[3H]Azidodantrolene: synthesis and use in identification of a putative skeletal muscle dantrolene binding site in sarcoplasmic reticulum.

Article abstract of DOI:10.1021/jm9805079

Dantrolene sodium is a medically important hydantoin derivative that interferes with release of Ca2+ from intracellular stores of skeletal muscle by an unknown mechanism. Identification of the molecular target of dantrolene would greatly aid in understand

Full text of DOI:10.1021/jm9805079

1872
J . Med. Chem. 1999, 42, 1872-1880
[3H]Azid od a n tr olen e: Syn th esis a n d Use in Id en tifica tion of a P u ta tive Sk eleta l
Mu scle Da n tr olen e Bin d in g Site in Sa r cop la sm ic Reticu lu m )
Sanjay S. Palnitkar,|, Bin Bin,§, Leslie S. J imenez,§ Hiromi Morimoto,∇ Philip G. Williams,∇,†
Kalanethee Paul-Pletzer,| and J erome Parness*,|,‡
Departments of Anesthesia, Pharmacology, and Pediatrics, UMDNJ -Robert Wood J ohnson Medical School,
New Brunswick, New J ersey 08901, Department of Chemistry, Rutgers University, Piscataway, New J ersey 08855,
National Tritium Labelling Facility and Physical Biosciences Division, E. O. Lawrence Berkeley National Laboratory,
One Cyclotron Road, Berkeley, California 94720, and Department of Pharmaceutical Chemistry, School of Pharmacy,
University of California, San Francisco, California 94143
Received September 2, 1998
Dantrolene sodium is a medically important hydantoin derivative that interferes with release
of Ca2+ from intracellular stores of skeletal muscle by an unknown mechanism. Identification
of the molecular target of dantrolene would greatly aid in understanding both the mechanism
of action of the drug and the dynamics of intracellular Ca2+ release in muscle. [3H]-
Azidodantrolene was designed and synthesized as a photoaffinity analogue in order to identify
a putative dantrolene receptor in skeletal muscle. Introduction of 1 mole-atom of tritium into
aldehyde 5b was required during radioligand synthesis in order to ensure high enough specific
activity for detection of photo-cross-linked proteins by fluorographic methods. This was
accomplished by reduction of ester 3 with custom synthesized, 100% tritium-labeled lithium
triethylborotritide, followed by oxidation to 5b by manganese(IV) oxide. Compound 6b was
demonstrated to be g95% tritium-labeled at the imine position by NMR spectroscopy, and the
specific radioactivity of [3H]azidodantrolene sodium was empirically determined by HPLC and
liquid scintillation counting to be 24.4 Ci/mmol, ∼85% of theoretical maximum. [3H]Azido-
dantrolene was found to be pharmacologically active in ligand-receptor binding studies with
skeletal muscle sarcoplasmic reticulum membranes. Photo-cross-linking experiments analyzed
by SDS-PAGE and tritium fluorography have identified a ∼160-kDa specifically labeled protein
as the putative, intracellular, skeletal muscle dantrolene receptor. This photolabeled protein
comigrates with a protein in Western blots immunologically cross-reactive to a polyclonal anti-
rabbit skeletal muscle ryanodine receptor antibody. Thus, the putative dantrolene receptor
may be related to the skeletal muscle ryanodine receptor.
In tr od u ction
inhibit the function of the primary Ca2+ release channel
of SR, also known as the ryanodine receptor. The
Dantrolene (hydrated 1-(((5-(4-nitrophenyl)-2-fura-
nyl)methylene)amino)-2,4-imidazolidinedione sodium salt)
(Figure 1) is the only drug currently available for the
therapy of a life-threatening, genetic sensitivity to
volatile anesthetics and depolarizing skeletal muscle
relaxants known as malignant hyperthermia (MH).
Once MH is triggered, it results in massive, intracellular
release of Ca2+ from stores in the sarcoplasmic reticu-
lum (SR) of skeletal muscle.1,2 Although the molecular
mechanism by which dantrolene acts is not known, its
therapeutic effects parallel an inhibition of anesthetic-
triggered Ca2+ release.3,4 Dantrolene is presumed to
) We dedicate this paper to the memory of Dr. Tom Schwan
(deceased) of Proctor & Gamble, Norwich, NY, for providing us with
many dantrolene congeners and synthetic intermediates and for his
abiding interest in dantrolene chemistry and pharmacology.
* To whom correspondence should be addressed at: Department of
Anesthesia, UMDNJ -Robert Wood J ohnson Medical School, Clinical
Academic Building/Suite 3100, 125 Paterson St, New Brunswick, NJ
08901. Tel: (732) 235-4824. E-mail: parness@umdnj.edu.
| Department of Anesthesia, UMDNJ -Robert Wood J ohnson Medi-
cal School.
‡ Departments of Pharmacology and Pediatrics, UMDNJ -Robert
Wood J ohnson Medical School.
§ Rutgers University.
∇ E. O. Lawrence Berkeley National Laboratory.
† University of California.
These authors contributed equally to this paper.
F igu r e 1. Dantrolene and its congeners.
10.1021/jm9805079 CCC: $18.00 © 1999 American Chemical Society
Published on Web 05/14/1999

[3H]Azidodantrolene
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 11 1873
Sch em e 1. Proposed Mechanism of Hydrogen-Tritium
Exchange at the 2′′ Position in [3H]Dantrolene
ryanodine receptor is a homotetrameric protein (mono-
mer Mr ) 565 kDa), whose channel function is known
to be regulated by a number of proteins, enzymes, and
small molecules, including the neutral plant alkaloid,
ryanodine.2,5-7 Deciphering whether dantrolene inter-
acts directly with the channel or an accessory/regulatory
protein has profound implications for how a skeletal
muscle cell is able to regulate intracellular Ca2+ release
in both normal and pathological states. The identifica-
tion of a molecular target for dantrolene, therefore,
acquires particular significance.
We have recently synthesized [2′′-3H]dantrolene and,
using a drug binding assay, identified the presence of
specific dantrolene binding sites in SR of porcine
skeletal muscle.8 Due to the reversibility of classical
ligand-receptor interactions, drug binding studies allow
only the demonstration that discrete drug binding sites
exist. This technique is useful in identifying active
fractions during receptor purification and molecular
identification only in cases of high-affinity ligand-
receptor interactions measurable with high signal-to-
noise ratios. Dantrolene, however, possesses interme-
diate affinity for its receptor (Kd ) 200-300 nM), and
previous binding studies demonstrate a poor signal-to-
noise ratio of ∼0.2.8 An experimental protocol designed
to enhance the signal-to-noise ratio of the [3H]dant-
rolene binding assay is therefore necessary to increase
the likelihood of receptor identification.
A direct approach to the molecular identification of a
drug receptor involves the synthesis and use of radio-
actively labeled, photoactive congeners in photoaffinity
labeling experiments.9-11 Specific photoaffinity labeling
of a receptor-ligand binding site results when a photo-
and pharmacologically active ligand bound to its recep-
tor binding site is irradiated. The resultant photoacti-
vated compound attacks chemically susceptible groups
in its immediate vicinity by any of a variety of mecha-
nisms, leading to the formation of a covalent bond(s)
with an amino acid residue(s) in or near the ligand
binding site. The specifically cross-linked, putative
receptor is then identified using a detection system
appropriate to the ligand label. Though many photoac-
tivatable and radioactive groups are available for con-
jugation to a ligand, our search was limited by the
relatively stringent requirements of structure-activity
relationships for dantrolene.12 Small changes in charge
distribution and molecular configuration of dantrolene
result in large decrements in biological activity. Our
aim, therefore, was to use photoactivatable groups and
radioactive atoms that would be expected to minimally
perturb the chemical structure and pharmacological
activity of this ligand.
resulting material (8.92 Ci/mmol, ChemSyn Laborato-
ries, Lenexa, KS) was subject to substantial tritium
exchange and underwent significant radiochemical de-
cay over a period of 2 years (data not shown). Indeed,
the tritium at this position is expected to undergo
hydrogen/tritium exchange because of the adjacent
electron-rich furan ring (Scheme 1). Conversely, hydro-
gen/tritium exchange at either the 3′′ position (ortho to
the electron-withdrawing nitro group) or the imine
hydrogen would not be expected to occur. The relative
ease of synthesis of the proposed imine-tritiated conge-
ner versus the 3′′-tritiated derivative led us to concen-
trate on synthesizing the former. Preliminary experi-
ments with dantrolene in basic deuterium oxide revealed
no hydrogen-deuterium exchange at the imine position
(data not shown).
This manuscript reports an improved synthesis of [3H]-
dantrolene (Scheme 2), the successful synthesis and
tritiation (Scheme 3) of the photoaffinity analogue of
dantrolene, azidodantrolene (Figure 1), and the identi-
fication of a putative molecular target of the drug.
Resu lts a n d Discu ssion
After examination of structure-activity relationships
described in the first report on dantrolene synthesis and
biological activity,12 we reasoned that replacing the
strongly electron-withdrawing nitro group of dantrolene
with the more weakly electron-withdrawing azido group
would still result in a pharmacologically active, photo-
activatable derivative, azidodantrolene (Figure 1). More-
over, examination of the structure of dantrolene reveals
only two potential sites for a nonexchangeable tritium
atom. Interestingly, our first synthesis of [3H]dantrolene
placed the tritium atom at the 2′′ position on the phenyl
ring because of the relative ease of its synthesis.8 The
Ch em istr y. 1. Im p r oved Syn th esis of [3H]Da n t-
r olen e. The nonexchangeable imine position of dant-
rolene was tritiated by reacting a tritide-reducing agent
with the formyl group of 1a , an intermediate in the
synthesis of dantrolene, and reoxidation to the tritiated
analogue 1b. This adds only two reaction steps to the
overall synthetic scheme of unlabeled dantrolene. Dan-
trolene was tritiated (Vitrax, Inc., Placentia, CA) by
reduction of the aldehyde 1a with commercially avail-
able sodium borotritide (∼75% tritium labeled), followed
by reoxidation with manganese(IV) oxide to the tritiated

1874 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 11
Sch em e 2. Synthesis of [3H]Dantrolene
Palnitkar et al.
Sch em e 3. Synthesis of Azidodantrolene
aldehyde 1b (Scheme 2). Theoretically, using the above
source of tritium, reduction of the formyl group with
tritride to its corresponding alcohol, and reoxidation to
the tritiated formyl group should introduce at best only
0.375 mole-atom of tritium. In fact, condensation with
1-aminohydantoin followed by deprotonation with so-
dium methoxide resulted in [3H]dantrolene with a
specific activity of 10 Ci/mmol. This is ∼93% of the
theoretical value if isotope effects are ignored, since the
specific activity of 1 mole-atom of tritium is 28.76 Ci/
mmol.13
2. Non r a d ioa ctive Azid od a n tr olen e. The synthesis
of azidodantrolene followed a route analogous to that
for dantrolene itself.12 It began with the coupling of the
aryldiazonium tetrafluoroborate 2 with 2-furaldehyde
using cupric chloride as the catalyst (Scheme 3). A
modification14,15 of the procedure reported by Snyder et
al.12 raised the yield of aldehyde 5a from 14% to 59%.
Condensation of 5a with aminohydantoin hydrochloride
in 2:3 acetonitrile:water occurred readily to give a 75%
yield of 6a . Reaction of 6a with 1.1 equiv of sodium
methoxide in methanol gave the corresponding sodium
salt in a 91% yield. Unreacted 6a was cleanly separated
from the sodium salt by removing methanol in vacuo
and then washing the resulting residue with absolute
ethanol. Only the sodium salt is soluble in ethanol;
therefore, it can be isolated in a pure form by concen-
trating the ethanol filtrate under reduced pressure.
3. [3H]Azid od a n tr olen e. A photoaffinity label with
the highest possible specific activity was desired to aid
in the identification of a putative dantrolene receptor,
a low abundance cellular protein. To synthesize [3H]-
azidodantrolene with the highest possible specific activ-
ity, we devised a reaction scheme in which 1 mole-atom
of tritium would be inserted into the imine position
(Scheme 3). The reduction of ester 3 (Scheme 3) with
custom synthesized 100%-labeled lithium triethylboro-
tritide (Super Tritide)16 was proposed to give the
completely labeled alcohol 4b, which could then be
reoxidized as above to yield the corresponding aldehyde
5b. In theory, this should allow the introduction of 1
mole-atom of tritium into the imine position.
To accomplish this goal, 5-(4-azidophenyl)-2-furoate
(3) was a necessary intermediate. The synthesis of 3 was
carried out using the same reaction conditions as for
5a except that methyl 2-furoate was substituted for
2-furaldehyde (Scheme 3). The yield in this reaction was
substantially lower, a mere 11%. Reduction of 3 with
lithium triethylborohydride cleanly reduced the methyl
ester in the presence of the azido group, resulting in a
73% yield of 4a . Oxidation with manganese(IV) oxide
in tetrahydrofuran gave a 72% yield of 5a . The synthesis
of [3H]azidodantrolene was performed as outlined in
Scheme 3.16 Reduction of 3 with lithium triethylboro-
tritide gave an approximately 2:3 ratio of 3:4b as judged
by the 1H NMR spectra. However, oxidation of the
alcohol 4b (as a mixture with 3) with manganese(IV)

[3H]Azidodantrolene
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 11 1875
oxide was considerably slower than the reaction rate
observed for the unlabeled alcohol 4a . A significant
isotope effect is to be expected, since a carbon-tritium
bond must be broken during the course of the reaction.
In the future, an alternative method such as tetrapro-
pylperruthenate (TPAP)/4-methylmorpholine N-oxide
(NMO) oxidation17 may be more useful since this
method has been previously used successfully in the
rapid oxidation of deuterated alcohols.18 In the present
situation, the unreacted alcohol 4b was carried forward
as a mixture with 5b, since only 5b will undergo
condensation with 1-aminohydantoin to give 6b. Depro-
tonation with sodium methoxide followed by HPLC
purification gave [3H]azidodantrolene. Theoretically,
this method should result in 100% tritium labeling. A
specific activity of only 24.4 ( 4 Ci/mmol was deter-
mined by HPLC for this sample of [3H]azidodantrolene.
However, the 1H and 3H NMR spectra of the [3H]-
azidodantrolene showed no sign of hydrogen in the
imine position (Figure 2). Although NMR spectroscopy
is a relatively insensitive technique, it can be concluded
that the imine position is g95% tritium-labeled. Both
[3H]dantrolene and [3H]azidodantrolene, prepared as
described above, have shown no sign of hydrogen/tritium
exchange during a period of over 1 year. Reduction of
an ester with lithium triethylborotritide followed by
oxidation appears to be a good general method for
synthesizing nonexchangeable, ∼100% tritium-labeled
aldehydes (eq 1).
1
F igu r e 2. Determination of the specific activity of 6b by H
and 3H NMR spectroscopy: (A) 3H NMR spectrum of 6b in
THF-d8, (B) H NMR spectrum of 6b in THF-d8, (C) H NMR
spectrum of 6a in THF-d8.
1
1
P h ar m acological Activity. 1. Non r adioactive Azi-
d od a n tr olen e. The pharmacological utility of unla-
beled azidodantrolene was evaluated using a modifica-
tion of the [3H]dantrolene binding inhibition assay
reported previously (see Experimental Section).8 Pre-
liminary experiments revealed that [3H]dantrolene bind-
ing in a buffer containing 500 µM-3 mM â,γ-methyle-
neadenosine 5′-triphosphate (AMP-PCP), a physiologically
nonhydrolyzable analogue of adenosine triphosphate
(ATP), is enhanced 3-4-fold over our earlier results, and
the signal-to-noise ratio increased from 0.2 to 0.6 (data
not shown). The Kd for [3H]dantrolene binding in this
buffer system is ∼210 nM (data not shown) and does
not differ significantly from that obtained in the absence
of AMP-PCP (∼275 nM).8 Therefore, AMP-PCP was
included in all binding experiments reported herein.
In the experiment shown, [3H]dantrolene, 250 nM,
was incubated with SR membranes and increasing
concentrations of azidodantrolene in the dark, for 1 h
at 37 °C. After filtration and washing of assay aliquots,
the glass fiber filters were processed and assayed for
radioactivity by liquid scintillation counting. The re-
sults, shown in Figure 3, reveal that azidodantrolene
specifically inhibits [3H]dantrolene binding to SR mem-
branes in a concentration-dependent manner (Ki ∼ 1.5
µM). Analysis of binding inhibition (Inplot 4.0) reveals
that the data are best fit by a curve mathematically
consistent with ligand competition for binding at a
single site. These results confirm that azidodantrolene
competes with [3H]dantrolene at the same ligand bind-
ing site.
F igu r e 3. Inhibition of [3H]dantrolene binding to SR by
azidodantrolene. SR membranes were incubated with [3H]-
dantrolene in the presence of increasing concentrations of
azidodantrolene as explained in the Experimental Section.
Azidodantrolene dose-dependently inhibited the specific bind-
ing of [3H]dantrolene to SR membranes.
As expected, azidodantrolene is extremely sensitive
to ambient and UV light. When irradiated at 366 nm
(2-cm distance, 20 °C), azidodantrolene undergoes com-
plete photolysis within 5 s, as determined by scanning
UV spectrophotometry (200-700 nm, λmax ) 360 nm).
Indeed, even under ambient light, degradation is evi-
dent within 2 min (data not shown).
2. [3H]Azid od a n tr olen e. The specific binding activ-
ity of [3H]azidodantrolene was determined using an
inhibition binding assay reciprocal to the one described

1876 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 11
Palnitkar et al.
F igu r e 4. Inhibition of [3H]azidodantrolene binding to SR by
dantrolene and its congeners. SR membranes were incubated
in the presence of 200 nM [3H]azidodantrolene and increasing
concentrations of dantrolene (2), azumolene (b), or aminod-
antrolene (9). Both dantrolene and azumolene were equipotent
in their inhibitory activity, whereas aminodantrolene was
inactive.
above for unlabeled azidodantrolene. SR membranes
were incubated in the dark with 200 nM [3H]azidodan-
trolene and increasing concentrations of dantrolene,
azumolene, and the physiologically inactive congener,
aminodantrolene, as described (see Experimental Sec-
tion). After incubation, the samples were harvested via
rapid filtration through glass fiber filters, and radioac-
tivity was determined by liquid scintillation counting.
The results, shown in Figure 4, demonstrate that both
dantrolene and azumolene are equipotent in their
specific competition of [3H]azidodantrolene binding (Ki
∼175 nM), while aminodantrolene is inactive. Once
again, nonlinear regression analysis of the inhibition
binding data is consistent with a single-site model of
ligand competition, and the data agree with the known
pharmacological activity of these congeners. Data from
experiments involving inhibition of [3H]azidodantrolene
binding by unlabeled azidodantrolene were uninterpret-
able due to scatter of data at total ligand concentrations
g 1.0 µM. This phenomenon may be due to dark
reactions of this azido compound with membrane lipids
(see below).
Photoincorporation of [3H]azidodantrolene into SR
membrane proteins was carried out as described in the
Experimental Section. Briefly, SR membranes were
incubated with 250 nM [3H]azidodantrolene in buffer
containing AMP-PCP in the dark for 1 h at 37 °C, in
the absence or presence of dantrolene (20 µM), azumolene
(100 µM), or atropine (100 µM), and then immediately
irradiated at 366 nm, as described. The irradiated
proteins were resolved by SDS-PAGE and blotted onto
PVDF membranes, and the tritium-labeled proteins
were detected by fluorography. As depicted in the
fluorogram (Figure 5A), there is only one specifically
labeled protein band at Mr ∼ 160 kDa. Both dantrolene
and azumolene inhibit the specific photoincorporation
of [3H]azidodantrolene into this ∼160-kDa protein, while
the chemically unrelated compound, atropine, has no
effect. This cross-linking is a result of photoactivation
of [3H]azidodantrolene, as it does not occur in the
absence of sample irradiation, even at radioligand
F igu r e 5. (A) Fluorographic detection of [3H]azidodantrolene
cross-linked SR proteins. Fluograph of SDS-PAGE resolved
proteins cross-linked with [3H]azidodantrolene in the presence
or absence of dantrolene (20 µM), azumolene (100 µM), or
atropine (100 µM) reveals specific photoincorporation into a
single protein band denoted by the arrowhead (Mr ∼ 160 kDa).
(B) Western blot of SR proteins photo-cross-linked with [3H]-
azidodantrolene. The same PVDF membrane used to generate
the fluorograph in panel A was probed with a polyclonal
antibody to rabbit RyR and visualized with a secondary
antibody coupled to alkaline phosphatase, as described in the
Experimental Section. Note the reactivity of both the RyR
doublet and the 160-kDa band. (C) Coomassie blue staining
of SR proteins separated by 7.5% SDS-PAGE. The RyR,
myosin, and Ca2+-ATPase are labeled for comparison.
concentrations as high as 2 µM (data not shown). It
proved impossible to use aminodantrolene as a negative
control in this experiment, as an irradiated [3H]azidod-
antrolene/aminodantrolene mixture in the absence of
membrane protein became trapped on the glass fiber
filters, presumably due to photochemical interaction
between one of the four known reactive species of the
aryl azide probe11 and aminodantrolene.
As can be seen from Figure 5A, no specific [3H]-
azidodantrolene cross-linking to the ryanodine receptor
monomers resolved in our SDS-PAGE system was
detected in any of these experiments (n ) 15). In an
attempt to determine if the cross-linked band could be
related to the RyR, the proteins from this gel were
electroblotted onto a PVDF membrane, and the mem-
brane was probed with a polyclonal antibody to the RyR,
as described in the Experimental Section. As can be seen
from Figure 5C, both the RyR doublet at 565 and 410
kDa and the ∼160-kDa band react with the antibody.
No antibody reaction occurred with secondary antibody
alone (data not shown).
The efficiency of [3H]azidodantrolene incorporation
into the 160-kDa band was determined. [3H]Azidodan-

[3H]Azidodantrolene
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 11 1877
trolene was incubated with SR protein in the presence
or absence of ∼100-fold molar excess of unlabeled
dantrolene or azumolene for 90 min at 37 °C in the dark,
as described in the Experimental Section. The reaction
mixtures were split into two, one portion was filtered
in the dark, and specific radioligand binding to SR
protein was determined. The second portion was ex-
posed to UV light, the SR membrane vesicles were
sedimented at 14 000 rpm for 10 min in a microfuge,
and the pellet was fractionated by SDS-PAGE (7.5%),
as described. The proteins in the gels were then elec-
troblotted onto PVDF membranes, and the specifically
labeled 160-kDa protein was identified by Western blots
(see above) and fluorography. This band was then cut
out of the membrane and specific radioactivity deter-
mined by liquid scintillation counting. The resultant
specific photoincorporation of radioactivity was normal-
ized to starting protein and then divided by the specific
radioactivity in the binding assay, yielding a specific
incorporation of ∼1.5%.
ryanodine receptor (565 kDa) or myosin (200 kDa), the
two major high-molecular-weight proteins in our SR
preparations. Western blot analysis of this ∼160-kDa
protein band using a polyclonal antibody raised against
the intact RyR monomer reveals immunologic cross-
reactivity. Though the definitive identity of this ∼160-
kDa protein is unknown at this time, our findings
suggest a number of possibilities. The first and most
likely possibility is that the dantrolene binding site is
on a proteolytic cleavage fragment of the RyR. Indeed,
a ∼150-160-kDa rabbit skeletal muscle RyR fragment
is among a number of fragments of different molecular
weights known to be cleaved from the intact monomer
by both exogenously added calpain and a specific, SR
membrane-bound, n-calpain.24-26 Moreover, the endog-
enous n-calpain of SR has been shown to cleave rabbit
skeletal muscle RyR to primarily 375- and 150-kDa
fragments.25 Given the species differences between our
muscle source (pig) and those of previous studies (rab-
bit), our 160-kDa fragment could easily be equivalent
to the previously reported 150-kDa fragment. Second,
the dantrolene binding site may be on a splice variant
of the RyR, thereby accounting for the immunologic
reactivity with the polyclonal anti-RyR antibody. Ex-
perimental support for this suggestion is to be found in
reports of alternative splicing of both murine RyR1 and
human RyR3 ryanodine receptors, though none of these
splice variants have been shown to give rise to a ∼160-
kDa protein.27,28 Third, dantrolene may specifically
interact with a hitherto unidentified, epitopically cross-
reactive, ∼160-kDa protein of presumed RyR regulatory
function. Unidentified proteins epitopically cross-reac-
tive with anti-RyR antibodies have been demonstrated
in osteoclasts and thymomas.29,30 Finally, we cannot
exclude the possibility of there being more than one
protein migrating at 160 kDa in our gel system, one of
which cross-reacts with the polyclonal, anti-RyR anti-
body.
An accretion curve for [3H]azidodantrolene binding to
SR could not be performed since the scatter of radioac-
tive signal among replicates was high at radioligand
concentrations above 500 nM, precluding us from de-
termining binding saturation. The scatter of radioactive
counts may be attributable to nonspecific dark-phase
reactions of [3H]azidodantrolene with the sarcoplasmic
reticulum membrane lipids, since, as described above,
no dark-phase reactivity toward proteins is noted. Given
the hydrophobic nature of this compound, such a result
is not surprising.
Evidence for the molecular site of action of dantrolene
has been circumstantial and contradictory. We had
suggested that the binding sites for [3H]dantrolene and
[3H]ryanodine might lie on separate molecular targets.
This proposal was based on our ability to separate
overlapping peaks of binding for the two drugs in
porcine skeletal muscle SR membranes fractionated by
linear sucrose gradient centrifugation and by lack of any
pharmacological interaction between dantrolene and
ryanodine in our binding studies.21 On the other hand,
recent reports present evidence suggesting that dant-
rolene acts directly at the level of the ryanodine receptor
in skeletal muscle. Nelson and colleagues demonstrated
a biphasic effect of dantrolene on partially purified
ryanodine receptor channel activity in lipid bilayers and
concluded that the action of dantrolene was directly on
the channel itself. 19 A similar purification strategy was
employed by Fruen et al. who reported the inhibitory
actions of dantrolene on both [3H]ryanodine binding to
and Ca2+ release from SR membrane vesicles.20 These
authors also concluded that dantrolene acts directly and
specifically on the ryanodine receptor itself. In an
attempt to resolve the controversy, we synthesized a
pharmacologically active, photoreactive, radiolabeled
dantrolene analogue, [3H]azidodantrolene, and directly
identified the skeletal muscle dantrolene binding pro-
tein by specific photo-cross-linking.
In summary, we have synthesized [3H]azidodant-
rolene and, for the first time, demonstrated the presence
of a specific dantrolene binding protein with immuno-
logic cross-reactivity to the RyR in skeletal muscle SR
membranes. Attempts to purify and characterize this
dantrolene binding protein are ongoing in our labora-
tory.
Exp er im en ta l Section
Gen er a l. Elemental analyses were processed by Quantita-
tive Technologies Inc., Whitehouse, NJ . Mass spectra were
processed by the Center for Advanced Food Technology,
Rutgers University, New Brunswick, NJ . Acetonitrile was
distilled from calcium hydride. Tetrahydrofuran was freshly
distilled from sodium and benzophenone. Triethylborane,
tetramethylethylenediamine (TMEDA), and anhydrous metha-
nol were purchased from Aldrich Chemical Co. Melting points
were determined on a Thomas-Hoover melting point apparatus
and were not corrected. Proton magnetic resonance spectra
were recorded on a Varian Gemini 200-MHz or J EOL GSX
400-MHz NMR spectrometer. Tritium labeling was conducted
at the National Tritium Labelling Facility, E. O. Lawrence
Berkeley National Laboratory, Berkeley, CA.22 Lithium tri-
ethylborotritide was prepared according to Andres et al.16
Proton and tritium NMR spectroscopy experiments were
carried out on an IBM Instruments, Inc. AF-300 spectrometer
(3H at 320 MHz, 1H at 300 MHz) using a 3H/1H 5-mm dual
probe. All potentially light-sensitive chemicals were handled
under red light. Materials used in the assay of biological
The data above demonstrate that [3H]azidodant-
rolene, under conditions designed to minimize nonspe-
cific receptor-ligand interactions,10 specifically labels
one protein band that migrates at ∼160 kDa in our
SDS-PAGE system. Under these conditions, the radio-
ligand does not specifically cross-link to the intact

1878 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 11
Palnitkar et al.
activity of azidodantrolene were purchased from Sigma Chemi-
cal Co., St. Louis, MI, or ICN Pharmaceuticals Inc., Costa
Mesa, CA, except for [3H]dantrolene (10.0 Ci/mmol), which was
custom synthesized by Vitrax, Placentia, CA. EtOAc, ethyl
acetate; PE, petroleum ether, bp 35-60 °C.
dissolved in 4 mL of CH3CN and then added to a 25-mL round-
bottom flask. A solution of 25 mg (0.16 mmol) of 1-amino-2,4-
imidazolidinedione hydrochloride in 3 mL of water was then
added to the flask, 3 mL of water was added, and the solution
was stirred at room temperature for 1 h. After some precipitate
formed, 10 mL of water was added to the suspension and the
flask was chilled in an ice bath for 5 min. The solid was
collected by filtration, washed with water several times, rinsed
with methanol once, and then air-dried. A total of 22 mg (75%)
of a yellow solid was obtained. Mp: >320 °C dec. 1H NMR
(DMSO-d6): δ 11.26 (s, 1H), 7.80 (d, 2H, J ) 8.4 Hz), 7.71 (s,
1H), 7.21 (d, 2H, J ) 8.4 Hz), 7.11 (d, 1H, J ) 3.5 Hz), 6.94 (d,
1H, J ) 3.5 Hz), 4.34 (s, 2H). MS (FAB+): m/e 311 (M + H+).
Anal. (C14H10N6O3) C, H, N.
1-{[5-(4-Azid op h en yl)fu r fu r ylid in e]a m in o}-2,4-im id a -
zolid in ed ion e Sod iu m Sa lt . To a slurry of 7.2 mg (0.023
mmol) of 6a in 1.0 mL of anhydrous methanol under a nitrogen
atmosphere was added 5.3 mg (0.024 mmol) of NaOCH3 (25
wt % solution in methanol). The mixture was stirred at room
temperature in the dark overnight. The solution was still clear,
and the solvent was removed in vacuo resulting in a yellow
solid. The solid was washed with acetone twice and was then
dissolved in absolute ethanol. The ethanol solution was
filtered, the solid residue was washed with ethanol three times,
and the filtrate was concentrated in vacuo giving 7.0 mg (91%)
of a yellow solid. Mp: >320 °C dec. 1H NMR (DMSO-d6) δ 7.77
(d, 2H, J ) 8.4 Hz), 7.40 (s, 1H), 7.18 (d, 2H, J ) 8.4 Hz), 7.02
(d, 1H, J ) 3.3 Hz), 6.71 (d, 1H, J ) 3.3 Hz), 3.66 (s, 2H). MS
(FAB-): m/e 309 (M-), 281 (M- - N2). HRMS (FAB-): calcd
for C14H9N6O3, 309.0736; obsd, 309.0733. Anal. (C14H10N6O3‚
1.5H2O) C, H; N: calcd, 23.39; found, 22.44.
[3H]-5-(4-Azid op h en yl)-2-fu r a n m eth a n ol (4b). Lithium
triethylborotritide (0.075 mmol) was prepared and dissolved
in dry THF (500 µL) and cooled to 0 °C. Methyl 5-(4-
azidophenyl)-2-furoate (3) (8.6 mg, 0.035 mmol) was added,
and the reaction was allowed to proceed for 30 min. After
quenching with 2 drops of ammonium chloride in methanol,
the solvent was removed and the residue was extracted with
ether. The ether layer was washed with water, dried by
passage through magnesium sulfate, and removed by a stream
of nitrogen gas. Yield: 825 mCi. 1H NMR (THF-d8): δ 7.65 (d,
J ) 8.2 Hz, 2H), 7.04 (d, J ) 8.2 Hz, 2H), 6.62 (d, J ) 2.4 Hz,
1H), 6.27 (d, J ) 2.4 Hz, 1H). 3H NMR (THF-d8): δ 4.41 (s,
C3H2OH).
Meth yl 5-(4-Azid op h en yl)-2-fu r oa te (3). To a solution of
400 mg (2.34 mmol) of 4-azidoaniline hydrochloride in 1.5 mL
of water was added 860 mg (4.69 mmol) of fluoroboric acid (48
wt % in water) dropwise. The reaction mixture was cooled in
an ice bath, and a solution of 170 mg (2.46 mmol) of NaNO2
in 0.5 mL of water (cooled in an ice bath) was added dropwise.
The reaction mixture was stirred at 2-5 °C for 40 min; some
white precipitate formed. A solution of 886 mg (7.03 mmol) of
methyl 2-furoate in 2 mL of acetone was added, followed by
the addition of 27 mg of CuCl2 in 0.3 mL of water. The reaction
mixture was allowed to warm to room temperature and stirred
in the dark for 2 days. The solvents was removed in vacuo;
the residue was diluted with CH2Cl2, washed with water twice,
1 N NaHCO3 once, and brine once, dried over Na2SO4, filtered,
and concentrated in vacuo. The residue was dried with an oil
pump overnight and was then chromatographed on silica using
PE:EtOAc (4:1) as eluent. A total of 65 mg (11%) of a brown
1
solid was obtained. Mp: 67-69 °C. H NMR (CDCl3): δ 7.70
(d, J ) 8.7 Hz, 2H), 7.24 (d, J ) 3.8 Hz, 1H), 7.07 (d, J ) 8.7
Hz, 2H), 6.70 (d, J ) 3.8 Hz, 1H), 3.92 (s, 3H). 13C NMR
(CDCl3): δ 159.2, 156.8, 143.6, 140.6, 126.4, 120.1, 119.5, 106.7,
51.92. MS (EI): m/e 243 (M+), 215 (M+ - N2). Anal. (C12H9N3O3)
C, H, N.
5-(4-Azid op h en yl)-2-fu r a n m eth a n ol (4a ). To a solution
of 9.8 mg (0.04 mmol) of 3 in 1 mL of THF at 0 °C was added
101 µL (0.10 mmol) of 1 M LiBEt3H in THF solution. After
the addition, the reaction mixture was stirred at 0 °C for 30
min and was then quenched with several drops of saturated
NH4Cl in MeOH solution. The solvents were removed in vacuo;
the residue was dissolved in ethyl ether, washed with water
twice and brine once, dried over Na2SO4, filtered, and concen-
trated in vacuo. The residue was then chromatographed on
silica using 4:1 PE/EtOAc as eluent. A total of 6.3 mg (73%) of
1
a pale solid was obtained. Mp: 86-88 °C. H NMR (CDCl3):
δ 7.64 (d, J ) 8.5 Hz, 2H), 7.03 (d, J ) 8.5 Hz, 2H), 6.55 (d, J
) 3.4 Hz, 1H), 6.37 (d, J ) 3.4 Hz, 1H), 4.66 (d, J ) 4.7 Hz,
2H), 1.85 (broad s, 1H). 13C NMR (CDCl3): δ 153.6, 153.3,
139.0, 127.6, 125.3, 119.3, 110.1, 105.6, 57.6; MS (EI): m/e 215
(M+), 187 (M+ - N2). Anal. (C11H9N3O2) C, H, N.
5-(4-Azid op h en yl)-2-fu r a ld eh yd e (5a ). P r oced u r e A: A
total of 300 mg (1.76 mmol) of 4-azidoaniline hydrochloride
was dissolved in 2 mL of water, 650 mg of 48% fluoroboric
acid was added, and the mixture was cooled in an ice bath. A
solution of 128 mg (1.85 mmol) of NaNO2 in 1 mL of water
was added dropwise, and the resulting solution was stirred
at 2-5 °C for 40 min. Some precipitation formed. A solution
of 520 mg (5.41 mmol) of 2-furaldehyde in 3 mL of acetone
was then added dropwise, followed by the addition of 20 mg
of CuCl2 in 0.2 mL of water, and the mixture was allowed to
stir at room temperature in the dark for 3 days. Some brownish
solid formed. The solid was then filtered and washed with
water twice. The solid residue was dissolved in a minimum
amount of CH2Cl2 and chromatographed on silica using PE:
EtOAc (3:1) as eluent. A total of 220 mg (59%) of a light-gray
solid was obtained.
[3H]-5-(4-Azid op h en yl)-2-fu r a ld eh yd e (5b). The THF-d8
was removed by a stream of nitrogen gas and replaced with
dry THF. Manganese(IV) oxide (100 mg) was added and the
reaction mixture stirred for 4.5 h. The MnO2 was removed by
filtration through GF/C borosilicate microfiber filter (MFS,
Dublin, CA) and rinsed with ethyl acetate. Yield: 530 mCi,
20% conversion to the aldehyde. 1H NMR (THF-d8): δ 7.85
(d, J ) 8.4 Hz, 2H), 7.35 (d, J ) 3.8 Hz, 1H), 7.14 (d, J ) 8.4
3
Hz, 2H), 6.98 (d, J ) 3.8 Hz, 1H). H NMR (THF-d8): δ 9.61
(s, C3HO), 4.41 residual alcohol (s, C3H2OH).
[3H ]-1-{[5-(4-Azid op h en yl)fu r fu r ylid en e]a m in o}-2,4-
im id a zolid in ed ion e (6b). Ethyl acetate was removed by a
stream of nitrogen gas, the residue was dissolved in acetoni-
trile (1 mL), and 1-amino-2,4-imidazolidenedione hydrochloride
(6.9 mg, 1 mL H2O) was added. After stirring overnight, the
solvent was removed and the residue suspended in methanol
(1 mL). The cloudy suspension was centrifuged in a clinical
centrifuge and the pellet dissolved in THF-d8 (750 µL). Yield:
184 mCi. The product was purified by HPLC using a Vydac
LC 18 column at 3 mL/min using a linear gradient of 0-100%
acetonitrile over 40 min. Yield: 20 mCi. 1H NMR (THF-d8): δ
7.76 (d, J ) 8.5 Hz, 2H), 7.09 (d, J ) 8.5 Hz, 2H), 6.84 (s, 2H),
P r oced u r e B: To a solution of 4.0 mg (0.019 mmol) of 4a
in 1 mL of THF was added MnO2 (16 mg, 0.19 mmol). The
reaction mixture was stirred at room temperature under a
nitrogen atmosphere for 5 h. The reaction mixture was then
filtered through a Celite pad, and the Celite cake was rinsed
with THF three times. The filtrate was concentrated and
chromatographed on silica using 20% EtOAc in hexane as
eluent. A total of 2.9 mg (72%) of a light-gray solid was
3
4.20 (s, 2H). H NMR (THF-d8): δ 7.89 (s, C3HdN).
[3H]-1-{[5-(4-Azid op h en yl)fu r fu r ylid in e]a m in o}-2,4-im -
id a zolid in ed ion e Sod iu m Sa lt. To a methanol solution of
the [3H]-1-{[5-(4-azidophenyl)furfurylidine]amino}-2,4-imida-
zolidinedione was added 25% sodium methoxide in methanol
(20 µL, 87 mmol), and the reaction was stirred overnight.
HPLC purification was performed on Vydac LC-18, as previ-
1
obtained. Mp: 104-105 °C. H NMR (CDCl3): δ 9.64 (s, 1H),
7.82 (d, 2H, J ) 8.5 Hz), 7.32 (d, 1H, J ) 3.8 Hz), 7.10 (d, 2H,
J ) 8.5 Hz), 6.81 (d, 1H, J ) 3.8 Hz). MS (CI): m/e 214 (M +
H+). Anal. (C11H7N3O2) C, H, N.
1-{[5-(4-Azid op h en yl)fu r fu r ylid en e]a m in o}-2,4-im id a -
zolid in ed ion e (6a ). A total of 20 mg (0.09 mmol) of 5a was
1
3
ously. Yield: 12 mCi. The H and H NMR spectra in THF-d8

[3H]Azidodantrolene
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 11 1879
showed 6b, so the sodium salt apparently becomes protonated
under these conditions.
Wester n Blottin g of P h oto-Cr oss-Lin k ed P r otein s.
After SDS-PAGE and electroblotting of resolved photo-cross-
linked proteins described above, the PVDF membrane was
blocked with 5% nonfat dry milk in Tris-buffered saline (TBS)
for 1 h at room temperature and washed three times with TBS
and the membrane probed with a polyclonal sheep anti-rabbit
skeletal muscle RyR antibody (a gift of Dr. Kevin Campbell)
at a 1:5000 dilution in TBS containing 5% nonfat dry milk for
1 h at room temperature. After being washed three more times
with TBS, the membrane was incubated with the secondary
antibody, donkey anti-sheep IgG conjugated to alkaline phos-
phatase (Sigma Immunochemicals), at 1:5000 dilution over-
night at 4 °C. Color was developed using 5-bromo-4-chloro-3-
indolyl phosphate in 100% dimethylformamide and p-nitro
blue tetrazolium chloride in 70% dimethylformamide using
standard procedures.
P r ep a r a tion of SR Mem br a n es fr om P or cin e Sk eleta l
Mu scle. Freshly dissected porcine fast twitch skeletal muscle
(Longissimus dorsi), a gift of Drs. Sheila Muldoon and Paul
Mongan of the Uniformed Services University of the Health
Sciences, Bethesda, MD, was immediately frozen in liquid
nitrogen and maintained at -72 °C until use. SR membranes
were prepared essentially as described previously.8 Briefly,
defrosted skeletal muscle was homogenized using a Waring
blender and a crude membrane preparation derived by remov-
ing the cell debris and nuclear material using differential
centrifugation. The crude microsomes were further fraction-
ated by ultracentrifugation on discontinuous sucrose density
gradients to separate fractions enriched in sarcolemma, trans-
verse tubules, and both light and heavy SR. Light SR are
enriched in Ca2+-ATPase (SERCA1), and the heavy SR are
enriched in the ryanodine receptor. Heavy SR were used
exclusively in the experiments reported herein, as this fraction
contains the greatest concentration of both dantrolene and
ryanodine binding sites.8 The SR membranes were stored at
-72 °C until use.
In h ibition Assa y for [3H]Azid od a n tr olen e Bin d in g to
SR Mem br a n es. All experiments with azidodantrolene, la-
beled or unlabeled, were performed under red light or in the
dark. Porcine skeletal muscle SR membranes (50 µg of protein)
were incubated with 200 nM [3H]dantrolene (10.2 Ci/mmol)
or [3H]azidodantrolene (24.4 Ci/mmol) and increasing concen-
trations of unlabeled congener for 60 min at 37 °C, in a buffer20
containing AMP-PCP (500 µM), MgCl2 (500 µM), CaCl2 (100
nM), calmodulin (1 µM), potassium propionate (150 mM), and
Na-PIPES (20 mM, pH 7.0). After incubation, the assay
mixture was rapidly filtered through Whatman GF/C filters
using a Hoefer filtration unit (model FH225V) and washed
with 5 mL of ice-cold buffer to remove free [3H]dantrolene. The
filters were then transferred into scintillation vials and
incubated overnight in the presence of hyamine hydroxide to
hydrolyze the proteins. CytoScint-ES was added to the vials,
and radioactive counts were determined using an LKB-
RacBeta scintillation counter. Statistical analysis of the bind-
ing data was carried out using Inplot (version 4.0, GraphPad
Inc.).
P h otoin cor p or a tion of [3H]Azid od a n tr olen e in SR
Mem br a n e P r otein s. One hundred micrograms of SR mem-
brane protein was incubated in the dark (1 h at 37 °C) with
250 nM [3H]azidodantrolene in the absence or presence of 25
µM dantrolene or 100 µM azumolene, as positive controls, or
100 µM atropine, as negative control, in the binding buffer
described above. The aqueous solubility limits of dantrolene
and azumolene at room temperature are ∼30 and 300 µM,
respectively.8 The samples were then irradiated with a hand-
held UV lamp (366 nm) at a distance of 2 cm for 2 × 1 min at
room temperature (at 4 °C, the [3H]azidodantrolene/dantrolene
mixture precipitates out of solution).
SDS-P AGE, Electr oblottin g, a n d F lu or ogr a p h y of
P h oto-Cr oss-Lin k ed P r otein s. Photo-cross-linked proteins
were resolved using sodium dodecyl sulfate-polyacrylamide
gel electrophoresis (SDS-PAGE).23 Briefly, Laemmli sample
buffer containing 2% SDS was added to the reaction mixture
containing the [3H]azidodantrolene photo-cross-linked proteins
and allowed to stand at room temperature for 1 h. Samples
(∼50 µg of protein) were loaded onto 7.5% SDS-polyacryla-
mide gels and run at 175 V until the dye front was within 1
cm of the bottom. Resolved proteins in the gel were then
electroblotted onto poly(vinyl difluoride) (PVDF) membranes
using a wet protein-transfer apparatus (Idea Scientific, Min-
neapolis, MN) following the manufacturer’s protocol. Following
protein transfer, the PVDF membrane was air-dried and
exposed to BioMax-MS film (Kodak) using a TranScreen LE
intensifying screen (Kodak) at -80 °C. The film was developed
after 5 days using standard film development technique. The
resultant fluorogram was digitally scanned (UMAX V-12,
Umax Corp.) and visualized using Photoshop (version 4.0,
Adobe Systems, Inc.).
Ack n ow led gm en t. L.S.J . thanks the donors of the
Petroleum Research Fund, administered by the Ameri-
can Chemical Society, the National Science Foundation
(CHE-9502149), and the Charles and J ohanna Busch
Memorial Fund for support of this work. J .P. is indebted
to the Foundation for Anesthesia Education and Re-
search for an Anesthesiology Young Investigator Award,
to the National Institutes of Health (NIAMS) for Grant
AR45593-01, and to the Department of Anesthesia,
UMDNJ -Robert Wood J ohnson Medical School, for
financial support. P.G.W. and H.M. thank the Biomedi-
cal Technology Area, National Center for Research
Resources, U.S. National Institutes of Health, for their
support under Grant P41 RR01237, through Depart-
ment of Energy Contract DE-AC03-76SF00098 with the
University of California.
Refer en ces
(1) MacLennan, D. H.; Phillips, M. S. Malignant Hyperthermia.
Science 1992, 256, 789-794.
(2) Mickelson, J . R.; Louis, C. F. Malignant Hyperthermia: Excita-
tion-Contraction Coupling, Ca2+ Release Channel, and Cell Ca2+
Regulation Defects. Physiol. Rev. 1996, 76, 537-592.
(3) Van Winkle, W. B. Calcium Release from Skeletal Muscle
Sarcoplasmic Reticulum: Site of Action of Dantrolene Sodium?
Science 1976, 193, 1130-1131.
(4) Harrison, G. G. Dantrolene - Dynamics and Kinetics. Br. J .
Anaesth. 1988, 60, 279-286.
(5) Meissner, G. Ryanodine Receptor/Ca2+ Release Channels and
their Regulation by Endogenous Effectors Annu. Rev. Physiol.
1994, 56, 485-508.
(6) Coronado, R.; Morrissette, J .; Sukhareva, M.; Vaughan, D. M.
Structure and Function of Ryanodine Receptors. Am. J . Physiol.
1994, 266, C1485-C1504.
(7) Sutko, J . L.; Airey, J . A. Ryanodine Receptor Ca2+ Release
Channels: Does Diversity in Form Equal Diversity in Function?
Physiol. Rev. 1996, 76, 1027-1071.
(8) Parness, J .; Palnitkar, S. S. Identification of Dantrolene Binding
Sites in Porcine Skeletal Muscle Sarcoplasmic Reticulum. J . Biol.
Chem. 1995, 270, 18465-18472.
(9) Bayley, H. Photogenerated Reagents in Biochemistry and Mo-
lecular Biology. Laboratory Techniques in Biochemistry and
Molecular Biology; Elsevier: Amsterdam, 1983; Vol. 12, pp
1-187.
(10) Ruoho, A. E.; Rashidbaigi, A.; Roeder, P. E. In Membranes,
Detergents, and Receptor Solubilization; Venter, J . C., Harrison,
L. C., Eds.; Alan R. Liss: New York, 1984; pp 119-160.
(11) Brunner, J . New photolabeling and cross-linking methods. Annu.
Rev. Biochem. 1993, 62, 483-514.
(12) Snyder, H. R., J r.; Davis, C. S.; Bickerton, R. K.; Halliday, R. P.
1-[(5-Arylfurfurylidene)amino]hydantoins. A new class of Muscle
Relaxants. J . Med. Chem. 1967, 10, 807-810.
(13) Evans, E. A. In Tritium and Its Compounds; Wiley: New York,
1974; pp 1-822.
(14) Starkey, E. B. p-Dinitrobenzene. Org. Synth. 1943, II, 225-227.
(15) Doyle, M. P.; Siegfried, B.; Dellaria, J . F., J r. Alkyl Nitrite-Metal
Halide Deamination Reactins. 2. Substitutive Deamination of
Arylamines by Alkyl Nitrites and Copper(II) Halides. J . Org.
Chem. 1977, 42, 2426-2431.
(16) Andres, H.; Morimoto, H.; Williams, P. G. Preparation and Use
of LiEt3BT and LiAlT4 at Maximum Specific Activity. J . Chem.
Soc., Chem. Commun. 1990, 627-628.

1880 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 11
Palnitkar et al.
(17) Cherif, A.; Farquhar, D. J . N-(5,5-diacetoxypent-1-yl)doxorubi-
cin: a New Intensely Potent Doxorubicin Analogue. J . Med.
Chem. 1992, 35, 3208-3214.
(18) Elmore, C. Investigation of the Stereochemistry of the Methyl-
to-Methylene Elimination in the Biosynthesis of Several Terpe-
nes Through the Use of Chiral Methyl Labeled Compounds.
Ph.D. Thesis, Department of Chemistry, University of Illinois
at Urbana-Champaign, 1997.
(19) Nelson, T. E.; Lin, M.; Zapata-Sudo, G.; Sudo, R. T. Dantrolene
Sodium can Increase or Attenuate Activity of Skeletal Muscle
Ryanodine Receptor Calcium Release Channel. Anesthesiology
1996, 84, 1368-1379.
(20) Fruen, B. R.; Mickelson, J . R.; Louis, C. F. Dantrolene Inhibition
of Sarcoplasmic Reticulum Ca2+ Release by Direct and Specific
Action at Skeletal Muscle Ryanodine Receptors. J . Biol. Chem.
1997, 272, 26965-26971.
(21) Palnitkar, S. S.; Mickelson, J . R.; Louis, C. F.; Parness, J .
Pharmacological Distinction between Dantrolene and Ryanodine
Binding Sites: Evidence from Normal and Malignant Hyper-
thermia-Susceptible Porcine Skeletal Muscle. Biochem. J . 1997,
326, 847-852.
(22) Morimoto, H.; Williams, P. G. Design and Operations at the
National Labeling Facility. Fusion Technol. 1992, 21, 256-261.
(23) Laemmli, U. K. Cleavage of Structural Proteins during the
Assembly of the Head of Bacteriophage T4. Nature 1970, 227,
680-685.
(24) Brandt, N. R.; Caswell, A. H.; Brandt, T.; Brew, K.; Mellgren,
R. L. Mapping of the calpain proteolysis products of the
junctional foot protein of the skeletal muscle triad junction. J .
Membr. Biol. 1992, 127, 35-47.
(25) Shevchenko, S.; Feng, W.; Varsanyi, M.; Shoshan-Barmatz, V.
Identification, characterization and partial purification of a thiol-
protease which cleaves specifically the skeletal muscle ryanodine
receptor/Ca2+ release channel. J . Membr. Biol. 1998, 161, 33-
43.
(26) Wu, Y.; Aghdasi, B.; Dou, S. J .; Zhang, J . Z.; Liu, S. Q.; Hamilton,
S. L. Functional interactions between cytoplasmic domains of
the skeletal muscle Ca2+ release channel. J . Biol. Chem. 1997,
272, 25051-25061.
(27) Futatsugi, A.; Kuwajima, G.; Mikoshiba, K. Tissue-specific and
developmentally regulated alternative splicing in mouse skeletal
muscle ryanodine receptor mRNA. Biochem. J . 1995, 305, 373-
378.
(28) Leeb, T.; Brenig, B. cDNA cloning and sequencing of the human
ryanodine receptor type 3 (RYR3) reveals a novel alternative
splice site in the RYR3 gene. FEBS Lett. 1998, 423, 367-370.
(29) Zaidi, M.; Shankar, V. S.; Tunwell, R.; Adebanjo, O. A.; Mackrill,
J .; Pazianas, M.; O’Connell, D.; Simon, B. J .; Rifkin, B. R.;
Venkitaraman, A. R.; Huang, C. L. H.; Lai, F. A. A ryanodine
receptor-like molecule expressed in the osteoclast plasma mem-
brane functions in extracellular Ca2+. J . Clin. Invest. 1995, 73,
1582-1590.
(30) Mygland, A.; Kuwajima, G.; Mikoshiba, K.; Tysnes, O. B.; Aarli,
J . A.; Gilhus, N. E. Thymomas express epitopes shared by the
ryanodine receptor. J . Neuroimmunol. 1995, 62, 79-83.
J M9805079