U.S. patent application number 13/649311 was filed with the patent office on 2013-10-17 for novel inhibitors of bacterial biofilms and related methods.
This patent application is currently assigned to SEQUOIA SCIENCES, INC.. The applicant listed for this patent is Sequoia Sciences, Inc.. Invention is credited to Ronald Neil Buckle, John Edward, Gary Eldridge, Michael Ellis, Zhongping Huang.
Application Number | 20130274256 13/649311 |
Document ID | / |
Family ID | 47226667 |
Filed Date | 2013-10-17 |
United States Patent
Application |
20130274256 |
Kind Code |
A1 |
Eldridge; Gary ; et
al. |
October 17, 2013 |
NOVEL INHIBITORS OF BACTERIAL BIOFILMS AND RELATED METHODS
Abstract
Certain multi-cyclic compounds and compositions thereof are
useful for reducing or inhibiting the growth of bacterial biofilms
and for controlling bacterial biofilm infections. Such compounds
and compositions are also useful in methods for reducing or
inhibiting the growth of biofilms and for controlling bacterial
biofilm infections involving biofilms.
Inventors: |
Eldridge; Gary; (St. Louis,
MO) ; Buckle; Ronald Neil; (Delmar, NY) ;
Ellis; Michael; (Clifton Park, NY) ; Huang;
Zhongping; (Voorheesville, NY) ; Edward; John;
(Voorheesville, NY) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Sequoia Sciences, Inc.; |
|
|
US |
|
|
Assignee: |
SEQUOIA SCIENCES, INC.
S. Louis
MO
|
Family ID: |
47226667 |
Appl. No.: |
13/649311 |
Filed: |
October 11, 2012 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
13188790 |
Jul 22, 2011 |
8324264 |
|
|
13649311 |
|
|
|
|
Current U.S.
Class: |
514/232.8 ;
435/245; 514/253.09; 514/254.06; 514/256; 514/307; 514/338;
514/363; 514/364; 514/379; 514/381; 514/384; 514/393; 514/407;
514/410; 514/529; 514/557; 514/561; 514/729; 544/131; 544/140;
544/333; 544/364; 544/371; 546/147; 546/272.4; 546/275.7; 548/139;
548/143; 548/241; 548/251; 548/263.8; 548/301.7; 548/358.1;
548/416; 558/429; 560/116; 562/498; 568/817 |
Current CPC
Class: |
A61K 31/191 20130101;
C07D 209/58 20130101; C07D 401/08 20130101; C07D 417/08 20130101;
C07D 413/08 20130101; C07D 403/08 20130101; A61K 31/215 20130101;
C07C 255/47 20130101; C07D 261/20 20130101; C07D 235/02 20130101;
C07D 231/54 20130101; A61K 31/047 20130101 |
Class at
Publication: |
514/232.8 ;
548/358.1; 514/407; 546/275.7; 514/338; 544/140; 544/371;
514/254.06; 546/272.4; 568/817; 514/729; 562/498; 514/557; 560/116;
514/529; 548/416; 514/410; 548/301.7; 514/393; 548/241; 514/379;
558/429; 514/561; 544/333; 514/256; 546/147; 514/307; 544/131;
544/364; 514/253.09; 548/251; 514/381; 548/143; 514/364; 548/263.8;
514/384; 548/139; 514/363; 435/245 |
International
Class: |
C07D 231/54 20060101
C07D231/54; A61K 31/047 20060101 A61K031/047; A61K 31/191 20060101
A61K031/191; A61K 31/215 20060101 A61K031/215; C07D 417/08 20060101
C07D417/08; C07D 235/02 20060101 C07D235/02; C07D 261/20 20060101
C07D261/20; C07C 255/47 20060101 C07C255/47; C07D 403/08 20060101
C07D403/08; C07D 413/08 20060101 C07D413/08; C07D 401/08 20060101
C07D401/08; C07D 209/58 20060101 C07D209/58 |
Claims
1-62. (canceled)
63. A compound corresponding to the following chemical structure:
##STR00279## wherein R1 is selected from the group consisting of
hydrogen, methyl, halide, lower haloalkyl, nitrile, lower alkyl
nitrile, lower alkyl, substituted lower alkyl, lower alkenyl,
substituted lower alkenyl, lower alkynyl, substituted lower
alkynyl, lower cycloalkyl, lower cycloalkenyl, aryl, substituted
aryl, heteroaryl, and substituted heteroaryl; wherein R.sup.2 is
selected from the group consisting of carboxyl, amide,
hydroxyamide, methylamide, --CH.sub.2N(CH.sub.3).sub.2,
--CH.sub.2NR.sup.6R.sup.7, ##STR00280## wherein R.sup.3 is selected
from the group consisting of hydrogen and methyl; wherein one of
R.sup.4 and R.sup.5 is hydrogen and the other is methyl; wherein
R.sup.6 and R.sup.7 are independently selected from the group
consisting of hydrogen, methyl, ethyl, propyl, isopropyl, and
butyl; wherein R.sup.8 is selected from the group consisting of
hydroxyl, amino, --N(CH.sub.3).sub.2, and --NHCH.sub.3; wherein
R.sup.9 is selected from the group consisting of hydrogen, halide,
lower alkyl, lower alkenyl, lower alkynyl, morpholinyl,
piperazinyl, lower alkyl piperazinyl, heterocycloalkyl, lower
cycloalkyl, lower cycloalkenyl; and lower alkyl, lower alkenyl, and
lower alkynyl optionally substituted with moieties selected from
the group consisting of hydroxyl, amino, lower aminoalkyl, halide,
lower alkoxy, lower alkoxyalkyl, lower alkoxycarbonyl, lower
alkylcarbonylamino, carboxyl, amide, hydroxyamide, --CONHCH.sub.3,
--NHCONH.sub.2, --SO.sub.2NH.sub.2, --SO.sub.2CH.sub.3,
--NHCOCH.sub.3, --NHCSNH.sub.2, and --NHSO.sub.2CH.sub.3; and
wherein R.sup.10 and R.sup.11 together with the carbon atoms to
which they are attached form an aromatic N-heterocycle which is
substituted or not selected from the group consisting of indole,
imidazole, quinoline, isoquinoline, pyridine, triazine, pyrazine,
pyrimidine, pyridazine, isoindole, pyrrole, benzimidazole, purine,
pyrazole, benzopyrazole, indazole, quinoxaline, acridine,
quinazoline, indolizine, carbazole and cinnoline; and salts
thereof.
64. A compound according to claim 1, wherein the compound
corresponds to the following chemical structure: ##STR00281##
wherein R.sup.1 is selected from the group consisting of hydrogen,
methyl, halide, lower haloalkyl, nitrile, lower alkyl nitrile,
lower alkyl, substituted lower alkyl, lower alkenyl, substituted
lower alkenyl, lower alkynyl, substituted lower alkynyl, lower
cycloalkyl, lower cycloalkenyl, aryl, substituted aryl, heteroaryl,
and substituted heteroaryl; wherein R.sup.2 is selected from the
group consisting of carboxyl, amide, hydroxyamide, methylamide,
--CH.sub.2N(CH.sub.3).sub.2, --CH.sub.2NR.sup.6R.sup.7,
##STR00282## wherein R.sup.3 is selected from the group consisting
of hydrogen and methyl; wherein one of R.sup.4 and R.sup.5 is
hydrogen and the other is methyl; wherein R.sup.6 and R.sup.7 are
independently selected from the group consisting of hydrogen,
methyl, ethyl, propyl, isopropyl, and butyl; wherein R.sup.8 is
selected from the group consisting of hydroxyl, amino,
--N(CH.sub.3).sub.2, and --NHCH.sub.3; and wherein R.sup.9 is
selected from the group consisting of hydrogen, halide, lower
alkyl, lower alkenyl, lower alkynyl, morpholinyl, piperazinyl,
lower alkyl piperazinyl, heterocycloalkyl, lower cycloalkyl, lower
cycloalkenyl; and lower alkyl, lower alkenyl, and lower alkynyl
optionally substituted with moieties selected from the group
consisting of hydroxyl, amino, lower aminoalkyl, halide, lower
alkoxy, lower alkoxyalkyl, lower alkoxycarbonyl, lower
alkylcarbonylamino, carboxyl, amide, hydroxyamide, --CONHCH.sub.3,
--NHCONH.sub.2, --SO.sub.2NH.sub.2, --SO.sub.2CH.sub.3,
--NHCOCH.sub.3, --NHCSNH.sub.2, and --NHSO.sub.2CH.sub.3; and salts
thereof.
65. A compound according to claim 63 wherein R.sup.1 is selected
from the group consisting of methyl, halide, lower haloalkyl,
nitrile, lower alkyl nitrile, lower alkyl, substituted lower alkyl,
lower alkenyl, substituted lower alkenyl, lower alkynyl,
substituted lower alkynyl, lower cycloalkyl, lower cycloalkenyl,
aryl, substituted aryl, heteroaryl, and substituted heteroaryl,
preferably R.sup.1 is selected from the group consisting of
pyrrolyl, pyrazolyl, imidazolyl, oxazolyl, isoxazolyl, oxadiazolyl,
thiazolyl, isothiazolyl, thienyl, furanyl, furazanyl, pyridinyl,
pyrimidinyl, pyridazinyl, indolyl, 3H-indolyl, isoindolyl,
indolinyl, indolizinyl, indazolyl, dihydroindolyl,
tetrahydroindolyl, purinyl, pyrazinyl, quinolinyl, isoquinolinyl,
tetrahydroisoquinolinyl, quinoxalinyl, quinazolinyl, cinnolinyl,
pteridinyl, benzimidazolyl, benzopyranyl, benzoxazolyl,
benzisoxazolyl, benzofuranyl, isobenzofuranyl, benzothiazolyl,
benzisothiazolyl, benzothienyl, furopyridinyl, phthalazinyl,
napthyridinyl, pyrazolopyridyl, pyrazolopyrimidinyl,
pyrrolopyridinyl, and tetrahydropyrrolopyridinyl optionally
substituted from moieties selected from the group consisting of
lower alkyl, halide, lower haloalkyl, hydroxyl, amino, lower
aminoalkyl, lower hydroxyalkyl, nitrile, lower alkyl nitrile, lower
alkyl ethers, lower alkoxy, lower alkoxyalkyl, lower
alkoxycarbonyl, lower alkylcarbonylamino, and thioalkyl.
66. A compound according to claim 64 wherein the compound
corresponds to the following chemical structure: ##STR00283##
wherein R.sup.1 is selected from the group consisting of hydrogen,
halide, lower alkyl, substituted lower alkyl, lower alkenyl,
substituted lower alkenyl, lower cycloalkyl, lower cycloalkenyl,
aryl, substituted aryl, heteroaryl, and substituted heteroaryl,
preferably R.sup.1 is hydrogen.
67. A compound according to claim 66 wherein R.sup.1 is selected
from the group consisting of halide, lower alkyl, substituted lower
alkyl, lower alkenyl, substituted lower alkenyl, lower
cycloalkenyl, aryl, substituted aryl, heteroaryl, and substituted
heteroaryl, preferably R.sup.1 is selected from the group
consisting of pyrrolyl, pyrazolyl, imidazolyl, oxazolyl,
isoxazolyl, oxadiazolyl, thiazolyl, isothiazolyl, thienyl, furanyl,
furazanyl, pyridinyl, pyrimidinyl, pyridazinyl, indolyl,
3H-indolyl, isoindolyl, indolinyl, indolizinyl, indazolyl,
dihydroindolyl, tetrahydroindolyl, purinyl, pyrazinyl, quinolinyl,
isoquinolinyl, tetrahydroisoquinolinyl, quinoxalinyl, quinazolinyl,
cinnolinyl, pteridinyl, benzimidazolyl, benzopyranyl, benzoxazolyl,
benzisoxazolyl, benzofuranyl, isobenzofuranyl, benzothiazolyl,
benzisothiazolyl, benzothienyl, furopyridinyl, phthalazinyl,
napthyridinyl, pyrazolopyridyl, pyrazolopyrimidinyl,
pyrrolopyridinyl, and tetrahydropyrrolopyridinyl optionally
substituted from moieties selected from the group consisting of
lower alkyl, halide, lower haloalkyl, hydroxyl, amino, lower
aminoalkyl, lower hydroxyalkyl, nitrile, lower alkyl nitrile, lower
alkyl ethers, lower alkoxy, lower alkoxyalkyl, lower
alkoxycarbonyl, lower alkylcarbonylamino, and thioalkyl.
68. A compound according to claim 64 wherein R.sup.1 is selected
from the group consisting of hydrogen, halide, lower alkyl,
substituted lower alkyl, lower alkenyl, substituted lower alkenyl,
substituted aryl, lower cycloalkenyl, aryl, heteroaryl, and
substituted heteroaryl; and wherein R.sup.2 is selected from the
group consisting of amide, hydroxyamide, methylamide,
--CH.sub.2N(CH.sub.3).sub.2, --CH.sub.2NR.sup.6R.sup.7,
##STR00284##
69. A compound according to claim 68 wherein R.sup.1 is selected
from the group consisting of pyrrolyl, pyrazolyl, imidazolyl,
oxazolyl, isoxazolyl, oxadiazolyl, thiazolyl, isothiazolyl,
thienyl, furanyl, furazanyl, pyridinyl, pyrimidinyl, pyridazinyl,
indolyl, 3H-indolyl, isoindolyl, indolinyl, indolizinyl, indazolyl,
dihydroindolyl, and tetrahydroindolyl optionally substituted from
moieties selected from the group consisting of lower alkyl, halide,
lower haloalkyl, hydroxyl, amino, lower aminoalkyl, lower
hydroxyalkyl, nitrile, lower alkyl nitrile, lower alkyl ethers,
lower alkoxy, lower alkoxyalkyl, lower alkoxycarbonyl, lower
alkylcarbonylamino, and thioalkyl.
70. A compound according to claim 64 wherein R.sup.2 is
##STR00285## wherein R.sup.8 is selected from the group consisting
of hydroxyl and amino; and wherein R.sup.9 is selected from the
group consisting of hydrogen, halide, lower alkyl, lower alkenyl,
lower alkynyl, morpholinyl, piperazinyl, lower alkyl piperazinyl,
heterocycloalkyl, lower cycloalkyl, lower cycloalkenyl; and lower
alkyl, lower alkenyl, and lower alkynyl optionally substituted with
moieties selected from the group consisting of hydroxyl, amino,
lower aminoalkyl, halide, lower alkoxy, lower alkoxyalkyl, lower
alkoxycarbonyl, lower alkylcarbonylamino, carboxyl, amide,
hydroxyamide, --CONHCH.sub.3, --NHCONH.sub.2, --SO.sub.2NH.sub.2,
--SO.sub.2CH.sub.3, --NHCOCH.sub.3, --NHCSNH.sub.2, and
--NHSO.sub.2CH.sub.3 preferably R.sup.9 is hydrogen; and salts
thereof.
71. A compound according to claim 70 wherein R.sup.1 is selected
from the group consisting of heteroaryl and substituted heteroaryl,
wherein R.sup.2 is ##STR00286## wherein R.sup.8 is selected from
the group consisting of hydroxyl and amino; and wherein R.sup.9 is
hydrogen.
72. A compound according to claim 64 wherein the compound
corresponds to the following chemical structure: ##STR00287##
wherein R.sup.1 is selected from the group consisting of hydrogen,
methyl, halide, lower alkyl, substituted lower alkyl, lower
alkenyl, substituted lower alkenyl, aryl, substituted aryl,
heteroaryl, and substituted heteroaryl, preferably R.sup.1 is
selected from the group consisting of heteroaryl and substituted
heteroaryl.
73. A compound according to claim 63, wherein the compound
corresponds to the following chemical structure: ##STR00288##
wherein R1 is selected from the group consisting of hydrogen,
methyl, halide, nitrile, lower alkyl, substituted lower alkyl,
lower alkenyl, substituted lower alkenyl, lower alkynyl,
substituted lower alkynyl, aryl, substituted aryl, heteroaryl, and
substituted heteroaryl; wherein R3 is selected from the group
consisting of hydrogen and methyl; and wherein one of R4 and R5 is
hydrogen and the other is methyl; and salts thereof.
74. A compound according to claim 63 wherein the compound
corresponds to the following chemical structure: ##STR00289##
75. A method for inhibiting or reducing the formation or growth of
a biofilm comprising contacting said biofilm or a cell capable of
biofilm formation or growth with an effective amount of a compound
or a composition comprising the compound corresponding to the
following chemical structure: ##STR00290## wherein R1 is selected
from the group consisting of hydrogen, methyl, halide, lower
haloalkyl, nitrile, lower alkyl nitrile, lower alkyl, substituted
lower alkyl, lower alkenyl, substituted lower alkenyl, lower
alkynyl, substituted lower alkynyl, lower cycloalkyl, lower
cycloalkenyl, aryl, substituted aryl, heteroaryl, and substituted
heteroaryl; wherein R.sup.2 is selected from the group consisting
of carboxyl, amide, hydroxyamide, methylamide,
--CH.sub.2N(CH.sub.3).sub.2, --CH.sub.2NR.sup.6R.sup.7,
##STR00291## wherein R.sup.3 is selected from the group consisting
of hydrogen and methyl; wherein one of R.sup.4 and R.sup.5 is
hydrogen and the other is methyl; wherein R.sup.6 and R.sup.7 are
independently selected from the group consisting of hydrogen,
methyl, ethyl, propyl, isopropyl, and butyl; wherein R.sup.8 is
selected from the group consisting of hydroxyl, amino,
--N(CH.sub.3).sub.2, and --NHCH.sub.3; wherein R.sup.9 is selected
from the group consisting of hydrogen, halide, lower alkyl, lower
alkenyl, lower alkynyl, morpholinyl, piperazinyl, lower alkyl
piperazinyl, heterocycloalkyl, lower cycloalkyl, lower
cycloalkenyl; and lower alkyl, lower alkenyl, and lower alkynyl
optionally substituted with moieties selected from the group
consisting of hydroxyl, amino, lower aminoalkyl, halide, lower
alkoxy, lower alkoxyalkyl, lower alkoxycarbonyl, lower
alkylcarbonylamino, carboxyl, amide, hydroxyamide, --CONHCH.sub.3,
--NHCONH.sub.2, --SO.sub.2NH.sub.2, --SO.sub.2CH.sub.3,
--NHCOCH.sub.3, --NHCSNH.sub.2, and --NHSO.sub.2CH.sub.3; and
wherein R.sup.10 and R.sup.11 together with the carbon atoms to
which they are attached form an aromatic N-heterocycle which is
substituted or not selected from the group consisting of indole,
imidazole, quinoline, isoquinoline, pyridine, triazine, pyrazine,
pyrimidine, pyridazine, isoindole, pyrrole, benzimidazole, purine,
pyrazole, benzopyrazole, indazole, quinoxaline, acridine,
quinazoline, indolizine, carbazole and cinnoline; and salts
thereof.
76. A method according to claim 75 wherein the compound corresponds
to the following chemical structure: ##STR00292## wherein R.sup.1
is selected from the group consisting of hydrogen, methyl, halide,
lower haloalkyl, nitrile, lower alkyl nitrile, lower alkyl,
substituted lower alkyl, lower alkenyl, substituted lower alkenyl,
lower alkynyl, substituted lower alkynyl, lower cycloalkyl, lower
cycloalkenyl, aryl, substituted aryl, heteroaryl, and substituted
heteroaryl; wherein R.sup.2 is selected from the group consisting
of carboxyl, amide, hydroxyamide, methylamide,
--CH.sub.2N(CH.sub.3).sub.2, --CH.sub.2NR.sup.6R.sup.7,
##STR00293## wherein R.sup.3 is selected from the group consisting
of hydrogen and methyl; wherein one of R.sup.4 and R.sup.5 is
hydrogen and the other is methyl; wherein R.sup.6 and R.sup.7 are
independently selected from the group consisting of hydrogen,
methyl, ethyl, propyl, isopropyl, and butyl; wherein R.sup.8 is
selected from the group consisting of hydroxyl, amino,
--N(CH.sub.3).sub.2, and --NHCH.sub.3; and wherein R.sup.9 is
selected from the group consisting of hydrogen, halide, lower
alkyl, lower alkenyl, lower alkynyl, morpholinyl, piperazinyl,
lower alkyl piperazinyl, heterocycloalkyl, lower cycloalkyl, lower
cycloalkenyl; and lower alkyl, lower alkenyl, and lower alkynyl
optionally substituted with moieties selected from the group
consisting of hydroxyl, amino, lower aminoalkyl, halide, lower
alkoxy, lower alkoxyalkyl, lower alkoxycarbonyl, lower
alkylcarbonylamino, carboxyl, amide, hydroxyamide, --CONHCH.sub.3,
--NHCONH.sub.2, --SO.sub.2NH.sub.2, --SO.sub.2CH.sub.3,
--NHCOCH.sub.3, --NHCSNH.sub.2, and --NHSO.sub.2CH.sub.3; and salts
thereof.
77. A method according to claim 75 wherein the compound corresponds
to the following chemical structure: ##STR00294## wherein R1 is
selected from the group consisting of hydrogen, methyl, halide,
nitrile, lower alkyl, substituted lower alkyl, lower alkenyl,
substituted lower alkenyl, lower alkynyl, substituted lower
alkynyl, aryl, substituted aryl, heteroaryl, and substituted
heteroaryl; wherein R3 is selected from the group consisting of
hydrogen and methyl; and wherein one of R4 and R5 is hydrogen and
the other is methyl; and salts thereof.
78. A method according to claim 75, wherein the compound
corresponds to the followings chemical structure: ##STR00295##
Description
FIELD OF THE INVENTION
[0001] The present invention generally relates to compounds and
compositions useful for reducing or inhibiting the growth of a
biofilm. The present invention also relates to compounds useful for
reducing or inhibiting the formation of a biofilm and for
controlling or treating a chronic bacterial infection involving
biofilms.
BACKGROUND
[0002] Bacterial biofilms exist in natural, medical, and
engineering environments. The biofilms offer a selective advantage
to a microorganism to ensure its survival, or allow it a certain
amount of time to exist in a dormant state until growth conditions
arise. Unfortunately, this selective advantage poses serious
threats to animal health, especially human health.
[0003] Chronic infections involving biofilms are serious medical
problems throughout the world. For example, biofilms are involved
in 65% of human bacterial infections. Biofilms are involved in
prostatitis, biliary tract infections, urinary tract infections,
cystitis, lung infections, sinus infections, ear infections, acne,
rosacea, dental caries, periodontitis, nosocomial infections, open
wounds, and chronic wounds.
[0004] Compounds that modify biofilm formation would have a
substantial medical impact by treating many chronic infections,
reducing catheter- and medical device-related infections, and
treating lung and ear infections. The potential market for biofilm
inhibitors could be enormous given the sheer number of cases in
which biofilms contribute to medical problems. The inhibitors may
be used to cure, treat, or prevent a variety of conditions, such
as, but are not limited to, arterial damage, gastritis, urinary
tract infections, pyelonephritis, cystitis, otitis media, otitis
externa, leprosy, tuberculosis, benign prostatic hyperplasia,
chronic prostatitis, chronic lung infections of humans with cystic
fibrosis, osteomyelitis, bloodstream infections, skin infections,
open or chronic wound infections, cirrhosis, and any other acute or
chronic infection that involves or possesses a biofilm.
[0005] In the United States, the market for antibiotics is greater
than $10 billion. The antibiotic market is fueled by the continued
increase in resistance to conventional antibiotics. Approximately
70% of bacteria found in hospitals resist at least one of the most
commonly prescribed antibiotics. Because biofilms appear to reduce
or prevent the efficacy of antibiotics, co-administration of
biofilm inhibitors could significantly boost the antibiotic
market.
[0006] Using the protection of biofilms, microbes can resist
antibiotics at a concentration ranging from 1 to 1.5 thousand times
higher than the amount used in conventional antibiotic therapy.
During an infection, bacteria surrounded by biofilms are rarely
resolved by the immune defense mechanisms of the host. It has been
proposed that in a chronic infection, a biofilm gives bacteria a
selective advantage by reducing the penetration of an antibiotic
into the depths of the tissue needed to completely eradicate the
bacteria's existence (Costerton, J. W. et al., Science. 1999 May
21; 284(5418):1318-22).
[0007] Traditionally, antibiotics are discovered using the
susceptibility test methods established by the Clinical Laboratory
and Standards Institute (CLSI). The methods identify compounds that
specifically affect growth or death of bacteria. These methods
involve inoculation of a bacterial species into a growth medium,
followed by the addition of a test compound, and then plot of the
bacterial growth over a time period post-incubation. Unfortunately
these antibiotics derived from the CLSI methods would not be
effective therapeutics against chronic infections involving
biofilms because the methods do not test compounds against bacteria
in a biofilm. Consistently, numerous publications have reported a
difference in gene transcription in bacteria living in biofilms
from bacteria in suspension, which further explains the failure of
conventional antibiotics to eradicate biofilm infections (Sauer, K.
et al. J. Bacteriol. 2001, 183: 6579-6589).
[0008] Biofilm inhibitors can provide an alternative treatment
approach for certain infections. Biofilm inhibitors, on the other
hand, act on the biological mechanisms that provide bacteria
protection from antibiotics and from a host's immune system.
Biofilm inhibitors may be used to "clear the way" for the
antibiotics to penetrate the affected cells and eradicate the
infection. Traditionally, treatment of nosocomial infections
requires an administration of a combination of products, such as
amoxicillin/clavulanate and quinupristin/dalfopristin, or an
administration of two antibiotics simultaneously. In one study of
urinary catheters, rifampin was unable to eradicate
methicillin-resistant Staphylococcus aureus in a biofilm but was
effective against planktonic, or suspended cells (Jones, S. M., et.
al., "Effect of vancomycin and rifampicin on methicillin-resistant
Staphylococcus aureus biofilms", Lancet 357:40-41, 2001).
[0009] Bacteria have no known resistance to biofilm inhibitors.
Biofilm inhibitors are not likely to trigger growth-resistance
mechanisms or affect the growth of the normal human flora. Thus,
biofilm inhibitors could potentially extend the product life of
antibiotics.
[0010] Pseudomonas aeruginosa in the lungs of cystic fibrosis (CF)
patients is resistant to high doses of antibiotics because it forms
biofilms. Biofilms are complex, heterogeneous communities of
bacterial cells encased in extrapolymeric substances (EPS). These
EPS are composed of polysaccharides, proteins, and extracellular
DNA. EPS provide frameworks for communities of bacteria to exist
and enhance attachment to themselves and surfaces. Bacteria within
biofilms differentiate into stratified communities of
phenotypically diverse cells that offer competitive advantages for
different environmental conditions. One of these advantages is
increased tolerance to antibiotics, which enables bacteria like P.
aeruginosa to persist in chronic infections despite antibiotic
therapy. While residing within these EPS, one hypothesis is that P.
aeruginosa reduces its metabolism, which prevents its death from
antibiotics. As antibiotic concentrations are reduced, specific
populations of P. aeruginosa within the EPS increase their
metabolism and spread. This cycle continuously repeats, enabling
the spread of P. aeruginosa bacteria within the lungs of CF
patients.
[0011] Chronic wound infection represents another illness that is
difficult to eradicate. Examples of the most common types of
chronic wounds are diabetic foot ulcers, venous leg ulcers,
arterial leg ulcers, and pressure ulcers. Diabetic foot ulcers
appear to be the most prevalent. These wounds are typically
colonized by multiple species of bacteria including Staphylococcus
spp., Streptococcus spp., Pseudomonas spp. and Gram-negative
bacilli (Lipsky, B. Medical Treatment of Diabetic Foot Infections.
Clin. Infect. Dis. 2004, 39, p. S104-14).
[0012] Based on clinical evidence, microorganisms cause or
contribute to chronic wound infections. Only recently have biofilms
been implicated in these infections (Harrison-Balestra, C. et al. A
Wound-isolated Pseudomonas aeruginosa Grow a Biofilm In Vitro
Within 10 Hours and Is Visualized by Light Microscopy, Dermatol
Surg 2003, 29; 631-635; Edwards, R. et al. Bacteria and wound
healing. Curr Opin Infect Dis, 2004, 17; 91-96). Approximately
140,000 amputations occur each year in the United States due to
chronic wound infections that could not be treated with
conventional antibiotics. Unfortunately, treating these infections
with high doses of antibiotics over long periods of time
contributes to the development of antibiotic resistance
(Howell-Jones, R. S., et al. A review of the microbiology,
antibiotic usage and resistance in chronic skin wounds. J.
Antimicrob. Ther. January 2005). Biofilm inhibitors in a
combination therapy with antibiotics may provide an effective
alternative to the treatment of chronic wounds.
[0013] Recent publications describe the cycles of the pathogenesis
of numerous species of bacteria involving biofilms. For example,
Escherichia coli, which cause recurrent urinary tract infections,
undergo a cycle of binding to and then invading a host's bladder
epithelial cells. E. coli form a biofilm intracellularly, modify
its morphology, and then burst out of the host cells to repeat the
cycle of pathogenesis (Justice, S. et al. Differentiation and
development pathways of uropathogenic Escherichia coli in urinary
tract pathogenesis, PNAS 2004, 101(5): 1333-1338). The authors
suggest that this repetitive cycle of pathogenesis of E. coli may
explain the recurrence of the infection.
[0014] In 1997, Finlay, B. et al. reported that numerous bacteria,
including Staphylococci, Streptococci, Bordetella pertussis,
Neisseria spp., Helicobactor pylori, and Yersinia spp., adhere to
mammalian cells during their pathogenesis. The authors hypothesized
that the adherence would lead to an invasion of the host cell.
Later publications confirm this hypothesis (Cossart, P. Science,
2004, 304; 242-248; see additional references infra). Other
publications presented similar hypotheses to Mulvey, M. et al.
(Mulvey, M. et al. "Induction and Evasion of Host Defenses by Type
1-Piliated Uropathogenic E. coli" Science 1998, 282 p. 1494-1497).
In particular, Mulvey, M. et al. stated invasion of E. coli into
epithelial cells provide protection from the host's immune response
to allow a build up of a large bacterial population.
[0015] Cellular invasion and biofilm formation appear to be
integral to the pathogenesis of most, if not all bacteria. P.
aeruginosa have been shown to invade epithelial cells during lung
infections (Leroy-Dudal, J. et al. Microbes and Infection, 2004, 6,
p. 875-881). P. aeruginosa are the principal infectious organisms
found in the lungs of cystic fibrosis patients, and the bacteria
exist within a biofilm. Antibiotics like tobramcyin, and other
current antibacterial compounds, do not provide effective treatment
against biofilms of chronic infections, perhaps because antibiotic
therapy fails to eradicate the biofilm.
[0016] The pathogenesis of cellular invasion and biofilm formation
gram-negative bacteria follow conserved mechanisms. For example,
Haemophilus influenzae invade epithelial cells and form biofilms
(Hardy, G. et al., Methods Mol. Med., 2003, 71; 1-18; Greiner, L.
et al., Infection and Immunity, 2004, 72(7); 4249-4260).
Burkholderia spp. invade epithelial cells and form biofilm
(Utaisincharoen, P. et al., Microb Pathog. 2005, 38(2-3); 107-112;
Schwab, U. et al. Infection and Immunity, 2003, 71(11); 6607-6609).
Klebsiella pneumoniae invade epithelial cells and form biofilm
(Cortes, G et al. Infection and Immunity. 2002, 70(3); 1075-1080;
Lavender, hours. et al., Infection and Immunity. 2004, 72(8);
4888-4890). Salmonella spp. invade epithelial cells and form
biofilms (Cossart, P. Science, 2004, 304; 242-248; Boddicker, J. et
al., Mol. Microbiol. 2002, 45(5); 1255-1265). Yersinia pestis
invade epithelial cells and form biofilms (Cossart, P. Science,
2004, 304; 242-248; Jarrett, C. et al. J. Infect. Dis., 2004, 190;
783-792). Neisseria gonorrhea invade epithelial cells and form
biofilms (Edwards, J. et al., Cellular Micro., 2002, 4(9); 585-598;
Greiner, L. et al., Infection and Immunity. 2004, 73(4);
1964-1970). Burkholderia spp. are another important class of
gram-negative bacterial pathogens. Chlamydia spp., including
Chlamydia pneumoniae is an intracellular, gram-negative pathogen
implicated in respiratory infections and chronic diseases such as
atherosclerosis and Alzheimer's disease (Little, C. S. et al.,
Infection and Immunity. 2005, 73(3); 1723-34).
[0017] These Gram-negative bacteria cause lung, ear, and sinus
infections, gonorrhoeae, plague, diarrhea, typhoid fever, and other
infectious diseases. E. coli and P. aeruginosa are two of the most
widely studied Gram-negative pathogens. Researchers believe that
the pathogenesis of these bacteria involves invasion of host cells
and formation of biofilms. These models have enabled those skilled
in the art to understand the pathogenesis of other Gram-negative
bacteria.
[0018] Accordingly, for the reasons discussed above and others,
there exists an unmet need for compounds that serve as biofilm
inhibitors and/or that would be useful for reducing or inhibiting
the formation or growth of bacterial biofilms and bacterial
infections involving biofilms.
SUMMARY OF INVENTION
[0019] The present invention provides compounds of the following
chemical Structure I
##STR00001##
wherein R.sup.1 is selected from the group consisting of hydrogen,
methyl, halide, lower haloalkyl, nitrile, lower alkyl nitrile,
lower alkyl, substituted lower alkyl, lower alkenyl, substituted
lower alkenyl, lower alkynyl, lower cycloalkyl, lower cycloalkenyl,
substituted lower alkynyl, aryl, substituted aryl, heteroaryl, and
substituted heteroaryl; R.sup.2 is selected from the group
consisting of carboxyl, amide, hydroxyamide, methylamide,
--CH.sub.2N(CH.sub.3).sub.2, --CH.sub.2NR.sup.6R.sup.7,
##STR00002##
[0020] R.sup.3 is selected from the group consisting of hydrogen or
methyl; one of R.sup.4 and R.sup.5 is hydrogen and the other is
methyl; R.sup.6 and R.sup.7 are independently selected from the
group consisting of hydrogen, methyl, ethyl, propyl, isopropyl, and
butyl; R.sup.8 selected from the group consisting of hydroxyl,
amino, --N(CH.sub.3).sub.2, and --NHCH.sub.3; and wherein R.sup.9
is selected from the group consisting of hydrogen, halide, lower
alkyl, lower alkenyl, lower alkynyl, morpholinyl, piperazinyl,
lower alkyl piperazinyl, heterocycloalkyl, lower cycloalkyl, lower
cycloalkenyl; and lower alkyl, lower alkenyl, and lower alkynyl
substituted with moieties selected from the group consisting of
hydroxyl, amino, lower aminoalkyl, halide, lower alkoxy, lower
alkoxyalkyl, lower alkoxycarbonyl, lower alkylcarbonylamino,
carboxyl, amide, hydroxyamide, --CONHCH.sub.3, --NHCONH.sub.2,
--SO.sub.2NH.sub.2, --SO.sub.2CH.sub.3, --NHCOCH.sub.3,
--NHCSNH.sub.2, and --NHSO.sub.2CH.sub.3. Salts are also
contemplated by the present invention as described in the
examples.
[0021] A number of the compounds of the invention and intermediates
may exist in different tautomeric forms. All such tautomeric forms
are within the scope of the invention. The depiction of any
tautomer herein is not intended to limit the scope of the invention
to one specific tautomer. For example, the following is within the
scope of the invention.
[0022] Those skilled in the art will understand that Structure I
may exist as a tautomer envisioned as the following. In any
respect, 3-aminopyrazole of any structure of the invention may be
as follows:
##STR00003##
[0023] Or a 3-aminopyrazole of any structure of the invention may
be as follows:
##STR00004##
[0024] These tautomeric depictions are within the scope of the
invention.
[0025] Furthermore, the hydroxypyrazole of compound 68 as described
herein may exist as tautomers. These tautomers are within the scope
of the invention.
[0026] Compositions containing the compounds described above and a
pharmaceutically acceptable carrier are also contemplated by this
invention. Such compositions containing the compounds described
above optionally include an antimicrobial agent. As demonstrated
herein such compositions are useful in reducing or inhibiting the
formation or growth of biofilms.
[0027] This invention also provides methods for reducing or
inhibiting the formation or growth of biofilms comprising
contacting the biofilm or cell capable of biofilm formation with an
effective amount of a composition or a compound of the preceeding
chemical Structure I wherein R.sup.1 is selected from the group
consisting of hydrogen, methyl, halide, lower haloalkyl, nitrile,
lower alkyl nitrile, lower alkyl, substituted lower alkyl, lower
alkenyl, substituted lower alkenyl, lower alkynyl, substituted
lower alkynyl, lower cycloalkyl, lower cycloalkenyl, aryl,
substituted aryl, heteroaryl, and substituted heteroaryl; R.sup.2
is selected from the group consisting of carboxyl, amide,
hydroxyamide, methylamide, --CH.sub.2N(CH.sub.3).sub.2,
--CH.sub.2NR.sup.6R.sup.7,
##STR00005##
[0028] R.sup.3 is selected from the group consisting of hydrogen or
methyl; one of R.sup.4 and R.sup.5 is hydrogen and the other is
methyl; R.sup.6 and R.sup.7 are independently selected from the
group consisting of hydrogen, methyl, ethyl, propyl, isopropyl, and
butyl; R.sup.8 selected from the group consisting of hydroxyl,
amino, --N(CH.sub.3).sub.2, and --NHCH.sub.3; and R.sup.9 is
selected from the group consisting of hydrogen, halide, lower
alkyl, lower alkenyl, lower alkynyl, morpholinyl, piperazinyl,
lower alkyl piperazinyl, heterocycloalkyl, lower cycloalkyl, lower
cycloalkenyl; and lower alkyl, lower alkenyl, and lower alkynyl
substituted with moieties selected from the group consisting of
hydroxyl, amino, lower aminoalkyl, halide, lower alkoxy, lower
alkoxyalkyl, lower alkoxycarbonyl, lower alkylcarbonylamino,
carboxyl, amide, hydroxyamide, --CONHCH.sub.3, --NHCONH.sub.2,
--SO.sub.2NH.sub.2, --SO.sub.2CH.sub.3, --NHCOCH.sub.3,
--NHCSNH.sub.2, and --NHSO.sub.2CH.sub.3. Salts are also
contemplated by the present invention as described in the
examples.
[0029] Inhibition or reduction of the formation or growth of
biofilms may be effected either in vivo or in vitro. Compositions
used to inhibit or reduce the formation or growth of biofilms may
further include an antimicrobial agent, biocide, or antibiotic. The
methods also provide for inhibiting or reducing the formation or
growth of biofilms on a variety of substrates.
[0030] Inhibition or reduction of the formation or growth of
biofilms reduces virulence of gram-negative bacteria. Adhesion,
biofilm growth, invasion, and the secretion of enzymes or toxins
contribute to virulence of gram-negative bacteria. It is well known
to those skilled in the art that biofilms increase the virulence of
gram-negative bacteria and an upregulation of virulence factors
(i.e. enzymes, toxins) has been demonstrated in gram-negative
bacterial biofilms. The compounds of the invention reduce virulence
of gram-negative bacteria.
[0031] Compound 1 as described herein modulates cysB and genes
under its control as described in U.S. Pat. No. 7,604,978
incorporated herein in its entirety by this reference. It is
anticipated that all of the compounds described herein modulate
cysB and genes under its control.
BRIEF DESCRIPTION OF THE DRAWINGS
[0032] FIG. 1 is a set of photographs of the Biofilm Growth Assay
for Compound 1, as described in Example I, below, wherein the round
plates on the left are negative controls, in which the agar in the
plates does not contain Compound 1, and wherein, as shown in the
negative control plates, P. aeruginosa spread out from the
inoculation point in the center of the plate during overnight
incubation at 37.degree. C. and covered the entire plate, whereas
the agar in the plates on the right contained 0.5 .mu.g/ml of
Compound 1 of the invention, which prevented the growth of the
spreading P. aeruginosa biofilm.
[0033] FIG. 2 is a set of photographs of the Biofilm Growth Assay
for Compound 1 in the presence of colistin (antibiotic) disks as
described in Example VIII, below, wherein the top left plate was a
negative control plate with paper disks without antibiotic and the
top right plate had 0.125 .mu.g/ml of Compound 1 in the agar on the
right side of the plate with paper disks without antibiotic (and
0.125 .mu.g/ml of Compound 1 was two dilutions below its optimal
working concentration of 0.5 .mu.g/ml), and the bottom left plate
had colistin 10 .mu.g disks, while the bottom right plate had 0.125
.mu.g/ml of Compound 1 in the agar on the right side of the plate
with colistin 10 .mu.g disks, the inhibition of the growth of P.
aeruginosa biofilm in the bottom right plate demonstrating that
Compound 1 and colistin (antibiotic) are synergistic at preventing
the growth of P. aeruginosa biofilms.
DESCRIPTION OF THE INVENTION
Definitions
[0034] "Acceptable carrier" refers to a carrier that is not
deleterious to the other ingredients of the composition and is not
deleterious to material to which it is to be applied.
[0035] "Pharmaceutically acceptable carrier" refers to a carrier
that is not deleterious to the other ingredients of the composition
and is not deleterious to the human or other animal recipient
thereof. "Agriculturally acceptable carrier" refers to a carrier
that is not deleterious to the other ingredients of the composition
and is not deleterious to the plant recipient thereof. In the
context of the other ingredients of the composition, "not
deleterious" means that the carrier will not react with or degrade
the other ingredients or otherwise interfere with their efficacy.
Interference with the efficacy of an ingredient does not encompass
mere dilution of the ingredient. In the context of the animal or
plant host, "not deleterious" means that the carrier is not
injurious or lethal to the plant or animal.
[0036] "Administration" refers to any means of providing a compound
or composition to a subject. Non-limiting examples of
administration means include oral, topical, rectal, percutaneous,
parenteral injection, intranasal and inhalation delivery.
[0037] "Biofilm" refers to an extracellular matrix in which
microorganisms are dispersed and/or form colonies. The biofilm
typically is made of polysaccharides and other macromolecules.
[0038] "Biofilm Growth Assay" refers to an assay performed on
semi-solid agar surface where the bacteria collectively move out
from an inoculation point. To those skilled in the art, the act of
bacteria collectively moving out from an inoculation point on a
semi-solid surface may be referred to as swarming or a spreading
biofilm. (Anderson, et al Microbiology, 2003, 149, 37-46; Daniels,
et al. FEMS Microbiology Reviews, 2004, 28, 261-289). The procedure
to perform this assay is described in detail in the Examples. This
type of surface motility by bacteria has been reported in the
literature by those skilled in the art. (Shrout, et al. Molecular
Microbiology, 2006, 62(5), 1264-1277; Kim, et al. J. Bacteriology,
2003, 185(10), 3111-3117; Lai, et al. Environmental Microbiology,
2009, 11(1), 126-136; Overhage, et al. J. Bacteriology, 2008,
190(8), 2671-2679) These literature references also demonstrate a
link to this type of surface motility and increased antibiotic
tolerance. These references are incorporated herein by reference in
their entirety.
[0039] "Commercial source" refers to a vendor that provides the
desired compound.
[0040] "Direct synthesis" refers to production of the desired
compound by reacting appropriate compound precursors under
appropriate conditions to obtain the desired compound.
[0041] "Effective amount" refers to the amount of compound or
composition that, in the case of biofilm formation, will reduce the
size or volume of existing biofilms; reduce the rate at which
bacteria are capable of producing biofilm; or will inhibit or
prevent the formation of biofilm by one or more microorganisms. In
the context of treating a bacterial infection, an "effective
amount" refers the amount of a compound or composition that will
reduce the degree of an existing infection or will inhibit or
prevent an infection from occurring.
[0042] "Essentially pure preparation" refers to a preparation in
which the concentration of the desired ingredient is at least 95%
or more of the preparation by weight. In the context of this
processes used in this invention, the antimicrobial agents and
compounds of the invention typically and preferably make up 99% or
more by weight of the preparation and are referred to herein as
"highly pure" preparations.
[0043] "In vivo", in the context of biofilm formation, refers to
effects mediated in or upon living organisms or subjects. Effects
mediated on biofilms associated with medical devices such as
central venous catheters, urinary catheters, endotracheal tubes,
mechanical heart valves, pacemakers, vascular grafts, stents, and
prosthetic joints located within a living organism or subject are
considered as "in vivo" uses of the compounds and compositions
described herein.
[0044] "In vitro", in the context of biofilm formation, refers to
effects mediated on substrates located outside of an organism that
are potential sites of biofilm formation. Non-limiting examples of
substrates include vessel hulls, cars, airplanes, industrial
equipment, devices, membranes, filters, microtiter plates,
continuous flow chambers, bioreactors, fermentors, chemostats and
machinery.
[0045] "Is one that permits" as it relates to a pharmaceutically
acceptable carrier that has characteristics that enable the
preparation to be used for a given mode of administration of the
composition. For example, pharmaceutically acceptable carriers that
permit parenteral administration to an animal are liquids that are
not injurious or lethal to the animals when so injected. Such
carriers often comprise sterile water, which may be supplemented
with various solutes to increase solubility. Sterile water or
sterile water supplemented with solutes is thus a pharmaceutically
acceptable carrier that permits parental administration.
[0046] As used herein, all agar percentages are expressed in terms
of weight/volume; all formulation percentages are expressed in
terms of weight/weight.
[0047] "Reducing or inhibiting" in reference to a biofilm refers to
the inhibiting or reducing of biofilm formation or growth, a
reduction in the rate of biofilm formation or growth, reduction or
removal of preformed or existing biofilm, as well as the partial or
complete inhibition of biofilm formation or growth. This definition
includes but is not limited to the biofilm growth that also occurs
on semi-solid surfaces like 0.4% to 1.0% agar, but is not limited
to these surfaces, as described in the Examples of the invention.
This type of surface motility, which is also referred to as
swarming or spreading biofilms and conducted on the same semi-solid
agar plates independent of terminology, demonstrates mechanistic
relationships between attached biofilms and gram-negative bacteria
spreading across semi-solid agar plates as known to those skilled
in the art. Michael Givskov and colleagues defined the movement of
a Pseudomonas spp. across a 0.6% Bacto agar plate as surface
motility with biofilm structures instead of swarming (Anderson, et
al Microbiology, 2003, 149, 37-46). Jan Michiels and colleagues
defined swarming as a biofilm spreading over a semi-solid surface
(Daniels, et al. FEMS Microbiology Reviews, 2004, 28, 261-289).
Even though terminology is evolving as more investigations are
reported, all of these data have been generated examining the
movement of gram-negative bacteria on 0.4% to 0.7% agar
surfaces.
[0048] "Subject in need thereof" refers to living organism that
would benefit from either prevention or reductions in the degree of
a bacterial infection. Subjects may include animals or more
specifically, mammals or humans. Subjects may also include
plants.
[0049] "Substrate" refers to any material to which the compound or
a composition containing the compound may be applied.
[0050] The term "lower alkyl" as used herein refers to a saturated
hydrocarbon chain having one, two, three, four, or five carbon
atoms. Lower alkyl groups may be optionally substituted with one or
more substituents as defined herein to form substituted lower alkyl
groups. Lower alkyl groups may be straight or branched. Examples of
lower alkyl groups include methyl, ethyl, propyl, isopropyl, butyl,
isobutyl, t-butyl, pentyl, and isopentyl.
[0051] The phrase "substituted lower alkyl" as used herein, refers
to a lower alkyl group, as previously defined, substituted by
independent replacement of one, two, or three of the hydrogen atoms
thereon with substituents including halide, nitrile, aryl,
heteroaryl, substituted heteroaryl, lower cycloalkyl, lower
cycloalkenyl, --SH, and lower thioalkyl.
[0052] The term "lower alkenyl" as used herein refers to an
unsaturated hydrocarbon chain having one, two, three, four, or five
carbon atoms and having one or more carbon-carbon double bonds
within the chain. The lower alkenyl groups may be straight or
branched and may be optionally substituted with one or more
substituents as defined herein to form substituted lower anlkenyl
groups.
[0053] The phrase "substituted lower alkenyl" as used herein,
refers to a lower alkenyl group, as previously defined, substituted
by independent replacement of one, two, or three of the hydrogen
atoms thereon with substituents including halide, nitrile, aryl,
heteroaryl, substituted heteroaryl, lower cycloalkyl, lower
cycloalkenyl, --SH, and lower thioalkyl.
[0054] The term "lower alkynyl" as used herein refers to an
unsaturated hydrocarbon chain having one, two, three, four, or five
carbon atoms and having one or more carbon-carbon triple bonds
within the chain. The lower alkynyl groups may be straight or
branched and may be optionally substituted with one or more
substituents as defined herein to form substituted lower alkynyl
groups.
[0055] The phrase "substituted lower alkynyl" as used herein refers
to a lower alkynyl group, as previously defined, substituted by
independent replacement of one, two, or three of the hydrogen atoms
thereon with substituents including halide, nitrile, aryl,
heteroaryl, substituted heteroaryl, lower cycloalkyl, lower
cycloalkenyl, --SH, and lower thioalkyl.
[0056] The phrase "lower alkyl ethers" as used herein refers to
ethers of the formula R'OR'', wherein R' is a lower alkyl, lower
alkenyl or lower alkynyl, and R'' is a lower alkyl, lower alkenyl,
lower alkynyl, or aryl, heteroaryl or heterocycloalkyl.
[0057] The term "lower alkoxy," as used herein by itself or as part
of another substituent, means a radical of the formula --OR,
wherein R is a lower alkyl, lower alkenyl, lower cycloalkyl, or
lower cycloalkenyl group as defined herein. Representative examples
of lower alkoxy groups include, but are not limited to, methoxy,
ethoxy, propoxy, isopropoxy, butoxy, tert-butoxy, cyclopropyloxy,
cyclopentyloxy, and the like.
[0058] The term "lower alkoxyalkyl" as used herein refers to a
lower alkyl moiety as defined herein with one carbon atom replaced
with an oxygen atom. Examples include --CH.sub.2CH.sub.2OCH.sub.3
and --CH.sub.2OCH.sub.2CH.sub.2CH.sub.3.
[0059] The term "lower alkoxycarbonyl" as used herein by itself or
as part of another substituent, refers to a radical of the formula
--C(O)-(lower alkoxy), wherein lower alkoxy is as defined
herein.
[0060] The phrase "lower alkyl nitrile" as used herein refers to
lower alkyl or lower alkenyl with one nitrile group replacing one
terminal carbon atom in the unbranched or branched chain. Lower
alkyl nitrile includes, but is not limited to, --CH.sub.2CN and
--CH.sub.2CH.sub.2CN.
[0061] The term "lower aminoalkyl" as used herein refers to lower
alkyl or lower alkenyl with one nitrogen atom replacing one carbon
atom in the unbranched or branched chain. Lower aminoalkyl
includes, but is not limited to, --NHCH.sub.2CH.sub.2CH.sub.3,
--CH.sub.2CH.sub.2--NHCH.sub.3, --CH.sub.2N(CH.sub.3).sub.2,
--N(CH.sub.3).sub.2, and --CH.sub.2CH.sub.2NH.sub.2.
[0062] The term "lower alkylcarbonylamino" as used herein refers to
lower alkyl with a carbonylamino or aminocarbonyl replacing two
carbon atoms in the unbranched or branched chain. Lower
alkylcarbonylamino includes, but is not limited to,
--NHCOCH.sub.2CH.sub.3, --CH.sub.2CONHCH.sub.3,
--CON(CH.sub.3).sub.2, and --CONHCH.sub.3 (methylamide).
[0063] The terms "halo" and "halogen," as used herein, mean an atom
selected from fluorine, chlorine, bromine and iodine and the term
"halide" and used herein means the corresponding anion.
[0064] The term "lower haloalkyl" as used herein refers to a lower
alkyl group wherein one or more hydrogen atoms attached to a member
atom within the lower alkyl group is replaced with 1, 2, 3, or 4
halide atoms. Lower haloalkyl includes, but are not limited to,
fluoromethyl, --CF.sub.3, difluoroethyl, and trifluoromethyl.
[0065] The term "lower hydroxyalkyl" as used herein refers to lower
alkyl or lower alkenyl wherein one or more hydrogen atoms attached
to a member atom within the lower alkyl or lower alkenyl group is
replaced with one or two hydroxyls. Lower hydroxyalkyl includes,
but is not limited to, --CH.sub.2CH.sub.2--OH and
--CH.sub.2CH(--OH)CH.sub.3, and the like.
[0066] The term "aryl" as used herein, means mono- or bicyclic
carbocyclic ring systems comprising 6 to 12 carbon atoms, which
consist of one or two aromatic rings, specifically including
phenyl, naphthyl, tetrahydronaphthyl, indanyl, and idenyl; and
specifically substitutions to these aryls by independent
replacement of one two, or three of the hydrogen atoms thereon with
substituents specifically selected from the group consisting of
halide, lower haloalkyl, amino, hydroxyl, lower alkoxy, lower
aminoalkyl, lower hydroxyalkyl, nitrile, lower alkyl nitrile, lower
alkyl, lower alkenyl, lower alkynyl, nitro, carboxyl, amide,
hydroxyamide, lower alkyl ethers, lower alkoxyalkyl, lower
alkoxycarbonyl, lower alkylcarbonylamino, --SH, and lower
thioalkyl.
[0067] The phrase "substituted aryl," as used herein, means a aryl
group, as previously defined, substituted by independent
replacement or one or two of the hydrogen atoms thereon with
substituents specifically selected from the group consisting of
--(CH.sub.2).sub.0-3-lower cycloalkyl, --(CH.sub.2).sub.0-3-aryl,
--(CH.sub.2).sub.0-3-lower cycloalkenyl,
--(CH.sub.2).sub.0-3-heteroaryl,
--(CH.sub.2).sub.0-3-heterocycloalkyl, --NH-lower cycloalkyl,
--NH-aryl, --NH-lower cycloalkenyl, --NH-heteroaryl,
--NH-heterocycloalkyl, --O-aryl, --O-heteroaryl,
--O-heterocycloalkyl, --C(O)-lower alkyl, --C(O)-lower alkenyl,
--C(O)-lower alkynyl, --C(O)-lower cycloalkyl, --C(O)-aryl,
--C(O)-lower cycloalkenyl, --C(O)-heteroaryl,
--C(O)-heterocycloalkyl, --CONH.sub.2, --CONH-lower alkyl,
--CONH-lower alkenyl, --CONH-lower alkynyl, --CONH-lower
cycloalkyl, --CONH-aryl, --CONH-lower cycloalkenyl,
--CONH-heteroaryl, --CONH-heterocycloalkyl, --OCO.sub.2-lower
alkyl, --OCO.sub.2-lower alkenyl, --OCO.sub.2-lower alkynyl,
--OCO.sub.2-lower cycloalkyl, --OCO.sub.2-aryl, --OCO.sub.2-lower
cycloalkenyl, --OCO.sub.2-heteroaryl, --OCO.sub.2-heterocycloalkyl,
--OCONH.sub.2, --OCONH-lower alkyl, --OCONH-lower alkenyl,
--OCONH-lower alkynyl, --OCONH-lower cycloalkyl, --OCONH-aryl,
--OCONH-lower cycloalkenyl, --OCONH-heteroaryl,
--OCONH-heterocycloalkyl, --NHC(O)-lower alkyl, --NHC(O)-lower
alkenyl, --NHC(O)-lower alkynyl, --NHC(O)-lower cycloalkyl,
--NHC(O)-aryl, --NHC(O)-lower cycloalkenyl, --NHC(O)-heteroaryl,
--NHC(O)-heterocycloalkyl, --NHCO.sub.2-lower alkyl,
--NHCO.sub.2-lower alkenyl, --NHCO.sub.2-lower alkynyl,
--NHCO.sub.2-lower cycloalkyl, --NHCO.sub.2-aryl,
--NHCO.sub.2-lower cycloalkenyl, --NHCO.sub.2-heteroaryl,
--NHCO.sub.2-heterocycloalkyl, --NHC(O)NH.sub.2, --NHC(O)NH-lower
alkyl, --NHC(O)NH-lower alkenyl, --NHC(O)NH-lower alkynyl,
--NHC(O)NH-lower cycloalkyl, --NHC(O)NH-aryl, --NHC(O)NH-lower
cycloalkenyl, --NHC(O)NH-heteroaryl, --NHC(O)NH-heterocycloalkyl,
--NHC(S)NH.sub.2, --NHC(S)NH-lower alkyl, --NHC(S)NH-lower alkenyl,
--NHC(S)NH-lower alkynyl, --NHC(S)NH-lower cycloalkyl,
--NHC(S)NH-aryl, --NHC(S)NH-lower cycloalkenyl,
--NHC(S)NH-heteroaryl, --NHC(S)NH-heterocycloalkyl, --S(O)-lower
alkyl, --S(O)-lower alkenyl, --S(O)-lower alkynyl, --S(O)-lower
cycloalkyl, --S(O)-aryl, --S(O)-lower cycloalkenyl,
--S(O)-heteroaryl, --S(O)-heterocycloalkyl,
--CH.sub.2SO.sub.2CH.sub.3, --SO.sub.2NH.sub.2, --SO.sub.2NH-lower
alkyl, --SO.sub.2NH-lower alkenyl, --SO.sub.2NH-lower alkynyl,
--SO.sub.2NH-lower cycloalkyl, --SO.sub.2NH-aryl,
--SO.sub.2NH-lower cycloalkenyl, --SO.sub.2NH-heteroaryl,
--SO.sub.2NH-heterocycloalkyl, --NHSO.sub.2-lower alkyl,
--NHSO.sub.2-lower alkenyl, --NHSO.sub.2-lower alkynyl,
--NHSO.sub.2-lower cycloalkyl, --NHSO.sub.2-aryl,
--NHSO.sub.2-lower cycloalkenyl, --NHSO.sub.2-heteroaryl,
--NHSO.sub.2-heterocycloalkyl, --S-lower alkenyl, --S-lower
alkynyl, --S-lower cycloalkyl, --S-aryl, --S-heteroaryl,
--S-heterocycloalkyl, or methylthiomethyl.
[0068] The phrase "lower cycloalkyl," as used herein, means a
saturated carbocyclic ring compound specifically including
cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, norbornane, and
adamantine; and specifically substitutions to these lower
cycloalkyls by independent replacement of one or two of the
hydrogen atoms thereon with substituents specifically selected from
the group consisting of halide, lower haloalkyl, amino, hydroxyl,
lower alkoxy, lower aminoalkyl, lower hydroxyalkyl, nitrile, lower
alkyl nitrile, lower alkyl, lower alkenyl, lower alkynyl, nitro,
carboxyl, amide, hydroxyamide, lower alkyl ethers, lower
alkoxyalkyl, lower alkoxycarbonyl, lower alkylcarbonylamino, --SH,
and thioalkyl.
[0069] The term "lower cycloalkenyl" as used herein specifically
refers to an unsaturated hydrocarbon ring with five or six carbons;
and specifically substitutions to these lower cycloalkenyls by
independent replacement of one or two of the hydrogen atoms thereon
with substituents specifically selected from the group consisting
of halide, lower haloalkyl, amino, hydroxyl, lower alkoxy, lower
aminoalkyl, lower hydroxyalkyl, nitrile, lower alkyl nitrile, lower
alkyl, lower alkenyl, lower alkynyl, nitro, carboxyl, amide,
hydroxyamide, lower alkyl ethers, lower alkoxyalkyl, lower
alkoxycarbonyl, lower alkylcarbonylamino, --SH, and thioalkyl.
[0070] The term "heteroaryl" as used herein specifically refers to
pyrrolyl, pyrazolyl, imidazolyl, oxazolyl, isoxazolyl, oxadiazolyl,
thiazolyl, isothiazolyl, thienyl, furanyl, furazanyl, pyridinyl,
pyrimidinyl, pyridazinyl, indolyl, 3H-indolyl, isoindolyl,
indolinyl, indolizinyl, indazolyl, dihydroindolyl,
tetrahydroindolyl, purinyl, pyrazinyl, quinolinyl, isoquinolinyl,
tetrahydroisoquinolinyl, quinoxalinyl, quinazolinyl, cinnolinyl,
pteridinyl, benzimidazolyl, benzopyranyl, benzoxazolyl,
benzisoxazolyl, benzofuranyl, isobenzofuranyl, benzothiazolyl,
benzisothiazolyl, benzothienyl, furopyridinyl, phthalazinyl,
napthyridinyl, pyrazolopyridyl, pyrazolopyrimidinyl; specifically
their partially reduced forms as known to those skilled in the art
like as tetrahydroisoquinolinyl is to isoquinolinyl; and
specifically a heteroaryl (as defined herein) of 6 members or less
fused with an aryl (as defined herein) of 6 members or less OR
separately two heteroaryls (as defined herein) of 6 members or less
fused together as known to those skilled in the art like as in
pyrrolopyridinyl and its partially reduced form
dihydropyrrolopyridinyl; and specifically substitutions to these
heteroaryls by independent replacement of one two, or three of the
hydrogen atoms thereon with substituents specifically selected from
the group consisting of halide, lower haloalkyl, amino, hydroxyl,
lower alkoxy, lower aminoalkyl, lower hydroxyalkyl, nitrile, lower
alkyl nitrile, lower alkyl, lower alkenyl, lower alkynyl, nitro,
carboxyl, amide, hydroxyamide, lower alkyl ethers, lower
alkoxyalkyl, lower alkoxycarbonyl, lower alkylcarbonylamino, --SH,
and thioalkyl; and heteroaryls as defined herein with substitutions
by independent replacement of a hydrogen atom on a ring nitrogen
specifically selected from the group consisting of lower alkyl,
lower alkenyl, and lower haloalkyl.
[0071] The phrase "substituted heteroaryl," as used herein, means a
heteroaryl group as previously defined, substituted by independent
replacement or one or two of the hydrogen atoms thereon with
substituents specifically selected from the group consisting of
--(CH.sub.2).sub.0-3-lower cycloalkyl, --(CH.sub.2).sub.0-3-aryl,
--(CH.sub.2).sub.0-3-lower cycloalkenyl,
--(CH.sub.2).sub.0-3-heteroaryl,
--(CH.sub.2).sub.0-3-heterocycloalkyl, --NH-lower cycloalkyl,
--NH-aryl, --NH-lower cycloalkenyl, --NH-heteroaryl,
--NH-heterocycloalkyl, --O-aryl, --O-heteroaryl,
--O-heterocycloalkyl, --C(O)-lower alkyl, --C(O)-lower alkenyl,
--C(O)-lower alkynyl, --C(O)-lower cycloalkyl, --C(O)-aryl,
--C(O)-lower cycloalkenyl, --C(O)-heteroaryl,
--C(O)-heterocycloalkyl, --CONH.sub.2, --CONH-lower alkyl,
--CONH-lower alkenyl, --CONH-lower alkynyl, --CONH-lower
cycloalkyl, --CONH-aryl, --CONH-lower cycloalkenyl,
--CONH-heteroaryl, --CONH-heterocycloalkyl, --OCO.sub.2-lower
alkyl, --OCO.sub.2-lower alkenyl, --OCO.sub.2-lower alkynyl,
--OCO.sub.2-lower cycloalkyl, --OCO.sub.2-aryl, --OCO.sub.2-lower
cycloalkenyl, --OCO.sub.2-heteroaryl, --OCO.sub.2-heterocycloalkyl,
--OCONH.sub.2, --OCONH-lower alkyl, --OCONH-lower alkenyl,
--OCONH-lower alkynyl, --OCONH-lower cycloalkyl, --OCONH-aryl,
--OCONH-lower cycloalkenyl, --OCONH-heteroaryl,
--OCONH-heterocycloalkyl, --NHC(O)-lower alkyl, --NHC(O)-lower
alkenyl, --NHC(O)-lower alkynyl, --NHC(O)-lower cycloalkyl,
--NHC(O)-aryl, --NHC(O)-lower cycloalkenyl, --NHC(O)-heteroaryl,
--NHC(O)-heterocycloalkyl, --NHCO.sub.2-lower alkyl,
--NHCO.sub.2-lower alkenyl, --NHCO.sub.2-lower alkynyl,
--NHCO.sub.2-lower cycloalkyl, --NHCO.sub.2-aryl,
--NHCO.sub.2-lower cycloalkenyl, --NHCO.sub.2-heteroaryl,
--NHCO.sub.2-heterocycloalkyl, --NHC(O)NH.sub.2, --NHC(O)NH-lower
alkyl, --NHC(O)NH-lower alkenyl, --NHC(O)NH-lower alkynyl,
--NHC(O)NH-lower cycloalkyl, --NHC(O)NH-aryl, --NHC(O)NH-lower
cycloalkenyl, --NHC(O)NH-heteroaryl, --NHC(O)NH-heterocycloalkyl,
--NHC(S)NH.sub.2, --NHC(S)NH-lower alkyl, --NHC(S)NH-lower alkenyl,
--NHC(S)NH-lower alkynyl, --NHC(S)NH-lower cycloalkyl,
--NHC(S)NH-aryl, --NHC(S)NH-lower cycloalkenyl,
--NHC(S)NH-heteroaryl, --NHC(S)NH-heterocycloalkyl, --S(O)-lower
alkyl, --S(O)-lower alkenyl, --S(O)-lower alkynyl, --S(O)-lower
cycloalkyl, --S(O)-aryl, --S(O)-lower cycloalkenyl,
--S(O)-heteroaryl, --S(O)-heterocycloalkyl,
--CH.sub.2SO.sub.2CH.sub.3, --SO.sub.2NH.sub.2, --SO.sub.2NH-lower
alkyl, --SO.sub.2NH-lower alkenyl, --SO.sub.2NH-lower alkynyl,
--SO.sub.2NH-lower cycloalkyl, --SO.sub.2NH-aryl,
--SO.sub.2NH-lower cycloalkenyl, --SO.sub.2NH-heteroaryl,
--SO.sub.2NH-heterocycloalkyl, --NHSO.sub.2-lower alkyl,
--NHSO.sub.2-lower alkenyl, --NHSO.sub.2-lower alkynyl,
--NHSO.sub.2-lower cycloalkyl, --NHSO.sub.2-aryl,
--NHSO.sub.2-lower cycloalkenyl, --NHSO.sub.2-heteroaryl,
--NHSO.sub.2-heterocycloalkyl, --S-lower alkenyl, --S-lower
alkynyl, --S-lower cycloalkyl, --S-aryl, --S-heteroaryl,
--S-heterocycloalkyl, or methylthiomethyl.
[0072] The term "heterocycloalkyl" as used herein specifically
refers to azetidinyl, [1,3]dioxolane, pyrazolinyl, pyrrolidinyl,
2-pyrrolinyl, 3-pyrrolinyl, tetrahydrofuranyl, dihydrofuranyl,
pyranyl, tetrahydropyranyl, dihydropyranyl, tetrahydrothienyl,
imidazolinyl, imidazolidinyl, pyrazolidinyl, oxazolidinyl,
isoxazolidinyl, thiazolidinyl, isothiazolidinyl, piperidinyl,
homopiperidinyl, quinuclidinyl, piperazinyl, lower alkyl
piperazinyl, morpholinyl, thiamorpholinyl, 1-pyrazolidinyl,
azepinyl; and heterocycloalkyls as defined herein with
substitutions by independent replacement of a hydrogen atom on a
ring nitrogen specifically selected from the group consisting of
lower alkyl, lower alkenyl, and lower haloalkyl; and specifically
substitutions to these heterocycloalkyls by independent replacement
of one or two of the hydrogen atoms thereon with substituents
specifically selected from the group consisting of halide, lower
haloalkyl, amino, hydroxyl, lower alkoxy, lower aminoalkyl, lower
hydroxyalkyl, nitrile, lower alkyl nitrile, lower alkyl, lower
alkenyl, lower alkynyl, carboxyl, amide, hydroxyamide, lower
alkoxyalkyl, lower alkoxycarbonyl, lower alkylcarbonylamino, --SH,
and thioalkyl. For example, substitutions to pyrrolidinyl may
include hydroxyl-pyrrolidinyl, chloropyrrolidinyl,
methoxy-pyrrolidinyl, nitrile-pyrrolidinyl, methyl-pyrrolidinyl,
and amino-pyrrolidinyl.
[0073] The term "heteroatom" as used herein refers to a nitrogen,
sulfur, or oxygen atom.
[0074] The term "lower thioalkyl" as used herein by itself or as
part of another substituent, means a radical of the formula --SR,
wherein R is a lower alkyl or lower cycloalkyl group as defined
herein. Examples of lower alkylthio groups include, but are not
limited to, methylthio, ethylthio, propylthio, isopropylthio,
butylthio, and tert-butylthio.
[0075] The term "carboxy," as used herein, means a group of formula
--COOH.
[0076] The term "hydroxy," as used herein, means a group of formula
--OH.
[0077] The phrase "hydroxy protecting group," as used herein, means
a labile chemical moiety which is known in the art to protect a
hydroxyl group against undesired reactions during synthetic
procedures. Following such procedures, the hydroxy protecting group
may be selectively removed. Examples of hydroxy protecting groups
include, but are not limited to, methylthiomethyl,
tert-butyldimethylsilyl, tertbutyldiphenylsilyl, acyl substituted
with an aromatic group, and the like.
[0078] The phrase "protected hydroxy," as used herein, means a
hydroxy group protected with a hydroxy protecting group, as defined
above, including benzoyl, acetyl, trimethylsilyl, triethylsilyl,
methoxymethyl groups, for example.
[0079] The phrase "amino protecting group," as used herein, means a
labile chemical moiety which is known in the art to protect an
amino group against undesired reactions during synthetic
procedures. Following such procedures, the amino protecting group
may be selectively removed. Examples of amino protecting groups
include, but are not limited to, t-butoxycarbonyl,
9-fluorenylmethoxycarbonyl, benzyloxycarbonyl, and the like.
[0080] The phrase "protected amino," as used herein, means an amino
group protected with an amino protecting group as defined
above.
[0081] In accordance with the present invention, compounds as
disclosed herein are surprisingly effective at inhibiting or
reducing the formation or growth of biofilms. Furthermore, it is
also disclosed that the co-administration to a bacterial biofilm of
compounds described herein with an antimicrobial agent, antibiotic,
or biocide provides increased susceptibility of the bacteria within
the biofilm to the antimicrobial agent, antibiotic, or biocide. The
instant invention thus provides for novel compounds, compositions,
compositions comprising biofilm inhibitors and antimicrobial agents
or antibiotics or biocides, and various methods of using the
compositions containing the biofilm inhibitors of the invention to
reduce or inhibit the formation or growth of bacterial
biofilms.
[0082] The compounds of the invention may be prepared by the
techniques described in the examples below, starting from the
ursane or oleanane triterpene scaffolds like ursolic acid and
oleanolic acid. While a typical starting chemical compound in the
semi-synthetic preparation of the compounds of the invention may be
ursolic acid, oleanolic acid, corosolic acid, asiatic acid, or
madecassic acid, oleanolic acid and ursolic acid are preferred
staring compounds. In designing semi-synthetic strategies to
prepare analogs of the starting chemical compound, modifications at
certain positions of the scaffold of the basic chemical compound
prove to be important for modulating biofilm inhibition, while
other modifications at positions can improve the bioavailability of
the compound. Many of these modifications or optimizations are
taught in the literature known to those skilled in the art,
including The Organic Chemistry of Drug Design and Drug Action,
2.sup.nd Edition, by Richard B. Silverman, incorporated herein in
its entirety by this reference. Improvement of the bioavailability
of the compound expands the therapeutic range of the compounds by
reducing certain cellular toxicities in the subject.
[0083] Compounds of this invention include:
TABLE-US-00001 Com- pound Chemical Structure 1 ##STR00006## 2
##STR00007## 3 ##STR00008## 4 ##STR00009## 5 ##STR00010## 6
##STR00011## 7 ##STR00012## 8 ##STR00013## 9 ##STR00014## 10
##STR00015## 11 ##STR00016## 12 ##STR00017## 62 ##STR00018## 64
##STR00019## 65 ##STR00020## 68 ##STR00021## 84 ##STR00022## 88
##STR00023## 90 ##STR00024## 91 ##STR00025## 92 ##STR00026##
[0084] The invention especially teaches the remarkable discovery as
demonstrated by the examples that the following Structure II
##STR00027##
may contain a diverse variety of moieties and structures at R.sup.1
and retain the ability to inhibit or reduce the formation or growth
of biofilms. The examples demonstrate that a diverse group of aryl,
substituted aryl, heteroaryl, and substituted heteroaryl moieties
and structures may be added at R.sup.1 and retain potent activity
to reduce or inhibit the formation or growth of biofilms. A few
representative chemical structures prepared semi-synthetically and
described herein including the examples are as follows:
##STR00028## ##STR00029##
[0085] The present invention also includes compounds of the
following chemical Structure III:
##STR00030##
wherein R.sup.1 is selected from the group consisting of hydrogen,
methyl, halide, nitrile, lower alkyl, substituted lower alkyl,
lower alkenyl, substituted lower alkenyl, lower alkynyl,
substituted lower alkynyl, aryl, substituted aryl, heteroaryl, and
substituted heteroaryl, R.sup.3 is selected from the group
consisting of hydrogen or methyl, and one of R.sup.4 and R.sup.5 is
hydrogen and the other is methyl. Salts are also contemplated by
the present invention as described in the examples.
[0086] Based upon the teachings described herein, those skilled in
the art appreciate that Structure III substituted with any of the
following moieties at R.sup.1 will exhibit potent activities at
inhibiting the formation of biofilms.
##STR00031## ##STR00032##
[0087] The present invention provides compounds of the following
chemical Structure IV
##STR00033##
wherein R.sup.1 is selected from the group consisting of hydrogen,
methyl, halide, nitrile, lower alkyl, substituted lower alkyl,
lower alkenyl, substituted lower alkenyl, lower alkynyl,
substituted lower alkynyl, aryl, substituted aryl, heteroaryl, and
substituted heteroaryl, wherein R.sup.3 is selected from the group
consisting of hydrogen or methyl; wherein one of R.sup.4 and
R.sup.5 is hydrogen and the other is methyl. Salts are also
contemplated by the present invention as described in the
examples.
[0088] Based upon the teachings described herein, those skilled in
the art appreciate that Structure IV substituted with the following
moieties at R.sup.1 would exhibit potent activities at inhibiting
the formation of biofilms:
##STR00034## ##STR00035##
[0089] Various pharmaceutical compositions contemplated by the
present invention, including the compounds of the invention and the
specific examples described herein, further including
pharmaceutically acceptable derivable prodrugs or prodrugs thereof.
A "pharmaceutically acceptable derivable prodrug or prodrug" means
any pharmaceutically acceptable salt, ester, salt of an ester, or
other derivable prodrug of a compound of this invention which, upon
administration to a patient, is capable of providing (directly or
indirectly) a compound used in this invention. It will be
recognized that the efficacy of the compounds of this invention is
related to localized reaction sites on the compounds. Accordingly,
as illustrated by the examples below, a wide variety of
substitutions therefore may be made at various sites on the
compounds spacially remote from the localized reaction sites that
do not significantly interfere with the efficacy of the compounds
Likewise, substitutions to form pharmaceutically acceptable salts,
esters, salts of esters, and other such derivable prodrugs of the
compounds of this invention are are contemplated herein as well.
Thus, compounds with such innocuous substitutions do no depart from
the scope of the invention.
[0090] On the other hand, however, certain moieties have been found
to be so significant in size or reactivity as to interfere
significantly with the efficacy of the compounds. Thus, highly
reactive, polar, ionic or large substituents such as those shown in
the examples below as having a deleterious affect on the activity
of the compound are are excluded from the most preferred
embodiments of the invention.
[0091] Compounds useful in the present invention may, optionally,
be converted to their therapeutically-active non-toxic acid salt
forms by treatment with appropriate acids. Such acids include
inorganic acids, e.g., hydrochloric and hydrobromic acids, sulfuric
acid, nitric acid, phosphoric acid and like acids; or organic
acids, such as acetic, propanoic, hydroxyacetic,
2-hydroxypropanoic, 2-oxo-propanoic, ethanedioic, propanedioic and
like acids. Of course, the salt forms may be converted into the
free base form by treatment with alkali. The
pharmaceutically-acceptable acid salts of the present invention
also comprise the solvates that the compositions of the present
invention may form, which, of course, are included within the scope
of the present invention. Non-limiting examples of such solvates
are hydrates, alcoholates and the like.
[0092] Such pharmacologic compositions may be formulated in various
ways known in the art for administration purposes. Pharmaceutical
compositions of the present invention can be prepared by combining
an effective amount of the particular compound of this invention,
typically in base or acid salt form, as the active ingredient with
one or more pharmaceutically-acceptable carriers and delivery
vehicles. Numerous pharmaceutically acceptable carriers and
delivery vehicles exist that are readily accessible and well-known
in the art, which may be employed to generate the preparation
desired (i.e. that permit administration of the pharmaceutical
composition orally, topically, rectally, percutaneously, by
parenteral injection, intranasally or by inhalation).
Representative examples of pharmaceutically acceptable carriers and
delivery vehicles include aluminum stearate, lecithin, serum
proteins, such as human serum albumin; buffer substances such as
the various phosphates, glycine, sorbic acid, potassium sorbate,
partial glyceride mixtures of saturated vegetable fatty acids;
water, salts or electrolytes, such as protamine sulfate, disodium
hydrogen phosphate, potassium hydrogen phosphate, sodium chloride,
and zinc salts; colloidal silica, magnesium trisilicate, polyvinyl
pyrrolidone, cellulose-based substances, polyethylene glycol,
sodium carboxymethylcellulose, polyarylates, waxes, polyethylene,
polyoxypropylene-block polymers, polyethylene glycol and wool fat,
and the like. Other constituents, such as aids for taste, color,
tableting, and so forth, may be combined with the active ingredient
and carrier for any of the many known purposes of such additives.
Examples of such additives are discussed bleow.
[0093] The pharmacologic compositions described herein may further
be prepared in unitary dosage form for administration orally,
percutaneously, by parenteral injection (including subcutaneous,
intramuscular, intravenous and intradermal), topically,
intranasally, by inhalation, or for application to a medical
device, such as an implant, catheter, or other device. In preparing
the compositions that permit administration of an oral dosage, for
example, any of the pharmaceutically acceptable carriers known in
the art may be used, such as water, glycols, oils, alcohols and the
like in the case of carriers that permit oral delivery of liquid
preparations such as suspensions, syrups, elixirs and solutions.
When solid pharmaceutically acceptable carriers are desired that
permit oral or rectal administration, starches, sugars, kaolin,
lubricants, binders, cellulose and its derivable prodrugs, and
disintegrating agents and the like may be used to prepare, for
example, powders, pills, capsules and tablets.
[0094] For pharmaceutically acceptable carriers that permit
parenteral administration, the pharmaceutically acceptable carriers
often comprise sterile water, which may be supplemented with
various solutes to, for example, increase solubility. Injectable
solutions may be prepared in which the pharmaceutically acceptable
carrier comprises saline solution, glucose solution, or a mixture
thereof, which may include certain well-known anti-oxidants,
buffers, bacteriostats, and other solutes that render the
formulation isotonic with the blood of the intended patient.
[0095] For pharmaceutically acceptable carriers that permit
intranasal administration, the pharmaceutically acceptable carriers
often comprise poly acrylic acids such as Carbopol.RTM. 940, a
hydrogenated castor oil such as Cremophor.RTM. RH40, glycerol,
vinylpyrrolidones such as PVP-K90.RTM. or PVP K30.RTM.,
polyethylene glycols such as PEG 1450.RTM., benzyl alcohol, Edetate
sodium, hydroxycellulose, potassium chloride, potassium phosphate,
and sodium phosphate. Compositions used for intranasal
administration also commonly include benzalkonium chloride as an
anti-microbial preservative.
[0096] For pharmaceutically acceptable carriers that permit
administration by inhalation, the pharmaceutically acceptable
carriers often comprise solvent/carrier/water mixtures that are
easily dispersed and inhaled via a nebulizer or inhaler. For
example, a mixture of ethanol/propylene glycol/water in the ratio
of about 85:10:5 (parts ethanol: parts propylene glycol: parts
water) can be used to administer the compounds and compositions of
the invention via inhalation. Ratios as expressed herein are based
on parts by weight.
[0097] For pharmaceutically acceptable carriers that permit
percutaneous administration, the pharmaceutically acceptable
carrier may, optionally, comprise a penetration enhancing agent
and/or a wetting agent.
[0098] Dosage forms that permit topical or transdermal
administration of a compound of this invention include ointments,
pastes, creams, lotions, gels, powders, solutions, sprays,
inhalants or patches. The active compound or compounds is/are mixed
under sterile conditions with a pharmaceutically acceptable carrier
and optionally one or more preservatives and/or buffers. In the
context of certain embodiments of this invention, the active
compound is a pentacyclic acid triterpene. In the context of other
embodiments of this invention, the pentacyclic acid triterpene is
combined in the composition with another active compound that is an
antimicrobial agent or antibiotic.
[0099] The ointments, pastes, creams and gels may contain, in
addition to an active compound or compounds according to the
present invention, pharmaceutically acceptable carriers that permit
topical or transdermal administration such as animal and vegetable
fats, oils, waxes, paraffins, starch, tragacanth, cellulose
derivable prodrugs, polyethylene glycols, silicones, bentonites,
silicic acid, talc and zinc oxide, or mixtures thereof.
[0100] In some cases, the pH of the pharmaceutical formulations
contemplated herein may be adjusted with acceptable acids, bases or
buffers to enhance the stability of one or more of the active
compounds present or their delivery forms. In the context of
certain embodiments of this invention, the active compound is a
pentacyclic acid triterpene. In the context of other embodiments of
this invention, the pentacyclic acid triterpene is combined in the
composition with another active compound that is an antimicrobial
agent or antibiotic.
[0101] Still further, in order to prolong the anti-bacterial effect
of a compound disclosed herein, it may be desirable to slow the
absorption of the compound from subcutaneous or intramuscular
injection. This may be accomplished using a liquid suspension of
crystalline or amorphous material with poor water solubility. The
rate of absorption of the compound then depends upon its rate of
dissolution, which, in turn, may depend upon crystal size and
crystalline form. Alternatively, delayed absorption of a
parenterally administered drug form may be accomplished by
dissolving or suspending the compound in an oil vehicle.
[0102] Injectable depot forms are made, e.g., by forming
microencapsule matrices of one or more compounds of the present
invention in biodegradable polymers such as
polylactide-polyglycolide. Depending upon the ratio of active(s) to
polymer and the nature of the particular polymer employed, the rate
at which such active(s) is released may be controlled. Examples of
other biodegradable polymers include poly(orthoesters) and
poly(anhydrides). Depot injectable formulations are also prepared
by entrapping the compound in liposomes or microemulsions that are
compatible with body tissues.
[0103] The pharmaceutical composition may also be a dentifrice. In
the present invention, "dentifrice" is understood to broadly
include compositions suitable for administering to the oral cavity,
especially, for example, to the gingival/mucosal tissue or to the
teeth. Thus, the dentifrice may include toothpastes, toothpowders,
liquid dentifrices, mouth detergents, mouthwashes, troches, chewing
gums, dental or gingival massage creams, dental strips, dental
gels, and gargle tablets.
[0104] When the pharmaceutical composition of this invention is a
dentifrice such as tooth paste, a tooth or gum adherence promoting
substance selected from the group consisting of copolymers of
methyl vinyl ether and maleic anhydride, copolymers of vinyl
pyrrolidone and vinyl acetate, and cyclodextrins may also be
included in the composition. Copolymers of methyl vinyl ether and
maleic anhydride useful in this invention may have molecular
weights ranging from 200,000 to 2,000,000 kD and may be free acids,
mixed sodium and calcium salts, or half ester derivable prodrugs.
Representative commercial sources of the copolymers of methyl vinyl
ether and maleic anhydride include GANTREZ.RTM. AN(CAS #9011-16-9)
GANTREZ.RTM. S (CAS #25153-40-69) GANTREZ.RTM. MS (CAS#62386-95-2)
GANTREZ.RTM. ES (CAS#25087-06-3 or CAS#25119-68-0) and can be
obtained from International Specialty Products Wayne, N.J.
Copolymers of vinyl pyrrolidone and vinyl acetate useful in the
invention typically have a molecule weight of approximately 27,000
kD and are water soluble. Representative commercial sources of the
copolymers of vinyl pyrrolidone and vinyl acetate PLASDONE.RTM.
S-630 and can be obtained from International Specialty Products
Wayne, N.J. Cyclodextrins useful in the invention are cyclic
oligosaccharides composed of either 6, 7 or 8 glucose units (a-, b-
and g-cyclodextrin, respectively). Representative commercial
sources of the cyclodextrins useful in this invention include
CAVAMAX.RTM. W6 Pharma, CAVAMAX.RTM. W7 Pharma and CAV AMAXW8
Pharma (a-, b- and g-cyclodextrin, respectively) and can be
obtained from International Specialty Products Wayne, N.J.
[0105] When the composition of this invention is a dentifrice, an
antimicrobial agent is selected from the group consisting of
triclosan, metronidazole, tetracyclines, quinolones, plant
essential oils, camphor, thymol, carvacrol, menthol, eucalyptol,
and methyl salicylate may also be included. Pharmaceutically
acceptable carriers that permit administration of the pentacyclic
acid triterpene compounds of this application as dentifrices
include sorbitol, glycerin, silica, sodium lauryl sulfate and
Xanthum gum. The dentifrices of this invention may also include
sodium fluoride.
Reducing or Inhibiting Bacterial Biofilms in Plants
[0106] Finally, bacterial biofilms also may be reduced or inhibited
by the application or administration of compositions containing
compounds disclosed herein when the subject is a plant. Thus, the
compound or a composition containing an active compound described
herein may be administered to a plant, such as a surface of a plant
to reduce or inhibit the formation of a biofilm on the plant.
[0107] It is believed that the methods described herein are
applicable to reducing or inhibiting a variety of bacterial
biofilms in plants. Pseudomonadaceae, Rhizobiaceae,
Enterobacteriaceae, Corynebacteriaceae and Streptomycetaceae
bacteria are all economically significant plant pathogens that may
be controlled by the present invention. Non-limiting examples of
specific plant pathogens involving biofilms that may be effectively
inhibited by the methods described herein include: Xanthomonas
species, such as, for example, Xanthomonas campestris pv. oryzae;
Pseudomonas species, such as, for example, Pseudomonas syringae pv.
lachrymans; and Erwinia species, such as, for example, Erwinia
amylovora. It is also believed that the compositions used in the
methods of reducing or inhibiting bacterial biofilms of plants
described herein can further comprise antimicrobial agents such as
bronopol, dichlorophen, nitrapyrin, nickel dimethyldithiocarbamate,
kasugamycin, octhilinone, furancarboxyl, oxytetracyclin,
probenazole, streptomycin, tecloftalam, copper sulphate and other
copper preparations.
[0108] Methods of reducing or inhibiting bacterial biofilms
described herein can be used to treat all plants and parts of
plants. By reference to "plants," what is meant here is all plants
and plant populations such as desired and undesired wild plants or
crop plants (including naturally occurring crop plants). Crop
plants can be plants obtainable by conventional breeding and
optimization methods or by biotechnological and genetic engineering
methods or combinations of these methods, and include the
transgenic plants and the plant varieties that can or cannot be
protected by varietal property rights. The phrase "parts of plants"
as used herein is to be understood as meaning all above-ground and
below-ground parts and organs of plants, such as shoot, leaf,
flower and root, examples which may be mentioned being leaves,
needles, stems, trunks, flowers, fruit-bodies, fruits and seeds and
also roots, tubers and rhizomes. Parts of plants also include
harvested plants and vegetative and generative propagation
material, for example, seedlings, tubers, rhizomes, cuttings and
seeds.
[0109] The treatment of the plants and the parts of plants with the
active compounds according to the invention is carried out directly
or by action on their surroundings, habitat or storage space,
according to customary treatment methods, for example by dipping,
spraying, evaporating, atomizing, broadcasting, spreading-on and,
in the case of propagation material, in particular in the case of
seeds, furthermore by one- or multi-layer coating.
Agriculturally Acceptable Carriers and Compositions
[0110] Depending on their particular physical and/or chemical
properties, the compounds and compositions described herein can be
converted to the customary formulations, such as solutions,
emulsions, suspensions, powders, foams, pastes, granules, aerosols
and microencapsulations in polymeric substances and in coating
compositions for seeds, and ULV cool and warm fogging
formulations.
[0111] The following examples illustrate various aspects of the
present invention and are not intended to limit the scope of the
present invention. Those of skill in the art should, in light of
the present disclosure, appreciate that many changes can be made in
the specific embodiments which are disclosed and still obtain a
like or similar result without departing from the spirit and scope
of the invention.
Example I
Specificity of the Biofilm Growth Assay
[0112] Table 1, below, shows experimental results illustrating
remarkable properties of Compound 1 to inhibit the spreading of
clinical isolates of P. aeruginosa and E. coli bioflms as
determined in a biofilm growth assay. The related scaffolds and
analogs shown in Table 1 have been found to exhibit significantly
less inhibitory activities than does Compound 1 in the biofilm
growth assay even though certain of the compounds consist of
chemical structures similar to that of Compound 1. These data
demonstrate the novel and unique aspects of Compound 1 and its
discovery and the specificity of the biofilm growth assay to
identify Compound 1 as a potent inhibitor of spreading
gram-negative bacterial biofilms.
[0113] The biofilm growth assay was carried out to measure swarming
or biofilm spreading on semi-solid agar media in round plates (for
example, 100.times.15 mm), also referred to as Petri dishes. For E.
coli, the plates contained LB medium with 0.6% agar and 0.5%
glucose. P. aeruginosa plates were made with M8 agar supplemented
with 0.2% glucose, 100 .mu.M CaCl.sub.2, 1 mM MgSO.sub.4, and 0.5%
Casamino acids. The agar plates of this assay consisted of 0.4% to
0.7% agar (Bacto or Noble agar), although other media may also be
used as described in the literature and known to those skilled in
the art. Test compounds at the desired concentration were added to
the cooled, autoclaved media. Portions of the media were then
poured into the plates (20 mL/plate), and the plates allowed to dry
at room temperature for approximately 3 to 4 hours. Alternatively,
however, plates may be dried in a laminar flow hood for
approximately fifteen to twenty minutes. Overnight cultures were
grown in a 37.degree. C. shaker in LB (E. coli) or TSB (P.
aeruginosa). Plates were inoculated by placing 5 .mu.L of the
appropriate overnight culture in the centers of the plates. The
plates then were incubated overnight at 37.degree. C. The area of
the zone of spreading bacteria was then measured and replicates are
averaged. Percent inhibition was calculated as 100.times.(area
without compound-area with compound)/(area without compound). An
active compound was considered that which reduces the area of the
spreading biofilm compared to negative controls by greater than or
equal to 85%. The values shown in Table 1 are the concentrations of
compounds (.mu.g/ml) tested in the biofilm growth assay that reduce
the area of the spreading biofilm compared to negative controls by
greater than or equal to 85% unless noted differently in Table 1. A
>("greater than" symbol) as used in Table 1 is well understood
to those skilled in the art as an indication that the listed
concentration was not effective at this concentration.
[0114] This example demonstrates the reduction of virulence of
gram-negative bacteria. Inhibition or reduction of the growth of
biofilms reduces virulence of gram-negative bacteria. Adhesion,
biofilm growth, invasion, and the secretion of enzymes or toxins
contribute to virulence of gram-negative bacteria. It is well known
to those skilled in the art that biofilms increase the virulence of
gram-negative bacteria and an upregulation of virulence factors
(i.e. enzymes, toxins) has been demonstrated in gram-negative
bacterial biofilms. The compounds of the invention reduce virulence
of gram-negative bacteria.
TABLE-US-00002 TABLE 1 Related Scaffolds and Analogs of Compound 1
P. aeruginosa E. coli COMPOUND STRUCTURE (.mu.g/ml) (.mu.g/ml)
Compound 1 ##STR00036## 0.5 1 Uvaol ##STR00037## >128 >8
alpha-Amyrin ##STR00038## >32 Not tested Lupeol ##STR00039##
>16 >8 Betulin ##STR00040## 16 >16 Betulinic acid
##STR00041## >16 >16 Oleanolic acid ##STR00042## 4 >8
Ursolic acid ##STR00043## 8 8 Oleanolic acid methyl ester
##STR00044## >16 >16 Corosolic acid ##STR00045## 4 Not tested
Maslinic acid ##STR00046## 4 Not tested A ##STR00047## 8 >8 B
##STR00048## 4 8 C ##STR00049## 0.5 0.5 D ##STR00050## 1 4 E
##STR00051## 2 >16 F ##STR00052## 4 >8 G ##STR00053## >8
>8 H ##STR00054## 8 >8 I ##STR00055## >16 8 J ##STR00056##
>16 >16 K ##STR00057## >8 >8 L ##STR00058## >16
>16 M ##STR00059## 8 8 N ##STR00060## 8 8
Example II
[0115] All of the compounds shown in Tables 2, 3, 4, and 5, below,
were prepared semi-synthetically from oleanolic acid, except for
Compounds 2 and 3. Compound 2 was prepared from ursolic acid.
Compound 3 was prepared from hederagenin, but could also be
prepared from an oleanolic acid analog with a hydroxyl at C.sub.23
or C.sub.24. These compounds were tested in the spreading biofilm
assay according to the methods detailed in Example I, above. The
values shown in Tables 2, 3, 4, and 5 are the concentrations of
compounds tested in the biofilm growth assay that reduce the area
of the spreading biofilm compared to negative controls by greater
than or equal to 85%.
TABLE-US-00003 TABLE 2 Compound 1 and its Analogs and their
inhibitory concentrations in the biofilm growth assay as detailed
in Example I, above P. aeruginosa E. coli COMPOUND Chemical
Structure (.mu.g/ml) (.mu.g/ml) 1 ##STR00061## 0.5 1 2 ##STR00062##
0.5 1 3 ##STR00063## 0.5 1 4 ##STR00064## 0.25 1 5 ##STR00065## 0.5
1 6 ##STR00066## 0.125 0.25 7 ##STR00067## 0.25 1 8 ##STR00068##
0.25 1 9 ##STR00069## 0.5 1 10 ##STR00070## 0.5 1 11 ##STR00071##
0.5 0.5 12 ##STR00072## 0.5 1
TABLE-US-00004 TABLE 3 Analogs of Compound 1 ##STR00073## COM- P.
aeruginosa E. coli POUND R.sub.1 (.mu.g/ml) (.mu.g/ml) 13
--CH.dbd.CH.sub.2 0.5 1 14 --CH.sub.2CH.sub.3 0.5 2 15 ##STR00074##
0.25 1 16 ##STR00075## 1 2 17 ##STR00076## 0.5 >2 18
##STR00077## 0.5 1 19 ##STR00078## 1 1 20 ##STR00079## 0.5 2 21
##STR00080## 0.5 >2 22 ##STR00081## 1 >2 23 ##STR00082## 1 2
24 --Cl 0.5 1 25 --Br 0.5 >2 26 ##STR00083## 0.25 1 27
##STR00084## 1 >2 28 ##STR00085## 1 2 29 ##STR00086## 1 1 30
##STR00087## 2 1 31 ##STR00088## 0.5 1 32 ##STR00089## 0.5 1 33
##STR00090## 2 4 34 ##STR00091## 1 1 35 ##STR00092## >1 1 36
##STR00093## >1 2 37 ##STR00094## >1 1 38 ##STR00095## 1 2 39
##STR00096## 1 2 40 ##STR00097## 0.5 2 41 ##STR00098## 1 2 42
##STR00099## 0.25 1 43 ##STR00100## 1 1 44 ##STR00101## 0.5 2 45
##STR00102## 0.5 1 46 ##STR00103## 0.5 1 47 ##STR00104## 0.5 1 48
##STR00105## 0.5 1 49 ##STR00106## 0.5 >1 50 ##STR00107## 2 1 51
##STR00108## 0.5 2 52 ##STR00109## 1 2 53 ##STR00110## 0.5 2 54
##STR00111## 1 2 55 ##STR00112## >1 2 56 ##STR00113## 1 2 57
##STR00114## >2 1 58 ##STR00115## 1 2 59 ##STR00116## 0.5
>1
TABLE-US-00005 TABLE 4 Analogs of Compound 1 ##STR00117## COM- P.
aeruginosa E. coli POUND R.sub.1 (.mu.g/ml) (.mu.g/ml) 60
##STR00118## 0.5 1 61 ##STR00119## 0.5 0.5 62 ##STR00120## 1 1 63
##STR00121## 4 1 64 ##STR00122## 0.5 1 65 ##STR00123## 0.25 0.25 66
##STR00124## 0.25 1 67 ##STR00125## 1 2 68 ##STR00126## 0.5 0.5 69
##STR00127## 1 1 70 ##STR00128## 0.5 2 71 ##STR00129## 0.5 2 72
##STR00130## 1 >2 73 ##STR00131## 1 1 74 ##STR00132## 1 >2 75
##STR00133## 0.5 1 76 ##STR00134## 0.5 2 77 ##STR00135## 1 2 78
##STR00136## >1 1 79 ##STR00137## 0.25 0.5 80 ##STR00138## 1 1
81 ##STR00139## 0.5 1 82 ##STR00140## 0.5 1 83 ##STR00141## 0.25
0.5 84 ##STR00142## 0.125 0.5 85 ##STR00143## 0.5 1 86 ##STR00144##
0.5 2 87 ##STR00145## 0.5 1 88 ##STR00146## 0.25 0.5 89
##STR00147## 1 1 90 ##STR00148## 0.5 1
TABLE-US-00006 TABLE 5 Analogs of Compound 1. Compounds 91 and 92
demonstrate that combined modifications at the R.sup.1 and R.sup.2
positions can be prepared and retain potent inhibitory activities
against biofilms. P. aeruginosa E. coli COMPOUND CHEMICAL STRUCTURE
(.mu.g/ml) (.mu.g/ml) 91 ##STR00149## 1 1 92 ##STR00150## 0.5 2
Example III
Semi-Synthesis Procedures for the Compounds Shown in Example II
[0116] Preparative HPLC:
[0117] Preparative HPLC was conducted using a SunFire Prep C18 OBD
Column, 5 .mu.m, 19.times.100 mm eluting with a gradient from 90:10
(water:acetonitrile, both with 0.05% trifluoroacetic acid) to 10:90
(water:acetonitrile, both with 0.05% trifluoroacetic acid) over 14
minutes followed by a 2 minute hold at 10:90 (water:acetonitrile,
both with 0.05% trifluoroacetic acid) with a flow rate at 25
mL/min.
[0118] CMA:
[0119] CMA=80:18:2 Methylene Chloride/Methanol/concentrated
Ammonium Hydroxide
Preparation of Common Intermediate I
##STR00151##
[0120] (i) Preparation of Ib:
(4aS,6aS,6bR,12aR)-2,2,6a,6b,9,9,12a-Heptamethyl-10-oxo-1,2,3,4,4a,5,6,6a-
,6b,7,8,8a,9,10,11,12,12a,12b,13,14b-icosahydropicene-4-a-carboxylic
acid
[0121] To a mixture of oleanolic acid (Ia, 5.0 g, 10.9 mmol) and
CH.sub.2Cl.sub.2 (200 mL) was added the Dess-Martin reagent (6.0 g,
14.2 mmol) under nitrogen at room temperature. After stirring at
room temperature for 1 hour, the starting material was consumed, as
indicated by TLC (1:1 hexanes:diethyl ether). The reaction mixture
was quenched with the addition of a solution of sodium thiosulfate
and NaHCO.sub.3 (50 g sodium thiosulfate in 200 mL saturated
NaHCO.sub.3 solution). The mixture was stirred at room temperature
for 10 minutes. The layers were separated and the aqueous layer was
extracted with EtOAc (3.times.250 mL).
[0122] The combined organics were dried (Na.sub.2SO.sub.4),
filtered and concentrated under reduced pressure. The residue was
purified by column chromatography (silica, 3:1 hexanes/diethyl
ether) to provide the sub-title compound (4.9 g, 99%) as a white
foam.
[0123] .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 0.81 (s, 3H),
0.90-2.05 (m, 39H), 2.42-2.44 (m, 1H), 2.59-2.61 (m, 1H), 2.83-2.85
(m, 1H), 5.29-5.31 (m, 1H). ESI MS m/z 455
[C.sub.30H.sub.46O.sub.3+H].sup.+.
(ii) Preparation of Ic: (4aS,6aS,6bR,12aR)-Benzyl
2,2,6a,6b,9,9,12a-heptamethyl-10-oxo-1,2,3,4,4a,5,6,6a,6b,7,8,8a,9,10,11,-
12,12a,12b,13,14b-icosahydropicene-4-a-carboxylate
[0124] A mixture of Ib (4.9 g, 10.8 mmol), benzyl bromide (1.9 mL,
16.2 mmol), K.sub.2CO.sub.3 (2.2 g, 16.2 mmol) and DMF (185 mL) was
stirred at room temperature for 2.5 hours. The solvent then was
removed under reduced pressure and the residue was partitioned
between H.sub.2O (250 mL) and EtOAc (250 mL). The layers were
separated and the aqueous layer was extracted with EtOAc (200 mL).
The combined organics were dried (Na.sub.2SO.sub.4) and filtered.
The solvent was removed under reduced pressure and the residue was
purified by column chromatography (silica, 3:1 hexanes/diethyl
ether) to provide the sub-title compound (4.7 g, 81%) as a white
solid.
[0125] .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 0.65 (s, 3H), 0.89
(s, 3H), 0.92 (s, 3H), 1.09-2.05 (m, 32H), 2.36-2.38 (m, 1H),
2.49-2.51 (m, 1H), 2.87-2.89 (m, 1H), 5.07-5.09 (m, 2H), 5.29-5.31
(m, 1H), 7.29-7.38 (m, 5H). ESI MS m/z 545
[C.sub.37H.sub.52O.sub.3+H].sup.+.
(iii) Preparation of Id: (4aS,6aS,6bR,12aR)-Benzyl
11-cyano-2,2,6a,6b,9,9,12a-heptamethyl-10-oxo-1,2,3,4,4a,5,6,6a,6b,7,8,8a-
,9,10,11,12,12a,12b,13,14b-icosahydropicene-4-a-carboxylate
[0126] A solution of diisopropylamine (31.8 mL, 227 mmol) and THF
(250 mL) was cooled to -78.degree. C. under nitrogen. A solution of
n-butyllithium (2.5 M in hexanes, 100 mL, 251 mmol) was slowly
added, maintaining the internal temperature below -70.degree. C.
The solution was allowed to stir for 30 min and was then slowly
added to a solution of Ic (65.0 g, 120 mmol) and THF (1.4 L) at
-78.degree. C. under nitrogen. This solution was stirred for 30 min
after which time a suspension of p-toluenesulfonyl cyanide (43.3 g,
239 mmol) and THF (200 mL) was added over 45 min. The solution was
stirred for 10 min and then quenched by the addition of saturated
ammonium chloride solution (250 mL) at -78.degree. C. The mixture
was allowed to warm to room temperature overnight. The organic
layer was separated and the aqueous layer was extracted with EtOAc.
The combined organic layers were dried (Na.sub.2SO.sub.4) and
filtered. The solvent was removed under reduced pressure and the
residue was purified by column chromatography (silica, 1:1
hexanes/EtOAc) to provide the sub-title compound (58 g, 85%) as a
white solid.
[0127] .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 0.65 (s, 3H),
2.14-2.16 (m, 39H), 2.93-2.95 (m, 1H), 5.06-5.08 (m, 2H), 5.29-5.31
(m, 1H), 7.29-7.37 (m, 5H).
(iv) Preparation of I: (4aS,6aS,6bR,13aR)-Benzyl
12-amino-2,2,6a,6b,9,9,13a-heptamethyl-2,3,4,4a,5,6,6a,6b,7,8,8a,9,11,13,-
13a,13b,14,15b-octadecahydro-1H-chryseno[1,2-f]indazole-4-a-carboxylate
[0128] A solution of Id (2.0 g, 3.5 mmol) and hydrazine (0.33 mL,
10.6 mmol) in EtOH (18 mL) was heated at reflux for 16 hours. The
solvent and excess hydrazine were removed under reduced pressure.
The residue was purified by column chromatography (silica, 0-7%
MeOH in CH.sub.2Cl.sub.2) to provide the title compound (1.8 g,
88%) as an off-white solid.
[0129] .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 0.69 (s, 3H),
0.85-2.03 (m, 37H), 2.24-2.35 (m, 1H), 2.92-2.98 (m, 1H), 5.06-5.08
(m, 2H), 5.22-5.24 (m, 1H), 7.28-7.37 (m, 5H). APCI MS m/z 584
[C.sub.38H.sub.53N.sub.3O.sub.2+H].sup.+.
Preparation of Common Intermediate II
##STR00152## ##STR00153## ##STR00154##
[0130] (i) Preparation of IIb: (4aS,6aS,6bR,10S,12aR)-Methyl
10-hydroxy-2,2,6a,6b,9,9,12a-heptamethyl-1,2,3,4,4a,5,6,6a,6b,7,8,8a,9,10-
,11,12,12a,12b,13,14b-icosahydropicene-4-a-carboxylate
[0131] Oleanolic acid (20 g, 43.79 mmol) was dissolved in DMF (200
mL). Potassium carbonate (9.06 g, 65.68 mmol) and methyl iodide
(3.3 mL, 52.55 mmol) were added. The mixture was stirred at room
temperature overnight and then poured into H.sub.2O (500 mL). The
precipitate was collected by filtration. The precipitate was
dissolved in EtOAc (500 mL). The solution was washed with brine
then dried (Na.sub.2SO.sub.4), filtered and concentrated to give
the sub-title compound (20.9 g, 100%) which was used without
further purification.
[0132] .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 0.70-1.97 (m,
42H), 2.83-2.84 (m, 1H), 3.19-3.22 (m, 1H), 3.62 (s, 3H), 5.28 (t,
J=3.6 Hz, 1H).
(ii) Preparation of IIc: (4aS,6aS,6bR,10S,12aR)-Methyl
10-acetoxy-2,2,6a,6b,9,9,12a-heptamethyl-1,2,3,4,4a,5,6,6a,6b,7,8,8a,9,10-
,11,12,12a,12b,13,14b-icosahydropicene-4-a-carboxylate
[0133] To a solution of IIb (20.9 g, 44.47 mmol) in pyridine (120
mL) was added DMAP (100 mg, 0.81 mmol) and acetic anhydride (5.04
mL, 53.36 mmol). The mixture was stirred at room temperature for 20
hours and poured into H.sub.2O (300 mL). The precipitate was
collected by filtration. The precipitate was dried in a vacuum oven
overnight to obtain the sub-title compound (21.33 g, 94%) which was
used without further purification.
[0134] .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 0.72-1.97 (m,
43H), 2.04 (s, 3H), 2.82-2.88 (m, 1H), 3.62 (s, 3H), 4.46-4.51 (m,
1H), 5.27 (t, J=3.6 Hz, 1H).
(iii) Preparation of IId: (4aR,6aS,6bR,10S,12aS)-Methyl
10-acetoxy-13,14,14b-tribromo-2,2,6a,6b,9,9,12a-heptamethyl-1,2,3,4,4a,5,-
6,6a,6b,7,8,8a,9,10,11,12,12a,12b,13,14b-icosahydropicene-4-a-carboxylate
[0135] To a solution of IIc (35 g, 68.35 mmol) in AcOH (330 mL) and
CCl.sub.4 (160 mL) was added bromine (14.05 mL, 273.4 mmol) slowly.
The mixture was stirred at room temperature for 17 hours. The
CCl.sub.4 was removed under reduced pressure and the reaction
mixture was poured into H.sub.2O (1 L). The precipitate was
collected by filtration and then dissolved in EtOAc (1 L). The
solution was washed with brine then dried (Na.sub.2SO.sub.4),
filtered and concentrated to give the sub-title compound (50.5 g,
98%) which was used without further purification.
[0136] .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 0.75-1.75 (m,
38H), 2.04 (s, 3H), 2.40-2.43 (m, 2H), 2.57 (t, J=12.6 Hz, 1H),
3.63 (s, 3H), 4.44-4.50 (m, 1H), 4.58-4.64 (m, 1H).
(iv) Preparation of IIe: (4aS,6aS,6bR,10S,12aR)-Methyl
10-acetoxy-14-bromo-2,2,6a,6b,9,9,12a-heptamethyl-1,2,3,4,4a,5,6,6a,6b,7,-
8,8a,9,10,11,12,12a,12b,13,14b-icosahydropicene-4-a-carboxylate
[0137] Zinc dust (21.8 g, 335.7 mmol) was added to a solution of
IId (50.5 g, 67.15 mmol) and AcOH (450 mL). The mixture was stirred
at room temperature overnight and then heated at reflux for 2
hours. The reaction mixture was cooled to room temperature and then
poured into H.sub.2O (600 mL). The precipitate was collected by
filtration and then dissolved in EtOAc (1 L). The solution was
washed with brine then dried (Na.sub.2SO.sub.4), filtered and
concentrated. The residue was purified by column chromatography
(silica, 0-10% EtOAc in hexanes) to obtain the sub-title compound
(21.5 g, 54%).
[0138] .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 0.75-1.78 (m,
40H), 1.93-1.95 (m, 1H), 2.04 (s, 3H), 2.41 (m, 2H), 3.54 (m, 1H),
3.63 (s, 3H), 4.48 (m, 1H).
(v) Preparation of IIf:
(4aS,6aS,6bR,10S,12aR)-10-Acetoxy-14-bromo-2,2,6a,6b,9,9,12a-heptamethyl--
1,2,3,4,4a,5,6,6a,6b,7,8,8a,9,10,11,12,12a,12b,13,14b-icosahydropicene-4-a-
-carboxylic acid
[0139] To a solution of IIe (21.5 g, 36.31 mmol) in 2,6-lutidine
(300 mL) was added lithium iodide (72.9 g, 544.7 mmol). The mixture
was heated at 143.degree. C. overnight and then cooled to room
temperature. The mixture was acidified (pH.apprxeq.4) with aqueous
HCl and extracted with EtOAc (300 mL.times.3). The combined
extracts were washed with H.sub.2O and brine then dried
(Na.sub.2SO.sub.4), filtered and concentrated to afford the
sub-title compound (22.8 g, 109%) which was used without further
purification.
[0140] .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 0.80-1.82 (m,
42H), 2.04 (s, 3H), 2.41 (m, 2H), 3.51 (m, 1H), 4.48 (m, 1H).
(vi) Preparation of IIg: (4aS,6aS,6bR,10S,12aR)-Benzyl
10-acetoxy-14-bromo-2,2,6a,6b,9,9,12a-heptamethyl-1,2,3,4,4a,5,6,6a,6b,7,-
8,8a,9,10,11,12,12a,12b,13,14b-icosahydropicene-4-a-carboxylate
[0141] To a solution of IIf (21.2 g, 36.67 mmol) and DMF (350 mL)
was added K.sub.2CO.sub.3 and BnBr (as used herein, "Bn" represents
benzyl). The reaction mixture was stirred overnight at room
temperature and then poured into H.sub.2O (1 L). The precipitate
was collected by filtration and then dissolved in EtOAc (1 L). The
solution was washed with H.sub.2O and brine then dried
(Na.sub.2SO.sub.4), filtered and concentrated to afford the
sub-title compound (24.5 g, 100%) which was used without further
purification.
[0142] .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 0.57-2.00 (m,
41H), 2.04 (s, 3H), 2.34 (d, J=8.7 Hz, 2H), 3.59 (dd, J=4.8, 4.2
Hz, 1H), 4.49 (m, 1H), 5.08 (s, 2H), 7.29-7.38 (m, 5H).
(vii) Preparation of IIh: (4aS,6aS,6bR,10S,12aR)-Benzyl
14-bromo-10-hydroxy-2,2,6a,6b,9,9,12a-heptamethyl-1,2,3,4,4a,5,6,6a,6b,7,-
8,8a,9,10,11,12,12a,12b,13,14b-icosahydropicene-4-a-carboxylate
[0143] To a solution of IIg (24.5 g, 36.67 mmol) in MeOH (500 mL)
was added potassium hydroxide (8.2 g, 146.7 mmol) and the mixture
was heated at reflux for 2.5 hours. The resulting mixture was
concentrated and the residue was dissolved in EtOAc (1 L). The
solution was washed with H.sub.2O and brine then dried
(Na.sub.2SO.sub.4), filtered and concentrated. The residue was
purified by column chromatography (silica, 0-30% EtOAc in hexanes)
to afford the sub-title compound (21.3 g, 93%).
[0144] .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 0.58-1.81 (m,
41H), 2.01 (m, 1H), 2.37 (d, J=9.3 Hz, 2H), 3.21 (m, 1H), 3.58 (dd,
J=4.8, 4.1 Hz, 1H), 5.08 (s, 2H), 7.29-7.38 (m, 5H).
(viii) Preparation of IIi: (4aS,6aS,6bR,12aR)-Benzyl
14-bromo-2,2,6a,6b,9,9,12a-heptamethyl-10-oxo-1,2,3,4,4a,5,6,6a,6b,7,8,8a-
,9,10,11,12,12a,12b,13,14b-icosahydropicene-4-a-carboxylatelate
[0145] To a solution of IIh (21.3 g, 34.02 mmol) in
CH.sub.2Cl.sub.2 (400 mL) was added the Dess-Martin reagent (18.76
g, 44.23 mmol). The mixture was stirred at room temperature for 5
hours, quenched with aqueous sodium thiosulfate and NaHCO.sub.3 and
then extracted with EtOAc (3.times.300 mL). The organic solution
was washed with H.sub.2O and brine then dried (Na.sub.2SO.sub.4),
filtered and concentrated. The residue was purified by column
chromatography (silica, 0-15% EtOAc in hexanes) to afford the
sub-title compound (19.3 g, 90%).
[0146] .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 0.24-2.27 (m,
40H), 2.94 (d, J=8.7 Hz, 1H), 3.44 (dd, J=4.8, 4.2 Hz, 1H),
5.13-5.15 (m, 2H), 7.33-7.35 (m, 5H).
(ix) Preparation of IIj: (4aS,6aS,6bR,12aR)-Benzyl
14-bromo-11-cyano-2,2,6a,6b,9,9,12a-heptamethyl-10-oxo-1,2,3,4,4a,5,6,6a,-
6b,7,8,8a,9,10,11,12,12a,12b,13,14b-icosahydropicene-4-a-carboxylate
[0147] To a solution of diisopropylamine (0.64 mL, 4.5 mmol) in THF
(15 mL) was added n-butyl lithium (2.5 M; 1.9 mL, 4.8 mmol) at
-78.degree. C. The mixture was stirred at -78.degree. C. for 10
min. The LDA solution was added to a solution of IIi (1.5 g, 2.4
mmol) in THF (40 mL) pre-cooled to -78.degree. C. The mixture was
stirred at -78.degree. C. for 1 hour. A solution of
4-methylbenzenesulfonyl cyanide (815 mg, 4.5 mmol) in THF (5 mL)
was added and stirred for 30 min at -78.degree. C. The reaction
mixture was warmed to -20.degree. C. over 1 hour. The reaction
mixture was quenched with saturated NH.sub.4Cl solution and
extracted with EtOAc (3.times.50 mL). The organic layer was washed
with H.sub.2O and brine then dried (Na.sub.2SO.sub.4), filtered and
concentrated. The residue was purified by column chromatography
(silica, 0-20% EtOAc in hexanes) to afford the sub-title compound
(1.2 g, 77%).
[0148] .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 0.56-2.42 (m,
41H), 3.61-3.89 (m, 2H), 5.08 (s, 2H), 7.35 (m, 5H).
(x) Preparation of II: (4aS,6aS,6bR,13aR)-Benzyl
12-amino-15-bromo-2,2,6a,6b,9,9,13a-heptamethyl-2,3,4,4a,5,6,6a,6b,7,8,8a-
,9,11,13,13a,13b,14,15b-octadecahydro-1H-chryseno[1,2-f]indazole-4-a-carbo-
xylate
[0149] To a solution of IIj (6.4 g, 9.89 mmol) and EtOH (30 mL) was
added hydrazine (0.62 mL, 19.78 mmol). The solution was heated at
reflux overnight. The reaction mixture was concentrated and then
the residue was purified by column chromatography (silica, 0-10%
MeOH in CH.sub.2Cl.sub.2) to afford the title compound (5.0 g,
76%).
[0150] .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 0.63-2.06 (m,
38H), 2.29 (d, J=14.7 Hz, 1H), 2.45 (d, J=9.0 Hz, 2H), 3.65 (dd,
J=4.4, 4.2 Hz, 1H), 5.08 (s, 2H), 7.29-7.39 (m, 5H). APCI MS
(Positive Mode) m/z 662
[C.sub.38H.sub.52BrN.sub.3O.sub.2+H].sup.+.
Preparation of Common Intermediate III
##STR00155##
[0151] (i) Preparation of III:
(4aS,6aS,6bR,13aR)-12-Amino-2,2,6a,6b,9,9,13a-heptamethyl-2,3,4,4a,5,6,6a-
,6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecahydro-1H-chryseno[1,2-f]indazole-
-4-a-carbonyl chloride
[0152] To a suspension of 1 (as defined above) (3.0 g, 6.08 mmol)
in CH.sub.2Cl.sub.2 was added thionyl chloride (4.4 mL, 60.85 mmol)
at room temperature. The mixture was stirred for 2.5 hours, after
which time the solvent was removed under reduced pressure. The
residue was dried in a high vacuum to provide the title compound
(3.2 g, >100%). The material was used without further
purification. APCI MS (Positive Mode) m/z 512
[C.sub.31H.sub.46ClN.sub.3O+H].sup.+.
##STR00156##
(i) Preparation of 1:
(4aS,6aS,6bR,13aR)-12-Amino-2,2,6a,6b,9,9,13a-heptamethyl-2,3,4,4a,5,6,6a-
,6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecahydro-1H-chryseno[1,2-f]indazole-
-4-a-carboxylic acid
[0153] A suspension of I (as defined above) (1.8 g, 3.1 mmol), 10%
Pd/C (1.0 g) and MeOH (100 mL) was stirred under hydrogen at
atmospheric pressure for 2 hours. The catalyst was removed by
filtration through diatomaceous earth and the filtrate was
concentrated under reduced pressure to provide the title compound
(1.4 g, 88%) as an off-white solid.
[0154] R.sub.f 0.37 (9:1 Methylene Chloride/Methanol) m.p.
263-266.degree. C.
[0155] .sup.1H NMR (500 MHz, CD.sub.3OD) .delta. 0.82-0.98 (m, 9H),
1.12-1.31 (m, 15H), 1.39-1.87 (m, 13H), 2.01-2.10 (m, 3H),
2.35-2.42 (m, 1H), 2.87-2.91 (m, 1H), 5.28-5.33 (m, 1H). ESI MS m/z
494 [C.sub.31H.sub.47N.sub.3O.sub.2+H].sup.+. HPLC 98.8% (area %),
t.sub.R=16.2 min.
Example 2
##STR00157## ##STR00158##
[0156] (i) Preparation of 2b:
(4aS,6aS,6bR,12aR)-1,2,6a,6b,9,9,12a-Heptamethyl-10-oxo-1,2,3,4,4a,5,6,6a-
,6b,7,8,8a,9,10,11,12,12a,12b,13,14b-icosahydropicene-4-a-carboxylic
acid
[0157] To a mixture of ursolic acid (2a, 1.0 g, 2.18 mmol) and
CH.sub.2Cl.sub.2 (55 mL) was added the Dess-Martin reagent (1.2 g,
2.83 mmol) under nitrogen at room temperature. After stirring at
room temperature for 2.5 hours, the starting material was consumed
as indicated by TLC (1:1 hexanes:diethyl ether). The reaction
mixture was quenched with the addition of a solution of sodium
thiosulfate and NaHCO.sub.3 (6.3 g sodium thiosulfate in 25 mL
saturated NaHCO.sub.3 solution). The mixture was stirred at room
temperature for 10 minutes. The layers were separated and the
aqueous layer was extracted with EtOAc (3.times.250 mL). The
combined organics were dried (Na.sub.2SO.sub.4), filtered and
concentrated under reduced pressure. The residue was purified by
column chromatography (silica, 3:1 hexanes/diethyl ether) to
provide the sub-title compound (924 mg, 92%) as a white foam
solid.
(ii) Preparation of 2c: (4aS,6aS,6bR,12aR)-Benzyl
1,2,6a,6b,9,9,12a-heptamethyl-10-oxo-1,2,3,4,4a,5,6,6a,6b,7,8,8a,9,10,11,-
12,12a,12b,13,14b-icosahydropicene-4-a-carboxylate
[0158] A mixture of 2b (924 mg, 2.03 mmol), benzyl bromide (0.31
mL, 0.64 mmol), K.sub.2CO.sub.3 (393 mg, 2.84 mmol) and DMF (50 mL)
was stirred at room temperature for 12 hours. The solvent was then
removed under reduced pressure and the residue was partitioned
between H.sub.2O (150 mL) and EtOAc (150 mL). The layers were
separated and the aqueous layer was extracted with EtOAc (50 mL).
The combined organics were dried (Na.sub.2SO.sub.4) and filtered.
The solvent was removed under reduced pressure and the residue was
purified by column chromatography (silica, 1:1 hexanes/diethyl
ether) to provide the sub-title compound (1.0 g, 92%) as a white
solid.
[0159] .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 0.68 (s, 3H), 0.84
(d, J=6.3 Hz, 3H), 0.92 (d, J=6.0 Hz, 3H), 1.02 (s, 3H), 1.03 (s,
3H), 1.08 (s, 6H), 1.20-2.05 (m, 20H), 2.26 (d, J=15.0 Hz, 1H),
2.30 (m, 1H), 2.50 (m, 1H), 4.96 (m, 2H), 5.24 (m, 1H), 7.30-7.35
(m, 5H).
(iii) Preparation of 2d: (4aS,6aS,6bR,12aR)-Benzyl
11-cyano-1,2,6a,6b,9,9,12a-heptamethyl-10-oxo-1,2,3,4,4a,5,6,6a,6b,7,8,8a-
,9,10,11,12,12a,12b,13,14b-icosahydropicene-4-a-carboxylate
[0160] A solution of diisopropylamine (0.45 mL, 3.24 mmol) and THF
(5 mL) was cooled to -78.degree. C. under nitrogen. A solution of
n-butyllithium (2.5 M in hexanes, 1.43 mL, 3.56 mmol) was slowly
added, maintaining the internal temperature below -70.degree. C.
The solution was allowed to stir for 30 min and was then slowly
added to a solution of 2c (880 mg, 1.62 mmol) and THF (10 mL) at
-78.degree. C. under nitrogen. This solution was stirred for 30 min
after which time a suspension of p-toluenesulfonyl cyanide (587 mg,
3.24 mmol) and THF (2 mL) was added over 45 min. The solution was
stirred for 10 min and then quenched by the addition of saturated
ammonium chloride solution (3 mL) at -78.degree. C. The mixture was
allowed to warm to room temperature overnight. The organic layer
was separated and the aqueous layer was extracted with EtOAc. The
combined organic layers were dried (Na.sub.2SO.sub.4) and filtered.
The solvent was removed under reduced pressure and the residue was
purified by column chromatography (silica, 3:1 hexanes/Et.sub.2O)
to provide the sub-title compound (600 mg, 65%) as a white foam
solid.
(iv) Preparation of 2e: (4aS,6aS,6bR,13aR)-Benzyl
12-amino-1,2,6a,6b,9,9,13a-heptamethyl-2,3,4,4a,5,6,6a,6b,7,8,8a,9,11,13,-
13a,13b,14,15b-octadecahydro-1H-chryseno[1,2-f]indazole-4-a-carboxylate
[0161] A solution of 2d (303 mg, 0.53 mmol) and hydrazine (0.050
mL, 1.59 mmol) in EtOH (5 mL) was heated at reflux for 12 hours.
The solvent and excess hydrazine were removed under reduced
pressure. The residue was purified by column chromatography
(silica, 0-10% MeOH in CH.sub.2Cl.sub.2) to provide the sub-title
compound (234 mg, 75%) as a white foam solid.
[0162] .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 0.69 (s, 3H), 0.86
(d, J=5.7 Hz, 3H), 0.88 (s, 3H), 0.93 (d, J=6.0 Hz, 3H), 1.09 (s,
3H), 1.13 (s, 3H), 1.22 (s, 3H), 1.38-2.05 (m, 19H), 2.28 (m, 1H),
2.30 (d, J=14.7 Hz, 1H), 4.96 (q, J=12.6 Hz, 2H), 5.29 (s, 1H),
7.32 (m, 5H).
(v) Preparation of 2:
(4aS,6aS,6bR,13aR)-12-Amino-2,2,6a,6b,9,9,13a-heptamethyl-2,3,4,4a,5,6,6a-
,6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecahydro-1H-chryseno[1,2-f]indazole-
-4-a-carboxylic acid
[0163] A suspension of 2e (234 mg, 0.40 mmol), 10% Pd/C (60 mg) and
MeOH (6 mL) was stirred under hydrogen at atmospheric pressure for
9 hours. The catalyst was removed by filtration and the filtrate
was concentrated under reduced pressure. The residue was purified
by column chromatography (silica, 0-15% MeOH in CH.sub.2Cl.sub.2)
to provide the title compound (103 mg, 52%) as an off-white
solid.
[0164] R.sub.f 0.16 (89:18:2 Methylene
Chloride/Methanol/concentrated Ammonium Hydroxide).
[0165] m.p. 258-278.degree. C. .sup.1H NMR (300 MHz, CD.sub.3OD)
.delta. 0.89 (s, 9H), 0.91 (s, 3H), 1.15 (s, 6H), 1.19 (s, 3H),
2.05-2.10 (m, 19H), 2.22 (m, 1H), 2.40 (m, 1H), 5.30 (s, 1H). ESI
MS m/z 494 [C.sub.31H.sub.47N.sub.3O.sub.2+H].sup.+.
Example 3
##STR00159## ##STR00160##
[0166] (i) Preparation of 3b:
(4aS,6aS,6bR,8aR,9R,10S,12aR,14bS)-Benzyl
10-hydroxy-9-(hydroxymethyl)-2,2,6a,6b,9,12a-hexamethyl-1,2,3,4,4a,5,6,6a-
,6b,7,8,8a,9,10,11,12,12a,12b,13,14b-icosahydropicene-4-a-carboxylate
[0167] A mixture of 3a (2.0 g, 4.2 mmol), benzyl bromide (0.6 mL,
5.0 mmol) and K.sub.2CO.sub.3 (580 mg, 6.3 mmol) in DMF (20 mL) was
stirred at room temperature for 12 hours. The reaction mixture was
diluted with EtOAc (300 mL). The organic phase was washed with
brine then dried (MgSO.sub.4), filtered and concentrated to
dryness. The residue was purified by column chromatography (silica,
0-50% EtOAc in hexanes) to afford the sub-title compound (2.3 g,
95%).
(ii) Preparation of 3c: (4aS,6aS,6bR,8aR,9R,12aR,14bS)-Benzyl
9-(hydroxymethyl)-2,2,6a,6b,9,12a-hexamethyl-10-oxo-1,2,3,4,4a,5,6,6a,6b,-
7,8,8a,9,10,11,12,12a,12b,13,14b-icosahydropicene-4-a-carboxylate
[0168] To a solution of 3b (400 mg, 0.71 mmol) in benzene (20 mL)
was added RuCl.sub.2(PPh.sub.3).sub.3 (682 mg, 0.71 mmol). The
mixture was stirred at room temperature for 12 hours. The reaction
was not complete. An additional amount of
RuCl.sub.2(PPh.sub.3).sub.3 (341 mg, 0.36 mmol) was added and the
reaction mixture was continued to stir for 8 hours. The reaction
mixture was diluted with EtOAc (150 mL). The organic phase was
washed with brine then dried (MgSO.sub.4), filtered and
concentrated to dryness. The residue was purified by column
chromatography (silica, 0-50% EtOAc in hexanes) to afford the
sub-title compound (150 mg, 38%).
(iii) Preparation of 3d: (4aS,6aS,6bR,8aS,9S,12aS,14bS)-Benzyl
2,2,6a,6b,9,12a-hexamethyl-10-oxo-1,2,3,4,4a,5,6,6a,6b,7,8,8a,9,10,11,12,-
12a,12b,13,14b-icosahydropicene-4-a-carboxylate
[0169] A mixture of 3c (150 mg, 0.26 mmol) and K.sub.2CO.sub.3 (44
mg, 0.32 mmol) in MeOH (10 mL) was stirred at room temperature for
24 hours. The reaction mixture was diluted with EtOAc (150 mL) and
washed with brine then dried (MgSO.sub.4), filtered and
concentrated to dryness. The residue was purified by column
chromatography (silica, 0-30% EtOAc in hexanes) to afford the
sub-title compound (70 mg, 49%).
(iv) Preparation of 3e: (4aS,6aS,6bR,8aS,9S,12aS,14bS)-Benzyl
11-cyano-2,2,6a,6b,9,12a-hexamethyl-10-oxo-1,2,3,4,4a,5,6,6a,6b,7,8,8a,9,-
10,11,12,12a,12b,13,14b-icosahydropicene-4-a-carboxylate
[0170] To a solution of diisopropylamine (0.073 mL, 0.52 mmol) in
THF (4 mL) was added n-butyllithium (0.22 mL, 2.5 M in hexanes,
0.54 mmol) at -78.degree. C. The solution was stirred for 30 min.
The LDA solution was added to 3d (150 mg, 0.27 mmol) in THF (5 mL).
The mixture was allowed to warm to -40.degree. C. for 5 min and
cooled to -78.degree. C. A suspension of p-toluene sulfonyl cyanide
(98 mg, 0.54 mmol) in THF (2 mL) was added. The reaction mixture
was allowed to warm to -40.degree. C. over 1.5 hours. The reaction
was quenched by saturated NH.sub.4Cl (3 mL) and extracted with
EtOAc (100 mL). The organic phase was washed with brine then dried
(MgSO.sub.4), filtered and concentrated to dryness. The residue was
purified by column chromatography (silica, 0-30% EtOAc in hexanes)
to afford the sub-title compound (90 mg, 58%).
[0171] .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 0.65 (s, 3H), 0.86
(s, 3H), 0.88 (s, 3H), 1.0-2.0 (m, 28H), 2.35 (m, 2H), 2.92 (m,
1H), 3.61 (m, 1H), 5.15 (s, 2H), 5.30 (m, 1H), 7.30 (s, 5H).
(v) Preparation of 3f: (4aS,6aS,6bR,8aS,9S,13aS,15bS)-Benzyl
12-amino-2,2,6a,6b,9,13a-hexamethyl-2,3,4,4a,5,6,6a,6b,7,8,8a,9,11,13,13a-
,13b,14,15b-octadecahydro-1H-chryseno[1,2-f]indazole-4-a-carboxylate
[0172] A mixture of 3e (90 mg, 0.15 mmol) and hydrazine (0.025 mL)
in EtOH (3 mL) was heated at reflux overnight. The reaction mixture
was concentrated to dryness under reduced pressure. The residue was
purified by column chromatography (silica, 0-10% MeOH in
CH.sub.2Cl.sub.2) to afford the sub-title compound (60 mg,
65%).
(v) Preparation of 3:
(4aS,6aS,6bR,8aS,9S,13aS,15bS)-12-Amino-2,2,6a,6b,9,13a-hexamethyl-2,3,4,-
4a,5,6,6a,6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecahydro-1H-chryseno[1,2-f-
]indazole-4-a-carboxylic acid
[0173] A mixture of 3f (60 mg, 0.10 mmol) and 10% Pd(OH).sub.2/C
(60 mg) in MeOH (5 mL) and EtOAc (5 mL) was stirred under hydrogen
balloon for 5 hours. The reaction mixture was filtered through a
pad of diatomaceous earth and washed with CMA (25 mL). The filtrate
was concentrated to dryness under reduced pressure. The residue was
purified by column chromatography (silica, 0-60% CMA in
CH.sub.2Cl.sub.2) to afford the title compound (32 mg, 68%) as a
brown solid.
[0174] R.sub.f 0.40 (80:18:2 Methylene
Chloride/Methanol/concentrated Ammonium Hydroxide).
[0175] .sup.1H NMR (300 MHz, DMSO-d.sub.6) .delta. 0.78 (s, 3H),
0.80 (s, 3H), 0.91 (s, 6H), 1.05 (s, 3H), 1.10-2.0 (m, 25H), 2.15
(m, 1H), 2.25 (d, J=15.0 Hz, 1H), 2.80 (m, 1H), 5.24 (s, 1H), 11.4
(bs, 1H). mp>300.degree. C. ESI MS (Positive Mode) m/z 478
[C.sub.30H.sub.45N.sub.3O.sub.2+H].sup.+.
Example 4
##STR00161##
[0176] (i) Preparation of 4b: (4aS,6aS,6bR,8aR,12aR,14bS)-Benzyl
2,2,6a,6b,9,9,12a,14-octamethyl-10-oxo-1,2,3,4,4a,5,6,6a,6b,7,8,8a,9,10,1-
1,12,12a,12b,13,14b-icosahydropicene-4-a-carboxylate
[0177] A mixture of IIi (300 mg, 0.48 mmol), trimethylboroxine (0.3
mL, 2.15 mmol), Pd(PPh.sub.3).sub.4 (60 mg, 0.048 mmol) and
K.sub.2CO.sub.3 (300 mg, 2.17 mmol) in DMF (9 mL) was heated at
100.degree. C. for 12 hours. The reaction mixture was diluted with
EtOAc (100 mL). The organic phase was washed with brine then dried
(MgSO.sub.4), filtered and concentrated to dryness. The residue was
purified by column chromatography (silica, 0-10% EtOAc in hexanes)
to afford the sub-title compound (220 mg, 82%).
(ii) Preparation of 4c: (4aS,6aS,6bR,8aR,12aR,14bS)-Benzyl
11-cyano-2,2,6a,6b,9,9,12a,14-octamethyl-10-oxo-1,2,3,4,4a,5,6,6a,6b,7,8,-
8a,9,10,11,12,12a,12b,13,14b-icosahydropicene-4-a-carboxylate
[0178] To a solution of diisopropylamine (0.15 mL, 1.07 mmol) in
THF (5 mL) was added n-butyllithium (0.45 mL, 2.5 M in hexanes, 1.1
mmol) at -78.degree. C. The solution was stirred for 30 min. The
LDA solution was added to 4b (315 mg, 0.56 mmol) in THF (5 mL). The
mixture was allowed to warm to -40.degree. C. for 5 min and cooled
to -78.degree. C. A suspension of p-toluene sulfonyl cyanide (203
mg, 1.1 mmol) in THF (2 mL) was added. The reaction mixture was
allowed to warm to -40.degree. C. over 1.5 hours. The reaction was
quenched by saturated NH.sub.4Cl (3 mL) and extracted with EtOAc
(100 mL). The organic phase was washed with brine then dried
(MgSO.sub.4), filtered and concentrated to dryness. The residue was
purified by column chromatography (silica, 0-20% EtOAc in hexanes)
to afford the sub-title compound (215 mg, 66%).
(iii) Preparation of 4d: (4aS,6aS,6bR,8aR,13aR,15bS)-Benzyl
12-amino-2,2,6a,6b,9,9,13a,15-octamethyl-2,3,4,4a,5,6,6a,6b,7,8,8a,9,11,1-
3,13a,13b,14,15b-octadecahydro-1H-chryseno[1,2-f]indazole-4-a-carboxylate
[0179] A mixture of 4c (215 mg, 0.36 mmol) and hydrazine (0.060 mL,
1.85 mmol) in EtOH (3 mL) was heated at reflux overnight. The
reaction mixture was concentrated to dryness under reduced
pressure. The residue was purified by column chromatography
(silica, 0-10% MeOH in CH.sub.2Cl.sub.2) to afford the sub-title
compound (180 mg, 84%). ESI MS (Positive Mode) m/z 598
[C.sub.39H.sub.55N.sub.3O.sub.2+H].sup.+.
(iv) Preparation of 4:
(4aS,6aS,6bR,8aR,13aR,15bS)-12-Amino-2,2,6a,6b,9,9,13a,15-octamethyl-2,3,-
4,4a,5,6,6a,6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecahydro-1H-chryseno[1,2-
-f]indazole-4-a-carboxylic acid
[0180] A mixture of 4d (180 mg, 0.30 mmol) and 10% Pd(OH).sub.2/C
(100 mg) in MeOH (15 mL) and EtOAc (2 mL) was stirred under
hydrogen balloon for 12 hours. The reaction mixture was filtered
through a pad of diatomaceous earth and washed with CMA (25 mL).
The filtrate was concentrated to dryness under reduced pressure.
The residue was purified by column chromatography (silica, 0-50%
CMA in CH.sub.2Cl.sub.2) followed by preparative HPLC to provide
the title compound (28 mg, 18%) as an off-white solid.
[0181] R.sub.f 0.62 (80:18:2 Methylene
Chloride/Methanol/concentrated Ammonium Hydroxide).
[0182] .sup.1H NMR (300 MHz, CD.sub.3OD) .delta. 0.85 (s, 3H), 0.90
(s, 3H), 0.93 (s, 3H), 0.99 (s, 3H), 1.20 (s, 3H), 1.23 (s, 3H),
1.38 (s, 3H), 1.40-2.05 (m, 23H), 2.39 (d, J=14.7 Hz, 1H).
mp>300.degree. C. ESI MS (Positive Mode) m/z 508
[C.sub.32H.sub.49N.sub.3O.sub.2+H].sup.+.
Example 5
##STR00162## ##STR00163##
[0183] (i) Preparation of 5b:
(4aS,6aS,6bR,8aR,10S,12aR,14bR)-Methyl
14-bromo-10-hydroxy-2,2,6a,6b,9,9,12a-heptamethyl-1,2,3,4,4a,5,6,6a,6b,7,-
8,8a,9,10,11,12,12a,12b,13,14b-icosahydropicene-4-a-carboxylate
[0184] A mixture of IIE (36.0 g, 61.0 mmol) and KOH (13.6 g, 243
mmol) in MeOH (500 mL) was heated at reflux for 3 hours. The
mixture was cooled to room temperature and concentrated to remove
MeOH. The residue was acidified with HCl (2 M) to pH 5 and
extracted with EtOAc (500 mL). The organic phase was washed with
brine then dried (MgSO.sub.4), filtered and concentrated to afford
the sub-title compound (34.0 g, 94%) which was used for the next
step without further purification.
[0185] .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 0.78 (s, 3H), 0.80
(s, 3H), 0.95 (s, 3H), 0.98 (s, 3H), 1.01-2.35 (m, 33H), 3.20 (m,
1H), 3.62 (s, 3H).
(ii) Preparation of 5c: (4aS,6aS,6bR,8aR,12aR,14bR)-Methyl
14-bromo-2,2,6a,6b,9,9,12a-heptamethyl-10-oxo-1,2,3,4,4a,5,6,6a,6b,7,8,8a-
,9,10,11,12,12a,12b,13,14b-icosahydropicene-4-a-carboxylate
[0186] A mixture of 5b (32.0 g, 61.0 mmol) and Dess-Martin reagent
(31.0 g, 73.0 mmol) in CH.sub.2Cl.sub.2 (500 mL) was stirred at
room temperature for 4 hours. The mixture was quenched with
saturated sodium thiosulfate (50 mL) and NaHCO.sub.3 (50 mL) and
extracted with EtOAc (500 mL). The organic phase was washed with
brine then dried (MgSO.sub.4), filtered and concentrated to dryness
under reduced pressure. The residue was purified by column
chromatography (silica, 5-10% EtOAc in hexanes) to afford the
sub-title compound (26.0 g, 81%).
[0187] .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 0.80 (s, 3H), 0.90
(s, 3H), 0.98 (s, 3H), 1.02 (s, 3H), 1.05 (s, 6H), 1.06-2.55 (m,
26H), 3.62 (s, 3H).
(iii) Preparation of 5d: (4a'R,6a'R,6b'S,8a'S,12a'R,14b'R)-Methyl
13'-bromo-4',4',6a',6b',11',11',14b'-heptamethyl-2',4',4a',5',6',6a',6b',-
7',8',8a',9',10',11',12',12a',14',14a',14b'-octadecahydro-1'H-spiro[[1,3]d-
ioxolane-2,3'-picene]-8a'-carboxylate
[0188] A flask equipped with a Dean-Stark trap was charged 5c (26.0
g, 47.4 mmol), ethylene glycol (7.9 mL, 142.3 mmol) and
p-toluenesulfoinc acid monohydrate (894 mg, 4.7 mmol) in benzene
(500 mL). The mixture was heated at reflux overnight. The reaction
mixture was cooled to room temperature and diluted with EtOAc (500
mL). The organic phase was washed with saturated NaHCO.sub.3 (50
mL) and brine (200 mL) then dried (MgSO.sub.4), filtered and
concentrated to dryness. The residue was purified by column
chromatography (silica, 0-10% EtOAc in hexanes) to afford the
sub-title compound (18.0 g, 64%).
[0189] .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 0.74 (s, 3H), 0.83
(s, 3H), 0.92 (s, 3H), 0.94 (s, 3H), 0.97 (s, 3H), 0.99 (s, 3H),
1.14 (s, 3H), 1.15-2.05 (m, 21H), 2.39 (d, J=9.0 Hz, 1H), 3.57 (m,
1H), 3.63 (s, 3H), 3.93 (m, 4H).
(iv) Preparation of 5e: (4a'R,6a'R,6b'S,8a'S,12a'S,14b'R)-Methyl
13'-formyl-4',4',6a',6b',11',11',14b'-heptamethyl-2',4',4a',5',6',6a',6b'-
,7',8',8a',9',10',11',12',12a',14',14a',14b'-octadecahydro-1'H-spiro[[1,3]-
dioxolane-2,3'-picene]-8a'-carboxylate
[0190] To a solution of 5d (3.0 g, 5.1 mmol) in THF (60 mL) was
added tert-butyllithium (10 mL, 1.5 M in heptane, 15.0 mmol) at
-78.degree. C. The mixture was stirred for 20 min.
n-Methylformanilide (1.8 mL, 15.2 mmol) was added. The reaction
mixture was allowed to warm to -10.degree. C. over 1 hour. The
reaction mixture was quenched by saturated NH.sub.4Cl (10 mL) and
extracted with EtOAc (200 mL). The organic phase was washed with
brine (200 mL) then dried (MgSO.sub.4), filtered and concentrated
to dryness. The residue was purified by column chromatography
(silica, 0-10% EtOAc in hexanes) to afford the sub-title compound
(2.0 g, 73%).
[0191] .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 0.65 (s, 3H), 0.81
(s, 3H), 0.90 (s, 6H), 0.92 (s, 3H), 0.99 (s, 3H), 1.12 (s, 3H),
1.25-2.18 (m, 19H), 2.65 (s, 3H), 3.85 (m, 4H), 10.3 (s, 1H).
(v) Preparation of 5f: (4a'R,6a'R,6b'S,8a'S,12a'S,14b'R)-Methyl
4',4',6a',6b',11',11',14b'-heptamethyl-13'-(prop-1-enyl)-2',4',4a',5',6',-
6a',6b',7',8',8a',9',10',11',12',12a',14',14a',14b'-octadecahydro-1'H-spir-
o[[1,3]dioxolane-2,3'-picene]-8a'-carboxylate
[0192] To a suspension of ethyltriphenylphosphonium iodide (1.2 g,
2.96 mmol) in benzene (20 mL) was added potassium tert-butoxide
(332 mg, 2.96 mmol). The mixture was stirred at room temperature
for 1 hour. A solution of 5e (400 mg, 0.74 mmol) in THF (5 mL) was
added. The mixture was stirred at room temperature for 1.5 hours.
The reaction mixture was diluted with EtOAc (100 mL). The organic
phase was washed with brine then dried (MgSO.sub.4), filtered and
concentrated to dryness. The residue was purified by column
chromatography (silica, 0-10% EtOAc in hexanes) to afford the
sub-title compound (380 mg, 93%) as a mixture of cis- and
trans-isomers.
(vi) Preparation of 5g: (4aS,6aS,6bR,8aR,12aR,14bS)-Methyl
2,2,6a,6b,9,9,12a-heptamethyl-10-oxo-14-(prop-1-enyl)-1,2,3,4,4a,5,6,6a,6-
b,7,8,8a,9,10,11,12,12a,12b,13,14b-icosahydropicene-4-a-carboxylate
[0193] A mixture of 5f (571 mg, 0.92 mmol) and p-toluenesulfonic
acid monohydrate (176 mg, 0.92 mmol) in acetone (20 mL) and
H.sub.2O (2 mL) was stirred at room temperature for 1.5 hours. The
reaction mixture was concentrated to remove acetone under reduced
pressure and extracted with EtOAc (100 mL). The organic phase was
washed with brine then dried (MgSO.sub.4), filtered and
concentrated to dryness. The residue was purified by column
chromatography (silica, 0-10% EtOAc in hexanes) to afford the
sub-title compound (500 mg, 95%).
[0194] .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 0.72 (s, 1.8H),
0.81 (s, 1.2H), 0.89 (s, 3H), 0.91 (s, 3H), 1.0-2.50 (m, 38H), 3.65
(s, 3H), 5.40-5.70 (m, 1H), 6.05 (d, J=12.0 Hz, 0.4H), 6.53 (d,
J=15.0 Hz, 0.6H).
(vii) Preparation of 5h: (4aS,6aS,6bR,8aR,12aR,14bS)-Methyl
1'-cyano-2,2,6a,6b,9,9,12a-heptamethyl-10-oxo-14-(prop-1-enyl)-1,2,3,4,4a-
,5,6,6a,6b,7,8,8a,9,10,11,12,12a,12b,13,14b-icosahydropicene-4-a-carboxyla-
te
[0195] To a solution of diisopropylamine (0.26 mL, 1.87 mmol) in
THF (5 mL) was added n-butyllithium (0.78 mL, 2.5 M in hexanes,
1.96 mmol) at -78.degree. C. The solution was stirred for 30 min.
The LDA solution was added to 5g (500 mg, 0.98 mmol) in THF (5 mL).
The mixture was allowed to warm to -40.degree. C. for 5 min and
cooled to -78.degree. C. A suspension of p-toluene sulfonyl cyanide
(339 mg, 1.87 mmol) in THF (2 mL) was added. The reaction mixture
was allowed to warm to -40.degree. C. over 1.5 hours. The reaction
was quenched by saturated NH.sub.4Cl (3 mL) and extracted with
EtOAc (100 mL). The organic phase was washed with brine then dried
(MgSO.sub.4), filtered and concentrated to dryness. The residue was
purified by column chromatography (silica, 0-20% EtOAc in hexanes)
to afford the sub-title compound (210 mg, 40%).
[0196] .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 0.65-2.01 (m,
44H), 2.42-2.80 (m, 1H), 3.60 (s, 3H), 4.21 (m, 1H), 5.41-5.70 (m,
1H), 6.01 (m, 1H).
(viii) Preparation of 5i: (4aS,6aS,6bR,8aR,13aR,15bS)-Methyl
12-amino-2,2,6a,6b,9,9,13a-heptamethyl-15-((E)-prop-1-enyl)-2,3,4,4a,5,6,-
6a,6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecahydro-1H-chryseno[1,2-f]indazo-
le-4-a-carboxylate
[0197] A mixture of 5h (200 mg, 0.37 mmol) and hydrazine (0.014 mL,
0.45 mmol) in EtOH (3 mL) was heated at reflux overnight. The
reaction mixture was concentrated to dryness under reduced
pressure. The residue was purified by column chromatography
(silica, 0-10% MeOH in CH.sub.2Cl.sub.2) to afford the sub-title
compound (130 mg, 64%).
[0198] .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 0.71 (s, 1.8H),
0.81 (s, 1.2H), 0.88 (s, 3H), 0.89 (s, 6H), 0.98 (s, 1.8H), 1.02
(s, 1.2H), 1.05 (s, 3H), 1.12 (s, 3H), 1.16-2.35 (m, 27H), 3.65 (s,
3H), 5.50 (m, 0.4H), 5.70 (m, 0.6H), 6.05 (d, J=12.0 Hz, 0.4H),
6.55 (d, J=15.0 Hz, 0.6H).
(ix) Preparation of 5:
(4aS,6aS,6bR,8aR,13aR,15bS)-12-Amino-2,2,6a,6b,9,9,13a-heptamethyl-15-((E-
)-prop-1-enyl)-2,3,4,4a,5,6,6a,6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecahy-
dro-1H-chryseno[1,2-f]indazole-4-a-carboxylic acid
[0199] A mixture of 5h (90 mg, 0.16 mmol) and lithium iodide (330
mg, 2.47 mmol) in 2,6-lutidine (3 mL) was heated to 144.degree. C.
for 6 hours. The reaction mixture was cooled to room temperature
and neutralized with HCl (2 M) and extracted with
CH.sub.2Cl.sub.2/i-PrOH (3:1). The organic phase was dried
(MgSO.sub.4), filtered and concentrated to dryness under reduced
pressure. The residue was purified by column chromatography
(silica, 0-40% CMA in CH.sub.2Cl.sub.2) followed by preparative
HPLC to provide the title compound (20 mg, 23%) as an off-white
solid.
[0200] R.sub.f 0.85 (80:18:2 Methylene
Chloride/Methanol/concentrated Ammonium Hydroxide).
[0201] .sup.1H NMR (300 MHz, CD.sub.3OD) .delta. 0.80 (s, 3H), 0.90
(s, 6H), 1.02 (s, 3H), 1.18 (s, 3H), 1.20 (s, 3H), 1.28 (s, 3H),
1.30-2.30 (m, 22H), 2.52 (d, J=15.0 Hz, 1H), 3.63 (m, 1H), 5.68
(dd, J=6.0, 15.0 Hz, 1H), 6.59 (d, J=15.0 Hz, 1H).
mp>300.degree. C. ESI MS (Positive Mode) m/z 534
[C.sub.34H.sub.51N.sub.3O.sub.2+H].sup.+.
Example 6
##STR00164##
[0202] wherein TIPS refers to triisopropylsilyl.
(i) Preparation of 6b: (4aS,6aS,6bR,8aR,13aR,15bS)-Benzyl
12-amino-2,2,6a,6b,9,9,13a-heptamethyl-15-(1-(triisopropylsilyl)-1H-pyrro-
l-3-yl)-2,3,4,4a,5,6,6a,6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecahydro-1H--
chryseno[1,2-f]indazole-4-a-carboxylate
[0203] A mixture of II (250 mg, 0.37 mmol),
1-(triisopropylsilyl)-1H-pyrrol-3-ylboronic acid (303 mg, 1.13
mmol), Pd(PPh.sub.3).sub.4 (43 mg, 0.037 mmol) and K.sub.2CO.sub.3
(203 mg, 1.47 mmol) in benzene (3.5 mL) and EtOH (1.5 mL) was
sealed and heated to 120.degree. C. by microwave for 1 hour. The
reaction mixture was diluted with EtOAc (100 mL). The organic phase
was washed with brine then dried (MgSO.sub.4), filtered and
concentrated to dryness. The residue was purified by column
chromatography (silica, 0-10% MeOH in CH.sub.2Cl.sub.2) to afford
the sub-title compound (130 mg, 43%). APCI MS (Positive Mode) m/z
805 [C.sub.51H.sub.76N.sub.4O.sub.2Si+H].sup.+.
(ii) Preparation of 6c: (4aS,6aS,6bR,8aR,13aR,15bS)-Benzyl
12-amino-2,2,6a,6b,9,9,13a-heptamethyl-15-(1H-pyrrol-3-yl)-2,3,4,4a,5,6,6-
a,6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecahydro-1H-chryseno[1,2-f]indazol-
e-4-a-carboxylate
[0204] To a solution of 6b (130 mg, 0.16 mmol) in THF (2 mL) was
added tetrabutylammonium fluoride (0.49 mL, 1 M in THF, 0.49 mmol).
The mixture was stirred at room temperature for 2 hours. The
reaction mixture was diluted with EtOAc (100 mL). The organic phase
was washed with brine then dried (MgSO.sub.4), filtered and
concentrated to dryness. The residue was purified by column
chromatography (silica, 0-10% MeOH in CH.sub.2Cl.sub.2) to afford
the sub-title compound (90 mg, 87%). APCI MS (Positive Mode) m/z
649 [C.sub.42H.sub.56N.sub.4O.sub.2+H].sup.+.
(iii) Preparation of 6:
(4aS,6aS,6bR,8aR,13aR,15bS)-12-Amino-2,2,6a,6b,9,9,13a-heptamethyl-15-(1H-
-pyrrol-3-yl)-2,3,4,4a,5,6,6a,6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecahyd-
ro-1H-chryseno[1,2-f]indazole-4-a-carboxylic acid
[0205] A mixture of 6c (90 mg, 0.13 mmol) and 10% Pd(OH).sub.2/C
(50 mg) in MeOH (12 mL) and EtOAc (3 mL) was stirred under a
hydrogen balloon for 5 hours. The reaction mixture was filtered
through a pad of diatomaceous earth and washed with CMA (25 mL).
The filtrate was concentrated to dryness under reduced pressure.
The residue was purified by column chromatography (silica, 0-50%
CMA in CH.sub.2Cl.sub.2) to afford the title compound (18 mg, 25%)
as a brown solid.
[0206] R.sub.f -0.26 (9:1 Methylene Chloride/Methanol).
[0207] .sup.1H NMR (400 MHz, CD.sub.3OD) .delta. 0.50 (s, 3H), 0.80
(s, 3H), 0.86 (s, 3H), 0.95 (s, 3H), 1.14 (s, 3H), 1.21 (s, 3H),
1.24 (s, 3H), 1.25-2.18 (m, 19H), 2.26 (s, 1H), 2.36 (d, J=14.8 Hz,
1H), 3.62 (m, 1H), 6.08 (s, 1H), 6.65 (s, 1H), 9.91 (s, 1H).
mp>300.degree. C. APCI MS (Positive Mode) m/z 559
[C.sub.35H.sub.50N.sub.4O.sub.2+H].sup.+.
Example 7
##STR00165##
[0208] (i) Preparation of 7b: (4aS,6aS,6bR,13aR)-Benzyl
12-amino-15-(furan-2-yl)-2,2,6a,6b,9,9,13a-heptamethyl-2,3,4,4a,5,6,6a,6b-
,7,8,8a,9,11,13,13a,13b,14,15b-octadecahydro-1H-chryseno[1,2-f]indazole-4--
a-carboxylate
[0209] To a mixture of II (250 mg, 0.377 mmol) and potassium
2-furantrifluoroborate (196 mg, 1.12 mmol) in toluene (4.5 mL) and
H.sub.2O (0.5 mL) was added K.sub.2CO.sub.3 (207 mg, 1.5 mmol). The
mixture was sparged with nitrogen and then Pd(PPh.sub.3).sub.4 (86
mg, 0.074 mmol) was added. The reaction mixture was heated at
120.degree. C. for 1 hour using microwave irradiation. The solvent
was concentrated under reduced pressure. The residue was taken up
in EtOAc (40 mL) and washed with brine (3.times.15 mL). The organic
layer was dried (Na.sub.2SO.sub.4), filtered and concentrated. The
residue was purified by column chromatography (silica, 0-10% MeOH
in CH.sub.2Cl.sub.2) to afford the sub-title compound (200 mg,
81%). APCI MS m/z 650 [C.sub.42H.sub.55N.sub.3O.sub.3+H].sup.+.
(ii) Preparation of 7:
(4aS,6aS,6bR,13aR)-12-Amino-15-(furan-2-yl)-2,2,6a,6b,9,9,13a-heptamethyl-
-2,3,4,4a,5,6,6a,6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecahydro-1H-chrysen-
o[1,2-f]indazole-4-a-carboxylic acid
[0210] A solution of 7b (200 mg, 0.308 mmol) and MeOH (15 mL) was
flushed with nitrogen and then 10% Pd/C (200 mg) was added. The
mixture was flushed with nitrogen followed by hydrogen. The mixture
was stirred under hydrogen at atmospheric pressure overnight.
[0211] The catalyst was removed by filtration through diatomaceous
earth and the filtrate was concentrated. The residue was purified
by column chromatography (silica, 0-10% MeOH in CH.sub.2Cl.sub.2)
followed by preparative HPLC to afford the title compound (19 mg,
11%).
[0212] R.sub.f 0.20 (90:9:1 Methylene
Chloride/Methanol/concentrated Ammonium Hydroxide).
[0213] .sup.1H NMR (400 MHz, CD.sub.3OD) .delta. 0.78 (s, 3H),
0.86-2.50 (m, 42H), 3.74 (d, J=12.1 Hz, 1H), 6.38 (s, 2H), 7.41 (s,
1H). APCI MS m/z 560 [C.sub.39H.sub.49N.sub.3O.sub.3+H].sup.+. m.p.
250-270.degree. C. dec. HPLC (Method A) 98.3% (214 nm) t.sub.R=16.6
min.
Example 8
##STR00166##
[0214] (i) Preparation of 8b: (4aS,6aS,6bR,8aR,13aR,15bS)-Benzyl
12-amino-2,2,6a,6b,9,9,13a-heptamethyl-15-(1-(phenylsulfonyl)-1H-indol-3--
yl)-2,3,4,4a,5,6,6a,6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecahydro-1H-chry-
seno[1,2-f]indazole-4-a-carboxylate
[0215] A mixture of II (250 mg, 0.37 mmol),
1-(phenylsulfonyl)-1H-indol-3-ylboronic acid (341 mg, 1.13 mmol),
Pd(PPh.sub.3).sub.4 (43 mg, 0.037 mmol) and cesium carbonate (491
mg, 1.50 mmol) in toluene (4.0 mL) and H.sub.2O (1.0 mL) was sealed
and heated to 120.degree. C. by microwave for 1 hour. The reaction
mixture was diluted with EtOAc (100 mL). The organic phase was
washed with brine then dried (MgSO.sub.4), filtered and
concentrated to dryness. The residue was purified by column
chromatography (silica, 0-5% MeOH in CH.sub.2Cl.sub.2) to afford
the sub-title compound (264 mg, 83%). APCI MS (Positive Mode) m/z
839 [C.sub.52H.sub.62N.sub.4O.sub.4S+H].sup.+.
(ii) Preparation of 8c: (4aS,6aS,6bR,8aR,13aR,15bS)-Benzyl
12-amino-15-(1H-indol-3-yl)-2,2,6a,6b,9,9,13a-heptamethyl-2,3,4,4a,5,6,6a-
,6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecahydro-1H-chryseno[1,2-f]indazole-
-4-a-carboxylate
[0216] A mixture of 8b (260 mg, 0.30 mmol) and NaOH (15 mL, 2.0 M)
in MeOH (10 mL) and EtOH (30 mL) was heated at reflux for 24 hours.
The reaction mixture was extracted with EtOAc (100 mL). The organic
phase was washed with brine then dried (MgSO.sub.4), filtered and
concentrated to dryness. The residue was purified by column
chromatography (silica, 0-5% MeOH in CH.sub.2Cl.sub.2) to afford
the sub-title compound (115 mg, 53%).
(iii) Preparation of 8:
(4aS,6aS,6bR,8aR,13aR,15bS)-12-Amino-15-(1H-indol-3-yl)-2,2,6a,6b,9,9,13a-
-heptamethyl-2,3,4,4a,5,6,6a,6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecahydr-
o-1H-chryseno[1,2-f]indazole-4-a-carboxylic acid
[0217] A mixture 8c (115 mg, 0.16 mmol) and 10% Pd(OH).sub.2/C (60
mg) in MeOH (15 mL) and EtOAc (5 mL) was stirred under a hydrogen
balloon for 12 hours. The reaction mixture was filtered through a
pad of diatomaceous earth and washed with CMA (25 mL). The filtrate
was concentrated to dryness under reduced pressure. The residue was
purified by column chromatography (silica, 0-40% CMA in CMA) to
afford the title compound (56 mg, 76%) as a brown solid.
[0218] R.sub.f 0.75 (80:18:2 Methylene
Chloride/Methanol/concentrated Ammonium Hydroxide).
[0219] .sup.1H NMR (400 MHz, CD.sub.3OD) .delta. 0.14 (s, 3H), 0.65
(s, 3H), 0.88 (s, 3H), 1.09 (s, 3H), 1.16 (s, 3H), 1.26 (s, 3H),
1.34 (s, 3H), 1.35-2.20 (m, 19H), 2.30 (d, J=14.4 Hz, 1H), 2.40 (m,
1H), 6.96 (t, J=8.0 Hz, 1H), 7.05 (t, J=8.0 Hz, 1H), 7.07 (s, 1H),
7.31 (d, J=8.0 Hz, 1H), 7.42 (d, J=8.0 Hz, 1H). mp>300.degree.
C. dec. APCI MS (Positive Mode) m/z 609
[C.sub.39H.sub.52N.sub.4O.sub.2+H].sup.+.
Example 9
##STR00167##
[0220] (i) Preparation of 9b:
4-((1H-Pyrrol-2-yl)methyl)morpholine
[0221] To a solution of 1H-pyrrole-2-carbaldehyde (5.0 g, 52.5
mmol) and morpholine (5.0 mL, 57.8 mmol) in CH.sub.2Cl.sub.2 (160
mL) was added sodium triacetoxyborohydride (12.2 g, 57.8 mmol). The
mixture was stirred at room temperature for 4 hours. The reaction
mixture was diluted with EtOAc (200 mL). The organic phase was
washed with aqueous NaHCO.sub.3 and brine then dried (MgSO.sub.4),
filtered and concentrated to dryness. The residue was purified by
column chromatography (silica, 0-10% MeOH in CH.sub.2Cl.sub.2) to
afford the sub-title compound (5.3 g, 61%).
[0222] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 2.40 (s, 4H), 3.42
(s, 2H), 3.70 (m, 4H), 6.03 (s, 1H), 6.09 (s, 1H), 6.74 (s, 1H),
8.30 (bs, 1H).
(ii) Preparation of 9c:
4-(1-(Triisopropylsilyl)-1H-pyrrol-2-yl)methyl)morpholine
[0223] To a solution of 9b (5.3 g, 31.9 mmol) in DMF (60 mL) was
added sodium hydride (60% in mineral oil, 1.4 g, 35.1 mmol) in an
ice bath. The mixture was stirred for 5 min.
Chlorotriisopropylsilane (7.4 mL, 35.1 mmol) was added. The
reaction mixture was stirred at room temperature for 12 hours. The
reaction mixture was diluted with EtOAc (200 mL). The organic phase
was washed with brine then dried (MgSO.sub.4), filtered and
concentrated to dryness. The residue was purified by column
chromatography (silica, 0-20% EtOAc in hexanes) to afford the
sub-title compound (10.0 g, 98%).
[0224] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 1.10 (d, J=2.5 Hz,
18H), 1.62 (m, 3H), 2.40 (s, 4H), 3.42 (s, 2H), 3.70 (m, 4H), 6.20
(s, 2H), 6.80 (s, 1H).
(iii) Preparation of 9d:
4-((4-Bromo-1-(triisopropylsilyl)-1H-pyrrol-2-yl)methyl)morpholine
[0225] To a solution of 9c (1.0 g, 3.1 mmol) in THF (10 mL) was
added N-bromosuccinimide (552 mg, 3.1 mmol) at -78.degree. C. The
mixture was stirred at -78.degree. C. for 2 hours and warmed to
room temperature for 1 hour. The reaction was quenched with
H.sub.2O (5 mL) and extracted with EtOAc (100 mL). The organic
phase was washed with brine then dried (MgSO.sub.4), filtered and
concentrated to dryness. The residue was purified by column
chromatography (silica, 0-10% EtOAc in hexanes) to afford the
sub-title compound (1.0 g, 83%).
[0226] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 1.15 (d, J=2.5 Hz,
18H), 1.58 (m, 3H), 2.40 (m, 4H), 3.32 (s, 2H), 3.68 (m, 4H), 6.20
(s, 1H), 6.72 (s, 1H).
(iv) Preparation of 9e:
5-(Morpholinomethyl)-1-(triisopropylsilyl)-1H-pyrrol-3-ylboronic
acid
[0227] To a solution of 9d (200 mg, 0.49 mmol) in THF (5 mL) was
added n-butyllithium (2.5 M in hexanes, 0.30 mL, 0.74 mmol) at
-78.degree. C. The mixture was stirred at -78.degree. C. for 20
min. Trimethyl borate (0.28 mL, 2.45 mmol) was added. The reaction
mixture was stirred at -78.degree. C. for 1 hour and quenched with
MeOH (4 mL). The reaction mixture was warmed to room temperature
for 1 hour and extracted with EtOAc (100 mL). The organic phase was
washed with brine then dried (MgSO.sub.4), filtered and
concentrated to dryness. The residue was purified by column
chromatography (silica, 0-10% MeOH in CH.sub.2Cl.sub.2) to afford
the sub-title compound (70 mg, 39%).
[0228] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 1.15 (d, J=2.5 Hz,
18H), 1.58 (m, 3H), 2.40 (m, 4H), 3.32 (s, 2H), 3.68 (m, 4H), 6.35
(s, 1H), 7.15 (s, 1H).
(v) Preparation of 9f: (4aS,6aS,6bR,8aR,13aR,15bS)-Benzyl
12-amino-2,2,6a,6b,9,9,13a-heptamethyl-15-(5-(morpholinomethyl)-1-(triiso-
propylsilyl)-1H-pyrrol-3-yl)-2,3,4,4a,5,6,6a,6b,7,8,8a,9,11,13,13a,13b,14,-
15b-octadecahydro-1H-chryseno[1,2-f]indazole-4-a-carboxylate
[0229] A mixture of II (220 mg, 0.33 mmol), 9e (310 mg, 0.84 mmol),
Pd(PPh.sub.3).sub.4 (60 mg, 0.052 mmol) and K.sub.2CO.sub.3 (184
mg, 1.32 mmol) in toluene (4.0 mL) and H.sub.2O (0.5 mL) was sealed
and heated to 120.degree. C. by microwave for 1 hour. The reaction
mixture was diluted with EtOAc (100 mL). The organic phase was
washed with brine then dried (MgSO.sub.4), filtered and
concentrated to dryness. The residue was purified by column
chromatography (silica, 0-10% MeOH in CH.sub.2Cl.sub.2) to afford
the sub-title compound (250 mg, 83%).
[0230] .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 0.42 (s, 3H), 0.73
(s, 3H), 0.78 (s, 3H), 0.85 (s, 3H), 1.11-2.40 (m, 55H), 3.43 (s,
3H), 3.60 (m, 6H), 5.02 (m, 2H), 6.08 (s, 1H), 6.68 (s, 1H), 7.33
(m, 5H). APCI MS (Positive Mode) m/z 904
[C.sub.56H.sub.85N.sub.5O.sub.3Si+H].sup.+.
(vi) Preparation of 9g: (4aS,6aS,6bR,8aR,13aR,15bS)-Benzyl
12-amino-2,2,6a,6b,9,9,13a-heptamethyl-15-(5-(morpholinomethyl)-1H-pyrrol-
-3-yl)-2,3,4,4a,5,6,6a,6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecahydro-1H-c-
hryseno[1,2-f]indazole-4-a-carboxylate
[0231] To a solution of 9f (250 mg, 0.27 mmol) in THF (5 mL) was
add tetrabutylammonium fluoride (0.83 mL, 1 M in THF, 0.83 mmol).
The mixture was stirred at room temperature for 2 hours. The
reaction mixture was diluted with EtOAc (100 mL). The organic phase
was washed with brine then dried (MgSO.sub.4), filtered and
concentrated to dryness. The residue was purified by column
chromatography (silica, 0-10% MeOH in CH.sub.2Cl.sub.2) to afford
the sub-title compound (176 mg, 87%). APCI MS (Positive Mode) m/z
748 [C.sub.47H.sub.65N.sub.5O.sub.3+H].sup.+.
(vii) Preparation of 9:
(4aS,6aS,6bR,8aR,13aR,15bS)-12-Amino-2,2,6a,6b,9,9,13a-heptamethyl-15-(5--
(morpholinomethyl)-1H-pyrrol-3-yl)-2,3,4,4a,5,6,6a,6b,7,8,8a,9,11,13,13a,1-
3b,14,15b-octadecahydro-1H-chryseno[1,2-f]indazole-4-a-carboxylic
acid
[0232] A mixture of 9g (176 mg, 0.23 mmol) and 10% Pd(OH).sub.2/C
(100 mg) in MeOH (15 mL) and EtOAc (5 mL) was stirred under a
hydrogen balloon for 12 hours. The reaction mixture was filtered
through a pad of diatomaceous earth and washed with CMA (25 mL).
The filtrate was concentrated to dryness under reduced pressure.
The residue was purified by column chromatography (silica, 0-60%
CMA in CH.sub.2Cl.sub.2) to afford the title compound (25 mg, 17%)
as an off-white solid.
[0233] R.sub.f 0.50 (80:18:2 Methylene
Chloride/Methanol/concentrated Ammonium Hydroxide).
[0234] .sup.1H NMR (400 MHz, CD.sub.3OD) .delta. 0.55 (s, 3H), 0.78
(s, 3H), 0.86 (s, 3H), 0.94 (s, 3H), 1.12 (s, 3H), 1.21 (s, 3H),
1.24 (s, 3H), 1.25-2.18 (m, 19H), 2.26 (m, 1H), 2.36 (d, J=14.8 Hz,
1H), 3.49 (m, 4H), 3.52 (m, 3H), 3.67 (m, 6H), 6.03 (s, 1H), 6.63
(s, 1H). mp 270-280.degree. C. dec. ESI MS (Positive Mode) m/z 659
[C.sub.40H.sub.59N.sub.5O.sub.3+H].sup.+.
Example 10
##STR00168##
[0235] (i) Preparation of 10b: (4aS,6aS,6bR,13aR)-Benzyl
12-amino-15-(4-(dimethylamino)phenyl)-2,2,6a,6b,9,9,13a-heptamethyl-2,3,4-
,4a,5,6,6a,6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecahydro-1H-chryseno[1,2--
f]indazole-4-a-carboxylate
[0236] To a solution of II (300 mg, 0.45 mmol),
4-(dimethylamino)phenylboronic acid (223 mg, 1.35 mmol), benzene (4
mL) and EtOH (1 mL) was added K.sub.2CO.sub.3 (249 mg, 1.80
mmol).
[0237] The mixture was sparged with nitrogen and then
Pd(PPh.sub.3).sub.4 (104 mg, 0.08 mmol) was added. The reaction
mixture was heated at 120.degree. C. for 1 hour using microwave
irradiation and then concentrated under reduced pressure. The
residue was dissolved in EtOAc (20 mL) and the solution was washed
with brine then dried (Na.sub.2SO.sub.4), filtered and
concentrated. The residue was purified (silica, 0-5% MeOH in
CH.sub.2Cl.sub.2) to afford the sub-title compound (254 mg,
79%).
[0238] .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 0.32-2.31 (m,
41H), 2.89 (s, 6H), 3.15 (m, 1H), 5.08 (m, 2H), 6.63 (d, J=8.7 Hz,
2H), 7.03 (d, J=8.7 Hz, 2H), 7.34-7.37 (m, 5H). APCI MS m/z 703
[C.sub.46H.sub.62N.sub.4O.sub.2+H].sup.+.
(ii) Preparation of 10:
(4aS,6aS,6bR,13aR)-12-Amino-15-(4-(dimethylamino)phenyl)-2,2,6a,6b,9,9,13-
a-heptamethyl-2,3,4,4a,5,6,6a,6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecahyd-
ro-1H-chryseno[1,2-f]indazole-4-a-carboxylic acid
[0239] A solution of 10b (248 mg, 0.35 mmol) and MeOH (20 mL) was
flushed with nitrogen and then 10% Pd/C (150 mg) was added. The
mixture was flushed with nitrogen followed by hydrogen. The mixture
was stirred under hydrogen at atmospheric pressure overnight. The
catalyst was removed by filtration and the filtrate was
concentrated. Purification by column chromatography (silica, 0-50%
CMA in CH.sub.2Cl.sub.2) afforded the title compound (40 mg,
19%).
[0240] R.sub.f 0.32 (90:9:1 Methylene
Chloride/Methanol/concentrated Ammonium Hydroxide).
[0241] .sup.1H NMR (300 MHz, CD.sub.3OD) .delta. 0.31-2.37 (m,
41H), 2.88 (s, 6H), 3.18 (m, 1H), 6.81 (d, J=8.7 Hz, 2H), 7.13 (d,
J=8.4 Hz, 2H). APCI MS m/z 613
[C.sub.39H.sub.56N.sub.4O.sub.2+H].sup.+. m.p.>300.degree. C.
HPLC (Method A) 97.0% (214 nm) t.sub.R=13.2 min.
Example 11
##STR00169##
[0242] (i) Preparation of 11b: (4aS,6aS,6bR,13aR)-Benzyl
12-amino-15-(1H-indol-5-yl)-2,2,6a,6b,9,9,13a-heptamethyl-2,3,4,4a,5,6,6a-
,6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecahydro-1H-chryseno[1,2-f]indazole-
-4-a-carboxylate
[0243] To a solution of II (200 mg, 0.302 mmol),
1H-indol-5-ylboronic acid (145 mg, 0.90 mmol), toluene (4.5 mL) and
H.sub.2O (0.5 mL) was added cesium carbonate (391 mg, 1.20 mmol).
The mixture was sparged with nitrogen and then Pd(PPh.sub.3).sub.4
(69 mg, 0.06 mmol) was added. The reaction mixture was heated at
120.degree. C. for 1 hour using microwave irradiation and then
concentrated under reduced pressure. The residue was dissolved in
EtOAc (30 mL) and the solution was washed with brine then dried
(Na.sub.2SO.sub.4), filtered and concentrated. The residue was
purified by column chromatography (silica, 0-5% MeOH in
CH.sub.2Cl.sub.2) to afford the title compound (195 mg, 92%). APCI
MS m/z 699 [C.sub.46H.sub.58N.sub.4O.sub.2+H].sup.+.
(ii) Preparation of 11:
(4aS,6aS,6bR,13aR)-12-Amino-15-(indolin-5-yl)-2,2,6a,6b,9,9,13a-heptameth-
yl-2,3,4,4a,5,6,6a,6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecahydro-1H-chrys-
eno[1,2-f]indazole-4-a-carboxylic acid
[0244] A solution of 11b (192 mg, 0.274 mmol), MeOH (12 mL) and
EtOAc (3 mL) was flushed with nitrogen and then 10% Pd/C (228 mg)
was added. The mixture was flushed with nitrogen followed by
hydrogen. The mixture was stirred under hydrogen at atmospheric
pressure overnight. The catalyst was removed by filtration and the
filtrate was concentrated. The residue was purified by column
chromatography (silica, 0-50% CMA in CH.sub.2Cl.sub.2) followed by
preparative HPLC to afford the title compound (7.5 mg, 5%) as a
solid.
[0245] R.sub.f 0.15 (90:9:1 Methylene
Chloride/Methanol/concentrated Ammonium Hydroxide).
[0246] .sup.1H NMR (400 MHz, CD.sub.3OD) .delta. 0.30 (s, 3H),
0.77-2.39 (m, 43H), 2.99-3.03 (m, 1H), 3.34 (t, J=7.75 Hz, 2H),
3.85 (t, J=7.75 Hz, 2H), 7.35-7.44 (m, 3H). APCI MS m/z 611
[C.sub.39H.sub.54N.sub.4O.sub.2+H].sup.+. m.p. 170-190.degree. C.
dec. HPLC (Method A) 97.7% (214 nm) t.sub.R=12.6 min.
Example 12
##STR00170##
[0247] (i) Preparation of 12b: (4aS,6aS,6bR,8aR,12aR,14bS)-Benzyl
2,2,6a,6b,9,9,12a-heptamethyl-10-oxo-14-(pyridin-3-yl)-1,2,3,4,4a,5,6,6a,-
6b,7,8,8a,9,10,11,12,12a,12b,13,14b-icosahydropicene-4-a-carboxylate
[0248] A mixture of IIi (400 mg, 0.64 mmol), pyridin-3-ylboronic
acid (237 mg, 1.93 mmol), Pd(PPh.sub.3).sub.4 (74 mg, 0.064 mmol)
and cesium carbonate (629 mg, 1.93 mmol) in benzene (15 mL) and
EtOH (5 mL) was heated at reflux for 12 hours. The reaction mixture
was diluted with EtOAc (100 mL). The organic phase was washed with
brine then dried (MgSO.sub.4), filtered and concentrated to
dryness. The residue was purified by column chromatography (silica,
0-50% EtOAc in hexanes) to afford the sub-title compound (200 mg,
48%).
[0249] .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 0.25 (s, 3H), 0.75
(s, 3H), 0.83 (s, 3H), 1.02 (s, 3H), 1.04 (s, 3H), 1.09 (s, 3H),
1.22 (s, 3H), 1.23-2.02 (m, 20H), 2.30-2.50 (m, 2H), 2.90 (m, 1H),
5.05 (m, 2H), 7.14 (m, 1H), 7.37 (m, 5H), 7.48 (m, 1H), 8.42 (m,
2H).
(ii) Preparation of 12c: (4aS,6aS,6bR,8aR,12aR,14bS)-Benzyl
11-cyano-2,2,6a,6b,9,9,12a-heptamethyl-10-oxo-14-(pyridin-3-yl)-1,2,3,4,4-
a,5,6,6a,6b,7,8,8a,9,10,11,12,12a,12b,13,14b-icosahydropicene-4-a-carboxyl-
ate
[0250] To a solution of diisopropylamine (0.085 mL, 0.61 mmol) in
THF (3 mL) was added n-butyllithium (0.26 mL, 2.5 M in hexanes,
0.64 mmol) at -78.degree. C. The solution was stirred for 30 min.
The LDA solution was added to 12b (200 mg, 0.32 mmol) in THF (5
mL). The mixture was allowed to warm to -40.degree. C. for 5 min
and cooled to -78.degree. C. A suspension of p-toluene sulfonyl
cyanide (116 mg, 0.64 mmol) in THF (2 mL) was added. The reaction
mixture was allowed to warm to -40.degree. C. over 1.5 hours. The
reaction was quenched by saturated NH.sub.4Cl (3 mL) and extracted
with EtOAc (100 mL). The organic phase was washed with brine then
dried (MgSO.sub.4), filtered and concentrated to dryness. The
residue was purified by column chromatography (silica, 0-50% EtOAc
in hexanes) to afford the sub-title compound (80 mg, 39%).
[0251] .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 0.25 (s, 3H), 0.75
(s, 3H), 0.83 (s, 3H), 1.08 (s, 3H), 1.13 (s, 3H), 1.15 (s, 3H),
1.21 (s, 3H), 1.23-2.25 (m, 20H), 2.90 (m, 1H), 3.82 (m, 1H), 5.04
(m, 2H), 7.14 (m, 1H), 7.37 (m, 5H), 7.48 (m, 1H), 8.43 (m,
2H).
(iii) Preparation of 12d: (4aS,6aS,6bR,8aR,13aR,15bS)-Benzyl
12-amino-2,2,6a,6b,9,9,13a-heptamethyl-15-(pyridin-3-yl)-2,3,4,4a,5,6,6a,-
6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecahydro-1H-chryseno[1,2-f]indazole--
4-a-carboxylate
[0252] A mixture of 12c (80 mg, 0.12 mmol) and hydrazine (0.020 mL)
in EtOH (2 mL) was heated at reflux overnight. The reaction mixture
was concentrated to dryness under reduced pressure. The residue was
purified by column chromatography (silica, 0-50% CMA in
CH.sub.2Cl.sub.2) to afford the sub-title compound (74 mg, 93%).
ESI MS (Positive Mode) m/z 661
[C.sub.43H.sub.56N.sub.4O.sub.2+H].sup.+.
(iv) Preparation of 12:
(4aS,6aS,6bR,8aR,13aR,15bS)-12-Amino-2,2,6a,6b,9,9,13a-heptamethyl-15-(py-
ridin-3-yl)-2,3,4,4a,5,6,6a,6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecahydro-
-1H-chryseno[1,2-f]indazole-4-a-carboxylic acid
[0253] A mixture of 12d (74 mg, 0.11 mmol) and 10% Pd(OH).sub.2/C
(60 mg) in MeOH (10 mL) and EtOAc (2 mL) was stirred under a
hydrogen balloon for 5 hours. The reaction mixture was filtered
through a pad of diatomaceous earth and washed with CMA (25 mL).
The filtrate was concentrated to dryness under reduced pressure.
The residue was purified by column chromatography (silica, 0-70%
CMA in CH.sub.2Cl.sub.2) to afford the title compound (25 mg, 40%)
as a brown solid.
[0254] R.sub.f 0.30 (80:18:2 Methylene
Chloride/Methanol/concentrated Ammonium Hydroxide).
[0255] .sup.1H NMR (300 MHz, CD.sub.3OD) .delta. 0.55 (s, 3H), 0.81
(s, 3H), 0.87 (s, 3H), 0.97 (s, 3H), 1.03 (s, 3H), 1.08 (s, 6H),
1.25-2.10 (m, 19H), 2.10 (d, J=14.8 Hz, 1H), 2.75 (m, 1H), 7.18 (t,
J=7.8 Hz, 1H), 7.56 (d, J=7.2 Hz, 1H), 8.16 (d, J=4.8 Hz, 1H), 8.26
(s, 1H). mp>300.degree. C. APCI MS (Positive Mode) m/z 571
[C.sub.36H.sub.50N.sub.4O.sub.2+H].sup.+.
Example 13
##STR00171## ##STR00172##
[0256] (i) Preparation of 13b:
(4a'R,6a'R,6b'S,8a'S,12a'S,14b'R)-Methyl
4',4',6a',6b',11',11',14b'-heptamethyl-13'-vinyl-2',4',4a',5',6',6a',6b',-
7',8',8a',9',10',11',12',12a',14',14a',14b'-octadecahydro-1'H-spiro[[1,3]d-
ioxolane-2,3'-picene]-8a'-carboxylate
[0257] To a suspension of methyltriphenylphosphonium iodide (808
mg, 2.0 mmol) in benzene (30 mL) was added potassium tert-butoxide
(224 mg, 2.0 mmol). The mixture was stirred at room temperature for
1 hour. A solution of 5c (600 mg, 1.11 mmol) in THF (5 mL) was
added. The mixture was stirred at room temperature for 1.5 hours.
The reaction mixture was diluted with EtOAc (100 mL). The organic
phase was washed with brine then dried (MgSO.sub.4), filtered and
concentrated to dryness. The residue was purified by column
chromatography (silica, 0-15% EtOAc in hexanes) to afford the
sub-title compound (490 mg, 82%).
[0258] .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 0.63 (s, 3H), 0.85
(s, 3H), 0.89 (s, 3H), 0.91 (s, 3H), 0.94 (s, 3H), 0.97 (s, 3H),
0.99 (s, 3H), 1.11 (s, 3H), 1.15-2.05 (m, 22H), 3.60 (s, 3H), 3.95
(m, 4H), 4.95 (m, 1H), 4.95 (d, J=9.3 Hz, 2H), 5.09 (d, J=16.2 Hz,
1H).
(ii) Preparation of 13c: (4aS,6aS,6bR,8aR,12aR,14bS)-Methyl
2,2,6a,6b,9,9,12a-heptamethyl-10-oxo-14-vinyl-1,2,3,4,4a,5,6,6a,6b,7,8,8a-
,9,10,11,12,12a,12b,13,14b-icosahydropicene-4-a-carboxylate
[0259] A mixture of 13b (600 mg, 1.11 mmol) and p-toluenesulfonic
acid monohydrate (211 mg, 1.11 mmol) in acetone (20 mL) and
H.sub.2O (2 mL) was heated at reflux for 4 hours. The reaction
mixture was concentrated to remove acetone under reduced pressure
and extracted with EtOAc (100 mL). The organic phase was washed
with brine then dried (MgSO.sub.4), filtered and concentrated to
dryness. The residue was purified by column chromatography (silica,
0-15% EtOAc in hexanes) to afford the sub-title compound (500 mg,
91%).
[0260] .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 0.72 (s, 1.8H),
0.81 (s, 1.2H), 0.89 (s, 3H), 0.91 (s, 3H), 1.0-2.50 (m, 38H), 3.65
(s, 3H), 5.40-5.70 (m, 1H), 6.05 (d, J=12.0 Hz, 0.4H), 6.53 (d,
J=15.0 Hz, 0.6H).
(iii) Preparation of 13d: (4aS,6aS,6bR,8aR,12aR,14bS)-Methyl
11-cyano-2,2,6a,6b,9,9,12a-heptamethyl-10-oxo-14-vinyl-1,2,3,4,4a,5,6,6a,-
6b,7,8,8a,9,10,11,12,12a,12b,13,14b-icosahydropicene-4-a-carboxylate
[0261] To a solution of diisopropylamine (0.27 mL, 1.92 mmol) in
THF (6 mL) was added n-butyllithium (0.81 mL, 2.5 M in hexanes,
2.02 mmol) at -78.degree. C. The solution was stirred for 30 min.
The LDA solution was added to 13c (500 mg, 1.01 mmol) in THF (10
mL). The mixture was allowed to warm to -40.degree. C. for 5 min
and cooled to -78.degree. C. A suspension of p-toluene sulfonyl
cyanide (329 mg, 1.82 mmol) in THF (3 mL) was added. The reaction
mixture was allowed to warm to -40.degree. C. over 1.5 hours. The
reaction was quenched by saturated NH.sub.4Cl (3 mL) and extracted
with EtOAc (100 mL). The organic phase was washed with brine then
dried (MgSO.sub.4), filtered and concentrated to dryness. The
residue was purified by column chromatography (silica, 0-15% EtOAc
in hexanes) to afford the sub-title compound (200 mg, 38%).
(iv) Preparation of 13e: (4aS,6aS,6bR,8aR,13aR,15bS)-Methyl
12-amino-2,2,6a,6b,9,9,13a-heptamethyl-15-vinyl-2,3,4,4a,5,6,6a,6b,7,8,8a-
,9,11,13,13a,13b,14,15b-octadecahydro-1H-chryseno[1,2-f]indazole-4-a-carbo-
xylate
[0262] A mixture of 13d (200 mg, 0.38 mmol) and hydrazine (0.015
mL, 0.46 mmol) in EtOH (3 mL) was heated at reflux overnight. The
reaction mixture was concentrated to dryness under reduced
pressure. The residue was purified by column chromatography
(silica, 0-10% MeOH in CH.sub.2Cl.sub.2) to afford the sub-title
compound (180 mg, 89%). ESI MS (Positive Mode) m/z 534
[C.sub.34H.sub.51N.sub.3O.sub.2+H].sup.+.
(v) Preparation of 13:
(4aS,6aS,6bR,8aR,13aR,15bS)-12-Amino-2,2,6a,6b,9,9,13a-heptamethyl-15-vin-
yl-2,3,4,4a,5,6,6a,6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecahydro-1H-chrys-
eno[1,2-f]indazole-4-a-carboxylic acid
[0263] A mixture of 13e (80 mg, 0.15 mmol) and lithium iodide (300
mg, 2.25 mmol) in 2,6-lutidine (3 mL) was heated to 144.degree. C.
for 5 hours. The reaction mixture was cooled to room temperature
and neutralized with HCl (2 M) and extracted with
CH.sub.2Cl.sub.2/i-PrOH (3:1). The organic phase was dried
(MgSO.sub.4), filtered and concentrated to dryness under reduced
pressure. The residue was purified by column chromatography
(silica, 0-50% CMA in CH.sub.2Cl.sub.2) followed by preparative
HPLC to provide the title compound (6 mg, 8%) as an off-white
solid.
[0264] R.sub.f 0.35 (80:18:2 Methylene
Chloride/Methanol/concentrated Ammonium Hydroxide).
[0265] .sup.1H NMR (300 MHz, CD.sub.3OD) .delta. 0.81 (s, 3H), 0.91
(s, 3H), 0.94 (s, 3H), 1.01 (s, 3H), 1.17 (s, 3H), 1.22 (s, 3H),
1.25 (m, 3H), 1.30-2.30 (m, 19H), 2.53 (d, J=15.0 Hz, 1H), 3.53 (m,
1H), 4.98 (d, J=12.3 Hz, 1H), 5.18 (d, J=19.2 Hz, 1H), 6.95 (dd,
J=12.3, 19.2 Hz, 1H). mp>300.degree. C. ESI MS (Positive Mode)
m/z 520 [C.sub.33H.sub.49N.sub.3O.sub.2+H].sup.+.
Example 14
##STR00173##
[0266] (i) Preparation of 14b: (4aS,6aS,6bR,8aR,13aR,15bS)-Methyl
12-amino-15-ethyl-2,2,6a,6b,9,9,13a-heptamethyl-2,3,4,4a,5,6,6a,6b,7,8,8a-
,9,11,13,13a,13b,14,15b-octadecahydro-1H-chryseno[1,2-f]indazole-4-a-carbo-
xylate
[0267] A mixture of 13e (80 mg, 0.15 mmol) and 10% Pd(OH).sub.2/C
(30 mg) in MeOH (10 mL) was stirred under a hydrogen balloon for 12
hours. The reaction mixture was filtered through a pad of
diatomaceous earth and washed with CMA (20 mL). The filtrate was
concentrated to dryness and the residue purified by column
chromatography (silica, 0-10% MeOH in CH.sub.2Cl.sub.2) to afford
the sub-title compound (70 mg, 87%). ESI MS (Positive Mode) m/z 536
[C.sub.34H.sub.53N.sub.3O.sub.2+H].sup.+.
(ii) Preparation of 14:
(4aS,6aS,6bR,8aR,13aR,15bS)-12-Amino-15-ethyl-2,2,6a,6b,9,9,13a-heptameth-
yl-2,3,4,4a,5,6,6a,6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecahydro-1H-chrys-
eno[1,2-f]indazole-4-a-carboxylic acid
[0268] A mixture of 14b (70 mg, 0.13 mmol) and lithium iodide (263
mg, 1.96 mmol) in 2,6-lutidine (3 mL) was heated to 144.degree. C.
for 5 hours. The reaction mixture was cooled to room temperature
and neutralized with HCl (2 M) and extracted with
CH.sub.2Cl.sub.2/i-PrOH (3:1). The organic phase was dried
(MgSO.sub.4), filtered and concentrated to dryness under reduced
pressure. The residue was purified by column chromatography
(silica, 0-50% CMA in CH.sub.2Cl.sub.2) followed by preparative
HPLC to provide the title compound (13 mg, 19%) as an off-white
solid.
[0269] R.sub.f 0.80 (80:18:2 Methylene
Chloride/Methanol/concentrated Ammonium Hydroxide).
[0270] .sup.1H NMR (300 MHz, CD.sub.3OD) .delta. 0.83 (s, 3H), 0.92
(s, 6H), 0.99 (s, 3H), 1.11 (s, 3H), 1.21 (s, 3H), 1.29 (s, 3H),
1.30-2.30 (m, 25H), 2.44 (d, J=15.0 Hz, 1H). mp>300.degree. C.
ESI MS (Positive Mode) m/z 522
[C.sub.33H.sub.51N.sub.3O.sub.2+H].sup.+.
Example 15
##STR00174##
[0271] (i) Preparation of 15b: (4aS,6aS,6bR,8aR,12aR,14bS)-Benzyl
1443-aminophenyl)-2,2,6a,6b,9,9,12a-heptamethyl-10-oxo-1,2,3,4,4a,5,6,6a,-
6b,7,8,8a,9,10,11,12,12a,12b,13,14b-icosahydropicene-4-a-carboxylate
[0272] A mixture of IIi (600 mg, 0.96 mmol), 3-aminophenylboronic
acid (362 mg, 2.66 mmol), Pd(PPh.sub.3).sub.4 (104 mg, 0.09 mmol)
and K.sub.2CO.sub.3 (353 mg, 2.56 mmol) in benzene (16 mL) and EtOH
(5 mL) was heated at reflux for 12 hours. The reaction mixture was
diluted with EtOAc (100 mL). The organic phase was washed with
brine then dried (MgSO.sub.4), filtered and concentrated to
dryness. The residue was purified by column chromatography (silica,
0-40% EtOAc in hexanes) to afford the sub-title compound (400 mg,
65%).
[0273] .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 0.30 (s, 3H), 0.76
(s, 3H), 0.86 (s, 3H), 1.04 (s, 3H), 1.06 (s, 3H), 1.08 (s, 3H),
1.19 (s, 3H), 1.20-2.50 (m, 24H), 3.12 (m, 1H), 5.02 (m, 2H), 6.48
(m, 3H), 7.01 (m, 1H), 7.33 (m, 5H).
(ii) Preparation of 15c: (4aS,6aS,6bR,8aR,12aR,14bS)-Benzyl
14-(3-aminophenyl)-11-cyano-2,2,6a,6b,9,9,12a-heptamethyl-10-oxo-1,2,3,4,-
4a,5,6,6a,6b,7,8,8a,9,10,11,12,12a,12b,13,14b-icosahydropicene-4-a-carboxy-
late
[0274] To a solution of diisopropylamine (0.22 mL, 1.57 mmol) in
THF (5 mL) was added n-butyllithium (0.64 mL, 2.5 M in hexanes,
1.61 mmol) at -78.degree. C. The solution was stirred for 30 min.
The LDA solution was added to 15b (400 mg, 0.62 mmol) in THF (5
mL). The mixture was allowed to warm to -40.degree. C. for 5 min
and cooled to -78.degree. C. A suspension of p-toluene sulfonyl
cyanide (285 mg, 1.57 mmol) in THF (3 mL) was added. The reaction
mixture was allowed to warm to -40.degree. C. over 1.5 hours. The
reaction was quenched by saturated NH.sub.4Cl (3 mL) and extracted
with EtOAc (100 mL). The organic phase was washed with brine then
dried (MgSO.sub.4), filtered and concentrated to dryness. The
residue was purified by column chromatography (silica, 0-20% EtOAc
in hexanes) to afford the sub-title compound (100 mg, 25%).
[0275] .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 0.32 (s, 3H), 0.76
(s, 3H), 0.86 (s, 3H), 0.87-2.30 (m, 32H), 3.12 (m, 1H), 3.30 (s,
2H), 3.82 (m, 1H), 5.02 (m, 2H), 6.48 (m, 3H), 7.01 (m, 1H), 7.33
(m, 5H).
(iii) Preparation of 15d: (4aS,6aS,6bR,8aR,13aR,15bS)-Benzyl
12-amino-15-(3-aminophenyl)-2,2,6a,6b,9,9,13a-heptamethyl-2,3,4,4a,5,6,6a-
,6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecahydro-1H-chryseno[1,2-f]indazole-
-4-a-carboxylate
[0276] A mixture of 15c (100 mg, 0.15 mmol) and hydrazine (0.020
mL) in EtOH (2 mL) was heated at reflux overnight. The reaction
mixture was concentrated to dryness under reduced pressure. The
residue was purified by column chromatography (silica, 0-30% CMA in
CH.sub.2Cl.sub.2) to afford the sub-title compound (70 mg, 69%).
APCI MS (Positive Mode) m/z 675
[C.sub.44H.sub.58N.sub.4O.sub.2+H].sup.+.
(iv) Preparation of 15:
(4aS,6aS,6bR,8aR,13aR,15bS)-12-Amino-15-(3-aminophenyl)-2,2,6a,6b,9,9,13a-
-heptamethyl-2,3,4,4a,5,6,6a,6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecahydr-
o-1H-chryseno[1,2-f]indazole-4-a-carboxylic acid
[0277] A mixture of 15d (70 mg, 0.10 mmol) and 10% Pd(OH).sub.2/C
(35 mg) in MeOH (12 mL) was stirred under a hydrogen balloon for 12
hours. The reaction mixture was filtered through a pad of
diatomaceous earth and washed with CMA (25 mL). The filtrate was
concentrated to dryness under reduced pressure. The residue was
purified by column chromatography (silica, 0-80% CMA in
CH.sub.2Cl.sub.2) followed by preparative HPLC to provide the title
compound (32 mg, 55%) as a brown solid.
[0278] R.sub.f 0.70 (80:18:2 Methylene
Chloride/Methanol/concentrated Ammonium Hydroxide).
[0279] .sup.1H NMR (300 MHz, DMSO-d.sub.6) .delta. 0.30 (s, 3H),
0.78 (s, 3H), 0.85 (s, 3H), 0.96 (s, 3H), 1.18 (s, 3H), 1.31 (s,
6H), 1.32-2.28 (m, 19H), 2.26 (d, J=14.7 Hz, 1H), 3.0 (m, 1H), 7.16
(m, 4H). mp>300.degree. C. APCI MS (Positive Mode) m/z 585
[C.sub.37H.sub.52N.sub.4O.sub.2+H].sup.+.
Example 16
##STR00175##
[0280] (i) Preparation of 16b: (4aS,6aS,6bR,13aR)-Benzyl
12-amino-15-(3-methoxyphenyl)-2,2,6a,6b,9,9,13a-heptamethyl-2,3,4,4a,5,6,-
6a,6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecahydro-1H-chryseno[1,2-f]indazo-
le-4-a-carboxylate
[0281] To a mixture of II (200 mg, 0.30 mmol) and
3-methoxyphenylboronic acid (92 mg, 0.60 mmol) in benzene (4 mL)
and EtOH (1 mL) was added K.sub.2CO.sub.3 (125 mg, 0.90 mmol). The
mixture was sparged with nitrogen and then Pd(PPh.sub.3).sub.4 (70
mg, 0.06 mmol) was added. The reaction mixture was heated at
120.degree. C. for 1 hour using microwave irradiation and then
concentrated. The residue was dissolved in EtOAc (20 mL) and the
solution was washed with brine then dried (Na.sub.2SO.sub.4),
filtered and concentrated under reduced pressure. The residue was
purified by column chromatography (silica, 0-30% CMA in
CH.sub.2Cl.sub.2) to afford the sub-title compound (179 mg,
86%).
[0282] .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 0.30-2.25 (m,
39H), 3.11 (m, 1H), 3.40 (s, 2H), 3.82 (s, 3H), 5.08 (d, J=12.6 Hz,
1H), 5.20 (d, J=12.3 Hz, 1H), 6.75 (m, 1H), 6.78 (s, 1H), 7.15 (t,
J=7.8 Hz, 1H), 7.31-7.36 (m, 5H). APCI MS m/z 690
[C.sub.45H.sub.59N.sub.3O.sub.3+H].sup.+.
(ii) Preparation of 16:
(4aS,6aS,6bR,13aR)-12-Amino-15-(3-hydroxyphenyl)-2,2,6a,6b,9,9,13a-heptam-
ethyl-2,3,4,4a,5,6,6a,6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecahydro-1H-ch-
ryseno[1,2-f]indazole-4-a-carboxylic acid
[0283] A mixture of 16b (179 mg, 0.25 mmol) and HBr (2 mL, 33% in
AcOH) was heated at 120.degree. C. for 1 hour. The reaction mixture
was concentrated under reduced pressure and the residue was
purified by column chromatography (silica, 0-80% CMA in
CH.sub.2Cl.sub.2) to afford the title compound (20 mg, 13%).
[0284] R.sub.f 0.30 (40:9:1 Methylene
Chloride/Methanol/concentrated Ammonium Hydroxide).
[0285] .sup.1H NMR (300 MHz, CD.sub.3OD) .delta. 0.34-2.37 (m,
41H), 3.15 (d, J=10.8 Hz, 1H), 6.61-6.70 (m, 3H), 7.11 (t, J=7.8
Hz, 1H). APCI MS m/z 586 [C.sub.37H.sub.51N.sub.3O.sub.3+H].sup.+.
m.p. 260-280.degree. C. dec. HPLC (Method A) 92.4% (214 nm)
t.sub.R=15.6 min.
Example 17
##STR00176##
[0286] (i) Preparation of 17b: (4aS,6aS,6bR,8aR,12aR,14bS)-Benzyl
14-(3-fluorophenyl)-2,2,6a,6b,9,9,12a-heptamethyl-10-oxo-1,2,3,4,4a,5,6,6-
a,6b,7,8,8a,9,10,11,12,12a,12b,13,14b-icosahydropicene-4-a-carboxylate
[0287] A mixture of IIi (300 mg, 0.48 mmol), 3-fluorophenylboronic
acid (100 mg, 0.72 mmol), Pd(PPh.sub.3).sub.4 (55 mg, 0.048 mmol)
and cesium carbonate (469 mg, 1.44 mmol) in benzene (15 mL) and
EtOH (5 mL) was heated at reflux for 12 hours. The reaction mixture
was diluted with EtOAc (100 mL). The organic phase was washed with
brine then dried (MgSO.sub.4), filtered and concentrated to
dryness. The residue was purified by column chromatography (silica,
0-10% EtOAc in hexanes) to afford the sub-title compound (250 mg,
82%).
[0288] .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 0.25 (s, 3H), 0.78
(s, 3H), 0.83 (s, 3H), 1.05 (s, 3H), 1.07 (s, 3H), 1.12 (s, 3H),
1.21 (s, 3H), 1.23-2.55 (m, 22H), 3.01 (m, 1H), 5.04 (m, 2H), 6.87
(m, 3H), 7.15 (m, 1H), 7.34 (m, 5H).
(ii) Preparation of 17c: (4aS,6aS,6bR,8aR,12aR,14bS)-Benzyl
11-cyano-14-(3-fluorophenyl)-2,2,6a,6b,9,9,12a-heptamethyl-10-oxo-1,2,3,4-
,4a,5,6,6a,6b,7,8,8a,9,10,11,12,12a,12b,13,14b-icosahydropicene-4-a-carbox-
ylate
[0289] To a solution of diisopropylamine (0.17 mL, 1.22 mmol) in
THF (6 mL) was added n-butyllithium (0.51 mL, 2.5 M in hexanes,
1.28 mmol) at -78.degree. C. The solution was stirred for 30 min.
The LDA solution was added to 17b (408 mg, 0.64 mmol) in THF (4
mL). The mixture was allowed to warm to -40.degree. C. for 5 min
and cooled to -78.degree. C. A suspension of p-toluene sulfonyl
cyanide (232 mg, 1.28 mmol) in THF (3 mL) was added at -78.degree.
C. The reaction mixture was allowed to warm to -40.degree. C. over
1.5 hours. The reaction was quenched by saturated NH.sub.4Cl (3 mL)
and extracted with EtOAc (100 mL). The organic phase was washed
with brine then dried (MgSO.sub.4), filtered and concentrated to
dryness. The residue was purified by column chromatography (silica,
0-20% EtOAc in hexanes) to afford the sub-title compound (280 mg,
66%).
[0290] .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 0.25 (s, 3H), 0.78
(s, 3H), 0.83 (s, 3H), 1.05 (s, 3H), 1.07 (s, 3H), 1.12 (s, 3H),
1.22 (s, 3H), 1.23-2.30 (m, 20H), 3.01 (m, 1H), 3.83 (m, 1H), 5.04
(m, 2H), 6.87 (m, 3H), 7.15 (m, 1H), 7.34 (m, 5H).
(iii) Preparation of 17d: (4aS,6aS,6bR,8aR,13aR,15bS)-Benzyl
12-amino-15-(3-fluorophenyl)-2,2,6a,6b,9,9,13a-heptamethyl-2,3,4,4a,5,6,6-
a,6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecahydro-1H-chryseno[1,2-f]indazol-
e-4-a-carboxylate
[0291] A mixture of 17c (280 mg, 0.42 mmol) and hydrazine (0.053
mL) in EtOH (5 mL) was heated at reflux overnight. The reaction
mixture was concentrated to dryness under reduced pressure. The
residue was purified by column chromatography (silica, 0-10% MeOH
in CH.sub.2Cl.sub.2) to afford the sub-title compound (230 mg,
81%). ESI MS (Positive Mode) m/z 678
[C.sub.44H.sub.56FN.sub.3O.sub.2+H].sup.+.
(iv) Preparation of 17:
(4aS,6aS,6bR,8aR,13aR,15bS)-12-Amino-15-(3-fluorophenyl)-2,2,6a,6b,9,9,13-
a-heptamethyl-2,3,4,4a,5,6,6a,6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecahyd-
ro-1H-chryseno[1,2-f]indazole-4-a-carboxylic acid
[0292] A mixture of 17d (230 mg, 0.34 mmol) and 10% Pd(OH).sub.2/C
(100 mg) in MeOH (6 mL) and EtOAc (6 mL) was stirred under a
hydrogen balloon for 12 hours. The reaction mixture was filtered
through a pad of diatomaceous earth and washed with CMA (25 mL).
The filtrate was concentrated to dryness under reduced pressure.
The residue was purified by column chromatography (silica, 0-50%
CMA in CH.sub.2Cl.sub.2) to afford the title compound (110 mg, 55%)
as a white solid.
[0293] R.sub.f 0.75 (4:1 Methylene Chloride/Methanol).
[0294] .sup.1H NMR (300 MHz, CD.sub.3OD) .delta. 0.30 (s, 3H), 0.75
(s, 3H), 0.87 (s, 3H), 0.99 (s, 3H), 1.16 (s, 3H), 1.19 (s, 3H),
1.25 (s, 3H), 1.26-2.25 (m, 19H), 2.30 (d, J=14.7 Hz, 1H), 3.15 (m,
1H), 6.91 (t, J=8.4 Hz, 1H), 7.01 (m, 2H), 7.28 (q, J=8.1 Hz, 1H).
mp>300.degree. C. APCI MS (Positive Mode) m/z 588
[C.sub.37H.sub.50FN.sub.3O.sub.2+H].sup.+.
Example 18
##STR00177##
[0295] (i) Preparation of 18b: (4aS,6aS,6bR,13aR)-Benzyl
12-amino-15-(3-(aminomethyl)phenyl)-2,2,6a,6b,9,9,13a-heptamethyl-2,3,4,4-
a,5,6,6a,6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecahydro-1H-chryseno[1,2-f]-
indazole-4-a-carboxylate
[0296] To a mixture of II (200 mg, 0.30 mmol) and
3-(aminomethyl)phenylboronic acid (169 mg, 0.90 mmol) in benzene (4
mL) and EtOH (1 mL) was added K.sub.2CO.sub.3 (166 mg, 1.20 mmol).
The mixture was sparged with nitrogen and then Pd(PPh.sub.3).sub.4
(70 mg, 0.06 mmol) was added. The reaction mixture was heated at
120.degree. C. for 1 hour using microwave irradiation and then
concentrated. The residue was dissolved in EtOAc (20 mL) and the
organic layer was washed with brine then dried (Na.sub.2SO.sub.4),
filtered and concentrated under reduced pressure. The residue was
purified by column chromatography (silica, 0-40% CMA in
CH.sub.2Cl.sub.2) to afford the sub-title compound (170 mg,
82%).
[0297] .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 0.23-2.25 (m,
45H), 3.05 (m, 1H), 3.40 (s, 1H), 3.82 (s, 2H), 5.06 (d, J=12.6 Hz,
1H), 5.23 (d, J=12.3 Hz, 1H), 7.06-7.20 (m, 4H), 7.33-7.38 (m, 5H).
APCI MS m/z 689 [C.sub.45H.sub.60N.sub.4O.sub.2+H].sup.+.
(ii) Preparation of 18:
(4aS,6aS,6bR,13aR)-12-Amino-15-(3-(aminomethyl)phenyl)-2,2,6a,6b,9,9,13a--
heptamethyl-2,3,4,4a,5,6,6a,6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecahydro-
-1H-chryseno[1,2-f]indazole-4-a-carboxylic acid
[0298] A solution of 18b (160 mg, 0.23 mmol) and MeOH (20 mL) was
flushed with nitrogen and then 10% Pd/C (100 mg) was added. The
mixture was flushed with nitrogen followed by hydrogen. The mixture
was stirred under hydrogen at atmospheric pressure overnight. The
catalyst was removed by filtration and the filtrate was
concentrated under reduced pressure. The residue was purified by
column chromatography (silica, 0-70% CMA in CH.sub.2Cl.sub.2)
followed by preparative HPLC to afford the title compound (48 mg,
35%) as a solid.
[0299] R.sub.f 0.13 (40:9:1 Methylene
Chloride/Methanol/concentrated Ammonium Hydroxide).
[0300] .sup.1H NMR (300 MHz, CD.sub.3OD) .delta. 0.27-2.34 (m,
41H), 2.99 (d, J=11.1 Hz, 1H), 4.13 (s, 2H), 7.30-7.44 (m, 4H).
APCI MS m/z 599 [C.sub.38H.sub.54N.sub.4O.sub.2+H].sup.+. m.p.
270-290.degree. C. dec. HPLC (Method A)>99% (214 nm)
t.sub.R=12.4 min.
Example 19
##STR00178##
[0301] (i) Preparation of 19b: (4aS,6aS,6bR,13aR)-Benzyl
12-amino-15-(3-carbamoylphenyl)-2,2,6a,6b,9,9,13a-heptamethyl-2,3,4,4a,5,-
6,6a,6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecahydro-1H-chryseno[1,2-f]inda-
zole-4-a-carboxylate
[0302] To a mixture of II (200 mg, 0.30 mmol) and
3-carbamoylphenylboronic acid (99 mg, 0.64 mmol) in benzene (4 mL)
and EtOH (1 mL) was added K.sub.2CO.sub.3 (125 mg, 0.90 mmol). The
mixture was sparged with nitrogen and then Pd(PPh.sub.3).sub.4 (69
mg, 0.06 mmol) was added. The reaction mixture was heated at
120.degree. C. for 1 hour using microwave irradiation and then
concentrated. The residue was dissolved in EtOAc (20 mL) and the
solution was washed with brine then dried (Na.sub.2SO.sub.4),
filtered and concentrated under reduced pressure. The residue was
purified by column chromatography (silica, 0-40% CMA in
CH.sub.2Cl.sub.2) to afford the sub-title compound (170 mg, 80%).
APCI MS m/z 703 [C.sub.45H.sub.58N.sub.4O.sub.3+H].sup.+.
(ii) Preparation of 19:
(4aS,6aS,6bR,13aR)-12-Amino-15-(3-carbamoylphenyl)-2,2,6a,6b,9,9,13a-hept-
amethyl-2,3,4,4a,5,6,6a,6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecahydro-1H--
chryseno[1,2-f]indazole-4-a-carboxylic acid
[0303] A solution of 19b (170 mg, 0.24 mmol) and MeOH (20 mL) was
flushed with nitrogen and then 10% Pd/C (100 mg) was added. The
mixture was flushed with nitrogen followed by hydrogen. The mixture
was stirred under hydrogen at atmospheric pressure overnight. The
catalyst was removed by filtration through diatomaceous earth and
the filtrate was concentrated under reduced pressure. The residue
was purified by column chromatography (silica, 0-60% CMA in
CH.sub.2Cl.sub.2) to afford the title compound (60 mg, 40%).
[0304] R.sub.f 0.22 (40:9:1 Methylene
Chloride/Methanol/concentrated Ammonium Hydroxide).
[0305] .sup.1H NMR (300 MHz, CD.sub.3OD) .delta. 0.24-2.38 (m,
41H), 3.02 (d, J=11.4 Hz, 1H), 7.42-7.45 (m, 2H), 7.73-7.76 (m,
2H). APCI MS m/z 613 [C.sub.38H.sub.52N.sub.4O.sub.3+H].sup.+.
m.p.>300.degree. C. HPLC (Method A)>99% (214 nm) t.sub.R=14.1
min.
Example 20
##STR00179##
[0306] (i) Preparation of 20b: (4aS,6aS,6bR,13aR)-Benzyl
12-amino-2,2,6a,6b,9,9,13a-heptamethyl-15-(4-methyl-3-nitrophenyl)-2,3,4,-
4a,5,6,6a,6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecahydro-1H-chryseno[1,2-f-
]indazole-4-a-carboxylate
[0307] To a mixture of II (250 mg, 0.37 mmol) and
4-methyl-3-nitrophenylboronic acid (136 mg, 0.75 mmol) in benzene
(4 mL) and EtOH (1 mL) was added K.sub.2CO.sub.3 (156 mg, 1.13
mmol). The mixture was sparged with nitrogen and then
Pd(PPh.sub.3).sub.4 (87 mg, 0.07 mmol) was added. The reaction
mixture was heated at 120.degree. C. for 1 hour using microwave
irradiation. The solvent was removed under reduced pressure and
then the residue was dissolved in EtOAc (20 mL). The solution was
washed with brine then dried (Na.sub.2SO.sub.4), filtered and
concentrated under reduced pressure. The residue was purified by
column chromatography (silica, 0-40% CMA in CH.sub.2Cl.sub.2) to
afford the sub-title compound (186 mg, 69%).
[0308] .sup.1H NMR (300 MHz, CD.sub.3OD) .delta. 0.26-2.34 (m,
41H), 2.51 (s, 3H), 3.01 (m, 1H), 5.05 (d, J=12.0 Hz, 1H), 5.23 (d,
J=12.0 Hz, 1H), 7.22-7.37 (m, 7H), 7.80 (s, 1H). APCI MS m/z 719
[C.sub.45H.sub.58N.sub.4O.sub.4+H].sup.+.
(ii) Preparation of 20:
(4aS,6aS,6bR,13aR)-12-Amino-15-(3-amino-4-methylphenyl)-2,2,6a,6b,9,9,13a-
-heptamethyl-2,3,4,4a,5,6,6a,6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecahydr-
o-1H-chryseno[1,2-f]indazole-4-a-carboxylic acid
[0309] A solution of 20b (180 mg, 0.25 mmol) and MeOH (20 mL) was
flushed with nitrogen and then 10% Pd/C (180 mg) was added. The
mixture was flushed with nitrogen followed by hydrogen. The mixture
was stirred under hydrogen at atmospheric pressure overnight. The
catalyst was removed by filtration through diatomaceous earth and
the filtrate was concentrated under reduced pressure. The residue
was purified by column chromatography (silica, 0-40% CMA in
CH.sub.2Cl.sub.2) followed by preparative HPLC to afford the title
compound (16 mg, 10%) as a solid.
[0310] R.sub.f 0.23 (90:9:1 Methylene
Chloride/Methanol/concentrated Ammonium Hydroxide).
[0311] .sup.1H NMR (300 MHz, CD.sub.3OD) .delta. 0.31-2.27 (m,
41H), 2.36 (s, 3H), 2.99 (d, J=10.5 Hz, 1H), 7.14-7.32 (m, 3H).
APCI MS m/z 599 [C.sub.38H.sub.54N.sub.4O.sub.2+H].sup.+. m.p.
220-240.degree. C. dec. HPLC (Method A)>99% (214 nm)
t.sub.R=12.4 min.
Example 21
##STR00180##
[0312] (i) Preparation of 21b: (4aS,6aS,6bR,12aR)-Benzyl
11-cyano-14-(3-(dimethylamino)phenyl)-2,2,6a,6b,9,9,12a-heptamethyl-10-ox-
o-1,2,3,4,4a,5,6,6a,6b,7,8,8a,9,10,11,12,12a,12b,13,14b-icosahydropicene-4-
-a-carboxylate
[0313] To a mixture of IIj (300 mg, 0.46 mmol) and
3-(dimethylamino)phenylboronic acid (458 mg, 2.78 mmol) in benzene
(10 mL) and EtOH (5 mL) was added K.sub.2CO.sub.3 (511 mg, 3.70
mmol). The mixture was sparged with nitrogen and then
Pd(PPh.sub.3).sub.4 (107 mg, 0.09 mmol) was added. The mixture was
heated at 85.degree. C. overnight and then concentrated under
reduced pressure. The residue was dissolved in EtOAc (50 mL) and
the solution was washed with brine then dried (Na.sub.2SO.sub.4),
filtered and concentrated under reduced pressure. The residue was
purified by column chromatography (silica, 0-10% EtOAc in hexanes)
to afford the sub-title compound (270 mg, 84%).
[0314] .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 0.29-2.30 (m,
41H), 2.95 (s, 6H), 3.13 (d, J=10.5 Hz, 1H), 3.81-3.91 (m, 1H),
5.07 (d, J=12.3 Hz, 1H), 5.17 (d, J=15.3 Hz, 1H), 6.51 (d, J=6.9
Hz, 1H), 6.51 (d, J=6.9 Hz, 1H), 7.03-7.09 (m, 1H), 7.34-7.36 (m,
5H).
(ii) Preparation of 21c: (4aS,6aS,6bR,13aR)-Benzyl
12-amino-15-(3-(dimethylamino)phenyl)-2,2,6a,6b,9,9,13a-heptamethyl-2,3,4-
,4a,5,6,6a,6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecahydro-1H-chryseno[1,2--
f]indazole-4-a-carboxylate
[0315] To a solution of 21b (266 mg, 0.38 mmol) and EtOH (4 mL) was
added hydrazine (60 .mu.L, 1.93 mmol). The mixture was heated at
90.degree. C. overnight and then concentrated under reduced
pressure. The residue was purified by column chromatography (0-30%
EtOAc in hexanes and 50% CMA in CH.sub.2Cl.sub.2) to afford the
sub-title compound (191 mg, 70%).
[0316] .sup.1H NMR (300 MHz, CD.sub.3OD) .delta. 0.27-2.36 (m,
42H), 2.89 (s, 6H), 5.05 (m, 1H), 5.22 (m, 1H), 6.49-7.06 (m, 4H),
7.35-7.7.36 (m, 5H). APCI MS m/z 703
[C.sub.46H.sub.62N.sub.4O.sub.2+H].sup.+.
(iii) Preparation of 21:
(4aS,6aS,6bR,13aR)-12-Amino-15-(3-(dimethylamino)phenyl)-2,2,6a,6b,9,9,13-
a-heptamethyl-2,3,4,4a,5,6,6a,6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecahyd-
ro-1H-chryseno[1,2-f]indazole-4-a-carboxylic acid
[0317] A solution of 21c (180 mg, 0.25 mmol) and MeOH (20 mL) was
flushed with nitrogen and then 10% Pd/C (90 mg) was added. The
mixture was flushed with nitrogen followed by hydrogen. The mixture
was stirred under hydrogen at atmospheric pressure overnight. The
catalyst was removed by filtration through diatomaceous earth and
the filtrate was concentrated. The residue was purified by column
chromatography (silica, 0-40% CMA in CH.sub.2Cl.sub.2) followed by
preparative HPLC to afford the title compound (36 mg, 23%) as a
solid.
[0318] R.sub.f 0.44 (90:9:1 Methylene
Chloride/Methanol/concentrated Ammonium Hydroxide).
[0319] .sup.1H NMR (300 MHz, CD.sub.3OD) .delta. 0.27-2.43 (m,
41H), 3.04 (d, J=9.6 Hz, 1H), 3.18 (s, 6H), 7.16-7.47 (m, 4H). APCI
MS m/z 613 [C.sub.39H.sub.56N.sub.4O.sub.2+H].sup.+.
m.p.>300.degree. C. HPLC (Method A) 98.1% (214 nm) t.sub.R=12.0
min.
Example 22
##STR00181##
[0320] (i) Preparation of 22b: (4aS,6aS,6bR,8aR,13aR,15bS)-Benzyl
12-amino-15-(2-fluoropyridin-4-yl)-2,2,6a,6b,9,9,13a-heptamethyl-2,3,4,4a-
,5,6,6a,6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecahydro-1H-chryseno[1,2-f]i-
ndazole-4-a-carboxylate
[0321] A mixture of II (500 mg, 0.75 mmol),
2-fluoropyridin-4-ylboronic acid (210 mg, 1.50 mmol),
Pd(PPh.sub.3).sub.4 (80 mg, 0.075 mmol) and K.sub.2CO.sub.3 (310
mg, 2.25 mmol) in benzene (4.0 mL) and EtOH (1.0 mL) was sealed and
heated to 120.degree. C. by microwave for 1 hour. The reaction
mixture was diluted with EtOAc (100 mL). The organic phase was
washed with brine then dried (MgSO.sub.4), filtered and
concentrated to dryness. The residue was purified by column
chromatography (silica, 0-10% MeOH in CH.sub.2Cl.sub.2) to afford
the sub-title compound (448 mg, 88%) as a brown solid. APCI MS
(Positive Mode) m/z 679
[C.sub.43H.sub.55FN.sub.4O.sub.2+H].sup.+.
(ii) Preparation of 22:
(4aS,6aS,6bR,8aR,13aR,15bS)-12-Amino-15-(2-(dimethylamino)pyridin-4-yl)-2-
,2,6a,6b,9,9,13a-heptamethyl-2,3,4,4a,5,6,6a,6b,7,8,8a,9,11,13,13a,13b,14,-
15b-octadecahydro-1H-chryseno[1,2-f]indazole-4-a-carboxylic
acid
[0322] A mixture of 22b (150 mg, 0.22 mmol) and dimethylamine (4.0
mL, 2 M in MeOH, 8.0 mmol) was sealed and heated to 140.degree. C.
by microwave for 5 hours. The mixture was concentrated to dryness.
The residue and 10% Pd(OH).sub.2/C (50 mg) in MeOH (12 mL) was
stirred under a hydrogen balloon for 5 hours. The reaction mixture
was filtered through a pad of diatomaceous earth and washed with
CMA (25 mL). The filtrate was concentrated to dryness under reduced
pressure. The residue was purified by column chromatography
(silica, 0-10% MeOH in CH.sub.2Cl.sub.2) followed by preparative
HPLC to provide the title compound (25 mg, 19%) as an off-white
solid.
[0323] R.sub.f 0.65 (80:18:2 Methylene
Chloride/Methanol/concentrated Ammonium Hydroxide).
[0324] .sup.1H NMR (300 MHz, CD.sub.3OD) .delta. 0.50 (s, 3H), 0.82
(s, 3H), 0.90 (s, 3H), 0.98 (s, 3H), 1.22 (s, 3H), 1.31 (s, 6H),
1.35-2.20 (m, 24H), 2.28 (m, 1H), 2.31 (d, J=14.8 Hz, 1H), 3.05 (m,
1H), 6.90 (s, 1H), 7.31 (s, 1H), 7.90 (s, 1H). mp>300.degree. C.
APCI MS (Positive Mode) m/z 614
[C.sub.38H.sub.55N.sub.5O.sub.2+H].sup.+.
Example 23
##STR00182##
[0325] (i) Preparation of 23b: 2-Aminophenylboronic acid
[0326] To a solution of 2-nitrophenylboronic acid (500 mg, 2.99
mmol) and MeOH (10 mL) was added 10% Pd/C (250 mg). The mixture was
stirred under hydrogen at atmospheric pressure for 2 hours. The
catalyst was removed by filtration through diatomaceous earth and
the filtrate was concentrated under reduced pressure. The residue
was purified by column chromatography (silica, 0-50% CMA in
CH.sub.2Cl.sub.2) to afford the sub-title compound (182 mg,
44%).
[0327] .sup.1H NMR (300 MHz, CD.sub.3OD) .delta. 6.69-7.75 (m,
4H).
(ii) Preparation of 23c: (4aS,6aS,6bR,13aR)-Benzyl
12-amino-15-(2-aminophenyl)-2,2,6a,6b,9,9,13a-heptamethyl-2,3,4,4a,5,6,6a-
,6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecahydro-1H-chryseno[1,2-f]indazole-
-4-a-carboxylate
[0328] To a mixture of II (150 mg, 0.22 mmol) and 23b (77 mg, 0.56
mmol) in benzene (4 mL) and EtOH (1 mL) was added K.sub.2CO.sub.3
(124 mg, 0.90 mmol). The mixture was sparged with nitrogen and then
Pd(PPh.sub.3).sub.4 (52 mg, 0.04 mmol) was added. The reaction
mixture was heated at 120.degree. C. for 1 hour using microwave
irradiation and then concentrated under reduced pressure. The
residue was dissolved in EtOAc (20 mL). The organic solution was
washed with brine then dried (Na.sub.2SO.sub.4), filtered and
concentrated. The residue was purified by column chromatography
(silica, 0-40% CMA in CH.sub.2Cl.sub.2) to afford the sub-title
compound (190 mg, 75%).
[0329] APCI MS m/z 675
[C.sub.44H.sub.58N.sub.4O.sub.2+H].sup.+.
(iii) Preparation of 23:
(4aS,6aS,6bR,13aR)-12-Amino-15-(2-aminophenyl)-2,2,6a,6b,9,9,13a-heptamet-
hyl-2,3,4,4a,5,6,6a,6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecahydro-1H-chry-
seno[1,2-f]indazole-4-a-carboxylic acid
[0330] A solution of 23c (180 mg, 0.26 mmol) and MeOH (20 mL) was
flushed with nitrogen and then 10% Pd/C (150 mg) was added. The
mixture was flushed with nitrogen followed by hydrogen. The mixture
was stirred under hydrogen at atmospheric pressure overnight. The
catalyst was removed by filtration through diatomaceous earth and
the filtrate was concentrated under reduced pressure. The residue
was purified by column chromatography (silica, 0-100% CMA in
CH.sub.2Cl.sub.2) followed by preparative HPLC to afford the title
compound (17 mg, 10%) as a solid.
[0331] R.sub.f 0.28 (40:9:1 Methylene
Chloride/Methanol/concentrated Ammonium Hydroxide).
[0332] .sup.1H NMR (300 MHz, CD.sub.3OD) .delta. 0.20-2.47 (m,
48H), .delta. 7.29 (d, J=7.2 Hz, 1H), .delta. 7.41-7.42 (m, 3H).
APCI MS m/z 585 [C.sub.37H.sub.52N.sub.4O.sub.2+H].sup.+. m.p.
210-230.degree. C. dec. HPLC (Method A)>99% (214 nm)
t.sub.R=12.7 min.
Example 24
##STR00183## ##STR00184##
[0333] (i) Preparation of 24b: (4aS,6aS,6bR,10S,12aR)-Benzyl
10-hydroxy-2,2,6a,6b,9,9,12a-heptamethyl-1,2,3,4,4a,5,6,6a,6b,7,8,8a,9,10-
,11,12,12a,12b,13,14b-icosahydropicene-4-a-carboxylate
[0334] A mixture of oleanolic acid (1a, 6.0 g, 13.1 mmol), benzyl
bromide (2.8 g, 16.4 mmol), K.sub.2CO.sub.3 (2.7 g, 19.7 mmol) and
DMF (120 mL) was stirred at room temperature for 15 hours. The
solvent was removed under reduced pressure and the residue was
purified by column chromatography (silica, 30% EtOAc in hexanes) to
provide the sub-title compound (6.6 g, 92%) as a white solid.
[0335] .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 0.61 (s, 3H),
0.65-0.72 (m, 1H), 0.78 (s, 3H), 0.87-1.00 (m, 10H), 1.05-1.46 (m,
12H), 1.49-1.72 (m, 12H), 1.81-2.05 (m, 3H), 2.86-2.91 (m, 1H),
3.15-3.27 (m, 1H), 5.02-5.11 (m, 2H), 5.43-5.45 (m, 1H), 7.31-7.49
(m, 5H).
(ii) Preparation of 24c: (4aS,6aS,6bR,10S,12aR)-Benzyl
10-acetoxy-2,2,6a,6b,9,9,12a-heptamethyl-1,2,3,4,4a,5,6,6a,6b,7,8,8a,9,10-
,11,12,12a,12b,13,14b-icosahydropicene-4-a-carboxylate
[0336] To a solution of 24b (6.6 g, 12.1 mmol), DMAP (200 mg) and
pyridine (65 mL) was slowly added acetic anhydride (1.5 g, 14.5
mmol) at 0.degree. C. under nitrogen. The mixture was allowed to
slowly warm to room temperature overnight and was quenched by
pouring into one liter of H.sub.2O. The precipitate was collected
by filtration and dried in a vacuum oven at 40.degree. C. to
provide the sub-title compound (6.6 g, 93%) as a white solid.
[0337] .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 0.61 (s, 3H),
0.77-0.94 (m, 16H), 0.98-1.75 (m, 21H), 1.84-2.02 (m, 3H), 2.04 (s,
3H), 2.85-2.95 (m, 1H), 4.46-4.51 (m, 1H), 5.01-5.08 (m, 2H),
5.27-5.29 (m, 1H), 7.29-7.35 (m, 5H).
(iii) Preparation of 24d: (4aS,6aS,6bR,10S,12aR)-Benzyl
10-acetoxy-14-chloro-2,2,6a,6b,9,9,12a-heptamethyl-1,2,3,4,4a,5,6,6a,6b,7-
,8,8a,9,10,11,12,12a,12b,13,14b-icosahydropicene-4-a-carboxylate
[0338] To a suspension of 24c (3.0 g, 5.13 mmol) and pyridine (65
mL) at 0.degree. C. was added chlorine gas which was generated by
addition of nitric acid to sodium chloride. After 20 min at
0.degree. C., the mixture was poured into H.sub.2O and the solid
was collected by filtration. The crude material was purified by
column chromatography (silica, 20% diethyl ether in hexanes) to
provide the sub-title compound (1.3 g, 40%).
[0339] .sup.1H NMR (500 MHz, CDCl.sub.3) .delta. 0.82-0.91 (m,
12H), 1.00 (s, 3H), 1.12-1.79 (m, 22H), 1.88-2.33 (m, 9H),
3.63-3.74 (m, 1H), 4.45-4.52 (m, 1H), 5.11-5.21 (m, 2H), 7.31-7.44
(m, 5H).
(iv) Preparation of 24e: (4aS,6aS,6bR,12aR)-Benzyl
14-chloro-2,2,6a,6b,9,9,12a-heptamethyl-10-oxo-1,2,3,4,4a,5,6,6a,6b,7,8,8-
a,9,10,11,12,12a,12b,13,14b-icosahydropicene-4-a-carboxylate
[0340] A mixture of 24d (1.0 g, 1.62 mmol), KOH (360 mg, 4.86 mmol)
and MeOH (70 mL) was heated at reflux for 96 hours. The solvent was
removed and the residue was partitioned between H.sub.2O (50 mL)
and CH.sub.2Cl.sub.2 (50 mL). The organic layer was separated then
dried (Na.sub.2SO.sub.4), filtered and concentrated under reduced
pressure. The crude product was dissolved in CH.sub.2Cl.sub.2 (40
mL) and the Dess-Martin reagent (900 mg, 2.1 mmol) was added at
room temperature. The mixture was stirred for 14 hours before
quenching with a solution of sodium thiosulfate (12.5 g) and
saturated NaHCO.sub.3 (50 mL). After stifling 30 min, the layers
were separated and the aqueous layer was extracted with
CH.sub.2Cl.sub.2 (50 mL). The combined organic layers were dried
(Na.sub.2SO.sub.4), filtered and concentrated under reduced
pressure. The residue was purified by column chromatography
(silica, 0-25% EtOAc in hexanes) to provide the sub-title compound
(785 mg, 100%) as a white solid.
[0341] .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 0.64 (s, 3H),
0.89-1.18 (m, 21H), 1.22-2.07 (m, 15H), 2.23-2.41 (m, 3H),
2.47-2.61 (m, 1H), 3.62-3.75 (m, 1H), 5.05-5.16 (m, 2H), 7.29-7.41
(m, 5H).
(v) Preparation of 24f: (4aS,6aS,6bR,12aR)-Benzyl
14-chloro-11-cyano-2,2,6a,6b,9,9,12a-heptamethyl-10-oxo-1,2,3,4,4a,5,6,6a-
,6b,7,8,8a,9,10,11,12,12a,12b,13,14b-icosahydropicene-4-a-carboxylate
[0342] A solution of diisopropylamine (0.2 mL, 1.46 mmol) and THF
(7 mL) was cooled to -78.degree. C. under nitrogen. A solution of
n-butyllithium (2.5 M in hexanes, 0.65 mL, 1.63 mmol) was slowly
added, maintaining the internal temperature below -70.degree. C.
The solution was allowed to stir for 45 min and was then slowly
added to a solution of 24e (500 mg, 0.86 mmol) and THF (10 mL) at
-78.degree. C. under nitrogen. This solution was stirred for 30 min
after which time a suspension of p-toluenesulfonyl cyanide (310 mg,
1.72 mmol) and THF (3 mL) was added over 15 min. The solution was
stirred for 30 min and then quenched by addition of saturated
ammonium chloride solution (10 mL) at -78.degree. C. The mixture
was allowed to warm to room temperature overnight. The organic
layer was separated and the aqueous layer was extracted with EtOAc.
The combined organic layers were dried (Na.sub.2SO.sub.4) and
filtered. The solvent was removed under reduced pressure and the
residue was purified by column chromatography (silica, 0-25% EtOAc
in hexanes) to provide the sub-title compound (350 mg, 67%).
[0343] .sup.1H NMR (500 MHz, CD.sub.3OD) .delta. 0.59 (s, 3H),
0.82-2.29 (m, 39H), 3.63-3.74 (m, 1H), 5.03-5.15 (m, 2H),
7.31-2.7.45 (m, 5H).
(vi) Preparation of 24g: (4aS,6aS,6bR,13aR)-Benzyl
12-amino-15-chloro-2,2,6a,6b,9,9,13a-heptamethyl-2,3,4,4a,5,6,6a,6b,7,8,8-
a,9,11,13,13a,13b,14,15b-octadecahydro-1H-chryseno[1,2-f]indazole-4-a-carb-
oxylate
[0344] A solution of 24f (350 mg, 0.58 mmol) and hydrazine (74 mg,
2.32 mmol) in EtOH (10 mL) was heated at reflux for 16 hours. The
solvent and excess hydrazine were removed under reduced pressure.
The residue was purified by column chromatography (silica, 0-50%
CMA in CH.sub.2Cl.sub.2) to provide the sub-title compound (175 mg,
50%).
[0345] .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 0.61 (s, 3H), 0.88
(s, 3H), 0.91 (s, 3H), 0.98 (s, 3H), 1.09-2.06 (m, 27H), 2.23-2.35
(m, 3H), 3.66-3.78 (m, 1H), 5.02-5.14 (m, 2H), 7.29-7.41 (m,
5H).
(vii) Preparation of 24:
(4aS,6aS,6bR,13aR)-12-Amino-15-chloro-2,2,6a,6b,9,9,13a-heptamethyl-2,3,4-
,4a,5,6,6a,6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecahydro-1H-chryseno[1,2--
f]indazole-4-a-carboxylic acid
[0346] A suspension of 24g (150 mg, 0.24 mmol), 10% Pd/C (100 mg)
and EtOAc/MeOH (20% MeOH, 25 mL) was stirred under hydrogen at
atmospheric pressure for 23 hours. The catalyst was removed by
filtration through diatomaceous earth and the filtrate was
concentrated under reduced pressure. The residue was purified by
column chromatography (silica, 50-100% CMA in CH.sub.2Cl.sub.2) to
provide the title compound (48 mg, 38%) as an off-white solid.
[0347] .sup.1H NMR (300 MHz, CD.sub.3OD) .delta. 0.89-1.01 (m,
12H), 1.12-1.30 (m, 12H), 1.34-2.10 (m, 15H), 2.33-2.41 (m, 3H),
3.62-3.70 (m, 1H). APCI MS m/z 528
[C.sub.31H.sub.46ClN.sub.3O.sub.2+H].sup.+. HPLC>99% (area %),
t.sub.R=15.9 min.
Example 25
##STR00185##
[0348] (i) Preparation of 25:
(4aS,6aS,6bR,8aR,13aR,15bR)-12-Amino-15-bromo-2,2,6a,6b,9,9,13a-heptameth-
yl-2,3,4,4a,5,6,6a,6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecahydro-1H-chrys-
eno[1,2-f]indazole-4-a-carboxylic acid
[0349] A mixture of II (75 mg, 0.11 mmol) and 10% Pd(OH).sub.2/C
(75 mg) in EtOAc (10 mL) and MeOH (2 mL) was stirred under a
hydrogen balloon for 12 hours. The reaction mixture was filtered
through a pad of diatomaceous earth and washed with CMA (25 mL).
The filtrate was concentrated to dryness under reduced pressure.
The residue was purified by column chromatography (silica, 0-60%
CMA in CH.sub.2Cl.sub.2) to afford the title compound (20 mg, 32%)
as a brown solid.
[0350] R.sub.f 0.40 (80:18:2 Methylene
Chloride/Methanol/concentrated Ammonium Hydroxide).
[0351] .sup.1H NMR (300 MHz, CD.sub.3OD) .delta. 0.91 (s, 6H), 0.93
(s, 3H), 1.0 (s, 3H), 1.12 (s, 3H), 1.15 (s, 3H), 1.21 (s, 3H),
1.32-2.28 (m, 20H), 2.35 (d, J=14.4 Hz, 1H), 2.53 (d, J=8.7 Hz,
2H), 3.53 (m, 1H). mp 268-270.degree. C. ESI MS (Positive Mode) m/z
572 [C.sub.31H.sub.46BrN.sub.3O.sub.2+H].sup.+.
Example 26
##STR00186##
[0352] (i) Preparation of 26b: (4aS,6aS,6bR,8aR,12aR,14bS)-benzyl
14-(furan-3-yl)-2,2,6a,6b,9,9,12a-heptamethyl-10-oxo-1,2,3,4,4a,5,6,6a,6b-
,7,8,8a,9,10,11,12,12a,12b,13,14b-icosahydropicene-4-a-carboxylate
[0353] A mixture of IIi (360 mg, 0.57 mmol), furan-3-ylboronic acid
(194 mg, 1.73 mmol), Pd(PPh.sub.3).sub.4 (66 mg, 0.057 mmol) and
cesium carbonate (564 mg, 1.73 mmol) in benzene (15 mL) and EtOH (4
mL) was heated at reflux for 12 hours. The reaction mixture was
diluted with EtOAc (100 mL). The organic phase was washed with
brine then dried (MgSO.sub.4), filtered and concentrated to
dryness. The residue was purified by column chromatography (silica,
0-20% EtOAc in hexanes) to afford the sub-title compound (340 mg,
97%).
[0354] .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 0.57 (s, 3H), 0.77
(s, 3H), 0.81 (s, 3H), 1.05 (s, 3H), 1.07 (s, 3H), 1.12 (s, 3H),
1.22 (s, 3H), 1.23-2.55 (m, 22H), 3.31 (m, 1H), 5.04 (m, 2H), 6.32
(s, 1H), 7.20 (s, 1H), 7.31 (m, 6H).
(ii) Preparation of 26c: (4aS,6aS,6bR,8aR,12aR,14bS)-benzyl
11-cyano-14-(furan-3-yl)-2,2,6a,6b,9,9,12a-heptamethyl-10-oxo-1,2,3,4,4a,-
5,6,6a,6b,7,8,8a,9,10,11,12,12a,12b,13,14b-icosahydropicene-4-a-carboxylat-
e
[0355] To a solution of diisopropylamine (0.12 mL, 0.84 mmol) in
THF (5 mL) was added n-butyllithium (0.35 mL, 2.5 M in hexanes,
0.88 mmol) at -78.degree. C. The solution was stirred for 30 min.
The LDA solution was added to 26b (270 mg, 0.44 mmol) in THF (5
mL). The mixture was allowed to warm to -40.degree. C. for 5 min
and cooled to -78.degree. C. A suspension of p-toluene sulfonyl
cyanide (203 mg, 1.12 mmol) in THF (2 mL) was added. The reaction
mixture was allowed to warm to -40.degree. C. over 1.5 hours. The
reaction was quenched by saturated NH.sub.4Cl (3 mL) and extracted
with EtOAc (100 mL). The organic phase was washed with brine then
dried (MgSO.sub.4), filtered and concentrated to dryness. The
residue was purified by column chromatography (silica, 0-20% EtOAc
in hexanes) to afford the sub-title compound (150 mg, 54%).
[0356] .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 0.51 (s, 3H), 0.62
(s, 3H), 0.65 (s, 3H), 1.03 (s, 3H), 1.04 (s, 3H), 1.07 (s, 3H),
1.16 (s, 3H), 1.23-2.55 (m, 21H), 3.32 (m, 1H), 5.02 (m, 2H), 6.32
(s, 1H), 7.20 (s, 1H), 7.31 (m, 6H).
(iii) Preparation of 26d: (4aS,6aS,6bR,8aR,13aR,15bS)-Benzyl
12-amino-15-(furan-3-yl)-2,2,6a,6b,9,9,13a-heptamethyl-2,3,4,4a,5,6,6a,6b-
,7,8,8a,9,11,13,13a,13b,14,15b-octadecahydro-1H-chryseno[1,2-f]indazole-4--
a-carboxylate
[0357] A mixture of 26c (150 mg, 0.23 mmol) and hydrazine (0.030
mL) in EtOH (3 mL) was heated at reflux overnight. The reaction
mixture was concentrated to dryness under reduced pressure. The
residue was purified by column chromatography (silica, 0-10% MeOH
in CH.sub.2Cl.sub.2) to afford the sub-title compound (110 mg,
71%).
[0358] ESI MS (Positive Mode) m/z 650
[C.sub.42H.sub.55N.sub.3O.sub.3+H].sup.+.
(iv) Preparation of 26:
(4aS,6aS,6bR,8aR,13aR,15bS)-12-Amino-15-(furan-3-yl)-2,2,6a,6b,9,9,13a-he-
ptamethyl-2,3,4,4a,5,6,6a,6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecahydro-1-
H-chryseno[1,2-f]indazole-4-a-carboxylic acid
[0359] A mixture of 26d (110 mg, 0.17 mmol) and 10% Pd(OH).sub.2/C
(50 mg) in MeOH (6 mL) and EtOAc (6 mL) was stirred under a
hydrogen balloon for 12 hours. The reaction mixture was filtered
through a pad of diatomaceous earth and washed with CMA (25 mL).
The filtrate was concentrated to dryness under reduced pressure.
The residue was purified by column chromatography (silica, 0-50%
CMA in CH.sub.2Cl.sub.2) to afford the title compound (20 mg, 21%)
as a brown solid.
[0360] R.sub.f 0.45 (80:18:2 Methylene
Chloride/Methanol/concentrated Ammonium Hydroxide).
[0361] .sup.1H NMR (300 MHz, DMSO-d.sub.6) .delta. 0.53 (s, 3H),
0.78 (s, 3H), 0.80 (s, 3H), 0.84 (s, 3H), 1.05 (s, 3H), 1.16 (s,
6H), 1.26-2.25 (m, 19H), 2.25 (d, J=14.7 Hz, 1H), 6.48 (s, 1H),
7.48 (s, 1H), 7.59 (s, 1H). mp>300.degree. C. APCI MS (Positive
Mode) m/z 560 [C.sub.35H.sub.49N.sub.3O.sub.3+H].sup.+.
Example 27
##STR00187##
[0362] (i) Preparation of 27b: (4aS,6aS,6bR,8aR,12aR,14bS)-Benzyl
14-(1-benzyl-1H-pyrazol-4-yl)-2,2,6a,6b,9,9,12a-heptamethyl-10-oxo-1,2,3,-
4,4a,5,6,6a,6b,7,8,8a,9,10,11,12,12a,12b,13,14b-icosahydropicene-4-a-carbo-
xylate carboxylate
[0363] A mixture of IIi (300 mg, 0.48 mmol),
1-benzyl-1H-pyrazol-4-ylboronic acid (291 mg, 1.44 mmol),
Pd(PPh.sub.3).sub.4 (60 mg, 0.048 mmol) and K.sub.2CO.sub.3 (198
mg, 1.44 mmol) in DMF (9 mL) was heated at 100.degree. C. for 24
hours. The reaction mixture was diluted with EtOAc (100 mL). The
organic phase was washed with brine then dried (MgSO.sub.4),
filtered and concentrated to dryness. The residue was purified by
column chromatography (silica, 0-20% EtOAc in hexanes) to afford
the sub-title compound (280 mg, 83%).
[0364] .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 0.43 (s, 3H), 0.76
(s, 3H), 0.77 (s, 3H), 0.78-2.53 (m, 37H), 3.32 (m, 1H), 5.02 (m,
4H), 7.15 (m, 4H), 7.31 (m, 8H).
(ii) Preparation of 27c: (4aS,6aS,6bR,8aR,12aR,14bS)-Benzyl
14-(1-benzyl-1H-pyrazol-4-yl)-11-cyano-2,2,6a,6b,9,9,12a-heptamethyl-10-o-
xo-1,2,3,4,4a,5,6,6a,6b,7,8,8a,9,10,11,12,12a,12b,13,14b-icosahydropicene--
4-a-carboxylate
[0365] To a solution of diisopropylamine (0.13 mL, 0.95 mmol) in
THF (5 mL) was added n-butyllithium (0.4 mL, 2.5 M in hexanes, 1.0
mmol) at -78.degree. C. The solution was stirred for 30 min. The
LDA solution was added to 27b (350 mg, 0.50 mmol) in THF (5 mL).
The mixture was allowed to warm to -40.degree. C. for 5 min and
cooled to -78.degree. C. A suspension of p-toluene sulfonyl cyanide
(181 mg, 1.0 mmol) in THF (2 mL) was added. The reaction mixture
was allowed to warm to -40.degree. C. over 1.5 hours. The reaction
was quenched by saturated NH.sub.4Cl (3 mL) and extracted with
EtOAc (100 mL). The organic phase was washed with brine then dried
(MgSO.sub.4), filtered and concentrated to dryness. The residue was
purified by column chromatography (silica, 0-20% EtOAc in hexanes)
to afford the sub-title compound (160 mg, 44%).
(iii) Preparation of 27d: (4aS,6aS,6bR,8aR,13aR,15bS)-Benzyl
12-amino-15-(1-benzyl-1H-pyrazol-4-yl)-2,2,6a,6b,9,9,13a-heptamethyl-2,3,-
4,4a,5,6,6a,6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecahydro-1H-chryseno[1,2-
-f]indazole-4-a-carboxylate
[0366] A mixture of 27c (160 mg, 0.22 mmol) and hydrazine (0.10 mL,
0.69 mmol) in EtOH (3 mL) was heated at reflux overnight. The
reaction mixture was concentrated to dryness under reduced
pressure. The residue was purified by column chromatography
(silica, 0-40% CMA in CH.sub.2Cl.sub.2) to afford the sub-title
compound (60 mg, 62%). APCI MS (Positive Mode) m/z 740
[C.sub.41H.sub.55N.sub.5O.sub.2+H].sup.+.
(iv) Preparation of 27:
(4aS,6aS,6bR,8aR,13aR,15bS)-12-Amino-2,2,6a,6b,9,9,13a-heptamethyl-15-(1H-
-pyrazol-4-yl)-2,3,4,4a,5,6,6a,6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecahy-
dro-1H-chryseno[1,2-f]indazole-4-a-carboxylic acid
[0367] A mixture of 27d (60 mg, 0.082 mmol) and 10% Pd(OH).sub.2/C
(60 mg) in MeOH (12 mL) and EtOAc (2 mL) was stirred under a
hydrogen balloon for 12 hours. The reaction mixture was filtered
through a pad of diatomaceous earth and washed with CMA (25 mL).
The filtrate was concentrated to dryness under reduced pressure.
The residue was purified by column chromatography (silica, 0-80%
CMA in CH.sub.2Cl.sub.2) to afford the title compound (8 mg, 18%)
as a brown solid.
[0368] R.sub.f 0.20 (80:18:2 Methylene
Chloride/Methanol/concentrated Ammonium Hydroxide).
[0369] .sup.1H NMR (300 MHz, CD.sub.3OD+CDCl.sub.3) .delta. 0.52
(s, 3H), 0.77 (s, 3H), 0.88 (s, 3H), 0.95 (s, 3H), 1.16 (s, 3H),
1.24 (s, 6H), 1.32-2.28 (m, 20H), 2.32 (d, J=14.7 Hz, 1H), 7.57 (s,
1H), 7.82 (s, 1H). mp>300.degree. C. APCI MS (Positive Mode) m/z
560 [C.sub.34H.sub.49N.sub.5O.sub.2+H].sup.+.
Example 28
##STR00188##
[0370] (i) Preparation of 28b: (4aS,6aS,6bR,8aR,12aR,14bS)-Benzyl
11-cyano-2,2,6a,6b,9,9,12a-heptamethyl-14-(1-methyl-1H-pyrazol-4-yl)-10-o-
xo-1,2,3,4,4a,5,6,6a,6b,7,8,8a,9,10,11,12,12a,12b,13,14b-icosahydropicene--
4-a-carboxylate
[0371] A mixture of IIj (100 mg, 0.15 mmol),
1-methyl-1H-pyrazol-4-ylboronic acid (75.0 mg, 0.60 mmol),
Pd(PPh.sub.3).sub.4 (34 mg, 0.030 mmol) and K.sub.2CO.sub.3 (184
mg, 1.33 mmol) in benzene (3.5 mL) and EtOH (1.5 mL) was sealed and
heated to 120.degree. C. by microwave for 1 hour. The reaction
mixture was diluted with EtOAc (100 mL). The organic phase was
washed with brine then dried (MgSO.sub.4), filtered and
concentrated to dryness. The residue was purified by column
chromatography (silica, 0-30% EtOAc in hexanes) to afford the
sub-title compound (60 mg, 61%).
[0372] .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 0.54 (s, 3H), 0.76
(s, 3H), 0.80 (s, 3H), 0.81-2.05 (m, 32H), 3.32 (m, 1H), 3.77 (s,
3H), 3.85 (m, 1H), 5.02 (m, 2H), 7.10 (s, 1H), 7.33 (m, 6H).
(ii) Preparation of 28c: (4aS,6aS,6bR,8aR,13aR,15bS)-Benzyl
12-amino-2,2,6a,6b,9,9,13a-heptamethyl-15-(1H-pyrrol-3-yl)-2,3,4,4a,5,6,6-
a,6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecahydro-1H-chryseno[1,2-f]indazol-
e-4-a-carboxylate
[0373] A mixture of 28b (60 mg, 0.09 mmol) and hydrazine (0.05 mL)
in EtOH (1.5 mL) was heated at reflux overnight. The reaction
mixture was concentrated to dryness under reduced pressure. The
residue was purified by column chromatography (silica, 0-30% CMA in
CH.sub.2Cl.sub.2) to afford the sub-title compound (33 mg, 54%).
APCI MS (Positive Mode) m/z 664
[C.sub.42H.sub.57N.sub.5O.sub.2+H].sup.+.
(iii) Preparation of 28:
(4aS,6aS,6bR,8aR,13aR,15bS)-12-Amino-2,2,6a,6b,9,9,13a-heptamethyl-15-(1--
methyl-1H-pyrazol-4-yl)-2,3,4,4a,5,6,6a,6b,7,8,8a,9,11,13,13a,13b,14,15b-o-
ctadecahydro-1H-chryseno[1,2-f]indazole-4-a-carboxylic acid
[0374] A mixture of 28c (33 mg, 0.05 mmol) and 10% Pd(OH).sub.2/C
(16 mg) in MeOH (12 mL) was stirred under a hydrogen balloon for 5
hours. The reaction mixture was filtered through a pad of
diatomaceous earth and washed with CMA (25 mL). The filtrate was
concentrated to dryness under reduced pressure. The residue was
purified by column chromatography (silica, 0-50% CMA in
CH.sub.2Cl.sub.2) to afford the title compound (20 mg, 71%) as a
brown solid.
[0375] R.sub.f -0.64 (9:1 Methylene Chloride/Methanol).
[0376] .sup.1H NMR (300 MHz, CD.sub.3OD) .delta. 0.55 (s, 3H), 0.81
(s, 3H), 0.87 (s, 3H), 0.93 (s, 3H), 1.15 (s, 3H), 1.23 (s, 3H),
1.24 (s, 3H), 1.25-2.18 (m, 19H), 2.36 (d, J=14.8 Hz, 1H), 3.34 (m,
1H), 7.45 (s, 1H), 7.52 (s, 1H). mp>300.degree. C. APCI MS
(Positive Mode) m/z 574
[C.sub.35H.sub.51N.sub.5O.sub.2+H].sup.+.
Example 29
##STR00189##
[0377] (i) Preparation of 29b: (4aS,6aS,6bR,8aR,12aR,14bS)-Benzyl
2,2,6a,6b,9,9,12a-heptamethyl-10-oxo-14-(pyridin-4-yl)-1,2,3,4,4a,5,6,6a,-
6b,7,8,8a,9,10,11,12,12a,12b,13,14b-icosahydropicene-4-a-carboxylate
[0378] A mixture of IIi (400 mg, 0.64 mmol), pyridine-4-ylboronic
acid (395 mg, 3.21 mmol), Pd(PPh.sub.3).sub.4 (222 mg, 0.19 mmol)
and K.sub.2CO.sub.3 (443 mg, 3.21 mmol) in DMF (10 mL) was heated
at 100.degree. C. for 24 hours. The reaction mixture was diluted
with EtOAc (100 mL). The organic phase was washed with brine then
dried (MgSO.sub.4), filtered and concentrated to dryness. The
residue was purified by column chromatography (silica, 0-30% EtOAc
in hexanes) to afford the sub-title compound (185 mg, 46%).
[0379] .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 0.32 (s, 3H), 0.75
(s, 3H), 0.89 (s, 3H), 1.02 (s, 3H), 1.04 (s, 3H), 1.10 (s, 3H),
1.23 (s, 3H), 1.32-2.05 (m, 20H), 2.30 (m, 1H), 2.45 (m, 1H), 2.98
(m, 1H), 5.10 (m, 2H), 7.10 (d, J=5.7 Hz, 2H), 7.30 (m, 5H), 8.35
(d, J=5.7 Hz, 2H).
(ii) Preparation of 29c: (4aS,6aS,6bR,8aR,12aR,14bS)-Benzyl
1'-cyano-2,2,6a,6b,9,9,12a-heptamethyl-10-oxo-14-(pyridin-4-yl)-1,2,3,4,4-
a,5,6,6a,6b,7,8,8a,9,10,11,12,12a,12b,13,14b-icosahydropicene-4-a-carboxyl-
ate
[0380] To a solution of diisopropylamine (0.16 mL, 1.16 mmol) in
THF (5 mL) was added n-butyllithium (0.48 mL, 2.5 M in hexanes, 1.2
mmol) at -78.degree. C. The solution was stirred for 30 min. The
LDA solution was added to 29b (326 mg, 0.52 mmol) in THF (5 mL).
The mixture was allowed to warm to -40.degree. C. for 5 min and
cooled to -78.degree. C. A suspension of p-toluene sulfonyl cyanide
(188 mg, 1.0 mmol) in THF (2 mL) was added. The reaction mixture
was allowed to warm to -40.degree. C. over 1.5 hours. The reaction
was quenched by saturated NH.sub.4Cl (3 mL) and extracted with
EtOAc (100 mL). The organic phase was washed with brine then dried
(MgSO.sub.4), filtered and concentrated to dryness. The residue was
purified by column chromatography (silica, 0-30% EtOAc in hexanes)
to afford the sub-title compound (171 mg, 51%).
[0381] .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 0.25 (s, 3H), 0.75
(s, 3H), 0.82 (s, 3H), 1.09 (s, 3H), 1.13 (s, 3H), 1.15 (s, 3H),
1.17 (s, 3H), 1.18-2.25 (m, 20H), 2.95 (m, 1H), 3.85 (m, 1H), 5.04
(m, 2H), 7.06 (m, 2H), 7.37 (m, 5H), 8.44 (m, 2H).
(iii) Preparation of 29d: (4aS,6aS,6bR,8aR,13aR,15bS)-Benzyl
12-amino-2,2,6a,6b,9,9,13a-heptamethyl-15-(pyridin-4-yl)-2,3,4,4a,5,6,6a,-
6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecahydro-1H-chryseno[1,2-f]indazole--
4-a-carboxylate
[0382] A mixture of 29c (171 mg, 0.26 mmol) and hydrazine (0.025
mL, 0.80 mmol) in EtOH (3 mL) was heated at reflux overnight. The
reaction mixture was concentrated to dryness under reduced
pressure. The residue was purified by column chromatography
(silica, 0-10% MeOH in CH.sub.2Cl.sub.2) to afford the sub-title
compound (143 mg, 83%).
[0383] ESI MS (Positive Mode) m/z 661
[C.sub.43H.sub.56N.sub.4O.sub.2+H].sup.+.
(iv) Preparation of 29:
(4aS,6aS,6bR,8aR,13aR,15bS)-12-Amino-2,2,6a,6b,9,9,13a-heptamethyl-15-(py-
ridin-4-yl)-2,3,4,4a,5,6,6a,6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecahydro-
-1H-chryseno[1,2-f]indazole-4-a-carboxylic acid
[0384] A mixture of 29d (143 mg, 0.21 mmol) and 10% Pd(OH).sub.2/C
(50 mg) in MeOH (15 mL) was stirred under a hydrogen balloon for 12
hours. The reaction mixture was filtered through a pad of
diatomaceous earth and washed with CMA (25 mL). The filtrate was
concentrated to dryness under reduced pressure. The residue was
purified by column chromatography (silica, 0-50% CMA in
CH.sub.2Cl.sub.2) to afford the title compound (85 mg, 72%) as a
brown solid.
[0385] R.sub.f 0.24 (80:18:2 Methylene
Chloride/Methanol/concentrated Ammonium Hydroxide).
[0386] .sup.1H NMR (300 MHz, CD.sub.3OD) .delta. 0.32 (s, 3H), 0.75
(s, 3H), 0.89 (s, 3H), 0.98 (s, 3H), 1.10 (s, 3H), 1.19 (s, 3H),
1.25 (s, 3H), 1.32-2.28 (m, 20H), 2.32 (d, J=14.7 Hz, 1H), 3.0 (m,
1H), 7.40 (d, J=5.7 Hz, 2H), 8.47 (d, J=5.7 Hz, 1H).
mp>300.degree. C. ESI MS (Positive Mode) m/z 571
[C.sub.36H.sub.50N.sub.4O.sub.2+H].sup.+.
Example 30
##STR00190##
[0387] (i) Preparation of 30b: (4aS,6aS,6bR,8aR,12aR,14bS)-Benzyl
11-cyano-2,2,6a,6b,9,9,12a-heptamethyl-10-oxo-14-(pyrimidin-5-yl)-1,2,3,4-
,4a,5,6,6a,6b,7,8,8a,9,10,11,12,12a,12b,13,14b-icosahydropicene-4-a-carbox-
ylate
[0388] A mixture of IIj (200 mg, 0.30 mmol), pyrimidin-5-ylboronic
acid (115 mg, 0.91 mmol), Pd(PPh.sub.3).sub.4 (34 mg, 0.030 mmol)
and K.sub.2CO.sub.3 (184 mg, 1.33 mmol) in benzene (3.5 mL) and
EtOH (1.5 mL) was sealed and heated to 120.degree. C. by microwave
for 1 hour. The reaction mixture was diluted with EtOAc (100 mL).
The organic phase was washed with brine then dried (MgSO.sub.4),
filtered and concentrated to dryness. The residue was purified by
column chromatography (silica, 0-40% EtOAc in hexanes) to afford
the sub-title compound (90 mg, 40%).
(ii) Preparation of 30c: (4aS,6aS,6bR,8aR,13aR,15bS)-Benzyl
12-amino-2,2,6a,6b,9,9,13a-heptamethyl-15-(pyrimidin-5-yl)-2,3,4,4a,5,6,6-
a,6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecahydro-1H-chryseno[1,2-f]indazol-
e-4-a-carboxylate
[0389] A mixture of 30b (90 mg, 0.13 mmol) and hydrazine (0.04 mL)
in EtOH (1.5 mL) was heated at reflux overnight. The reaction
mixture was concentrated to dryness under reduced pressure. The
residue was purified by column chromatography (silica, 0-30% CMA in
CH.sub.2Cl.sub.2) to afford the sub-title compound (65 mg,
71%).
[0390] APCI MS (Positive Mode) m/z 664
[C.sub.42H.sub.55N.sub.5O.sub.2+H].sup.+.
(iii) Preparation of 30:
(4aS,6aS,6bR,8aR,13aR,15bS)-12-Amino-2,2,6a,6b,9,9,13a-heptamethyl-15-(py-
rimidin-5-yl)-2,3,4,4a,5,6,6a,6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecahyd-
ro-1H-chryseno[1,2-f]indazole-4-a-carboxylic acid
[0391] A mixture of 30c (65 mg, 0.098 mmol) and 10% Pd(OH).sub.2/C
(30 mg) in MeOH (10 mL) was stirred under a hydrogen balloon for 5
hours. The reaction mixture was filtered through a pad of
diatomaceous earth and washed with CMA (25 mL). The filtrate was
concentrated to dryness under reduced pressure. The residue was
purified by column chromatography (silica, 0-60% CMA in
CH.sub.2Cl.sub.2) to afford the title compound (32 mg, 57%) as a
brown solid.
[0392] R.sub.f 0.50 (80:18:2 Methylene
Chloride/Methanol/concentrated Ammonium Hydroxide).
[0393] .sup.1H NMR (300 MHz, CD.sub.3OD) .delta. 0.31 (s, 3H), 0.80
(s, 3H), 0.89 (s, 3H), 1.02 (s, 3H), 1.20 (s, 3H), 1.25 (s, 3H),
1.31 (s, 3H), 1.32-2.30 (m, 21H), 2.33 (d, J=14.8 Hz, 1H), 2.88 (m,
1H), 8.78 (s, 1H), 9.04 (s, 1H). mp>300.degree. C. APCI MS
(Positive Mode) m/z 572
[C.sub.35H.sub.49N.sub.5O.sub.2+H].sup.+.
Example 31
##STR00191##
[0394] (i) Preparation of 31b: (4aS,6aS,6bR,13aR)-Benzyl
12-amino-15-(4-(benzyloxycarbonylamino)-3-fluorophenyl)-2,2,6a,6b,9,9,13a-
-heptamethyl-2,3,4,4a,5,6,6a,6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecahydr-
o-1H-chryseno[1,2-f]indazole-4-a-carboxylate
[0395] To a mixture of II (200 mg, 0.30 mmol) and
4-(benzyloxycarbonylamino)-3-fluorophenylboronic acid (262 mg, 0.90
mmol) in benzene (4 mL) and EtOH (1 mL) was added K.sub.2CO.sub.3
(166 mg, 1.20 mmol). The mixture was sparged with nitrogen and then
Pd(PPh.sub.3).sub.4 (69 mg, 0.06 mmol) was added. The reaction
mixture was heated at 120.degree. C. for 1 hour using microwave
irradiation and then solvent was removed under reduced pressure.
The residue was dissolved in EtOAc (20 mL) and washed with brine.
The organic layer was dried (Na.sub.2SO.sub.4), filtered and
concentrated. The residue was purified by column chromatography
(silica, 0-10% MeOH in CH.sub.2Cl.sub.2) to afford the sub-title
compound (190 mg, 76%).
[0396] APCI MS m/z 827
[C.sub.52H.sub.63N.sub.4O.sub.4+H].sup.+.
(ii) Preparation of 31:
(4aS,6aS,6bR,13aR)-12-Amino-15-(4-amino-3-fluorophenyl)-2,2,6a,6b,9,9,13a-
-heptamethyl-2,3,4,4a,5,6,6a,6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecahydr-
o-1H-chryseno[1,2-f]indazole-4-a-carboxylic acid
[0397] A solution of 31b (190 mg, 0.22 mmol) and MeOH (15 mL) was
flushed with nitrogen and then 10% Pd/C (300 mg) was added. The
mixture was flushed with nitrogen followed by hydrogen. The mixture
was stirred under hydrogen at atmospheric pressure overnight. The
catalyst was removed by filtration through diatomaceous earth and
the filtrate was concentrated under reduced pressure. The residue
was purified by column chromatography (silica, 0-30% CMA in
CH.sub.2Cl.sub.2) followed by preparative HPLC to afford the title
compound (35 mg, 21%) as as solid.
[0398] R.sub.f 0.74 (40:9:1 Methylene
Chloride/Methanol/concentrated Ammonium Hydroxide).
[0399] .sup.1H NMR (300 MHz, CD.sub.3OD) .delta. 0.37-2.43 (m,
41H), 3.07 (dd, J=3.4, 6.5 Hz, 1H), 6.99-7.10 (m, 3H). APCI MS m/z
603 [C.sub.37H.sub.51FN.sub.4O.sub.2+H].sup.+. m.p. 180-200.degree.
C. dec. HPLC (Method A)>99% (214 nm) t.sub.R=13.4 min.
Example 32
##STR00192##
[0400] (i) Preparation of 32b:
4-((3-Bromofuran-2-yl)methyl)morpholine
[0401] To a solution of 3-bromofuran-2-carbaldehyde (500 mg, 2.85
mmol) and morpholine (0.5 mL, 5.71 mmol) in CH.sub.2Cl.sub.2 (10
mL) was added sodium triacetoxyborohydride (1.2 g, 5.71 mmol). The
mixture was stirred at room temperature for 12 hours. The reaction
mixture was diluted with EtOAc (100 mL). The organic phase was
washed with aqueous NaHCO.sub.3 and brine then dried (MgSO.sub.4),
filtered and concentrated to dryness. The residue was purified by
column chromatography (silica, 0-10% MeOH in CH.sub.2Cl.sub.2) to
afford the sub-title compound (670 mg, 89%).
[0402] .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 2.50 (s, 4H), 3.62
(s, 2H), 3.70 (m, 4H), 6.43 (s, 1H), 7.35 (s, 1H).
(ii) Preparation of 32c: 2-(Morpholinomethyl)furan-3-ylboronic
acid
[0403] To a solution of 32b (670 mg, 2.54 mmol) in THF (15 mL) was
added n-butyllithium (2.5 M in hexanes, 1.30 mL, 3.31 mmol) at
-78.degree. C. The mixture was stirred at -78.degree. C. for 20
min. Triisopropyl borate (2.9 mL, 12.7 mmol) was added. The
reaction mixture was stirred at -78.degree. C. for 1 hour and
quenched with HCl (2 M, 4 mL). The reaction mixture was stirred for
5 min and neutralized by NaOH (2 M) and extracted with EtOAc (100
mL). The organic phase was washed with brine then dried
(MgSO.sub.4), filtered and concentrated to dryness. The residue was
purified by column chromatography (silica, 0-10% MeOH in
CH.sub.2Cl.sub.2) to afford the sub-title compound (450 mg,
83%).
[0404] .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 2.40 (s, 4H), 3.65
(s, 2H), 3.68 (m, 4H), 6.52 (s, 1H), 7.30 (s, 1H).
(iii) Preparation of 32d: (4aS,6aS,6bR,8aR,13aR,15bS)-Benzyl
12-amino-2,2,6a,6b,9,9,13a-heptamethyl-15-(2-(morpholinomethyl)furan-3-yl-
)-2,3,4,4a,5,6,6a,6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecahydro-1H-chryse-
no[1,2-f]indazole-4-a-carboxylate
[0405] A mixture of II (200 mg, 0.30 mmol), 32c (250 mg, 1.18
mmol), Pd(PPh.sub.3).sub.4 (35 mg, 0.030 mmol) and K.sub.2CO.sub.3
(207 mg, 1.50 mmol) in benzene (3.5 mL) and EtOH (1.5 mL) was
sealed and heated to 120.degree. C. by microwave for 1 hour. The
reaction mixture was diluted with EtOAc (100 mL). The organic phase
was washed with brine then dried (MgSO.sub.4), filtered and
concentrated to dryness. The residue was purified by column
chromatography (silica, 0-10% MeOH in CH.sub.2Cl.sub.2) to afford
the sub-title compound (135 mg, 60%).
[0406] APCI MS (Positive Mode) m/z 749
[C.sub.47H.sub.64N.sub.4O.sub.4+H].sup.+.
(iv) Preparation of 32:
(4aS,6aS,6bR,8aR,13aR,15bS)-12-Amino-2,2,6a,6b,9,9,13a-heptamethyl-15-(2--
(morpholinomethyl)furan-3-yl)-2,3,4,4a,5,6,6a,6b,7,8,8a,9,11,13,13a,13b,14-
,15b-octadecahydro-1H-chryseno[1,2-f]indazole-4-a-carboxylic
acid
[0407] A mixture of 32d (135 mg, 0.18 mmol) and 10% Pd(OH).sub.2/C
(100 mg) in MeOH (12 mL) and EtOAc (3 mL) was stirred under a
hydrogen balloon for 6 hours. The reaction mixture was filtered
through a pad of diatomaceous earth and washed with CMA (25 mL).
The filtrate was concentrated to dryness under reduced pressure.
The residue was purified by column chromatography (silica, 0-50%
CMA in CMA) to afford the title compound (35 mg, 30%) as a brown
solid.
[0408] R.sub.f 0.63 (80:18:2 Methylene
Chloride/Methanol/concentrated Ammonium Hydroxide).
[0409] .sup.1H NMR (300 MHz, CD.sub.3OD) .delta. 0.55 (s, 3H), 0.81
(s, 3H), 0.89 (s, 3H), 0.99 (s, 3H), 1.14 (s, 3H), 1.28 (s, 6H),
1.35-2.28 (m, 20H), 2.61 (m, 4H), 2.90 (m, 1H), 3.53 (m, 6H), 6.33
(s, 1H), 7.45 (s, 1H). mp>300.degree. C. dec. APCI MS (Positive
Mode) m/z 659 [C.sub.40H.sub.58N.sub.4O.sub.4+H].sup.+.
Example 33
##STR00193## ##STR00194##
[0410] (i) Preparation of 33b:
1-(Triisopropylsilyl)-1H-pyrrole-3-carbaldehyde
[0411] To a solution of 3-bromo-1-(triisopropylsilyl)-1H-pyrrole
(2.0 g, 6.61 mmol) in THF (30 mL) was added n-butyllithium (2.5 M
in hexanes, 3.4 mL, 8.60 mmol) at -78.degree. C. The mixture was
stirred at -78.degree. C. for 10 min then DMF (0.76 mL, 9.92 mmol)
was added and the mixture was warmed to 0.degree. C. over 1 hour.
The reaction mixture was quenched by saturated NH.sub.4Cl and
extracted with EtOAc (200 mL). The organic phase was washed with
brine then dried (MgSO.sub.4), filtered and concentrated to
dryness. The residue was purified by column chromatography (silica,
0-10% EtOAc in hexanes) to afford the sub-title compound (1.65 g,
100%).
[0412] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 2.40 (m, 4H), 3.42
(s, 2H), 3.70 (m, 4H), 6.03 (s, 1H), 6.09 (s, 1H), 6.74 (s, 1H),
8.30 (bs, 1H).
(ii) Preparation of 33c:
4-Bromo-1-(triisopropylsilyl)-1H-pyrrole-3-carbaldehyde
[0413] To a solution of 33b (1.45 g, 5.8 mmol) in THF (40 mL) was
added N-bromosuccinimide (1.03 g, 5.8 mmol) at room temperature.
The mixture was stirred for 2 hours and quenched with H.sub.2O (5
mL) and extracted with EtOAc (100 mL). The organic phase was washed
with brine then dried (MgSO.sub.4), filtered and concentrated to
dryness. The residue was purified by column chromatography (silica,
0-10% EtOAc in hexanes) to afford the sub-title compound (1.4 g,
74%).
[0414] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 1.15 (d, J=2.5 Hz,
18H), 1.58 (m, 3H), 6.70 (s, 1H), 7.32 (s, 1H), 9.82 (s, 1H).
(iii) Preparation of 33d:
4-((4-Bromo-1-(triisopropylsilyl)-1H-pyrrol-3-yl)methyl)morpholine
[0415] To a solution of 33c (1.5 g, 4.5 mmol) and morpholine (0.79
mL, 9.0 mmol) in CH.sub.2Cl.sub.2 (30 mL) was added sodium
triacetoxyborohydride (1.9 g, 9.0 mmol). The mixture was stirred at
room temperature for 12 hours. The reaction mixture was diluted
with EtOAc (200 mL). The organic phase was washed with aqueous
NaHCO.sub.3 and brine then dried (MgSO.sub.4), filtered and
concentrated to dryness. The residue was purified by column
chromatography (silica, 0-10% EtOAc in hexanes) to afford the
sub-title compound (1.8 g, 91%).
[0416] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 1.15 (d, J=2.5 Hz,
18H), 1.58 (m, 3H), 2.40 (m, 4H), 3.40 (s, 2H), 3.72 (m, 4H), 6.61
(s, 1H), 6.65 (s, 1H).
(iv) Preparation of 33e:
4-((4-(4,4,5,5-Tetramethyl-1,3-dioxolan-2-yl)-1-(triisopropylsilyl)-1H-py-
rrol-3-yl)methyl)morpholine
[0417] To a solution of 33d (400 mg, 1.0 mmol) was added
n-butyllithium (2.5 M in hexanes, 0.52 mmol, 1.3 mmol) at
-78.degree. C. The mixture was stirred for 20 min and
2-isopropoxy-4,4,5,5-tetramethyl-1,3,2-dioxaborolane (0.24 mL, 1.2
mmol) was added. The mixture was stirred at -78.degree. C. for 30
min and warmed to room temperature over 1 hour. The reaction was
quenched with H.sub.2O and extracted with EtOAc (100 mL). The
organic phase was washed with brine then dried (MgSO.sub.4),
filtered and concentrated to dryness. The residue was subjected to
column chromatography (silica, 0-50% EtOAc in hexanes) to afford
the still impure sub-title compound (448 mg, 100%) which was used
without further purification.
(v) Preparation of 33f: (4aS,6aS,6bR,8aR,13aR,15bS)-Benzyl
12-amino-2,2,6a,6b,9,9,13a-heptamethyl-15-(4-(morpholinomethyl)-1-(triiso-
propylsilyl)-1H-pyrrol-3-yl)-2,3,4,4a,5,6,6a,6b,7,8,8a,9,11,13,13a,13b,14,-
15b-octadecahydro-1H-chryseno[1,2-f]indazole-4-a-carboxylate
[0418] A mixture of II (200 mg, 0.30 mmol), 33e (500 mg, 1.06
mmol), Pd(PPh.sub.3).sub.4 (35 mg, 0.030 mmol) and cesium carbonate
(390 mg, 1.20 mmol) in toluene (4.5 mL) and H.sub.2O (0.3 mL) was
sealed and heated to 140.degree. C. by microwave for 1 hour. The
reaction mixture was diluted with EtOAc (100 mL). The organic phase
was washed with brine then dried (MgSO.sub.4), filtered and
concentrated to dryness. The residue was purified by column
chromatography (silica, 0-10% MeOH in CH.sub.2Cl.sub.2) to afford
the sub-title compound (176 mg, 65%).
(vi) Preparation of 33g: (4aS,6aS,6bR,8aR,13aR,15bS)-Benzyl
12-amino-2,2,6a,6b,9,9,13a-heptamethyl-15-(4-(morpholinomethyl)-1H-pyrrol-
-3-yl)-2,3,4,4a,5,6,6a,6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecahydro-1H-c-
hryseno[1,2-f]indazole-4-a-carboxylate
[0419] To a solution of 33f (176 mg, 0.19 mmol) in THF (3 mL) was
added tetrabutylammonium fluoride (0.38 mL, 1 M in THF, 0.38 mmol).
The mixture was stirred at room temperature for 2 hours. The
reaction mixture was diluted with EtOAc (100 mL). The organic phase
was washed with brine then dried (MgSO.sub.4), filtered and
concentrated to dryness. The residue was purified by column
chromatography (silica, 0-10% MeOH in CH.sub.2Cl.sub.2) to afford
the sub-title compound (60 mg, 42%).
[0420] APCI MS (Positive Mode) m/z 748
[C.sub.47H.sub.65N.sub.5O.sub.3+H].sup.+.
(vii) Preparation of 33:
(4aS,6aS,6bR,8aR,13aR,15bS)-12-Amino-2,2,6a,6b,9,9,13a-heptamethyl-15-(4--
(morpholinomethyl)-1H-pyrrol-3-yl)-2,3,4,4a,5,6,6a,6b,7,8,8a,9,11,13,13a,1-
3b,14,15b-octadecahydro-1H-chryseno[1,2-f]indazole-4-a-carboxylic
acid
[0421] A mixture of 33g (60 mg, 0.08 mmol) and 10% Pd(OH).sub.2/C
(30 mg) in MeOH (12 mL) and EtOAc (3 mL) was stirred under a
hydrogen balloon for 6 hours. The reaction mixture was filtered
through a pad of diatomaceous earth and washed with CMA (25 mL).
The filtrate was concentrated to dryness under reduced pressure.
The residue was purified by column chromatography (silica, 0-70%
CMA in CMA) to afford the title compound (35 mg, 67%) as a brown
solid.
[0422] R.sub.f 0.54 (80:18:2 Methylene
Chloride/Methanol/concentrated Ammonium Hydroxide).
[0423] .sup.1H NMR (400 MHz, CD.sub.3OD) .delta. 0.55 (s, 3H), 0.85
(s, 3H), 0.92 (s, 3H), 1.12 (s, 3H), 1.15 (s, 3H), 1.38 (m, 6H),
1.39-2.20 (m, 21H), 2.26 (d, J=14.4 Hz, 1H), 2.90 (m, 1H), 3.12 (m,
3H), 3.75 (m, 6H), 6.55 (s, 1H), 7.92 (s, 1H). mp>300.degree. C.
dec. APCI MS (Positive Mode) m/z 658
[C.sub.40H.sub.59N.sub.5O.sub.3+H].sup.+.
Example 34
##STR00195##
[0424] (i) Preparation of 34b:
4-((3-Bromothiophen-2-yl)methyl)morpholine
[0425] To a solution of 3-bromothiophene-2-carbaldehyde (500 mg,
2.62 mmol) and morpholine (0.68 mL, 7.85 mmol) and CH.sub.2Cl.sub.2
(10 mL) was added sodium triacetoxyborohydride (1.11 g, 5.24 mmol).
The mixture was stirred at room temperature overnight. The
resulting mixture was diluted with EtOAc (100 mL) and the organic
layer was washed with brine then dried (Na.sub.2SO.sub.4), filtered
and concentrated. The residue was purified by column chromatography
(silica, 0-5% MeOH in CH.sub.2Cl.sub.2) to afford the sub-title
compound (580 mg, 84%).
[0426] .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 2.47-2.54 (m, 4H),
3.68-3.71 (m, 4H), 6.90 (d, J=5.2 Hz, 1H), 7.23 (d, J=5.2 Hz,
1H).
(ii) Preparation of 34c: 2-(Morpholinomethyl)thiophen-3-ylboronic
acid
[0427] To a solution of 34b (500 mg, 1.90 mmol) in THF (10 mL) was
added n-butyllithium (1.14 mL, 2.86 mmol) at -78.degree. C. After
stifling for 20 min, triisopropyl borate (1.99 mL, 8.69 mmol) was
added at -78.degree. C. The mixture was slowly warmed to room
temperature and then quenched with aqueous HCl (2.0 M, 2 mL). The
reaction mixture was neutralized with 2 M NaOH solution and
extracted with EtOAc (3.times.10 mL) followed by
i-PrOH/CH.sub.2Cl.sub.2 (1:2, 10 mL.times.2). The combined extracts
were dried (Na.sub.2SO.sub.4), filtered and concentrated under
reduced pressure. The residue was purified by column chromatography
(silica, 0-10% MeOH in CH.sub.2Cl.sub.2) to afford the sub-title
compound (220 mg, 51%).
[0428] .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 2.47-2.49 (m, 4H),
3.52-3.76 (m, 6H), 6.91-6.98 (m, 1H), 7.22-7.24 (m, 1H).
(iii) Preparation of 34d: (4aS,6aS,6bR,13aR)-Benzyl
12-amino-2,2,6a,6b,9,9,13a-heptamethyl-15-(2-(morpholinomethyl)thiophen-3-
-yl)-2,3,4,4a,5,6,6a,6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecahydro-1H-chr-
yseno[1,2-f]indazole-4-a-carboxylate
[0429] To a mixture of II (250 mg, 0.38 mmol) and 34c (220 mg, 0.96
mmol) in benzene (4 mL) and EtOH (1 mL) was added K.sub.2CO.sub.3
(182 mg, 1.32 mmol). The mixture was sparged with nitrogen and then
Pd(PPh.sub.3).sub.4 (87 mg, 0.075 mmol) was added. The reaction
mixture was heated at 120.degree. C. for 1 hour using microwave
irradiation. The solvent was concentrated under reduced pressure.
The residue was taken up in EtOAc (20 mL) and washed with brine.
The organic layer was dried (Na.sub.2SO.sub.4), filtered and
concentrated. The residue was purified by column chromatography
(silica, 0-10% MeOH in CH.sub.2Cl.sub.2) to afford the sub-title
compound (190 mg, 66%).
[0430] APCI MS m/z 765
[C.sub.47H.sub.64N.sub.4O.sub.3S+H].sup.+.
(iv) Preparation of 34:
(4aS,6aS,6bR,13aR)-12-Amino-2,2,6a,6b,9,9,13a-heptamethyl-15-(2-(morpholi-
nomethyl)thiophen-3-yl)-2,3,4,4a,5,6,6a,6b,7,8,8a,9,11,13,13a,13b,14,15b-o-
ctadecahydro-1H-chryseno[1,2-f]indazole-4-a-carboxylic acid
[0431] A solution of 34d (190 mg, 0.24 mmol), EtOH (2 mL) and MeOH
(8 mL) was flushed with nitrogen and then 10% Pd/C (400 mg) was
added. The mixture was flushed with nitrogen followed by hydrogen.
The mixture was stirred under hydrogen at atmospheric pressure
overnight. The catalyst was removed by filtration through
diatomaceous earth and the filtrate was concentrated. The residue
was purified by column chromatography (silica, 0-50% CMA in
CH.sub.2Cl.sub.2) followed by preparative HPLC to afford the title
compound (18 mg, 11%) as a solid.
[0432] R.sub.f 0.13 (90:9:1 Methylene
Chloride/Methanol/concentrated Ammonium Hydroxide).
[0433] .sup.1H NMR (300 MHz, CD.sub.3OD) .delta. 0.40 (s, 3H), 0.80
(s, 3H), 0.92 (s, 3H), 1.05 (s, 3H), 1.10-2.67 (m, 32H), 3.30 (s,
3H), 3.97 (s, 4H), 4.57-4.62 (m, 1H), 6.95 (d, J=4.95 Hz, 1H), 7.67
(d, J=4.95 Hz, 1H). APCI MS m/z 675
[C.sub.40H.sub.58N.sub.4O.sub.3S+H].sup.+. m.p. 280-300.degree. C.
dec. HPLC (Method A) 98.7% (214 nm) t.sub.R=12.9 min.
Example 35
##STR00196##
[0434] (i) Preparation of 35b: tert-Butyl
4-((3-bromofuran-2-yl)methyl)piperazine-1-carboxylate
[0435] To a solution of 3-bromofuran-2-carbaldehyde (500 mg, 2.85
mmol) and tert-butyl piperazine-1-carboxylate (1.1 g, 5.71 mmol) in
CH.sub.2Cl.sub.2 (10 mL) was added sodium triacetoxyborohydride
(1.2 g, 5.71 mmol). The mixture was stirred at room temperature for
12 hours. The reaction mixture was diluted with EtOAc (100 mL). The
organic phase was washed with aqueous NaHCO.sub.3 and brine then
dried (MgSO.sub.4), filtered and concentrated to dryness. The
residue was purified by column chromatography (silica, 0-10% MeOH
in CH.sub.2Cl.sub.2) to afford the sub-title compound (910 mg,
92%).
[0436] .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 1.42 (s, 9H), 2.40
(m, 4H), 3.42 (m, 4H), 3.60 (s, 2H), 6.35 (s, 1H), 7.35 (s,
1H).
(ii) Preparation of 35c:
2-((4-(tert-Butoxycarbonyl)piperazin-1-yl)methyl)furan-3-ylboronic
acid
[0437] To a solution of 35b (910 mg, 2.63 mmol) in THF (20 mL) was
added n-butyllithium (2.5 M in hexanes, 1.4 mL, 3.43 mmol) at
-78.degree. C. The mixture was stirred at -78.degree. C. for 20
minutes. Triisopropyl borate (3.0 mL, 13.2 mmol) was added. The
reaction mixture was stirred at -78.degree. C. for 1 hour and
quenched with HCl (2 M, 4 mL). The reaction mixture was stirred for
5 min then neutralized by NaOH (2 M) and extracted with EtOAc (100
mL). The organic phase was washed with brine then dried
(MgSO.sub.4), filtered and concentrated to dryness. The residue was
purified by column chromatography (silica, 0-10% MeOH in
CH.sub.2Cl.sub.2) to afford the sub-title compound (560 mg,
68%).
[0438] .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 1.42 (s, 9H), 2.40
(m, 4H), 3.65 (m, 4H), 3.68 (s, 2H), 6.52 (s, 1H), 7.31 (s,
1H).
(iii) Preparation of 35d: (4aS,6aS,6bR,8aR,13aR,15bS)-Benzyl
12-amino-15-(2-((4-(tert-butoxycarbonyl)piperazin-1-yl)methyl)furan-3-yl)-
-2,2,6a,6b,9,9,13a-heptamethyl-2,3,4,4a,5,6,6a,6b,7,8,8a,9,11,13,13a,13b,1-
4,15b-octadecahydro-1H-chryseno[1,2-f]indazole-4-a-carboxylate
[0439] A mixture of II (300 mg, 0.45 mmol), 35c (418 mg, 1.35
mmol), Pd(PPh.sub.3).sub.4 (52 mg, 0.045 mmol) and K.sub.2CO.sub.3
(248 mg, 1.80 mmol) in benzene (3.5 mL) and EtOH (1.5 mL) was
sealed and heated to 120.degree. C. by microwave for 1 hour. The
reaction mixture was diluted with EtOAc (100 mL). The organic phase
was washed with brine then dried (MgSO.sub.4), filtered and
concentrated to dryness. The residue was purified by column
chromatography (silica, 0-10% MeOH in CH.sub.2Cl.sub.2) to afford
the sub-title compound (400 mg, 100%).
[0440] APCI MS (Positive Mode) m/z 848
[C.sub.52H.sub.73N.sub.5O.sub.5+H].sup.+.
(iv) Preparation of 35e: (4aS,6aS,6bR,8aR,13aR,15bS)-Benzyl
12-amino-2,2,6a,6b,9,9,13a-heptamethyl-15-(2-(piperazin-1-ylmethyl)furan--
3-yl)-2,3,4,4a,5,6,6a,6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecahydro-1H-ch-
ryseno[1,2-f]indazole-4-a-carboxylate
[0441] To a solution of 35d (400 mg, 0.45 mmol) in MeOH (3 mL) and
CH.sub.2Cl.sub.2 (2 mL) was added HCl (2.3 mL, 2 M in Et.sub.2O,
4.6 mmol). The mixture was stirred at room temperature for 12
hours. The reaction mixture was concentrated to dryness. The
residue was purified by column chromatography (silica, 0-10% MeOH
in CH.sub.2Cl.sub.2) to afford the sub-title compound (250 mg,
74%).
[0442] APCI MS (Positive Mode) m/z 748
[C.sub.47H.sub.65N.sub.5O.sub.3+H].sup.+.
(v) Preparation of 35:
(4aS,6aS,6bR,8aR,13aR,15bS)-12-Amino-2,2,6a,6b,9,9,13a-heptamethyl-15-(2--
(piperazin-1-ylmethyl)furan-3-yl)-2,3,4,4a,5,6,6a,6b,7,8,8a,9,11,13,13a,13-
b,14,15b-octadecahydro-1H-chryseno[1,2-f]indazole-4-a-carboxylic
acid
[0443] A mixture of 35e (250 mg, 0.33 mmol) and 10% Pd(OH).sub.2/C
(110 mg) in MeOH (15 mL) and EtOAc (5 mL) was stirred under a
hydrogen balloon for 12 hours. The reaction mixture was filtered
through a pad of diatomaceous earth and washed with CMA (25 mL).
The filtrate was concentrated to dryness under reduced pressure.
The residue was purified by column chromatography (silica, 0-10%
MeOH in CMA) to afford the title compound (160 mg, 75%) as a brown
sold.
[0444] R.sub.f 0.20 (80:28:2 Methylene
Chloride/Methanol/concentrated Ammonium Hydroxide).
[0445] .sup.1H NMR (300 MHz, CD.sub.3OD) .delta. 0.55 (s, 3H), 0.78
(s, 3H), 0.86 (s, 3H), 0.94 (s, 3H), 1.12 (s, 3H), 1.21 (s, 3H),
1.24 (s, 3H), 1.35-2.18 (m, 19H), 2.36 (d, J=14.8 Hz, 1H), 2.73 (m,
4H), 3.0 (m, 1H), 3.18 (m, 4H), 3.52 (m, 1H), 3.90 (m, 1H), 6.33
(s, 1H), 7.44 (s, 1H). mp>300.degree. C. APCI MS (Positive Mode)
m/z 658 [C.sub.40H.sub.59N.sub.5O.sub.3+H].sup.+.
Example 36
##STR00197##
[0446] (i) Preparation of 36b:
1-((3-Bromofuran-2-yl)methyl)-4-methylpiperazine
[0447] To a solution of 3-bromofuran-2-carbaldehyde (500 mg, 2.85
mmol), 1-methylpiperazine dihydrochloride (986 mg, 5.71 mmol) and
triethylamine (1.5 mL, 11.4 mmol) in CH.sub.2Cl.sub.2 (10 mL) was
added sodium triacetoxyborohydride (1.2 g, 5.71 mmol). The mixture
was stirred at room temperature for 12 hours. The reaction mixture
was diluted with EtOAc (100 mL). The organic phase was washed with
aqueous NaHCO.sub.3 and brine then dried (MgSO.sub.4), filtered and
concentrated to dryness. The residue was purified by column
chromatography (silica, 0-10% MeOH in CH.sub.2Cl.sub.2) to afford
the sub-title compound (570 mg, 78%).
[0448] .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 2.25 (s, 3H), 2.40
(m, 4H), 2.60 (m, 4H), 3.60 (s, 2H), 6.35 (s, 1H), 7.35 (s,
1H).
(ii) Preparation of 36c:
2-((4-Methylpiperazin-1-yl)methyl)furan-3-ylboronic acid
[0449] To a solution of 36b (570 mg, 2.20 mmol) in THF (15 mL) was
added n-butyllithium (2.5 M in hexanes, 1.1 mL, 2.80 mmol) at
-78.degree. C. The mixture was stirred at -78.degree. C. for 20
minutes. Triisopropyl borate (2.5 mL, 11.0 mmol) was added. The
reaction mixture was stirred at -78.degree. C. for 1 hour and
quenched with HCl (2 M, 4 mL). The reaction mixture was stirred for
5 min then neutralized by NaOH (2 M) and extracted with EtOAc (100
mL). The organic phase was washed with brine then dried
(MgSO.sub.4), filtered and concentrated to dryness. The residue was
purified by column chromatography (silica, 0-15% MeOH in
CH.sub.2Cl.sub.2) to afford the sub-title compound (200 mg,
41%).
[0450] .sup.1H NMR (300 MHz, CD.sub.3OD) .delta. 2.32 (s, 3H), 2.40
(m, 4H), 2.80 (m, 4H), 3.95 (s, 2H), 6.46 (s, 1H), 7.38 (s,
1H).
(iii) Preparation of 36d: (4aS,6aS,6bR,8aR,13aR,15bS)-Benzyl
12-amino-2,2,6a,6b,9,9,13a-heptamethyl-15-(2-((4-methylpiperazin-1-yl)met-
hyl)furan-3-yl)-2,3,4,4a,5,6,6a,6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecah-
ydro-1H-chryseno[1,2-f]indazole-4-a-carboxylate
[0451] A mixture of II (200 mg, 0.30 mmol), 36c (200 mg, 0.89
mmol), Pd(PPh.sub.3).sub.4 (35 mg, 0.030 mmol) and K.sub.2CO.sub.3
(166 mg, 1.20 mmol) in benzene (3.5 mL) and EtOH (1.5 mL) was
sealed and heated to 120.degree. C. by microwave for 1 hour. The
reaction mixture was diluted with EtOAc (100 mL). The organic phase
was washed with brine then dried (MgSO.sub.4), filtered and
concentrated to dryness. The residue was purified by column
chromatography (silica, 0-50% CMA in CH.sub.2Cl.sub.2) to afford
the sub-title compound (200 mg, 87%).
[0452] APCI MS (Positive Mode) m/z 762
[C.sub.48H.sub.67N.sub.5O.sub.3+H].sup.+.
(iv) Preparation of 36:
(4aS,6aS,6bR,8aR,13aR,15bS)-12-Amino-2,2,6a,6b,9,9,13a-heptamethyl-15-(2--
((4-methylpiperazin-1-yl)methyl)furan-3-yl)-2,3,4,4a,5,6,6a,6b,7,8,8a,9,11-
,13,13a,13b,14,15b-octadecahydro-1H-chryseno[1,2-f]indazole-4-a-carboxylic
acid
[0453] A mixture of 36d (200 mg, 0.26 mmol) and 10% Pd(OH).sub.2/C
(100 mg) in MeOH (15 mL) and EtOAc (5 mL) was stirred under a
hydrogen balloon for 12 hours. The reaction mixture was filtered
through a pad of diatomaceous earth and washed with CMA (25 mL).
The filtrate was concentrated to dryness under reduced pressure.
The residue was purified by column chromatography (silica, 0-70%
CMA in CH.sub.2Cl.sub.2) to afford the title compound (22 mg, 13%)
as a brown solid.
[0454] R.sub.f 0.30 (80:28:2 Methylene
Chloride/Methanol/concentrated Ammonium Hydroxide).
[0455] .sup.1H NMR (300 MHz, CD.sub.3OD) .delta. 0.55 (s, 3H), 0.78
(s, 3H), 0.86 (s, 3H), 0.99 (s, 3H), 1.12 (s, 3H), 1.28 (s, 6H),
1.35-2.18 (m, 16H), 2.28 (d, J=14.8 Hz, 1H), 2.29 (m, 4H), 2.40 (m,
9H), 2.90 (m, 1H), 3.52 (m, 2H), 3.80 (m, 1H), 6.33 (s, 1H), 7.47
(s, 1H). mp>300.degree. C. APCI MS (Positive Mode) m/z 672
[C.sub.41H.sub.61N.sub.5O.sub.3+H].sup.+.
Example 37
##STR00198##
[0456] (i) Preparation of 37b:
1-((3-Bromofuran-2-yl)methyl)pyrrolidine
[0457] To a solution of 3-bromofuran-2-carbaldehyde (1.5 g, 8.57
mmol) and pyrrolidine (1.42 mL, 17.14 mmol) and CH.sub.2Cl.sub.2
(30 mL) was added sodium triacetoxyborohydride (3.63 g, 17.14
mmol). The mixture was stirred at room temperature for 5 hours. The
resulting mixture was diluted with EtOAc (300 mL) and the organic
layer was washed with saturated NaHCO.sub.3 and brine then dried
(Na.sub.2SO.sub.4), filtered and concentrated. The residue was
purified by column chromatography (silica, 0-10% MeOH in
CH.sub.2Cl.sub.2) to afford the sub-title compound (1.26 g,
65%).
[0458] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 1.74-1.80 (m, 4H),
2.56-2.59 (m, 4H), 3.67 (s, 2H), 6.37 (d, J=2.0 Hz, 1H), 7.34 (d,
J=2.0 Hz, 1H).
(ii) Preparation of 37c: 2-(Pyrrolidin-1-ylmethyl)furan-3-ylboronic
acid
[0459] To a solution of 37b (1.29 g, 5.60 mmol) in THF (20 mL) was
added n-butyllithium (4.26 mL, 10.65 mmol) at -78.degree. C. After
stirring for 20 min, triisopropylborate (1.99 mL, 8.69 mmol) was
added at -78.degree. C. The mixture was slowly warmed to room
temperature and then quenched with H.sub.3PO.sub.4 (85% in
H.sub.2O, 3 mL). The reaction mixture was neutralized with 2 N NaOH
solution and extracted with EtOAc (3.times.10 mL) followed by
i-PrOH/CH.sub.2Cl.sub.2 (2:1, 3.times.10 mL). The combined extracts
were dried (Na.sub.2SO.sub.4), filtered and concentrated under
reduced pressure. The residue was purified by column chromatography
(silica, 0-50% CMA in CH.sub.2Cl.sub.2) to afford the sub-title
compound (515 mg, 47%).
[0460] .sup.1H NMR (400 MHz, CD.sub.3OD) .delta. 2.04-2.07 (m, 4H),
3.15-3.18 (m, 4H), 3.29-3.31 (m, 2H), 4.19 (s, 2H), 6.41 (d, J=1.6
Hz, 1H), 7.32 (d, J=1.6 Hz, 1H).
(iii) Preparation of 37d: (4aS,6aS,6bR,13aR)-Benzyl
12-amino-2,2,6a,6b,9,9,13a-heptamethyl-15-(2-(pyrrolidin-1-ylmethyl)furan-
-3-yl)-2,3,4,4a,5,6,6a,6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecahydro-1H-c-
hryseno[1,2-f]indazole-4-a-carboxylate
[0461] To a mixture of II (500 mg, 0.75 mmol) and 37c (441 mg, 2.26
mmol) in benzene (4 mL) and EtOH (1 mL) was added K.sub.2CO.sub.3
(416 mg, 3.02 mmol). The mixture was sparged with nitrogen and then
Pd(PPh.sub.3).sub.4 (174 mg, 0.15 mmol) was added. The reaction
mixture was heated at 130.degree. C. for 1 hour using microwave
irradiation. The solvent was concentrated under reduced pressure.
The residue was dissolved in EtOAc (40 mL) and washed with brine
(3.times.15 mL). The organic layer was dried (Na.sub.2SO.sub.4),
filtered and concentrated. The residue was purified by column
chromatography (silica, 0-10% MeOH in CH.sub.2Cl.sub.2 followed by
30% CMA in CH.sub.2Cl.sub.2) to afford the sub-title compound (260
mg, 47%).
[0462] .sup.1H NMR (400 MHz, CD.sub.3OD) .delta. 0.51-2.66 (m,
50H), 3.05-3.08 (m, 1H), 3.61-3.87 (m, 3H), 5.0 (d, J=12.20 Hz,
1H), 5.21 (d, J=12.20 Hz, 1H), 6.23 (d, J=1.56 Hz, 1H), 7.31-7.52
(m, 7H). APCI MS m/z 733
[C.sub.47H.sub.64N.sub.4O.sub.3+H].sup.+.
(iv) Preparation of 37:
(4aS,6aS,6bR,13aR)-12-Amino-2,2,6a,6b,9,9,13a-heptamethyl-15-(2-(pyrrolid-
in-1-ylmethyl)furan-3-yl)-2,3,4,4a,5,6,6a,6b,7,8,8a,9,11,13,13a,13b,14,15b-
-octadecahydro-1H-chryseno[1,2-f]indazole-4-a-carboxylic acid
[0463] A solution of 37d (260 mg, 0.35 mmol), EtOAc (5 mL) and MeOH
(10 mL) was flushed with nitrogen and then 10% Pd/C (130 mg) was
added. The mixture was flushed with nitrogen followed by hydrogen.
The mixture was stirred under hydrogen at atmospheric pressure
overnight. The catalyst was removed by filtration through
diatomaceous earth and the filtrate was concentrated. The residue
was purified by column chromatography (silica, 0-50% CMA in
CH.sub.2Cl.sub.2) to afford the title compound (85 mg, 37%).
[0464] R.sub.f 0.24 (90:9:1 Methylene
Chloride/Methanol/concentrated Ammonium Hydroxide).
[0465] .sup.1H NMR (300 MHz, CD.sub.3OD) .delta. 0.59 (s, 3H), 0.85
(s, 3H), 0.89 (s, 3H), 1.04-2.32 (m, 36H), 2.75-2.79 (m, 1H),
3.11-3.15 (m, 2H), 3.48 (s, 2H), 4.08-4.19 (m, 2H), 6.38 (d, J=1.5
Hz, 1H), 7.60 (d, J=1.5 Hz, 1H). APCI MS m/z 643
[C.sub.40H.sub.58N.sub.4O.sub.3+H].sup.+. m.p. 260-280.degree. C.
dec. HPLC (Method A) 98.4% (214 nm) t.sub.R=13.1 min
Example 38
##STR00199##
[0466] (i) Preparation of 38b:
1-((4-Bromofuran-2-yl)methyl)pyrrolidine
[0467] To a solution of 4-bromofuran-2-carbaldehyde (500 mg, 2.85
mmol) and pyrrolidine (0.47 mL, 5.74 mmol) and CH.sub.2Cl.sub.2 (10
mL) was added sodium triacetoxyborohydride (1.2 g, 5.71 mmol). The
mixture was stirred at room temperature overnight. The resulting
mixture was diluted with EtOAc (100 mL) and the organic layer was
washed with brine then dried (Na.sub.2SO.sub.4), filtered and
concentrated. The residue was purified by column chromatography
(silica, 0-5% MeOH in CH.sub.2Cl.sub.2) to afford the sub-title
compound (400 mg, 61%).
[0468] .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 1.77-1.82 (m, 4H),
2.53-2.58 (m, 4H), 3.62 (s, 2H), 6.25 (s, 1H), 7.35 (s, 1H).
(ii) Preparation of 38c: 5-(Pyrrolidin-1-ylmethyl)furan-3-ylboronic
acid
[0469] To a solution of 38b (400 mg, 1.73 mmol) in THF (10 mL) was
added n-butyllithium (0.90 mL, 2.26 mmol) at -78.degree. C. After
stifling for 20 min, triisopropylborate (1.99 mL, 8.69 mmol) was
added at -78.degree. C. The mixture was slowly warmed to room
temperature and then quenched with phosphoric acid. The reaction
mixture was neutralized with 2 M NaOH solution and extracted with
EtOAc (3.times.10 mL) followed by i-PrOH/CH.sub.2Cl.sub.2 (1:2,
2.times.10 mL). The combined extracts were dried
(Na.sub.2SO.sub.4), filtered and concentrated under reduced
pressure. The residue was purified by column chromatography
(silica, 0-100% CMA in CH.sub.2Cl.sub.2) to afford the sub-title
compound (197 mg, 58%).
[0470] .sup.1H NMR (300 MHz, CD.sub.3OD) .delta. 1.84 (d, J=3.9 Hz,
4H), 2.74 (m, 4H), 3.81 (d, J=4.2 Hz, 2H), 6.46 (d, J=4.2 Hz, 1H),
7.62 (d, J=3.9 Hz, 1H).
(iii) Preparation of 38d: (4aS,6aS,6bR,13aR)-Benzyl
12-amino-2,2,6a,6b,9,9,13a-heptamethyl-15-(5-(pyrrolidin-1-ylmethyl)furan-
-3-yl)-2,3,4,4a,5,6,6a,6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecahydro-1H-c-
hryseno[1,2-f]indazole-4-a-carboxylate
[0471] To a mixture of II (200 mg, 0.30 mmol) and 38c (176 mg, 0.96
mmol) in benzene (4 mL) and EtOH (1 mL) was added K.sub.2CO.sub.3
(166 mg, 1.20 mmol). The mixture was sparged with nitrogen and then
Pd(PPh.sub.3).sub.4 (69 mg, 0.06 mmol) was added. The reaction
mixture was heated at 120.degree. C. for 1 hour using microwave
irradiation. The solvent was concentrated under reduced pressure.
The residue was taken up in EtOAc (20 mL) and washed with brine.
The organic layer was dried (Na.sub.2SO.sub.4), filtered and
concentrated. The residue was purified by column chromatography
(silica, 0-10% MeOH in CH.sub.2Cl.sub.2) to afford the sub-title
compound (200 mg, 90%).
[0472] .sup.1H NMR (300 MHz, CD.sub.3OD) .delta. 0.59-3.69 (m,
52H), 5.03 (d, J=12.0 Hz, 1H), 5.21 (d, J=12.3 Hz, 1H), 6.22 (s,
1H), 7.21 (s, 1H), 7.58-7.67 (m, 5H). APCI MS m/z 733
[C.sub.47H.sub.64N.sub.4O.sub.3+H].sup.+.
(iv) Preparation of 38:
(4aS,6aS,6bR,13aR)-12-Amino-2,2,6a,6b,9,9,13a-heptamethyl-15-(5-(pyrrolid-
in-1-ylmethyl)furan-3-yl)-2,3,4,4a,5,6,6a,6b,7,8,8a,9,11,13,13a,13b,14,15b-
-octadecahydro-1H-chryseno[1,2-f]indazole-4-a-carboxylic acid
[0473] A solution of 38d (200 mg, 0.27 mmol) and MeOH (15 mL) was
flushed with nitrogen and then 10% Pd/C (300 mg) was added. The
mixture was flushed with nitrogen followed by hydrogen. The mixture
was stirred under hydrogen at atmospheric pressure overnight. The
catalyst was removed by filtration through diatomaceous earth and
the filtrate was concentrated. The residue was purified by column
chromatography (silica, 0-80% CMA in CH.sub.2Cl.sub.2) followed by
preparative HPLC to afford the title compound (16 mg, 9%) as a
solid.
[0474] R.sub.f 0.30 (40:9:1 Methylene
Chloride/Methanol/concentrated Ammonium Hydroxide).
[0475] .sup.1H NMR (300 MHz, CD.sub.3OD) .delta. 0.62-2.19 (m,
46H), 2.41 (d, J=15.0 Hz, 2H), 3.53 (m, 2H), 4.45 (s, 2H), 6.64 (s,
1H), 7.57 (s, 1H). APCI MS m/z 643
[C.sub.40H.sub.58N.sub.4O.sub.3+H].sup.+. m.p. 220-240.degree. C.
dec. HPLC (Method A) 98.6% (214 nm) t.sub.R=12.3 min.
Example 39
##STR00200##
[0476] (i) Preparation of 39b: tert-Butyl
4-((4-bromofuran-2-yl)methyl)piperazine-1-carboxylate
[0477] To a solution of 4-bromofuran-2-carbaldehyde (500 mg, 2.85
mmol) and CH.sub.2Cl.sub.2 (10 mL) was added tert-butyl
piperazine-1-carboxylate (1.06 g, 5.74 mmol) and sodium
triacetoxyborohydride (1.2 g, 5.74 mmol). The mixture was stirred
at room temperature overnight. The resultant mixture was diluted
with EtOAc (100 mL), washed with brine then dried
(Na.sub.2SO.sub.4), filtered and concentrated under reduced
pressure. The residue was purified by column chromatography
(silica, 0-5% MeOH in CH.sub.2Cl.sub.2) to afford the sub-title
compound (900 mg, 91%).
[0478] .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 1.45 (s, 9H),
2.39-2.42 (m, 4H), 3.42-3.45 (m, 4H), 3.52 (s, 2H), 6.27 (s, 1H),
7.38 (s, 1H).
(ii) Preparation of 39c:
5-((4-(tert-Butoxycarbonyl)piperazin-1-yl)methyl)furan-3-ylboronic
acid
[0479] To a solution of 39b (900 mg, 2.60 mmol) and THF (10 mL) was
added n-butyllithium (1.35 mL, 3.39 mmol) at -78.degree. C. After
stifling for 10 min, triisopropylborate (2.99 mL, 13.04 mmol) was
added at -78.degree. C. The mixture was slowly warmed to room
temperature and quenched with phosphoric acid. The reaction mixture
was neutralized with 2 M NaOH solution and extracted with EtOAc
(3.times.10 mL). The extracts were dried (Na.sub.2SO.sub.4),
filtered and concentrated under reduced pressure. The residue was
purified by column chromatography (silica, 0-5% CMA in
CH.sub.2Cl.sub.2) to afford the sub-title compound (180 mg,
58%).
[0480] .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 1.44 (s, 9H), 3.44
(m, 4H), 3.56 (s, 2H), 3.73 (m, 4H), 6.39 (s, 1H), 7.87 (s,
1H).
(iii) Preparation of 39d: (4aS,6aS,6bR,13aR)-Benzyl
12-amino-15-(5-((4-(tert-butoxycarbonyl)piperazin-1-yl)methyl)furan-3-yl)-
-2,2,6a,6b,9,9,13a-heptamethyl-2,3,4,4a,5,6,6a,6b,7,8,8a,9,11,13,13a,13b,1-
4,15b-Octadecahydro-1H-chryseno[1,2-f]indazole-4-a-carboxylate
[0481] To a mixture of II (300 mg, 0.45 mmol) and 39c (420 mg, 1.35
mmol) in benzene (4 mL) and EtOH (1 mL) was added K.sub.2CO.sub.3
(249 mg, 1.80 mmol). The mixture was sparged with nitrogen and then
Pd(PPh.sub.3).sub.4 (105 mg, 0.09 mmol) was added. The reaction
mixture was heated at 120.degree. C. for 1 hour using microwave
irradiation. The solvent was removed under reduced pressure and the
residue was dissolved in EtOAc (20 mL). The solution was washed
with brine then dried (Na.sub.2SO.sub.4), filtered and
concentrated. The residue was purified by column chromatography
(silica, 0-10% MeOH in CH.sub.2Cl.sub.2) to afford the sub-title
compound (260 mg, 68%).
[0482] .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 0.57-2.41 (m,
65H), 3.42-3.48 (m, 6H), 5.04 (d, J=14.7 Hz, 1H), 5.14 (d, J=12.3
Hz, 1H), 6.18 (s, 1H), 7.21 (s, 1H), 7.33-7.35 (m, 5H). APCI MS m/z
848 [C.sub.52H.sub.73N.sub.5O.sub.5+H].sup.+.
(iv) Preparation of 39e: (4aS,6aS,6bR,13aR)-Benzyl
12-amino-2,2,6a,6b,9,9,13a-heptamethyl-15-(5-(piperazin-1-ylmethyl)furan--
3-yl)-2,3,4,4a,5,6,6a,6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecahydro-1H-ch-
ryseno[1,2-f]indazole-4-a-carboxylate
[0483] To a solution of 39d (260 mg, 0.30 mmol) and MeOH (2 mL) was
added HCl (1.0 M in diethyl ether, 10 mL, 10.0 mmol). After
stifling overnight at room temperature, the reaction mixture was
concentrated. Purification of the residue by column chromatography
(silica, 0-80% CMA in CH.sub.2Cl.sub.2) afforded the sub-title
compound (190 mg, 83%).
[0484] .sup.1H NMR (300 MHz, CD.sub.3OD) .delta. 0.57-2.37 (m,
42H), 2.44 (s, 4H), 2.80 (s, 4H), 3.43 (s, 2H), 4.98-5.03 (m, 1H),
5.15-5.21 (m, 1H), 6.20 (s, 1H), 7.21 (s, 1H), 7.36-7.37 (m, 5H).
APCI MS m/z 748 [C.sub.47H.sub.65N.sub.5O.sub.3+H].sup.+.
(v) Preparation of 39:
(4aS,6aS,6bR,13aR)-12-Amino-2,2,6a,6b,9,9,13a-heptamethyl-15-(5-(piperazi-
n-1-ylmethyl)furan-3-yl)-2,3,4,4a,5,6,6a,6b,7,8,8a,9,11,13,13a,13b,14,15b--
octadecahydro-1H-chryseno[1,2-f]indazole-4-a-carboxylic acid
[0485] A solution of 39e (190 mg, 0.25 mmol) and MeOH (10 mL) was
flushed with nitrogen and then 10% Pd/C (190 mg) was added. The
mixture was flushed with nitrogen followed by hydrogen. The mixture
was stirred under hydrogen at atmospheric pressure overnight. The
catalyst was removed by filtration through diatomaceous earth and
the filtrate was concentrated under reduced pressure. The residue
was purified by column chromatography (silica, 0-70% CMA in
CH.sub.2Cl.sub.2) followed by preparative HPLC to afford the title
compound (34 mg, 20%) as a solid.
[0486] R.sub.f 0.10 (32:17:1 Methylene
Chloride/Methanol/concentrated Ammonium Hydroxide).
[0487] .sup.1H NMR (300 MHz, CD.sub.3OD) .delta. 0.60-2.44 (m,
42H), 3.14 (s, 4H), 3.39 (s, 4H), 4.06 (s, 2H), 6.64 (s, 1H), 7.48
(s, 1H).
[0488] APCI MS m/z 658 [C.sub.40H.sub.59N.sub.5O.sub.3+H].sup.+.
m.p. 220-240.degree. C. dec. HPLC (Method A)>99% (214 nm)
t.sub.R=13.7 min.
Example 40
##STR00201##
[0489] (i) Preparation of 40b:
4-((4-Bromofuran-2-yl)methyl)morpholine
[0490] To a solution of 4-bromofuran-2-carbaldehyde (500 mg, 2.85
mmol) and morpholine (0.5 mL, 5.71 mmol) in CH.sub.2Cl.sub.2 (10
mL) was added sodium triacetoxyborohydride (1.2 g, 5.71 mmol). The
mixture was stirred at room temperature for 12 hours. The reaction
mixture was diluted with EtOAc (100 mL). The organic phase was
washed with aqueous NaHCO.sub.3 and brine then dried (MgSO.sub.4),
filtered and concentrated to dryness. The residue was purified by
column chromatography (silica, 0-10% MeOH in CH.sub.2Cl.sub.2) to
afford the sub-title compound (600 mg, 86%).
[0491] .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 2.42 (m, 4H), 3.62
(s, 2H), 3.72 (m, 4H), 6.27 (s, 1H), 7.32 (s, 1H).
(ii) Preparation of 40c: 5-(Morpholinomethyl)furan-3-ylboronic
acid
[0492] To a solution of 40b (300 mg, 1.20 mmol) in THF (6 mL) was
added n-butyllithium (2.5 M in hexanes, 0.64 mL, 1.60 mmol) at
-78.degree. C. The mixture was stirred at -78.degree. C. for 20
min. Triisopropyl borate (1.3 mL, 6.0 mmol) was added. The reaction
mixture was stirred at -78.degree. C. for 1 hour and quenched with
HCl (2 M, 4 mL). The reaction mixture was stirred for 5 min and
neutralized by NaOH (2 M) and extracted with EtOAc (100 mL). The
organic phase was washed with brine then dried (MgSO.sub.4),
filtered and concentrated to dryness. The residue was purified by
column chromatography (silica, 0-10% MeOH in CH.sub.2Cl.sub.2) to
afford the sub-title compound (120 mg, 47%).
[0493] .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 2.40 (s, 4H), 3.65
(s, 2H), 3.68 (m, 4H), 6.58 (s, 1H), 7.95 (s, 1H).
(iii) Preparation of 40d: (4aS,6aS,6bR,8aR,13aR,15bS)-Benzyl
12-amino-2,2,6a,6b,9,9,13a-heptamethyl-15-(5-(morpholinomethyl)furan-3-yl-
)-2,3,4,4a,5,6,6a,6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecahydro-1H-chryse-
no[1,2-f]indazole-4-a-carboxylate
[0494] A mixture of II (100 mg, 0.18 mmol), 40c (120 mg, 0.56
mmol), Pd(PPh.sub.3).sub.4 (20 mg, 0.018 mmol) and K.sub.2CO.sub.3
(100 mg, 0.72 mmol) in benzene (3.5 mL) and EtOH (1.5 mL) was
sealed and heated to 120.degree. C. by microwave for 1 hour. The
reaction mixture was diluted with EtOAc (100 mL). The organic phase
was washed with brine then dried (MgSO.sub.4), filtered and
concentrated to dryness. The residue was purified by column
chromatography (silica, 0-10% MeOH in CH.sub.2Cl.sub.2) to afford
the sub-title compound (120 mg, 89%).
[0495] APCI MS (Positive Mode) m/z 749
[C.sub.47H.sub.64N.sub.4O.sub.4+H].sup.+.
(iv) Preparation of 40:
(4aS,6aS,6bR,8aR,13aR,15bS)-12-Amino-2,2,6a,6b,9,9,13a-heptamethyl-15-(5--
(morpholinomethyl)furan-3-yl)-2,3,4,4a,5,6,6a,6b,7,8,8a,9,11,13,13a,13b,14-
,15b-octadecahydro-1H-chryseno[1,2-f]indazole-4-a-carboxylic
acid
[0496] A mixture of 40d (120 mg, 0.16 mmol) and 10% Pd(OH).sub.2/C
(40 mg) in MeOH (13 mL) and EtOAc (2 mL) was stirred under a
hydrogen balloon for 6 hours. The reaction mixture was filtered
through a pad of diatomaceous earth and washed with CMA (25 mL).
The filtrate was concentrated to dryness under reduced pressure.
The residue was purified by column chromatography (silica, 0-60%
CMA in CH.sub.2Cl.sub.2) followed by preparative HPLC to provide
the title compound (25 mg, 24%) as an off-white solid.
[0497] R.sub.f 0.25 (80:18:2 Methylene
Chloride/Methanol/concentrated Ammonium Hydroxide).
[0498] .sup.1H NMR (300 MHz, CD.sub.3OD) .delta. 0.60 (s, 3H), 0.84
(s, 3H), 0.89 (s, 3H), 0.94 (s, 3H), 1.22 (s, 3H), 1.25 (s, 3H),
1.30 (s, 3H), 1.35-2.28 (m, 20H), 2.38 (d, J=15.0 Hz, 1H), 3.85 (m,
3H), 4.44 (s, 2H), 6.86 (s, 1H), 7.60 (s, 1H). mp>300.degree. C.
APCI MS (Positive Mode) m/z 659
[C.sub.40H.sub.58N.sub.4O.sub.4+H].sup.+.
Example 41
##STR00202##
[0499] (i) Preparation of 41b: (4aS,6aS,6bR,8aR,13aR,15bS)-Benzyl
12-amino-15-(6-fluoropyridin-3-yl)-2,2,6a,6b,9,9,13a-heptamethyl-2,3,4,4a-
,5,6,6a,6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecahydro-1H-chryseno[1,2-f]i-
ndazole-4-a-carboxylate
[0500] A mixture of II (400 mg, 2.85 mmol),
6-fluoropyridin-3-ylboronic acid (400 mg, 2.85 mmol),
Pd(PPh.sub.3).sub.4 (105 mg, 0.090 mmol) and K.sub.2CO.sub.3 (600
mg, 4.34 mmol) in benzene (4.0 mL) and EtOH (1.0 mL) was sealed and
heated to 120.degree. C. by microwave for 1 hour. The reaction
mixture was diluted with EtOAc (100 mL). The organic phase was
washed with brine then dried (MgSO.sub.4), filtered and
concentrated to dryness. The residue was purified by column
chromatography (silica, 0-10% MeOH in CH.sub.2Cl.sub.2) to afford
the sub-title compound (500 mg, 81%) as a brown solid.
[0501] APCI MS (Positive Mode) m/z 679
[C.sub.43H.sub.55FN.sub.4O.sub.2+H].sup.+.
(ii) Preparation of 41:
(4aS,6aS,6bR,8aR,13aR,15bS)-12-Amino-15-(6-aminopyridin-3-yl)-2,2,6a,6b,9-
,9,13a-heptamethyl-2,3,4,4a,5,6,6a,6b,7,8,8a,9,11,13,13a,13b,14,15b-octade-
cahydro-1H-chryseno[1,2-f]indazole-4-a-carboxylic acid
[0502] A mixture of 41b (100 mg, 0.14 mmol) and hydrazine (0.5 mL)
in MeOH (4 mL) was sealed and heated to 140.degree. C. by microwave
for 5 hours. The mixture was concentrated to dryness. The residue
was dissolved in MeOH (20 mL), transferred to a Parr hydrogenation
flask and Raney Ni (70 mg) was added. The mixture was placed on a
Parr shaker under 40 psi of hydrogen for 12 hours. The mixture was
filtered through a pad of diatomaceous earth and the filter cake
washed with CMA. The filtrate was concentrated to dryness and the
residue dissolved in MeOH (10 mL), transferred to a Parr
hydrogenation flask and 10% Pd(OH).sub.2/C (70 mg) was added and
the mixture placed on a Parr Shaker under 30 psi of hydrogen for 12
hours. The reaction mixture was filtered through a pad of
diatomaceous earth and washed with CMA (25 mL). The filtrate was
concentrated to dryness under reduced pressure. The residue was
purified by column chromatography (silica, 0-60% CMA in
CH.sub.2Cl.sub.2) to afford the title compound (8 mg, 7%) as a
brown solid.
[0503] R.sub.f 0.30 (80:18:2 Methylene
Chloride/Methanol/concentrated Ammonium Hydroxide).
[0504] .sup.1H NMR (300 MHz, CD.sub.3OD) .delta. 0.50 (s, 3H), 0.82
(s, 3H), 0.90 (s, 3H), 0.98 (s, 3H), 1.22 (s, 3H), 1.31 (s, 6H),
1.35-2.30 (m, 21H), 6.61 (d, J=8.1 Hz, 1H), 7.47 (d, J=8.1 Hz, 1H),
7.80 (s, 1H). mp>300.degree. C. dec. APCI MS (Positive Mode) m/z
586 [C.sub.36H.sub.51N.sub.5O.sub.2+H].sup.+.
Example 42
##STR00203##
[0505] (i) Preparation of 42b: (4aS,6aS,6bR,8aR,13aR,15bS)-Benzyl
12-amino-15-(6-methoxypyridin-3-yl)-2,2,6a,6b,9,9,13a-heptamethyl-2,3,4,4-
a,5,6,6a,6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecahydro-1H-chryseno[1,2-f]-
indazole-4-a-carboxylate
[0506] A mixture of II (200 mg, 0.30 mmol),
6-methoxypyridin-3-ylboronic acid (138 mg, 0.90 mmol),
Pd(PPh.sub.3).sub.4 (34 mg, 0.030 mmol) and K.sub.2CO.sub.3 (208
mg, 1.50 mmol) in benzene (4.0 mL) and EtOH (1.0 mL) was sealed and
heated to 120.degree. C. by microwave for 1 hour. The reaction
mixture was diluted with EtOAc (100 mL). The organic phase was
washed with brine then dried (MgSO.sub.4), filtered and
concentrated to dryness. The residue was purified by column
chromatography (silica, 0-10% MeOH in CH.sub.2Cl.sub.2) to afford
the sub-title compound (200 mg, 96%) as a brown solid.
[0507] APCI MS (Positive Mode) m/z 691
[C.sub.44H.sub.58N.sub.4O.sub.3+H].sup.+.
(ii) Preparation of 42:
(4aS,6aS,6bR,8aR,13aR,15bS)-12-Amino-15-(6-methoxypyridin-3-yl)-2,2,6a,6b-
,9,9,13a-heptamethyl-2,3,4,4a,5,6,6a,6b,7,8,8a,9,11,13,13a,13b,14,15b-octa-
decahydro-1H-chryseno[1,2-f]indazole-4-a-carboxylic acid
[0508] To a solution of 42b (107 mg, 0.15 mmol) in CH.sub.2Cl.sub.2
(2 mL) was added boron tribromide (1 M in THF, 0.93 mL, 0.93 mmol)
at -78.degree. C. The mixture was warmed to 0.degree. C. for 6
hours. The reaction mixture was quenched with MeOH (2 mL) and
concentrated to dryness under reduced pressure. The residue was
purified by column chromatography (silica, 0-4% CMA in
CH.sub.2Cl.sub.2) to afford the title compound (34 mg, 38%).
[0509] R.sub.f 0.75 (80:18:2 Methylene
Chloride/Methanol/concentrated Ammonium Hydroxide).
[0510] .sup.1H NMR (300 MHz, CD.sub.3OD) .delta. 0.32 (s, 3H), 0.78
(s, 3H), 0.88 (s, 3H), 1.0 (s, 3H), 1.16 (s, 3H), 1.25 (s, 3H),
1.28 (s, 3H), 1.30-2.25 (m, 19H), 2.32 (d, J=14.7 Hz, 1H), 3.04 (m,
1H), 3.89 (s, 3H), 6.78 (d, J=8.7 Hz, 1H), 7.62 (d, J=10.2, 1H),
8.02 (s, 1H). mp>300.degree. C. APCI MS (Positive Mode) m/z 601
[C.sub.37H.sub.52N.sub.4O.sub.3+H].sup.+.
Example 43
##STR00204##
[0511] (i) Preparation of 43b: (4aS,6aS,6bR,8aR,13aR,15bS)-Benzyl
12-amino-2,2,6a,6b,9,9,13a-heptamethyl-15-(6-(methylamino)pyridin-3-yl)-2-
,3,4,4a,5,6,6a,6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecahydro-1H-chryseno[-
1,2-f]indazole-4-a-carboxylate
[0512] A mixture of 41b (100 mg, 0.14 mmol) and methylamine (1.5
mL, 1 M in THF, 1.5 mmol) in i-PrOH (2 mL) was sealed and heated to
160.degree. C. by microwave for 5 hours. The mixture was
concentrated to dryness. The residue was purified by column
chromatography (silica, 0-30% CMA in CH.sub.2Cl.sub.2) to afford
the sub-title compound (41 mg, 42%) as a brown solid.
[0513] APCI MS (Positive Mode) m/z 690
[C.sub.44H.sub.59N.sub.5O.sub.2+H].sup.+.
(ii) Preparation of 43:
(4aS,6aS,6bR,8aR,13aR,15bS)-12-Amino-2,2,6a,6b,9,9,13a-heptamethyl-15-(6--
(methylamino)pyridin-3-yl)-2,3,4,4a,5,6,6a,6b,7,8,8a,9,11,13,13a,13b,14,15-
b-octadecahydro-1H-chryseno[1,2-f]indazole-4-a-carboxylic acid
[0514] A mixture of 43b (32 mg, 0.046 mmol) and 10% Pd(OH).sub.2/C
(32 mg) in MeOH (20 mL) was stirred under a hydrogen balloon for 5
hours. The reaction mixture was filtered through a pad of
diatomaceous earth and washed with CMA (25 mL). The filtrate was
concentrated to dryness under reduced pressure. The residue was
purified by column chromatography (silica, 0-70% CMA in
CH.sub.2Cl.sub.2) followed by preparative HPLC to provide the title
compound (10 mg, 37%) as an off-white solid.
[0515] R.sub.f 0.60 (80:18:2 Methylene
Chloride/Methanol/concentrated Ammonium Hydroxide).
[0516] .sup.1H NMR (300 MHz, CD.sub.3OD) .delta. 0.50 (s, 3H), 0.82
(s, 3H), 0.90 (s, 3H), 0.98 (s, 3H), 1.22 (s, 3H), 1.28 (s, 3H),
1.31 (s, 3H), 1.35-2.30 (m, 21H), 3.02 (m, 1H), 3.03 (s, 3H), 7.05
(d, J=9.3 Hz, 1H), 7.83 (s, 1H), 7.90 (d, J=8.4 Hz, 1H).
mp>300.degree. C. APCI MS (Positive Mode) m/z 600
[C.sub.37H.sub.53N.sub.5O.sub.2+H].sup.+.
Example 44
##STR00205##
[0517] (i) Preparation of 44b: (4aS,6aS,6bR,8aR,13aR,15bS)-Benzyl
12-amino-15-(6-(dimethylamino)pyridin-3-yl)-2,2,6a,6b,9,9,13a-heptamethyl-
-2,3,4,4a,5,6,6a,6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecahydro-1H-chrysen-
o[1,2-f]indazole-4-a-carboxylate
[0518] A mixture of 41b (150 mg, 0.22 mmol) and dimethylamine (2.0
mL, 1 M in MeOH, 2.0 mmol) in MeOH (1 mL) was sealed and heated to
140.degree. C. by microwave for 7 hours. The mixture was
concentrated to dryness. The residue was purified by column
chromatography (silica, 0-10% MeOH in CH.sub.2Cl.sub.2) to afford
the sub-title compound (88 mg, 58%) as a brown solid.
[0519] APCI MS (Positive Mode) m/z 690
[C.sub.45H.sub.61N.sub.5O.sub.2+H].sup.+.
(ii) Preparation of 44:
(4aS,6aS,6bR,8aR,13aR,15bS)-12-Amino-15-(6-(dimethylamino)pyridin-3-yl)-2-
,2,6a,6b,9,9,13a-heptamethyl-2,3,4,4a,5,6,6a,6b,7,8,8a,9,11,13,13a,13b,14,-
15b-octadecahydro-1H-chryseno[1,2-f]indazole-4-a-carboxylic
acid
[0520] A mixture of 44b (88 mg, 0.12 mmol) and 10% Pd(OH).sub.2/C
(40 mg) in MeOH (15 mL) was stirred under a hydrogen balloon for 12
hours. The reaction mixture was filtered through a pad of
diatomaceous earth and washed with CMA (25 mL). The filtrate was
concentrated to dryness under reduced pressure. The residue was
purified by column chromatography (silica, 0-40% CMA in
CH.sub.2Cl.sub.2) to afford the title compound (35 mg, 26%) as an
off-white solid.
[0521] R.sub.f 0.75 (80:18:2 Methylene
Chloride/Methanol/concentrated Ammonium Hydroxide).
[0522] .sup.1H NMR (300 MHz, CD.sub.3OD) .delta. 0.38 (s, 3H), 0.78
(s, 3H), 0.87 (s, 3H), 0.99 (s, 3H), 1.15 (s, 3H), 1.25 (s, 3H),
1.26 (s, 3H), 1.35-2.30 (m, 19H), 2.33 (d, J=14.4 Hz, 1H), 3.06 (s,
6H), 3.07 (m, 1H), 6.66 (d, J=8.7 Hz, 1H), 7.50 (d, J=8.7 Hz, 1H),
7.94 (s, 1H). mp>300.degree. C. dec. APCI MS (Positive Mode) m/z
614 [C.sub.38H.sub.55N.sub.5O.sub.2+H].sup.+.
Example 45
##STR00206##
[0523] (i) Preparation of 45b: (4aS,6aS,6bR,13aR)-Benzyl
12-amino-2,2,6a,6b,9,9,13a-heptamethyl-15-(6-(pyrrolidin-1-yl)pyridin-3-y-
l)-2,3,4,4a,5,6,6a,6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecahydro-1H-chrys-
eno[1,2-f]indazole-4-a-carboxylate
[0524] To a solution of 41b (150 mg, 0.22 mmol) in MeOH (4 mL) was
added pyrrolidine (1 mL, 12.09 mmol). The mixture was heated at
140.degree. C. for 5 hours using microwave irradiation. The solvent
was removed under reduced pressure and the residue was dissolved in
EtOAc (20 mL). The solution was washed with brine then dried
(Na.sub.2SO.sub.4), filtered and concentrated to afford the
sub-title compound (162 mg, 100%) which was used without further
purification.
[0525] APCI MS m/z 730
[C.sub.47H.sub.63N.sub.5O.sub.2+H].sup.+.
(ii) Preparation of 45:
(4aS,6aS,6bR,13aR)-12-Amino-2,2,6a,6b,9,9,13a-heptamethyl-15-(6-(pyrrolid-
in-1-yl)pyridin-3-yl)-2,3,4,4a,5,6,6a,6b,7,8,8a,9,11,13,13a,13b,14,15b-oct-
adecahydro-1H-chryseno[1,2-f]indazole-4-a-carboxylic acid
[0526] A solution of 45b (162 mg, 0.22 mmol) and MeOH (10 mL) was
flushed with nitrogen and then 10% Pd/C (200 mg) was added. The
mixture was flushed with nitrogen followed by hydrogen. The mixture
was stirred under hydrogen at atmospheric pressure overnight. The
catalyst was removed by filtration through diatomaceous earth and
the filtrate was concentrated. The residue was purified by column
chromatography (silica, 0-40% CMA in CH.sub.2Cl.sub.2) to afford
the title compound (52 mg, 37%).
[0527] R.sub.f 0.48 (43:6:1 Methylene
Chloride/Methanol/concentrated Ammonium Hydroxide).
[0528] .sup.1H NMR (300 MHz, CD.sub.3OD) .delta. 0.39 (s, 3H), 0.78
(s, 3H), 0.87-2.27 (m, 38H), 2.38 (d, J=14.6 Hz, 1H), 3.08 (m, 1H),
3.43 (m, 4H), 6.50 (d, J=8.7 Hz, 1H), 7.51 (dd, J=2.1 Hz, 2.9 Hz,
1H), 7.90 (s, 1H). m.p. 280-300.degree. C. dec. APCI MS m/z 640
[C.sub.40H.sub.57N.sub.5O.sub.2+H].sup.+.
[0529] HPLC (Method A)>99% (214 nm) t.sub.R=12.2 min.
Example 46
##STR00207##
[0530] (i) Preparation of 46b: (4aS,6aS,6bR,13aR)-Benzyl
12-amino-15-(isoquinolin-5-yl)-2,2,6a,6b,9,9,13a-heptamethyl-2,3,4,4a,5,6-
,6a,6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecahydro-1H-chryseno[1,2-f]indaz-
ole-4-a-carboxylate
[0531] To a mixture of II (200 mg, 0.30 mmol) and
isoquinolin-5-ylboronic acid (156 mg, 0.90 mmol) in benzene (4 mL)
and EtOH (1 mL) was added K.sub.2CO.sub.3 (166 mg, 1.20 mmol). The
mixture was sparged with nitrogen and then Pd(PPh.sub.3).sub.4 (70
mg, 0.06 mmol) was added. The reaction mixture was heated at
120.degree. C. for 1 hour using microwave irradiation and then
concentrated. The residue was dissolved in EtOAc (20 mL) and washed
with brine. The organic solution was dried (Na.sub.2SO.sub.4),
filtered and concentrated under reduced pressure. The residue was
purified by column chromatography (silica, 0-10% MeOH in
CH.sub.2Cl.sub.2) to afford the sub-title compound (180 mg,
84%).
[0532] APCI MS m/z 711
[C.sub.47H.sub.58N.sub.4O.sub.2+H].sup.+.
(ii) Preparation of 46:
(4aS,6aS,6bR,13aR)-12-Amino-15-(isoquinolin-5-yl)-2,2,6a,6b,9,9,13a-hepta-
methyl-2,3,4,4a,5,6,6a,6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecahydro-1H-c-
hryseno[1,2-f]indazole-4-a-carboxylic acid
[0533] A solution of 46b (180 mg, 0.25 mmol) and MeOH (15 mL) was
flushed with nitrogen and then 10% Pd/C (220 mg) was added. The
mixture was flushed with nitrogen followed by hydrogen. The mixture
was stirred under hydrogen at atmospheric pressure overnight. The
catalyst was removed by filtration through diatomaceous earth and
the filtrate was concentrated under reduced pressure. The residue
was purified by column chromatography (silica, 0-80% CMA in
CH.sub.2Cl.sub.2) followed by preparative HPLC to afford the title
compound (13 mg, 8%) as a solid.
[0534] R.sub.f 0.73 (40:9:1 Methylene
Chloride/Methanol/concentrated Ammonium Hydroxide).
[0535] .sup.1H NMR (300 MHz, CD.sub.3OD) .delta. -0.17-2.33 (m,
41H), 2.95 (d, J=11.1 Hz, 1H), 7.94-7.99 (m, 1H), 8.13 (d, J=6.3
Hz, 1H), 8.19 (d, J=7.2 Hz, 1H), 8.32 (d, J=8.4 Hz, 1H), 8.54 (d,
J=6.3 Hz, 1H), 9.61 (s, 1H). APCI MS m/z 621
[C.sub.40H.sub.52N.sub.4O.sub.2+H].sup.+. m.p.>300.degree. C.
HPLC (Method A) 93.4% (214 nm) t.sub.R=11.6 min.
Example 47
##STR00208##
[0536] (i) Preparation of 47b: (4aS,6aS,6bR,13aR)-Benzyl
12-amino-2,2,6a,6b,9,9,13a-heptamethyl-15-(1,2,3,4-tetrahydroisoquinolin--
5-yl)-2,3,4,4a,5,6,6a,6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecahydro-1H-ch-
ryseno[1,2-f]indazole-4-a-carboxylate
[0537] To a solution of 46b (150 mg, 0.21 mmol) and THF (5 mL) was
added lithium triethylborohydride (1.26 mL, 1.26 mmol, 1.0 M in
THF). The mixture was heated at 60.degree. C. overnight and then
cooled to room temperature. The mixture was taken up in EtOAc (40
mL) and washed with brine (3.times.15 mL). The organic layer was
dried (Na.sub.2SO.sub.4), filtered and concentrated. The residue
was purified by column chromatography (silica, 0-80% CMA in
CH.sub.2Cl.sub.2) to afford the sub-title compound (40 mg,
26%).
[0538] APCI MS m/z 715
[C.sub.47H.sub.62N.sub.4O.sub.2+H].sup.+.
(ii) Preparation of 47:
(4aS,6aS,6bR,13aR)-12-Amino-2,2,6a,6b,9,9,13a-heptamethyl-15-(1,2,3,4-tet-
rahydroisoquinolin-5-yl)-2,3,4,4a,5,6,6a,6b,7,8,8a,9,11,13,13a,13b,14,15b--
octadecahydro-1H-chryseno[1,2-f]indazole-4-a-carboxylic acid
[0539] A solution of 47b (40 mg, 0.055 mmol) and MeOH (10 mL) was
flushed with nitrogen and then 10% Pd/C (50 mg) was added. The
mixture was flushed with nitrogen followed by hydrogen. The mixture
was stirred under hydrogen at atmospheric pressure overnight. The
catalyst was removed by filtration through diatomaceous earth and
the filtrate was concentrated. The residue was purified by column
chromatography (silica, 0-80% CMA in CH.sub.2Cl.sub.2) to afford
the title compound (19 mg, 56%).
[0540] R.sub.f 0.28 (40:9:1 Methylene
Chloride/Methanol/concentrated Ammonium Hydroxide).
[0541] .sup.1H NMR (400 MHz, CD.sub.3OD) .delta. 0.24 (s, 3H),
0.75-2.36 (m, 38H), 2.86-3.44 (m, 5H), 4.10-4.20 (m, 2H), 6.98 (d,
J=7.42 Hz, 1H), 7.19 (d, J=7.42 Hz, 1H), 7.40 (d, J=7.42 Hz, 1H).
APCI MS m/z 625 [C.sub.40H.sub.56N.sub.4O.sub.2+H].sup.+.
m.p.>300.degree. C. dec. HPLC (Method A) 99.1% (214 nm)
t.sub.R=12.4 min
Example 48
##STR00209##
[0542] (i) Preparation of 48b: (4aS,6aS,6bR,13aR)-Benzyl
12-amino-15-(1H-indol-4-yl)-2,2,6a,6b,9,9,13a-heptamethyl-2,3,4,4a,5,6,6a-
,6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecahydro-1H-chryseno[1,2-f]indazole-
-4-a-carboxylate
[0543] To a mixture of II (200 mg, 0.302 mmol) and
1H-indol-4-ylboronic acid (146 mg, 0.906 mmol) in toluene (4.5 mL)
and H.sub.2O (0.5 mL) was added cesium carbonate (393 mg, 1.208
mmol). The mixture was sparged with nitrogen and then
Pd(PPh.sub.3).sub.4 (70 mg, 0.060 mmol) was added. The reaction
mixture was heated at 120.degree. C. for 1 hour using microwave
irradiation. The solvent was concentrated under reduced pressure.
The residue was taken up in EtOAc (20 mL) and washed with brine
(3.times.15 mL). The organic layer was dried (Na.sub.2SO.sub.4),
filtered and concentrated. The residue was purified by column
chromatography (silica, 0-10% MeOH in CH.sub.2Cl.sub.2) to afford
the sub-title compound (180 mg, 85%).
[0544] APCI MS m/z 699
[C.sub.46H.sub.58N.sub.4O.sub.2+H].sup.+.
(ii) Preparation of 48:
(4aS,6aS,6bR,13aR)-12-Amino-15-(1H-indol-4-yl)-2,2,6a,6b,9,9,13a-heptamet-
hyl-2,3,4,4a,5,6,6a,6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecahydro-1H-chry-
seno[1,2-f]indazole-4-a-carboxylic acid
[0545] A solution of 48b (180 mg, 0.26 mmol), EtOAc (2 mL) and MeOH
(8 mL) was flushed with nitrogen and then 10% Pd/C (280 mg) was
added. The mixture was flushed with nitrogen followed by hydrogen.
The mixture was stirred under hydrogen at atmospheric pressure
overnight. The catalyst was removed by filtration through
diatomaceous earth and the filtrate was concentrated. The residue
was purified by column chromatography (silica, 0-10% MeOH in
CH.sub.2Cl.sub.2) followed by preparative HPLC to afford the title
compound (29 mg, 18%) as a solid.
[0546] R.sub.f 0.30 (90:9:1 Methylene
Chloride/Methanol/concentrated Ammonium Hydroxide).
[0547] .sup.1H NMR (400 MHz, CD.sub.3OD) .delta. -0.08 (s, 3H),
0.22 (s, 3H), 0.54 (s, 3H), 0.78-2.56 (m, 32H), 6.28 (s, 1H),
7.01-7.24 (m, 5H). APCI MS m/z 609
[C.sub.39H.sub.52N.sub.4O.sub.2+H].sup.+. m.p. 280-300.degree. C.
dec. HPLC (Method A) 99.3% (214 nm) t.sub.R=16.6 min
Example 49
##STR00210##
[0548] (i) Preparation of 49b: (4aS,6aS,6bR,13aR)-Benzyl
12-amino-15-(1H-indol-5-yl)-2,2,6a,6b,9,9,13a-heptamethyl-2,3,4,4a,5,6,6a-
,6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecahydro-1H-chryseno[1,2-f]indazole-
-4-a-carboxylate
[0549] To a mixture of II (200 mg, 0.302 mmol) and
1H-indol-5-ylboronic acid (146 mg, 0.91 mmol) in toluene (4.5 mL)
and H.sub.2O (0.5 mL) was added cesium carbonate (393 mg, 1.21
mmol). The mixture was sparged with nitrogen and then
Pd(PPh.sub.3).sub.4 (70 mg, 0.06 mmol) was added. The reaction
mixture was heated at 120.degree. C. for 1 hour using microwave
irradiation. The solvent was concentrated under reduced pressure.
The residue was taken up in EtOAc (20 mL) and washed with brine
(3.times.15 mL). The organic layer was dried (Na.sub.2SO.sub.4),
filtered and concentrated. The residue was purified by column
chromatography (silica, 0-10% CMA in CH.sub.2Cl.sub.2) to afford
the sub-title compound (178 mg, 84%).
[0550] APCI MS m/z 699
[C.sub.46H.sub.58N.sub.4O.sub.2+H].sup.+.
(ii) Preparation of 49:
(4aS,6aS,6bR,13aR)-12-Amino-15-(1H-indol-5-yl)-2,2,6a,6b,9,9,13a-heptamet-
hyl-2,3,4,4a,5,6,6a,6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecahydro-1H-chry-
seno[1,2-f]indazole-4-a-carboxylic acid
[0551] A solution of 49b (175 mg, 0.25 mmol), EtOAc (5 mL) and MeOH
(10 mL) was flushed with nitrogen and then 10% Pd/C (175 mg) was
added. The mixture was flushed with nitrogen followed by hydrogen.
The mixture was stirred under hydrogen at atmospheric pressure
overnight. The catalyst was removed by filtration through
diatomaceous earth and the filtrate was concentrated. The residue
was purified by column chromatography (silica, 0-6% MeOH in
CH.sub.2Cl.sub.2) followed by preparative HPLC to afford the title
compound (18 mg, 12%) as a solid.
[0552] R.sub.f 0.30 (90:9:1 Methylene
Chloride/Methanol/concentrated Ammonium Hydroxide).
[0553] .sup.1H NMR (400 MHz, CD.sub.3OD) .delta. 0.10 (s, 3H), 0.70
(s, 3H), 0.89-2.43 (m, 35H), 3.21-3.25 (m, 1H), 6.38-6.39 (m, 1H),
7.04 (s, 1H), 7.18 (s, 1H), 7.32 (d, J=8.26 Hz, 1H), 7.39 (s, 1H).
APCI MS m/z 609 [C.sub.39H.sub.52N.sub.4O.sub.2+H].sup.+. m.p.
230-250.degree. C. dec. HPLC (Method A) 96.2% (214 nm) t.sub.R=17.1
min
Example 50
##STR00211##
[0554] (i) Preparation of 50b: tert-Butyl
4-(4-bromophenyl)piperazine-1-carboxylate
[0555] To a slurry of 1-(4-bromophenyl)piperazine hydrochloride
(3.0 g, 10.8 mmol), DMAP (135 mg, 1.1 mmol) and triethylamine (4.5
mL, 32.4 mmol) in CH.sub.3CN (25 mL) was added di-tert-butyl
dicarbonate (2.6 g, 11.9 mmol). The mixture was stirred at room
temperature overnight. Water (25 mL) was added to the resultant
mixture and stirred for 30 min. The resultant solid was collected
by filtration. The solid was washed with H.sub.2O and dried in a
vacuum oven at 40.degree. C. to afford the sub-title compound (3.2
g, 87%).
[0556] .sup.1H NMR ((300 MHz, DMSO-d.sub.6) .delta. 1.48 (s, 9H),
3.15 (m, 4H), 3.54 (m, 4H), 6.95 (d, J=9.0 Hz, 2H), 7.37 (d, J=9.0
Hz, 2H).
(ii) Preparation of 50c:
4-(4-(tert-Butoxycarbonyl)piperazin-1-yl)phenylboronic acid
[0557] To a solution of 50b (500 mg, 1.46 mmol) and THF (10 mL) was
added n-butyllithium (0.76 mL of 2.5 M in hexanes, 1.90 mmol) at
-78.degree. C. After stirring for 30 min, triisopropylborate (1.68
mL, 7.33 mmol) was added at -78.degree. C. The mixture was slowly
warmed to room temperature and quenched with phosphoric acid. The
reaction mixture was neutralized with saturated NaHCO.sub.3 and
extracted with EtOAc (10 mL.times.3). The extracts were dried
(Na.sub.2SO.sub.4), filtered and concentrated under reduced
pressure. The residue was purified by column chromatography
(silica, 0-10% MeOH in CH.sub.2Cl.sub.2) to afford the sub-title
compound (180 mg, 58%).
[0558] .sup.1H NMR (300 MHz, CD.sub.3OD) .delta. -0.50 (s, 9H),
0.93-0.96 (m, 4H), 1.15-1.19 (m, 4H), 4.86-4.89 (m, 4H).
(iii) Preparation of 50d: (4aS,6aS,6bR,13aR)-Benzyl
12-amino-15-(4-(4-(tert-butoxycarbonyl)piperazin-1-yl)phenyl)-2,2,6a,6b,9-
,9,13a-heptamethyl-2,3,4,4a,5,6,6a,6b,7,8,8a,9,11,13,13a,13b,14,15b-octade-
cahydro-1H-chryseno[1,2-f]indazole-4-a-carboxylate
[0559] To a mixture of II (130 mg, 0.19 mmol) and 50c (180 mg, 0.58
mmol) in benzene (4 mL) and EtOH (1 mL) was added K.sub.2CO.sub.3
(108 mg, 0.78 mmol). The mixture was sparged with nitrogen and then
Pd(PPh.sub.3).sub.4 (45 mg, 0.06 mmol) was added. The reaction
mixture was heated at 120.degree. C. for 1 hour using microwave
irradiation and then concentrated under reduced pressure. The
residue was dissolved in EtOAc (20 mL) and the solution was washed
with brine then dried (Na.sub.2SO.sub.4), filtered and
concentrated. The residue was purified by column chromatography
(silica, 0-5% MeOH in CH.sub.2Cl.sub.2) to afford the sub-title
compound (100 mg, 60%).
[0560] .sup.1H NMR (300 MHz, CD.sub.3OD) .delta. 0.28-3.59 (m,
59H), 5.18 (d, 2H), 6.70-7.07 (m, 9H).
[0561] APCI MS m/z 844
[C.sub.53H.sub.73N.sub.5O.sub.4+H].sup.+.
(iv) Preparation of 50e: (4aS,6aS,6bR,13aR)-Benzyl
12-amino-2,2,6a,6b,9,9,13a-heptamethyl-15-(4-(piperazin-1-yl)phenyl)-2,3,-
4,4a,5,6,6a,6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecahydro-1H-chryseno[1,2-
-f]indazole-4-a-carboxylate
[0562] To a solution of 50d (220 mg, 0.26 mmol) in MeOH (3 mL) was
added HCl (1.0 M in diethyl ether, 6 mL, 6.0 mmol). The mixture was
stirred at room temperature for 62 hours and then concentrated
under reduced pressure. The residue was purified by column
chromatography (silica, 0-50% CMA in CH.sub.2Cl.sub.2) to afford
the sub-title compound (65 mg, 33%).
[0563] .sup.1H NMR (300 MHz, CD.sub.3OD) .delta. 0.27-2.34 (m,
42H), 2.98-3.06 (m, 8H), 5.02 (d, J=12.3 Hz, 1H), 5.22 (d, J=12.0
Hz, 1H), 6.78 (d, J=8.7 Hz, 2H), 6.99 (d, J=8.7 Hz, 2H), 7.39 (m,
5H). APCI MS m/z 744 [C.sub.48H.sub.65N.sub.5O.sub.2+H].sup.+.
(v) Preparation of 50:
(4aS,6aS,6bR,13aR)-12-Amino-2,2,6a,6b,9,9,13a-heptamethyl-15-(4-(piperazi-
n-1-yl)phenyl)-2,3,4,4a,5,6,6a,6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecahy-
dro-1H-chryseno[1,2-f]indazole-4-a-carboxylic acid
[0564] A solution of 50e (65 mg, 0.087 mmol) and MeOH (15 mL) was
flushed with nitrogen and then 10% Pd/C (90 mg) was added. The
mixture was flushed with nitrogen followed by hydrogen. The mixture
was stirred under hydrogen at atmospheric pressure overnight. The
catalyst was removed by filtration through diatomaceous earth and
the filtrate was concentrated. The residue was purified by column
chromatography (silica, 0-70% CMA in CH.sub.2Cl.sub.2) followed by
preparative HPLC to afford the title compound (11 mg, 20%) as a
solid.
[0565] R.sub.f 0.10 (40:9:1 Methylene
Chloride/Methanol/concentrated Ammonium Hydroxide).
[0566] .sup.1H NMR (300 MHz, CD.sub.3OD) .delta. 0.28-2.42 (m,
41H), 3.10 (d, J=10.2 Hz, 1H), 3.37 (m, 8H), 7.03 (d, J=9.0 Hz,
2H), 7.24 (d, J=7.8 Hz, 2H). APCI MS m/z 654
[C.sub.41H.sub.59N.sub.5O.sub.2+H].sup.+. m.p. 270-290.degree. C.
dec. HPLC (Method A)>99% (214 nm) t.sub.R=13.2 min.
Example 51
##STR00212## ##STR00213##
[0567] (i) Preparation of 51b: (4aS,6aS,6bR,12aR)-Benzyl
11-cyano-2,2,6a,6b,9,9,12a-heptamethyl-14-(4-morpholinophenyl)-10-oxo-1,2-
,3,4,4a,5,6,6a,6b,7,8,8a,9,10,11,12,12a,12b,13,14b-icosahydropicene-4-a-ca-
rboxylate
[0568] To a mixture of IIj (300 mg, 0.46 mmol) and
4-morpholinophenylboronic acid (575 mg, 2.78 mmol) in benzene (10
mL) and EtOH (5 mL) was added K.sub.2CO.sub.3 (511 mg, 3.70 mmol).
The mixture was sparged with nitrogen and then Pd(PPh.sub.3).sub.4
(107 mg, 0.09 mmol) was added. The mixture was heated at 85.degree.
C. overnight and concentrated under reduced pressure. The residue
was dissolved in EtOAc (50 mL) and the solution was washed with
brine then dried (Na.sub.2SO.sub.4), filtered and concentrated. The
residue was purified by column chromatography (silica, 0-30% EtOAc
in hexanes) to afford the sub-title compound (200 mg, 59%).
[0569] .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 0.28-2.29 (m,
43H), 3.09-3.10 (m, 4H), 3.85-3.86 (m, 4H), 5.05 (m, 1H), 5.17 (m,
1H), 6.75-6.78 (m, 2H), 7.02-7.06 (m, 2H), 7.25-7.36 (m, 5H).
(ii) Preparation of 51c: (4aS,6aS,6bR,13aR)-Benzyl
12-amino-2,2,6a,6b,9,9,13a-heptamethyl-15-(4-morpholinophenyl)-2,3,4,4a,5-
,6,6a,6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecahydro-1H-chryseno[1,2-f]ind-
azole-4-a-carboxylate
[0570] To a solution of 51b (196 mg, 0.26 mmol) and EtOH (5 mL) was
added hydrazine (42 .mu.L, 1.34 mmol). The mixture was heated at
90.degree. C. for 60 hours and then concentrated under reduced
pressure. The residue was purified by column chromatography
(silica, 0-20% CMA in CH.sub.2Cl.sub.2) to provide the sub-title
compound (109 mg, 55%).
[0571] .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 0.30-2.24 (m,
43H), 3.07-3.12 (m, 4H), 3.40-3.47 (m, 2H), 3.85-3.88 (m, 4H), 5.10
(d, J=12.3 Hz, 1H), 5.22 (d, J=12.6 Hz, 1H), 6.78 (d, J=8.7 Hz,
1H), 7.06 (d, J=8.4 Hz, 1H), 7.36-7.39 (m, 5H). APCI MS m/z 745
[C.sub.48H.sub.64N.sub.4O.sub.3+H].sup.+.
(iii) Preparation of 51:
(4aS,6aS,6bR,13aR)-12-Amino-2,2,6a,6b,9,9,13a-heptamethyl-15-(4-morpholin-
ophenyl)-2,3,4,4a,5,6,6a,6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecahydro-1H-
-chryseno[1,2-f]indazole-4-a-carboxylic acid
[0572] A solution of 51c (108 mg, 0.14 mmol) and MeOH (15 mL) was
flushed with nitrogen and then 10% Pd/C (55 mg) was added. The
mixture was flushed with nitrogen followed by hydrogen. The mixture
was stirred under hydrogen at atmospheric pressure overnight. The
catalyst was removed by filtration through diatomaceous earth and
the filtrate was concentrated. The residue was purified by column
chromatography (silica, 0-10% MeOH in CH.sub.2Cl.sub.2) to afford
the title compound (26 mg, 27%).
[0573] R.sub.f 0.35 (90:9:1 Methylene
Chloride/Methanol/concentrated Ammonium Hydroxide).
[0574] .sup.1H NMR (300 MHz, CD.sub.3OD) .delta. 0.30-2.37 (m,
42H), 3.10 (m, 4H), 3.84 (m, 4H), 6.95 (d, J=7.8 Hz, 2H), 7.16 (d,
J=7.5 Hz, 2H). APCI MS m/z 655
[C.sub.41H.sub.58N.sub.4O.sub.3+H].sup.+. m.p. 250-270.degree. C.
dec. HPLC (Method A)>99% (214 nm) t.sub.R=13.3 min.
Example 52
##STR00214## ##STR00215##
[0575] (i) Preparation of 52b: (4aS,6aS,6bR,13aR)-Benzyl
12-amino-15-(4-formylphenyl)-2,2,6a,6b,9,9,13a-heptamethyl-2,3,4,4a,5,6,6-
a,6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecahydro-1H-chryseno[1,2-f]indazol-
e-4-a-carboxylate
[0576] To a solution of II (250 mg, 0.37 mmol),
4-formylphenylboronic acid (170 mg, 1.13 mmol), benzene (4 mL) and
EtOH (1 mL) was added K.sub.2CO.sub.3 (208 mg, 1.50 mmol). The
mixture was sparged with nitrogen and then Pd(PPh.sub.3).sub.4 (87
mg, 0.07 mmol) was added. The reaction mixture was heated at
120.degree. C. for 1 hour using microwave irradiation and then
concentrated under reduced pressure. The residue was dissolved in
EtOAc (20 mL) and the solution was washed with brine. The organic
layer was dried (Na.sub.2SO.sub.4), filtered and concentrated. The
residue was purified by column chromatography (silica, 0-10% MeOH
in CH.sub.2Cl.sub.2) to afford the sub-title compound (224 mg,
86%).
[0577] APCI MS m/z 688
[C.sub.45H.sub.57N.sub.3O.sub.3+H].sup.+.
(ii) Preparation of 52c: (4aS,6aS,6bR,13aR)-Benzyl
12-amino-2,2,6a,6b,9,9,13a-heptamethyl-15-(4-(morpholinomethyl)phenyl)-2,-
3,4,4a,5,6,6a,6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecahydro-1H-chryseno[1-
,2-f]indazole-4-a-carboxylate
[0578] To a solution of 52b (220 mg, 0.32 mmol), morpholine (56
.mu.L, 0.64 mmol) and CH.sub.2Cl.sub.2 (10 mL) was added sodium
triacetoxyborohydride (136 mg, 0.64 mmol). The mixture was stirred
at room temperature overnight. The resultant mixture was diluted
with EtOAc (100 mL) and the solution was washed with brine then
dried (Na.sub.2SO.sub.4), filtered and concentrated under reduced
pressure. The residue was purified by column chromatography
(silica, 0-20% MeOH in CH.sub.2Cl.sub.2) to afford the sub-title
compound (80 mg, 33%).
[0579] .sup.1H NMR (300 MHz, CD.sub.3OD) .delta. 0.28-2.24 (m,
41H), 2.39-2.42 (m, 4H), 3.03 (m, 1H), 3.45 (s, 2H), 3.69-3.70 (m,
4H), 5.03 (d, J=12.6 Hz, 1H), 5.23 (d, J=12.3 Hz, 1H), 7.15-7.18
(m, 4H), 7.37 (s, 5H).
(iii) Preparation of 52:
(4aS,6aS,6bR,13aR)-12-Amino-2,2,6a,6b,9,9,13a-heptamethyl-15-(4-(morpholi-
nomethyl)phenyl)-2,3,4,4a,5,6,6a,6b,7,8,8a,9,11,13,13a,13b,14,15b-octadeca-
hydro-1H-chryseno[1,2-f]indazole-4-a-carboxylic acid
[0580] A solution of 52c (75 mg, 0.09 mmol) and MeOH (10 mL) was
flushed with nitrogen and then 10% Pd/C (100 mg) was added. The
mixture was flushed with nitrogen followed by hydrogen. The mixture
was stirred under hydrogen at atmospheric pressure overnight. The
catalyst was removed by filtration through diatomaceous earth and
the filtrate was concentrated. The residue was purified by
preparative HPLC to afford the title compound (8 mg, 12%).
[0581] R.sub.f 0.75 (40:9:1 Methylene
Chloride/Methanol/concentrated Ammonium Hydroxide).
[0582] .sup.1H NMR (300 MHz, CD.sub.3OD) .delta. 0.25-2.39 (m,
45H), 2.99 (m, 1H), 3.73 (s, 2H), 4.02 (s, 2H), 4.38 (s, 2H),
7.47-7.52 (m, 4H). APCI MS m/z 669
[C.sub.42H.sub.60N.sub.4O.sub.3+H].sup.+. m.p.>300.degree. C.
dec. HPLC (Method A)>99% (214 nm) t.sub.R=12.1 min.
Example 53
##STR00216##
[0583] (i) Preparation of 53b:
1-(4-Bromophenyl)-4-methylpiperazine
[0584] A mixture of 1-(4-bromophenyl)piperazine hydrochloride (1.0
g, 3.61 mmol), formaldehyde (37% aqueous, 3 mL, 39.71 mmol), AcOH
(0.23 mL, 3.97 mmol) and MeOH (30 mL) was heated at 80.degree. C.
under nitrogen. After 1.5 hours, the mixture was cooled to
0.degree. C. in an ice bath. Methylene chloride (5 mL) was added
followed by a slow addition of sodium borohydride (1.91 g, 50.54
mmol) under nitrogen. The mixture was stirred for 1 hour and then
poured into a seperatory funnel containing a saturated solution of
NH.sub.4Cl (25 mL) and CH.sub.2Cl.sub.2 (50 mL). The layers were
separated and the aqueous layer was extracted with CH.sub.2Cl.sub.2
(2.times.50 mL). The extracts were dried (Na.sub.2SO.sub.4),
filtered and concentrated to to give the sub-title compound (900
mg, 97%) which was used without further purification.
[0585] .sup.1H NMR ((300 MHz, CDCl.sub.3) .delta. 2.34 (s, 3H),
2.55 (m, 4H), 3.17 (m, 4H), 6.79 (d, J=9.0 Hz, 2H), 7.32 (d, J=9.0
Hz, 2H).
(ii) Preparation of 53c: 4-(4-Methylpiperazin-1-yl)phenylboronic
acid
[0586] To a solution of 53b (390 mg, 1.52 mmol) and THF (10 mL) was
added n-butyllithium (0.79 mL of 2.5 M in hexanes, 1.98 mmol) at
-78.degree. C. and stirred for 10 min. Triisopropylborate (1.75 mL,
7.64 mmol) was added at -78.degree. C. The mixture was slowly
warmed to room temperature and then quenched with phosphoric acid.
The reaction mixture was neutralized with saturated NaHCO.sub.3
solution and extracted with EtOAc (3.times.10 mL). The solution was
dried (Na.sub.2SO.sub.4), filtered and concentrated under reduced
pressure. The residue was purified by column chromatography
(silica, 0-10% MeOH in CH.sub.2Cl.sub.2) to afford the sub-title
compound (180 mg, 58%).
[0587] .sup.1H NMR ((300 MHz, CD.sub.3OD) .delta. 2.32 (s, 3H),
2.55 (m, 4H), 3.03 (s, 1H), 3.21 (m, 3H), 6.69-7.59 (m, 4H).
(iii) Preparation of 53d: (4aS,6aS,6bR,13aR)-Benzyl
12-amino-2,2,6a,6b,9,9,13a-heptamethyl-15-(4-(4-methylpiperazin-1-yl)phen-
yl)-2,3,4,4a,5,6,6a,6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecahydro-1H-chry-
seno[1,2-f]indazole-4-a-carboxylate
[0588] To a mixture of II (160 mg, 0.24 mmol) and 53c (159 mg, 0.72
mmol) in benzene (4 mL) and EtOH (1 mL) was added K.sub.2CO.sub.3
(133 mg, 0.96 mmol). The mixture was sparged with nitrogen and then
Pd(PPh.sub.3).sub.4 (55 mg, 0.04 mmol) was added. The reaction
mixture was heated at 120.degree. C. for 1 hour using microwave
irradiation and then concentrated. The residue was dissolved in
EtOAc (20 mL) and the solution was washed with brine then dried
(Na.sub.2SO.sub.4), filtered and concentrated under reduced
pressure. The residue was purified by column chromatography
(silica, 0-10% MeOH in CH.sub.2Cl.sub.2) to afford the sub-title
compound (80 mg, 44%).
[0589] .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 0.29-3.46 (m,
53H), 5.06 (d, J=12.6 Hz, 1H), 5.22 (d, J=12.6 Hz, 1H), 6.80 (d,
J=8.4 Hz, 2H), 7.05 (d, J=8.7 Hz, 2H), 7.35-7.44 (m, 5H). APCI MS
m/z 758 [C.sub.49H.sub.67N.sub.5O.sub.2+H].sup.+.
(iv) Preparation of 53:
(4aS,6aS,6bR,13aR)-12-Amino-2,2,6a,6b,9,9,13a-heptamethyl-15-(4-(4-methyl-
piperazin-1-yl)phenyl)-2,3,4,4a,5,6,6a,6b,7,8,8a,9,11,13,13a,13b,14,15b-oc-
tadecahydro-1H-chryseno[1,2-f]indazole-4-a-carboxylic acid
[0590] A solution of 53d (75 mg, 0.09 mmol) and MeOH (10 mL) was
flushed with nitrogen and then 10% Pd/C (70 mg) was added. The
mixture was flushed with nitrogen followed by hydrogen. The mixture
was stirred under hydrogen at atmospheric pressure overnight. The
catalyst was removed by filtration through diatomaceous earth and
the filtrate was concentrated. The residue was purified by column
chromatography (silica, 0-40% CMA in CH.sub.2Cl.sub.2) followed by
preparative HPLC to afford the title compound (6 mg, 5%) as a
solid.
[0591] R.sub.f 0.37 (40:9:1 Methylene
Chloride/Methanol/concentrated Ammonium Hydroxide).
[0592] .sup.1H NMR (300 MHz, CD.sub.3OD) .delta. 0.28-2.42 (m,
34H), 2.97 (s, 3H), 3.12-3.77 (m, 8H), 3.37 (s, 8H), 7.02 (d, J=8.7
Hz, 2H), 7.24 (d, J=8.1 Hz, 2H). APCI MS m/z 668
[C.sub.42H.sub.61N.sub.5O.sub.2+H].sup.+. m.p.>300.degree. C.
dec. HPLC (Method A)>99% (214 nm) t.sub.R=13.9 min.
Example 54
##STR00217##
[0593] (i) Preparation of 54:
(4aS,6aS,6bR,8aR,13aR,15bS)-12-Amino-2,2,6a,6b,9,9,13a-heptamethyl-15-(6--
morpholinopyridin-3-yl)-2,3,4,4a,5,6,6a,6b,7,8,8a,9,11,13,13a,13b,14,15b-o-
ctadecahydro-1H-chryseno[1,2-f]indazole-4-a-carboxylic acid
[0594] A mixture of 41b (100 mg, 0.14 mmol) and morpholine (0.5 mL)
in i-PrOH (2 mL) was sealed and heated to 160.degree. C. by
microwave for 2 hours. The mixture was concentrated to dryness. The
residue was purified by column chromatography (silica, 0-30% CMA in
CH.sub.2Cl.sub.2) followed by preparative HPLC to afford the
sub-title compound (10 mg, 11%) as an off-white solid.
[0595] R.sub.f 0.75 (80:18:2 Methylene
Chloride/Methanol/concentrated Ammonium Hydroxide).
[0596] .sup.1H NMR (300 MHz, CD.sub.3OD) .delta. 0.46 (s, 3H), 0.82
(s, 3H), 0.90 (s, 3H), 0.99 (s, 3H), 1.22 (s, 3H), 1.25 (s, 3H),
1.29 (s, 3H), 1.35-2.30 (m, 19H), 2.37 (d, J=14.4 Hz, 1H), 3.01 (s,
1H), 3.62 (m, 4H), 3.84 (m, 4H), 7.34 (d, J=9.6 Hz, 1H), 8.00 (s,
2H). mp>300.degree. C. APCI MS (Positive Mode) m/z 656
[C.sub.40H.sub.57N.sub.5O.sub.3+H].sup.+.
Example 55
##STR00218##
[0597] (i) Preparation of 55:
(4aS,6aS,6bR,13aR)-12-Amino-2,2,6a,6b,9,9,13a-heptamethyl-15-(6-(piperazi-
n-1-yl)pyridin-3-yl)-2,3,4,4a,5,6,6a,6b,7,8,8a,9,11,13,13a,13b,14,15b-octa-
decahydro-1H-chryseno[1,2-f]indazole-4-a-carboxylic acid
[0598] To 41b (150 mg, 0.22 mmol) in MeOH (5 mL) was added
piperazine (1.38 g, 20.89 mmol). The reaction mixture was heated at
140.degree. C. for 5 hours and then at 150.degree. C. for an
additional 5 hours using microwave irradiation. The solvent was
concentrated and then the residue was dissolved in
EtOAc/i-PrOH/CH.sub.2Cl.sub.2 (1:1:1, 30 mL). The solution was
washed with brine, dried (Na.sub.2SO.sub.4), filtered and
concentrated. The residue was purified by column chromatography
(silica, 0-40% CMA in CH.sub.2Cl.sub.2) followed by preparative
HPLC to afford the title compound (15 mg, 10%) as a solid.
[0599] R.sub.f 0.10 (40:9:1 Methylene
Chloride/Methanol/concentrated Ammonium Hydroxide).
[0600] .sup.1H NMR (300 MHz, CD.sub.3OD) .delta. 0.40-2.43 (m,
41H), 2.98 (m, 1H), 3.39-3.41 (m, 4H), 3.85-3.87 (m, 4H), 7.23 (d,
J=9.0 Hz, 1H), 7.89 (d, J=3.4 Hz, 1H), 8.12 (s, 1H). APCI MS m/z
655 [C.sub.40H.sub.58N.sub.6O.sub.2+H].sup.+. m.p. 240-260.degree.
dec. HPLC (Method A) 96.2% (214 nm) t.sub.R=10.7 min.
Example 56
##STR00219##
[0601] (i) Preparation of 56b: (4aS,6aS,6bR,13aR)-Benzyl
12-amino-2,2,6a,6b,9,9,13a-heptamethyl-15-(6-(4-methylpiperazin-1-yl)pyri-
din-3-yl)-2,3,4,4a,5,6,6a,6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecahydro-1-
H-chryseno[1,2-f]indazole-4-a-carboxylate
[0602] To a solution of 41b (210 mg, 0.30 mmol) in MeOH (4 mL) was
added 1-methylpiperazine dihydrochloride (1.5 g, 8.67 mmol). The
reaction mixture was heated at 140.degree. C. for 10 hours using
microwave irradiation and then concentrated. The residue was
dissolved in EtOAc (20 mL) and the solution was washed with brine
then dried (Na.sub.2SO.sub.4), filtered and concentrated under
reduced pressure. The residue was purified by column chromatography
(silica, 0-30% CMA in CH.sub.2Cl.sub.2) to afford the sub-title
compound (90 mg, 38%).
[0603] .sup.1H NMR (300 MHz, CD.sub.3OD) .delta. 0.34-2.16 (m,
43H), 2.34 (s, 4H), 2.56 (s, 4H), 3.01 (m, 1H), 3.49 (s, 4H), 5.03
(d, J=12.1 Hz, 2H), 5.20 (d, J=12.0 Hz, 2H), 6.65 (d, J=8.4 Hz,
1H), 6.65 (d, J=4.8 Hz, 1H), 7.36-7.38 (m, 6H), 7.98 (s, 1H). APCI
MS m/z 759 [C.sub.48H.sub.66N.sub.6O.sub.2+H].sup.+.
(ii) Preparation of 56:
(4aS,6aS,6bR,13aR)-12-Amino-2,2,6a,6b,9,9,13a-heptamethyl-15-(6-(4-methyl-
piperazin-1-yl)pyridin-3-yl)-2,3,4,4a,5,6,6a,6b,7,8,8a,9,11,13,13a,13b,14,-
15b-octadecahydro-1H-chryseno[1,2-f]indazole-4-a-carboxylic
acid
[0604] A solution of 56b (70 mg, 0.09 mmol) and MeOH (10 mL) was
flushed with nitrogen and then 10% Pd/C (70 mg) was added. The
mixture was flushed with nitrogen followed by hydrogen. The mixture
was stirred under hydrogen at atmospheric pressure overnight. The
catalyst was removed by filtration through diatomaceous earth and
the filtrate was concentrated under reduced pressure. The residue
was purified by column chromatography (silica, 0-60% CMA in
CH.sub.2Cl.sub.2) to afford the title compound (42 mg, 69%).
[0605] R.sub.f 0.25 (40:9:1 Methylene
Chloride/Methanol/concentrated Ammonium Hydroxide).
[0606] .sup.1H NMR (300 MHz, CD.sub.3OD) .delta. 0.35-2.24 (m,
40H), 2.37 (s, 3H), 2.62 (s, 4H), 3.07 (m, 1H), 3.53 (s, 4H), 4.53
(s, 1H), 6.86 (d, J=8.4 Hz, 1H), 7.75 (d, J=8.1 Hz, 1H), 8.03 (s,
1H). APCI MS m/z 669 [C.sub.41H.sub.60N.sub.6O.sub.2+H].sup.+. m.p.
280-300.degree. C. dec. HPLC (Method A)>99% (214 nm)
t.sub.R=10.3 min.
Example 57
##STR00220## ##STR00221##
[0607] (i) Preparation of 57b: (4aS,6aS,6bR,13aR)-Benzyl
12-amino-15-(6-(4-(tert-butoxycarbonylamino)piperidin-1-yl)pyridin-3-yl)--
2,2,6a,6b,9,9,13a-heptamethyl-2,3,4,4a,5,6,6a,6b,7,8,8a,9,11,13,13a,13b,14-
,15b-octadecahydro-1H-chryseno[1,2-f]indazole-4-a-carboxylate
[0608] To 41b (200 mg, 0.29 mmol) in MeOH (4 mL) was added
tert-butyl piperidin-4-ylcarbamate (1.5 g, 7.50 mmol). The reaction
mixture was heated at 140.degree. C. for 7 hours using microwave
irradiation and then concentrated. The residue was dissolved in
EtOAc (20 mL) and the solution was washed with brine then dried
(Na.sub.2SO.sub.4), filtered and concentrated under reduced
pressure. The residue was purified by column chromatography (0-10%
MeOH in CH.sub.2Cl.sub.2) to afford the sub-title compound (200 mg,
79%).
[0609] APCI MS m/z 859
[C.sub.53H.sub.74N.sub.6O.sub.4+H].sup.+.
(ii) Preparation of 57c: (4aS,6aS,6bR,13aR)-Benzyl
12-amino-15-(6-(4-aminopiperidin-1-yl)pyridin-3-yl)-2,2,6a,6b,9,9,13a-hep-
tamethyl-2,3,4,4a,5,6,6a,6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecahydro-1H-
-chryseno[1,2-f]indazole-4-a-carboxylate
[0610] To a solution of 57b (190 mg, 0.22 mmol) in MeOH (10 mL) was
added HCl (1.0 M in diethyl ether; 10 mL, 10.0 mmol). The mixture
was stirred at room temperature for 32 hours and then concentrated
under reduced pressure. Purification of the residue by column
chromatography (silica, 0-70% CMA in CH.sub.2Cl.sub.2) afforded the
sub-title compound (100 mg, 59%).
[0611] APCI MS m/z 759
[C.sub.48H.sub.66N.sub.6O.sub.2+H].sup.+.
(iii) Preparation of 57:
(4aS,6aS,6bR,13aR)-12-Amino-15-(6-(4-aminopiperidin-1-yl)pyridin-3-yl)-2,-
2,6a,6b,9,9,13a-heptamethyl-2,3,4,4a,5,6,6a,6b,7,8,8a,9,11,13,13a,13b,14,1-
5b-octadecahydro-1H-chryseno[1,2-f]indazole-4-a-carboxylic acid
[0612] A solution of 57c (100 mg, 0.13 mmol) and MeOH (10 mL) was
flushed with nitrogen and then 10% Pd/C (100 mg) was added. The
mixture was flushed with nitrogen followed by hydrogen. The mixture
was stirred under hydrogen at atmospheric pressure overnight. The
catalyst was removed by filtration through diatomaceous earth and
the filtrate was concentrated under reduced pressure. The residue
was purified by column chromatography (silica, 0-50% CMA in
CH.sub.2Cl.sub.2) to afford the title compound (25 mg, 29%).
[0613] R.sub.f 0.11 (40:9:1 Methylene
Chloride/Methanol/concentrated Ammonium Hydroxide).
[0614] .sup.1H NMR (300 MHz, CD.sub.3OD) .delta. 0.36-2.35 (m,
46H), 3.07 (m, 2H), 3.10 (dd, J=3.4 Hz, 3.9 Hz, 1H), 4.29 (d,
J=13.2 Hz, 2H), 6.87 (d, J=8.7 Hz, 1H), 7.61 (m, 1H), 8.03 (s, 1H).
APCI MS m/z 669 [C.sub.41H.sub.60N.sub.6O.sub.2+H].sup.+. m.p.
260-270.degree. C. dec. HPLC (Method A) 95.3% (214 nm) t.sub.R=10.8
min.
Example 58
##STR00222## ##STR00223##
[0615] (i) Preparation of 58b: (4aS,6aS,6bR,13aR)-Benzyl
12-amino-15-(6-(3-(tert-butoxycarbonylamino)pyrrolidin-1-yl)pyridin-3-yl)-
-2,2,6a,6b,9,9,13a-heptamethyl-2,3,4,4a,5,6,6a,6b,7,8,8a,9,11,13,13a,13b,1-
4,15b-octadecahydro-1H-chryseno[1,2-f]indazole-4-a-carboxylate
[0616] To a solution of 41b (246 mg, 0.36 mmol) in MeOH (4 mL) was
added tert-butyl pyrrolidin-3-ylcarbamate (1.0 g, 5.36 mmol). The
reaction mixture was heated at 140.degree. C. for 6 hours using
microwave irradiation. The solvent was removed under reduced
pressure and the residue was dissolved in EtOAc (20 mL). The
solution was washed with brine then dried (Na.sub.2SO.sub.4),
filtered and concentrated. The residue was purified by column
chromatography (silica, 0-10% MeOH in CH.sub.2Cl.sub.2) to afford
the sub-title compound (306 mg, 100%).
[0617] APCI MS m/z 845
[C.sub.52H.sub.72N.sub.6O.sub.4+H].sup.+.
(ii) Preparation of 58c: (4aS,6aS,6bR,13aR)-Benzyl
12-amino-15-(6-(3-aminopyrrolidin-1-yl)pyridin-3-yl)-2,2,6a,6b,9,9,13a-he-
ptamethyl-2,3,4,4a,5,6,6a,6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecahydro-1-
H-chryseno[1,2-f]indazole-4-a-carboxylate
[0618] To a solution of 58b (300 mg, 0.35 mmol) in MeOH (10 mL) was
added HCl (1.0 M in diethyl ether, 7.8 mL, 7.80 mmol). The reaction
mixture was stirred at room temperature for 24 hours and then
concentrated. The residue was purified by column chromatography
(silica, 0-40% CMA in CH.sub.2Cl.sub.2) to afford the sub-title
compound (50 mg, 19%).
[0619] .sup.1H NMR (300 MHz, CD.sub.3OD) .delta. 0.36-2.35 (m,
42H), 3.08-3.68 (m, 7H), 5.05 (d, J=12.0 Hz, 1H), 5.21 (d, J=12.0
Hz, 1H), 6.32 (d, J=8.7 Hz, 1H), 7.30-7.39 (m, 6H), 7.89 (s,
1H).
[0620] APCI MS m/z 745
[C.sub.47H.sub.64N.sub.6O.sub.2+H].sup.+.
(iii) Preparation of 58:
(4a,6aS,6bR,13aR)-12-Amino-15-(6-(3-aminopyrrolidin-1-yl)pyridin-3-yl)-2,-
2,6a,6b,9,9,13a-heptamethyl-2,3,4,4a,5,6,6a,6b,7,8,8a,9,11,13,13a,13b,14,1-
5b-octadecahydro-1H-chryseno[1,2-f]indazole-4-a-carboxylic acid
[0621] A solution of 58c (50 mg, 0.067 mmol) and MeOH (15 mL) was
flushed with nitrogen and then 10% Pd/C (50 mg) was added. The
mixture was flushed with nitrogen followed by hydrogen. The mixture
was stirred under hydrogen at atmospheric pressure overnight. The
catalyst was removed by filtration through diatomaceous earth and
the filtrate was concentrated. The residue was purified by column
chromatography (silica, 0-70% CMA in CH.sub.2Cl.sub.2) to afford
the title compound (33 mg, 76%).
[0622] R.sub.f 0.14 (40:9:1 Methylene
Chloride/Methanol/concentrated Ammonium Hydroxide).
[0623] .sup.1H NMR (300 MHz, CD.sub.3OD) .delta. 0.37-2.36 (m,
44H), 3.10-3.80 (m, 5H), 6.53 (d, J=8.7 Hz, 1H), 7.61 (d, J=8.4 Hz,
1H), 7.93 (s, 1H). APCI MS m/z 655
[C.sub.40H.sub.58N.sub.6O.sub.2+H].sup.+. m.p.>300.degree. C.
HPLC (Method A) 97.4% (214 nm) t.sub.R=10.8 min.
Example 59
##STR00224##
[0624] (i) Preparation of 59b: (4aS,6aS,6bR,8aR,13aR,15bS)-benzyl
12-amino-15-(2-formylphenyl)-2,2,6a,6b,9,9,13a-heptamethyl-2,3,4,4a,5,6,6-
a,6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecahydro-1H-chryseno[1,2-f]indazol-
e-4-a-carboxylate
[0625] A mixture of II (500 mg, 0.75 mmol), 2-formylphenylboronic
acid (337 mg, 2.24 mmol) Pd(PPh.sub.3).sub.4 (80 mg, 0.069 mmol)
and K.sub.2CO.sub.3 (414 mg, 3.0 mmol) in benzene (3.5 mL) and EtOH
(1.5 mL) was sealed and heated to 120.degree. C. by microwave for 1
hour. The reaction mixture was diluted with EtOAc (100 mL). The
organic phase was washed with brine then dried (MgSO.sub.4),
filtered and concentrated to dryness. The residue was purified by
column chromatography (silica, 0-10% MeOH in CH.sub.2Cl.sub.2) to
afford the sub-title compound (540 mg) as a brown solid.
[0626] APCI MS (Positive Mode) m/z 688
[C.sub.45H.sub.57N.sub.3O.sub.3+H].sup.+.
(ii) Preparation of 59c: (4aS,6aS,6bR,8aR,13aR,15bS)-Benzyl
12-amino-2,2,6a,6b,9,9,13a-heptamethyl-15-(2-(morpholinomethyl)phenyl)-2,-
3,4,4a,5,6,6a,6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecahydro-1H-chryseno[1-
,2-f]indazole-4-a-carboxylate
[0627] To a solution of 59b (140 mg, 0.20 mmol) and morpholine
(0.035 mL, 0.40 mmol) in CH.sub.2Cl.sub.2 (5 mL) was added sodium
triacetoxyborohydride (85 mg, 0.40 mmol). The mixture was stirred
at room temperature for 12 hours. The reaction mixture was diluted
with EtOAc (150 mL). The organic phase was washed with brine then
dried (MgSO.sub.4), filtered and concentrated to dryness. The
residue was purified by column chromatography (silica, 0-10% MeOH
in CH.sub.2Cl.sub.2) to afford the sub-title compound (71 mg, 47%)
as a brown solid.
[0628] APCI MS (Positive Mode) m/z 759
[C.sub.49H.sub.66N.sub.4O.sub.3+H].sup.+.
(iii) Preparation of 59:
(4aS,6aS,6bR,8aR,13aR,15bS)-12-Amino-2,2,6a,6b,9,9,13a-heptamethyl-15-(2--
(morpholinomethyl)phenyl)-2,3,4,4a,5,6,6a,6b,7,8,8a,9,11,13,13a,13b,14,15b-
-octadecahydro-1H-chryseno[1,2-f]indazole-4-a-carboxylic acid
[0629] A mixture of 59c (71 mg, 0.093 mmol) and 10% Pd(OH).sub.2/C
(50 mg) in MeOH (12 mL) and EtOAc (3 mL) was stirred under a
hydrogen balloon for 12 hours. The reaction mixture was filtered
through a pad of diatomaceous earth and washed with CMA (25 mL).
The filtrate was concentrated to dryness under reduced pressure.
The residue was purified by column chromatography (silica, 0-70%
CMA in CH.sub.2Cl.sub.2) to afford the title compound (20 mg, 33%)
as a mixture of rotamers.
[0630] R.sub.f 0.80 (80:18:2 Methylene
Chloride/Methanol/concentrated Ammonium Hydroxide).
[0631] .sup.1H NMR (400 MHz, CD.sub.3OD) .delta. 0.25 (s, 3H), 0.78
(s, 3H), 0.89 (s, 3H), 1.08 (s, 3H), 1.12 (s, 3H), 1.28 (s, 6H),
1.35-2.30 (m, 23H), 2.28 (d, J=14.8 Hz, 1H), 2.40 (m, 1H), 2.90 (m,
1H), 3.95-4.50 (m, 5H), 7.20-7.62 (m, 4H). mp>300.degree. C.
dec. APCI MS (Positive Mode) m/z 669
[C.sub.42H.sub.60N.sub.4O.sub.3+H].sup.+.
Example 60
##STR00225##
[0632] (i) Preparation of 60b:
3-((4aS,6aS,6bR,13aR)-12-Amino-2,2,6a,6b,9,9,13a-heptamethyl-2,3,4,4a,5,6-
,6a,6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecahydro-1H-chryseno[1,2-f]indaz-
ol-4-a-yl)-3-oxopropanenitrile
[0633] To a suspension of 1 (150 mg, 0.30 mmol) and
CH.sub.2Cl.sub.2 (7 mL) was added thionyl chloride (0.22 mL, 3.0
mmol) at room temperature. The mixture was stirred for 1 hour,
after which time the solvent was removed under reduced pressure.
The residue was taken up in toluene (5 mL), concentrated and placed
under vacuum at room temperature. A solution of CH.sub.3CN (0.16
mL, 3.0 mmol) and THF (5 mL) was cooled to -78.degree. C. under
nitrogen. The solution was treated with n-butyllithium (2.5 M in
hexanes, 1.27 mL, 3.2 mmol) while maintaining the internal
temperature below -70.degree. C. The previously prepared acid
chloride was taken up in THF (2 mL), cooled to -78.degree. C. and
slowly added to the lithium salt mixture. The reaction mixture was
stirred for 80 minutes and then quenched with 1 N HCl (10 mL). The
layers were separated and the aqueous layer was extracted with
EtOAc (15 mL) and the combined organic layers were dried
(Na.sub.2SO.sub.4) and filtered. The solvent was removed under
reduced pressure and the residue was purified by column
chromatography (silica, 0-50% CMA in CH.sub.2Cl.sub.2) to provide
the sub-title compound (115 mg, 74%) as an off-white solid.
[0634] .sup.1H NMR (300 MHz, CD.sub.3OD) .delta. 0.80 (s, 3H),
0.91-0.96 (m, 9H), 1.12-1.17 (m, 12H), 1.31-1.91 (m, 15H),
2.08-2.12 (m, 3H), 2.39-2.45 (m, 1H), 2.71-2.81 (m, 1H), 5.43-5.45
(m, 1H). ESI MS m/z 517 [C.sub.33H.sub.48N.sub.4O+H].sup.+. HPLC
97.6% (area %), t.sub.R=16.2 min.
(ii) Preparation of 60:
(4aS,6aS,6bR,13aR)-4-a-(5-Amino-1H-pyrazol-3-yl)-2,2,6a,6b,9,9,13a-heptam-
ethyl-2,3,4,4a,5,6,6a,6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecahydro-1H-ch-
ryseno[1,2-f]indazol-12-amine
[0635] A solution of 60b (100 mg, 0.19 mmol), hydrazine (31 mg,
0.97 mmol) and EtOH (5 mL) was heated at reflux under nitrogen for
16 hours. A second portion of hydrazine (91 mg, 2.85 mmol) was
added and the reaction continued for 72 hours at reflux. The
solvent was removed under reduced pressure and the residue was
purified by column chromatography (silica, 0-50% CMA in
CH.sub.2Cl.sub.2) to provide the title compound (43 mg, 43%) as an
off-white solid.
[0636] .sup.1H NMR (300 MHz, CD.sub.3OD) .delta. 0.58 (s, 3H), 0.82
(s, 3H), 0.91-1.35 (m, 18H), 1.39-1.91 (m, 15H), 2.13-2.35 (m, 2H),
2.73-2.81 (m, 1H), 5.43-5.48 (m, 2H). ESI MS m/z 531
[C.sub.33H.sub.50N.sub.6+H].sup.+. HPLC 97.9% (area %),
t.sub.R=14.2 min.
Example 61
##STR00226##
[0637] (i) Preparation of 61:
(4aS,6aS,6bR,13aR)-12-Amino-2,2,6a,6b,9,9,13a-heptamethyl-N-(1H-tetrazol--
5-yl)-2,3,4,4a,5,6,6a,6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecahydro-1H-ch-
ryseno[1,2-f]indazole-4-a-carboxamide
[0638] To a solution of 1H-tetrazol-5-amine hydrate (103 mg, 1.0
mmol) and triethylamine (0.27 mL, 2.0 mmol) in THF (3 mL) was added
III (100 mg, 0.20 mmol). The mixture was stirred at room
temperature overnight. The reaction mixture was diluted with EtOAc
(60 mL). The organic phase was washed with brine, dried
(MgSO.sub.4), filtered and concentrated to dryness under reduced
pressure. The residue was purified by column chromatography
(silica, 0-80% CMA in CH.sub.2Cl.sub.2) followed by preparative
HPLC to afford the title compound (35 mg, 32%) as an off-white
solid.
[0639] R.sub.f 0.40 (80:18:2 Methylene
Chloride/Methanol/concentrated Ammonium Hydroxide).
[0640] .sup.1H NMR (300 MHz, CD.sub.3OD) .delta. 0.73 (s, 3H), 0.85
(s, 3H), 0.92 (s, 3H), 0.97 (2, 3H), 1.15 (s, 3H), 1.20 (s, 3H),
1.25 (s, 3H), 1.30-1.90 (m, 16H), 2.10 (m, 2H), 2.20 (m, 1H), 2.41
(d, J=15.0 Hz, 1H), 3.05 (m, 1H), 5.46 (s, 1H). mp>300.degree.
C. ESI MS (Positive Mode) m/z 561
[C.sub.32H.sub.48N.sub.8O+H].sup.+.
Example 62
##STR00227##
[0641] (i) Preparation of 62:
(4aS,6aS,6bR,13aR)-12-Amino-2,2,6a,6b,9,9,13a-heptamethyl-2,3,4,4a,5,6,6a-
,6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecahydro-1H-chryseno[1,2-f]indazole-
-4-a-carboxamide
[0642] To a suspension of 1 (250 mg, 0.51 mmol) and
CH.sub.2Cl.sub.2 (5 mL) was added thionyl chloride (0.4 mL, 5.1
mmol) at room temperature. The mixture was stirred for 3 hours,
after which time the solvent was removed under reduced pressure.
The residue was taken up in toluene (5 mL), concentrated and placed
under vacuum at room temperature for 1 hour. The residue was taken
up in THF (5 mL) and concentrated NH.sub.4OH (1.4 mL) was added.
The mixture was stirred for 1 hour and another portion of
NH.sub.4OH (1.4 mL) was added. The mixture was stirred at room
temperature over night before being quenched with saturated
NaHCO.sub.3 solution (10 mL). The mixture was extracted with
CH.sub.2Cl.sub.2 (3.times.15 mL), dried (Na.sub.2SO.sub.4) and
filtered. The solvent was removed under reduced pressure and the
residue was purified by column chromatography (silica, 0-3% MeOH in
CH.sub.2Cl.sub.2) to provide the title compound (144 mg, 58%) as an
off-white solid.
[0643] .sup.1H NMR (500 MHz, CD.sub.3OD) .delta. 0.89-0.98 (m,
12H), 1.02-1.31 (m, 12H), 1.39-1.91 (m, 12H), 2.03-2.11 (m, 3H),
2.42-2.45 (m, 1H), 2.76-2.83 (m, 1H), 5.43-5.45 (m, 1H).
[0644] ESI MS m/z 493 [C.sub.31H.sub.48N.sub.4O+H].sup.+. HPLC
97.9% (area %), t.sub.R=14.2 min.
Example 63
##STR00228##
[0645] (i) Preparation of 63:
(4aS,6aS,6bR,13aR)-4-a-(Aminomethyl)-2,2,6a,6b,9,9,13a-heptamethyl-2,3,4,-
4a,5,6,6a,6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecahydro-1H-chryseno[1,2-f-
]indazol-12-amine
[0646] To a solution of 62 (300 mg, 0.61 mmol) and THF (20 mL) was
added lithium aluminum hydride (230 mg, 6.1 mmol) at room
temperature. The mixture was heated at reflux for 24 hours, cooled
to room temperature, carefully quenched with H.sub.2O and EtOAc
then extracted with CH.sub.2Cl.sub.2 (3.times.20 mL). The organics
were dried (Na.sub.2SO.sub.4) and filtered. The solvent was removed
under reduced pressure and the residue was purified by column
chromatography (silica, 0-60% CMA in CH.sub.2Cl.sub.2) to provide
the title compound (94 mg, 30%).
[0647] .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 0.82-1.03 (m,
12H), 1.15-1.85 (m, 14H), 1.91-2.11 (m, 15H), 2.52-2.60 (m, 2H),
2.83-2.91 (m, 1H), 3.25-3.30 (m, 1H), 3.56-3.63 (m, 1H), 5.23-5.39
(m, 2H), 7.92-7.94 (m, 2H). ESI MS m/z 479
[C.sub.31H.sub.50N.sub.4+H].sup.+. HPLC 96.6% (area %),
t.sub.R=11.3 min.
Example 64
##STR00229##
[0648] (i) Preparation of 64b:
(4aS,6aS,6bR,13aR)-12-Amino-N,N,2,2,6a,6b,9,9,13a-nonamethyl-2,3,4,4a,5,6-
,6a,6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecahydro-1H-chryseno[1,2-f]indaz-
ole-4-a-carboxamide
[0649] To a suspension of 1 (200 mg, 0.41 mmol) and
CH.sub.2Cl.sub.2 (4 mL) was added thionyl chloride (0.3 mL, 4.1
mmol) at room temperature. The mixture was stirred for 0.5 hours,
after which time the solvent was removed under reduced pressure.
The residue was taken up in toluene (5 mL), concentrated and placed
under vacuum at room temperature for 1 hour. The residue was taken
up in THF (4 mL) and N,N-diisopropylethylamine (0.23 mL, 1.2 mmol)
was added followed by diethylamine solution (2.0 M in THF, 2.05 mL,
4.1 mmol). The solution was stirred overnight and then quenched
with a 2% solution of citric acid. The layers were separated and
the aqueous solution was extracted with CH.sub.2Cl.sub.2
(2.times.10 mL). The combined extracts were dried
(Na.sub.2SO.sub.4) and filtered. The solvent was removed under
reduced pressure and the residue was purified by column
chromatography (silica, 0-7% MeOH in CH.sub.2Cl.sub.2) to provide
the sub-title compound (153 mg, 73%) as an off-white solid.
[0650] .sup.1H NMR (300 MHz, CD.sub.3OD) .delta. 0.83 (s, 3H),
0.91-0.98 (m, 9H), 1.17-1.26 (m, 13H), 1.39-2.25 (m, 16H),
3.01-3.20 (m, 7H), 5.29 (m, 1H). ESI MS m/z 521
[C.sub.33H.sub.52N.sub.4O+H].sup.+. HPLC 98.4% (area %),
t.sub.R=17.2 min.
(ii) Preparation of 64:
(4aS,6aS,6bR,13aR)-4-a-((Dimethylamino)methyl)-2,2,6a,6b,9,9,13a-heptamet-
hyl-2,3,4,4a,5,6,6a,6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecahydro-1H-chry-
seno[1,2-f]indazol-12-amine
[0651] A solution of 64b (129 mg, 0.25 mmol) and THF (12.5 mL) was
slowly added to a suspension of lithium aluminum hydride (94 mg,
2.5 mmol) and THF (6.5 mL) at room temperature. The mixture was
stirred overnight and then quenched with EtOAc followed by
H.sub.2O. The layers were separated and the aqueous layer was
extracted with EtOAc (3.times.15 mL). The organics were combined,
dried (Na.sub.2SO.sub.4) and filtered. The solvent was removed
under reduced pressure and the residue was purified by column
chromatography (silica, 0-7% MeOH in CH.sub.2Cl.sub.2) to provide
the title compound (54 mg, 43%) as a white solid.
[0652] .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 0.83-0.95 (m,
12H), 1.02-1.33 (m, 20H), 1.35-2.10 (m, 16H), 2.30-2.41 (m, 3H),
5.21-5.25 (m, 1H). ESI MS m/z 507
[C.sub.33H.sub.54N.sub.4+H].sup.+. HPLC 96.4% (area %),
t.sub.R=11.6 min
Example 65
##STR00230##
[0653] (i) Preparation of 65:
(4aS,6aS,6bR,13aR)-12-Amino-N-hydroxy-2,2,6a,6b,9,9,13a-heptamethyl-2,3,4-
,4a,5,6,6a,6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecahydro-1H-chryseno[1,2--
f]indazole-4-a-carboxamide
[0654] To a suspension of 1 (75 mg, 0.15 mmol) in CH.sub.2Cl.sub.2
(5 mL) was added thionyl chloride (0.1 mL, 1.5 mmol) at room
temperature. The mixture was stirred for 0.5 hours, after which
time the solvent was removed under reduced pressure. The residue
was taken up in toluene (5 mL), concentrated and placed under
vacuum at room temperature overnight. The residue was taken up in
CH.sub.2Cl.sub.2 (5 mL) at room temperature.
N,N-Diisopropylethylamine (0.2 mL, 1.05 mmol) was added followed by
hydroxylamine hydrochloride (52 mg, 0.75 mmol). The mixture was
stirred at room temperature for 14 hours. A solution of citric acid
(2%, 5 mL) was added and the mixture was extracted with
CH.sub.2Cl.sub.2 (2.times.5 mL). The combined organic layers were
dried (Na.sub.2SO.sub.4), filtered and concentrated under reduced
pressure. The residue was purified by column chromatography
(silica, 0-6% MeOH in CH.sub.2Cl.sub.2) to provided the title
compound (144 mg, 58%) as an off-white solid.
[0655] .sup.1H NMR (500 MHz, CD.sub.3OD) .delta. 0.87-0.95 (m,
12H), 1.05-1.33 (m, 12H), 1.38-1.88 (m, 13H), 2.08-2.15 (m, 3H),
2.43-2.46 (m, 1H), 2.75-2.82 (m, 1H), 5.43-5.45 (m, 1H). APCI MS
m/z 509 [C.sub.31H.sub.48N.sub.4O+H].sup.+. HPLC 97.4% (area %),
t.sub.R=13.5 min.
Example 66
##STR00231##
[0656] (i) Preparation of 66:
(4aS,6aS,6bR,13aR)-12-Amino-N-(5-hydroxy-1H-pyrazol-3-yl)-2,2,6a,6b,9,9,1-
3a-heptamethyl-2,3,4,4a,5,6,6a,6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecahy-
dro-1H-chryseno[1,2-f]indazole-4-a-carboxamide
[0657] To a solution of III (250 mg, 0.48 mmol) and THF (2 mL) was
added triethylamine (0.68 mL, 4.88 mmol) and the solution was
stirred for 5 min. To the resultant mixture was added a solution of
3-amino-5-hydroxypyrazole (241 mg, 2.44 mmol) in DMF (6 mL) and the
mixture was heated at 50.degree. C. for 3 hours. The mixture was
poured into H.sub.2O (20 mL) and the precipitate was removed by
filtration. The filtrate was concentrated and the residue was
purified by column chromatography (silica, 0-50% CMA in
CH.sub.2Cl.sub.2) followed by preparative HPLC to afford the title
compound (48 mg, 17%) as a solid.
[0658] R.sub.f 0.28 (90:9:1 Methylene
Chloride/Methanol/concentrated Ammonium Hydroxide).
[0659] .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 0.49-2.97 (m,
42H), 5.36 (s, 1H), 5.76 (s, 1H). APCI MS m/z 575
[C.sub.34H.sub.50N.sub.6O.sub.2+H].sup.+. m.p. 275-295.degree. C.
dec. HPLC (Method A) 97.7% (214 nm) t.sub.R=11.5 min.
Example 67
##STR00232## ##STR00233##
[0660] (i) Preparation of 67b:
(4aS,6aS,6bR,12aR)-11-Cyano-2,2,6a,6b,9,9,12a-heptamethyl-10-oxo-1,2,3,4,-
4a,5,6,6a,6b,7,8,8a,9,10,11,12,12a,12b,13,14b-icosahydropicene-4-a-carboxy-
lic acid
[0661] A solution of diisopropylamine (1.4 mL, 10.2 mmol) and THF
(20 mL) was cooled to -78.degree. C. under nitrogen. A solution of
n-butyllithium (2.5 M in hexanes, 4.4 mL, 11.0 mmol) was slowly
added, maintaining the internal temperature below -70.degree. C.
The solution was allowed to stir for 30 min and was then slowly
added to a solution of Ib (2.0 g, 4.4 mmol) and THF (250 mL) at
-78.degree. C. under nitrogen. This solution was stirred for 30 min
after which time a suspension of p-toluenesulfonyl cyanide (1.6 g,
8.8 mmol) and THF (25 mL) was added over 45 min. The solution was
stirred for 1 hour and then quenched by the addition of saturated
ammonium chloride solution (100 mL) at -78.degree. C. The mixture
was allowed to warm to room temperature overnight. The organic
layer was separated and the aqueous layer was extracted with EtOAc
(3.times.100 mL). The combined organic layers were dried
(Na.sub.2SO.sub.4) and filtered. The solvent was removed under
reduced pressure and the residue was purified by column
chromatography (silica, 0-35% EtOAc in hexanes) to provide the
sub-title compound (1.3 g, 60%) as a white solid.
[0662] .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 0.77-0.82 (m, 9H),
1.06-1.11 (m, 13H), 1.12-2.03 (m, 19H), 2.09-2.38 (m, 1H),
2.81-2.89 (m, 1H), 5.26-5.35 (m, 1H).
(ii) Preparation of 67c:
(4aS,6aS,6bR,13aR)-12-Amino-11-benzyl-2,2,6a,6b,9,9,13a-heptamethyl-2,3,4-
,4a,5,6,6a,6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecahydro-1H-chryseno[1,2--
f]indazole-4-a-carboxylic acid (and N-benzyl structural isomer)
[0663] A solution of 67b (1.3 g, 2.6 mmol), benzylhydrazine
dihydrochloride (1.2 g, 6.2 mmol) and EtOH (15 mL) was heated at
reflux for 58 hours. The solvent was removed under reduced pressure
and the residue was purified by column chromatography (silica, 0-5%
MeOH in CH.sub.2Cl.sub.2) to provide the sub-title compound (1.1 g,
72%) as yellow solid.
[0664] .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 0.78-0.95 (m,
15H), 1.05-2.03 (m, 25H), 2.25-2.35 (m, 1H), 2.81-2.91 (m, 1H),
5.21-5.36 (m, 3H), 7.04-7.11 (m, 2H), 7.21-7.35 (m, 3H).
[0665] ESI MS m/z 584 [C.sub.38H.sub.53N.sub.3O.sub.2+H].sup.+.
(iii) Preparation of 67d:
(4aS,6aS,6bR,13aR)-1'-Benzyl-12-(benzyloxycarbonylamino)-2,2,6a,6b,9,9,13-
a-heptamethyl-2,3,4,4a,5,6,6a,6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecahyd-
ro-1H-chryseno[1,2-f]indazole-4-a-carboxylic acid (and N-benzyl
structural isomer)
[0666] To a solution of 67c (560 mg, 0.96 mmol) in THF (50 mL) were
added triethylamine (0.27 mL, 1.92 mmol) and benzyl chloroformate
(0.16 mL, 1.15 mmol) under nitrogen.
[0667] The mixture was stirred at room temperature for 16 hours and
then quenched with brine (40 mL). The layers were separated and the
aqueous layer was extracted with EtOAc (2.times.20 mL). The
solution was dried (Na.sub.2SO.sub.4), filtered and concentrated to
afford the sub-title compound (108 mg, quant) which was used
without further purification.
[0668] ESI MS m/z 718 [C.sub.46H.sub.59N.sub.3O.sub.4+H].sup.+.
(iv) Preparation of 67e: Benzyl
(4aS,6aS,6bR,13aR)-11-benzyl-4-a-(hydrazinecarbonyl)-2,2,6a,6b,9,9,13a-he-
ptamethyl-2,3,4,4a,5,6,6a,6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecahydro-1-
H-chryseno[1,2-f]indazol-12-ylcarbamate (and N-benzyl structural
isomer)
[0669] To a solution of 67d (744 mg, 1.03 mmol) in CH.sub.2Cl.sub.2
(100 mL) was added thionyl chloride (0.76 mL, 10.37 mmol). The
mixture was stirred at room temperature for 1.5 hours and then the
solvent was removed under reduced pressure. The residue was
azeotroped with toluene and then dried under vacuum overnight to
afford the intermediate acid chloride (687 mg, 93%). The crude acid
chloride (364 mg, 0.49 mmol) was dissolved in THF (10 mL) and then
triethylamine (0.34 mL, 2.47 mmol) and hydrazine (0.15 mL, 4.94
mmol) were added. The mixture was stirred at room temperature for
20 hours and concentrated under reduced pressure. The residue was
partitioned between EtOAc (20 mL) and H.sub.2O (10 mL). The layers
were separated and the organic layer was washed with H.sub.2O and
brine. The solution was dried (Na.sub.2SO.sub.4), filtered and
concentrated. The residue was purified by column chromatography
(silica, 0-40% CMA in CH.sub.2Cl.sub.2) to afford the sub-title
compound (224 mg, 62%).
[0670] ESI MS (m/z 733
[C.sub.46H.sub.61N.sub.5O.sub.3+H].sup.+.
(v) Preparation of 67f: Benzyl
(4aS,6aS,6bR,13aR)-11-benzyl-2,2,6a,6b,9,9,13a-heptamethyl-4-a-(1,3,4-oxa-
diazol-2-yl)-2,3,4,4a,5,6,6a,6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecahydr-
o-1H-chryseno[1,2-f]indazol-12-ylcarbamate (and N-benzyl structural
isomer)
[0671] A mixture of 67e (100 mg, 0.13 mmol), trimethoxymethane
(0.22 mL, 2.04 mmol) and p-toluene sulfonic acid monohydrate (4 mg,
0.02 mmol) was heated at 120.degree. C. overnight. The mixture was
concentrated under reduced pressure and the residue was purified by
column chromatography (silica, 2:1 hexanes/EtOAc) to afford the
sub-title compound (75 mg, 74%).
[0672] ESI MS m/z 651
[C.sub.47H.sub.59N.sub.5O.sub.3--C.sub.7H.sub.7+H].sup.+.
(vi) Preparation of 67:
(4aS,6aS,6bR,13aR)-2,2,6a,6b,9,9,13a-Heptamethyl-4-a-(1,3,4-oxadiazol-2-y-
l)-2,3,4,4a,5,6,6a,6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecahydro-1H-chrys-
eno[1,2-f]indazol-12-amine
[0673] A solution of 67d (70 mg, 0.09 mmol), acetic acid (0.2 mL)
and MeOH (10 mL) was flushed with nitrogen. To the resultant
mixture was added 10% Pd/C (150 mg) and the mixture was flushed
with nitrogen followed by hydrogen. The mixture was stirred under
hydrogen at atmospheric pressure for 23 hours. The catalyst was
removed by filtration through diatomaceous earth and the filtrate
was concentrated under reduced pressure. The residue was purified
by column chromatography (silica, 0-40% CMA in CH.sub.2Cl.sub.2) to
afford the title compound (6 mg, 12%).
[0674] R.sub.f 0.42 (90:9:1 Methylene
Chloride/Methanol/concentrated Ammonium Hydroxide).
[0675] .sup.1H NMR (300 MHz, CD.sub.3OD) .delta. 0.45-2.41 (m,
41H), 3.14 (d, J=10.8 Hz, 1H), 5.44 (s, 1H), 8.84 (s, 1H). ESI MS
m/z 518 [C.sub.32H.sub.47N.sub.5O+H].sup.+. m.p. 255-270.degree. C.
dec. HPLC (Method A) 89.0% (214 nm) t.sub.R=16.1 min.
Example 68
##STR00234##
[0676] (i) Preparation of 68b: Ethyl
3-((4aS,6aS,6bR,13aR)-12-amino-2,2,6a,6b,9,9,13a-heptamethyl-2,3,4,4a,5,6-
,6a,6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecahydro-1H-chryseno[1,2-f]indaz-
ol-4-a-yl)-3-oxopropanimidate hydrochloride
[0677] A solution of 60b (200 mg, 0.39 mmol) and EtOH (15 mL) was
saturated with anhydrous HCl at -5.degree. C. The solution was kept
at that temperature for 10 d before removal of the solvent and
excess HCl under reduced pressure. The residue was coevaporated
with EtOH followed by diethyl ether to provide the sub-title
compound (220 mg, 100%) as a tan solid.
[0678] APCI MS m/z 563
[C.sub.33H.sub.54N.sub.4O.sub.2+H].sup.+.
(ii) Preparation of 68c: Ethyl
3-((4aS,6aS,6bR,13aR)-12-amino-2,2,6a,6b,9,9,13a-heptamethyl-2,3,4,4a,5,6-
,6a,6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecahydro-1H-chryseno[1,2-f]indaz-
ol-4-a-yl)-3-oxopropanoate hydrochloride
[0679] To a solution of 68b (220 mg, 0.39 mmol) and EtOH (3 mL) was
added 1 N HCl (2 mL) at room temperature. After 24 hours, the
solvent was removed under reduced pressure and the residue was
purified by column chromatography (silica, 0-65% CMA in
CH.sub.2Cl.sub.2) to provide the sub-title compound (135 mg,
58%).
[0680] .sup.1H NMR (500 MHz, CDCl.sub.3) .delta. 0.71-0.95 (m,
12H), 1.13-1.31 (m, 16H), 1.34-2.15 (m, 17H), 2.29-2.33 (m, 1H),
2.71-2.74 (m, 1H), 3.49-3.71 (m, 2H), 4.13-4.21 (m, 2H), 5.43-5.45
(m, 1H).
(iii) Preparation of 68:
3-((4aS,6aS,6bR,13aR)-12-Amino-2,2,6a,6b,9,9,13a-heptamethyl-2,3,4,4a,5,6-
,6a,6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecahydro-1H-chryseno[1,2-f]indaz-
ol-4-a-yl)-1H-pyrazol-5-ol
[0681] A solution of 68c (135 mg, 0.24 mmol), hydrazine (38 mg, 1.2
mmol) and EtOH (5 mL) was heated at reflux under nitrogen for 66
hours and then cooled to room temperature. The solvent was removed
under reduced pressure and the residue was purified by column
chromatography (silica, 0-55% CMA in CH.sub.2Cl.sub.2) to provide
the title compound (11 mg, 9%) as a solid.
[0682] .sup.1H NMR (300 MHz, CD.sub.3OD) .delta. 0.62 (s, 3H),
0.86-1.01 (m, 9H), 1.03-1.39 (m, 15H), 1.41-1.99 (m, 11H),
2.03-2.11 (m, 2H), 2.25-2.45 (m, 2H), 2.74-2.81 (m, 1H), 5.43-5.45
(m, 1H). APCI MS m/z 532 [C.sub.33H.sub.49N.sub.5O+H].sup.+. HPLC
98.6% (area %), t.sub.R=10.2 min.
Example 69
##STR00235##
[0683] (i) Preparation of 69:
(4aS,6aS,6bR,13aR)-12-amino-N-(3-amino-1H-pyrazol-5-yl)-2,2,6a,6b,9,9,13a-
-heptamethyl-2,3,4,4a,5,6,6a,6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecahydr-
o-1H-chryseno[1,2-f]indazole-4-a-carboxamide
[0684] To a solution of 1H-pyrazole-3,5-diamine (70 mg, 0.54 mmol)
prepared by the procedure described in the literature (US82902,
2007) and pyridine (2.0 mL) was added III (100 mg, 0.20 mmol). The
mixture was stirred at room temperature overnight. The reaction
mixture was concentrated to dryness under reduced pressure. The
residue was purified by column chromatography (silica, 0-40% CMA in
CH.sub.2Cl.sub.2) followed by preparative HPLC to afford the title
compound (25 mg, 25%) as an off-white solid.
[0685] R.sub.f 0.20 (180:18:2 Methylene
Chloride/Methanol/concentrated Ammonium Hydroxide).
[0686] .sup.1H NMR (300 MHz, DMSO-d.sub.6) .delta. 0.83 (s, 3H),
0.92 (s, 3H), 0.95 (s, 3H), 1.0 (s, 3H), 1.25 (s, 6H), 1.30 (s,
3H), 1.30-1.90 (m, 23H), 2.06 (m, 3H), 2.25 (m, 1H), 2.44 (d,
J=15.0 Hz, 1H), 2.62 (m, 1H), 5.35 (s, 1H). mp>300.degree. C.
APCI MS (Positive Mode) m/z 574
[C.sub.34H.sub.51N.sub.7O+H].sup.+.
Example 70
##STR00236##
[0687] (i) Preparation of 70:
(4aS,6aS,6bR,13aR)--S-5-Amino-4H-1,2,4-triazol-3-yl
12-amino-2,2,6a,6b,9,9,13a-heptamethyl-2,3,4,4a,5,6,6a,6b,7,8,8a,9,11,13,-
13a,13b,14,15b-octadecahydro-1H-chryseno[1,2-f]indazole-4-a-carbothioate
[0688] To a solution of 5-amino-4H-1,2,4-triazole-3-thiol (1.42 g,
12.2 mmol) in pyridine (30 mL was added III (2.0 g, 3.91 mmol). The
mixture was stirred at room temperature for 3 hours. The reaction
mixture was concentrated to remove pyridine under reduced pressure.
The residue was purified by column chromatography (silica, 0-15%
MeOH in CH.sub.2Cl.sub.2) to afford the title compound (1.41 g,
59%) as an off-white solid.
[0689] R.sub.f 0.12 (89:10:1 Methylene
Chloride/Methanol/concentrated Ammonium Hydroxide).
[0690] .sup.1H NMR (300 MHz, CD.sub.3OD) .delta. 0.87 (s, 6H), 0.90
(s, 3H), 0.95 (s, 3H), 1.10 (s, 3H), 1.15 (s, 3H), 1.20 (s, 3H),
1.30-2.20 (m, 19H), 2.38 (d, J=15.0 Hz, 1H), 2.88 (m, 1H), 5.39 (s,
1H). mp 278-296.degree. C. ESI MS (Positive Mode) m/z 59
[C.sub.33H.sub.49N.sub.7OS--H].sup.-.
Example 71
##STR00237##
[0691] (i) Preparation of 71:
(4aS,6aS,6bR,13aR)-12-Amino-N-(5-amino-1,3,4-thiadiazol-2-yl)-2,2,6a,6b,9-
,9,13a-heptamethyl-2,3,4,4a,5,6,6a,6b,7,8,8a,9,11,13,13a,13b,14,15b-octade-
cahydro-1H-chryseno[1,2-f]indazole-4-a-carboxamide
[0692] To a solution of III (500 mg, 0.97 mmol) and pyridine (4 mL)
was added 1,3,4-thiadiazole-2,5-diamine (340 mg, 2.92 mmol). The
reaction mixture was heated at 60.degree. C. for 20 hours and then
poured into H.sub.2O (50 mL). The precipitate was collected by
filtration and dissolved in CH.sub.2Cl.sub.2/i-PrOH (2:1, 20 mL).
The aqueous layer was extracted with CH.sub.2Cl.sub.2/i-PrOH (2:1,
2.times.25 mL). The combined extracts were dried
(Na.sub.2SO.sub.4), filtered and concentrated under reduced
pressure. The residue was purified by column chromatography
(silica, 0-40% CMA in CH.sub.2Cl.sub.2) followed by preparative
HPLC to afford the title compound (32 mg, 6%) as a solid.
[0693] R.sub.f 0.20 (90:9:1 Methylene
Chloride/Methanol/concentrated Ammonium Hydroxide)
[0694] .sup.1H NMR (300 MHz, DMSO-d.sub.6) .delta. 0.77-2.43 (m,
41H), 2.94 (d, J=12 Hz, 1H), 5.32 (s, 1H), 7.46 (s, 1H), 11.50 (s,
1H), 13.20 (s, 1H).
[0695] APCI MS m/z 592 [C.sub.33H.sub.49N.sub.7OS+H].sup.+. m.p.
280-300.degree. C. dec. HPLC (Method A) 95.4% (214 nm) t.sub.R=10.2
min.
Example 72
##STR00238##
[0696] (i) Preparation of 72:
(4aS,6aS,6bR,8aR,13aR,15bS)-4-a-(5-Amino-1-methyl-1H-pyrazol-3-yl)-2,2,6a-
,6b,9,9,13a-heptamethyl-2,3,4,4a,5,6,6a,6b,7,8,8a,9,11,13,13a,13b,14,15b-o-
ctadecahydro-1H-chryseno[1,2-f]indazol-12-amine
[0697] A mixture of 60b (282 mg, 0.54 mmol) and methylhydrazine
(0.14 mL, 2.73 mmol) in EtOH (4 mL) was sealed and heated to
160.degree. C. by microwave for 3 hours. The reaction mixture was
concentrated to dryness. The residue was purified by column
chromatography (silica, 0-10% MeOH in CH.sub.2Cl.sub.2) followed by
preparative HPLC to afford title compound (35 mg, 12%) as a white
solid.
[0698] R.sub.f 0.34 (89:10:1 Methylene
Chloride/Methanol/concentrated Ammonium Hydroxide).
[0699] .sup.1H NMR (300 MHz, CD.sub.3OD) .delta. 0.65 (s, 3H), 0.88
(s, 3H), 0.95 (s, 3H), 0.97 (s, 3H), 1.15 (s, 3H), 1.20 (s, 3H),
1.24 (s, 3H), 1.25-2.10 (m, 19H), 2.38 (m, 2H), 2.78 (m, 1H), 3.68
(s, 3H), 5.47 (m, 1H). mp 195-210.degree. C. dec. APCI MS (Positive
Mode) m/z 545 [C.sub.34H.sub.52N.sub.6+H].sup.+.
Example 73
##STR00239##
[0700] (i) Preparation of 73:
(2-Amino-1H-imidazol-1-yl)((4aS,6aS,6bR,13aR)-12-amino-2,2,6a,6b,9,9,13a--
heptamethyl-2,3,4,4a,5,6,6a,6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecahydro-
-1H-chryseno[1,2-f]indazol-4-a-yl)methanone
[0701] To a solution of III (200 mg, 0.39 mmol) and pyridine (2 mL)
was added 1H-imidazol-2-amine (70 mg, 0.84 mmol). The reaction
mixture was stirred at room temperature for 3.5 hours and then
poured into H.sub.2O (20 mL). The precipitate was collected by
filtration and dissolved in CH.sub.2Cl.sub.2/i-PrOH (2:1, 10 mL).
The aqueous layer was extracted with CH.sub.2Cl.sub.2/i-PrOH (2:1,
2.times.10 mL). The combined organics were dried
(Na.sub.2SO.sub.4), filtered and concentrated under reduced
pressure. The residue was purified by column chromatography
(silica, 0-30% CMA in CH.sub.2Cl.sub.2) to afford the title
compound (96 mg, 44%).
[0702] R.sub.f 0.50 (90:9:1 Methylene
Chloride/Methanol/concentrated Ammonium Hydroxide).
[0703] .sup.1H NMR (300 MHz, DMSO-d.sub.6) .delta. 0.91-2.49 (m,
41H), 3.00 (d, J=11.1 Hz, 1H), 4.12 (s, 2H), 5.29 (s, 1H), 6.46 (s,
1H), 6.64 (s, 2H), 7.39 (s, 1H), 10.95 (s, 1H). APCI MS m/z 559
[C.sub.34H.sub.50N.sub.6O+H].sup.+. m.p. 230-250.degree. C. dec.
HPLC (Method A) 97.6% (214 nm) t.sub.R=9.9 min.
Example 74
##STR00240##
[0704] (i) Preparation of 74:
(4aS,6aS,6bR,13aR)-12-Amino-N-(5-mercapto-1,3,4-thiadiazol-2-yl)-2,2,6a,6-
b,9,9,13a-heptamethyl-2,3,4,4a,5,6,6a,6b,7,8,8a,9,11,13,13a,13b,14,15b-oct-
adecahydro-1H-chryseno[1,2-f]indazole-4-a-carboxamide
[0705] To a solution of 5-amino-1,3,4-thiadiazole-2-thiol (200 mg,
2.1 mmol) prepared by the procedure described in the literature
(US3940409, 1976) and pyridine (10.0 mL) was added III (215 mg,
0.42 mmol). The mixture was stirred at room temperature overnight.
The reaction mixture was concentrated to dryness under reduced
pressure. The residue was purified by column chromatography
(silica, 0-70% CMA in CH.sub.2Cl.sub.2) followed by preparative
HPLC to afford the title compound (30 mg, 12%) as an off-white
solid.
[0706] R.sub.f 0.60 (80:18:2 Methylene
Chloride/Methanol/concentrated Ammonium Hydroxide).
[0707] .sup.1H NMR (300 MHz, CD.sub.3OD) .delta. 0.89 (s, 3H), 0.90
(s, 3H), 0.92 (s, 3H), 0.97 (s, 3H), 1.29 (s, 6H), 1.36 (s, 3H),
1.43-2.30 (m, 19H), 2.43 (d, J=14.7 Hz, 1H), 2.84 (m, 1H), 5.42 (s,
1H). mp>300.degree. C. ESI MS (Positive Mode) m/z 609
[C.sub.33H.sub.48N.sub.6OS.sub.2+H].sup.+.
Example 75
##STR00241##
[0708] (i) Preparation of 75b:
2-((4aS,6aS,6bR,13aR)-12-Amino-2,2,6a,6b,9,9,13a-heptamethyl-2,3,4,4a,5,6-
,6a,6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecahydro-1H-chryseno[1,2-f]indaz-
ole-4-a-carbonyl)hydrazinecarbothioamide
[0709] To a solution of hydrazinecarbothioamide (914 mg, 10.0 mmol)
and pyridine (50.0 mL) was added III (1.0 g, 2.0 mmol). The mixture
was stirred at room temperature for 2 hours. The reaction mixture
was poured into H.sub.2O. Brown precipitates were collected by
filtration and washed with H.sub.2O, dried in an oven at 40.degree.
C. to provide the sub-title compound (900 mg). The crude compound
was used without further purification.
[0710] ESI MS (Positive Mode) m/z 567
[C.sub.32H.sub.50N.sub.6OS+H].sup.+.
(ii) Preparation of 75:
5-((4aS,6aS,6bR,13aR)-12-Amino-2,2,6a,6b,9,9,13a-heptamethyl-2,3,4,4a,5,6-
,6a,6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecahydro-1H-chryseno[1,2-f]indaz-
ol-4-a-yl)-4H-1,2,4-triazole-3-thiol
[0711] A mixture of 75b (100 mg) and NaOH (2 M, 2.0 mL) was heated
at reflux for 7 d. The reaction mixture was concentrated to dryness
under reduced pressure. The residue was triturated with MeOH and
CH.sub.2Cl.sub.2. The filtrate was concentrated and purified by
preparative HPLC to provide the title compound (61 mg, 66%) as an
off-white solid.
[0712] R.sub.f 0.60 (180:18:2 Methylene
Chloride/Methanol/concentrated Ammonium Hydroxide).
[0713] .sup.1H NMR (300 MHz, CD.sub.3OD) .delta. 0.60 (s, 3H), 0.82
(s, 3H), 0.95 (s, 3H), 0.98 (s, 3H), 1.21 (s, 3H), 1.25 (s, 3H),
1.46 (s, 3H), 1.50-2.05 (m, 18H), 2.25 (m, 1H), 2.40 (d, J=15.0 Hz,
1H), 2.98 (m, 1H), 5.40 (s, 1H). mp>300.degree. C. ESI MS
(Positive Mode) m/z 549 [C.sub.32H.sub.48N.sub.6S+H].sup.+.
Example 76
##STR00242##
[0714] (i) Preparation of 76:
((4aS,6aS,6bR,13aR)-12-Amino-2,2,6a,6b,9,9,13a-heptamethyl-2,3,4,4a,5,6,6-
a,6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecahydro-1H-chryseno[1,2-f]indazol-
-4-a-yl)(3-mercapto-1H-1,2,4-triazol-1-yl)methanone
[0715] To a solution of III (300 mg, 0.58 mmol) and pyridine (3 mL)
was added 1H-1,2,4-triazole-3-thiol (177 mg, 1.75 mmol). The
reaction mixture was stirred at room temperature for 1 hour and
then poured into H.sub.2O (30 mL). The precipitate was dissolved in
CH.sub.2Cl.sub.2/i-PrOH (2:1, 20 mL). The aqueous layer was
extracted with CH.sub.2Cl.sub.2/i-PrOH (2:1, 3.times.10 mL) and the
combined organics were dried (Na.sub.2SO.sub.4), filtered and
concentrated under reduced pressure. The residue was purified by
column chromatography (silica, 0-8% MeOH in CH.sub.2Cl.sub.2) to
afford the title compound (110 mg, 33%).
[0716] R.sub.f 0.27 (90:9:1 Methylene
Chloride/Methanol/concentrated Ammonium Hydroxide)
[0717] .sup.1H NMR (300 MHz, CD.sub.3OD) .delta. 0.89-2.23 (m,
40H), 2.41 (d, J=14.7 Hz, 1H), 3.00 (dd, J=3.6, 12.9 Hz, 1H), 5.40
(s, 1H), 8.46 (s, 1H). APCI MS m/z 577
[C.sub.33H.sub.48N.sub.6OS+H].sup.+. m.p. 260-280.degree. C. dec.
HPLC (Method A) 96.2% (214 nm) t.sub.R=14.3 min.
Example 77
##STR00243##
[0718] (i) Preparation of 77:
(4aS,6aS,6bR,13aR)-12-Amino-N-(3-mercapto-5-methyl-4H-1,2,4-triazol-4-yl)-
-2,2,6a,6b,9,9,13a-heptamethyl-2,3,4,4a,5,6,6a,6b,7,8,8a,9,11,13,13a,13b,1-
4,15b-octadecahydro-1H-chryseno[1,2-f]indazole-4-a-carboxamide
[0719] To a solution of III (300 mg, 0.58 mmol) and pyridine (3 mL)
was added 4-amino-5-methyl-4H-1,2,4-triazole-3-thiol (228 mg, 1.75
mmol). The reaction mixture was stirred at room temperature for 1
hour and then poured into H.sub.2O (30 mL). The precipitate was
collected by filtration and dried under vacuum overnight. The
residue was purified by column chromatography (silica, 0-30% CMA in
CH.sub.2Cl.sub.2) followed by preparative HPLC to afford the title
compound (30 mg, 8%) as a solid.
[0720] R.sub.f 0.25 (90:9:1 Methylene
Chloride/Methanol/concentrated Ammonium Hydroxide)
[0721] .sup.1H NMR (300 MHz, CD.sub.3OD) .delta. 0.89-2.50 (m,
44H), 2.93-2.97 (m, 1H), 5.36 (s, 1H). APCI MS m/z 606
[C.sub.34H.sub.51N.sub.7OS+H].sup.+. m.p. 250-270.degree. C. dec.
HPLC (Method A) 92.7% (214 nm) t.sub.R=13.2 min.
Example 78
##STR00244##
[0722] (i) Preparation of 78b:
(4aS,6aS,6bR,13aR)-12-Amino-2,2,6a,6b,9,9,13a-heptamethyl-2,3,4,4a,5,6,6a-
,6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecahydro-1H-chryseno[1,2-f]indazole-
-4-a-carbohydrazide
[0723] To a solution of hydrazine (0.15 mL, 4.5 mmol) and
triethylamine (0.77 mL) was added III (500 mg, 0.91 mmol). The
mixture was stirred at room temperature overnight. The reaction
mixture was diluted with EtOAc (100 mL). The organic phase was
washed with brine then dried (MgSO.sub.4), filtered and
concentrated to dryness. The residue was purified by column
chromatography on (silica, 0-50% CMA in CH.sub.2Cl.sub.2) to afford
the sub-title compound (360 mg, 78%) as a brown solid.
(ii) Preparation of 78:
5-((4aS,6aS,6bR,13aR)-12-Amino-2,2,6a,6b,9,9,13a-heptamethyl-2,3,4,4a,5,6-
,6a,6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecahydro-1H-chryseno[1,2-f]indaz-
ol-4-a-yl)-1,3,4-thiadiazol-2-ol
[0724] To a solution of 78b (140 mg, 0.27 mmol) in CHCl.sub.3 (5
mL) was added carbon disulfide (0.58 mL, 0.96 mmol). The mixture
was stirred at room temperature for 4 hours. The reaction mixture
was concentrated to dryness under reduced pressure. The residue was
dissolved in EtOH (5 mL) and NaOH (2 M, 5 mL) was added. The
mixture was heated at reflux for 12 hours. The reaction mixture was
concentrated to remove EtOH. Brown solids were collected by
filtration and washed with H.sub.2O. The residue was purified by
column chromatography (silica, 0-50% CMA in CH.sub.2Cl.sub.2)
followed by preparative HPLC to afford the title compound (30 mg,
12%) as an off-white solid.
[0725] R.sub.f 0.30 (10:1 Methylene Chloride/Methanol).
[0726] .sup.1H NMR (300 MHz, DMSO-d.sub.6) .delta. 0.58 (s, 3H),
0.75 (s, 3H), 0.92 (s, 6H), 1.16 (s, 3H), 1.21 (s, 3H), 1.26 (s,
3H), 1.31-1.97 (m, 22H), 2.22 (m, 1H), 2.43 (d, J=15.0 Hz, 1H),
2.83 (m, 1H), 5.35 (s, 1H). mp>300.degree. C. ESI MS (Positive
Mode) m/z 550 [C.sub.32H.sub.47N.sub.5OS+H].sup.+.
Example 79
##STR00245##
[0727] (i) Preparation of 79:
(4aS,6aS,6bR,13aR)-12-Amino-N-(3-mercapto-1H-pyrazol-5-yl)-2,2,6a,6b,9,9,-
13a-heptamethyl-2,3,4,4a,5,6,6a,6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecah-
ydro-1H-chryseno[1,2-f]indazole-4-a-carboxamide
[0728] To a solution of 5-amino-1H-pyrazole-3-thiol (459 mg, 4.0
mmol) prepared by the procedure described in the literature (J.
Med. Chem. 2008, 51(15), 4672-4684) and pyridine (10.0 mL) was
added III (490 mg, 0.95 mmol). The mixture was stirred at room
temperature overnight. The reaction mixture was concentrated to
dryness under reduced pressure. The residue was purified by column
chromatography (silica, 0-70% CMA in CH.sub.2Cl.sub.2) followed by
preparative HPLC to afford the title compound (115 mg, 17%) as an
off-white solid.
[0729] R.sub.f 0.75 (80:18:2 Methylene
Chloride/Methanol/concentrated Ammonium Hydroxide).
[0730] .sup.1H NMR (300 MHz, CD.sub.3OD) .delta. 0.89 (s, 6H), 0.91
(s, 3H), 0.95 (s, 3H), 1.28 (s, 3H), 1.40 (s, 3H), 1.48 (s, 3H),
1.55-2.20 (m, 19H), 2.43 (d, J=14.4 Hz, 1H), 2.93 (m, 1H), 5.39 (m,
1H), 5.57 (m, 1H). mp>300.degree. C. ESI MS (Positive Mode) m/z
591 [C.sub.34H.sub.50N.sub.6OS+H].sup.+.
Example 80
##STR00246##
[0731] (i) Preparation of 80b:
5-Amino-1,3,4-thiadiazole-2-sulfonamide
[0732] To a solution of
N-(5-sulfamoyl-1,3,4-thiadiazol-2-yl)acetamide (1.0 g, 4.50 mmol)
and MeOH (15 mL) was added HCl (2 N, 10 mL). The reaction mixture
was heated at reflux for 18 hours and then concentrated under
reduced pressure. The residue was purified by column chromatography
(silica, 0-10% MeOH in CMA) to afford the sub-title compound (800
mg, 98%).
(ii) Preparation of 80:
(4aS,6aS,6bR,13aR)-12-Amino-2,2,6a,6b,9,9,13a-heptamethyl-N-(5-sulfamoyl--
1,3,4-thiadiazol-2-yl)-2,3,4,4a,5,6,6a,6b,7,8,8a,9,11,13,13a,13b,14,15b-oc-
tadecahydro-1H-chryseno[1,2-f]indazole-4-a-carboxamide
[0733] To a solution of III (400 mg, 0.78 mmol), pyridine (15 mL)
and DMF (1 mL) was added 80b (281 mg, 1.56 mmol). The reaction
mixture was stirred at room temperature for 24 hours and then
poured into H.sub.2O (15 mL). The precipitate was collected by
filtration and dried under vacuum overnight. The residue was
purified by column chromatography (silica, 0-60% CMA in
CH.sub.2Cl.sub.2) followed by preparative HPLC to afford the title
(8 mg, 2%) as a solid.
[0734] R.sub.f 0.35 (90:9:1 Methylene
Chloride/Methanol/concentrated Ammonium Hydroxide).
[0735] .sup.1H NMR (300 MHz, Acetone-d.sub.6) .delta. 0.68-2.27 (m,
40H), 2.45 (d, J=15 Hz, 1H), 3.05-3.08 (m, 1H), 5.47 (s, 1H). APCI
MS m/z 656 [C.sub.33H.sub.49N.sub.7O.sub.3S.sub.2+H].sup.+. m.p.
280-300.degree. C. dec.
[0736] HPLC (Method A)>99% (214 nm) t.sub.R=14.0 min.
Example 81
##STR00247##
[0737] (i) Preparation of 81b:
3-((4aS,6aS,6bR,13aR)-12-Amino-2,2,6a,6b,9,9,13a-heptamethyl-2,3,4,4a,5,6-
,6a,6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecahydro-1H-chryseno[1,2-f]indaz-
ole-4-a-carbonyl)dihydrofuran-2(3H)-one
[0738] To a solution of lithium bis(trimethylsilyl)amide (2.3 mL, 1
M in THF, 2.3 mmol) was added dihydrofuran-2(3H)-one (0.15 mL, 1.9
mmol) at -78.degree. C. The mixture was stirred for 15 min. The
anion solution was added to a solution of III (100 mg, 0.19 mmol)
in THF (7 mL) precooled to -78.degree. C. The mixture was stirred
at -78.degree. C. for 30 min and deemed incomplete. To a second
solution of lithium bis(trimethylsilyl)amide (1.1 mL, 1 M in THF,
1.1 mmol) was added dihydrofuran-2(3H)-one (0.075 mL, 1.0 mmol) at
-78.degree. C. The second anion mixture was stirred for 15 min. The
anion solution was added to the above reaction mixture at
-78.degree. C. The mixture was continued to stir at -78.degree. C.
for 30 min. The reaction mixture was quenched with saturated
NH.sub.4Cl (60 mL) and extracted with EtOAc (100 mL). The organic
phase was washed with brine, dried (MgSO.sub.4), filtered and
concentrated to dryness. The residue was purified by column
chromatography (silica, 0-30% CMA in CH.sub.2Cl.sub.2) to afford
the sub-title compound (150 mg) as a brown solid.
[0739] ESI MS (Positive Mode) m/z 562
[C.sub.35H.sub.51N.sub.3O.sub.3+H].sup.+.
(ii) Preparation of 81:
3-((4aS,6aS,6bR,13aR)-12-Amino-2,2,6a,6b,9,9,13a-heptamethyl-2,3,4,4a,5,6-
,6a,6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecahydro-1H-chryseno[1,2-f]indaz-
ol-4-a-yl)-4-(2-hydroxyethyl)-1H-pyrazol-5-ol
[0740] A mixture of 81b (150 mg) and hydrazine (0.037 mL) in EtOH
(2 mL) was heated at reflux overnight. The reaction mixture was
concentrated to dryness under reduced pressure. The residue was
purified by column chromatography (silica, 0-60% CMA in
CH.sub.2Cl.sub.2) followed by preparative HPLC to afford the title
compound (12 mg, 8%) as an off-white solid.
[0741] R.sub.f 0.70 (80:18:2 Methylene
Chloride/Methanol/concentrated Ammonium Hydroxide).
[0742] .sup.1H NMR (300 MHz, CD.sub.3OD) .delta. 0.50 (s, 3H), 0.86
(s, 3H), 0.95 (s, 3H), 0.98 (s, 3H), 1.23 (s, 3H), 1.28 (s, 3H),
1.32 (s, 3H), 1.35-2.08 (m, 18H), 2.25 (m, 1H), 2.36 (d, J=15.0 Hz,
1H), 2.74 (m, 2H), 2.82 (m, 1H), 3.72 (t, J=6.6 Hz, 2H), 5.51 (s,
1H). mp 251-255.degree. C. APCI MS (Positive Mode) m/z 576
[C.sub.35H.sub.53N.sub.5O.sub.2+H].sup.+.
Example 82
##STR00248##
[0743] (i) Preparation of 82b: Ethyl
3-((4aS,6aS,6bR,13aR)-12-amino-2,2,6a,6b,9,9,13a-heptamethyl-2,3,4,4a,5,6-
,6a,6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecahydro-1H-chryseno[1,2-f]indaz-
ol-4-a-yl)-2-methyl-3-oxopropanoate
[0744] To a solution of lithium bis(trimethylsilyl)amide (3.8 mL, 1
M in THF, 3.8 mmol) was added ethyl propionate (0.22 mL, 1.9 mmol).
The mixture was stirred for 30 min. The anion solution was added to
a solution of III (100 mg, 0.19 mmol) in THF (3 mL) precooled to
-78.degree. C. The mixture was stirred at -78.degree. C. for 1 hour
and the reaction was deemed incomplete. To a second solution of
lithium bis(trimethylsilyl)amide (1.9 mL, 1 M in THF, 1.9 mmol) was
added ethyl propionate (0.33 mL, 1.9 mmol). The second anion
mixture was stirred for 30 min. The anion solution was added to the
above reaction mixture at -78.degree. C. The mixture was continued
to stir at -78.degree. C. for 30 min. The reaction mixture was
quenched with saturated NH.sub.4Cl (20 mL) and extracted with EtOAc
(100 mL). The organic phase was washed brine, dried (MgSO.sub.4),
filtered and concentrated to dryness. The residue was purified by
column chromatography (silica, 0-30% CMA in CH.sub.2Cl.sub.2) to
afford the sub-title compound (100 mg) as a brown solid.
[0745] APCI MS (Positive Mode) m/z 578
[C.sub.36H.sub.55N.sub.3O.sub.3+H].sup.+.
(ii) Preparation of 82:
3-((4aS,6aS,6bR,13aR)-12-Amino-2,2,6a,6b,9,9,13a-heptamethyl-2,3,4,4a,5,6-
,6a,6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecahydro-1H-chryseno[1,2-f]indaz-
ol-4-a-yl)-4-methyl-1H-pyrazol-5-ol
[0746] A mixture of 82b (100 mg) and hydrazine (0.06 mL) in EtOH (2
mL) was heated at reflux overnight. The reaction mixture was
concentrated to dryness under reduced pressure. The residue was
purified by column chromatography (silica, 0-50% CMA in
CH.sub.2Cl.sub.2) followed by preparative HPLC to the title
compound (7 mg, 7%) as an off-white solid.
[0747] R.sub.f 0.70 (80:18:2 Methylene
Chloride/Methanol/concentrated Ammonium Hydroxide).
[0748] .sup.1H NMR (300 MHz, CD.sub.3OD) .delta. 0.55 (s, 3H), 0.85
(s, 3H), 0.96 (s, 3H), 1.00 (s, 3H), 1.19 (s, 3H), 1.23 (s, 3H),
1.28 (s, 3H), 1.32-2.08 (m, 21H), 2.39 (m, 1H), 2.44 (d, J=15.0 Hz,
1H), 2.90 (m, 1H), 5.48 (s, 1H). mp>300.degree. C. APCI MS
(Positive Mode) m/z 546 [C.sub.34H.sub.51N.sub.5O+H].sup.+.
Example 83
##STR00249##
[0749] (i) Preparation of 83b: Ethyl
3-((4aS,6aS,6bR,13aR)-12-amino-2,2,6a,6b,9,9,13a-heptamethyl-2,3,4,4a,5,6-
,6a,6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecahydro-1H-chryseno[1,2-f]indaz-
ol-4-a-yl)-2-fluoro-3-oxopropanoate
[0750] To a solution of lithium bis(trimethylsilyl)amide (5.9 mL, 1
M in THF, 5.9 mmol) was added ethyl 2-bromo-2-fluoroacetate (0.46
mL, 3.9 mmol) at -78.degree. C. The mixture was stirred for 10 min.
The anion solution was added to a solution of III (200 mg, 0.39
mmol) in THF (4 mL) precooled to -78.degree. C. The mixture was
stirred at -78.degree. C. for 1 hour and the reaction was deemed
incomplete. To a second solution of n-butyllithium (1.8 mL, 2.5 M
in hexanes, 5.9 mmol) was added ethyl 2-bromo-2-fluoroacetate (0.46
mL, 3.9 mmol) at -78.degree. C. The second anion mixture was
stirred for 10 min. The anion solution was added to the above
reaction mixture at -78.degree. C. The mixture was stirred at
-78.degree. C. for 1 hour and warmed to -20.degree. C. over 1 hour.
The reaction mixture was quenched with saturated NH.sub.4Cl (20 mL)
and extracted with EtOAc (100 mL). The organic phase was washed
with brine, dried (MgSO.sub.4), filtered and concentrated to
dryness. The residue was purified by column chromatography (silica,
0-30% CMA in CH.sub.2Cl.sub.2) to afford the sub-title compound
(110 mg) as a brown solid.
[0751] APCI MS (Positive Mode) m/z 582
[C.sub.35H.sub.52FN.sub.3O.sub.3+H].sup.+.
(ii) Preparation of 83:
3-((4aS,6aS,6bR,13aR)-12-Amino-2,2,6a,6b,9,9,13a-heptamethyl-2,3,4,4a,5,6-
,6a,6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecahydro-1H-chryseno[1,2-f]indaz-
ol-4-a-yl)-4-fluoro-1H-pyrazol-5-ol
[0752] A mixture of 83b (110 mg) and hydrazine (0.05 mL) in EtOH (2
mL) was heated at reflux overnight. The reaction mixture was
concentrated to dryness under reduced pressure. The residue was
purified by column chromatography (silica, 0-50% CMA in
CH.sub.2Cl.sub.2) followed by preparative HPLC to afford the title
compound (9 mg, 10%) as an off-white solid.
[0753] R.sub.f 0.50 (80:18:2 Methylene
Chloride/Methanol/concentrated Ammonium Hydroxide).
[0754] .sup.1H NMR (300 MHz, CD.sub.3OD) .delta. 0.52 (s, 3H), 0.85
(s, 3H), 0.96 (s, 3H), 1.00 (s, 3H), 1.19 (s, 3H), 1.23 (s, 3H),
1.28 (s, 3H), 1.32-2.08 (m, 18H), 2.39 (m, 1H), 2.40 (d, J=15.0 Hz,
1H), 2.90 (m, 1H), 5.42 (s, 1H). mp>300.degree. C. APCI MS
(Positive Mode) m/z 550 [C.sub.33H.sub.48FN.sub.5O+H].sup.+.
Example 84
##STR00250##
[0755] (i) Preparation of 84b:
3-((4aS,6aS,6bR,13aR)-12-Amino-2,2,6a,6b,9,9,13a-heptamethyl-2,3,4,4a,5,6-
,6a,6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecahydro-1H-chryseno[1,2-f]indaz-
ole-4-a-carbonyl)-1-vinylpyrrolidin-2-one
[0756] To a solution of lithium bis(trimethylsilyl)amide (38 mL, 1
M in THF, 38.0 mmol) was added 1-vinyl-2-pyrrolidinone (2.1 mL,
19.0 mmol) at -78.degree. C. The mixture was stirred for 10 min.
The anion solution was added to a solution of III (1.0 g, 1.9 mmol)
in THF (20 mL) precooled to -78.degree. C. The mixture was stirred
at -78.degree. C. for 1 hour. The reaction mixture was quenched
with saturated NH.sub.4Cl (60 mL) and extracted with EtOAc (100
mL). The organic phase was washed with brine, dried (MgSO.sub.4),
filtered and concentrated to dryness. The residue was purified by
column chromatography (silica, 0-10% MeOH in CH.sub.2Cl.sub.2) to
afford the sub-title compound (350 mg) as a brown solid.
[0757] APCI MS (Positive Mode) m/z 587
[C.sub.37H.sub.54N.sub.4O.sub.2+H].sup.+.
(ii) Preparation of 84:
3-((4aS,6aS,6bR,13aR)-12-Amino-2,2,6a,6b,9,9,13a-heptamethyl-2,3,4,4a,5,6-
,6a,6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecahydro-1H-chryseno[1,2-f]indaz-
ol-4-a-yl)-4-(2-aminoethyl)-1H-pyrazol-5-ol
[0758] A mixture of 84b (100 mg) and hydrazine (0.05 mL) in EtOH (2
mL) was sealed and heated to 160.degree. C. by microwave for 3
hours. The reaction mixture was concentrated to dryness under
reduced pressure. The residue was purified by column chromatography
(silica, 0-80% CMA in CH.sub.2Cl.sub.2) to afford the title
compound (50 mg, 30%) as a brown solid.
[0759] R.sub.f 0.27 (80:18:2 Methylene
Chloride/Methanol/concentrated Ammonium Hydroxide).
[0760] .sup.1H NMR (300 MHz, CD.sub.3OD) .delta. 0.50 (s, 3H), 0.86
(s, 3H), 0.95 (s, 3H), 0.98 (s, 3H), 1.09 (s, 3H), 1.21 (s, 6H),
1.35-2.40 (m, 21H), 2.72 (m, 2H), 2.96 (m, 2H), 5.47 (s, 1H). mp
250-260.degree. C. dec. APCI MS (Positive Mode) m/z 575
[C.sub.35H.sub.54N.sub.6O+H].sup.+.
Example 85 and Example 86
##STR00251##
[0761] (i) Preparation of 85 and 86:
3-((4aS,6aS,6bR,13aR)-12-Amino-2,2,6a,6b,9,9,13a-heptamethyl-2,3,4,4a,5,6-
,6a,6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecahydro-1H-chryseno[1,2-f]indaz-
ol-4-a-yl)-4-(2-(dimethylamino)ethyl)-1H-pyrazol-5-ol and
3-((4aS,6aS,6bR,13aR)-12-Amino-2,2,6a,6b,9,9,13a-heptamethyl-2,3,4,4a,5,6-
,6a,6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecahydro-1H-chryseno[1,2-f]indaz-
ol-4-a-yl)-4-(2-(methylamino)ethyl)-1H-pyrazol-5-ol
[0762] To a solution of 84 (100 mg, 0.17 mmol) in MeOH (8 mL) was
added 37% formaldehyde (0.021 mL, 0.36 mmol) in an ice bath. The
mixture was stirred for 5 min. Sodium borohydride (32 mg, 0.84
mmol) was added. The reaction mixture was stirred at 0.degree. C.
for 1 hour and concentrated to dryness. The residue was purified by
column chromatography (silica, 0-80% CMA in CH.sub.2Cl.sub.2) to
afford 85 (35 mg, 20%) as an off-white solid and 86 (18 mg, 20%) as
an off-white solid.
Data for 85:
[0763] R.sub.f 0.35 (80:18:2 Methylene
Chloride/Methanol/concentrated Ammonium Hydroxide).
[0764] .sup.1H NMR (300 MHz, CD.sub.3OD) .delta. 0.50 (s, 3H), 0.86
(s, 3H), 0.95 (s, 3H), 0.98 (s, 3H), 1.15 (s, 3H), 1.21 (s, 6H),
1.35-2.35 (m, 20H), 2.35 (s, 6H), 2.67 (m, 4H), 2.90 (m, 1H), 5.48
(s, 1H). mp 260-270.degree. C. dec. APCI MS (Positive Mode) m/z 603
[C.sub.37H.sub.58N.sub.6O+H].sup.+.
Data for 86:
[0765] R.sub.f 0.30 (80:18:2 Methylene
Chloride/Methanol/concentrated Ammonium Hydroxide).
[0766] .sup.1H NMR (300 MHz, CD.sub.3OD) .delta. 0.50 (s, 3H), 0.86
(s, 3H), 0.95 (s, 3H), 0.98 (s, 3H), 1.15 (s, 3H), 1.21 (s, 6H),
1.35-2.08 (m, 20H), 2.30 (s, 2H), 2.71 (m, 3H), 2.83 (m, 3H), 5.49
(s, 1H). mp 260-270.degree. C. dec. APCI MS (Positive Mode) m/z 589
[C.sub.36H.sub.56N.sub.6O+H].sup.+.
Example 87
##STR00252##
[0767] (i) Preparation of 87:
1-(2-(3-((4aS,6aS,6bR,13aR)-12-Amino-2,2,6a,6b,9,9,13a-heptamethyl-2,3,4,-
4a,5,6,6a,6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecahydro-1H-chryseno[1,2-f-
]indazol-4-a-yl)-5-hydroxy-1H-pyrazol-4-yl)ethyl)urea
[0768] To a solution of 84 (66 mg, 0.11 mmol) and acetic acid (0.2
mL) in THF (2 mL) and benzene (3 mL) was added potassium cyanate
(22 mg, 0.26 mmol). The mixture was stirred at room temperature for
12 hours. Sodium hydroxide (2 mL, 2.0 M) was added. The reaction
mixture was stirred at room temperature for 3 hours and
concentrated to dryness. The residue was purified by column
chromatography (silica, 0-10% MeOH in CMA) to afford the title
compound (7 mg, 10%) as a brown solid.
[0769] R.sub.f 0.50 (80:28:2 Methylene
Chloride/Methanol/concentrated Ammonium Hydroxide).
[0770] .sup.1H NMR (300 MHz, CD.sub.3OD) .delta. 0.51 (s, 3H), 0.87
(s, 3H), 0.96 (s, 3H), 1.00 (s, 3H), 1.12 (s, 3H), 1.28 (s, 3H),
1.32 (s, 3H), 1.35-2.12 (m, 20H), 2.28 (s, 1H), 2.30 (d, J=15.0 Hz,
1H), 2.60 (m, 2H), 2.85 (m, 1H), 5.52 (s, 1H). mp>300.degree. C.
APCI MS (Positive Mode) m/z 618
[C.sub.36H.sub.55N.sub.7O.sub.2+H].sup.+.
Example 88
##STR00253##
[0771] (i) Preparation of 88b: Methyl
3-((4aS,6aS,6bR,8aR,13aR,15bS)-12-amino-2,2,6a,6b,9,9,13a-heptamethyl-2,3-
,4,4a,5,6,6a,6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecahydro-1H-chryseno[1,-
2-f]indazol-4-a-yl)-2-morpholino-3-oxopropanoate
[0772] To a solution of lithium bis(trimethylsilyl)amide (15.6 mL,
1 M in THF, 15.6 mmol) was added methyl morpholinoacetate (1.8 g,
11.7 mmol) at -78.degree. C. The mixture was stirred for 10 min.
The anion solution was added to a solution of III (400 mg, 0.78
mmol) in THF (10 mL) precooled to -10.degree. C. The mixture was
stirred at -10.degree. C. for 1 hour. The reaction mixture was
quenched with saturated NH.sub.4Cl (60 mL) and extracted with EtOAc
(100 mL). The organic phase was washed with brine, dried
(MgSO.sub.4), filtered and concentrated to dryness. The residue was
purified by column chromatography (silica, 0-20% CMA in
CH.sub.2Cl.sub.2) to afford the sub-title compound (120 mg) as a
brown solid.
[0773] APCI MS (Positive Mode) m/z 635
[C.sub.38H.sub.58N.sub.4O.sub.4+H].sup.+.
(ii) Preparation of 88:
3-((4aS,6aS,6bR,13aR)-12-Amino-2,2,6a,6b,9,9,13a-heptamethyl-2,3,4,4a,5,6-
,6a,6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecahydro-1H-chryseno[1,2-f]indaz-
ol-4-a-yl)-4-morpholino-1H-pyrazol-5-ol
[0774] A mixture of 88b (120 mg) and hydrazine (0.05 mL) in n-BuOH
(2 mL) was heated at reflux overnight. The reaction mixture was
concentrated to dryness under reduced pressure. The residue was
purified by column chromatography (silica, 0-30% CMA in
CH.sub.2Cl.sub.2) to afford the title compound (6 mg, 5%) as a
brown solid.
[0775] R.sub.f 0.42 (80:18:2 Methylene
Chloride/Methanol/concentrated Ammonium Hydroxide).
[0776] .sup.1H NMR (300 MHz, CD.sub.3OD) .delta. 0.53 (s, 3H), 0.84
(s, 3H), 0.94 (s, 3H), 1.12 (s, 3H), 1.21 (s, 3H), 1.23 (s, 6H),
1.32-2.37 (m, 24H), 2.53 (m, 1H), 3.68 (m, 4H), 5.42 (s, 1H).
mp>300.degree. C. ESI MS (Positive Mode) m/z 617
[C.sub.37H.sub.56N.sub.6O.sub.2+H].sup.+.
Example 89
##STR00254##
[0777] (i) Preparation of 89b: tert-Butyl
2-(4-methylpiperazin-1-yl)acetate
[0778] To a suspension of 1-methylpiperazine dihydrochloride (8.5
g, 49.2 mmol) and triethylamine (22.8 mL, 164.0 mmol) in THF (150
mL) was added tert-butyl 2-bromoacetate (8.0 g, 41.0 mmol). The
mixture was stirred at room temperature for 12 hours. The reaction
mixture was diluted with EtOAc (200 mL) and H.sub.2O (100 mL). The
organic phase was washed with brine, dried (MgSO.sub.4), filtered
and concentrated to dryness. The residue was purified by column
chromatography (silica, 0-10% MeOH in CH.sub.2Cl.sub.2) to afford
the sub-title compound (6.0 g, 69%).
[0779] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 1.42 (s, 9H), 2.30
(s, 3H), 2.41-2.62 (m, 8H), 3.10 (s, 2H).
(ii) Preparation of 89c: tert-Butyl
3-((4aS,6aS,6bR,13aR)-12-amino-2,2,6a,6b,9,9,13a-heptamethyl-2,3,4,4a,5,6-
,6a,6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecahydro-1H-chryseno[1,2-f]indaz-
ol-4-a-yl)-2-(4-methylpiperazin-1-yl)-3-oxopropanoate
[0780] To a solution of lithium bis(trimethylsilyl)amide (15.6 mL,
1 M in THF, 15.6 mmol) was added 89b (2.5 g, 11.7 mmol) at
-78.degree. C. The mixture was stirred for 10 min. The anion
solution was added to a solution of III (400 mg, 0.78 mmol) in THF
(10 mL) precooled to -10.degree. C. The mixture was stirred at
0.degree. C. for 1.5 hours. The reaction mixture was quenched with
saturated NH.sub.4Cl (60 mL) and extracted with EtOAc (100 mL). The
organic phase was washed with brine, dried (MgSO.sub.4), filtered
and concentrated to dryness. The residue was purified by column
chromatography (silica, 0-20% CMA in CH.sub.2Cl.sub.2) to afford
the sub-title compound (200 mg) as a brown solid.
[0781] APCI MS (Positive Mode) m/z 690
[C.sub.42H.sub.67N.sub.5O.sub.3+H].sup.+.
(ii) Preparation of 89:
3-((4aS,6aS,6bR,13aR)-12-Amino-2,2,6a,6b,9,9,13a-heptamethyl-2,3,4,4a,5,6-
,6a,6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecahydro-1H-chryseno[1,2-f]indaz-
ol-4-a-yl)-4-(4-methylpiperazin-1-yl)-1H-pyrazol-5-ol
[0782] A mixture of 89c (100 mg) and hydrazine (0.02 mL) in n-BuOH
(2 mL) was heated at reflux overnight. The reaction mixture was
concentrated to dryness under reduced pressure. The residue was
purified by column chromatography (silica, 0-40% CMA in
CH.sub.2Cl.sub.2) to afford the title compound (16 mg, 18%) as a
brown solid.
[0783] R.sub.f 0.70 (80:18:2 Methylene
Chloride/Methanol/concentrated Ammonium Hydroxide).
[0784] .sup.1H NMR (400 MHz, CD.sub.3OD) .delta. 0.54 (s, 3H), 0.84
(s, 3H), 0.91 (s, 3H), 0.95 (s, 3H), 1.01 (s, 3H), 1.10 (s, 3H),
1.11-2.06 (m, 16H), 2.20 (m, 1H), 2.41 (m, 1H), 2.84 (s, 3H),
2.91-3.70 (m, 14H), 5.45 (s, 1H). mp>300.degree. C. APCI MS
(Positive Mode) m/z 630 [C.sub.38H.sub.59N.sub.7O+H].sup.+.
Example 90
##STR00255##
[0785] (i) Preparation of 90b: tert-Butyl
4-(2-tert-butoxy-2-oxoethyl)piperazine-1-carboxylate
[0786] To a suspension of tert-butyl piperazine-1-carboxylate (3.0
g, 16.1 mmol) and triethylamine (4.5 mL, 32.2 mmol) in THF (80 mL)
was added tert-butyl 2-bromoacetate (2.4 mL, 16.1 mmol). The
mixture was stirred at room temperature for 12 hours. The reaction
mixture was diluted with EtOAc (200 mL) and H.sub.2O (100 mL). The
organic phase was washed with brine, dried (MgSO.sub.4), filtered
and concentrated to dryness. The residue was purified by column
chromatography (silica, 0-5% MeOH in CH.sub.2Cl.sub.2) to afford
the sub-title compound (4.2 g, 87%).
[0787] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 1.42 (s, 18H),
2.50 (m, 4H), 3.10 (s, 2H), 3.47 (m, 4H).
(ii) Preparation of 90c: tert-Butyl
4-(1-((4aS,6aS,6bR,13aR)-12-amino-2,2,6a,6b,9,9,13a-heptamethyl-2,3,4,4a,-
5,6,6a,6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecahydro-1H-chryseno[1,2-f]in-
dazol-4-a-yl)-3-tert-butoxy-1,3-dioxopropan-2-yl)piperazine-1-carboxylate
[0788] To a solution of lithium bis(trimethylsilyl)amide (19.4 mL,
1 M in THF, 19.4 mmol) was added 90b (3.5 g, 11.7 mmol) at
-78.degree. C. The mixture was stirred for 10 min. The anion
solution was added to a solution of III (500 mg, 0.97 mmol) in THF
(10 mL) precooled to -78.degree. C. The mixture was stirred at
0.degree. C. for 1.5 hours. The reaction mixture was quenched with
saturated NH.sub.4Cl (60 mL) and extracted with EtOAc (100 mL). The
organic phase was washed brine, dried (MgSO.sub.4), filtered and
concentrated to dryness. The residue was purified by column
chromatography (silica, 0-20% CMA in CH.sub.2Cl.sub.2) to afford
the sub-title compound (237 mg) as a brown solid.
[0789] ESI MS (Positive Mode) m/z 776
[C.sub.46H.sub.73N.sub.5O.sub.5+H].sup.+.
(iii) Preparation of 90d: tert-Butyl
4-(3-((4aS,6aS,6bR,13aR)-12-amino-2,2,6a,6b,9,9,13a-heptamethyl-2,3,4,4a,-
5,6,6a,6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecahydro-1H-chryseno[1,2-f]in-
dazol-4-a-yl)-5-hydroxy-1H-pyrazol-4-yl)piperazine-1-carboxylate
[0790] A mixture of 90c (237 mg) and hydrazine (0.059 mL) in n-BuOH
(5 mL) was heated at reflux overnight. The reaction mixture was
concentrated to dryness under reduced pressure. The residue was
purified by column chromatography (silica, 0-40% CMA in
CH.sub.2Cl.sub.2) to afford the sub-title compound (15 mg, 7%) as a
brown solid.
(iv) Preparation of 90:
3-((4aS,6aS,6bR,13aR)-12-Amino-2,2,6a,6b,9,9,13a-heptamethyl-2,3,4,4a,5,6-
,6a,6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecahydro-1H-chryseno[1,2-f]indaz-
ol-4-a-yl)-4-(piperazin-1-yl)-1H-pyrazol-5-ol
[0791] To a solution of 90d (15 mg) in MeOH (2 mL) was added HCl
(0.8 mL, 2 M in diethyl ether) and stirred at room temperature for
5 hours. The reaction mixture was concentrated and purified by
preparative HPLC to provide the title compound (4.5 mg, 30%) as an
off-white solid.
[0792] R.sub.f 0.12 (80:18:2 Methylene
Chloride/Methanol/concentrated Ammonium Hydroxide).
[0793] .sup.1H NMR (400 MHz, CD.sub.3OD) .delta. 0.55 (s, 3H), 0.86
(s, 3H), 0.89 (s, 3H), 0.91 (s, 3H), 1.14 (s, 3H), 1.19 (s, 3H),
1.28 (s, 3H), 1.30-2.06 (m, 23H), 2.20 (m, 1H), 2.41 (d, J=15.0 Hz,
1H), 2.85-3.65 (m, 4H), 5.45 (s, 1H). mp>300.degree. C. APCI MS
(Positive Mode) m/z 616 [C.sub.37H.sub.57N.sub.7O+H].sup.+.
Example 91
##STR00256##
[0794] (i) Preparation of 91:
(4aS,6aS,6bR,8aR,13aR,15bS)--S-5-Amino-4H-1,2,4-triazol-3-yl
12-amino-2,2,6a,6b,9,9,13a-heptamethyl-15-(pyridin-3-yl)-2,3,4,4a,5,6,6a,-
6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecahydro-1H-chryseno[1,2-f]indazole--
4-a-carbothioate
[0795] To a solution of 12 (150 mg, 0.26 mmol) in CH.sub.2Cl.sub.2
(10 mL) was added thionyl chloride (0.19 mL, 2.6 mmol). The mixture
was stirred at room temperature for 2 hours. The reaction mixture
was concentrated to dryness under reduced pressure. The residue was
dissolved in pyridine (10 mL) and DMF (2 mL).
5-Amino-4H-1,2,4-triazole-3-thiol (71 mg, 0.60 mmol) was added and
stirred at room temperature for 1 hour. The reaction mixture was
concentrated to dryness under reduced pressure. The residue was
purified by column chromatography (silica, 0-50% CMA in
CH.sub.2Cl.sub.2) followed by preparative HPLC to afford the title
compound (19 mg, 14%) as an off-white solid.
[0796] R.sub.f 0.20 (80:18:2 Methylene
Chloride/Methanol/concentrated Ammonium Hydroxide).
[0797] .sup.1H NMR (300 MHz, CD.sub.3OD) .delta. 0.30 (s, 3H), 0.82
(s, 3H), 0.90 (s, 3H), 0.98 (s, 3H), 1.22 (s, 3H), 1.32 (s, 3H),
1.34 (s, 3H), 1.35-2.30 (m, 19H), 2.35 (d, J=15.0 Hz, 1H), 2.75 (m,
1H), 7.91 (dd, J=5.7, 7.5 Hz, 1H), 8.40 (d, J=7.8 Hz, 1H), 8.68 (d,
J=4.8 Hz, 1H), 8.79 (s, 1H). mp>300.degree. C. ESI MS (Positive
Mode) m/z 669 [C.sub.38H.sub.52N.sub.8OS+H].sup.+.
Example 92
##STR00257##
[0798] (i) Preparation of 92b:
3-((4aS,6aS,6bR,8aR,13aR,15bS)-12-Amino-2,2,6a,6b,9,9,13a-heptamethyl-15--
(2-(morpholinomethyl)furan-3-yl)-2,3,4,4a,5,6,6a,6b,7,8,8a,9,11,13,13a,13b-
,14,15b-octadecahydro-1H-chryseno[1,2-f]indazole-4-a-carbonyl)-1-vinylpyrr-
olidin-2-one
[0799] To a solution of 32 (250 mg, 0.37 mmol) in CH.sub.2Cl.sub.2
(10 mL) was added thionyl chloride (0.27 mL, 3.7 mmol). The mixture
was stirred at room temperature for 2 hours. The reaction mixture
was concentrated to dryness under reduced pressure. The residue was
dissolved in THF (5 mL) and added to a solution of
1-vinylpyrrolidin-2-one (0.39 mL, 3.7 mmol) and lithium
bis(trimethylsilyl)amide (7.4 mL, 1 M in THF, 7.4 mmol) in THF (5
mL) at -78.degree. C. The reaction mixture was stirred at
-78.degree. C. for 1 hour. The reaction mixture was quenched with
saturated NH.sub.4Cl (10 mL) and extracted with EtOAc (100
mL.times.2). The organic phase was washed with brine then dried
(MgSO.sub.4), filtered and concentrated to dryness under reduced
pressure. The residue was purified by column chromatography
(silica, 0-10% MeOH in CH.sub.2Cl.sub.2) to afford the sub-title
compound (110 mg, 40%).
[0800] .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 0.51 (s, 3H), 0.81
(s, 3H), 0.87 (s, 6H), 1.10 (s, 3H), 1.18 (s, 3H), 1.20 (s, 3H),
1.21-2.60 (m, 29H), 2.95 (m, 1H), 3.35-3.75 (m, 8H), 4.05 (m, 1H),
4.45 (m, 2H), 6.40 (m, 1H), 7.01 (m, 1H), 7.37 (m, 1H).
(ii) Preparation of 92:
3-((4aS,6aS,6bR,8aR,13aR,15bS)-12-Amino-2,2,6a,6b,9,9,13a-heptamethyl-15--
(2-(morpholinomethyl)furan-3-yl)-2,3,4,4a,5,6,6a,6b,7,8,8a,9,11,13,13a,13b-
,14,15b-octadecahydro-1H-chryseno[1,2-f]indazol-4-a-yl)-4-(2-aminoethyl)-1-
H-pyrazol-5-ol
[0801] A mixture of 92b (110 mg, 0.14 mmol) and hydrazine (0.5 mL)
in EtOH (2 mL) was sealed and heated to 160.degree. C. by microwave
for 3 hours. The reaction mixture was concentrated to dryness. The
residue was purified by column chromatography (silica, 0-70% CMA in
CH.sub.2Cl.sub.2) to afford the title compound (25 mg, 25%).
[0802] R.sub.f 0.54 (80:28:2 Methylene
Chloride/Methanol/concentrated Ammonium Hydroxide).
[0803] .sup.1H NMR (300 MHz, CD.sub.3OD) .delta. 0.60 (s, 6H), 0.84
(s, 3H), 0.86 (s, 3H), 1.12 (s, 3H), 1.22 (s, 3H), 1.28 (s, 3H),
1.35-2.0 (m, 19H), 2.18 (m, 2H), 2.27 (d, J=15.0 Hz, 2H), 2.61-2.72
(m, 6H), 2.97 (m, 2H), 3.50-3.70 (m, 7H), 6.41 (s, 1H), 7.51 (s,
1H). mp 238-240.degree. C. APCI MS (Positive Mode) m/z 740
[C.sub.44H.sub.65N.sub.7O.sub.3+H].sup.+.
Example IV
[0804] Table 6, below, lists the inhibitory concentrations of
select compounds and antibiotics in the biofilm growth assay
against various gram-negative bacterial biofilms. "N" means the
number of isolates of bacteria tested. The biofilm growth assay
procedure detailed in Example I, above, was followed. Burkholderia
cepacia used media consisting of M9 salts, 100 .mu.M CaCl2, 1 mM
MgSO4, and 0.7% citrate in 0.4% Noble agar. Salmonella spp. used
media consisting of Nutrient Broth with 0.5% glucose in 0.5% Noble
agar. Compound 1 of the invention is the most active compound in
Table 6. Compound 1 is superior to tobramycin, ceftazidime, and
azithromycin in the biofilm growth assay.
[0805] A successful research lead optimization strategy requires
examining multiple clinical isolates in parallel based upon the
inherent risks of antibacterial development and heterogeneity of
biofilms. Past medicinal chemistry efforts focused on identifying
superior analogs of approved classes of antibiotics have typically
demonstrated that closely related analogs can exhibit varying
degrees of antibacterial activities against different isolates and
species of bacteria in an unpredictable trend. Hence, accurately
selecting the next set of synthetic targets requires
microbiological activities from a broad group of bacterial isolates
and species of bacteria that would be encountered in clinical and
community settings. In addition, significant variability of biofilm
formation among different clinical isolates of P. aeruginosa has
been shown to exist. Therefore, the lead optimization of a biofilm
inhibitor may be detrimentally misguided if an unrepresentative
group of clinical isolates not exhibiting these different biofilms
are used to generate structure activity relationships.
TABLE-US-00007 TABLE 6 P. aeruginosa E. coli B. cepacia Salmonella
Compound (n = 10) (n = 10) (n = 10) spp. (n = 6) Oleanolic Acid 8
16 0.5 >64 C644 2 >16 0.5 16 C649 16 >16 1 >16 Compound
1 1 1 0.25 1 Tobramycin 4 16 >64 8 Ceftazidime 4 0.25 >8 0.5
Azithromycin >16 -- >16 >16
Example V
[0806] The bioavailabilities of certain compounds were examined in
mice. Administration of the compounds was performed orally and by
intraperitoneal (IP) injection using a vehicle as known to those
skilled in the art. Many vehicles can be used to examine
bioavailability. Prior to administration, each vehicle was
optimized based on compound solubility according to the formulation
research conducted by Uckun et al. (Arznelmittel-Forschung (Drug
Research) 2007; 57(4):218-226). Based upon this publication,
vehicles containing approximate ratios of 2:1:1 of propylene
glycol:Tween20:PEG400 (Tween20 being a common emulsier used in
formulations and food products) and less than 5% ethanol upon
administration exhibit good solubility properties and increase
serum bioavailability. During these experiments, PEG400
demonstrated a critical role in serum bioavailability. The
concentration of PEG400 is modulated depending upon solubility of
the compounds and the amount of aqueous phase added (0.02 M citrate
and 0.9% NaCl).
[0807] Compounds 1, 12, 64, and 68 demonstrated good
bioavailability when administered orally or via IP injection at
approximately 20 mg/kg to 50 mg/kg exceeding approximately 5
.mu.g/ml in the serum of mice at 30 minutes or 1 hour after
administration. Based upon these results, many of the compounds
described herein would yield bioavailability including the
compounds in Example II, Tables 2 and 3. This example demonstrates
that the compounds of the invention can be formulated into tablets,
capsules, suppositories, and sterile liquids for parenteral
administration as known to those skilled in the art.
Example VI
[0808] The biofilm growth assay as described in Example I, as
amended by the details that follow, was performed using Pseudomonas
syringae, Xanthomonas campestris, and Pectobacterium atrosepticum
(gram-negative bacterial plant pathogens). A freezer stock of each
plant pathogen was grown separately overnight on 1.5% agar plates
containing TSB at 30.degree. C. The next day a pipette tip was used
to inoculate a round plate composed of M8 salts and 0.7% glucose on
0.5% Noble agar from the overnight TSB plates. Plates were allowed
to incubate at 30.degree. C. for approximately 24 to 48 hours.
Compound 1 of the invention was examined against these plant
pathogens and found to inhibit the spreading biofilms at 0.03
.mu.g/ml against Pseudomonas syringae, 0.5 .mu.g/ml against
Xanthomonas campestris, and 0.06 .mu.g/ml against Pectobacterium
atrosepticum. These data demonstrate that the compounds of the
invention inhibit spreading biofilms of plant pathogens.
Example VII
[0809] INHIBITION OF THE GROWTH OF PREFORMED BIOFILMS. The Biofilm
Growth Assay described in Example I was performed in round plates
with the compounds of the invention only in agar on half of the
plate. The agar not containing compound was inoculated with
bacteria and the formed biofilm moved toward the agar containing
compound on the other half of the round plate. Once the spreading
biofilm reached the agar containing compound, the biofilm was
inhibited from moving or spreading onto the agar with compound.
This Biofilm Growth Assay performed on these agar plates
demonstrates that the compounds of the invention inhibit the growth
of preformed biofilms.
[0810] Approximately 0.5% agar not containing a compound of the
invention was poured and allowed to dry. Agar on half of the plate
was removed and then agar containing a compound of the invention
was poured on the empty half of the plate and allowed to dry.
Bacteria is inoculated onto the agar not containing compound and
allowed in incubate overnight as described in Example I. During
incubation bacteria spread as a biofilm until they reach the agar
containing compound. The compounds of the invention inhibit the
spreading biofilm at the same concentrations at shown in Examples
I, II, and III, above.
Example VIII
[0811] INHIBITION OF THE GROWTH OF PREFORMED BIOFILMS. The Biofilm
Growth Assay as described in Example VII was performed in round
plates with the compounds of the invention in agar on half of the
plate and antibiotic disks placed onto the agar containing the
compounds of the invention. This assay demonstrates that the
compounds of the invention are synergistic with antibiotics like
tobramycin and colistin at inhibiting spreading biofilms across the
agar.
[0812] The Biofilm Growth Assay was performed as described in
Example VIII. 2 to 4 antibiotic disks were placed onto the agar
containing a compound of the invention in a line parallel to the
line that separates the two agars in one round plate. Appropriate
negative and positive control plates were performed as known to
those skilled in the art. This assay demonstrates that the
compounds of the invention in combination with antibiotics inhibit
spreading biofilms at approximately 4 times less the concentration
than when performed alone as described in Example I.
Example IX
Evaluation of Salts
[0813] Evaluation of Compound 62 (Table 4) salt formation was
performed. HCl, HBr, H.sub.3PO.sub.4, p-toluenesulfonic acid,
benzenesulfonic acid, methanesulfonic acid, L-tartaric acid, acetic
acid, xinafoic acid, L-lactic acid, benzoic acid, adipic acid,
oxalic acid, pamoic acid, maleic acid, and laurylsulfuric acid
indicated crystallinity. p-toluenesulfonic acid (mono) and
H.sub.3PO.sub.4 (bis) provided the most desirable physical
properties compared to freebase. As stated herein, the invention
also includes compounds and their salts.
[0814] Evaluation of Compound 64 (Table 4) salt formation was
performed. HCl (mono and bis), p-TSA (bis), BSA (bis), MSA (bis),
H.sub.2SO.sub.4 (mono), and H.sub.3PO.sub.4 (mono) provided salts
with crystallinity. Bis-MSA and mono-H.sub.3PO.sub.4 increased
aqueous solubility compared to freebase. As stated herein, the
invention also includes compounds and their salts.
Example X
[0815] A topical gel containing 2% by weight of the compound of the
invention with azithromycin for use in treating skin infections can
be prepared.
[0816] 0.25 gram of the compound of the invention is dissolved in
6.75 grams of ethanol. 0.2 grams of azithromycin is dissolved in
this solution. 0.25 grams of hydroxypropyl methylcellulose is added
with gentle stifling until a homogenous solution is obtained. 4.8
grams of water is then added with gentle shaking.
[0817] A formulation without antibiotic can also be prepared using
this same procedure.
Example XI
Pharmaceutical Formulation for Nebulization of a Compound of the
Invention
[0818] Solutions were prepared comprising 2 mg/ml and 10 mg/ml of
the compound of the invention in ethanol/propylene glycol/water
(85:10:5). These solutions were nebulized separately by a ProNeb
Ultra nebulizer manufactured by PARI. The nebulized solutions were
collected in a cold trap, processed appropriately, and were
detected by mass spectrometry. The compound of the invention is
expected to be recovered from both formulations to demonstrate that
nebulization can be used to deliver this compound to patients with
lung infections.
Example XII
[0819] The compounds shown in the table below were prepared
semi-synthetically and tested in the biofilm growth assay as
described herein. These compounds with substitutions at R.sup.1 and
R.sup.2 did not inhibit the biofilms of P. aeruginosa or E. coli at
1 or 2 .mu.g/ml sufficiently to warrant further investigation
relative to the compounds noted above.
TABLE-US-00008 ##STR00258## R.sup.1 ##STR00259## R.sup.2
##STR00260## ##STR00261## ##STR00262## ##STR00263## ##STR00264##
##STR00265## ##STR00266## ##STR00267## ##STR00268## ##STR00269##
##STR00270## ##STR00271## ##STR00272## ##STR00273## ##STR00274##
##STR00275## ##STR00276## ##STR00277## ##STR00278##
[0820] All references, including without limitation all papers,
publications, presentations, texts, reports, manuscripts,
brochures, internet postings, journal articles, periodicals, and
the like, cited in this specification are hereby incorporated by
reference. The discussion of the references herein is intended
merely to summarize the assertions made by their authors and no
admission is made that any reference constitutes prior art. The
inventors reserve the right to challenge the accuracy and
pertinence of the cited references.
[0821] It is intended that all patentable subject matter disclosed
herein be claimed and that no such patentable subject matter be
dedicated to the public. Thus, it is intended that the claims be
read broadly in light of that intent. In addition, unless it is
otherwise clear to the contrary from the context, it is intended
that all references to "a" and "an" and subsequent corresponding
references to "the" referring back to the antecedent basis denoted
by "a" or "an" are to be read broadly in the sense of "at least
one." Similarly, unless it is otherwise clear to the contrary from
the context, the word "or," when used with respect to alternative
named elements is intended to be read broadly to mean, in the
alternative, any one of the named elements, any subset of the named
elements or all of the named elements.
[0822] In view of the above, it will be seen that the several
advantages of the invention are achieved and other advantageous
results obtained. It should be understood that the aforementioned
embodiments are for exemplary purposes only and are merely
illustrative of the many possible specific embodiments that can
represent applications of the principles of the invention. Thus, as
various changes could be made in the above methods and compositions
without departing from the scope of the invention, it is intended
that all matter contained in the above description as shown in the
accompanying drawings shall be interpreted as illustrative and not
in a limiting sense.
[0823] Moreover, one of ordinary skill in the art can make various
changes and modifications to the invention to adapt it to various
usages and conditions, including those not specifically laid out
herein, without departing from the spirit and scope of this
invention. Accordingly, those changes and modifications are
properly, equitably, and intended to be, within the full range of
equivalents of the invention disclosed and described herein.
* * * * *