Note: Descriptions are shown in the official language in which they were submitted.
CA 02327188 2000-10-03
WO 99/51241 PCT/US99/07760
CALCILYTIC COMPOUNDS AND METHOD OF USE
The present invention relates to novel calcilytic compounds,
pharmaceutical compositions containing these compounds and their use as
calcium
receptor antagonists.
In mammals, extracellular Ca2+ is under rigid homeostatic control and
regulates various processes such as blood clotting, nerve and muscle
excitability,
and proper bone formation. Extracellular Ca2+ inhibits the secretion of
parathyroid
hormone ("PTH"} from parathyroid cells, inhibits bone resorption by
osteoclasts,
and stimulates secretion of calcitonin from C-cells. Calcium receptor proteins
enable certain specialized cells to respond to changes in extracellular Ca2+
concentration.
PTH is the principal endocrine factor regulating Ca2+ homeostasis in the
blood and extracellular fluids. P'TH, by acting on bone and kidney cells,
increases
the level of Ca2+ in the blood. This increase in extracellular Ca2+ then acts
as a
negative feedback signal, depressing PTH secretion. The reciprocal
relationship
between extracellular Ca2+ and P'TH secretion forms an important mechanism
maintaining bodily Ca2+ homeostasis.
~~c~llular Ca2+ acts directly on parathyroid cells to regulate P'Ix
secretion. The existence of a parathyroid cell surface protein which detects
changes in extracellular Ca2+ has been confirmed. See Brown et al., Nature
366:574, 1993. In parathyroid cells, this protein, the calcium receptor, acts
as a
receptor for extracellular Ca2+, detects changes in the ion concentration of
extracellular Ca2+ , and initiates a functional cellular response, PTH
secretion.
Extracellular Ca2+ influences various cell functions, reviewed in Nemeth
et al., Cell Calcium 11:319, 1990. For example, extracellular Ca2+ plays a
role in
parafollicuiar (C-cells) and parathyroid cells. See Nemeth, Cell Calcium
11:323,
1990. The role of extracellular Ca2+ on bone osteoclasts has also been
studied. See
7~di, Bioscienee Reports 10:493, 1990.
Various compounds are known to mimic the effects of extra-cellular Ca2+
on a calcium receptor molecule. Calcilytics are compounds able to inhibit
calcium
receptor activity, thereby causing a decrease in one or more calcium receptor
1
CA 02327188 2000-10-03
WO 99/51241 PCT/US99/07760
activities evoked by extracellular Ca2+ . Calcilytics are useful as lead
molecules in
the discovery, development, design, modification and/or construction of useful
calcium modulators which are active at Ca2+ receptors. Such calcilytics are
useful
in the treatment of various disease states characterized by abnormal levels of
one or
more components, e.g., polypeptides such as hormones, enzymes or growth
factors,
the expression and/or secretion of which is regulated or affected by activity
at one
or more Ca2+ receptors. Target diseases or disorders for calcilytic compounds
include diseases involving abnormal bone and mineral homeostasis.
Abnormal calcium homeostasis is characterized by one or more of the
following activities: an abnormal increase or decrease in serum calcium; an
abnormal increase or decrease in urinary excretion of calcium; an abnormal
increase or decrease in bone calcium levels (for example, as assessed by bone
mineral density measurements); an abnormal absorption of dietary calcium; an
abnormal increase or decrease in the production and/or release of messengers
which affect serum calcium levels such as PTH and calcitonin; and an abnormal
change in the response elicited by messengers which affect serum calcium
levels.
Thus, calcium receptor antagonists offer a unique approach towards the
pharmacotherapy of diseases associated with abnormal bone or mineral
homeostasis, such as hypoparathyroidism, osteosarcoma, periodontal disease,
fracture healing, osteoarthritis, rheumatoid arthritis, Paget's disease,
humoral
hypercalcemia associated with malignancy and fracture healing, and
osteoporosis.
SUMMARY OF THE iNVENT10N
The present invention comprises novel calcium receptor antagonists
represented by Formula (I) hereinbelow and their us in the treatment of a
variety of
diseases associated with abnormal bone or mineral homeostasis, including but
not
limited to hypoparathyroidism, osteosarcoma, periodontal disease, fracture
healing,
osteoarthrids, rheumatoid arthritis, Paget's disease, humoral hypercalcemia
associated with malignancy and fracture healing, and osteoporosis.
The present invention further provides a method for antagonizing calcium
receptors in an animal, including humans, which comprises administering to an
2
CA 02327188 2000-10-03
WO 99/51241 PCT/US99/07760
animal in need thereof an effective amount of a compound of Formula (I),
indicated
hereinbelow.
The present invention further provides a method for increasing serum
parathyroid levels in an animal, including humans, which comprises
administering
to an animal in need thereof an effective amount of a compound of Formula (n,
indicated hereinbelow.
DETAILEDpESCRII'TION OF THE INVENTION
The compounds of the present invention are selected from Formula (n
hereinbelow:
Re
Y Y2wN~Gip'wB~Rs
H
Y~ ~~R$
Formula (I)
wherein:
Y1 is a covalent bond, alkylene or alkenylene of up to 4 carbon atoms,
unsubstituted or substituted by C1~ alkyl or O;
Y2 is methylene, unsubstituted or substituted by C 1 ~ alkyl or haloalkyl;
Y3 is covalent bond or O, S, N-RN or C 1 ~ alkylene-O, C 1 _q alkylene-S, C 1
~
alkylene-N-R~ ;
RN is selected from the group consisting of H, C1~ alkyl, C3_6 cycioalkyl;
R3 and R4 are, independently, methyl or ethyl, or, together, form cyclopropyl;
Rg is heteroaryl or fused heteroaryl; wherein the hetero-ring contains N, O or
S,
and is aromatic, dihydro or tetrahydro, unsubstituted or substituted with any
substituents being selected from the group consisting of OH, OCH3, CH(CH3)2,
halogen, C1..4 alkyl, C1_4 allcoxy, Cg_6 cycloalkyl, OS02RN, CN, N02, OCF3,
CF3, CH2CF3, (CH2)n C02H, (CH~n C02RN, and O-(CH~n C02RN;
n is an integer from 0 to 3;
G is a covalent bond, CHR6 or C-R6 ,wherein R6 is H, OH or O (forming a
ketone);
3
CA 02327188 2000-10-03
WO 99/51241 PCT/US99/07760
R~ is H, OH, or O-C 1 ~ allryl;
Rg is H or C1~ alkyl; or R~ and Rg together form a ketone;
A and B are, independently, selected from the group consisting of a bond, CH2,
NH, O, S and C=O, provided that either A or B is selected from CH2 and NH; or
A
and B together form a bond; or the A-B moiety is represented by CH=CH or CSC;
X is selected from sub formulas (Ia) to (Ie) hereinbelow:
Ri
YY
(la)
C
./
R' Rz .
(Ib)
x
o~
R' ' Rz
(Ic)
4
CA 02327188 2000-10-03
WO 99/51241
n
(D).
awl
R~
(Id)
x
rrn
4
(le)
wherein
PCT/US99/07760
W is selected from the group consisting of R1, S02R1, C(O)R1, S02NR1R1',
C(O)NR1R1', C(O)ORI, SOgRI', wherein R1 and R1' are independently selected
from the group consisting of hydrogen, C1~ alkyl, Cg_6 cycloalkyl, C2_5
alkenyl,
C2_g alkynyl, heterocycloalkyl, aryl and aryl C1~ alkyl; or R1 and R1'together
form a 3 to 7 membered optionally substituted heterocyclic ring; wherein any
substituents are selected from the group consisting of CN, aryl, C02R, C02NHR,
OH, OR, NH2, halo, CF3, OCF3 and N02; wherein R represents C1~ alkyl, or C3_
6 cycloalkyl;
Xl is selected from the group consisting of CN, N02, CI, F, Br, I, H, R', OR',
CF3,
OCF3 and OS02R', wherein R'represents C1~ alkyl, or Cg_6 cycloalkyl;
X2 , Xg and X4 are, independently, selected from the group consisting of CN,
N02, Cl, F, Br, I, H, R", OR", CFg, OCFg and OS02R", provided that either X1
or
X3 is H, wherein R" is C1~ alkyl or haloalkyl; or Xl and X2 together form an
aryl
or heteroaryl ring, substituted or unsubstituted; wherein the heteroatom is
selected
from N, S and O; and any substituents are selected from the group consisting
of
halo, C1~ alkyl, OCFg, CF3, OMe, CN, OS02R' and N02; or X3 and X4
independently represent C(O)R1; and
5
CA 02327188 2000-10-03
WO 99/51241 PCT/US99/07760
R2 is selected from the group consisting of hydrogen, C1_4 alkyl, C3_6
cycloallcyl,
C2_g allcenyl, C2_5 alkynyl, heterocycloalkyl aryl and aryl-C1_4 alkyl;
X1" is selected from the group consisting of CN, N02, Cl, F, Br, I, H, R, OR,
CF3,
OCF3 and OS02R, wherein R represents C1~ alkyl, or Cg_6 cycloalkyl;
X2", X3" and X4" are, independently, selected from the group consisting of CN,
N02, Cl, F, Br, I, H, R', OR', CF3, OCF3 and OS02R', provided that either X"1
or
X"3 is H, wherein R' is C1~ alkyl or haloalkyl; or Xl" and X2' together form
an
aryl or heteroaryl ring, substituted or unsubstituted; wherein the heteroatom
is
selected from N, S and O and any substituents are selected from the group
consisting of halo, C1~ alkyl, OCF3, CF3, OMe, CN, OS02-C1~ alkyl, OS02-
Cg_6 cycloalkyl and N02;
or X3" and Xq." independently represent C(O)Rl; and
R1" and R2" are, independently, selected from the group consisting of
hydrogen,
C1_4 alkyl, C3_6 cycloalkyl, C2_~ alkenyl, C2_g allrynyl, heterocycloallcyl
and
aryl; or Rl" and R2" together form a 3 to 7 membered optionally substituted
heterocyclic ring; wherein any substituents are selected from the group
consisting
of CN, aryl, C02R", C02NHR", OH, OR", NH2, halo, CF3, OCF3 and N02;
wherein R" represents C1~ alkyl, or C3_6 cycloaikyl;
X 1 ~' is selected from the group consisting of CN, N02, Cl, F, Br, I, H, R,
OR, CF3,
OCF3 and OS02R, wherein R represents C1~ alkyl, or C3_6 cycioallcyl;
X2'", X3"', and X4"' are, independently, selected from the group consisting of
CN,
N02, Cl, F, Br, I, H, R', OR', CF3, OCF3 and OS02R', provided that either X"'1
or
X'"3 is H, wherein R' is C1~ alkyl or haloalkyl;
or X1'" and X2~' together form an aryl or heteroaryl ring, substituted or
unsubstituted; wherein the heteroatom is selected from N, S and O and the
substituents are selected from the group consisting of halo, C1~ alkyl, OCF3,
CF3,
OMe, CN, OS02-C1_4 allryl, OS02-C3_6 cycloalkyl and N02;
or X3'" and X4 "' independently represent C(O)Rl;
R1'" and R2"' are, independently, selected from the group consisting of
hydrogen,
C1~ alkyl, Cg_6 cycloallryl, C2_g alkenyl, C2_5 alkynyl, heterocycioalkyl and
aryl;
or Rl'"and R2"' together form a 3 to 7 membered optionally substituted
heterocyclic ring; wherein the substituents are selected from the group
consisting of
6
CA 02327188 2000-10-03
WO 99/51241
PCT/US99/07760
CN, aryl, C02R", C02NHR", OH, OR", NH2, halo, CF3, OCF3 and N02; wherein
R" represents C1~ alkyl, or Cg_6 cycloallcyl;
D is selected from the group consisting of H, CN, N02, Cl, F, Br, I, R, OR,
SR,
CF3, OCF3 and OS02R, wherein R represents C1..4 alkyl, C3_6 cycloalkyl, or C1_
aT3'1 or heteroaryl wherein the heteroatom is selected from N, S and O and
substituents are selected from the group consisting of halo, C 1..4 alkyl,
OCF3, CF3,
OMe, CN, OS02-C1~ alkyl, OS02-
C3_6 cycloalkyl and N02;
n is the integer 1 or 2;
10 each E is independently C or N, provided that no more than two E moieties
are N;
further provided that when n is 2, each E is C;
a and b are optionally present bonds;
R1 "' is selected from the group consisting of (CH2)nC02R', (CH2)nC02H,
(CH2)nCONR 2, (CH2)nCH20R', OR', SR', CN, N02, Cl, F, Br, I, H, CF3, OCF3,
OS02R', R' and H; wherein R'represents C1~ alkyl, or C3_6 cycloalkyl;
or R1 rv is O, foaming a ketone such that Y R1 "' represents -C=O;
R2"' is selected from the group consisting of hydrogen, CN, N02 Cl, F, Br, I,
H,
R", OR", CF3, OCF3, and OS02R"; wherein R" represents C1~ alkyl, or C3_6
cycloalkyl.
Y is selected from the group consisting of C, CH, O, N and S; provided that
when
Y is S, Rl'° is O or not present; further provided that when Y is O,
R1'~ is not
present;
X' is selected from the group consisting of CH2, NH, O and S.
R9 is selected from the group consisting of O-alkyl, O-CH2-aryl, and O- aryl;
X1"" is selected from the group consisting of CN, N02, Cl, F, Br, I, H, R, OR,
CF3, OCF3 and OS02R, wherein R represents C1_4 alkyl, or C3_6 cycloallcyl;
X2"", X3'"', and X4'"' are, independently, selected from the group consisting
of CN,
N02, Cl, F, Br, I, H, R', OR', CF3, OCF3 and OS02R', provided that either
X'"'1 or
X'"'3 is H, wherein R' is Cl_4 alkyl or haloalkyl;
or X1'"' and X2"" together form an aryl or heteroaryl ring, substituted or
unsubstituted; wherein the heteroatom is selected from N, S and O and the
substituents are selected from the group consisting of halo, C1_4 alkyl, OCF3,
CF3,
OMe, CN, OS02-C1~ alkyl, OS02-Cg_6 cycloalkyl and N02;
7
CA 02327188 2000-10-03
WO 99/51241 PCT/US99/07760
or X2"" and X4 "" independently represent C(O)R1;
and pharmaceutically acceptable salts and complexes thereof.
Preferably, the compounds of the present invention have a structure
according to Formula (In:
AwB~Rs
X H
~O pH R6
Formula (Il7
wherein:
Rg is heteroaryl or fused heteroaryl; wherein the hereto-ring contains N, O or
S,
and is aromatic, dihydro or tetrahydro, unsubstituted or substituted with any
substituents being selected from the group consisting of OH, OCH3, CH(CH3)2,
halogen, C 1 ~ alkyl, C 1 ~ alkoxy, C3_6 cycloalkyl, OS02RN, CN, N02, OCF3,
CF3, CH2CF3, (CH~n C02H, (CH~n C02RN, and O-(CH2)n C02RN; and
A and B are, independently, selected from the group consisting of a bond, CH2,
15 NH, O, S and C=O, provided that either A or B is selected from CH2 and NH;
or A
and B together form a bond; or the A-B moiety is represented by CH=CH or CSC.
More preferably, RS is heteroaryl or fused heteroaryl, wherein the hereto
ring contains N, O or S and is aromatic, dihydro or tetrahydro, unsubstituted
or
substituted with any substituents being selected from the group consisting of
OCH3, halogen, C1~ alkyl, , CN, N02, OCF3, CF3, CH2CF3;
R6 is H; and
A and B are, independently, selected from the group consisting of a bond, CH2,
NH, O, S and C=O, provided that either A or B is selected from CH2 and NH, or
A
and B together form a bond.
Most preferably, RS is heteroaryl or fused heteroaryl, wherein the hetero-ring
contains N, O or S and is aromatic, dihydro or tetrahydro, unsubstituted or
substituted with any substituents being selected from the group consisting of
OCH3, halogen, C1_4 allcyl, , CN, N02, OCF3, CF3, CH2CF3;
R6 is H; and
A and B are, independently, selected from the group consisting of a bond, CH2,
O,
or A and B together form a bond.
8
CA 02327188 2000-10-03
WO 99/51241
PCT/US99/07760
In sub-formula (Ia), preferably, Xl is selected from the group consisting of
CN, N02, Cl, F, Br, I and H. Preferably, X2, X3 and X4 are, independently,
selected from the group consisting of Cl, F, Br, I and H, provided Xl and X3
is H.
Preferably, Rl, Rl'and R2 are, independently, selected from the group
consisting of
Cl~ alkyl, Cg_6 cycloalkyl, heterocycloallcyl, aryl or arylalkyl.
In sub-formula (Ia), more preferably, Rl, Rl'and R2 are, independently, H,
alkyl, or aryl. More preferably, Xl is selected from the group consisting of
CN,
N02, Cl, F, Br, I and H. More preferably, X2, X3 and X4 are, independently,
selected from the group consisting of Cl, F, Br, I and H provided Xl and X3 is
H.
In sub-formula (Ia), more preferably still, Rl, Rl'and R2 are,
independently, Cl~ alkyl, or aryl. More preferably still, Xl is CN, N02, or
Cl.
More preferably still, X2 is Cl, F or H. More preferably stilt, X3 and X4 are
H.
In sub-formula (Ia), most preferably, Xl is CN, or N02. Most preferably,
X2 is Cl.
In sub-formula (Ib), preferably, Xl" is selected from the group consisting
of CN, N02, Cl, F, Br, I and H. Preferably, X2', X3" and X4" are,
independently,
selected from the group consisting of Cl, F, Br, I and H. Preferably, Rl" and
R2"
are, independently, selected from the group consisting of C1~ alkyl, C3_6
cycloallcyl, heterocycloalkyl or aryl; or Rl" and RZ' together form an
optionally
substituted 3-7 membered ring, optionally containing an additional heteroatom
selected from O, S, and N.
In sub-formula (Ib), more preferably, R1" and R2" are, independently, H,
Cl~ alkyl, or aryl; or Rl" and R2' together form an optionally substituted 4-7
membered ring, optionally containing a heteroatom selected from O, S, and N.
More preferably, XI" is selected from the group consisting of CN, N02, Cl, F,
Br, I
and H. More preferably, X2' is selected from the group consisting of Cl, F,
Br, I
and H.
In sub-formula (Ib), more preferably still, R1"and R2" are, independently,
C1~ alkyl, or aryl; or Rl" and R2" together form a 4-7 membered ring as
described
hereinabove. More preferably still, Xl" is CN, N02, or Cl. More preferably
still,
X2' is Cl, F or H.
9
CA 02327188 2000-10-03
WO 99/51241 PCT/US99/077G0
In sub-formula (Ib), most preferably, Rl" and R2" together form a 4-7
membered ring as described hereinabove. Most preferably, X1" is CN, or N02.
Most preferably, X2 "is Cl.
In sub-formula {Ic), preferably, X1'" is selected from the group consisting
of CN, N02, Cl, F, Br, I and H. Preferably, X2"', X3"' and X4"' are,
independently,
selected from the group consisting of Cl, F, Br, I and H. provided either X1
"' or
X3'" is H. Preferably, Rl "' and R2'" are, independently, selected from the
group
consisting of C1~ alkyl, C3-6 cycloalkyl, heterocycloallcyl or aryl; or R1 ~'
and R2"'
together form an optionally substituted 3-7 membered ring, optionally
containing
an additional heteroatom selected from O, S, and N.
In sub-formula (Ic), more preferably, R1'", and R2"' are, independently, H,
C 1 ~ alkyl, or aryl; or R 1 "' and R2'" together form an optionally
substituted 4-7
membered ring, optionally containing a heteroatom selected from O, S, and N.
More preferably, X1 "' is selected from the group consisting of CN, N02, Cl,
F, Br,
I and H. More preferably, X2"', X3'~ and X4"' are, independently, selected
from the
group consisting of Cl, F, Br, I and H provided either X1 "' or Xg'" is H.
In sub-formula (Ic), more preferably still, Rl'"and R2'" are, independently,
C1~ alkyl, or aryl; or R1'" and R2'" together form a 4-7 membered ring as
described hereinabove. More preferably still, X1 "' is CN, N02, or Cl. More
preferably still, X2'~ is Cl or H. More preferably still, X3'" and X4"' are H.
In sub-formula (Ic), most preferably, R 1 ~' and R2"' together form a 4-7
membered ring as described hereinabove. Most preferably, Xl'" is CN or N02.
Most preferably, X2 "' is Cl.
In $ub-formula (Id), preferably, each D is selected from the group
consisting of F, Br, Cl, I, R, OR, SR, and H. Preferably, R1'" is selected
from the
group consisting of (CH2)nC02R', (CH~nC02H, (CH2)nCONR 2,
{CH~nCH20R', OR', SR', R' and H; wherein R' is as R hereinabove; or Rl'" is O,
forming a ketone such that Y R 1 "' represents -C=O. Preferably, R2 '" is
selected
from the group consisting of hydrogen, CN, N02~ Cl, Br, F and I;
In sub-formula (Id), more preferably, n is O. More preferably, each E is C.
More preferably, X' is CH2, O, or NH. More preferably, Y is C or N. More
preferably, R1'" is CH2C02R', SR', or O forming a ketone.
CA 02327188 2000-10-03
WO 99/51241 PCT/US99/07760
In subformula (Id), more preferably still, X' is CH2 or O. More preferably
still, R1'" is CH2C02R' or SR'. More preferably still, R2" is H, CN, or N02.
In subformula (Id), most preferably, X' is CH2. Most preferably, Y is C.
Most preferably, R2'" is CN or N42.
In subformula (Ie), preferably R9 is selected from the group consisting of
O_(CH2)n-aryl, and O- aryl;
X1 "" is selected from the group consisting of CN, N02, Cl, F, Br, H, R, and
OS02R, wherein R represents C1~ allryl, or C3_6 cycloalkyl;
X2'"', X3"", and X4"" are, independently, selected from the group consisting
of CN,
N02, CI, F, Br, Hand OS02R; provided that either X""1 or X""3 is H, wherein R'
is C1~ alkyl or haloallcyl;
or X I "" and X2"" together form an aryl or heteroaryl ring, substituted or
unsubstituted; wherein the heteroatom is selected from N, S and O and the
substituents are selected from the group consisting of halo, C1~ alkyl, OCF3,
CF3,
OMe, CN, OS02-C1~ alkyl, OS02-Cg_6 cycloalkyl and N02;
or X2'"' and X4 '"' independently represent C(O)Rl;
In subformula (Ie), more preferably Rg is selected from the group
consisting of O-(CH~n-aryl, and O- aryl;
XI "" is selected from the group consisting of CN, N02, and CI
X2"", X3"", and X4'"' are, independently, selected from the group consisting
of Cl,
F, and H, provided that either X""I or X""3 is H,
or X2"" and X4 "" independently represent C(O)Rl;
In subformula (Ie), most preferably R9 is selected from the group
consisting of O-(CH2)n-aryl, and O- aryl;
X1"" is CN or N02,
X2" " is Cl, X3'~' and X4'"' are, independently F, and H.
Preferred heteroaryls useful in the present invention include unsubstituted
and substituted quinolines, isoquinolines, benzofurans, dihydrobenzofurans,
benzothiophenes, dihydrobenzothiophenes and pyridines.
As used herein "cycloallcyl" refers to optionally substituted 3-7 membered
carbocyclic rings wherein any substituents are selected from the group
consisting
of, F, CI, Br, I, N(RI)2, SR1 and ORI, unless otherwise indicated.
lI
CA 02327188 2000-10-03
WO 99/51241 PCT/US99/07760
As used herein "hetetrocycloallryl" refers to optionally substituted 4, 5, 6
or
7 membered heterocyclic rings containing 1 to 2 heteroatoms selected from N,
O,
and S.
As used herein, "aryl" refers to an optionally substituted aromatic group
with at least one ring having a conjugated pi-electron system, containing up
to two
conjugated or fused ring systems. Aryl includes carbocyclic aryl, and biaryl
groups, all of which may be optionally substituted. Preferred aryl include
phenyl
and naphthyl. More preferred aryl include phenyl. Preferred substituents are
selected from the group consisting of halo, C1_4 alkyl, OCF3~ CFg~ OMe, CN,
OS02 R and N02~ wherein R represents C1~ alkyl or C3_6 cycloalkyl.
As used herein, "acyl" refers to C1~ alkylcarbonyl.
As used herein, "alkenyl" refers to an optionally substituted hydrocarbon
group containing at least one carbon-carbon double bond and containing upto 5
carbon atoms joined together. The alkenyl hydrocarbon chain may be straight,
branched or cyclic. Any substituents are selected from the group consisting of
halo, C1_4 alkyl, OCF3~ CFg~ OMe, CN, OS02 R and N02~ wherein R represents
C1~ alkyl or C3_6 cycloalkyl.
As used herein, "alkynyl" refers to an optionally substituted hydrocarbon
group containing at least one carbon-carbon triple bond between the carbon
atoms
and containing up to 5 carbon atoms joined together. The alkynyl hydrocarbon
group may be straight-chained, branched or cyclic. Any substituents are
selected
from the group consisting of halo, C1~ alkyl, OCF3, CF3, OMe, CN, OS02 R and
N02, wherein R represents C1~ allcyl or C3_6 cycloalkyl.
The compounds of the present invention may contain one or more
asymmetric carbon atoms and may exist in racemic and optically active forms.
All
of these compounds and diastereomers are contemplated to be within the scope
of
the present invention.
Preferred compounds of the present invention are selected from the group
consisting of:
(R)-N-[2-Hydroxy-3-(3-chloro-2-cyanophenoxy)propyl]-1,1-dimethyl-2-(2,3-
dihydrobenzo[b]furan-5yl)ethylamine;
(R)-N-[2-Hydroxy-3-(3-chioro-2-cyanophenoxy)propyl]-1,1-dimethyl-2-(quinolin-
3-yl~thylamine;
12
CA 02327188 2000-10-03
WO 99/51241 PCT/US99/07760
(R)-N-[2-Hydroxy-3-{3-chloro-2-cyanophenoxy)propyl]-1,1-dimethyl-2-(quinolin-
2-yl)ethylamine;
(R)-N-[2-Hydroxy-3-(3-chloro-2-cyanophenoxy)propyl]-1,1-dimethyl-2-
(isoquinolin-3-yl)ethylamine;
(R)-N-[2-Hydroxy-3-(3-chloro-2-cyanophenoxy)propyl]-1,1-dimethyl-4-(2-
pyridyl)butylamine;
(RAN-[2-Hydroxy-3-(3-chloro-2-cyano-4-
morpholinosulfonamidophenoxy)propyl]-1,1-dimethyl-4-(2-pyridyl)butylamine;
(R)-N-[2-Hydroxy-3-(3-chloro-2-cyanophenoxy)propyl]-1,1-dimethyl-4-(3-
pyridyl)butylamine;
(R)-N-[2-Hydroxy-3-(3-chloro-2-cyano-4-
morphoiinosulfonamidophenoxy)propyl]-1,1-dimethyl-4-(3-pyridyl)butylamine;
(R)-N-[2-Hydroxy-3-(3-chloro-2-cyanophenoxy)propyl]-1,1-dimethyl-4-(4-
carbethoxyphenyl)butylamine;
(R)-N-[2-Hydroxy-3-(3-chloro-2-cyanophenoxy)propyl]-1,1-dimethyl-2-(4-
ethylpyrid-2-yl)ethylamine;
(R)-N-[2-Hydroxy-3-(3-chloro-2-cyanophenoxy)propyl]-1,1-dimethyl-2-
benzamidoethylamine;
(R)-N-[2-Hydroxy-3-(3-chloro-2-cyanophenoxy)propyl]-4-phenylbutylamine;
(R)-N-[2-Hydroxy-3-(3-chloro-2-cyanophenoxy)propyI]-1,1-dimethyl-4-phenylbut-
2-ynylamine;
and pharmaceutically acceptable salts and complexes thereof. Preferred salts
include hydrochloride and dihydrochloride.
More preferred compounds useful in the present invention include:
(R)-N-[2-Hydroxy-3-(3-chloro-2-cyanophenoxy)propyl]-1,1-dimethyl-2-(2,3-
dihydrobenzo[b]furan-Syl)ethylamine;
(R)-N-(2-Hydroxy-3-(3-chloro-2-cyanophenoxy)propyl]-1,1-dimethyl-2-(quinolin-
3-yl)ethylamine;
(R)-N-[2-Hydroxy-3-(3-chloro-2-cyanophenoxy)propyl]-1,1-dimethyl-2-(quinolin-
2-yl~thylamine;
(R)-N-[2-Hydroxy-3-(3-chloro-2-cyanophenoxy)propyl]-1,1-dimethyl-2-
(isoquinolin-3-yl)~thylamine;
13
CA 02327188 2000-10-03
WO 99/51241 PCT/US99/07760
(R)-N-[2-Hydroxy-3-(3-chloro-2-cyanophenoxy)propyl]-1,1-dimethyl-4-(2-
pyridyl)butylamine;
(R)-N-[2-Hydroxy-3-(3-chloro-2-cyano-4-
morpholinosulfonamidophenoxy)propyl]-1,1-dimethyl-4-(2-pyridyl)butylamine;
(R)-N-[2-Hydroxy-3-(3-chloro-2-cyanophenoxy)propyl]-I,1-dimethyl-4-(3-
pyridyl)butylamine;
(R)-N-[2-Hydroxy-3-(3-chloro-2-cyano-4-
morpholinosulfonamidophenoxy)propyl]-1,1-dimethyl-4-(3-pyridyl)butylamine;
(R)-N-[2-Hydroxy-3-(3-chloro-2-cyanophenoxy)propyl]-1,1-dimethyl-2-(4-
IO ethylpyrid-2-ylkthylamine;
(R)-N-[2-Hydroxy-3-(3-chloro-2-cyanophenoxy)propyl]-1,1-dimethyl-2-
benzamidoethylamine;
(R)-N-[2-Hydroxy-3-(3-chloro-2-cyanophenoxy)propyl]-4-phenylbutylamine;
and pharmaceutically acceptable salts and complexes thereof.
The most preferred compounds useful in the present invention include:
(R)-N-[2-Hydroxy-3-(3-chloro-2-cyanophenoxy)propyl]-1,1-dimethyl-2-(2,3-
dihydrobenzo[b]furan-Syl)ethylamine;
(RAN-[2-Hydroxy-3-(3-chloro-2-cyanophenoxy)propyl]-1,1-dimethyl-2-(quinolin-
3-yl)ethylamine;
(R)-N-[2-Hydroxy-3-(3-chloro-2-cyanophenoxy)propyl]-1,1-dimethyl-2-(quinolin-
2-yl)ethylamine;
(R)-N-[2-Hydroxy-3-(3-chloro-2-cyanophenoxy)propyl]-1,1-dimethyl-2-
(isoquinolin-3-yl)ethylamine;
(R)-N-[2-Hydroxy-3-(3-chloro-2-cyanophenoxy)propyl]-1,1-dimethyl-4-(2-
pyridyl)butylamine;
(R)-N-[2-Hydroxy-3-(3-chioro-2-cyano-4-
morpholinosulfonamidophenoxy)propyl]-I,1-dimethyl-4-(2-pyridyl)butylamine;
(RrN-[2-Hydroxy-3-(3-chloro-2-cyanophenoxy)propyl]- I,1-dimethyl-4-(3-
pyridyl)butylamine;
(R)-N-[2-Hydroxy-3-(3-chloro-2-cyano-4-
morpholinosulfonamidophenoxy)propyl]-1,1-dimethyl-4-(3-pyridyl)butylamine;
(R)-N-[2-Hydroxy-3-(3-chloro-2-cyanophenoxy)propyl]-1,1-dimethyl-2-(4-
ethylpyrid-2-yl)ethylamine;
14
CA 02327188 2000-10-03
WO 99/51241 PCT/US99/07760
and pharmaceutically acceptable salts and complexes thereof.
Pharmaceutically acceptable salts are non-toxic salts in the amounts and
concentrations at which they are administered.
Pharmaceutically acceptable salts include acid addition salts such as those
containing sulfate, hydrochloride, fumarate, maleate, phosphate, sulfamate,
acetate,
citrate, lactate, tartrate, methanesulfonate, ethanesulfonate,
benzenesulfonate, p-
toluenesulfonate, cyclohexylsulfamate and quinate. A preferred salt is a
hydrochloride. Pharmaceutically acceptable salts can be obtained from acids
such
as hydrochloric acid, malefic acid, sulfuric acid, phosphoric acid, sulfamic
acid,
acetic acid, citric acid, lactic acid, tartaric acid, maionic acid,
methanesulfonic acid,
ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid,
cyclohexylsulfamic acid, fumaric acid, and quinic acid.
Pharmaceutically acceptable salts also include basic addition salts such as
those containing benzathine, chloroprocaine, choline, diethanolamine,
ethylenediamine, meglumine, procaine, aluminum, calcium, lithium, magnesium,
potassium, sodium, ammonium, alkylamine, and zinc, when acidic functional
groups, such as carboxylic acid or phenol are present.
The present invention provides compounds of Formula (>) above, which
can be prepared using standard techniques. An overall strategy for preparing
preferred compounds described herein can be carried out as described in this
section. The examples which follow illustrate the synthesis of specific
compounds.
Using the protocols described herein as a model, one of ordinary skill in the
art can
readily produce other compounds of the present invention.
~hgme__1
as
~~O ~ H~~~B/ ~' Aw iR5
O R6 H T B
X '~ ~ OH pg
KxCOa~ acetone X OOH X
He~l
15
CA 02327188 2000-10-03
WO 99/51241 PCT/US99/07760
S eme 2
o'
N~
\ \ H Me0-NHz \ \ H ZnlfFA
N ~ / N
I
O
II.
N H ~~ I N ~ _
s I o 'I ~ o
\ \ eF, \ \ Zn/TFA
/ N 2. ~ /
N~ _
Na+ O
\ \ Nz
N
,~eme
DPPA, Benzyl
O I //~\\y~ /~Z 9-BBN,THF
N
OH
H
1.
~er
i
~N ~ PdCl2(dppf) \
H KzC03, HZO N v v ~NH2
2. Pd(OH)~/C,
EtOH
16
CA 02327188 2000-10-03
WO 99/51241 PCT/US99/07760
e4
O 1. NaH, DMSO
H ~-PPh3 ~ 1. SOW A~cOH
w
O ~ ~ 2. NaOH, EtOH
p ~ reflex 24h
NHZ
O
General Preparation
A general procedure used to synthesize many of the compounds can be
carried out as described in Scheme 1: A solution of aryl alcohol in acetone
was
treated with an appropriate base such as K2C03, heated for 15 min. R-glycidyl
nosylate was added and the reaction continued overnight to give the
corresponding
glycidyl ether (Scheme 1 ). In the case of an alkyl alcohol, a stronger base,
e.g.
NaH in DMF was used. This method can also be used for aryl alcohols. A
solution
of the substituted glycidyl ether and excess amine (typically 1,1-dimethyl-2-
(4-
methyloxyphenyl)ethylamine) in absolute ethanol, acetonitrile, THF or any
other
similar solvent in the presence of a suitable catalyst such as LiC104 is
stirred
overnight at reflex. The product is purified by normal phase chromatography.
Hydrochloride salts are prepared by treatment of the corresponding free base
with
HCl either in gas phase or 4M dioxane solution, or any other standard method.
The synthesis of various corresponding amines is described in Scheme 2,
3, 4 and 5. The synthesis of 3-(2-amino-2-methylpropyl)quinoline illustrates
the
general procedure to obtain these amines, and it is described in Scheme 2. The
reduction of the oxime obtained from 3-quinolinecarboxaldehyde leads to the
corresponding benzylic amine. Reaction of the aforementioned amine with 2,4,6-
triphenylpyrylium tetrafluoroborate followed by nucleophilic displacement of
the
17
CA 02327188 2000-10-03
WO 99151241 PCT/US99/07760
pyridinium salt thus formed with the anion of 2-nitropropane, leads to the
formation of the corresponding vitro compound which, after reduction, leads to
the
title compound.
The synthesis of 2-(4-amino-4-methylpentyl)pyridine illustrates the general
procedure to obtain pentyl amines, and it is described in Scheme 3. The
Curtius
rear angement of 2,2-dimethyl-4-pentenoic acid leads to the corresponding Cbz
protected amine. Addition of 9-BBN to the terminal olefin of the protected
amine
leads to the corresponding boronate. Palladium catalyzed coupling reaction
between the boronate and the corresponding aryl bromide (2-bromopyridine in
Scheme 3) leads to the formation of the corresponding amine after the removal
of
the protecting group.
The synthesis of 5-(2-amino-2-methylpmpyl)-2,3-dihydrobenzo[b]furan
illusuates the general procedure to obtain these amines, and it is described
in
Scheme 4. Wittig reaction between 2,3-dihydrobenzo[b]furan-5-carboxaldehyde
and the anion formed from isopropyltriphenylphosphonium leads to the
corresponding olefin. Ritter reaction on the olefin followed by hydrolysis
leads to
the corresponding amine.
Nuclear magnetic resonance spectra were recorded at either 250 or 400
MHz using, respectively, a Broker AM 250 or Broker AC 400 spectrometer.
CDCI3 is deuteriochloroform, DMSO-d6 is hexadeuteriodimethylsulfoxide, and
CD30D is tetradeuteriomethanol. Chemical shifts are reported in parts per
million
(~) downfieId from the internal standard tetramethylsilane. Abbreviations for
NMR
data are as follows: s=singlet, d=doublet, t=triplet, q=quartet, m=multiplet,
dd=doublet of doublets, dt=doublet of triplets, app=apparent, br-broad. J
indicates
the NMR coupling constant measured in Hertz. Continuous wave infrared (IR)
spectra were recorded on a Perkin-Elmer 683 infrared spectrometer; and Fourier
transform infrared (FIZR) spectra were recorded on a Nicolet Impact 400 D
infrared spectrometer. IR and FTIR spectra were recorded in transmission mode,
and band positions are reported in inverse wavenumbers (cm-1). Mass spectra
were taken on either VG 70 FE, PE Syx API III, or VG ZAB HF instruments, using
fast atom bombardment (FAB) or electrospray (ES) ionization techniques.
LC/MS/MS was obtained on a Perkin Elmer Sciex API 365 Instrument. Elemental
analyses were obtained using a Perkin-Elmer 2400 elemental analyzer. Melting
18
CA 02327188 2000-10-03
WO 99/51241 PCT/US99/07760
points were taken on a Thomas-Hoover melting point apparatus and are
uncorrected. All temperatures are reported in degrees Celsius.
Analtech Silica Gel GF and E. Merck Silica Gel 60 F-254 thin layer plates
were used for thin layer chromatography. Both flash and gravity chromatography
were carried out on E. Merck Kieselgel 60 (230-400 mesh) silica gei.
Analytical
and preparative HPLC were carried out on Raisin or Beckman chromatographs.
ODS refers to an octadecylsilyl derivatized silica gel chromatographic
support. 5 fl
Apex-ODS indicates an octadccylsilyl derivatized silica gel chromatographic
support having a nominal particle size of 5 E.i, made by Jones Chromatography,
Littleton, Colorado. YMC ODS-AQ~ is an ODS chromatographic support and is a
registered trademark of YMC Co. Ltd.,.Kyoto, Japan. PRP-10 is a polymeric
(styrene-divinylbenzene) chromatographic support, and is a registered
trademark of
Hamilton Co., Reno, Nevada) Celite~ is a filter aid composed of acid-washed
diatomaceous silica, and is a registered trademark of Manville Corp., Denver,
Colorado.
All reagents and solvents were obtained from commercial vendors. Starting
materials (e.g., amines and epoxides) were synthesized using standard
techniques
and procedures.
With appropriate manipulation and protection of any chemical
functionality, synthesis of the remaining compounds of Formula (I) is
accomplished by methods analogous to those above and to those described in the
Experimental section.
In order to use a compound of Formula (I) or a pharmaceutically acceptable
salt thereof for the treatment of humans and other mammals, it is normally
formulated in accordance with standard pharmaceutical practice as a
pharmaceutical composition.
The calcilytic compounds can be administered by different routes including
intravenous, intraperitoneal, subcutaneous, intramuscular, oral, topical
(transdermal), or transmucosal administration. For systemic administration,
oral
administration is preferred. For oral administration, for example, the
compounds
can be formulated into conventional oral dosage fonms such as capsules,
tablets,
and liquid preparations such as syrups, elixirs, and concentrated drops.
19
CA 02327188 2000-10-03
WO 99/51241 PCTNS99/07760
Alternatively, injection (parenteral administration) may be used, e.g.,
intramuscular, intravenous, intraperitoneal, and subcutaneous. For injection,
the
compounds of the invention are formulated in liquid solutions, preferably, in
physiologically compatible buffers or solutions, such as saline solution,
Hank's
solution, or Ringer's solution. In addition, the compounds may be formulated
in
solid form and redissolved or suspended immediately prior to use. Lyophilized
forms can also be produced.
Systemic administration can also be by transmucosal or transdermaI means.
For transmucosal or transdenmal administration, penetrants appropriate to the
barrier to be permeated are used in the formulation. Such penetrants are
generally
known in the art, and include, for example, for transmucosal administration,
bile
salts and fusidic acid derivatives. In addition, detergents may be used to
facilitate
permeation. Transmucosal administration, for example, may be through nasal
sprays, rectal suppositories, or vaginal suppositories.
For topical administration, the compounds of the invention can be
formulated into ointments, salves, gels, or creams, as is generally known in
the att.
The amounts of various calcilytic compounds to be administered can be
determined by standard procedures taking into account factors such as the
compound ICgO, ECgO, the biological half life of the compound, the age, size
and
weight of the patient, and the disease or disorder associated with the
patient. The
importance of these and other factors to be considered are known to those of
ordinary skill in the art.
Amounts administered also depend on the routes of administration and the
degree of oral bioavailability. For example, for compounds with low oral
bioavailability, relatively higher doses will have to be administered.
Preferably the composition is in unit dosage form. For oral application, for
example, a tablet, or capsule may be administered, for nasal application, a
metered
aerosol dose may be administered, for transdermal application, a topical
formulation or patch may be administered and for transmucosal delivery, a
buccal
patch may be administered. In each case, dosing is such that the patient may
administer a single dose.
Each dosage unit for oral administration contains suitably from 0.01 to 500
mg/Kg, and preferably from 0.1 to 50 mg/Kg, of a compound of Formula (I) or a
CA 02327188 2000-10-03
WO 99/51241 PCT/US99/07760
pharmaceutically acceptable salt thereof, calculated as the free base. The
daily
dosage for parenteral, nasal, oral inhalation, transmucosal or transdeimal
routes
contains suitably from 0.01 mg to 100 mg/Kg, of a compound of Formula(n. A
topical formulation contains suitably 0.01 to 5.0% of a compound of Fonmula
{I).
The active ingredient may be administered, for example, from 1 to 6 times per
day,
preferably once, sufficient to exhibit the desired activity, as is readily
apparent to
one skilled in the art.
As used herein, "treatment" of a disease includes, but is not linuted to
prevention, retardation and prophylaxis of the disease.
Diseases and disorders which might be treated or prevented, based upon the
affected cells, include bone and mineral-related diseases or disorders;
hypoparathyroidism; those of the central nervous system such as seizures,
stroke,
head trauma, spinal cord injury, hypoxia-induced nerve cell damage, such as
occurs
in cardiac arrest or neonatal distress, epilepsy, neurodegenerative diseases
such as
Alzheimer's disease, Huntington's disease and Parkinson's disease, dementia,
muscle tension, depression, anxiety, panic disorder, obsessive-compulsive
disorder,
post-traumatic stress disorder, schizophrenia, neuroleptic malignant syndrome,
and
Tourette's syndrome; diseases involving excess water reabsorption by the
kidney,
such as syndrome of inappropriate ADH secretion (SIADH), cirrhosis, congestive
heart failure, and nephrosis; hypertension; preventing and/or decreasing renal
toxicity from cationic antibiotics (e.g., aminoglycoside antibiotics); gut
motility
disorders such as diarrhea and spastic colon; GI ulcer diseases; GI diseases
with
excessive calcium absorption such as sarcoidosis; autoimmune diseases and
organ
transplant rejection; squamous cell carcinoma; and pancreatitis.
In a preferred embodiment of the present invention, the present compounds
are used to increase serum parathyroid hormone ("PTH") levels. Increasing
serum
>rTH levels can be helpful in treating diseases such as hypoparathyroidism,
osteosarcoma, periodontal disease, fracture, osteoarthritis, rheumatoid
arthritis,
Paget's disease, humoral hypercalcemia malignancy and osteoporosis.
Another aspect of the present invention describes a method of treating a
patient comprising administering to the patient an amount of a present
compound
sufficient to increase the serum PTH level. Preferably, the method is carried
out by
21
CA 02327188 2000-10-03
WO 99/51241 PC'TNS99/07760
administering an amount of the compound effective to cause an increase in
duration
and/or quantity of serum PTH level sufficient to have a therapeutic effect.
In various embodiments, the compound administered to a patient causes an
increase in serum P'TH having a duration of up to one hour, about one to about
twenty-four hours, about one to about twelve hours, about one to about six
hours,
about one to about five hours, about one to about four hours, about two to
about
five hours, about two to about four hours, or about three to about six hours.
In an alterate embodiment of the present invention, the compound
administered causes an increase in serum PTH of longer than about twenty-four
hours, but the compound is co-administered with an anti-resorptive agent.
In additional different embodiments, the compound administered to a
patient causes an increase in serum PTTI of up to two fold, two to five fold,
five to
ten fold, and at least 10 fold, greater than peak serum PTH in the patient.
The peak
serum level is measured with respect to a patient not undergoing treatment.
Composition of Formula (I) and their pharmaceutically acceptable salts,
which are active when given orally, can be formulated as syrups, tablets,
capsules
and lounges. A syrup formulation will generally consist of a suspension or
solution of the compound or salt in a liquid carrier for example, ethanol,
peanut oil,
olive oil, glycerine or water with a flavoring or coloring agent. Where the
composition is in the form of a tablet, any pharmaceutical carrier routinely
used for
preparing solid formulations may be used. Examples of such carriers include
magnesium stearate, terra albs, talc, gelatin, acacia, stearic acid, starch,
lactose and
sucrose. Where the composition is in the form of a capsule, any routine
encapsulation is suitable, for example using the aforementioned carriers in a
hard
gelatin capsule shell. Where the composition is in the form of a soft gelatin
shell
capsule any pharmaceutical carrier routinely used for preparing dispersions or
suspensions may be considered, for example aqueous gums, celluloses, silicates
or
oils, and are incorporated in a soft gelatin capsule shell.
Typical parenteral compositions consist of a solution or suspension of a
compound or salt in a sterile aqueous or non-aqueous carrier optionally
containing
a parenterally acceptable oil, for example polyethylene glycol,
polyvinylpyrrolidone, lecithin, arachis oil or sesame oil.
22
CA 02327188 2000-10-03
WO 99/51241 PCT/US99/07760
Typical compositions for inhalation are in the form of a solution,
suspension or emulsion that may be administered as a dry powder or in the form
of
an aerosol using a conventional propellant such as dichlorodifluoromethane or
trichlorofluoromethane.
A typical suppository formulation comprises a compound of Formula (I) or
a pharmaceutically acceptable salt thereof which is active when administered
in
this way, with a binding and/or lubricating agent, for example polymeric
glycols,
gelatins, cocoa-butter or other low melting vegetable waxes or fats or their
synthetic analogs.
Typical dermal and transdermal formulations comprise a conventional
aqueous or non-aqueous vehicle, for example a cream, ointment, lotion or paste
or
are in the form of a medicated plaster, patch or membrane.
Preferably the composition is in unit dosage fonm, for example a tablet,
capsule or metered aerosol dose, so that the patient may administer a single
dose.
No unacceptable toxological effects are expected when compounds of the
present invention are administered in accordance with the present invention.
The biological activity of the compounds of Formula (I) are demonstrated
by the following tests:
(I) Calcium Receptor Inhibitor Assay
Calcilytic activity was measured by determining the IC50 of the test
compound for blocking increases of intracellular Ca2+ elicited by
extracellular
Ca2+ in HEK 293 4.0-7 cells stably expressing the human calcium receptor. HEK
293 4.0-7 cells were constructed as described by Rogers et al., J. Bone Miner.
Res.
10 Suppl. 1:5483, 1995 (hereby incorporated by reference herein).
Intracellular
~2+ increases were elicited by increasing extracellular Ca2+ from 1 to 1.75
n~IVI.
Intracellular Ca2f was measured using fluo-3, a fluorescent calcium indicator.
The procedure was as follows:
1. Cells were maintained in T-150 flasks in selection media (DMEM
supplemented with 109fo fetal bovine serum and 200 ug/mL hygromycin B), under
5% C02:95°.b air at
37 oC and were grown up to 90% confluency.
2. The medium was decanted and the cell monolayer was washed twice
with phosphate-buffered saline (PBS) kept at 37 oC. After the second wash, 6
mL
23
CA 02327188 2000-10-03
WO 99/51241 PCT/US99/07760
of 0.02% EDTA in PBS was added and incubated for 4 minutes at 37 oC.
Following the incubation, cells were dispersed by gentle agitation.
3. Cells from 2 or 3 flasks were pooled and pelleted (100 x g). The
cellular pellet was resuspended in 10-15 mL of SPRPCB+ and pelleted again by
centrifugation. This washing was done twice.
Sulfate- and phosphate-free parathyroid cell buffer (SPF-PCB) contains 20
mM Na-Hepes, pH 7.4, 126 mM NaCI, 5 mM KCI, and 1 mM MgCl2. SPF-PCB
was made up and stored at 4 oC. On the day of use, SPF-PCB was supplemented
with 1 mg/mL of D-glucose and 1 mM CaCl2 and then split into two fractions. To
one fraction, bovine serum albumin (BSA; fraction V, ICN) was added at 5 mg/mL
(SPF-PCB+). This buffer was used for washing, loading and maintaining the
cells.
The BSA-free fraction was used for diluting the cells in the cuvette for
measurements of fluorescence.
4. The pellet was resuspended in 10 mL of SPF-PCB+ containing 2.2 uM
fluo-3 (Molecular Probes) and incubated at room temperature for 35 minutes.
5. Following the incubation period, the cells were pelleted by
centrifugation. The resulting pellet was washed with SPF-PCB+. After this
washing, cells were resuspended in SPF-PCB+ at a density of 1-2 x 106
cells/nnL.
6. For recording fluorescent signals, 300 uL of cell suspension were
diluted in 1.2 mL of SPF buffer containing 1 mM CaCl2 and 1 mg/mL of
D-glucose. Measurements of fluorescence were performed at 37 oC with constant
stirring using a spectrofluorimeter. Excitation and emission wavelengths were
measured at 485 and 535 nm, respectively. To calibrate fluorescence signals,
digitonin (5 mg/mL in ethanol) was added to obtain Fmax, and the apparent Fmin
was determined by adding Tris-EGTA (2.5 M Tris-Base, 0.3 M EGTA). The
concentration of intracellular calcium was calculated using the following
equation:
Intracellular calcium = (F F~n/Fm~) x Kd; where Kd = 400 nM.
7. To determine the potential calcilytic activity of test compounds, cells
were incubated with test compound (or vehicle as a control) for 90 seconds
before
increasing the concentration of extracellular Ca2+ from 1 to 2mM. Calcilytic
compounds were detected by their ability to block, in a concentration-
dependent
manner, increases in the concentration of intracellular Ca2+ elicited by
extracellular Ca2+ .
24
CA 02327188 2000-10-03
WO 99/51241 PCT/US99/07760
In general, those compounds having lower IC50 values in the Calcium
Receptor Inhibitor Assay are more preferred compounds. Compounds having an
IC50 greater than 50 uM were considered to be inactive. Preferred compounds
are
those having an IC50 of lOuM or lower, more preferred compounds have an IC50
of luM, and most preferred compounds have an ICSp of O.IuM or lower.
(II) Calcium Receptor Binding Assay
HEK 293 4.0-7 cells stably transfected with the Human Parathyroid
Calcium Receptor("HuPCaR") were scaled up in T180 tissue culture flasks.
Plasma membrane is obtained by polytron homogenization or glass douncing in
buffer {50mM Tris-HCl pH 7.4, 1mM EDTA, 3mM MgCl2) in the presence of a
protease inhibitor cocktail containing luM Leupeptin, 0.04 uM Pepstatin, and 1
mM PMSF. Aliquoted membrane was snap frozen and stored at -80oC. 3H labeled
compound was radiolabeled to a radiospecific activity of 44Ci/mmole and was
aliquoted and stored in liquid nitrogen for radiochemical stability.
A typical reaction mixture contains 2 nM 3H compound ((R,R)-N-4'-
Methoxy-t-3-3=methyl-1=ethylphenyl-1-(1-naphthyl)ethylamine), or 3H compound
(R)-N-[2-Hydroxy-3-(3-chloro-2-cyanophenoxy)propyl]-1,1-dimethyl-2-(4-
methoxyphenyl~thylamine 4-10 ug membrane in homogenization buffer
containing 0.1 % gelatin and 10°k EtOH in a reaction volume of 0.5 mL.
Incubation
is performed in 12 x 75 polyethylene tubes in an ice water bath. To each tube
25
uL of test sample in 100% EtOH is added, followed by 400 uL of cold incubation
buffer, and 25 uL of 40 nM 3H-compound in 100% EtOH for a final concentration
of 2nM. The binding reaction is initiated by the addition of 50 uL of 80-200
ug/mL
HEK 293 4.0-7 membrane diluted in incubation buffer, and allowed to incubate
at
4oC for 30 min. Wash buffer is 50 mM Tris-HCl containing 0.1 % PEI.
Nonspecific binding is determined by the addition of 100-fold excess of
unlabeled
homologous ligand, and is generally 20% of total binding. The binding reaction
is
terminated by rapid filtration onto 1 % PEI pretreated GF/C filters using a
Brandel
Harvestor. Filters are placed in scintillation fluid and radioactivity
assessed by
liquid scintillation counting.
The following examples are illustrative, but not limiting of the
embodiments of the present invention.
CA 02327188 2000-10-03
WO 99/51241 PCT/US99/07760
I~~ple 1_
S Preparation of (R)-N-f2-Hvdroxy-3-(3-chloro-2-cyanophenoxY)propvl]-1 1
dimethyl-2-12.3-dihvdrobenzo[]~lfuran-Syl~tlwlamine Hydrochloride
5-(2-Amino-2-methylprQ,pv1)-2 3-dihydrob~nzo 1[~
Sodium hydride (0.898, 37.1 mmole) was added to 45 mL of DMSO and
stirred for 30 min at room temperature. Isopropyltriphenylphosphonium iodide
(16.OSg, 37.1 mmole) was then added and stirred for 1.5 hours followed by the
addition of 2,3-dihydrobenzo[b]furan-5-carboxaldehyde (S.Og, 33.75 mmole).
This
mixture was stirred for 18 hours at room temperature then poured into water
(300
mL) / conc. HCl (5 mL) and extracted with ether. The crude product was
chromatographed on silca gel in 1% MeOH/CHC13 to afford 5.1 g (87%) of 5-(2-
methylpropenyl)-2,3-dihydrobenzo[b]furan , which was 100% pure by GC-MS.
To a OoC suspension of sodium cyanide ( 1.44g, 29.3 mmole) in 6 mL of acetic
acid
was slowly added a 0°C solution of sulfuric acid (3.2 mL) in acetic
acid (3.2 mL).
After stirring for 45 min at OoC, 5-(2-methylpropenyl)-2,3-
dihydrobenzo[b]furan
(S.lg, 29.3 mmole) was added, and the mixture allowed to warm to room
temperature while stirring for 18 hours. The reaction was poured into ice/
NaOH
and extracted with ether. The ether layer was dried over sodium sulfate, then
concentrated in vacuo. The crude amidated product was taken up in EtOH/NaOH
and refluxed for 24 hours. The ethanol was removed in vacuo, and the residue
taken up in ether and water. The ether layer was separated, dried over sodium
sulfate and concentrated in vacuo to yield the crude amine as a dark oil. The
product was purified by short-path distillation at reduced pressure.
(R)-N-[2-Hydroxy-3-(3-chloro-2-cyanophenoxy)propyl]-1,1-dimethyl-2-(2,3-
dihydrobenzo[b]furan-Syl)ethylamine Hydrochloride
Using previously described methods, (R)-3-chloro-2-cyanophenyl glycidyl
ether (0.398 g, 1.9 mmol) and 5-(2-amino-2-methylpropyl)-2,3-
dihydrobenzo[b]furan (0.382 g, 2.0 mmol) were used to prepare 100 mg of the
title
compound as a white solid. 1H-NMR (CDC13) ~ 9.65 (1H, m), 8.13 (1H, m), 7.4
26
CA 02327188 2000-10-03
WO 99/51241 PGT/US99/077b4
(1H, t}, 7.05 (2H, d), 6.92 (2H, d), 6.65 (1H, d), 5.7 (1H, d), 4.77 (1H, br
m), 4.53
(2H, t), 4.25 (2H, d), 3.4 (2H, m), 3.1 (4H, m), 1.4 (6H, d).
Example _2
S
Pre aration,~~ (R)-N-[2-~y,~~-3-(3-chloro-2-cyanophenoxvlpropyll 1 1
dimethvl-~-(auin~lin-3-yl)ethvlamine Dihvdrochloride
3-f2-Amino-2-methyl~pylZquinoline
To a solution of 3-quinolinecarboxaldehyde ( 10.27g, 65.3 mmole) in 10
mL of pyridine and 30 mL of ethanol was added methoxylamine hydrochloride (6.0
g, 71.9 mmole). After stirring for 2 hours the solvents were removed under
reduced pressure, and the residue taken up in ether and water. The ether layer
was
separated, dried over sodium sulfate and concentrated in vacuo. The crude
oxime
(11.91 g, 63.9 mmole) was dissolved in 120 mL of trifluoroacetic acid and
treated
with zinc powder ( 13.0 g, 199 mmole). After 10 min, the reaction refluxed
spontaneously for a few seconds, and the mixture was stirred for another 3
hours.
The mixture was poured into water, and washed with ether. The aqueous layer
was
then made basic with NaOH, and the amine extracted into ether. The ether layer
was separated, dried over sodium sulfate and concentrated in vacuo to yield
8.68 g
of 3-(aminomethyl)quinoline. To this amine (8.68 g, 54.9 rnmole), dissolved in
200
mL of dichloromethane, was added 2,4,6-triphenylpyrylium tetrafluoroborate
( 19.56 g, 49.4 mmoIe), and the reaction stirred at room temperature for 48
hours.
The solids were filtered off, and the resulting solution concentrated in vacuo
to give
25.4 g ($6.3°x) of the crude N-(3-quinolinylmethyi)-2,4,6-
triphenylpyridinium
tetrafluoroborate salt. A solution of this salt (25.4 g, 47.4 mmole) in 100 mL
of
DMSO was added to the sodium salt of 2-nitropropane ( 142.1 mmole) (made by
adding sodium hydride (3.41 g, 142 mmole) to 50 mL of methanol followed by
addition of 2-nitropropane (12.66 g, 142.1 mmole), then removing the methanol
in
vacuo). The reaction was stirred for 24 hours at 100 C then cooled and diluted
with
ether and aqueous HCI. The aqueous layer was separated, made basic with NaOH,
and extracted with ether. The ether layer was dried over sodium sulfate, and
concentrated in vacuo to give after purification on silica gel (in chloroform)
10.7 g
27
CA 02327188 2000-10-03
WO 99/51241 PCTNS99/077b0
(98°!0) of 3-(2-vitro-2-methylpropyl)quinoline. To this vitro compound
(10.7 g,
47.2 mmole) dissolved in 100 mL of trifluoroacetic acid was slowly added zinc
powder (9.3 g, 142 mmole). Stirred for 24 hours at room temperature. The
reaction mixture was then poured into water and washed with ether. The aqueous
layer was separated, made basic with NaOH, and extracted with ether. The ether
layer was dried over sodium~sulfate, and concentrated in vacuo to give 4.5 g
(4886)
of 3-(2-amino-2-methylpropyl)quinoline. GC/EI-MS, m/z, (rel. int.) 185 (M' -
15,
3), 143 (58), 115 (1I), 58 (100), 42 (7).
(R)-N-[2-Hydroxy-3-(3-chloro-2-cyanophenoxy)propyl]-1,1-dimethyl-2-(quinolin-
3-yl)ethylamine Dihydrochloride
Using previously described methods, (R)-3-chloro-2-cyanophenyl glycidyl
ether (0.398g, 1.9 mmol) and 3-(2-amino-2-methylpropyl)quinoline (0.401 g, 2.0
mmol) were used to prepare 130 mg of the title compound as a white solid. 1H-
NMR
(CDC13) ~ 9.7 ( 1 H, br t), 9.25 ( 1 H, s), 9.0 (2H, br s), 8.42 ( 1 H, d),
8.37 ( 1 H, d), 8.1
(1H, dd), 7.93 (1H, dd), 7.65 (1H, dd), 7.35 ( 1H, d), 7.28 (1H, d), 4.3 (3H,
m), 3.45
(2H, s), 3.3 (2H, m), 2.5 ( 1H, s), 1.4 (6H, s).
Examu~
Py,~ation of (Rl-N-f2-Hydroxv-~3-chloro-2-cyanophenoxx)oropvl] 1 1
dimethy,~-2-(auinolin-2-yl)ethyrlamine Dihvdrochloride
2-(2-Amino-2-meth3rlpropv~u' olive
Using the method of Example 2, supra, 2-(2-amino-2-
methylpropyl)quinoline was prepared from quinoline-2-carboxaldehyde. GC/EI-
MS, m/z, (rel. int.) 185 (M' - 15, 5), 143 (42), 115 (13), 58 (100), 42 (6).
(R)-N-[2-Hydroxy-3-(3-chloro-2-cyanophenoxy)propyl]-1,1-dimethyl-2-(quinolin-
2-yl~thylamine Dihydrochloride
Using previously described methods (R)-3-chloro-2-cyanophenyl glycidyl
ether (0.21 g, 1.0 mmol) and 2-(2-amino-2-methylpropyl)quinoline (0.24 g, 1.2
mmol) were used to prepare 24 mg of the title compound as a white solid. 1H-
NMR
(CDC13) ~ 9.7 (1H, m), 9.3 (1H, m), 8.55 (1H, d), 8.39 (1H, d), 7.83 (1H, d),
7.7
28
CA 02327188 2000-10-03
WO 99/51241 PCT/US99/07760
(2H, m), 7.53 ( 1 H, m), 7.15 { 1 H, t), 6.75 (2H, m), 4.3 ( 1 H, m), 4.0 (2H,
m), 3.65
(2H, dd), 3.15 (2H, m), 1.3 (6H, d).
~~le 4
~reep~ lion of ,$ -) Nf2-Hydroxv-3 y3-ch~oro-2-cyano~hgnoxy~oro~vl]-1 1-
dimethy~-2-(is~o uinolin-3-yjkthylamine Dihydrochloride
3-l2-Amino-2-meth~nrooyl)is~quinoline
Using the method of Example 2, supra, 3-(2-amino-2-
methylpropyl)isoquinoline was prepared from isoquinoline-3-carboxaldehyde.
GC/EI-MS, rnlz, (rel. int.) 185 (M' - 15, 7), 144 (13), 143 (100), 116 (9),
115 (22),
58 (47), 42 (8).
(R)-N-[2-Hydroxy-3-(3-chloro-2-cyanophenoxy)propyI]-1,1-dimethyl-2-
(isoquinolin-3-yi)ethylamine Dihydrochloride
Using previously described methods {R)-3-chloro-2-cyanophenyl glycidyl ether
(0.478, 2.24 mmol) and 3-(2-amino-2-methylpropyl)isoquinoline (0.49 g, 2.45
mmol) were used to prepare 200 mg of the title compound as a light yellow
solid.
1 H-NMR (CDCI3) ~ 9.7 (2H, s on top of m), 9.25 ( 1 H, m), 8.45 ( 1 H, d),
8.27 ( 1 H,
s), 8.2 (1H, d), 8.1 (1H, t), 7.9 (1H, t), 7.68 (1H, t), 7.35 (1H, d), 7.25
(1H, d), 4.39
(1H, m), 4.3 (2H, s), 3.57 (2H, dd), 3.3 (2H, m), 1.4 (6H, d).
Facample 5
P,~e,~aration of,~R)-N-f2-1[~,vdroxy-3 l -chloro-2-c,~ranophr~o~y)proRvll-1 1-
dimet~, I~Qy~~,vl)but~lamine Di~,vdrochloride
4-Benzvloxvcarbonyiamino-4-methyl~gnt- j -ene
The 2,2-dimethyl-4-pentenoic acid (20.7g, 162 mmoles) was dissolved in
300 mL of benzyl alcohol followed by addition of triethylamine ( 17.988, 178
mmoles). biphenyl phosphorylazide (46.678, 170 mmoles) was added and the
reaction heated to 100oC overnight undcr nitrogen. The product was separated
from
29
CA 02327188 2000-10-03
WO 99151241 PCT/US99/07760
the excess benzyl alcohol by distillation. The product distilled at approx.
130°C C~?
0.01 mm.
9-(4-Benzyloxycarbonylamino-4-methylpentyl)-9-borabicyclo[3.3.1 ]nonane
To a 0.5 M solution of 9-BBN in THF (100 mL, 50 mmole) was added 4-
benzyloxycatbonylamino-4-methylpent-1-ene (I 1.b7 g, 50 mmole). The reaction
was allowed to stand for 24 hours at room temperature. Analysis by GC-MS
showed no starting alkene left. The solution, which was approximately 0.439 M
in
the borane, was used without purification.
2-(4-Amino-4-methylpentyl)pyridine
To 2-bromopyridine (0.948 g, 6 mmole) was added 9-(4-benzyloxycarbonylamino-
4-methylpentyl~9-borabicyclo[3.3.1]nonane (12 mL, 5 mmole of a THF solution)
in a nitrogen flushed reaction tube. To this solution was added 0.122 g (0.15
mmole) of [1,1'-bis(diphenylphosphino)ferrocene]dichloropalladium(II)
dichloromethane complex ( 1:1 ), 1.38 g ( 10 mmole) of potassium carbonate,
and
1.25 mL of water. The reaction was stirred for 18 hours at 65 C, then poured
into
aqueous NaOH, and extracted with ether. The ether layer was separated, washed
with brine, dried over sodium sulfate, and concentrated in vacuo. The crude
product was taken up in 20 mL of ethanol to which 1 g of palladium hydroxide
on
carbon (i0%) was added. The mixture was stirred for 18 hours under a hydrogen
balloon. The reaction mixture was filtered and concentrated in vacuo. The
residue
was taken up in aqueous HCI, and extracted with ether. The aqueous layer was
separated, made basic with NaOH, and extracted with ether. The ether layer was
dried over sodium sulfate, and concentrated in vacuo to give 0.63 g of 2-(4-
Amino-
4-methylpentyl)pyridine.
(R)-N-[2-Hydroxy-3-(3-chloro-2-cyanophenoxy)propyi]-1,1-dimethyl-4-(2-
pyridyl)butylamine Dihydrochloride
Using previously described methods {R)-3-chloro-2-cyanophenyl glycidyl
ether (0.21g, 1.0 mmol) and 2-(4-Amino-4-methylpentyl)pyridine (0.196 g, 1.1
mmol) were used to prepare 19 mg of the title compound as a light yellow
solid.
LGMS1MS* (In-Source ISD technique), m/z, 388 (M~, 227, 162, 106.
CA 02327188 2000-10-03
WO 99/51241 PCT/US99l07760
Examp~6
Preparation of (R)-N-f2-Hydroxy-3-f3-chloro-2-cy~no-4-
m_ orpholinosulfonamidophenoxy)propvll-1.1-dimethyl-4-(2-pvridyl)butylamine
Dihvdrochlori a
Using the method of Example 5, supra, (R)-3-chloro-2-cyano-4-
morpholinosulfonamidophenyl glycidyl ether (0.3g, 0.83 mmol) and 2-(4-Amino-4-
methylpentyl)pyridine (0.156 g, 0.87 mmol) were used to prepare 150 mg of the
title compound as an off white solid. 1H-NMR (CDC13) ~~9.3 (1H, m), 8.8 (2H, d
on top of m), 8.55 (1H, t), 8.18 (1H, d), 8.02 (1H, d), 7.95 (1H, dd), 7.5
(1H, d), 4.4
(3H, m), 3.6 (4H, br s), 3.1 (6H, m), 2.5 (2H* s), 1.8 (4H, m}, 1.3 (6H, s).
am I
~naration of lR)-N-f2-H droxv-3-f3-chloro-2-cxanophenoxv)oropyl~-1.1-
dimethyl-4-(3-R r~ l~lbutylamine i~ydrochloride
3-l4-Amino-4-methylpen~R n'
Using the method of Example 5, supra, 0.66 g of 3-(4-Amino-4-
methylpentyl)pyridine was prepared, starting with 6 mmoles of 3-bromopyridine.
(R)-N-[2-Hydroxy-3-(3-chloro-2-cyanophenoxy)propyl]-1, I-dimethyl-4-(3-
pyridyl)butylamine Dihydrochloride
Using previously described methods, (R)-3-chloro-2-cyanophenyl glycidyl
ether (0.218, 1.0 mmol) and 3-(4-Amino-4-methylpentyl)pyridine (0.196 g, 1.1
mmol) were used to prepare 25 mg of the title compound as a light yellow
glassy
solid. LGMS/MS* (In-Source ISD technique), m/z, 388 {M'), 227, 162, 106.
31
CA 02327188 2000-10-03
WO 99/51241 PCT/US99/077b0
Example _8
Prevaration of (R)-N-f 2-Hydroxy-3 J3-chloro-2-cyano-4-
~o~pholinosulfonamidophenoxv)nropyll-1.1-dimethy~~pvridyl)butylamine
Dihydrochloride
Using the method of Example 5, supra, (R)-3-chloro-2-cyano-4-
morpholinosulfonamidophenyl glycidyl ether (0.3g, 0.83 mmol) and 3-(4-Amino-4-
methylpentyl)pyridine (0.156 g, 0.87 mmol) were used to prepare 30 mg of the
title compound as an off white solid. 1H-NMR (CDCl3) ~ 9.3 (IH, m), 8.9 (1H,
s),
8.8 (2H, d on top of m), 8.56 ( 1 H, d), 8.16 ( 1 H, d), 8.04 ( 1 H, dd), 7.5
( 1 H, d), 4.4
(3H, m), 3.6 (4H, br s), 3.1 (4H, m), 2.8 (2H, br s), 2.5 (2H, s), 1.7 (4H,
m), 1.3
(6H, s).
Exam le
Preparation of lR)-N-f2-Hvdro~-3-(3-chloro-2-cvanoRhenoxy)propyll-4-
phenylbu~r_~,amine Hydrochloride
Using previously described methods, (R)-3-chloro-2-cyanophenyl glycidyl
ether (0.21g, 1.0 mmol) and 4-phenylbutylamine (0.164 g, 1.1 mmol) were used
to
prepare 250 mg of the title compound as a white solid. 'H NMR {CDCl3) d 10.09
(1H, s), 9.38 (1H, s), 9.12 (IH, s), 7.53 (2H, m), 7.19 (7H, m), 6.08 (1H, m),
5.98
1H, m), 4.63 (1H, m), 4.52 (1H, m), 4.23 (3H, m), 3.95 (1H, m), 3.43 (1H, m),
3.20
(1H, m), 3.00 (1H, m), 2.65 (3H, m), 1.86 (2H, m), 1.71 (2H, m).
Example 10
Preparation of (R)-N-f2-Hydroxv-3-(3-~hloro-2-cvanophenoxy)propvll-1 1-
d~mettlyl-~2-carbethoxyphenyl)butyla'mine Hydrochloride
Ethyl 2-(4-Amino-4-meth~nentvl)benzoate
32
CA 02327188 2000-10-03
WO 99/51241 PCT/US99/07760
To ethyl 2-bromobenzoate (0.504 g, 2.2 mmole) in a nitrogen flushed
reaction tube was added 0.049 g (0.06 mmole) of [1,1'-
bis(diphenylphosphino)ferrocene]dichloropalladium(II) dichloromethane complex
( 1:1 ) dissolved in 2 mL of DMF. To this solution was added 1.3 g {4 mmoles)
of
cesium carbonate, followed by 9-(4-benzyloxycarbonylamino-4-methylpentyl)-9-
borabicyclo[3.3.1]nonane {4.56 mL, 2.0 mmole of a THF solution). The reaction
was stirred for 16.5 hours at 50 C, then poured into aqueous NaOH, and
extracted
with ether. The ether layer was separated, washed with brine, dried over
sodium
sulfate, and concentrated in vacuo. The crude product was taken up in 10 mL of
ethanol to which 0.3 g of palladium hydroxide on carbon (10°!0) was
added. The
mixture was stirred for 18 hours under a hydrogen balloon. The reaction
mixture
was filtered and concentrated in vacuo. The residue was taken up in aqueous
HCI,
and extracted with ether. The aqueous layer was separated, made basic with
NaOH,
and extracted with ether. The ether layer was dried over sodium sulfate, and
concentrated in vacuo to give crude ethyl 2-(4-amino-4-methylpentyl)benzoate.
The crude product was purified by reversed-phase HPLC on a C-18 column using a
gradient of 0.1 % HCl to 40% acetonitrile in 0.1 % HCI.
(R)-N-[2-Hydroxy-3-(3-chioro-2-cyanophenoxy)propyl]-1,1-dimethyl-4-(2-
carbethoxyphenyl)butylamine Hydrochloride
Using previously described methods, (R)-3-chloro-2-cyanophenyl glycidyl
ether (0.21g, 1.0 mmol) and 1,1-dimethyl-4-(2-carbethoxyphenyl)butylamine
(0.274 g, 1.1 mmol) were used to prepare 260 mg of the title compound as a
white
solid. 'H NMR (CDCl3) ~ 9.54 (1H, s), 8.17 (1H, m), 7.85 (1H, dd), 7.43 (2H,
m),
7.24 (2H, m), 7.06 (1H, d), 6.97 (1H, d), 6.00 (1H, d), 4.71 (1H, s), 4.33
(2H, q),
4.26 (2H, d), 3.27 (2H, m), 2.60 (2H, m), 1.85 (2H, m), 1.69 (2H, m), 1.46
(6H, s),
1.37 (3H, t).
Example 11
f~eoaration of (R)-N-f2-Hydroxv-3-(3-chloro-2-cvanop noxy)~Ryll-1 1-
dimethvl-4-(3-carbethoxyphenvl)butylamine Hy~ochloride
33
CA 02327188 2000-10-03
WO 99/51241 PCTNS99/07760
Using previously described methods, (R)-3-chloro-2-cyanophenyl glycidyl
ether (0.21g, 1.0 mmol) and 1,1-dimethyl-4-(3-carbethoxyphenyl)butylamine
(0.274 g, 1.1 mmol) were used to prepare 230 mg of the title compound as a
white
solid. 'H NMR (CDCI,) ~ 9.56 (1H, m), 8.21 (1H, m), 7.86 (2H, m), 7.37 (3H,
m),
7.07 ( 1 H, d), 6.94 ( 1 H, d), 5.59 ( 1 H, d), 4.70 ( 1 H, m), 4.35 (2H, q),
4.24 (2H, d), '
3.25 (2H, m), 2.71 (2H, m), 1.82 (4H, m), 1.49 (6H, s), 1.39 (3H, t).
Example 12
PreQaration of (R)-N-f2-~rdroxy-3-(,3-chloro-2-cvano hn enox,y)propyll 1 1
dimethvl-4-(4-carbethoxyphenvl)butvlamine Hydrochloride
Using the method of Example 5, supra, (R)-3-chloro-2-cyanophenyl
glycidyl ether (0.21g, 1.0 mmol) and 1,1-dimethyl-4-(4-
carbethoxyphenyi)butylamine (0.274 g, 1.1 mmol) were used to prepare 250 mg of
the title compound as a white solid. 'H NMR (CDCI,) ~ 9.56 (1H, m), 8.19 (1H,
m), 7.95 (2H, d), 7.43 ( 1 H, ddd), 7.26 (2H, d), 7.07 ( 1 H, d), 6.93 ( 1 H,
d), 5.58
(1H, d), 4.69 (1H, m), 4.33 (2H, q), 4.22 (2H, d), 3.23 (2H, m), 2.71 (2H, m),
1.48
(3H, s), 1.47 (3H, s), 1.37 (3H, t).
E~ple 13
Preparation of (RAN-f2-Hy~l~roxy-3-(3-chloro-2-cyan henoxy)propyll-1 1
di~e~h~l-2-(4-ethylp, ny 'd-Z-yllethylamine Dihydrochloride
1 1-Dimet yl-2-l4-ethvlpyrid-2- lv )eth~yl~r_r~ine
4 mmoles 5-ethyl-2-methyl pyridine in 4 mL dry ether was treated with
4.32 mmoles of phenyl lithium ( 1.8 M solution in cyclohexane/ether) at
0°C. After
reaction at RT for 1 h the solution was added dropwise to a chilled (ice bath)
solution of 2 mmoles isopropylidene-3-nitrobenzene sulfenamide in 2 mL dry
ether. After reaction at RT for 1 hr and at reflux for 0.5 hrs the cooled
reaction
mixture was quenched with 5 mls water. The organic layer was extracted three
34
CA 02327188 2000-10-03
WO 99/51241 PCT/US99l07760
times with 6 Molar HCl . The pooled HCl extracts were evaporated to an oil,
made
basic with 10 N NaOH and extracted with ether. The ether was extracted twice
with
pH 7 Phosphate Buffer, buffer extracts made basic with NaOH and extracted with
chloroform. Removal of the chloroform resulted in the title compound in 24%
yield.
(R)-N-[2-Hydroxy-3-(3-chloro-2-cyanophenoxy)propyl]-1,1-dimethyl-2-(4-
ethylpyrid-2-yl)ethylamine Dihydrochloride
Using previously described methods, (R}-3-chloro-2-cyanophenyl glycidyl
ether (0.21g, 1.0 mmol) and 1,1-Dimethyl-2-(4-ethylpyrid-2-yl)ethylamine (0.25
g,
1.4 mmol) were used to prepare 314 mg of the title compound as a white solid.
1H-
NMR (CDC13) ~ 9.95 ( 1 H, br s), 9.05 ( 1 H, br s), 8.62 ( 1 H,s), 8.27 ( 1 H,
br s), 8.02
(1H, br s), 7.44 (1H, t), 7.0 (2H, d), 5.6 (2H, br s), 4.7 (1H, m), 4.34 (2H>
br s), 3.9
(2H, br s), 3.53 (2H, br s) , 2.86 (2H, q), 1.62 (6H, s), 1.32 (3H, t).
Exam lp a 14
(R)-N-f2-Hvdroxy-3-(3-chloro-2-cyanophenoxy,~p~,pyll 1 1-dimethyl 2
b~nzamidoethylamine Hy rochlo
Using previously described methods, (R)-3-chloro-2-cyanophenyl glycidyl
ether (O.OOg, 00 mmol) and 1,1-Dimethyl-2-benzamidoethylamine (00 g, 00 mmol)
were used to prepare 000 mg of the title compound as a white solid. 1H-NMR
(CDCIf) ~ 9.52 (1H, m), 8.40 (1H, m), 8.20 (1H, s) 7.95 (1H, d), 7.36 (4H, m),
7.02
( 1 H, d), 6.82 ( 1 H, d), 4.70 ( 1 H, m), 4.16 (2H, m), 3.82 (3H, m), 3.37
(2H, m), 1.51
(3H, s), 1.46 (3H, s); "C NMR (CDCh) ~~169.2, 161.3, 137.7, 134.7, 133.1,
131.9,
128.5, 127.8, 122.3, 113.8, 110.8, 103.2, 77.2, 70.8, 65.5, 61.4, 46.4, 44.8,
22.0,
21.6.
Example 15
(R)-N-12-Hvdroxv-3-(3-chloro-2-cvano henoxy)pro~vll-1 1-dimethvl-4-phenyl ut-
2-ynylamine Hydrochloride
CA 02327188 2000-10-03
WO 99/51241 PCT/t3S99/07760
Using previously described methods, (R)-3-chloro-2-cyanophenyl glycidyl
ether (O.OOg, 00 mmol) and 1,1-Dimethyl-4-phenylbut-2-ynylamine (00 g, 00
mmol) were used to prepare 000 mg of the title compound as a white solid. 1H-
NMR (CDCI,) ~~9.97 (1H, m), 8.76 (1H, m), 7.41 (1H, ddd), 7.30 (3H, m), 7.18
(2H, m), 7.04 ( 1 H, d), 6.92 ( 1 H, d), 5.63 ( 1 H, m), 4.76 ( 1 H, m), 4.23
(2H, m), 3.51
(2H, m), 1.85 (3H, s), 1.83 (3H, s); "C NMR (CDC13) ~ 161.4, 137.8, 135.7,
134.5,
128.2, 127.9, 126.8, 122.2, 113.5, 110.9, 103.4, 86.8, 79.0, 70.9, 65.3, 55.5,
46.8,
26.8, 26.7, 24.7.
Exam lie 16
Preparation of 1 I-dimethvl-2-ftethyl-4ioxl~~atel-nh~wllethylamine
A mixture of 1-nitro-1,1-dimethyl-2-(4-hydroxyphenyl]ethane (3.9g,
0.02mole), K2C03 (2.76g, 0.02mole) and ethylbromoacetate (3.06g, 0.02mole)
were refluxed in 75m1 of acetone for 18h. The reaction was cooled to room
temperature and filtered. The filtrate was concentrated in vacuo to yield 5.5g
of an
oil. This oil was dissolved in 75 ml of EtOH and 1 large spatula-full of
washed
Raney-nickel was added under argon. The nnixture was hydrogenated at room
temperature and 55 psi for 18 H. The reaction was filtered and the filtrate
concentrated in vacuo to an oil which was filtered through a pad of silica gel
eluting with 10% MeOH-CH2C12 (v/v). The first 200 ml were combined and
concentrated in vacuo to yield 3.2g of a pale yellow oil. MS, mlz 252 (M+H),
503
(2M+H).
Formulations for pharmaceutical use incorporating compounds of the
present invention can be prepared in various forms and with numerous
excipients.
Examples of such formulations are given below.
Example 17
Inhalant ~ormula,~tion
A compound of Formula (I) ( 1 mg to 100 mg) is aerosolized from a metered dose
inhaler to deliver the desired amount of drug per use.
36
CA 02327188 2000-10-03
WO 99/51241 PCT/US99/07760
Exam~~g 18
Tablet Formulation
TabletsJlngr~dients Per let
1. Active ingredient 40 mg
(Cmp. of Formula(I))
2. Corn Starch 20 mg
3. Alginic acid 20 mg
4. Sodium Alginate 20 mg
5. Mg stearate 13 mg
Procedure for tablet formulation
Ingredients 1, 2, 3 and 4 are blended in a suitable mixerlblender. Sufficient
water is added portion-wise to the blend with careful mixing after each
addition
until the mass is of a consistency to permit its conversion to wet granules.
The wet
mass is converted to granules by passing it through an oscillating granulator
using a
No. 8 mesh (2.38 mm) screen. The wet granules are then dried in an oven at
140°F
(60°C) until dry. The dry granules are lubricated with ingredient No.
5, and the
lubricated granules are compressed on a suitable tablet press.
Example 19
Parenteral Formulation
A pharmaceutical composition for parenteral administration is prepared by
dissolving an appropriate amount of a compound of Formula (I) in polyethylene
glycol with heating. This solution is then diluted with water for injections
(to 100
mi). The solution is then rendered sterile by filtration through a 0.22 micron
membrane filter and sealed in sterile containers.
All publications, including but not limited to patents and patent
applications cited in this specification are herein incorporated by reference
as if
each individual publication were specifically and individually indicated to be
incorporated by reference as though fully set forth.
37
