Note: Descriptions are shown in the official language in which they were submitted.
i
! Wo 95a~~1~ 218 6 2 2 4 PCT~TS95103589
1
Backaro and of th- r_nv n ,on
The present invention relates to novel amidino
derivates, pharmaceutical compositions containing these
novel compounds, and to their use in therapy, in
particular their use as nitric oxide synthase inhibitors.
It has been known since the early 1980~s that the
vascular relaxation brought about by acetycholine is
dependent on the presence of the endothelium and this
activity was ascribed to a labile humoral factor termed
endothelium-derived relaxing factor (EDRF). The activity
of nitric oxide (NO) as a vasodilator has been known for
well over 100 years and NO is the active component of
amylnitrite, glyceryltrinitrite and other
nitrovasodilators. The recent identification of EDRF as
NO has coincided with the discovery of a biochemical
pathway by which NO is synthesized-from the amino acid L-
arginine by the enzyme NO synthase.
NO is the endogenous stimulator of the soluble
guanylate cyclase arid is involved in a number of
biological actions in addition to endothelium-dependent
relaxation including cytotoxicity of phagocytic cells and
cell-to-cell communication in the central nervous system
(see Moncada et al. B'o h ms~aT pharmacnlOrn,,. '~R, 1709-
1715 (1989) and Moncada a . ha~a oloasnal Ravipwa~
a ~, 109-142 (1991). It is now thought that excess No
production may be involved in a number of conditions,
particularly conditions which involve systemic
~UBSTITL~TE SHEET (RULE 26~
WO 95125717 2 I 8 6 2 2 4 PCT~595/03589
2
hypertension such as toxic shock and therapy with certain
cytokines. -
The synthesis of No from-L-arginine can be inhibited
by the L-arginin~ analogue, L-N-monomethyl-arginine_(L-
NMMA) and the therapeutic use of L-NMMA for the treatment
of toxic shock and other types- of systemic hypertension
has been proposed-(WO 91/04024 and GB-A-2240041). The
therapeutic use of certain-other NO synthase inhibitors
apart from L-NMMA for the same purpose has also been
proposed in WO 91104024 and in EP-A-0446699.
It has recently become apparent that there- are at-
least two types of NO synthase as follows:
(i) a constitutive, Ca++lcalmodulin dependent
enzyme that releases NO inresponse to receptor-cr
physical stimulation.
(ii) a Ca+f independent-enzyme which is induced
after activation -of vascular smooth muscle, macrophages,
endothelial cells, and a number of other cells by
endotoxin and eytokines. Once expressed this inducible
NO synthase synthesizes NO for long periods.
The N0-released by the constitutive enzyme acts as a
transduction mechanism underlying several physiologicab
responses. The NO produced by the inducible enzyme is a
cytotoxic molecule for tumor cells and. invading
microorganisms. .It also appears that the adver-se effects
of excess N0 production, in particular pathological-
vasodilation and tissue damage, may result largely from
the effects of NO synthesized by the induci7~le NO -
synthase.
Some of the NO synthase inhibitors proposed for .
therapeutic use.so far, and in particular L-NMMA, are
non-selective in that they inhibit both the constitutive
and the inducible NO synthase_ Use of such a non-
BUBST(TUTE SHEET (RUSE Z8~
WO 951257f7 _, . 21 ~ 6 2 2 4 PCT/US95/03589
3
selective NO synthase inhibitor reguires that great care
be taken in order to avoid the potentially serious
consequences of over-inhibition of the constitutive NO-
synthase including hypertension and possible thrombosis
,' 5 and tissue damage. In particular, in the case of the
therapeutic use of L-NMMA for the treatment of toxic
shock it has been recommended-that-the patient must be
subject to continuous blood pressure monitoring
throughout the treatment. Thus, while non-selective NO
synthase inhibitors have therapeutic utility provided
that appropriate precautions are taken, NO synthase
inhibitors which are selective in the sense that they
inhibit the inducible NO synthase to a considerably
greaterextent than the constitutive N0 synthase would be
of even greater therapeutic benefit and easier to use.
WO 93/13055, W093/24126 and LT S. Patent No:
5,132,453 disclose compounds that inhibit nitric oxide
synthesis and preferentially inhibit the inducible
isoform of nitric oxide .synthase.
SUBSTITUTE SHEEP (RW.E ~j
WO 95!25717 2 i 8 b 2 2 4 PCT~S95/03589
4
The present invention provides amidino derivatives of the
formula-(I): -
R2 p
/~ X
HN~N~ R3
H \
H2N R~
(I)
salts, pharmaceutically acceptable esters and prodrugs
thereof, wherein
X is lower alkyl, lower alkenyl, lower alkynyl,
-(CH2)kQ(CH2)1- where k is 2 or 3, 1 is 1 or 2 and Q is
0, Se, SiY2 where Y is lower alkyl, S(0)Z where.a is D, 1
or 2, NR where R is hydrogen or lower alkyl, or
-(CH2)mA(CH2)n- where m is 0, 1 or 2, n is 0, 1 or 2, A
is a 3 to 6 membered aromatic hydrocarbon radical or 3 to
6 membered heterocyclic radical wherein 1 to 3 hetro
atoms are oxygen, sulfur Dr nitrogen and wherein all said
radicals may optionally be substituted by one or more-
substituents such as lower alkyl, lower alkoxy, hydroxy,
halogen, vitro, cyano, trifluoroalkyl and amino;
,
R1 maybe a bond tp x, or a radical selected-from the
group consisting of hydrogen (when Q is Se),
hydroxyalkyl, lower alkyl, lower alkenyl, lower alkynyl,
or haloalkyl and-wherein all said radicals may
optionally be substituted by one or more substituents
such as lower alkyl, lower alkoxy, hydroxy, halogen,
vitro, cyano, trifluoroalkyl and amino;
SUBSTITUTE SHEET ~RU~.E 26)
W O 951257 f7 218 6 2 2 4 PCT~S95/113589
R2 is lower alkyl, lower-alkenyl, lower alkynyl,
aromatic hydrocarbon radical, haloalkyl, hydroxylamine or
heterocyclic radical wherein 1 to 3 hetro atoms are
oxygen, sulfur-or nitrogen and wherein all said radicals
S may optionally be substituted by one or more
substituents such as lower alkyl, lower alkoxy, hydroxy,
halogen, vitro, cyano, trifluoroalkyl and amino; and
R3 is amino, alkylamine, amino acid, hydroxy, lower
alkoxy, tetrazole, or tetrazoloamine.
The invention further relates to. pharmaceutical
compositions comprising a compound of formula (I).
Compounds and compositions defined_above have usefulness
as inhibitors of nitric oxide synthase. These compounds
also prefentially inhibit the inducible farm over the
constitutive form of nitric oxide synthase and in the
most prefered form inhibit the inducible form over the
constitutive form of nitric-oxide synthase by at least 3
fold.
Conditions in which there is an advantage in
inhibiting NO production from L-arginine include systemic
hypotension associated with septic and/or toxic shock
induced by a wide variety of agents; therapy with
cytokines such as TNF, IL-1 and IL-2; and as an adjuvant
to short term immunosuppression in transplant therapy.
Thereis also a growing body of evidence that NO may
be involved in the degeneration of cartilage which takes
place in certain conditions such as arthritis and it -is
~ also known that NO synthesis is increased in rheumatoid
arthritis. Accordingly, further conditions in which there
is an advantage in inhibiting NO production from L-
arginine include autoimmune and/or inflammatory
conditions affecting the joints, for example arthritis.
SUBSTITi UIrE SHEET (RU~.E 28j
wo 9sns~ W 218 6 2 2 4 P~~S9s103S89
6
Still.further conditions in which there is an
advantage in inhibiting NO production-from L-arginine
include inflammatory bowel disease, cardiovascular -.
ischemia, diabetes, cerebral ischemia and other=CNS
disorders mediated by NO. ,
A preferred-embodiment of-the present invention is a
compound of-the formula (I):
Rz O
/~ X
HN~N~ R3
H \
HzN Ri
.. _ -(I)
and salts, and pharmaceutically acceptable ester and
prodrugs thereof, wherein:
X is lower alkyl, lower alkenyl, lower alkynyl,
-(CH2)kQ(CH2)1- where k is 2 or 3, 1 is-1 or 2 and Q is
0, Se, SiY2 where Y is lower alkyl, S(O)Z where--z is-0, 1
or 2, NR where A is hydrogen or lower alkyl, or -
-(CH2)mA(CH2)n- where m is 0, 1 or-2, n is 0, 1 or 2, A
is a 3 to 6 membered aromatic hydrocarhon radicalor
heterocyclic radical wherein-1-to 3 hetro atoms are
oxygen, sulfur or-nitrogen andwherein all said-radicals
may optionally be substituted by one or more _
substituents such as lower alkyl, lower alkoxy,-=hydroxy, ,
halogen,- nitro, cyano, trifluoroalkyl and amino;
R1-maybe a bond to X, or -a radical selected from the
group consisting of hydrogen (when Q is Se),
hydroxyalkyl, lower alkyl or haloalkyl;
~U$STITUTE SHEET (RW.E 26)
WO 95125717 - , . ~ ~ g ~ 2 2 ~ PCTlUS95/03589
7
R2 is lower alkyl, lower alkenyl, lower alkynyl,
aromatic hydrocarbon radical , haloalkyl, or
hydroxylamine; and
R3 is amino, alkylamine, natural amino acid,
hydroxy, lower alkoxy, tetrazole, or tetrazoloamine.
Another preferred embodiment of the present
invention is a compound of the formula (I)
R2 O
/~ X
HN~N~ R3
H \
H2N Ri
(I)
and salts, and pharmaceutically acceptable ester and
prodrugs thereof, wherein:
X is a lower alkyl of 1 to about 6carbon atoms, lower
alkenyl of 1 to about 6 carbon atoms, lower alkynyl of 1
toabout 6carbon atoms, -(CH2)kQ(CH2)1- where k is 2 or
3, 1 is 1 or 2 and Q is 0, Se, SiY2 whereY is lower
alkyl, S(O)z where z is 0, 1 or 2, or NR where R is H or
lower alkyl; or -(CH2)mA(CH2)n- where m is 0, 1 or 2, n
is 0, 1 or 2, A is a 3 to 6 membered aromatic hydrocarbon
radical or heterocyclic radical wherein 1 to 3 hetero
atoms are oxygen, sulfur or nitrogen and wherein all said
radicals may optionally be substituted by one or more
substituents such as lower alkyl, lower alkoxy, hydroxy,
halogen, nitro, cyano, trifluoroalkyl and amino;
R1 is hydrogen (when Q is Se), a hydroxyalkyl group
of 1 to about 4 carbon atoms, a lower alkyl group of 1 to
suBS~nr~ sHE~r c~u~.E ~s~
WO 95125717 , ~ ~ 4 PCTlU595103589
8
about 4 carbon atoms or a haloalkyl group of 1 to about 4
carbon atoms;
A2 is lower alkyl of,:1 to about 4- carbon atoms, -
lower alkenyl of 1 to about 4 carbon atoms alkynyl of-1 ,
to about 4 carbon atoms, aromatic hydrocarbon radical of
3 to about 6 carbon atoms and haloalkyl of 1 to about -4-
carbon atoms; and
R3 is an amino, alkylamine of 1 to about 9 carbon
atoms, hydroxy, lower alkoxy of 1 to about 4 carbon
atoms, tetrazole, tetrazoloamine, or natural amino acid.
Still another preferred-embodiment-of formula (I) is
X is an alkylerie group-having 3 to 5 carbon atoms and
which may optionally be substituted by one or more C1-3
alkyl; a group of formula -(CH2)kQ(CH2)1- where:k is 2-or
3, 1 is 1 or 2 and Q is O, Se, S(0)z-where z is 0, 1 or
2; or a group of formula -(CH2)mA(CH2)n- where m is 0, 1
or 2, n us 0;-1 or 2, A is-a-3-to 6 membered carbocyclic
or heterocyclic ring;
Rl is hydrogen (when Q is Se), a hydroxyalkyl grog
of-1 to.about 4 carbon atoms, a lower alkyl radical of-1
to about 4 carbon atoms; -
R2 is lower alkyl radical. of l.to about 4 carbon
atoms;
R3 is an amino, alkylamine of 1 to about 4, carbon
atoms, hydroxy, lower-alkoxy group of 1 to about 4 carbon -
atoms.
The Fresent invention includes compounds of formula
(I) in the form of--salts, in-particular acid addition -
salts. Suitable--salts include those formed with both
organic and inorganic acids. Such acid addition salts.
~UgSTITU'~E SHEET (RULE ~)
W 0 95125717 PCT/UB95~03589
9
will normally be pharmaceutically acceptable although
salts of non-phaxinaceutically acceptable salts may be of
utility in the preparation and purification of the
compound in question. Thus, preferred salts include
those formed from hydrochloric, hydrobromic, sulphuric,
citric, tartaric, phosphoric, lactic, pyruvic, acetic,
succinic, oxalic, fumaric, malefic, oxaloacetic,
methanesulphonic, ethanesulphonic, p-toluenesulphonic,
benzenesulphonic and isethionic acids. Salts of the
compounds of formula (I) can be made by reacting the
appropriate compound in the form of the free base with
the appropriate acid.
While it may be possible for the compounds of
15-- formula (I) to be administered as the raw chemical, it is
pref-_erable to present them as a pharmaceutical
formulation. According to a further aspect, the present
invention provides a pharmaceutical formulation
comprising a compound of formula (I) or a
pharmaceutically acceptable salt orsolvate thereof,
together with one or more pharmaceutically acceptable
carriers thereof and optionally one or more other
therapeutic ingredients. The carriers) must be
"acceptable" in the sense of being compatible with the
other ingredients of the formulation and not deleterious
to the recipient thereof.-
The formulations include those suitable for aral,
parenteral (including subcutaneous, intradermal,
intramuscular, intravenous and iiitraarticular), rectal
and topical (including dermal, buccal, sublingual and
- intraocular) administration although the most suitable
route may depend upon for example the condition and
disorder of-the recipient. The formulations may
conveniently be presented in unit dosage form and may be
prepared by any of the methods well known in the art of
pharmacy. All methods include the step of bringing into -
SUBSTITUTE SHEET' (RULE 28)
W0 95125717 PCTIUS95103589
2186224
association a compound of formula (I) or a
pharmaceutically acceptable salt or solvate thereof -. '.
("active ingredient") with the carrier-which constitutes
one or more accessory ingredients. In general,the
5 formulations are prepared by uniformly and,intimately .
bringing into association the active ingredient with
liquid carriers. or finely divided solid carriers or both
and then, if necessary, shaping the product into the
desired-foz'mulation:
Formulations of the present invention-suitable for-
oral administration may be presented as discrete units
such as capsules, cachets or -tablets--each containing a-:
predetermined amount of the active ingredient; as a
powder or granules; as a solution or a suspension in an-
aqueousliquid o-r a non-aqueous liquid; or-~s ari oil-in-
water liquid emulsion or a water-in-oil liquid emulsion:
The active ingredient may also-be presented as a bolus~_
electuary or paste.
A tablet may be made by comp-ression or-moulding, -
optionally with one or more accessory ingredients.
Compressed- tablets may be prepared by compressing- in a
suitable machine the active ingredient in a free-flowing
form such as a powder,Dr-granules, optionally mixed with
a binder, lubricant, inert diluent, lubricating, surface
active or dispersing agent. Moulded tablets may be made
by moulding in a suitable machine a mixture of the
powdered compound moistened with an inert liquid diluent.
The tablets may optionally be coated or scored and may-be
formulated so as-~o provide slow or controlled release of
the active ingredient therein.
Formulations for parenter-al administration-include
aqueous and non-aqueous sterile injection solutions which
may contain antioxidants, buffers, bacteriostats and
solutes which render the formulation isotonic with the
SUBSTITUTE SHEET (RULE 26~
W O 95/2571'7 218 6 2 2 4 P~~S95/03589
11
blood of the intended recipient; and aqueous and non-
aqueous sterile suspensions which may include suspending
~ agents and thickening agents.., The formulations may be
presented in unit-dose or multi-dose containers, for
_ 5 example sealed ampoules and vials, and may be stored in a
freeze-dried (lyophilized) condition requiring only the
addition of the sterile liquid carrier, for example,
saline, water-for-injection, immediately prior to use.
Extemporaneous injection so-lutions and suspensions may be
prepared from sterile powders, granules and tablets of
the kind previously described.
Formulations for rectal administration may be
presented as a suppository with the usual carriers such
as cocoa butter or polyethylene glycol.
Formulations for topical administration in the
mouth, for example buccally or sublingually, include
lozenges comprising the active ingredient in a flavored
basis such as sucrose and acacia or tragacanth, and
pastilles comprising the active ingredient in a basis
such as gelatin and glycerin or sucrose and acacia.
Preferred unit dosage formulations are-those
containing an effective dose, as hereinbelow recited, or
an appropriate fraction thereof, of the active
ingredient.
It should be understood that in addition to the
ingredients particularly mentioned above, the
formulations of this invention may include other agents
conventional in the art having regard to the type of
formulation in question, for example those suitable for
oral administration may include flavoring agents.
The compounds of the invention may be administered
' orally or via injection at a dose of from 0.1 to 500
SUBSTITUTE SHEET (RULE 26)
WO 95/25717 218 6 2 2 4 PCT~S95103589
12
mg/kg per day. The dose xange for-adult humans'is -
generally from Smg to 2g/day. :Tablets or other-.forms of
presentation provided..in_discrete units may conveniently
contain an amount of compound of -the invention which is-
effective at su-ch-dosage or as-a multiple of the same,--.
for instance, units containing Smg to 500mg, usually
around lOmg to 200mg.
The compounds of formula -(I) are preferably
administered orally or by injection (intravenous or
subcutaneous). The precise-amount of compound
administered to a patient will-be the responsibility of
the attendant physician. However, the dose employed will
depend on a number of -factors, including the age and sex
of-the patient, the precise disorder being treated, and
its severity. Also, the route of administration may vary
depending on the--condition and its severity.
As utilized herein, the t-erm °lower alkyl", alone or
in combination, means an acyclic alkyl radical containing
from 1 to about-10, preferably from 1 to about 8 carbon
atoms and more-preferably 1-to about 6 carbon atoms.
Examples of such-radicals'incl-ude methyl, ethyl, n-
propyl, isopropyl, n-butyl, isobutyl, sec-butyl,tert-
butyl, pentyl, isn-amyl, hexyl, octyl and the like.
The term °lower alkenyl~~ refers to an unsaturated
acyclic-hydrocarbon radical in so much as it contains at
least one double bond. Such radicals containing from -
about 2 to about10-carbon atoms, preferably from about Z
to about 8 carbon atoms and morE preferably 2 to about 6
carbon atoms. Examples of-suitable alkenyl radicals -
include-propylenyl, buten-1-yl, isobutenyl, pentenylen-1-
yl, 2-2-methylbuten-1-yl, 3-methylbuten-1-yl, hexen-1-yl,
hepten-1-yl, and octen-1-yl, and the like.
SUBSTITUTE SHEET ~RW.E ~)
WO 95125717 ~ PCT/US95/03589
13
The term "lower alkynyl" refers to an unsaturated
acyclic hydrocarbon radicals in so much as it contains
one or more triple bonds, such radicals containing about
2 to about 10 carbon atoms, preferably having from about
2 to about 8 carbon atoms and more preferably having 2 to
about 6 carbon atoms. Examples of suitable alkynyl
radicals include ethynyl, propynyl, butyn-1-yl, butyn-2-
yl, pentyn-1-yl, pentyn-2-yl, 3-methylbutyn-1-yl, hexyn-
1-yl, hexyn-2-yl, hexyn-3-yl, 3,3-dimethylbutyn-1-yl
radicals and the like,
The term "aromatic hydrocarbon radical" means 4 to
about 16 carbon atoms, preferably 6 to about.l2 carbon
atoms, more-preferably 6 to about 10 carbon atoms.
Examples of suitable aromatic hydrocarbon radicals
include phenyl,naphthyl, and the like.
The term °heterocyclyl radical-° means a saturated or
unsaturated cyclic hydrocarbon radical with 4 to about 10
carbon atoms, preferably about 5 to about 6; wherein 1 to
about 3 carbon atoms are replaced by nitrogen, oxygen or
sulfur. The "heterocyclic radical" may be fused to an
aromatic hydrocarbon radical. Suitable examples include
pyrrolyl, pyridinyl, pyrazolyl, triazolyl,
pyrimidinyl, pyridazinyl, oxazolyl, thiazolyl,
imidazolyl, indolyl, thiophenyl, furanyl, tetrazolyl, 2-
pyrrolinyl, 3-pyrrolinyl, pyrrolindinyl, 1,3-dioxolanyl,
2-imidazonlinyl, imidazolidinyl, 2-pyrazolinyl,
pyrazolidinyl, isoxazolyl, isothiazolyl, 1,2,3-
oxadiazolyl, 1,2,3-triazolyl, 1,3,4-thiadiazolyl, 2H
pyranyl, 4H-pyranyl, piperidinyl, 1,4-dioxanyl,
- morpholinyl, 1,4-dithianyl, thiomorpholinyl, pyrazinyl,
piperazinyl, 1,3,5-triazinyl, 1,3,5-trithianyl,
benzo(b)thiophenyl, benzimidazolyl, quinolinyl, and the
like.
~UBSTITUl'E SHEET (RULE ~
WO 95/25717 2 ~ PCTIUS95I03589
14
The term lower alkoxy", alone or in combination,
means an alkyl ether radical wherein the term alkyl is as
defined above and most preferably containing 1 to about 4
carbon atoms. Examples-of suitable alkyl ether radicals
include methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy,
iso-butoxy, sec-butoxy, tert-butoxy and the like.
The term "halogen" means fluorine, chlorine; bromine
or iodine.
The term amino acid-means an amino-alkanoic wherein
the amino can be postioned anywhere on the alkyl group.-
The alkyl is as is defined above.
The term "prodrug" refers to a compound that is made
more -active in vivo.
As used herein, reference to °treatment" af-a
patient is intended to include prophylaxis.
All references, patents or applications, U.S. or
foreign, cited in the-application are hereby incorporated
by reference as if written herein.
Disclosed-i.s a general synthetic processes useful in
the preparation of the compounds of the present
invention.
SUBSTITUTE SHEEP (RU~.E 26)
WO 95125717 218 6 2 ~ 4 PCT~S95/03589
sc~sme x
8aN' ~ ~ ' COaC83 O
Cl
H
MgS04
O
C1 ~ ~ ~N
OCH3
N
C1
1. NaN(Si(CH313y
2 . CHy I
3. HCl
HyN CH;
~ \J' 'ox
BaN~~/\~ ~~
O
1. C1iC03
2. ethyl acetimidate
~'Ia HaN C8;
~~J OH
H3C' N
H
O
SUBS~'ITUTE SHEET (RU~.E 25j
W 0 95/25717 PCTIUS95l03589
16 -.
SCHEME dI
° ~s NaBH4 ~x
,s. ox ~~ aso~oa
xo~so v ~~
Na= o 0
HZN-CHZ-CHz-Br
~1
Sa~ OH
HyN v ICI
O
I. L~C03
2. ethyl acetunidate
NH NHy
~ se~ oa
x3c v ~N
a
0
RUBSTITUTE SHEET (RU~.E 26~
218 6 2 2 4 PCT/US95/03589
17
SCHEr~ III
- C (CxHs)zNSi(CH3)y o
82N' x ~ LSi(CH3)x7xN
'~ ~OH ~)2SQ! OSi(CHj)3
- R1 RS
mo'pfCF NaNISi(CII;)31x
~ ~ Ri ~a l.Boc-NH(CT-I~aBr C
H N~~~ ~fSiICH3)x7xN
1 OSi(CHj)j
O 2. 4N HCl/dioxane g1 ~
1. CuC03
2 ethyl aceiimidate
N'IH R'i /NHx
1 ~ J~ -OH
Bj~N~~
H 'I0
~UBST(TUTE SHEET ~RU~.E 28~
WO 95125717 ~ ~ PCTIUS95103589
18
SCHEMEIV
O NHS _ p
OH
O N
~H O d
NaOH
JQ~ HN v
O" N~~
H O
AcaO, 90-100 eC
l \ ~ \
0 339' HCHO
0
0
pyridine p
Q n
O
SN HCl
~H I. CuCO; NHz H, CHaOH
C~-~ ~ ~~ N ~ OH
HiN
O 2. ethyl acetimidate H
O
SUBSTITUTE SHEET (RULE 26~
WO 95!2571T 218 6 2 2 4 PCT~S95/03589
19
SCH>;ME V
[Si(CH~]zN
. O (C2H5)2NSi(CH3)3 OSi(CH3)3
HZN
~ (NH4)2SOa
N[Si(CH~3]z
NHz
NaN[Si(CH3)312
R~ NHz RtX, THF, 40 ~C
HzN~~OH ~~
I' OSi(CH3)a
O
1. CuC03
N[Si(CH~3]z
2. ethyl acetimidate
NH Ri NHz
~ OH
H3C' - N
H
O
SUgST(TUTE SHEET (RUtE 28)
CA 02186224 2004-05-26
The invention is further illustrated Y7y the following
examples:
EXAMPLE 1
5
a-Methyl-NS-iminoethyl-ornithine
CH3 O
HN N ~ OOH
H \
NiiZ ~CH~s
10 a-Methyl-D,L-ornithine hydrochloride (250 mg; 1.37 mmoles
purchased from Sigma) was dissolved in 5 ml of water and
0.5 g of cupric carbonate was added. The mixture was
stirred at 60 OC for 1 hr and filtered. The pH of the
filtrate was adjusted to pH 9.5 with 0.5 N NaOH solution.
15 Ethyl acetimidate hydrochloride (178 mg; 1.45 mmoles) was
added and the pH was maintained at 9.0-9.5 for one hour.
The solution was then adjusted to a pH of 7.5 and kept at
room temperature for 12 hours. The solution was
acidified with 1 N HC1 to pH 4 and applied to a DowexTM 50
20 X 4 (hydrogen form ). The column was washed with water and
then with 10 ~ pyridine. a-Methyl-N8-iminoethyl-
ornithine was eluted from the column with 1 N NH~OH. The
ninhydrin positive fractions were combined and
lyophilized. The residue was dissolved in 0.5 N of
aqueous hydrochloric acid and re-lyophilized. a-Methyl-
N8-iminoethyl-ornithine hydrochloride appears as white
solids. (MH+ = 188); 1H-NMR LD20): d 1.4 (s, 3H); 1.45-
1.9 (m. 4 H) ; 2.15 (s, 3 H) ; 3 .3 (t, 2 H1
WO 951257f7 2 i 8 6 2 2 4 P~~S95/03589
21
EXAMPLE 2
a-Methyl-NS-iminoethyl-lysine
CH3 NH2 CH3
~ OH
HN' 'N
H I
O
A suspension of lysine ethyl ester dihydrochloride (33 g;
0.14 mole) and MgS04 (34 g; 0.28 moles) in a solution of
4-chloro-benzaldehyde (39 g; 0.28 moles) and acetonitrile
1500 m1) was stirred while N,N-diisopropylethylamine (36
g; 0.28 moles) was added in portions over 1/2 h. The
mixture was stirred for 12 h, filtered, concentrated to a
sma7.l volume, and diluted with 500 ml of ethyl ether. The .
ether solution was washed with 0.1$ aqueous NaHC03,
aqueous 2 N NaOH containing 2g/100 m1 of NH20H.HC1, again
with 0.1$ aqueous NaHC03 and saturated aqueous NaCl.
After drying with MgS04 and removal of the solvent in
vacuo, ethyl N, N'-di(4-chloro-phenylmethylene)-L-alanine
was obtained as a clear liquid. The liquid was
triturated with hexanes and the resulting solid was
washed with hexanes for several times. This partially
purified intermediate was dissolved in 200 ml of THF and
stirred in an acetone/dry ice bath. Sodium bis-
(trimethylsilyl)amide in THF (11 ml, 1 M solution) was
added dropwisely over 30 min. After one hour, methyl
iodide (0.8-g; 13 mmoles) in THF was added dropwisely.
The reaction mixture was slowly warmed up to room
temperature and stirred overnight. The mixture was
diluted with water, and extracted with ethyl ether. The
ether extract was iaashed with 0.1~ aqueous NaHC03 and
saturated aqueous NaCl and concentrated to yield crude
ethyl N.N'-di(4-chloro-phenylmethylene)-a-methyl-D,L-
alanine (M + H= 434). This material (4 g) dissolved in
SU~ST(TUTE SHEET (RU~.E 2~)
I 1
CA 02186224 2004-05-26
22
ethyl .ether (100 ml) was stirred vigorously with 1 N HC1
(50 ml) fo.r 2 h, the layer :.;as separated and the aqueous
phase was washed with ethyl ether. The aqueous solution
was further acidified by the addition of concentrated HC1.
to 6 N and was =Bated to ref hax for 15 h. The solut_on
was cooled to room temperature, and rotary evaporated to
dryness. The residue was dissolved in water and applied
to a DowexTM 50 x 4 (hydrogen form). The column was washed
with water, and then 10% pyridine. a-Methyl-D,L-lysine.
~0 !MH+ H = 161) :aas eluted from the column with 1 M NH40H.
a-Methyl-D,L-lysine 1300 mg) was dissolved in water and
0.5 g of cupric carbonate was added. The suspension was
stirred at 600 C for 1 h and filtered. The filtrate was
adjusted to pH °.5 and ethyl acetimidate was added
portionwise in 15 min. The pH was maintained at °.0 to
9.5. After 1 h cf stirring at room temperature, the
solution was adjusted to pH 7 and the stirring was
continued over~ight. a-Methyl-NE-iminoethyl-lysine caas
purl f led by DowexTM 50 x 4 'hydrogen f orm ) chromatography
similarly described for a-Methyl-N8-iminoethyl-ornithine.
MH+ = 202; 1T_-i-rrMR (D20) : d 1.4 (s, 3H) ; 1.5-1:8 (m, o H) ;
2.05 (s, 3 H) : ~ .1 (t, 2 H) .
EXAMPLE 3
gist-iminoethyl-aminoethylselenocysteine
CH3 NH2 H
Se OH
HN N
H
O
Seleno-D,L-cystine (117 mg; 0.5 mmoles, purchased from
Sigma) was suspended in 15 ml of nitrogen gas-purged
water. Sodium borohydride (38 mg; 1 mole) was added. The
reaction mixture became clear in a few minutes. After 2 h
W0 95f25717 ' PCT/US95/03589
23
at room temperature, 2-bromoethylamine HC1 (1.2 g; 6
mmoles) was added and the reaction mixture was stirred
~ for 12 h. The reaction was applied on to a Dowex 50 X 4
(hydrogen form)- column. The column was washed with water
. ' S and 10~ pyridine. 2-Aminoethyl-D,L-seleno-cysteine was
eluted with 1 M NH40H and, subsequently, treated with
cupric carbonate and ethyl acetimidate as described for
N8-iminoethyl-ornithine. NE-iminoethyl-
aminoethylselenocysteine-was obtained as pale yellow
solids. MH+ = 254.
EXAMPLE 4
cx-Hydroxymethyl-Ne-iminoethyl-lysine
NH2 CHpOH
OH
HN N
H
s
O
To an ice-cold stirred mixture of Ne-Cbz-L-lysine (14 g;
0.05 moles, purchased from Sigma) in 2.5 N NaOH (24 ml),
benzoyl chloride (10 g) was added gradually. The pH of
the solution was maintained at.10.5-10.9 by addition of 2
N Na.OH. The mixture was stirred at room temperature for 1
h anal filtered. The filtrate was extracted with a small
amount of ethyl acetate and the organic layer was dried
over sodium sulfate. The solid was removed by filtration
and the filtrate was evaporated to dryness. The remaining
crude oily Ns-Cbz-Na-benzoyl-lysine (6 g) was heated at
90-1000 C with acetic anhydride (100 ml) for 30 min. The
mixture was then evaporated. The residue was dissolved in
pyridine and treated with aqueous formaldehyde (35~
solution, Fisher). The mixture was stirred for 8 hr, then
diluted. The reaction mixture was kept at 100C overnight
the precipitated crude material was hydrolyzed in boiling
SUBSTITUTE SHEEN' (RU~.E
CA 02186224 2004-05-26
24
N HC1 during 5 h. The reaction mixture was cooled and
filtered before being evaporated. The solid residue was
dissolved in water and passed through DowexTM 50 x 4
(hydrogen form) column. a-Hydroxymethyl-D,L-lysine ~MFi+
5 _ 1771 was eluted with 1 N NH40H and, subsec_ruently,
treated with cupric carbonate and ethyl acetimidate to
form a-hydroxymethyl-NE-iminoethyl-lysine similarly
described for a-Methyl-N8-iminoethyl-ornithine. MH+ _
218. 1H-NMR (D20): d 1.1-1.8 (m, 6H1; 2.1 (s. 3H); 3:1
(t, 2H); 3.6-3.9 (a(a,b), 2H).
The activity of the above listed compounds as NO
synthase inhibitors have been determined in the following
assays:
~~rr"lline-Assav fob Nitric Oxide Svnthase
Nitric oxide synthase activity was measured by monitoring
the conversion of [3H]-arginine to (3H)-citrulline. Mouse
inducible nitric oxide synthase (miNOS) was prepared from
an extract of LPS-treated RAw 264.7 cells and partially
purified by DEAF-SepharoseTM chromatography. Rat brain
constitutive nitric oxide synthase lrnNOS) was prepared
from an extract of rat cerebellum and partially purified
by DEAF-SepharoseTM chromatography. Enzyme and inhibitors
were incubated at 37°C for 15 minutes in a reaction
volume of 100 ~L with the following components added to
start the reaction: 50 mM Tris (pH ~.6), 1 mg/ml bovine
s erum a lbumi n , 1 mM DTT , ~ 2 mM C aC 12 , 10 ~.tM FAD , 10 ~t.M
30. tetrahydrobiopterin, 30 ~1M L-arginine containing L-[2,3-
3H~-arginine at 300 cpm/pmole and 1 mM NADPH. For
constitutive NOS, 50 nM calmodulin was also added. The
reaction was terminated by addition of cold stop buffer
containing 10 mM EGTA, 100 mM HEPES, pH 5.5 and 1 mM
citrulline. [3H]-Cit~rulline was separated by
chromatography on DowexTM 50W X-8 ration exchange resin and
W095I25717 ~ ~ PCT/US95/03589
radioactivity determined with a liquid scintillation
counter.
Recombinant human inducible nitric oxide synthase (h1N05)
5 was extracted from Sf9 insect cells (infected with
recombinant baculoviruses encoding a cDNA for human
inducible nitric oxide synthase which has been isolated
and purified from a lambda Zapll cDNA library made from
RNA isolated from a colon sample from a patient with
10 ulcerative colitis) and partially purified by DEAE-
chromatography.
15 RAw 264.7 cells are plated to confluency on a 96-well
tissue culture plate grown overnight (17h) in the
presence of LPS to induce NOS. A row of 3-6 wells were
left untreated and served as controls for subtraction of
nonspecific background. The media was removed from each
20 well and the cells are washed twice with Krebs-Ringers-
Hepes (25mM, pH 7.4) with 2 mg/ml glucose. The cells are
then placed on ice and incubated with 50 ~L of buffer
containing L-arginine (30 ELM) +/- inhibitors for 1h. The
assay is initiated by warming the plate tc 37°C in a
25 water bath for 1h. Production of nitrite by intracellular -
iNOS is linear with time. To terminate the cellular
assay, the plate of cells is placed on ice and the
nitrite-containing buffer removed and analyzed for
nitrite using a previously published fluorescent
determination for-nitrite.-All values are the average of
triplicate wells and are compared to a background-
subtracted induced set of cells (100 value).
LPS is the abbreviation ~or lipopolysaccharide.
~UBSTI'rU'CE SHEET (RU~.E Z~j
WO 95/25717 , ~ ~ ~ ~ PCT/US95103589
26
TABLE I -
CompoundhiNOS miNOS rnNOS Raw -
Cell _ .. :: __ .._ _._. . . _._ _. . _ __. . _.__ . __. . _
ICSp L~l IC50
LELMI
Example 1 73.5 39.9-- 355.9
25.0
_
Example 2 26.6 7.2 192.8
52.5
Example.3 35.5 5.0 - 229.0
0.37
Example 4 39~ Inh. 8.6~ Inh.
@ 10 ELM @ 10 ELM
From the foregoing description, one skilled in the
art can easily ascertain the essential characteristics of
this invention, and without departing from the spirit and
scope thereof, can make various changes and modifications
of the invention to adapt it to- various usages and
conditions.
sugs~rru~ sHF~r (RUB
