U.S. patent application number 14/796301 was filed with the patent office on 2015-11-05 for new steroids having increased water solubility and resistance against metabolism and methods for their production.
This patent application is currently assigned to Umecrine AB. The applicant listed for this patent is Umecrine AB. Invention is credited to Torbjorn Backstrom, Gianna Ragagnin.
Application Number | 20150315231 14/796301 |
Document ID | / |
Family ID | 39429970 |
Filed Date | 2015-11-05 |
United States Patent
Application |
20150315231 |
Kind Code |
A1 |
Backstrom; Torbjorn ; et
al. |
November 5, 2015 |
New Steroids Having Increased Water Solubility and Resistance
Against Metabolism and Methods For Their Production
Abstract
Steroid compounds having increased resistance against metabolism
and increased water solubility are disclosed, together with methods
for their production. These substances are suitable for the
manufacture of pharmaceuticals for the treatment of steroid related
or steroid induced CNS disorders and for use in methods of
prevention, alleviation or treatment of such disorders.
Inventors: |
Backstrom; Torbjorn; (Umea,
SE) ; Ragagnin; Gianna; (Umea, SE) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Umecrine AB |
Umea |
|
SE |
|
|
Assignee: |
Umecrine AB
|
Family ID: |
39429970 |
Appl. No.: |
14/796301 |
Filed: |
July 10, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13788074 |
Mar 7, 2013 |
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14796301 |
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11943555 |
Nov 20, 2007 |
8580983 |
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13788074 |
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60860658 |
Nov 21, 2006 |
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Current U.S.
Class: |
514/182 ;
552/603 |
Current CPC
Class: |
A61P 25/00 20180101;
C07J 1/0011 20130101; C07J 41/0016 20130101; C07J 7/0065 20130101;
C07J 7/00 20130101; C07J 7/007 20130101; A61P 3/02 20180101; A61P
19/08 20180101; A61P 15/00 20180101; A61P 25/02 20180101; A61P
21/00 20180101; C07J 5/0015 20130101; A61P 25/14 20180101; C07J
7/009 20130101; A61P 25/26 20180101; A61P 27/02 20180101; A61P
25/28 20180101; A61P 27/00 20180101; A61P 25/24 20180101; C07J
7/002 20130101; A61P 5/24 20180101; C07J 41/005 20130101; A61P
25/22 20180101; A61P 37/08 20180101; A61P 19/00 20180101; A61P 5/00
20180101; C07J 41/00 20130101; A61P 43/00 20180101; A61P 25/08
20180101; C07J 1/00 20130101; A61P 25/20 20180101; A61P 25/10
20180101; A61P 27/16 20180101; A61P 3/04 20180101 |
International
Class: |
C07J 7/00 20060101
C07J007/00 |
Claims
1. The compound 3.alpha.-ethynyl,
5.alpha.-pregnan-3.beta.,20(R)-diol, or a pharmaceutically
acceptable salt thereof.
2-14. (canceled)
15. A pharmaceutical composition comprising a pharmaceutically
effective amount of the compound or a salt thereof according to
claim 1 and a pharmaceutically acceptable carrier.
16-25. (canceled)
Description
RELATED APPLICATIONS
[0001] This application claims priority to U.S. Provisional
Application Ser. No. 60/860,658 filed Nov. 21, 2006, which is
hereby incorporated by reference in its entirety.
FIELD OF THE INVENTION
[0002] The present invention concerns novel steroid compounds that
act on the gamma-aminobutyric acid receptor-chloride ionophore
(GABA.sub.A-R) complex, and which can be used in the treatment of
GABA and GABA-steroid related and/or steroid induced disorders of
the central nervous system (CNS).
BACKGROUND OF THE INVENTION
[0003] The metabolites of progesterone, desoxycorticosterone,
testosterone, androstenedione cortisone and cortisol known as
androstanolones and pregnanolones have been the subject of various
studies, at least partially elucidating their role in the
neurological signal system in mammals. The nomenclature differs in
the field and therefore the IUPAC nomenclature will be used
throughout this application. The steroids inducing CNS symptoms and
disorders of interest in the present application all share a common
feature in comprising a 3.alpha.-hydroxy group, a 5.alpha. or
5.beta. pregnane steroid body, and a ketone on position 20.
Examples of such steroids are given in table 1:
TABLE-US-00001 TABLE 1 Nomenclature of the pregnanolone group IUPAC
- nomenclature CAS Number 3.alpha.-hydroxy-5.alpha.-pregnan-20-one
516-54-1 3.alpha.-hydroxy-5.beta.-pregnan-20-one 128-20-1
3.alpha.,21-dihydroxy-5.alpha.-pregnan-20-one 567-02-2
3.alpha.,21-dihydroxy-5.beta.-pregnan-20-one 567-03-3
3.alpha.,11.beta.,17.alpha.,21-tetrahydroxy-5.beta.-pregnan-20-one
53-02-1
3.alpha.-11.beta.,17.alpha.,21-tetrahydroxy-5.alpha.-pregnan-20-one
302-91-0
3.alpha.-17.alpha.,21-trihydroxy-5.alpha.-pregnan-11,20-dione
547-77-3
3.alpha.-17.alpha.,21-trihydroxy-5.beta.-pregnan-11,20-dione
53-05-4
[0004] To the best knowledge of the inventors, all compounds
described as novel in the description and examples are previously
non-disclosed. Other steroids for the treatment of CNS disorders
have however been disclosed, for example in the following
documents:
[0005] U.S. Pat. No. 5,232,917 (Bolger et al.) and U.S. Pat. Nos.
5,925,630; 5,939,545; 6,143,736; 6,277,838, (Upasani et al.)
disclose a number of 3.alpha.-hydroxy steroids and some 3.beta.
steroids. These patents concern the agonistic modulation of the
GABA-A receptor. In other words, the patents are focused on
3.alpha.-hydroxy-steroids and their benzodiazepine like effect. All
steroids that are modulators of the GABA-A receptor have the common
feature of one 3.alpha.-hydroxy structure.
[0006] WO 99/45931 (Backstrom & Wang) discloses the
antagonistic effect of one steroid, namely
3.beta.-OH-5.alpha.-pregnan-20-one, but is silent about the
steroids described in this application.
[0007] WO 03/059357 (Backstrom et al.) discloses several
3bets-hydroxy steroids and their antagonistic effect on the GABA-A
receptor but is silent about the steroids described in this
application.
[0008] The antagonistic effects of
3.beta.-OH-5.alpha.-pregnan-20-one and other
3.beta.-OH-5.alpha./.beta. pregnan-steroids are disclosed by Wang
et al. (Wang M. D., Backstrom T. and Landgren S. (2000) The
inhibitory effects of allopregnanolone and pregnanolone on the
population spike, evoked in the rat hippocampal CA1 stratum
pyramidale in vitro, can be blocked selectively by
epiallo-pregnanolone, Acta Physiol Scand 169, 333-341 and Wang M,
He Y, Eisenman L N, Fields C, Zeng C M, Mathews J et al.,
3.beta.-hydroxypregnane steroids are pregnenolone sulfate-like
GABA(A) receptor antagonists, J Neurosci 2002; 22 (9):3366-75). In
those papers, dose dependent antagonistic effect of
3.beta.-OH-5.alpha./.beta.-pregnan-steroids and sulfatated-steroids
are described. However the compounds in the present invention are
not mentioned.
[0009] The present invention relates to the field of medicinal
chemistry and is intended for producing compounds and compositions
useful for modulation of the mammal brain excitability via the
gamma-aminobutyric acid receptor-chloride ionophore (GABA.sub.A-R)
complex and other neurotransmitter systems which are, directly or
indirectly, correlated to the GABA.sub.A-R complex. It has been
shown that a variety of steroidal molecules are effective in the
modulation and stimulation of GABA signaling, displaying a variety
of physiologic effects. The steroids comprising the components
3.alpha.-hydroxy-5.alpha./.beta.-pregnan-20-one have been shown to
be specific GABA-A {gamma-aminobutyric acid (A)} receptor
enhancers. Due to these properties, these naturally occurring
stress- and sex steroids also have adverse effects and cause
certain disorders. The adverse effects of the
3.alpha.-hydroxy-pregnan-20-one-steroids are the basis for the
negative CNS effects induced by these steroids. Examples of the
adversive compounds are
3.alpha.-hydroxy-5.alpha./.beta.-pregnanolone steroids listed in
table 1. Some of these steroids are very potent and have e.g. been
shown to have an ability to induce anesthesia at a high
pharmacological dose.
[0010] As the 3.alpha.-hydroxy-pregnane-steroids are endogenously
produced and are metabolites of steroid hormones essential for
life, their production cannot easily be interrupted. These steroids
are produced in high amounts during several days to weeks during
acute and chronic stress, the luteal phase of the menstrual cycle
and during pregnancy. They are also produced within the brain.
Specific blockers are therefore needed as therapy.
[0011] It has earlier been shown that certain 3.beta. hydroxy
pregnanolone steroids can block the negative brain effect of the
aversive stress and sex steroids. A problem with the earlier
discovered compounds is that they are easily metabolized in the
body at the critical 3 position and that they are difficult to
dissolve in water solution.
[0012] The direct mechanism at the receptor site has not yet been
fully elucidated, due to the structural complexity of the
GABA.sub.A-R complex. The GABA receptor family includes several
subunit compositions, of which some are known to be related to
specific functions and disorders in the CNS. One aim of the present
invention is thus to find new compounds that are useful in the
treatment of anomaly in the excitability of GABA receptors or other
neurotransmitters related to GABA receptors, in a manner which can
be general or specific for some subunit compositions and functions.
Disorders that are caused by the action of endogenously produced
3.alpha.-hydroxy-5.alpha. steroids or 3.alpha.-hydroxy-5.beta.
steroids on the GABA-A receptor are well characterized and
understood. It is also known that
3.alpha.-hydroxy-5.alpha./.beta.-steroids can induce tolerance to
themselves and other similar substances after exposure, and that
abstinence effects occur at withdrawal of the
3.alpha.-hydroxy-5.alpha./.beta.-steroids. This will be further
elucidated in the following:
[0013] Diseases Caused by 3.alpha.-hydroxy-pregna(e)n-steroids
[0014] a) Direct Action
[0015] It is established that
3.alpha.-hydroxy-5.alpha./.beta.-steroids can directly cause
inhibition of CNS functions. Examples of disorders and symptoms
caused by the direct action of
3.alpha.-hydroxy-5.alpha./.beta.-steroids are premenstrual
dysphoric disorder, premenstrual syndrome, dementia, Alzheimer's
disease, sedation, tiredness, chronic fatigue syndrome, memory
disturbance, learning disturbance, disturbance of motor function,
fractures, clumsiness, increased appetite and food cravings,
obesity, negative mood as tension, irritability, depression,
decreased hearing and eye sight, worsening of Petit Mal epilepsy,
burn out syndrome.
[0016] b) Tolerance
[0017] Continuous and long exposure to
3.alpha.-hydroxy-5.alpha./.beta.-steroids causes a malfunctioning
of the GABA-A receptor system. A tolerance develops and this
tolerance is the initial step in a process that ultimately leads to
stress sensitivity, concentration difficulties, and loss of impulse
control and depression. The action of
3.alpha.-hydroxy-5.alpha./.beta.-steroids have been found to be a
factor which reinforces drug dependency. This has been the focus of
extensive research.
[0018] c) Abstinence
[0019] A continuous but shorter exposure to
3.alpha.-hydroxy-5.alpha./.beta.-steroids results in a withdrawal
effect when the exposure is ended. This phenomenon occurs i.e.
during menstruation when the production of
3.alpha.-hydroxy-5.alpha./.beta.-steroids by the corpus luteum of
the ovary is interrupted. This withdrawal phenomenon also occurs
after giving birth (post partum) when the
3.alpha.-hydroxy-5.alpha./.beta.-steroid production by the placenta
is interrupted. The same phenomenon is also noted when a period of
stress is ended. As a response to stress, the adrenals have
produced 3a-hydroxy-5.alpha./.beta.-steroids. When this production
is interrupted, withdrawal symptoms may occur. Examples of
conditions that are influenced by this withdrawal/abstinence
phenomenon are partial epilepsy where the patient has an epileptic
focus in the cerebral cortex where a worsening occurs at the
withdrawal period during menstruation. This phenomenon is called
"catamenial epilepsy". Other examples are menstrual related
migraine and stress related migraine, mood changes post partum and
weekend headache. Abstinence is a sign of an earlier developed
tolerance.
[0020] In view of the above, it is clear that steroids are
important drug candidates. Naturally occurring steroids are however
subject to intense metabolism and are therefore usually not
suitable for oral administration. Also in other routes of
administration the metabolism is high and makes it impossible to
use the compounds as medication and treatments as the active parts
of the compounds are first destroyed by the metabolism.
[0021] A second problem with steroid compounds is that they are
difficult to solute in water solutions and therefore difficult to
administer in vivo.
[0022] These problems and others are solved by compounds according
to the present invention.
SUMMARY OF THE INVENTION
[0023] The present inventors have synthesized new compounds that
are protected against metabolism in the 3 position of the steroid.
Surprisingly, these compounds also have increased water solubility,
due to their modified structural features. The inventors do not
wish to be bound to any theory, but it is assumed that the
advantageous properties of the novel compounds are due to the
presence of a double bond in the steroid core, and the replacement
of a keto group with an oxime group at positions 20, 21 or 17
respectively in the mentioned scaffolds of pregnane, pregnene,
androstane and androstene series.
[0024] In summary, it is known that 3.alpha.-hydroxy-delta 4-5,
5.alpha./.beta.-steroids cause CNS disorders through the above
mentioned three possible mechanisms: a) direct action, b) tolerance
induction, and c) withdrawal effect. The compounds made available
through the embodiments of the present invention belong to the
pregnane-, pregnene-, androstane- androstene- series, with the
addition of suitable functionalities. The compounds can be used
alone or as prodrugs and/or in combination with formulations and
other compositions in order to enhance and modulate the effects on
CNS. Compositions within the scope of this invention include all
compositions wherein the compounds of this invention are contained
in an amount that is effective to achieve the intended
purposes.
[0025] To the best knowledge of the present inventors, this is the
first time steroid compounds with increased resistance against
metabolism and increased water solubility are disclosed. In
addition, these substances are suggested for the manufacture of
pharmaceuticals for the treatment of many specific steroid related
or steroid induced CNS disorders and for use in methods of
treatment, according to the attached claims which are incorporated
herein by reference.
DETAILED DESCRIPTION OF THE INVENTION
[0026] The invention concerns new compounds, with protection
against metabolism and with increased water solubility, and methods
of producing such compounds with antagonistic and blocking effects
of the 3.alpha.-hydroxy-pregna(e)n-steroid induced CNS disorders.
The present invention is based on the discovery that steroids
represented by the formulae I or II have an effect as modulators of
the GABA receptor signaling as agonists, antagonists or inverse
agonists.
[0027] The inventors have shown that the presence of a tertiary
alcohol moiety in position 3 prolongs the half-life of a steroidal
compound within the body through preventing metabolic oxidations or
degradation in the body. The presence of a hydrogen-bond
acceptor/donator group bonded to the D ring of a steroidal molecule
influences the ability of the steroid to modulate the GABA receptor
signaling.
[0028] The present invention relates to novel steroids represented
by the formulae I or II, and pharmaceutically acceptable
derivatives, salts, prodrugs or solvates thereof:
##STR00001##
[0029] wherein
[0030] R.sub.1 is chosen among an ethynyl, ethenyl, ethyl, or other
saturated or unsaturated alkyl groups; hydroxyl, in its free form
or combined with carboxylic acid residues, sugars, alkyl groups to
form esters or ether or glycosylated compounds; fluorine or other
halogens; a proton;
[0031] R.sub.2 is chosen among an ethynyl, ethenyl, ethyl, or other
saturated or unsaturated alkyl groups; hydroxyl, in its free form
or combined with carboxylic acid residues, sugars, alkyl groups to
form esters or ether or glycosylated compounds; fluorine or other
halogens; a proton;
[0032] R.sub.3 is 5.alpha.- or 5.beta.-H
[0033] R.sub.4 is a nitro, hydroxyl, free or bonded with an ester,
ether, sugar; and
[0034] R.sub.5 is a proton.
[0035] According to an embodiment of the invention, the compounds
are steroids represented by the above formulae I or II, and
pharmaceutically acceptable derivatives, salts, prodrugs or
solvates thereof, wherein
[0036] R.sub.4, R.sub.5 is .dbd.O or N as oxime .dbd.NOH, or a
homo- or heterocycle;
[0037] R.sub.6 is a methyl, an alkyl group or --CH.sub.2OR where R
is H, a carboxylic acid residue, an alkyl group or a sugar;
--CH.sub.2X where X is fluorine or another halogen;
[0038] R.sub.7, R.sub.10 is OH, CH.sub.3 or H at the seven
position.
[0039] R.sub.8, R.sub.9 or R.sub.11, R.sub.12 represent two
Me-groups, or Me- and H, respectively, or two --H.
[0040] According to an embodiment R.sub.7, R.sub.10 is OH or
CH.sub.3 at the seven position.
[0041] According to an embodiment of the invention, the compounds
are steroids represented by the above formula I, or a
pharmaceutically acceptable salt thereof, wherein R.sub.1 is an
ethynyl group; hydroxyl, in its free form or combined with
carboxylic acid residues; fluorine; or a proton; R.sub.2 is an
ethynyl group; hydroxyl, in its free form or combined with
carboxylic acid residues; fluorine; or a proton; R.sub.3 is
5.alpha.- or 5.beta.-H; R.sub.4 is hydroxyl and R.sub.5 a proton,
or R.sub.4, R.sub.5 together is O or N as oxime .dbd.NOH; R.sub.6
is a methyl group; R.sub.7 is H; and R.sub.8.dbd.R.sub.9=methyl or
H.
[0042] According to an embodiment of the invention, the compounds
are steroids represented by the above formula II, or a
pharmaceutically acceptable salt thereof, wherein R.sub.1 is an
ethynyl group or hydroxyl; R.sub.2 is an ethynyl group or hydroxyl;
R.sub.3 is 5.alpha.- or 5.beta.-H; R.sub.4 and R.sub.5 together is
O or N as oxime .dbd.NOH; R.sub.10 is H; and
R.sub.11=R.sub.12.dbd.H.
[0043] According to an embodiment, an unsaturation can be present
between C4-C5 or C5-C6 or at other positions in the molecule.
R.sub.8, R.sub.9 or R.sub.11, R.sub.12 represent two Me-groups, or
Me- and H, respectively, or two --H, or if said unsaturation is
between C4-C5, then one of R.sub.8, R.sub.9 or R.sub.11, R.sub.12,
respectively, is Me- or H while the other one is absent. According
to one aspect of this embodiment, the compounds are steroids
represented by the above formula I, wherein R.sub.1 is an ethynyl
group or hydroxyl; R.sub.2 is an ethynyl group or hydroxyl; R.sub.3
is absent; R.sub.4 is hydroxyl; R.sub.5 a proton; or R.sub.4 and
R.sub.5 is together .dbd.O or N as oxime .dbd.NOH; R.sub.6 is a
methyl group; R.sub.7 is H; and R.sub.8, R.sub.11 is methyl or H;
and R.sub.9, R.sub.12 is methyl, H, or, if said unsaturation is
between C4-C5, absent.
[0044] The compounds of formula I and II may exist as optical
isomers; consequently the invention includes all individual isomers
that may be separated by common chromatographic techniques as well
as other known separation methods known by those skilled in the
art.
[0045] The present invention also encompasses all functionally
equivalent derivatives and prodrugs where esters and ethers have
been added to the hydroxyl groups of the steroids. Examples of
suitable derivatives include, but are not limited to, sulphates,
formates, acetates, propionates, glycosilations with sugars or
oligosaccarides, methylates, ethylates. One skilled in the art can
recognize that other functional groups not included in this list of
examples can be employed.
[0046] Tables 2 and 3 show examples of the structure of a series of
compounds according to the invention, were the 3-hydroxy position
of the pregnane, pregnene, androstane or androstene steroid
structure is protected against metabolism in 3-position with an
ethynyl, ethenyl or acetate structure added to the steroid molecule
or the hydroxyl group replaced with a fluorine atom.
TABLE-US-00002 TABLE 2 New Compounds with metabolism protection
based on formula I UC2005 ##STR00002## 3.alpha.-ethynyl,
3.beta.-hydroxyl, 5.beta.-pregnan- 20-one UC2007 ##STR00003##
3.alpha.-ethynyl, 5.beta.-pregnan- 3.beta.,20(R)- diol UC2009
##STR00004## 3.beta.-ethenyl, 5.alpha.-pregnan- 3.alpha.,20(R)-
diol UC2012 ##STR00005## 3.beta.-fluor, 5.beta.-pregnan- 20-one
UC2013 ##STR00006## 3.beta.-fluor, 5.beta.-pregnan- 20-(R)-ol
UC2014 ##STR00007## 3.beta.-fluor, 5.beta.-pregnan- 20-(S)-ol
UC2016 ##STR00008## 3.beta.-fluor, 5.beta.-pregnan- 20-one UC2017
##STR00009## 3.alpha.-fluor, 5.alpha.-pregnan- 20-(R)-ol UC2018
##STR00010## 3.alpha.-ethynyl, 5.alpha.-pregnan- 3.beta.,20(R)-
diol UC2019 ##STR00011## 3.beta.-ethynyl, 3.alpha.-acetyl,
5.alpha.-pregnan- 20-one UC2024 ##STR00012## 3.beta.-ethynyl,
3.alpha.-hydroxyl, .DELTA.4-pregnen- 20-one UC2026 ##STR00013##
3.beta.-ethynyl, 3.alpha.-hydroxyl, 5.alpha.-pregnan- 20-one oxime
UC2029 ##STR00014## 3.beta.-ethynyl, 3.alpha.-hydroxyl,
.DELTA.4-pregnen- 20-one UC2030 ##STR00015## 3.alpha.-fluor,
5.alpha.- pregnan-20- one oxime UC2032 ##STR00016## 3-dimethyl,
.DELTA.5-pregnen- 3.beta.,20(R)- diol UC2034 ##STR00017##
3.beta.-fluor, 5.alpha.-pregnan- 20-one oxime
TABLE-US-00003 TABLE 3 New Compounds with metabolism protection
based on formula II UC2021 ##STR00018## 3.beta.-ethynyl,
3.alpha.-hydroxyl, androstan-17- one UC2025 ##STR00019##
3.beta.-ethynyl, 3.alpha.-hydroxyl, androstan-17- one oxime UC2027
##STR00020## 3.alpha.-ethynyl, 3.beta.-hydroxyl, androstan-17- one
oxime
[0047] In Table 4 are shown examples of structures of a series of
compounds where the water solubility is increased relative to the
5-saturated compound (UC2024) and where the water solubility is
increased by adding an oxime group relative to a keto group or
simple hydroxy group (UC2027, UC2029).
TABLE-US-00004 TABLE 4 Examples of new compounds with increased
water solubility UC2013 ##STR00021## 3.beta.-fluor, 5.beta.-
pregnan-20- (R)-ol UC2014 ##STR00022## 3.beta.-fluor, 5.beta.-
pregnan-20- (S)-ol UC2017 ##STR00023## 3.alpha.-fluor, 5.alpha.-
pregnan-20- (R)-ol UC2018 ##STR00024## 3.alpha.-ethynyl,
5.alpha.-pregnan- 3.beta.,20(R)- diol UC2024 ##STR00025##
3.beta.-ethynyl, 3.alpha.-hydroxyl, .DELTA.4-pregnen- 20-one UC2027
##STR00026## 3.alpha.-ethynyl, 3.beta.-hydroxyl, androstan-
17-oxime UC2029 ##STR00027## 3.beta.-ethynyl, 3.alpha.-hydroxyl,
.DELTA.4-pregnen- 20-oxime UC2030 ##STR00028## 3.alpha.-fluor,
5.alpha.- pregnan-20- oxime UC2032 ##STR00029## 3-dimethyl,
.DELTA.5-pregnen- 3.beta.,20(R)- diol UC2034 ##STR00030##
3.beta.-fluor, 5.alpha.- pregnan-20- one oxime
[0048] HPLC Retention Times [Min] for Compounds According to the
Invention
[0049] HPLC Conditions:
[0050] HPLC System (Waters) Column Symmetry.RTM. C.sub.18 3.5 .mu.m
4.6.times.75 mm (Waters); T: 45.degree. C.; flow 1.0 mL/min;
isocratic eluent conditions 40:60 v/v H.sub.2O:MeOH. Injection
volume: 100 .mu.L.
[0051] Solvents used for eluent are of HPLC grade, water filtered
through a Millipore apparatus; all solvents were filtered through a
0.45 .mu.m Millipore filter and degassed by N.sub.2 stream prior
use.
[0052] Since the analyses are carried in reverse phase, shorter
retention times correspond to higher hydrophilicity.
TABLE-US-00005 TABLE 5 Reference substances: retention time [min]
3.beta.-5.beta.-pregnan-20-one 24.4 3.beta.-5.alpha.-pregnan-20-one
30.0
TABLE-US-00006 TABLE 6 Inventive compounds: retention time [min] UC
2021 7.8 UC 2024 14.0 UC 2025 13.9 UC 2026 16.9
[0053] As seen above in Tables 5 and 6 the retention times for the
inventive compounds are shorter than for the reference compounds,
indicating that the former have higher hydrophilicity than the
latter.
[0054] The synthesis, the separation and the identification of the
compounds in this invention can be performed using method steps
known as such by those skilled in the art.
[0055] The present inventors have surprisingly found that the
reaction of the ethynyl Grignard reagent with 3, 20/17 diketone
steroids is in the most cases selective for the position 3 and no
protection/deprotection for the other ketone functionality was
required. Both .alpha. and .beta. isomers are formed, which can be
separated by chromatographic methods and recrystallization.
[0056] The starting materials for the compounds described in the
present invention are the corresponding steroids with a 3-hydroxyl
substituent and a keto group in positions 20 or 17. They can be
converted into the respective diones by oxidation with IBX reagent.
The reaction proceeds smoothly and with complete conversion. Other
suitable steroids can be employed as starting material when
required.
[0057] The reactions are carried out in a suitable solvent such as
methanol, ethanol, water, THF, diethyl ether, dichloromethane or
other solvents or mixture of solvents that one skilled in the art
can recognize as suitable.
[0058] The reactants are chosen in order to avoid, when possible,
use of reactants, such as heavy metals, which are toxic even in
traces or are difficult to be completely removed in the workup
procedure.
[0059] Reactions involving air or moisture sensitive reagents or
products are carried out under inert atmosphere, such as nitrogen
or argon gas, in the presence of dry solvents. Diethyl ether and
tetrahydrofuran are dried over Na in the presence of benzophenone.
Syringes purged with inert gas were used for the transfer of
reagents and dry solvents. Optimized reaction conditions, such as
time and temperature, were determined by monitoring the formation
of products and the loss of starting material using a suitable
chromatographic technique such as TLC or GC/MS.
[0060] Purifications are carried out by using chromatographic
techniques such as flash silica chromatography or preparative high
performance liquid chromatography (HPLC) by using a HPLC apparatus.
Those skilled in the art can recognize that alternative
purification methods can be employed, and laboratory
chromatographic techniques can be adapted to industrial scale by
using chromatographic columns for scaled preparations.
[0061] Identification of the products is carried out by using
suitable analytical techniques such as 1H-NMR, 13C-NMR, mass
spectrometry, IR spectroscopy and any other assay that one skilled
in the art would recognize as suitable for structural
identification and purity determination of the compounds in the
present invention.
[0062] The compounds according to the invention have inter alia the
advantage of being both protected against metabolism, and more
easily water soluble. The method of synthesis has the advantage of
consisting of steps known as such, and is comparatively easy--once
disclosed--to use.
[0063] The following examples of steroids in the present invention
are provided. The examples are illustrative, but not limiting, of
the methods and compositions of the present invention. One skilled
in the art will recognize that similar reagents, solvents,
conditions and parameters can be used in the reactions, depending
on the substrate. NMR data were recorded using a Bruker 400 MHz
spectrometer.
EXAMPLES
Examples Based on formula I
Example 1
UC2016-3.beta.-fluor,5.alpha.-pregnan-20-one
[0064] 3.alpha.-OH 5.alpha.-pregnan-20-one (3 mmol), was dissolved
in 20 mL dry dichloromethane under N.sub.2 atmosphere. DAST (700
mg, 4.33 mmol) was added slowly dropwise at room temperature (rt)
and the resulting yellowish solution was left stirring at rt for 1
h. Reaction was followed by TLC. The solution was quenched by slow
addition of a 5% NaHCO.sub.3 solution (60 mL). The water phase was
extracted with dichloromethane (3.times.20 mL), the organic phases
collected dried over MgSO.sub.4 and the solvent removed under
reduced pressure, yielding a yellowish oil which was purified by
silica flash column chromatography (pentane:ethyl acetate 9:1).
Products are, in the order: elimination of H.sub.2O at 2,3
positions (yield: 67%); fluorination at 3-OH with inversion of
configuration 30% (UC2016); fluorination at 3-OH with retention of
configuration (3%--traces). 1H NMR (400 MHz, CDCl3-d6): .delta.
4.57-4.37 (dm, 1H); 2.51 (t, 1H); 2.12 (m, 1H); 2.11 (s, 3H);
2.02-1.99 (m, 2H); 0.83 (s, 3H); 0.67 (m, 1H); 0.61 (s, 3H).
Example 2
UC2018-3.alpha.-ethynyl, 5.alpha.-pregnan-3.beta.,20(R)-diol
[0065] 3.alpha.-ethynyl, 3.beta.-hydroxy, 5.alpha.-pregnan-20-one
(0.3 mmol) was dissolved in a solution of 2 mL dichloromethane and
5 mL MeOH at rt, in a flask with outlet to air. NaBH.sub.4 (2.1
equiv.) was added in one portion and the suspension left stirring
for 3 h at rt. The colorless solution was evaporated in vacuum,
yielding a white residue which was extracted with 20+20 mL H.sub.2O
diethyl ether. The aqueous phase was extracted with 30 mL
dichloromethane:diethyl ether 1:1, the organic phases collected,
dried over MgSO.sub.4, and the solvents removed under vacuum. The
white solid was purified by silica flash column chromatography (1:4
diethyl ether:dichloromethane), Quantitative overall yield. The
product with (R)-configuration at 20-C is the main product (90%),
as determined by NMR measurements.
[0066] .sup.1H NMR (400 MHz, CDCl.sub.3-d.sub.6): .delta. 3.72 (m,
1H); 2.41 (s, 1H); 2.02 (m, 1H); 1.86 (2m, 2H); 1.12 (d, 3H); 0.80
(s, 3H); 0.74 (s, 3H).
Example 3
UC2019-3.beta.-ethynyl, 5.alpha.-pregnan-20-one
3.alpha.-acetate
[0067] 3.beta.-ethynyl, 3.alpha.-hydroxy, 5.alpha.-pregnan-20-one
(0.25 mmol) and pyridine (2 equiv.) were dissolved in dry
dichloromethane, followed by the dropwise addition of acetic
anhydride (4 equiv.) at rt under nitrogen atmosphere.
[0068] The mixture was left stirring at 40.degree. C. under three
days. The dark mixture was quenched by the addition of 50 mL HCl
10%, then washed with a NaHCO.sub.3 10% aqueous solution
(2.times.30 mL) until pH=7. The organic phase was collected, dried
over MgSO.sub.4, and concentrated. The yellowish residue was
purified by silica flash column chromatography (1:4 diethyl ether:
dichloromethane) to afford the ester in 87% yield.
[0069] 1H NMR (400 MHz, CDCl3-d6): .delta. 2.60 (s, 1H); 2.51 (t,
1H); 2.45 (m, 1H); 2.11 (s, 3H); 2.03 (s, 3H); 0.82 (s, 3H); 0.60
(s, 3H).
Example 4
UC2024-3.beta.-ethynyl, 3.alpha.-hydroxyl,
.DELTA.-4-pregnen-20-one
[0070] Progesterone (1 mmol) was dissolved in 25 mL dry THF at rt
under nitrogen. Ethynyl magnesium bromide (1.1 equiv.) was added
dropwise at rt under stirring and the solution was left stirring
overnight at rt under nitrogen. The yellowish solution was then
quenched with saturated NH.sub.4Cl.sub.(aq) and the aqueous phase
extracted with dichloromethane (3.times.30 mL). The collected
organic phases were evaporated under reduced pressure, the
resulting yellow oil dissolved in dichloromethane, washed with
brine and dried over MgSO.sub.4. The solution was reduced under
vacuum, and the residue purified by silica flash column
chromatography (1:4 diethylether:dichloromethane), typical yields
30%.
[0071] 1H NMR (400 MHz, CDCl3-d6): .delta. 5.32 (s, 1H); 2.51 (m,
2H); 2.14 (m, 2H); 2.11 (s, 3H); 1.05 (s, 3H); 0.64 (s, 3H).
Example 5
UC2026-3.beta.-ethynyl, 3.alpha.-hydroxyl, 5.alpha.-pregnan-20-one
oxime
3.beta.-ethynyl, 3.alpha.-hydroxyl, 5.alpha.-pregnan-20-one
[0072] 3,20-5.alpha.-pregnandione (1.580 g, 5.0 mmol) was dissolved
in 50 mL dry THF at rt under nitrogen. Ethynyl magnesium bromide
(1.1 equiv) was added dropwise at rt under stirring and the
solution was left stirring overnight at rt under nitrogen flow. The
yellowish solution was then quenched with saturated NH.sub.4Cl(aq)
and the aqueous phase extracted with dichloromethane (3.times.30
mL). The collected organic phases were evaporated under reduced
pressure, the resulting yellow oil dissolved in dichloromethane,
washed with brine and dried over MgSO.sub.4. The solution was
reduced under vacuum, and the residue purified by silica flash
column chromatography (1:4 diethylether:dichloromethane), typical
yields 72%. Eventual traces of byproducts can be eliminated by
further recrystallization from diethylether.
[0073] 1H NMR (400 MHz, CDCl3-d6): .delta. 2.51 (t, 1H); 2.47 (s,
3H); 2.14 (m, 1H); 2.11 (s, 3H); 0.81(s, 1H); 0.60 (s, 3H).
3.alpha.-ethynyl, 3.beta.-hydroxyl, 5.alpha.-pregnan-20-one
[0074] Was obtained as a byproduct from the above described
reaction and separated by silica flash column chromatography.
Typical yield 13%.
[0075] 1H NMR (400 MHz, CDCl3-d6): .delta. 2.52 (t, 1H); 2.43 (s,
1H); 2.11 (s, 3H); 0.80 (s, 3H), 0.60 (s, 3H).
3.beta.-ethynyl, 3.alpha.-hydroxyl, 5.alpha.-pregnan-20-one
oxime
[0076] 3.beta.-ethynyl, 3.alpha.-hydroxyl, 5.alpha.-pregnan-20-one
(10 mmol) is dissolved in dichloromethane 5 mL and ethanol 50 mL at
rt and air atmosphere, in a 250 mL round bottom flask. 4 equiv. of
NH.sub.2OH chlorhydrate and 4 equiv. of sodium acetate are
dissolved in 5 mL H.sub.2O and then added to the steroid solution.
20 mL of ethanol are added and the mixture put on reflux overnight.
The mixture is then cooled and the solvent removed under reduced
pressure. The white residue is then treated with 50 mL H.sub.2O and
50 mL dichloromethane, the aqueous phase extracted with 3.times.30
mL dichloromethane. The collected organic phases are then dried
over MgSO.sub.4, filtrated and the solvent removed under reduced
pressure. The final residue is purified by silica flash column
chromatography dichloromethane:diethyl ether 4:1, typical yields
95-100%.
[0077] 1H NMR (400 MHz, CDCl3-d6): .delta. 2.47 (s, 1H); 2.22 (t,
1H); 2.05 (m, 1H); 1.88 (s, 3H); 1.86 (m, 1H); 0.81 (s, 3H), 0.62
(s, 3H).
Example 6
UC2029-3.beta.-ethynyl, 3.alpha.-hydroxyl, .DELTA.4-pregnen-20-one
oxime
[0078] 3.beta.-ethynyl, 3.alpha.-hydroxyl, .DELTA.4-pregnen-20-one
(10 mmol) is dissolved in dichloromethane 5 mL and ethanol 50 mL at
rt and air atmosphere, in a 250 mL round bottom flask. 4 equiv. of
NH.sub.2OH chlorhydrate and 4 equiv. of sodium acetate are
dissolved in 5 mL H.sub.2O and then added to the steroid solution.
20 mL of ethanol are added and the mixture put on reflux overnight.
The mixture is then cooled and the solvent removed under reduced
pressure. The white residue is then treated with 50 mL H.sub.2O and
50 mL dichloromethane, the aqueous phase extracted with 3.times.30
mL dichloromethane. The collected organic phases are then dried
over MgSO.sub.4, filtrated and the solvent removed under reduced
pressure. The final residue is purified by silica flash column
chromatography dichloromethane:diethyl ether 4:1, typical yields
85%.
[0079] 1H NMR (400 MHz, CDCl3-d6): .delta. 5.32 (s, 1H); 2.51 (s,
1H); 2.19 (m, 2H); 2.06 (m, 1H); 1.88 (s, 3H); 2.03 (s, 3H); 1.05
(s, 3H); 0.65 (s, 3H).
Example 7
UC2030-3.alpha.-fluor, 5.alpha.-pregnan-20-one oxime
3.alpha.-fluor, 5.alpha.-pregnan-20-one
[0080] 3.alpha.-OH 5.alpha.-pregnan-20-one (3 mmol), was dissolved
in 20 mL dry dichloromethane under N.sub.2 atmosphere. DAST (700
mg, 4.33 mmol) was added slowly dropwise at -78.degree. C. and the
resulting yellowish solution was left stirring at rt for 1 h.
Reaction was followed by TLC. The solution was quenched by careful
addition of a 5% NaHCO.sub.3 solution (60 mL). The water phase was
extracted with dichloromethane (3.times.20 mL), the organic phases
collected dried over MgSO.sub.4 and the solvent removed under
reduced pressure, yielding a yellowish oil which was purified by
silica flash column chromatography (pentane:ethyl acetate 9:1).
Products are, in the order: elimination of H.sub.2O at 2,3
positions (yield: 67%); fluorination at 3-OH with inversion of
configuration 30%; fluorination at 3-OH with retention of
configuration (3%--traces).
[0081] 1H NMR (400 MHz, CDCl3-d6): .delta. 4.87-4.75 (d, 1H); 2.53
(t, 1H); 2.11 (s, 3H); 2.00 (m, 1H); 0.95 (m, 1H); 0.80 (m, 1H);
0.78 (s, 3H); 0.60 (s, 3H).
3.alpha.-fluor, 5.alpha.-pregnan-20-one oxime
[0082] 3.alpha.-fluor, 5.alpha.-pregnan-20-one (10 mmol) is
dissolved in dichloromethane 5 mL and ethanol 50 mL at rt and air
atmosphere, in a 250 mL round bottom flask. 4 equiv. of NH.sub.2OH
chlorhydrate and 4 equiv. of sodium acetate are dissolved in 5 mL
H.sub.2O and then added to the steroid solution. 20 mL of ethanol
are added and the mixture put on reflux overnight. The mixture is
then cooled and the solvent removed under reduced pressure. The
white residue is then treated with 50 mL H.sub.2O and 50 mL
dichloromethane, the aqueous phase extracted with 3.times.30 mL
dichloromethane. The collected organic phases are then dried over
MgSO.sub.4, filtrated and the solvent removed under reduced
pressure. The final residue is purified by silica flash column
chromatography (dichloromethane:diethyl ether 4:1). Quantitative
yield.
[0083] 1H NMR (400 MHz, CDCl3-d6): .delta. 4.90-4.78 (d, 1H); 2.26
(t, 1H); 2.10 (m, 1H); 1.90 (s, 3H); 0.98 (m, 1H); 0.82 (s, 3H);
0.65 (s, 3H).
Example 8
UC2034-3.beta.-fluor, 5.alpha.-pregnan-20-one oxime
[0084] was obtained by using the same synthetic protocol as Example
7 for UC2030 starting from the corresponding 3.beta.-fluor,
5.alpha.-pregnan-20-one isomer.
Example 9
UC2032-3-dimethyl, .DELTA.5-pregnen-3.beta.,20(R)-diol
3-dimethyl, .DELTA.5-pregnen-3,20-dione
[0085] 525 mg progesterone are dissolved in 10 mL dry toluene at
rt. 3.4 mL (2 equiv.) of a 1.0 M solution of sodium t-buthylate in
dry toluene is added dropwise to the progesterone solution, under
stirring and N.sub.2 atmosphere. The yellowish solution is let
stirring in 20 min 2 equiv. of MeI are then added dropwise to the
mixture, which is stirred overnight at rt under N.sub.2.
[0086] The mixture is quenched with 10 mL water and 10 mL
dichloromethane, the aqueous phase extracted with 2.times.30 mL
dichloromethane. The organic phases are collected, dried over
MgSO4, the solvent removed under vacuum yielding a yellowish
residue, which is purified by silica flash column chromatography
(1:4 diethyl ether:dichloromethane). A further purification of the
desired fraction is performed by silica flash column chromatography
(1:9 ethyl acetate:pentane). Yield: 25%.
[0087] .sup.1H NMR (400 MHz, CDCl.sub.3-d.sub.6): .delta. 55.56 (m,
1H); 2.54 (m, 3H); 2.13 (s, 3H); 1.23 (s, 6H); 0.86 (s, 3H); 0.64
(s, 3H).
3-dimethyl, .DELTA.5-pregnen-3.beta.,20(R)-diol
[0088] 91 mg of 3-dimethyl, .DELTA.5-pregnen-3,20-dione are
dissolved in 3.0 mL dichloromethane and 15 mL MeOH at rt, in a
flask with outlet to air. NaBH.sub.4 (2.1 equiv.) is added in one
portion and the suspension left stirring for 6 h at rt. The
colorless solution was evaporated in vacuum, yielding a white
residue which was extracted with 20+20 mL H.sub.2O diethyl ether.
The aqueous phase was extracted with 30 mL dichloromethane:diethyl
ether 1:1, the organic phases collected, dried over MgSO.sub.4, and
the solvents removed under vacuum. The white solid was purified by
silica flash column chromatography (1:4 diethyl
ether:dichloromethane), 95% yield.
[0089] .sup.1H NMR (400 MHz, CDCl.sub.3-d.sub.6): .delta. 5.60 (m,
1H); 3.75 (m, 1H); 3.26 (m, 1H); 2.09-2.13 (m, 2H); 1.18 (s, 6H);
1.21 (s, 3H); 1.10 (s, 3H); 0.80 (s, 3H).
Examples Based on formula II
Example 10
UC2021-3.beta.-ethynyl, 3.alpha.-hydroxyl, androstan-17-one
3.beta.-ethynyl, 3.alpha.-hydroxyl, androstan-17-one
[0090] 3, 17 androstandione (5.0 mmol) was dissolved in 50 mL dry
THF at rt under nitrogen. Ethynyl magnesium bromide (1.1 equiv) was
added dropwise at rt under stirring and the solution was left
stirring overnight at rt under nitrogen flow.
[0091] The solution was then quenched with saturated NH.sub.4Cl(aq)
and the aqueous phase extracted with dichloromethane (3.times.30
mL). The collected organic phases were evaporated under reduced
pressure, the resulting yellow oil dissolved in dichloromethane,
washed with brine and dried over MgSO.sub.4. The solution was
reduced under vacuum, and the residue purified by silica flash
column chromatography (1:4 diethylether:dichloromethane), typical
yields 65%. Eventual traces of byproducts can be eliminated by
further recrystallization from diethylether.
[0092] 1H NMR (400 MHz, CDCl3-d6): .delta. 2.47 (s, 1H); 2.42 (m,
1H); 2.10-2.04 (m, 2H); 1.02 (m, 1H); 0.86 (s, 3H); 0.83 (s,
3H).
3.beta.-ethynyl, 3.alpha.-hydroxyl, androstane-17-one
[0093] Was obtained as a byproduct from the above described
reaction and separated by preparative HPLC chromatography. Typical
yield 8%.
[0094] 1H NMR (400 MHz, CDCl.sub.3-d.sub.6): .delta. 2.43 (s, 1H);
0.86 (s, 3H), 0.83 (s, 3H).
Example 11
UC2025-3.beta.-ethynyl, 3.alpha.-hydroxyl, androstan-17-one
oxime
[0095] 3.beta.-ethynyl, 3.alpha.-hydroxyl, androstan-17-one (10
mmol) is dissolved in dichloromethane 5 mL and ethanol 50 mL at rt
and air atmosphere, in a 250 mL round bottom flask. 4 equiv. of
NH.sub.2OH chlorhydrate and 4 equiv. of sodium acetate are
dissolved in 5 mL H.sub.2O and then added to the steroid solution.
20 mL of ethanol are added and the mixture put on reflux overnight.
The mixture is then cooled and the solvent removed under reduced
pressure. The white residue is then treated with 50 mL H.sub.2O and
50 mL dichloromethane, the aqueous phase extracted with 3.times.30
mL dichloromethane. The collected organic phases are then dried
over MgSO.sub.4, filtrated and the solvent removed under reduced
pressure. The final residue is purified by silica flash column
chromatography dichloromethane:diethyl ether 4:1, typical yields
95-100% (quantitative).
[0096] 1H NMR (400 MHz, CDCl3-d6): .delta. 2.56-2.41 (m, 2H); 2.48
(s, 1H); 1.87 (m, 2H); 1.00 (m, 1H); 0.80 (m, 1H); 0.90 (s, 3H),
0.83 (s, 3H).
Example 12
UC2027-3.alpha.-ethynyl, 3.beta.-hydroxyl, androstan-17-one
oxime
[0097] The title compound is obtained with the same procedure
described for UC2025, starting from the corresponding
3.beta.-ethynyl, 3.alpha.-hydroxyl, androstan-17-one, which is
obtained as a byproduct from the reaction described for the
synthesis of UC2021.
[0098] .sup.1H NMR (400 MHz, CDCl.sub.3-d.sub.6): .delta. 2.51-2.47
(m, 2H); 2.43 (s, 1H); 1.00 (m, 1H); 0.80 (m, 1H); 0.90 (s, 3H),
0.83 (s, 3H).
* * * * *