139.34, 129.16, 128.24, 126.90, 80.75, 76.44, 74.29, 70.58,
69.88, 69.75, 62.47, 58.27, 46.66, 46.47, 42.75, 42.63, 42.51,
42.35, 39.77, 36.87, 35.73, 35.04, 33.77, 32.90, 30.38, 28.71,
27.70, 27.32, 24.89, 24.09, 23.53, 23.36, 23.25, 18.24, 12.62;
HRFAB-MS (thioglycerol+Na.sup.4 matrix) m/e: ([M+H].sup.+) 627.5199
(23.3%), calcd. 627.5213.
[0174] Compound CSA-7: To a solution of 23 (0.18 g, 0.28 mmol) in
dry DMF (4 mL) were added NaH (0.224 g, 60% in mineral oil, 5.60
mmol) and 1-bromo octane (0.48 mL, 2.80 mmol). The suspension was
stirred under N.sub.2 at 65.degree. C. overnight followed by the
introduction of H.sub.2 O (60 mL) and extraction with ether
(4.times.20 mL). The combined extracts were washed with brine and
dried over Na.sub.2 SO.sub.4. SiO.sub.2 chromatography (hexanes and
5% EtOAc in hexanes) afforded the desired product (0.169 g, 80%
yield) as a pale yellowish oil. IR (neat) 2927, 2865, 2099, 1478,
1462, 1451, 1350, 1264, 1105; .sup.1H NMR (CDCl.sub.3, 300 MHz)
.delta. 3.69-3.35 (series of multiplets, 15H), 3.26-3.02 (series of
multiplets, 4H), 2.19-2.02 (m, 3H), 1.97-1.16 (series of
multiplets, 37H), 1.12-0.99 (m, 2H), 0.92-0.86 (m, 9H), 0.65 (s,
3H); .sup.13C NMR (CDCl.sub.3, 75 MHz) .delta. 80.69, 79.84, 76.13,
71.57, 71.15, 65.07, 64.49, 64.39, 49.08, 48.99, 48.80, 46.68,
46.45, 42.72, 42.05, 39.88, 35.74, 35.49, 35.36, 35.14, 32.42,
32.03, 30.01, 29.85, 29.81, 29.76, 29.67, 29.48, 29.14, 27.92,
27.80, 27.70, 26.58, 26.42, 23.59, 23.09, 22.92, 22.86, 18.11,
14.31, 12.65; HRFAB-MS (thioglycerol+Na.sup.+ matrix) m/e:
([M+Na].sup.+) 778.5685 (22.1%), calcd. 778.5683. The triazide
(0.169 g, 0.224 mmol) and LiAlH.sub.4 (0.025 g, 0.67 mmol) were
suspended in anhydrous THF (10 mL) and stirred under N.sub.2 at
room temperature overnight followed by careful introduction of
Na.sub.2 SO.sub.4 hydrate. After the grey color disappeared,
anhydrous Na.sub.2 SO.sub.4 was added and stirred. The white
precipitate was removed by filtration and washed with THF. After
removal of solvent, the residue was dissolved in 1 M hydrochloric
acid and the aqueous solution was extracted with ether (5 mL) once.
The aqueous solution was then made basic by adding 20% aqueous NaOH
solution followed by extraction with Et.sub.2 O (4.times.5 mL). The
combined extracts were washed, dried and concentrated. The residue
was then subject to SiO.sub.2 chromatography (MeOH/CH.sub.2Cl.sub.2
(1:1) followed by MeOH/CH.sub.2Cl.sub.2/NH.sub.3. H.sub.2 O
(4:4:1)) to afford the desired product (0.091 g, 60% yield) as a
colorless oil. ER (neat) 3361, 2927, 2855, 1576, 1465, 1351, 1105
cm.sup.-1; .sup.1H NMR (CD.sub.3OD, 300 MHz) .delta. 4.86 (bs, 6H),
3.77-3.72 (m, 1 H), 3.70-3.61 (m, 1H), 3.57-3.53 (m, 3H), 3.43-3.38
(m, 4H), 3.34-3.27 (m, 2H), 3.18-3.10 (m, 2H), 2.84-2.71 (m, 6H),
2.22-2.07 (m, 3H), 2.00-1.02 (series of multiplets, 39H), 097-0.88
(m, 9H), 0.71 (s, 3H); .sup.13C NMR (CD.sub.3 OD, 75 MHz) .delta.
82.20, 81.00, 77.62, 72.52, 72.06, 68.00, 67.92, 67.39, 48.20,
47.53, 44.26, 43.40, 41.42, 41.15, 40.84, 40.35, 36.88, 36.73,
36.42, 36.11, 34.24, 34.05, 33.94, 33.67, 33.17, 30.95, 30.72,
30.62, 29.81, 29.35, 28.87, 28.79, 27.51, 24.57, 23.90, 23.83,
23.44, 18.76, 14.62, 13.07; HRFAB-MS (thioglycerol matrix) m/e:
([M+H].sup.+) 678.6133 (100%), calcd. 678.6149.
[0175] Compound CSA-8: A suspension of 23 (0.126 g, 0.196 mmol) and
LiAlH.sub.4 (0.037 g, 0.98 mmol) in THF (40 mL) was stirred at room
temperature under N.sub.2 overnight followed by careful addition of
Na.sub.2SO.sub.4.10H.sub.2O. After the grey color in the suspension
disappeared, anhydrous Na.sub.2SO.sub.4 was added and stirred until
organic layer became clear. The white precipitate was removed by
filtration and washed with twice THF. The THF was removed in vacuo,
and the residue was subject to SiO.sub.2 chromatography
(MeOH/CH.sub.2Cl.sub.2/NH.sub.3/H.sub.2O (4:4:1)) to afford the
desired product (0.066 g, 60% yield) as a colorless oil. IR (neat)
3365, 2933, 2865, 1651, 1471, 1455, 1339, 1103 cm.sup.-1; .sup.1H
NMR (CDCl.sub.3/30% CD.sub.3OD, 300 MHz) .delta. 4.43 (bs, 7H),
3.74-3.68 (m, 1H), 3.66-3.60 (m, 1H), 3.57-3.50 (m, 5H), 3.34-3.25
(M, 2H), 3.17-3.06 (M, 2H), 2.84-2.74 (M, 6H), 2.19-2.01 (M, 3H),
1.97-0.96 (series of multiplets, 27H), 0.94 (d, J=7.2 Hz, 3H), 0.92
(s, 3H), 0.69 (s, 3H); .sup.13C NMR (CDCl.sub.3, 75 MHz) .delta.
80.44, 79.27, 75.77, 66.59, 66.53, 65.86, 62, 51, 46.21, 45.84,
42.55, 41.53, 40.09, 39.43, 39.31, 39.02, 35.16, 34.93, 34, 86,
34.57, 32.93, 32.71, 31.57, 28.66, 28.33, 27.64, 27.22, 23.04,
22.40, 22.29, 17.60, 11.98; HRFAB-MS (thioglycerol+Na.sup.+ matrix)
m/e: ([M+H].sup.+) 566.4889 (8.9%), calcd. 566.4897.
Example 5
[0176] This example includes a description of one or more exemplary
synthestic procedures for obtaining Compounds CSA-11 and 43-47.
[0177] Compound 43: Precursor compound 41 was prepared following
the method reported by D. H. R. Barton, J. Wozniak, S. Z. Zard,
Tetrahedron, 1989, vol. 45, 3741-3754. A mixture of 41 (1.00 g,
2.10 mmol), ethylene glycol (3.52 mL, 63 mmol) and p-TsOH (20 mg,
0.105 mmol) was refluxed in benzene under N.sub.2 for 16 hours.
Water formed during the reaction was removed by a Dean-Stark
moisture trap. The cooled mixture was washed with NaHCO.sub.3
solution (50 mL) and extracted with Et.sub.2O (50 mL, 2.times.30
mL). The combined extracts were washed with brine and dried over
anhydrous Na.sub.2 SO.sub.4. Removal of the solvent gave the
product (1.09 g, 100%) as a white glass. IR (neat) 2939, 2876,
1735, 1447, 1377, 1247, 1074, 1057, 1039 cm.sup.-1; .sup.1H NMR
(CDCl.sub.3, 300 MHz) .delta. 5.10 (t, J=2.70 Hz, 1H), 4.92 (d,
J=2.69 Hz, 1H), 4.63-4.52 (m, 1H), 3.98-3.80 (m, 4H), 2.32 (t,
J=9.51 Hz, 1H), 2.13 (s, 3H), 2.08 (s, 3H), 2.05 (s, 3H), 2.00-1.40
(series of multiplets, 15H), 1.34-0.98 (m, 3H), 1.20 (s, 3H), 0.92
(s, 3H), 0.82 (s, 3H); .sup.13C NMR (CDCl.sub.3, 75 MHz)
.delta.170.69, 170.63, 170.47, 111.38, 75.07, 74.23, 70.85, 64.95,
63.43, 49.85, 44, 73, 43.39, 41.11, 37.37, 34.84, 34.80, 34.52,
31.42, 29.18, 27.02, 25.41, 24.16, 22.72, 22.57, 22.44, 21.73,
21.63, 13.40; HRFAB-MS (thioglycerol+Na.sup.+ matrix) m/e:
([M+H].sup.+) 521.3106 (38.6%), calcd. 521.3114. The triacetate
(1.09 g, 2.10 mmol) was dissolved in MeOH (50 mL). NaOH (0.84 g, 21
mmol) was added to the solution. The suspension was then refluxed
under N.sub.2 for 24 hours. MeOH was then removed in vacuo and the
residue was dissolved in Et.sub.2 O (100 mL) and washed with
H.sub.2O, brine, and then dried over anhydrous Na.sub.2 SO.sub.4.
The desired product (0.80 g, 96% yield) was obtained as white solid
after removal of solvent. m.p. 199-200.degree. C. IR (neat) 3396,
2932, 1462, 1446, 1371, 1265, 1078, 1055 cm.sup.-1; .sup.1H NMR
(10% CD.sub.3 OD in CDCl.sub.3, 300 MHz) .delta. 4.08-3.83 (series
of multiplets, 9H), 3.44-3.34 (m, 1H), 2.41 (t, J=9.28 Hz, 1H),
2.22-2.10 (m, 2 H), 1.96-1.50 (series of multiplets, 12H),
1.45-0.96 (series of multiplets, 4H), 1.32 (s, 3H), 0.89 (s, 3H),
0.78 (s, 3H); .sup.13C NMR (10% CD.sub.3OD in CDCl.sub.3, 75 MHz)
.delta. 112.11, 72.35, 71.57, 68.09, 64.54, 63.24, 49.36, 45.90,
41.48, 41.45, 39.18, 38.79, 35.29, 34.71, 34.45, 29.90, 27.26,
26.60, 23.65, 22.54, 22.44, 22.35, 13.46; HRFAB-MS
(thioglycerol+Na.sup.+ matrix) m/e: ([M+Na].sup.+) 417.2622
(87.3%), calcd. 417.2617.
[0178] Compound 44: To a round-bottom flask were added 43 (0.80 g,
2.03 mmol) and dry THF (100 mL) followed by the addition of NaH
(60% in mineral oil, 0.81 g, 20.3 mmol). The suspension was
refluxed under N.sub.2 for 30 minutes before the addition of allyl
bromide (1.75 mL, 20.3 mmol). After 48 hours of reflux, another 10
eq. of NaH and allyl bromide were added. After another 48 hours,
TLC showed no intermediates left. Cold water (50 mL) was added to
the cooled suspension. The resulted mixture was extracted with
Et.sub.2O (60 mL, 2.times.30 mL). The combined extracts were washed
with brine and dried over anhydrous Na.sub.2SO.sub.4. SiO.sub.2
column chromatography (6% EtOAc in hexanes) gave the desired
product (0.94 g, 90% yield) as a pale yellow oil. IR (neat) 3076,
2933, 2866, 1645, 1446, 1423, 1408, 1368, 1289, 1252, 1226, 1206,
1130, 1080, 1057 cm.sup.-1; .sup.1H NMR (CDCl.sub.3, 300 MHz)
.delta. 6.02-5.84 (m, 3H), 5.31-5.04 (m, 6H), 4.12-4.05 (m, 2H),
4.01-3.81 (m, 7H), 3.70 (dd, J=12.94, 5.62 Hz, 1H), 3.55 (t, J=2.56
Hz, 1H), 3.33 (d, J=2.93 Hz, 1H), 3.18-3.08 (m, 1H), 2.65 (t,
J=10.01 Hz, 1H), 2.32-2.14 (m, 3H), 1.84-1.45 (series of
multiplets, 10H), 1.41-1.22 (m, 3H), 1.27 (s, 3H), 1.14-0.92 (m,
2H), 0.89 (s, 3H), 0.75 (s, 3H); .sup.13C NMR (CDCl.sub.3, 75 MHz)
.delta. 136.38, 136.07, 136.00, 116.31, 115.54, 115.38, 112.34,
80.07, 79.22, 75.05, 69.83, 69.34, 68.82, 65.14, 63.24, 48.80,
45.96, 42.47, 42.15, 39.40, 35.55, 35, 16, 35.15, 29.04, 28.22,
27.52, 24.21, 23.38, 23.11, 22.95, 22.58, 13.79; HRFAB-MS
(thioglycerol+Na.sup.+ matrix) m/e: ([M+Na].sup.+) 537.3549 (100%),
calcd. 537.3556.
[0179] Compound 45: To the solution of 44 (0.94 g, 1.83 mmol) in
dry THF (50 mL) was added 9-BBN (0.5 M solution in THF, 14.7 mL,
7.34 mmol) and the mixture was stirred under N.sub.2 at room
temperature for 12 hours before the addition of 20% NaOH solution
(4 mL) and 30% H.sub.2 O.sub.2 solution (4 mL). The resulted
mixture was then refluxed for an hour followed by the addition of
brine (100 mL) and extracted with EtOAc (4.times.30 mL). The
combined extracts were dried over anhydrous Na.sub.2SO.sub.4. After
the removal of solvent, the residue was purified by SiO.sub.2
column chromatography (EtOAc followed by 10% MeOH in
CH.sub.2Cl.sub.2) to give the product (0.559 g, 54% yield) as a
colorless oil. IR (neat) 3410, 2933, 2872, 1471, 1446, 1367, 1252,
1086 cm.sup.-1; .sup.1H NMR (CDCl.sub.3, 300 MHz) .delta. 4.02-3.52
(series of multiplets, 17H), 3.41-3.35 (m, 1H), 3.29 (d, J=2.44 Hz,
1H), 3.22-3.15 (m, 3H), 2.58 (t, J=10.01 Hz, 1H), 2.27-1.95 (m,
3H), 1.83-1.48 (series of multiplets, 16H), 1.40-0.93 (series of
multiplets, 5H), 1.27 (s, 3H), 0.90 (s, 3H), 0.75 (s, 3H); .sup.13C
NMR (CDCl.sub.3, 75 MHz) .delta. 112.41, 80.09, 79.09, 76.31,
66.70, 66.02, 65.93, 64.80, 63.26, 61.53, 61.25, 60.86, 48.59,
45.80, 42.51, 41.72, 39.10, 35.36, 35.02, 34.98, 32.87, 32.52,
32.40, 28.88, 27.94, 27.21, 24.33, 23.02, 22.84 (2 C's), 22.44,
13.69; HRFAB-MS (thioglycerol+Na.sup.+ matrix) m/e: ([M+Na].sup.+)
591.3881 (100%), calcd. 591.3873.
[0180] Compound 46: To a solution of 45 (0.559 g, 0.98 mmol) in
acetone (40 mL) and water (4 mL) was added PPTS (0.124 g, 0.49
mmol) and the solution was refluxed under N.sub.2 for 16 hours. The
solvent was removed under reduced pressure. Water (40 mL) was then
added to the residue and the mixture was extracted with EtOAc (40
mL, 2.times.20 mL). The combined extracts were washed with brine,
dried and evaporated to dryness. SiO.sub.2 column chromatography
(8% MeOH in CH.sub.2Cl.sub.2) of the residue afforded the desired
product (0.509 g, 98% yield) as clear oil. IR (neat) 3382, 2941,
2876, 1699, 1449, 1366, 1099 cm.sup.+1; .sup.1H NMR (CDCl.sub.3,
300 MHz) .delta. 3.83-3.72 (m, 8H), 3.66 (t, J=5.62 Hz, 2H), 3.54
(bs, 2H), 3.43-3.28 (m, 4H), 3.24-3.12 (m, 2H), 2.26-2.00 (m, 4H),
2.08 (s, 3H), 1.98-1.50 (series of multiplets, 15H), 1.42-0.96
(series of multiplets, 6H), 0.90 (s, 3H), 0.62 (s, 3H); .sup.13C
NMR (CDCl.sub.3, 75 MHz) .delta. 210.49, 78.87 (2 C's), 76.30,
66.86, 66.18, 65.69, 61.74, 61.43, 60.71, 55.31, 48.05, 43.02,
41.58, 39.53, 35.28, 35.09, 34.96, 32.77, 32.70, 32.31, 31.12,
28.72, 27.88, 27.14, 23.47, 22.75, 22.47, 22.34, 13.86; HRFAB-MS
(thioglycerol+Na.sup.+ matrix) m/e: ([M+Na].sup.+) 547.3624 (100%),
calcd. 547.3611.
[0181] Compound 47: To a solution of 46 (0.18 g, 0.344 mmol) in dry
CH.sub.2Cl.sub.2 (10 mL) at 0.degree. C. was added Et.sub.3 N
(0.168 mL, 1.20 mmol) followed by the addition of mesyl chloride
(0.088 mL, 1.13 mmol). After 10 minutes, H.sub.2O (3 mL) and brine
(30 mL) were added. The mixture was extracted with EtOAc (30 mL,
2.times.10 mL) and the extracts were washed with brine and dried
over anhydrous Na.sub.2 SO.sub.4. After removal of solvent, the
residue was dissolved in DMSO (5 mL) and NaN.sub.3 (0.233 g, 3.44
mmol). The suspension was heated up to 50.degree. C. under N.sub.2
for 12 hours. H.sub.2 O (50 mL) was added to the cool suspension
and the mixture was extracted with EtOAc (30 mL, 2.times.10 mL) and
the extracts were washed with brine and dried over anhydrous
Na.sub.2 SO.sub.4. SiO.sub.2 column chromatography (EtOAc/hexanes
1:5) afforded the product (0.191 g, 88% yield for two steps) as a
pale yellow oil. IR (neat) 2933, 2872, 2096, 1702, 1451, 1363,
1263, 1102 cm.sup.-1; .sup.1H NMR (CDCl.sub.3, 300 MHz) .delta.
3.72-3.64 (m, 2H), 3.55-3.24 (series of multiplets, 11H), 3.18-3.02
(m, 2H), 2.22-2.02 (m, 4H), 2.08 (s, 3H), 1.95-1.46 (series of
multiplets, 15H), 1.38-0.96 (series of multiplets, 6H), 0.89 (s,
3H), 0.62 (s, 3H); .sup.13C NMR (CDCl.sub.3, 75 MHz) S 210.36,
79.69, 79.22, 75.98, 65.08, 64.80, 64.53, 55.31, 48.93, 48.86,
48.76, 48.06, 43.03, 41.91, 39.66, 35.44, 35.31, 35.12, 31.04,
29.77, 29.69, 29.67, 28.99, 28.10, 27.65, 23.60, 22.99, 22.95,
22.50, 14.00; HRFAB-MS (thioglycerol+Na.sup.+ matrix) m/e:
([M+Na].sup.+) 622.3820 (100%), calcd. 622.3805.
[0182] Compound CSA-11: Compound 47 (0.191 g, 0.319 mmol) was
dissolved in dry THF (20 mL) followed by the addition of
LiAlH.sub.4 (60.4 mg, 1.59 mmol). The grey suspension was stirred
under N.sub.2 at room temperature for 12 hours.
Na.sub.2SO.sub.4.10H.sub.2O powder was carefully added. After the
grey color in the suspension disappeared, anhydrous
Na.sub.2SO.sub.4 was added and the precipitate was filtered out.
After the removal of solvent, the residue was purified by column
chromatography (silica gel, MeOH/CH.sub.2Cl.sub.2/28%
NH.sub.3.H.sub.2 O 3:3:1). After most of the solvent was rotavapped
off from the fractions collected, 5% HCl solution (2 mL) was added
to dissolve the milky residue. The resulted clear solution was then
extracted with Et.sub.2O (2.times.10 mL). 20% NaOH solution was
then added until the solution became strongly basic.
CH.sub.2Cl.sub.2 (20 mL, 2.times.10 mL) was used to extract the
basic solution. The combined extracts were dried over anhydrous
Na.sub.2SO.sub.4 and removal of solvent gave the desired product
(0.115 g, 69% yield) as a colorless oil. From .sup.1H NMR it
appears that this compound was a mixture of two stereoisomers at
C20 with a ratio of approximately 9:1. The stereoisomers were not
separated, but used as recovered. Spectra for the most abundant
isomer: IR (neat) 3353, 2926, 2858, 1574, 1470, 1366, 1102
cm.sup.-1; .sup.1H NMR (20% CDCl.sub.3 in CD.sub.3 OD, 300 MHz)
.delta. 4.69 (bs, 7H), 3.76-3.69 (m, 1H), 3.63-3.53 (m, 5 H),
3.50-3.40 (m, 1H), 3.29 (bs, 1H), 3.18-3.07 (m, 2H), 2.94-2.83 (m,
1H), 2.81-2.66 (m, 5H), 2.23-2.06 (m, 4H), 1.87-1.50 (series of
multiplets, 15H), 1.39-0.96 (series of multiplets, 6H), 1.11 (d,
J=6.10 Hz, 3H), 0.93 (s, 3H), 0.75 (s, 3H); .sup.13C NMR (20%
CDCl.sub.3 in CD.sub.3 OD, 75 MHz) .delta. 81.46, 80.67, 77.32,
70.68, 67.90, 67.66, 67.18, 50.32, 47.17, 43.30, 43.06, 40.74,
40.64, 40.38, 40.26, 36.31, 36.28, 35.93, 34.30, 34.02, 33.29,
29.63, 29.31, 28.43, 26.10, 24.67, 24.09, 23.96, 23.50, 13.30 for
the major isomer; HRFAB-MS (thioglycerol+Na.sup.+ matrix) m/e:
([M+H].sup.+) 524.4431 (64.2%), calcd. 524.4427.
Example 6
[0183] This example includes a description of one or more exemplary
synthestic procedures for obtaining Compounds CSA-10 and 48-497
[0184] Compound 48: To a solution of 23 (0.15 g, 0.233 mmol) in dry
CH.sub.2 Cl.sub.2 (15 mL) at 0.degree. C. was added Et.sub.3 N
(48.8 .mu.L, 0.35 mmol) followed by the addition of
CH.sub.3SO.sub.2Cl (21.7 .mu.L, 0.28 mmol). The mixture was stirred
for 15 minutes before H.sub.2O (3 mL) was added. Saturated NaCl
solution (20 mL) was then added, and the mixture was extracted with
EtOAc (40 mL, 2.times.20 mL). The combined extracts were washed
with brine and dried over anhydrous Na.sub.2SO.sub.4. The solvent
was rotovapped off and to the residue were added NaBr (0.12 g, 1.17
mmol) and DMF (10 mL). The suspension was heated up to 80.degree.
C. under N.sub.2 for 2 hours. DMF was removed under vacuum and the
residue was chromatographed on silica (EtOAc/hexanes 1:10) to give
the desired product (0.191 g, 97% yield) as a pale yellow oil.
.sup.1H NMR (CDCl.sub.3, 300 MHz) .delta. 3.69-3.35 (series of
multiplets, 13H), 3.28-3.02 (series of multiplets, 4H), 2.18-2.04
(m, 3H), 2.00-1.60 (series of multiplets, 16H), 1.58-0.96 (series
of multiplets, 11H), 0.92 (d, J=6.34 Hz, 3H), 0.89 (s, 3H), 0.66
(s, 3H); .sup.13C NMR (CDCl.sub.3, 75 MHz) .delta. 80.62, 79.81,
76.08, 65.07, 64.50, 64.34, 49.03, 48.98, 48.79, 46.49, 46.46,
42.73, 42.02, 39.85, 35.47, 35.34, 35.12, 34.79, 34.72, 29.82,
29.80, 29.74, 29.11, 27.91, 27.78, 27.69, 23.55, 23.07, 22.88,
18.10, 12.62; HRFAB-MS (thioglycerol+Na.sup.+ matrix) m/e:
([M-H].sup.+) 706.3609 (63.1%), calcd. 706.3591; 704.3616 (52.8%),
calcd. 704.3611.
[0185] Compound 49: Compound 48 (0.191 g, 0.269 mmol) and 23 (0.295
g, 0.459 mmol) was dissolved in DMF (3 mL, distilled over BaO at 6
mm Hg before use) followed by the addition of NaH (0.054 g, 60% in
mineral oil). The suspension was stirred under N.sub.2 at room
temperature for 24 hours. H2 O (100 mL) was added to quench excess
NaH and the mixture was then extracted with Et.sub.2O (40 mL,
3.times.20 mL) and the combined extracts were washed with brine and
dried over anhydrous Na.sub.z SO.sub.4. The desired product (0.177
g, 52% yield based on compound 23) was obtained as a pale yellow
oil after SiO.sub.2 chromatography (EtOAc/hexanes 1:6, then 1:2).
IR (neat) 2940, 2862, 2095, 1472, 1456, 1362, 1263, 1113 cm.sup.-1;
.sup.1H NMR (CDCl.sub.3, 300 MHz) .delta. 3.68-3.35 (series of
multiplets, 26H), 3.28-3.02 (series of multiplets, 8H), 2.20-2.04
(m, 6H), 1.96-1.60 (series of multiplets, 30H), 1.52-0.98 (series
of multiplets, 12H), 0.91 (d, J=6.59 Hz, 6H), 0.89 (s, 6H), 0.65
(s, 6H); .sup.13C NMR (CDCl.sub.3, 75 MHz) .delta. 80.68, 79.83,
76.13, 71.71, 65.06, 64.48, 64.39, 49.08, 48.98, 48.80, 46.64,
46.44, 42.71, 42.04, 39.88, 35.73, 35.49, 35.36, 35.14, 32.41,
29.84, 29.81, 29.76, 29.14, 27.92, 27.78, 27.69, 26.58, 23.59,
23.08, 22.92, 18.12, 12.64.
[0186] Compound CSA-10: Compound 49 (0.219 g, 0.173 mmol) was
dissolved in dry THF (10 mL) followed by the addition of
LiAlH.sub.4 (65 mg, 1.73 mmol). The grey suspension was stirred
under N.sub.2 at room temperature for 12 hours.
Na.sub.2SO.sub.4.10H.sub.2O powder was carefully added. After the
grey color in the suspension disappeared, anhydrous
Na.sub.2SO.sub.4 was added and the precipitate was filtered out.
After the removal of solvent, the residue was purified by column
chromatography (silica gel, MeOH/CH.sub.2Cl.sub.2/28%
NH.sub.3.H.sub.2O 2.5:2.5:1). After most of the solvent was
rotavapped off from the fractions collected, 5% HCl solution (2 mL)
was added to dissolve the milky residue. The resulted clear
solution was then extracted with Et.sub.2O (2.times.10 mL). 20%
NaOH solution was then added until the solution became strongly
basic. CH.sub.2Cl.sub.2 (20 mL, 2.times.10 mL) was used to extract
the basic solution. The combined extracts were dried over anhydrous
Na.sub.2SO.sub.4 and removal of solvent gave the desired product
(0.147 g, 76% yield) as a white glass. 1R (neat) 3364, 3287, 2934,
2861, 1596, 1464, 1363, 1105 cm.sup.-1; .sup.1H NMR (20% CDCl.sub.3
in CD.sub.3OD, 500 MHz) .delta. 4.74 (bs, 12H), 3.75-3.70 (m, 2H),
3.65-3.61 (m, 2H), 3.57-3.52 (m, 6H), 3.40 (t, J=3.60 Hz, 4H), 3.30
(bs, 4H), 3.16-3.10 (m, 4H), 2.84-2.73 (m, 12H), 2.18-2.07 (m, 6H),
1.97-1.61 (series of multiplets, 30H), 1.58-0.98 (series of
multiplets, 24H), 0.95 (d, Hz, 6H), 0.94 (s, 6H), 0.70 (s, 6H);
.sup.13C NMR (20% CDCl.sub.3 in CD.sub.3OD, 125 MHz) .delta. 81.70,
80.52, 77.09, 72.34, 67.75 (2 C's), 67.07, 47.80, 47.13, 43.76,
42.87, 41.20, 40.65, 40.58, 40.14, 36.43, 36.25, 36.08, 35.77,
34.15, 33.87 (2 C's), 33.18, 29.55, 28.92, 28.47, 28.42, 27.25,
24.27, 23.54, 23.41, 18.70, 13.07; HRFAB-MS (thioglycerol+Na.sup.+
matrix) m/e: ([M+H].sup.+) 1113.9625 (68.8%), calcd. 1113.9610.
Example 7
[0187] This example includes a description of one or more exemplary
synthestic procedures for obtaining Compounds 111-113 and
116a-d.
[0188] Compounds 116a-d: Representative procedure: preparation of
116b. NaH (0.06 g, 60% in mineral oil, 1.49 mmol) and propyl
bromide (0.136 mL, 1.49 mmol) were added to a DMF solution of
compound 23 (described in Li et al., J. Am. Chem. Soc. 1998, 120,
2961) (0.096 g, 0.149 mmol). The suspension was stirred under
N.sub.2 for 24 hr. H.sub.2O (20 mL) was added, and the mixture was
extracted with hexanes (3.times.10 mL). The combined extracts were
dried over Na.sub.2SO.sub.4 and concentrated in vacuo. Silica gel
chromatography (10% EtOAc in hexanes) afforded the desired product
(92 mg, 90% yield) as a pale yellow oil. .sup.1H NMR (CDCl.sub.3,
500 MHz) .delta. 3.68-3.64 (m, 1H), 3.61-3.57 (m, 1H), 3.52 (t,
J=6.1 Hz, 2H), 3.49 (bs, 1H), 3.46-3.35 (m, 10H), 3.25 (d, J=2.4
Hz, 1H), 3.23-3.19 (m, 1H), 3.16-3.11 (m, 1H), 3.09-3.03 (m, 1H),
2.17-2.03 (m, 3H), 1.95-1.55 (m, 17H), 1.51-1.40 (m, 4H), 1.38-1.17
(m, 5H), 1.11-0.96 (m, 3H), 0.93-0.89 (m, 9H), 0.65 (s, 3H);
.sup.13C NMR (CDCl.sub.3, 75 MHz) .delta. 80.64, 79.79, 76.08,
72.67, 71.59, 65.01, 64.44, 64.33, 49.04, 48.94, 48.75, 46.61,
46.40, 42.68, 42.00, 39.83, 35.72, 35.45, 35.30, 35.10, 32.38,
29.81, 29.77, 29.72, 29.09, 27.88, 27.76, 27.65, 26.52, 23.55,
23.12, 23.04, 22.87, 18.06, 12.60, 10.79; HRFAB-MS
(thioglycerol+Na.sup.+ matrix) m/e ([M+Na].sup.+) 708.4910 (23.5%),
calcd. 708.4920.
[0189] Compounds 111, CSA-17, and 113: Representative procedure:
preparation of CSA-17. Compound 116b (0.092 g, 0.134 mmol) was
dissolved in THF (10 mL) followed by the addition of LiAlH.sub.4
(0.031 g, 0.81 mmol). The suspension was stirred under N2 for 12
hr. Na.sub.2SO.sub.4.10H.sub.2O (about 1 g) was then carefully
added. After the gray color in the suspension dissipated, anhydrous
Na.sub.2SO.sub.4 was added, and the precipitate was removed by
filtration. Concentration and silica gel chromatography
(CH.sub.2Cl.sub.2/MeOH/28% NH.sub.3.H.sub.2O 12:6:1, then 10:5:1)
yielded a glass which was dissolved in 1 M HCl (2 mL). The
resulting clear solution was washed with Et.sub.2O (2.times.10 mL).
20% NaOH solution was added to the aqueous phase until the solution
became strongly basic. CH.sub.2Cl.sub.2 (3.times.10 mL) was used to
extract the basic solution. The combined extracts were dried over
anhydrous Na.sub.2SO.sub.4 and concentrated in vacuo to give the
desired product (0.045 g, 55% yield) as a white glass. .sup.1H NMR
(about 20% CDCl.sub.3 in CD.sub.3OD, 500 MHz) .delta. 4.73 (bs,
6H), 3.74-3.70 (m, 1H), 3.65-3.61 (m, 1H), 3.55 (t, J=6.3 Hz, 2H),
3.42-3.38 (m, 4H), 3.33-3.30 (m, 2H), 3.16-3.10 (m, 2H), 2.83-2.73
(m, 6H), 2.18-2.06 (m, 3H), 1.96-1.20 (series of multiplets, 26H),
1.12-0.98 (m, 3H), 0.95-0.92 (m, 9H), 0.70 (s, 3H); .sup.13C NMR
(about 20% CDCl.sub.3 in CD.sub.3OD, 75 MHz) .delta. 81.67, 80.49,
77.04, 73.44, 72.28, 67.77, 67.71, 67.06, 47.74, 47.08, 43.75,
42.82, 41.21, 40.60, 40.56, 40.12, 36.47, 36.19, 36.04, 35.74,
34.09, 33.82, 33.78, 33.16, 29.49, 28.87, 28.43, 27.18, 24.22,
23.66, 23.49, 23.40, 18.64, 13.04, 11.03; HRFAB-MS
(thioglycerol+Na.sup.+ matrix) m/e: ([M+H].sup.+) 608.5348 (100%),
calcd. 608.5330. 111: .sup.1H NMR (about 20% CDCl.sub.3 in
CD.sub.3OD, 500 MHz) .delta. 4.79 (bs, 6H), 3.74-3.71 (m, 1H),
3.66-3.62 (m, 1H), 3.55 (t, J=6.1 Hz, 2H), 3.52 (bs, 1H), 3.38-3.28
(series of multiplets, 4H), 3.33 (s, 3H), 3.16-3.10 (m, 2H),
2.83-2.72 (m, 6H), 2.19-2.07 (m, 3H), 1.97-1.62 (series of
multiplets, 15H), 1.58-1.20 (series of multiplets, 9H), 1.13-0.98
(m, 3H), 0.95 (d, J=6.3 Hz, 3H), 0.93 (s, 3H), 0.70 (s, 3H);
.sup.13C NMR (about 20% CDCl.sub.3 in CD.sub.3OD, 75 MHz) .delta.
81.82, 80.65, 77.20, 74.43, 67.85, 67.18, 58.90, 47.80, 47.22,
43.91, 43.01, 41.31, 40.78, 40.69, 40.22, 36.63, 36.35, 36.18,
35.86, 34.27, 33.97, 33, 26, 29.60, 29.03, 28.58, 28.53, 27.14,
24.33, 23.61, 23.45, 18.68, 13.06; HRFAB-MS (thioglycerol+Na.sup.+
matrix) rule ([M+Na].sup.4) 602.4855 (100%), calcd. 602.4873. 113:
.sup.1H NMR (about 50% CDCl.sub.3 in CD.sub.3OD, 500 MHz) .delta.
4.08 (bs, 6H), 3.71-3.67 (m, 1H), 3.62-3.58 (m, 1H), 3.53 (t, Hz,
2H), 3.49 (bs, 1H), 3.43-3.38 (m, 4H), 3.31-3.27 (m, 2H), 3.14-3.07
(m, 2H), 2.83-2.73 (m, 6H), 2.16-2.03 (m, 3H), 1.93-1.17 (series of
multiplets, 30H), 1.10-0.96 (m, 3H), 0.93-0.89 (m, 9H), 0.67 (s,
3H); .sup.13C NMR (about 50% CDCl.sub.3 in CD.sub.3OD, 75 MHz)
.delta. 80.51, 79.35, 75.85, 71.29, 70.83, 66.73, 66.62, 65.96,
46.68, 45.98, 42.59, 41.63, 40.20, 39.53, 39.43, 39.21, 35.34,
35.04, 35.00, 34.71, 33.11, 32.90, 32.82, 32.00, 29, 15, 28.49,
28.15, 27.75, 27.35, 26.22, 23.18, 22.60, 22.45, 22.34, 17.77,
13.75, 12.22; HRFAB-MS (thioglycerol+Na.sup.+ matrix) m/e
([M+H].sup.+) 636.5679 (100%), calcd. 636.5669.
Example 8
[0190] This example includes a description of one or more exemplary
synthestic procedures for obtaining Compounds 106 and 124.
[0191] Compound 124: Compound 47 (0.256 g, 0.489 mmol) was
dissolved in CH.sub.2Cl.sub.2 (10 mL), and cooled to 0.degree. C.
followed by the addition of Na.sub.2HPO.sub.4 (0.69 g, 4.89 mmol)
and urea-hydrogen peroxide complex (UHP) (0.069 g, 0.733 mmol).
Trifluoroacetic anhydride (TFAA) (0.138 mL, 0.977 mmol) was then
added dropwise. The suspension was stirred for 12 hr, and
additional UHP (23 mg, 0.25 mmol) and TFAA (0.069 mL, 0.49 mmol)
were added. After another 12 hr, H.sub.2O (30 mL) was added, and
the resulting mixture was extracted with EtOAc (3.times.20 mL). The
combined extracts were washed with brine (50 mL), dried over
anhydrous Na.sub.2SO.sub.4, and concentrated in vacuo. SiO.sub.2
chromatography (EtOAc/hexanes 1:5) afforded the desired product
(0.145 g, 55% yield) as a colorless oil. .sup.1H NMR (CDCl.sub.3,
300 MHz) .delta. 5.21 (dd, J=9.3 and 7.3 Hz, 1H), 3.70-3.57 (m,
2H), 3.55 (t, J=6.0 Hz, 2H), 3.43-3.37 (m, 6H), 3.32-3.25 (m, 3H),
3.17-3.02 (m, 2H), 2.28-2.05 (m, 4H), 2.03 (s, 3H), 1.86-1.19
(series of multiples, 19H), 0.97 (dd, J=14.5 and 3.3 Hz, 1H), 0.90
(s, 3H), 0.78 (s, 3H); .sup.13C NMR (CDCl.sub.3, 75 MHz) .delta.
171.08, 79.71, 78.03, 75.72, 75.53, 65.41, 65.04, 64.53, 48.79,
48.70, 46.49, 41.92, 39.44, 37.81, 35.45, 35.22, 35.10, 29.73,
29.63, 28.89, 28.33, 27.50, 27.34, 23.39, 22.97, 22.92, 21.28,
12.72; HRFAB-MS (thioglycerol+Na.sup.+ matrix) m/e: ([M-H].sup.+)
614.3798 (24.5%), calcd. 614.3778.
[0192] Compound 106: Compound 124 (0.145 g, 0.236 mmol) was
dissolved in CH.sub.2Cl.sub.2 (2 mL) and MeOH (1 mL). 20% NaOH
solution (0.2 mL) was added. The mixture was stirred for 12 hr, and
anhydrous Na.sub.2SO.sub.4 was used to remove water. After
concentration in vacuo, the residue was purified by silica gel
chromatography (EtOAc/hexanes 1:3) to afford the desired product
(0.124 g, 92% yield) as a colorless oil. .sup.1H NMR (CDCl.sub.3,
300 MHz) .delta. 4.29 (bs, 1H), 3.69-3.60 (m, 2H), 3.52 (t, J=6.0
Hz, 2H), 3.45-3.32 (m, 8H), 3.26 (d, J=2.7 Hz, 1H), 3.17-3.02 (m,
2H), 2.19-1.94 (m, 4H), 1.90-1.62 (series of multiplets, 13H),
1.57-1.20 (series of multiplets, 7H), 0.97 (dd, J=14.3 and 3.1 Hz,
1H), 0.90 (s, 3H), 0.73 (s, 3H); .sup.13C NMR (CDCl.sub.3, 75 MHz)
.delta. 79.69, 78.03, 75.47, 73.38, 65, 46, 65.00, 64.47, 48.87,
48.68, 46.83, 41.93, 39.71, 37.87, 35.43, 35.20, 35.09, 29.96,
29.69, 29.59, 29.53, 28.89, 28.44, 27.48, 23.72, 22.91, 22.71,
11.77. The alcohol (0.124 g, 0.216 mmol) was dissolved in dry THF
(20 mL) followed by the addition of LiAlH.sub.4 (33 mg, 0.866
mmol). The gray suspension was stirred under N.sub.2 for 12 hr.
Na.sub.2SO.sub.4.10 H.sub.2O (about 2 g) was carefully added. After
the gray color in the suspension dissipated, anhydrous
Na.sub.2SO.sub.4 was added and the precipitate was removed by
filtration. After the removal of solvent, the residue was purified
by column chromatography (SiO.sub.2, MeOH/CH.sub.2Cl.sub.2/28%
NH.sub.3.H.sub.2O 2.5:2.5:1). After concentration of the relevant
fractions, 1 M HCl (2 mL) was added to dissolve the milky residue.
The resulting clear solution was washed with Et.sub.2O (2.times.10
mL). To the aqueous phase, 20% NaOH solution was added until the
solution became strongly basic. CH.sub.2Cl.sub.2 (20 mL, 2.times.10
mL) was used to extract the basic solution. The combined extracts
were dried over anhydrous Na.sub.2SO.sub.4 and removal of solvent
gave the desired product (0.050 g, 47% yield) as a colorless oil.
.sup.1H NMR (20% CDCl.sub.3 in CD.sub.3OD, 300 MHz) .delta. 4.77
(s, 7H), 4.25 (t, J=8.5 Hz, 1H), 3.75-3.68 (m, 1H), 3.66-3.58 (m,
1H). 3.55 (t, J=6.1 Hz, 2H), 3.48-3.41 (m, 1H), 3.34 (bs, 1H), 3.30
(d, J=3.6 Hz, 1H), 3.17-3.08 (m, 2H), 2.86-2.70 (m, 6H), 2.20-1.91
(m, 4H), 1.88-1.16 (series of multiplets, 19H), 1.00 (dd, J=-14.2
and 3.0 Hz, 1H), 0.93 (s, 3H), 0.73 (s, 3H); .sup.13C NMR (20%
CDCl.sub.3 in CD.sub.3OD, 75 MHz) .delta. 80.62, 79.12, 76.74,
73.77, 68.50, 67.79, 67.17, 47.69, 43.04, 40.76, 40.64, 40.62,
40.22, 39.01, 36.32, 36.25, 35.94, 34.27, 33, 97, 33.72, 30.13,
29.53, 28.43, 24.48, 23.58, 23.40, 12.38; HRFAB-MS
(thioglycerol+Na.sup.+ matrix) m/e: ([M+H].sup.+) 496.4108 (100%),
calcd. 496.4114.
Example 9
[0193] This example includes a description of one or more exemplary
synthestic procedures for obtaining Compounds 109 and 126-129.
[0194] Compound 126: Compound 125 (2.30 g, 3.52 mmol) was dissolved
in MeOH (50 mL) and CH.sub.2Cl.sub.2 (100 mL). A small amount of
Et.sub.3N was added, and the solution was cooled to -78.degree. C.
Ozone was bubbled through the solution until a blue color
persisted. Me.sub.2S (4 mL) was introduced followed by the addition
of NaBH.sub.4 (0.266 g, 0.703 mmol) in MeOH (10 mL). The resulting
solution was allowed to warm and stir overnight. The solution was
concentrated in vacuo, and brine (60 mL) was added. The mixture was
extracted with EtOAc (40 ml, 2.times.30 mL), and the combined
extracts were washed with brine and dried over anhydrous
Na.sub.2SO.sub.4. Silica gel chromatography (EtOAc) afforded the
product (1.24 g, 76% yield) as a white solid. m.p. 219-220 C.;
.sup.1H NMR (CDCl.sub.3, 300 MHz) .delta. 5.10 (t, J=2.8 Hz, 1H),
4.90 (d, J=2.7 Hz, 1H), 3.73-3.59 (m, 2H), 3.56-3.44 (m, 1H), 2.13
(s, 3H), 2.09 (s, 3H), 2.07-0.95 (series of multiplets, 23H), 0.91
(s, 3H), 0.83 (d, J=6.3 Hz, 3H), 0.74 (s, 3H); .sup.13C NMR
(CDCl.sub.3, 75 MHz) .delta. 170.84, 170.82, 75.63, 71.77, 71.03,
60.73, 48.10, 45.26, 43.54, 41.16, 38.78, 37.89, 35.00, 34.43,
32.26, 31.50, 30.60, 29.07, 27.50, 25.70, 22.96, 22.71, 21.81,
21.63, 18.18, 12.35; HRFAB-MS (thioglycerol+Na.sup.+ matrix) m/e:
([M+H].sup.+) 465.3197 (20%), calcd. 465.3216.
[0195] Compound 127: Compound 126 (1.24 g, 2.67 mmol) was dissolved
in MeOH (30 mL), and NaOH (0.54 g, 13.4 mmol) was added. The
suspension was refluxed under N.sub.2 for 24 hr. The MeOH was
removed in vacuo followed by the addition of H.sub.2O (50 mL). The
precipitate was filtered, washed with H.sub.2O and then dried in
vacuo to give a white solid (1.02 g). This solid was dissolved in
DMF (40 mL) followed by the sequential addition of NEt.sub.3 (1.12
mL, 8.02 mmol), DMAP (16.3 mg, 0.13 mmol) and trityl chloride (1.49
g, 5.34 mmol). The suspension was stirred under N.sub.2 for 12 hr
and then heated up to 50.degree. C. for 24 hr. H.sub.2O (100 mL)
was added to the cooled suspension, and the mixture was extracted
with EtOAc (3.times.50 mL). The combined extracts were washed with
brine (100 mL), dried over anhydrous Na.sub.2SO.sub.4, and
concentrated in vacuo. Silica gel chromatography (EtOAc) afforded
the product (1.20 g, 72% yield) as a pale yellow glass. To this
glass was added dry THF (80 mL) and NaH (60% in mineral oil, 0.77
g, 19.3 mmol). The suspension was refluxed under N.sub.2 for half
an hour before the introduction of allylbromide (1.67 mL, 19.3
mmol). After 48 hr at reflux, another 10 eq. of Nail and
allylbromide were introduced. After another 48 hr, the reaction
mixture was cooled and H.sub.2O (100 mL) was slowly added. The
resulting mixture was extracted with hexanes (3.times.50 mL), and
the combined extracts were washed with brine (100 mL) and dried
over anhydrous Na.sub.2SO.sub.4. Silica gel chromatography (5%
EtOAc in hexanes) afforded the product (1.27 g, 64% yield for all
three steps) as a clear glass. .sup.1H NMR (CDCl.sub.3, 300 MHz)
.delta. 7.46-7.43 (m, 6H), 7.29-7.16 (m, 9H), 5.98-5.81 (m, 3H),
5.29-5.18 (m, 3H), 5.14-5.03 (m, 3H), 4.11-3.97 (m, 4H), 3.75-3.67
(m, 2H), 3.49 (bs, 1H), 3.32-3.13 (d, J=2.4 Hz, 1H), 3.20-3.13 (m,
2H), 3.00 (m, 1H), 2.33-2.12 (m, 3H), 2.03-0.92 (series of
multiplets, 19H), 0.88 (s, 3H), 0.78 (d, J=6.6 Hz, 3H), 0.65 (s,
3H); .sup.13C NMR (CDCl.sub.3, 75 MHz) .delta. 144.71, 136.08,
136.04, 135.94, 128.80, 127.76, 126.86, 116.30, 115.57, 86.53,
80.77, 79.20, 74.96, 69.42, 69.34, 68.81, 62.00, 46.87, 46.48,
42.67, 42.11, 39.90, 36.15, 35.50, 35.14, 35.10, 33.23, 28.99,
28.09, 27.75, 27.56, 23.36, 23.32, 23.12, 18.24, 12.66; HRFAB-MS
(thioglycerol+Na.sup.+ matrix) m/e: ([M+Na].sup.+) 765.4875 (100%),
calcd. 765.4859.
[0196] Compound 128: To a THF (40 mL) solution of 127 (1.27 g, 1.71
mmol) was added 9-BBN (0.5 M solution in THF, 17.1 mL). The mixture
was stirred for 12 hr before the addition of NaOH (20% solution, 10
mL) and H.sub.2O.sub.2 (30% solution, 10 mL). The resulted mixture
was refluxed for 1 hr followed by the addition of brine (100 mL)
and extraction with EtOAc (4.times.30 mL). The combined extracts
were dried over anhydrous Na.sub.2SO.sub.4 and concentrated in
vacuo. Silica gel chromatography (5% MeOH in CH.sub.2Cl.sub.2)
afforded the product (1.26 g, 93% yield) as a clear glass. .sup.1H
NMR (5% CD.sub.3OD in CDCl.sub.3, 300 MHz) .quadrature. 7.46-7.43
(m, 6H), 7.32-7.20 (m, 9H), 3.94 (s, 3H), 3.78-3.56 (m, 10H), 3.48
(bs, 1H), 3.32-3.26 (m, 2H), 3.24-3.12 (m, 3H), 3.00 (dd, J=8.2 and
6.1 Hz, 1H), 2.23-1.96 (m, 3H), 1.90-0.95 (series of multiplets,
25H), 0.90 (s, 3H), 0.77 (d, J=6.6 Hz, 3H), 0.66 (s, 3H); .sup.13C
NMR (5% CD.sub.3OD in CDCl.sub.3, 75 MHz) .delta. 144.52, 128.64,
127.64, 126.76, 86.43, 80.55, 79.31, 77.65, 77.23, 76.80, 76.06,
66.17, 66.01, 65.41, 61.93, 61, 20, 60.73, 60.39, 47.29, 46.08,
42.65, 41.62, 39.49, 36.02, 35.10, 34.89, 34.77, 32.89, 32.71,
32.41, 32.26, 28.68, 27.70, 27.51, 27.19, 23.26, 22.66, 22.50,
18.23, 12.34; HRFAB-MS (thioglycerol+Na.sup.+ matrix) m/e:
([M+Na].sup.+) 819.5169 (100%), calcd. 819.5099.
[0197] Compound 129: To a CH.sub.2Cl.sub.2 (50 mL) solution of
compound 128 (1.26 g, 1.58 mmol) at 0.degree. C. was added
Et.sub.3N (0.92 mL, 6.60 mmol) followed by mesyl chloride (0.47 mL,
6.05 mmol). After 15 minutes, H.sub.2O (10 mL) was followed by
brine (80 mL). The mixture was extracted with EtOAc (60 mL,
2.times.30 mL) and the combined extracts were dried over anhydrous
Na.sub.2SO.sub.4. After removal of solvent in vacuo, the residue
was dissolved in DMSO (10 mL) and NaN.sub.3 (1.192 g, 18.3 mmol)
was added. The suspension was heated to 60.degree. C. under N.sub.2
overnight. H.sub.2O (100 mL) was added, and the mixture was
extracted with EtOAc (3.times.40 mL). The combined extracts were
washed with brine and dried over anhydrous Na.sub.2SO.sub.4.
Removal of the solvent in vacuo afforded a pale yellow oil. The oil
was dissolved in MeOH (10 mL) and CH.sub.2Cl.sub.2 (20 mL) and TsOH
(17.4 mg, 0.092 mmol) was added. After 12 hr, saturated aqueous
NaHCO.sub.3 (20 mL) and brine (50 mL) were added and the mixture
was extracted with EtOAc (3.times.40 mL). The combined extracts
were washed with brine (50 mL) and dried over anhydrous
Na.sub.2SO.sub.4. Silica gel chromatography (EtOAc/hexanes 1:3)
afforded the desired product (0.934, 94%) as a pale yellow oil.
.sup.1H NMR (CDCl.sub.3, 500 MHz) .delta. 3.75-3.70 (m, 1H),
3.68-3.63 (m, 2H), 3.62-3.57 (m, 1H), 3.53 (t, J=6.1 Hz, 2H), 3.50
(bs, 1H), 3.46-3.38 (m, 6H), 3.26 (d, J=2.4 Hz, 1H), 3.24-3.20 (m,
1H), 3.16-3.12 (m, 1H), 3.10-3.04 (m, 1H), 2.17-2.04 (m, 3H),
1.96-1.63 (m, 14H), 1.53-1.45 (m, 3H), 1.35-1.20 (m, 7H), 1.08-1.00
(m, 1H), 0.97-0.88 (m, 1H), 0.94 (d, J=6.8 Hz, 3H), 0.89 (s, 3H),
0.67 (s, 3H); .sup.13C NMR (CDCl.sub.3, 75 MHz) .delta. 80.64,
79.81, 76.06, 65.05, 64.49, 64.34, 61.03, 49.02, 48.98, 48.78,
46.93, 46.53, 42.76, 42.01, 39.83, 39.14, 35.46, 35.33, 35.12,
32.97, 29.79, 29.73, 29.10, 27.90, 27.68, 23.56, 23.06, 22.88,
18.24, 12.60; HRFAB-MS (thioglycerol+Na.sup.+ matrix) m/e:
([M+Na].sup.+) 652.4285 (100%), calcd. 652.4295.
[0198] Compound 109: Compound 129 (0.245 g, 0.391 mmol) was
dissolved in THF (30 mL) followed by the addition of LiAlH.sub.4
(59 mg, 1.56 mmol). The gray suspension was stirred under N.sub.2
12 hr. Na.sub.2SO.sub.4.10H.sub.2O powder (about 1 g) was carefully
added. After the gray color in the suspension dissipated, anhydrous
Na.sub.2SO.sub.4 was added and the precipitate was removed by
filtration. After the removal of solvent, the residue was purified
by silica gel chromatography (CH.sub.2Cl.sub.2/MeOH/28%
NH.sub.3.H.sub.2O 10:5:1 then 10:5:1.5). The solvent was removed
from relevant fractions, and 1 M HCl (4 mL) was added to dissolve
the residue. The resulting clear solution was extracted with
Et.sub.2O (3.times.10 mL). 20% NaOH solution was added until the
solution became strongly basic. CH.sub.2Cl.sub.2 (4.times.10 mL)
was used to extract the basic solution. The combined extracts were
dried over anhydrous Na.sub.2SO.sub.4, and removal of solvent in
vacuo gave the desired product (0.15 g, 71% yield) as a colorless
oil. .sup.1H NMR (about 20% CD.sub.3OD in CDCl.sub.3, 500 MHz)
.delta. 4.73 (bs, 7H), 3.74-3.70 (m, 1H), 3.65-3.60 (m, 2H),
3.56-3.52 (m, 4H), 3.31-3.28 (m, 2H), 3.16-3.09 (m, 2H), 2.82-2.71
(m, 6H), 2.19-2.06 (m, 3H), 1.97-1.66 (series of multiplets, 15H),
1.58-1.48 (m, 3H), 1.38-0.98 (m, 7H), 0.96 (d, J=6.8 Hz, 3H), 0.93
(s, 3H), 0.71 (s, 3H); .sup.13C NMR (about 20% CD.sub.3OD in
CDCl.sub.3, 75 MHz) .delta. 81.80, 80.60, 77.17, 67.88, 67.86,
67.18, 60.73, 48, 11, 47.28, 43.93, 42.99, 41, 34, 40.76, 40.72,
40.24, 39.70, 36.33, 36.18, 35.86, 34.29, 33.99, 33.96, 33.83,
29.60, 29.00, 28.57, 28.54, 24.33, 23.59, 23.48, 18.86, 13.04;
HRFAB-MS (thioglycerol+Na.sup.+ matrix) m/e: ([M+H].sup.+) 552.4756
(100%), calcd. 552.4772.
Example 10
[0199] This example includes a description of one or more exemplary
synthestic procedures for obtaining Compounds 108 and 130.
[0200] Compound 130: o-NO.sub.2C6H.sub.4SeCN (0.094 g, 0.21 mmol)
and Bu.sub.3P (0.095 mL, 0.38 mmol) were stirred in dry THF (5 mL)
at 0.degree. C. for 1/2 hr followed by the addition of compound 129
(0.10 g, 0.159 mmol) in THF (2 mL). The suspension was stirred for
1 hr followed by the addition of H.sub.2O.sub.2 (30% aqueous
solution, 2 mL). The mixture was stirred for 12 hr followed by
extraction with hexanes (4.times.10 mL). The combined extracts were
dried over anhydrous Na.sub.2SO.sub.4. The desired product (0.035
g, 36% yield) was obtained as pale yellowish oil after silical gel
chromatography (10% EtOAc/hexanes). .sup.1H NMR CDCl.sub.3, 500
MHz) .delta. 5.73-5.66 (ddd, J=17.1, 10.2, 8.3 Hz, 1H), 4.90 (dd,
J=17.1, 2.0 Hz, 1H), 4.82 (dd, J=10.2 Hz, 1.96 Hz, 1H), 3.68-3.64
(m, 1H), 3.62-3.58 (m, 1H), 3.54-3.26 (m, 9H), 3.25-3.22 (m, 2H),
3.15-3.11 (m, 1H), 3.10-3.04 (m, 1H), 2.17-1.62 (series of
multiplets, 18H), 1.51-1.43 (m, 2H), 1.35-1.18 (m, 4H), 1.06-0.91
(m, 2H), 1.02 (d, J=6.3 Hz, 3H), 0.90 (s, 3H), 0.68 (s, 3H);
.sup.13C NMR (CDCl.sub.3, 75 MHz) .delta. 145.50, 111.72, 80.60,
79.82, 76.09, 65.06, 64.50, 64.45, 49.05, 48.97, 48.79, 46.43,
46.13, 42.76, 42.03, 41.30, 39.84, 35.49, 35.34, 35.15, 29.82,
29.80, 29.75, 29.11, 28.00, 27.84, 27.68, 23.56, 23.08, 22.95,
19.79, 12.87; HRFAB-MS (thioglycerol+Na.sup.+ matrix) m/e:
([M+Na].sup.+) 634.4167 (90.6%), calcd. 634.4169.
[0201] Compound 108: Compound 130 (0.105 g, 0.172 mmol) was
dissolved in CH.sub.2Cl.sub.2 (5 mL) and MeOH (5 mL) at -78.degree.
C. O.sub.3 was bubbled into the solution for ca. 20 min. Me.sub.2S
(1 mL) was added followed, and the solvent was removed in vacuo.
The residue was dissolved in THF (15 mL), and LiAlH.sub.4 (0.033 g,
0.86 mmol) was added. The suspension was stirred for 12 hr.
Na.sub.2SO.sub.4.10H.sub.2O (about 2 g) was carefully added. After
the gray color of the suspension dissipated, anhydrous
Na.sub.2SO.sub.4 was added and the precipitate was removed by
filtration. Concentration and silica gel chromatography
(CH.sub.2Cl.sub.2/MeOH/28% NH.sub.3.H.sub.2O 10:5:1.5 then 9:6:1.8)
yielded a white glass. To this material was added 1 M HCl (4 mL).
The resulting clear solution was washed with Et.sub.2O (3.times.10
mL). 20% NaOH solution was added to the aqueous phase until the
solution became strongly basic. CH.sub.2Cl.sub.2 (4.times.10 mL)
was used to extract the basic solution. The combined extracts were
dried over anhydrous Na.sub.2SO.sub.4 and removal of solvent gave
the desired product (0.063 g, 68% yield) as a colorless oil.
.sup.1H NMR (about 10% CD.sub.3OD in CDCl.sub.3, 500 MHz) .delta.
4.76 (bs, 7H), 3.75-3.71 (m, 1H), 3.66-3.62 (m, 1H), 3.58-3.52 (m,
4H), 3.33-3.29 (m, 2H), 3.22 (dd, J=10.5 and 7.6 Hz, 1H), 3.15-3.09
(m, 2H), 2.81 (t, J=6.8 Hz, 2H), 2.76-2.71 (m, 4H), 2.19-2.08 (m,
3H), 2.00-1.66 (series of multiplets, 14H), 1.58-1.45 (m, 3H),
1.40-1.08 (m, 5H), 1.03 (d, J=6.8 Hz, 3H), 1.02-0.96 (m, 1H), 0.93
(s, 3H), 0.72 (s, 3H); .sup.13C NMR (about 10% CD.sub.3OD in
CDCl.sub.3, 75 MHz) .delta. 81.74, 80.64, 77.23, 67.95, 67.87,
67.18, 47.32, 44.59, 43.72, 43.01, 41.26, 40.80, 40.71, 40.23,
40.02, 36.36, 36.20, 35.87, 34.27, 33.99, 33.90, 29.60, 29.05,
28.58, 28.08, 24.49, 23.62, 23.46, 16.84, 13.12; HRFAB-MS
(thioglycerol+Na.sup.+ matrix) m/e: ([M+H].sup.+) 538.4578 (4.7%),
calcd. 538.4584.
Example 11
[0202] This example includes a description of one or more exemplary
synthestic procedures for obtaining Compounds CSA-21, 133-134 and
CSA-15.
[0203] Compound CSA-21: Compound 115 (0.118 g, 0.183 mmol) was
dissolved in dry CH.sub.2Cl.sub.2 (10 mL), and SO.sub.3 pyridine
complex (0.035 g, 0.22 mmol) was added. The suspension was stirred
for 12 hr. The solvent was removed in vacuo to give white powder.
To the white powder was added 1 M HCl (10 mL) and the resulting
mixture was extracted with CH.sub.2Cl.sub.2 (4.times.10 mL). The
combined extracts were dried over anhydrous Na.sub.2SO.sub.4. The
desired product (0.11 g, 84%) was obtained as a pale yellow oil
after silica gel chromatography (10% MeOH in CH.sub.2Cl.sub.2).
.sup.1H NMR (about 10% CD.sub.3OD in CDCl.sub.3, 500 MHz) .delta.
4.03 (t, J=6.8 Hz, 2H), 3.69-3.65 (m, 1H), 3.62-3.58 (m, 1H), 3.55
(t, J=6.1 Hz, 2H), 3.51 (bs, 1H), 3.46-3.38 (m, 6H), 3.27 (d, J=2.4
Hz, 1H), 3.26-3.21 (m, 1H), 3.18-3.07 (m, 2H), 2.18-2.03 (m, 3H),
1.95-1.47 (series of multiplets, 19H), 1.40-0.96 (series of
multiplets, 9H), 0.92 (d, J=6.8 Hz, 3H), 0.91 (s, 3H), 0.66 (s,
3H); .sup.13C NMR (about 10% CD.sub.3OD in CDCl.sub.3, 75 MHz)
.delta. 80.43, 79.68, 75.87, 69.30, 64.82, 64.32, 64.14, 48.78,
48.73, 48.50, 46.44, 46.21, 42.49, 41.76, 39.61, 35.36, 35.17,
35.06, 34.85, 31.73, 29.53, 29.46, 29.44, 28.84, 27.68, 27.48,
27.38, 25.91, 23.30, 22.75, 22.66, 17.70, 12.32; HRFAB-MS
(thioglycerol+Na.sup.+ matrix) m/e: ([M-H+2Na].sup.+) 768.3831
(100%), calcd. 768.3843. The azides were reduced by treating the
triazide (0.11 g, 0.15 mmol) with Ph.sub.3 P (0.20 g, 0.77 mmol) in
THF (10 mL) and H.sub.2O (1 mL). The mixture was stirred for 3
days. The solvent was removed in vacuo, and the residue was
purified by silica gel chromatography (CH.sub.2Cl.sub.2/MeOH/28%
NH.sub.3.H.sub.2O 12:6:1 then 10:5:1.5) to afford the desired
product (0.077 g, 78% yield) as a glass. HCl in Et.sub.2O (1 M, 0.5
mL) was added to the glass to give the corresponding HCl salt.
.sup.1H NMR (about 10% CDCl.sub.3 in CD.sub.3OD, 500 MHz) .delta.
4.81 (s, 10H), 4.07-3.97 (m, 2H), 3.82 (bs, 1H), 3.71 (bs, 1H),
3.65 (t, J=5.2 Hz, 2H), 3.57 (bs, 1H), 3.37-3.30 (m, 2H), 3.22-3.02
(m, 8H), 2.12-1.71 (series of multiplets, 17H), 1.65-1.01 (series
of multiplets, 13H), 0.97 (d, J=6.8 Hz, 3H), 0.94 (s, 3H), 0.73 (s,
3H); .sup.13C NMR (about 10% CDCl.sub.3 in CD.sub.3OD, 75 MHz)
.delta. 81.89, 80.58, 77.50, 70.04, 66.71, 66.56, 66.02, 47, 11,
46.76, 44.20, 42.66, 40.50, 39.60, 39.40, 36.24, 36.11, 35.89,
35.67, 32.28, 29.38, 29.23, 29.10, 28.94, 28.49, 26.06, 24.21,
23.46, 23.30, 18.50, 12.86; HRFAB-MS (thioglycerol+Na.sup.+ matrix)
m/e: ([M+Na].sup.+) 668.4271 (100%), calcd. 668.4258.
[0204] Compound CSA-13: The mesylate derived from 23 (0.19 g, 0.264
mmol) was stirred with excess octyl amine (2 mL) at 80.degree. C.
for 12 hr. After removal of octylamine in vacuo, the residue was
chromatographed (silica gel, EtOAc/hexanes 1:4 with 2% Et.sub.3 N)
to afford the desired product (0.19 g, 95% yield) as a pale yellow
oil. NMR (CDCl.sub.3, 300 MHz) .delta. 3.69-3.37 (series of
multiplets, 11H), 3.26-3.00 (m, 4H), 2.61-2.53 (m, 4H), 2.20-2.02
(m, 3H), 1.98-0.99 (series of multiplets, 40H), 0.92-0.85 (m, 9H),
0.65 (s, 3H); .sup.13C NMR (CDCl.sub.3, 75 MHz) .delta. 80.60,
79.74, 76.05, 64.97, 64.40, 64.28, 50.79, 50.25, 49.00, 48.90,
48.71, 46.47, 46.34, 42.65, 41.96, 39.80, 35.77, 35.41, 35.27,
35.05, 33.73, 31.96, 30.25, 29.76, 29, 74, 29.67, 29.39, 29.05,
27.84, 27.61, 27.55, 26.70, 23.50, 23.00, 22.82, 22.79, 18.06,
14.23, 12.54; HRFAB-MS (thioglycerol+Na.sup.+ matrix) m/e:
([M+H].sup.+) 755.6012 (100%), calcd. 755.6024. The triazide (0.18
g, 0.239 mmol) was dissolved in THF (10 mL) and EtOH (10 mL).
Lindlar catalyst (44 mg) was added, and the suspension was shaken
under H.sub.2 (50 psi) for 12 hr. After removal of the solvent in
vacuo, the residue was purified by silica gel chromatography
(CH.sub.2Cl.sub.2/MeOH/28% NH.sub.3.H.sub.2O 10:5:1, then
10:5:1.5). To the product, 1 M HCl (2 mL) and the resulting clear
solution was extracted with Et.sub.2O (2.times.10 mL). 20% NaOH
solution was added until the solution became strongly basic.
CH.sub.2Cl.sub.2 (20 mL, 2.times.10 mL) was used to extract the
basic solution. The combined extracts were dried over anhydrous
Na.sub.2SO.sub.4, and removal of solvent in vacuo gave the desired
product (0.114 g, 68% yield) as a clear oil. .sup.1H NMR (about 20%
CDCl.sub.3 in CD.sub.3OD, 500 MHz) .delta. 4.79 (bs, 7H), 3.74-3.70
(m, 1H), 3.66-3.61 (m, 1H), 3.56-3.51 (m, 3H), 3.31-3.29 (m, 2H),
3.16-3.09 (m, 2H), 2.88-2.72 (m, 6H), 2.59-2.51 (m, 4H), 2.18-2.07
(m, 3H), 1.97-1.66 (series of multiplets, 14H), 1.62-0.97 (series
of multiplets, 25H), 0.95 (d, J=6.3 Hz, 3H), 0.93 (s, 3H), 0.89 (t,
J=6.8 Hz, 3H), 0.70 (s, 3H); .sup.13C NMR (about 20% CDCl.sub.3 in
CD.sub.3OD, 75 MHz) .delta. 81.82, 80.63, 77.23, 67.85, 67.19,
51.20, 50.69, 47.82, 47.24, 43.92, 43.01, 41.30, 40.80, 40.68,
40.22, 36.74, 36.38, 36.20, 35.87, 34.66, 34.15, 33.87, 32.90,
30.54, 30.39, 30.30, 29.64, 29.03, 28.59, 28.41, 26.96, 24.37,
23.65, 23, 48, 18.75, 14.63, 13.09; HRFAB-MS (thioglycerol+Na.sup.+
matrix) m/e: ([M+H].sup.+) 677.6309 (46.6%), calcd. 677.6309.
[0205] Compound CSA-46: Compound CSA-46 was prepared using the
methods of CSA-13, substituting 7-deoxycholic steroid backbone
precursor in place of cholic acid.
[0206] Compound 134: Compound CSA-13 (0.08 g, 0.12 mmol) was
dissolved in CHCl.sub.3 (5 mL) and MeOH (5 mL), aminoiminosulfonic
acid (0.045 g, 0.36 mmol) was added, and the suspension was stirred
for 12 hr. The solvent was removed in vacuo, and the residue was
dissolved in 1 M HCl (6 mL) and H.sub.2O (10 mL). The solution was
washed with Et.sub.2O (3.times.5 mL), and 20% NaOH solution was
then added dropwise until the solution became strongly basic. The
basic mixture was extracted with CH.sub.2Cl.sub.2 (4.times.5 mL).
The combined extracts were dried over anhydrous Na.sub.2SO.sub.4
and concentrated in vacuo to give the desired product (0.087 g, 91%
yield) as a white glass. .sup.1H NMR (about 20% CDCl.sub.3 in
CD.sub.3OD, 500 MHz) .delta. 4.96 (bs, 13H), 3.74-3.68 (m, 1H),
3.65-3.50 (m, 4H), 3.38-3.18 (series of multiplets, 10H), 2.60-2.50
(m, 4H), 2.15-1.99 (m, 3H), 1.88-1.72 (m, 14H), 1.60-0.99 (series
of multiplets, 25H), 0.94 (bs, 6H), 0.89 (1, J=6.6 Hz, 3H), 0.71
(s, 3H); .sup.13C NMR (about 20% CDCl.sub.3 in CD.sub.3OD, 75 MHz)
.delta. 159.00, 158.87, 158.72, 81.68, 79.93, 76.95, 66.59, 65.93,
65.45, 50.82, 50.40, 47.64, 46.94, 43.67, 42.27, 40.18, 39.25,
36.19, 35.66, 35.40, 34.21, 32.45, 30.51, 30.26, 30.18, 30.10,
29.86, 29.35, 28.71, 28.15, 28.00, 26.87, 23.94, 23.44, 23.23,
23.12, 18.61, 14.42, 12.98; HRFAB-MS (thioglycerol+Na.sup.+ matrix)
m/e: ([M+H].sup.+) 803.6958 (18.4%), calcd. 803.6953.
[0207] Compound CSA-15: The mesylate derived from 23 (0.092 g,
0.128 mmol) was dissolved in DMSO (2 mL) followed by the addition
of NaN.sub.3 (0.0167 g, 0.256 mmol). The suspension was heated to
70.degree. C., for 12 hr. H.sub.2O (20 mL) was added to the cooled
suspension, and the mixture was extracted with EtOAc/hexanes (1:1)
(20 mL, 3.times.10 mL). The combined extracts were washed with
brine (30 mL), dried over anhydrous Na.sub.2SO.sub.4, and
concentrated in vacuo to give the product (0.081 g, 95% yield) as a
pale yellow oil. .sup.1H NMR (CDCl.sub.3, 300 MHz) .delta.
3.69-3.36 (m, 11H), 3.25-3.02 (m, 6H), 2.20-2.02 (m, 3H), 1.97-1.60
(m, 15H), 1.55-0.98 (m, 13H), 0.92 (d, J=6.3 Hz, 3H), 0.89 (s, 3H),
0.66 (s, 3H); .sup.13C NMR (CDCl.sub.3, 75 MHz) .delta. 80.59,
79.77, 76.03, 65.01, 64.46, 64.30, 52.12, 48.99, 48.95, 48.76,
46.44, 46.42, 42.70, 41.99, 39.82, 35.56, 35.44, 35.31, 35.09,
33.09, 29.79, 29.77, 29.71, 29.08, 27.88, 27.78, 27.66, 25.65,
23.53, 23.03, 22.85, 18.00, 12.58; HRFAB-MS (thioglycerol+Na.sup.+
matrix) m/e: ([M+Na].sup.+) 691.4512 (100%), calcd. 691.4496. The
tetraazide (0.081 g, 0.12 mmol) was dissolved in THF (5 mL) and
EtOH (10 mL). Lindlar catalyst (30 mg) was added, and the
suspension was shaken under H.sub.2 (50 psi) for 12 hr. After
removal of the solvent in vacuo, the residue was purified by silica
gel chromatography (CH.sub.2Cl.sub.2/MeOH/28% NH.sub.3.H.sub.2O
5:3:1, then 2:2:1). To the product, 1M HCl (2 mL) was added, and
the resulting solution was washed with Et.sub.2O (2.times.10 mL).
20% NaOH solution was added to the aqueous phase until the solution
became strongly basic. CH.sub.2Cl.sub.2 (10 mL, 2.times.5 mL) was
used to extract the basic solution. The combined extracts were
dried over anhydrous Na.sub.2SO.sub.4, and concentration in vacuo
gave the desired product (0.044 g, 64% yield) as a colorless oil.
.sup.1H NMR (about 20% CDCl.sub.3 in CD.sub.3OD, 500 MHz) .delta.
4.79 (bs, 8H), 3.74-3.70 (m, 1H), 3.66-3.62 (m, 1H), 3.56-3.52 (m,
3H), 3.31-3.27 (m, 2H), 3.16-3.10 (m, 2H), 2.82-2.70 (m, 6H),
2.64-2.54 (m, 2H), 2.19-2.07 (m, 3H), 1.99-1.66 (series of
multiplets, 14H), 1.58-0.96 (series of multiplets, 13H), 0.96 (d,
J=6.6 Hz, 3H), 0.93 (s, 3H), 0.70 (s, 3H); .sup.13C NMR (about 20%
CDCl.sub.3 in CD.sub.3OD, 75 MHz) .delta. 81.96, 90.76, 77.33,
67.92, 67.26, 47.84, 47.33, 44.04, 43.24, 43.15, 41.40, 40.91,
40.78, 40.29, 36.82, 36.48, 36.28, 35.96, 34.39, 34.11, 30.59,
29.69, 29.13, 28.68, 28.64, 24.43, 23.69, 23.48, 18.77, 13.06;
HRFAB-MS (thioglycerol+Na.sup.+ matrix) m/e: ([M+H].sup.+) 565.5041
(100%), calcd. 565.5057.
Example 12
[0208] This example includes a description of one or more exemplary
synthestic procedures for obtaining Compounds 203a-b, 207a-c,
209a-c, 210a-b and CSA-31.
[0209] Compounds 203a-b, 207a-c, 208a-c, 209a-c, and 210a-b:
BOC-glycine was reacted with DCC, DMAP and cholic acid derivative
201 (Scheme 11) to give triester 202a in good yield. A similar
reaction incorporating BOC-.beta.-alanine was also successful,
giving 202b. Deprotection of 202a and 202b with HCl in dioxane,
followed by purification (SiO.sub.2 chromatography with a
CH.sub.2Cl.sub.2 MeOH/NH.sub.4OH eluent), gave triesters 203a and
203b in good yield.
[0210] Triamides of glycine and (3-alanine (207a and 207b,
respectively) were formed using the same reaction conditions
(Scheme 12). Triamides with .alpha.-branched amino acids could also
be formed. For example, under the conditions described, a triamide
with bis-BOC-lysine side chains was formed (compound 207c). The C24
esters of 207a-c were hydrolyzed with LiOH in THF and methanol to
give alcohols 208a-c. Deprotection using HCl in dioxane (208a-c)
gave triamides 209a-c in good yield. In addition, alcohols 208a and
208b were mesylated and reacted with benzylmethyl amine.
Deprotection of the resulting compounds with HCl in dioxane gave
triamides 210a and 210b (Scheme 12). Compound CSA-31 was prepared
by analogy to compounds 210a and 210b.
Example 13
[0211] This example includes a description of one or more exemplary
synthestic procedures for obtaining Compounds 302, 312-321,
324-326, 328-331 and 341-343.
[0212] Compound 302: Compound 308 (5.beta.-cholanic acid
3,7,12-trione methyl ester) was prepared from methyl cholate and
pyridinium dichromate in near quantitative yield from methyl
cholate. Compound 308 can also be prepared as described in Pearson
et al., J. Chem. Soc. Perkins Trans. 1 1985, 267; Mitra et al., J.
Org. Chem. 1968, 33, 175; and Takeda et al., J. Biochem. (Tokyo)
1959, 46, 1313. Compound 308 was treated with hydroxyl amine
hydrochloride and sodium acetate in refluxing ethanol for 12 hr (as
described in Hsieh et al., Bioorg. Med. Chem. 1995, 3, 823), giving
309 in 97% yield.
[0213] A 250 ml three neck flask was charged with glyme (100 ml);
to this was added 309 (1.00 g, 2.16 mmol) and sodium borohydride
(2.11 g, 55.7 mmol). TiCl.sub.4 (4.0 mL, 36.4 mmol) was added to
the mixture slowly under nitrogen at 0.degree. C. The resulting
green mixture was stirred at room temperature for 24 hours and then
refluxed for another 12 h. The flask was cooled in an ice bath, and
ammonium hydroxide (100 mL) was added. The resulting mixture was
stirred for 6 hours at room temperature. Conc. HCl (60 mL) was
added slowly, and the acidic mixture was stirred for 8 hours. The
resulting suspension was made alkaline by adding solid KOH. The
suspension was filtered and the solids were washed with MeOH. The
combined filtrate and washings were combined and concentrated in
vacuo. The resulting solid was suspended in 6% aqueous KOH (100 mL)
and extracted with CH.sub.2Cl.sub.2 (4.times.75 mL). The combined
extracts were dried over Na.sub.2SO.sub.4 and solvent was removed
in vacuo to give 1.14 g of a white solid. The mixture was
chromatographed on silica gel (CH.sub.2Cl.sub.2/MeOH/NH.sub.4OH
12:6:1) giving 302 (0.282 g, 33% yield), 3 (0.066 g, 8% yield), 4
(0.118 g, 14% yield).
[0214] Compound 302: m.p. 200-202.degree. C.; NMR (about 10%
CDCl.sub.3 in CD.sub.3OD, 300 MHz) .delta. 4.81 (bs, 7H), 3.57-3.49
(m, 2H), 3.14 (t, J=3.2 Hz, 1H), 2.97 (bs, 1H), 2.55-2.50 (m, 1H),
2.15-2.10 (m, 1H), 1.95-1.83 (m, 3H), 1.74-0.99 (series of
multiplets, 20H), 1.01 (d, J=6.4 Hz, 3H), 0.95 (s, 3H), 0.79 (s,
3H); .sup.13C NMR (10% CDCl.sub.3 in CD.sub.3OD, 75 MHz) .delta.
63.28, 55.01, 52.39, 49.20, 48.69, 47.00, 43.24, 42.77, 41.03,
40.27, 36.82, 36.35, 35.75, 35.12, 32.77, 31.36, 30.10, 28.54,
27.88, 26.96, 24.35, 23.38, 18.18, 14.23, HRFAB-MS
(thioglycerol+Na.sup.+ matrix) m/e; ([M+H].sup.+) 392.3627 (100%);
calcd. 392.3641.
[0215] Octanyl cholate (328): Cholic acid (3.14 g, 7.43 mmol) and
10-camphorsulfonic acid (0.52 g, 2.23 mmol) were dissolved in
octanol (3.5 mL, 23.44 mmol). The solution was warmed to
40-50.degree. C. in oil bath under vacuum (about 13 mm/Hg). After
14 h, the remaining octanol was evaporated under high vacuum. The
crude product was purified via chromatography (silica gel, 5% MeOH
in CH.sub.2Cl.sub.2) to afford the desired product (2.81 g, 73%
yield) as a white powder. .sup.1H NMR (CDCl.sub.3, 500 MHz) .delta.
4.06 (t, J=6.7 Hz, 2H), 3.98 (s, 1H), 3.86 (s, 1H), 3.48-3.44 (m,
1H), 2.41-2.34 (m, 1H), 2.28-2.18 (m, 3H), 1.98-1.28 (series of
multiplets, 35H), 0.99 (d, J=3.3 Hz, 3H), 0.90 (s, 3H), 0.89 (t,
J=7 Hz, 3H), 0.69 (s, 3H); .sup.13C NMR (CDCl.sub.3, 75 MHz)
.delta. 154.38, 73.18, 72.14, 68.63, 56.07, 50.02, 49, 32, 47.07,
46, 74, 41.96, 41.67, 39.84, 39.76, 35.66, 35.45, 34.95, 34.86,
34.15, 32.97, 32.91, 31.65, 31.11, 30.68, 28.39, 27.78, 26.66,
26.52, 25.82, 25.70, 25.54, 25.15, 24.95, 23.45, 22.69, 17.77,
12.71; HRFAB-MS (thioglycerol+Na.sup.+ matrix) m/e; ([M+Na].sup.+)
543.4015 (100%), calcd. 543.4026.
[0216] Representative synthesis of compounds 329-331: Octanyl
cholate (328) (0.266 g, 0.511 mmol), N-t-Boc-glycine (0.403 g,
2.298 mmol), DCC (0.474 g, 2.298 mmol) and DMAP (0.0624 g, 0.051
mmol) were mixed in CH.sub.2Cl.sub.2 (15 mL) for 3 h. The resulting
white precipitate was removed by filtration. The filtrate was
concentrated, and the product was purified by chromatography
(silica gel, EtOAc/Hexane 1:2) to afford the desired product (0.481
g, 95% yield) as a white powder. Compound 329 .sup.1H NMR
(CDCl.sub.3, 300 MHz) 5.18 (br, 3H), 5.01 (s, 1H), 4.61 (m, 1H),
4.04 (t, J=6.5 Hz, 2H), 3.97-3.88 (series of multiplets, 6H),
2.39-2.15 (series of multiplets, 2H), 2.06-1.02 (series of
multiplets, 35H), 1.46 (s, 18H), 1.45 (s, 9H), 0.93 (s, 3H), 0.88
(t, J=6.7 Hz, 3H), 0.81 (d, J=6 Hz, 3H), 0.74 (s, 3H); .sup.13C NMR
(CDCl.sub.3, 75 MHz) .delta.174.26, 170.19, 169.9, 169.78, 155.87,
155.67, 79.95, 76.47, 75.167, 72.11, 64.55, 47.40, 45.28, 43.17,
42.86, 40.82, 37.94, 34.71, 34.63, 34, 43, 31.86, 31.340, 31.20,
30.76, 29.29, 29.25, 28.80, 28.72, 28.42, 28.06, 27.96, 27.19,
26.81, 26.29, 26.012, 25.66, 22.87, 22.71, 22.57, 17.55, 14.18,
12.27; HRFAB-MS (thioglycerol+Na.sup.+ matrix) m/e: ([M+Na].sup.+)
1014.6261 (100%), calcd. 1014.6242. Compound 330: .sup.1H NMR
(CDCl.sub.3, 500 MHz) .delta. 5.10 (s, 1H), 4.92 (d, J=2.44 Hz,
1H), 4.55 (m, 1H), 4.00 (t, J=6.8 Hz, 2H), 3.39-3.33 (series of
multiplets, 6H), 2.595-2.467 (series of multiplets, 6H), 2.31-2.12
(series of multiplets, 2H), 2.01-1.00 (series of multiplets, 37H),
1.39 (s, 27H), 0.88 (s, 3H), 0.84 (t, J=6.8 Hz, 3H), 0.76 (d, J=6.3
Hz, 3H), 0.69 (s, 3H); .sup.13C NMR (CDCl.sub.3, 75 MHz) .delta.
174.16, 172.10, 171.78, 171.67, 155.95, 79.45, 75.67, 74.21, 71.10,
64.63, 47.79, 45.27, 43.52, 40.97, 37.92, 36.35, 35.14, 35.05,
34.90, 34.71, 34.46, 31.91, 31.45, 30.95, 29, 35, 29.31, 28.96,
28.78, 28.56, 28.55, 27, 22, 26.98, 26.269, 25.71, 23.00, 22.77,
22.64, 17.75, 14.24, 12.39; HRFAB-MS (thioglycerol+Na.sup.+ matrix)
m/e: ([M+Na].sup.+) 1056.6702 (100%), calcd. 1056.6712. Compound
331 .sup.13C NMR (CDCl.sub.3, 125 MHz) .delta.174.00, 172.75,
172.41, 172.30, 156.03, 79.00, 75.28, 73.79, 70.77, 64.39, 47.43,
45.04, 43.21, 40.76, 40.00, 39.93, 37.78, 34.74, 34.62, 34.23,
32.19, 32.01, 31.70, 31.24, 30.77, 29.13, 29.10, 28.67, 38.58,
28.38, 25.86, 25, 37, 22.56, 22.38, 17.51, 14.05, 12.13; HRFAB-MS
(thioglycerol+Na.sup.+ matrix) m/e: ([M+Na].sup.+) 1098.7181
(100%), calcd. 1098.7181.
[0217] Representative synthesis of compounds 341-343: To compound
329 (0.463 g, 0.467 mmol) was added HCl in dioxane (0.3 mL, 4.0 M).
After stirring the mixture for 30 min, the excess HCl and solvent
were removed in vacuo. The product was isolated, after
chromatography (silica gel, CH.sub.2Cl.sub.2/MeOH/NH.sub.3,H.sub.2O
10:1.2:0.1) as a (0.271 g, 84%) pale oil. The trihydrochloride salt
of 341 was prepared by addition of HCl in dioxane and evaporation
of excess HCl and dioxane in vacuo giving a white powder. Compound
341: .sup.1H NMR (CDCl.sub.3 with about 10% CD.sub.3OD, 500 MHz)
.delta. 5.16 (s, 1H), 4.99 (t, J=3.6 Hz, 1H), 4.61 (m, 1H), 4.04
(t, J=6.8 Hz, 2H), 3.51-3.36 (m, 6H), 2.34-2.15 (m, 2H), 2.00-1.05
(series of multiplets, 40H), 0.93 (s, 3H), 0.88 (t, J=7.1 Hz, 3H),
0.80 (d, J=3.2 Hz, 3H), 0.74 (s, 3H); .sup.13C NMR (CDCl.sub.3 and
about 10% CD.sub.3OD, 75 MHz) .delta. 174.32, 173.92, 173.81,
76.08, 74.67, 71.61, 64.73, 47.64, 45.39, 44.41, 43.49, 40, 97,
37.99, 34.99, 34.77, 34.71, 34.52, 31.96, 31.54, 31.35, 30.96,
29.39, 29, 36, 29.02, 28.82, 27.32, 27.11, 26.11, 25.83, 23.01,
22.82, 22.69, 17.79, 14.28, 12.41; HRFAB-MS (thioglycerol+Na.sup.+
matrix) mile: ([M+Na].sup.+) 714.4651 (100%), calcd. 714.4669.
Compound 342: .sup.1H NMR (CDCl.sub.3 and about 10% CD.sub.3OD, 300
MHz) .delta. 5.142 (s, 1H), 4.96 (d, J=2.7 Hz, 1H), 4.60, (m, 1H),
4.04 (t, J=6.6 Hz, 2H), 3.07-2.95 (series of multiplets, 6H),
2.56-2.43 (series of multiplets, 6H), 2.38-2.13 (series of
multiplets, 2H), 2.07-1.02 (series of multiplets, 36H), 0.92 (s,
3H), 0.88 (t, J=6.6 Hz, 3H), 0.82 (d, J=6.6 Hz, 3H), 0.73 (s, 3H);
.sup.13C NMR (CDCl.sub.3 and CD.sub.3OD, 75 MHz) .delta. 174.29,
172.29, 171.98, 171.92, 75.52, 74.09, 70.98, 64.67, 47.78, 45.26,
43.52, 40.98, 38.73, 38.62, 38.35, 38.07, 38.03, 37.99, 35.01,
34.81, 34.77, 34.49, 31.92, 31.50, 31.40, 30.99, 29.36, 29.33,
28.93, 28.80, 27.43, 26.96, 26.08, 25.56, 23.07, 22.79, 22.62,
17.73, 14.25, 12.34; HRFAB-MS (thioglycerol+Na.sup.+ matrix) m/e:
([M+Na].sup.+) 714.4651 (100%), calcd. 714.4669. Compound 343:
.sup.1H NMR (CDCl.sub.3 and CD.sub.3OD, 500 MHz) .delta. 5.12 (s,
1H) 4.93 (s, 1H), 4.59 (m, 1H), 4.04 (t, J=7 Hz, 2H), 2.79-2.69
(series of multiplets, 6H), 2.4621-2.2999 (series of multiplets,
6H), 2.2033-1.0854 (series of multiplets, 42H), 0.94 (s, 2H), 0.91
(s, 1H), 0.88 (t, J=7 Hz, 3H), 0.82 (d, J=6.4 Hz, 3H), 0.75 (s,
3H); .sup.13C NMR (CDCl.sub.3 and CD.sub.3OD, 75 MHz) .delta.
174.70, 171.97, 171.86, 171.75, 76.10, 74.55, 71.56, 64.85, 47.96,
45.31, 43.37, 40.87, 38.09, 34.86, 34.80, 34.73, 34.46, 32.84,
32.62, 32.27, 31.87, 31.75, 31.42, 31.08, 29.31, 29.28, 29.26,
28.78, 28.73, 27.38, 26.91, 26.05, 25.37, 23.24, 23.15, 22.95,
22.74, 22.71, 22.43, 17.78, 14.11, 12.28; HRFAB-MS
(thioglycerol+Na.sup.+ matrix) m/e: ([M+Na].sup.+) 798.5624 (100%),
calcd. 798.5609.
[0218] Benzyl cholate (312): Cholic acid (4.33 g, 10.62 mmol) and
10-caphorsulfonic acid (0.493 g, 2.21 mmol) were dissolved in
benzyl alcohol (1.97 mL, 19.3 mmol). The suspension was heated to
50.degree. C., in oil bath and stirred under vacuum (about 13
mm/Hg) for 16 h. Excess benzyl alcohol was removed in vacuo, and
the crude product was chromatographed (silica gel, 5% MeOH in
CH.sub.2Cl.sub.2) to give the desire product as a white powder
(4.23 g, 81% yield). .sup.1H NMR (CDCl.sub.3, 500 MHz) .delta.
7.34-7.33 (m, 5H), 5.10 (d, J=1.5 Hz, 2H), 3.92 (s, 1H), 3.81 (s,
1H), 3.42 (s, 1H), 3.40 (br, m, 3H), 2.44-2.38 (m, 1H), 2.31-2.25
(m, 1H), 2.219 (t, J=12 Hz, 2H), 0.96 (d, J=5.5 Hz, 3H), 0.86 (s,
3H), 0.63 (s, 3H); .sup.13C NMR (CDCl.sub.3, 125 MHz)
.delta.174.25, 136.30, 128.66, 128.63, 128.32, 128.28, 128.24,
73.18, 71.98, 68.54, 66.18, 47.14, 46.56, 41.69, 39.65, 35.51,
35.37, 34.91, 34.84, 31.49, 31.08, 30.50, 28.31, 27.62, 26.47,
23.35, 22.65, 22.60, 17.42, 12.63, 12.57; HRFAB-MS
(thioglycerol+Na.sup.+ matrix) m/e: ([M+Na].sup.+) 521.3235 (100%),
calcd. 521.3242.
[0219] Representative synthesis of compounds 313-315: Benzyl
cholate (312) (0.248 g, 0.499 mmol), N-t-Boc-glycine (0.404 g, 2.30
mmol), DCC (0.338 g, 1.49 mmol) and DMAP (0.051 g, 0.399 mmol) were
added to CH.sub.2Cl.sub.2 (15 mL), and the suspension was stirred
for 16 h. The resulting white precipitate was removed by
filtration, and the filtrate was concentrated. The product was
obtained after chromatorgraphy (silica gel, EtOAc/Hexane 0.6:1) as
a white powder (0.329 g, 68%). Compound 313: .sup.1H NMR
(CDCl.sub.3, 300 MHz) .delta. 7.34-7.33 (m, 5H), 5.16 (s, 1H), 5.08
(dd, J=22.5 Hz, 12.3 Hz, 4H), 5.00 (s, 1H), 4.60 (m, 1H), 4.04-3.81
(series of multiplets, 6H), 2.43-1.01 (series of multiplets, 25H),
1.46 (s, 9H), 1.44 (s, 18H), 0.92 (s, 3H), 0.797 (d, J=5.7 Hz, 3H),
0.69 (s, 1H); .sup.13C NMR (CDCl.sub.3, 75 MHz) .delta. 173.99,
170.25, 170.05, 169.85, 155.73, 136.19, 128.69, 128.45, 128.35,
80.06, 77.65, 77.23, 76.80, 76.53, 75.24, 72.19, 66.29, 47.46,
45.35, 43.24, 42.91, 40.89, 38.00, 34.79, 34.66, 34.49, 31.43,
31.25, 30.77, 28.88, 28.40, 27.23, 26.89, 25.74, 22.94, 22.65,
17.61, 12.32; FAB-MS (thioglycerol+Na.sup.+ matrix) m/e:
([M+Na].sup.+) 992.5468 (100%), calcd. 992.5460.
[0220] Representative synthesis of compounds 316-318: Compound 313
(0.505 g, 0.520 mmol) and Pd (5 wt. % on active carbon, 0.111 g,
0.0521 mmol) were added to MeOH (5 mL). The suspension was stirred
under H.sub.2 (50 psi) for 20 hours. The solids were removed by
filtration and the filtrate was concentrated. Purification of the
product via chromatography (silica gel, 5% MeOH in
CH.sub.2Cl.sub.2) gave a white powder (0.450 g, 98% yield).
Compound 316: .sup.1H NMR (CDCl.sub.3, 500 MHz) .delta. 5.20 (s,
1H), 5.12 (br., 2H), 4.92 (s, 1H), 4.55 (m, 1H), 3.98-3.83 (series
of multiplets, 6H), 2.30-2.13 (series of multiplets, 2H), 1.96-0.98
(series of multiplets, 30H), 1.40 (s, 9H), 1.39 (s, 18H), 0.87 (s,
3H), 0.76 (d, Hz, 3H), 0.68 (s, 3H); .sup.13C NMR (CDCl.sub.3 75
MHz) .delta.174.11, 165.60, 165.41, 165.22, 151.28, 151.14, 75.48,
75.26, 71.81, 70.57, 67.50, 45.95, 42.58, 40.65, 38.52, 38.16,
36.17, 33.28, 30.01, 29.78, 26.71, 26.42, 25.95, 24.16, 23.78,
23.40, 23.31, 22.55, 22.16, 21.03, 18.23, 17.93, 12.91, 7.61;
FAB-MS (thioglycerol+Na.sup.+ matrix) m/e: ([M+Na].sup.+) 902.4997
(21%), calcd. 902.4990.
[0221] Representative synthesis of compounds 319-321: Compound 316
(0.375 g, 0.427 mmol), DCC (0.105 g, 0.512 mmol) and DMAP (0.062 g,
0.512 mmol) and N,N-dimethylethanolamine (0.09 ml, 0.896 mmol) were
added to CH.sub.2Cl.sub.2 (15 mL). The mixture for 16 h, and
solvent and excess N,N-dimethylethanolamine were removed in vacuo.
The product was purified via chromatography (silica gel
EtOAc/hexane/Et.sub.3 N, 12:10:0.6) giving a white powder (0.330 g,
82% yield). .sup.1H NMR (CDCl.sub.3 and about 10% CD.sub.3OD, 500
MHz) .delta. 5.18 (s, 1H), 5.00 (s, 1H), 4.19 (t, J=5.0 Hz, 2H),
3.92 (s, 3H), 3.81 (s, 3H), 2.62 (t, J=10 Hz, 2H), 2.30 (s, 6H),
1.47 (s, 9H), 1.47 (s, 1H), 1.45 (s, 1H), 2.12-1.05 (series of
multiplets, 27H), 0.96 (s, 3H), 0.84 (d, J=10.5 Hz, 3H), 0.78 (s,
3H); .sup.13C NMR (CDCl.sub.3 and about 10% CD.sub.3OD, 125 MHz)
.delta.174.19, 170.05, 169.87, 156.21, 79.36, 79.27, 76.06, 76.90,
71.80, 61.19, 57.04, 46.88, 44.87, 44.67, 44.53, 42, 78, 42.15,
42.01, 40.43, 37.47, 34.32, 34.11, 33.92, 33.35, 33.25, 30.74,
30.56, 30.16, 28.40, 27.67, 27.62, 26.73, 26.19, 25.18, 25, 10,
24.72, 24.49, 22.29, 21.81, 16.76, 11.56; FAB-MS
(thioglycerol+Na.sup.+ matrix) m/e: ([M+Na].sup.+) 973.5723 (100%),
calcd, 973.5725. The white solid from the previous reaction (0.680
g, 0.714 mmol) and MeI (1 M in CH.sub.2Cl.sub.2, 1.5 mL) were
stirred together for 2 h. The solvent and excess MeI were removed
in vacuo giving a white solid (0.812 g about 100%). The product was
carried on without further purification.
[0222] Representative synthesis of compounds 324-326: Compound 319
(0.812 g, 0.714 mmol) was dissolved in CH.sub.2Cl.sub.2 (5 mL) and
trifluoroacetic acid (0.5 mL) was added. The mixture was stirred
for 16 min. The solvent and excess acid were removed in vacuo, and
the resulting oil was chromatographed (silica gel,
CH.sub.2Cl.sub.2/MeOH/NH.sub.3.H.sub.2O 4:4:1) to give the desired
product as a pale glass (0.437 g, 90% yield). Addition of HCl (2 M
in ethyl ether, 2.5 mL) gave the trihydrochloride salt of 324 as a
pale yellow powder. Compound 324: .sup.1H NMR (50% CDCl.sub.3, 50%
CD.sub.3OD, 300 MHz) .delta. 5.43 (s, 1H), 5.24 (s, 1H), 4.84 (m,
1H), 4.66 (m, 2H), 4.16-3.96 (series of multiplets, 6H), 3.88 (m,
2H), 3.37 (s, 9H), 0.67 (s, 3H), 0.59 (d, J=6.3 Hz, 3H), 0.56 (s,
3H); .sup.13C NMR (50% CDCl.sub.3, 50% CD.sub.3OD, 75 MHz)
.quadrature. 173.47, 167.06, 167.01, 166.70, 78.01, 76.49, 73.78,
64.98, 57.67, 53.36, 47.49, 46.99, 45.61, 43.28, 40.83, 40.23,
40.10, 37.69, 34.80, 34.48, 34.28, 31.03, 30.63, 30.44, 28.94,
27.05, 26.56, 25.50, 22.53, 21.56, 16.95, 11.37; FAB-MS
(thioglycerol+Na.sup.+ matrix) m/e: ([M-I].sup.+) 665.4475 (85.6%),
calcd 665.4489. Compounds 325 and 326 proved too unstable to
chromatograph using the basic eluent used for the purification of
324. Consequently, 325 and 326 were prepared by deprotection of 320
and 321 using HCl (2 M in diethyl ether), followed by trituration
with ethyl acetate. The compounds were then used without further
purification. .sup.1H NMR spectroscopy indicated that compounds 325
and 326 were >95% pure. Compound 325: .sup.1H NMR (50%
CDCl.sub.3, 50% CD.sub.3OD, 500 MHz) .delta. 5.21 (s, 1H), 5.02 (d,
J=4 Hz, 1H), 4.64 (m, 1H), 4.53 (m, 2H), 3.74 (m, 2H), 3.31-3.01
(series of multiplets, 6H), 3.23 (s, 9H), 2.96-2.73 (series of
multiples, 6H), 2.51-2.44 (m, 1H), 2.35-2.29 (m, 1H), 2.14-1.09
(series of multiplets, 26H), 0.99 (s, 3H), 0.85 (d, J=6.5 Hz, 3H),
0.80 (s, 3H); .sup.13C NMR (50% CDCl.sub.3, 50% CD.sub.3OD, 125
MHz) .delta. 172.77, 169.88, 169.56, 169.50, 75.94, 74.44, 71.57,
64.31, 56.94, 52.92, 46.78, 44.59, 42.70, 40.21, 37.16, 34.80,
34.72, 34.66, 34.05, 34.00, 33.78, 33.62, 30.95, 30.91, 30.81,
30.41, 29.96, 29.81, 28.20, 26.37, 26.06, 24.74, 24.24, 22.04,
21.13, 16.54, 10.97; FAB-MS (thioglycerol+Na.sup.+ matrix) m/e:
([M-I].sup.+) 707.4958 (25.6%), calcd 707.4958. Compound 326:
.sup.1H NMR (50% CDCl.sub.3, 50% CD.sub.3OD, 500 MHz) .delta. 5.12
(s, 1H), 4.94 (d, J=2.5 Hz, 1H), 4.56 (m. 1H), 4.51 (t, J=2.3 Hz,
2H), 3.74 (m, 2H), 3.23 (s, 9H), 3.05-3.01 (m, 4H), 2.98 (t, J=7.5
Hz, 2H), 2.63-2.43 (series of multiplets, 6H), 2.31-2.24 (series of
multiplets, 2H), 2.07-1.87 (series of multiplets, 12H), 1.17-1.05
(series of multiplets, 23H), 0.94 (s, 3H), 0.82 (d, J=6.0 Hz, 3H),
0.76 (s, 3H); .sup.13C NMR (50% CDCl.sub.3, 50% CD.sub.3OD, 125
MHz) .delta.171.87, 169.79, 169.59, 169.50, 76.12, 74.70, 71.65,
65.57, 65.08, 64.40, 57.68, 53.74, 52.78, 45, 33, 43.54, 41.04,
39.12, 37.92, 43.85, 34.72, 34.56, 34, 34, 32.30, 31.47, 31.27,
30.87, 30.58, 29.03, 27.053, 26.84, 25.51, 24.95, 24.91, 22.87,
22.82, 22.65, 21.93, 17.31, 11.81; FAB-MS (thioglycerol+Na.sup.4
matrix) m/c: ([M-I].sup.+) 749.5432 (100%), calcd 749.5436.
Example 14
[0223] This example includes data indicating the stability of
Compounds 352-354 under acidic, neutral and basic conditions.
[0224] Compounds 352-354 were dissolved in 50 mM phosphate buffered
water (pH 2.0, 7.0 or 12.0) at approximately 10 mM concentrations.
The structures of compounds 352-354 are given in FIG. 9.
Decomposition of the compounds was observed via HPLC (cyano-silica
column, 0.15% TFA water-acetonitrile gradient elution). Table 15
shows the stabilities (half-lives) of compounds 352-354 in
phosphate buffer at room temperature, pH 2.0, pH 7.0 and pH 12.0.
These compounds were used since they contain a chromophore that
facilitated monitoring of decomposition by absorption methods
common in the HPLC apparatus used.
[0225] At low pH, the amines are expected to be protonated and the
compounds showed relative stability. At higher pH, the amines were
less strongly protonated and became involved in ester hydrolysis.
The .gamma.-aminobutyric acid-derived compound was especially
susceptible to hydrolysis, presumably yielding pyrrolidone. In
general, the compounds are believed to hydrolyse to give cholic
acid, choline or octanol, and glycine, beta-alanine, or
pyrrolidone, depending on the particular compound.
[0226] Decomposition through ester hydrolysis yielded compounds
that were less polar and easily separable from the starting
compounds. Initially, only one benezene-containing decomposition
product was observed; at longer reaction times, two other
decomposition products were observed which presumably corresponded
to sequential ester hydrolysis.
Example 15
[0227] This example includes a description of additional exemplary
synthetic procedures for producing compounds of formula I. In one
example, hydroxyl groups on cholic acid can be converted into amine
groups as described in in Hsieh et al. (Synthesis and DNA Binding
Properties of C3-, C12-, and C24-Substituted Amino-Steroids Derived
from Bile Acids, Biorganic and Medicinal Chemistry, 1995, vol. 6,
823-838).
[0228] Compounds of formula I prepared as shown in the following
Scheme.
##STR00026## ##STR00027##
Example 16
[0229] This example includes various materials and methods. This
example also includes data indicating that the CSAs have
anti-herpesvirus activity.
Preparation of Virus
[0230] Human Herpes Simplex Virus Type 2 (HSV) was grown and
passaged in Human Embryonic Lung Fibroblasts in Eagles Minimum
Essential Media (E-MEM) culture media with 2.5% fetal calf serum
(FCS), and 1% penicillin/streptomycin, Freshly trypsinized lung
fibroblasts were grown 3 days to confluence and inoculated with
approximately 1 plaque-forming unit (PFU) per cell in culture
medium. Cells were checked daily for cytopathic effects. The
supernatant was harvested after 48-72 hours of incubation at
37.degree. C. in 5% CO.sub.2, freeze thawed five times and
centrifuged 15 minutes at 1000 RPM. For HSV titration, ten-fold
dilutions of stock were made and 0.1 ml of each dilution was added
to the fibroblast cell sheets in 24 well tissue culture plates.
Virus adsorption took place for 1 hour at 37.degree. C. in 5%
CO.sub.2 and was followed by the addition of E-MEM with 2.5% FCS.
After 48 hours of incubation, cytopathic effects were observed,
media was removed and cells fixed with formalin-crystal violet.
Plaques were visualized on an Inverted Nikon Microscope under
1.3.times.10 magnification. Virus stocks were stored at -70.degree.
C. until us.
[0231] Viral Killing Assay:
[0232] BS-C-1 (African Green Monkey Kidney Cells, ATCC CCL-26)
cells were seeded at 2.times.10.sup.5 cells/well in 24 well plates
and allowed to grow to confluence overnight at 37.degree. C., 5%
CO.sub.2 in E-MEM with 10% FCS and 1% penicillin/streptomycin.
CSA-8, CSA-13, CSA-31 and CSA-54 were used in 7 dilutions from
1-100 .mu.M. (FIG. 1). Each of the CSAs were added to an eppendorf
tube containing 1.times.10.sup.3 PFU HSV and incubated for 24 hours
at 37.degree. C. in a volume not to exceed 0.1 ml. Growth media was
removed from the cell sheet and rinsed once using E-MEM with 2.5%
FCS. The virus:CSA solution was added to the cells and adsorbed for
1 hour at 37.degree. C. and 5% CO.sub.2. Growth media was added to
0.5 ml and incubated for 48 hours. Media was then removed and cells
fixed with formalin-crystal violet. The antiviral activity of these
CSA was determined by counting viral forming plaques within the
wells and multiplying by the dilution factors used (FIG. 11).
* * * * *