U.S. patent application number 11/376117 was filed with the patent office on 2006-07-27 for treatment of neovascularization disorders with squalamine.
This patent application is currently assigned to Genaera Corporation. Invention is credited to Steven Jones, William Kinney, Ann Shinnar, Michael Zasloff.
Application Number | 20060166950 11/376117 |
Document ID | / |
Family ID | 23935748 |
Filed Date | 2006-07-27 |
United States Patent
Application |
20060166950 |
Kind Code |
A1 |
Zasloff; Michael ; et
al. |
July 27, 2006 |
Treatment of neovascularization disorders with squalamine
Abstract
Aminosterol compounds are described that are useful as
inhibitors of the sodium/proton exchanger (NHE). Methods of using
such aminosterols compounds are also enclosed, including those
employing compounds that are inhibitors of a spectrum of NHEs as
well as those using compounds that are inhibitors of only one
specific NHE. Advantageous screening techniques and assays for
evaluating a compound's therapeutic activity are also
disclosed.
Inventors: |
Zasloff; Michael; (Merion
Station, PA) ; Shinnar; Ann; (Teaneck, NJ) ;
Kinney; William; (Churchville, PA) ; Jones;
Steven; (West Chester, PA) |
Correspondence
Address: |
MORGAN LEWIS & BOCKIUS LLP
1111 PENNSYLVANIA AVENUE NW
WASHINGTON
DC
20004
US
|
Assignee: |
Genaera Corporation
|
Family ID: |
23935748 |
Appl. No.: |
11/376117 |
Filed: |
March 16, 2006 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
11046906 |
Feb 1, 2005 |
|
|
|
11376117 |
Mar 16, 2006 |
|
|
|
09985417 |
Nov 2, 2001 |
6962909 |
|
|
11046906 |
Feb 1, 2005 |
|
|
|
09198486 |
Nov 24, 1998 |
|
|
|
09985417 |
Nov 2, 2001 |
|
|
|
08487443 |
Jun 7, 1995 |
5847172 |
|
|
09198486 |
Nov 24, 1998 |
|
|
|
Current U.S.
Class: |
514/169 ;
552/521 |
Current CPC
Class: |
G01N 33/5011 20130101;
G01N 33/5044 20130101; A61K 31/56 20130101; G01N 33/5088 20130101;
C07J 41/0005 20130101; G01N 33/5061 20130101; G01N 33/505 20130101;
G01N 33/5091 20130101; G01N 33/5064 20130101; G01N 33/5008
20130101; G01N 2500/00 20130101; C07J 9/00 20130101 |
Class at
Publication: |
514/169 ;
552/521 |
International
Class: |
A61K 31/56 20060101
A61K031/56; C07J 41/00 20060101 C07J041/00 |
Claims
1-3. (canceled)
4. A method of treating retinopathy in a mammal, comprising
administering to the mammal a composition comprising an amount of
squalamine or a pharmaceutically acceptable salt thereof effective
to treat the retinopathy.
5. The method of claim 4, wherein the effective amount is about 0.1
to about 10 mg/kg body weight.
6. The method of claim 4, wherein the mammal is a human.
7. The method of claim 4, wherein the composition is administered
intravenously.
8. The method of claim 4, wherein the retinopathy is diabetic
retinopathy.
9. The method of claim 4, wherein the retinopathy is retinopathy of
prematurity.
10. A method of treating retinopathy in a human, comprising
administering systemically to the human in need of treatment, a
composition comprising an amount of squalamine or a
pharmaceutically acceptable salt thereof effective to treat the
retinopathy.
11. The method of claim 10, wherein the systemic administration is
selected from the group consisting of intravenous, subcutaneous and
intramuscular injection.
12. The method of claim 10, wherein the retinopathy is diabetic
retinopathy.
13. The method of claim 10, wherein the retinopathy is retinopathy
of prematurity.
14. The method of claim 10, wherein the method consists essentially
of administering to the human in need treatment, a composition
comprising an amount of squalamine or a pharmaceutically acceptable
salt thereof effective to treat the retinopathy.
15. The method of claim 14, wherein the retinopathy is diabetic
retinopathy.
16. The method of claim 10, wherein the method consists of
administering to the human in need of treatment, a composition
comprising an amount of squalamine or a pharmaceutically acceptable
salt thereof effective to treat the retinopathy.
17. The method of claim 16, wherein the retinopathy is diabetic
retinopathy.
18. The method of claim 4 or claim 10, wherein the composition
consists essentially of squalamine.
19. The method of claim 4 or claim 10, wherein the composition
consists of squalamine.
20. The method of claim 4 or claim 10, wherein the composition
further comprises one or more pharmaceutically acceptable
excipients.
21. The method of claim 4 or claim 10, wherein the composition is
used in combination with at least one other therapy for treating
retinopathy.
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] This application is related to U.S. Ser. No. 08/416,883,
which is the U.S. national phase of International Application No.
PCT/US94/10265, filed Sep. 13, 1994.
FIELD OF THE INVENTION
[0002] The present invention relates to aminosterol compounds
useful as inhibitors of the sodium/proton exchanger (NHE). The
invention is also directed to pharmaceutical compositions
containing such compounds, and the use of such compounds for
inhibiting NHE. The invention is further directed to assaying
techniques for screening compounds for their efficacy as NHE
inhibitors.
BACKGROUND OF THE INVENTION
[0003] Each of the body's cells must maintain its acid-base balance
or, more specifically, its hydrogen ion or proton concentration.
Only slight changes in hydrogen ion concentration cause marked
alterations in the rates of chemical reactions in the cells--some
being depressed and others accelerated. In very broad and general
terms, when a person has a high concentration of hydrogen ions
(acidosis), that person is likely to die in a coma, and when a
person has a low concentration of hydrogen ions (alkalosis), he or
she may die of tetany or convulsions. In between these extremes is
a tremendous range of diseases and conditions that depend on the
cells involved and level of hydrogen ion concentration experienced.
Thus, the regulation of hydrogen ion concentration is one of the
most important aspects of homeostasis.
[0004] A shorthand method of expressing hydrogen ion concentration
is pH: pH=log 1/(H.sup.+ concentration)=-log (H.sup.-
concentration). The normal cell pH is 7.4, but a person can only
live a few hours with a pH of less than 7.0 or more than 7.7. Thus,
the maintenance of pH is critical for survival.
[0005] There are several mechanisms of maintaining pH balance. For
example, during quiescence and constitutive growth, cells appear to
utilize the chloride/bicarbonate exchanger, a well-studied device
which provides for proton exchange across cells such as the red
cell.
[0006] In addition, during accelerated periods of growth, which are
induced by mitogens, growth factors, sperm, etc., cells engage
another piece of cellular equipment to handle the impending
metabolic burst. This is the sodium/proton (Na.sup.+/H.sup.+)
exchanger--the "NHE," which is also called an "antiporter." Because
the NHE functions in a number of roles and in a number of tissues,
the body has developed a family of NHEs, and recent work has
elucidated a family of NHE "isoforms" that are localized in certain
tissues and associated with various functions. The NHE isoforms
listed below are most likely to be significant.
[0007] NHE1 is a housekeeping exchanger and is believed to be
unregulated in hypertension. It is thought to play a role in
intracellular pH conduct. Also, it is believed that control of this
exchanger will protect a patient from ischemic injury.
[0008] NHE1 is associated genetically with diabetes and, thus,
inhibition might alter evolution of diabetes through effects on
beta cells in the pancreas. In addition, vascular smooth muscle
proliferation, responsive to glucose, is associated with increased
expression of NHE1a.
[0009] NHE1.beta. is present on nucleated erythrocytes. It is
inhibited by high concentrations of amiloride. This NHE isoform is
regulated by adrenergic agents in a cAMP-dependent fashion.
[0010] NHE2 is associated with numerous cells of the GI tract and
skeletal muscle. Inhibition could alter growth of hyperplastic
states or hypertrophic states, such as vascular smooth muscle
hypertrophy or cardiac hypertrophy. Cancers of muscle origin such
as rhabdomyosarcoma and leiomyoma are reasonable therapeutic
targets.
[0011] NHE3 is associated with the colon. The work described below
shows it to be associated with endothelial cells. Inhibition would
affect functions such as water exchange in the colon (increase
bowel fluid flux, which is the basis of, e.g., constipation),
colonic cancer, etc. On endothelial cells, normal growth would be
inhibited through inhibition of the exchanger.
[0012] NHE4 is associated with certain cells of the kidney. It
appears to play a role in cellular volume regulation. Specific
inhibitors might affect kidney function, and hence provide
therapeutic benefit in hypertension.
[0013] NHE5 is associated with lymphoid tissue and cells of the
brain. Inhibition of NHE5 should cause inhibition of proliferative
disorders involving these cells. NHE5 is a likely candidate for the
proliferation of glial cells in response to HIV and other viral
infections.
[0014] As indicated by the above, although the NHE functions to
assist the body, the inhibition of NHE function should provide
tremendous therapeutic advantages. For example, although the NHE,
normally operates only when intracellular pH drops below a certain
level of acidity, upon growth factor stimulation the cell's NHEs
are turned on even though the cell is poised at a "normal" resting
pH. As a consequence, the NHEs begin to pump protons from the cell
at a pH at which they would normally be inactive. The cell
undergoes a progressive loss of protons, increasing its net
buffering capacity or, in some cases, actually alkalinizing. In
settings where the pump is prevented from operating, the growth
stimulus does not result in a cellular effect. Thus, inhibitors of
the NHE family are likely to exert growth-inhibitory effects.
[0015] During severe acid stress--the condition that a tissue might
find itself in when deprived of oxygen (or a blood supply)--the NHE
family is believed to contribute to subsequent irreversible damage.
For example, when blood flow to the heart is impaired, local
acidosis occurs. Heart muscle cells develop a profound internal
acidity. The acidity, in turn, activates otherwise dormant NHEs.
These exchangers readily eliminate protons from the cell, but in
exchange for sodium. As a consequence, intracellular sodium
concentrations rise. Subsequently, the sodium-calcium exchanger is
activated, exchanging internal sodium for external calcium. The
rise in internal Ca.sup.+ concentrations leads to cell death,
decreased contractility, and arrhythmias. Thus, post ischemic
myocardial damage and associated arrhythmias are-believed to arise
from an NHE-dependent mechanism, and inhibition of this NHE should
therefore prevent such occurrences. If the NHE inhibited the
internalization of Na.sup.+ and slowed down metabolic activity as a
consequence of the depressed pH, damage of the cell could be
avoided. Hence, there is an interest in the development of NHE
inhibitors for use in cardiac ischemia.
[0016] Other members of the NHE family appear to play a more
classical role in water and sodium transport across epithelial
surfaces. Specifically, the NHE3 isoform found in the colon is
believed to play a role in regulating the fluid content of the
colonic lumen. This pump is inhibited in cases of diarrhea. The
NHE3 isoform present on the proximal tubules of the kidney is
believed to play a similar role with respect to renal salt and acid
exchange. Accordingly, inhibitors of the NHE family have been
regarded as therapeutic modalities for the treatment of
hypertension.
[0017] In view of the expected value of the inhibition of NHE
action, scientists have sought out NHE inhibitors. The most widely
studied inhibitor of NHE is amiloride, a guanidine-modified
pyrazine used clinically as a diuretic. A number of derivatives
have been generated, incorporating various alkyl substitutions.
These derivatives have been studied with the several isoforms of
NHE that are known and described above, except for NHE5, for which
there is no known inhibitor.
[0018] The activities of these inhibitors against these specific
exchangers have been previously determined. As seen in Table A
below, each exchanger exhibits a different spectrum of response to
each inhibitor: TABLE-US-00001 TABLE A Amiloride DMA MPA K.sub.i
(.mu.M) K.sub.i (.mu.M) K.sub.i (.mu.M) NHE1 3 0.1 0.08 NHE2 3 0.7
5.0 NHE3 100 11 10 Notes: DMA = dimethylamiloride; MPA =
methylpropylamiloride.
[0019] See Counillon et al., Molecular Pharmacology 44, 1993,
1041-1045.
[0020] The NHE inhibitors described by Counillon et al. exhibit
specificity for NHE1. They therefore serve a therapeutic value in
the treatment of conditions where inhibition of this isoform is
beneficial. However, these inhibitors do not target the other known
NHE isoforms--e.g., NHE3 is unaffected.
[0021] NHE3, as is demonstrated below, is expressed on endothelial
cells, and its inhibition results in anti-angiogenic effects. The
spectrum of NHE isoforms inhibited by the aminosterol compounds in
accordance with the invention are different from those inhibited by
the amiloride or the Counillon et al. compounds, and have
different, distinct pharmacological effects.
[0022] In addition, Counillon et al. also reported that certain
benzoylguanidine derivatives inhibit other NHE isoforms. In
particular, (3-methylsulfonyl-4-piperidinobenzoyl)guanidine
methanesulfonate exhibits particular selectivity to the NHE1 as
shown in the table below. TABLE-US-00002 TABLE B NHE Isoform Ki
(.mu.M) NHE1 0.16 NHE2 5.0 NHE3 650
[0023] These benzoylguanadine compounds, which are based on the
chemical structure of amiloride, exhibit greatest specificity for
inhibiting NHE1 while retaining considerable activity against NHE2
and NHE3. To achieve pharmacological inhibition of NHE1, the widely
distributed "housekeeping" isoform, undesirable inactivation of
NHE2 and NHE3 would occur.
[0024] Those in the art have therefore continued to search for NHE
inhibitors that exhibit selective action against a single, specific
NHE. Such inhibitors would permit more precise inhibition of a
tissue by perturbing the effect of the NHE on its growth.
[0025] Thus, artisans have recognized that the development of
various NHE-specific inhibitors would allow for the development of
new therapies for a whole host of diseases or conditions,
including: treating arrhythmias; treating and preventing cardiac
infarction; treating and preventing angina pectoris and ischemic
disorders of the heart; treating and preventing ischemic disorders
of the peripheral and central nervous system; treating and
preventing ischemic disorders of peripheral organs and limbs;
treating shock; providing anti-arteriosclerotic agents; treating
diabetic complications; treating cancers; treating fibrotic
diseases, including fibroses of lung, liver and kidney; and
treating prostatic hyperplasia. Other therapeutic targets include:
treatment of viral disease, such as HIV, HPV and HSV; prevention of
malignancies; prevention of diabetes (i.e., islet cell injury);
prevention of vascular complications of diabetes; treatment of
disorders of abnormal neovascularization, e.g., macular
degeneration, rheumatoid arthritis, psoriasis, cancer, malignant
hemangiomas; prevention of vascular retenosis; prevention of
hypertension-associated vascular damage; immunosuppression; and
treatment of collagen vascular disorders.
[0026] Inhibitors of NHEs of bacteria fungi and protozoa would also
be valuable as specific antimicrobials. It is known that all living
cells use an NHE of one form or another to maintain intracellular
Na.sup.+ and pH homeostasis. NHEs have been cloned from numerous
bacteria and fungi, and bear some sequence homology to the
mammalian isoforms. Using a highly specific bacterial or fungal NHE
as a target, it should be possible to develop a highly specific
inhibitor of such an exchanger, one that is particularly
advantageous or that lacks activity against the mammalian isoforms.
Such compounds would be useful as antibiotics of a different
mechanism.
[0027] Thus, there is a need in the art for specific inhibitors of
NHEs. There is further a need to develop NHE inhibitors for various
therapeutic uses.
SUMMARY OF THE INVENTION
[0028] The present invention fulfills needs felt in the art by
providing various aminosterol compounds that inhibit various NHEs.
The invention is directed to aminosterol compounds that exhibit
inhibitory action on NHEs, and to compositions containing such
compounds.
[0029] Thus, the invention is directed to newly isolated and
synthesized aminosterol compounds that are useful as inhibitors of
NHEs, such as compounds FX1A, FX1B; 1360, 1361, 371, 1437, and 353.
Some of these steroid compounds have been found to inhibit a
spectrum of NHEs, while others have been found to advantageously
inhibit a single, specific NHE.
[0030] Further, the invention is directed to pharmaceutical uses
and therapies employing the compounds of the invention. The
invention is also directed to new uses for squalamine, which had
been previously isolated and characterized.
[0031] Additionally, the invention is directed to advantageous
screening methods for evaluating a compound's therapeutic efficacy.
In particular, a tadpole assay has been developed, which has been
found to be a convenient tool for screening compounds for NHE
inhibition and therapeutic effects.
[0032] An especially preferred compound of the invention is
compound 1436 (or a pharmaceutically acceptable salt thereof). The
invention is directed to a pharmaceutical composition comprising an
effective amount of this compound and a pharmaceutically acceptable
vehicle or carrier. The invention is further directed to a method
of inhibiting the proliferation of cells, comprising administering
an effective amount of compound 1436, and particularly to such a
method where the cells are malignant cells, vascular smooth muscle
cells, bronchial'smooth muscle cells, fibroblasts, lymphocytes or
lymphoid tissue, muscle, bone, cartilage, epithelium, hematopoietic
tissue, or neural tissue. Furthermore, the invention is directed to
a method of inhibiting the proliferation of cells, comprising
administering an effective amount of a combination comprising
compound 1436 and squalamine. The invention also relates to a
method for suppressing the immune system by inhibiting the
proliferation of lymphocytes, comprising administering an effective
amount of compound 1436. In addition, the invention involves
suppressing the growth of a vertebrate, comprising administering an
effective amount of compound 1436. The invention also relates to
treating a viral infection by suppressing the growth of a viral
target cell, comprising administering an effective amount of
compound 1436. A method of controlling arterial pressure,
comprising administering an effective amount of compound 1436, is
also preferred. Also, the invention is directed to a method of
protecting against cardiac ischemia, comprising administering an
effective amount of compound 1436. The invention also relates to a
method of preserving transplanted organs, comprising the
administration of an effective amount of compound 1436.
Furthermore, the invention is directed to a method of treating an
infection caused by an microbial agent, such as bacteria, viruses,
fungi, and protozoa, comprising the administration of an effective
amount of compound 1436. The invention also pertains to the
administration of an effective amount of this compound to inhibit
an NHE.
[0033] The invention is also directed to a method of inhibiting
NHE3, preferably to a method of specifically inhibiting this NHE
isoform that is expressed in a pathological process, comprising the
administration of an effective amount of squalamine (or its
pharmaceutically acceptable salt). Another method according to the
invention involves inhibiting the growth of endothelial cells,
especially ones of new capillaries, comprising administering an
effective amount of squalamine.
[0034] The invention also pertains to a or method for evaluating a
compound for NHE-inhibiting activity or anti-angiogenic activity,
comprising performing a tadpole assay comprising the steps of: (i)
preparing an aqueous solution containing a compound to be assayed
(e.g., at a concentration of 10 .mu.g/ml); (ii) introducing a
tadpole into the solution; and (iii) after at least one interval of
time (e.g., about an hour), observing the tadpole (e.g., its tail
and/or hands and feet) under a microscope. Preferably, the tadpole
is a Xenopus tadpole, more preferably a stage 59-60 Xenopus
tadpole. Such an assay can be used alone or in combination with
another assay, e.g., a chick chorioallantoic membrane assay and/or
a chick embryo vitelline capillary regression assay.
[0035] Other aspects, objects and advantages will be apparent from
the detailed disclosure below, which illustrates preferred features
and embodiments of the invention in conjunction with the appended
drawing figures.
BRIEF DESCRIPTION OF DRAWING FIGURES
[0036] FIGS. 1A and 1B show the inhibition of rabbit sodium/proton
exchanger isoform 3 (NHE3) by squalamine. FIG. 1A is a plot of the
rate of pH recovery (y-axis) as a function of restored
extracellular sodium ion concentration (x-axis) for cells
acid-preloaded by exposure to 40 mM NH.sub.4Cl, with the curve
marked by "+" being for control (no drug) and the curve marked by
"A" being for squalamine. FIG. 1B shows the actual internal pH
(y-axis) as a function of time (x-axis) following addition of 5
.mu.g/ml of squalamine for cells not acid-preloaded.
[0037] FIG. 2A shows the lack of inhibition of rabbit sodium/proton
exchanger isoform 1 (NHE1) by squalamine. FIG. 2B shows the lack of
inhibition of human NHE1 by squalamine. In these plots of internal
pH vs. time, the curve marked by lo" is for squalamine and that
marked by "+" is the control (cells incubated in the absence of
squalamine).
[0038] FIGS. 3A, 3B and 3C illustrate that endothelial cells
exhibit greater sensitivity to squalamine (bar above 3 on the
x-axis) than to other membrane-active agents, and that endothelial
cells are more sensitive to squalamine than are epithelial cells
and fibroblasts. FIG. 3A is for the administration of 1 .mu.g/ml of
the agent against bovine pulmonary endothelial cells, whereas FIGS.
3B and 3C are for administration of 10 .mu.g/ml of the
membrane-active agents to human epithelial cells and to human
foreskin fibroblasts, respectively.
[0039] FIGS. 4A, 4B and 4C show the suppression of the growth of
murine melanoma, respectively through the subcutaneous,
intraperitoneal and oral administration of squalamine.
[0040] FIG. 5 demonstrates the suppression of the growth of human
melanoma 1205Lu in immunocompromised (RAG-1) mice by squalamine at
various dosages (".smallcircle."=10 mg/kg/d, "+"=20 mg/kg/d,
".smallcircle."=40 mg/kg/d; d=day).
[0041] FIG. 6 illustrates the suppression of murine melanoma in
mice by intraperitoneal administration of compound 319.
[0042] FIG. 7 shows the pharmacokinetic clearance of compound 319
from a mouse IV PK study.
[0043] FIG. 8 shows the pharmacokinetic clearance of squalamine
from a mouse IV PK study.
[0044] FIG. 9 is an HPLC profile for aminosterols derived from the
liver of the dogfish shark, illustrating the diversity of these
compounds.
[0045] FIG. 10 illustrates the inhibitory effect of compound 1436
on NHE3.
[0046] FIG. 11 illustrates the effect of compound 1436 on survival
in mice bearing L1210 leukemia.
[0047] FIG. 12 illustrates that squalamine and compound 1436
exhibit synergy in suppressing growth of murine melanoma in
mice.
[0048] FIGS. 13 and 14 show the in vitro suppression of the growth
of human coronary artery smooth muscle by compound 1436 (FIG. 13)
and squalamine (FIG. 14), with absorbance plotted vs. concentration
in .mu.g/ml.
[0049] FIG. 15 is a expanded plot of the data shown in FIGS. 14A
and 14B, evidencing that both compound 1436 and squalamine suppress
in vitro the growth of human coronary artery smooth muscle.
[0050] FIG. 16 illustrates that compound 1436 suppresses the growth
of mice in a dose-dependent fashion.
[0051] FIGS. 17A and 17B show the effect of compound 353 versus
squalamine on human melanoma.
DETAILED DESCRIPTION OF THE INVENTION
Syntheses of Aminosterol Compounds
[0052] The steroid known as squalamine is the subject of U.S. Pat.
No. 5,192,756 to Zasloff et al., the disclosure of which is herein
incorporated by reference. This compound is a broad-spectrum
antibiotic, killing bacteria, fungi and protozoa. The absolute
stereochemistry for squalamine, compound 1256, is shown below. The
total chemical synthesis of squalamine was reported in 1994.
EXAMPLE 1
Preparation of Shark Liver Isolates
[0053] In addition to squalamine, at least ten other distinctly
different aminosterols have been recovered from extracts of dogfish
shark liver. To prepare the aminosterols, shark liver was extracted
in methanol:acetic acid. The aqueous extract was adsorbed to C18
silica and eluted with 70% acetonitrile, and the eluate was
adsorbed to SP-sephadex and eluted with 1.5 M NaCl. The eluate was
adjusted to 5M NaCl, and the steroids salted out. The precipitate
was filtered over Celite and eluted with hot water, followed by
methanol. The eluate was reduced in volume and applied to a 1-inch
C18 column, and subjected to chromatography utilizing an increasing
gradient in acetonitrile. Fractions were collected, concentrated by
evaporation, and analyzed separately by thin layer chromatography
(TLC).
[0054] The HPLC profile of the aminosterols isolated from 40 kg of
shark liver is shown in FIG. 9. Final HPLC purification was
performed as described in Moore et al., Proc. Natl. Acad. Sci. 90,
1993, 1354-1358. HPLC fractions were resolved individually by
silica thin layer chromatography (6:3:1
CH.sub.2Cl.sub.2:MeOH:NH.sub.4OH) visualized in iodine vapor.
Fraction 40 represents the more hydrophilic portion of the elution
profile, and fraction 66 represents the more hydrophobic
portion.
[0055] Squalamine elutes beginning at about fraction 62 and
continues until fraction 80. In addition, other steroids can be
seen eluting between fractions 43-47 (R.sub.f 82), 53-55 (R.sub.f
1.02), 56-59 (R.sub.f 0.51), 57-62 (R.sub.f 0.96), 60-64 (R.sub.f
0.47) and 61-66 (R.sub.f 1.06), as described below in Table 1.
TABLE-US-00003 TABLE 1 Chemical and Structural Characterization of
Aminosterols Isolated From Shark Liver FRACTION TLC Rf Compound No.
Mass 43-47 0.82 FX 1A 664.5 FX 1B 641.5 53-55 1.02 1360 641.49
56-59 0.51 FX 3 57-62 0.96 1437 657.52 60-64 0.47 1436 684.52 61-66
1.05 1361 543.48 63-80 1.0 1256 627.98
[0056] The structures for some of these compounds are shown below.
##STR1## ##STR2##
[0057] Each of these entities was isolated, purified,
characterized, and the structure determined by NMR as described
below.
Compound 1360:
[0058] This compound, the major steroid in Fraction 2 from
preparative C18 RP--HPLC, was purified by strong cation exchange
using sulfoethylaspartamide HPLC eluted with an increasing NaCl
gradient. Steroid fractions were assayed by silica TLC developed in
CH.sub.2Cl.sub.2:CH.sub.3OH:NH.sub.4OH (6:3:1) and visualized with
iodine. Steroid fractions were then pooled and re-chromatographed
by C18 RP--HPLC with a gradient of increasing CH.sub.3CN in aqueous
0.1% TFA. TLC analysis of the purified compound showed a single
spot with R.sub.f=1.02 with respect to squalamine (R.sub.f
squalamine=1.0).
[0059] For subsequent isolations of compound 1360, strong cation
exchange chromatography was not performed. Instead, pooled
fractions from preparative C18 RP--HPLC were subjected directly to
C18 or C8 RP--HPLC using a shallower CH.sub.3CN gradient. Fractions
were assayed both by TLC and also by .sup.1H-NMR for samples
redissolved in D.sub.2O.
[0060] When analyzed by FAB-MS, the compound typically showed only
a very weak (M+H)+ signal at 642.5 and an intense fragment at 562.5
daltons in the positive ion mode. In negative ion FAB-MS, (M-H)-
was observed at 640.4. Subsequent electrospray ionization (ESI-MS)
analysis exhibited strong (M+H)+ and (M-H)- signals consistent with
a parent mass of 641.4, along with many TFA adducts. This suggested
that lability of sulfate in compound 1360 was more pronounced under
FAB conditions.
[0061] Since the parent ion in FAB positive ion mode was very weak
in intensity, accurate mass determination was conducted on the
dessulfate fragment. An accurate mass of 561.49325 was observed. An
accurate parent mass of 641.49 daltons was then calculated by
adding the mass of SO.sub.4--, which matches with the molecular
formula C.sub.34H.sub.63N.sub.3O.sub.6S. This molecular formula,
with one additional oxygen and two fewer hydrogens when compared to
squalamine (C.sub.34H.sub.65N.sub.3O.sub.5S), was suggestive of a
compound bearing a carbonyl moiety.
[0062] .sup.1H-NMR of compound 1360 in D.sub.2O (300 MHz) revealed
several features distinguishing it from squalamine. For compound
1360, the resonance appearing furthest downfield at .delta.=4.15
ppm showed a splitting pattern of at least 7 signals with an
integration of two protons; for squalamine, the most deshielded
resonance at .delta.=4.2 ppm was the sulfate position (H24),
resolved at 300 MHz as a multiplet of 5 signals with an integration
of one proton. At 2.6 ppm, compound 1360 also showed a poorly
resolved multiplet, attributed to 2 protons. It was suspected that
these resonances could be attributed to methylene protons alpha to
a carbonyl, based on comparison to the literature. In squalamine
the region from 2.2-2.75 exhibited no resonances. Compound 1360
showed two distinct methyl doublets, one at 0.95 ppm and the other
at 1.1 ppm; this contrasts with squalamine, where three methyl
groups split as doublets overlapping in the 0.9-1.05 ppm region.
Other resonance characteristics were quite similar for the two
steroids. In the upfield methyl region, both compound 1360 and
squalamine showed annular singlet methyl signals at 0.85 and 0.65
ppm, positions 19 and 18 respectively. The resonances from the
steroid nucleus (1.0-2.1 ppm) and from spermidine also showed the
same characteristic pattern for both compound 1360 and squalamine.
H7, at the alcohol position in the ring, resonates at 3.85 ppm for
both steroids in D.sub.2O
[0063] A COSY (correlated spectroscopy) spectrum conducted in
D.sub.2O (300 MHz) established the sulfate at position 27,
--CH.sub.2OSO.sub.3--. Diagnostic crosspeak patterns in the
2-dimensional frequency (2D) plot indicated that the doublet peak
at .delta.=1.1 ppm was methyl 26, which then connected to H25 at
3.05 ppm (hidden under the polyamine resonances), which in turn was
the nearest neighbor to the complex multiplet at 4.15 ppm.
[0064] The polyamine region in the 2D map was consistent with the
splitting pattern of spermidine, confirming that compound 1360 had
the same polyamine as squalamine. Otherwise, the 2D map in D.sub.2O
was deficient in many crosspeak signals, particularly in the
steroid nucleus region, and could not be used to established the
complete primary structure.
[0065] Compound 1360 was dried in-vacuo and dissolved in DMSO-d6
under nitrogen, and then sealed under nitrogen using cycles of
freeze-thaw-pump. Both 1D and 2D .sup.1H-NMR spectra were conducted
at 300 MHz and at 600 MHz. All of the proton resonances of the
compound in DMSO were sharp (except for the polyamine region
(2.8-3.1)) and shifted with respect to assignments in D.sub.2O. For
example, the multiplet for position 27 was shifted upfield to 3.77
ppm and the H7 proton shifted to 3.60 ppm. In addition, a new
doublet signal (integration=1 H) appeared at 4.17 ppm. This new
resonance was identified as the hydroxyl at position 7, based on
its 2D connectivity to H7, which was unambiguous at 600 MHz. This
resonance could not be observed in D.sub.2O due to its rapid
exchange with solvent. The 2D map of compound 1360 in DMSO
reconfirmed the location of the sulfate group at position 27, which
was first deduced from the 2D COSY map in D.sub.2O.
[0066] A careful comparison of 2D COSY maps for compound 1360 and
squalamine suggested the presence of a carbonyl at position 24. The
multiplet centered at 2.5 ppm (2.6 ppm in D.sub.2O) with
integration of 2 protons gave strong crosspeaks that identified
these resonances as H23a,b (located alpha to a carbonyl at position
24) and with nearest neighbor connections to H22a,b. In a total
correlation spectroscopy experiment (TOCSY), a new crosspeak was
discernible as H22/H21, due to propagation of magnetization along
the tail of the steroid. Noticeably absent from the 2D COSY and
TOCSY maps were signals that would allow propagation of the
magnetization from positions 23 to 24 and then from 24 to 25.
Unlike the COSY of squalamine, which shows nearest neighbor
crosspeaks for 22-23-24-25-26/27, compound 1360 showed an
interruption in connectivity, suggesting that a functional group at
position 24 blocked transfer of the proton signal. The structure
can be verified by reducing C.dbd.O to an alcohol, however, since
an alcoholic group at position 24 would allow for complete proton
connectivity from positions 21-26 and 27.
[0067] .sup.13C-NMR of compound 1360 in DMSO indicated 34 carbon
signals. In comparison to squalamine, compound 1360 has one
carbonyl at 212 ppm (C24). C27 resonated at 67 ppm, which is in
good agreement with values reported for scymnol sulfate.
Compound 1361:
[0068] Compound 1361 was isolated from shark liver preparations in
two different ways: first, as a degradation product of compound
1360; and, subsequently, as a minor aminosterol component
fractionating with slightly faster retention time than squalamine
during preparative C18 RP--HPLC (component of Fraction VI). In
early attempts to purify each aminosterol to homogeneity, pooled
fractions from C18 RP--HPLC were subjected to silica gel flash
chromatography with CH.sub.2Cl.sub.2:CH.sub.3OH:NH.sub.4OH 6:3:1,
and the aminosterols were assayed on TLC plates. The pools of free
base steroids were then re-subjected to C18 RP--HPLC, using an
analytical column. The RP--HPLC elution profile showed two major
steroids--compound 1360 and a second, more hydrophobic steroid
eluting at higher % CH.sub.3CN. The lability of the sulfate at
position 27 in compound 1360 led to the formation of compound 1361,
apparently through base-catalyzed elimination.
[0069] Hallmark features of the H spectrum for compound 1361 in
D.sub.2O (400 MHz) included the absence of multiplet at
.delta.=4.15 ppm corresponding to methylene protons at position 27,
bearing the sulfate. Two new singlet protons at .delta.=5.95 and
6.15 in D.sub.2O, each with integration values of 1H, were
identified as vinyl protons. Also,.in contrast to the methyl
doublet at .delta.=0.9 ppm in compound 1360, the new steroid showed
a singlet methyl at .delta.=1.8 ppm, characteristic of an allylic
methyl. The chemical shifts for the vinyl groups and for the
allylic methyl compare favorably with literature values. Otherwise,
the .sup.1H spectrum showed features characteristic of compound
1360, including the presence of methylene signals at .delta.=2.75
ppm, alpha to carbonyl at position 24. The polyamine regions
exhibited splitting patterns like that of squalamine, confirming
the spermidine adduct.
[0070] An accurate mass of 543.4823 was measured by FAB-MS
(positive ion mode). The molecular formula
C.sub.34H.sub.61N.sub.3O.sub.2 has a calculated mass of 543.4842,
in good agreement with the experimentally observed value. This
molecular formula for compound 1361 is consistent with elimination
of sulfate from the parent molecule, compound 1360. Moreover, the
molecular formula for compound 1361 has a double bond equivalent
(DBE) of 5.5, in comparison to 5.0 for compound 1360 and 4.0 for
compound 1256; this DBE value is consonant with the additional
unsaturation in compound 1361.
Compound 1436:
[0071] This compound and the steroids described below were purified
by subjecting fractions from preparative C18 RP--HPLC to shallower
CH3CN gradient conditions on smaller C18 columns. Although strong
cation exchange chromatography and silica gel (SG) flash
chromatography followed by RP--HPLC had been used in the
purification of compounds 1360 and 1361, these protocols were not
used when the pH lability was recognized.
[0072] Although compound 1436 elutes from C18 RP--HPLC with
retention time only slightly faster than squalamine, its
R.sub.f=0.47 on TLC hints of a chemical structure with
significantly greater polarity than squalamine under alkaline
conditions (CH.sub.2Cl.sub.2:CH.sub.3OH:NH.sub.4OH 6:3:1).
[0073] The .sup.1H NMR spectrum in D.sub.2O (400 MHz) revealed the
polyamine regions differing from that of squalamine. Both the
splitting pattern and integration resembled spermine rather than
spermidine, i.e. N,N'-bis-3-aminopropyl-1,4-butane-diamine rather
than N-(3-aminopropyl)-1,4-butanediamine. Otherwise, the .sup.1H
spectrum appeared identical to that of squalamine: one proton at
.delta.=4.15, the 24 sulfate position; one proton at .delta.=3.85,
corresponding to H7 alcoholic ring position; three overlapping
doublets in 0.85-0.95 ppm corresponding to methyl 21 and methyls 26
and 27. The identity of spermine was supported by performing COSY
in D.sub.2O, comparing crosspeak patterns to that of reference
standards of spermine (C.sub.10H.sub.26N.sub.4) and spermidine
(C.sub.7H.sub.19N.sub.3) as well as to that of squalamine in
D.sub.2O. Although COSY spectra of the aminosterols generally do
not give a complete 2-dimensional map of crosspeaks in D.sub.2O and
therefore cannot be relied on for complete nearest neighbor
assignments, the polyamine region did produce a complete set of
off-diagonal crosspeaks, which served reliably as the signature
pattern for discerning between spermidine and spermine.
[0074] The .sup.13C spectrum of compound 1436 showed 3 additional
signals in D.sub.2O, but otherwise the carbon skeleton of the
steroid was the same as for squalamine in D.sub.2O. DEPT-135
(distortionless enhanced polarization transfer) was conducted such
that methyl and methine signals were phased as positive signals,
methylene groups as negative signals, and quaternary carbons gave
zero intensity. DEPT-135 of the compound demonstrated that these 3
additional signals were methylenes (negative).
[0075] The molecular formula of C.sub.37H.sub.72N.sub.4O.sub.5S has
a calculated mass of 684.53017, in good agreement with the
experimentally observed accurate mass of 684.5216 measured by high
resolution FAB-MS (positive ion mode). The additional mass of 58
daltons (684.5 versus 627.5 for squalamine) was consistent with the
presence of an extra 3-aminopropyl group attributed to spermine.
Furthermore, the even number mass for the parent ion is consonant
with the nitrogen rule, which predicts a compound having an even
number of nitrogens. FAB-MS also showed fragmentation into species
both 80 and 98 mass units less than the (M+H)+ parent at 685 amu
(atomic mass units). These fragments represent loss of sulfate
followed by dehydration, paralleling the structural lability of
squalamine under FAB-MS conditions.
[0076] Compound 1436 was also synthesized from compound 1256
(squalamine) according to the following reaction scheme:
##STR3##
[0077] Into a round-bottom flask was introduced 95 mg (0.106 mmol)
of squalamine (TFA salt), which was dissolved in 800 .mu.l of
anhydrous methanol. To the mixture was added 118 .mu.l (6.848 mmol)
of triethyl amine, followed by 100 .mu.l (0.106 mmol) of diluted
acrylonitrile solution (70 .mu.l acrylonitrile diluted to 1000
.mu.l in methanol). After 6 hours, a further 40 .mu.l (0.042 mmol)
of the dilute acrylonitrile solution was added. After 24 hours, TLC
showed the presence of starting material and a product with
R.sub.f=0.7 (squalamine R.sub.f=0.5). The reaction was stopped, and
the product isolated by flash chromatography (12:3:1 to 6:3:1
CH.sub.2Cl.sub.2:MeOH:NH.sub.4OH).
[0078] The product, although homogenous by TLC, appeared to be a
mixture by NMR spectroscopy. The product thus obtained was added to
a hydrogenation flask along with 10 mg of Raney nickel, 7.3 mg of
sodium hydroxide and 5 ml of absolute ethanol, and hydrogenated at
40 psi for 17 hours. Two products, now separable (flash
chromatography, 6:3:1 CH.sub.2Cl.sub.2:MeOH:NH.sub.4OH), were seen
on TLC, wherein the lower spot co-spotted with the reference
(naturally isolated compound 1436). This product was separated by
reverse-phase chromatography to yield 1.5 mg of pure material. This
compound had a positive mass (M+1) ion of 685, and its .sup.1H and
.sup.13C NMR spectra were identical to those of the naturally
isolated material, thus confirming its characterization and
structure.
Compound 1437:
[0079] This steroid, eluting immediately after compound 1360 in
preparative C18, exhibits R.sub.f=0.96 on TLC, reflecting a more
polar character than squalamine itself. .sup.1H NMR (400 MHz) in
D.sub.2O appeared essentially identical to squalamine for the
methyl region, steroid nucleus and spermidine region, and 7H at the
ring hydroxyl position. Conspicuously absent from the .sup.1H
spectrum was the multiplet at .delta.=4.15 ppm corresponding to one
proton at the 24-sulfate position. Instead, a new signal centered
at .delta.=3.95 was observed with the characteristic gem alcohol
coupling and integration for 2 protons.
[0080] When compared to squalamine, the .sup.13C spectrum of
compound 1437 in D.sub.2O revealed only two noticeable changes. One
new signal appeared at .delta.=72 ppm, which was subsequently
identified as a --CH.sub.2OH group since its DEPT-135 signal was
negative. In squalamine, the sulfate position was identified at
.delta.=86 ppm as a primary carbon (positive DEPT-135 signal). For
compound 1437, however, this carbon resonance for the sulfate
position shifted to 76 ppm and gave no DEPT-135 signal, thereby
identifying it as a quaternary carbon. The aminosterol structure
consistent with these data has the carbon skeleton of ergostanol,
with carbon 24 bearing the sulfate and carbon 24' being the
alcohol.
[0081] FAB-MS in positive ion mode indicated (M+H)+ at 658.6,
fragmenting to 578.6 due to loss of sulfate; negative ion mode
analysis confirmed a pseudomolecular parent ion (M-H)- at 656.4. An
accurate mass of 578.5264 was determined on the dessulfate
fragment, on account of the low intensity of the parent signal. The
accurate mass of the parent ion could then be calculated as 657.526
(by adding the mass of sulfate). Compound 1437 is thus 30 daltons
greater than squalamine, which could be explained by an additional
--CH.sub.2OH moiety.
Steroids in Fraction I:
[0082] Fraction I (FX1) is the earliest steroid fraction eluting
from preparative C18 RP--HPLC. TLC analysis typically showed a
single major spot with R.sub.f=0.80-0.84 (with respect to
squalamine, R.sub.f=1.0) and protein which stayed at the TLC
origin. If the TLC plates were run with concentrated samples
(.gtoreq.3 mg/ml), hints of additional spots, with R.sub.f values
either slightly greater than or less than the major component, were
discernable.
[0083] When subjected to high resolution RP--HPLC using C18 columns
with 60-100 .ANG. pore size and very shallow CH.sub.3CN gradients,
Fraction I could be separated into as many as 7 components,
designated I-1, I-2, I-3, I-4, I-5, I-6, and I-7. Steroids FX1A,
FX1B, FX1C, FX1D are presented as possible structures: ##STR4##
EXAMPLE 2
Synthesis of Aminosterols
[0084] In addition to the above compounds, which were isolated from
shark liver, synthetic aminosterol compounds have been developed.
Various polyaminosterol compounds, including those specified in
Examples A-G, are described in U.S. Ser. No. 08/416,883, which is
the U.S. national phase of International Application No.
PCT/US94/10265, filed Sep. 13, 1994, the disclosure of which is
herein incorporated by reference. Compounds exemplified therein
include the following: ##STR5##
[0085] Additional aminosterol compounds have now been developed.
Preferred compounds of the invention include those exemplified
below.
EXAMPLE H
[0086] Preparation of compound 353 and compound 354: ##STR6##
[0087] The above compounds were prepared by reductive coupling of
5.alpha.-cholestan-3-one to spermine (4 equivalents) with sodium
cyanoborohydride in a manner analogous to the preparation of
compound 303. Purification was achieved on silica gel (gradient
elution with 9:3:1 to 3:3:1 chloroform:methanol:isopropylamine).
Compound 353 (more polar) and compound 354 (less polar) were
converted to their hydrochloride salts in the same manner as for
compound 303. .alpha.-Amino compound 354: .sup.1H NMR (200 MHz,
CD.sub.3OD) .delta.: 3.47 (m, 1H), 3.3-2.9 (m, 12H), 2.3-1.0 (m,
39H), 1.0-0.8 (m, 12H), 0.70 (s, 3H); IR (KBr, cm.sup.-1): 3396,
2934, 1594, 1457, 1383; MS(+FAB): 573.6 (M+1); Anal. calcd. for
C.sub.37H.sub.72N.sub.4-4HCl--H.sub.2O: C=60.31, H=10.67, N=7.60;
Found: C=60.01, H=10.83, N=7.67. .beta.-Amine compound 353: .sup.1H
NMR (200 MHz, CD.sub.3OD) .delta.: 3.3-3.0 (m, 13H), 2.2-1.0 (m,
39H), 1.0-0.8 (m, 12H), 0.70 (s, 3H); IR (KBr, cm.sup.-1): 2945,
1596, 1466, 1383; MS exact mass (+FAB) calcd.: 573.5835; Found:
573.5801; Anal. calcd. for C.sub.37H.sub.72N.sub.4-4HCl--H.sub.2O:
C=58.87, H=10.68, N=7.42; Found: C=58.49, H=10.94, N=7.94.
[0088] Compound 353 is a simple adduct of spermine and cholestanol,
representing a very inexpensive compound. It can be synthesized
like compound 354 in the following straightforward manner:
##STR7##
EXAMPLE I
[0089] Preparation of compound 458and compound 459: ##STR8##
[0090] The above compounds were prepared from methyl
3-oxo-5.alpha.-cholanoate and spermine (1.35 equivalents) as in the
synthesis of compound 353. Purification on silica gel (gradient
elution with 6:3:1 to 3:5:2 chloroform:methanol:isopropylamine)
afforded the less polar .alpha.-amino compound 458 and the more
polar .beta.-amino compound 459. These compounds were converted to
their hydrochloride salts as done as for compound 303. Compound
458: .sup.1H NMR (400 MHz, CD.sub.3OD) .delta.: 3.64 (s, 3H), 3.45
(m, 1H), 3.25-3.05 (m, 12H), 2.4-1.0 (m, 36H), 0.93 (d, J=6 Hz,
3H), 0.87 (s, 3H), 0.70 (s, 3H); IR (KBr, cm.sup.-1): 2943, 1741,
1458, 1169; MS(+FAB): 575.6 (M+1); Anal. calcd. for
C.sub.35H.sub.66H.sub.4O.sub.2-4HCl-1.2H.sub.2O: C=56.63, H=9.83,
N=7.55; Found: C=56.58, H=9.46, N=7.29. Compound 459: .sup.1H NMR
(400 MHz, CD.sub.3OD) .delta.: 3.63 (s, 3H), 3.2-3.0 (m, 13H),
2.4-1.0 (m, 36H), 0.92 (d, J=6 Hz, 3H), 0.86 (s, 3H), 0.69 (s, 3H);
IR (KBr, cm.sup.-1): 42942, 1739, 1595, 1459, 1382, 1170; MS(+FAB):
575.6 (M+1); Anal. calcd. for
C.sub.35H.sub.66H.sub.4O.sub.2-4HCl-1.4H.sub.2O: C=56.35, H=9.84,
N=7.51; Found: C=56.35, H=9.26, N=7.67.
EXAMPLE J
[0091] Preparation of compounds 380, 381, 382 and 394: ##STR9##
[0092] The steroid methyl
7.alpha.-hydroxy-3-oxo-5.alpha.-cholanoate was prepared according
to the method of Iida et al., Chem. Pharm. Bull. 41(4), 1993,
763-765. This steroid was coupled to the polyamine compound 301
with sodium cyanoborohydride, the BOC groups were removed with
trifluoroacetic acid, and the ester was hydrolyzed as in the
preparation of compound 319, except that lithium hydroxide was used
as the base. Purification was achieved on silica gel (15:4:1 to
10:4:1 chloroform:methanol:isopropylamine). Compounds 381 and 382
were treated with 2 M ammonia in methanol and evaporated
(3.times.20 ml) to drive off isopropylamine. The hydrochloride salt
was prepared as for compound 303.
[0093] Compound 380, C.sub.32H.sub.59N.sub.3O.sub.3: .sup.1H NMR
(200 MHz, CDCl.sub.3) .delta.: 3.83 (br s, 1H), 3.66 (s, 3H),
2.8-2.4 (m, 9H), 2.3-1.0 (m, 32H), 0.92 (d, J=6 Hz, 3H), 0.78 (s,
3H), 0.65 (s, 3H); IR (KBr, cm.sup.-): 3278, 2928, 1736, 1447,
1163; MS(+FAB): 534 (M+1).
[0094] Compound 381, C.sub.31H.sub.57N.sub.3O.sub.3-1.7 H.sub.2O;
.sup.1H NMR (200 MHz, CD.sub.3OD) .delta.: 3.80 (br s, 1H), 3.0-2.5
(m, 9H), 2.2-1.1 (m, 32H), 0.94 (d, J=6 Hz, 3H), 0.84 (s, 3H), 0.69
(s, 3H); IR (KBr, cm.sup.-): 3380, 2929, 1560, 1395; MS(+FAB)
calcd.: 520.4478 (M+1); Found: 520.4506; Anal. calcd.: C=67.64,
H=11.06, N=7.63; Found: C=67.64, H=10.24, N=7.83.
[0095] Compound 382, C.sub.31H.sub.57N.sub.3O.sub.3-2H.sub.2O:
.sup.1H NMR (200 MHz, CD.sub.3OD) .delta.: 3.80 (br s, 1H), 3.15
(br s, 1H), 3.1-2.6 (m, 8H), 2.2-1.1 (m, 32H), 0.96 (d, J=6 Hz,
3H), 0.85 (s, 3H), 0.69 (s, 3H); IR (KBr, cm.sup.-1): 3416, 2930,
1560, 1395; MS(+FAB) calcd.: 520.4478 (M+1); Found: 520.4489; Anal.
calcd: C=66.99, H=11.06, N=7.56; Found: C=66.93, H=10.16,
N=7.28.
[0096] Compound 394,
C.sub.32H.sub.59N.sub.3O.sub.3-3HCl-0.5H.sub.2O: .sup.1H NMR (200
MHz, CD.sub.3OD) .delta.: 3.83 (br s, 1H), 3.64 (s, 3H), 3.48 (br
s, 1H), 3.3-2.9 (m, 8H), 2.4-1.1 (m, 32 H), 0.94 (d, J=6 Hz, 3H),
0.87 (s, 3H), 0.70 (s, 3H); MS(+FAB): 535 (M+1); Anal. calcd.:
C=58.93, H=9.74, N=6.44; Found: C=58.71, H=10.13, N=6.39.
EXAMPLE K
[0097] Preparation of compounds 395, 396 and 397: ##STR10##
[0098] Methyl 7.alpha.-hydroxy-3-oxo-5.alpha.-cholanoate was
coupled to spermine (2 equivalents) with sodium cyanoborohydride,
and the ester was hydrolyzed as in the preparation of compound 319,
except that lithium hydroxide was used as the base. Purification of
compounds 395 and 396 was achieved on silica gel (15:5:1 to 5:5:1
chloroform:methanol:isopropylamine). Purification of compound 397
was achieved on silica gel (2:6:1 benzene:methanol:isopropylamine),
followed by treatment with 2 M ammonia in methanol (3.times.20 ml)
to drive off isopropylamine. The hydrochloride salts of compounds
395 and 396 were prepared in the same manner as for compound
303.
[0099] Compound 395, C.sub.35H.sub.66N.sub.4O.sub.3-4HCl-2H.sub.2O:
.sup.1H NMR (200 MHz, CD.sub.3OD) .delta.: 3.80 (br s, 1H), 3.64
(s, 3H), 3.3-3.0 (m, 13H), 2.4-1.0 (m, 34H), 0.94 (d, J=6 Hz, 3H),
0.87 (s, 3H), 0.70 (s, 3H); Anal. calcd.: C=54.40, H=9.65, N=7.25;
Found: C=54.16, H=9.31, N=7.12.
[0100] Compound 396,
C.sub.35H.sub.66H.sub.4O.sub.3-4HCl-0.5H.sub.2O: MS(+FAB): 592
(M+1); Anal. calcd.: C=56.37, H=9.60, N=7.51; Found: C=56.43,
H=9.83, N=7.27.
[0101] Compound 397, C.sub.34H.sub.64H.sub.4O.sub.3: .sup.1H NMR
(200 MHz, CD.sub.3OD) .delta.: 3.78 (br s, 1H), 2.9-2.5 (m, 13H),
2.2-1.1 (m, 34H), 0.95 (d, J=6 Hz, 3H), 0.87 (s, 3H), 0.70 (s, 3H);
MS(+FAB): 577.3 (M+1).
EXAMPLE L
[0102] Preparation of compound 393: ##STR11##
[0103] Compound 304 (210 mg, 0.41 mmol) was dissolved in methanol
(10 ml) under nitrogen, and treated with o-methylisourea
hydrochloride (50 mg, 0.45 mmol) and 1 N sodium hydroxide solution
(0.45 ml, 0.45 mmol). After 23 hours, additional o-methylisourea
was added (102 mg, 0.92 mmol), and the reaction was continued for 7
hours, quenched with 1 N hydrochloric acid solution (pH<7), and
evaporated. The residue was partitioned between 1 N sodium
hydroxide solution (50 ml) and chloroform (100 ml). After washing
with additional chloroform (50 ml), the combined organic layers
were dried (Na.sub.2SO.sub.4) and concentrated. Purification by
flash chromatography on silica gel (2-cm diameter, gradient elution
with 5 to 15% methanol in methylene chloride) afforded a white
solid (32 mg). This material was dissolved in chloroform (3 ml),
cooled in an ice bath, treated with 1 M hydrogen chloride in ether
(1 ml), and concentrated in vacuo to afford compound 393 (37 mg,
14% yield). C.sub.35H.sub.64N.sub.4-2HCl-2H.sub.2O: .sup.1H NMR
(200 MHz, CD.sub.3OD) .delta.: 3.5-3.3 (m, 5H), 3.2-3.0 (m, 4H),
2.2-1.0 (m, 37H), 0.95-0.86 (m, 9H), 0.70 (s, 3H); IR (KBr,
cm.sup.-1): 3306, 3153, 2934, 1654, 1586, 1445, 1383; MS(+FAB):
541.4 (M+1); Anal. calcd.: C=64.69, H=10.86, N=8.62, Found:
C=65.06, H=10.98, N=8.83.
EXAMPLE M
[0104] Preparation of compounds 370 and 371: ##STR12##
[0105] Preparation of compound 1010: ##STR13##
[0106] To a suspension of pyridinium chlorochromate (6.85 g, 31.8
mmol) in dichloromethane (130 ml) was added a solution of compound
1006 (5.95 g, 13.3 mmol) in dichloromethane (70 ml). After stirring
for 3 hours at room temperature, the reaction mixture was diluted
with ether (100 ml), filtered, and washed with ether. The organic
layer was washed with 5% sodium hydroxide solution, 5% hydrochloric
acid solution, saturated sodium bicarbonate and brine. The dried
ethereal layer was evaporated and purified by flash chromatography
(6 cm, gradient elution with 0-20% ethyl acetate in hexane) to
yield pure compound 1010 (5.25 g, 77% yield). .sup.1H NMR (200 MHz,
CDCl.sub.3) .delta.: 4.92 (m, 1H), 2.5-1.0 (m, 29H), 2.06 (s, 3H),
1.03 (s, 3H), 0.91 (d, J=6.5 Hz, 3H), 0.87 (d, J=6.5 Hz, 6H), 0.67
(s, 3H); IR (KBr, cm.sup.-1): 2949, 1736, 1468, 1372, 1244, 1023;
MS(ES+): 467.8 (M+Na).
[0107] Preparation of compounds 370 and 371: The steroid 1010 was
coupled to polyamine 301 with sodium cyanoborohydride, the BOC
groups were removed with trifluoroacetic acid, and the acetate was
hydrolyzed as in the preparation of compound 319, except that
sodium hydroxide was used as the base. Purification was achieved on
silica gel (2:6:1 benzene:methanol:isopropylamine). Compound 370
(.sup.1H NMR (200 MHz, CD.sub.3OD) .delta.: 3.80 (m, 1H), 2.97 (m,
1H), 2.9-2.6 (m, 8H), 2.1-1.0 (m, 35H), 0.94 (d, J 6.5 Hz, 3H),
0.88 (d, J=6.5 Hz, 6H), 0.87 (s, 3H), 0.70 (s, 3H)) and compound
371 (.sup.1H NMR (200 MHz, CD.sub.3OD) .delta.: 3.77 (m, 1H),
2.8-2.5 (m, 9H), 2.1-1.0 (m, 35H), 0.93 (d, J=6.5 Hz, 3H), 0.88 (d,
J=6.5 Hz, 6H), 0.83 (s, 3H), 0.69 (s, 3H)) were converted to their
hydrochloride salts as in the preparation of compound 303. Compound
370: IR (KBr, cm.sup.-1): 3415, 2948, 1595, 1467, 1382, 1031;
MS(+FAB): 532.4 (M+1); Anal. calcd. for
C.sub.34H.sub.65N.sub.3O-3HCl-2H.sub.2O: C=60.29, H=10.71, N=6.20;
Found: C=60.01, H=11.09, N=6.3. Compound 371: IR (KBr, cm.sup.-1):
3414, 2953, 1596, 1468, 1381, 1033; MS(+FAB): 532.4 (M+1); Anal.
calcd. for C.sub.34H.sub.65N.sub.3O.3HCl.2H.sub.2O: C=60.29,
H=10.71, N=6.20; Found: C=60.49, H=11.00, N=6.47.
EXAMPLE N
[0108] Preparation of compound 470: ##STR14##
[0109] Preparation of precursors: ##STR15## ##STR16##
[0110] Preparation of compounds 1011 and 1012: A solution of methyl
3-oxo-5.alpha.-cholanoate (compound 310, 2.00 g, 5.15 mmol),
p-toluenesulfonic acid (250 mg), and ethylene glycol (25 ml) in
benzene (160 ml) was heated to reflux with the removal of water for
6 hours. After cooling to room temperature, saturated sodium
bicarbonate (30 ml) was added, and the aqueous phase was extracted
with benzene and ethyl acetate. The organic layers were washed with
water and brine, dried over sodium sulfate, and evaporated to yield
compound 1011, which was used for the next step without
purification.
[0111] A solution of 1 M lithium aluminum hydride (25 ml, 25 mmol)
in ether under nitrogen was treated with a solution of compound
1011 in anhydrous ether (80 ml) and heated to reflux for 5 hours.
After stirring overnight, the reaction mixture was quenched at
0.degree. C. with water and 2 N sodium hydroxide solution. The
aqueous layer was extracted with ether, followed by washing with
brine, drying over magnesium sulfate, and evaporating to afford
compound 1012 (1.80 g, 86% yield). .sup.1H NMR (400 MHz,
CDCl.sub.3) .delta.: 3.94 (s, 4H), 3.62 (m, 2H), 2.0-1.0 (m, 28H),
0.92 (d, J=6 Hz, 3H), 0.81 (s, 3H), 0.66 (s, 3H).
[0112] Preparation of compounds 1013 and 1014: A solution of
compound 1012 (3.63 g, 8.97 mmol) in anhydrous pyridine (16 ml) was
treated with p-toluenesulfonyl chloride (2.3 g, 12.1 mmol) at room
temperature, and left overnight. Ice water was added, and the
reaction mixture was left for 30 minutes with stirring. Then 6 N
hydrochloric acid was added (70 ml), and the aqueous layer was
extracted with dichloromethane and ether. The organic layers were
washed with 2 N hydrogen chloride, saturated sodium bicarbonate and
brine, dried, and evacuated to yield crude compound 1013. Compound
1013 was dissolved in dimethylsulfoxide (40 ml) and treated with
sodium cyanide (1.4 g, 28 mmol) at 90.degree. C. for 2.5 hours
under nitrogen. After cooling, the reaction mixture was treated
with ice water and extracted into ether and dichloromethane. The
organic layers were washed with brine, dried over sodium sulfate,
and purified by chromatography (4-cm diameter, gradient elution
with 0-25% ethyl acetate in hexane) to yield pure compound 1014.
.sup.1H NMR (400 MHz, CDCl.sub.3) .delta.: 3.94 (s, 4H), 2.32 (m,
2H), 2.0-1.0 (m, 28H), 0.93 (d, J=6 Hz, 3H), 0.81 (s, 3H), 0.66 (s,
3H); IR (KBr, cm.sup.-1): 2930, 2247, 1445, 1381, 1357, 1133, 1091,
928, 899; MS(+FAB): 414.4 (M+1).
[0113] Preparation of compound 1015: A solution of compound 1014
(480 mg, 1.16 mmol) in acetic acid (35 ml) and concentrated
hydrochloric acid (25 ml) was refluxed for 25 hours. After
evaporating the solvent, the residue was partitioned between water
and ethyl acetate. After drying and evaporating, the crude
carboxylic acid was dissolved in methanol (25 ml), treated with
concentrated hydrochloric acid (1 ml), and brought to reflux for 20
minutes. After evaporation of solvent, the product was dissolved in
ethyl acetate and water and extracted again with ethyl acetate. The
organic layers were washed with brine, dried over sodium sulfate,
and purified by flash chromatography (2-cm diameter, gradient
elution with. 0-25% ethyl acetate in hexane) to afford pure
compound 1015 (298 mg, 64% yield), m.p. 147-148.degree. C. .sup.1H
NMR (400 MHz, CDCl.sub.3) .delta.: 3.67 (s, 3H), 2.4-1.0 (m, 30H),
1.01 (s, 3H), 0.93 (d, J=6 Hz, 3H), 0.68 (s, 3H); .sup.13C NMR (400
MHz, CDCl.sub.3) .delta.: 212.3, 174.5, 56.5, 56.1, 54.0, 51.6,
46.9, 44.9, 42.8, 40.1, 38.8, 38.4, 35.8, 35.7, 35.6, 34.7, 31.9,
29.2, 28.4, 24.4, 21.7, 21.6, 18.8, 12.2, 11.7; MS(+FAB): 403.3
(M+1); Anal. calcd. for C.sub.26H.sub.42O.sub.3: C=77.56, H=10.51;
Found: C=77.49, H=10.52.
[0114] Preparation of compound 470: Steroid 1015 was coupled to
polyamine 301 with sodium cyanoborohydride, the BOC groups were
removed with trifluoroacetic acid, and the ester was hydrolyzed as
in the preparation of compound 319, except that lithium hydroxide
was used as the base. Purification was achieved on silica gel
(gradient elution with 14:4:1 to 4:4:1
chloroform:methanol:isopropylamine). After evaporation from
methanol:chloroform (3.times.), the compound was treated with 2 M
ammonia in methanol and evaporated (3.times.20 ml) to drive off
isopropylamine. .sup.1H NMR (400 MHz, CD.sub.3OD) .delta.: 2.8-2.6
(m, 9H), 2.2-1.0 (m, 36H), 0.92 (d, J=6 Hz, 3H), 0.80 (s, 3H), 0.66
(s, 3H); MS(+FAB): 518.4 (M+1); Anal. calcd.: C=71.73, H=11.47,
N=7.84; Found: C=2.03, H=11.06, N=7.53.
EXAMPLE O
[0115] Preparation of compounds 431, 432, 433, 465, 466, 467, and
469. ##STR17## ##STR18##
[0116] Preparation of precursors: ##STR19## ##STR20##
[0117] Preparation of compound 1016: The methyl ester of
hyodeoxycholic acid was prepared by acid-catalyzed esterification
of hyodeoxycholic acid in methanol. To a magnetically stirred
500-ml round-bottom flask containing absolute methanol (200 ml) was
added hyodeoxycholic acid (10 g, 25.5 mmol) and concentrated
sulfuric acid (5 ml) dropwise. The reaction was stirred overnight
and then treated with dichloromethane (250 ml), followed by washing
with sodium bicarbonate solution (2.times.100 ml) and brine (100
ml). The organic layer was then dried over anhydrous sodium
sulfate, filtered, and dried under vacuum to yield compound 1016
(10.1 g, 97% yield) (see Organic Preparations and Procedures Int.
19(2-3), 1987, 197-208).
[0118] Preparation of compound 1017: The 3,6-dioxo sterol was
prepared by oxidation of methyl hyodeoxycholic acid with pyridinium
chlorochromate. Compound 1016 (10.1 g, 25 mmol) was dissolved in
dichloromethane (200 ml). To a magnetically stirred flask in an ice
water bath was added pyridinium chlorochromate (33 g, 150 mmol).
The reaction was allowed to warm to room temperature and to proceed
for 8 hours, until the product was the only visible TLC spot. A
major portion of the dichloromethane was removed under vacuum, and
ethyl acetate (250 ml) was then added to the flask. The chromium
crust in the bottom of the flask was broken up with a spatula, and
the contents of the flask were filtered through a Celite column.
The elutant from the column was then reduced in volume under vacuum
and filtered through a florisil column (elution with ethyl
acetate). The elutant was again reduced in volume to approximately
200 ml, and diethyl ether (100 ml) was added, followed by washing
with sodium bicarbonate solution (2.times.250 ml) and then brine
(250 ml). The organic layer was dried over anhydrous sodium
sulfate, filtered, and dried under vacuum. The total yield of
methyl 3,6-dioxo-5.beta.-cholan-24-oate 1017 without
recrystallization was 9.6 g (24 mmol, 96%) (see Organic
Preparations and Procedures Int. 19(2-3), 1987, 197-208). The
product can be recrystallized from a number of solvents (absolute
methanol, ethyl acetate in hexanes, or diethyl ether in hexanes) if
any chromium remains.
[0119] Preparation of compound 1018: The 3,6-dioxo-5.alpha. sterol
was prepared by acid-catalyzed isomerization of the 5.beta. sterol.
To methanol (250 ml) was added the 3,6-dioxo-5.beta. sterol 1017
(9.6 g, 24 mmol) and tetrahydrofuran (25 ml) to dissolve the sterol
completely: Concentrated hydrochloric acid (12.5 ml) was added, and
the reaction was allowed to proceed overnight. The solvent was then
removed under vacuum to yield 9.6 g (100% yield) of methyl
3,6-dioxo-5.alpha.-cholan-24-oate 1018 (see Organic Preparations
and Procedures Int. 19(2-3), 1987, 197-208; authors used
base-catalyzed isomerization using sodium methoxide rather than
HCl).
[0120] Preparation of compound 1019: The mono-protection of methyl
3,6-dioxo-5.alpha.-cholan-24-oate 1018 may be accomplished using a
variety of techniques. One technique involved refluxing compound
1018 (9.6 g, 23.8 mmol) in toluene (250 ml) with ethylene glycol
(1.77 g, 28.5 mmol) in the presence of catalytic p-toluenesulfonic
acid. A Dean Stark trap was used for removing the toluene/water
azeotrope. The reaction was judged to be complete by TLC after
approximately 20 minutes. The reaction was worked up by pouring the
toluene over sodium bicarbonate solution (500 ml) and ice slurry.
The organic layer was washed with additional sodium bicarbonate
(200 ml) and brine (200 ml), dried over anhydrous sodium sulfate,
filtered, and dried under vacuum. The crude product was
chromatographed on silica gel (4 cm.times.25 cm, elution with 33%
ethyl acetate in hexanes). Methyl
3-dioxolane-6-oxo-5.alpha.-cholan-24-oate 1019 (8.9 g, 81%) was the
second band off the column; the only other product present was the
less polar di-dioxolane. Subsequent techniques yielded better
results by substituting benzene for toluene and following the
reaction by TLC, which apparently allows for greater selectivity.
The reaction can be stopped before significant di-protection occurs
in the lower boiling solvent. Compound 1019: m.p. 124-126.degree.
C.; .sup.1H NMR (200 MHz, CDCl.sub.3) .delta.: 4.04-3.93 (m, 4H),
3.68 (s, 3H), 0.95 (d, J=6 Hz, 3H), 0.78 (s, 3H), 0.69 (s, 3H); IR
(KBr, cm.sup.-1): 2945, 1742, 1709, 1439, 1381, 1313, 1162, 1090;
MS(FD): 446 (M.sup.+), 388.
[0121] Preparation of compound 1020: The 6.beta.-hydroxy sterol was
prepared in good yield from the mono-protected diketone by
reduction with sodium borohydride. The 3-dioxolane-6-oxo sterol
1019 (5 g, 11 mmol) was dissolved in tetrahydrofuran (10 ml) and
added to absolute methanol (200 ml) and sodium borohydride (2.5 g,
66 mmol). The sodium borohydride was dissolved and stirred for
approximately 20-30 minutes before the addition of the sterol.
After stirring overnight, the reaction mixture was treated with
chloroform (500 ml), and washed with distilled water (2.times.200
ml) and then brine (100 ml). The organic layer was then dried over
sodium sulfate, filtered, concentrated under vacuum, and purified
by flash chromatography on silica gel (4 cm.times.25 cm, elution
with 2:1:1 hexanes:ethyl acetate:methylene chloride) to yield
methyl 3-dioxolane-6.beta.-hydroxy-5.alpha.-cholan-24-oate 1020
(4.35 g, 87% yield). Alternatively, the crude product can be
recrystallized from benzene in hexanes, ethyl acetate in hexanes,
or chloroform in hexanes (2.times.) to yield a product of high
purity without need for column chromatography. Compound 1020: m.p.
164.degree. C.; .sup.1H NMR (200 MHz, CDCl.sub.3) .delta.:
4.04-3.93 (m, 4H), 3.77 (br s, 1H), 3.66 (s, 3H), 1.03 (s, 3H),
0.92 (d, J=6 Hz, 3H), 0.69 (s, 3H); IR (KBr, cm.sup.-1): 3533,
2937, 1726, 1438, 1379, 1255, 1191, 1096; X-ray diffraction
revealed the expected structure.
[0122] Preparation of compound 1021: The 3-dioxolane was
deprotected using acidic acetone solution. The
3-dioxolane-6.beta.-hydroxy-sterol 1020 (4.0 g, 8.9 mmol) was
dissolved in acetone (200 ml) and treated with concentrated
hydrochloric acid solution (10 ml). After approximately 1 hour, the
reaction mixture was poured into a sodium bicarbonate solution. The
solution was extracted with dichloromethane (3.times.200 ml),
washed with distilled water (100 ml) and then brine (100 ml), dried
over anhydrous sodium sulfate, filtered, and evaporated under
vacuum to yield methyl
3-oxo-6.beta.-hydroxy-5.alpha.-cholan-24-oate 1021 (3.45 g, 100%
yield): .sup.1H NMR (200 MHz, CDCl.sub.3) .delta.: 3.8 (br m, 1H),
3.69 (s, 3H), 1.24 (s, 3H), 0.95 (d, J=6 Hz, 3H), 0.74 (s, 3H); IR
(KBr, cm.sup.-1): 3447, 2954, 1742, 1707, 1431.
[0123] Preparation of compounds 431 and 432: The ethylene-diamine
compounds were prepared as follows. A magnetically stirred solution
of 50:50 methanol:tetrahydrofuran (100 ml) and ethylenediamine (2
ml) was treated with acetic acid to lower the pH to approximately
6. The 3-oxo sterol 1021 (1.5 g, 3.7 mmol) was added, and the
mixture was stirred for 15 minutes. Sodium cyanoborohydride (1 g,
16 mmol) was dissolved in 10 ml methanol and added to the reaction
vessel, and the pH was again adjusted to 6 by the addition of
acetic acid. The reaction was stirred for 1 hour, and the contents
of the flask were poured into a pH 10.5 carbonate-buffer ice slurry
(250 ml). The solution was extracted with chloroform (5.times.150
ml). The organic layers were combined, dried over anhydrous sodium
sulfate, filtered, dried under vacuum, and purified by flash
chromatography on silica gel (4 cm.times.25 cm, elution with 8:2:1
chloroform:methanol:isopropylamine) to afford the less polar
.alpha.-isomer 431 (260 mg, 15% yield) and the more polar
.beta.-isomer 432 (840 mg, 49% yield). Compound 431: .sup.1H NMR
(400 MHz, CD.sub.3OD) .delta.: 3.74 (m, 1H), 3.65 (s, 3H), 3.53 (m,
1H), 1.06 (s, 3H), 0.94 (d, J=6 Hz, 3H), 0.74 (s, 3H); IR (KBr,
cm.sup.-1): 3426, 2943, 1740, 1590, 1438, 1379, 1258, 1168, 1027;
MS(+FAB): 449.5 (M+1); Anal. calcd. for
C.sub.27H.sub.48N.sub.2O.sub.3-2HCl-0.7H.sub.2O: C=60.70, H=9.70,
N=5.24; Found: C=60.97, H=9.68, N=5.34. Compound 432: .sup.1H NMR
(400 MHz, CD.sub.3OD) .delta.: 3.75 (m, 1H), 3.64 (s, 3H), 1.02 (s,
3H), 0.94 (d, J=6 Hz, 3H), 0.73 (s, 3H); IR (KBr, cm.sup.-1): 3560,
3366, 3257, 2936, 1726, 1648, 1605, 1438, 1376, 1166, 1047;
MS(+FAB): 449.5 (M+1); Anal. calcd. for
C.sub.27H.sub.48N.sub.2O.sub.3-0.4H.sub.2O: C=71.13, H=10.79,
N=6.14; Found: C=71.15, H=10.71, N=6.28.
[0124] Preparation of compounds 465 and 466: To a magnetically
stirred flask containing anhydrous methanol (100 ml) was added
compound 1021 (1.5 g, 3.7 mmol), spermine (2 g, 9.9 mmol), powdered
3 .ANG. sieves (2 g), and acetic acid until the pH was 6. The flask
was sealed, the contents stirred overnight, and then sodium
cyanoborohydride (1 g, 16 mmol) in methanol (10 ml) was added. The
pH was again adjusted with acetic acid, and the reaction mixture
was stirred for 8 hours. The workup was similar to the workup for
the ethylenediamine compounds. The crude product was purified by
flash chromatography (5 cm.times.25 cm, elution with 4:5:1
chloroform:methanol:isopropylamine), affording less polar
.alpha.-amino isomer 465 and more polar .beta.-amino isomer 466.
The total yield of amino sterol was 1.3 g (58% yield). Compound
465: .sup.1H NMR (400 MHz, CD.sub.3OD) .delta.: 3.75 (m, 1H), 3.65
(s, 3H), 3.54 (m, 1H), 1.06 (s, 3H), 0.95 (d, J=6 Hz, 3H), 0.74 (s,
3H); IR (KBr, cm.sup.-1): 3406, 2944, 1740, 1596, 1466, 1168, 1049,
1027; MS(+FAB): 591.4 (M+1); Anal. calcd. for
C.sub.35H.sub.66N.sub.4O.sub.3-4HCl-1.2H.sub.2O: C=55.43, H=9.62,
N=7.39; Found: C=55.70, H=9.15, N=7.12. Compound 466: .sup.1H NMR
(400 MHz, CD.sub.3OD) .delta.: 3.79 (m, 1H), 3.65 (s, 3H), 1.06 (s,
3H), 0.95 (d, J=6 Hz, 3H), 0.74 (s, 3H); IR (KBr, cm.sup.-1): 3406,
2944, 1740, 1595, 1459, 1381, 1167, 1051, 1026; MS(+FAB): 591.4
(M+1); Anal. calcd. for
C.sub.35H.sub.66N.sub.4O.sub.3-4HCl-1.2H.sub.2O: C=55.43, H=9.62,
N=7.39; Found: C=55.48, H=9.03, N=7.33.
[0125] Preparation of compound 469: This compound was prepared in a
manner analogous to that used for compound 466, but using polyamine
1023: ##STR21## The polyamine was prepared from piperazine by
double addition of acrylonitrile to yield compound 1022, which was
reduced by Raney nickel catalyzed hydrogenation. .beta.-amino
isomer 469: .sup.1H NMR (400 MHz, CD.sub.3OD) .delta.: 3.78 (m,
1H), 3.64 (s, 3H), 3.5-3.3 (m, 8H), 3.2-3.0 (m, 9H), 2.4-1.0 (m,
30H), 1.03 (s, 3H), 0.92 (d, J=6.5 Hz, 3H), 0.71 (s, 3H); IR (KBr,
cm.sup.-1): 3406, 2943, 1736, 1594, 1443, 1165; MS(+FAB): 589.4
(M+1); Anal. calcd. for
C.sub.35H.sub.64H.sub.4O.sub.3-4HCl-3H.sub.2O: C=53.29, H=9.46,
N=7.10; Found: C=53.06, H=8.90, N=8.43.
[0126] Preparation of compounds 433 and 467: An amount of
aminosterol methyl ester (1 mmol) as the free base was weighed into
a 25-ml round-bottom flask. The aminosterol was dissolved in a
minimal amount of tetrahydrofuran (2 ml), treated with 1 N
potassium hydroxide solution (10 ml), and magnetically stirred for
1 hour. The solution was then neutralized with 1N HCl, and the
solvent was removed under vacuum. The residue was redissolved in a
minimal amount of deionized water and applied to an
octadecyl-functionalized silica gel column (Aldrich, 2.times.10 cm,
gradient elution of acetonitrile in 2% trifluoroacetic acid in
water). The fractions containing aminosterol were pooled, and the
solvent was removed under vacuum. The aminosterol was redissolved
in 0.1N HCl, and the solvent was removed under vacuum (2.times.) to
insure the removal of trifluoroacetate. Benzene was added to the
resulting hydrochloride salts, followed by evaporation overnight to
remove as much water as possible.
[0127] Ethylenediamine .beta.-amino isomer 433 was not treated with
HCl, but isolated as the trifluoroacetate salt. Compound 433:
.sup.1H NMR (400 MHz, CD.sub.3OD) .delta.: 3.78 (m, 1H), 1.06 (s,
3H), 0.95 (d, J=6.5 Hz, 3H), 0.74 (s, 3H); IR (KBr, cm.sup.-1)
3533, 3488, 2941, 1716, 1679, 1615, 1489, 1431, 1191; MS(+FAB):
435.5 (M+1), 531.5 (likely a trace of the trifluoroacetamide);
Anal. calcd. for C.sub.26H.sub.46N.sub.2O.sub.3-2TFA-0.7H.sub.2l O:
C=53.36, H=7.37, N=4.15; Found: C=54.36, H=7.45, N=4.40.
[0128] Spermine .beta.-amino isomer 467: .sup.1H NMR (400 MHz,
CD.sub.3OD) .delta.: 3.80 (m, 1H), 1.05 (s, 3H), 0.95 (d, J=6.5 Hz,
3H), 0.73 (s, 3H); IR (KBr, cm.sup.-1): 3406, 2944, 1718, 1637,
1458; MS(+FAB): 577.4 (M+1); Anal. calcd. for
C.sub.34H.sub.64H.sub.4O.sub.3-4HCl-4H.sub.2O: C=51.38, H=9.64,
N=7.05; Found: C=51.40, H=8.77, N=7.01.
EXAMPLE P
[0129] Preparation of bile acid methyl esters 409, 410, 411, 355,
356, 416, 448, 414, 415, 412, 413, 417 and 449: ##STR22## ##STR23##
##STR24##
[0130] Preparation of precursors: The methyl esters of
chenodeoxycholic acid and deoxycholic acid, which are structurally
depicted below, were prepared by the same procedure as used to
esterify hyodeoxycholic acid to compound 1016. ##STR25##
[0131] Silver carbonate oxidations of bile acid esters to prepare
3-keto steroids: Both chenodeoxycholic and deoxycholic acid
derivatives were prepared by reductive aminations of the 3-oxo
sterols with the appropriate amines. The 3-oxo sterols were
prepared by similar procedures. Silver carbonate on Celite was
prepared by dissolving 4 equivalents of silver nitrate in deionized
water and adding sufficient Celite to result in 50% silver
carbonate on Celite. To the magnetically stirred solution was added
2.2 equivalents of sodium carbonate dissolved in deionized water,
with continued vigorous stirring. The resulting silver carbonate
precipitated on Celite was filtered through a glass-fritted funnel,
washed with tetrahydrofuran, and allowed to dry in a vacuum
desiccator. The methyl ester of the bile acid to be oxidized was
dissolved in toluene, treated with 2 equivalents of silver
carbonate on Celite, and heated to reflux using a Dean Stark
apparatus for azeotropic removal of water. The oxidation was
complete in less than 6 hours for both sterols. The only product in
both cases was the desired 3-oxo sterol. The solution was filtered
and the solvent removed under vacuum. The product in both cases
recrystallized readily from ethyl acetate in hexanes to give the
3-oxo sterol in excellent yield (>89% in both cases).
[0132] Preparation of compounds 409 and 410: The 3-oxo sterol
methyl ester of chenodeoxycholic acid (1.5 g, 3.7 mmol) was
dissolved in methanol, to which a ten-fold excess of
ethylenediamine (2.5 ml) was added. The pH was lowered with acetic
acid to approximately 6, NaBH.sub.3CN (1 g, 15.9 mmol) dissolved in
methanol was added, and the pH was again adjusted with acetic acid.
The solution was stirred for 1 hour, and then worked up and
purified in the same manner as compound 431. The total yield of
aminosterol was 58%, with an approximate ratio of .alpha.-amino
isomer to the less polar .beta.-amino isomer of 7:3. .beta.-Amino
isomer 409: .sup.1H NMR (400 MHz, CD.sub.3OD) .delta.: 3.81 (m,
1H), 3.68 (s, 3H), 3.42 (m, 1H), 1.04 (s, 3H), 0.95 (d, J=6.5 Hz,
3H), 0.72 (s, 3H); IR (KBr, cm.sup.-1): 3428, 2940, 2055, 1740,
1591, 1440, 1377, 1169, 1077, 984; MS(+FAB): 449.3 (M+1); Anal.
calcd. for C.sub.27H.sub.48N.sub.2O.sub.3-2HCl-1.2H.sub.2O:
C=59.70, H=9.72, N=5.16; Found: C=59.59, H=9.49, N=5.15.
.alpha.-Amino isomer 410: .sup.1H NMR (400 MHz, CD.sub.3OD)
.delta.: 3.82 (m, 1H), 3.65 (s, 3H), 3.05 (br m, 1H), 1.00 (s, 3H),
0.94 (d, J=6.5 Hz, 3H), 0.72 (s, 3H); IR (KBr, cm.sup.-1): 3522,
2944, 2017, 1718, 1619, 1448, 1377, 1314, 1282, 1260, 1163, 1018;
MS(+FAB): 449.3 (M+1); Anal. calcd. for
C.sub.27H.sub.48N.sub.2O.sub.3-2HCl-3.7H.sub.2O: C=55.13, H=9.84,
N=4.76; Found: C=55.03, H=9.32, N=4.78.
[0133] Preparation of compound 411: This spermine compound was
prepared by the same procedure as the ethylenediamine compounds,
except for the following modification. One gram of the 3-oxo sterol
methyl ester of chenodeoxycholic acid and 1 g of spermine (approx.
2 equiv.) were used, and the chromatography required a more polar
solvent system (i.e., 5:4:1 CHCl.sub.3:methanol:isopropylamine).
The total yield of aminosterol was 48%. The ratio of .alpha.-amino
isomer to .beta.-amino isomer 411 was not determined due to
incomplete separation. Compound 411: .sup.1H NMR (400 MHz,
CD.sub.3OD) .delta.: 3.83 (m, 1H), 3.65 (s, 3H), 3.42 (m, 1H), 1.04
(s, 3H), 0.95 (d, J=6.5 Hz, 3H), 0.70 (s, 3H); IR (KBr, cm.sup.-1):
3404, 2946, 2059, 1739, 1595, 1458, 1378, 1168, 1073, 1012, 985,
759; MS(+FAB): 591.4 (M+1); Anal. calcd. for
C.sub.36H.sub.66N.sub.4O.sub.3-4HCl-4H.sub.2O: C=51.97, H=9.72,
N=6.93; Found: C=51.65, H=8.53, N=6.77.
[0134] Preparation of compounds 355 and 356: The 3-oxo sterol
methyl ester of chenodeoxycholic acid was coupled to polyamine 301
with sodium cyanoborohydride, the BOC groups were removed with
trifluoroacetic acid, and the ester was hydrolyzed as in the
preparation of compound 319. Purification was achieved on silica
gel (15:4:1 to 10:4:1 chloroform:methanol:isopropylamine). Less
polar .beta.-amino isomer 355, C.sub.32H.sub.59N.sub.3O.sub.3:
.sup.1H NMR (400 MHz, CDCl.sub.3) .delta.: 3.87 (m, 1H), 3.68 (s,
3H), 3.15 (m, 1H), 3.0-2.7 (m, 8H), 2.4-1.0 (m, 32H), 0.99 (s, 3H),
0.91 (d, J=6 Hz, 3H), 0.66 (s, 3H); MS(DCI): 534 (M+1). More polar
.alpha.-amino isomer 356, C.sub.32H.sub.59N.sub.3O.sub.3-3HCl:
.sup.1H NMR (400 MHz, CD.sub.3OD) .delta.: 3.82 (m, 1H), 3.25-2.95
(m, 9H), 2.5-1.0 (m, 32H), 0.97 (S, 3H), 0.94 (d, J=6 Hz, 3H), 0.69
(s, 3H); MS (DCI): 534 (M+1).
[0135] Preparation of compound 1416: The procedures used for the
preparation of deoxycholic acid derivatives were the same as those
used in the preparation of the chenodeoxycholic acid derivatives.
For the ethylenediamine compound, the total yield of aminosterol
was 62%, with the ratio of .alpha.-amino isomer 416 to
.delta.-amino isomer being 4:1. Compound 416: .sup.1H NMR (400 MHz,
CD.sub.3OD) .delta.: 3.97 (m, 1H), 3.68 (s, 3H), 3.22 (br m, 1H),
1.02 (d, J=6.5 Hz, 3H), 1.01 (s, 3H), 0.73 (s, 3H); IR (KBr, cm
.sup.1): 3418, 2940, 1739, 1616, 1456, 1379, 1253, 1169, 1036;
MS(+FAB): 449.4 (M+1);
C.sub.27H.sub.48N.sub.2O.sub.3-2HCl-0.5H.sub.2O: C=61.12, H=9.69,
N=5.28; Found: C=61.20, H=9.50, N=5.07.
[0136] Preparation of compound 448: For the spermine derivatives of
deoxycholic acid, the total yield of aminosterol was 46%
(difficulty in the workup was likely responsible for the lower
yield). The ratio of .alpha.-amino isomer 448 to .beta.-amino
isomer was not determined due to incomplete separation. Compound
448: .sup.1H NMR (400 MHz, CD.sub.3OD) .delta.: 3.98 (m, 1H), 3.67
(s, 3H), 1.01 (d, J=6 Hz, 3H), 1.01 (s, 3H), 0.73 (s, 3H); IR (KBr,
cm.sup.-1): 2944, 1738, 1594, 1451, 1378, 1169, 1038, 758;
MS(+FAB): 591.5 (M+1); Anal. calcd. for
C.sub.35H.sub.66N4O.sub.3-4HCl-2.3H.sub.2O: C=54.02, H=9.66,
N=7.20; Found: C=54.00, H=8.64, N=7.22.
[0137] Preparation of compounds 414 and 415: The free acids were
prepared from the methyl esters as in the preparation of
6.beta.-hydroxy 433. .alpha.-Amino isomer 414: .sup.1H NMR (400
MHz, CD.sub.3OD) .delta.: 3.83 (m, 1H), 3.06 (br m, 1H), 1.04 (s,
3H), 0.96 (d, J=6 Hz, 3H), 0.73 (s, 3H); IR (KBr, cm.sup.-1): 2940,
2053, 1709, 1452, 1378, 1167, 1076, 1007, 975; MS(+FAB): 435.5
(M+1); Anal. calcd. for
C.sub.26H.sub.46N.sub.2O.sub.3-2HCl-1.5H.sub.2O: C=58.41, H=9.62,
N=5.24; Found: C=58.24, H=9.40, N=5.47. .beta.-Amino isomer 415:
.sup.1H NMR (400 MHz, CD.sub.3OD) .delta.: 3.83 (m, 1H), 3.47 (m,
1H), 1.06 (s, 3H), 0.95 (d, J=6 Hz, 3H), 0.73 (s, 3H); IR (KBr,
cm.sup.-1): 3488, 2935, 2054, 1709, 1593, 1499, 1450, 1246, 1168,
1077, 1022, 984; MS(+FAB): 435.5 (M+1); Anal. calcd. for
C.sub.26H.sub.46N.sub.2O.sub.3-2HCl-1.5H.sub.2O: C=58.41, H=9.62,
N=5.24; Found: C=58.59, H=9.35, N=5.43.
[0138] Preparation of compounds 412, 413, 417 and 449: These
compounds were produced using procedures analogous to those
above.
[0139] .alpha.-Amino 412: .sup.1H NMR (400 MHz, CD.sub.3OD)
.delta.: 3.83 (m, 1H), 3.00 (br m, 1H), 1.04 (s, 3H), 0.96 (d, J=6
Hz, 3H), 0.74 (s, 3H); IR (KBr, cm.sup.-1): 3413, 2942, 2061, 1710,
1594, 1460, 1377, 1167, 1074; MS(+FAB): 577.7 (M+1); Anal. calcd.
for C.sub.34H.sub.64H.sub.4O.sub.3-4HCl-2.5H.sub.2O: C=53.19,
H=9.58, N=7.30; Found: C=53.27, H=9.47, N=7.32.
[0140] .beta.-Amino 413: .sup.1H NMR (400 MHz, CD.sub.3OD) .delta.:
3.8 (m, 1H), 3.4 (m, 1H), 1.05 (s, 3H), 0.96 (d, J=6 Hz, 3H), 0.73
(s, 3H); MS(+FAB): 577.7 (M+1).
[0141] Deoxycholic acid ethylenediamine 417 (.alpha.-amino isomer):
.sup.1H NMR (400 MHz, CD.sub.3OD) .delta.: 4.03 (m, 1H), 3.22 (br
m, 1H), 1.03 (d, J=6 Hz, 3H), 1.00 (s, 3H), 0.74 (s, 3H); IR (KBr,
cm.sup.-1) 2940, 1706, 1456, 1379, 1254, 1034; MS(+FAB): 435.4
(M+1); Anal. calcd. for
C.sub.26H.sub.46N.sub.2O.sub.3-2HCl-2H.sub.2O: C=57.45, H=9.64,
N=5.15; Found: C=57.32, H=9.22, N=5.13.
[0142] Deoxycholic acid spermine 449 (.alpha.-amino isomer):
.sup.1H NMR (400 MHz, CD.sub.3OD) .delta.: 4.02 (m, 1H), 1.04 (d,
J=6 Hz, 3H), 1.00 (s, 3H), 0.75 (s, 3H); IR (KBr, cm.sup.-1): 2941,
1716, 1448, 1038; MS(+FAB): 577.4 (M+1); Anal. calcd. for
C.sub.34H.sub.64N.sub.4O.sub.3-4HCl-1.5H.sub.2O: C=54.57, H=9.54,
N=7.47; Found: C=54.31, H=8.71, N=7.80.
EXAMPLE Q
[0143] Preparation of monoamine compounds 363 and 364: ##STR26##
##STR27## ##STR28##
[0144] Preparation of compound 1002: To a suspension of chromium
trioxide (72.6 g, 660 mmol) in dichloromethane (1000 ml) at
-78.degree. C., was added 3,5-dimethylpyrazole (63.4 g, 660 mmol).
After 20 minutes, cholesteryl acetate (compound 1001, 24 g, 56
mmol) was added, and the mixture-was allowed to warm to room
temperature slowly and stirred overnight. To the reaction mixture
(0.degree. C.) was added 5 N sodium hydroxide solution (280 ml),
and the mixture was stirred for 1 hour. The organic phase was
washed with 2 N HCl, water and brine. After removing the solvent,
the crude product was purified by chromatography (6 cm, gradient
elution with 10% to 30% ethyl acetate in hexane) to afford starting
material (6.78 g) and compound 1002 (12.78 g, 52%).
[0145] .sup.1H NMR (200 MHz, CDCl.sub.3) .delta.: 5.71 (s, 1H), 4.7
(br m, 1H), 2.5-1.0 (m, 27 H), 2.05 (s, 3H), 1.21 (s, 3H), 0.92 (d,
J=6.5 Hz, 3H), 0.86 (d, J=7 Hz, 6H), 0.68 (s, 3H).
[0146] Preparation of compound 1003: A solution of compound 1002
(14.32 g, 32.3 mmol) in ethyl acetate (1.4 l) was purged with
nitrogen, treated with platinum (IV) oxide (263 mg), and hydrogen
gas (atmospheric) for 3 hours at room temperature. After filtration
through Celite, the solution was evaporated and purified by flash
chromatography (6 cm, gradient elution with 0-20% ethyl acetate in
hexane) to yield pure compound 1003 (10.86 g, 76% yield). .sup.1H
NMR (200 MHz, CDCl.sub.3) .delta.: 4.67 (br m, 1H), 2.4-1.0 (m,
29H), 2.02 (s, 3H), 1.10 (s, 3H), 0.90 (d, J=6.5 Hz, 3H), 0.86 (d,
J=6.5 Hz, 6H), 0.65 (s, 3H); IR (KBr, cm.sup.-1): 2950, 1730, 1707,
1471, 1373, 1264, 1032; MS(+ES): 445.7 (M+1).
[0147] Preparation of compounds 1004 and 1005: To absolution of
compound 1003 (11.62 g, 26.1 mmol) in tetrahydrofuran (THF) (500
ml) was added 1 M K-Selectride.RTM. (80 ml, 80 mmol) in THF at room
temperature. After 5 hours at 50.degree. C., the reaction mixture
was cooled in an-ice bath, and then treated with 30% hydrogen
peroxide (45 ml) and saturated ammonium chloride solution (200 ml).
The organic phase was separated and the aqueous phase was extracted
with ether (3.times.100 ml), and the combined organic layers were
washed with saturated sodium bicarbonate, ammonium chloride and
water. After drying, the crude product was purified by
chromatography (6 cm, gradient elution with 0-30% ethyl acetate in
hexane) to afford compound 1004 (10.95 g, 24.5 mmol), which was
dissolved in dichloromethane (200 ml) with dimethylaminopyridine
(30.30 g, 248 mmol) and treated with acetic anhydride (40 ml, 424
mmol) for 19 hours. To this solution was added methanol (150 ml),
and the solvent was evaporated. The residue was dissolved in ethyl
acetate, washed with 2 N hydrochloric acid solution (3.times.150
ml), water (100 ml), saturated sodium carbonate solution
(3.times.100 ml) and brine (2.times.100 ml). The organic layer was
dried, evaporated, and purified by flash chromatography (6 cm,
gradient elution with 0-20% ethyl acetate in hexane) to yield
compound 1005 (11.47 g, 90% yield). .sup.1H NMR (200 MHz,
CDCl.sub.3) .delta.: 4.88 (m, 1H), 4.71 (br m, 1H), 2.08 (s, 3H),
2.02 (s, 3H), 2.0-1.0 (m, 29H), 0.92-0.83 (m, 12H), 0.64 (s, 3H);
IR (KBr, cm.sup.-1): 2954, 2867, 1730, 1468, 1367, 1257, 1025;
Anal. calcd. for C.sub.31H.sub.52O.sub.4: C=76.18, H=10.72; Found:
C=76.09, H=10.56.
[0148] Preparation of compound 1006: A solution of compound 1005
(10.85 g, 22.2 mmol) and sodium cyanide (1.20 g, 24.4 mmol) in
methanol (420 ml) was stirred overnight at room temperature, and
then refluxed for 10 hours. The solvent was evaporated, and the
residue was dissolved in dichloromethane and water, which was
acidified with 2 N hydrochloric acid solution. After another
dichloromethane extraction, the organic layer was washed with
brine, dried over magnesium sulfate, and evaporated to afford
compound 1006 (9.22 g, 92% yield). .sup.1H NMR (200 MHz,
CDCl.sub.3) .delta.: 4.89 (m, 1H), 3.62 (br m, 1H), 2.07 (s, 3H),
2.0-1.0 (m, 29H), 0.90 (d, J=6 Hz, 3H), 0.87 (d, J=6.5 Hz, 6H),
0.82 (s, 3H), 0.64 (s, 3H); IR (KBr, cm.sup.-1): 3446, 2935, 1735,
1469, 1375, 1245, 1042, 941; MS(+ES): 470 (M+Na).
[0149] Preparation of compounds 1007, 1008 and 1009: To a solution
of compound 1006 (892 mg, 2.0 mmol) in anhydrous dichloromethane
(20 ml) under nitrogen (-10 to -5.degree. C.) was added
triethylamine (3 ml, 22 mmol) and methanesulfonyl chloride (0.40
ml, 5.2 mmol) in dichloromethane (4 ml). After 40 minutes, the
mixture was poured into 1 N hydrochloric acid solution (100 ml),
and the organic phase was separated. After extracting with more
dichloromethane (3.times.20 ml), the organic phase was washed with
1 N hydrochloric acid solution (30 ml), saturated sodium
bicarbonate solution (30 ml) and brine (2.times.30 ml). After
drying over sodium sulfate, the solvent was evaporated to yield
compound 1007, which was used for the next step without
purification.
[0150] Crude compound 1007 was dissolved in dimethylformamide (50
ml), treated with sodium azide (2.0 g, 31 mmol), and heated to
100.degree. C. for 18 hours. After cooling, the reaction mixture
was diluted with water (250 ml), extracted with dichloromethane
(3.times.150 ml), washed with water (3.times.100 ml), dried
(Na.sub.2SO.sub.4), filtered, and evaporated to yield compound
1008, which was used in the next step without purification.
[0151] A solution of compound 1008 in anhydrous tetrahydrofuran (60
ml) was treated with 1 M lithium aluminum hydride (20 ml, 20 mmol)
and heated to reflux for 5 hours. After cooling in an ice bath, to
the mixture was added water (50 ml) and then 2 M sodium hydroxide
solution (200 ml). The aqueous phase was extracted with
dichloromethane (3.times.150 ml), followed by washing with brine
(2.times.100 ml) and water (50 ml). The dried organic layer was
evaporated to afford compound 1009, which was used without
purification in the next step.
[0152] Preparation of compounds 1363 and 1364: Crude compound 1009
was dissolved in methanol (40 ml) and treated with 2 N sodium
hydroxide solution (40 ml) at 80.degree. C. for 12 hours. After
evaporation, water was added (40 ml), followed by extraction with
dichloromethane (3.times.60 ml). After washing with brine
(3.times.50 ml), the organic layer was dried (Na.sub.2SO.sub.4),
filtered and evaporated. Purification by flash chromatography (2-cm
diameter, elution with 95:4.5:0.5 dichloromethane:methanol:ammonium
hydroxide) afforded compound 1363 (slower eluting; .sup.1H NMR (200
MHz, CDCl.sub.3) .delta.: 3.82 (m, 1H), 3.19 (m, 1H), 2.0-1.0 (m,
29H), 0.91 (d, J=6.5 Hz, 3H), 0.87 (d, J=6.5 Hz, 6H), 0.78 (s, 3H),
0.65 (s, 3H); IR (KBr, cm.sup.-1): 3362, 2931, 1575, 1467, 1382; MS
(+FAB): 404.4 (M+1)) and compound 1364 (faster eluting; .sup.1H NMR
(200 MHz, CDCl.sub.3) .delta.: 3.4 (br m, 1H), 3.2 (m, 1H), 2.0-1.0
(m, 29H), 0.91 (d, J=6.5 Hz, 3H), 0.86 (d, J=6.5 Hz, 6H), 0.80 (s,
3H), 0.68 (s, 3H)). Each compound was dissolved in methanol,
treated with excess 1 N hydrogen chloride in ether, and evaporated
to yield the hydrochloride salt. Compound 1363 (646 mg, 74% overall
yield for 4 steps): Anal. calcd. for
C.sub.27H.sub.49NO--HCl-0.5H.sub.2O: C=72.20, H=11.44, N=3.12;
Found: C=72.40, H=11.44, N=3.26. Compound 1364 (50 mg, 6% overall
yield for 4 steps): MS(+FAB): 404.4 (M+1); Anal. calcd. for
C.sub.27H.sub.49NO--HCl--H.sub.2O: C=70.78, H=11.44, N=3.06; Found:
C=71.02, H=11.33, N=3.35.
EXAMPLE R
[0153] Preparation of compounds 388 and 387: ##STR29## ##STR30##
##STR31## ##STR32## ##STR33##
[0154] .sup.1H-NMR spectra were obtained on a Varian XL-200 (200
MHz) or a Varian Unity-500 (500 MHz) NMR spectrometer. Infrared
spectra were recorded on a Perkin Elmer 298 spectrometer. Direct
insertion probe (DIP) chemical ionization mass spectral data were
obtained on a Hewlett Packard HP 5087 GC-MS. Melting points were
determined on a Thomas Hoover capillary melting point apparatus and
are uncorrected. Elemental analyses were performed by Quantitative
Technologies Inc., Whitehouse, N.J. FAB mass spectral data (low and
high resolution) were obtained from M-Scan Inc., West Chester,
Pa.
[0155] 5-Cholenic acid was obtained from Steraloids and used as
received. The following reagents were purchased from Aldrich
Chemical Company and were used as received unless otherwise
indicated: dihydropyran (distilled prior to use), p-toluenesulfonic
acid, lithium aluminum hydride, t-butyldimethylsilyl chloride,
imidazole, 3,5-dimethylpyrazole, platinum (IV) oxide, potassium
tri-sec-butylborohydride (K-Selectride.RTM., 1M in THF), hydrogen
peroxide (30%), sodium hydride (60% in mineral oil), benzyl bromide
(distilled prior to use), tetrabutylammonium fluoride (1M in THF),
oxalyl chloride, diisopropylethylamine, dimethylsulfoxide
(distilled prior to use), isopropylmagnesium chloride (2M in THF),
magnesium bromide, sodium cyanoborohydride (1M in THF), 10%
palladium on carbon, and sulfur trioxide pyridine complex. THF and
Et.sub.2O were distilled from sodium/benzophenone ketyl. Pyridine
was distilled from KOH. Methylene chloride and pentane were
distilled from CaH.sub.2. DMF was distilled from BaO under reduced
pressure. Methanol was dried over 3 .ANG. molecular sieves prior to
use. PPTS was prepared via the method of Miyashita et al., J. Org.
Chem. 42, 1977, 3772). Molecular sieves were dried in an oven
(170.degree. C.) overnight prior to use. Silica gel (EM Science
Silica Gel 60, 230-400 mesh) was used for all flash
chromatography.
[0156] Preparation of 3.beta.-Tetrahydropyranyloxychol-5-en-24-oic
acid 24-tetrahydropyranyl ester (2001):
[0157] 5-Cholenic acid (7.58 g, 20 mmol) was suspended in a
solution of dry CH.sub.2Cl.sub.2 (300 ml). Distilled dihydropyran
(19.0 ml, 200 mmol) was added, followed by a catalytic amount of
pyridinium p-toluene sulfonate (1.1 g, 4.0 mmol). The suspension
was stirred at room temperature overnight under argon. During this
period of time, the steroid went into solution. The resultant
solution was washed with a aqueous saturated NH.sub.4Cl solution
(2.times.), aqueous saturated NaHCO.sub.3 solution (2.times.), and
aqueous saturated NaCl solution. The organic layer was dried over
anhydrous MgSO.sub.4, filtered, and the solvent was removed in
vacuo. The crude solid was purified by flash chromatography
(SiO.sub.2, hexanes/EtOAc (10:1), giving compound 2001 as a white
solid (9.8 g, 18.5 mmol, 92%). .sup.1H NMR (500 MHz, CDCl.sub.3)
.delta.: 5.96 (brs, 1H, THP ester methine H), 5.37-5.32 (m, 1H, C-6
H), 4.72 (brs, 1H, THP ether methine H), 3.95-3.87 (m, 2H, THP
CH.sub.2O), 3.71-3.64 (m, 1H, THP CH.sub.2O), 3.58-3.44 (m, 2H, THP
CH.sub.2O & C-3 H), 1.01 (s, 3H, C-19 H), 0.94 (d, J=6.3 Hz,
3H, C-21 H), 0.68 (s, 3H, C-18 H).
[0158] Preparation of 3.beta.-Tetrahydropyranyloxychol-5-en-24-ol
(2002):
[0159] Compound 2001 (16.1 g, 30 mmol) in dry tetrahydrofuran (THF,
150 ml) was added to a suspension of LiAlH.sub.4 (5.5 g, 145 mmol)
in dry THF (200 ml). The suspension was stirred at 0.degree. C.
with a mechanical stirrer under argon overnight. The resultant gray
slurry was quenched with EtOAc, followed by aqueous saturated
Na.sub.2SO.sub.4 solution. During the addition of the
Na.sub.2SO.sub.4 solution, a white precipitate formed and the
solution became clear. Anhydrous Na.sub.2SO.sub.4 was added, the
mixture was stirred for 15 minutes, and then filtered. The filter
cake was washed well with ethyl acetate and the filtrate was
concentrated in vacuo. The resulting solid was purified by flash
chromatography (SiO.sub.2, hexanes:EtOAc 5:1) giving compound 2002
as a white solid (12.3 g, 27.7 mmol, 92%). .sup.1H NMR (500 MHz,
CDCl.sub.3) .delta. 5.37-5.32 (m, 1H, C-6 H), 4.72 (brs, 1H, THP
methine H), 3.95-3.88 (m, 1H, THP CH.sub.2O), 3.62-3.47 (m, 4H, THP
CH.sub.2O & C-3 H & C-24 H), 1.01 (s, 3H, C-19 H), 0.93 (d,
J=6.6 Hz, C-21 H), 0.68 (s, 3H, C-18 H); IR (CHCl.sub.3) 3610, 2900
cm.sup.-1; MS (CI/isobutane) m/z 445 (M+1, 2%), 343 (M+1-THPOH,
100%); m.p. 130-131 .degree. C.
[0160] Preparation of
24-t-Butyldimethylsilyloxy-3.beta.-tetrahydropyranyloxychol-5-ene
(2003):
[0161] Compound 2002 (7.6 g, 17 mmol) in dry CH.sub.2Cl.sub.2 (300
ml ) was treated with a solution of t-butyldimethylsilylchloride
(TBDMSCl, 1.0 M) and imidazole (0.5 M) in dry CH.sub.2Cl.sub.2
(38.0 ml, 38,0 mmol TBDMSCl). The solution was stirred at room
temperature under argon overnight. The resultant solution was
poured into an aqueous saturated NaHCO.sub.3 solution and the
mixture extracted with CH.sub.2Cl.sub.2 (3.times.). The combined
organic layers were washed with saturated sodium chloride, dried
over anhydrous MgSO.sub.4, filtered, and the solvent removed in
vacuo. The resultant solid was purified by flash chromatography
(SiO.sub.2, hexanes:EtOAc gradient from 20:1 to 5:1) giving
compound 2003 (9.4 g, 17 mmol, 98%). .sup.1H NMR (500 MHz,
CDCl.sub.3) .delta.: 5.38-5.32 (m, 1H, C-6 H), 4.72 (brs, 1H, THP
methine H), 3.95-3.88 (m, 1H, THP CH.sub.2O), 3.60-3.46 (m, 4H THP
CH.sub.2O & C-3 H & C-24 H), 1.01 (s, 3H, C-19 H), 0.93 (d,
J=6.6 Hz, C-21 H), 0.89 (s, 9H, t-Bu), 0.67 (s, 3H, C-18 H), 0.05
(s, 6H, TBDMS CH.sub.3); IR (CHCl.sub.3) 2900 cm.sup.-1; MS
(CI/isobutane) m/z 559 (M+1, 1%), 474 (M+1-THP, 12%), 457
(M+1-THPOH, 18%), 343 (M+1-THP-TBDMSOH, 6%), 325
(M+1-THPOH-TBDMSOH, 100%); m.p. 116-118.degree. C.; Anal. calcd.
for C.sub.35H.sub.62O.sub.3Si: C=75.21, H=11.18; Found: C=75.37,
H=11.24.
[0162] Preparation of
24-t-Butyldimethylsilyloxy-3.beta.-tetrahydropyranyloxychol-5-en-7-one
(2004):
[0163] Chromium trioxide (6.43 g, 64.4 mmol) was suspended in dry
CH.sub.2Cl.sub.2 (100 ml). The mechanically stirred suspension
under argon was cooled to -78.degree. C. via a dry-ice/acetone
bath. 3,5-Dimethylpyrazole (6.18 g, 64.4 mmol) was added to the
suspension as a solid and the mixture was allowed to stir for 25
minutes at -78.degree. C. to ensure complete formation of the
complex. Compound 2003 (3.10 g, 5.37 mmol) was then added to the
mixture as a solid, and the reaction mixture was allowed to slowly
warm to room temperature and stirred overnight. The mixture was
then transferred to a one-neck 500 ml round-bottom flask and silica
gel (flash grade) was introduced. The slurry was concentrated to a
free-flowing solid which was introduced onto the top of a wet
packed flash column (SiO.sub.2) and the product was eluted with
hexanes:ethyl acetate (gradient 30:1 to 15:1 to 6:1 to 3:1). The
desired product, compound 2004 (1.80 g, 59%) was obtained as a
white solid. .sup.1H NMR (500 MHz, CDCl.sub.3) .delta.: 5.65 &
5.63 (2S, 1H, C-6 H), 4.70-4.64 (m, 1H, THP methine), 3.90-3.81 (m,
1H, THP CH.sub.2O), 3.70-3.62 (m, 1H, C-3H or THP CH.sub.2O), 3.57
(t, J=6.6 Hz, 2H, C-24 H), 3.52-3.46 (m, 1H, C-3 H or THP
CH.sub.2O), 1.19 (s, 3H, C-19 H), 0.93 (d, J=6.3 Hz, C-21 H), 0.90
(s, 9H, t-butyl), 0.68 (s, 3H, C-18 H), 0.05 (s, 6H, TBDMS
CH.sub.3); IR (CHCl.sub.3) 2900, 1650 cm.sup.-1; MS (CI/isobutane)
m/z 573 (M+1, 11%), 489 (M+1-THP, 100%); m.p. 118-120.degree.
C.
[0164] Preparation of
24-t-Butyldimethylsilyloxy-3.beta.-tetrahydropyranyloxy-5.alpha.-cholan-7-
-one (2005):
[0165] Compound 2004 (1.0 g, 1.75 mmol) was dissolved in EtOAc (75
ml) and platinum (IV) oxide (0.012 g, 0.049 mmol) was added. The
mixture was placed on a hydrogenation apparatus (atmospheric). The
set-up was evacuated to remove the dissolved oxygen and then
hydrogen was introduced. The evacuation and introduction of
hydrogen process was repeated 2 times. The reaction was stirred
under hydrogen at atmospheric pressure for 2.5 hours. The reaction
mixture was filtered through Celite and concentrated in vacuo. The
crude product was purified by flash chromatography (SiO.sub.2,
hexanes:EtOAc gradient starting with 20:1) giving compound 2005 as
a white solid (0.70 g, 71%).
24-t-Butyldimethylsilyloxy-3.beta.-tetrahydropyranyloxy-5.alpha.-cholan-7-
.beta.-ol was obtained as a by-product (21%). (Note: This
by-product could be converted to the desired ketone 2005 with
Collin's reagent in 64% yield.) Compound 2005: .sup.1H NMR (500
MHz, CDCl.sub.3) .delta.: 4.73-4.66 (m, 1H, THP methine H),
3.95-3.85 (m, 1H, THP CH.sub.2O), 3.66-3.52 (m, 3H, THP CH.sub.2O
& C-24 H), 3.50-3.45 (M, 1H, C-3 H), 1.08 (s, 3H, C-19 H), 0.91
(d, J=6.6 Hz, C-21 H), 0.89 (s, 9H, t-Bu), 0.64 (s, 3H, C-18 H),
0.04 (s, 6H, TBDMS CH.sub.3); IR (CHCl.sub.3) 2900, 1685 cm.sup.-1;
MS (CI/isobutane) m/z 575 (M+1, 85%), 491 (M+1-THP, 100%); m.p.
166-170.degree. C.; Anal. calcd. for C.sub.35H.sub.62O.sub.4Si:
C=73.12, H=10.87; Found: C=72.88, H=10.78.
[0166] Preparation of
24-t-Butyldimethyulsilyloxy-3.beta.-tetrahydropyranyloxy-5.alpha.-cholan--
7.alpha.-ol (2006):
[0167] K-Selectride.RTM. (potassium tri-sec-butylborohydride) (8.9
ml, 1 M in THF, 8.9 mmol) was added dropwise via syringe to a
solution of ketone 200.5 (1.7 g, 3.0 mmol) in dry THF (50 ml) at
room temperature under argon. The reaction mixture was heated to
50.degree. C. in an oil bath and stirred for 5 hours. The mixture
was allowed to cool to room temperature and then quenched by adding
30% H.sub.2O.sub.2 dropwise until the evolution of gas ceased.
Saturated aqueous NH.sub.4Cl solution was added and the aqueous
solution was extracted (3.times.) with Et.sub.2O. The combined
organic extracts were washed with aqueous saturated NaHCO.sub.3
solution (2.times.), distilled H.sub.2O (2.times.), and aqueous
saturated NaCl solution, dried over anhydrous MgSO.sub.4, filtered,
and the solvent was removed in vacuo. The crude product was
purified by flash chromatography (SiO.sub.2, hexanes:EtOAc 10:1)
giving alcohol 2006 as a white solid (1.6 g, 94%).
[0168] .sup.1H NMR (500 MHz, CDCl.sub.3) .delta.: 4.73-4.66 (m, 1H,
THP methine H), 3.95-3.85 (m, 1H, THP CH.sub.2O), 3.82 (s, 1H, C-7
H), 3.66-3.52 (m, 3H, THP CH.sub.2O & C-24 H), 3.50-3.45 (m,
1H, C-3 H), 1.08 (s, 3H, C-19 H), 0.91 (d, J=6.6 Hz, C-21 H), 0.89
(s, 9H, t-Bu), 0.64 (s, 3H, C-18 H), 0.04 (s, 6H, TBDMS CH.sub.3);
MS (CI/isobutane) m/z 577 (M+1, 5%), 493 (M+1-THP, 22%), 475
(M+1-THPOH, 26%), 458 (M+1-THPOH-H.sub.2O, 38%), 343
(M+1-THPOH-TBDMSOH, 80%), 325 (M+1-THPOH-TBDMSOH-H.sub.2O, 100%);
IR (CHCl.sub.3) 3430, 2860 cm.sup.-1; m.p. 130-133.degree. C.;
Anal. calcd. for C.sub.35H.sub.64O.sub.4Si: C=72.86, H=11.18;
Found: C=72.69, H=11.32.
[0169] Preparation of
7.alpha.-Benzyloxy-24-t-butyldimethylsilyloxy-3.beta.-tetrahydropyranylox-
y-5.alpha.-cholane (2007):
[0170] A flame-dried round-bottom flask with stirring bar was
charged with sodium hydride (60% in mineral oil, 28 mg, 0.69 mmol),
equipped with a septum and a gas-needle inlet and flushed with
argon. The mineral oil was removed by washing (3.times.) with dry
pentane, and the pentane was removed to provide the sodium hydride
as a powder. Dry DMF (2.0 ml) was added. A solution of alcohol 2006
(40 mg, 0.069 mmol) in dry THF (2 ml) was added dropwise via
syringe. The reaction mixture was stirred overnight and then heated
to 40.degree. C. in an oil bath over a 20-minute period. Freshly
distilled benzyl bromide (0.165 ml, 1.38 mmol) was added dropwise,
and the reaction mixture was stirred at 40.degree. C. for 10 hours.
The reaction was allowed to cool to room temperature, and the
solvent was removed under reduced pressure. The flask was placed
under vacuum overnight to remove any residual DMF. The crude
material was purified by flash chromatography (SiO.sub.2,
hexanes:EtOAc.50:1) giving compound 2007 as a white solid (40 mg,
0.060 mmol, 87%). A gradient of increasing EtOAc concentration
provided other components, including the 7.alpha.-formate (1 mg,
1%) as well as recovered starting material (3 mg, 8%). Compound
2007: .sup.1H NMR (500 MHz, CDCl.sub.3) .delta.: 7.35-7.20 (m, 5H,
benzyl Ar--CH.sub.2), 4.73-4.66 (m, 1H, methine H), 4.535 (d,
J=12.0 Hz, 1/2H, benzyl-CH.sub.2), 4.53 (d, J=12.0 Hz, 1/2 H,
benzyl-CH.sub.2), 4.26 (d, J=12.2 Hz, 1/2H, benzyl-CH.sub.2), 4.245
(d, J=11.8 Hz, 1/2H, benzyl-CH.sub.2), 3.95-3.85 (m, 1H, THP
CH.sub.2O), 3.66-3.52 (m, 3H, THP CH.sub.2O & C-24 H),
3.50-3.45 (m, 1H, C-3 H), 1.08 (s, 3H, C-19 H), 0.91 (d, J=6.6 Hz,
C-21 H), 0.89 (s, 9H, t-Bu), 0.64 (s, 3H, C-18 H), 0.04 (s, 6H,
TBDMS CH.sub.3); MS (CI/isobutane) m/z 668 (M+1, 6%), 584 (M+1-THP,
18%), 475 (M+1-THPOH, 30%), 457 (M+1-THPOH-HOBn, 58%), 343
(M+1-THP--HOBn-TBDMSOH, 100%), 325 (M+1-THPOH-TBDMSOH-HOBn,
83%).
[0171] Preparation of
7.alpha.-Benzyloxy-3.beta.-tetrahydropyranyloxy-5.alpha.-cholan-24-ol
(2008):
[0172] Compound 2007 (0.0527 g, 0.079 mmol) in anhydrous THF (4 ml)
under Ar was treated with tetrabutylammonium fluoride (TBAF) (0.237
ml, 1 M in THF, 0.237 mmol). The solution was stirred until no
starting material remained by TLC. The solvent was removed in
vacuo, the residue taken up in 5 ml CH.sub.2Cl.sub.2, washed with 5
ml aqueous saturated NaHCO.sub.3 solution, and the aqueous layer
was extracted 2.times. with 5 ml CH.sub.2Cl.sub.2. The combined
organic layers were dried over anhydrous MgSO.sub.4, filtered, and
the solvent removed in vacuo. Flash chromatography (SiO.sub.2, 8:1
hexanes:EtOAc) gave compound 2008 (0.0395 g, 90%) as a white solid
foam. .sup.1H NMR (500 MHz, CDCl.sub.3) .delta.: 7.35-7.34 (m, 5H,
benzyl Ar--H), 4.71-4.69 (m, 1H, THP ether methine H), 4.585 (d,
J=11.8 Hz, 1/2H, benzyl-CH.sub.2), 4.58 (d, J=11.8 Hz, 1/2H,
benzyl-CH.sub.2), 4.315 (d, J=12.0 Hz, 1/2H, benzyl-CH.sub.2), 4.29
(d, J=12.0 Hz, 1/2H, benzyl-CH.sub.2), 3.94-3.90 (m, 1H, THP
OCH.sub.2), 3.62-3.58 (m, 3H, C-24 H & THP OCH.sub.2),
3.50-3.48 (m, 1H, C-3 H), 3.45 (s, 1H, Z-H), 0.92 (d, J=6.6 Hz, 3H,
C-21 H), 0.81 (s, 3H, C-19 H), 0.63 (s, 3H, C-18 H) (Note: product
is a mixture of diastereomers); IR (CHCl.sub.3) 3600, 2900
cm.sup.-1; MS (CI/isobutane) m/z 554 (M+1, 2%), 361 (M+1-THP--HOBn,
42%), 343 (M+1-THP--HPBn, H.sub.2O, 100%); m.p. 52-56.degree. C.;
Anal. calcd. for C.sub.36H.sub.56O.sub.4: C=78.21, H=10.21; Found:
C=77.93, H=10.39.
[0173] Preparation of
7.alpha.-Benzyloxy-3.beta.-tetrahydropyranyloxy-5.alpha.-cholan-24-al
(2009):
[0174] DMSO (0.01 ml, 0.14 mmol) in CH.sub.2Cl.sub.2 (0.1 ml) was
added dropwise to a stirred solution of oxalyl chloride (0.008 ml,
0.0917 mmol) in anhydrous CH.sub.2Cl.sub.2 (2 ml) at -78.degree. C.
under anhydrous conditions (drying tube). This solution was stirred
at -78.degree. C. for 15 minutes. Steroid 2008 (0.0234 g, 0.0423
mmol) in dry CH.sub.2Cl.sub.2 (0.5 ml) was then added dropwise and
the solution stirred for 40 minutes at -78.degree. C.
Diisopropylethylamine (DIPEA) (0.08 ml, 0.458 mmol) was added and
the solution allowed to warm to 0.degree. C. with stirring over a
30-minute period. Aqueous saturated NaHCO.sub.3 solution (5 ml) was
added and the solution extracted 3.times. with 5 ml
CH.sub.2Cl.sub.2 The combined organic extracts were washed 2.times.
with 5 ml of aqueous-saturated NaCl solution, dried over anhydrous
MgSO.sub.4, filtered, and the solvent removed in vacuo. Flash
chromatography (SiO.sub.2, 10:1 hexanes:EtOAc) gave compound 2009
(0.0226 g, 97%) as a white solid foam. .sup.1H NMR (500 MHz,
CDCl.sub.3) .delta.: 9.76 (s, 1H, C-24 H), 7.35-7.34 (m, 5H, benzyl
Ar--H), 4.71-4.69 (m, 1H, THP ether methine H), 4.59 (d, J=11.8 Hz,
1/2H, benzyl-CH.sub.2), 4.585 (d, J=11.8 Hz, 1/2H,
benzyl-CH.sub.2), 4.30 (d, J=12.0 Hz, 1/2H, benzyl-CH.sub.2), 4.29
(d, J=12.0 Hz, 1/2H, benzyl-CH.sub.2), 3.95-3.89 (m, 1H, THP
OCH.sub.2), 3.63-3.58 (m, 3H, C-24 H & THP OCH.sub.2),
3.50-3.47 (m, 1H, C-3 H), 3.45 (s, 1H, 7-H), 2.49-2.42 (m, 1H,
C-23H), 2.37-2.31 (m, 1H, C-23 H), 0.958 (d, J=6.5 Hz, 3H, C-21 H),
0.81 (s, 3H, C-19 H), 0.63 (s, 3H, C-18 H) (Note: product is a
mixture of diastereomers); IR (CHCl.sub.3) 2900, 1700 cm.sup.-1; MS
(CI/isobutane) m/z 552 (M+1, 0.4%), 465 (M+1-THP, 3%), 449
(M+1-THPO, 14%), 375 (M+1-THP-Bn, 7%), 359 (M+1-THP--HOBn, 68%),
341 (M+1-THP--HOBn-H.sub.2O, 100%); m.p. 50-54.degree. C.; Anal.
calcd. for C.sub.36H.sub.54O.sub.4: C=78.50, H=9.88; Found:
C=78.11, H=10.04.
[0175] Preparation of
7.alpha.-Benzyloxy-3.beta.-tetrahydropyranyloxycholestan-24.xi.-ol
(2010):
[0176] A solution of compound 2009 (0.374 g, 0.679 mmol) in
anhydrous THF (10 ml) under argon was treated with
isopropylmagnesium chloride (2 ml, 2M in THF, 5.43 mmol) at room
temperature. The reaction was stirred until no starting material
remained by TLC. Aqueous NH.sub.4Cl solution (10%, 15 ml) was added
to quench the reaction and the THF was removed in vacuo. Distilled
H.sub.2O (5 ml) was added and the solution extracted 3.times. with
15 ml CH.sub.2Cl. The combined organic layers were washed with
aqueous saturated NaCl solution (15 ml), dried over anhydrous
MgSO.sub.4, filtered and the solvent removed in vacuo. Flash
chromatography (SiO.sub.2, 12:1 hexanes:EtOAc) gave compound 2010
(0.3117 g, 77%) as a white foam. .sup.1H NMR (500 MHz, CDCl.sub.3)
.delta.: 7.35-7.34 (m, 5H, benzyl Ar--H), 4.71-4.69 (m, 1H, THP
ether methine H), 4.585 (d, J=11.9 Hz, 1H, benzyl-CH.sub.2), 4.31
(d, J=12.0 Hz, 1/2H, benzyl-CH.sub.2), 4.295 (d, J=12.0 Hz, 1/2H,
benzyl-CH.sub.2), 3.94-3.91 (m, 1H, THP, OCH.sub.2), 3.62-3.58 (m,
1H, THP OCH.sub.2), 3.50-3.48 (m, 1H, C-3 H), 3.45 (s, 1H, C-7 H),
3.32-3.31 (m, 1H, C-24 H), 0.81 (s, 3H, C-19 H), 0.63 (d J=2.4 Hz,
3H, C-18 H) (Note: product is a mixture of diastereomers); IR
(CHCl.sub.3) 3605, 2900 cm.sup.-1; MS (CI/isobutane) m/z 595 (M+1,
10%), 401 (M+1-THP-Bn-H.sub.2O, 25%), 385 (M+1-THP--HOBn, H.sub.2O,
100%); m.p. 55-59.degree. C.; Anal. calcd. for
C.sub.39H.sub.62O.sub.4: C=78.74, H=10.50; Found: C=78.65,
H=10.54.
[0177] Preparation of
7.alpha.-Benzyloxy-24.xi.-t-butyldimethylsilyloxy-3.beta.-tetrahydropyran-
yl-oxycholestane (2011):
[0178] Compound 2010 (0.050 g, 0.084 mmol) in dry CH.sub.2Cl.sub.2
(1 ml) was treated with a solution of t-butyldimethylsilylchloride
(TBDMSCl, 0.5 M) and imidazole (1.0 M) in dry CH.sub.2Cl.sub.2
(0.80 ml, 0.40 mmol TBDMSCl). The reaction was stirred at room
temperature under argon for 24 hours. Aqueous saturated NaHCO.sub.3
solution (5 ml) was added and the solution extracted 3.times. with
10 ml CH.sub.2Cl.sub.2. The combined organic layers were washed
with 10 ml aqueous saturated NaCl solution and dried over anhydrous
Na.sub.2SO.sub.4. Filtration and removal of solvent in vacuc
followed by flash chromatography (SiO.sub.2, 20:1 hexanes:EtOAc)
gave the desired product 2011 (0.057 g, 96%) as a white solid. 1H
NMR (500 MHz, CDCl.sub.3) .delta.: 7.35-7.34 (m, 5H, benzyl Ar--H),
4.70-4.69 (m, 1H, THP ether methine H), 4.59 (d, J=12.0 Hz, 1H,
benzyl-CH.sub.2), 4.315 (d, J=12.0 Hz, 1/2H, benzyl-CH.sub.2), 4.31
(d, J=12.0 Hz, 1/2H, benzyl-CH.sub.2), 3.94-3.91 (m, 1H, THP
OCH.sub.2), 3.62-3.58 (m, 1H, THP OCH.sub.2), 3.50-3.48 (m, 1H, C-3
H), 3.45 (s, 1H, C-7 H), 3.37-3.35 (m, 1H, C-24 H), 0.89 (d, J=1
Hz, 9H, SiC(CH.sub.3).sub.3, diastereomeric at C-24), 0.81 (s, 3H,
C-19 H), 0.62 (s, 3H, C-18 H), 0.04 & 0.03 (2s, 6H,
Si(CH.sub.3).sub.2, diastereotopic and/or diastereomeric) (Note:
product is a mixture of diastereomers); IR (CHCl.sub.3) 2900
cm.sup.-1; MS (CI/isobutane) m/z 709 (M+1, 20%), 367
(M+1-THPOH--HOBn-TBDMSOH, 100%); m.p. 52-58.degree. C.; Anal.
calcd. for C.sub.45H.sub.76O.sub.4S: C=76.21, H=10.80; Found:
C=76.11, H=10.81.
[0179] Preparation of
7.alpha.-Benzyloxy-24.xi.-t-butyldimethylsilyloxycholestan-3.beta.-ol
(2012):
[0180] Compound 2011 (0.057 g, 0.0803 mmol) was dissolved in dry
Et.sub.2O (3 ml) under argon. MgBr.sub.2 (0.142 g, 0.771 mmol) was
added quickly as a solid and the reaction was stirred until no
starting material remained by TLC. H.sub.2O (10 ml) was added and
the mixture was extracted 3.times. with 10 ml Et.sub.2O. The
combined organic layers were dried over anhydrous MgSO.sub.4,
filtered and the solvent removed in vacuo. Flash chromatography
(SiO.sub.2, 7:1 hexanes:EtOAc) gave compound 2012 (0.0493 g, 98%)
as a white foam. .sup.1H NMR (500 MHz, CDCl.sub.3) .delta.:
7.36-7.35 (m, 5H, benzyl-Ar H), 4.59 (d, J=12.0 Hz, 1H,
benzyl-CH.sub.2), 4.34 (d, J=12.0 Hz, 1H, benzyl-CH.sub.2),
3.65-3.60 (m, 1H, C-3 H), 3.475 (d, J=2.4 Hz, 1H, C-7 H), 3.40-3.36
(m, 1H, C-24 H), 0.91 (d, J=0.9 Hz, 9H, SiC(CH.sub.3).sub.3,
diastereomeric at C-24), 0.82 (s, 3H, C-19 H), 0.65 (s, 3H, C-18
H), 0.05 & 0.04 (s, 6H, Si(CH.sub.3).sub.2, diastereotopic
and/or diastereomeric) (Note: product is a mixture of
diastereomers); IR (CHCl.sub.3) 3600, 2900 cm.sup.-1; MS
(CI/isobutane) m/z 624 (M+1, 3%), 501 (M+1-OTHP, 6%), 385
(M+1-OTHP-TBDMS, 68%), 367 (M+1-THPOH-TBDMSOH, 100%); m.p.
55-58.degree. C.; Anal. calcd. for C.sub.40H.sub.68O.sub.3Si:
C=76.86, H=10.97; Found: C=76.69, H=10.87.
[0181] Preparation of
7.alpha.-Benzyloxy-24.epsilon.-t-butyldimethyl-silyloxycholest-3-one
(2013a) and
7.alpha.-Benzoyloxy-24.epsilon.-t-butyldimethylsilyloxycholestan-3-one
(2013b):
[0182] A solution of compound 2012 (0.229 g, 0.3664 mmol) in dry
CH.sub.2Cl.sub.2 (30 ml) was treated with Collin's reagent (0.385
g, 1.49 mmol). The mixture was stirred at room temperature
overnight under argon. At this time no starting material remained
by TLC. Celite was added and the mixture was stirred for 20 minutes
and then filtered through a pad of Celite. The cake was rinsed well
with CH.sub.2Cl.sub.2. The solvent was removed in vacuo. Flash
chromatography (SiO.sub.2, 20:1 hexanes:EtOAc) gave the desired
product 2013a (0.198 g, 87%) as a white solid along with the
7.alpha.-benzoate 2013b (0.015 g, 6.4%) as a white foam. Note: If
the reaction was run at higher concentration, a higher yield of the
benzoate was obtained. Compound 2013a: .sup.1H NMR (500 MHz,
CDCl.sub.3) .delta.: 7.35-7.27 (m, 5H, benzyl Ar--H), 4.55 (d,
J=11.7 Hz, 1H, benzyl-CH.sub.2), 4.32 (d, J=11.7 Hz,
benzyl-CH.sub.2), 3.495 (d, J=2.0 Hz, 1H, C-7 H), 3.38-3.35 ( H,
1H, C-24 H), 1.02 (s, 3H, C-19 H), 0.90 (d, J=0.8 Hz, 9H,
SiC(CH.sub.3).sub.3, diastereomeric at C-24), 0.67 (s, 3H, C-18 H),
0.04 & 0.03 (s, 6H, Si(CH.sub.3).sub.3, diastereotopic and/or
diastereomeric) (Note: product is a mixture of diastereomers); IR
(CHCl.sub.3) 2900, 1690 cm.sup.-1; MS (CI/isobutane) m/z 624 (M+1,
50%), 534 (M+1-Bn, 7%), 518 (M+1-OBn, 36%), 492
(M+1-HOSi(Me).sub.2t-Bu, 28%), 383 (M+1-C.sub.14H.sub.30OSi, 100%).
Compound 2013b: .sup.1H NMR (500 MHz, CDCl.sub.3) .delta.: 8.03 (d,
J=7.3 Hz, 2H, benzoate Ar H), 7.59 (t, J=7.4 Hz, 1H, Ar H), 7.48
(t, J=7.7 Hz, 2H, Ar H), 5.20 (br s, 1H, C-7 H), 3.35-3.31 (m, 1H,
C-24 H), 1.08 (s, 3H, C-19 H), 0.86 (d, J=3.7, 9H,
SiC(CH.sub.3).sub.3), 0.71 (s, 3H, C-18 H) (Note: product is a
mixture of diastereomers); IR (CHCl.sub.3) 2900, 1690 cm.sup.-1; MS
(CI/isobutane) m/z 637 (M+1, 3%), 516 (M+1-OBz, 16%), 382
(M+1-OBz-TBDMSOH, 100%); m.p. 62-65.degree. C.
[0183] Preparation of
7.alpha.-Benzyloxy-3.epsilon.-(5,10-di-t-butoxycarbonyl-1,5,10-triazadecy-
l)-24.epsilon.-t-butyldimethylsilyloxycholestane (2014a):
[0184] A mixture of compound 2013a (0.07 g, 0.11 mmol),
approximately 2 equivalents of amino compound 301 (based on 60%
yield for the reduction of compound 2018 to compound 301), and 3
.ANG. molecular sieves (0.5 g) in MeOH (6 ml, dried over 3 .ANG.
sieves) was stirred for 12 hours at room temperature under argon.
NaCNBH.sub.3 (0.33 ml, 1 M in THF, 0.33 mmol) was added and the
solution stirred for 24 hours at room temperature under argon. The
mixture was filtered through Celite and the cake was washed well
with MeOH and CH.sub.2Cl.sub.2 and the solvents were removed in
vacuo. The residue was dissolved in CH.sub.2Cl.sub.2 (10 ml),
washed 2.times. with 5 ml H.sub.2O made basic with aqueous NaOH
solution (5%), and washed 2.times. with 5 ml aqueous saturated NaCl
solution. The combined aqueous layers were back-extracted with
CH.sub.2Cl.sub.2, and the combined organic layers were dried over
anhydrous MgSO.sub.4. Filtration, removal of the solvent in vacuo,
and flash chromatography (SiO.sub.2, gradient of increasing
polarity from 2% MeOH in CH.sub.2Cl.sub.2 to 10% MeOH in
CH.sub.2Cl.sub.2) gave the desired product 2014a (0.07 g, 66%) and
a more polar product, compound 2014b, which is missing one t-BOC
group and is contaminated with excess amine. Compound 2014a:
.sup.1H NMR (500 MHz, CDCl.sub.3) .delta.: 7.36-7.28 (m, H,
benzyl-Ar H), 4.63 (d, J=12.0 Hz, 1/2H, benzyl-CH.sub.2), 4.58 (d,
J=12.0 Hz, 1/2H, benzyl-CH.sub.2), 4.33 (t, J=1.25 Hz, 1H,
benzyl-CH.sub.2), 3.49 (s, 1H, C-7 H), 3.46-3.14 (m, 8H,
N(BOC)CH.sub.2 & C-24 H), 2.91-2.86 (m, 2H, NCH.sub.2),
1.47-1.41 (m, 18H, 2.times.COC(CH.sub.3).sub.3), 0.90 (s, 9H,
SiC(CH.sub.3).sub.3), 0.84 (s, 3H, C-19 H), 0.64 (s, 3H, C-18 H),
0.05 & 0.04 (s, 6H, Si(CH.sub.3).sub.2, diastereotopic and/or
diastereomeric) (Note: This product is a mixture of
diastereomers).
[0185] Preparation of
7.alpha.-Benzyloxy-3.beta.-(1,5,10-triazadecyl)cholestan-24.epsilon.-ol
(2015.beta.) and
7.alpha.-benzyloxy-3.alpha.-(1,5,10-triazadecyl)cholestan-24.epsilon.-ol
(2015.alpha.):
[0186] TFA (1.8 ml, 24 mmol) was added to a solution of compound
2014a (0.386 g, 0.4 mmol) in CHCl.sub.3 (15 ml) at room
temperature. The reaction was stirred until no starting material
remained by TLC. The solvent was removed in vacuo and the residue
purified by preparative TLC (SiO.sub.2, 2000 .mu.m, 6:3:1
CH.sub.2Cl.sub.2:MeOH:NH.sub.4OH, R.sub.f=0.46) to give the desired
3.beta. product 2015.beta. (0.122 g, 48%) and the 3.alpha. isomer
2015.alpha. (0.109 g, 43%). Compound 2014b could be treated with
TFA under the same conditions to give compounds 2015.alpha. and
2015.beta.. Compound 2015.beta.:
[0187] .sup.1H NMR (500 MHz, CD.sub.3OD) .delta.: 7.32-7.35 (m, 5H,
benzyl-Ar H), 4.57 (d, J=11.7 Hz, 1H, benzyl-CH.sub.2), 4.31 (d,
J=11.7 Hz, 1H, benzyl-CH.sub.2), 3.52 (s, 1H, C-7 H) 3.22-3.21 (m,
2H, C-24 H & NCH.sub.2), 2.86 (t, J=7.1 Hz, 2H, NCH.sub.2),
2.81 (t, J=6.6 Hz, 2H, NCH.sub.2), 2.74 (t, J=7.0 Hz, 2H,
NCH.sub.2), 2.67 (t, J=6.3 Hz, 2H, NCH.sub.2), 0.85 (s, 3H, C-19
H), 0.683 (s, 1.5H, C-18, diastereomeric at C-34), 0.678 (s, 1.5H,
C-18, diastereomeric at C-24); MS (pos. FAB) m/z 638.6 (M+1, 100%).
Compound 2015.alpha.: .sup.1H NMR (500 MHz, CD.sub.3OD) .delta.:
7.35-7.22 (m, 5H, benzyl-Ar H), 4.61 (d, J=11.4 Hz, 1H,
benzyl-CH.sub.2), 4.28 (d, J=11.5 Hz, 1HA, benzyl-CH.sub.2), 3.53
(s, 1H, C-7 H), 3.43 (s, 1H, C-3H), 3.24-3.20 (m, 2H, C-24 H &
NCH.sub.2), 3.11 (t, J=7.1 Hz, 2H, NCH.sub.2), 3.08-3.02 (m, 2H,
NCH.sub.2), 2.96 (t, J=6.9 Hz, 2H, NCH.sub.2), 0.85 (s, 3H, C-19
H), 0.691 (s, 1.5H, C-18, diastereomeric at C-24), 0.686 (s, 1.5H,
C-18, diastereomeric at C-24); MS (pos. FAB) m/z 638.6 (M+1,
100%).
[0188] Preparation of
3.beta.-(1,5,10-Triazadecyl)cholesta-7.alpha.,24.epsilon.-diol
(2016):
[0189] To a solution of compound 2015.beta. (0.0128 g, 0.02 mmol)
in absolute EtOH (8 ml) was added a catalytic amount of 10% Pd/C
and 2 drops of concentrated hydrochloric acid. The mixture was
placed on a Parr hydrogenation apparatus and shaken under 55 psi
(H.sub.2) for 24 hours. The solution was filtered through a pad of
Celite, and the cake was washed well with EtOH and MeOH, and the
solvents removed in vacuo. The desired product 2016 (0.0074 g, 68%)
was obtained. If the product was pure by TLC, it was used without
further purification. If impurities were observed by TLC, the
material was purified by flash chromatography (SiO.sub.2, 15:4:1
CH.sub.2Cl.sub.2:MeOH:NH.sub.4OH). .sup.1H NMR (500 MHz,
CD.sub.3OD) .delta.: 3.79 (s, 1H, C-7 H) 3.22-3.13 (m, 6H,
2.times.CH.sub.2N & C-24 H & C-3 H), 3.09 (t, J=7.4, 2H,
CH.sub.2N), 2.99 (t, J=7.3 Hz, 2H, CH.sub.2N), 0.87 (s, 3H, C-19
H), 0.694 (s, 1.5H, C-18 H, diastereomeric at C-24), 0.691 (s,
1.5H, C-18 H, diastereomeric at C-24).
[0190] Preparation of squalamine (compound 1256):
[0191] Compound 2016 (0.0176 g, 0.032 mmol) was dissolved in a
solution of concentrated hydrochloric acid in MeOH (1 ml
concentrated hydrochloric acid in 10 ml MeOH). The solution was
stirred for 15 minutes and the solvent removed in vacuo. To the
crude, dried product was added SO.sub.3-pyridine complex (0.010 g,
0.064 mmol) and the flask was flushed with argon. Dry pyridine (1
ml) was added, the solution was warmed to 80.degree. C. in an oil
bath and stirred for 2 hours. MeOH (2 ml) was added. The flask was
removed from the oil bath and the mixture was stirred for 15
minutes. The solvent was removed in vacuo, and the residue was
resuspended in MeOH and filtered through a pad of Celite. The cake
was washed well with MeOH. Flash chromatography (SiO.sub.2, 12:4:1
CH.sub.2Cl.sub.2:MeOH:NH.sub.4OH) gave the desired product 1256
(0.0113 g, 56%) as a white solid. .sup.1H NMR (500 MHz, CD.sub.3OD)
.delta.: 4.13-4.10 (m, 1H, C-24 H), 3.79 (s, 1H, C-7 H), 3.22-3.10
(m, 5H CH.sub.2N), 3.08 (t, J=6.7 Hz, 2H, CH.sub.2N), 2.98 (t,
J=6.8 Hz, 2H, CH.sub.2N), 0.87 (s, 3H, C-19 H), 0.70 (s, 3H, C-18
H); MS (pos. FAB) m/z 628.4 (M+1, 57%), 548.5 (M+1-SO.sub.3, 23%),
530.5 (M+1-H.sub.2SO.sub.4, 100%); high resolution MS (pos. FAB)
m/z 628.4669 (calcd.: 628.4723).
[0192] Preparation of 5,10-Di-t-butoxycarbonyl-1,5,10-triazadecane
(301):
[0193] Nitrile 2018 (0.0624 g, 0.181 mmol) in dry Et.sub.2O (0.30
ml) was added to a suspension of LiAlH.sub.4 (0.024 g, 0.63 mmol)
in dry diethyl ether (1 ml) at 0.degree. C. The mixture was stirred
at 0.degree. C. for 30 minutes. Aqueous NaOH solution (1M) was
added to quench excess LiAlH.sub.4, and the resulting white
suspension was filtered through a pad of Celite. The cake was
washed well with Et.sub.2O, and the combined organic layers were
washed with H.sub.2O. The H.sub.2O layer was extracted with
Et.sub.2O and the combined ether layers were washed with aqueous
saturated NaCl solution, dried over anhydrous MgSO.sub.4, filtered
and the solvent removed in vacuo. The .sup.1H NMR spectrum (500
MHz) of the crude product 301 (0.056 g, 88%) matched that reported
in the literature (Tetrahedron 46, 1990, 3267-3286), and the
material was used crude.
[0194] Preparation of
7.alpha.-Benzoyloxy-3.epsilon.-(5,10-di-t-butoxycarbonyl-1,5,10-triazadec-
yl)-24.epsilon.-t-butyldimethylsilyloxycholestane (2020):
[0195] Compound 2013b (0.110 g, 0.1726 mmol) was converted to
compound 2020 (0.166 g, 99%) using the previously described
procedure for the conversion of compound 2013a to compound 2014a.
.sup.1H NMR (500 MHz, CDCl.sub.3) .delta.: 8.19 (d, J=7.6 Hz, 1/2H,
benzoate-Ar H), 8.05 (d, J=7.4 Hz, 3/2H, benzoate-Ar H), 7.61-7.58
(m, 1H, benzoate-Ar H), 7.55-7.45 (m, 2H, benzyl-Ar H), 5.23 (s,
1/4H, C-7 H), 5.16 (s, 3/4H, C-7 H), 4.78-4.64 (m, 1H), 3.40-3.22
(m, 2H), 3.20-3.06 (m, 3H, NCH.sub.2), 2.98-2.8 (m, 4H, NCH.sub.2),
0.673 (s, 1.5H, C-18, diastereomeric at C-24), 0.667 (s, 1.5H,
C-18, diastereomeric at C-24).
[0196] Preparation of
7.alpha.-Benzoyloxy-3.alpha.-(1,5,10-triazadecyl)cholestan-24.epsilon.-ol
(2021.alpha.) and
7.alpha.-benzoyloxy-3.beta.-(1,5,10-triazadecyl)cholestan-24.epsilon.-ol
(2021.beta.)
[0197] Compound 2020 (0.166 g, 0.1717 mmol) was converted to
compounds 2021.alpha. and 2021.beta. (quantitative yield of a 1:1
mixture of the 3.alpha. and 3.beta. products) in the same manner as
described previously for the conversion of compound 2014a to
compounds 2015.alpha. and 2015.beta.. Compound 2021.beta.: .sup.1H
NMR (500 MHz, CD.sub.3OD) .delta.: 8.01 (d, J=8.3 Hz, 2H,
benzoate-Ar H), 7.61-7.59 (m, 1H, benzoate-Ar H), 7.51-7.45 (m, 2H,
benzyl-Ar H), 5.14 (s, 1H, C-7 H), 3.20-3.15 (m, 1H), 2.90-2.75 (m,
4H, NCH.sub.2), 2.72 (t, J=6.9 Hz, 2H, NCH.sub.2), 2.65 (t, J=6.7
Hz, 2H, NCH.sub.2), 0.85 (s, 3H, C-19 H), 0.726 (s, 1.5H, C-18,
diastereomeric at C-24), 0.723 (s, 1.5H, C-18, diastereomeric at
C-24); MS (pos. FAB) m/z 652.5 (M+1, 100%), 530.5 (M+1-HOBz, 6%).
Compound 2021.alpha.: .sup.1H NMR (500 MHz, CD.sub.3OD) .delta.:
8.01 (d, J=8.3 Hz, 2H, benzoate-Ar H), 7.61-7.59 (m, 1H,
benzoate-Ar H), 7.51-7.45 (m, 2H, benzyl-Ar H), 5.12 (s, 3H, C-7 ),
3.19-3.15 (m, 1H), 2.86 (s, 1H), 2.70-2.60 (m, 4H, NCH.sub.2),
2.60-2.54 (m, 2H, NCHhd 2), 2.54-2.49 (m, 2H, NCH.sub.2), 0.73 (s,
3H, C-18, diastereomeric at C-24); MS (pos. FAB) m/z 652.5 (M+1,
100%), 530.5 (M+1-HOBz, 10%).
[0198] Preparation of
7.alpha.-Benzoyloxy-3.alpha.-(1,5,10-triazadecyl)cholestan-24.epsilon.-su-
lfate (2022):
[0199] Compound 2021.alpha. (0.0214 g, 0.0328 mmol) was converted
to compound 2022 (0.0190 g, 79%) as previously described for the
conversion of compound 2016 to compound 2017. .sup.1H NMR (500 MHz,
CD.sub.3OD) .delta.: 8.21-8.14 (m, 2H, benzoate-Ar H), 7.62-7.50
(m, 2H, benzoate-Ar H), 5.18-5.09 (m, 1H, C-7 H), 4.22-4.16 (m,
1/2H, C-24 H), 4.10-4.06 (m, 1/2H, C-24 H), 3.43 (br 8, 1H, C-3 H),
3.22-3.10 (m, 5H, CH.sub.2N), 3.09 (t, J=7.5 Hz, 2H, CH.sub.2N),
3.04 (br s, 2H, CH.sub.2N), 2.99-2.96 (m, 2H, CH.sub.2N), 0.60 (s,
3/2H, C-18 H), 0.52 (s, 3/2H, C-18 H) (Note: compound is a mixture
of diastereomers at C-24).
[0200] Preparation of 3-Episqualamine (388):
[0201] Compound 2022 (0.066 g, 0.085 mmol) was dissolved in
methanolic KOH (5%, 5 ml) and refluxed for 7 hours. No starting
material remained by TLC. Neutralization with 5% (v/v) concentrated
hydrochloric acid in methanol followed by removal of the solvent
and flash chromatography (SiO.sub.2, 12:4:1
CH.sub.2Cl.sub.2:MeOH:NH.sub.4OH) gave the desired product 2023
(0.0365 g, 67%). .sup.1H NMR (500 MHz, CD.sub.3OD) .delta.:
4.14-4.09 (m, 1H, C-24 H), 3.80 (s, 1H, C-7 H), 3.48 (s, 1H, C-3
H), 3.24-3.15 (m, 4H, CH.sub.2N), 3.10 (t, J=7.4 Hz, 2H,
CH.sub.2N), 3.01 (t, J=7.1 Hz, 2H, CH.sub.2N), 0.86 (s, 3H, C-19
H), 0.69 (s, 3H, C-18 H); MS (pos. FAB) m/z 628.5 (M+1, 18%), 548.5
(M+1-SO.sub.3, 65%), 530.4 (M+1-H.sub.2SO.sub.4, 100%); high
resolution MS (pos. FAB) m/z 628.4713 (calcd.: 628.4723).
[0202] Preparation of 3-Episqualamine Dessulfate
(3.alpha.-(1,5,10-Triazadecyl)cholestan-7.alpha.,24.epsilon.-diol,
387):
[0203] Compound 2015.alpha. (0.089 g, 0.1397 mmol) was converted to
compound 387 (0.0372 g, 49%) as described for the conversion of
compound 2015.beta. to compound 2016. .sup.1H NMR (500 MHz,
CD.sub.3OD) .delta.: 3.80 (s, 1H, C-7 H), 3.48 (s, 1H, C-3 H),
3.24-3.15 (m, 4H, CH.sub.2N), 3.10 (t, J=7.4 Hz, 2H, CH.sub.2N),
3.00 (t, J=7.3 Hz, 2H, CH.sub.2N), 0.86 (s, 3H, C-19H), 0.69 (2s,
3H, C-18 H), MS (pos. FAB) m/z 548.5 (M+1, 100%); high resolution
MS (pos. FAB) 548.5162 (calcd.: 548.5155).
EXAMPLE S
[0204] Preparation of compound 399: ##STR34## ##STR35##
[0205] Preparation of 3-oxo-4-cholenic acid methyl ester 3002:
[0206] A solution of 3.beta.-hydroxy-5-cholenic acid methyl ester
3001 (24.16 g, 57.11 mmol), aluminum tri-t-butoxide (56.27 g,
228.43 mmol) and isopropylmethylketone (50 ml) in dry toluene (150
ml) was stirred and heated to 120.degree. C. (oil bath) for 6
hours. The reaction mixture was then cooled to room temperature
diluted with toluene (100 ml) and acidified with 2NHCl (70 ml). The
organic layer was separated, and the aqueous layer extracted with
toluene (3.times.50 ml). The combined organic extracts were washed
with water (1.times.50 ml), saturated NaHCO.sub.3 (2.times.50 ml),
water (1.times.50 ml), brine (1.times.50 m), dried (MgSO.sub.4),
filtered and evaporated in vacuo to get the crude product. Flash
chromatography of the crude product using toluene followed by a
gradient of ethyl acetate/hexane (5, 10, 20 and 40%) solvent
systems gave a pure white solid, 3-oxo-4-cholenic acid methyl ester
3002 (13.43 g, 60%). .sup.1H NMR (400 MHz, CDCl.sub.3) .delta.:
0.71 (3H, s, 18-CH.sub.3), 0.90 (3H, d, 21-CH.sub.3), 1.17 (3H, S,
19-CH.sub.3), 3.66 (3H, s, CO.sub.2CH.sub.3) and 5.71 (1H, 9,
4-H).
[0207] Preparation of 3-oxo-5.alpha.-cholanic acid methyl ester
3003:
[0208] Into a solution of 3-oxo-4-cholenic acid methyl ester 3002
(13.0 g, 23.68 mmol) in dry ether (50 ml) was added distilled
liquid ammonia (70 ml) at -78.degree. C. Lithium (1.0 g, 144.1
mmol) was added in small portions until a blue coloration persisted
for 10 minutes, after which the solution was quenched with solid
NH.sub.4Cl (50 g). Ammonia was evaporated, and the resulting
residue was partitioned between water (100 ml) and ether (150 ml).
The aqueous solution was extracted further with ether (3.times.50
ml). The combined extracts were washed with brine (1.times.70 ml),
dried (MgSO.sub.4), filtered and concentrated in vacuo to get the
crude product. Flash chromatography of the crude product in silica
gel using ethyl acetate:hexane (2:8) gave pure
3-oxo-5.alpha.-cholanic acid methyl ester 3003 (3.85 g, 29w).
.sup.1H NMR (400 MHz, CDCl.sub.3) .delta.: 0.69 (3H, s,
18-CH.sub.3, 0.91 (3H, d, 21-CH ), 1.02 (3H, s, 19-CH.sub.3) and
3.66 (3H, s, CO.sub.2CH.sub.3).
[0209] Preparation of
N-(3'-Aminipropyl)-N,N'-(di-tert-butoxycarbonyl)-1,4-diaminobutane
301:
[0210] (a) To a solution of 1,4-diaminobutane (8.6 g, 97.6 mmol) in
methanol (3.0 ml) was added a solution of acrylonitrile (6.2 g,
116.8 mmol) in methanol (3.0 ml) at 0.degree. C., and the mixture
was stirred for 12 hours. Evaporation of the solvent in vacuo
afforded N-(2'-cyanoethyl)-1,4-diaminobutane as a colorless oil
(11.0 g, 80%). .sup.1H NMR (400 MHz, CDCl.sub.3) .delta.: 1.45 (4H,
br, --CH.sub.2CH.sub.2--), 2.46 (2H, t), 2.58 (2H, t), 2.62 (2H, t)
and 2.84 (2H, t).
[0211] (b) To a solution of N-(2'-cyanoethyl)-1,4-diaminobutane
(5.6 g, 40 mmol) in dichloromethane (140 ml) was added dropwise a
solution of di-t-butyldicarbonate (19.2 g, 88 mmol) in
dichloromethane (20 ml) at room temperature, and the mixture was
stirred for 12 hours. The organic solvent was removed in vacuo and
the residual oil was dissolved in ethyl acetate (150 ml), and
washed with saturated NaHCO.sub.3 (2.times.75 ml), water
(2.times.75 ml), brine (75 ml), dried (MgSO.sub.4), filtered and
evaporated to get the crude viscous oil. The crude product was
purified by flash chromatography in silica gel to give pure
(N-(2'-cyanoethyl)-N,N'-(di-t-butoxycarbonyl)-1,4-diaminobutane as
a colorless viscous oil (8.4 g, 75%). .sup.1H NMR (400 MHz,
CDCl.sub.3) .delta.: 1.44 (9H, s, t-Boc), 1.46 (9H, merged s,
t-Boc), 2.60 (2H, m), 3.15 (2H, m) 3.28 (2H, t) and 3.45 (2H, t);
CIMS (m/e): 342 (M+1, 62.7%), 239 (100%), 186 (83.1%).
[0212] (c) To a suspension of lithium aluminum hydride (1.8 g, 48.9
mmol) in dry ether (300 ml) was added a solution of
N-(2'-cyanoethyl)-N,N'-(di-t-butoxycarbonyl)-1,4-diaminobutane (4.8
g, 13.8 mmol) in dry ether (150 ml) dropwise at 0.degree. C., and
the mixture was stirred for 30 minutes. The excess lithium aluminum
hydride was quenched with 1 N NaOH at 0.degree. C. and the
resulting white suspension wads filtered through Celite and washed
with ether, and the ether extract was washed with brine, dried
(MgSO.sub.4), filtered and evaporated in vacuo to get a crude oil.
The crude product was purified by flash chromatography in silica
gel to give pure
N-(3'-aminopropyl)-N,N'-(di-t-butoxycarbonyl)-1,4-diaminobutane 301
(3.3 g. 68%) as a colorless oil. .sup.1H NMR (400 MHz, CDCl.sub.3)
.delta.: 1.44 (18H, s, 2(t-Boc)), 2.68 (2H, t), 3.05-3.25 (6H, br),
and 4.65 (1H, br); CIMS (m/e): 346 (M.sup.++1, 100%), 290 (3.1%),
246 (32.2%).
[0213] Preparation of
3.beta.-N-1-{N[3-(4-Aminobutyl)]-1,3-diaminopropane}-24-hydroxy-5.alpha.--
cholane trihydrochloride 3005:
[0214] To a solution of 3-oxo-5.dbd.-cholanic acid methyl ester
3003 (3.0 g, 7.73 mmol) and
N-(3'-aminopropyl)-N,N'-(di-t-butoxycarbonyl)-1,4-diaminobutane 301
(4.01 g, 11.60 mmol) in methanol (150 ml) was added 3 .ANG.
molecular sieves (10 g) and NaCNBH.sub.3 (0.73 g, 11.61 mmol). The
reaction mixture was stirred at room temperature for 16 hours.
After filtering through Celite, methanol was removed in vacuo. The
residue was dissolved in methanol (50 ml) and then methanol
pre-saturated with HCl gas (15 ml) was added. The reaction mixture
was stirred at room temperature for 6 hours. After removing
methanol in vacuo, the crude product was dissolved in
tetrahydrofuran (100 ml) and then lithium aluminum hydride (1.50 g,
39.52 mmol) was added in one portion. The reaction mixture was
gently refluxed for 8 hours. The reaction mixture was cooled to
0.degree. C. (ice bath), then a solution of 2N NaOH was added
dropwise until white solid granulates were formed. Tetrahydrofuran
was decanted and the residue further extracted with toluene
(3.times.50 ml), and the combined organic extracts were dried
(K.sub.2CO.sub.3), filtered and evaporated in vacuo to get the
residue. The residue was dissolved in dry methanol (50 ml) and then
methanol pre-saturated with HCl gas (20 ml) was added. After one
hour, excess methanol was removed in vacuo to get white solid. The
crude product was purified by flash chromatography in silica gel
using chloroform:methanol:isopropylamine (15:1:1) to get pure
3.beta.-N-1-{N[3-(4-aminobutyl)]-1,3-diaminopropane}-24-hydroxy-5.alpha.--
cholane trihydrochloride 3005 (1.10 g, 24%). .sup.1H NMR (400 MHz,
CD.sub.3OD) .delta.: 0.71 (3H, s, 18-CH.sub.3), 0.89 (3H, 8,
19-CH.sub.3), 096 (3H, d, 21-CH.sub.3), 2.90-3.40 (9H, m) and 3.51
(2H, br t, CH.sub.2O); MS--FAB (positive): 490 (M++1, 100%), 419
(8%) and 360 (7.5%)
[0215] Preparation of
3.beta.-N-1-{N[3-(4-Trifluoroacetyl)aminobutyl)]-1,3-di(trifluoroacetyl)d-
iaminopropane}-24-hydroxy-5.alpha.-cholane 3006:
[0216] To a solution of
3.beta.-N-1-{N[3-(4-aminobutyl)]-1,3-diaminopropane}-24-hydroxy-5.alpha.--
cholane trihydrochloride 3005 (0.95 g, 1.58 mmol) in dry methanol
(20 ml) was added dry triethylamine (2.29 ml, 15.8 mmol) followed
by ethyl trifluoroacetate (2.80 ml, 23.53 mmol) at room
temperature, and the mixture was stirred for 20 hours. After
removal of excess methanol and low boiling organic reagents in
vacuo produced a white residue. The residue was dissolved in ethyl
acetate (50 ml) and then washed with 2 N HCl (3.times.20 ml), water
(2.times.20 ml), saturated NaHCO.sub.3 (3.times.20 ml) and brine
(1.times.20 ml), dried (MgSO.sub.4), filtered and evaporated in
vacuo to get an almost pure white solid,
3.beta.-N-1-{N[3-(4-trifluoroacetyl)aminobutyl)]-1,3-di(trifluoroacetyl)d-
iaminopropane}-24-hydroxy-5.alpha.-cholane 3006 (0.77 g, 73%).
.sup.1HNMR (400 MHz, CDCl.sub.3) .delta.: 0.71 (3H, 8,
18-CH.sub.3), 0.89 (3H, s, 19-CH.sub.3), 0.96 (3H, d, 21-CH.sub.3),
3.01-3.57 (1H, m, 4.times.CH.sub.2N+1.times.CHN+CH.sub.2O).
[0217] Preparation of
3.beta.-N-1-{N[3-[4-Trifluoroacetyl)aminobutyl)]-1,3-di(trifluoroacetyl)d-
iaminopropane}-24-hydroxy-5.alpha.-cholane 24-pyridinium sulfate
3007:
[0218] To a solution of compound 3006 (0.70 g, 1.05 mmol) in dry
pyridine (20 ml) was added sulfur trioxide pyridine complex (0.75
g, 4.71 mmol) at room temperature, and the mixture was stirred for
6 hours. The excess pyridine was removed in vacuo to get solid
residue, from which the sulfated compound was extracted with
chloroform (5.times.20 ml). Removal of chloroform gave white solid,
3.beta.-N-1-{N[3-(4-trifluoro-acetyl)aminobutyl)]-1,3-di(trifluoroacetyl)-
diaminopropane}-24-hydroxy-5.alpha.-cholane24-pyridinium sulfate
3007 along with excess reagent (1.0 g). The crude product was used
in the next step without further purification.
[0219] Preparation of
3.beta.-N-1-{N[3-(4-Aminobutyl)]-1,3-diaminopropane}-24-hydroxy-5.alpha.--
cholane24-potassium sulfate (399):
[0220] To a solution of crude compound 3007 (1.0 g) in methanol (25
ml) was added a solution of potassium carbonate in water (10 ml) at
room temperature, and the mixture was stirred overnight. After 18
hours, the excess methanol and water were removed in vacuo to get
the residue. The residue was extracted with methanol (3.times.30
ml). The combined methanol extracts were concentrated in vacuo to
get crude product. Flash chromatography of the crude product in
silica gel using dichloromethane:methanol:ammonium hydroxide
(7:2:1) (dried over MgSO.sub.4 before use) gave white solid,
3.beta.-N-1-{N[3-(4-aminobutyl)]-1,3-diaminopropane}-24-hydroxy-5.alpha.--
cholane24-potassium sulfate or compound 399 (0.22 g, 35% based on
compound 3006). .sup.1H NMR (400 MHz, CD.sub.3OD) .delta.: 0.74
(3H, 9, 18-CH.sub.3), 0.92 (3H, s, 19-CH.sub.3), 1.0 (3H, d,
21-CH.sub.3), 2.95-3.24 (9H, m) and 4.00 (2H, br t,
CH.sub.2OSO.sub.3); MS--FAB (positive) (m/e): 570 (M.sup.++2, 85%),
490 (44%), 430 (15%), 402 (16%); MS--FAB (negative) (m/e): 568
(M.sup.+, 3.7%), 495 (10%), 452 (7%), 438 (17%), 423 (14%).
EXAMPLE T
[0221] Preparation of compound 1436:
[0222] This compound can be readily prepared from squalamine
through the coupling of .beta.-alanine aldehyde, followed by
reduction, as shown in the following scheme: ##STR36##
[0223] The above approach permits the ready conversion of
squalamine, present in large amounts in shark liver, to compound
1436, present in about 5% the quantity of squalamine.
[0224] Additional aminosterol compounds such as those shown in
Tables I and II herein can be prepared in manners analogous to
those given above.
Therapeutic Activities and Utilities
[0225] Aminosterol compounds such as squalamine have been
discovered to be effective inhibitors of NHE. In seeking to
elucidate the antimicrobial mechanism of action for squalamine,
squalamine has been found to advantageously inhibit a specific NHE
isoform--the compound inhibits NHE3, but not NHE1. In addition,
squalamine has been determined to inhibit the exchanger through a
special mechanism. The special and advantageous effects and
utilities of squalamine and other aminosterols are further evident
from the results of the experimental tests discussed below.
[0226] Specific Inhibition of NHE3:
[0227] To determine the specificity of squalamine's inhibition of
NHEs, squalamine was assayed against a cell line expressing either
human NHE1 or human NHE3 following procedures outlined in Tse et
al., J. Biol. Chem. 268, 1993, 11917-11924. Internal pH was
measured either following acid loading or in the absence of an
acid-loading challenge, with the results shown in FIGS. 1A and
1B.
[0228] Specifically, PS120 fibroblasts transfected with rabbit NHE3
were grown in supplemented Dulbecco's-modified Eagle's medium as
described by Levine et al., J. Biol. Chem. 268, 1993, 25527-25535.
Transfected cells grown on glass coverslips were then assayed for
internal pH changes following treatment with 5 .mu.g/ml squalamine
using the fluorescent dye BCECF-AM
(2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein-acetoxymethyl
ester) as a pH indicator as described by Levine et al. For cells
acid-preloaded by exposure to 40 mM NH.sub.4Cl, the rate of pH
recovery as a function of restored extracellular sodium ion
concentration was monitored, with the results being shown in FIG.
1A. For cells not acid-preloaded, the actual internal pH value was
monitored as a function of time following addition of squalamine,
with the results depicted in FIG. 1B.
[0229] As seen in FIGS. 1A and 1B, squalamine inhibited NHE3 with
respect to proton concentration at both K.sub.m and V.sub.max
levels. In contrast, existing agents such as amiloride affected
only V.sub.max.
[0230] Thus, the aminosterol squalamine not only reduces the
absolute number of protons that can be secreted by the cell (the
V.sub.max effect), but also forces the cell to fall to a lower pH
in the presence of this inhibitor (the K.sub.m effect). As a
consequence, the sodium/proton exchanger is more profoundly
inactivated by squalamine than by amiloride.
[0231] In contrast to its effects on NHE3 shown in FIGS. 1A and 1B,
squalamine exhibited no inhibitory activity against human NHE1 or
rabbit NHE1 as shown in FIGS. 2A and 2B. PS120 fibroblasts
transfected with rabbit or human NHE1 were grown as described
above. Transfected cells expressing rabbit NHE1 (FIG. 2A) or human
NHE1 (FIG. 2B) grown on glass coverslips were then assayed for
internal pH changes following treatment with 5 .mu.g/ml squalamine
using the fluorescent dye BCECF-AM with cells acid-preloaded by
exposure to 40 mM NH.sub.4Cl. The rate of pH recovery as a function
of restored extracellular sodium ion concentration was
monitored.
[0232] In addition, as demonstrated by FIG. 1B, the resting pH of
these cells was also inhibited. Thus, squalamine's effect on proton
exchange causes the cell to drop to a lower pH in its presence
before activation of the pump occurs.
[0233] Through these studies, squalamine has been discovered to be
a distinct inhibitor with specificity for NHE3 over NHE1. Moreover,
squalamine has been identified as an inhibitor that causes a cell
to drop to a lower pH before the pump is activated. The results
shown in FIGS. 1A, 1B, 2A and 2B demonstrate that squalamine
exhibits a unique NHE specificity.
[0234] The Expression of NHE3:
[0235] Such a specific effect for NHE3 is important for several
reasons. NHE3, being present on apical surfaces of a limited number
of cell types, is more specialized than NHE1. A cell of particular
interest therapeutically is the endothelial cell.
[0236] Using PCR, the expression of this antiporter in both human.
microvascular and human pulmonary artery endothelial cells has now
been demonstrated. Total RNA was isolated from primary human
pulmonary artery endothelial cells (HPAEC), human melanoma cell
line wm1617, and human microvascular endothelial cells (HMVEC) by a
modification of the method of Chomczynski et al., Anal. Biochem.
162, 1987, 156, and then reverse-transcribed with MMLV reverse
transcriptase using a first strand cDNA synthesis kit (Clontech
Laboratories, Palo Alto, Calif.). Human small-intestine total RNA
obtained from Clontech was also reverse-transcribed in the same
fashion.
[0237] Approximately 80 ng of the cDNA product were then amplified
in a 50-.mu.l reaction mixture using reagents from the AmpliTaq DNA
Polymerase kit (Perkin Elmer, Norwalk, Conn.) and containing 400 ng
each of two oligonucleotides specific for human NHE3 (B13:
5'-CATCTGGACCTGGAACACG-3'; B14: 5'-CGTAGCTGATGGCATCCTTC-3') using
one thermal cycle of 5', 94.degree. C., and then 38 cycles of 50'',
94.degree. C., 1', 57.degree. C., 2', 71.degree. C., and finally
10', 72.degree. C. before cooling to 4.degree. C. Twenty .mu.l of
this sample was analyzed on a 1.7% agarose gel. The expected NHE3
PCR band of about 550 bp was seen in most instances, as indicated
in Table 2 below.
[0238] One .mu.l of each PCR reaction was then further analyzed by
nested PCR in a 50-.mu.l reaction mix using two internal primers
(B15: 5'-CTGGTCTTCATCTCCGTGTAC-3'; B16: 5'-AGCTCGTGGAA-GACATTCAGG)
with a 5', 94.degree. C. program of thermal cycling, then 35 cycles
of 50'', 94.degree. C., 1', 58.degree. C., 2', 71.degree. C., and
finally 10', 72.degree. C. before cooling to 4.degree. C. About 20
.mu.l of this reaction was analyzed on another 1.7% agarose gel.
The expected NHE3 PCR band of about 490 bp was seen in all cases as
noted in the table below. DNA sequencing of the HPAEC and HMVEC
nested PCR bands from both ends confirmed they had the expected
human NHE3 sequences. TABLE-US-00004 TABLE 2 EXPRESSION OF HUMAN
NHE3 IN HUMAN ENDOTHELIAL CELL LINES Visible Detection of Human
NHE3 by PCR Total RNA Source 1 PCR Round 2 Nested PCR Rounds Small
Intestine - + Human Melanoma + + HPAEC + + HMVEC +/- (multiple
bands) +
[0239] Thus, a variety of endothelial cell growth/shape related
events are inhibited by squalamine and functionally related
compounds. The experimental tests discussed below were conducted to
assess this aminosterol's effects.
[0240] Growth Inhibition of Endothelial Cells,
[0241] Fibroblasts and Epithelial Cells In Vitro:
[0242] When non-transformed human cells are grown in the presence
of increasing concentrations of squalamine, endothelial cells
exhibit a particular sensitivity to squalamine, as shown by the
following experiment. Bovine pulmonary endothelial cells, human
epithelial cell line MCF 10A, and human foreskin fibroblasts were
incubated in the presence of 12 different membrane-active agents,
including peptides and squalamine.
[0243] Specifically, bovine pulmonary endothelial cells, human
epithelial cell line MCF 10A, and human foreskin fibroblasts were
incubated in the presence of the following twelve membrane-active
agents: (1) RGD[KIAGKIA].sub.3-NH.sub.2; (2) d-[KKLLKKL]2-NH.sub.2;
(3) squalamine; (4) SWLSKTAKKLENSAKKRISEGIAIAIQGGPR; (5)
FLGGLIKIVPAMICAVTKKC; (6) Magainin 2; (7) PGLA; (8)
GFASFLGKALKAALKIGANLLGGTPQQ; (9) PR-39; (10)
1-[KKLLKKL].sub.2-NH.sub.2 (11) Cecropin B; and (12)
[KIAGKIA].sub.3-NH.sub.2. Cell growth was measured by absorbance at
600 nm. Results are shown in FIGS. 3A-3C.
[0244] As evident from FIG. 3A, squalamine inhibited the growth of
bovine pulmonary artery endothelial cells (BPE) at 1 .mu.g/ml. In
contrast, at 10 .mu.g/ml it exerted no effect on the growth of
either epithelial (FIG. 3B) or fibroblast (FIG. 3C) lines. However,
peptides that inhibited the growth of epithelial cells exhibited no
effect on BPE. Thus, endothelial cells are more sensitive to
squalamine than are either fibroblasts or epithelial cells.
[0245] Inhibition of Endothelial Cell Cord Formation In Vitro:
[0246] Endothelial cells have the capacity in vitro to form tubular
aggregates resembling capillaries in various early stages of
formation. This conversion occurs under relatively specific
conditions, in which essential growth factors along with an
effective substratum are provided. It has been shown that both the
interaction of growth factors with the endothelial cell and its
attachment to a substratum activate the NHE. The activation of this
exchanger is believed to be required for subsequent morphologic
transformation of the endothelial cell into a multicellular tubular
structure.
[0247] To assess the effect of compounds on the cord-like
structures formed by human microvascular cells when plated in the
presence of VGEF (Vascular Endothelial Growth Factor) and basic
fibroblast growth factor on a collagen matrix, a standard cord
formation assay was used. The results are shown in the table below.
TABLE-US-00005 TABLE 3 EFFECT OF VARIOUS AMINOSTEROLS ON
ENDOTHELIAL CORD FORMATION .mu.g/ml 0.01 0.1 1.0 10.0 Fumagillin -
+/- + Squalamine - + + + Compound 319 - + + + Compound 353 + + +
Compound 410 - + +* Compound 411 - - + Compound 412 - - + Compound
413 - - + Compound 415 - - +/T Compound 371 T T Compound 432 - -
Compound 449 - +/- Compound 467 - - Notes: + = Inhibition of
angiogenesis; - = No inhibition of angiogenesis; T = Toxic; *= cell
rounding @ 10 .mu.g/ml.
[0248] As shown in Table 3, squalamine inhibits cord formation at
about 0.1 .mu.g/ml, compared with fumagillin, which exhibits
comparable activity at 10 .mu.g/ml. At these concentrations,
squalamine does not appear to profoundly affect cell viability or
proliferation. This property in vitro roughly correlates with
anti-angiogenic activity in more complex in vivo models (see Goto
et al., Lab Investigation 69, 1993, 508-518).
[0249] LPS-Induced Neutrophil Adherence to Human
[0250] Umbilical Venous Endothelial Cells:
[0251] When endothelial cells are exposed to certain stimuli,
including lipopolysaccharide (LPS) and certain cytokines, specific
adhesion molecules are induced on the plasma membrane that enhance
the binding of leukocytes. These leukocyte-endothelial cell
interactions are believed to be necessary to localize leukocytes to
sites of bacterial invasion and to facilitate extravasation of the
leukocytes from the capillary into the surrounding tissue space.
Leukocyte-adhesion molecules include the Selectins and ICAM-1.
[0252] To determine if squalamine inhibited this particular
endothelial cell function, standard adhesion assays were performed
as outlined in Gamble et al., J. Imm. Methods 109, 1988, 175-184.
The expression of cell surface ligands in an endothelial-based
system has been shown to effect adherence to granulocytes with a
system using human umbilical venous endothelial cells, purified
neutrophils, and inducers of cell surface ligands such as LPS (100
ng/ml) and TNF-.alpha. (40 ng/ml). In these experiments,
approximately 2.times.10.sup.5 human umbilical venous cells
(passage 2-6) were plated per well. The cells were grown in
serum-free media overnight. For induction, either TNF-.alpha. (40
ng/ml) was added to endothelial cells for 6 hours prior to adding
neutrophils or LPS (100 ng/ml) was added for 4-6 hours. It was
found that the LPS response was increased by adding 1 FBS to the
wells to provide a source of LPS-binding protein. After activation
of the endothelial cells, approximately 50.times.10.sup.6
neutrophils were added per well. The plates were gently rocked for
30 minutes at room temperature, followed by removal of the media
and washing in serum-free-media three times and then photographing
of each well. Experiments to test the effects of squalamine were
performed by adding squalamine at 10 .mu.g, 1.0 .mu.g, or 0.1 .mu.g
at the time of adding LPS or TNF-.alpha.. A second repeat dose of
squalamine was added at the time of adding neutrophils. A
monoclonal Ab to ICAM-1 was a positive control.
[0253] Using three different subjects, there was no inhibition of
squalamine on neutrophil adherence using activated human
endothelial cells. There was approximately 50% inhibition of
adherence when adding 40 .mu.g/ml of a monoclonal Ab to ICAM-1
prior to adding neutrophils.
[0254] These results indicate that inhibition of the endothelial SE
by squalamine affects both growth and capillary formation in vitro,
but does not inhibit all signal transduction pathways in this cell.
Thus, certain "housekeeping" functions, such as the capacity of the
endothelial cell to attract leukocytes to the site of an infection,
should not be impaired by squalamine. This demonstrates that
squalamine can be used to inhibit angiogenesis but will not
otherwise disrupt certain important endothelial cell functions,
such as leukocyte recruitment to sites of infection or
inflammation.
[0255] Anti-Proliferative Activity:
[0256] The Chorioallantoic Membrane Model:
[0257] Using the classical chorioallantoic membrane model, it has
been found that squalamine is an inhibitor of capillary growth. The
growing capillaries within the chorioallantoic membrane model (CAM
model) have been used as a system in which to evaluate the effect
of agents on their potential to inhibit new vessel growth.
Neovascularization occurs most aggressively over the first week of
embryonic development. Thereafter capillary growth is characterized
by principally "elongation" rather than "de novo" formation.
[0258] In the standard assay, agents are applied locally to a
region of the embryo over which neovascularization will occur.
Agents are assessed by their ability to inhibit this process, as
evaluated by visual examination about 7 days after application.
Agents which disrupt vascular growth during the period of de novo
capillary formation, but do not interfere with subsequent capillary
growth, are generally regarded as "specific" inhibitors of
neovascularization, as distinguished from less specific toxic
substances. The assay utilized is described in detail in Auerbach
et al., Pharm. Ther. 51, 1991, 1-11. Results are tabulated below.
TABLE-US-00006 TABLE 4 INHIBITION OF CAPILLARY GROWTH IN CAM MODEL
3-Day Squalamine Percentage positive Embryo: Applied (.mu.g) Assay
1 Assay 2 Mean 0.65 28 1.25 18 18 18 2.5 35 18 27 5.0 91 57 74 20
52* 58* 55 40 50* 13* 32 13-Day Squalamine Embryo Applied (.mu.g)
Percentage positive 5.0 0/26 Note: *= Some vascular irritation
noted.
[0259] As seen from Table 4, applying as little as 0.65 .mu.g
squalamine to a 3-day CAM resulted in inhibition of CAM vessel
neovascularization. In contrast, applying ten times that amount of
squalamine onto a 13-day old chick exerted no inhibitory
effect.
[0260] Thus, in a classical angiogenesis assay, squalamine
exhibited potent but specific inhibitory activity, equal in potency
to the most active compounds described to date in the literature.
The effect is compatible with suppression of neovascularization
rather than toxic inhibition of capillary growth.
[0261] The Vitelline Capillaries of 3-5 Day Chick Embryo Model:
[0262] In the course of evaluating squalamine in the "classical"
chick chorioallantoic membrane model, it was noted that this
steroid exerted a dramatic and rapid effect on capillary vessel
integrity in the three- to five-day old chick embryo. Using the
chick embryo vitelline capillaries assay, compounds were tested for
their ability to induce capillary regression. Each compound was
applied in 0.1 ml of 15% Ficol 400 and PBS onto the embryo, and
vascular regression was assessed after 60 minutes.
[0263] Squalamine was found to disrupt vitelline capillaries in 3-
to 5-day chick embryos. The 3-day chick embryo consists of an
embryonic disc from which numerous vessels emerge and return,
forming a "FIG. 8"-shaped structure--the embryo in the center with
vascular loops extending outward over both poles. Application of
squalamine onto the embryonic structure (0.1 ml in 15% Ficol in
PBS) resulted in progressive "beading up" of the vitelline vessels,
with the finest capillaries being the first to exhibit these
changes. Following a lag period of around 15 minutes, the
constriction of continuity between capillary and secondary vessels,
generally on the "venous" side, was observed. Continued pulsatile
blood flow progressed, resulting in a "swelling" of the blind tube,
followed by a pinching off of the remaining connection and
formation of an enclosed vascular sac resembling a "blood island."
This process progressed until only the largest vessels remained
intact. The embryonic heart continued to beat vigorously. No
hemorrhage was seen, reflecting the integrity of the capillary
structure. In addition, no obvious disruption of circulating red
cells was observed microscopically, demonstrating the absence of
hemolysis.
[0264] Utilizing this assay, which appears to demonstrate what is
commonly called capillary "regression," a minimum concentration of
squalamine required to observe an effect in 60 minutes can be
determined. Results are summarized in the table below.
TABLE-US-00007 TABLE 5 EFFECTS OF VARIOUS AMINOSTEROLS IN CHICK
EMBRYO VITELLINE CAPILLARY REGRESSION ASSAY Amount of Compound
Applied (.mu.g) Compound 10 1 0.1 0.01 0.001 Compound 1436 + + + +
+/- Compound 319 + + + + +/- squalamine + + + + 0 Compound 415 + +
+ 0 Compound 410 + +/- +/- 0 Compound 412 + 0 0 0 Compound 411 +/-
0 0 0 Compound 382 + + 0 0 0 Compound 1364 + + 0 0 0 Compound 371 +
+/- 0 0 0 Compound 396 + 0 0 0 Compound 353 +/- Compound 413 0
Compound 414 0 Compound 381 0 Compound 303 0 Compound 318 0
Compound 409 0 Compound 1360 0 Vehicle 0 0 0 0 0 Notes: + =
Vascular reactivity; 0 = No vascular reactivity; +/- = Equivocal
reactivity; Vehicle = 15% (w/w) Ficol in phosphate-buffered
saline.
[0265] As apparent from Table 5, 0.1-0.01 .mu.g of squalamine in
0.1 ml medium can induce changes. Compounds having various ranges
of activities were found, with squalamine, compound 319 and
compound 415 being especially active. This experiment demonstrates
that the steroids tested can dramatically restructure capillaries
over a time interval amounting to several minutes. The results
reflect that squalamine exerts this effect through inhibition of
NHE.
[0266] Tadpole Assay:
[0267] A newly developed assay employing tadpoles, preferably
Xenopus laevis Stages 59-60, were employed to study the effect of a
compound by monitoring capillary occlusion in the tadpole's tail.
Animals at these stages were used because they represent the period
of transition through metamorphosis at which time the animals
possess both embryonic and adult stage tissues. The compounds of
the invention affect the shape, viability and integrity of the
embryonic tissues while not affecting the adult tissues, providing
a powerful, highly specific screen. For example, substances that
destroy all of the animal's epithelium, both adult and embryonic,
could be regarded as toxic. Substances that destroy only the
embryonic tissues exhibit a very unique specificity.
[0268] In this assay, tadpoles are introduced into Petri dishes
containing a solution of the test compound in distilled water,
preferably about 100 ml. The preferred concentration of the test
compound is from about 1 .mu.g/ml to about 10 .mu.g/ml. The volume
of liquid is sufficient for the animal to swim freely and drink
from the solution. Thus, the effect observed results from oral
absorption and subsequent systemic distribution of the agent. If
the volume of liquid is not sufficient to permit oral intake, the
effects that are observed would result from absorption through the
surface epithelium. Thus, this simple assay can identify if an
compound has characteristics of oral availability.
[0269] In another embodiment of this assay, a solution of a
compound in water can be injected directly into the abdomen of the
animal using standard techniques. Concentrations of the compound
from about 0.05 mg/ml to about 0.5 mg/ml in about 0.05 ml of water
are preferred.
[0270] After an amount of time, typically about 60 minutes, the
occlusion of blood flow through capillaries in the tadpole's tail
are observed under an inverted microscope at a magnification of
roughly 100.times..
[0271] When the tadpoles were introduced into distilled water
containing squalamine at 10 .mu.g/ml, it was observed that blood
flow through the capillaries of the tail shut down. The process
occurred from the caudal to cranial direction. Blood flow within
the most distal vessels stopped initially, followed by the larger
vessels. During this period, it was observed that the
cardiovascular system was otherwise robust, as evidenced by a
continued heartbeat, pulsatile expansion of the great vessels, and,
most curiously, unaltered blood flow through the fine capillaries
of the hands and feet. Thus, selective cessation of blood flow was
seen in localized regions. If the animals are maintained in
squalamine for several days, enhanced regression of the most distal
aspects of the tail, as well as the peripheral aspects of the tail
fin are observed, corresponding to regions of the animal perfused
by the occluded vasculature.
[0272] This effect apparently results from selective change in the
resting diameter of the capillaries of the tail. Inhibition of the
endothelial cell NHE evidently leads to a change in shape of the
cell making up the capillary, resulting in diminished flow. The
continued functioning of capillary beds in the "adult" portions of
the tadpole (the limbs) indicates that squalamine is selective for
certain capillaries. From the results of the tadpole tail capillary
occlusion assay, compound 319, squalamine and compound 1436 were
found to induce a common vascular occlusive effect.
[0273] Suppression of Melanoma Growth:
[0274] Suppression of Growth of Melanomas in Mice by
[0275] Oral and Parenteral Routes of Administration:
[0276] The growth of B16 melanoma in C57B mice is dependent upon
neovascularization. Hence, this is a recognized model for
evaluating the impact of inhibitors of angiogenesis on the growth
of cancer.
[0277] Using the growth of B16 melanoma cells in C57B mice, a
recognized model for the evaluation of inhibitors of angiogenesis
on the growth of cancers, the effects of subcutaneous,
intraperitoneal and oral administration of squalamine were
evaluated. An inoculum of B16 melanoma cells was implanted
subcutaneously on the dorsum of the C57B mouse, which resulted in
the progressive growth of melanoma lesions over 30-40 days as shown
in FIGS. 4A-4C.
[0278] In this model, there was observed little evidence of
metastasis with or without treatment with chemotherapeutic agents.
When animals were treated with squalamine either subcutaneously
(FIG. 4A), intraperitoneally (FIG. 4B) or orally (FIG. 4C), a
dose-dependent suppression of tumor volume was observed.
Measurement of both body weight and hematologic parameters
demonstrated no significant depression. Since squalamine itself
shows minimal cytostatic activity against B16 in culture, except at
very high concentrations, this response of the tumor was
interpreted to be secondary to interference with capillary
development.
[0279] Suppression of Growth of Human
[0280] Melanoma in Immunocompromised Mice:
[0281] As apparent from FIG. 5, melanoma 1205Lu develops
aggressively in RAG-1 mice after implantation. Squalamine has been
found t6 suppress the growth of melanoma 1205Lu in RAG-1 mice in a
dose-dependent fashion.
[0282] Squalamine was administered after tumors had reached about
0.1 ml, and clear suppression of tumor growth in a dose-dependent
fashion was found as evidenced by FIG. 5. After cessation of
treatment, tumor growth continued at a rate similar to untreated
controls, suggesting that the impact of squalamine in this setting
is reversible.
[0283] Suppression of Tumor-Induced Corneal
[0284] Neovascularization in Rabbits:
[0285] The implantation of VX2 carcinoma into the rabbit cornea
results in the induction of new blood vessels within several days
(Tamargo et al., Cancer Research 51, 1991, 672-675). It is believed
that this carcinoma secretes growth factors that stimulate new
blood-vessel growth. Thus, this model is indicative in vivo
evidence of therapeutic utility in the treatment of pathological
disorders of vascularization, including the metastatic spread of
tumors, diabetic retinopathy, macular degeneration, and rheumatoid
arthritis.
[0286] This experiment followed the published protocol--tumor was
implanted adjacent to a polymer containing a concentration of the
agent to be evaluated. The polymer releases the agent slowly in the
immediate neighborhood of the tumor, providing sustained high local
concentrations of the agent. In this experiment, squalamine
introduced into a pellet of ELVAX 40 P (DuPont, Wilmington, Del.)
inhibited new blood vessel formation by about 60% at days 7 and 14,
and by about 25% at day 21.
[0287] As demonstrated by the experiments described above,
squalamine provides a potent inhibitor of NHE3. Squalamine
therefore should provide invaluable therapeutic intervention
wherever new blood vessel formation in vivo is implicated.
[0288] Indeed, any pathological processes dependent on new blood
vessel formation can be treated through inhibition of NHE3. As an
agent that interferes with the process of neovascularization,
squalamine has therapeutic utility in the treatment of diseases or
disorders dependent on continued neovascularization where
interruption of neovascularization diminishes the intensity of the
pathological process. Thus, squalamine has utility for treating
disorders including solid tumor growth and metastasis, rheumatoid
arthritis, psoriasis, diabetic retinopathy, macular degeneration,
neovascular glaucoma, papilloma, retrolental fibroplasia, and organ
rejection.
[0289] Moreover, other aminosterols have shown anti-angiogenic
activity. Compounds were subjected to a various assays, including
the chick embryo capillary regression assay, the tadpole assay, the
assay for inhibition of endothelial cord formation, and the assay
for direct inhibition of NHE, as described above, to determine
their utilities. As evident from the above data, correlation among
the results from the chick, tadpole and in vitro inhibition of
endothelial cell cord formation assays is excellent.
[0290] Through the application of these assays, compound 319
emerged as an attractive alternative to squalamine. In fact, it has
been found to be superior to squalamine in the following
characteristics: potency as an NHE inhibitor; simpler synthetic
route; specificity--i.e, no CNS effects. Further-properties of
compound 319 are discussed below.
[0291] Melanoma Growth Suppression:
[0292] Compound 319 has been found to exhibit activity against B16
melanoma in vivo. As seen in FIG. 6, which illustrates the results
from the murine melanoma assay described above, subcutaneous
administration of the compound achieved control of B16 in C57B mice
to an extent almost comparable to squalamine (FIG. 4B).
[0293] Pharmakokinetic Clearance:
[0294] Compound 319 also has a more rapid pharmacokinetic clearance
than squalamine. To assess clearance, a mouse IV PK study was
performed for compound 319 and squalamine. The compound was
administered i.v. and blood samples were taken every 10 minutes.
The concentration of the administered steroid was determined by
HPLC analysis. As shown in FIG. 7, after i.v. administration, the
compound was cleared from the blood stream of the mouse with a
half-life of about 14 minutes. In comparison, squalamine was
cleared with about a 35-minute half-life, as reflected by FIG.
8.
[0295] It should be possible to achieve further reductions in
clearance in vivo through derivatives of compound 319. It is
frequently of value to extend the lifetime of an agent in the
bloodstream, to achieve higher blood levels with a given dose of
drug and to reduce the frequency of administration. Polyamines are
readily metabolized by a variety of oxidases, which degrade the
free terminal amino group of the polyamine moiety. See Seller et
al., Prog. Drug. Res. 43, 1994, 88-126. Alkylation of the primary
amine generally retards this metabolic pathway. Seller et al., id.
Thus, through simple alkylation of the primary amine on compound
319 or on any of the steroids bearing a metabolizable polyamine,
straightforward modifications of this type would be expected to
extend biological lifetime.
[0296] Xenopus Tadpole Assay:
[0297] The tadpole assay described above provides an advantageous
way to determine the pharmacological targets of each steroid when
introduced into a mammal, and to determine pharmacological
categories into which synthetic compounds belong. In the assay,
each of the steroids was dissolved in 100 ml of distilled water at
a concentration of 10 .mu.g/ml. Stage 59-60 Xenopus tadpoles were
introduced and evaluated by light microscopy and gross observation
1 hour later.
[0298] The steroids tested were observed as producing different and
distinctive pharmacological responses in this animal: [0299]
Compound 1256 (Squalamine): Vascular occlusion in fine capillaries
of tail. No effect on vascular flow through hands or feet.
Inactivity and death occurred within 2 hours. [0300] FX1: Increased
passage of fecal material within 1 hour. By 12 hours, solution
contained considerable fecal debris. Circulatory system of animal
appeared hyperemic, suggestive of hematopathic stimulation. [0301]
Compound 1360: Swelling and lysis of certain erythrocytes occurred,
resulting in accumulation of nuclei within certain small vessels of
the tail. Subsequent tissue breakdown occurred in areas of tail
surrounding these nuclear plugs. [0302] Compound 1361: Similar to
compound 1360. [0303] Compound 1436: Gradual reduction in overall
activity.
[0304] Heart beat remained strong, suggesting nervous system
depression. Melanocytes over head and tail began to swell, first
exhibiting visibly distinct nuclei, followed by rupture into
fragments. Animal died by about 2 hours. [0305] Compound 1437:
Epithelium covering the embryonic portions of the animal, such as
the tail and antennae, began to slough off in sheets. Sheets of
cells remain intact initially, but gradually detach from one
another. Trypan Blue staining demonstrates that cell death
occurred. Animal was otherwise active, with little tissue breakdown
noted. [0306] FX 3: Muscular bundle within the tail began to leak
myoglobin. Striations of the skeletal muscle grew less distinct.
Segments of muscle began to separate.
[0307] Inhibition of Mitogen-Stimulated Growth of Human
T-cells:
[0308] Specific assays were used to identify steroids with a
particular biological activity, such as an assay for inhibition of
mitogen-stimulated growth of human T-cells. Mitogen-induced cell
proliferation has been reported to be dependent on the activation
of the NHE. Thus, to determine which steroids act on particular
cells, one need only determine which steroids inhibit mitogen--(or
growth factor)--activated cellular proliferation.
[0309] The T lymphocyte is the lymphoid cell which serves as the
host of HIV infection. A steroid that inhibits transformation of
human lymphocytes is, in principle, acting on an NHE probably
activated during HIV infection. Indeed, since GP120 activates
hippocampal cell NHE upon binding to its cellular receptor (Benos
et al., J. Biol. Chem. 269, 1954, 13811-13816), the assumption that
a similar event follows early viral interaction with the lymphocyte
is reasonable. This formed the basis of the next assay.
[0310] Human heparinized blood, freshly collected, was introduced
into tissue culture flasks containing 10 .mu.g/ml
phytohaemagglutinin (PHA) in RPMI medium with 10% FCS. Various
purified steroids were introduced subsequently at concentrations of
1, 5, and 10 .mu.g/ml. Cultures were incubated for 72 hours, after
which time colcemid was added to 1 .mu.g/ml. Cultures were
maintained for an additional 2 hours, and cells collected. Mitotic
figures were estimated using standard cytochemical techniques,
following Giemsa staining. Results are tabulated below.
TABLE-US-00008 TABLE 6 INHIBITION OF PHA-STIMULATED HUMAN
LYMPHOCYTES (% control) Compound 1 .mu.g/ml 5 .mu.g/ml 10 .mu.g/ml
1256 3 8 10 1360 5 5 5 1436 20 50 80 1437 10 FX 3 5
[0311] As seen from the above table, compound 1436 inhibited blast
transformation most potently, with greater than 75% inhibition
observed at 10 .mu.g/ml. Some effect was observed for squalamine at
this concentration, but the other steroids were considerably less
active. By using this simple assay, compound 1436 was identified
for use in the treatment of T-cell lympho-proliferative diseases,
including viral infections which actively propagate on these
cells.
[0312] Assays of similar design, employing a cell of interest and
an appropriate growth factor, can be readily constructed. Thus, to
determine which steroid might be most useful in inhibiting
proliferation of vascular smooth muscle cells following
angioplasty, one need only set up a culture with human coronary
smooth muscle and determine which steroid inhibits the
PDGF-stimulated growth of these cells, as discussed below.
[0313] The Inhibition of a Spectrum of Cells:
[0314] Using the assay of Baker et al., Cancer Res. 53, 1993,
3052-3057, compound 1436 was observed to inhibit the growth of a
very broad spectrum of cells. As set forth in Table 7 below, all
malignant tumors evaluated in tissue culture, endothelial cells and
vascular smooth muscle cells were sensitive to inhibition. Thus,
compound 1436 has application in the control of growth factor
dependent proliferation of many types of tissue. TABLE-US-00009
TABLE 7 CANCER CELLS INHIBITED IN VITRO BY COMPOUND 1436 (10
.mu.g/ml): Human colon carcinoma SW948 Human colon carcinoma HT29
Human ovarian carcinoma SKOV3 Human melanoma WM 1617 Lewis lung
carcinoma Murine B16 melanoma Murine L1210 leukemia NON-TRANSFORMED
CELLS INHIBITED IN VITRO BY COMPOUND 1436 (10 .mu.g/ml): Bovine
pulmonary endothelial cells Human microvascular endothelial cells
Human umbilical venous endothelial cells Human coronary artery
smooth muscle cells
[0315] Inhibition of NHE3:
[0316] Compound 1436 was also found to inhibit rabbit NHE3. PS120
fibroblasts transfected with rabbit NHE3 were grown and acid
preloaded by exposure to 40 mM NH.sub.4Cl as described above in
conjunction with FIGS. 1A and 1B. Internal cellular pH changes
expressed as the rate of pH recovery as a function of restored
extracellular sodium ion concentration following exposure to 10
.mu.g/ml of the compound were assayed with the fluorescent dye
BCECF-AM as described above. The results are presented in FIG.
10.
[0317] Thus, compound 1436 is an inhibitor of NHE3. The inhibition
of NHE3 caused by compound 1436, however, does not adequately
explain the very different pharmacologic effects of squalamine and
compound 1436 when assessed on cells in culture and several in vivo
models, as described above. This suggested that compound 1436 was
inhibiting another NHE in addition to NHE3. A reasonable candidate
NHE is NHE5 (recently cloned, see Klanke et al., Genomics 25, 1995,
615-622), which is expressed at least in lymphoid cells, brain, and
testes.
[0318] Inhibition of NEE5-Expressing Cells:
[0319] To determine whether NHE5 was the other NHE affected by
compound 1436, a test was performed to evaluate whether cells
inhibited by this compound expressed NHE5. Using the method of
Klanke et al. and appropriate PCR primers as described in Klanke et
al., it was found that NHE5 was expressed in all cell types which
exhibit sensitivity to compound 1436 (see table below). Total cDNA
was prepared from isolated total RNA or total RNA or polyA+ RNA
purchased from Clontech Laboratories (Palo Alto, Calif.). Initial
PCR cycle reactions were carried out as described in conjunction
with Table 2 above, with primers specific for human NHE1, human
NHE3 or human NHE5 with 80 ng cDNA or, in the case of polyA.sup.+
RNA as the cDNA source, with 1.5 ng cDNA. The annealing
temperatures were 57.degree. C. in all instances. Hemi-nested PCR
reactions were then carried out for human NHE1 and NHE5 and nested
PCR reactions for human NHE3 in second-cycle PCR reactions, with
the conditions as described above in conjunction with Table 2,
except that the annealing temperature for the second PCR round for
the primers to detect NHE5 was 65.degree. C. Results are tabulated
below. TABLE-US-00010 TABLE 8 Antiporter: NHE1 NHE3 NHE5 Assayed
cell line Rounds of PCR: or tissue 1 2 1 2 1 2 adrenal gland + + -
+ + + brain, whole + + +* + + + small intestine - + - + skeletal
muscle + + - -/+* - + HPAEC (endothelial) + + +* + + + HMVEC
(endothelial) + + - + + + CaCO.sub.2 (epithelial) + + +* + + +
melanoma (WM1617) + + +* + + + colon carcinoma + + +* + - -
(polyA.sup.+ RNA) leukemia HL-60 + + - + - + leukemia MOLT4 + + - +
- + (polyA.sup.+ RNA) astrocytoma + + + glioblastoma + + + Note: *=
multiple PCR bands observed.
[0320] It is believed that NHE5, which is similar in sequence to
NHE3, is the more effectively inhibited target of compound 1436.
Cells which exhibit both NHE3 and NHE5 would experience both NHE
isoforms shut down by compound 1436, but only NHE3 would be
inhibited in the presence of squalamine.
[0321] Inhibition of Mouse Leukemia:
[0322] Because of its inhibitory activity on the growth of numerous
cancer cells, compound 1436 was evaluated in a classical mouse
model of leukemia (Baker et al., Cancer Res. 53, 1993, 3052-3057).
C57B mice were inoculated with L1210 lymphoblastic leukemia cells
at an inoculum that causes leukemia in 100% of animals. Mice
received compound 1436 at 1, 5, 10 mg/kg every 3 days
intraperitoneally. As shown in FIG. 11, significant prolongation of
life was achieved with the highest dose of compound 1436.
[0323] Of particular interest is the hematological profile
determined during the course of treatment. Animals were treated
with cisplatin and compound 1436. As apparent from Table 9 below,
animals treated with cisplatin developed a profound anemia by day
28, due to a suppression of marrow erythroid precursors. In
contrast, animals treated with compound 1436 exhibited a near
normal hematocrit, with evidence of a robust granulocyte count.
TABLE-US-00011 TABLE 9 RBC WBC (10.sup.6/mm.sup.3)
(10.sup.3/mm.sup.3) Early Late Early Late Agent Treatment Time Time
Time Time Cisplatin Inoculate mice 9.4 1.5 8.1 18.1 5 .times.
10.sup.5 L1210 cells ip, d 1 inject cisplatin 8 mg/kg ip Cmpd. 1436
Inoculate mice 4.8 8.8 3.3 3.7 5 .times. 10.sup.5 L1210 cells ip,
inject compound 1436 10 mg/kg ip q4 d
[0324] Synergistic Inhibition of Tumor Growth:
[0325] Based on the idea that tumor growth involves both the clonal
expansion of a malignant cell along with the development of a
supporting vascular supply, a combination of compound 1436 with
squalamine was tested to determine whether it would achieve a
synergistic effect on solid tumor growth. This concept was
evaluated in the B16 melanoma model.
[0326] Animals were implanted with B16 melanoma followed by
treatment with compound 1436 or 1256 administered in a combined
schedule or separately. As apparent from FIG. 12, when squalamine
was administered at 5 mg/kg/day or compound 1436 was administered
at 10 mg/kg/every 3 days, no significant impact on tumor volume was
observed. In contrast, when both agents were administered together,
a significant reduction in tumor growth was noted. Neither
administration of squalamine at 15 mg/kg/day nor compound 1436
alone in a tolerable schedule could achieve this effect. Thus, a
combination of these two compounds achieves a therapeutic benefit
in tumors dependent on neovascularization that may prevent
metastatic spread.
[0327] Effect of Aminosterols on Lymphotropic Viruses:
[0328] Since compound 1436 inhibits the PHA-stimulated
proliferation of human T cells and controls the proliferation of a
lymphoblastic leukemia in mice without unfavorable toxicity as
shown above, it seemed a reasonable candidate for evaluation in
vitro as an inhibitor of HIV. PHA-stimulated lymphocytes were
inoculated with a clinical isolate of HIV at a multiplicity of
infection of 10. Fresh lymphocytes were obtained and stimulated
with PHA and IL-2. After 3 days, 1000 TCID of virus--(HIV clinical
isolate) were applied for 1 hour and there was a M.O.I. of 1:10.
The cells were washed and in a dose response fashion, and drug in
media was applied. After 3 days, the supernatant was exchanged with
1/2 volume of fresh media and 1/2 the volume of fresh drug. After 7
days, detergent was added, and HIV P-24 Antigen was determined by
Elisa. Viability of the lymphocytes was evaluated along with
appearance of the viral gene product p24. Results are tabulated
below. TABLE-US-00012 TABLE 10 INHIBITION OF HIV REPLICATION BY
COMPOUND 1436 Conc. .mu.M P-24 Elisa % Viability 0.5 40561 91 1
7464 -- 5 3426 -- 10 421 95 20 9 90.1
[0329] A seen above, at 10 .mu.g/ml compound 1436 effectively
inhibited antigen synthesis by 97%, while retaining lymphocyte
viability to 95%.
[0330] The above experiments clearly support the utility of
compound 1436 in the treatment of lymphotropic viral diseases.
Based on these studies, the identification of the specific NHE
inhibitors of specific cellular targets of specific virus should
permit the rational development of an effective antiviral therapy
for a given infectious agent. Thus, the NHE inhibitor from the
aminosterols that acts on the respiratory epithelial cell should be
effective in the treatment of respiratory viruses which propagate
on these cells, such as Herpes, influenza and RSV. The concept can
be generalized to viruses infecting the CNS (herpes) and liver
(hepatitis). The approach prevents infection by the virus of the
cellular target by preventing activation of the cellular NHE,
required for cellular proliferation and effective intracellular
viral multiplication.
[0331] Effect on Insulin Secretion:
[0332] In studying additional roles for the aminosterols of this
invention, it was noted that the release of insulin from the islet
cell of the pancreas requires activation of the islet cell's NHE,
ultimately activated through a mechanism triggered by glucose. It
is believed that overstimulation of the islet cell might play a
role in the depletion of islet cell function in Type II disease. In
addition, suggestions have been presented that genetic mechanisms
leading to hyperactivity of the islet cell NHE may play a role in
Type I disease.
[0333] Thus, the onset of diabetes in individuals genetically
susceptible, or placed into conditions of risk through acquired
processes (obesity), might be delayed or allayed if islet cell
function could be dampened. Inhibition of the NHE responsible for
secretion of insulin could provide therapeutic benefit in these
settings.
[0334] To study the effect of steroid administrator or the NHE
responsible for secretion of insulin, several of the aminosterols
from shark liver were administered to fasting mice. Male CD-1 mice
were assigned to one of four treatment groups. Whole blood glucose
was tested using glucometer (Lifescan Glucometer II and One Touch
test strips). Statistical analysis was via one-way analysis of
variance (ANOVA) followed by subsequent Bonferonni's t-test.
Results are tabulated below. TABLE-US-00013 TABLE 11 EFFECT OF
COMPOUND ON FASTING BLOOD GLUCOSE IN MICE Fasting Blood Total
Glucose Com- Dose Mean .+-. SEM Group n pound mg/kg Treatment
(mg/dl) 1 5 -- -- Overnight fast, 38 .+-. 5.2 blood obtained 2 4
1437 20 10 mg/kg i.v. 82 .+-. 15.3 (in Dy 0 PM, overnight H.sub.2O)
fast, 10 mg/kg iv. Dy 1 AM, blood obtained 30 min. after 2 d dose 3
4 1256 20 10 mg/kg i.v. 65 .+-. 7.3 (in Dy 0 PM, overnight
H.sub.2O) fast, 10 mg/kg iv. Dy 1 AM, blood obtained 30 min. after
2 d dose 4 3 1436 20 10 mg/kg i.v. 105 .+-. 8.0 (in Dy 0 PM,
overnight D5W) fast, 10 mg/kg iv. Dy 1 AM, blood obtained 30 min.
after 2 d dose
[0335] As apparent from the data above, blood glucose levels were
elevated between 2-3 fold normal after administration of these
steroids. The fasting blood glucose of Group 2 was significantly
elevated compared to Group 1 (p<0.05). The fasting blood glucose
of Group 4 was significantly elevated compared to Group 1. Thus, it
appears that the intravenous administration of compound 1436 in D5W
(5% glucose) or compound 1437 in water caused hyperglycemia in
mice. It is assumed that the observed hyperglycemic response
results from inhibition of insulin secretion, as suggested from
basic physiological principles. Thus, the long-term chronic
administration of a compound such as compound 1436 may be of value
in preventing or delaying the onset of both Type I and Type II
diabetes.
[0336] Effect on Growth of Arterial Smooth Muscle:
[0337] Aminosterols of the invention may also have utility in
inhibiting the growth factor mediated proliferation of smooth
muscle within the artery. Following coronary angioplasty,
reocclusion commonly occurs, secondary to reparative proliferation
of the vascular smooth muscle within the wall of the surgically
manipulated blood vessel. This process generally takes place over
the course of 7-10 days. To evaluate whether an agent could prevent
the growth factor mediated proliferation of smooth muscle within
the artery, compound 1436 was evaluated in vitro for its effect on
the proliferation of human coronary artery smooth muscle. Results
for compound 1436 are shown in FIG. 13, and those for squalamine
are shown in FIG. 14, with FIG. 15 being a composite logarithmic
plot.
[0338] As seen from FIG. 13, at about 10-12 .mu.g/ml compound 1436
was effective in suppressing growth of these cells. For example,
cells could be maintained in a quiescent state in the presence of
this steroid at about 11 .mu.g/ml without loss of viability. This
experiment suggests that, for several days following angioplasty,
local administration of compound 1436 to the site of angioplasty
via slow-release administration in a proximal vascular placement
could reduce muscle proliferation during the period over which the
vessel's endothelium reestablishes continuity and the cellular
events surrounding acute injury have subsided.
[0339] Effect on Growth and Weight Gain:
[0340] During evaluation of the physiological effects of compound
1436 in normal growing mice, it became evident that this steroid
suppresses both linear growth and weight gain in growing mice in a
dose-dependent fashion. Animals were dosed QTD (i.p.) starting on
Day 1. FIG. 16 shows that C57B mice treated with 10 mg/kg, QTD
i.p., 5 mg/kg QTD i.p., and 1 mg/kg QTD i.p. exhibited a dose
dependent reduction in growth. After 6 doses, growth of the animals
receiving 10 mg/kg QTD had been suppressed to a degree that growth
was almost completely inhibited over about 1 month from the
initiation of treatment. Animals receiving 5 mg/kg QTD experienced
about a 50% reduction in growth, compared to untreated controls,
while animals receiving 1 mg/kg QTD were affected by about 10%.
Striking was the apparent health of the treated animals--all were
active, normally proportioned, not-cachectic, and in excellent
apparent clinical health. They appeared very much like
hypophysectamized animals might appear.
[0341] Compound 1436 clearly inhibits the growth of many different
types of cell and tissue and this, to some extent, explains the
profound suppression of growth observed. However, the extraordinary
good health of these animals suggests that an additional mechanism
must be involved--one involving inhibition of pituitary function.
Compound 1436 is believed to partially inhibit secretion of
anterior pituitary hormones, resulting in the observed growth
suppression.
[0342] This property of compound 1436, regardless of its precise
mechanism, suggests that it can produce an unprecedented form of
antiproliferative effect when administered to an animal. It will
not only inhibit growth-factor induced cellular proliferation by
acting on the proliferating cell, but also inhibit growth-promoting
hormone secretion at a central, endocrine level. Thus, compound
1436 places the animal in a "growth-inhibited" state. In such a
state, malignant cells will not receive optimal exogeneous hormonal
stimulation from hormones such as growth hormone, and perhaps LH
and FSH. Secretion of hormones such as estrogen and progesterone,
as well as insulin, are likely to be dysregulated. Virally infected
cells will be placed under physiologically unfavorable conditions,
and the efficiency of viral infection should be dramatically
reduced. Immunologically foreign cells, suppressed in growth,
should be cleared by existing immune systems, now giving a chance
to catch up kinetically to these "foreign" cells.
[0343] Effect on Arterial Resistance:
[0344] Compound 1436 has also been found to reduce arterial
resistance in the rat after intravenous (i.v.) administration. A
200-g rat was catheterized in the right carotid artery, and the
compound was introduced over a ten-second period to a total dosage
of 5 mg/kg. Within 30 seconds, the mean arterial pressure had
fallen from about 100 mm Hg to about 70 mm Hg, with a marked
reduction in pulse pressure from about 40 mm to about 10 mm.
Despite the fall in blood pressure, no significant increase in
heart rate was observed. If cardiac output was basically
unaffected, reduction in blood pressure would have resulted
principally from a reduction in system resistance.
[0345] The effect was followed for 30 minutes, without significant
change. At that time, 40 .mu.g of noradrenaline was introduced. An
almost immediate increase in blood pressure was observed, with an
associated increase in pulse pressure. This data demonstrate that
the effect of compound 1436 is readily reversible by standard
pharmacological practice.
[0346] The ability of compound 1436 to reduce arterial resistance
and arterial blood pressure indicates its application as an
antihypertensive agent. Because it does not appear to induce a
compensatory tachycardia, the net effect is to reduce cardiac
afterload. A physiological consequence of this type of
cardiovascular effect would be to slow the process of cardiac
hypertroph and arteriolar smooth muscle proliferation. Because of
these properties, compound 1436 should be an effective treatment of
congestive heart failure, where reduction in afterload would be
desired. Its rapidity of action and ready reversibility, along with
minimal tachycardic effect, make the compound a valuable
therapeutic agent.
[0347] Thus, compound 1436 represents an antiproliferative and
therapeutic agent with previously unknown and valuable properties
and utilities. It clearly can be utilized in disorders where
suppression of growth of specific tissues or entire organ systems
is desired.
[0348] Suppression of Cardiotoxic Effects of Ischemia:
[0349] It has been suggested that inhibitors of the NHE family
could play a therapeutic role in the treatment of cardiac ischemic
states. These states occur after heart attacks, during heart
failure, and in the course of transplantation of an organ from
donor to recipient.
[0350] To determine if compound 1436 has such utility, the
following experiment was performed. The heart of a juvenile Xenopus
laevis frog was dissected from the living animal. The heart was
placed into a Petri dish containing Krebs-Ringer buffer with
adrenaline 50 .mu.g/ml, and examined with the naked eye. At room
temperature, the heart continued beating in a coordinated fashion
(atrial beat followed by ventricular beat) for about one hour. In
the presence of 10 .mu.g/ml of compound 1436, spontaneous beating
persisted up to 24 hours. The atrial pacemaker and the conduction
of the atrial beat to the ventricle remained vigorous over this
period.
[0351] Although the precise mechanism explaining this phenomenon of
persistence of cardiac activity ex vivo is not fully understood, it
is believed that compound 1436, by inhibiting NHE3 and NHE5,
prevents accumulation of intracardiac calcium by blocking these
NHEs. It is the current understanding in the art that intracellular
acid accumulating during ischemia is exchanged by the NHE for
extracellular sodium. In turn, the sodium driven into the cell is
subsequently excreted in exchange for extracellular calcium through
the action of a sodium/calcium exchanger. It is the calcium
entering via this route that leads to cardiac death and cardiac
arrhythmia. By blocking the NHE, compound 1436 prevents protons or
acid from leaving the cardiac cell, reducing energy consumption and
work output, effects which are protective to the cell, along with
preventing the ultimate entry of damaging calcium.
[0352] Anti-Proliferative Assays and Tumor Growth
[0353] Suppression Assays as characterizing Assays:
[0354] As above, the tadpole assay was used to screen for
additional compounds. In the presence of 10 .mu.g/ml of compound
1436, the stage 59-60 Xenopus tadpole experiences dramatic
disruption of melanocytes over its head, trunk, and tail, along
with depression of its nervous system. No effects are observed on
epithelial cell integrity, vascular flow, erythrocyte volume,
tissue integrity, muscle fiber striation, or GI tract activity.
[0355] Using the tadpole assay to screen for functionally similar
compounds, compounds 353, 371 and 413 were found to produce effects
like those produced by compound 1436. Of these, compound 353 is
especially preferred because of its ease of synthesis as described
above. This compound was also found to exhibit other advantageous
properties.
[0356] Using the growth suppression methods set forth above, it was
determined that compound 353 exhibits potent activity against
melanoma and a number of cancer cells as set forth below:
TABLE-US-00014 TABLE 12 CYTOTOXIC ACTIVITY OF COMPOUND 353 AGAINST
CANCER CELLS Cell IC.sub.50 (.mu.g/ml) Human melanoma WM 1167 3.0
Lewis Lung carcinoma 1.9
[0357] In addition, using the method of Baker et al., Cancer Res.
53, 1994, 3052-3057, it was observed that the effect of compound
353 on the growth of melanoma is most pronounced over 48 hours,
with less of an effect noted within the first 12 hours of
incubation, as shown in FIG. 17A. For comparative purposes, the
effect of squalamine in human melanoma is shown in FIG. 17B.
[0358] Analysis of the cells exposed to compound 353 reveals that
apoptotic cell death has been induced. Thus, this aminosterol
exhibits the same highly selective mechanism of killing as does
compound 1436.
[0359] Although as set forth above, compound 1436 exhibits
melanocyte disruptive activity in the tadpole, it causes vitelline
capillary regression with about the same potency as squalamine. In
contrast, compound 353 exhibits no effect on the chick embryo
capillary bed. Thus, it appears that compound 353 inhibits NHE5 to
a greater extent than NHE3, with even greater selectivity than
compound 1436. Compound 353 demonstrates that it is possible to
create aminosterols that exhibit greater specificity than naturally
occurring molecules.
[0360] Compound 1437 (Fraction 4) contains an unusual
ergosterol-like side chain. This molecule can be distinguished
readily from all other steroids extracted from shark on the basis
of its dramatic effect on the embryonic epithelium covering the
tadpole tale.
[0361] Using the tadpole assay described above, within 60 minutes
of exposure to this steroid at 10 .mu.g/ml, the larval skin was
observed to shed off in a sheet. The speeded appearance of the
process suggests that an NHE expressed by this epithelial tissue is
the target. Since NHE activity and cell membrane proteins involved
in adhesion cross-communicate (Schwartz et al., Proc. Nat'l. Acad.
Sci. 888, 7849-7853), it is proposed that inhibition of NHE on the
epithelium results in disruption of cellular contacts between the
epithelium and its substratum, leading to a shedding effect.
[0362] Using the assay described above, the anticancer effects of
compound 1437 against several cancer lines was assessed. Compound
1437 was found to exhibit anticancer activity against the human
ovarian carcinoma, SKOV3. Thus, compound 1437 should find use in
the treatment of carcinomas exhibiting a sensitive phenotype.
[0363] As the study above demonstrates, compound 1437 targets a
"mesothelium-like" epithelial layer, a skin layer that is comprised
of only one cellular layer. Such a layer resembles epithelial
surfaces such as the human peritoneum, synovium, pericardium and
ependyma. Accordingly, compound 1437 should exhibit
antiproliferative effects on these tissues and malignancies which
derive from them. In addition, these tissues can support viral
infections, and therefore in these instances the compound should
provide therapeutic antiviral benefit.
[0364] By use of the Xenopus tadpole assay, it is possible to
identify compounds that exhibit little chemical resemblance to
compound 1437, but produce the same pharmacological effect with
respect to epithelial shedding. Using such a method, it was found
that compounds 409, 410, 416, 431, 432 and 433 are functionally
similar to compound 1437.
[0365] Steroid 1360 (Fraction 2) contains a side chain bearing a
keto group on carbon 24 and a sulfate on the C.sub.27 hydroxyl.
Although somewhat similar in structure to squalamine, it exhibits a
dramatically different pharmacologic profile in both the tadpole
and chick embryo assays.
[0366] When stage 59-60 tadpoles were introduced into a 10 .mu.g/ml
solution of compound 1360, extensive vasocclusion occurred within
60 minutes throughout the tail--the distal portions to a greater
extent than the proximal. Occlusion occurred due to the visible
swelling of erythrocytes followed by rupture and release of nuclei.
Nuclei were shuttled by the design of the vascular bed into distal
arteries and veins, which can be analogized to a coin separating
machine separating coins of different size and weight into specific
collecting tubes. As the nuclei pooled within these vessels, blood
flow stopped proximally. Within 20-30 minutes after nuclear plugs
formed, tissue surrounding these plugs began to break down. It
appeared as if the nuclei were releasing hydrolytic enzymes that
were essentially dissolving the ground substance holding these
tissues.
[0367] In the chick embryo assay, application of compound 1360
produced a different effect than seen in the tadpole. Within 20
minutes, the blood circulating through the embryonic vessels
exhibited a brighter red color, reflecting a higher degree of
oxygenation than the red cells not exposed to the compound.
Although many numerous mechanisms might explain this effect, it is
believed that the red cell of the chick is experiencing a more
alkaline internal pH after exposure to compound 1360. This could
arise through activation of the NHE of this cell. Furthermore,
activation of the exchanger would also cause cellular swelling--a
phenomenon observed in tadpole.
[0368] It is known that the nucleated erythrocytes of amphibia and
fish (and probably birds) express a specific NHE, termed NHE1-beta.
Unlike all others characterized, this exchanger is activated by
cAMP and is influenced by the state of hemoglobin oxygenation. This
data suggest that compound 1360 will be shown to activate this
exchanger. Furthermore, the chemical structure of the compound
makes it ideally suited to function in the suggested fashion. It
has been discovered that, under slightly alkaline conditions,
compound 1360 undergoes a dehydration of the 27 hydroxyl, loss of
sulfate, and generation of the corresponding 27-ene, as set forth
in the scheme below: ##STR37##
[0369] Thus, as the alkalinity of the interior of a cell increases,
the lifetime of compound 1360 decreases, thereby providing an
extraordinary form of "feedback." It is possible that compound
1361, the product of this hydrolytic conversion, is inhibitory to
the same NHE.
[0370] The data demonstrate that compound 1360 clearly interacts
with a NHE present on embryonic stage blood cells. Since the human
fetus generates nucleated red cells comparable in size to those of
the birds, fish, and amphibia, it is thought that certain human
blood cells, perhaps fetal, will also represent cellular targets of
this compound. Activation of the fetal NHE might find use in
strategies designed to protect the fetus from hypoxic damage.
[0371] The full scope of applications for compound 1360 awaits
description of the distribution of the erythrocyte NHE isoform in
man. However, it appears to be stimulatory activity in some
settings, rather than an inhibitor of an NHE. In any event,
compound 1360 could be used for the following: antibacterial,
antifungal, antiviral, etc.; fetal distress treatment; and
hematologic malignancies treatment.
[0372] Although the chemical structure of Fraction 3 (FX 3) is yet
to be fully determined, from its thin layer chromatographic
properties it has a spermine associated with the steroid, much like
compound 1436. This steroid has a profound effect on the embryonic
skeletal muscles of the tadpole.
[0373] In the tadpole assay, within 1 hour after exposure, leakage
of brown pigment from the tail muscle bundles of stage 59-60
tadpole was observed. Cross striations became fuzzy and obscure.
Heartbeat and other functions, including muscle activity in the
limbs, were unaffected. These observations suggest that FX 3 is
targeting primitive mesenchyme, including muscle.
[0374] If the observations of the tadpole extend to man, then FX 3
should profoundly affect the proliferation of certain mesenchymal
cells. Thus, it would have use in the treatment of a variety of
cancers of mesenchymal origin, such as cancers of striated muscle,
cartilage, fibroblastic tissues, bone, and fatty tissue.
[0375] In addition, if proliferation of fibroblasts is affected,
then FX 3 would have application in the control of fibroblastic
proliferation in settings where this process is unwanted. Thus,
scarring after CNS injury might be prevented. Unwanted scarring
after surgery at sites complicated by fibrosis would be serious
therapeutic targets. Generalized conditions of fibroblastic
proliferation, such as seen in heart, kidney and liver disease,
might be allayed.
[0376] If proliferation of muscle is inhibited, FX 3 could find use
in the inhibition of hyperplastic diseases of muscle, such as in
certain forms of cardiac disease.
[0377] Through use of the Xenopus tadpole assay, several
aminosterols have been identified as exhibiting pharmacologic
activity similar to that seen for FX 3. These compounds include
compounds 370, 412, 458, 459, 465 and 466. These compounds in
general share the spermine moiety. They are simpler chemically than
squalamine and offer a less expensive route to drug design than the
naturally occurring steroids.
[0378] The structure of the Fraction 1 (FX 1A) steroid is shown
above. It appears to undergo conversion to another molecule (FX 1B)
rapidly in water. FX 1A exerts a distinctive pharmacologic effect
on the Xenopus tadpole using the assay set forth above.
[0379] Within several hours of introduction of this steroid into
the water surrounding the tadpole, fecal elimination is
dramatically increased. Since the GI tract of a number of
vertebrates utilizes NHE in the control of gut fluid secretion, it
is believed that Fraction 1 acts on such an NHE. The increase in
fecal material could correspond to "diarrhea," a condition which
occurs in man when the colonic NHE is inhibited. Since this steroid
has little effect on overall activity, muscle integrity or
viability of any visible tissue, it might serve a physiological
function such as regulation of sodium-water exchange.
[0380] Although the uses will be clearer after the steroid and
target are better characterized, the tadpole data suggest that
Fraction 1 will find use in the modulation of sodium/proton
exchange in certain physiological derangements. These include
treatment of hypertension, cystic fibrosis and constipation.
[0381] Because of its effects on bowel fluid dynamics, this agent
may be as a antimicrobial--one which would effect killing of
susceptible bacteria, parasites, fungi, etc., while promoting the
discharge of the infectious load from the gut. Fraction 1 may also
find use as an effective antibacterial, antiparasitic or antifungal
agent.
[0382] Through the use of the Xenopus tadpole assay, aminosterols
with pharmacologic activity similar to Fraction 1 have been
identified. Surprisingly, these compounds have been found to
include compound 1363 and compound 414. Although these compounds
exhibit potencies comparable to that of Fraction 1, they have
chemically simpler structures.
[0383] Preliminary data has revealed the presence of a least two
hydrophilic steroids eluting slightly ahead of Fraction 1 on the
reversed-phase separation depicted in FIG. 9. The structures of
these steroids are presented below. ##STR38##
[0384] FX1C and FX1D are seen to be steroids with a single sulfate
like sclualamine and an additional hydroxyl, resembling compound
1437.
[0385] Additional Aminosterol Structures:
[0386] From the diverse aminosteroids isolated from Squalus
acanthias, it is possible to predict the existence of related
aminosterols not as yet isolated from this animal's tissues. These
sterols can be deduced to exist in vertebrate tissues based on the
structures determined to date and the-known biochemical
transformations the cholesterol side chain can undergo (Tammar,
"Bile Salts in Fishes," Chemical Zoology, (eds. Florkin et al.),
Academic Press, 1974, 595-612).
[0387] Thus, based on the existence of squalamine, which bears a 24
sulfated hydroxyl, one should be able to find other derivatives
with the squalamine steroidal nucleus and aminosterol portion, but
differing in the position of the sulfated hydroxyl on the side
chain as shown below. Since hydroxylation can occur on carbons 25,
26 or 27, and since each would represent a stereospecific chemical
entity, it is reasonable to expect their existence in nature and to
assume they would exhibit distinct pharmacological properties.
[0388] Similarly, the existence of steroids bearing a single
sulfate along with a second hydroxyl in the cholesterol side chain
suggests potential diversity in the pattern of side-chain sulfation
and single-site hydroxylation. Thus, aminosterols likely exist in
nature where sulfation is found on carbons 24, 25, 26 or 27. In
turn, each of these four sulfated aminosterols can be hydroxylated
at available carbons 24, 25, 26 and 27. At least the following
steroids may be isolated from natural products, based on inductive
logic and the data revealed herein: ##STR39## ##STR40##
[0389] Structure-Activity Considerations for NHE Inhibitors:
[0390] Based on the information given above, key structural
elements of the aminosterol inhibitors of the sodium/proton
exchangers can now be deduced. The key core structure contains a
steroid nucleus and a distinctive side chain. An aminosterol
portion specifies interaction of the molecule with a NHE. The side
chain, bearing free or sulfated hydroxyl groups, determines the
specificity for a specific NHE isoform. In addition, the presence
of spermine or spermidine attached to the steroid extends the
spectrum of activity. Based on this generalization it can be
readily seen that the structure of the side chain imparts great
specificity.
[0391] Thus, other synthetic steroidal NHE inhibitors can be
designed with great pharmacological specificity by considering the
modular nature of the molecule. Chemical entities which mimic the
aminosterol in shape and molecular surface characteristics will
interact with the NHE family. Such chemical mimics of the steroidal
nuclei are known and widely used in the synthesis of non-steroidal
estrogen agonists and antagonists. Coupling of specific cholesterol
side chains to these steroidomimetic structures will in turn
establish specificity for individual NHE isoforms.
[0392] Antimicrobial Activity:
[0393] The aminosterol NHE inhibitors represent a class of
antibiotics based on mechanism of action. Because these agents also
interact with specific NHE isoforms in human tissues, prudent
selection of an antibiotic of this class can eliminate undesirable
side effects, due to host NHE inhibition, or potentiate the
therapeutic intent. Thus, use of an agent like compound 1436 would
suppress lymphoid proliferation during active treatment of an
infection. Oral administration of Fraction 1 may increase bowel
fluid transit as it kills parasitic targets. An effective
antifungal agent can be designed further to increase specificity
for its pathogenic target over sensitive vertebrate isoforms.
[0394] As seen in Table I at the end of this specification, the
antibacterial/antifungal spectrum differs from compound to
compound. Thus, it is possible to achieve an antimicrobial steroid
with or without squalamine-like pharmacological activity.
[0395] As set forth in Table II, which follows Table I, the
activities of natural and synthetic aminosterols in the different
assays vary. In light of the foregoing, it is now possible to
screen for steroids with or without squalamine-like pharmacological
activity.
[0396] Selection of NHE Isoform:
[0397] Through the use of molecular biological techniques, it is
possible to determine which NHE isoforms are expressed in specific
cells, such as malignancies. Human melanoma expresses NHE1, NHE3
and NHE5, and human adenocarcinoma expresses principally NHE3 (see
Table 8).
[0398] Thus, treatment of this type of adenocarcinoma might most
effectively be accomplished with the use of a more specific NHE3
inhibitor, such as squalamine or compound 319. In contrast,
melanoma expresses considerable amounts of NHE5 along with NHE3.
Hence, treatment of this malignancy should include an inhibitor of
both NHE3 and NHE5, such as compound 1436, alone or in combination
with squalamine.
[0399] In summary, the invention allows for the utilities of the
aminosterol NHE inhibitors to be established through diagnostic
evaluation of the NHE isoforms expressed in the target tissues.
Diagnostic approaches can include immunological detection of the
specific NHE isoform protein or a molecular biological procedure
such as PCR, utilizing specific sequence information, and standard
techniques.
[0400] Thus, other embodiments of the invention will be apparent to
those skilled in the art from consideration of the specification
and practice of the invention. The embodiments and preferred
features described above should be considered as exemplary, with
the invention being defined by the appended claims. TABLE-US-00015
TABLE I ANTIBIOTIC ACTIVITY OF NATURAL AND SYNTHETIC AMINOSTEROLS
MIC Values (.mu.g/mL) S. aureus E. coli ##STR41## 4 >256
##STR42## 16 >256 ##STR43## 4-16 32 ##STR44## 8-16 64 ##STR45##
8 128-256 ##STR46## 0.5-1 2-4 ##STR47## 2-4 128 ##STR48## 64 32-64
##STR49## 128 32 ##STR50## 16 128 ##STR51## 8 64-128 ##STR52##
16-32 128 ##STR53## 2-4 4-8 ##STR54## 4-8 32 ##STR55## 32 64
##STR56## 16 64 ##STR57## 2 >256 ##STR58## 4 64 ##STR59## 4 32
##STR60## 2-4 64 ##STR61## 16 32-64 ##STR62## 4 32-64 ##STR63## 16
64 ##STR64## 64 256 ##STR65## 4 32-64 ##STR66## 8 128 ##STR67## 4
32 ##STR68## 2 >256 ##STR69## 2 >256 ##STR70## 4 32 ##STR71##
4 64 ##STR72## 32 64 ##STR73## 16 16 ##STR74## 8 64 ##STR75## 8 64
##STR76## 8 256 ##STR77## 32 64 ##STR78## 128 128 ##STR79## 8 8
##STR80## 16-32 64 ##STR81## 16 32-64 ##STR82## 1 8-16 ##STR83##
>256 >256 ##STR84## ##STR85## ##STR86## 2 16 ##STR87## 4 8-16
##STR88## 4 32 ##STR89## 1-2 32 ##STR90## 2-4 32 ##STR91## 2 32
##STR92## 16 16 MIC Values (.mu.g/mL) C. P. aerug albicans
##STR93## >256 64 ##STR94## >256 >256 ##STR95## 128 64
##STR96## 128 64 ##STR97## 128 256 ##STR98## 16 8 ##STR99## 128 128
##STR100## 32 8 ##STR101## 64 >256 ##STR102## 64 32 ##STR103##
64 16-32 ##STR104## 256 64 ##STR105## 16 16 ##STR106## 64 32
##STR107## 32 128 ##STR108## 32 32 ##STR109## >256 32 ##STR110##
64 32 ##STR111## 64 64 ##STR112## 128 16 ##STR113## 16 32
##STR114## 256 64 ##STR115## 128 128 ##STR116## >256 256
##STR117## 64 64 ##STR118## 128 32 ##STR119## 128 4 ##STR120##
>256 2 ##STR121## >256 2 ##STR122## 64 2 ##STR123## 64 2
##STR124## 128 16 ##STR125## 32 16 ##STR126## 64 2 ##STR127## 8 8
##STR128## 64 32 ##STR129## 64 64 ##STR130## 256 256 ##STR131##
16-32 32 ##STR132## 128 32 ##STR133## 128 8 ##STR134## 64 2-4
##STR135## 128 >256 ##STR136## ##STR137## ##STR138## 8 8
##STR139## 4 4 ##STR140## 64 2 ##STR141## 64 2 ##STR142## 128 4
##STR143## 32 2 ##STR144## 8 4
[0401] TABLE-US-00016 TABLE II ACTIVITY OF NATURAL AND SYNTHETIC
AMINOSTEROLS IN CHICK EMBRYO AND TADPOLE ASSAYS Minimum Effective
Concentration (.mu.g/mL) Chick embryo diss. Tadpole assay (10
.mu.g/mL) Structure (.mu.g) V M E ##STR145## >10 - ##STR146##
>10 - - - ##STR147## 0.001 + - - ##STR148## >1 - + -
##STR149## >10 - - - ##STR150## + - - ##STR151## + - -
##STR152## >10 - - - ##STR153## 1-10 - + - ##STR154## 1 + - .+-.
##STR155## 10 - - - ##STR156## 1 - - - ##STR157## >10 - - -
##STR158## >1 - - + ##STR159## 0.01 .+-. - + ##STR160## >10 -
- - ##STR161## 0.01 - - - ##STR162## >10 - .+-. - ##STR163##
>10 - ##STR164## >10 - - - ##STR165## >1 - - .+-.
##STR166## - - .+-. ##STR167## 1 - - .+-. ##STR168## - - -
##STR169## >1 .+-. - + ##STR170## >1 - - + ##STR171## 1 - - +
##STR172## 1 .+-. - + ##STR173## >1 - - .+-. ##STR174## >1 -
- .+-. ##STR175## >1 .+-. - - ##STR176## 1 - - - ##STR177##
>1 - + - ##STR178## >1 - - - ##STR179## >1 - - -
##STR180## >1 .+-. - + ##STR181## >1 - - - ##STR182## 0.01 +
- - ##STR183## >10 - - + ##STR184## - - - ##STR185## 1 + + .+-.
##STR186## >1 - - + Minimum Effective Concentration (.mu.g/mL)
Tadpole (10 .mu.g/mL) Structure TB GI Mus Tpx ##STR187## ##STR188##
- - - - ##STR189## - - - - ##STR190## - - - + ##STR191## - - - -
##STR192## - - - .+-. ##STR193## - - - + ##STR194## - - + +
##STR195## - - - + ##STR196## - - - + ##STR197## - - - + ##STR198##
- - - + ##STR199## + - - - ##STR200## + - - + ##STR201## + - - +
##STR202## - - - + ##STR203## - - - + ##STR204## - - - + ##STR205##
##STR206## - - - + ##STR207## - - + + ##STR208## - - + + ##STR209##
- - + + ##STR210## - - - + ##STR211## + - - + ##STR212## - - - +
##STR213## - .+-. - - ##STR214## + - - + ##STR215## .+-. - - +
##STR216## - - + + ##STR217## - - - + ##STR218## - - + + ##STR219##
- - - + ##STR220## - + - - ##STR221## - - - - ##STR222## + - - +
##STR223## - - - - ##STR224## - - - + ##STR225## + - - - ##STR226##
- + - - ##STR227## - - - + ##STR228## - - - - Minimum Effective
Concentration (.mu.g/mL) HM Cord MTT Formation assay Structure
(.mu.g/mL) (.mu.g/mL) ##STR229## ##STR230## ##STR231## 13.8
##STR232## 10 3.0 ##STR233## ##STR234## ##STR235## ##STR236## 4.0
##STR237## ##STR238## ##STR239## ##STR240## ##STR241## ##STR242##
##STR243## 10 2.6 ##STR244## ##STR245## 58.6 ##STR246## ##STR247##
##STR248## ##STR249## 5.0 ##STR250## ##STR251## ##STR252##
##STR253## ##STR254## ##STR255##
##STR256## ##STR257## ##STR258## 6.8 ##STR259## 10 ##STR260##
>10 18.1 ##STR261## >10 ##STR262## ##STR263## >10
##STR264## 2.4 ##STR265## ##STR266## 0.01-0.1 7.8 ##STR267##
##STR268## ##STR269## 6.9 ##STR270## Mini- mum Eff- ective Con-
cen- tration (.mu.g/mL) LLC MTT Structure (.mu.g/mL) ##STR271##
##STR272## ##STR273## ##STR274## 1.9 ##STR275## ##STR276##
##STR277## ##STR278## ##STR279## ##STR280## ##STR281## ##STR282##
##STR283## ##STR284## ##STR285## ##STR286## ##STR287## ##STR288##
##STR289## ##STR290## ##STR291## ##STR292## ##STR293## ##STR294##
##STR295## ##STR296## ##STR297## ##STR298## ##STR299## ##STR300##
##STR301## ##STR302## ##STR303## ##STR304## ##STR305## ##STR306##
##STR307## ##STR308## 13.2 ##STR309## ##STR310## ##STR311## 16.7
##STR312## V = vascular M = menocytes E =epirea TB = GI =
gastrointestinal Mus = muscle Tox = ethanty at
* * * * *