Note: Descriptions are shown in the official language in which they were submitted.
CA 02501228 2005-04-05
WO 2004/037251 PCT/GB2003/004590
INHIBITORS OF 11-BETA-HYDROXY STEROID DEHYDROGENASE TYPE 1 AND TYPE 2
FIELD OF INVENTION
The present invention relates to a compound. In particular the present
invention
provides compounds capable of inhibiting 11 ~i-hydroxysteroid dehydrogenase
(11 (3-
HSD).
Introduction
The role of glucocorticoids
Glucocorticoids are synthesised in the adrenal cortex from cholesterol. The
principle
glucocorticoid in the human body is cortisol, this hormone is synthesised and
secreted in
response to the adrenocortictrophic hormone (ACTH) from the pituitary gland in
a
circadian, episodic manner, but the secretion of this hormone can also be
stimulated by
stress, exercise and infection. Cortisol circulates mainly bound to
transcortin (cortisol
binding protein) or albumin and only a small fraction is free (5-10%) for
biological
processes [7 ].
Cortisol has a wide range of physiological effects, including regulation of
carbohydrate,
protein and lipid metabolism, regulation of normal growth and development,
influence on
cognitive function, resistance to stress and mineralocorticoid activity.
Cortisol works in
the opposite direction compared to insulin meaning a stimulation of hepatic
gluconeogenesis, inhibition of peripheral glucose uptake and increased blood
glucose
concentration. Glucocorticoids are also essential in the regulation of the
immune
response. When circulating at higher concentrations glucocorticoids are
immunosuppressive and are used pharmacologically as anti-inflammatory agents.
Glucocorticoids like other steroid hormones are lipophilic and penetrate the
cell
membrane freely. Cortisol binds, primarily, to the intracellular
glucocorticoid receptor
(GR) that then acts as a transcription factor to induce the expression of
glucocorticoid
responsive genes, and as a result of that protein synthesis.
CA 02501228 2005-04-05
WO 2004/037251 PCT/GB2003/004590
2
The role of the 11 j3-HSD enzyme
The conversion of cortisof (F) to its inactive metabolite cortisone (E) by 11
[3-HSD was
first described in the 1950's, however it was not until later that the
biological importance
for this conversion was suggested [2]. In 1983 Krozowski et al. showed that
the
mineralocorticoid receptor (MR) has equal binding affinities for
glucocorticoids and
mineralocorticoids [3]. Because the circulating concentration of cortisol is a
100 times
higher than that of aldosterone and during times of stress or high activity
even more, it
was not clear how the MR remained mineralocorticoid specific and was not
constantly
occupied by glucocorticoids. Earlier Ulick et al. j4] had described the
hypertensive
condition known as, "apparent mineralocorfiicoid excess" (AME), and observed
that
whilst secretion of aldosterone from the adrenals was in fact fow the
peripheral
' metabolism of cortisol was disrupted. These discoveries lead to the
suggestion of a
protective role for the enzymes. By converting cortisol to cortisone in
mineralocorticoid
dependent tissues 11 (3-HSD enzymes protects the MR from occupation by
glucocorticoids and allows it to be mineralcorticoid specific. Aldosterone
itself is
protected from the enzyme by the presence of an aldehyde group at the C-18
position.
Congenital defects in the 19 [3-HSD enzyme results in over occupation of the
MR by
cortisol and hypertensive and hypokalemic symptoms seen in AME.
Localisation of the 11 [3-HSD showed that the enzyme and its activity is
highly present in
the MR dependent tissues, kidney and parotid. However in tissues where the MR
is not
mineralocorticoid specific and is normally occupied by glucocorticoids, 11 J3-
HSD is not
present in these tissues, for example in the heart and hippocampus [5]. This
research
also showed that inhibition of 11 (3-HSD caused a loss of the aldosterone
specificity of
the MR in these mineralocorticoid dependent tissues.
It has been shown that two iso-enzymes of 11 [i-HSD exist. Both are members of
the
sHort chain alcohol dehydrogenase (SCAD) superfamily which have been widely
conserved throughout evolution. 11 ji-HSD type 2 acts as a dehydrogenase to
convert
the secondary alcohol group at the C-11 position of cortisol to a secondary
ketone, so
producing the less active metabolite cortisone. 11 a-HSD type 1 is thought to
act mainly
in vivo as a reductase, that is in the opposite direction to type 2 [6] jsee
below]. 11 [3-
HSD type 1 and type 2 have only a 30% amino acid homology.
CA 02501228 2005-04-05
WO 2004/037251 PCT/GB2003/004590
3
Fi.aUH (:Li~Uf~t
C01~C1SOI COt'~ISOII~
~,..~-0 C-=:=n
~i~_ '~ __.~;~; 11 f3 HSD '~"'yl~e2 ~ ...<~~i
/ 11(3 .f~S~ TVtat'l ;~~''
11 [3-HSD enzyme activity
The intracellular activity of cortisol is dependent on the concentration of
glucocorticoids
and can be modified and independently controlled without involving the overall
secretion
and synthesis of the hormone.
The role of 11 (3-HSD Type 1
The direction of 11 (3-HSD type 1 reaction in vivo is generally accepted to be
opposite to
the dehydrogenation of type 2. In vivo homozygous mice with a disrupted type 1
gene
are unable to convert cortisone to cortisol, giving further evidence for the
reductive
activity of the enzyme [7]. 11 (3-HSD type 1 is expressed in many key
glucocorticoid
regulated tissues like the fiver, pituitary, gonad, brain, adipose and
adrenals ,however,
the function of the enzyme in many of these tissues is poorly understood [8].
The concentration of cortisone in the body is higher than that of cortisol ,
cortisone also
binds poorly to binding globulins, making cortisone many times more
biologically
available. Although cortisol is secreted by the adrenal cortex, there is a
growing amount
of evidence that the intracellular conversion of E to F may be an important
mechanism in
regulating the action of glucocorticoids [9].
It may be that 11 [3-HSD type 1 allows certain tissues to convert cortisone to
cortisol to
increase local glucocorticoid activity and potentiate adaptive response and
counteracting
the type 2 activity that could result in a fall in active glucocorticoids
[10]. Potentiation of
the stress response would be especially important in the brain and high levels
of 11 [i-
HSD type 1 are found around the hippocampus, further proving the role of the
enzyme.
11 ~i-HSD type 1 also seems to play an important role in hepatocyte maturation
[8].
Another emerging role of the 11 (3-HSD type 1 enzyme is in the detoxification
process of
CA 02501228 2005-04-05
WO 2004/037251 PCT/GB2003/004590
4
many non-steroidal carbonyl compounds, reduction of the carbonyl group of many
toxic
compounds is a common way to increase solubility and therefore increase their
excretion. The 11 (3-HSD type1 enzyme has recently been shown to be active in
lung
tissue [11]. Type 1 activity is not seen until after birth, therefore mothers
who smoke
during pregnancy expose their children to the harmful effects of tobacco
before the child
is able to metabolically detoxify this compound.
The role of 11 (3-HSD Type 2
As already stated earlier the 11 [i-HSD type 2 converts cortisol to cortisone,
thus
protecting the MR in many key regulatory tissues of the body. The importance
of
protecting the MR from occupation by glucocorticoids is seen in patients with
AME or
liquorice intoxification. Defects or inactivity of the type 2 enzyme results
in hyperterisive
syndromes and research has shown that patients with an hypertensive syndrome
have
an increased urinary excretion ratio of cortisol : cortisone. This along with
a reported
increase in the half life of radiolabelled cortisol suggests a reduction of 11
(3-HSD type 2
activity [12].
Rationale for the development of 11 [3-HSD inhibitors
As said earlier cortisol opposes the action of insulin meaning a stimulation
of hepatic
gluconeogenesis, inhibition of peripheral glucose uptake and increased blood
glucose
concentration. The effects of cortisol appear to be enhanced in patients
suffering from
glucose intolerance or diabetes mellitus. Inhibition of the enzyme 11 (3-HSD
type 1
would increase glucose uptake and inhibit hepatic gluconeogenesis, giving a
reduction in
circulatory glucose levels. The development of a potent 11 [3-HSD type 1
inhibitor could
therefore have considerable therapeutic potential for conditions associated
with elevated
blood glucose levels.
An excess in glucocorticoids can result in neuronal dysfunctions and also
impair
cognitive functions. A specific 11 [3-HSD type 1 inhibitor might be of some
importance
by reducing neuronal dysfunctions and the loss of cognitive functions
associated with
ageing, by blocking the conversion of cortisone to cortisol.
Glucocorticoids also have an important role in regulating part of the immune
response
CA 02501228 2005-04-05
WO 2004/037251 PCT/GB2003/004590
[13]. Glucocorticoids can suppress the production ofi cytokines and regulate
the receptor
levels. They are also involved in determining whether T-helper (Th)
lymphocytes
progress into either Th1 or Th2 phenotype. These two different types of Th
cells secrete
a dififerent profile of cytokines, Th2 is predominant in a glucocorticoid
environment. By
5 inhibiting 11 [i-HSD type 1, Th1 cytokine response would be favoured. It is
also possible
to inhibit 11 ~i-HSD type 2 , thus by inhibiting the inactivation ofi
cortisol, it may be
possible to potentiate the anti-inflammatory effects of glucocorticoids.
Aspects of the invention are defined in the appended claims.
SUMMARY ASPECTS OF THE PRESENT INVENTION
In one aspect the present invention provides a compound having Formula I
R~ N Formula I
R3
~X
R2
wherein one of R, and R2 is a group ofi the formula
O
R5
O N
Ra
wherein R4 is selected from H and hydrocarbyl, R5 is a hydrocarbyl group and L
is an
optional linker group,
or R, and RZ together form a ring substituted with the group
O
R5 Il~ ~~~
O N
R4
wherein R3 is H or a substituent, and wherein X is selected from S, O, NR6 and
C(R,)(R$), wherein R6 is selected from H and hydrocarbyl groups, wherein each
of R,
and Ra are independently selected from H and hydrocarbyl groups.
CA 02501228 2005-04-05
WO 2004/037251 PCT/GB2003/004590
6
In one aspect the present invention provides a pharmaceutical composition
comprising
(i) a compound having Formula 1
R~ N Formula I
R3
~X
R2
wherein one of R, and R2 is a group of the formula
O
R5
O N
R~
wherein R4 is selected from H and hydrocarbyl, R5 is a hydrocarbyl group and L
is an
optional linker group, or R, and RZ together form a ring substituted with the
group
O
R5
O N
Ra
wherein R3 is H or a substituent, and wherein X is selected from S, O, NR6 and
C(R,)(R8), wherein R6 is selected from H and hydrocarbyl groups, wherein each
of R,
and R$ are independently selected from H and hydrocarbyl groups.
(ii) optionally admixed with a pharmaceutically acceptable carrier, diluent,
excipient or
adjuvant.
In one aspect the present invention provides a compound having Formula I
R~ N Formula I
R3
~X
R2
wherein one of R~ and RZ is a group of the formula
CA 02501228 2005-04-05
WO 2004/037251 PCT/GB2003/004590
7
O
R5
O N
R4
wherein R4 is selected from H and hydrocarbyl, R5 is a hydrocarbyl group and L
is an
optional linker group, or R, and Ra together form a ring substituted with the
group
O
R5
O N
Ra
wherein R3 is H or a substituent, and wherein X is selected from S, O, NR6 and
C(R,)(R8), wherein R6 is selected from H and hydrocarbyl groups, wherein each
of R,
and R8 are independently selected from H and hydrocarbyl groups, for use in
medicine.
In one aspect the present invention provides a use of a compound in the
manufacture of
a medicament for use in the therapy of a condition or disease associated with
11 (3-HSD,
wherein the compound has Formula I
R~ N Formula I
R3
~X
R2
wherein one of R, and R2 is a group of the formula
O
R5
O N
R4
wherein R4 is selected from H and hydrocarbyl, R5 is a hydrocarbyl group and L
is an
optional linker group, or R, and R2 together form a ring substituted with the
group
CA 02501228 2005-04-05
WO 2004/037251 PCT/GB2003/004590
8
O
R5
O N
R4
wherein R3 is H or a substituent, and wherein X is selected from S, O, NR6 and
C(R,)(R$), wherein R6 is selected from H and hydrocarbyl groups, wherein each
of R,
and R$ are independently selected from H and hydrocarbyl groups.
SOME ADVANTAGES
One key advantage of the present invention is that the compounds of the
present
invention can act as 11 (3-HSD inhibitors. The compounds may inhibit the
interconversion
of inactive 11-keto steroids with their active hydroxy equivalents. Thus
present invention
provides methods by which the conversion of the inactive to the active form
may be
controlled, and to useful therapeutic effects which may be obtained as a
result of such
control. More specifically, but not exclusively, the invention is concerned
with
interconversion between cortisone and cortisol in humans.
Another advantage of the compounds of the present invention is that they may
be potent
11 (3-HSD inhibitors in vivo.
Some of the compounds of the present invention are also advantageous in that
they may
be orally active.
The present invention may provide for a medicament for one or more of (i)
regulation of
carbohydrate metabolism, (ii) regulation of protein metabolism, (iii)
regulation of lipid
metabolism, (iv) regulation of normal growth and/or development, (v) influence
on
cognitive function, (vi) resistance to stress and mineralocorticoid activity.
Some of the compounds of the present invention may also be useful for
inhibiting hepatic
gluconeogenesis. The present invention may also provide a medicament to
relieve the
effects of endogenous glucocorticoids in diabetes mellitus, obesity (including
centripetal
obesity), neuronal loss and/or the cognitive impairment of old age. Thus, in a
further
aspect, the invention provides the use of an inhibitor of 11 (3-HSD in the
manufacture of a
medicament for producing one or more therapeutic effects in a patient to whom
the
CA 02501228 2005-04-05
WO 2004/037251 PCT/GB2003/004590
9
medicament is administered, said therapeutic effects selected from inhibition
of hepatic
gluconeogenesis, an increase in insulin sensitivity in adipose tissue and
muscle, and the
prevention of or reduction in neuronal loss/cognitive impairment due to
glucocorticoid-
potentiated neurotoxicity or neural dysfunction or damage.
From an alternative point of view, the invention provides a method of
treatment of a
human or animal patient suffering from a condition selected from the group
consisting of:
hepatic insulin resistance, adipose tissue insulin resistance, muscle insulin
resistance,
neuronal loss or dysfunction due to glucocorticoid potentiated neurotoxicity,
and any
combination of the aforementioned conditions, the method comprising the step
of
administering to said patient a medicament comprising a pharmaceutically
active amount
of a compound in accordance with the present invention.
Some of the compounds of the present invention may be useful for the treatment
of
cancer, such as breast cancer, as well as (or in the alternative) non-
malignant
conditions, such as the prevention of auto-immune diseases, particularly when
pharmaceuticals may need to be administered from an early age.
DETAILED ASPECTS OF THE PRESENT INVENTION
In one aspect the present invention provides a compound having Formula I
R~ N Formula I
R3
~X
R2
wherein one of R, and R~ is a group of the formula
O
R5
O N
R~
wherein R4 is selected from H and hydrocarbyl, R5 is a hydrocarbyl group and L
is an
optional linker group, or R, and R~ together form a ring substituted with the
group
CA 02501228 2005-04-05
WO 2004/037251 PCT/GB2003/004590
O
R5
O N
R4
wherein R3 is H or a substituent, and wherein X is selected from S, O, NR6 and
C(R,)(R8), wherein R6 is selected from H and hydrocarbyl groups, wherein each
of R,
and R$ are independently selected from H and hydrocarbyl groups.
5
In one aspect the present invention provides a pharmaceutical composition
comprising
(i) a compound having Formula I defined above
(ii) optionally admixed with a pharmaceutically acceptable carrier, diluent,
excipient or
adjuvant.
In one aspect the present invention provides a compound having Formula I
defined
above, for use in medicine.
In one aspect the present invention provides a use of a compound having
Formula I
defined above in the manufacture of a medicament for use in the therapy of a
condition
or disease associated with 11 (3-HSD.
In one aspect the present invention provides a use of a compound having
Formula I
defined above in the manufacture of a medicament for use in the therapy of a
condition
or disease associated with adverse 11 ~i-HSD levels.
In one aspect the present invention provides a use of a compound having
Formula I
defined above in the manufacture of a pharmaceutical for inhibiting 11 ~i-HSD
activity.
In one aspect the present invention provides a use of a compound having
Formula I
defined above in the manufacture of a pharmaceutical for inhibiting 11 ~i-HSD
activity.
In one aspect the present invention provides a method comprising (a)
performing a 11(3-
HSD assay with one or more candidate compounds having Formula I defined above;
(b)
determining whether one or more of said candidate compounds is/are capable of
modulating 11 ~3-HSD activity; and (c) selecting one or more of said candidate
compounds that is/are capable of modulating 11 ~i-HSD activity.
CA 02501228 2005-04-05
WO 2004/037251 PCT/GB2003/004590
11
In one aspect the present invention provides a method comprising (a)
performing a 11 ~i-
HSD assay with one or more candidate compounds having Formula I defined above;
(b)
determining whether one or more of said candidate compounds islare capable of
inhibiting 11 (3-HSD activity; and (c) selecting one or more of said candidate
compounds
that is/are capable of inhibiting 11 (3-HSD activity.
In one aspect the present invention provides
~ a compound identified by the above method,
~ the use of the said compound in medicine,
~ a pharmaceutical composition comprising the said compound, optionally
admixed with
a pharmaceutically accepfiable carrier, diluent, excipient or adjuvant,
~ use of the said compound in the manufacture of a medicament for use in the
therapy
of a condition or disease associated with 11 ~i-HSD, and
~ use of the said compound in the manufacture of a medicament for use in the
therapy
of a condition or disease associated with adverse 11 ~i-HSD levels.
For ease of reference, these and further aspects of the present invention are
now
discussed under appropriate section headings. However, the teachings under
each
section are not necessarily limited to each particular section.
PREFERABLE ASPECTS
In one preferred aspect the compound of the present invention has Formula II
R~ N Formula il
Rs
S
R2
In one preferred aspect L is not present. In this aspect the present invention
provides a
compound having Formula I
CA 02501228 2005-04-05
WO 2004/037251 PCT/GB2003/004590
12
R~ N Formula I
R3
~X
R2
wherein one of R, and R2 is a group of the formula
O
R5 ~~\N~
O
R4
wherein R4 is selected from H and hydrocarbyl, and R5 is a hydrocarbyl group;
or R, and
R2 together form a ring substituted with the group
O
R5 ~~\N~
O
Ra
wherein R3 is H or a substituent
In ane preferred aspect the compound of the present invention R, and R2
together form
a ring substituted with the group
O
R5
O N
1
Ra
In one preferred aspect the compound of the present invention R~ and Rz
together form
a carbocyclic ring.
In one preferred aspect the compound of the present invention R, and R~
together form
a six membered ring.
In one preferred aspect the compound of the present invention R1 and RZ
together form
a six membered carbocyciic ring.
CA 02501228 2005-04-05
WO 2004/037251 PCT/GB2003/004590
13
In one preferred aspect the compound of the present invention wherein R~ and
R2
together form an aryl ring.
Preferred compounds of the present invention are those having one of the
following
formulae.
O ' N Formula III
R II L I R
II~N~ ~ ~ S 3
w
O
R4
O \ N Formula IV
R II L I ~ R
5 I~~N~ ~ ~ S 3
w
O
R4
O Formula V
R II ~ N
5
I ~ 'N I ~ Rs
O I I ~ S
R4
O Formula VI
R5 I I ~ N
O R4 ~ R3
S
O Formula Vla
N
R5 I I I L
O R4 ~ R3
S
CA 02501228 2005-04-05
WO 2004/037251 PCT/GB2003/004590
14
N Formula VII
O ~ R3
R (I N ~ S
0 R4
N Formula Vlla
O ~ R3
R II N ~ ~ S
5
O R4
In preferred aspects of the present invention R3 is selected from H,
hydrocarbyl, -S-
hydrocarbyl, -S-H, halogen and N(R9)(R,o), wherein each of R9 and R,o are
independently selected from H and hydrocarbyl groups.
5
In preferred aspects of the present invention R3 is selected from H, hydroxy,
alkyl
especially C,-C,o alkyl groups, C,-Cs alkyl, e.g. C~-C3 alkyl group, methyl,
ethyl, n-propyl,
isopropyl, n-butyl, sec-butyl, tert-butyl, n-pentyl and other pentyl isomers,
and n-hexyl and
other hexyl isomers, alkoxy especially C,-C,o alkoxy groups, C,-C6 alkoxy,
e.g. C~-C3
alkoxy group, methoxy, ethoxy, propoxy etc., alkinyl, e.g. ethinyl, or
halogen, e.g. fluoro
substituents.
When R3 is -S-hydrocarbyl, preferably R3 is selected from -S-alkyl, -S-
carboxylic acid,
S-ether, and -S-amide, preferably selected from -S-C,-,oalkyl, -S- C,-
,ocarboxcylic acid,
-S- C,-,nether, and -S- C,-,oamide.
In preferred aspects of the present invention R3 is -CH3.
Further preferred compounds of the present invention are those having one of
the
following formulae.
R~ N Formula la
R3
~X
R2
CA 02501228 2005-04-05
WO 2004/037251 PCT/GB2003/004590
O \ N Formula VIII
R ~ I ~ I Rs
5
II\N/ I ~ S
R4
O Formula IX
R II ~ N
5
~I~N ~ R3
S
4
O Formula X
R5 I I ~ N
O R4 ~ R3
S
O Formula Xa
R5 II I L ~ N
O R4 ~ R3
S
N Formula XI
O ~ a Rs
R II N ~ S
5
O R4
N Formula Xla
O R3
R II N ~ ~ S
5
O R4
In further preferred aspects of the present invention, such as when the
compound has
Formula la, Formula VIII, Formula IX, Formula X, Formula Xa, Formula XI, or
Formula
CA 02501228 2005-04-05
WO 2004/037251 PCT/GB2003/004590
16
Xla, R3 is selected from O, hydrocarbyl, and N(R9) wherein Rg is selected from
H and
hydrocarbyl groups. More preferably R3 is selected from O, C,-Coo alkenyl
groups, such
as C~-C6 alkenyl group, and C,-C3 alkenyl group, NH and N-C,-C,o alkyl groups,
such as
N-C,-C6 alkyl group, and N-C,-C3 alkyl groups.
In further preferred aspects of the present invention R4 is selected from H
and C,-Coo
alkyl groups, such as C,-C6 alkyl group, and C,-C3 alkyl group. Preferably R4
is H.
In further preferred aspects of the present invention R4 is a group of the
formula.
O S O
Rs
In these aspects the group shown above as
O
R5
O N
Ra
may be of the formula
O
R5
O N
O S O
R5
wherein each R5 °is independently selected from hydrocarbyl groups.
Each R5 may be the
same of different to the other R5. In one aspect the two R5 groups are the
same.
In some preferred aspects of the invention R5 is a cyclic hydrocarbyl group.
Preferably R5
is a cyclic hydrocarbyl group comprising a hydrocarbon ring.
R5 may be a substituted ring or an unsubstituted ring. In some preferred
aspects of the
invention R5 is substituted ring.
CA 02501228 2005-04-05
WO 2004/037251 PCT/GB2003/004590
17
Preferably R5 is a carbocyclic ring.
Preferably R5 is a six membered ring.
Preferably R5 is a six membered carbocyclic ring. More preferably R5 is a
substituted six
membered carbocyclic ring.
In some preferred aspects of the invention R5 is an aryl ring. Preferably R5
is a
substituted aryl ring.
In one highly preferred aspect R5 is a group having the formula
R,a
R~s
wherein each of R", R,z, R13, R,4 and R~5 are independently selected from H,
halogen, and
hydrocarbyl groups.
Preferably each of R~,, R,z, R13, R~4 and R,5 are independently selected from
H, halogen,
alkyl, such as C,_6 alkyl, phenyl, O-alkyl, O-phenyl, nitrite, haloaikyl, such
as CF3, CCi3
and CBr3, carboxyalkyl, -C02H, COzalkyl, and NH-acetyl groups..
Two or more of R", R~z, R,3, R,4 and R,5 may join to form a ring. The two or
more of R",
R,z, R,3, R~4 and R,5 may or may not be adjacent. The ring may be carbocyclic
or
heterocyclic ring. The ring may be optionally substituted by any of the R,~,
R,z, Ry3, R,4
and R,5 substituents listed above. When two or more of R", R,z, R~3, R,4 and
R,5 may join
to form a ring the group
CA 02501228 2005-04-05
WO 2004/037251 PCT/GB2003/004590
18
R1d
R~s
R12
may provide a naphthyl, quinolyl, tetrahydroquinolyl, or
benzothtrahydropyranyl, each of
which may be substituted or unsubstituted.
SUBSTITUENTS
The compound of the present invention may have substituents other than those
of the
ring systems show herein. Furthermore the ring systems herein are given as
general
formulae and should be interpreted as such. The absence of any specifically
shown
substituents on a given ring member indicates that the ring member may
substituted with
any moiety of which H is only one example. The ring system may contain one or
more
degrees of unsaturation, for example is some aspects one or more rings of the
ring
system is aromatic. The ring system may be carbocyclic or may contain one or
more
hetero atoms.
The compound of the invention, in particular the ring system compound of the
invention
of the present invention may contain substituents other than those show
herein. By way
of example, these other substituents may be one or more of: one or more halo
groups,
one or more O groups, one or more hydroxy groups, one or more amino groups,
one or
more sulphur containing group(s), one or more hydrocarbyl groups) - such as an
oxyhydrocarbyl group.
In general terms the ring system of the present compounds may contain a
variety of non-
interfering substituents. In particular, the ring system may contain one or
more hydroxy,
alkyl especially lower (C~-C6) alkyl, e.g. methyl, ethyl, n-propyl, isopropyl,
n-butyl, sec-
butyl, tert-butyl, n-pentyl and other pentyl isomers, and n-hexyl and other
hexyl isomers,
alkoxy especially lower (C,-C6) alkoxy, e.g. methoxy, ethoxy, propoxy etc.,
alkinyl, e.g.
ethinyl, or halogen, e.g. fluoro substituents.
CA 02501228 2005-04-05
WO 2004/037251 PCT/GB2003/004590
19
For some compounds of the present invention, the compound may be substituted
with a
hydrocarbylsulphanyl group. The term "hydrocarbylsulphanyl" means a group that
comprises at least hydrocarbyl group (as herein defined) and sulphur,
preferably -S-
hydrocarbyl, more preferably -S-hydrocarbon. That sulphur group may be
optionally
oxidised.
Preferably the hydrocarbylsulphanyl group is -S-C~_10 alkyl, more preferably -
S-C,_5 alkyl,
more preferably -S-C,_3 alkyl, more preferably -S-CHaCH~CH3, -S-CHZCH~ or -
SCH3
FURTHER ASPECTS
For some applications, preferably the compounds have a reversible action.
For some applications, preferably the compounds have an irreversible action.
In one embodiment, the compounds of the present invention are useful for the
treatment
of breast cancer.
The compounds of the present invention may be in the form of a salt.
The present invention also covers novel intermediates that are useful to
prepare the
compounds of the present invention. For example, the present invention covers
novel
alcohol precursors for the compounds. By way of further example, the present
invention
covers bis protected precursors for the compounds. Examples of each of these
precursors are presented herein. The present invention also encompasses a
process
comprising each or both of those precursors for the synthesis of the compounds
of the
present invention.
STEROID DEHYDROGENASE
11 f3 Steroid dehydrogenase may be referred to as "11 f3-HSD" or "HD" for
short
In some aspects of the invention 11 (3-HSD is preferably 11 (3-HSD Type 1.
In some aspects of the invention 11 (3-HSD is preferably 11 (3-HSD Type 2.
CA 02501228 2005-04-05
WO 2004/037251 PCT/GB2003/004590
STEROID DEHYDROGENASE INHIBITION
It is believed that some disease conditions associated with HD activity are
due to
conversion of a inactive, cortisone to an active, cortisol. In disease
conditions
5 associated with HD activity, it would be desirable to inhibit HD activity.
Here, the term "inhibit" includes reduce and/or eliminate and/or mask and/or
prevent the
detrimental action of HD.
10 HD INHIBITOR
In accordance with the present invention, the compound of the present
invention is
capable of acting as an HD inhibitor.
15 Here, the term "inhibitor" as used herein with respect to the compound of
the present
invention means a compound that can inhibit HD activity - such as reduce
and/or
eliminate and/or mask and/or prevent the detrimental action of HD. The HD
inhibitor
may act as an antagonist.
20 The ability of compounds to inhibit steroid dehydrogenase activity can be
assessed
using the suitable Assay Protocol presented in the Examples section.
It is to be noted that the compound of the present invention may have other
beneficial
properties in addition to or in the alternative to its ability to inhibit HD
activity.
HYDROCARBYL
The term "hydrocarbyl group" as used herein means a group comprising at least
C and
H and may optionally comprise one or more other suitable substituents.
Examples of
such substituents may include halo, alkoxy, nitro, an alkyl group, a cyclic
group etc. In
addition to the possibility of the substituents being a cyclic group, a
combination of
substituents may form a cyclic group. If the hydrocarbyl group comprises more
than one
C then those carbons need not necessarily be linked to each other. For
example, at
least two of the carbons may be linked via a suitable element or group. Thus,
the
hydrocarbyl group may contain hetero atoms. Suitable hetero atoms will be
apparent to
those skilled in the art and include, for instance, sulphur, nitrogen and
oxygen. A non-
CA 02501228 2005-04-05
WO 2004/037251 PCT/GB2003/004590
21
limiting example of a hydrocarbyl group is an acyl group.
A typical hydrocarbyl group is a hydrocarbon group. Here the term
"hydrocarbon"
means any one of an alkyl group, an alkenyl group, an alkynyl group, which
groups may
be linear, branched or cyclic, or an aryl group. The term hydrocarbon also
includes
those groups but wherein they have been optionally substituted. If the
hydrocarbon is a
branched structure having substituent(s) thereon, then the substitution may be
on either
the hydrocarbon backbone or on the branch; alternatively the substitutions may
be on
the. hydrocarbon backbone and on the branch.
In some aspects of the present invention, one or more hydrocarbyl groups is
independently selected from optionally substituted alkyl group, optionally
substituted
haloalkyl group, aryl group, alkylaryl group, alkylarylakyl group, and an
alkene group.
In some aspects of the present invention, one or more hydrocarbyl groups is
independently selected from C1-C1o alkyl group, such as C1-C6 alkyl group, and
C1-C3
alkyl group. Typical alkyl groups include C1 alkyl, C2 alkyl, C3 alkyl, C4
alkyl, C5 alkyl, C,
alkyl, and Ca alkyl.
In some aspects of the present invention, one or more hydrocarbyl groups is
independently selected from C1-C1o haloalkyl group, C1-C6 haloalkyl group, C1-
C3
haloalkyl group, C1-C1o bromoalkyl group, C1-C6 bromoalkyl group, and C1-C3
bromoalkyl
group. Typical haloalkyl groups include C1 haloalkyl, C2 haloalkyl, C3
haloalkyl, C4
haloalkyl, C5 haloalkyl, C7 haloalkyl, C$ haloalkyl, C1 bromoalkyl, C2
bromoalkyl, C3
bromoalkyl, C4 bromoalkyl, C5 bromoalkyl, C, bromoalkyl, and C$ bromoalkyl.
In some aspects of the present invention, one or more hydrocarbyl groups is
independently selected from aryl groups, alkylaryl groups, alkylarylakyl
groups, -(CH2)1_
1o-aryl, -(CH~)1-1o-Ph, (CHZ)1-1o-Ph-C1_1o alkyl, -(CH2)1-5 Ph, (CH2)1-s-Ph-
C1_5 alkyl, -(CH~)1-s-
Ph, (CH2)1.~-Ph-C1~ alkyl, -CHZ-Ph, and -CHZ Ph-C(CH3)3. The aryl groups may
contain a
hetero atom. Thus the aryl group or one or more of the aryl groups may be
carbocyclic
or more may heterocyclic. Typical hetero atoms include O, N and S, in
particular N.
In some aspects of the present invention, one or more hydrocarbyl groups is
independently selected from -(CH2)1_1o-cycloalkyl, -(CH~)1_10-Ca-locYcloalkyl,
-(CHZ)1_,-C3_
,cycloalkyl, -(CHZ)1_5-Ca-scYcloalkyl, -(CH~)1_3-C3_5cycloalkyl, and -CHZ-
C3cycloalkyl.
CA 02501228 2005-04-05
WO 2004/037251 PCT/GB2003/004590
22
In some aspects of the present invention, one or more hydrocarbyl groups is
independently selected from alkene groups. Typical alkene groups include C,-
C,o alkene
group, C,-C6 alkene group, C,-C3 alkene group, such as C~, C2, C3, C4, C5, C6,
or C,
alkene group. In a preferred aspect the alkene group contains 1, 2 or 3 C=C
bonds. In
a preferred aspect the alkene group contains 1 C=C bond. In some preferred
aspect at
least one C=C bond or the only C=C bond is to the terminal C of the alkene
chain, that is
the bond is at the distal end of the chain to the ring system.
In some aspects of the present invention, one or more hydrocarbyl groups is
independently selected from oxyhydrocarbyl groups.
OXYHYDROCARBYL
The term "oxyhydrocarbyl" group as used herein means a group comprising at
least C, H
and O and may optionally comprise one or more other suitable substituents.
Examples
of such substituents may include halo-, alkoxy-, nitro-, an alkyl group, a
cyclic group etc.
In addition to the possibility of the substituents being a cyclic group, a
combination of
substituents may form a cyclic group. If the oxyhydrocarbyl group comprises
more than
one C then those carbons need not necessarily be linked to each other. For
example, at
least two of the carbons may be linked via a suitable element or group. Thus,
the
oxyhydrocarbyl group may contain hetero atoms. Suitable hetero atoms will be
apparent
to those skilled in the art and include, for instance, sulphur and nitrogen.
In one embodiment of the present invention, the oxyhydrocarbyl group is a
oxyhydrocarbon group.
Here the term "oxyhydrocarbon" means any one of an alkoxy group, an oxyalkenyl
group, an oxyalkynyl group, which groups may be linear, branched or cyclic, or
an
oxyaryl group. The term oxyhydrocarbon also includes those groups but wherein
they
have been optionally substituted. If the oxyhydrocarbon is a branched
structure having
substituent(s) thereon, then the substitution may be on either the hydrocarbon
backbone
or on the branch; alternatively the substitutions may be on the hydrocarbon
backbone
and on the branch.
Typically, the oxyhydrocarbyl group is of the formula C,~O (such as a C,_3O).
CA 02501228 2005-04-05
WO 2004/037251 PCT/GB2003/004590
23
ANIMAL ASSAY MODEL FOR DETERMINING OESTROGENIC ACTIVITY
(PROTOCOL 1 )
Lack of in vivo oestrogenicity
The compounds of the present invention may be studied using an animal model,
in
particular in ovariectomised rats. In this model, compounds which are
oestrogenic
stimulate uterine growth.
The compound (10 mgiKgiday for five days) was administered orally to rats with
another
group of animals receiving vehicle only (propylene glycol). A further group
received the
estrogenic compound EMATE subcutaneously in an amount of 10pglday for five
days.
At the end of the study uteri were obtained and weighed with the results being
expressed
as uterine weightiwhole body weight x 100.
Compounds having no significant effect on uterine growth are not oestrogenic.
REPORTERS
A wide variety of reporters may be used in the assay methods (as well as
screens) of the
present invention with preferred reporters providing conveniently detectable
signals (e.g.
by spectroscopy). By way of example, a reporter gene may encode an enzyme
which
catalyses a reaction which alters light absorption properties.
Other protocols include enzyme-linked immunosorbent assay (ELISA),
radioimmunoassay (RIA) and fluorescent activated cell sorting (FACS). A two-
site,
monoclonal-based immunoassay utilising monoclonal antibodies reactive to two
non-
interfering epitopes may even be used. These and other assays are described,
among
other places, in Hampton R et al (1990, Serological Methods, A Laboratory
Manual, APS
Press, St Paul MN) and Maddox DE et al (1983, J Exp Med 15 8:121 1 ).
Examples of reporter molecules include but are not limited to ((3-
galactosidase,
invertase, green fluorescent protein, luciferase, chloramphenicol,
acetyltransferase, (-
glucuronidase, exo-glucanase and glucoamylase. Alternatively, radiolabelled or
fluorescent tag-labelled nucleotides can be incorporated into nascent
transcripts which
CA 02501228 2005-04-05
WO 2004/037251 PCT/GB2003/004590
24
are then identified when bound to oligonucleotide probes.
By way of further examples, a number of companies such as Pharmacia Biotech
(Piscataway, NJ), Promega (Madison, WI), and US Biochemical Corp (Cleveland,
OH)
supply commercial kits and protocols for assay procedures. Suitable reporter
molecules
or labels include those radionuclides, enzymes, fluorescent, chemiluminescent,
or
chromogenic agents as well as substrates, cofactors, inhibitors, magnetic
particles and
the like. Patents teaching the use of such labels include US-A-3817837; US-A-
3850752;
US-A-3939350; US-A-3996345; US-A-4277437; US-A-4275149 and US-A-4366241.
HOST CELLS
The term "host cell" - in relation to the present invention includes any cell
that could
comprise the target for the agent of the present invention.
IS
Thus, a further embodiment of the present invention provides host cells
transformed or
transfected with a polynucleotide that is or expresses the target of the
present invention.
Preferably said polynucleotide is carried in a vector for the replication and
expression of
polynucleotides that are to be the target or are to express the target. The
cells will be
chosen to be compatible with the said vector and may for example be
prokaryotic (for
example bacterial), fungal, yeast or plant cells.
The gram negative bacterium E. coli is widely used as a host for heterologous
gene
expression. However, large amounts of heterologous protein terid to accumulate
inside
the cell. Subsequent purification of the desired protein from the bulk of
E.coli
intracellular proteins can sometimes be difficult.
In contrast to E.coli, bacteria from the genus Bacillus are very suitable as
heterologous
hosts because of their capability to secrete proteins into the culture medium.
Other
bacteria suitable as hosts are those from the genera Streptomyces and
Pseudomonas.
Depending on the nature of the polynucleotide encoding the polypeptide of the
present
invention, and/or the desirability for further processing of the expressed
protein,
eukaryotic hosts such as yeasts or other fungi may be preferred. In general,
yeast cells
are preferred over fungal cells because they are easier to manipulate.
However, some
proteins are either poorly secreted from the yeast cell, or in some cases are
not
CA 02501228 2005-04-05
WO 2004/037251 PCT/GB2003/004590
processed properly (e.g. hyperglycosylation in yeast). In these instances, a
different
fungal host organism should be selected.
Examples of suitable expression hosts within the scope of the present
invention are fungi
5 such as Aspergillus species (such as those described in EP-A-0184438 and EP-
A-
0284603) and Trichoderma species; bacteria such as Bacillus species (such as
those
described in EP-A-0134048 and EP-A-0253455), Streptomyces species and
Pseudomonas species; and yeasts such as ICluyveromyces species (such as those
described in EP-A-0096430 and EP-A-0301670) and Saccharomyces species. By way
10 of example, typical expression hosts may be selected from Aspergillus
niger, Aspergillus
niger var. tubigenis, Aspergillus niger var. awamori, Aspergillus aculeatis,
Aspergillus
nidulans, Aspergillus orvzae, Trichoderma reesei, Bacillus subtilis, Bacillus
licheniformis,
Bacillus amyloliquefaciens, Kluyveromyces lactis and Saccharomyces cerevisiae.
15 The use of suitable host cells - such as yeast, fungal and plant host cells
- may provide
for post-translational modifications (e.g. myristoylation, glycosylation,
truncation,
lapidation and tyrosine, serine or threonine phosphorylation) as may be needed
to confer
optimal biological activity on recombinant expression products of the present
invention.
20 ORGANISM
The term "organism" in relation to the present invention includes any organism
that could
comprise the target according to the present invention and/or products
obtained
therefrom. Examples of organisms may include a fungus, yeast or a plant.
The term "transgenic organism" in relation to the present invention includes
any
organism that comprises the target according to the present invention andlor
products
obtained.
TRANSFORMATION OF HOST CELLS/HOST ORGANISMS
As indicated earlier, the host organism can be a prokaryotic or a eukaryotic
organism.
Examples of suitable prokaryotic hosts include E. coli and Bacillus subtilis.
Teachings
on the transformation of prokaryotic hosts is well documented in the art, for
example see
Sambrook et al (Molecular Cloning: A Laboratory Manual, 2nd edition, 1989,
Cold Spring
Harbor Laboratory Press) and Ausubel et al., Current Protocols in Molecular
Biology
CA 02501228 2005-04-05
WO 2004/037251 PCT/GB2003/004590
26
(1995), John Wiiey & Sons, lnc.
If a prokaryotic host is used then the nucleotide sequence may need to be
suitably
modified before transformation - such as by removal of introns.
In another embodiment the transgenic organism can be a yeast. in this regard,
yeast
have also been widely used as a vehicle for heterologous gene expression. The
species
Saccharomyces cerevisiae has a long history of industrial use, including its
use for
heterologous gene expression. Expression of heterologous genes in
Saccharomyces
cerevisiae has been reviewed by Goodey et al (1987, Yeast Biotechnology, D R
Berry et
al, eds, pp 401-429, Allen and Unwin, London) and by King et al (1989,
Molecular and
Cell Biology of Yeasts, E F Walton and G T Yarronton, eds, pp 107-133,
Blackie,
Glasgow).
For several reasons Saccharomyces cerevisiae is well suited for heterologous
gene
expression. First, it is non-pathogenic to humans and it is incapable of
producing certain
endotoxins. Second, it has a long history of safe use following centuries of
commercial
exploitation for various purposes. This has led to wide public acceptability.
Third, the
extensive commercial use and research devoted to the organism has resulted in
a
wealth of knowledge about the genetics and physiology as well as large-scale
fermentation characteristics of Saccharomyces cerevisiae.
A review of the principles of heterologous gene expression in Saccharomyces
cerevisiae
and secretion of gene products is given by E Hinchciiffe E Kenny (1993, "Yeast
as a
vehicle for the expression of heterologous genes", Yeasts, Vol 5, Anthony H
Rose and
J Stuart Harrison, eds, 2nd edition, Academic Press Ltd.).
Several types of yeast vectors are available, including integrative vectors,
which require
recombination with the host genome for their maintenance, and autonomously
replicating
plasmid vectors.
In order to prepare the transgenic Saccharomyces, expression constructs are
prepared
by inserting the nucleotide sequence into a construct designed for expression
in yeast.
Several types of constructs used for heterologous expression have been
developed.
The constructs contain a promoter active in yeast fused to the nucleotide
sequence,
usually a promoter of yeast origin, such as the GAL1 promoter, is used.
Usually a signal
CA 02501228 2005-04-05
WO 2004/037251 PCT/GB2003/004590
27
sequence of yeast origin, such as the sequence encoding the SUC2 signal
peptide, is
used. A terminator active in yeast ends the expression system.
For the transformation of yeast several transformation protocols have been
developed.
For example, a transgenic Saccharomyces according to the present invention can
be
prepared by following the teachings of Hinnen et al (1978, Proceedings of the
National
Academy of Sciences of the USA 75, 1929); Beggs, J D (1978, Nature, London,
275,
104); and Ito, H et al (1983, J Bacteriology 153, 163-168).
The transformed yeast cells are selected using various selective markers.
Among the
markers used for transformation are a number of auxotrophic markers such as
LEU2,
HIS4 and TRP1, and dominant antibiotic resistance markers such as
aminoglycoside
antibiotic markers, e.g. G418.
Another host organism is a plant. The basic principle in the construction of
genetically
modified plants is to insert genetic information in the plant genome so as to
obtain a
stable maintenance of the inserted genetic material. Several techniques exist
for
inserting the genetic information, the two main principles being direct
introduction of the
genetic information and introduction of the genetic information by use of a
vector system.
A review of the general techniques may be found in articles by Potrykus (Annu
Rev Plant
Physiol Plant Mol Biol [1991] 42:205-225) and Christou (Agro-Food-Industry Hi-
Tech
March/April 1994 17-27). Further teachings on plant transformation may be
found in EP-
A-0449375.
Thus, the present invention also provides a method of transforming a host cell
with a
nucleotide sequence that is to be the target or is to express the target. Host
cells
transformed with the nucleotide sequence may be cultured under conditions
suitable for
the expression of the encoded protein. The protein produced by a recombinant
cell may
be displayed on the surface of the cell. If desired, and as will be understood
by those of
skill in the art, expression vectors containing coding sequences can be
designed with
signal sequences which direct secretion of the coding sequences through a
particular
prokaryotic or eukaryotic cell membrane. Other recombinant constructions may
join the
coding sequence to nucleotide sequence encoding a polypeptide domain which
will
facilitate purification of soluble proteins (Kroll DJ et al (1993) DNA Cell
Biol 12:441-53).
CA 02501228 2005-04-05
WO 2004/037251 PCT/GB2003/004590
28
VARIANTS/HOMOLOGUES/DERIVATIVES
In addition to the specific amino acid sequences and nucleotide sequences
mentioned
herein, the present invention also encompasses the use of variants, homologue
and
derivatives thereof. Here, the term "homology" can be equated with "identity".
In the present context, an homologous sequence is taken to include an amino
acid
sequence which may be at least 75, 85 or 90% identical, preferably at least 95
or 98%
identical. Although homology can also be considered in terms of similarity
(i.e. amino
acid residues having similar chemical properties/functions), in the context of
the present
invention it is preferred to express homology in terms of sequence identity.
Homology comparisons can be conducted by eye, or more usually, with the aid of
readily
available sequence comparison programs. These commercially available computer
programs can calculate % homology between two or more sequences.
homology may be calculated over contiguous sequences, i.e. one sequence is
aligned with the other sequence and each amino acid in one sequence is
directly
compared with the corresponding amino acid in the other sequence, one residue
at a
time. This is called an "ungapped" alignment. Typically, such ungapped
alignments are
performed only over a relatively short number of residues.
Although this is a very simple and consistent method, it fails to take into
consideration
that, for example, in an otherwise identical pair of sequences, one insertion
or deletion
will cause the following amino acid residues to be put out of alignment, thus
potentially
resulting in a large reduction in % homology when a global alignment is
performed.
Consequently, most sequence comparison methods are designed to produce optimal
alignments that take into consideration possible insertions and deletions
without
penalising unduly the overall homology score. This is achieved by inserting
"gaps" in the
sequence alignment to try to maximise local homology.
However, these more complex methods assign "gap penalties" to each gap that
occurs
. in the alignment so that, for the same number of identical amino acids, a
sequence
alignment with as few gaps as possible - reflecting higher relatedness between
the two
compared sequences - will achieve a higher score than one with many gaps.
"Affine gap
CA 02501228 2005-04-05
WO 2004/037251 PCT/GB2003/004590
29
costs" are typically used that charge a relatively high cost for the existence
of a gap and
a smaller penalty for each subsequent residue in the gap. This is the most
commonly
used gap scoring system. High gap penalties will of course produce optimised
alignments with fewer gaps. Most alignment programs allow the gap penalties to
be
modified. However, it is preferred to use the default values when using such
software
for sequence comparisons. For example when using the GCG Wisconsin Bestfit
package (see below) the default gap penalty for amino acid sequences is -12
for a gap
and -4 for each extension.
Calculation of maximum % homology therefore firstly requires the production of
an
optimal alignment, taking into consideration gap penalties. A suitable
computer program
for carrying out such an alignment is the GCG Wisconsin Bestfit package
(University of
Wisconsin, U.S.A.; Devereux et al., 1984, Nucleic Acids Research 12:387).
Examples of
other software than can perform sequence comparisons include, but are not
limited to,
the BLAST package (see Ausubel et al., 1999 ibid - Chapter 18), FASTA (Atschul
et al.,
1990, J. Mol. Biol., 403-410) and the GENEWORfCS suite of comparison tools.
Both
BLAST and FASTA are available for offline and online searching (see Ausubel et
al.,
1999 ibid, pages 7-58 to 7-60). However it is preferred to use the GCG Bestfit
program.
A further useful reference is that found in FEMS Microbiol Lett 1999 May
15;174(2):247-
50 (and a published erratum appears in FEMS Microbiol Lett 1999 Aug
1;177(1):187-8).
Although the final % homology can be measured in terms of identity, the
alignment
process itself is typically not based on an all-or-nothing pair comparison.
Instead, a
scaled similarity score matrix is generally used that assigns scores to each
pairwise
comparison based on chemical similarity or evolutionary distance. An example
of such a
matrix commonly used is the BLOSUM62 matrix - the default matrix for the BLAST
suite
of programs. GCG Wisconsin programs generally use either the public default
values or
a custom symbol comparison table if supplied (see user manual for further
details). It is
preferred to use the public default values for the GCG package, or in the case
of other
software, the default matrix, such as BLOSUM62.
Once the software has produced an optimal alignment, it is possible to
calculate
homology, preferably % sequence identity. The software typically does this as
part of
the sequence comparison and generates a numerical result.
CA 02501228 2005-04-05
WO 2004/037251 PCT/GB2003/004590
The sequences may also have deletions, insertions or substitutions of amino
acid
residues which produce a silent change and result in a functionally equivalent
substance.
Deliberate amino acid substitutions may be made on the basis of similarity in
polarity,
charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic
nature of the
5 residues as long as the secondary binding activity of the substance is
retained. For
example, negatively charged amino acids include aspartic acid and glutamic
acid;
positively charged amino acids include lysine and arginine; and amino acids
with
uncharged polar head groups having similar hydrophilicity values include
leucine,
isoleucine, valine, glycine, alanine, asparagine, glutamine, serine,
threonine,
10 phenylalanine, and tyrosine.
Conservative substitutions may be made, for example according to the Table
below.
Amino acids in the same block in the second column and preferably in the same
line in
the third column may be substituted for each other:
Table 1
ALIPHATIC Non-polar G A P
ILV
Polar - uncharged C S T M
NQ
Polar - charged D E
IC R
AROMATIC H F W Y
EXPRESSION VECTORS
The nucleotide sequence for use as the target or for expressing the target can
be
incorporated into a recombinant replicable vector. The vector may be used to
replicate
and express the nucleotide sequence in and/or from a compatible host cell.
Expression
may be controlled using control sequences which include promoters/enhancers
and
other expression regulation signals. Prokaryotic promoters and promoters
functional in
eukaryotic cells may be used. Tissue specific or stimuli specific promoters
may be used.
Chimeric promoters may also be used comprising sequence elements from two or
more
different promoters described above.
The protein produced by a host recombinant cell by expression of the
nucleotide
CA 02501228 2005-04-05
WO 2004/037251 PCT/GB2003/004590
31
sequence may be secreted or may be contained intraceflularly depending on the
sequence and/or the vector used. The coding sequences can be designed with
signal
sequences which direct secretion of the substance coding sequences through a
particular prokaryotic or eukaryotic cell membrane.
FUSION PROTEINS
The target amino acid sequence may be produced as a fusion protein, for
example to aid
in extraction and purification. Examples of fusion protein partners include
glutathione-S-
transferase (GST), 6xHis, GAL4 (DNA binding and/or transcriptional activation
domains)
and (-galactosidase. It may also be convenient to include a proteolytic
cleavage site
between the fusion protein partner and the protein sequence of interest to
allow removal
of fusion protein sequences. Preferably the fusion protein will not hinder the
activity of
the target.
The fusion protein may comprise an antigen or an antigenic determinant fused
to the
substance of the present invention. In this embodiment, the fusion protein may
be a
non-naturally occurring fusion protein comprising a substance which may act as
an
adjuvant in the sense of providing a generalised stimulation of the immune
system. The
antigen or antigenic determinant may be attached to either the amino or
carboxy
terminus of the substance.
In another embodiment of the invention, the amino acid sequence may be ligated
to a
heterologous sequence to encode a fusion protein. For example, for screening
of
peptide libraries for agents capable of affecting the substance activity, it
may be useful to
encode a chimeric substance expressing a heterologous epitope that is
recognised by a
commercially available antibody.
35
THERAPY
The compounds of the present invention may be used as therapeutic agents -
i.e. in
therapy applications.
The term "therapy" includes curative effects, alleviation effects, and
prophylactic effects.
The therapy may be on humans or animals, preferably female animals.
CA 02501228 2005-04-05
WO 2004/037251 PCT/GB2003/004590
32
PHARMACEUTICAL COMPOSITIONS
In one aspect, the present invention provides a pharmaceutical composition,
which
comprises a compound according to the present invention and optionally a
pharmaceutically acceptable carrier, diluent or excipient (including
combinations
thereof).
The pharmaceutical compositions may be for human or animal usage in human and
veterinary medicine and will typically comprise any one or more of a
pharmaceutically
acceptable diluent, carrier, or excipient. Acceptable carriers or diluents for
therapeutic
use are well known in the pharmaceutical art, and are described, for example,
in
Remington's Pharmaceutical Sciences, Mack Publishing Co. (A. R. Gennaro edit.
1985). The choice of pharmaceutical carrier, excipient or diluent can be
selected with
regard to the intended route of administration and standard pharmaceutical
practice.
The pharmaceutical compositions may comprise as - or in addition to - the
carrier,
excipient or diluent any suitable binder(s), lubricant(s), suspending
agent(s), coating
agent(s), solubilising agent(s).
Preservatives, stabilisers, dyes and even flavouring agents may be provided in
the
pharmaceutical composition. Examples of preservatives include sodium benzoate,
sorbic acid and esters of p-hydroxybenzoic acid. Antioxidants and suspending
agents
may be also used.
There may be different composition/formulation requirements dependent on the
different
delivery systems. By way of example, the pharmaceutical composition of the
present
invention may be formulated to be delivered using a mini-pump or by a mucosal
route,
for example, as a nasal spray or aerosol for inhalation or ingestable
solution, or
parenterally in which the composition is formulated by an injectable form, for
delivery, by,
for example, an intravenous, intramuscular or subcutaneous route.
Alternatively, the
formulation may be designed to be delivered by both routes.
Where the agent is to be delivered mucosally through the gastrointestinal
mucosa, it
should be able to remain stable during transit though the gastrointestinal
firact; for
example, it should be resistant to proteolytic degradation, stable at acid pH
and resistant
to the detergent effects of bile.
CA 02501228 2005-04-05
WO 2004/037251 PCT/GB2003/004590
33
Where appropriate, the pharmaceutical compositions can be administered by
inhalation,
in the form of a suppository or pessary, topically in the form of a lotion,
solution, cream,
ointment or dusting powder, by use of a skin patch, orally in the form of
tablets
containing excipients such as starch or lactose, or in capsules or ovules
either alone or
in admixture with excipients, or in the form of elixirs, solutions or
suspensions containing
flavouring or colouring agents, or they can be injected parenterally, for
example
intravenously, intramuscularly or subcutaneously. For parenteral
administration, the
compositions may be best used in the form of a sterile aqueous solution which
may
contain other substances, for example enough salts or monosaccharides to
make,the
solution isotonic with blood. For buccal or sublingual administration the
compositions
may be administered in the form of tablets or lozenges which can be formulated
in a
conventional manner.
COMBINATION PHARMACEUTICAL
The compound of the present invention may be used in combination with one or
more
other active agents, such as one or more other pharmaceutically active agents.
By way of example, the compounds of the present invention may be used in
combination
with other 11 ~i-HSD inhibitors and/or other inhibitors such as an aromatase
inhibitor (such
as for example, 4hydroxyandrostenedione (4-OHA)), and/or a steroid sulphatase
inhibitors such as EMATE and/or steroids - such as the naturally occurring
sterneurosteroids dehydroepiandrosterone sulfate (RHEAS) and pregnenolone
sulfate
(PS) and/or other structurally similar organic compounds.
In addition, or in the alternative, the compound of the present invention may
be used in
combination with a biological response modifier.
The term biological response modifier ("BRM") includes cytokines, immune
modulators,
growth factors, haematopoiesis regulating factors, colony stimulating factors,
chemotactic, haemolytic and thrombolytic factors, cell surFace receptors,
ligands,
leukocyte adhesion molecules, monoclonal antibodies, preventative and
therapeutic
vaccines, hormones, extracellular matrix components, fibronectin, etc. For
some
applications, preferably, the biological response modifier is a cytokine.
Examples of
cytokines include: interleukins (IL) - such as IL-1, IL-2, IL-3, IL-4, IL-5,
IL-6, IL-7, IL-8, IL-
CA 02501228 2005-04-05
WO 2004/037251 PCT/GB2003/004590
34
9, IL-10, IL-11, IL-12, IL-19; Tumour Necrosis Factor (TNF) - such as TNF-a;
Interferon
alpha, beta and gamma; TGF-(i. For some applications, preferably the cytokine
is
tumour necrosis factor (TNF). For some applications, the TNF may be any type
of TNF -
such as TNF-a, TNF-(3, including derivatives or mixtures thereof. More
preferably the
cytokine is TNF-a. Teachings on TNF may be found in the art - such as WO-A-
98/08870
and WO-A-98/13348.
ADMINISTRATION
Typically, a physician will determine the actual dosage which will be most
suitable for an
individual subject and it will vary with the age, weight and response of the
particular
patient. The dosages below are exemplary of the average case. There can, of
course,
be individual instances where higher or lower dosage ranges are merited.
The compositions of the present invention may be administered by direct
injection. The
composition may be formulated for parenteral, mucosal, intramuscular,
intravenous,
subcutaneous, intraocular or transdermal administration. Depending upon the
need, the
agent may be administered at a dose of from 0.01 to 30 mg/kg body weight, such
as
from 0.1 to 10 mg/kg, more preferably from 0.1 to 1 mg/kg body weight.
By way of further example, the agents of the present invention may be
administered in
accordance with a regimen of 1 to 4 times per day, preferably once or twice
per day.
The specific dose level and frequency of dosage for any particular patient may
be varied
and will depend upon a variety of factors including the activity of the
specific compound
employed, the metabolic stability and length of action of that compound, the
age, body
weight, general health, sex, diet, mode and time of administration, rate of
excretion, drug
combination, the severity of the particular condition, and the host undergoing
therapy.
Aside from the typical modes of delivery - indicated above - the term
"administered"
also includes delivery by techniques such as lipid mediated transfection,
liposomes,
immunoliposomes, lipofectin, cationic facial amphiphiles (CFAs) and
combinations
thereof. The routes for such delivery mechanisms include but are not limited
to mucosal,
nasal, oral, parenteral, gastrointestinal, topical, or sublingual routes.
The term "administered" includes but is not limited to delivery by a mucosal
route, for
example, as a nasal spray or aerosol for inhalation or as an ingestable
solution; a
CA 02501228 2005-04-05
WO 2004/037251 PCT/GB2003/004590
parenteral route where delivery is by an injectable form, such as, for
example, an
intravenous, intramuscular or subcutaneous route.
Thus, for pharmaceutical administration, the compounds of the present
invention can be
5 formulated in any suitable manner utilising conventional pharmaceutical
formulating
techniques and pharmaceutical carriers, adjuvants, excipients, diluents etc.
and usually
for parenteral administration. Approximate effective dose rates may be in the
range from
1 to 1000 mg/day, such as from 10 to 900 mg/day or even from 100 to 800 mg/day
depending on the individual activities of the compounds in question and for a
patient of
10 average (70Kg) bodyweight. More usual dosage rates for the preferred and
more active
compounds will be in the range 200 to 800 mg/day, more preferably, 200 to 500
mg/day,
most preferably from 200 to 250 mg/day. They may be given in single dose
regimes,
split dose regimes and/or in multiple dose regimes lasting over several days.
For oral
administration they may be formulated in tablets, capsules, solution or
suspension
15 containing from 100 to 500 mg of compound per unit dose. Alternatively and
preferably
the compounds will be formulated for parenteral administration in a suitable
parenterally
administrable carrier and providing single daily dosage rates in the range 200
to 800 mg,
preferably 200 to 500, more preferably 200 to 250 mg. Such effective daily
doses will,
however, vary depending on inherent activity of the active ingredient and on
the
20 bodyweight of the patient, such variations being within the skill and
judgement of the
physician.
CELL CYCLING
25 The compounds of the present invention may be useful in the method of
treatment of a
cell cycling disorder.
As discussed in "Molecular Cell Biology" 3rd Ed. Lodish et al, pages 177-181
different
eukaryotic cells can grow and divide at quite different rates. Yeast cells,
for example,
30 can divide every 120 min., and the first divisions of fertilised eggs in
the embryonic cells
of sea urchins and insects take only 1530 min. because one large pre-existing
cell is
subdivided. However, most growing plant and animal cells take 10-20 hours to
double in
number, and some duplicate at a much slower rate. Many cells in adults, such
as nerve
cells and striated muscle cells, do not divide at all; others, like the
fibroblasts that assist
35 in healing wounds, grow on demand but are otherwise quiescent.
CA 02501228 2005-04-05
WO 2004/037251 PCT/GB2003/004590
36
Still, every eukaryotic cell that divides must be ready to donate equal
genetic material to
two daughter cells. DNA synthesis in eukaryotes does not occur throughout the
cell
division cycle but is restricted to a part of it before cell division.
The relationship between eukaryotic DNA synthesis and cell division has been
thoroughly analysed in cultures of mammalian cells that were all capable of
growth and
division. In contrast to bacteria, it was found, eukaryotic cells spend only a
part of their
time in DNA synthesis, and it is completed hours before cell division
(mitosis). Thus a
gap of time occurs after DNA synthesis and before cell division; another gap
was found
to occur after division and before the next round of DNA synthesis. This
analysis led to
the conclusion that the eukaryotic cell cycle consists of an M (mitotic)
phase, a G, phase
(the first gap), the S (DNA synthesis) phase, a GZ phase (the second gap), and
back to
M. The phases between mitoses (G~, S, and G2) are known collectively as the
interphase.
Many nondividing cells in tissues (for example, all quiescent fibroblasts)
suspend the
cycle after mitosis and just prior to DNA synthesis; such "resting" cells are
said to have
exited from the cell cycle and to be in the Go state.
It is possible to identify cells when they are in one of the three interphase
stages of the
cell cycle, by using a fluorescence-activated cell sorter (FRCS) to measure
their relative
DNA content: a cell that is in G, (before DNA synthesis) has a defined amount
x of DNA;
during S (DNA replication), it has between x and 2x; and when in G2 (or M), it
has 2x of
DNA.
The stages of mitosis and cytokinesis in an animal cell are as follows
(a) Interphase. The GZ stage of interphase immediately precedes the beginning
of
mitosis. Chromosomal DNA has been replicated and bound to protein during the S
phase, but chromosomes are not yet seen as distinct structures. The nucleolus
is the
only nuclear substructure that is visible under light microscope. In a diploid
cell before
DNA replication there are two morphologic chromosomes of each type, and the
cell is
said to be 2n. In G2, after DNA replication, the cell is 4n. There are four
copies of each
chromosomal DNA. Since the sister chromosomes have not yet separated from each
other, they are called sister chromatids.
CA 02501228 2005-04-05
WO 2004/037251 PCT/GB2003/004590
37
b) Early prophase. Centrioles, each with a newly formed daughter centriole,
begin
moving toward opposite poles of the cell; the chromosomes can be seen as long
threads. The nuclear membrane begins to disaggregate into small vesicles.
(c) Middle and late prophase. Chromosome condensation is completed; each
visible
chromosome structure is composed of two chromatids held together at their
centromeres. Each chromatid contains one of the two newly replicated daughter
DNA
molecules. The microtubular spindle begins to radiate from the regions just
adjacent to
the centrioles, which are moving closer to their poles. Some spindle fibres
reach from
pole to pole; most go to chromatids and attach at kinetochores.
(d) Metaphase. The chromosomes move toward the equator of the cell, where they
become aligned in the equatorial plane. The sister chromatids have not yet
separated.
(e) Anaphase. The two sister chromatids separate into independent chromosomes.
Each contains a centromere that is linked by a spindle fibre to one pole, to
which it
moves. Thus one copy of each chromosome is donated to each daughter cell.
Simultaneously, the cell elongates, as do the pole-to-pole spindles.
Cytokinesis begins
as the cleavage furrow starts to form.
(f) Telophase. New membranes form around the daughter nuclei; the chromosomes
uncoil and become less distinct, the nucleolus becomes visible again, and the
nuclear
membrane forms around each daughter nucleus. Cytokinesis is nearly complete,
and
the spindle disappears as the microtubules and other fibres depolymerise.
Throughout
mitosis the "daughter" centriole at each pole grows until it is full-length.
At telophase the
duplication of each of the original centrioles is completed, and new daughter
centrioles
will be generated during the next interphase.
(g) Interphase. Upon the completion of cytokinesis, the cell enters the G,
phase of
the cell cycle and proceeds again around the cycle.
It will be appreciated that cell cycling is an extremely important cell
process. Deviations
from normal cell cycling can result in a number of medical disorders.
Increased and/or
unrestricted cell cycling may result in cancer. Reduced cell cycling may
result in
degenerative conditions. Use of the compound of the present invention may
provide a
means to treat such disorders and conditions.
CA 02501228 2005-04-05
WO 2004/037251 PCT/GB2003/004590
3~
Thus, the compound of the present invention may be suitable for use in the
treatment of
cell cycling disorders such as cancers, including hormone dependent and
hormone
independent cancers.
In addition, the compound of the present invention may be suitable for the
treatment of
cancers such as breast cancer, ovarian cancer, endometrial cancer, sarcomas,
melanomas, prostate cancer, pancreatic cancer etc. and other solid tumours.
For some applications, cell cycling is inhibited and/or prevented and/or
arrested,
preferably wherein cell cycling is prevented and/or arrested. In one aspect
cell cycling
may be inhibited and/or prevented and/or arrested in the GZ/M phase. In one
aspect cell
cycling may be irreversibly prevented and/or inhibited and/or arrested,
preferably wherein
cell cycling is irreversibly prevented and/or arrested.
By the term "irreversibly prevented and/or inhibited andlor arrested" it is
meant after
application of a compound of the present invention, on removal of the compound
the
effects of the compound, namely prevention andlor inhibition and/or arrest of
cell cycling,
are still observable. More particularly by the term "irreversibly prevented
and/or inhibited
and/or arrested" it is meant that when assayed in accordance with the cell
cycling assay
protocol presented herein, cells treated with a compound of interest show less
growth after
Stage 2 of the protocol I than control cells. Details on this protocol are
presented below.
Thus, the present invention provides compounds which: cause inhibition of
growth of
oestrogen receptor positive (ER+) and ER negative (ER-) breast cancer cells in
vitro by
preventing and/or inhibiting and/or arresting cell cycling; andlor cause
regression of
nitroso-methyl urea (NMU)-induced mammary tumours in intact animals (i.e. not
ovariectomised), and/or prevent and/or inhibit and/or arrest cell cycling in
cancer cells;
and/or act in vivo by preventing and/or inhibiting and/or arresting cell
cycling and/or act as
a cell cycling agonist.
CELL CYCLING ASSAY
f PROTOCOL 2)
Procedure
Stage 1
CA 02501228 2005-04-05
WO 2004/037251 PCT/GB2003/004590
39
MCF-7 breast cancer cells are seeded into multi-well culture plates at a
density of 105
cells/well. Cells were allowed to attach and grown until about 30% confluent
when they
are treated as follows:
Control - no treatment
Compound of Interest (COI) 20p,M
Cells are grown for 6 days in growth medium containing the COl with changes of
medium/COI every 3 days. At the end of this period cell numbers were counted
using a
Coulter cell counter.
Stage 2
After treatment of cells for a 6-day period with the COI cells are re-seeded
at a density of
104 cells/well. No further treatments are added. Cells are allowed to continue
to grow
for a further 6 days in the presence of growth medium. At the end of this
period cell
numbers are again counted.
CANCER
As indicated, the compounds of the present invention may be useful in the
treatment of a
cell cycling disorder. A particular cell cycling disorder is cancer.
Cancer remains a major cause of mortality in most Western countries. Cancer
therapies
developed so far have included blocking the action or synthesis of hormones to
inhibit
the growth of hormone-dependent tumours. However, more aggressive chemotherapy
is currently employed for the treatment of hormone-independent tumours.
Hence, the development of a pharmaceutical for anti-cancer treatment of
hormone
dependent and/or hormone independent tumours, yet lacking some or all of the
side-
effects associated with chemotherapy, would represent a major therapeutic
advance.
We believe that the compound of the present invention provides a means for the
treatment of cancers and, especially, breast cancer.
In addition or in the alternative the compound of the present invention may be
useful in
CA 02501228 2005-04-05
WO 2004/037251 PCT/GB2003/004590
the blocking the growth of cancers including leukaemias and solid tumours such
as
breast, endometrium, prostate, ovary and pancreatic tumours.
OTHER THERAPIES
5
It is also to be understood that the compound/composition of the present
invention may
have other important medical implications.
For example, the compound or composition of the present invention may be
useful in the
10 treatment of the disorders listed in WO-A-99152890 - viz:
In addition, or in the alternative, the compound or composition of the present
invention
may be useful in the treatment of the disorders listed in WO-A-98/05635. For
ease of
reference, part of that list is now provided: diabetes including Type fl
diabetes, obesity,
15 cancer, inflammation or inflammatory disease, dermatological disorders,
fever,
cardiovascular effects, haemorrhage, coagulation and acute phase response,
cachexia,
anorexia, acute infection, HIV infection, shock states, graft-versus-host
reactions,
autoimmune disease, reperfusion injury, meningitis, migraine and aspirin-
dependent
anti-thrombosis; tumour growth, invasion and spread, angiogenesis, metastases,
20 malignant, ascites and malignant pleural effusion; cerebral ischaemia,
ischaemic heart
disease, osteoarthritis, rheumatoid arthritis, osteoporosis, asthma, multiple
sclerosis,
neurodegeneration, Alzheimer's disease, atherosclerosis, stroke, vasculitis,
Crohn's
disease and ulcerative colitis; periodontitis, gingivitis; psoriasis, atopic
dermatitis, chronic
ulcers, epidermolysis bullosa; corneal ulceration, retinopathy and surgical
wound
25 healing; rhinitis, allergic conjunctivitis, eczema, anaphylaxis;
restenosis, congestive heart
failure, endometriosis, atherosclerosis or endosclerosis.
!n addition, or in the alternative, the compound or composition of the present
invention
may be useful in the treatment of disorders fisted in WO-A-98/07859. For ease
of
30 reference, part of that list is now provided: cytokine and cell
proliferation/differentiation
activity; immunosuppressant or immunostimulant activity (e.g. for treating
immune
deficiency, including infection with human immune deficiency virus; regulation
of
lymphocyte growth; treating cancer and many autoimmune diseases, and to
prevent
transplant rejection or induce tumour immunity); regulation of haematopoiesis,
e.g.
35 treatment of myeloid or lymphoid diseases; promoting growth of bone,
cartilage, tendon,
ligament and nerve tissue, e.g. for healing wounds, treatment of burns, ulcers
and
CA 02501228 2005-04-05
WO 2004/037251 PCT/GB2003/004590
41
periodontal disease and neurodegeneration; inhibition or activation of
follicle-stimulating
hormone (modulation of fertility); chemotactic/chemokinetic activity (e.g. for
mobilising
specific cell types to sites of injury or infection); haemostatic and
thrombolytic activity
(e.g. for treating haemophilia and stroke); antiinflammatory activity (for
treating e.g.
septic shock or Crohn's disease); as antimicrobials; modulators of e.g.
metabolism or
behaviour; as analgesics; treating specific deficiency disorders; in treatment
of e.g.
psoriasis, in human or veterinary medicine.
In addition, or in the alternative, the composition of the present invention
may be useful
in the treatment of disorders listed in WO-A-98/09985. For ease of reference,
part of
that list is now provided: macrophage inhibitory and/or T cell inhibitory
activity and thus,
anti-inflammatory activity; anti-immune activity, i.e. inhibitory effects
against a cellular
and/or humoral immune response, including a response not associated with
inflammation; inhibit the ability of macrophages and T cells to adhere to
extracellular
matrix components and fibronectin, as well as up-regulated fas receptor
expression in T
cells; inhibit unwanted immune reaction and inflammation including arthritis,
including
rheumatoid arthritis, inflammation associated with hypersensitivity, allergic
reactions,
asthma, systemic lupus erythematosus, collagen diseases and other autoimmune
diseases, inflammation associated with atherosclerosis, arteriosclerosis,
atherosclerotic
heart disease, reperfusion injury, cardiac arrest, myocardial infarction,
vascular
inflammatory disorders, respiratory distress syndrome or other cardiopulmonary
diseases, inflammation associated with peptic ulcer, ulcerative colitis and
other diseases
of the gastrointestinal tract, hepatic fibrosis, liver cirrhosis or other
hepatic diseases,
thyroiditis or other glandular diseases, glomerulonephritis or other renal and
urologic
diseases, otitis or other oto-rhino-laryngological diseases, dermatitis or
other dermal
diseases, periodontal diseases or other dental diseases, orchitis or epididimo-
orchitis,
infertility, orchidal trauma or other immune-related testicular diseases,
placental
dysfunction, placental insufficiency, habitual abortion, eclampsia, pre-
eclampsia and
other immune and/or inflammatory-related gynaecological diseases, posterior
uveitis,
intermediate uveitis, anterior uveitis, conjunctivitis, chorioretinitis,
uveoretinitis, optic
neuritis, intraocular inflammation, e.g. retinitis or cystoid macular oedema,
sympathetic
ophthalmic, scleritis, retinitis pigmentosa, immune and inflammatory
components of
degenerative fondus disease, inflammatory components of ocular trauma, ocular
inflammation caused by infection, proliferative vitreo-retinopathies, acute
ischaemic optic
neuropathy, excessive scarring, e.g, following glaucoma filtration operation,
immune
and/or inflammation reaction against ocular implants and other immune and
CA 02501228 2005-04-05
WO 2004/037251 PCT/GB2003/004590
42
inflammatory-related ophthalmic diseases, inflammation associated with
autoimmune
diseases or conditions or disorders where, both in the central nervous system
(CNS) or
in any other organ, immune and/or inflammation suppression would be
beneficial,
Parkinson's disease, complication and/or side effects from treatment of
Parkinson's
disease, AIDS-related dementia complex HIV-related encephalopathy, Devic's
disease,
Sydenham chorea, Alzheimer's disease and other degenerative diseases,
conditions or
disorders of the CNS, inflammatory components of stokes, post-polio syndrome,
immune
and inflammatory components of psychiatric disorders, myelitis, encephalitis,
subacute
sclerosing pan-encephalitis, encephalomyelitis, acute neuropathy, subacute
neuropathy,
chronic neuropathy, Guillaim-Barre syndrome, Sydenham chora, myasthenia
gravis,
pseudo-tumour cerebri, Down's Syndrome, Huntington's disease, amyotrophic
lateral
sclerosis, inflammatory components of CNS compression or CNS trauma or
infections of
the CNS, inflammatory components of muscular atrophies and dystrophies, and
immune
and inflammatory related diseases, conditions or disorders of the central and
peripheral
nervous systems, post-traumatic inflammation, septic shock, infectious
diseases,
inflammatory complications or side effects of surgery, bone marrow
transplantation or
other transplantation complications and/or side effects, inflammatory and/or
immune
complications and side effects of gene therapy, e.g. due to infection with a
viral carrier,
or inflammation associated with AIDS, to suppress or inhibit a humoral and/or
cellular
immune response, to treat or ameliorate monocyte or leukocyte proliferative
diseases,
e.g. leukaemia, by reducing the amount of monocytes or lymphocytes, for the
prevention and/or treatment of graft rejection in cases of transplantation of
natural or
artificial cells, tissue and organs such as cornea, bone marrow, organs,
lenses,
pacemakers, natural or artificial skin tissue.
As previously mentioned, in one aspect the present invention provides use of a
compound as described herein in the manufacture of a medicament for use in the
therapy of a condition or disease associated with 11 (3-HSD.
Conditions and diseases associated with 11 ~i-HSD have been reviewed in
Walker, E. A,;
Stewart, P. M.; Trends in Endocrinology and Metabolism, 2003, 14 (7), 334-339.
In a preferred aspect, the condition or disease is selected from the list
consisting of:
~ metabolic disorders, such as diabetes and obesity
~ cardiovascular disorders, such as hypertension
~ glaucoma
CA 02501228 2005-04-05
WO 2004/037251 PCT/GB2003/004590
43
~ inflammatory disorders, such as arthritis or asthma
~ immune disorders
~ bone disorders, such as osteoporosis
~ cancer
~ intra-uterine growth retardation
~ apparent mineralocorticoid excess syndrome (AME)
~ polycystic ovary syndrome (PCOS)
~ hirsutism
~ acne
~ oligo- or amenorrhea
~ adrenal cortical adenoma and carcinoma
~ Cushing's syndrome
~ pituitary tumours
~ invasive carcinomas
~ breast cancer; and
~ endometrial cancer.
SUMMARY
In summation, the present invention provides compounds for use as steroid
dehydrogenase inhibitors, and pharmaceutical compositions for the same.
BRIEF DESCRIPTION OF THE FIGURES
The present invention will be described in further detail by way of example
only with
reference to the accompanying figures in which:-
Figure 1 is of graph 1 which shows the amount of protein per ~,L of rat liver
and rat
kidney.
Figure 2 is of graph 2 which shows the enzyme concentration and time-
dependency
course, E to F, in rat liver, 11 ~i-HSD type 1 activity.
Figure 3 is of graph 3 which shows the enzyme concentration and time-
dependency
course, F to E, in rat kidney, 11 ~3-HSD type 2 activity.
Figure 4 is a graph showing extraction efficiencies obtained with four
extraction
methods.
CA 02501228 2005-04-05
WO 2004/037251 PCT/GB2003/004590
44
Figure 5 is a graph showing a comparison of 11 ~i-HSD1 activity in rat and
human
hepatic microsomes.
Figure 6 is a series of graphs showing the effect of incubation time on human
microsomal 11 ~i-HSD1 activity.
Figure 7 is a series of graphs showing the effect of microsomal protein
concentration on
human microsomal 11 [3-HSD1 activity.
Figure 8 is a graph showing the substrate (cortisone) saturation curve for
human hepatic
microsomal 11 ~ HSD1.
Figure 9 is a Lineweaver-Burke plot of substrate saturation data for human
hepatic
microsomal 11 (3 HSD1.
Figure 10 is a graph showing the ICSO determination for glycyrrhetinic acid.
Figure 11 is a graph showing the ICSO determination for carbenoxolone.
Figures 12(A), 12(B) and 12(C) are graphs showing the 11 ~i-HSD1 activity
measured
by Immunoassay. Figure 12(A) shows the effect of protein; Figure 12(B) shows
the
effect of cortisone; and Figure 12(C) shows the effect of Tween-80.
Figure 13 is a graph showing the evaluation of the Assay Designs Cortisol
Immunoassay.
Figure 14 is a graph showing the effect of increasing microsomal protein on
measurement of 11 (3 HSD1 activity detected by Assay Designs Immunoassay.
Figure 15 is a graph showing the detection of 11 ~i HSD1 activity by RIA using
the
Immunotech anti-cortisol antibody.
Figure 16 is a graph showing the effect of lowering the Immunotech antibody
concentration on the signal to noise (microsome group compared to GA blank
group).
Figure 17 is a graph showing the Immunotech antibody saturation curve for
detection of
11 ~3 HSD1 activity by RIA.
Figure 18 is a graph showing the linearity of human hepatic microsomal 11 a
HSD1
activity detected by RIA.
Figure 19 is a graph showing the effect of Tween 80 on detection of human
hepatic
microsomal 11 a HSD1 activity by' RIA.
Figure 20 is a graph showing the effect of buffer systems on detection of
human hepatic
microsomal 11 ~3 HDS1 activity by RIA.
Figure 21 is a graph showing the linearity of human hepatic microsomal 11 ~i
HSD1
activity with incubation time detected by RIA.
Figure 22 is a graph showing the substrate saturation curve for human hepatic
microsomal 11 ~i HDS1 activity detected by RIA.
CA 02501228 2005-04-05
WO 2004/037251 PCT/GB2003/004590
Figure 23 is a Lineweaver-Burke plot of substrate saturation data for human
hepatic
microsomal 11 [i HDS1 activity detected by RlA.
Figure 24 is a graph showing the DMSO tolerance of human hepatic microsomal 11
[i
HSD1 activity.
5 Figure 25 is an ICSO curve for inhibition of human hepatic microsomal 11 p
HSD1 activity
by glycyrrhetinic acid.
EXAMPLES
10 The present invention will now be described only by way of example.
MATERIALS AND METHODS
Materials
Enzymes - Rat livers and rat kidneys were obtained from normal Wistar rats
(Harlan
Olac, Bicester, Oxon,UK). Both the kidneys and livers were homogenised on ice
in PBS-
sucrose buffer (1g/ 10 ml) using an Ultra-Turrax. After the livers and kidneys
were
homogenised the homogenate was centrifuged for five minutes at 4000 rpm. The
supernatant obtained was removed and stored in glass vials at -20°C.
The amount of
protein per pl of rat liver and kidney cytosol was determined using the
Bradford method
[14].
Apparatus
~ Incubator: mechanically shaken water bath, SW 20, Germany.
~ Evaporator, Techne Driblock DB 3A, UK
~ TLC aluminium sheets 20 x 20 cm silica gel 60 F254, Merck, Germany.
~ Scintillation vials: 20 ml polypropylene vials with caps, SARSTEDT, Germany.
~ Scintillation counter: Beckman LS 6000 SC, Beckman Instruments Inc., USA.
Solutions
~ Assay medium: PBS-sucrose buffer, Dulbecco's Phosphate Buffered Saline, 1
tablet/100 ml with 0,25 M sucrose, pH 7,4 BDH Laboratory supplies, UK.
~ Scintillation fluid: Ecoscint A (National Diagnostics, USA).
~ Radioactive compound solutions: [1,2,6,7 3H]-cortisol (Sp. Ac. 84 Ci/mmol)
NEN
Germany, [4-'4C]-cortisol (Sp. Ac. 53 mCi/mmol) NEN Germany.
CA 02501228 2005-04-05
WO 2004/037251 PCT/GB2003/004590
46
~ Cr03 and Acetic acid (Sigma Chemical Co., UK).
~ Extraction fluid: Di-ethylether, Fischer Chemicals, UK.
~ Bradford Reagent solution: Coomassie Brilliant Blue G-250, 100 mg in 95%
ethanol
with 100 ml of phosphoric acid (85% w/v) diluted to 1 litre.
Compounds
~ Inhibitors: compounds were synthesised in accordance with the synthetic
routes
below .
~ Cofactor: NADPH and NADP, Sigma Chemical Co., UK.
Methods
Synthesis of radio labelled cortisone
Labelled cortisol (F) (3H-F and '4C-F) was oxidised at the C-11 position with
Cr03 in
order to synthesize to the corresponding labelled cortisone (3H-E and'4C-E).
For this reaction F was oxidised in a 0,25% Cr(73 (w/v) dissolved in a 50%
acetic-
acid/distilled water (v/v) solution. The labelled F was then added to 1 ml of
the Cr03
solution, vortex mixed and put in an incubator for 20 minutes at 37°C.
The aqueous
reaction mixture was extracted twice with 4 ml of di-ethylether, the di-
ethylether was then
evaporated and the residue transferred to a TLC-plate, which was developed in
the
following system, chloroform : methanol 9:1 (v/v). Unlabelled cortisone (E)
was also run
on the TLC-plate to locate the position of the labelled steroids. After
locating the spot of
the labelled steroids this area is cut out from the TLC-plate and eluted with
0,5 ml of
methanol.
The amount of protein per p,L of rat liver and rat kidney
The amount of protein in rat liver and rat kidney needed to be determined. The
experiment was done according to the Bradford method [14]. The following
method was
used: first a BSA (protein) solution was prepared (1 mglml). Protein solutions
containing
10 to 100 p,g protein were pipetted into tubes and volumes adjusted with
distilled water.
Then 5 ml of protein reagent was added to the tubes and vortex mixed. The
absorbance
was measured at 595 nm after 15 minutes and before 1 hour in 3 ml cuvettes
against a
reagent blank. The weight of the protein was plotted against the corresponding
CA 02501228 2005-04-05
WO 2004/037251 PCT/GB2003/004590
47
absorbance resulting in a standard curve used to determine the protein
concentration in
rat liver and rat kidney cytosols.
Assay validation - Enzyme concentration and time-dependency of 11 ~3-HSD
activity
Before carrying out 11 (3-HSD assays to examine the conversion E to F and F to
E and
the influence that different inhibitors have on these conversions the amount
of rat liver
homogenate and rat kidney homogenate and their incubation time need to be
determined.
11 (i-HSD type 1 is the enzyme responsible for the conversion E to F and this
type of
enzyme is present in rat liver. The substrate solution used in this assay
contained
70,000 cpm/ml 3H-E in PBS-sucrose and 0.5 pM of unlabelled E and co-factor
NADPH (9
mg/10 ml of substrate solution). 1 ml of the substrate solution and the
different amounts
of rat liver homogenate was added to all tubes.
The amount of rat liver homogenate needed for an assay was determined by
incubating
the substrafie solution with 25, 50, 100 and 150 pl for 30, 60, 90 and 120
minutes at 37°C
in a water bath with the tubes being mechanically shaken. After the incubation
50 p,L of
recovery solution was added, containing about 8,000 cpm/ 50 wL of'4C-F and 50
~.g/50
p,L of unlabelled F for visualising the spot on the TLC-plate, to correct for
the losses
made in the next two steps. F was then extracted from the aqueous phase with 4
ml of
ether (2 x 30 sec cycle, vortex mix). The aqueous phase was then frozen using
dry-ice
and the organic layer was decanted and poured into smaller tubes and
evaporated. 6
drops of ether were then added to the small tubes to re-dissolve the residue
which was
transferred to an aluminium thin layer chromatography plate (TLC-plate). The
TLC-plate
was developed in a TLC tank under saturated conditions. The solvent system
used was
chloroform : methanol 9:1 (v/v). The F spots on the TLC-plate were visualised
under
UV- light and cut out from the TLC-plate (R~=0.45). The spots from the TLC-
plate were
then put into scintillation vials and 0.5 ml of methanol was added to all
vials to elute the
radioactivity from the TLC-plate for 5 minutes. 10 ml of Ecoscint was added to
the
scintillation vials and they were put into the scintillation counter to count
amount of
product formed.
CA 02501228 2005-04-05
WO 2004/037251 PCT/GB2003/004590
48
The same procedure was used for the 11 (i-HSD type 2 assay, the conversion F
to E, to
determine the amount of rat kidney to be used and the incubation time. Except
this time
the substrate solution contained 3H-F and unlabelled F and the recovery
contained '4C-E
and unlabelled E and cortisone has a Rf value of 0.65 on the TLC-plate.
Assay procedure - The 11 (3-HSD inhibitors
In these assays the influence of different inhibitors on the 11 (3-HSD
activity both in
reductive (type 1 ) and oxidative (type 2) directions were assessed. In the
reductive
direction E is the substrate and F the product and visa versa in the case of
oxidation.
The method described here is for the oxidative direction.
The substrate solution contained about 50,000 cpm/ml 3H-F in PBS-sucrose and
0.5 pM
F. 1 ml of the substrate solution was added to each tube, the inhibitors were
also added,
at a 10 ~M concentration, to each tube except to the "control" and "blank"
tubes. 150 wL
was added to all tubes except to the blanks, this was done to correct for the
amount of
3H-F spontaneously formed. The tubes were incubated for 60 minutes in a
mechanically
shaken water bath at 37°C. The amount of kidney liver homogenate and
incubation time
used resulted from the enzyme- and time-dependency assay. After incubation 50
p,L of
recovery was added to correct for the losses made in the next steps,
containing 5000
cpm/50 ~,L of '4C-E and 50~,g/50 ~L of unlabelled E (to visualise the spot on
the TLC-
plate). The aqueous mixture was then extracted with 4 ml of ether (2 x 30 sec
cycle,
vortex mix). After freezing the aqueous phase, the ether (upper) layer was
decanted into
smaller tubes and evaporated at 45°C until completely dry. The residue
was then re-
dissolved in 6 drops of ether and transferred to a TLC-plate. The TLC-plate
was
developed in chloroform : methanol (9:1 v/v) solvent system, the TLC-plate ran
for about
90 minutes until the solvent front had moved about 18 cm. The position of the
product E
was visualised under UV-light and cut out from the TLC-plate and put into
scintillation
vials. Radioactivity was eluted over 5 minutes with 0.5 ml methanol. 0.5 ml of
PBS-
sucrose and 10 ml of Ecoscint were then added and vortex mixed before counting
in the
scintillation counter. Before counting the samples, two total activity vials
were prepared.
These contained 0.5 ml of the substrate solution, 50 p,L of the recovery, 0.5
ml of
methanol and 10 ml of Ecoscint. These two total activity vials were needed to
determine
the amount of'4C-E and 3H-F added in the beginning to make the calculations.
CA 02501228 2005-04-05
WO 2004/037251 PCT/GB2003/004590
49
In case of the reductive direction, E to F ,the same method was used. Only the
substrate solution containing 3H-E and unlabelled E and the recovery
containing '4C-F
and unlabelled F are different to the method used in the oxidative direction.
After testing all the inhibitors at 10 p,M a dose-response experiment was done
for the
most potent 11 (i-HSD type 1 and type 2 inhibitors. To look at the percentage
of
inhibition four different concentrations, 1, 5, 10 and 20 p.M, were used. The
method for
both the rat liver, type 1 the reductive, and rat kidney, type 2 the
oxidative, stay the
same throughout the entire experiment.
RESULTS
The amount of protein per ~,L of rat liver and rat kidney
An initial experiment was carried out to determine the amount of protein in
rat liver
cytosol and rat kidney cytosol, to be added to each tube. Graph 1 in figure 1
shows the
standard curve from which the amount of protein used in both experiments was
calculated. The amount of protein added to each tube in the rat liver
experiment was
75.5 pg (per 25 p,L). In the rat kidney experiment the amount of protein added
to each
tube was 135.6 ~g (per 150~L).
Enzyme concentration and time-dependency of 11 [3-HSD activity
In this experiment the amount of rat liver homogenate and rat kidney
homogenate added
to each tube and the incubation time was determined. Graph 2 in figure 2 shows
the
enzyme concentration and time-dependency course of the rat liver experiment E
to F, 11
(3-HSD type 1 activity. Graph 3 in figure 3 shows the enzyme concentration and
time-
dependency course F to E, 11 (3-HSD type 2 activity. After drawing the graphs
the
optimal amount of rat liver cytosol and rat kidney cytosol and both their
incubation times
were selected. One important rule when selecting both variables, to select an
amount of
rat liver and rat kidney and incubation time on a linear part of the graph.
This is done to
avoid fluctuations in enzyme activity. The amount of rat liver cytosol
selected was 25 pL
and 90 minutes of incubation time, the amount of rat kidney cytosol selected
was 150 ~L
and 60 minutes of incubation time.
CA 02501228 2005-04-05
WO 2004/037251 PCT/GB2003/004590
The 11 ~3-HSD inhibitors
In this experiment the influence of different inhibitors on the conversion E
to F and F to E
was determined. The reason why inhibition in both directions was examined was
to
5 make a comparison between the inhibitors and which type of 11 ~-HSD they
inhibit
more. Compounds were screened for their ability to inhibit 11 [3-HSD type 1 (E
to F) and
type 2 (F to E). All the inhibitors were initially tested at a 10 p.M
concentration. The
percent of inhibition was calculated as the percentage of decrease in radio
labelled 3H-E
and 3H-F of product formed, compared with the control activity (the tubes
without an
10 inhibitor in it). All the results calculated are means, n=2.
Table 2: Inhibitory Effect
STX Structure % inhibition of 11~i % inhibition of 11(3
No. HSD1 @ 10 pM HSD2 @ 10 NM typical
t ical sd ~ 5% sd ~ 5%
412 cl \ % s oN 27 3
~o
cl
s
413 °' S3 n=2 ~ 0.2
~g'~NH Ipso = 6.6 pM
\ ~ 'o /
421 _ 60 n=2 0.9
g ~NH iCSp = 10 i.lM
424 \ , 0 24 0.7
g=NH
s
425 -40 0.0
,o
=N
SO
CI
S
469 cl 63 29
,O l
N
SO
S
CA 02501228 2005-04-05
WO 2004/037251 PCT/GB2003/004590
51
STX Structure % inhibition of 11(3 % inhibition of 11~i
No. HSD1 @ 10 NM HSD2 @ 10 NM typical
t ical sd ~ 5% sd ~ 5%
470 cl 39 30
,o ~
N
SO
~ N
S"
48 8
519
Br ~ / S oNH
~O
S
521 ~ _ 0.5 5
g ~NH
~O
N
S' _
522 cl 37 6
cl
g=NH
~O
CI
S
523 21 8
Br \ / S ~NH
~O
N
S- _
524 Sy 31 53
lIN
O
NH
CI \ / S ~NH
~Q
CI ~ \
S
552 0 18 24
g=NH
~O
N
S
553 0 0.7 18
g=NH
S"
CA 02501228 2005-04-05
WO 2004/037251 PCT/GB2003/004590
52
STX Structure % inhibition of 11 ~3 % inhibition of 11 a
No. HSD1 @ 10 pM HSD2 @ 10 NM typical
t ical sd ~ 5% sd ~ 5%
554 p 69 43
\ / g~ NH
\ ~O ~ ~ N
\/
s
575 0 62 1.6
OH
CI \ / S ~NH
~O
CI
S
580 0l 75 1.4
0
\ / g=NH
CI
S
581 0 77 32
/ g=NH
N o ~ ~ N
CF3
O S
582 0- 40 0.7
0
g=NH
CI
S
583 ° ~~ 29 0.4
0
\ / g=NH
~O
S
584 0- 48 10
0
~=NH
SO
S'
585 48 1.6
\ /
/ g°-~NH
~O
S
CA 02501228 2005-04-05
WO 2004/037251 PCT/GB2003/004590
53
STX - Structure % inhibition of 11 ~i % inhibition of 11 ~i
No. HSD1 @ 10 pM HSD2 @ 10 NM typical
t ical sd ~ 5% sd ~ 5%
701 0 34 36
0
g=NH
~O
S
703 35 4
0
g'-NH
~O
\
S
704 0 38 4
g=NH
~O
\ N
S"
705 0 6 6
SNH
O ~ \ N
O
S
706 29 7
0
gNH
O ~ \ N
S"
707 -0 21 11
O ~ ~ g~NH
~O \
S
708 39 11
O ~ ~ g~NH
~O \
S
709 ~~ 0 10 13
gNH
O ~ \ N
S"
710 55 10
0
g=NH
O ~ \ N
S-
CA 02501228 2005-04-05
WO 2004/037251 PCT/GB2003/004590
54
STX Structure % inhibition of 11 ~i % inhibition of 11 (3
No. HSD1 @ 10 pM HSD2 @ 10 pM typical
t ical sd ~ 5% sd ~ 5%
711 ~0 37 6
\H ~ ~ S~N
~0
N
S
712 F F 24 3
F ~ / g ~NH
F F O I \
S\
713 26 3
° / '~N
So
N
S
730 32 9
i
0 o\~s\\ ~ ~
s oN/ o
/ w
o / \ N
s~
731 N~~ 45 12
i
s
.._ o
=N O
/ S~
O I \ N
S"
750 CI 4 10
0
g=NH
o I ~ S
N
751 c~
5
~oH
~\ N
O ~ ~ S
i 'S
N
CA 02501228 2005-04-05
WO 2004/037251 PCT/GB2003/004590
STX Structure % inhibition of 11 ~i % inhibition of 11 (3
No. HSD1 @ 10 pM HSD2 @ 10 pM typical
t ical sd ~ 5% sd ~ 5%
752 °~ 5 1
0
sNH
0
N
O
O
753 ~~ 8 2
\ ~ ~ ON
O ~ ~ S
N"O
754 c~ 20 6
0
g=NH
\O
N S
O~
755 c~ 21 8
\_ o
~\ N
O ~ ~ S
N~S
BIOLOGICAL ASSAY DEVELOPMENT USING HUMAN 11 ~3-HYDROXYSTEROID
DEHYDROGENASE TYPE 1.
5
Standard Operating Procedure for the 11 ~3-Hydroxysteroid Dehydrogenase Type 1
cortisol Radioimmunoassay.
11 (3 HSD1 cortisol RIA
Reagents: Cortisone, Cortisol (Hydrocortisone), NADPH, Glucose-6-phosphate,
Glycyrrhetinic acid (GA), Dextran coated charcoal (C6197) and DMSO were
obtained
from Sigma Aldrich, Carbenoxolone was obtained from ICN Biomedicals, Product
215493001, 3H-cortisone was obtained from American Radiolabelled Componds Inc,
Product ART-743, 3H-cortisol was obtained from NEN, Product NET 396, '4C-
cortisol
CA 02501228 2005-04-05
WO 2004/037251 PCT/GB2003/004590
56
was obtained from NEN, Product NEC 163, human hepatic microsomes were obtained
from XenoTech, product H0610 / Lot 0210078, rat hepatic microsomes were
obtained
from XenoTech, SPA beads were obtained from Amersham, Product RPNQ0017, the
Immunoassay kit was obtained from Assay Designs, Product 900-071, the
Immunologicals Direct anti-cortisol antibody was Product OBT 0646, the Sigma
anti-
cortisol antibody was Product C8409 and the Immunotech antibody was supplied
by
Beckman, Product IMBULK3 6D6.
Buffer Solutions
Buffer 1, from Bart [15]: 30 mM Tris-HCL, pH 7.2, containing 1 mM EDTA
Buffer 2, from the Sterix protocol: PBS (pH 7.4) containing 0.25M sucrose
Buffer 3, from the Sigma RIA protocol: 50 mM Tris-HCL, pH 8, containing 0.1 M
NaCL
and 0.1 % gelatin
Stop solution, from Barf [15]: 1 mM glycyrrhetinic acid in 100 % DMSO
Enzyme assays were carried out in the presence of 181 ~,M NADPH, 1 mM Glucose-
6-
Phosphate and cortisone concentrations indicated for each experiment.
Enzyme assay buffer: 30 mM Tris-HCL, pH 7.2 containing 1 mM EDTA
Antibody binding buffer: 50 mM Tris-HCL, pH 8, containing 0.1 M NaCI and 0.1
gelatin
Compound preparation: Prepare 10 mM stock solutions in 100% DMSO at 100 times
the required assay concentration. Dilute into assay buffer 1 in 25. Also
dilute neat
DMSO 1 in 25 into assay buffer for controls.
Substrate preparation: Prepare a solution of cortisone in ethanol 600 times
the required
assay concentration (175 nM). Dilute this 1 in 50 into assay buffer.
Prepare NADPH as a 1.8 mg/ml solution in assay buffer.
Prepare G-6-P as a 3.65 mg/ml solution in assay buffer.
Mix these 3 solutions 1:1:1 to make a solution of sufficient volume for 25 ~,I
additions to
each sample. Add 0.5 ~,Ci tritiated cortisone per 25 ~,I and mix the solution
well.
Microsome preparation: Dilute stock 20 mg/ml solution 1 in 100 with assay
buffer.
CA 02501228 2005-04-05
WO 2004/037251 PCT/GB2003/004590
57
Antibody preparation: Dilute stock antibody solution to 17 p,g/ml in antibody
binding
buffer.
Dextran coated charcoal preparation: Make a 20 mg/ml solution in antibody
binding
buffer and chill on ice.
Enzyme assay: To a u-bottom polypropylene 96 well plate add:
25 p.l compound dilution or diluted DMSO to controls, NSB's and blanks
p,l 1 mM GA in DMSO (enzyme stop solution) to blanks
10 25 wl substrate mixture to all samples
50 ~I diluted microsomes to all samples
Incubate plate for 30 min at 37°C shaking
Add 10 pl enzyme stop solution to all wells except the blanks
Add 100 wl antibody solution to all wells except the NSB's, add antibody
binding buffer to
these wells
Incubate at 37°C for 1 h
Chill plate on ice for 15 min
Add 50 ~,I / well charcoal solution and mix with an 8-channel pipette (4 - 5
aspirations)
Chill the plate on ice
Centrifuge at 4°C, 2000 x g for 15 min
Transfer 100 p,l supernatant into an Optiplate, also add 25 ~,I substrate
mixture to 2
empty wells to indicate counting efficiency
Add 200 wl Microscint-40 to all wells and count on a Topcount
Radioimmunoassay
The 11 a HSD1 enzyme assay was carried out following the standard operating
procedure described above in u-bottom polypropylene 96 well plates or 1.5 ml
Eppendorf
tubes as indicated for each experiment. Subsequent to stopping the enzyme
reaction,
100 ~I antibody prepared in buffer 3 unless otherwise indicated was added to
test
samples and 100 p,l buffer 3 was added to the NSB samples. The samples were
incubated for 1 hour at 37°C and the chilled on ice for 15 mins.
Dextran coated charcoal
(50 wl / sample) prepared to the indicated concentration in buffer 3 was added
and the
samples were mixed (vortex for tubes and aspiration 5 times with an >3-channel
pipette
CA 02501228 2005-04-05
WO 2004/037251 PCT/GB2003/004590
58
for 96 well plates) and chilled for a further 10 min. The samples were
centrifuged at
2000 x g for 15 min at 4°C to pellet the charcoal. Aliquots of the
supernatant (100 pl)
were transferred to an Optiplate and counted on the Topcount in 150-200 ~,I
Microscint
40. In some experiments, aliquots of supernatant were transferred to
scintillation vials
and counted on the Tricarb LSC in 5 ml Ultima Gold scintillant.
11 (3 HSD1 ASSAY DEVELOPMENT
11 ~3 HSD1 TLC format assay
Separation of Cortisone and Cortisol
Prior to performing an enzyme assay, solvent systems reported in the
literature for
separation of cortisone from cortisol were investigated [16, 17]. Solutions of
cortisone
and cortisol at 10 mg/ml were prepared in methanol, and aliquots spotted onto
a silica
gel TLC plate. The plate was run in CH2Cla: IMS 92 : 8 "/" (2). The plate was
then air
dried and sprayed with 0.1% Rhodamine B in methanol to visualise the spots.
The table
below describes the separation obtained.
Table 3: Separation of cortisone from cortisoi by TLC
Steroid Distance run originSolvent front migration
from /
(cm) steroid migration
(cm)
Cortisone 7.5 2.3
Cortisol ~ 4.5 3.8
This separation was considered adequate for use in an enzyme assay.
The literature details several methods of extracting cortisol from aqueous
solution [16,
17]. In order to select a method for use, ['4C]-labelled cortisol was obtained
from NEN.
A stock was prepared in phosphate buffered saline (PBS) containing 4000 DPM in
50,1
with cold cortisol (1~.g) added as a carrier. The final ethanol concentration
was 0.4%.
Aliquots of this solution were added to glass tubes (100p,1) and the following
extractions
were carried out: 1. 1 ml CH2CI2, vortex and pass through phase separating
filter paper
(Whatman, IPS) 2. 1 ml ethyl acetate, vortex and pass through phase separating
filter
CA 02501228 2005-04-05
WO 2004/037251 PCT/GB2003/004590
59
paper 3. 1 ml CH2C12 and 200p,1 0.05% CaCl2, vortex, centrifuge (500g for 5
min) and
remove upper aqueous phase 4. 1 ml ethyl acetate and 200p,1 0.05 % CaCl2,
vortex,
centrifuge (500g for 5 min) and collect upper organic phase. The organic
phases were
dried and the residues were taken up in 100p,1 IMS. An aliquot of this was
spotted onto a
TLC plate and the plate run as before. Following visualisation with Rhodamine
B, the
spots were scraped into scintillation vials and counted on a liquid
scintillation counter
(Packard TriCarb) in 5ml Ultima gold scintillant. Extraction efficiencies were
calculated
and are given in Figure 4.
From these results it appears that 90 % of the cortisol is lost by phase
separating
filtration. Ethyl acetate appears to extract cortisol more efficiently than
CH2CI2, possibly
because the organic phase is easier to collect. Ethyl acetate appears to be a
suitable
method of extraction.
Human and Rat Helaatic Microsomal 11 j3-HSD1 Activity
11 (3-HSD1 activity in rat and human hepatic microsomes was evaluated, to
determine
the minimum microsomal protein concentrations required for measurement of
enzyme
activity. The experiment was done according to the Bradford Method [14]. The
assay
was performed in BufFer 2 and the cortisone concentration used was 2pM
containing
0.5wCi [3H]-cortisone per incubation. Microsomes were tested at concentrations
ranging
from 50p,g to 400~g protein per incubation in a final incubation volume of
100p1 in glass
tubes. Samples were incubated for 1 h in a shaking water bath at 37°C
and the assay
was stopped by addition of 1 ml ethyl acetate. To correct for recovery, 50,1
['4C]-cortisol
was added to the samples followed by 200p1 0.05 % CaCl2. The samples were
vortex
mixed and centrifuged as described above. The upper organic phase was removed
and
dried down, and the residue dissolved in 100p.1 methanol and 50,1 aliquots
were spotted
onto TLC plates, which were run as described above. Samples were counted on a
TriCarb liquid scintillation counter using a dual label programme. Recovery
efficiency
was determined from the DPM obtained in 50p,1 ['4C]-cortisol solution, which
was counted
with the samples. Results are shown in Figure 5.
The 11 [3-HSD1 activities in rat and human microsomes were similar,
0.7pmol/mg/min
and 0.5pmol/mg/min for rat and human microsomes respectively. The activity in
human
CA 02501228 2005-04-05
WO 2004/037251 PCT/GB2003/004590
microsomes is apparently not related to microsomal protein concentration,
which may
suggest that that the protein concentration range examined is too high.
Lower human microsome protein concentrations were evaluated; 3.7p,g to 100 pg
per
5 sample. The time course of activity was also determined, from 0 to 60
minutes at 37°C.
The extraction conditions were as described above. The results from these
experiments
are shown in figures 6 and 7.
The results shown in figures 6 and 7 demonstrate that enzyme activity is
linear at
10 incubation times up to 30 min at all the microsomal protein concentrations
tested, and
that enzyme activity is linear at microsomal protein concentrations below
30p,g per
sample.
The influence of substrate concentration on activity was examined. The [3H]-
cortisone
15 concentration was kept constant at 0.5p,Ci/sample, and unlabelled cortisone
varied from
44nM to 2pM. The assay was carried out with 10pg microsomal protein per sample
with
an incubation time of 30 minutes at 37°C. The results are shown in
figure 8. A double
reciprocal plot (Lineweaver-Burke) of these data gives an apparent Km for
cortisone of
660nM, figure 9.
The standard compounds glycyrrhetinic acid and carbenoxolone were examined in
this
assay system, as part of the validation process. The assay was performed using
175nM
cortisone substrate, with 10p,g microsomal protein and a 30 minute incubation
at 37°C,
as described by Barf [15]. Although the data in figures 8 and 9 above suggest
that this
substrate concentration is not saturating under these assay conditions.
Glycyrrhetinic
acid and carbenoxolone were tested at concentrations from 0.012wM to 3~M , the
DMSO
concentration was 1 % in all samples. The results are shown in figures 10 and
11.
Glycyrrhetinic acid and carbenoxolone give ICSO values of 40nM and 119nM
respectively.
The ICSO reported for carbenoxolone by Barf et al. using the SPA format and
recombinant 11 [i-HSD is 330nM [15], approximately three-fold less potent. The
difference in potency in the two assay systems is probably due to the
different assay
conditions, SPA compared to tlc end point, and also the enzyme source, native
hepatic
enzyme compared to recombinant enzyme.
CA 02501228 2005-04-05
WO 2004/037251 PCT/GB2003/004590
61
The assay conditions described above support good enzyme activity however,
which
should be transferable to a 96 well plate format.
Development of High Througihput 11 ~3 HSD1 Assays
Supply of the antibody used by Bart [15] in the Scintillation Proximity assay
(SPA)
proved problematic. A sample batch of the antibody (from Immunotech) was
tested for
suitability and a second order was placed for a larger quantity. A robust 96
well plate
assay using Radioimmunoassay (RIA) format was developed using the Immunotech
antibody available, this is described below.
Immunoassay Format
An Assay Designs enzyme immunoassay system was evaluated as a potential assay
format. The basis of the assay is competition for antibody binding between
sample
cortisol, generated by 11[i-HSD1, and labelled cortisol binding. The anti-
cortisol
detection antibody provided in the kit is a mouse monoclonal, reported to
cross react
less than 0.1 % with cortisone. The kit is designed for the analysis of
cortisol levels in
saliva, urine, serum and plasma and also in tissue culture media, rather than
for
determining enzyme activity however.
The 11 [i-HSD1 enzyme assay conditions described by Ba'rf et al [15] were
used; human
hepatic microsomes in Buffer 1 at protein concentrations from 25pg to 200~.g,
cortisone
at concentrations from 44nM to 700nM incubated for 60 minutes at 37°C.
The effect of
0.9% Tween 80 was also investigated, as this detergent is reported to improve
the
activity of enzymes involved in steroid metabolism. Results are shown in
Figure 12.
Figure 12(A) shows the effect of protein. Data taken from the 700~,M cortisone
group
tested in the presence of Tween-80.
Figure 12(B) shows the effect of cortisone. Data taken from the 25~.g
microsomal
protein group tested in the presence of Tween-80.
Figure 12(C) shows the effect of Tween-80. Data taken from the 25~g microsomal
protein group tested in the presence of 700 ~,M cortisone.
CA 02501228 2005-04-05
WO 2004/037251 PCT/GB2003/004590
62
The assay detected cortisol in the standard curve (313 pg/ml to 10,000 pg/ml)
as
expected but the signal obtained from the enzyme assay samples decreased with
increasing microsomal protein concentration, suggesting that the microsomal
protein
may interfere with the immunoassay, figure 12(A). Addition of exogenous
cortisone had
no effect on levels of cortisol detected in the enzyme assay samples,
suggesting the
antibody does not cross react with cortisone, figure 12(B). Inclusion of
detergent in the
enzyme assay buffer had little effect, figure 12(C).
The assay conditions were varied to determine if it was feasible to use the
immunoassay
system to detect 11 (3-HSD1 activity; 24~.g microsomal protein per sample and
2~.M
cortisone substrate in Buffer 2. Enzyme activity was also measured in samples
following
the addition of steroid displacement reagent; a kit component which releases
cortisol
from cortisol binding protein, if present in the sample. The assay detected
the cortisol in
the standard curve (313pg/ml to 10,OOOpg/ml). Figure 13 shows the absorbance
at
405m obtained for the different groups:
The lowest and highest concentrations of the cortisol standard have been
included in
Figure 13 as 313 pg/ml and 1000 pglml together with the NSB absorbance to show
the
dynamic range obtained in the assay.
Absorbance obtained in the presence of reaction mixture taken from samples
incubated
with microsomal protein ("Enzyme") are lower than those in the presence of
reaction
mixture not containing microsomal protein ("No enzyme") indicating increases
in levels of
cortisol.
In the presence of the kit steroid displacement reagent ("DR") these two
reaction
mixtures show the same pattern but the signal is depressed.
Glycyrrhetinic acid (GA) in the presence of the top concentration of cortisol
standard has
no effect on the ability of the kit to measure cortisol concentrations.
Although the signal to background ratio of 2.5 for the assay is rather poor,
these data
demonstrate that the antibody can bind the cortisoI:AP conjugate and that this
can be
displaced by cortisol. An experiment was carried out to examine the effect of
increasing
microsomal protein concentration, in an attempt to improve the signal to noise
obtained.
CA 02501228 2005-04-05
WO 2004/037251 PCT/GB2003/004590
63
Microsomal protein was tested from 100pg/incubation down to 5p,g/incubation
using 2~,M
cortisone in Buffer 2. All other conditions were identical to those detailed
above. The
results are shown in figure 14.
Decreasing microsomal protein from 10~g/incubation to 5pg/incubation results
in a
corresponding decrease in enzyme activity. Increasing microsomal protein above
10~g/incubation results in a quenching of signal which may be due to the
colour of the
microsomes. Therefore the dynamic range of this assay cannot be improved by
increasing the microsomal protein concentration.
RIA development using Immunotech Antibody
The 11 ~i HSD1 assay was carried out using 10~glwell human hepatic microsomal
protein. The Immunotech antibody was used in the RIA at concentrations from
15 6.25~,g/well to 25p,g/well, the results are shown in figure 15.
The Immunotech antibody worked well in the assay and gave good signal to
background
at all the concentrations tested. The signal to noise with 12.5 and 6.1 p,g
antibody per
well was similar suggesting it may be possible to reduce the antibody
concentration.
The antibody titre, at concentrations from 0.67~.glwell to 6.7p,g/well, was
examined. The
11 j3 HSD1 assay was carried out using human microsomal protein at 20~g/well,
to
generate the optimum signal to background. Each antibody concentration was
tested
against a "no enzyme" blank (buffer substituted for microsomes), a "GA blank"
(10 pl
stop solution added prior to microsomes) and a control group. The results are
shown in
figures 16 and 17.
The saturation curve indicates that there is no difFerence in the detection of
enzyme
activity above 1.68 p,g/well. The signal to background ratio with this
antibody
concentration is good, (6 fold).Consequently the antibody will be used at 1.7
~g/well in
future assays.
Linearity of enzyme activity with human hepatic microsomal protein
concentration using
RIA detection was examined. The 11 ~ HSD1 assay was carried with microsomal
protein
concentrations varying from 1 p.g/well to 40 ~g/well. 11 (3 HSD1 activity was
linear with
CA 02501228 2005-04-05
WO 2004/037251 PCT/GB2003/004590
64
protein up to concentrations of 20 p.g/well, figure 18,~ confirming the
results obtained with
the classical enzyme assay (figure 7).
The optimal concentration of human microsomal protein to use in the assay
appears to
be l0p.g/well.
The effect of including Tween 80 in the enzyme assay buffer was also
investigated. This
assay was carried out in parallel with the assay above and under the same
conditions
except that the enzyme assay buffer (Buffer 2) contained 0.05 % Tween 80.
Microsomal
20 protein was tested at four concentrations. Tween 80 was found to increase
the blank
CPM, reducing the signal to noise of the assay. Representative data, from the
group
tested 10pg/well microsomal protein, are shown in figure 19. Similar results
were
obtained with all the microsome protein concentrations examined, consequently
Tween
will not be used in future studies..
To simplify the protocol such that both enzyme assay and RIA stages are
carried out in
the same buffer, both phases were carried out in either enzyme assay buffer
(Buffer 2)
or Buffer 3 (RIA buffer). The microsomal protein concentration used was 10
p.g/well and
the cortisone concentration was 175 nM. Performing both enzyme assay and RIA
in
Buffer 3 appears to improve the data slightly, figure 20.
Linearity of enzyme activity with incubation time was investigated. The enzyme
assay
was carried out with 10~,g/well microsomal protein and with 175nM cortisone,
and
stopped at varying time points, the results are shown in figure 21.
With microsome protein concentrations of 10~,glwell and 175nM substrate, the
reacfiion
is linear at time points up to 30 minutes. These results indicate that a
substrate
concentration of 175nM is too low. The apparent Km observed in the classical
11 [3 HSD1
assay was 660 nM (figures 8 and 9), although these assays are end-point
measurement, hence it is not certain that initial rates were measured in the
low substrate
groups with a 30 minute incubation time. However, published Km values for
cortisone in
human hepatic microsomal 11 a HSD1 assays are in the micromolar range [18,
19].
Although 175nM substrate is well below the apparent Km, it may not be possible
to
increase the concentration significantly for two reasons:
CA 02501228 2005-04-05
WO 2004/037251 PCT/GB2003/004590
(i) If the compounds are competitive with cortisone, the measured inhibition
will
fall if the substrate is increased above the concentration used in Reference
1.
(ii) Increasing the substrate concentration will reduce the specific activity
of the
label, reducing the sensitivity of the assay. This could be overcome by adding
5 higher concentrations of [3H]-cortisone, but the protocol uses 0.5~,Ci/well
and
there is a cost implication if higher levels of radioactivity are used.
Substrate saturation was examined. The enzyme assay was carried out exactly
described in the methods section, in Buffer 3 with 10~g/well microsomal
protein and with
10 [cold cortisone] as indicated. [3H]-cortisone was 0.5~Ci/sample
fihroughout. The
reaction was stopped after 30 min by the addition of 10p1 stop solution. The
RIA was
carried out exactly as indicated in the methods section. The results are shown
in
figures 22 and 23.
15 The apparent Km (700 nM), determined from the Lineweaver-Burke plot of
these data
shown in figure 23 is very similar to that determined in the tlc format 11 (3
HSD1 assay
(Figure 9, apparent Km 660 nM). The data suggests that at 10p.g microsomal
protein,
the enzyme is not saturated at 175nM cortisone, over an incubation period of
30
minutes.
Lowering the microsomal protein concentration or the incubation time to bring
the
reaction within the linear range would partly overcome the problem. However
either of
these adjustment adjustments would decrease the assay sensitivity, and
decrease the
apparent potency of inhibitors. Consequently the initial experiments were
performed with
175nM cortisone.
11 [3-HSD1 Assa~i Validation
Prior to compound testing, the tolerance of the enzyme assay to DMSO was
determined.
inclusion of DMSO at 1 % in the enzyme assay does not affect total or blank
values, but
slightly increases enzyme activity and the signal to noise ratio (Table 4).
The experiment
was repeated over a range of DMSO concentrations from 0.3 to 10%, figure 24.
CA 02501228 2005-04-05
WO 2004/037251 PCT/GB2003/004590
66
Table 4:Control and blank CPM obtained in the Glycyrrhetinic acid
IC5° assay
showing effect of 1% DMSO and signal to noise ratio obtained,
Group 1 % DMSO ~~~ No DMSO
NSB 670 661
GA blank 640 660
Control 3515 2583
Signal to noise 5 fold 4 fold
There is a slight increase in microsomal enzyme activity in the presence of
0.3% and 1
DMSO. At DMSO concentrations above 1 %, there is a linear reduction in enzyme
activity. It is reported that DMSO can both increase and reduce microsomal
enzyme
activity, depending on the concentration, presumably due to effects on the
microsomal
membranes. On the basis of these data, it is intended that compounds be
screened in
the presence of 1 % DMSO.
An ICSO value was generated for the standard inhibitor glycyrrhetinic acid,
the compound
was tested at concentrations between 0.012~M and 3wM, with a final DMSO
concentration of 1 %, figure 25.
Glycyrrhetinic acid gives a concentration-related inhibition of the enzyme
with an ICSO of
41 nM, with good curve fit values (r2 = 0.962) and Hillslope. This is similar
to the value of
40nM generated using the tlc format assay, (see figure 10). An ICSO value of
30nM has
been reported for glycyrrhetinic acid inhibition of 11 (3 HSD1 in human
hepatic
microsomes, using dehydro-dexamethasone as the substrate [19]. However, these
values are lower than the value reported by Barf et al. [15].
Table 5: Inhibition Data
STX No. Structure % inhibition of Human 11 (3 HSD1
10 M ical sd ~ 5% N=2
976 - 110
CI ~~ ~N ~ N
'N
CA 02501228 2005-04-05
WO 2004/037251 PCT/GB2003/004590
67
STX No. Structure % inhibition of Human 11 (3 HSD1
M t ical sd ~ 5% N=2
993 ~ - - 89
cl
N\ ~/ ~ CI
//
c.
994 83
cl
/ N\ // \
//
1029 78
\ H O
/ N~ //
s
CI
984 73
o~
N
O
/ CI
H
995 68
\ /
H C~ 0//
CA 02501228 2005-04-05
WO 2004/037251 PCT/GB2003/004590
68
STX No. Structure % inhibition of Human 11~i HSD1
M t i_cal sd ~ 5% N=2
469 66
N ~~~~ CI
O
S
~N
1018 / 66
°
HN NH
986 65
p ~ ~ /\\\ '/ ci
N
H p
1020 64
N
N ~-
~S
996 63
N
HN
1
°~s-o
CA 02501228 2005-04-05
WO 2004/037251 PCT/GB2003/004590
69
STX No. Structure % inhibition of Human 11~i HSD1
987 _..~a 10 uM typical sd ~- 5% N=2
62
I
Ch
1030 61
/N
S
523 - 51
/N S
O /~
\O N' \
B
992 ~ - - 61
cl
N /
~/ \
s \
cl
985 61
CI ~N ~ N
/ \\
\ O / H
CA 02501228 2005-04-05
WO 2004/037251 PCT/GB2003/004590
STX No. Structure % inhibition of Human 11 ~i HSD1
____ a 10 M ical sd ~ 5% N=2
977 61
\ /
0
N i \ ~~
0
1019 61
ci
N
~i \\
521 60
S \N
O
N-
H
978 60
°
°
b \\
1017
I
0
NH
\N CI
CA 02501228 2005-04-05
WO 2004/037251 PCT/GB2003/004590
71
STX No. Structure % inhibition of Human 11~i HSD1
,_,- 10 M ical sd ~- 5% N=2
g98 5g
a
N
s ~N~ \\
H O
585 58
H
\\ \
w ~ \o w
o _
N
ggg 55
0
CI \~ ~" ~ S
N
CI
1021 54
s
" bi
/%
470 54
N
/y
°
554 53
_W~
s
°w ~ \ /
°
\ \
-N
CA 02501228 2005-04-05
WO 2004/037251 PCT/GB2003/004590
72
STX No. Structure % inhibition of Human 11~i HSD1
M ical sd ~ 5°I° N=2
53
H H
N \ N
~e
o ~ /
991 51
/ \
°I °OS a
/ Il_~~°
°
°I \ /
N
997 ~I/ 51
"N
/ \
CI
O
CI
575 50
H
O
N a Iwl/
/%
O OH
709 49
H
O
a
\ ~ ~ s
CA 02501228 2005-04-05
WO 2004/037251 PCT/GB2003/004590
73
STX No. Structure % inhibition of Human 11~i HSD1
M ical sd ~ 5% N=2
553 4g
% %
,o
N S
H
N
519 4g
\ ,,N
\%O
S
424 47
H
i~/%
s
°i
'~N
522 - 47
H C1
CI
S'
\Ilyy~/ N
552 45
- ~ ~i°
% s
H
N/
CA 02501228 2005-04-05
WO 2004/037251 PCT/GB2003/004590
74
STX No. Structure % inhibition of Human 11 ~i HSD1
M t ical sd ~ 5% N=2
975 45
ci
o~
s~
0
N NH
~N
989 45
ci
/
\ / /~
~N
0
S
704 \ 44
H
-~s ~ ~ //
i
524 44
°.
\
/ \ ° i
N N
~S ° / \
~N
S' \
CA 02501228 2005-04-05
WO 2004/037251 PCT/GB2003/004590
STX No. Structure °l° inhibition of Human 11~i HSD1
.- 10 M ical sd ~ 5% N=2
421 42
H
\~/
// a ~
a
~N
425 - 41
N
CI H
701 41
0
N ~~~ ~
~N
981 - 40
o H
~~ ~ N \
N~
H
703 37
N
S
a
_ // w
~~~N
H
CA 02501228 2005-04-05
WO 2004/037251 PCT/GB2003/004590
76
STX No. Structure % inhibition of Human 11 ~3 HSD1
M t ical sd ~ 5% N=2
412 36
cl
~N o
~s~ cl
s o
710 35
N
~S
//O
%//~N
582 34
\
N S
O\
-O
O
N
CI
580 30
N S
CI
~O
413 29
N
CI \~ ~ ~ N
\\
CA 02501228 2005-04-05
WO 2004/037251 PCT/GB2003/004590
77
STX No. Structure % inhibition of Human 11 ~i HSD1
M ical sd ~ 5% N=2
581 2g
H
O O
F ~~/~ \
s \~
F O ~S
F \
583 2g
N
H
O
N ~ ~~
S
~N
705 2g
N
S
/%
\ //\I \
O O H
831 2g
~ \ N ~~
iN
~S
~~O
751 22
\\ ,/N \ S
\
CA 02501228 2005-04-05
WO 2004/037251 PCT/GB2003/004590
78
STX No. Structure ~% inhibition of Human 11~i HSD1
M typical sd ~ 5% N=2
708 21
0
\ /
S~N
584 20
N O
O-
S O
\Fi
SULPHONAMIDE SYNTHESIS
Method A
5 To the amine (1 eq.) dissolved in pyridine (3 eq.) was added the
corresponding
sulphonyl chloride (1.2 eq.) and the reaction mixture was stirred at RT under
N~
overnight. The resulting mixture was poured into aq. HCI and the organic layer
was
extracted with ethyl acetate, dried (MgS04), filtered and concentrated under
reduced
pressure to give the desired sulphonamide as crystalline solid or as a thick
syrup. The
10 crude compound was then purified by flash chromatography using EtOAc/hexane
(3:2)
or CH2CI2/EtOAc (4:1 ) as eluent to give crystalline solid.
Method B
To the amine (1 eq.) dissolved in Et3N (5 eq.) was added the corresponding
sulphonyl
chloride (1.2 eq.) and the reaction mixture was stirred at RT under N~
overnight. The
resulting mixture was poured into water and the organic layer was extracted
with ethyl
acetate, dried (MgSO4), filtered and concentrated under reduced pressure to
give the
desired sulphonamide as crystalline solid or as a thick syrup. The crude
compound was
then purified by flash chromatography using EtOAc/hexane (3:2) or CH~CIz/EtOAc
(4:1 )
as eluent to give crystalline solid.
CA 02501228 2005-04-05
WO 2004/037251 PCT/GB2003/004590
79
Note : Insoluble amines and sulphonyl chlorides were dissolved in minimum
amount of
CH2CI2, THF or DMF.
Method C
To a solution Arylsulphonyl chloride (1.1 eq.) in DCM were added Pyridine (2.2
eq.) and
catalytic amount of DMAP. The solution was stirred at room temperature under
nitrogen
for 10 minutes. Then the amine (1 eq.) was added and the reaction mixture was
stirred
at room temperature under nitrogen for 416 hrs. The resulting mixture was
partitioned
between DCM and 5% sodium bicarbonate. The organic layer was washed with
brine,
dried over MgS04, and concentrated to give a solid or a thick syrup. The crude
compound was then purified by flash chromatography to give desired
arylsulphonamide
as crystalline solid.
DGS03020A (STX412)
Synthesised by method A. Off-white crystals of DGS03020A (186 mg; 55%). mp 189-
190 °C; TLC Rf : 0.68 EtOAc/Hexane (3:2);'H NMR (CDCI3) b 2.80 (s, 3H,
CH3), 7.13 (s,
1 H, N-H, exchanged with D20), 7.212 (dd,1 H, Ar-H, J = 2.34 Hz and 8.59 Hz),
7.27 (dd,
1 H, Ar-H, J = 1.95 Hz and 8.59 Hz), 7.51 (d, 1 H, Ar-H, J = 1.95 Hz), 7.65
(d, 1 H, Ar-H, J
= 1.95 Hz), 7.69 (d, 1 H, Ar-H, J = 8.59 Hz), 7.91 (d, 1 H, Ar-H, J = 8.59
Hz); MS (FAB+)
372.9 [100, (M+H)+]; HRMS m/z (FAB+) 372.9627, C,4H1035CI2N~O2S2 requires
372.9639,
376.9574, C~qH,°3'ChNZO~Sg requires 376.9580; HPLC t~ 3.65 min (92 : 08
= MeOH
HBO).
DGS03022A (STX413)
Synthesised by method A. Off-white crystals of DGS03022A (233 mg; 72%). mp 178
°C;
TLC Rf : 0.71 EtOAc/Hexane (3:2); 'H NMR (CDCI3) b 2.75 (s, 3H, CH3), 2.80 (s,
3H,
CH3), 6.75 (s, 1 H, N-H, exchanged with D20), 7.11 (dd, 1 H, Ar-H, J = 1.95 Hz
and 8.59
Hz), 7.17 - 7.21 (m, 1 H, Ar-H), 7.53 (d, 1 H, Ar-H, J = 1.17 Hz), 7.55 (d, 1
H, Ar-H, J =
1.95 Hz), 7.68 (d, 1 H, Ar-H, J = 8.20 Hz), 7.92 (dd, 1 H, Ar-H, J = 1.17 Hz
and 7.81 Hz);
MS (FAB+) 164.1 [35, (5-Amino-2-methyl benzothiazole)k], 353.0 [100, (M+H)+];
HRMS
m/z (FAB+) 353.0176, C,5H1435CIN202S~ requires 353.0185, 355.0155,
C,5H143'CINZOZS2
requires 355.0156; HPLC tr 3.78 min (92 : 08 = MeOH : H20).
DGS03024A (STX421)
Synthesised by method A. White crystals of DGS03024A (240 mg; 76%). mp 133-134
°CTLC Rf : 0.7 EtOAc/Hexane (3:2); 'H NMR (CDCI3) 5 0.90 (t, 3H,
CH3CHZCH2, J =
CA 02501228 2005-04-05
WO 2004/037251 PCT/GB2003/004590
7.42 Hz), 1.56 - 1.66 (m, 2H, CH3CH2CH~), 2.59 (t, 2H, CH3CHzCH2, J = 7.42
Hz), 2.80
(s, 3H, CH3), 6.71 (s, 1 H, N-H, exchanged with D20), 7.17 (d 1 H, Ar-H, J =
2.34 Hz and
8.59 Hz), 7.201 - 7.214 (m, 1 H, Ar-H), 7.218- 7.223 (m, 1 H, Ar-H), 7.57 (d,
1 H, Ar-H, J =
2.34 Hz), 7.76-7.69 (m, 3H, Ar-H); MS (FAB+) 347.1 [100, (M+H)+]; HRMS m/z
(FAB+)
S 347.0881, C,.,H,9N2OZSz requires 347.0887; HPLC tr 3.69 min (92 : 08 = MeOH
: Ha0).
DGS03034A (STX424)
Synthesised by method A. White crystals of DGS03034A (262 mg; 86%). mp 152
°C;
TLC Rf : 0.48 EtOAc/Hexane (3:2); 'H NMR (CDCI3) S 10.31 (s, 1 H, NH, Ex. With
DSO),
10 7.85 (d, 1 H, Ar-H, J = 8.59 Hz), 7.66 - 7.69 (m, 2H, Ar-H), 7.57 (d, 1 H,
Ar-H, J = 1.95
Hz), 7.11 (dd, 1 H, Ar-H, J = 2.34 Hz and 8.59 Hz), 7.02 - 7.05 (m, 2H, Ar-H),
3.76 (s,
3H, OCH3), 2.73 (s, 3H, CH3); MS (FAB+) 164.0[25 (Amine SM+)], 335.0 [100,
(M+H)+];
HRMS m/z (FAB+) 335.0519, C,5H,5N~O3S~ requires 335.0524; HPLC t~ 1.94 min (80
20 = MeOH : HBO).
1S
DGS03036A (STX425)
Synthesised by method A. White crystals of DGS03036A (136 mg; 42%). mp 295-296
°C; TLC Rf : 0.56 EtOAc/Hexane (3:2); 'H NMR (DMSO-ds) b 10.66 (s, 1 H,
NH, Ex. With
D2O), 7.87 (d, 1 H, Ar-H, J = 8.59 Hz), 7.52 (d, 1 H, Ar-H, J = 1.95 Hz), 7.32
- 7.44 (m,
20 3H, Ar-H), 7.12 (dd, 1 H, Ar-H, J = 2.3 Hz and 8.59 Hz), 2.73 (s, 3H, CH3),
2.64 (s, 3H,
CH3); MS (FAB+) 164.0 [40, (Starting amine)+], 353.0 [100, (M+H)+]; HRMS m/z
(FAB+)
353.0187, C,5H,4s5CIN2O2S2 requires 353.0185, 355.0165, C,5H143'CIN2O2S2
requires
355.0155; HPLC tr 1.94 min (80 : 20 = MeOH : HBO).
2S DGS03058A (STX519)
Synthesised by method A. White crystals of DGS03058A (199 mg; 57%). mp 172
°C;
TLC Rf : 0.56 EtOAc/Hexane (3:2); 'H NMR (DMSO-ds) 5 10.53 (s, 1 H, NH, Ex.
With
D20), 7.88 (d, 1 H, Ar-H, J = 8.59 Hz), 7.74 - 7.77 (m, 2H, Ar-H), 7.64 - 7.68
(m, 2H, Ar-
H), 7.58 (d, 1 H, Ar-H, J = 1.95 Hz), 7.11 (dd, 1 H, Ar-H, J = 1.95 Hz and
8.59 Hz), 2.74
30 (s, 3H, CH3); MS (FAB+) 384.9 [100, (M+H)+]; HRMS m/z (FAB+) 384.9494,
C,aH,28'BrN202S2 requires 384.9503, 382.9501, C,QH,Z'9BrN2OZS2 requires
382.9523;
HPLC t~ 2.64 min (90 : 10 = MeOH : HBO).
DGS03062B (STX469)
3S To a stirred solution of DGS03022A (50 mg, 0.14 mmol, 1 eq.) in anhy. DMF
(5 ml) and
NaH (7 mg, 0.16 mmol, 1.1 eq.) was added Mel (3 ml, 0.21 mmol, 1.5 eq.) and
the
CA 02501228 2005-04-05
WO 2004/037251 PCT/GB2003/004590
81
mixture was stirred for 1 h. The resulting mixture was poured into water and
the organic
layer was extracted with ethyl acetate, dried (MgS04), filtered and
concentrated under
reduced pressure to give a yellow suspension. The crude compound (70 mg) was
purified by flash chromatography using EtOAc/hexane (3:2) as eluent to give
white
crystals of DGS03062A (36 mg; 69%). mp 97-98 °C; TLC Rf : 0.61
EtOAc/Hexane (3:2);
'H NMR (CDCI3) b 7.73 (dd, 1 H, Ar-H, J = 1.17 Hz and 7.81 Hz), 7.37 (d, 1 H,
Ar-H, J =
8.59 Hz), 7.59 (d, 1 H, Ar-H, J = 1.95 Hz), 7.49 (dd, 1 H, Ar-H, J = 1.17 Hz
and 8.2 Hz),
7.24 (dd, 1 H, Ar-H, J = 2.34 Hz and 8.59 Hz), 7.11 - 7.15 (m, 1 H, Ar-H),
3.24 (s, 3H,
CH3), 2.76 (s, 3H, CH3), 2.35 (s, 3H, CH3); MS (FAB+) 366.9 (100, (M+H)~];
HRMS m/z
(FAB+) 366.0262, C,6H1535CIN~OZS2 requires 366.0262, 368.0300,
C,6H153'CIN~OZS2
requires 368,0234; HPLC tr 1.93 min (96 : 04 = MeOH : H20).
DGS03072A (STX470)
To a stirred solution of DGS03022A (50 mg, 0.14 mmol, 1 eq.) in anhy. DMF (5
ml) and
NaH (10 mg, 0.16 mmol, 1.1 eq.) was added Etl (23 mg, 0.21 mmol, 1.5 eq.) and
the
mixture was stirred for 1 h. The resulting mixture was poured into water and
the organic
layer was extracted with ethyl acetate, dried (MgSO4), filtered and
concentrated under
reduced pressure to give a yellow suspension. The crude compound (75 mg) was
purified by flash chromatography using EtOAc/hexane (3:2) as eluent to give a
pale
yellow thick syrup of DGS03072A (16 mg; 30%). TLC Rf : 0.71 EtOAc/Hexane
(3:2); 'H
NMR (CDCI3) b 7.76 - 7.78 (m, 2H, Ar-H), 7.66 (m, 1 H, Ar-H), 7.53 - 7.55 (m,
1 H, Ar-H),
7.27 - 7.28 (m, 1 H, Ar-H), 7,14 - 7.18 (m, 1 H, Ar-H), 7.11 - 7.18 (m, 1 H,
Ar-H), 5.30 (s,
1 H, NH, Ex. with D20), 3.74 (q, 2H, Ar-H, J = 7.42 Hz and 7.03 Hz), 2.83 (s,
3H, CH3),
2.53 (s, 3H, CH3), 1.12 (t, 3H, CH3, J = 7.03 Hz), MS (FAB+) 381.1 [100,
(M+H)+]; HRMS
mlz (FAB+) 381.1062, C~,H"35CIN2O2S2 requires 381.1058, 385.0952,
C"H~,~'CIN~O~SZ
requires 385.0949.
DGS03082A (STX529 )
Synthesised by method A. White crystals of DGS03082A (230 mg; 67%). mp 85-86
°C;
TLC Rf : 0.64 EtOAc/Hexane (3:2); 'H NMR (CDCI3) 5 10.54 (s, 1 H, NH, Ex. With
DZO),
7.87 (d, 1 H, Ar-H, J = 8.59 Hz), 7.84 (broad s, 4H, Ar-H), 7.67 - 7.69 (m,
2H, Ar-H), 7.62
(d, 1 H, Ar-H, J = 1.95 Hz), 7.39 - 7.49 (m, 3H, Ar-H), 7.17 (dd, 1 H, Ar-H, J
= 1.95 Hz
and 8.59 Hz), 2.73 (s, 3H, CH3); MS (FAB+) 381.2 [100, (M+H)+]; HRMS m/z
(FAB+)
381.0730, CZ°H"N202S2 requires 381.0731; HPLC tr 1.36 min (96 : 04 =
MeOH : H20).
CA 02501228 2005-04-05
WO 2004/037251 PCT/GB2003/004590
82
DGS03084A (STX522)
Synthesised by method A. Yellow crystals of DGS03084A (46 mg; 10%). mp 253-254
°C; TLC Rf : 0.74 EtOAc/Hexane (3:2); 'H NMR (DMSO-ds) S 11.09 (s, 1 H,
NH, Ex. with
D20), 7.91 (d, 1 H, Ar-H, J = 8.59 Hz), 7.86 (s, 2H, Ar-H), 7.59 (d, 1 H, Ar-
H, J = 2.34 Hz),
7.15 (dd, 1 H, Ar-H, J = 1.95 Hz and 8.59 Hz), 2.74 (s, 3H, CH3); MS (FAB+)
409.1 [100,
(M+H)~]; MS (FAB-) 407.0 [100, (M-H)+]; HRMS m/z (FAB+) 406.9176,
C,4H1935CI3NzO2S~
requires 406.9167, 408.9136, C,4H,g3'CI3NaO2S2 requires 408.9140.
DGS03086A (STX523)
Synthesised by method A. Pale yellow crystals of DGS03086A (101 mg; 57%). mp
219
°C; TLC Rf : 0.71 EtOAclHexane (3:2); 'H NMR (DMSO-ds) b 10.68 (s, 1 H,
NH, Ex. With
D20), 7.87 (d, 1 H, Ar-H, J = 8.59 Hz), 7.79 (d, 1 H, Ar-H, J = 8.59 Hz), 7.64
(d, 1 H, Ar-H,
J = 1.95 Hz), 7.54 - 7.57 (m, 2H, Ar-H), 7.11 (dd, 1 H, Ar-H, J = 2.34 Hz and
8.59 Hz),
2.73 (s, 3H, CH3), 2.59 (s, 3H, CH3); MS (FAB+) 399.0 [100, (M+H)+], 164.1
[50,
(Starting amine)+]; HRMS m/z (FAB+) 398.9663, C,SH138'BrN~O~S2 requires
398.9569,
396.9684, C,5H13'9BrN~O~S2 requires 396.9680; HPLC t~ 1.39 min (96 : 04 = MeOH
HBO).
DGS03064
2,4-Dichloro benzoic acid (10 g, 0.0523 mol, 1 eq.) was heated to 115
°C with excess
chlorosulphonic acid (10.5 mL, 0.1571 mol, 3 eq.) under N2 for 18 h. The
resulting
mixture was cooled and consciously poured into ice-water. The resulted white
precipitate
was filtered out, washed with plenty of water and dried under vacuum over
night. The
crude DGS03064 (11.5 g, 76 %) was used for the subsequent reaction without
further
purification. mp 173-174 °C; TLC Rf ; 0.48 (4:1, CH2Ch/EtOAc); 'H NMR
(CDCI3) b 8.28
(1 H, s, Ar-H), 7.65 (1 H, s, Ar-H); MS m/z (FAB+) 286.9 [100, (M+H)+]; HRMS
m/z
(FAB+) 287.8798, C,H3aeC1304S requires 287.8818, 291.8755, C,H33'CI3O4S
requires
291.8759.
DGS03088A (STX524)
Synthesised by method B. Two compounds were isolated - DGS03088A and
DGS03088A. White crystals of DGS03088A (48 mg; 13%). mp 153-155 °C;
TLC Rf
0.79 EtOAc/Hexane (3:2); 'H NMR (CDCI3) b 8.31 (s, 1 H, NH, Ex. With D20),
8.07 (s,
1 H, NH, Ex. With DZO), 8.07 (s, 1 H, Ar-H), 7.71 - 7.79 (m, 4H, Ar-H), 7.67
(d, 1 H, Ar-H,
J = 1.95 Hz), 7.58 (s, 1 H, Ar-H), 7.27 (dd, 1 H, Ar-H, J = 2.72 Hz and 8.59
Hz), 2.83 (s,
CA 02501228 2005-04-05
WO 2004/037251 PCT/GB2003/004590
83
3H, CH3), 2.79 (s, 3H, CH3); MS (FAB+) 562.9 [100, (M+H)+]; HRMS m/z (FAB+)
562.9825, C23H~,35ChN403S3 requires 562.9839, 566.9778, C~~H"3'CIzN4O3S3
requires
566.9781; HPLC t~ 1.33 min (96 : 04 = MeOH : H20).
DGS03088-1 (STX575)
White crystals of DGS03088-1 (31 mg; 12%). mp 147-948 °C; TLC Rf :
0.45
EtOAc/Hexane (3:2); 'H NMR (CDCI3) b 8.45 (s, 1 H, NH, Ex. With D20), 8.17 (d,
1 H, Ar-
H, J = 8.09 Hz), 8.04 (s, 1 H, Ar-H), 7.77 (s, 1 H, Ar-H), 7.50 (d, 1 H, Ar-H,
J = 1.83 Hz),
7.35 (dd, 1 H, Ar-H, J = 1.83 Hz and 8.05 Hz), 2.85 (s, 3H, CH3); LC-MS 418.1
[100,
(M~)]; HPLC tr 1.97 min (96 : 04 = MeOH : HBO).
DGS03100A (STX552)
Synthesised by method B. White crystals of DGS03100A (224 mg; 69%). mp 222-223
°C; TLC Rf : 0.56 CH2CI2/EtOAc (4:1 ); 'H NMR (DMSO-d6) S 10.27 (s, 1
H, NH, Ex. With
DSO), 9.16 - 9.17 (m, 1 H, Ar-H), 8.48 - 8.51 (m, 2H, Ar-H), 8.36 - 8.38 (m,
2H, Ar-H),
8.23 - 8.25 (m, 1 H, Ar-H), 7.67 - 7.34 (m, 3H, Ar-H), 7.51 - 7.12 (m, 1 H, Ar-
H), 7.09 -
7.12 (m, 1 H, Ar-H), 2.67 (s, 3H, CH3); LC-MS 355.7 [(M)+]; MS (FAB+) 356.0
[100,
(M+H)+]; HRMS m/z (FAB+) 356.0531, C"H,4N3O2S2 requires 356.0527; HPLC tr 1.86
min (96 : 04 = MeOH : Ha0).
DGS03102A (STX553)
Synthesised by method B. Pale yellow crystals of DGS03102A (170 mg; 52%). mp
89-90
°C; TLC Rf : 0.55 CH~GhIEtOAc (4:1 ); 'H NMR (DMSO-d6) 5 10.87 (s, 1 H,
NH, Ex. With
D20), 8.28 - 8.24 (m, 1 H, Ar-H), 8.06 - 8.22 (m, 2H, Ar-H), 8.05 (d, 1 H, Ar-
H, J = 8.20
Hz), 7.60 - 7.77 (m, 2H, Ar-H), 7.47 (d, 1 H, Ar-H, J = 1.95 Hz), 7.04 (dd, 1
H, Ar-H, J =
1.95 Hz and 8.59 Hz), 2.69 (s, 3H, CH3); MS (FAB+) 355.0 [100, (M+H)+]; HRMS
m/z
(FAB+) 355.0576, C,8H,5N202S2 requires 355.0575; HPLC tr 1.93 min (96 : 04 =
MeOH
H20).
DGS03104A (STX554)
Synthesised by method B. Yellow crystals of DGS03104A (230 mg; 63%). mp 85-86
°C;
TLC Rf : 0.65 CH2CI2/EtOAc (4:1 ); 'H NMR (DMSO-ds) b 10.84 (s, 1 H, NH, Ex.
With
DSO), 8.40 - 8.42 (m, 2H, Ar-H), 8.22 - 8.23 (m, 1 H, Ar-H), 7.76 - 7.78 (m, 1
H, Ar-H),
7.58 - 7.65 (m, 2H, Ar-H), 7.51 - 7.56 (m, 1 H, Ar-H), 7.23 - 7.25 (m, 1 H, Ar-
H), 7.05 -
7.07 (m, 1 H, Ar-H), 2.79 (s, 6H, 2xCH3), 2.69 (s, 3H, CH3); MS (FAB+) 398.1
[100,
CA 02501228 2005-04-05
WO 2004/037251 PCT/GB2003/004590
84
(M+H)+]; HRMS m/z (FAB+) 398.0978, C2°HZ°N302S2 requires
398.0997; HPLC tr 2.01
min (96 : 04 = MeOH : H20).
DGS03116A (STX580)
Synthesised by method B. Pale yellow crystals of DGS03116A (151 mg; 44%). mp
153
°C; TLC Rf : 0.55 CH2CI2/EtOAc (4:1 ); 'H NMR (DMSO-ds) c5 10.96 (s, 1
H, NH, Ex. With
DSO), 8.00 (d, 1 H, Ar-H, J = 2.34 Hz), 7.90 (d, 1 H, Ar-H, J = 8.59 Hz), 7.66
- 7.73 (m,
2H, Ar-H), 7.58 (d, 1 H, Ar-H, J = 2.34 Hz), 7.17 (dd, 1 H, Ar-H, J = 2.34 Hz
and 8.59 Hz),
2.74 (s, 3H, CH3); MS (FAB+) 372.8 [100, (M+H)+]; HRMS m/z (FAB+) 375.9599,
lO C,4H"3'CIZN2OZS2 requires 375.9502, 372.9606, C,4H1135CIZN2O2S2 requires
372.9639;
HPLC t~ 2.98 min (90 : 10 = MeOH : HZO).
DGS03118A (STX581)
Synthesised by method B. White crystals of DGS03118A (416 mg; 42%). mp 88-89
°C;
TLC Rf : 0.49 CH~Ch/EtOAc (4:1 ); 'H NMR (DMSO-d6) S 10.47 (s, 1 H, NH, Ex.
With
D20), 7.74 (d, 1 H, Ar-H, J = 8.59 Hz), 7.58 - 7.61 (m, 2H, Ar-H), 7.35 - 7.38
(m, 1 H, Ar-
H), 7.13 - 7.17 (m, 1 H, Ar-H), 4.76 - 4.78 (m, 2H, CHI), 3.75 - 3.79 (m, 2H,
CHZ), 2.90 -
2.93 (m, 2H, CHa), 2.73 (s, 3H, CH3); MS (FAB+) 456.0 [100, (M+H)+]; HRMS m/z
(FAB+) 456.0663, C~9H"F3N3O3S2 requires 456.0663; HPLC t~ 1.63 min (96 : 04 =
MeOH
: H20).
DGS03120A (STX582)
Synthesised by method B. Pale yellow crystals of DGS03120A (185 mg; 55%). mp
91-92
°C; TLC Rf : 0.51 CHaCl2/EtOAc (4:1 ); 'H NMR (DMSO-d6) S 10.35 (s, 1
H, NH, Ex. With
D20), 7.85 (d, 1 H, Ar-H, J = 8.98 Hz), 7.69 (d, 1 H, Ar-H, J = 2.34 Hz), 7.61
(dd, 1 H, Ar-
H, J = 2.73 Hz and 8.98 Hz), 7.55 (d, 1 H, Ar-H, J = 2.73 Hz), 7.20 (d, 1 H,
Ar-H, J = 8.98
Hz), 7.15 (dd, 1 H, Ar-H, J = 2.3 Hz and 8.59 Hz), 3.89 (s, 3H, OCH3), 2.73
(s, 3H, CH3);
MS (FAB+) 369.0 [100, (M+H)+]; HRMS m/z (FAB+) 371.0114, C,5H143'CIN2O3S2
requires
371.0105, 369.0135, C,5H,435CIN~O3S~ requires 369.0134; HPLC t~ 1.68 min (96 :
04 =
MeOH : H20).
DGS03122A (STX731)
Synthesised by method B. Two compounds were isolated - DGS03122A and
DGS03122B. Yellow crystals of DGS03122A (67 mg; 22%). mp 272-273 °C;
TLC Rf
0.59 CH2CI2/EtOAc (4:1 ); 'H NMR (DMSO-ds) b 8.15 (m, 5H, , Ar-H), 8.02 - 8.09
(m, 4H,
CA 02501228 2005-04-05
WO 2004/037251 PCT/GB2003/004590
Ar-H), 7.74 (d, 1 H, Ar-H, J = 2.3 Hz), 7.14 (dd, 1 H, Ar-H, J = 1.95 Hz and
8.59 Hz), 2.84
(s, 3H, CH3); MS (FAB+) 495.0 [100, (M+H)+]; HPLC t~ 1.79 min (90 : 10 = MeOH
: H20).
DGS03122B (STX583)
5 Yellow crystals of DGS03122B (47 mg; 16%). mp 204-206 °C; TLC Rf :
0.48
CH2CI2lEtOAc (4:1 ); 'H NMR (DMSO-ds) b 10.95 (s, 9 H, NH, Ex. With D20), 8.03
- 8.07
(m, 2H, Ar-H), 7.86 - 7.91 (m, 2H, Ar-H), 7.77 - 7.81 (m, 1 H, Ar-H), 7.55 (d,
1 H, Ar-H, J
= 1.95 Hz), 7.12 (dd, 1 H, Ar-H, J = 2.34 Hz and 8.59 Hz), 2.74 (s, 3H, CH3);
MS (FAB+)
330.0 [100, (M+H)+]; HRMS m/z (FAB+) 330.0370, C,SH~ZN3O2S2 requires 330.0371;
10 HPLC tr 1.84 min (90 : 10 = MeOH : HZO).
DGS03124A (STX584)
Synthesised by method B. Pale yellow crystals of DGS03124A (125 mg; 55%). mp
188-
189 °C; TLC Rf : 0.37 CHzCl2/EtOAc (4:1 ); 'H NMR (DMSO-d6) b 10.09 (s,
1 H, NH, Ex.
15 With DSO), 7.81 (d, 1 H, Ar-H, J = 8.59 Hz), 7.63 (d, 1 H, Ar-H, J = 8.20
Hz), 7.56 (d, 1 H,
Ar-H, J = 1.95 Hz), 7.14 (dd, 1 H, Ar-H, J = 1.95 Hz and 8.59 Hz), 6.96 (s, 1
H, Ar-H),
6.81 (d, 1 H, Ar-H, J = 8.59 Hz), 3.87 (s, 3H, OCH3), 2.72 (s, 3H, CH3), 2.28
(s, 3H, CH3);
MS (FAB+) 219.1 [20, (sufphonyl chloride-H)+], 349.0 [100, (M+H)+]; HRMS m/z
(FAB+)
349.0678, C,6H"NZO3S2 requires 349.0681; HPLC tr 1.80 min (96 : 04 = MeOH :
H20).
DGS03126A (STX585)
Synthesised by method B. Pale yellow crystals of DGS03126A (145 mg; 40%). mp
84-86
°C; TLC Rf : 0.71 CH~CI2/EtOAc (4:1 ); 'H NMR (DMSO-ds) b 10.42 (s, 1
H, NH, Ex. With
DzO), 7.88 (d, 1 H, Ar-H, J = 8.59 Hz), 7.73 - 7.77 (m, 2H, Ar-H), 7.59 (d, 1
H, Ar-H, J =
1.95 Hz), 7.41 - 7.46 (m, 2H, Ar-H), 7.22 - 7.26 (m, 2H, Ar-H), 7.13 (dd, 1 H,
Ar-H, J =
8.59 Hz and 2.34 Hz), 7.02 - 7.10 (m, 4H, Ar-H), 2.75 (s, 3H, CH3); MS (FAB+)
397.0
[100, (M+H)+]; HRMS m/z (FAB+) 397.0671, C~°H"N203S2 requires 397.0681;
HPLC t~
1.93 min (96 : 04 = MeOH : H20).
DGS03130A (STX730)
Synthesised by method B. Two compounds were isolated - DGS03130A and
DGS03130B. Synthesised by method B. Pale yellow crystals of DGS03130A (105 mg;
33%). mp 125-126 °C; TLC Rf : 0.55 CHZCh/EtOAc (4:1 ); 'H NMR (DMSO-ds)
S 8.21 -
8.24 (m, 4H, Ar-H), 8.13 (d, 1 H, Ar-H, J = 8.59 Hz), 7.99 - 8.03 (m, 4H, Ar-
H), 7.57 (d,
1 H, Ar-H, J = 1.95 Hz), 7.03 (dd, 1 H, Ar-H, J = 8.59 Hz and 1.95 Hz), 2.82
(s, 3H, CH3);
CA 02501228 2005-04-05
WO 2004/037251 PCT/GB2003/004590
86
2.69 (s, 6H, 2xCH3); MS (FAB+) 529.0 [100, (M+H)+]; MS (FAB-) 527.1 [70, (M-
H)+],
345.0 [100; (M-2-Acetyl sulphonyl chloride)+]; HPLC t~ 1.81 min (96 : 04 =
MeOH : H20).
DGS03130B (STX701)
Pale yellow crystals of DGS03130A (45 mg; 14%). mp 169 °C; TLC Rf
: 0.42
CH~CI~/EtOAc (4:1 ); 'H NMR (DMSO-ds) S 10.63 (s, 1 H, NH, Ex. With D2O), 8.04
- 8.07
(m, 2H, Ar-H), 7.86 - 7.89 (m, 3H, Ar-H), 7.59 (d, 1 H, Ar-H, J = 1.95 Hz),
7.13 (dd, 1 H,
Ar-H, J = 8.9 Hz and 2.3 Hz), 3.73 (s, 3H, CH3); 2.56 (s, 3H, CH3); MS (FAB+)
347.0
[100, (M+H)+], 219.1 [10, (sulphonyl chloride+H)+]; HRMS m/z (FAB+) 347.0522,
lO C,6H15N203Sa requires 347.0524; HPLC t~ 1.77 min (96 : 04 = MeOH : HBO).
DGS03134A (STX703)
Synthesised by method B. Pale yellow crystals of DGS03134A (91 mg; 23%). mp
206
207 °C; TLC Rf : 0.81 CH2CI2/EtOAc (4:1 ); 'H NMR (DMSO-ds) 8 10.37 (s,
1 H, NH, Ex.
With DSO), 7.87 (d, 1 H, Ar-H, J = 8.59 Hz), 7.46 (d, 1 H, Ar-H, J = 1.95 Hz),
7.19 (s, 2H,
Ar- -H), 7.07 (dd, 1 H, Ar-H, J = 8.59 Hz and 1.95 Hz), 4.13 - 4.20 (m, 2H,
2x(CH3)~H),
2.83 - 2.89 (m, 1 H, (CH3)ZH), 2.72 (s, 3H, CH3), 1.15 (d, 12H, 4x(CH3)2, J =
7.03 Hz),
1.11 (d, 9H, 2x(CH3)2, J = 6.64 Hz); LC-MS 429.72 (M)+; HPLC tr 2.84 min (90 :
10 =
MeOH : H20).
DGS03136A (STX704)
Synthesised by method B. Pale yellow crystals of DGS03136A (225 mg; 71 %). mp
54-55
°C; TLC Rf : 0.50 CH2Ch/EtOAc (4:1 ); ' H NMR (CDCI3) S 7.65 (m, 3H, Ar-
H), 7.58 (d, 1 H,
Ar-H, J = 2.34 Hz), 7.18 (dd, 1 H, Ar-H, J = 8.6 Hz and 1.95 Hz), 6.84 - 6.85
(m, 2H, Ar-
H); 6.82 (s, 1 H, NH, Ex. With DaO), 4.51 - 4.60 (m, 1 H, (CH3)2H), 2.80 (s,
3H, CH3), 1.31
(s, 6H, (CH3)Z); LC-MS 347.6 (M)+; HRMS m/z (FAB+) 347.0847, C"H,9N2O2S2
requires
347.0837; HPLC t~ 2.39 min (90 : 10 = MeOH : HBO).
DGS03138B (STX705)
Synthesised by method B. Pale yellow crystals of DGS03138B (24 mg; 7%). mp 248
°C;
TLC Rf : 0.52 CHZCh/EtOAc (4:1 ); 'H NMR (CDCI3) b 8.18 (d, 1 H, Ar-H, J =
8.59 Hz),
8.15 (d, 1 H, Ar-H, J = 1.95 Hz), 7.89 - 8.04 (m, 4H, Ar-H), 7.51 (dd, 1 H, Ar-
H, J = 8.20
Hz and 1.95 Hz), 7.27 (s, 1 H, NH, Ex. With DSO), 2.89 (s, 3H, CH3), 1.59 (s,
3H, CH3);
LC-MS 372.90 (M+CH3CN)+; HRMS m/z (FAB+) 371.2281, C,6H~5N2O4Sz requires
371.2278; HPLC t~ 2.22 min (90 : 10 = MeOH : HBO).
CA 02501228 2005-04-05
WO 2004/037251 PCT/GB2003/004590
87
DGS03140A (STX711 )
Synthesised by method B. Brown crystals of DGS03140A (85 mg; 26%). mp 73-75
°C;
TLC Rf : 0.59 CH2Ch/EtOAc (4:1 ); 'H NMR (CDCI3) c5 7.85 (d, 1 H, Ar-H, J =
8.59 Hz),
7.80 (d, 1 H, Ar-H, J = 8.59 Hz), 7.68 (s, 1 H, NH, Ex. With D20), 7.57 (d, 1
H, Ar-H, J =
1.95 Hz), 7.54 (s, 1 H, NH, Ex. With DSO), 7.24 (d, 1 H, Ar-H, J = 2.34 Hz),
7.18 (dd, 1 H,
Ar-H, J = 8.20 Hz and 1.95 Hz), 7.03 (dd, 1 H, Ar-H, J = 8.59 Hz and 2.34 Hz),
6.77 (dd,
1 H, Ar-H, J = 8.59 Hz and 2.34 Hz), 2.78 (s, 3H, CH3), 2.24 (s, 3H, CH3); LC-
MS 362.32
(M)+; HRMS m/z (FAB+) 361.0587, C~gH16N3~3'S2 requires 361.0636; HPLG t~ 2.09
min
(90 : 10 = MeOH : H20).
DGS03142A (STX706)
Synthesised by method B. Pale yellow crystals of DGS03142A (79 mg; 24%). mp 89-
91
°C; TLC R, : 0.65 CH~Ch/EtOAc (4:1 );'H NMR (CDCI3) 5 7.78 (d, 1 H, Ar-
H, J = 8.20 Hz),
7.61 (d, 1 H, Ar-H, J = 1.56 Hz), 6.98 (dd, 1 H, Ar-H, J = 1.95 Hz and 8.20
Hz), 6.93 (s,
1 H, Ar-H), 6.92 (s, 1 H, NH, Ex. With DSO), 3.99 (s, 6H, 2xCH3), 3.93 (s, 6H,
2xCH3),
2.85 (s, 3H, CH3); LC-MS 361.48 (M)+; HRMS m/z (FAB+) 361.1605, C,8H2,N~OzS~
requires 361.1606; HPLC t~ 2.26 min (90 : 10 = MeOH : H2O).
DGS03144A (STX707)
Synthesised by method B. Pale yellow crystals of DGS03144A (79 mg; 24%). mp 89-
91
°C; TLC Rf : 0.69 CHzCh/EtOAc (4:1 );'H NMR (CDCI3) b 7.70 (d, 1 H, Ar-
H, J = 8.59 Hz),
7.58 (d, 1 H, Ar-H, J = 2.34 Hz), 7.39 (dd, 1 H, Ar-H, J = 2.34 Hz and 8.59
Hz), 7.19 (d,
1 H, Ar-H, J = 1.95 Hz), 7.17 (t, 1 H, Ar-H, J = 1.95 Hz), 6.83 (d, 1 H, Ar-H,
J = 8.59 Hz),
6.59 (s, 1 H, NH, Ex. With D20), 3.89 (s, 3H, OCH3), 3.76 (s, 3H, OCH3), 2.81
(s, 3H,
CH3); LC-MS 363.02 (M)+; HRMS m/z (FAB+) 365.0642, C,sH~,N2O4Sa requires
365.0585; HPLC t~ 2.15 min (90 : 10 = MeOH : HZO).
DGS03146A (STX708)
Synthesised by method B. Pale yellow crystals of DGS03146A (181 mg; 51%). mp
175
°C; TLC Rf : 0.57 CH2CI2/EtOAc (4:1 ); 'H NMR (CDCI3) S 7.71 (dd, 1 H,
Ar-H, J = 2.3 Hz
and 8.98 Hz), 7.59 (d, 1 H, Ar-H, J = 1.95 Hz), 7.43 (d, 1 H, Ar-H, J = 8.98
Hz), 7.21 (dd,
1 H, Ar-H, J = 1.95 Hz and 8.59 Hz), 6.67 (s, 9 H, NH, Ex. With D20), 2.81 (s,
3H, OCH3),
1.59 (s, 6H, 2xCH3), 1.29 (s, 6H, 2xCH3); LC-MS 377.01 (M)+; HRMS m/z (FAB+)
377.0988, C,gH2~NZO3S2 requires 377.0994; HPLC tr 2.53 min (90 : 10 = MeOH :
H20).
CA 02501228 2005-04-05
WO 2004/037251 PCT/GB2003/004590
88
DGS03148A (STX709)
Synthesised by method B. Off-white crystals of DGS03148A (102 mg; 31%). mp 214
215 °C; TLC Rf : 0.62 CHZCIZ/EtOAc (4:1 ); 'H NMR (CDCI3) 5 7.71 - 7.73
(m, 2H, Ar-H),
7.60 - 7.61 (m, 1 H, Ar-H), 7.44 - 7.46 (m, 2H, Ar-H), 7.21 - 7.24 (m, 2H, Ar-
H), 6.61 (s,
1 H, NH, Ex. With Dz0), 2.83 (s, 3H, CH3), 1.31 (s, 9H, (CH3)3); LC-MS 360.12
(M)+;
HRMS m/z (FAB+) 361.1057, C~gHz,NzO3S2 requires 361.1044; HPLC t~ 2.67 min (90
= MeOH : Ha0).
10 ~ DGS03150A (STX710)
Synthesised by method B. Pale yellow crystals of DGS03150A (101 mg; 30%). mp
200-
201 °G; TLC Rf : 0.50 CH~Ci~/EtOAc (4:1 ); 'H NMR (CDCI3) b 7.65 (d, 1
H, Ar-H, J = 8.59
Hz), 7.44 (d, 1 H, Ar-H, J = 1.95 Hz), 7.09 (dd, 1 H, Ar-H, J = 1.95 Hz and
8.59 Hz), 6.75
(s, 1 H, NH, Ex. With D20), 2.79 (s, 3H, CH3), 2.57 (s, 6H, 2xCH3), 2.24 (s,
3H, CH3),
2.19 (s, 6H, 2xCH3); LC-MS 374.10 (M)+; HRMS m/z (FAB+) 375.1195, C,gH~3N2O~S2
requires 375.1201; HPLC t~ 3.15 min (80 : 20 = MeOH : HBO).
DGS03152A (STX712)
Synthesised by method B. Pale yellow crystals of DGS03152A (120 mg; 33%). mp
181-
182 °C; TLC Rf : 0.65 CHZCiZIEtOAc (4:1 ); 'H NMR (CDCI3) 5 7.63 (d, 1
H, Ar-H, J = 8.59
Hz), 7.59 (d, 1 H, Ar-H, J = 2.3 Hz), 7.22 (dd, 1 H, Ar-H, J = 2.3 Hz and 8.59
Hz), 4.91 (s,
1 H, NH, Ex. With DSO), 3.82 (s, 3H, CH3); LC-MS 392.96 (M)+; HRMS m/z (FAB+)
394.9941, C,4HaF5N202S~ requires 394.9947; HPLC t~ 2.49 min (90 : 10 = MeOH :
H20).
DGS03158A (STX713)
Synthesised by method B. Yellow crystals of DGS03158A (158 mg; 40%). mp 334-
335
°C; TLC Rf : 0.47 CH2CIZ/EtOAc (4:1 ); ' H NMR (CDCI3) b 7.65 (d, 1 H,
Ar-H, J = 8.59 Hz),
7.47 (d, 1 H, Ar-H, J = 2.3 Hz), 7.10 (dd, 1 H, Ar-H, J = 2.3 Hz and 8.59 Hz),
6.69 (s, 1 H,
NH, Ex. With DSO), 2.79 (s, 3H, CH3), 2.61 (t, 2H, CHz, J = 6.64 Hz), 2.55 (s,
3H, CH3),
2.51 (s, 3H, CH3), 2.08 (s, 3H, CH3), 1.79 (t, 2H, CH2, J = 7.03 Hz), 1.29 (s,
6H, 2xCH3);
LC-MS 431.11 (M)+; HPLC t~ 3.24 min (90 : 10 = MeOH : H20).
CA 02501228 2005-04-05
WO 2004/037251 PCT/GB2003/004590
89
Synthesis of Benzothiazole A~~Isuliphonamide Derivatives
N
a _ ~ N R b ~ I O ~ N R
I >-SH ~ ~~--S ~ ~ ,, I
HaN i S HEN i S CI OS'N i S
H
R = -C2Hs R = -C2Hs
R = -CzH4OCH3 R = -CZH40CH3
R = -CH2COOC2H5 R = -CH2COOC~H~
N
I ,O
CI ~ ,S'N I ~ S S O
I O ~ N ~ c O H
y
CI ~ ~S N I , S S O +
O H ~ I O ~N~N~
CI O,S'N I ~- S
H
~ N b O ~ N
I / ~~-R ----~ Ar~s\ I / ~>-R
H N S ~ N S
H
R=-H
R = -CH3 Ar = 3-CI-2-CH3-phenyl, R = -H
Ar = 3-CI-2-CH3-phenyl, R = -CH3,
Ar = 4-n-propylphenyl, R = -CH3,
Ar = 2,5-dichlorophenyl, R = -CH3,
H H R
N
H~N I ~ s ° -' cl ~ ~ ,s~ I o S ° ' cl ~ I ,s~ I ~- S
°
O H O R
R = -CH3
R = -CH2COOC~HS
a) RX, NaH, THF r.t. b) ArSO3Cl, DCM,Pyridine or ArS03Cl, DCM,Pyridine/DMAP
c) Diethylamine, DCM, AICI3 d) RX, IC~C03, Acetone, reflux
CA 02501228 2005-04-05
WO 2004/037251 PCT/GB2003/004590
I ~ N>--CI a OZN I ~ ~>---CI + I / ~CI
/ S / S OzN S
Ib
HEN \ N ~, I
~~-CI + ~ CI
/ S HEN / S
1~
H
4 ~N
/ S~-cl cl ~ I s° I % S cl
p N
CI H
d d
H
O~ N
\ ON I / S>--R CI ~ I ~S~N I / S R
CI H
R = -NHCH3 R = -NHCH3
R =-N~C2Hs)2 R = _N~CZHs)a
I ~ I ~ '~--- b _ I ~ '~- I
R~S R / S R / S R / S
N02 NH2 O~\ NH
R=-CH3 O R=-CH3
R = -OCH3 I / R = -OCH3
CI
NOZ NHS
S,
c NH
N a I S~ b I / S ~ I
I / S ~ / S
N ~ N c N
OzN I / S H2N I / S ArwS~O I / y
~S
O H
Ar = 3-CI-2-CH3-phenyl
Ar = 2,5-dichlorophenyl
a) HN03, HZS04 -5 - 0°C b) H2, 5°lo PdlC, C2H50H, c) ArS03Cl,
DCM,Pyridine or ArS03Cl,
DCM,Pyridine/DMAP d) amine, THF, reflux
CA 02501228 2005-04-05
WO 2004/037251 PCT/GB2003/004590
91
CI H CI
HaN ~ N b ~ ~S~N ~ N
- . ~ ,o ~ ,~-
HaN ~ N a CI ~ S ' CI ~ S
CI
S
CI H CI
HaN ~ N b ~ ,N N
Ar, SO
S ~ S
Ar = 3-CI-2-CH3-phenyl
Ar = 2,5-dichlorophenyl
Ar = 4-n-propylphenyl
O"O
S,
N
Br I ~ ~ ~ ~ , H
S ~ S
CI
S
i
O.~N I ~ N
CI S
a) N-chlorosuccinimide, IPA b) ArS03Cl, DCM,Pyridine or ArS03Cl,
DCM,Pyridine/DMAP
c) N-bromosucinimide, CCI4, benzoyl peroxide d) 3-chloro-
2methylbenzenesulphomamide, KaC03, CH3CN
CA 02501228 2005-04-05
WO 2004/037251 PCT/GB2003/004590
92
N \ ~ ~ °_
cl I so I \ ~~S I ~° ~ N ~ I ,~ ~ N
o ~N~S cl ,sue I ~ '~-S o cl ,s' I ~ s~-s
H O N S O N
H H
STX751, XDS01141 STX752, XDS01142 STX754, XDS01144
N
,o ~ N ~ I ,o
CI OS~ I / S~-S O CI ~ ,S~N~S N
N O
. , H O
STX755, XDS01145A STX763, XDS01145B
I
I ~ O I ~ ~ I ~ ,o I ~ ~ CI
CI ,S,N ~ S CI ,S~N ~ S
O H O H
STX750, XDS01139 STX886, XDS01187B
CI
STX887,
CI ~ N XDS01187A
N I
I ' I '~ OS\N / S \ ~ N
,S~N / S CI O=S=O I / S~ I ,
CI O H CI ~ N
H
STX888, XDS01188B CI I / STX890,
XDS01189
STX889,XDS01188A
H / ~ ~N O
N ~ ~ N I
CI I ~ ,S N I ~ S~° CI I ~ ,S N I / S~° CI QS~N O S
O H O
STX753, XDS01143 STX831, XDS01163 ~° STX764,
XDS01149
N H
I ~~ I \ ' CI ~ y N ~~ 'N y N
CI ,S~N~g~ I ,~ I '>---CI ~ SO I '>---CI
O ~ CI ,S~N~S I , ~S
O=S=O O H
CI
I STX768,XDS01151B STX834,XDS01168
CI
STX767, XDS01151A
CA 02501228 2005-04-05
WO 2004/037251 PCT/GB2003/004590
93
H
I \ N NH \ °SO ~ \ ~NH \ °SvN I \
cl ,s' ~s~ ' I ~ ~S ' I °
o N ~ s
H CI
CI
STX833, XDS01167 STX835, XDS01176 STX836, XDS01177
\ ~ \ N
I
\ I \ N N \O ~ S ~ ~ S
CI I ,S N,~S~ ~ CI ~S\ NH CI ~S~NH
O H I O ~O
I~
STX878, XDS01164 STX989, XDS02038 STX1021,
XDS02069
I \ ~O ~ ~O N
\ \
CI ~S CI ,S'N ~ S I O
O 'NH ~;S=O CI ~S'N I ~' S
I \ ~ , ~ ° H
i
S \ I CI STX998,
STX996, XDS02047 XDS02048B
CI STX997,XDS02048A
GI
\ \ N O H
N
cl I 'so ~ j ~ ~ =s~° cl
cl o s
H ~ CI \ ~S~ N I \ N
STX999, XDS02049 STX992, XDS02042B ~ ~ ° ~' S
STX991,
XDS02042A
H CI \
CI °\ N \ N I / ° H CI
I , S° I ~ S~ ° \S~° CI \ \SO \
~S..N \ N ~ ~ I ~ S
CI I \ v°
S
STX993, XDS02043B STX994,
STX995, XDS02044A XDS02044B
=N
S
H CI s S i
CI OS~ N \ N CI ~S.N \ I ~~ \ I ~ S
N
°I I ~ S~ I °o cl °s,N \ I
U I ,o
STX1017, XDS02055B STX1029, XDS02070A STX1030,
XDS02070B
CA 02501228 2005-04-05
WO 2004/037251 PCT/GB2003/004590
94
Genera! Method for the Preparation of N-Benzothiazole Benzenesulphonamide
derivatives:
To a solution arylsulphonyl chloride (1.1 eq.) in DCM (5-10 mL) were added
pyridine (2.2
eq.) and catalytic amount of DMAP. The solution was stirred at room
temperature under
nitrogen for 10 minutes. Then the amine (1 eq.) was added and the reaction
mixture was
stirred at room temperature under nitrogen for 4-16 hrs. The resulting mixture
was
partitioned between DCM and 5% sodium bicarbonate. The organic layer was
washed
with brine, dried over MgS04, and concentrated to give a yellow residue. The
crude
compound was then purified by flash chromatography to give desired
benzenesulphonamide as crystalline solid. (Yield 40-90%).
Synthesis of 2-Alkylsulfanyl-benzothiazol-6-yl-amine
To a solution of 6-amino-2-merceptobenzothiazole (273mg, 1.5 mmol) in
anhydrous THF
(10 mL) was added NaH (60% dispersion, 1.5 mmol), followed by alkyl halide
(1.5
mmol). The mixture was stirred at rt for 24h, partitioned between ethyl
acetate and 5%
sodium bicarbonate. The organic phase was washed with brine, dried over sodium
sulphate and concentrated in vacuo to a yellow solid, which was purified with
recrystallization or flash chromatography. (Yield 60-90%).
The following amines were synthesized with the method described above:
2-Ethylsulfanylbenzothiazol-6-ylamine
Yellow crystalline solid. mp 77-78°C (lit.77°C). TLC single spot
at Rf 0.78 (8%
methanol/DCM); 'H NMR (270 MHz, DMSO): b 7.50 (1 H, d, J = 8.5 Hz, 4-H), 6.98
(1 H,
d, J = 2.2 Hz, 7-H), 6.69 (1 H, dd, J = 8.5, 2.2 Hz, 5-H), 5.33 (2H, broad,
NH2), 3.23 (2H,
q, J = 7.3 Hz, SCHa), 1.35 (3H, t, J = 7.3 Hz, CH3).
(Francolor, S.A.; US 2500093; 1945)
2-(2-Methoxyethylsulfanyl)-benzothiazol-6-ylamine
Yellow thick syrup. TLC single spot at Rf 0.65 (30% ethyl acetate/DCM); 'H NMR
(270
MHz, DMSO): b 7.49 (1 H, d, J = 8.8 Hz, 4-H), 6.97 (1 H, d, J = 2.2 Hz, 7-H),
6.69 (1 H,
dd, J = 8.8, 2.2 Hz, 5-H), 5.34 (2H, broad, NHS), 3.63 (2H, f, J = 6.3 Hz,
CH2), 3.43 (2H,
t, J = 6.3 Hz, CHZ), 3.26 (3H, s, CH3).
CA 02501228 2005-04-05
WO 2004/037251 PCT/GB2003/004590
(6-aminobenzothiazol-2-ylmercapto)-acetic acid ethyl ester
Off white solid. mp 87-89°C (lit.92°C, [20]); TLC single spot
at Rf 0.72 (8%
methanol/DCM); 'H NMR (270 MHz, DMSO): S 7.48 (1 H, d, J = 8.7 Hz, 4-H), 7.01
(1 H,
5 d, J = 1.8 Hz, 7-H), 6.71 ( 1 H, dd, J = 8.7, 1.8 Hz, 5-H), 5.58 (2H, broad,
NH2), 4.17 (2H,
s, SCH~), 4.12 (2H, t, J = 7.3 Hz, CH2), 1.19 (3H, t, J = 7.3 Hz, CH3).
The following compounds were synthesized with the general method for N-
benzothiazole benzenesulphonamide:
3-Chloro-N-(2-ethylsulfanylbenzothiazol-6-yl)-2-methylbenzenesulphonamide
(STX751, XDS01141)
Off-white solid (220 mg; 55%). TLC single spot at Rf : 0.83 (17% EtOAc/DCM);
HPLC
purity 96% (tR 1.9 min in methanol);'HNMR (400MHz, DMSO-d6) b 10.8 (1H, s,
NH),
7.89 (1 H, dd, J = 8.0, 1.0 Hz, 6'-H of benzene), 7.71 (1 H, d, J = 8 Hz, 4-H
of
benzothiazole), 7.70 (1 H, d, J = 2 Hz, 7-H of benzothiazole), 7.70 (1 H, dd,
J = 8.0, 1.0
Hz, 4'-H of benzene), 7.36 (1H, t, J = 8 Hz, 5'-H of benzene), 7.15 (1H, dd, J
= 8.0, 2.0
Hz, 5-H of benzothiazole), 3.30 (2H, q, J = 7.0 Hz, SCHZ), 2.66 (3H, s, CH3),
1.38 (3H, t,
J = 7.0 Hz, CH3); APCI-MS 397.99 (M)+; FAB-HRMS calcd for C16H16CIN202S3 (MH+)
399.0062, found 399.0048.
[6-(3-Chloro-2-methylbenzenesulphonylamino)-benzothiazol-2-ylsulfanyl]-acetic
acid ethyl ester (STX752, XDS01142)
White crystalline solid (210 mg; 46%). TLC single spot at Rf : 0.69 (17%
EtOAc/DCM);
HPLC purity 99% (tR 2.9 min in 10% water-methanol); 'HNMR (400MHz, DMSO-d6) S
10.8 (1 H, s, S02NH), 7.88 (1 H, dd, J = 8.0, 1.0 Hz, 6'-H of benzene), 7.72
(1 H, d, J = 2.0
Hz, 7-H of benzothiazole), 7.69 (1 H, dd, J = 8.0, 1.0 Hz, 4'-H of benzene),
7.68 (1 H, d, J
= 8.0 Hz, 4-H of benzothiazole), 7.36 (1 H, t, J = 8.0 Hz, 5'-H of benzene),
7.15(1 H, dd, J
= 8.0, 2.0 Hz, 5-H of benzothiazole), 4.25 (2H, s, 2-SCH~-), 4.13 (2H, q, J =
7.1 Hz,
COOCHZ), 2.64 (3H, s, CH3), 1.17 (3H, t, J = 7.1 Hz, 2-COOCH2CH3); APCI-MS
456.0
(M)+; FAB-HRMS calcd for C18H18CIN204S3 (MH+) 457.0117, found 457.0109.
3-Chloro-N-[2-(2-methoxyethylsulfanyl)-benzothiazol-6-yl]-2-
methylbenzenesulphonamide (STX754, XDS01144)
Off-White solid (150 mg; 77%). TLC single spot at Rf 0.60 (17% EtOAc/DCM);
HPLC
purity 94% (tR 3.1 min in 10% water-methanol); 'HNMR (400MHz, DMSO-d6) b 10.8
CA 02501228 2005-04-05
WO 2004/037251 PCT/GB2003/004590
96
{1 H, s, SOZNH), 7.88 (1 H, dd, J = 8, 1 Hz, 6'-H of benzene), 7.71 (1 H, d, J
= 8 Hz , 4-H
of benzothiazole), 7.70 (1 H, dd, J = 8, 1 Hz, 4'-H of benzene), 7.69 {1 H, d,
J = 2 Hz, 7-H
of benzothiazole), 7.36 (1 H, t, J = 8 Hz, 5'-H of benzene), 7.15(1 H, dd, J =
8, 2 Hz, 5-H
of benzothiazole), 3.64 (2H, t, J = 6 Hz, CHZ), 3.50 (2H, t, J = 6 Hz, SCHz),
3.27 (3H, s,
CH3), 2.65 (3H, s, CH3); APCI-MS 428.0 (M)+; FAB-HRMS calcd for C17H18CIN203S3
(MH+) 429.0168, found 429.0159.
2-[6-(3-Chloro-2-methylbenzenesulphonylamino)-benzothiazol-2-ylsulfanyl~-N,N-
diethylacetamide (STX755, XDS01145) and 2-[6-(3-chloro-2-methyi-
benzenesuiphonylamino)-benzothiazol-2-yl]-N,N-diethylacetamide (STX763,
XDS01145B)
To a suspension of AICI3 (50 mg) in DCM (5 ml) was added diethylamine ( 0.4
ml). The
solution was stirred under nitrogen at room temperature for 10 minutes. [6-(3-
Chloro-2-
methyi-benzenesulphonyiamino)-benzothiazol-2-ylsulfanyl]-acetic acid ethyl
ester
(STX752, 100 mg) was added and the mixture was kept stirring at room
temperature for
30 minutes. The reaction was quenched with water, partitioned between DCM and
5%
NaHC03. The organic phase was washed with water, dried over MgSO4 and
evaporated
in vacuo to give a yellow residue, which was purified with flash column
chromatography
using 20-30% ethyl acetate-DCM as eluting solvent. STX755 (50 mg, 47%) was
obtained as white solid. TLC single spot at Rf 0.60 (25% EtOAc/DCM); HPLC
purity 89%
(tR 2.7 min in 10% water-methanol); 'HNMR (270MHz, DMSO-d6) b 10.7 (1H, s,
S02NH), 7.86 (1H, d, J = 8 Hz, 6'-H of benzene), 7.64-7.68 (3H, m, 4'-H of
benzene and
4,7-H of benzothiazole), 7.34 (1 H, t, J = 8 Hz, 5'-H of benzene), 7.12 (1 H,
dd, J = 8, 2
Hz, 5-H of benzothiazole), 4.42 (2H, s, 2-SCHZ-), 3.26-3.38 (4H, m, -N(CHa)~-
), 2.50 (3H,
s, 1'-CH3), 1.17 (3H, t, J = 7 Hz, -NCHaCH3), 1.00 (3H, t, J = 7 Hz, -
NCH~CH3); APCI-MS
484.0 (M)+; FAB-HRMS calcd for C2pH23CIN3O3S3 (MH+) 484.0590, found 484.0584.
STX763 (25 mg, 25%) was obtained as white solid. TLC single spot at Rf 0.39
(25%
EtOAc/DCM); LCMS purity 98% (tR 6.9 min in 10% water-CH3CN); 'HNMR {400 MHz,
DMSO-d6) b 10.8 (1 H, s, S02NH), 7.88 (1 H, dd, J = 8.1, 1.2 Hz, 6'-H of
benzene), 7.79
(1 H, d, J = 8.6 Hz, 4-H), 7.72 {1 H, d, J = 2.0 Hz, 7-H), 7.68 (1 H, dd, J =
8.1, 1.2 Hz, 4'-H
of benzene), 7.35 (1 H, t, J = 8.1 Hz, 5'-H of benzene), 7.17 (1 H, dd, J =
8.6, 2 Hz, 5-H of
benzothiazole), 4.2 (2H, s, 2-SCHZ-), 3.26-3.38 (4H, m, -N(CH2)2-), 2.65 (3H,
s, CH3),
1.10 (3H, t, J = 7 Hz, -NCH~GH3), 1.02 (3H, t, J = 7 Hz, -NCH2CH3); APCI-MS
451.0
(M)+; FAB-HRMS calcd for C2pH23CIN3O3S2 (MH+) 452.0869, found 452.0870.
3-Chloro-N-benzothiazol-6-yl-2-methylbenzenesulphonamide (STX750, XDS01139)
CA 02501228 2005-04-05
WO 2004/037251 PCT/GB2003/004590
97
Light pink needles (260 mg; 77%). TLC single spot at Rf 0.46 (17% EtOAc/DCM);
HPLC
purity 99% (tR 2.5 min in 10% water-methanol); 'HNMR (400MHz, DMSO-d6) b 10.9
(1 H, s, S02NH), 9.25 (1 H, s, 2-H of benzothiazole), 7.95 (1 H, d, J = 9 Hz,
4-H of
benzothiazole), 7.92 (1 H, dd, J = 8.0, 1.0 Hz, 6'-H of benzene), 7.84 (1 H,
d, J = 2 Hz, 7-
H of benzothiazole), 7.70 (1 H, dd, J = 8.0, 1.0 Hz, 4'-H of benzene), 7.37(1
H, t, J = 8 Hz,
5'-H of benzene), 7.25 (1 H, dd, J = 9.0, 2.0 Hz, 5-H of benzothiazole), 2.66
(3H, s, CH3);
APCI-MS 337.9 (M)+; FAB-HRMS calcd for C2pH23CIN3O3S2 (MH+) 452.0869, found
452.0870.
3-Chloro-N-(2-methylbenzothiazol-6-yl)-2-methylbenzenesulphonamide (STX886,
XDS01187B)
Off-white solid. TLC single spot at Rf 0.65 (10% methanol/DCM); HPLC purity
>99% (tR
2.4 min in 10% water-methanol); 'HNMR (270MHz, DMSO-d6) 5 10.7 (1 H, s, NH),
7.87 (1H, dd, J = 7.8, 1.9 Hz, ArH), 7.75 (1H, d, J = 8.8 Hz, ArH), 7.69 (1H,
d, J = 2.2
Hz, ArH), 7.68 (1 H, dd, J = 7.8, 1.9 Hz, ArH), 7.34 (1 H, t, J = 7.8 Hz,
ArH), 7.15 (1 H, dd,
J = 8.8, 2.2 Hz, ArH), 2.71 (3H, s, CH3), 2.63 (3H, s, CH3); APCI-MS 351 (M-
H)+; FAB-
HRMS calcd for C15H14CIN202S2 (MH+) 353.0185, found 353.0197.
N-(2-methylbenzothiazol-6-yl)-N-(3-chloro-2-methylphenylsulphonyl)-3-chloro-2-
methylbenzenesulphonamide (STX887, XDS01187A)
Off-white powder. TLC single spot at Rf 0.89 (10% methanol/DCM); HPLC purity
91 % (tR
3.1 min in 10% water-methanol); 'HNMR (270MHz, DMSO-d6) 5 8.09 (1 H, d, J =
2.2
Hz, ArH), 7.92 (1 H, d, J = 8.6 Hz, ArH), 7.85-7.90 (4H, m, ArH), 7.46 (2H, t,
J = 8.0 Hz,
ArH), 7.35 (1 H, dd, J = 8.8, 2.2 Hz, ArH), 2.81 (3H, s, CH3), 2.33 (6H, s, 2
x CH3); APCI-
MS 539 (M-H)~; FAB-HRMS calcd for C22H1gC12N204S3 (MH+) 540.9884, found
540.9897.
2,5-Dichloro-N-(2-methylbenzothiazoi-6-yl)-benzenesulphonamide (STX888,
XDS01188B)
White crystalline solid. TLC single spot at Rf 0.68 (10% methanol/DCM); HPLC
purity
>99% (tR 2.3 min in 10% water-methanol);'HNMR (270MHz, DMSO-d6) ~ 10.9 (1H,
s, NH), 7.98 (1 H, d, J = 2.3 Hz, ArH), 7.76 (1 H, d, J = 8.9 Hz, ArH), 7.74
(1 H, d, J = 2.3
Hz, ArH), 7.69 (1 H, dd, J = 8.6, 2.3 Hz, ArH), 7.66 (1 H, d, J = 8.6 Hz,
ArH), 7.18 (1 H, dd,
J = 8.9, 2.3 Hz, ArH), 2.71 (3H, s, CH3); APCI-MS 371 (M-H)+; FAB-HRMS calcd
for
C14H11CI2N202S2 (MH+) 372.9639, found 372.9651.
CA 02501228 2005-04-05
WO 2004/037251 PCT/GB2003/004590
98
N-(2-Methylbenzothiazol-6-yl)-N-(2,5-dichlorophenylsulphonyl)-2,5-dichloro-
benzenesulphonamide (STX889, XDS01188A)
Yellow solid. TLC single spot at Rf 0.72 (10% methanol/DCM); HPLC purity 94%
(tR 2.9
min in 10% water-methanol); 'HNMR (270MHz, DMSO-d6) 8 8.13 (1 H, d, J = 2.2
Hz,
ArH)" 7.99 (2H, d, J = 2.4 Hz, ArH), 7.90-7.94 (3H, m, ArH), 7.77 (2H, d, J =
8.4 Hz,
ArH), 7.29 (1 H, dd, J = 8.6, 2.2 Hz, ArH), 2.86 (3H, s, CH3); APCf-MS 581
(M)+; FAB
HRMS calcd for C2pH13C14N204S3 (MH+) 580.8792, found 580.8777.
N-(2-Methylbenzothiazol-6-yl)-4-propylbenzenesulphonamide (STX890, XDS01189)
OfF-white solid. TLC single spot at Rf 0.72 (10% methanol/DCM); HPLC purity
99% (tR
2.3 min in 10% water-methanol); 'HNMR (270MHz, DMSO-d6) b 10.3 (1 H, s, NH),
7.67 (1 H, d, J = 8.7 Hz, ArH) 7.64 (1 H, d, J = 1.5 Hz, ArH), 7.59 (2H, d, J
= 7.7 Hz, ArH),
7.27 (2H, d, J = 7.7 Hz, ArH), 7.09 (1 H, dd, J = 8.7, 1.5 Hz, ArH), 2.65 (3H,
s, CH3), 2.49
(2H, t, J = 7.9 Hz, CH2), 1.47 (2H, sextet, J = 7.9 Hz, CH2), 0.58 (3H, t, J =
7.9 Hz, CH3);
APCI-MS 345 (M-H)+; FAB-HRMS calcd for C17H1gN202S2 (MH+) 347.0888, found
347.0904.
3-Chloro-2-methyl-N-(2-oxo-2,3-dihydro-benzothiazol-6-yl)-benzenesulphonamide
(STX753, XDS01143)
White crystalline solid (160 mg; 45%). TLC single spot at Rf 0.42 (17%
EtOAc/DCM);
HPLC purity 98% (tR 2.3 min in 10% water-methanol); 'HNMR (400MHz, DMSO-d6) S
11.8 (1 H, s, 3-NH), 10.5 (1 H, s, SO~NH), 7.81 (1 H, dd, J = 8, 1 Hz, 6'-H of
benzene),
7.71 (1 H, dd, J = 8, 1 Hz, 4'-H of benzene), 7.36 (1 H, t, J = 8 Hz, 5'-H of
benzene), 7.28
(1 H, d, J = 2 Hz, 7-H of benzothiazole), 6.93-6.98 (2H, m, 4,5-H of
benzothiazole), 2.63
(3H, s, CH3); APCI-MS 353.7 (M)+; FAB-HRMS calcd for C14H12CIN2O3S2 (MH+)
354.9978, found 354.9980.
3-Chloro-N-methyl-N-(3-methyl-2-oxo-2,3-dihydro-benzothiazol-6-yl)-2-
methylbenzenesulphonamide (STX831, XDS01163)
To a solution of STX753 (66 mg, 0.19 mmol) in acetone (3 mL) was added
potassium
carbonate (66 mg), followed by methyl iodide (66 mg). The mixture was stirred
at rt for 2
h, extracted into DCM and washed with brine. After drying over sodium
sulphate; the
solvent was removed in vaeuo to give an oily residue that was purified with
flash
chromatography. Off white solid (59 mg, 80%) was obtained. TLC single spot at
Rf 0.37
(100% DCM); HPLC purity 99% (tR 2.0 min in 10% water-methanol);'HNMR (400MHz,
CA 02501228 2005-04-05
WO 2004/037251 PCT/GB2003/004590
99
DMSO-d6) b 7.73-7.80 (2H, m, ArH), 7.61 (1 H, d, J = 2.1 Hz, ArH), 7.42 (1 H,
t, J = 8.0
Hz, ArH), 7.29 (1H, d, J= 8.8 Hz, ArH), 7.21 (1H, dd, J= 8.1, 2.1 Hz, ArH),
3.38 (3H, s,
CH3), 3.32 (3H, s, CH3), 2.33 (3H, s, CH3); APCI-MS 383 (MH)~; FAB-HRMS calcd
for
C16H16CIN2O3S2 (MH+) 383.0291, found 383.0273.
[(3-Chloro-2-methylbenzenesulphonyl)-(3-ethoxycarbonylmethyl-2-oxo-2,3-
dihydro-benzothiazol-6-yl)-amino]-acetic acid ethyl ester (STX764, XDS01149)
To a solution of STX753 (20 mg, 0.056 mmol) in acetone (3 mL) was added
potassium
carbonate (20 mg), followed by methyl 2-bromoethyl acetate (50 pl). The
mixture was
stirred at rt for 4 h, extracted into EtOAc and washed with brine. Affier
drying over
sodium sulphate, the solvent was removed in vacuo to give an oily residue that
was
purified with flash chromatography. White crystalline solid (20 mg, 68%) was
obtained.
TLC single spot at Rf 0.51 (30% ethyl acetate/hexane); HPLC purity 98% (tR 2.6
min in
10% water-methanol); 'HNMR (400MHz, DMSO-d6) b 7.75-7.78 (2H, m, ArH), 7.70
(1 H, d, J = 2.3 Hz, ArH), 7.37 (1 H, t, J = 8.2 Hz, ArH), 7.30 (1 H, d, J =
8.6 Hz, ArH), 7.24
(1 H, dd, J = 8.6, 2.3 Hz, ArH), 4.81 (2H, s, CH2), 4.56 (2H, s, CHZ), 4.14
(2H, q, J = 7.0
Hz, CH2), 4.06 (2H, q, J = 7.0 Hz, CHZ), 2.44 (3H, s, CH3), 1.19 (3H, t, J =
7.0 Hz, CH3),
1.13 (3H, t, J - 7.0 Hz, CH3); FAB-MS 527 (MH)+; FAB-HRMS calcd for
C22H24CIN2O7S2 (MH+) 527.0713, found 527.0694.
Synthesis ofi 2-chlorobenzothiazol-6-yl-amine and 2-chloro-benzothiazol-5-yl-
amine
To a solution of 2-chlorobenzothiazole (12.0 g, 70.7 mmol) in concentrated
H2S04 (60
mL) was added HN03 (69% solution, 6 mL) dropwise at 0°C for 20 min. The
mixture
was stirred at 5°C for 3h, poured into ice-water (150 mL). The
precipitate was collected
and washed with 5% sodium bicarbonate and water, dried in vacuo. 'H NMR
analysis
showed the mixture contained 78% 6-nitro-2-chlorobenzothiazole and 8% 5-nitro-
2-
chlorobenzothiazole. Recrystallization from ethanol gave 6-nitro-2-
chlorobenzothiazole
as white crystalline solid (11 g, 72%). 3.5 g of the solid was dissolved in
refluxing
ethanol-acetic acid (150 : 15 mL), Iron powder was added in one portion.. The
mixture
was refluxed for 1.5h, filtered. The filtrate was concentrated in vacuo to
half volume and
neutralized with 10% NaOH to pH 7.5, extracted with ethyl acetate. The organic
phase
was washed with brine, dried over magnesium sulphate and evaporated to give a
residue, which was recrystallized from ethanol. Light purple crystals (2.5 g,
83%) were
obtained. Mp 160-164°G; TLC single spot at Rf 0.27 (30% EtOAc/hexane);
'HNMR
CA 02501228 2005-04-05
WO 2004/037251 PCT/GB2003/004590
100
(270 MHz, DMSO-d6) b 7.58 (1 H, d, J = 9.0 Hz, 4-H), 7.03 (1 H, d, J = 2.0 Hz,
7-H),
6.77 (1 H, dd, J = 9.0, 2.0 Hz, 5-H), 5.55 (2H, s, NH2).
The mother liquor from the recrystaliization of nitration product was
evaporated and
subjected to iron powder reduction as described above. The crude product was
purified
with flash chromatography (ethyl acetate-DCM gradient elution) to give 2-
chloro
benzothiazol-5-yl-amine as yellow solid. Mp 146-149°C; TLC single spot
at Rf 0.52 (10%
EtOAc/DCM);'HNMR (270 MHz, DMSO-d6) b 7.63 (1 H, d, J = 8.6 Hz, 7-H), 7.05 (1
H,
d, J = 2.3 Hz, 4-H), 6.78 (1 H, dd, J = 8.6, 2.3 Hz, 6-H), 5.40 (2H, s, NH2).
The following compounds were synthesized with the general method for N-
benzothiazole benzenesulphonamide;
N-(2-chlorobenzothiazol-6-yl)-N-(3-chloro-2-methylphenylsulphonyl)-3-chloro-2-
methylbenzenesulphonamide (STX767, XDS01151A)
White crystalline solid. TLC single spot at Rf 0.78 (33% EtOAc/DCM); HPLC
purity 95%
(tR 6.4 min in 10% water-methanol);'HNMR (270MHz, DMSO-d6) 8 8.21 (1H, d, J=
1.3 Hz, ArH), 8.00 (1 H, d, J = 8.8 Hz, ArH), 7.83-7.90 (4H, m, ArH), 7.46
(2H, t, J = 8.0
Hz, ArH), 7.37 (1 H, dd, J = 8.8, 1.8 Hz, ArH), 2.33 (6H, s, 2 x CH3); APCI-MS
560 (M)+;
FAB-HRMS calcd for C21 H16CI3N204S3 (MH+) 560.9338, found 560.9344.
3-Chloro-N-(2-chlorobenzothiazol-6-yl)-2-methylbenzenesulphonamide (STX768,
XDS01151 B)
Off-white crystalline solid. TLC single spot at Rf 0.68 (33% EtOAc/DCM); HPLC
purity
99% (tR 1.7 min in methanol); 'HNMR (270MHz, DMSO-d6) 5 10.9 (1 H, s, NH),
7.91
(1H, d, J = 8.1 Hz, ArH), 7.82 (1H, d, J = 8.8 Hz, ArH), 7.80 (1H, d, J = 3.1
Hz, ArH),
7.70 (1 H, d, J = 8,1 Hz, ArH), 7.36 (1 H, t, J = 8.1 Hz, ArH), 7.23 (1 H, dd,
J = 8.8, 3.0 Hz,
ArH), 2.63 (3H, s, CH3); APCI-MS 372 (M)+; FAB-HRMS calcd for C14H11CI2N202S2
(MH+) 372.9639, found 372.9651.
3-Chloro-N-(2-chlorobenzothiazol-5-yl)-2-methylbenzenesulphonamide (STX834,
XDS01168)
White crystalline solid. TLC single spot at Rf 0.52 (30% EtOAc/hexane); HPLC
purity
99% (tR 1.7 min in methanol);'HNMR (270MHz, DMSO-d6) b 10.9 (1H, s, NH), 7.91-
7.96 (2H, m, ArH), 7.71 (1 H, d, J = 8.1 Hz, ArH), 7.58 (1 H, d, J = 2.2 Hz,
ArH), 7.39 (1 H,
d, J = 8.1 Hz, ArH), 7.22 (1 H, dd, J = 8.1, 2.2 Hz, ArH), 2.64 (3H, s, CH3);
APCI-MS 371
(M-H)+; FAB-HRMS calcd for C14H11 CI2N202S2 (MH~) 372.9639, found 372.9656.
CA 02501228 2005-04-05
WO 2004/037251 PCT/GB2003/004590
101
3-Chloro-2-methyl-N-(2-methylaminobenzothiazol-6-yl)-benzenesulphonamide
(STX833, XDS01167)
The solution of 3-chloro-N-(2-chlorobenzothiazol-6-yl)-2-
methylbenzenesulphonamide
(STX768, 150 mg, 0.40 mmol) in CH3NH-THF (2M, 3 mL) was stirred at 82°C
in a sealed
tube for 24h, extracted with ethyl acetate. The organic phase was washed
brine, dried
over sodium sulphate and concentrated in vacuo to give a residue that was
purified with
flash chromatography (ethyl acetatelDCM gradient elution). White crystals (100
mg,
68%) were obtained. TLC single spot at Rf 0.27 (30% EtOAc/DCM); HPLC purity
99%
(tR 1.8 min in 4% water-methanol); 'HNMR (270MHz, DMSO-d6) b 10.2 (1 H, s,
NH),
7.82 (1 H, q, J = 4.8 Hz, NH), 7.72 (1 H, d, J = 7.7 Hz, ArH), 7.61 (1 H, d, J
= 7.7 Hz, ArH),
7.29 (1 H, d, J = 2.2 Hz, ArH), 7.26 (1 H, t, J = 8.0 Hz, ArH), 7.15 (1 H, d,
J = 8.7 Hz, ArH),
6.79 (1 H, dd, J = 8.7, 2.2 Hz, ArH), 2.80 (3H, d, J = 4.8 Hz, NCH3), 2.54
(3H, s, CH3);
APCI-MS 366 (M-H)+; FAB-HRMS calcd for C15H15CIN302S2 (MH+) 368.0294, found
368.0292.
3-Chloro-2-methyl-N-(2-methylaminobenzothiazol-5-yl)-benzenesulphonamide
(STX835, XDS01176)
The compound was prepared as described for STX833 using 3-chloro-N-(2
chlorobenzothiazol-5-yl)-2-methylbenzenesulphonamide (STX834, 80 mg, 0.21
mmol) as
starting material. White crystals (60 mg, 78%) were obtained. TLC single spot
at Rf
0.25 (30% EtOAc/DCM); HPLC purity 99% (tR 2.3 min in 10% water-methanol);
'HNMR
(270MHz, DMSO-d6) S 10.5 (1 H, s, NH), 7.96 (1 H, q, J = 4.7 Hz, NH), 7.86 (1
H, d, J =
8.1 Hz, ArH), 7.69 (1 H, d, J = 8.1 Hz, ArH), 7.47 (1 H, d, J = 8.0 Hz, ArH),
7.37 (1 H, t, J =
8.1 Hz, ArH), 7.05 (1 H, d, J = 1.9 Hz, ArH), 6.73 (1 H, dd, J = 8.0, 1.9 Hz,
ArH), 2.88 (3H,
d, J = 4.7 Hz, NCH3), 2.64 (3H, s, CH3); APCI-MS 368 (MH)+; FAB-HRMS calcd for
C15H15CIN302S2 (MH+) 368.0294, found 368.0292.
3-Chloro-N-(2-diethylaminobenzothiazol-5-yl)-2-methylbenzenesulphonamide
(STX836, XDS01177)
The compound was prepared as described for STX833 using 3-chloro-N-(2-
chlorobenzothiazol-5-yl)-2-methylbenzenesulphonamide (STX834, 70 mg, 0.18
mmol)
and diethylamine-THF (3 mL) as starting material. White crystals (50 mg, 68%)
were
obtained. TLC single spot at Rf 0.60 (30% EtOAc/DCM); HPLC purity 97% (tR 3.0
min in
10% water-methanol);'HNMR (270MHz, DMSO-d6) b 10.5 (1 H, s, NH), 7.85 (1 H, d,
J
= 7.9 Hz, ArH), 7.68 (1 H, d, J = 8.0 Hz, ArH), 7.52 (1 H, d, J = 8.4 Hz,
ArH), 7.36 (1 H, t, J
CA 02501228 2005-04-05
WO 2004/037251 PCT/GB2003/004590
102
= 8.0 Hz, ArH), 7.06 (1 H, d, J = 2.2 Hz, ArH), 6.75 (1 H, dd, J = 8.3, 2.2
Hz, ArH), 3.45
(4H, q, J = 7.0 Hz, N(CH2)~), 2.63 (3H, s, CH3), 1.15 (6H, t, J = 7.0 Hz, 2 x
CH3); APCI-
MS 410 (MH)~; FAB-HRMS calcd for C18H21CIN302S2 (MH+) 410.0764, found
410.0753.
3-Chloro-N-(2-diethylaminobenzothiazol-6-yl)-2-methylbenzenesulphonamide
(STX878, XDS01164)
The compound was prepared as described for STX833 using 3-chloro-N-(2
chlorobenzothiazol-6-yl)-2-methylbenzenesulphonamide (STX768, 240 mg, 0.64
mmol)
and diethylamine-IPA (3 mL) as starting material. Off-white crystalline solid
(128 mg,
49%) were obtained. TLC single spot at Rf 0.33 (30% EtOAc/hexane); HPLC purity
96%
(tR 2.2 min in 4% water-methanol);'HNMR (270MHz, DMSO-d6) b 7.56-7.62 (3H, m,
ArH), 7.18-7.27 (2H, m, ArH), 7.00 (1 H, dd, J = 8.5, 1.7 Hz, ArH), 5.52 (1 H,
s, NH), 3.54
(4H, q, J = 7.0 Hz, N(CH2)a), 2.25 (3H, s, CH3), 1.22 (6H, t, J = 7.0 Hz, 2 x
CH3); APCI-
MS 409 (M)~; FAB-HRMS calcd for C18H21CIN302S2 (MH+) 410.0764, found 410.0698.
Synthesis of 2,6-Dimethylbenzothiazol-7-ylamine
To a solution of 2,6-dimethylbenzothiazol (350 mg, 2.15 mmol) in Conc. HZS04
(4 mL)
was added HNO3 (69%, 0.3 mmol) at 0°C. After stirred at 0°C for
0.5h, the mixture was
poured over ice-water. The precipitate was collected and washed with 5% sodium
bicarbonate and water, recrystallized from ethanol to give 7-nitro-2,6-
dimethylbenzothiazol as yellow solid (160 mg). The product (150 mg) was
hydrogenated
over 5% Pd/C in ethanol-THF (10 : 2 mL) at atmosphere pressure to give 2,6-
dimethyl-
benzothiazol-7-ylamine as yellow solid (120 mg). TLC single spot at Rf 0.55
(10%
EtOAc/DCM); 'H NMR (270 MHz, DMSO): S 7.07 (2H, s, ArH), 5.23 (2H, s, NHz),
2.73
(3H, s, CH3), 2.20 (3H, s, CH3).
Synthesis of 6-Methoxy-2-methylbenzothiazol-7-ylamine
The compound was prepared as described above starting from 6-methoxy-2
methylbenzothiazol. Yellow solid was obtained. mp 117-119°C (lit.121-
122°C); TLC
single spot at Rf 0.55 (40% EtOAc/DCM);'H NMR (270 MHz, DMSO): b 7.14 (1 H, d,
J =
8.7 Hz, ArH), 7.05 (1 H, d, J = 8.7 Hz, ArH), 5.10 (2H, broad, NH2), 3.83 (3H,
s, OCH3),
2.71 (3H, s, CH3).
(Friedman, S.G. J Gen Chem USSR 31, 1961, 3162-3167)
CA 02501228 2005-04-05
WO 2004/037251 PCT/GB2003/004590
103
Synthesis of 2,5-Dimethylbenzothiazol-4-ylamine and 2,5-Dimethyfbenzothiazol-6-
ylamine
To a solution of 2,5-dimethylbenzothiazol (1.63 g, 10 mmol) in Conc. H2S04 (12
mL) was
added HNO3 (69%, 1 mmol) at -5°C. After stirred at -5 - 0°C for
2h, the mixture was
poured over ice-water (150 mL). The precipitate was collected and washed with
5%
sodium bicarbonate, water and 70% ethanol. The product (1.98 g) was a mixture
of 4-
nitro-2,5-dimethylbenzothiazol and 6-vitro-2,5-dimethylbenzothiazol in 1 : 1
ratio judged
by NMR. The product (998 mg) was hydrogenated over 5% Pd/C (600 mg) in ethanol-
THF (50 : 20 mL) at atmosphere pressure to give a yellow solid (880 mg).
Separation
with flash chromatography (EtOAc/DCM gradient elution) yielded 2,5-
dimethylbenzothiazol-4-ylamine as yellow crystals (400 mg). TLC single spot at
Rf 0.60
(15% EtOAc/DCM); 'H NMR (270 MHz, DMSO): b 7.05 (1 H, d, J = 8.0 Hz, ArH),
6.99
(1H, d, J.= 8.0 Hz, ArH), 5.26 (2H, s, NHS), 2.74 (3H, s, CH3), 2.18 (3H, s,
CH3); APCI-
MS 177 (M-H)+.
2,5-Dimethylbenzothiazol-6-ylamine was obtained as yellow solid (320 mg). TLC
single
spot at Rf 0.55 (15% EtOAc/DCM);'H NMR (270 MHz, DMSO): S 7.47 (1H, s, ArH),
7.05
(1 H, s, ArH), 5.05 (2H, s, NH2), 2.65 (3H, s, CH3), 2.16 (3H, s, CH3); APCI-
MS 177 (M-
H)+.
Synthesis of 4-chloro-2-methylbenzothiazol-5-ylamine and 4,6-dichloro-2-
methylbenzothiazol-5-ylamine
To a solution of 5-amino-2-methylbenzothiazole (818 mg, 4.99 mmol) in
isopropanol (12
mL) was added N-chlorosuccinimide (732 mg, 5.48 mmol). The mixture was stirred
at
60°C for 15 min., partitioned between DCM and 5% sodium bicarbonate.
The organic
phase was washed with brine, dried over sodium sulphate and concentrated in
vacuo to
give a residue that was purified with flash chromatography (EtOAc/DCM gradient
elution). 4-Chloro-2-methylbenzothiazol-5-ylamine was obtained as off-white
crystalline
solid (510 mg, 51 %). mp 121-122°C (lit.124°C); TLC single spot
at Rf 0.51 (20%
EtOAc/DCM); 'H NMR (270 MHz, DMSO): b 7.61 (1 H, d, J = 8.6 Hz, ArH), 6.91 (1
H, d, J
= 8.6 Hz, ArH), 5.49 (2H, s, NHZ), 2.75 (3H, s, CH3); APCI-MS 199 (MH)+.
4,6-Dichloro-2-methylbenzothiazol-5-ylamine was obtained as yellow solid (60
mg, 5%).
TLC single spot at Rf 0.57 (20% EtOAc/DCM); 'H NMR (270 MHz, DMSO): S 7.89 (1
H,
s, ArH), 5.26 (2H, s, NH2), 2.77 (3H, s, CH3); APCI-MS 233 (MH)+.
CA 02501228 2005-04-05
WO 2004/037251 PCT/GB2003/004590
104
The following compounds were synthesized with the general method for N-
benzothiazole benzenesulphonamide.
3-Chloro-N-(6-methoxy-2-methylbenzothiazol-7-yl)-2-methylbenzenesulphonamide
(STX989, XDS02038)
White crystalline solid. TLC single spot at Rf 0.71 (30% EtOAc/DCM); HPLC
purity 99%
(tR 2.3 min in 10% water-methanol);'H NMR (270 MHz, DMSO-d6) b 10.1 (1H, s,
NH),
7.76 (1 H, d, J = 8.9 Hz, ArH), 7.72 (1 H, d, J = 8.2 Hz, ArH), 7.53 (1 H, d,
J = 8.2 Hz,
ArH), 7.23 (1H, t, J = 8.2 Hz, ArH), 7.05 (1H, d, J = 8.9 Hz, ArH), 3.29 (3H,
s, OCH3),
2.74 (3H, s, CH3), 2.70 (3H, s, CH3); APCI-MS 381 (M-H)+; FAB-HRMS calcd for
C16H16CIN2O3S2 (MH+) 383.0291, found 383.0284.
3-Chloro-N-(2,6-dimethyl-benzothiazol-7-yl)-2-methyl-benzenesulphonamide
(STX1021, XDS02069)
Off-white crystalline solid. TLC single spot at Rf 0.49 (10% EtOAc/DCM); HPLC
purity
98% (tR 2.0 min in 20% water-methanol);'H NMR (270 MHz, DMSO-d6) b 10.3 (1H,
s,
NH), 7.78 (1 H, d, J = 7.9 Hz, ArH), 7.74 (1 H, d, J = 8.4 Hz, ArH), 7.64 (1
H, d, J = 7.9 Hz,
ArH), 7.34 (1 H, t, J = 7.9 Hz, ArH), 7.30 (1 H, d, J = 7.9 Hz, ArH), 2.68
(3H, s, CH3), 2.61
(3H, s, CH3), 2.03 (3H, s, CH3); FAB-MS 367 (MH)+; FAB-HRMS calcd for
C16H16CIN202S2 (MH+) 367.0342, found 367.0347.
3-Chloro-N-(2,5-dimethyl-benzothiazol-4-yl)-2-methyl-benzenesulphonamide
(STX996, XDS02047)
White crystalline solid. TLC single spot at Rf 0.76 (10% EtOAc/DCM); HPLC
purity
>99% (tR 2.9 min in 10% water-methanol);'H NMR (270 MHz, DMSO-d6) 8 9.98 (1H,
s, NH), 7.81 (1 H, d, J = 8.3 Hz, ArH), 7.64 (1 H, d, J = 7.9 Hz, ArH), 7.45
(1 H, d, J = 7.9
Hz, ArH), 7.31 (1 H, d, J = 8.3 Hz, ArH), 7.12 (1 H, t, J = 7.9 Hz, ArH), 2.73
(3H, s, CH3),
2.47 (3H, s, CH3), 2.44 (3H, s, CH3); APCI-MS 367 (MH)+; FAB-HRMS calcd for
ClgHIgCIN202S2 (MH+) 367.0342, found 367.0342.
N-(2,5-dimethylbenzothiazol-6-yl)-N-(3-chloro-2-methylphenylsulphonyl)-3-
chloro-
2-methylbenzenesulphonamide (STX997, XDS02048A)
Off-white syrup. TLC single spot at Rf 0.78 (10% EtOAcIDCM); HPLC purity 85%
(tR 4.2
min in 10% water-methanol);'H NMR (270 MHz, DMSO-d6) b 8.06 (1H, s, ArH),
7.86°
7.95 (5H, m, ArH), 7.68 (1 H, s, ArH), 7.52 (2H, t, J = 8.2 Hz, ArH), 2.83
(3H, s, CH3),
CA 02501228 2005-04-05
WO 2004/037251 PCT/GB2003/004590
105
2.29 (6H, s, 2 x CH3), 2.05 (3H, s, CH3); APCI-MS 555 (MH)+; FAB-HRMS calcd
for
C23H21 CI2N204S3 (MH+) 555.0040, found 555.0041.
3-Chloro-N-(2,5-dimethylbenzothiazol-6-yl)-2-methylbenzenesulphonamide
(STX998, XDS02048B)
White crystalline solid. TLC single spot at Rf 0.39 (10% EtOAc/DCM); HPLC
purity 96%
(tR 2.1 min in 10% water-methanol);'H NMR (270 MHz, DMSO-d6) b 10.0 (1H, s,
NH),
7.74 (1 H, d, J = 7.9 Hz, ArH), 7.70 (1 H, s, ArH), 7.67 (1 H, d, J = 7.9 Hz,
ArH), 7.63 (1 H,
s, ArH), 7.33 (1 H, t, J = 7.9 Hz, ArH), 2.75 (3H, s, CH3), 2.60 (3H, s, CH3),
2.13 (3H, s,
CH3); APCI-MS 367 (MH)+; FAB-HRMS calcd for ClgHIgCIN202S2 (MH+) 367.0342,
found 367.0350.
2,5-Dichloro-N-(2,5-dimethylbenzothiazol-6-yl)-benzenesulphonamide (STX999,
XDS02049)
White crystalline solid. TLC single spot at Rf 0.43 (10% EtOAc/DCM); HPLC
purity 98%
(tR 2.0 min in 10% water-methanol);'H NMR (270 MHz, DMSO-d6) b 10.3 (1H, s,
NH),
7.76 (3H, s, ArH), 7.72 (1 H, s, ArH), 7.67 (1 H, s, ArH), 2.75 (3H, s, CH3),
2.23 (3H, s,
CH3); APCI-MS 387 (MH)+; FAB-HRMS calcd for C15H13CI2N202S2 (MH+) 386.9795,
found 386.9806.
N-(4-Chloro-2-methyl-benzothiazol-5-yl)-N-(3-chloro-2-methylphenylsulphonyl)-3-
chloro-2-methyl-benzenesulphonamide (STX991, XDS02042A)
White powder. TLC single spot at Rf 0.75 (8% EtOAc/DCM); HPLC purity >99% (tR
4.4
min in 10% water-methanol);'H NMR (270 MHz, DMSO-d6) b 8.19 (1H, d, J= 8.7 Hz,
ArH), 7.93 (4H, d, J = 8.2 Hz" ArH), 7.59 (1 H, d, J = 8.7 Hz, ArH), 7.50 (2H,
d, J = 8.2
Hz, ArH), 2.85 (3H, s, CH3), 2.41 (6H, s, 2 x CH3); APCI-MS 575 (MH)+; FAB-
HRMS
calcd for C22H18CI3N204S3 (MH+) 574.9494, found 574.9492.
3-Chloro-N-(4-chloro-2-methylbenzothiazol-5-yl)-2-methylbenzenesulphonamide
(STX992, XDS02042B)
White crystalline solid. TLC single spot at Rf 0.69 (8% EtOAc/DCM); HPLC
purity 99%
(tR 2.5 min in 10% water-methanol);'H NMR (270 MHz, DMSO-d6) b 10.5 (1 H, s,
NH),
7.96 (1 H, d, J = 8.7 Hz, ArH), 7.73 (1 H, d, J = 7.9 Hz" ArH), 7.64 (1 H, d,
J = 7.9 Hz,
ArH), 7.30 (1 H, d, J = 8.7 Hz, ArH), 7.29 (1 H, t, J = 7.9 Hz" ArH), 2.79
(3H, s, CH3), 2.70
CA 02501228 2005-04-05
WO 2004/037251 PCT/GB2003/004590
106
(3H, s, CH3); APCI-MS 385 (M-H)+; FAB-HRMS calcd for C15H13C12N202S2 (MH+)
386.9795, found 386.9790.
2,5-Dichloro-N-(4-chloro-2-methylbenzothiazol-5-yl)-benzenesulphonamide
(STX993, XDS02043B)
White crystalline solid. TLC single spot at Rf 0.71 (8% EtOAc/DCM); HPLC
purity 99%
(tR 5.0 min in 10% water-methanol);'H NMR (270 MHz, DMSO-d6) 5 10.7 (1H, s,
NH),
7.97 (1H, d, J = 8.6 Hz, ArH), 7.71-7.78 (3H, m" ArH), 7.28 (1H, d, J = 8.6
Hz, ArH),
2.81 (3H, s, CH3); APCI-MS 407 (MH)+; FAB-HRMS calcd for C14H10CI3N202S2 (MH+)
406.9249, found 406.9234.
N-(4-Chloro-2-methylbenzothiazol-5-yl)-4-propylbenzenesulphonamide (STX994,
XDS02044B)
White crystalline solid. TLC single spot at Rf 0.70 (8% EtOAc/DCM); HPLC
purity 99%
(tR 2.7 min in 10% water-methanol);'H NMR (270 MHz, DMSO-d6) 5 10.1 (1H, s,
NH),
7.93 (1H, d, J = 8.6 Hz, ArH), 7.60 (2H, d, J = 8.2 Hz, ArH), 7.35 (2H, d, J =
8,2 Hz,
ArH), 7.28 (1 H, d, J = 8.4 Hz, ArH), 2.79 (3H, s, CH3), 2.69 (2H, t, J = 7.2
Hz, CHI), 1.59
(2H, m, CH2), 0.86 (3H, t, J = 7.2 Hz, CH3); APCI-MS 381 (MH)+; FAB-HRMS calcd
for
C17H18CIN2O2S2 (MH+) 381.0498, found 381.0484.
N-(4-Chloro-2-methylbenzothiazol-5-yl)-N-(4-propylphenylsulphonyl)-4-
propylbenzenesulphonamide (STX995, XDS02044A)
White powder. TLC single spot at Rf 0.70 (8% EtOAc/DCM); HPLC purity 99% (tR
3.8
min in 10% water-methanol); 'H NMR (270 MHz, DMSO-d6) b 8.10 (1 H, d, J = 8.4
Hz, ArH), 7.74 (4H, d, J = 8.1 Hz, ArH), 7.50 (4H, d, J = 8.1 Hz, ArH), 7.08
(1 H, d, J =
8.4 Hz, ArH), 2.91 (3H, s, CH3), 2.71 (4H, t, J = 7.1 Hz, 2 x CH2), 1.59 (4H,
m, CHI), 0.86
(6H, t, J = 7.1 Hz, 2 x CH3); APCI-MS 561 (M-H)+; FAB-HRMS calcd for
C26H28CIN204S3 (MH+) 563.0900, found 563.0886.
Synthesis of 3-Chloro-2-methyl-N-(2-methyl-benzothiazol-5-ylmethyl)-
benzenesulphonamide (STX1029, XDS02070A) and 3-Chloro-2-methyl-N,N-bis-(2-
methyl-benzothiazol-5-ylmethyl)-benzenesulphonamide (STX1030, XDS02070B)
To a solution of 3-chloro-2-methylbenzenesulphonamide (103 mg, 0.5 mmol) in
CH3CN
was added potassium carbonate (100 mg), followed 5-bromomethyl-2
methylbenzothiazole (121 mg, 0.5 mmol). The mixture was refluxed under NZ for
6h,
CA 02501228 2005-04-05
WO 2004/037251 PCT/GB2003/004590
107
partitioned between ethyl acetate and water. The organic phase was washed
brine,
dried over sodium sulphate and concentrated in vacuo to give a yellow residue,
which
was separated with flash chromatography (ethyl acetate/DCM, gradient elution).
STX1029 was obtained as white solid. TLC single spot at Rf 0.55 (10%
EtOAcIDCM);
HPLC purity >99% (tR 2.0 min in 10% water-methanol); 'HNMR (270 MHz, CDCI3) b
7.89 (1 H, d, J = 7.9 Hz, ArH), 7.66 (1 H, d, J = 7.9 Hz, ArH), 7.65 (1 H, d,
J = 1.3 Hz,
ArH), 7.49 (1 H, d, J = 7.9 Hz, ArH), 7.17 (1 H, t, J = 7.9 Hz, ArH), 7.13 (1
H, dd, J = 7.9,
1.5 Hz, ArH), 5.35 (1H, t, J = 5.9 Hz, NH), 4.24 (2H, d, J = 5.9 Hz, CH2),
2.79 (3H, s,
CH3), 2.62 (3H, s, CH3); APCI-MS 367 (MH)~'; FAB-HRMS calcd for C1gH16CIN2O2S2
(MH+) 367.0342, found 367.0330.
STX1030 was obtained as white solid. TLC single spot at Rf 0.50 (10%
EtOAc/DCM);
HPLC purity 99% (tR 6.1 min in 20% water-methanol); 'H NMR (270 MHz, DMSO-d6)
b
7.87 (3H, d, J = 8.1 Hz, ArH), 7.75 (1H, d, J = 8.0 Hz, ArH), 7.59 (2H, broad
w"2 = 1.1
Hz, ArH), 7.38 (1 H, t, J = 8.0 Hz, ArH), 7.11 (2H, dd, J = 8.1, 1.1 Hz, ArH),
4.56 (4H, s, 2
x NCHZ), 2.78 (6H, s, 2 x CH3), 2.58 (3H, s, CH3); APCI-MS 528 (MH)+; FAB-HRMS
calcd for C25H23C1N302S3 (MH+) 528.0641, found 528.0630.
S~rnthesis of N-Indole or N-Indoline Arysulfonamide Derivatives
H H H
\ ~S; N ~ ~S.N ~ ~' .N ~ N
I .~ \ ~ 'o ~ ~ ~ So ~ /
Ii Ii
H ~ H
CI CI CI
STX832, XDS01165 STX981, XDS02019 STX982, XDS02020
O H O H
~S,N ~ O~ ~ 'S~ N I ~ \ CI ~ 'S~ N I ~ \
~O I / \ I / O ~ N I s O ~ N
/ H O H CI H
CI CI
STX986, XDS0203Q STX1018, XDS02061 STX1019, XDS02062
~'.N O''~N ~ ~'~N
S ~ ~ \ \ SO ~ SO
\ 'O i N I , I r N I , I ~ N~HCI
H
CI ~C CI
STX1020, XDS02063 STX984, XDS02025 STX987, XDS02031
CA 02501228 2005-04-05
WO 2004/037251 PCT/GB2003/004590
108
General method for synthesis N-indole or N-indoline arylsulphonamide
derivatives
(STX832, STX981-982, STX984, STX986-987, STX1018-1020):
To a solution arylsulphonyl chloride (1.1 eq.) in DCM were added pyridine (2.2
eq.) and
catalytic amount of DMAP, followed by the corresponding amine (1 eq.). The
reaction
mixture was stirred at rt under nitrogen for 4-6 h, then partitioned between
ethyl acetate
and 5% sodium bicarbonate after TLC showed completion of the reaction. The
organic
layer was washed with brine, dried over sodium sulphate, and concentrated in
vacuo to
give crude product as solid or thick syrup. The compound was then purified by
flash
chromatography (methanol-DCM gradient elution) to give desired
arylsulphonamide as
crystalline solid. Yield ranges from 50-35%.
3-Chloro-2-methyl-N-(2-methyl-1H-indol-5-yl)-benzenesulphonamide (STX832,
XDS01165)
White crystalline solid. TLC single spot at Rf 0.68 (30% ethyl
acetate/hexane); HPLC
purity > 99 % (t~ 1.8 min in 4 % water-methanol); 'H NMR (270 MHz, DMSO): 8
10.9
(1 H, s, NH), 9.98 (1 H, s, NH), 7.72 (1 H, d, J = 8 Hz, ArH), 7.63 (1 H, d, J
= 8 Hz, ArH),
7.27 (1 H, t, J = 8 Hz, ArH), 7.07 (1 H, d, J = 8 Hz, ArH), 7.05 (1 H, d, J =
2 Hz, ArH), 6.67
(1 H, dd, J=8, 2 Hz, ArH), 5.99 (1 H, s, 3-H), 2.60 (3H, s, CH3), 2.29 (3H, s,
CH3); APCI
MS 334 (M+); FAB-HRMS calcd for ClgHIgCIN202S (MH+) 335.0621, found 335.0609
3-Chloro-2-methyl-N-(1H-indol-5-yl)-benzenesulphonamide (STX981, XDS02019)
White crystalline solid. TLC single spot at Rf 0.72 (6% methanol/DCM); HPLC
purity 98
(tR 2.1 min in 10 % water-methanol); 'H NMR (270 MHz, DMSO): 5 11.1 (1 H, s,
NH),
10.1 (1 H, s, NH), 7.76 (1 H, d, J = 7.9 Hz, ArH), 7.66 (1 H, d, J = 7.9 Hz,
ArH), 7.22-7.32
(4H, m, ArH), 6.81 (1H, dd, J=7.9, 1.2 Hz, ArH), 6.33 (1H, broad, 3-H), 2.64
(3H, s, CH3);
APCI-MS 319 (M-H+); FAB-HRMS calcd for C15H14CIN202S (MH+) 321.0465, found
321.0453.
3-Chloro-2-methyl-N-(1H-indol-6-yl)-benzenesulphonamide (STX982, XDS02020)
White crystalline solid. TLC single spot at Rf 0.88 (10% methanol/DCM); HPLC
purity 98
(t~ 2.5 min in 20 % water-methanol);'H NMR (270 MHz, DMSO): S 11.0 (1H, s,
NH),
10.3 (1 H, s, NH), 7.80 (1 H, d, J = 7.9 Hz, ArH), 7.67 (1 H, d, J = 7.9 Hz,
ArH), 7.31-7.38
(2H, m, ArH), 7.26 (1H, m, ArH), 7.80 (1H, d, J= 1.2 Hz, ArH), 6.75 (1H, dd,
J=7.9, 1.2
Hz, ArH), 6.31 (1 H, broad, 3-H), 2.65 (3H, s, CH3); APCI-MS 319 (M-H+); FAB-
HRMS
calcd for C15H14CIN202S (MH+) 321.0465, found 321.0446.
CA 02501228 2005-04-05
WO 2004/037251 PCT/GB2003/004590
109
5-(3-Ch(oro-2-methylbenzenesulfonylamino)-1H-indole-2-carboxylic acid ethyl
ester (STX986, XDS02030)
White crystalline solid. TLC single spot at Rf 0.82 (8% methanol/DCM); HPLC
purity >
99 % (tR 2.3 min in 10% wafer-metf~anol); 'H NMR (270 MHz, DMSO): 5 11.9 (1 H,
s,
NH), 10.3 (1 H, s, NH), 7.78 (1 H, d, J = 7.9 Hz, ArH), 7.67 (1 H, d, J = 7.9
Hz, ArH), 7.28
7.33 (3H, m, ArH), 7.06 (1 H, d, J = 2.2 Hz, ArH), 7.00 (1 H, dd, J= 8.2, 2.2
Hz, ArH), 4.32
(2H, q, J = 6.9 Hz, OCHZ), 2.63 (3H, s, CH3), 1.31 (3H, t, J = 6.9 Hz, CH3);
APCI-MS 391
(M-H+); FAB-HRMS calcd for C18H18CIN204S (MH+) 393.0676, found 393.0659
3-Chloro-2-methyl-N-(2,3-dimethyl-1H-indol-5-yl)-benzenesulphonamide (STX1018,
XDS02061 )
White crystalline solid. TLC single spot at Rf 0.83 (30% ethyl
acetate/hexane); HPLC
purity 97 % (tR 2.9 min in 20% water-methanol);'H NMR (270 MHz, DMSO): 5 10.6
(1H,
s, NH), 10.0 (1H, s, NH), 7.74 (1H, d, J = 7.5 Hz, ArH), 7.65 (1H, d, J = 7.5
Hz, ArH),
7.29 (1 H, f, J = 8.0 Hz, ArH), 7.05 (1 H, d, J = 8.6 Hz, ArH), 6.99 (1 H, d,
J = 1.7 Hz, ArH),
6.66 (1 H, dd, J= 8.6, 1.7 Hz, ArH), 2.62 (3H, s, CH3), 2.25 (3H, s, CH3),
2.03 (3H, s,
CHI); APCI-MS 349 (MH+); FAB-HRMS calcd for C17H18CIN202S (MH+) 349.0778,
found 349.0737.
2,5-Dichloro-N-(2,3-dimethyl-1H-indol-5-yl)-benzenesulphonamide (STX1019,
XDS02062)
White amorphous powder. TLC single spot at Rf 0.82 (10% ethyl acetate/hexane);
HPLC purity 98 % (tR 3.0 min in 20% water-methanol); 'H NMR (270 MHz, DMSO): S
10.7 (1 H, s, NH), 10.2 (1 H, s, NH), 7.80 (1 H, m, ArH), 7.67-7.70 (2H, m,
ArH), 7.07 (1 H,
d, J = 8.5 Hz, ArH), 7.05 ( 1 H, d, J = 1.7 Hz, ArH), 6.73 (1 H, dd, J= 8.5,
1.7 Hz, ArH),
2.25 (3H, s, CH3), 2.04 (3H, s, CH3); APCI-MS 367 (M-H~); FAB-HRMS calcd for
C16H14CI2N202S (M+) 368.0153, found 368.0146
4-n-Propyl-N-(2,3-dimethyl-1 H-indol-5-yl)-benzenesulphonamide (STX1020,
XDS02063)
Off-white crystalline solid. TLC single spot at Rf 0.82 (10% ethyl
acetate/hexane); HPLC
purity 97 % (tR 2.9 min in 20% wafer-methanol);'H NMR (270 MHz, DMSO): S 10.6
(1H,
s, NH), 9.6 (1 H, s, NH), 7.56 (2H, d, J = 8.3 Hz, ArH), 7.30 (2H, d, J = 8.3
Hz, ArH), 7.03
( 1 H, d, J = 8.3 Hz, ArH), 6.95 ( 1 H, d, J = 1.7 Hz, ArH), 6.68 ( 1 H, dd,
J= 8.3, 1.7 Hz,
ArH), 2.56 (2H, t, J = 7.3 Hz, CH2), 2.24 (3H, s, CH3), 2.01 (3H, s, CH3),
1.55 (2H, sextet,
CA 02501228 2005-04-05
WO 2004/037251 PCT/GB2003/004590
110
J = 7.3 Hz, CH2), 0.85 (3H, t, J = 7.3 Hz, CH3),; APCI-MS 343 (MH+); FAB-HRMS
calcd
for C1 gH22N202S (M+) 342.1402, found 342.1403
3-Chloro-2-methyl-N-(1-acetyl-2,3-dihydro-1 H-indol-5-yl)-benzenesulphonamide
(STX984, XDS02025)
White crystalline solid. TLC single spot at Rf 0.58 (5% methanol/DCM); HPLC
purity 95
(tR 2.2 min in 20% water-methanol); 'H NMR (270 MHz, DMSO): b 10.4 (1 H, s,
NH),
7.80-7.86 (2H, m, ArH), 7.71 (1 H, d, J = 7.8 Hz, ArH), 7.36 (1 H, t, J = 8.0
Hz, ArH), 6.93
(1 H, d, J = 1.8 Hz, ArH), 6.83 (1 H, dd, J = 8.2, 1.8 Hz, ArH), 4.01 (2H, t,
J = 8.3 Hz,
CH2), 3.03 (2H, t, J = 8.4 Hz, CH2), 2.62 (3H, s, CH3), 2.09 (3H, s, CH3);
APCI-MS 363
(M-Ht); FAB-HRMS calcd for C17H18CIN2O3S (MH+) 365.0727, found 365.0796.
5-(3-Chloro-2-methyl-benzenesulfonylamino)-1-ethyl-2,3-dihydro-1 H-indolium
chloride (STX987, XDS02031)
The free base of STX987 was synthesized as above. A purple amorphous powder
was
obtained; TLC single spot at Rf 0.79 (8% methanol/DCM); 'H NMR (270 MHz,
CDCI3): S
7.79 (1 H, d, J = 7.9 Hz, ArH), 7.53 (1 H, d, J = 7.9 Hz, ArH), 7.15 (1 H, t,
J = 8.0 Hz, ArH),
6.75 (1 H, d, J = 1.8 Hz, ArH), 6.58 (1 H, dd, J = 8.1, 1.8 Hz, ArH), 6.35 (1
H, s, NH), 6.22
( 1 H, d, J = 8.1 Hz, ArH), 3.30 (2H, t, J = 8.4 Hz, CH2), 3.05 (2H, q, J =
7.2 Hz, CH2), 2.84
(2H, t, J = 8.3 Hz, CH2), 2.67 (3H, s, CH3), 1.11 (3H, t, J = 7.2 Hz, CH3).
The free base
was treated with HCI-ether solution to give STX987 as light pink crystalline
solid. HPLC
purity 93 % (tR 3.6 min in 20 % water-methanol);'H NMR (270 MHz, DMSO): S 10.3
(1H,
s, NH), 7.82 (1 H, d, J = 8.1 Hz, ArH), 7.72 (1 H, d, J = 8.1 Hz, ArH), 7.38
(1 H, t, J = 8.1
Hz, ArH), 6.79-6.89 (3H, m, broad, ArH), 3.42 (2H, t, J = 8.4 Hz, CH2), 3.16
(2H, q, J =
7.0 Hz, CHz), 2.91 (2H, t, J = 8.4 Hz, CH2), 2.62 (3H, s, GH3), 1.10 (3H, t, J
= 7.0 Hz,
CH3); APCI-MS 349 (M-HCI-H+); FAB-HRMS calcd for C"HZ°CINzO2S (M-
HCI+H+)
351.0934, found 351.0941.
1-Acetyl-5-aminoindoline
The solution of 1-acetyl-5-nitroindoline (1.0 g, 4.85 mmol) in ethanol-THF
(100 mL : 30
mL) was hydrogenated over 5% PdIC (600 mg) at atmosphere pressure for 2h,
filtered
through Celite and concentrated in vacuo to give a white solid which was
recrystalllized
from ethanol. White crystalline solid (580 mg, 68%) was obtained. Mp 185-
186.5°C (lit
184-185°C, [21]); 'H NMR (270 MHz, DMSO): b 7.73 (1 H, d, J = 8.6 Hz,
ArH), 6.45 (1 H,
s broad, w1/2 = 1.8 Hz, ArH), 6.33 (1 H, dd, J = 8.6, 1.8 Hz, ArH), 4.82 (2H,
s, NHa), 3.97
CA 02501228 2005-04-05
WO 2004/037251 PCT/GB2003/004590
111
a
(2H, t, J = 8.4 Hz, CH2), 2.99 (2H, t, J = 8.4 Hz, CH2), 2.07 (3H, s, CH3);
APCI-MS 175
(M-H~).
1-Ethyl-5-aminoindoline
To a suspension of 1-acetyl-5-aminoindoline (130 mg, 0.74 mmol) in anhydrous
THF (10
mL) was added LiAIH4 (42 mg, 1.11 mmol). The mixture was stirred at rt for 6h,
quenched with saturated NH4CI and extracted with ethyl acetate. The organic
phase
was washed with brine, dried over sodium sulphate and concentrated in vacuo to
give a
purple residue (80 mg, 67%) that was used without further purification. 'H NMR
(270
MHz, DMSO): 5 6.56 (1 H, s, ArH), 6.47 (1 H, d broad, J = 8.1 Hz, ArH), 6.37
(1 H, d, J =
8.0 Hz, ArH), 3.29 (2H, s, NHa), 3.20 (2H, t, J = 7.6 Hz, CH2), 3.02 (2H, q, J
= 6.9 Hz,
CHI), 2.86 (2H, t, J = 7.6 Hz, CHZ), 1.17 (3H, t,J = 6.9 Hz, CH3).
Synthesis of 5-(3-chloro-2-methyl-benzenesulfonamino)-1H-indole-3-carboxylic
acid methyl ester, STX 1050 (KRB01132):
i N'
CI I ~ DSO ~ '
O H
O. O
5-amino-1H-indole-3-carboxylic acid methyl ester (KRB01131): To a solution of
5-
nitro-1 H indole-3-carboxylic acid methyl ester (206 mg, 0.940 mmol) in
methanol (40
mL) was added 5% palladium on carbon (40 mg) and the mixture was stirred under
1
atm HZ for 5h. The mixture was filtered through celite and the filtrate
evaporated to yield
a brown solid that was used without further purification (173 mg, 97%), single
spot at Rf
0.64 (ethyl acetate). 'H NMR (ds DMSO): 5 11.50 (1 H, s, N-H), 7.83 (1 H, d,
J=3.2 Hz),
7.17 (1 H, d, J=2.0 Hz), 7.14 (1 H, d, J=8.4 Hz), 6.56 {1 H, dd, J=8.6, 2.2
Hz), 4.77 (2H, s,
N-H2), 3.76 (3H, s).
To a solution of 3-chloro-2-methylbenzenesulphonyl chloride (124 mg, 0.552
mmol) in
dichloromethane (4 mL) was added pyridine (100 pL, 1.3 mmol) and the mixture
was
stirred under Nz for 5 min, after which time 5-amino-1H indole-carboxylic acid
methyl
ester (100 mg, 0.526 mmol) was added. The resulting mixture was stirred for
1.5 h at
room temperature, then saturated NaHC03 solution (15 mt_) was added .and the
mixture
was extracted into ethyl acetate (20 mL). The organic phase was washed with
brine,
CA 02501228 2005-04-05
WO 2004/037251 PCT/GB2003/004590
112
dried (Na2S04), filtered and evaporated to give a residue that was purified
using flash
chromatography to afford a white solid (129 mg, 65%), single spot at Rf 0.84
(ethyl
acetate). mp 216.8-219.3°C, [22] , HPLC purity 99+% (tR 2.07 min in 10%
water-
acetonitrile). 'H NMR (ds-DMSO): 5 11.91 (1 H, s), 10.32 (1 H, s), 8.03 (1 H,
d, J=3.0 Hz),
7.82 (1H, d, J=7.9 Hz), 7.70-7.67 (2H, m), 7.37-7.31 (2H, m), 6.95 (1H, dd,
J=8.6, 2.0
Hz), 3.77 (3H, s), 2.65 (3H, s). LCMS: 377.09. FAB-MS (MH+, C"H~5CIN2O4S):
calcd
378.0441, found 378.0439.
CA 02501228 2005-04-05
WO 2004/037251 PCT/GB2003/004590
113
,S~mthesis of Benzimidazole Ar)rsulphonamide Derivatives
02N ~ N a OZN ~ N \ N
I ~~- I ~~- + I
~N ~N 02N / N
R R
~b
H2N I ~ N~ ~ b
/ N
H HzN I ~ N~ I W N
Ic ~N HzN /
H i R R
. N I ~ N~ c
~N O H
v ,N
S'O Arrs~ I ~ N>--- Arm i,0 I ~ N~-
O / N ~Sw / N
N
R O H R
d CI
Ar = 2-Me-3-CI-phenyl R = -CH3 Ar = 2-Me-3-CI-phenyl
H
CI \ ~ ~N I \ N~ Ar = 2-Me-3-CI-phenyl R = -CzHS R = -CH3
~N Ar = 2-Me-3-CI-phenyl R = -CH2CH(CH3)z R = -CzHS
H R = -CHzCH(CH3)z
Ar = 2-Me-3-CI-phenyl R = -CH2COOCzHS
Ar = 2-Me-3-CI-phenyl R = -CH2C6H5 R = -CHzCOOCzHS
Ar = 4-n-propylphenyl R = -CH3 R = -CH2C6H5
Ar = 2,5-dichlorophenyl R = -CH3
Ar = 2,4-dichlorophenyl R = -CH3
Ar = 2-Me-4-Br-phenyl R = -CH3
Ar = 4-biphenyl R = -CH3
CI O H
HaN ~ N c ~~ .N ~ N
yCFa ~ \ S~ ~ yCFs
i N ~ O / N
H H
CI
CI H
N
HzN ~ N a HzN ~ N c CI ~ \ O ~~ N ~ v
N ---~ I / N~- ~ O I / N
H H H
a) RX, KzC03, Acetone ,rt or reflux b)Hz/ 5%Pd-C, Ethanol-THF r.t. or Fe, AcOH-
ethanol c) ArS03Cl,
DCM,Pyridine or ArS03Cl, DCM,Pyridine/DMAP d)HOBt, THF, r.t. e)N-
chlorosuccimide, IPA
CA 02501228 2005-04-05
WO 2004/037251 PCT/GB2003/004590
114
O H H CI
~S.N ~ N ~~ .N ~ N ~~ ,N
I ~ ,o I ~ N~--- I w Sb I ~ N~- I ~ So I ~ N
i \ i \
CI CI
CI
STX975, XDS02001 STX976, XDS02003 STX1121, XDS02102
H O H O H
o~ ,N
I ~ So I ~ N~ cl ~ ~so I ~ N~ w ~s~ I ~ N~--
~N I , ~N I , ~N
\ ~CI \ CI~CI \
STX1112, XDS02088 STX1113, XDS02089 STX1114, XDS02090
H O~ H N O H
I / ,so I ~ N~- I / ,so
N ~ \
Br \
CI
STX1115, XDS02091 STX1116, XDS02092 STX1110, XDS02084
H
~S.N ~ N p H ~ ~~ ,N N
W s0 I ~>--- vS.N ~ N ~ S~ I
'a ~N I w a0 I ~ ,~- I ~ p ~N
~N
CI CI
CI
STX1111, XDS02085 STX1119, XDS02100 STX1120, XDS02101
~O
H O~ O H
O ~~ .N N
~S~N N ~ S~
O I ~ ~~- I ~ p~N
N
CI O CI
CI
STX977, XDS02015 STX978, XDS02017 ( STX1117, XDS02098
H
~S'N~N O\ H ~~ .N
W v TI ~>- ~S~N~N S ~ N
O i N I W s0 T //\ ~~-CF3
N , N
CI ~ H H
CI CI
STX1118, XDS02099 STX879, XDS01173 STX985, XDS02026
Preparation of 1-Alkyl-5-amino-2-methylbenzimidazole and 1-alkyl-6-amino-2-
methylbenzimidazole
To a solution of 5-nitrobenzimidazole (1.0 g, 5.6 mmol) in acetone (50 mL) was
added
potassium carbonate (1.0 g), followed by alkyl halide (1.2 - 1.5 equivalents).
The
mixture was stirred under nitrogen at rt, then partitioned between ethyl
acetate and water
CA 02501228 2005-04-05
WO 2004/037251 PCT/GB2003/004590
115
after TLC showed completion of the reaction. The organic phase was washed with
brine, dried over sodium sulphate and concentrated in vacuo to give a mixture
of 1-alkyl-
5-nitro-2-methylbenzimidazoie and 1-alkyl-6-nitro-2-methylbenzimidazole, which
were
dissolved in ethanol-THF (100 mL, 2 : 1) and hydrogenated over 5% Pd-C under
atmosphere pressure for 8h. After filtration through celite~, the filtrate was
evaporated to
give a yellow solid that was separated with flash chromatography (Methanol-DCM
gradient elution). 1-Alkyl-5-aminobenzimidazole and 1-alkyl-6-
aminobenzimidazole were
obtained as yellow solid or thick syrup.
5-Amino-1,2-dimethylbenzimidazole (XDS01191B, XDS02082B):
Yellow solid, mp 126-127°C (lit.128°C, [23]). TLC single
spot at Rf 0.30
(5%methanol/DCM); 'H NMR (270 MHz, DMSO): b 7.08 (1 H, d, J = 8.7 Hz, 7-H),
7.65
(1 H, d, J = 1.5 Hz, 4-H), 6.50 (1 H, dd, J = 8.7, 1.5 Hz, 6-H), 4.63 (2H,
broad, NH2),
3.58(3H, s, NCH3), 2.39 (3H, s, CH3).
6-Amino-1,2-dimethylbenzimidazole (XDS01191A, XDS02082A):
Yellow solid. TLC single spot at Rf 0.33 (5%methanol/DCM); 'H NMR (270 MHz,
DMSO): S 7.13 (1 H, d, J = 8.4 Hz, 4-H), 6.48 (1 H, d, J = 2.0 Hz, 7-H), 6.43
(1 H, dd, J =
8.4, 2.0 Hz, 5-H), 4.83 (2H, broad, NH2), 3.53(3H, s, NCH3), 2.39 (3H, s,
CH3).
5-Amino-1-ethyl-2-methylbenzimidazole (XDS02079B):
Yellow syrup. TLC single spot at Rf 0.27 (5%methanol/DCM); 'H NMR (270 MHz,
DMSO): 5 7.12 (1 H, d, J = 8.3 Hz, 7-H), 6.68 (1 H, d, J = 2.0 Hz, 4-H), 6.51
(1 H, dd, J =
8.3, 2.0 Hz, 6-H), 4.68 (2H, broad, NH2), 4.08 (2H, q, J = 7.2 Hz, NCH2), 2.43
(3H, s,
CH3), 1.24(3H, t, J = 7.2 Hz, CH3); APCI-MS 175 (M+).
6-Amino-1-ethyl-2-methylbenzimidazole (XDS02079A):
Yellow solid. TLC single spot at Rf 0.30 (5%methanol/DCM); 'H NMR (270 MHz,
DMSO): i5 7.16 (1 H, d, J = 8.4 Hz, 4-H), 6.68 (1 H, d, J = 1.7 Hz, 7-H), 6.46
(1 H, dd, J =
8.4, 1.7 Hz, 5-H), 4.85 (2H, broad, NH2), 4.02 (2H, q, J = 7.9 Hz, NCH2), 2.42
(3H, s,
CH3), 1.24(3H, t, J = 7.9 Hz, CH3); APCI-MS 175 (M+).
5-Amino-1-i-butyl-2-methylbenzimidazole (XDS02093B):
Yellow syrup. TLC single spot at Rf 0.42 (10%methanol/DCM); 'H NMR {400 MHz,
DMSO): b 7.08 (1 H, d, J = 8.5 Hz, 7-H), 6.65 (1 H, d, J = 1.9 Hz, 4-H), 6.48
(1 H, dd, J =
CA 02501228 2005-04-05
WO 2004/037251 PCT/GB2003/004590
116
8.5, 1.9 Hz, 6-H), 4.63 (2H, broad, NH2), 3.82 (2H, d, J = 7.4 Hz, NCH), 2.41
(3H, s,
CH3), 2.07 (1 H, m, CH), 0.84 (6H, d, J = 7.0 Hz, 2 x CH3); APCI-MS 204 (MH+)
6-Amino-1-i-butyl-2-methylbenzimidazole (XDS02093A):
Yellow solid. TLC single spot at Rf 0.45 (10%methanol/DCM); 'H NMR (270 MHz,
DMSO): b 7.15 (1 H, d, J = 8.2 Hz, 4-H), 6.62 (1 H, d, J = 1.6 Hz, 7-H), 6.44
(1 H, dd, J =
8.2, 1.8 Hz, 5-H), 4.83 (2H, broad, NHZ), 3.79 (2H, d, J = 7.7 Hz, NCHz), 2.41
(3H, s,
CH3), 2.10 (1 H, m, CH), 0.87 (6H, d, J = 6.6 Hz, 2 x CH3); APCI-MS 204 (MH+)
(5-Aminobenzoimidazol-1-yl)-acetic acid ethyl ester (XDS02012B)
Yellow solid. TLC single spot at Rf 0.36 (5%methanol/DCM); 'H NMR (270 MHz,
DMSO): 5 7.08 (1 H, d, J = 8.4 Hz, 7-H), 6.69 (1 H, d, J = 2.2 Hz, 4-H), 6.49
(1 H, dd, J =
8.4, 2.2 Hz, 6-H), 5.02 (2H, s, NCH2), 4.68 (2H, s, NH2), 4.16 (2H, q, J = 7.2
Hz, CHa),
2.36 (3H, s, CH3), 1.21 (3H, t, J = 7.2 Hz, CH3); APCI-MS 234 (MH+).
(6-Aminobenzoimidazol-1-yl)-acetic acid ethyl ester (XDS02012A)
Yellow solid. TLC single spot at Rf 0.40 (5%methanol/DCM); 'H NMR (270 MHz,
DMSO): b 7.17 (1 H, d, J = 9.0 Hz, 4-H), 6.45-6.48 (2H, m, 5 and 7-H), 4.95
(2H, s,
NCH), 4.87 (2H, s, NH2), 4.17 (2H, q, J = 7.1 Hz, CH2), 2.36 (3H, s, CH3),
1.22 (3H, t, J
= 7.1 Hz, CH3); APCI-MS 234 (MH+).
5-Amino-1-benzyl-2-methylbenzimidazole (XDS02086B):
Yellow syrup. TLC single spot at Rf 0.27 (5%methanol/DCM); 'H NMR (400 MHz,
DMSO): b 7.26-7.32 (2H, m, ArH), 7.23 (1 H, tt, J = 7.5, 2.3 Hz, ArH), 7.05-
7.09 (3H, m,
ArH), 6.69 (1 H, d, J = 2.3 Hz, 4-H), 6.46 (1 H, dd, J = 8.2, 2.3 Hz, 6-H),
5.30 (2H, s, CH2),
4.68 (2H, broad, NHZ), 2.40 (3H, s, CH3); APCI-MS 238 (MH+).
6-Amino-1-benzyl-2-methylbenzimidazole (XDS02086A):
Yellow solid. TLC single spot at Rf 0.30 (5%methanol/DCM); 'H NMR (400 MHz,
DMSO): b 7.28-7.32 (2H, m, ArH), 7.23 (1 H, tt, J = 7.5, 2.3 Hz, ArH), 7.17 (1
H, d, J =
8.2, Hz, ArH), 7.06 (2H, m ArH), 6.43-6.46 (2H, m, ArH), 5.26 (2H, s, CHz),
4.63 (2H, s,
NH2), 2.40 (3H, s, CH3); APCI-MS 238 (MH+).
Preparation of 5-amino-4-chloro-1,2-dimethylbenzimidazole (XDS02096A)
To a solution of 5-amino-1,2-dimethylbenzimidazole (600 mg, 3.73 mmol) in IPA
(15 mL)
was added N-chlorosuccinimide (548 mg, 4.10 mmol). The mixture was stirred at
rt for
CA 02501228 2005-04-05
WO 2004/037251 PCT/GB2003/004590
117
20 min, diluted with DCM (80 mL) and washed with 5% sodium bicarbonate and
brine.
The dark brown solution was dried over sodium sulphate and concentrated in
vacuo to
give a brown residue, which was subjected to flash chromatography (methanol-
DCM
gradient elution). Yellow solid (220 mg, 33%) was obtained. TLC single spot at
Rf 0.69
(10%methanol/DCM); 'H NMR (270 MHz, DMSO): b 7.16 (1 H, d, J = 7.9 Hz, ArH),
6.72
(1 H, d, J = 7.9, Hz, ArH), 4.90 (2H, s, NHZ), 3.64 (3H, s, NCH3), 2.40 (3H,
s, CH3); APCI-
MS 196 (MH+)
General method for synthesis of N-benzimidazole arylsulphonamide derivatives:
To a solution arylsulphonyl chloride (1.1 eq.) in DCM were added pyridine (2.2
eq.) and
catalytic amount of DMAP, followed by the corresponding amine (1 eq.). The
reaction
mixture was stirred at rt under nitrogen for 4-16 h, then partitioned between
ethyl acetate
and 5% sodium bicarbonate after TLC showed completion of the reaction. The
organic
layer was washed with brine, dried over sodium sulphate, and concentrated in
vacuo to
give crude product as solid or thick syrup. The compound was then purified by
flash
chromatography (Methanol-DCM gradient elution) to give desired
arylsulphonamide as
crystalline solid. Yield ranges from 50-80%.
3-Chloro-N-(1,2-dimethyl-1 H-benzoimidazol-6-yl)-2-methylbenzenesulphonamide
(STX975, XDS02001 )
White crystalline solid. Mp 265-266°C; TLC single spot at Rf 0.43 (5%
methanol/DCM);
HPLC purity > 99% (tR 2. min in 10% water-methanol); 'H NMR (270 MHz, DMSO): b
10.4 (1 H, s, NH), 7.84 (1 H, d, J = 7.9 Hz, ArH), 7.67 (1 H, d, J = 7.9 Hz,
ArH), 7.34 (1 H,
d, J = 8.2 Hz, ArH), 7.32 (1 H, t, J = 7.9 Hz, ArH), 7.14(1 H, d, J = 2 Hz,
ArH ), 6.80 (1 H,
dd, J= 8.2, 2.0 Hz, ArH), 3.61 (3H, s, NCH3), 2.64 (3H, s, CH3), 2.46 (3H, s,
CH3); APCI-
MS 348 (M-H+); FAB-HRMS calcd for C16H17CIN3O2S (MH+) 350.0730, found
350.0749.
3-Chloro-N-(1,2-dimethyl-1 H-benzoimidazol-5-yi)-2-methylbenzenesuiphonamide
(STX976, XDS02003)
White crystalline solid. Mp 283-283.5°C; TLC single spot at Rf
0.38 (5%
methanol/DCM); HPLC purity >99% (tR 2.0 min in 10% water-methanol); 'H NMR
(270
MHz, DMSO): 5 10.3 (1 H, s, NH), 7.77 (1 H, d, J = 7.6 Hz, ArH), 7.66 (1 H, d,
J = 7.6 Hz,
ArH), 7.32 (1H, d, J = 8.4 Hz, ArH), 7.30 (1H, t, J = 7.6 Hz, ArH), 7.16(1H,
d, J = 2 Hz,
ArH ), 6.90 (1 H, dd, J= 8.4, 2.0 Hz, ArH), 3.64 (3H, s, NCH3), 2.64 (3H, s,
CH3), 2.44
CA 02501228 2005-04-05
WO 2004/037251 PCT/GB2003/004590
118
(3H, s, CH3); APCI-MS 348 (M-H~); FAB-HRMS calcd for C1gH17CIN3O2S (MH~)
350.0730, found 350.0747.
3-Chloro-N-(4-chloro-1,2-dimethyl-1 H-benzoimidazol-5-yl)-2-
methylbenzenesulphonamide (STX1121, XDS02102B)
Off-white crystalline solid. TLC single spot at Rf 0.50 (10% methanol/DCM);
HPLC purity
95% (tR 2.1 min in 20% water-methanol); 'H NMR (270 MHz, DMSO): S 10.1 (1 H,
s,
NH), 7.70 (1 H, dd, J = 7.7, 1.7 Hz, ArH), 7.56 (1 H, dd, J = 7.8, 1.7 Hz,
ArH), 7.39 (1 H, d,
J = 8.2 Hz, ArH), 7.25 (1 H, t, J = 7.7 Hz, ArH), 7.04 (1 H, d, J = 8.2 Hz,
ArH), 3.69 (3H, s,
NCH3), 2.67 (3H, s, CH3), 2.51 (3H, s, CH3); APCI-MS 384 (MH+).
N-(1,2-Dimethyl-1H-benzoimidazol-5-yl)-4-propyibenzenesulphonamide (STX1112,
XDS02088)
White crystalline solid. TLC single spot at Rf 0.38 (5% methanol/DCM); HPLC
purity
>99% (tR 2.1 min in 20% water-methanol); 'H NMR (270 MHz, DMSO): 5 9.90 (1H~
s,
NH), 7.58 (2H, d, J = 8.3 Hz, ArH), 7.29-7.32 (3H, m, ArH), 7.17 (1 H, d, J =
1.5 Hz, ArH),
6.91 (1 H, dd, J = 8.6, 2.0 Hz, ArH), 3.64 (3H, s, NCH3), 2.55 (2H, m, CHI),
2.50 (3H, s,
CH3), 1.55 (2H, sextet, J = 7.6 Hz, CH2), 0.84 (3H, t, J = 7.6 Hz, CH3); APCI-
MS 344
( M H+).
2,5-Dichloro-N-(1,2-dimethyl-1 H-benzoimidazol-5-yl)-benzenesulphonamide
(STX1113, XDS02089)
White crystalline solid. TLC single spot at Rf 0.67 (10% methanol/DCM); HPLC
purity
99% (tR 2.0 min in 20% water-methanol); 'H NMR (270 MHz, DMSO): b 10.5 (1 H,
s,
NH), 7.84 (1 H, t, J = 1.4 Hz, ArH), 7.68 (2H, d, J = 2.0 Hz, ArH), 7.35 (1 H,
d, J = 8.5 Hz,
ArH), 7.21 (1 H, d, J = 2.0 Hz, ArH), 6.97 (1 H, dd, J = 8.2, 2.0 Hz, ArH ),
3.64 (3H, s,
NCH3), 2.45 (3H, s, CH3); APCI-MS 370 (MH~).
2,4-Dichloro-N-(1,2-dimethyl-1 H-benzoimidazol-5-yl)-benzenesulphonamide
(STX1114, XDS02090)
Off-white crystalline solid. TLC single spot at Rf 0.59 (10% methanol/DCM);
HPLC purity
>99% (tR 2.0 min in 20% water-methanol); 'H NMR (270 MHz, DMSO): b 10.4 (1H,
s,
NH), 7.88 (1 H, d, J = 8.7 Hz, ArH), 7.84 (2H, d, J = 1.9 Hz, ArH), 7.52 (1 H,
dd, J = 8.7,
1.9 Hz, ArH), 7.33 (1 H, d, J = 8.5 Hz, ArH), 7.20 (1 H, d, J = 1.7 Hz, ArH ),
6.95 (1 H, dd,
J = 8.7, 1.7 Hz, ArH ), 3.63 (3H, s, NCH3), 2.45 (3H, s, CH3); APCI-MS 370
(MH+).
CA 02501228 2005-04-05
WO 2004/037251 PCT/GB2003/004590
119
4-Bromo-N-(1,2-dimethyl-1 H-benzoimidazol-5-yl)-2-methylbenzenesulphonamide
(STX1115, XDS02091)
White crystalline solid. TLC single spot at R, 0.67 (10% methanol/DCM); HPLC
purity
>99% (t~ 2.1 min in 20% water-methanol); ~H NMR (270 MHz, DMSO): i5 10.1 (1H,
s,
NH), 7.62-7.69 (2H, m, ArH), 7.50 (1 H, dd, J = 8.5, 2.2 Hz, ArH), 7.32 (1 H,
d, J = 8.5 Hz,
ArH), 7.14 (1H, d, J = 1.9 Hz, ArH), 6.89 (1H, dd, J = 8.5, 1.9 Hz, ArH), 3.63
(3H, s,
NCH3), 2.55 (3H, s, CH3), 2.45 (3H, s, CH3); APCI-MS 394 (MH+).
N-(1,2-Dimethyl-1 H-benzoimidazol-5-yl)-4-phenylbenzenesulphonamide (STX1116,
XDS02092)
White crystalline solid. TLC single spot at Rf 0.72 (5% methanol/DCM); HPLC
purity
>99% (tR 2.1 min in 20% water-methanol); 'H NMR (270 MHz, DMSO): b 10.0 (1 H,
s,
NH), 7.67-7.82 (6H, m, ArH), 7.41-7.50 (3H, m, ArH), 7.33 (1 H, d, J = 8.5 Hz,
ArH), 7.22
(1H, d, J = 1.9 Hz, ArH), 6.95 (1H, dd, J = 8.5, 1.9 Hz, ArH), 3.64 (3H, s,
NCH3), 2.44
IS (3H, s, CH3); APCI-MS 378 (MH+).
3-Chloro-N-(1-ethyl-2-methyl-1 H-benzoimidazol-6-yl)-2-
methylbenzenesulphonamide (STX1110, XDS02084)
Off-white solid. TLC single spot at Rf 0.45 (8% methanol/DCM); HPLC purity
>99% (tR
2.2 min in 20% water-methanol); 'H NMR (270 MHz, DMSO): b 10.4 (1 H, s, NH),
7.84
(1 H, d, J = 7.9 Hz, ArH), 7.67 (1 H, d, J = 7.8 Hz, ArH), 7.35 (1 H, d, J =
8.5 Hz, ArH),
7.32 (1 H, t, J = 7.9 Hz, ArH), 7.10 (1 H, d, J = 2.2 Hz, ArH ), 6.82 (1 H,
dd, J= 8.5, 2.1 Hz,
ArH), 4.09 (2H, q, J =7.1 Hz, CH2), 2.61 (3H, s, CH3), 2.46 (3H, s, CH3), 1.18
(3H, t, J =
7.1 Hz, CH3); APCI-MS 364 (MH+).
3-Chloro-N-(1-ethyl-2-methyl-1 H-benzoimidazol-5-yl)-2-
methylbenzenesulphonamide (STX1111, XDS02085)
Off-white solid. TLC single spot at Rf 0.42 (8% methanol/DCM); HPLC purity
>99% (tR
2.2 min in 20% water-methanol);'H NMR (270 MHz, DMSO): i5 10.3 (1H, s, NH),
7.78
(1 H, d, J = 7.9 Hz, ArH), 7.66 (1 H, d, J = 7.9 Hz, ArH), 7.36 (1 H, d, J =
8.2 Hz, ArH),
7.32 (1 H, t, J = 7.9 Hz, ArH), 7.16 (1 H, d, J = 1.9 Hz, ArH ), 6.90 (1 H,
dd, J= 7.9, 2.0 Hz,
ArH), 4.12 (2H, q, J =7.1 Hz, CH2), 2.64 (3H, s, CH3), 2.46 (3H, s, CH3), 1.23
(3H, t, J =
7.1 Hz, CH3); APCI-MS 364 (MH+).
CA 02501228 2005-04-05
WO 2004/037251 PCT/GB2003/004590
120
3-Chloro-N-(1-isobutyl-2-methyl-1 H-benzoimidazol-6-yl)-2-
methylbenzenesulphonamide (STX1119, XDS02100)
Off-white solid. TLC single spot at Rf 0.57 (8% methanol/DCM); HPLC purity 99%
(tR 2.2
min in 20% water-methanol); 'H NMR (270 MHz, DMSO): a 10.4 (1 H, s, NH), 7.80
(1 H,
d, J = 8.9 Hz, ArH), 7.66 (1H, d, J = 8.8 Hz, ArH), 7.36 (1H, d, J = 8.5 Hz,
ArH), 7.29
(1 H, t, J = 7.9 Hz, ArH), 7.03 (1 H, d, J = 1.9 Hz, ArH ), 6.84 (1 H, dd, J=
8.5, 1.8 Hz,
ArH), 3.85 (2H, d, J =7.2 Hz, NCHZ), 2.61 (3H, s, CH3), 2.45 (3H, s, CH3),
1.91 (1 H, m,
CH), 0.81 (6H, d, J = 7.0 Hz, 2 x CH3); APCI-MS 392 (MH+).
3-Chloro-N-(1-isobutyl-2-methyl-1 H-benzoimidazol-5-yl)-2-
methylbenzenesulphonamide (STX1120, XDS02101)
Off-white solid. TLC single spot at Rf 0.52 (8% methanol/DCM); HPLC purity 99%
(tR 2.3
min in 20% water-methanol); 'H NMR (270 MHz, DMSO): b 10.3 (1 H, s, NH), 7.80
(1 H,
d, J = 7.9 Hz, ArH), 7.68 (1 H, d, J = 7.9 Hz, ArH), 7.38 (1 H, d, J = 8.8 Hz,
ArH), 7.33
(1 H, t, J = 7.9 Hz, ArH), 7.16 (1 H, d, J = 1.9 Hz, ArH ), 6.90 (1 H, dd, J=
8.7, 1.9 Hz,
ArH), 3.91 (2H, d, J =7.3 Hz, NCH2), 2.62 (3H, s, CH3), 2.47 (3H, s, CH3),
2.05 {1 H, m,
CH), 0.83 (6H, d, J = 7.0 Hz, 2 x CH3); APCI-MS 392 (MH+)
[6-(3-Chloro-2-methylbenzenesulphonylamino)-2-methylbenzoimidazol-1-yl]-acetic
acid ethyl ester (STX977, XDS02015)
Off-white solid. TLC single spot at Rf 0.46 (6% methanol/DCM); HPLC purity
>99% (tR
2.0 min in 10% water-methanol); 'H NMR (270 MHz, DMSO): S 10.4 (1H, s, NH),
7.82
(1 H, d, J = 8.0 Hz, ArH), 7.67 (1 H, d, J = 8.0 Hz, ArH), 7.37 (1 H, d, J =
8.5 Hz, ArH),
7.30 (1 H, t, J = 8.0 Hz, ArH), 7.11 (1 H, d, J = 2.0 Hz, ArH ), 6.83 (1 H,
dd, J= 8.5, 2.0 Hz,
ArH), 5.09 (2H, s, NCH2), 4.16 (2H, q, J = 7.1 Hz, CHZ), 2.62 (3H, s, CH3),
2.45 (3H, s,
CH3), 1.21 (3H, t, J = 7.1 Hz, CH3); APCI-MS 420 (M-H+); FAB-HRMS calcd for
C1 gH21 CIN304S (MH+) 422.0941, found 422.0942.
[5-(3-Chloro-2-methylbenzenesulphonylamino)-2-methylbenzoimidazol-1-yl]-acetic
acid ethyl ester (STX9713, XDS02017)
Off white solid. TLC single spot at Rf 0.40 (6% methanol/DCM); HPLC purity 99%
(tR 2.0
min in 10% water-methanol); 'H NMR (270 MHz, DMSO): 5 10.3 (1 H, s, NH), 7.80
(1 H,
d, J = 7.9 Hz, ArH), 7.67 (1 H, d, J = 7.9 Hz, ArH), 7.29-7.34 (2H, m, ArH),
7.18 (1 H, d, J
= 1.7 Hz, ArH ), 6.90 (1 H, dd, J= 8.6, 1.7 Hz, ArH), 5.11 (2H, s, NCH2), 4.15
(2H, q, J =
7.1 Hz, CHZ), 2.64 (3H, s, CH3), 2.40 (3H, s, CH3), 1.19 (3H, t, J = 7.1 Hz,
CH3); APCI-
CA 02501228 2005-04-05
WO 2004/037251 PCT/GB2003/004590
121
MS 420 (M-H+); FAB-HRMS calcd for C1gH21CIN304S (MH~) 422.0941, found
422.0944.
3-Chloro-N-(1-benzyl-2-methyl-1 H-benzoimidazol-6-yl)-2-
methylbenzenesulphonamide (STX1117, XDS02098)
Off-white solid. TLC single spot at Rf 0.70 (10% methanol/DCM); HPLC purity
99% (tR
2.2 min in 20% water-methanol); 'H NMR (270 MHz, DMSO): b 10.4 (1 H, s, NH),
7.66
( 1 H, d, J = 7.9 Hz, ArH), 7.64 ( 1 H, d, J = 7.9 Hz, ArH), 7.30-7.39 (4H, m,
ArH), 7.21 ( 1 H,
t, J = 7.9 Hz, ArH), 7.11 (1H, d, J = 2.0 Hz, ArH), 7,02-7.06 (2H, m, ArH ),
6.83 (1H, dd,
J= 7.9, 2.0 Hz, ArH), 5.34 (2H, s, NCH2), 2.58 (3H, s, CH3), 2.45 (3H, s,
CH3); APCI-MS
426 (MH~).
3-Chloro-N-(1-benzyl-2-methyl-1 H-benzoimidazol-5-yl)-2-
methylbenzenesulphonamide (STX1118, XDS02099)
Off-white solid. TLC single spot at Rf 0.65 (10% methanol/DCM); HPLC purity
99% (tR
2.2 min in 10% water-methanol); 'H NMR (270 MHz, DMSO): 5 10.3 (1 H, s, NH),
7.80
(1 H, d, J = 7.9 Hz, ArH), 7.67 (1 H, d, J = 7.9 Hz, ArH), 7.25-7.35 (5H, m,
ArH), 7.19 (1 H,
d, J = 1.9 Hz, ArH), 7.06-7.09 (2H, m, ArH ), 6.87 (1H, dd, J= 8.5, 1,9 Hz,
ArH), 5.38
(2H, s, NCH2), 2.62 (3H, s, CH3), 2.45 (3H, s, CH3); APCI-MS 426 (MH+).
3-Chloro-N-(2-trifluromethyl-1 H-benzoimidazol-5-yl)-2-
methylbenzenesulphonamide (STX879, XDS01173)
White crystalline solid. TLC single spot at Rf 0.58 (20% ethyl acetate/DCM);
HPLC purity
99% (tR 2.4 min in 20% water-methanol); 'H NMR (270 MHz, DMSO): ~ 13.9 (1 H,
s,
NH), 10.7 (1 H, s, NH), 7.85 (1 H, d, J = 8.0 Hz, ArH), 7.68 (1 H, d, J = 8.0
Hz, ArH), 7.60
(1 H, d broad, J = 8.1 Hz, ArH), 7.34 (2H, m, ArH), 7.10(1 H, d, J = 8.3 Hz,
ArH ), 2.64
(3H, s, CH3); APCI-MS 388 (M-H~); FAB-HRMS calcd for C15H12CIF3N302S (MH+)
390.0291, found 390.0291.
Preparation of 3-chloro-N-(2-methyl-1 H-benzoimidazol-5-yl)-2-
methylbenzenesulphonamide (STX985, XDS02026)
The coupling reaction of 3-chloro-2-methyibenzenesulphonyi chloride (2 eq.)
with 2-
methylbenzimidazoel (1 eq.) under the condition described above yielded a
mixfiure of 3-
chloro-N-[1-(3-chloro-2-methylbenzenesulphonyl)-2-methyl-1 H-benzoimidazol-5-
yl]-2-
methyl-benzenesulphonamide and 3-chloro-N-[1-(3-chloro-2-
methylbenzenesulphonyl)-
2-methyl-1 H-benzoimidazol-6-yl]-2-methyl-benzenesulphonamide in 1 : 1 ratio
as judged
CA 02501228 2005-04-05
WO 2004/037251 PCT/GB2003/004590
122
by HNMR. 1H NMR (270 MHz, DMSO): 5 10.7 (2H, s, 2 x NH), 7.86-7.96 (3H, m,
ArH),
7.65-7.78 (5H, m, ArH), 7.52-7.58 (5H, m, ArH), 7.25-7.40 (3H, m, ArH), 7.07
(2H, t, J =
8.2 Hz, ArH), 2.61 (3H, s, CH3), 2.58 (3H, s, CH3), 2.54 (6H, s, 2 x CH3),
2.39 (3H, s,
CH3), 2.33 (3H, s, CH3). The mixture (200 mg) was dissolved in THF (15 mL), N-
hydroxybenzotriazole (200 mg) was added. After stirred at rt for 48h, the
mixture was
partitioned between ethyl acetate and 5% sodium bicarbonate. The organic phase
was
washed with brine, dried over sodium sulphate and concentrate in vacuo to give
a yellow
residue, which was purified with flash chromatography (methanol/DCM gradient
elution).
Off white amorphous powder was obtained. TLC single spot at Rf 0.38 (10%
methanol/DCM); HPLC purity 99% (tR 2.0 min in 10% wafer-methanol); 1H NMR (270
MHz, DMSO): b 12.1 (1 H, s, NH), 10.3 (1 H, s, NH), 7.78 (1 H, d, J = 7.9 Hz,
ArH), 7.68
(1 H, d, J = 7.9 Hz, ArH), 7.29-7.35 (2H, m, ArH), 7.12(1 H, s, ArH ), 6.83 (1
H, dd, J= 8.4,
1.8 Hz, ArH), 2.63 (3H, s, CH3), 2.41 (3H, s, CH3); APCI-MS 334 (M-H+); FAB-
HRMS
calcd for C15H15C1N3O2S (MH+) 336.0573, found 336.0583.
Synthesis of N-Benzimidazole Ark Isul~honamide Derivatives
H
OzN~NOa a HzN~NH2 b ~N N~ c H2N I ~ N~ d I ~ SO N I
oo' NH ~ ll~r' NH ~ nO I o N ~ / N '~ o i'
CI
STX1140,XDS02110
CI CI ~ H ~ H
o w N a o ~ N ~ S-N ~ N ~ ~ S-N
Q ~ O ~ O >-- O
S"N ON ~ S"- / r rN r rN
H 0 ~ H ~ CI 0 CI
,0 OH OH
STX977, XDSf'02015 STX1141, XDS02115 STX978, XDS02017 STX1142, XDS02116
a) Raney-Ni, NHpNHz H20, ethanol, rt b) AcaO, AcOH, 80°C c) 6N HCI,
75°C
d) 3-CI-2-Me-benzenesulphonyl chloride, DCM,Pyridine e) LiAIH4, THF,
0°C
N'-Phenyl-benzene-9,2,4-triamine:
To a solution of 2,4-dinitrophenylamine (1.5 g, 5.8 mmol) in ethanol-THF (150
: 50 mL)
were added hydrazine hydrate (2 mL, 65 mmol) and Raney Nickel (2.0 g). The
reaction
mixture was stirred at rt for 20 min, filtered through Celite. Evaporation of
the solvent
gave a black residue, which was purified by flash chromatography (methanol-DCM
gradient elution). A black crystalline solid (1.0 g, 87%) was obtained. Mp 128-
129°C;
TLC single spot at Rf 0.46 (8% methanol/DCM); 1H NMR (270 MHz, DMSO): 5 7.25
(2H,
CA 02501228 2005-04-05
WO 2004/037251 PCT/GB2003/004590
123
t, J = 7.5 Hz, ArH), 6.73 (1 H, s, NH), 6.61 (1 H, d, J = 8.3 Hz, ArH), 6.49-
6.55 (3H, m,
ArH), 5.99 (1 H, d, J = 2.5 Hz, ArH), 5.83 (1 H, dd, J = 8.2, 2.5 Hz, ArH),
4.66 (2H, s,
NHZ), 4.44 (2H, s, NH2); APCI-MS 198 (M-H+).
N-(2-Methyl-1-phenyl-1 H-benzimidazol-5-yl)-acetamide:
N'-Phenyl-benzene-1,2,4-triamine (800 mg, 4 mmol) was dissolved in acetic acid
(10
mL), acetic anhydride (1.0 mL) was added to the solution. The mixture was
stirred at
80°C for 6h, cooled to rt and neutralized with 5% sodium carbonate,
then extracted with
ethyl acetate. The organic phase was washed with brine, dried over magnesium
sulphate and concentrated to give a residue, which was crystallized from
ethanol. A
brown crystalline solid (0.85 g, 80%) was obtained. Mp 231-232°C; TLC
single spot at Rf
0.39 (10% methanol/DCM);'H NMR (270 MHz, DMSO): b 9.92 (1 H, s, NH), 7.97 (1
H, d,
J = 1.6 Hz, ArH), 7.51-7.67 (5H, m, ArH), 7.31 (1 H, dd, J = 8.3, 1.9 Hz,
ArH), 7.04 (1 H,
d, J = 8.3 Hz, ArH)" 2.41 (3H, s, CH3), 2.05 (3H, s, CH3); APCI-MS 264 (M-H+).
2-Methyl-1-phenyl-1 H-benzoimidazol-5-ylamine:
The solution of N-(2-methyl-1-phenyl-1 H-benzimidazol-5-yl)-acetamide (800 mg,
3
mmol) in 6N HCI (5 mL) was stirred at 75°C for 3h, cooled to rt and
neutralized with
sodium carbonate to pH 7, then extracted with ethyl acetate. The organic phase
was
washed with brine, dried over magnesium sulphate and concentrated to give a
dark
brown solid (600 mg, 90%). Mp 145-146°C; TLC single spot at R, 0.47
(10%
methanol/DCM); 'H NMR (270 MHz, DMSO): b 7.58-7.64 (2H, m, ArH), 7.46-7.53
(3H,
m, ArH), 6.82 (1 H, d, J = 8.5 Hz, ArH), 6.76 (1 H, d, J = 1.9 Hz, ArH), 6.51
(1 H, dd, J =
8.5, 1.9 Hz, ArH), 4.78 (2H, s, NH2), 2.36 (3H, s, CH3); APCI-MS 223 (M+).
3-Chloro-2-methyl-N-(2-methyl-1-phenyl-1 H-benzoimidazol-5-yl)-
benzenesulphonamide (STX1140, XDS02110)
The compound was prepared with general method of benzenesulphonamide
formation.
Light pink crystalline solid was obtained. Mp 254-256°C; TLC single
spot at Rf 0.62 (8%
methanol/DCM); HPLC purity > 99% (tR 2.6 min in 20% water-methanol); 'H NMR
(270
MHz, DMSO): S 10.4 (1 H, s, NH), 7.83 (1 H, dd, J = 8.6, 1.8 Hz, ArH), 7.69 (1
H, dd, J =
8.6, 1.7 Hz, ArH), 7.58-7.63 (5H, m, ArH), 7.34 (1 H, t, J = 8.0 Hz, ArH),
7.28 (1 H, d, J =
1.9 Hz, ArH), 6.90-7.00 (2H, m, ArH), 2.66 (3H, s, CH3), 2.36 (3H, s, CH3);
APCI-MS 412
(MH+)
CA 02501228 2005-04-05
WO 2004/037251 PCT/GB2003/004590
124
3-Chloro-N-[1-(2-hydroxyethyl)-2-methyl-1 H-benzoimidazol-6-yl]-2-methyl-
benzenesulfonamide (STX1141, XDS02115)
To a solution of [6-(3-Chloro-2-methyl-benzenesulfonylamino)-2-methyl-
benzoimidazol-
1-yl]-acetic acid ethyl ester (100 mg, 0.237 mmol) in anhydrous THF (10 mL)
was added
LiAIH4 (54 mg, 1.42 mmol) at 0°C. The mixture was stirred at 0°C
for 0.5h, quenched
with saturated ammonium chloride solution, neutralized with 6N HCI and
extracted with
ethyl acetate. The organic phase was washed with brine, dried over sodium
sulphate
and concentrated in vacuo to give a light pink crystalline solid (82 mg, 91
%). Mp 213-
214.5°C; TLC single spot at Rf 0.39 (12% methanol/DCM); HPLC purity >
99% (tR 2.0
min in 20% water-methanol); 'H NMR (270 MHz, DMSO): 5 10.4 (1 H, s, NH), 7.84
(1 H,
d, J = 7.7 Hz, ArH), 7.67 (1 H, d, J = 7.9 Hz, ArH), 7.28-7.35 (2H, m, ArH),
7.15 (1 H, d, J
= 1.9 Hz, ArH), 6.80 (1H, dd, J = 8.5, 1.9 Hz, ArH), 4.94 (1H, t, J = 5.2 Hz,
OH), 4.10
(2H, t, J = 5.0 Hz, NCH), 3.59 (2H, q, J = 5.2 Hz, CH2), 2.51 (3H, s, CH3),
2.47 (3H, s,
CH3); APCI-MS 380 (MH+).
3-Chloro-N-[1-(2-hydroxy-ethyl)-2-methyl-1 H-benzoimidazol-5-yl]-2-methyl-
benzenesulfonamide (STX1142, XDS02116)
The compound was prepared as above from [5-(3-Chloro-2-methyl-
benzenesulfonylamino)-2-methyl-benzoimidazol-1-yl]-acetic acid ethyl ester (35
mg,
0.083 mmol). White crystalline solid (22 mg, 89%) was obtained. Mp 245-
247°C; TLC
single spot at Rf 0.38 (12% methanol/DCM); HPLC purity > 99% (tR 2.0 min in
20%
water-methanol);'H NMR (270 MHz, DMSO): 5 10.3 (1H, s, NH), 7.79 (1H, d, J =
8.0
Hz, ArH), 7.67 (1 H, d, J = 8.0 Hz, ArH), 7.29-7.35 (2H, m, ArH), 7.16 (1 H,
s, ArH), 6.89
(1 H, d, J = 8.5, ArH), 4.90 (1 H, t, J = 5.0 Hz, OH), 4.14 (2H, t, J = 5.0
Hz, NCH), 3.63
(2H, q, J = 5.2 Hz, CHZ), 2.65 (3H, s, CH3), 2.48 (3H, s, CH3); APCI-MS 380
(MH+).
Synthesis of Benzoxazole Derivatives
R~ R~
R R2
O
R S. ~ R3 ~S~ ~N
3
R4 O H R4 O H
STX 839: R~=R2=H, R3=CI, R4=Me STX 842: R~=R2=H, R3=CI, R4=Me
STX 840: R~=R3=R4=H, R2=n-propyl STX 843: R~=R3=R4=H, R2=n-propyl
STX 841: R~=R4=CI, R2=R3=H STX 846: R~=R4=CI, R2=R3=H
CA 02501228 2005-04-05
WO 2004/037251 PCT/GB2003/004590
125
Synthesis of 3-chloro-2-methyl-N-(2-methyl-benzooxazol-6-yl)-
benzenesulfonamide, STX 839 (KRB01009):
w
ci I ~ ,s' w
N O
O H
To a solution of 3-chloro-2-methylbenzenesulphonyl chloride (163 mg, 0.723
mmol) in
dichloromethane (3 mL) was added pyridine (140 pL, 1.72 mmol) and the mixture
was
stirred under N2 for 5 min, after which time 6-amino-2-methylbenzoxazole (102
mg,
0.688 mmol) was added. The resulting mixture was stirred for 1 h at room
temperature,
then saturated NaHC03 solution was added (8 mL) and the mixture was extracted
into
ethyl acetate (15 mL). The organic phase was washed with brine, dried
(Na~S04),
filtered and evaporated to give a residue that was purified using flash
chromatography to
afford a white solid (151 mg, 65%), single spot at Rf 0.50 (60:40 hexane:ethyl
acetate),
mp 127.1-127.5°C, HPLC purity 97% (tR 2.05 min in 10% water-
acetonitrile). 'H NMR
(CDCI3): ~ 7.85 ( 1 H, dd, J=8.1, 1.1 Hz), 7.53 ( 1 H, dd, J=8.1, 1.3 Hz),
7.45 ( 1 H, d, J=8.4
Hz), 7.27 (1 H, d, J=2.2 Hz), 7.17 (1 H, f, J=7.9 Hz), 6.93 (1 H, s, N-H),
6.86 (1 H, dd,
J=8.4, 2.2 Hz), 2.71 (3H, s), 2.58 (3H, s). LCMS: 335.14 (M-). FAB-MS (MH+,
~'15H13C'IN2~3S): calcd 337.0413, found 337.0406.
Synthesis of N-(2-methyl-benzooxazol-6-yl)-4-propyl-benzenesulfonamide, STX
840 (KRB01010):
O
,S.
O
H
To a solution of 4n-propylbenzenesulphonyl chloride (163 mg, 0.744 mmol) in
dichloromethane (3 mL) was added pyridine (140 pL, 1.72 mmol) and the mixture
was
stirred under NZ for 5 min, after which time 6-amino-2-methylbenzoxazole (105
mg,
0.709 mmol) was added. The resulting mixture was stirred for 1 h at room
temperature,
then saturated NaHC03 solution was added (8 mL) and the mixture was extracted
into
ethyl acetate (15 mL). The organic phase was washed with brine, dried
(Na2S04),
filtered and evaporated to give a residue that was purified using flash
chromatography to
afford a pale pink solid (164 mg, 70%), single spot at Rf 0.49 (60:40
hexane:ethyl
CA 02501228 2005-04-05
WO 2004/037251 PCT/GB2003/004590
126
acetate). mp 101.7-102.3°C, HPLC purity 99% (tR 2.02 min in 10% water-
acetonitrile).
'H NMR (CDCI3): S 7.61 (2H, m), 7.43 (1H, d, J=8.4 Hz), 7.37 (1H, d, J=1.8
Hz), 7.19
(2H, m), 6.83 (2H, m), 2.57 (5H, m), 1.58 (2H, sextet, J=7.3 Hz), 0.88 (3H, t,
J=7.3 Hz).
LCMS: 329.21 (M-). FAB-MS (MH+, C~,H,gN2O3S): calcd 331.1116, found 331.1107.
Synthesis of 2,5-dichloro-(2-methyl-benzooxazol-6-yl)-benzenesulfonamide, STX
841 (KRB01011):
CI
N
,S.N W O
CI 0 H
To a solution of 2,5-dichlorobenzenesulphonyl chloride (174 mg, 0.709 mmol) in
dichloromethane (3 mL) was added pyridine (140 pL, 1.72 mmol) and the mixture
was
stirred under N2 for 5 min, after which time 6-amino-2-methylbenzoxazole (100
mg,
0.675 mmol) was added. The resulting mixture was stirred for 1 h at room
temperature,
then saturated NaHC03 solution was added (8 mL) and the mixture was extracted
into
ethyl acetate (15 mL). The organic phase was washed with brine, dried
(Na2S04),
filtered and evaporated to give a residue that was purified using flash
chromatography to
afford a white solid (154 mg, 64%), single spot at Rf 0.50 (60:40 hexane:ethyl
acetate).
mp 167.0-167.3°C, HPLC purity 97% (tR 1.97 min in 10% water-
acetonitrile). 'H NMR
(CDCI3): 6 7.93 (1 H, d, J=2.3 Hz), 7.47 (1 H, d, J=8.6 Hz), 7.46-7.40 (4H,
m), 7.00 (1 H,
dd, J=8.6, 2.0 Hz), 2.61 (3H, s). LCMS: 355.07 (M-). FAB-MS (MH+,
C,4H10CI2NZO3S):
calcd 356.9867, found 356.9875.
Synthesis of 3-chloro-2-methyl-N (2-methyl-benzooxazol-5-yl)-
benzenesulfonamide, STX 842 (KRB01014):
O
f ,o
CI ~r OS N w
H
To a solution of 3-chloro-2-methylbenzenesulphonyl chloride (96 mg, 0.43 mmol)
in
dichloromethane (2 mL) was added pyridine (80 pL, 1.0 mmol) and the mixture
was
stirred under Nz for 5 min, after which time 5-amino-2-methylbenzoxazole (60
mg, 0.40
CA 02501228 2005-04-05
WO 2004/037251 PCT/GB2003/004590
127
mmol) was added. The resulting mixture was stirred for 1 h at room
temperature, then
saturated NaHC03 solution was added (8 mL) and the mixture was extracted into
ethyl
acetate (15 mL). The organic phase was washed with brine, dried (Na2S04),
filtered and
evaporated to give a residue that was purified using flash chromatography to
afford a
white solid (89 mg, 64%), single spot at Rf 0.52 (1:1 hexane:ethyl acetate).
mp 180.2-
180.5°C, HPLC purity 99% (tR 2.32 min in 10% water-acetonitrile). 'H
NMR (CDCI3): b
7.82 (1 H, dd, J=8.1, 1.1 Hz), 7.52 (1 H, dd, J=7.7, 1.1 Hz), 7.32 (1 H, d,
J=8.4 Hz), 7.25
(1H, d, J~2.9 Hz (overlap with CHCI3)), 7.14 (1H, t, J=8.1 Hz), 6.99 (1H, dd,
J=8.4, 2.2
Hz), 6.67 (1H, s, N-H), 2.71 (3H, s), 2.58 (3H, s). LCMS: 335.01 (M-). FAB-MS
(MH+,
lO C,5H,3CIN2O3S): calcd 337.0413, found 337.0420.
Synthesis of N (2-methyl-benzooxazoi-5-yl)-4-propyl-benzenesulfonamide, STX
843 (KRB01015):
~O
~S, w N;
~ H
To a solution of 4n-propylbenzenesulphonyl chloride (93 mg, 0.43 mmol) in
dichloromethane (2 mL) was added pyridine (80 pL, 1.0 mmol) and the mixture
was
stirred under NZ for 5 min, after which time 5-amino-2-methylbenzoxazole (60
mg, 0.40
mmol) was added. The resulting mixture was stirred for 1 h at room
temperature, then
saturated NaHC03 solution was added (8 mL) and the mixture was extracted into
ethyl
acetate (15 mL). The organic phase was washed with brine, dried (Na~S04),
filtered and
evaporated to give a residue that was purified using flash chromatography to
afford a
pale pink oil (112 mg, 85%), single spot at Rf 0.53 (1:1 hexane:ethyl
acetate). HPLC
purity 99+% (tR 2.38 min in 10% water-acetonitrile) 'H NMR (CDCI3): 5 7.64
(2H, dt,
J=8.1, 1.8 Hz), 7.31 (2H, m), 7.18 (2H, d, J=8.4 Hz), 7.06 (1H, dd, J=8.6, 2.4
Hz), 2.59
(5H, m), 1.58 (2H, sextet, J=7.3 Hz), 0.89 (3H, t, J=7.3 Hz). LCMS: 329.15 (M-
). FAB-
MS (MH+, C"H~gN2O3S): calcd 331.1116, found 331.1118.
Synthesis of 2,5-dichloro-N-(2-methyl-benzooxazol-5-yl)-benzenesulfonamide,
STX
846 (KRB01016):
CA 02501228 2005-04-05
WO 2004/037251 PCT/GB2003/004590
128
CI
O
f
'N N
CI O H
To a solution of 2,5-dichlorobenzenesulphonyl chloride (52 mg, 0.21 mmol) in
dichloromethane (1.5 mL) was added pyridine (40 pL, 0.5 mmol) and the mixture
was
stirred under N2 for 5 min, after which time 5-amino-2-methylbenzoxazole (30
mg, 0.20
mmol) was added. The resulting mixture was stirred for 1 h at room
temperature, then
saturated NaHC03 solution was added (8 mL) and the mixture was extracted into
ethyl
acetate (15 mL). The organic phase was washed with brine, dried (Na2S04),
filtered and
evaporated to give a residue that was purified using flash chromatography to
afford a
pale pink solid (45 mg, 63%), single spot at Rf 0.53 (1:1 hexane:ethyl
acetate). mp
193.5-193.9°C, HPLC purity 98% (tR 2.27 min in 10% water-acetonitrile).
'H NMR
(CDCI3): b 7.86 (1 H, d, J=2.2 Hz), 7.39 (4H, m), 7.12 (1 H, dd, J=8.4, 1.8
Hz), 2.58 (3H,
s). LCMS: 355.07 (M-). FAB-MS (MH+, C~4H10CI2NaO3S): calcd 356.9867, found
356.9878.
All publications mentioned in the above specification are herein incorporated
by
reference. Various modifications and variations of the described methods and
system of
the invention will be apparent to those skilled in the art without departing
from the scope
and spirit of the invention. Although the invention has been described in
connection with
specific preferred embodiments, it should be understood that the invention as
claimed
should not be unduly limited to such specific embodiments. Indeed, various
modifications of the described modes for carrying out the invention which are
obvious to
those skilled in chemistry or related fields are intended to be within the
scope of the
following claims.
CA 02501228 2005-04-05
WO 2004/037251 PCT/GB2003/004590
129
REFERENCES
1. Hammond, GH ( 1990) : Molecular properties of corticosteroid binding
globulin and
sex-steroid binding proteins. Endocr. Rev. 11, 65- 79.
2. Gomez-Sanchez EP,Gomex-Sanchez CE (1997): First there was one, then two
..why
not more 11 (3-Hydroxysteroid Dehydrogenases? Endocrinology vol. 138, 12.
3. Krozowski ZS, Funder JW (1983): Renal mineralocorticosterone receptors and
hippocampal corticosterone binding species have identical intrinsic steroid
specificity .
Proc. Natl. Sci. USA 80: 6056-60
4. Ulick S, Levine LS, Gunczler P, Zanconato G, Rarnirez LC, Rauh W, Rosler A,
Bradlow HL, Mew MI (1979): A syndrome of apparent mineralocorticoid excess
associated with defects in the peripheral metabolism of cortisol. J. Clin.
Endo. And
Metab. 49: 757-64.
5. Edwards CRW, Stewart PM, Burt D, Brett L, Mclntyre MA, Sutanto WS, Kloet
ER,
Monder C (1998): Localisation of 11 (3-HSD-tissue specific protector of the
mineralocorticoid receptor. Lancet 2: 986-989.
6. Moore CCD, Melloh SH, Murai I, Siiteri PK, Miller WL (1993): Structure and
function
of the hepatic form of 11 ~i-HSD in the spuirrel monkey, an animal model of
glucocorticoid resistance. Endocrinology 133: 368-375.
7. Kotelevtsev YV, larnieson PM, Best R, Stewart F, Edwards CRW, Seckl JR,
Mullins II
( 1996): Inactivation of 11 (3-HSD type 1 by gene targeting in mice.
Endocrinology Res.
22: 791-792.
8. Ricketts ML, Verhaeg JM, Bujalska I, Howie AJ, Rainey WE, Stewart PM
(1998):
Irnmunohistochemicallocalisation of type 1 11 ~3-HSD in human tissues. I.
Clin. Endoc.
Metab. 83:1325-35.
9. Stewart PM, Sheppard MC (1992): Novel aspects of horrnone action:
intracellular
ligand supply and its control by a series of tissue specific enzymes.
Molecular and
Cellular Endocrinology 83: C13-C18.
10. Seckl JR, Chapman KE (1997): The 11 ~i-HSD system, a determinant of
glucocorticoid and mineralocorticoid action. Medical and physiological
aspects.
European 1. Biochem. 249: 361-364.
11. Maser E (1998): 11 ~i-HSD responsible for carbonyl reduction of the
tobacco-
specific nitrosoamine in mouse lung microsomes. Cancer Res. 58: 2996-3003.
12. Walker BR, Stewart PM, Shackleton C H L, Padfield PL, Edwards CRW (1993):
Deficient inactivation of cortisol by 11 ~i-HSD in essential hypertension.
Clin. Endocr.
38: 221-227.
CA 02501228 2005-04-05
WO 2004/037251 PCT/GB2003/004590
130
13. Daynes RA, Araneo BA (1998) : Contrasting effects of glucocorticoids on
the
capacity of T -cells to produce the growth factors interleukin-2 and
interleukin-4. Eur. J.
Immunol. 19: 2319-2324.
14. Bradford MM (1976): A rapid and sensitive method for the quantitation of
microgram
quantities of protein utilizing the principle ofprotein-dye binding. Anal.
Biochem. 72:
248-254.
15. Bart, T. et al., (2002), Arylsulfonamidothiazoles as a new class of
potential
antidiabetic drugs. Discovery of potent and selective inhibitors of the 11 (3-
Hydroxysteroid
Dehydrogenase Type 1. J. Med. Chem., 45, 3813-3815.
16. Stewart, P.M. and Mason, J.I., (1995), Cortisol to cortisone:
Glucocorticoid to
mineralocortcoid. Steriods, 60,143-146.
17. Escher, G, et al,, (1995), Furosemide inhibits 11 a-Hydroxysteroid
Dehydrogenase in
vitro and in vivo. Endocrinology, 136, 1759-1765.
18. Hult, M. et. al., (1998), Selective inhibition of human type 1 11 a-
hydroxysteroid
dehydrogenase by synthetic steroids and xenobiotics. FEBS Letters, 441, 25-28.
19. Diederich, S. et al., (2002), In the search for specific inhibitors of
human 11 (3-
hydroxysteroid-dehydrogenase (11 ~i-HSDs): chenodeoxycholic acid selectively
inhibits
11 (3-HSD-1. Eur. J. Endocrinol., 142, 200-207.
20. Takahashi; Okada; Yakugaku Zasshi; 73; 1953; 802, 804; Chem.Abstr.; 1954;
9364.
21. Hunt, Richard el.al. J Chem Soc C, 1966, 344, 184-185.
22. DeGraw, Joseph and Goodman, Leon; J. Med. Chem.; 7; 1964; 213.
23. Fries, et.al.; Justus Liebigs Ann. Chem.; 454; 1927; 204.
