Note: Descriptions are shown in the official language in which they were submitted.
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DESCRIPTION
Inorganic Ion Receptor-Active Compounds
FIELD OF THE INVENTION
This invention relates to compounds able to modulate one
or more inorganic ion receptor activities.
BACKGROUND OF THE INVENTION
The references provided herein are not admitted to be
prior art to the claimed invention.
Certain cells in the body respond not only to chemical
signals, but also to ions such as extracellular calcium ions
(Ca2'). Extracellular Caz' is under tight homeostatic control
and regulates various processes such as blood clotting, nerve
and muscle excitability, and proper bone formation.
Calcium receptor proteins enable certain specialized
cells to respond to changes in extracellular Caz
concentration. For example, 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.
PTH is the principal endocrine factor regulating Ca2'
homeostasis in the blood and extracellular fluids. PTH, 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 PTH
secretion forms an important mechanism maintaining bodily Ca2.
homeostasis.
Extracellular Caz' acts directly on parathyroid cells to
regulate PTH secretion. The existence of a parathyroid cell
surface protein which detects changes in extracellular Ca2.
has been confirmed. (Brown et al., Nature 366:574, 1993.)
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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' can exert effects on different cell
functions, reviewed in Nemeth et al., Cell Calcium 11:319,
1990. The role of extracellular Ca2' in parafollicular (C-
cells) and parathyroid cells is discussed in Nemeth, Cell
Calcium 11:323, 1990. These cells were shown to express
similar calcium receptors. (See Brown et al., Nature
366:574, 1993; Mithal et al., J. Bone Miner. Res. 9, Suppl.
1, s282, 1994; Rogers et al., J. Bone Miner. Res. 9, Suppl,
1, s409, 1994; Garrett et al., Endocrinology 136:5202-5211,
1995. )
The ability of various molecules to mimic extracellular
Ca2' in vitro is discussed in references such as Nemeth et
al., in ~~Calcium-Binding Proteins in Health and Disease,~~
1987, Academic Press, Inc., pp. 33-35; Brown et al.,
Endocrinology 128:3047, 1991; Chen et al., J. Bone Miner.
Res. 5:581, 1990; and Zaidi et al., Biochem. Biophys. Res.
Commun. 167:807, 1990.
Nemeth et al., PCT/US92/07175, International Publication
Number WO 93/04373, Nemeth et al., PCT/US93/01642,
International Publication Number WO 94/18959, and Nemeth et
al., PCT/US94/12117, International Publication Number WO
95/11211, describe various compounds which can modulate the
effect of an inorganic ion receptor.
SUMMARY OF THE INVENTION
The present invention features compounds able to
modulate one or more activities of an inorganic ion receptor
and methods for treating diseases or disorders using such
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compounds. Preferred compounds can mimic or block the effect
of extracellular calcium on a cell surface calcium receptor.
Inorganic ion receptor activities are those processes
brought about as a result of inorganic ion receptor
activation. Such processes include the production of
molecules which can act as intracellular or extracellular
messengers.
Inorganic ion receptor-modulating compounds include
ionomimetics, ionolytics, calcimimetics, and calcilytics.
l0 Ionomimetics are compounds which mimic (i.e., evoke or
potentiate) the effects of an inorganic ion at an inorganic
ion receptor. Preferably, the compound affects one or more
calcium receptor activities. Calcimimetics are ionomimetics
which affect one or more calcium receptor activities.
. Ionolytics are compounds which block (i.e., inhibit or
diminish) one or more activities caused by an inorganic ion
at an inorganic ion receptor. Preferably, the compound
affects one or more calcium receptor activities. Calcilytics
are ionolytics which block one or more calcium receptor
activities evoked by extracellular calcium.
Ionomicnetics and ionolytics may bind at the same
receptor site as the native inorganic ion ligand binds or can
bind at a different site (e.g., ~an allosteric site). For
example; NPS R-467 binding to a calcium receptor results in
.calcium receptor activity and, thus, NPS R-467 is classified
as a calcimimetic. However, NPS R-467 binds to the calcium
receptor at a different site (i.e., an allosteric site) than
extracellular calcium.
A measure of the effectiveness of a compound to modulate
receptor activity can be determined by calculating the ECso or
ICSO for that compound. The ECso is the concentration of a
compound which causes a half-maximal mimicking effect. The
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ICSO is the concentration of a compound which causes a half-
maximal blocking effect. ECso and ICso values for compounds at
a calcium receptor can be determined by assaying one or more
of the activities of extracellular calcium at a calcium
receptor. Examples of assays for measuring ECso and ICso
values are described Nemeth et al., PCT/US93/01642,
International Publication Number WO 94/18959, and Nemeth et
al., PCT/US92/07175, International Publication Number WO
93/04373 and below. Such assays include oacyte
expression assays and measuring increases in intracellular
calcium ion concentration ([Ca'~]i) due to calcium receptor
activity. Preferably, such assays measure the release or
inhibition of a particular hormone associated with activity
of a calcium receptor.
An inorganic ion receptor-modulating compound preferably
selectively targets inorganic ion receptor activity in a
particular cell. For example, selective targeting of a
calcium receptor activity is achieved by a compound exerting
a greater effect on a calcium receptor activity in one cell
type than at another~cell type for a given concentration of
compound. Preferably, the differential effect is 10-fold or
greater as measured in vivo or in vitxo. More preferably,
the differential effect is measured in v~ vo and the compound
concentration is measured as the plasma concentration or
extracellular fluid concentration and the measured effect is
the production of extracellular messengers such as plasma
calcitonin, parathyroid hormone, or plasma calcium. Fox
example, in a preferred embodiment, the compound selectively
targets PTH secretion over calcitonin secretion.
Preferably, the compound is either a calcimimetic or
calcilytic having an ECSO or an ICso at a calcium receptor of
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less than or equal to 5 ~M, and even more preferably less
than or equal to 1 ,uM, 100 nmolar, l0 nmolar, or 1 nmolar
using one of the assays described below. More preferably,
the assay measures intracellular Ca2+ in HEK 293 cells
5 transformed with nucleic acid expressing the human
parathyroid calcium receptor and loaded with fura-2. Lower
ECso or ICso values are advantageous since they allow lower
concentrations of compounds to be used in vivo or in vitro.
The discovery of compounds with low ECso and ICso values
enables the design and synthesis of additional compounds
having similar or improved potency, effectiveness, and/or
selectivity.
Thus, a first aspect the invention features an inorganic
ion receptor-modulating compound having the formula:
STRUCTURE I
N Ar2
Are ~ G
:.
R~ R2 Rs
wherein Arl is either optionally substituted naphthyl,
optionally substituted phenyl, or an optionally substituted
heterocyclic aryl, where up to 5 substituents may be present
and each substituent is independently selected from the group
consisting of: alkyl, alkenyl, halogen, alkoxy, thioalkyl,
methylene dioxy, haloalkyl, haloalkoxy, OH, CHzOH, CONHz, CN,
acetoxy, N(alkyl)z, phenyl, phenoxy, benzyl, benzyloxy, a,a-
dimethylbenzyl, N02, CHO, CH3CH(OH), acetyl, OCHZCOOH, and
ethylene dioxy;
Arz is either optionally substituted naphthyl,
optionally substituted phenyl, or an optionally substituted
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heterocyclic aryl, where up to 5 substituents may be present
and each substituent is independently selected from the group
consisting of: alkyl, alkenyl, halogen, alkoxy, thioalkyl,
methylene dioxy, haloalkyl, haloalkoxy, OH, CHzOH, CONHz, CN,
OCH2COOH, ethylene dioxy, and acetoxy;
q is 0, 1, 2, or 3;
Rl is either H or alkyl; and
RZ and R3 are each independently either hydrogen, alkyl,
or together cycloalkyl or cycloalkenyl;
and pharmaceutically acceptable salts and complexes
thereof.
Preferably, the compound is an ionomimetic which
modulates one or more inorganic ion receptor activities, more
preferably the compound is a calcimimetic.
"Alkenyl" refers to a hydrocarbon chain having 2-6
carbons and at least one double-bond which may be a straight
chain, branched, or non-aromatic cyclic. Preferably, the
alkenyl has 2-4 carbon atoms.
"Alkyl" refers to a saturated hydrocarbon having 1-6
carbons which may be a straight chain, branched, or cyclic.
Preferably, the alkyl has 1-4 carbon atoms.
"Alkoxy" refers to "O-alkyl," where "O" is an oxygen
joined to an alkyl.
"Cycloalkenyl" refers to a non-aromatic cyclic
hydrocarbon chain having 3-12 carbons and at least one
double-bond, and includes multiple ring structures.
Preferably, the cycloalkenyl has 3 to 6 carbon atoms.
"Cycloalkyl" refers to a saturated cyclic hydrocarbon
chain having 3-12 carbons, and includes multiple ring
structures. Preferably, the cycloalkyl has 3 to 6 carbon
atoms.
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"Thioalkyl" refers to "S-alkyl," where "S" is a sulfur
joined to an alkyl.
"Haloalkyl" refers to an alkyl substituted with at least
one halogen. Preferably, only the terminal carbon of the
haloalkyl is substituted with a halogen and 1 to 3 halogens
are present. More preferably, the haloalkyl contains 1
carbon. Preferably, the halogen substitutions are either C1
or F.
"Haloalkoxy" refers to "O-haloalkyl," where "O" is an
oxygen joined to a haloalkyl.
"Heterocyclic aryl" refers to an aryl ring system having
1 to 3 heteroatoms as ring atoms in a heteroaromatic ring
system and the remainder of the ring atoms are carbon atoms.
Suitable heteroatoms include oxygen, sulfur, and nitrogen.
Preferably, the heterocyclic ring system is mono- or
bicyclic. More preferably, the heterocyclic aryl is either
furanyl, thiofuranyl (also known as "thienyl"), benzofuranyl
or benzothiofuranyl (also known as "benzothienyl").
Another aspect of the present invention features an
inorganic ion receptor-modulating compound having the
formula:
STRUCTURE II
R8
H
Are ~ N Ar2
R~ Rs Rz Rs
Where Arl, Arz, RZ and R, are as described for Structure I
compounds;
- R-, is either hydrogen, alkyl or phenyl;
RB is either hydrogen, or alkyl;
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R9 is either hydrogen, alkyl or phenyl;
and pharmaceutically acceptable salts and complexes thereof.
Preferably, the compound is an ionomimetic modulating
one or more inorganic ion receptor activities, more
preferably the compound is a calcimimetic.
Another aspect of the present invention features a
pharmaceutical composition made up of an inorganic ion
receptor-modulating compound described herein and a
physiologically acceptable carrier. A ~~pharmacological
composition~~ refers to a composition in a form suitable for
administration into a mammal, preferably a human.
Preferably, the pharmaceutical composition contains a
sufficient amount of a calcium receptor-modulating compound
in a proper pharmaceutical form to exert a therapeutic effect
on a human.
Considerations concerning forms suitable for
administration are known in the art and include toxic
effects, solubility, route of administration, and maintaining
activity. For example, pharmacological compositions injected
into the blood stream should be soluble.
Pharmaceutical compositions can also be formulated as
pharmaceutically acceptable salts (e. g., acid addition salts)
and complexes thereof. The preparation of such salts can
facilitate the pharmacological use of a compound by altering
its physical characteristics without preventing it from
exerting a physiological effect.
Another aspect the present invention features a method
for treating a patient by using inorganic ion receptor-
modulating compounds described herein. The method involves
administering to the patient a pharmaceutical composition
- containing a therapeutically effective amount of an inorganic
ion receptor-modulating compound. In a preferred embodiment,
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the disease or disorder is treated by administering to the
patient a therapeutically effective amount of a calcium
receptor-modulating compound.
Inorganic ion receptor-modulating compounds, and
compositions containing such compounds, can be used to treat
different types of patients. A "patient" refers to a mammal
in which compounds able to modulate inorganic ion receptor
activity will have a beneficial effect including a beneficial
prophylactic effect. Suitable patients can be diagnosed
using standard techniques known to those in the medical
profession.
Preferably, a patient is a human having a disease or
disorder characterized by one more of the following: (1)
abnormal inorganic ion homeostasis, more preferably abnormal
calcium homeostasis; (2) an abnormal level of a messenger
whose production or secretion is affected by inorganic ion
receptor activity, more preferably affected by calcium
receptor activity; and (3) an abnormal level or activity of a
messenger whose function is affected by inorganic ion
receptor activity, more preferably affected by calcium
receptor activity.
Diseases characterized by abnormal calcium homeostasis
include hyperparathyroidism, osteoporosis and other bone and
mineral-related disorders, and the like (as described, e.g.,
in standard medical text books, such as "Harrison's
Principles of Internal Medicine"). Such diseases are treated
using calcium receptor-modulating compounds which mimic or
block one or more of the effects of extracellular Caz' on a
calcium receptor.
By "therapeutically effective amount" is meant an amount
of a compound which relieves to some extent one or more
symptoms of a disease or disorder in the patient; or returns
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to normal either partially or completely one or more
physiological or biochemical parameters associated with or
causative of the disease or disorder. Thus, a
therapeutically effective amount can be an amount effective
5 to prophylactically decrease the likelihood of the onset of a
disease or disorder.
In a preferred embodiment, the patient has a disease or
disorder characterized by an abnormal level of one or more
calcium receptor-regulated components and the compound is
10 active on a calcium receptor of a cell selected from the
group consisting of: parathyroid cell, bone osteoclast,
juxtaglomerular kidney cell, proximal tubule kidney cell,
distal tubule kidney cell, central nervous system cell,
peripheral nervous system cell, cell of the thick ascending
limb of Henle's loop and/or collecting duct, keratinocyte in
the epidermis, parafollicular cell in the thyroid (C-cell),
intestinal cell, platelet, vascular smooth muscle cell,
cardiac atrial cell, gastrin-secreting cell, glucagon-
secreting cell, kidney mesangial cell, mammary cell, beta
cell, fat/adipose cell, immune cell, GI tract cell, skin
cell, adrenal cell, pituitary cell, hypothalamic cell, and
cell of the subfornical organ.
More preferably, the cells are chosen from the group
consisting of: parathyroid cell, central nervous system cell,
peripheral nervous system cell, cell of the thick ascending
limb of Henle's loop and/or collecting duct in the kidney,
parafollicular cell in the thyroid (C-cell), intestinal cell,
GI tract cell, pituitary cell, hypothalamic cell, and cell of
the subfornical organ.
In a preferred embodiment, the compound reduces the
- level of parathyroid hormone in the serum of the patient.
More preferably, the level is reduced to a degree sufficient
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to cause a decrease in plasma Ca2+. Most preferably, the
parathyroid hormone level is reduced to that present in a
normal individual.
Patients in need of treatment using the compounds
described by the present invention can be diagnosed by
standard medical techniques, such as blood or urine analysis.
Examples of such medical techniques include detecting a
deficiency of protein whose production or secretion is
affected by changes in inorganic ion concentrations, and by
detecting abnormal levels of inorganic ions or hormones which
effect inorganic ion homeostasis.
In further aspects, the invention provides uses of
the compounds or compositions of the invention for treating
or for preparing medicaments for treating patients as
described above.
The invention also provides a commercial package
comprising a compound or composition of the invention and
associated therewith instructions for the use thereof in
treating patients as described above.
Various examples are used throughout the
application. These examples are not intended in any way to
limit the claimed invention.
Other features and advantages of the invention
will be apparent from the following figures, detailed
description of the invention, examples, and the claims.
BRIEF DESCRIPTION OF THE DRAWING
Figure 1 provides the chemical structures of
different ionomimetic compounds.
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lla
Figure 2 provides the chemical structures of
different ionomimetic compounds.
Figure 3 provides the chemical structures of
different ionomimetic compounds.
Figure 4 provides the chemical structures of
different ionomimetic compounds.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
The present invention features compounds able to
modulate one or more inorganic ion receptor activities.
Preferably, the compounds can mimic or block an effect of an
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extracellular ion on a cell having an inorganic ion receptor,
more preferably, the extracellular ion is Ca2' and the effect
is on a cell having a calcium receptor. Most preferably, the
compounds can mimic the effect of extracellular Caz' on a cell
having a calcium receptor.
While the compounds described herein are believed to be
able to act at an inorganic ion receptor, preferably a
calcium receptor, unless otherwise explicitly stated in the
claims that a compound exerts an effect by acting at a
receptor, there is no intention to limit the claimed methods
to those requiring modulation of receptor activity. Rather,
the compounds are characterized by their ability to modulate
inorganic ion receptor activity in vivo or in vitro.
I.
Calcium receptors are present in different cells. The
pharmacological effects of the following cells, in response
to extracellular Cap', is consistent with the presence of a
calcium receptor: parathyroid cell, bone osteoclast,
juxtaglomerular kidney cell, proximal tubule kidney cell,
distal tubule kidney cell, central nervous system cell,
peripheral nervous system cell, cell of the thick ascending
limb of Henle~s loop and/or collecting duct, keratinocyte in
the epidermis, parafollicular cell in the thyroid (C-cell),
intestinal cell, trophoblast in the placenta, platelet,
vascular smooth muscle cell, cardiac atrial cell, gastrin-
secreting cell, glucagon-secreting cell, kidney mesangial
cell, mammary cell, endocrine and exocrine cells in the
pancreas, fat/adipose cell, immune cell, GI tract cell, skin
cell, adrenal cell, pituitary cell, hypothalamic cell, and
cell of the subfornical organ.
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The presence of a calcium receptor on the following
cells have been confirmed using physical data, such as
hybridization with nucleic acid encoding a calcium receptor:
_ parathyroid cell, central nervous system cell, peripheral
nervous system cell, cell of the thick ascending limb of
Henle's loop and/or collecting duct in the kidney,
parafollicular cell in the thyroid (C-cell), intestinal cell,
GI tract cell, pituitary cell, hypothalamic cell, cell of the
subfornical organ, and endocrine and exocrine cells in the
pancreas.
The calcium receptor on these different cell types may
be different. It is also possible that a cell can have more
than one type of calcium receptor. Comparison of calcium
receptor activities and amino acid sequences from different
cells indicate that distinct calcium receptor types exist.
For example, calcium receptors can respond to a variety of
di- and trivalent cations. The parathyroid cell calcium
receptor responds to calcium and Gd3', while osteoclasts
respond to divalent cations such as calcium, but do not
respond to Gd3t. Thus, the parathyroid cell calcium receptor
is pharmacologically distinct from the calcium receptor on
the osteoclast.
On the other hand, the nucleic acid sequences encoding
calcium receptors present in parathyroid cells and C-cells
indicate that these receptors have a very similar amino acid
structure. Nevertheless, calcimimetic compounds exhibit
differential pharmacology and regulate different activities
at parathyroid cells and C-cells. Thus, pharmacological
properties of calcium receptors may vary significantly
depending upon the cell type or organ in which they are
expressed even though the calcium receptors may have similar
or even identical structures.
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Calcium receptors, in general, have a low affinity
for extracellular Caz* (apparent Kd generally greater than
about 0.5 mM). Calcium receptors may include a free or bound
effector mechanism as defined by Cooper, Bloom and Roth, ~~The
Biochemical Basis of Neuropharmacology~~, Ch. 4, and are thus
distinct from intracellular calcium receptors, e.g.,
calmodulin and the troponins.
Calcium receptors respond to changes in extracellular
calcium levels. The exact changes depend on the particular
receptor and cell line containing the receptor. For example,
the in vitro effect of calcium on the calcium receptor in a
parathyroid cell includes the following:
1. An increase in internal calcium. The
increase is due to the influx of external calcium and/or to
mobilization of internal calcium. Characteristics of the
increase in internal calcium include the following:
(a) A rapid (time to peak < 5 seconds) and
transient increase in [Ca2*]i that is refractory to inhibition
by 1 lcM La'* or 1 /.cM Gd3* and is abolished by pretreatment with
ionomycin (in the absence of extracellular Caz*);
(b) The increase is not inhibited by
dihydropyridines;
(c) The transient increase is abolished by
pretreatment for 10 minutes with 10 mM sodium fluoride;
(d) The transient increase is diminished by
pretreatment with an activator of protein kinase C (PKC),
such as phorbol myristate acetate (PMA), mezerein or (-)-
indolactam V. The overall effect of the protein kinase C
activator is to shift the concentration-response curve of
calcium to the right without affecting the maximal response;
and
.___..~ _......__.._._._._.._ ..__._,___........ T
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(e) Pretreatment with pertussis toxin (100
ng/ml for > 4 hours) does not affect the increase.
2. A rapid (< 30 seconds) increase in the
formation of inositol-1,4,5-triphosphate or diacylglycerol.
5 Pretreatment with pertussis toxin (100 ng/ml for > 4 hours)
does not affect this increase;
3. The inhibition of dopamine- and isopro-
terenol-stimulated cyclic AMP formation. This effect is
blocked by pretreatment with pertussis toxin (100 ng/ml for >
10 4 hours); and
4. The inhibition of PTH secretion.
Pretreatment with pertussis toxin (100 ng/ml for > 4 hours)
does not affect the inhibition in PTH secretion.
Using techniques known in the art, the effect of calcium
I5 on other calcium receptors in different cells can be readily
determined. Such effects may be similar in regard to the
increase in internal calcium observed in parathyroid cells.
However, the effect is expected to differ in other aspects,
such as causing or inhibiting the release of a hormone other
than parathyroid hormone.
II. INORGANIC ION RECEPTOR-MODULATING COMPOUNDS
Inorganic ion receptor-modulating compounds modulate one
or more inorganic ion receptor activities. Preferred
inorganic ion receptor-modulating compounds are calcimimetics
or calcilytics. Inorganic ion receptor-modulating compounds
can be identified by screening compounds which are modeled
after a compound shown to have a particular activity (i.e., a
lead compound).
A preferred method of measuring calcium receptor
activity is to measure changes in [Caz~] i. Changes in [Ca2~l i
can be measured using different techniques such as by using
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HEK 293 cells transduced with nucleic acid expressing the
human parathyroid calcium receptor and loaded with fura-2;
and by measuring an increase in C1- current in a Xenopus
oocyte injected with nucleic acid coding for a calcium
receptor. (See Nemeth et al., PCT/US93/01642, International
Publication Number 4V0 94/18959.) For example, poly(A)* mRNA
can be obtained from cells expressing a calcium receptor,
such as a parathyroid cell, bone osteoclast, juxtaglomerular
kidney cell, proximal tubule kidney cell, distal tubule
kidney cell, cell of the thick ascending limb of Henle's loop
and/or collecting duct, keratinocyte in the epidermis,
parafollicular cell in the thyroid (C-cell), intestinal cell,
central nervous cell, peripheral nervous system cell,
platelet, vascular smooth muscle cell, cardiac atrial cell,
gastrin-secreting cell, glucagon-secreting cell, kidney
mesangial cell, mammary cell, beta cell, fat/adipose cell,
immune cell, and GI tract cell. Preferably, the nucleic acid
is from a parathyroid cell, C-cell, or osteoclast. More
preferably, the nucleic acid encodes a calcium receptor and
is present on a plasmid or vector.
In a preferred embodiment, the compound has an ECSo or
ICso less than or equal to 5 /.cM at one or more, but not all
cells chosen from the group consisting of: parathyroid cell,
bone osteoclast, juxtaglomerular kidney cell, proximal tubule
kidney cell, distal tubule kidney cell, central nervous
system cell, peripheral nervous system cell, cell of the
thick ascending limb of Henle's loop and/or collecting duct,
keratinocyte in the epidermis, parafollicular cell in the
thyroid (C-cell), intestinal cell, platelet, vascular smooth
muscle cell, cardiac atrial cell, gastrin-secreting cell,
- glucagon-secreting cell, kidney mesangial cell, mammary cell,
beta cell, fat/adipose cell, immune cell, GI tract cell, skin
__~...~ ...__ . _ _._...__.. T
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cell, adrenal cell, pituitary cell, hypothalamic cell, and
cell of the subfornical organ. More preferably, the cells
are chosen from the group consisting of parathyroid cell,
central nervous system cell, peripheral nervous system cell,
cell of the thick ascending limb of Henle's loop and/or
collecting duct in the kidney, parafollicular cell in the
thyroid (C-cell), intestinal cell, GI tract cell, pituitary
cell, hypothalamic cell, and cell of the subfornical organ.
The presence of a calcium receptor in this group of cells has
been confirmed by physical data such as in situ hybridization
and antibody staining.
Preferably, inorganic ion receptor-modulating compounds
mimic or block the effects of an extracellular ion on a cell
having an inorganic ion receptor, such that the compounds
achieve a therapeutic effect. Inorganic ion receptor-
modulating compounds may have the same, or different, effects
on cells having different types of inorganic ion receptor
morphology (e.g., such as cells having normal inorganic ion
receptors, a normal number of inorganic ion receptors, an
abnormal inorganic ion receptor, and an abnormal number of
inorganic ion receptors).
Calcium receptor-modulating compounds preferably mimic
or block all of the effects of extracellular ion in a cell
having a-calcium receptor. However, calcimimetics need not
possess all the biological activities of extracellular Cap'.
Similarly, calcilytics need not block all of the activities
caused by extracellular calcium. Additionally, different
calcimimetics and different calcilytics do not need to bind
to the same site on the calcium receptor as does
3o extracellular Ca~~ to exert their effects.
Inorganic receptor-modulating compounds need not effect
inorganic receptor activity to the same extent or in exactly
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the same manner as the natural ligand. For example, a
calcimimetic may affect calcium receptor activity to a
different extent, to a different duration, by binding to a
different binding site, or by having a different affinity,
compared to calcium acting at a calcium receptor.
A. Ionomimetics
Different compound are described by Nemeth et al.,
PCT/US92/07175, International Publication Number WO 93/04373,
Nemeth et al., PCT/US93/01642, International Publication
Number WO 94/18959, Nemeth et al., PCT/US94/12117,
International Publication Number WO 95/11211, and Van Wagenen
et al. PCT/US95/13704, International Publication Number
WO 96/12697. Different generic groups are described herein,
preferably, these groups exclude each of the specific
compounds described in these prior international applications
(i.e., the specific compounds described in PCT/US92/07175,
PCT/US93/01642, PCT/US94/12117, and PCT/US95/13704, are
preferably excluded from the different generic and subgeneric
formula provided herein).
1. Structure I Compounds
Structure I compounds able to modulate calcium
receptor activity have the following formula:
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STRUCTURE I
H
' N Ar2
Are '
R~ R2 R3
Where Ar, is either optionally substituted naphthyl,
optionally substituted phenyl, or an optionally substituted
heterocyclic aryl, where up to 5 substituents may be present
and each substituent is independently selected from the group
consisting of: alkyl, alkenyl, halogen, alkoxy, thioalkyl,
methylene dioxy, haloalkyl, haloalkoxy, OH, CHZOH, CONHz, CN,
acetoxy, N(alkyl)2, phenyl, phenoxy, benzyl, benzyloxy, a,a-
dimethylbenzyl, NO2, CHO, CH3CH(OH), acetyl, OCHZCOOH, and
ethylene dioxy. In one embodiment of the present invention
Arl is either an optionally substituted naphthyl, or a
substituted phenyl, having 1 to 4 substituents, more
preferably Ar,is either an unsubstituted naphthyl or a
substituted phenyl; more preferably, Arl is a substituted
phenyl; preferably each Arl substituent is independently
selected from the group consisting of: isopropyl, CH,O, CF,
CH3S, CF30, Br, I, C1, F, and CH,. In another embodiment of
the present invention Arl is an optionally substituted
heterocyclic aryl. Preferred heterocyclic aryl substituents
are independently selected from the group consisting of:
isopropyl, CH30, CF3 CH3S, CF,O, Br, I, C1, F, and CH3.
Preferred heterocyclic aryls are either furanyl, thiofuranyl,
benzofuranyl, or benzothiophenyl;
Arz is either optionally substituted naphthyl,
optionally substituted phenyl, or an optionally substituted
_ heterocyclic aryl, where up to 5 substituents may be present
and each substituent is independently selected from the group
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consisting of: alkyl, alkenyl, halogen, alkoxy, thioalkyl,
methylene dioxy, haloalkyl, haloalkoxy, OH, CHzOH, CONHz, CN,
OCHzCOOH, ethylene dioxy, and acetoxy; In one embodiment Arz
is preferably either an optionally substituted naphthyl, or a
5 substituted phenyl having 1 to 4 substituents, more
preferably Arz is either an unsubstituted naphthyl or a
substituted phenyl; more preferably, Ar2 is a substituted
phenyl with a substituent in the meta position, even more
preferably, Are is mono substituted with a substituent in the
10 meta position; preferably each Ar2 substituent is
independently selected from the group consisting of:
isopropyl, CH30, CH3S, CF30, Br, I, C1, F, CF3, and CH3, more
preferably a CH30 is located in the meta position. In another
embodiment of the present invention Arz is an optionally
15 substituted heterocyclic aryl. Preferred heterocyclic aryl
substituents are independently selected from the group
consisting of : isopropyl, CH30, CF, CH3S, CF30, Br, I, Cl, F,
and CH3. Preferred heterocyclic aryls are either furanyl,
thiofuranyl, benzofuranyl, or benzothiophenyl;
20 q is 0, 1, 2, or 3; in alternative embodiments q is 0 or
2;
R, is either H or alkyl; when R1 is alkyl in alternative
embodiments the alkyl is methyl, or the alkyl has more than
one carbon atom, preferably 2 to 4 carbon atoms;
R2 and R3 are each independently either hydrogen, alkyl,
or together cycloalkyl or cycloalkenyl; preferably, RZ and R,
are each independently either hydrogen or alkyl, provided
that at least one of R2 and R3 is not hydrogen, preferably, Rz
is alkyl, more preferably Rz is methyl;
and pharmaceutically acceptable salts and complexes
thereof .
___.._~._ ...._..___.r._._r..T__
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21
In a more preferred embodiment the compound has
following formula:
STRUCTURE IA
H
Are N Ar2
;i
R~ R2 Rs
Where Arl, Arz, R1, Rz, and R3 are as described above for
Structure I compounds, including preferred embodiments.
In another more preferred embodiment the compound has
the formula:
structure IB
Xn
Zm
H
Ar~~N
R~ Rz R3
Where Arl, R1, R2, and R, is as described above for
Structure I compounds including preferred embodiments;
each X and Z is independently selected from the group
consisting of: alkyl, alkenyl, halogen, alkoxy, thioalkyl,
methylene dioxy, haloalkyl, haloalkoxy, OH, CHzOH, CONH2, CN,
OCHZCOOH, ethylene dioxy, and acetoxy; more preferably each X
and Z is independently selected from the group consisting of:
isopropyl, CH30, CH3S, CF30, Br, I, C1, F, CF3, and CH3;
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22
n and m are each independently 0, 1, 2, or 3, provided
that n and m together are no more than 5; preferably n and m
are each independently 0 or 1, more preferably, 0.
~ Structure II Compounds
Structure II compounds have the formula:
STRUCTURE II
R8
H
Are ~ N Arz
R I Rz Ra
7 9
Where Arl, Arz, R3 and R4 are as described above for
Structure I compounds, including preferred embodiments;
R, is either hydrogen, alkyl or phenyl; preferably
hydrogen;
Re is either hydrogen, or alkyl; preferably hydrogen;
R9 is either hydrogen, alkyl or phenyl; preferably
hydrogen or alkyl, when R9 is alkyl in alternative embodiments
the alkyl is methyl, or the alkyl has more than one carbon
atom, preferably 2 to 4 carbon atoms;
and pharmaceutically acceptable salts and complexes
thereof.
3. Calcimimetic Activity
The ability of compounds to mimic the activity of Caz~ at
calcium receptors can be determined using procedures known in
the art such as those described by Nemeth et al.,
- PCT/US93/01642, International Publication Number WO 94/18959.
For example, calcimimetics possess one or more and preferably
__,~.~,.__._T_ _..
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23
all of the following activities when tested on parathyroid
cells in vitro:
1. The compound causes a rapid (time to peak < 5
seconds) and transient increase in intracellular calcium
concentration that is refractory to inhibition by 1 /cM La'+ or
1 ~M Gd3'. The increase in [Ca2'] i persists in the absence of
extracellular Caz', but is abolished by pretreatment with
ionomycin (in the absence of extracellular Caz');
2. The compound potentiates increases in [Ca2~];
elicited by submaximal concentrations of extracellular Ca2+;
3 . The increase in [Caz~] ; elicited by
extracellular Caz' is not inhibited by dihydropyridines;
4. The transient increase in [Ca2~]; caused by
the compound is abolished by pretreatment for 10 minutes with
10 mM sodium fluoride;
5. The transient increase in [Ca2+]i caused by
the compound is diminished by pretreatment with an activator
of protein kinase C (PKC), such as phorbol myristate acetate
(PMA), mezerein or (-)-indolactam V. The overall effect of
the protein kinase C activator is to shift the concentration-
response curve of the compound to the right without affecting
the maximal response;
6. The compound causes a rapid (< 30 seconds)
increase in the formation of inositol-1,4,5-triphosphate
and/or diacylglycerol;
7. The compound inhibits dopamine- or isopro-
terenol-stimulated cyclic AMP formation;
8. The compound inhibits PTH secretion;
9. Pretreatment with pertussis toxin (100 ng/ml
for > 4 hours) blocks the inhibitory effect of the compound
on cyclic AMP formation, but does not effect increases in
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[Caz']i, inositol-1,4,5-triphosphate, or diacylglycerol, nor
decreases in PTH secretion;
10. The compound elicits increases in C1- current
in Xenopus oocytes injected with poly(A);-enriched mRNA from
bovine or human parathyroid cells, but is without effect in
Xenopus oocytes injected with water, or liver mRNA; and
11. Similarly, using a cloned calcium receptor
from a parathyroid cell, the compound will elicit a response
in Xenopus oocytes injected with the specific cDNA or mRNA
encoding the receptor.
Different calcium activities can be measured using
available techniques. Parallel definitions of compounds
mimicking Ca2' activity on other calcium responsive cell,
preferably at a calcium receptor, are evident from the
examples provided herein and Nemeth et al., PCT/US93/01642,
International Publication Number WO 94/18959.
Preferably, the compound as measured by the bioassays
described herein, or by Nemeth et al., PCT/US93/01642,
International Publication Number WO 94/18959, has one or
more, more preferably all of the following activities: evokes
a transient increase in internal calcium, having a duration
of less that 30 seconds (preferably by mobilizing internal
calcium); evokes a rapid increase in [Caz~)i, occurring within
thirty seconds; evokes a sustained increase (greater than
thirty seconds) in [Ca2')i (preferably by causing an influx of
external calcium); evokes an increase in inositol-1,4,5-
triphosphate or diacylglycerol levels, preferably within less
than 60 seconds; and inhibits dopamine- or isoproterenol-
stimulated cyclic AMP formation.
The transient increase in [Ca2']; is preferably abolished
by pretreatment of the cell for ten minutes with 10 mM sodium
fluoride, or the transient increase is diminished by brief
.. , ...___.... .. ..... ._...... ... ,. __.T.... ....._ ____..__....
.._._........__d..e__.~_. .. _ _... . _....
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pretreatment (not more than ten minutes) of the cell with an
activator of protein kinase C, preferably, phorbol myristate
acetate (PMA), mezerein or (-) indolactam V.
5 ~ ~alcilytics
The ability of a compound to block the activity of
extracellular calcium at a calcium receptor can be determined
using standard techniques based on the present disclosure.
(See, also Nemeth et al., PCT/US93/01642, International
10 Publication Number WO 94/18959.) For example, compounds
which block the effect of extracellular calcium, when used in
reference to a parathyroid cell, possess one or more, and
preferably all of the following characteristics when tested
on parathyroid cells in vitro:
15 1. The compound blocks, either partially or
completely, the ability of increased concentrations of
extracellular Caz+ to:
(a) increase [Caz'] i,
(b) mobilize intracellular Caz',
20 (c) increase the formation of inositol-1,4,5-
triphosphate,
(d) decrease dopamine- or isoproterenol-
stimulated cyclic AMP formation, and
(e) inhibit PTH secretion;
25 2. The compound blocks increases in C1- current
in Xenopus oocytes injected with poly(A)~-mRNA from bovine or
human parathyroid cells elicited by extracellular Ca2t or
calcimimetic compounds, but not in Xenopus oocytes injected
with water or liver mRNA;
3. Similarly, using a cloned calcium receptor
from a parathyroid cell, the compound will block a response
in Xenopus oocytes injected with the specific cDNA, mRNA or
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26
cRNA encoding the calcium receptor, elicited by extracellular
Ca2+ or a calcimimetic compound.
Parallel definitions of compounds blocking Ca2~ activity
on a calcium responsive cell, preferably at a calcium
receptor, are evident from the examples provided herein and
Nemeth et al., PCT/US93/01642, International Publication
Number WO 94/18959.
III. TREATMENT OF DISEASES OR DISORD RS
Diseases or disorders which can be treated using
compounds able to modulate inorganic ion receptor activity
include one or more of the following types: (1) those
characterized by abnormal inorganic ion homeostasis,
preferably calcium homeostasis; (2) those characterized by an
abnormal amount of an extracellular or intracellular
messenger whose production can be affected by inorganic ion
receptor activity, preferably calcium receptor activity; (3)
those characterized by an abnormal effect (e. g., a different
effect in kind or magnitude) of an intracellular or
extracellular messenger which can itself be ameliorated by
inorganic ion receptor activity, preferably calcium receptor
activity; and (4) other diseases or disorders in which
modulation of inorganic ion receptor activity, preferably
calcium receptor activity, will exert a beneficial effect,
for example, in diseases or disorders where the production of
an intracellular or extracellular messenger stimulated by
receptor activity compensates for an abnormal amount of a
different messenger. Examples of extracellular messengers
whose secretion and/or effect can be affected by modulating
inorganic ion receptor activity include inorganic ions,
hormones, neurotransmitters, growth factors, and chemokines.
_... __._... _ _ __ . _T _ _._.. __ _ _ _ _ _.. .__ _ _
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27
Examples of intracellular messengers include cAMP, cGMP, IP3,
and diacylglycerol.
In a preferred embodiment, the compound is used to treat
a disease or disorder characterized by abnormal bone and
mineral homeostasis, more preferably calcium homeostasis.
Extracellular Ca2+ is under tight homeostatic control and
controls various processes such as blood clotting, nerve and
muscle excitability, and proper bone formation. Abnormal
calcium homeostasis is characterized by one or more of the
following activities: (1) an abnormal increase or decrease in
serum calcium; (2) an abnormal increase or decrease in
urinary excretion of calcium; (3) an abnormal increase or
decrease in bone calcium levels, for example, as assessed by
bone mineral density measurements; (4) an abnormal absorption
of dietary calcium; (5) an abnormal increase or decrease in
the production and/or release of messengers which affect
serum calcium levels such as parathyroid hormone and
calcitonin; and (6) an abnormal change in the response
elicited by messengers which affect serum calcium levels.
The abnormal increase or decrease in these different aspects
of calcium homeostasis is relative to that occurring in the
general population and is generally associated with a disease
or disorder.
Diseases and disorders characterized by abnormal calcium
homeostasis can be due to different cellular defects such as
a defective calcium receptor activity, a defective number of
calcium receptors, or a defective intracellular protein acted
on by a calcium receptor. For example, in parathyroid cells,
the calcium receptor is coupled to the Gi protein which in
turn inhibits cyclic AMP production. Defects in Gi protein
can affect its ability to inhibit cyclic AMP production.
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28
Diseases or disorders which can be treated by modulating
calcium receptor activity are known in the art. For example,
diseases or disorders which can be treated by modulating
calcium receptor activity can be identified based on the
functional responses of cells regulated by calcium receptor
activity.
Functional responses of cells regulated by calcium
receptor are know in the art, including PTH secretion by
parathyroid cells, calcitonin secretion by C-cells, and bone
resorption by osteoclasts. Such functional responses are
associated with different diseases or disorders. For
example, hyperparathyroidism results in elevated levels of
PTH in the plasma. Decreasing the plasma levels of PTH
offers an effective means of treating hyperparathyroidism.
Likewise, increasing plasma levels of calcitonin is
associated with an inhibition of bone resorption. Inhibiting
bone resorption is an effective treatment for osteoporosis.
Thus, modulation of calcium receptor activity can be used to
treat diseases such as hyperparathyroidism, and osteoporosis.
Those compounds modulating inorganic ion receptor
activity, preferably calcium receptor activity, can be used
to confer beneficial effects to patients suffering from a
variety of diseases or disorders. For example, osteoporosis
is an age-related disorder characterized by loss of bone mass
and increased risk of bone fracture. Compounds can be used
to block osteoclastic bone resorption either directly (e. g.,
an osteoclast ionomimetic compound) or indirectly by
increasing endogenous calcitonin levels (e. g., a C-cell
calcimimetic). Alternatively, a calcilytic active on the
parathyroid cell calcium receptor will increase circulating
_ levels of parathyroid hormone, stimulating bone formation.
.-_..-...,.... T _.... ...
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29
All three of these approaches will result in beneficial
effects to patients suffering from osteoporosis.
In addition, it is known that intermittent low dosing
with PTH results in an anabolic effect on bone mass and
appropriate bone remodeling. Thus, compounds and dosing
regimens evoking transient increases in parathyroid hormone
(e. g., intermittent dosing with a parathyroid cell ionolytic)
can increase bone mass in patients suffering from
osteoporosis.
Additional diseases or disorders can be identified by
identifying additional cellular functional responses,
associated with a disease or disorder, which are regulated by
calcium receptor activity. Diseases or disorder which can be
treated by modulating other inorganic ion receptors can be
identified in an analogous manner.
Different diseases can be treated by the present
invention by targeting cells having a calcium receptor. For
example, primary hyperparathyroidism (HPT) is characterized
by hypercalcemia and abnormal elevated levels of circulating
PTH. A defect associated with the major type of HPT is a
diminished sensitivity of parathyroid cells to negative
feedback regulation by extracellular Ca2'. Thus, in tissue
from patients with primary HPT, the ~~set-point~~ for
extracellular Ca2' is shifted to the right so that higher than
normal concentrations of extracellular Ca2~ are required to
depress PTH secretion. Moreover, in primary HPT, even high
concentrations of extracellular Ca2i often depress PTH
secretion only partially. In secondary (uremic) HPT, a
similar increase in the set-point for extracellular Ca2~ is
observed even though the degree to which Ca2~ suppresses PTH
secretion is normal. The changes in PTH secretion are
paralleled by changes in [Ca2'];: the set-point for
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extracellular Ca2a-induced increases in [Ca2;] i is shifted to
the right and the magnitude of such increases is reduced.
Patients suffering from secondary HPT may also have
renal osteodystrophy. Calcimimetics appear to be useful for
5 treating both abnormal PTH secretion and renal osteodystrophy
in such patients.
Compounds that mimic the action of extracellular Ca2+ are
beneficial in the long-term management of both primary and
secondary HPT. Such compounds provide the added impetus
10 required to suppress PTH secretion which the hypercalcemic
condition alone cannot achieve and, thereby, help to relieve
the hypercalcemic condition. Compounds with greater efficacy
than extracellular Ca2r may overcome the apparent
nonsuppressible component of PTH secretion which is
15 particularly troublesome in the major form of primary HPT
caused by adenoma of the parathyroid gland. Alternatively,
or additionally, such compounds can depress synthesis of PTH,
as prolonged hypercalcemia has been shown to depress the
levels of preproPTH mRNA in bovine and human adenomatous
20 parathyroid tissue. Prolonged hypercalcemia also depresses
parathyroid cell proliferation in vitro, so calcimimetics can
also be effective in limiting the parathyroid cell
hyperplasia characteristic of secondary HPT.
Cells other than parathyroid cells can respond directly
25 to physiological changes in the concentration of
extracellular Ca2'. For example, calcitonin secretion from
parafollicular cells in the thyroid (C-cells) is regulated by
changes in the concentration of extracellular Caz'.
Isolated osteoclasts respond to increases in the
30 concentration of extracellular Cant with corresponding
increases in [Ca2']i that arise partly from the mobilization of
intracellular Ca2~. Increases in [Ca2T]i in osteoclasts are
.... .... _........ ...... T. .........__.~..~. ~...__.._..,_. .........
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31
associated with the inhibition of bone resorption. Release
of alkaline phosphatase from bone-forming osteoblasts is
directly stimulated by calcium.
Renin secretion from juxtaglomerular cells in the
kidney, like PTH secretion, is depressed by increased
concentrations of extracellular Ca2'. Extracellular Ca~f
causes the mobilization of intracellular Cap' in these cells.
Other kidney cells respond to calcium as follows: elevated
Ca'' inhibits formation of 1,25(OH)z-vitamin D by proximal
tubule cells, stimulates production of calcium-binding
protein in distal tubule cells, and inhibits tubular
reabsorption of Caz' and Mg's and the action of vasopressin on
the thick ascending limb of Henle's loop (MTAL), reduces
vasopressin action in the cortical collecting duct cells, and
affects vascular smooth muscle cells in blood vessels of the
renal glomerulus.
Calcium also promotes the differentiation of intestinal
goblet cells, mammary cells, and skin cells; inhibits atrial
natriuretic peptide secretion from cardiac atria; reduces
cAMP accumulation in platelets; alters gastrin and glucagon
secretion; acts on vascular smooth muscle cells to modify
cell secretion of vasoactive factors; and affects cells of
the central nervous system and peripheral nervous system.
Thus, there are sufficient indications to suggest that
Cap', in addition to its ubiquitous role as an intracellular
signal, also functions as an extracellular signal to regulate
the responses of certain specialized cells. Compounds of
this invention can be used in the treatment of diseases or
disorders associated with disrupted Ca2' responses in these
cells.
Specific diseases and disorders which might be treated
or prevented, based upon the affected cells, also include
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32
those of the central nervous system such as seizures, stroke,
head trauma, spinal cord injury, hypoxia-induced nerve cell
damage such as 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
I5 diarrhea, and spastic colon; GI ulcer diseases; GI diseases
with excessive calcium absorption such as sarcoidosis; and
autoimmune diseases and organ transplant rejection.
While calcium receptor-modulating compounds of the
present invention will typically be used in therapy for human
patients, they may also be used to treat similar or identical
diseases in other warm-blooded animal species such as other
primates, farm animals such as swine, cattle, and poultry;
and sports animals and pets such as horses, dogs and cats.
IV. ADMINISTRATION
The compounds described by the present invention can be
formulated for a variety of modes of administration,
including systemic and topical or localized administration.
. Techniques and formulations generally may be found in
Reminc~ton's Pharr_nacemt~~a~ ~ iencp~, ig°" ed., Mack Publishing
Co., Easton, PA, 1990.
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33
Suitable dosage forms, in part, depend upon the use or
the route of entry, for example, oral, transdermal, trans-
mucosal, or by injection (parenteral). Such dosage forms
should allow the compound to reach a target cell whether the
target cell is present in a multicellular host or in culture.
For example, pharmacological compounds or compositions
injected into the blood stream should be soluble. Other
factors are known in the art, and include considerations such
as toxicity and dosage forms which retard the compound or
to composition from exerting its effect.
Compounds can also be formulated as_pharmaceutically
acceptable salts and complexes thereof. Pharmaceutically
acceptable salts are non-toxic salts in the amounts and
concentrations at which they are administered. The
preparation of such salts can facilitate the pharmacological
use by altering the physical characteristics of the compound
without preventing it from exerting its physiological effect.
Useful alterations in physical properties include lowering
the melting point to facilitate transmucosal administration
and increasing the solubility to facilitate administering
higher concentrations of the drug.
The pharmaceutically acceptable salt of the different
compounds may be present as a complex. Examples of complexes
include an 8-chlorotheophylline complex (analogous to, e.g.,
dimenhydrinate:diphenhydramine e-chlorotheophylline (1:1)
complex; Dramamine) and various cyclodextrin inclusion
complexes.
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
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34
quinate. 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, malonic acid,
S 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,
l0 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. For. example, see Remington~s Pharmaceutj,~~al
15 sciences, 18th ed., Mack Publishing Co., &aston, PA, p. 1445,
1990. Such salts can be prepared using the appropriate
corresponding bases.
Pharmaceutically acceptable salts can be prepared by
standard techniques. For example, the free-base form of a
2o compound is dissolved in a suitable solvent, such as an
aqueous or aqueous-alcohol in solution containing the
appropriate acid and then isolated by evaporating the
solution. In another example, a salt is prepared by reacting
the free base and acid in an organic solvent. (See, e.g.,
25 PCT/US92/03736, International Publication Number WO 92/20642.)
Carriers or excipients can also be used to facilitate
administration of the compound. Examples of carriers include
calcium carbonate, calcium phosphate, various sugars such as
lactose, glucose, or sucrose, or types of starch, cellulose
30 derivatives, gelatin, vegetable oils, polyethylene glycols
and physiologically compatible solvents. Examples of
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physiologically compatible solvents include sterile solutions
of water.f or injection (WFI), saline solution and dextrose.
The compounds can be administered by different routes
including intravenous, intraperitoneal, subcutaneous,
5 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 forms such as capsules, tablets, and liquid
10 preparations such as syrups, elixirs, and concentrated drops.
Alternatively, injection (parenteral administration) may
be .used, for example, intramuscular, intravenous,
intraperitoneal, and/or subcutaneous administration. For
injection, the compounds of the invention are formulated in
15 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
20 produced.
Systemic administration can also be by transmucosal or
transdermal means. For transmucosal or transdermal
administration, penetrants appropriate to the barrier to be
permeated are used in the formulation. Such penetrants are
25 generally known in the art, and include, for example, for
transmucosal administration, bile salts and fusidic aci d
derivatives. In addition, detergents may be used to
facilitate permeation. Transmucosal administration, for
example, may be through nasal sprays, buccal or sublingual
30 tablets, rectal suppositories, or vaginal suppositories.
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36
For topical administration, the compounds of the
invention can be formulated into ointments, salves, gels, or
creams, as is generally known in the art.
The amounts of various compounds to be administered can
be determined by standard procedures taking into account
factors such as the compound ICso, ECso, 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
l0 considered are known to those of ordinary skill in the art.
Generally, it is an amount between about 0.01 and 50 mg/kg,
preferably 0.01 and 20 mg/kg of the animal to be treated.
V. EXAMPLES
Examples are provided below illustrating different
aspects and embodiments of the present invention. These
examples are not intended to limit the claimed invention.
Included in these examples are synthesis protocols
illustrating techniques which can be used to synthesize
different compounds described herein. Other compounds
falling within the generic groups described herein can be
prepared using standard techniques.
~~le 1. Assaying Calcium Receptor Activity
The ability of different compounds to modulate calcium
receptor activity are described in this example. Other
methods which can be used to measure calcium receptor
activity are known in the art.
Recombinant HEK 293 4.0-7 cells containing a calcium
receptor were constructed as described by Rogers et al., .T.
Bone Miner. Res. 10 Suppl. 1:5483, 1995.
The recombinant cells were loaded with
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fura-2 by incubating the cells in Dulbecco~s modified Eagle s
medium buffered with 20 mM HEPES containing about 5 ~.M fluo-
3/AM for one hour at room temperature. Cells were then
rinsed with Hank s balanced salt solution buffered with 20 mM
HEPES containing 1 mM CaCl2 and 1 mM MgCl2. Compounds to be
tested were then added to the cells and fluorescence was
measured (excitation and emission wavelengths of 340 and 510
nm, respectively). Table I provides results for different
compounds.
Table I
Compound ECso (nM)
26A ~ 52 ( 1 )
6X 286
26B 10900
26C 22000
26D 47 (3)
2 0 26E 77 (3)
26F 15 (3)
26G 11 (3)
26H 36 (1 )
261 126 ( 1 )
2 5 26J 47 ( 1 )
27E 12000
27F 230
27G 70
27H 2750
3 0 280 2500
27J 1100
27K 3800
27L >100000
27M 1800
35 27N 960
270 29
27P 1600
27Q 23
27R 2550
_ 40 27S 210
27T 2900
27U 210
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Compound ECso (nM)
27V 140
27W 1500
27X 22
27Y 12
27Z 16
28A 9.5
28B 24
28C 270
28D 7300
l0 28E 810
28F 660
28G 602
28H 3000
281 1200
28J 1100
28K 57
28L > 3000
28M 170
1 GVIV I
Example 2 - Synthesis of 26D (R R) -N- ( 1-Ethyl 4 1 iodo~henyl )
1-(1-naphthyl)ethylamine hydrochloride
The synthesis of the title compound (26D) was accomplished
in a one-pot, two-step reaction sequence by reductive amination
of the imine formed from the commercially available
41-iodoacetophenone and (R)-naphthyl-1-ethylamine. The
reduction of the imine diastereoselectively was conducted under
similar conditions as previously reported (Tetrahedron Lett.
(1985) 41, 6005-6011.).
A mixture of 41-iodoacetophenone (0.25 g, 1.0 mmol),
(R)-naphthyl-1-ethylamine (0.17 g, 1.0 mmol), and Ti(i-Pr0)9
(0.38 mL, 1.1 mmol) in abs. EtOH (5 mL) was refluxed for 18 h.
Diethyl-1,4-dihydro-2,6-dimethyl-3,5-pyridine decarboxylate
(0.25 g, 1.0 mmol) and Mg(C109)z (0.22 g, 1.0 mmol) were then
_ 35 added to the reaction mixture and the reflux was continued for
an additional 18 h. The reaction mixture was then cooled to
.. _ ._... _ _ _......__. .. . T _.. __. .__._ . _ _
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ambient temperature, H20 (3 mL) and diethyl ether (10 mL) were
added and the mixture was centrifuged (3000 rpm) to remove the
inorganic salts. The supernatant was decanted away from the
pellet and the volatiles were removed under reduced pressure.
S The resulting residue was chromatographed on silica gel (elution
with 1% MeOH/CHZC12) to provide the purified product as its free
base. This material was converted to its hydrochloride salt.
The salt was recrystallized from CHzClz/hexane to provide
GC/MS-pure material.
Example 3: S~mthesis of 26E, (R,R)-N-(1-Ethyl-4~-ethoxy-3~-
methylnhenyl)-1-(1-naphthyl)ethylamine hydrochloride
The synthesis of the title compound (26E) was accomplished
in a three-step, two-pot reaction sequence. Commercially
available 4-hydroxy-3-methylacetophenone was O-alkylated with
ethyl iodide/KzC03/acetone. This ketone was subsequently
reacted with (R}-naphthyl-1-ethylamine in the presence of
Ti(i-Pr0)q to provide the imine. This imine was reduced in high
diastereoselective yield by catalytic hydrogenation with
Raney-nickel.
A mixture of 4-ethoxy-3-methylacetophenone (2.0 g, 11.2
mmol), (R)-naphthyl-1-ethylamine (2.0 g, 11.2 mmol), Ti(i-Pro)9
(4.2 mL, 14.1 mmol), and EtOH (10 mL) were stirred at 60 °C for
18 h. The reaction mixture was then transferred to a Parr
hydrogenation flask, Raney-nickel (100 mg; washed with EtOH, 3 x
20 mL) was added, and the mixture was hydrogenated at 50 psig,
25 °C, for 4 h. The reaction mixture was then filtered
(Celite/fritted glass), the catalyst was washed (EtOH, 20 mL),
and the filtrate was evaporated under reduced pressure to
provide the crude product. This material was purified by silica
gel chromatography (elution with 2% MeOH/CHzClz). The free base
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was converted to its hydrochloride salt to provide 1.1 g (270)
of a white solid.
Example 4~ Synthesis of 26F (R R)-N-(1-Propyl-4~ methoxy 3~
5 meth~rlphen5rl) -1- (1-naphthyl) eth~rlamine h~rdrochlor' d
The synthesis of the title compound (26F) was accomplished
in a four-step, three-pot reaction sequence. Commercially
available 3-methyl-p-anisaldehyde was reacted with
ethylmagnesium bromide to provide its phenylpropanol derivative.
10 This alcohol was then oxidized to the corresponding ketone in
the usual manner with PCC. This ketone was subsequently reacted
with (R)-naphthyl-1-ethylamine in the presence of Ti(i-Pr0)4 to
provide the imine. This imine was reduced in high
diastereoselective yield by catalytic hydrogenation in the
15 presence of Raney-nickel.
In a manner similar to the synthesis of 26E, a mixture of
4-methoxy-3-methylpropiophenone (5.7 g, 31.7 mmol),
(R)-naphthyl-1-ethylamine (5.2 mL, 31.7 mmol), Ti(i-Pro)4 (11.8
mL, 39.6 mmol), and EtOH (30 mL) were reacted as above to form
20 the imine which was subsequently reduced under catalytic
hydrogenation conditions over Raney-nickel. The crude product
was purified by silica gel chromatography (elution with 10:1,
hexane/EtOAc). The free base was converted to its hydrochloride
salt to provide 0.50 g (4's) of a white solid.
Example 5: Synthesis of 26G. (R_R1-N-(1-Ethyl-4~-methoxy-3~
bromophenvl)-1-(1-naphthyl)ethylamine h~rdrochloride
The synthesis of the title compound (26G) was accomplished
in a four-step, three-pot reaction sequence. Commercially
available 3-bromo-4-methoxybenzaldehyde was reacted with
- methylmagnesium bromide to provide its phenylethanol derivative.
This alcohol was then oxidized to the corresponding ketone in
_.. . _. _ . . .. I _.._ _... __ _ _, . . .. .. . _ . _ .
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the usual manner with pyridinium chlorochromate (PCC). This
ketone was subsequently reacted with (R)-naphthyl-1-ethylamine
in the presence of Ti(i-Pr0)9 to provide the imine. This imine
was reduced in high diastereoselective yield using
diethyl-1,4-dihydro-2,6-dimethyl-3,5-pyridine decarboxylate.
In a manner similar to the synthesis of 26D, a mixture of
3-bromo-4-methoxyacetophenone (3.0 g, 13.1 mmol),
(R)-naphthyl-1-ethylamine (2.1 mL, 13.1 mmol), and Ti(i-Pr0)4
(4.7 mL, 15.7 mmol) in abs. EtOH (100 mL) was reduced with
diethyl-1,4-dihydro-2,6-dimethyl-3,5-pyridine decarboxylate in
the presence of Mg(C109)2. The resulting crude material was
converted to its hydrochloride salt. The salt was purified by
precipitation from diethyl ether/hexane to provide GC/MS-pure
material (0.6 g, 11%) as a white solid.
Example 6 ~ SSmthesis of 26H 26I and 26J (R) -N- (3-phen~rl -
2-pronenyl)-1-(1-na~hthyl)ethylamine hydrochloride (R)-N-(2
methyl-3-phenyl-2-propen~rl)-1-(1-nax~ht~yl)ethv~amine
hydrochloride and (R) -N- l2-methox~r-3-phenyl-2- ropeny~ ~ -
7-.(1-n~t~hthvl)ethvlamine hydrochloride
The syntheses of the title compounds were accomplished in
three, two-step, one-pot reaction sequences. Commercially
available cinnamaldehyde, 2-methyl-trans-cinnamaldehyde, and
2-methoxycinnamaldehyde, respectively, were reacted with
(R)-naphthyl-1-ethylamine in the presence of Ti(i-Pr0)4 to
provide the corresponding imine. These imines were reduced
using sodium cyanoborohydride to provide the title compounds in
high overall yields.
Example 7: Ph~rsical Data
Table II provides physical data for some of the compounds
described herein. Gas chromatographic and mass spectral data
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42
were obtained on a Hewlett-Packard 5890 Series II Gas
Chromatograph with a 5971 Series Mass Selective Detector
[Ultra-2 Ultra Performance Capillary Column (crosslinked 5% Ph
Me silicone); column length, 25 m, column i.d., 0.20 mm, film
thickness, 0.33 Vim; He flow rate, 60 mL/min; injector temp., 250
°C; temp. program, 20 °C/min from 125 to 325 °C for 10
min,
then held constant at 325 °C for 6 min].
TABLE II
Compound GC rc m/z
25Z 8.32 285
26A 8.75 286
26B 8.51 288
26C 9.60 346
26D 11.08 401
26E 10.71 333
26F 10.56 333
26G 9.09 385
--
26H 10.95 287
26I 10.98 301
[ 26J [ 11.79
317
Additional Gas chromatographic and mass spectral data were
obtained on a Hewlett-Packard 5890 Series II Gas Chromatograph
with a 5971 Series Mass Selective Detector [Ultra-2 Ultra
Performance Capillary Column (crosslinked 5% phenyl methyl
silicone); column length, 25 m, column i.d., 0.20 mm; He flow
rate, 60 mL/min; injector temp., 250 °C; gradient temperature
program, 20 °C/min from 125 to 325 °C for 10 min, then held
constant at 325 °C for 6 min].
Compound 26Z, rt = 10.22, m/z (rel. int.) 331 (M+,15),
- 316 (56), 182 (9), 168 (5), 156 (20), 155 (100), 154 (28), 153
__ .___._._...___..~.__... . ... _.._._... _. T ~__....___ .
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(18), 152 (8), 141 (11), 133 (43), 131 (5), 129 (11), 128 (18),
127 (15}, 117 (9),115 (13), 115 (13), 105 (8), 91 (7).
Compound 27A,rt = 10.13', m/z (rel. int.) 331 (M+,18),
316 (76) , 182 (10), 176 (5) , 168 (10) , 167 (5) , 156 (17)
, 155
(100) , 154 ) 53 (27) , 152 (14) , 141 (14) , 134 (7)
(57 , , 133
1
(58) , 133 (58), 1 (7) , 129 (14) , 128 (21) , 127 (23} ,
13 126 (5) ,
119 (5), 117 12),116 (5), 115 (18), 105 (10), 91 (12), 77
( (5).
Compound 27D,rt = 9.41', m/z (rel. int.) 292 (M+,5),
171
(7) , 160 (7) 157 (9) , 147 (6) , 146 (9) , 145 (66) , 143
, (7) , 134
(7), 133 (20), 132 (11), 131 (13), 129 (10), 119 (11), 117
(25),
116 (100), 115 (14 ), 115 (14}, 105 (10), 103 (5), 91 (16),
89
(17), 77 (8).
Compound 27E,rt = 7.81', m/z (rel. int.) 283 (M+,3),
268
(100), 176 (16 ),
150
{14),
149
(39),
148
(7),
135
(7),
134
(11),
121 (19) , 118 (6) 117 (6) , 115 (6) , 109 (10) , 105 (8) ,
, 104
(11), 103 (9), 92 (12), 91 (75), 79 (9), 78 (10), 77 {21),
77
(21) , 65 (15) 51 (5) , 42 (6) , 41 (6) .
.
Compound 27F,rt = 7.38', m/z (rel. int.) 365 (M+,1),
231
(6), 230 (31), 216 (28), 215 (59), 214 (17), 190 (15), 174
(25),
136 (41) , 135 (100)
,
134
(14)
,
129
(13)
,
128
(15)
,
127
(9)
,
119
(9), 117 (6), 114 (9), 109 (10), 105 (21), 104 (7}, 103 (18),
91
(21), 91 (10), 79 (11), 78 (7), 77 (19), 68 (12), 65 (6),
42
(9) . 0 (0)
.
Compound 27G,rt = 7.45', m/z (rel. int.) 365 (M+,4),
231
(8) , 230 (49) 216 (44) , 215 (86) , 213 (27) , 190 (23) ,
, 187 (6) ,
175 (6) , 174 31) 136 (37) , 135 (100) , 134 (14) , 130 (8)
( , , 129
(11), 128 (13) ,
127
(9),
120
(7),
120
{7),
116
(5),
115
(8),
109
(8), 105 (19), 103 (13), 92 (8), 91 (16), 79 (8), 77 (13),
68
(9) . 0 (0)
.
Compound 27H, rt = 10.44', m/z (rel. int.} 317 (M+,8), 170
(9) , 162 (5) , 155 (19) , 154 (28) , 153 (14) , 152 (9) , 148 (5) ,
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147 (13), 146 (100), 134-(7), 129 (6), 128 (18), 127 (21), 126
(7) , 115 (12) , 115 (12) , 103 (7) , 102 (6) , 89 {8) , 77 (8) .
Compound 27J, rt = 9.88', m/z (rel. int.) 337 (M+,2), 323
(22), 322 (100), 210 (26), 196 (9), 184 {12), 182 (11), 170
(13) , 169 (53) , 168 (31) , 167 (14) , 165 (10) , 154 (22) , 153
(41), 152 (32), 150 (9), 141 (53), 129 {27), 128 (34), 127 (62),
126 (20), 124 (98), 115 (24), 103 (23), 91 (15), 89 (18), 77
(23) , 42 (11) , 41 {9) , 0 (0) .
Compound 27K, rt = 9.03', m/z (rel. int.) 342 {M+,.1), 327
(40), 325 (41), 308 (14), 306 (21), 204 (17), 202 (31), 174
(43), 173 (26), 172 (66), 171 (26), 139 (11), 138 (15), 137
(20), 127 (33), 124 (100), 117 (10), 115 (12), 111 (11), 103
(37), 102 (41), 101 (30), 98 (12), 91 (11), 89 (28), 77 (35), 75
(21) , 63 (12) , 51 (10) , 0 (0) .
Compound 27L, rt = 8.84', m/z (rel. int.) 264 (M+,24), 145
(100) , 145 (7) , 119 (29) , 118 (26) , 118 (16) , 117 (7) , 116 (5) ,
102 (37), 92 (10), 91 (41), 90 (41), 77 (6), 76 (9), 75 {14), 75
(14), 65 (5), 64 (21), 63 (23), 51 (8).
Compound 27M, rt = 8.48', m/z (rel. int.) 305 (M+,.0), 291
(6), 290 (31), 164 (28}, 136 (17), 135 (100), 120 (6), 111 (7),
I11 (7) , 105 (16) , 103 (9) , 98 (7) , 92 {6) , 91 (13) , 79 (8) , 77
(12), 65 (5), 63 (5).
Compound 27N, rt = 8.81', m/z (rel. int.) 294 (M+,6), 279
(100), 187 (5), 164 (7), 144 (7), 136 (16), 135 (75), 135 (75),
134 (11}, 130 (15}, 121 (6), 120 (7), 117 (11), 116 (36), 115
(6) , 105 (18) , 104 (14) , 103 (30) , 102 (7) , 92 (9) , 91 (19) , 90
(6) , 89 (17) , 79 (10) , 78 (7) , 77 (23) , 65 {6) , 63 (6) .
Compound 270, rt = 9.33', m/z (rel, int.) 347 (M+,1), 304
(58), 192 (6), 156 (14), 156 (14), 155 (100), 154 (22), 153
(22), 152 (9), 150 (24), 149 (16}, 148 (23), 135 (28), 129 (9),
- 128 (14) , 127 (15) , 115 (9) , 91 {8) , 77 (6) .
_._.__ ... __..._.~....._. .. ~ . . ..._... . t
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Compound 27P, rt = 9.23', m/z (rel. int.) 347 (M+,.0), 304
(100) , 177 (3) , 156 (12) , 155 (87) , 154 (12) , 153 (15) , 152 (6) ,
150 (20) , 149 (10) 148 (12) , 128 (6) . 127 (6) .
,
Compound 27Q, rt = 9.64', m/z (rel. int.) 361 (M+,.1),
304
5 (54), 156 (17), 155 (100), 153 (17), 152 (7), 151 (5), 150
(40),
148 (12) , 135 (27) 129 (7) , 128 (9) , 127 (9) , 115 (7)
, , 91 (5) ,
91 (5) .
Compound 27R, rt = 9.16', m/z (rel, int.) 294 (M+,3),
279
{100), 187 (5), 164 (6), 136 (24), 135 (77), 121 (10), 120
(6),
10 117 (5), 116 (33), 105 {15), 104 (7), 103 (15), 92 (6), 91
(14),
91 (14) , 89 (10) 9 (8) , 78 (5) , 77 (14) , 65 (5) .
, 7
Compound 275, rt = 9.27', m/z (rel. int.) 338 (M+,.0),
323
(7), 322 (38), 164 (9), 162 (7), 160 (25), 158 (37), 136
(25),
136 (6) , 135 (100) 134 (16) , 124 (7) , 122 (6) , 120 (8)
, , 120
15 (7), 115 (8), 105 19), 104 (5), 103 (16), 102 (11), 101
( (9), 92
(10), 91 (19), 89 8), 79 (10), 78 (6), 77 (17), 65 (6),
( 63 (6),
0 (0) .
Compound 27U, rt = 8.65', m/z (rel. int.) 385 (M+,3),
230
(16), 230 (16), 216 (12), 215 (55), 214 (15), 210 (12), 174
20 (19), 156 (23), 155 (100), 154 (27), 153 (24), 152 (12), 140
(5) , 129 (15) , (25) , 127 (22) , 126 (5) , 115 (12) ,
128 109 (5) ,
68 (5) .
Compound 27V, rt = 8.59', m/z (rel. int.) 385 (M+,3),
230
(14), 216 (9), 215 (49), 214 (13), 210 (5), 174 (17), 156
(23),
25 155 (100) , 154 (25), 153 (26) , 152 (11) , 130 (5) , 129
(19) , 129
(19), 128 (27), 127 (26), 115 (14), 109 (6), 101 (5), 77 (5),
69
(7) .
Compound 27W, rt = 8.88', m/z (rel. int.) 371 (M+,2), 356
(100), 244 (20}, 184 (5), 182 (5), 170 (8), 169 (24), 168 (14),
30 167 (8), 160 (5), 159 (46), 154 (11), 153 (24), 153 (24), 152
(15), 150 (6), 141 (26), 133 (9), 129 (11), 128 (13), 127 (19),
126 (5) , 115 (6) , 109 (10) .
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Compound 27X, rt = 10.61', m/z (rel. int.) 419 (M+,.0),
406 (50), 404 (20), 403 (100), 402 (11), 401 (51), 263 (6), 250
(27), 248 (55), 246 (29), 169 (9), 167 (7), 156 (5), 155 (14),
154 (16) , 153 (12) , 153 (12) , 152 (6) , 128 (9) , 127 (9) .
Compound 27Y, rt = 10.21', m/z (rel. int.) 375 (M+,4), 361
(20), 360 (100), 359 (15), 358 (78), 279 (7}, 217 (11), 206
(23), 205 (7), 204 (93), 202 (74), 170 (13), 168 (8), 156 (12),
155 (38), 154 (53), 153 (37), 152 (21), 141 (11), 129 (16), 128
(37), 127 (41), 126 (21), 123 (20), 115 (14), 89 (28), 77 (10),
75 (10), 63 (8), 0 (0).
Compound 27Z, rt = 11.10', m/z (rel. int.) 466 (M+,.1),
451 (60), 450 (13), 449 (61), 311 (9), 309 (11), 296 (97), 295
(8), 294 {100), 169 (29), 168 (9), 167 (24}, 156 (20), 155 (56),
154 (74), 153 (45), 152 (27), 151 (8), 141 (13), 129 (21), 128
(52), 127 (61), 126 (18), 115 (18), 89 (43), 77 (13), 75 (14),
74 {9), 63 (16), 0 (0).
Compound 28A, rt = 10.73', m/z (rel. int.) 421 (M+,4), 408
(33), 407 (21), 407 (21), 406 (100), 279 (9), 265 (7), 252 (22),
251 (6} , 250 (70) , 156 (6) , 155 (20) , 154 (25) , 153 (19) , 152
(11), 141 (6), 129 (7), 128 (18), 127 (21), 126 (10), 123 (11),
115 (7) , 89 (16) .
Compound 28B, rt = 10.75', m/z (rel. int.) 417 (M+,3), 274
(5), 261 (16), 261 (16), 247 (10), 246 (100), 156 (7), 155 (29),
154 (35), 153 (19), 152 (11), 141 (6), 129 (8), 128 (23), 127
(23) , 126 (7) , 115 (8) , 105 (9) , 91 (7) , 90 (16) , 89 {9) , 77
(15) .
Compound 28C, rt = 8.73', m/z (rel. int.) 317 (M+,.1), 303
(12), 302 (62), 282 (9), 178 (6), 149 (22), 148 (100), 148 (7),
135 (9) , 131 (6) , 127 (16) , 124 (46) , 119 (12} , 117 (6) , 115
(8) , 104 (6} , 103 (24) , 102 (6) , 92 (9} , 91 (65) , 90 (7) , 89
(18) , 78 (6) , 77 (25) , 65 {19) , 63 (11) .
T _r_~ _ _ _._ .__... __ _.._ _ .
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Compound 28D, rt = 8.73, m/z (rel. int.) 317 (M+,.1), 303
(14), 302 (71), 282 (11), 178 (6), 149 (23), 149 (23), 148
(100), 135 (9), 131 (6), 127 (14), 124 (42}, 119 (10), 117 (5),
115 (7) , 103 (19) , 92 (8) , 91 (56) , 90 (5) , 89 (14) , 78 (6} , 77
(19) , 65 (16) , 63 (7) .
Compound 28E, rt = 9.33, m/z (rel. int.) 338 (M+,2), 325
(7), 324 {35), 323 (11), 323 (I1), 322 (54), 164 (9), 161 (15),
159 (23), 136 (30), 135 (100), 121 (15), 120 (5), 205 {14), 103
(10) , 92 (5) , 91 (11) , 79 (7) , 77 (11) .
Compound 28F, rt = 9.11, m/z (rel. int.) 338 (M+,1), 325
(7), 324 (39), 323 (11), 322 (59), 164 (10), 161 (19), 161 (19),
159 (29) , 136 (27) , 135 (100) , 121 (11) , 120 (6) , 115 (5) , 105
(17) , 103 (12) , 102 (7) , 101 (5) , 92 (6) , 91 (14) , 89 (6) , 79
{9) , 77 (14) , 65 (5) .
Compound 28G, rt = 7.18, m/z (rel. int.) 251 (M+,6), 236
(43) , 156 (6) , 155 (26) , 154 (32) , 153 (24) , 152 (18) , 152 (18) ,
151 (6), 141 (8), 129 (11), 128 (25), 127 (31), 126 (11), 115
(12) , 95 (12) , 82 (6) , 81 (100) , 77 (8) , 53 (27) , 51 (6) .
Compound 28H, rt = 7.31, m/z (rel. int.} 251 (M+,9), 236
(100), 208 (7), 170 (10), 168 (8), 156 (5), 155 (26), 154 (39),
153 (27) , 152 (19) , I52 (19) , 151 (6) , 141 (8) , 129 (9) , 128
(22) , 127 (29) , 126 (10) , 115 (9) , 94 (5) , 82 {5) , 81 (77) , 53
(13) .
Compound 2BI, rt = 8.20, m/z (rel, int.) 267 (M+,6), 252
(36), 156 (6), 155 (21), 154 {15), 153 (15), 152 (10), 141 {7),
129 (7) , 128 (15) , 127 (16) , 126 (5) , 115 (8) , 112 (16) , 98 (8) ,
98 (8), 98 (6), 96 (100), 53 (5), 44 (6}.
Compound 28J, rt = 8.23, m/z (rel. int.) 267 (M+,6), 251
(56) , 170 (11) , 155 (25) , 154 (31) , 153 (23) , 153 (23) , 152
(16) , 151 {5) , 141 (7) , 129 (9) , 128 (22) , 127 (26) , 126 (9) ,
- 115 (10), 111 (7), 110 (7), 98 (6), 97 (8), 96 (100), 85 (5), 77
(5) , 53 (6) , 44 (9) .
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Compound 28K, rt = 9.28', m/z (rel. int.) 315 (M+,42), 301
(5), 300 (23), 160 (19), 156 (19), 155 (78), 154 (42), 153 (27),
152 (15) , 146 (16} 145 (100) , 144 (19) , 141 (6) , 129 (11)
, , 128
(24) , 127 (31) , (31) , 126 (8) , 118 (7) , 117 (14) ,
127 116 (8) ,
115 (41) , 91 (12) 89 (9) , 77 (7) .
,
Compound 28L, rt = 7.41', m/z (rel. int.} 319 (M+,6),
318
(8) , 159 (15) , (12) , 146 (100) , 132 (6) , 131 (5) ,
147 130 (7) ,
119 (6) , 117 (13) 115 (10) , 109 (8) , 105 (6) , 104 (16)
, , 103
(11) , 91 (8) , ) , 77 (8) , 42 (8) .
78 (8
Compound rt = 10.76', m/z (rel. int.) 372 (M+,2),
28M, 360
(8), 359 (10), (44), 357 (16), 356 (68), 169 (6), 168
358 (29),
167 (8) , 160 (32) 158 (51) , 156 (17) , 155 (100) , 154
, (29) , 153
(34), 152 (18), (6), 141 (9), 129 (18), 128 (25), 127
151 (28),
126 (8) , 124 (7) 22 (9) , 115 (19) , 102 (6) , 101 (7)
, 1 , 89 (10) ,
77 (7), 0 (0).
Compound 28N, rt = 7.40', m/z (rel. int.) 270 (M+,6),
136
(62), 135 (100), 3 (20), 120 (12), 120 (8), 106 (5), 105
13 (34),
103 (18), 103 (18},103 (6), 91 (28), 91 (23), 79 (11), 79
(5),
78 (11), 77 (22), 6 (5), 64 (10), 63 (5), 62 (7).
7
Other embodiments are within the following claims.
Thus, while several embodiments have been shown and described,
various modifications may be made without departing from the
spirit and scope of the present invention.
__._~.~, _... . ._._. __.r
