Metal coordination-based inhibitors of adenylyl cyclase: Novel potent P-site antagonists

Article abstract of DOI:10.1021/jm0205604

The adenylyl cyclases (ACs) are a family of intracellular enzymes associated with signal transduction by virtue of their ability to convert ATP to cAMP. The catalytic mechanism of this transformation proceeds through initial binding of ATP to the so-calle

Full text of DOI:10.1021/jm0205604

J . Med. Chem. 2003, 46, 2177-2186
2177
Meta l Coor d in a tion -Ba sed In h ibitor s of Ad en ylyl Cycla se: Novel P oten t P -Site
An ta gon ists
Daniel E. Levy,*,† Ming Bao, Diana B. Cherbavaz, J ames E. Tomlinson, David M. Sedlock,
Charles J . Homcy, and Robert M. Scarborough*
Departments of Medicinal Chemistry and Biology, Millennium Pharmaceuticals, Inc., 256 East Grand Avenue,
South San Francisco, California 94080
Received December 13, 2002
The adenylyl cyclases (ACs) are a family of intracellular enzymes associated with signal
transduction by virtue of their ability to convert ATP to cAMP. The catalytic mechanism of
this transformation proceeds through initial binding of ATP to the so-called purine binding
site (P-site) of the enzyme followed by metal-mediated cyclization with loss of pyrophosphate.
Crystallographic analysis of ACs with known inhibitors reveals the presence of two metals in
the active site. Presently, nine isoforms of adenylyl cyclase are known, and unique isoform
combinations are expressed in a tissue-specific manner. The development of isoform-specific
inhibitors of adenylyl cyclase may prove to be a useful strategy toward the design of unique
signal transduction inhibitors. To develop novel AC inhibitors, we have chosen an approach to
inhibitor design utilizing an adenine ring system joined to a metal-coordinating hydroxamic
acid via various linkers. Previous work in our group has validated this approach and identified
novel inhibitors that possess an adenine ring joined to a metal-coordinating hydroxamic acid
through flexible acyclic linkers (Levy, D. E., et al. Bioorg. Med. Chem. Lett. 2002, 12, 3085-
3088). Subsequent studies have focused on the introduction of conformational restrictions into
the tether of the inhibitors with the goal of increasing potency (Levy, D. E., et al. Bioorg. Med.
Chem. Lett. 2002, 12, 3089-3092). Building upon the favorable spatial positioning of the adenine
and hydroxamate groups coupled with potentially favorable entropic factors, the unit joining
the carbocycle to the hydroxamate was explored further and a stereochemical-based SAR was
elucidated, leading to a new series of highly potent AC inhibitors.
In tr od u ction
â-Adrenergic signaling is a key process in cardiovas-
cular,1 CNS,2 and metabolic regulation.3 Figure 14
illustrates the regulation of cAMP production through
the â-adrenergic receptor. As shown, heterotrimeric
G-protein complexes interact with the â-adrenergic
receptor and release activated GsR when â-adrenergic
ligands are bound.5 Following dissociation, activated
GsR binds to and stabilizes adenylyl cyclase (AC),6,7
which catalyzes the conversion of ATP into cAMP.8 One
strategy for regulating â-adrenergic signaling that has
been pursued is prolonged use of â-agonists/antagonists.
This approach is unfortunately plagued by poor tissue
selectivity, sensitization/desensitization following therapy,
F igu r e 1. Interaction cascade leading to conversion of ATP
and dynamic changes to the â-adrenergic receptors that
are inconsistent among disease states.9
to cAMP.
Previous studies in our labs have demonstrated that
Successful crystallographic studies with known AC
AC P-site antagonists may be obtained by linking the
P-site inhibitors were reported utilizing a heterodimeric
adenine ring system to a metal chelating hydroxamic
crystal comprised of the C1 domain from type V AC and
acid via flexible linkers.10 Subsequent studies demon-
the C2 domain from type II AC. As noted previously,
strated the importance of stereochemistry and confor-
these reported crystallographic studies12,13 of adenylyl
mational rigidity in the tether.11 Figure 2 illustrates
cyclase/GsR complexes with bound P-site substrates
several representative inhibitors derived from this
confirmed our original inhibitor design hypothesis re-
approach.
garding the importance of metal chelating groups in
enzyme catalysis. On the basis of these studies, Figure
* Millennium Pharmaceuticals, Inc. 256 East Grand Avenue, South
3 illustrates the expected binding interactions between
one of our designed inhibitors and the AC active site
residues. Subsequently, having established the impor-
tance of stereochemistry and structural rigidity within
San Francisco, CA 94080. Tel: (650) 244-6822. Fax: (650) 244-9287.
E-mail: bob.scarborough@mpi.com.
† Current address: Scios, Inc. 820 West Maude Avenue, Sunnyvale,
CA 94085. Tel: (408) 616-8391. Fax: (408) 616-8317. E-mail:
levyd@sciosinc.com.
10.1021/jm0205604 CCC: $25.00 © 2003 American Chemical Society
Published on Web 04/19/2003

2178 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 11
Levy et al.
Sch em e 1
hydroxamic acids prepared in this study were compared
to the activities of the analogous carboxylic acids and
esters to confirm the importance of the metal-chelating
functionality in enzyme inhibition.
Ch em istr y. The compounds of the present study
were prepared in two groups. The first group possessed
the metal chelating functionality separated from the
cyclopentene/cyclopentane ring by a methylene unit.
This involved removal of the oxygen atom of our
previous inhibitor IV affording the cyclopentene/cyclo-
pentane acetic acid derived compounds V. The second
group consisted of compounds analogous to the first with
the methylene unit removed affording the cyclopentene/
cyclopentane carboxylic acid-derived compounds VI.
Beginning with the compounds of the first group, the
starting material for all structures was commercially
available (1R,3S)-4-cyclopentene-1,3-diol-1-acetate, 1.
As shown in Scheme 1, the hydroxyl group of compound
1 was converted into its corresponding silyl ether, and
the acetate group was subsequently cleaved giving the
allylic alcohol, 2. The allylic alcohol was converted to
the corresponding allylic chloride 3, on treatment with
mesyl chloride, with stereochemical inversion. Com-
pound 3 was then used to alkylate dimethylmalonate,
again with inversion of the stereochemistry at the
chloride center. The resulting diester 4 was monode-
carboxylated on treatment with lithium iodide,14,15 and
the silyl ether was subsequently cleaved under acidic
conditions affording the allylic alcohol, 5. Finally, utiliz-
ing Mitsunobu conditions,16,17 compound 5 was coupled
with adenine with stereochemical inversion at the
hydroxide center giving the cyclopenteneacetic acid ester
6a .
F igu r e 2. Representative synthetic P-site inhibitors.
F igu r e 3. Proposed interactions between ATP binding site
and a synthetic P-site inhibitor.
Conversion of 6a to its corresponding cyclopentene-
and cyclopentane-based carboxylic and hydroxamic
acids is shown in Scheme 2. Although Scheme 2 is
drawn with no inferred stereochemistry, there was no
compromise to the configurational purity of the starting
ester during any of the illustrated steps. As shown, the
ester was converted into the hydroxamic acid, 7a , on
treatment with hydroxylamine and potassium hydroxide
in methanol.18 Alternately, the ester group was hydro-
lyzed to the carboxylic acid, 8a , on treatment with
sodium hydroxide in aqueous methanol. Following
hydrogenation of the cyclopentene ester, 6a , into the
corresponding cyclopentane derivative 9a , the analogous
hydroxamic acid and carboxylic acid analogues (com-
pounds 10a and 11a , respectively) were prepared.
The cyclopentene products prepared according to
Scheme 1 possess the (1R, 3S) configuration as defined
F igu r e 4. Modification of unit joining hydroxamate to cyclic
tether.
the inhibitor tethers, the present study was initiated
to probe the spatial requirements of the P-site through
further modification of the unit joining the adenine ring
and the metal-chelating hydroxamic acid (Figure 4). As
noted in previous studies, there is a dependence upon
the stereochemical configuration around the cyclopen-
tene/cyclopentane ring tether which we continued to
probe through the synthesis of all possible stereoiso-
meric hydroxamic acids. The inhibitory activities of the

Metal Coordination-Based Inhibitors of AC
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 11 2179
Sch em e 2
Sch em e 4
Sch em e 5
Sch em e 3
mers was inversion of the hydroxyl group stereocenter
of compound 2. This was accomplished by utilizing the
Mitsunobu coupling of compound 2 with p-nitrobenzoic
acid19 followed by cleavage of the p-nitrobenzoate with
sodium methoxide. The resulting allylic alcohol, 16, was
converted to the corresponding allyl chloride, 17, on
treatment with mesyl chloride, with stereochemical
inversion. Compound 17 was then used to alkylate
dimethylmalonate with inversion of the stereochemistry
at the chloride center. The resulting compound 18 was
decarboxylated on treatment with lithium iodide and
the silyl ether was subsequently cleaved with tetrabu-
tylammonium fluoride giving the allylic alcohol 19.
Finally, utilizing Mitsunobu conditions, compound 19
and adenine were coupled with stereochemical inversion
at the hydroxide center giving compound 6c.
Applying the chemistry described in Scheme 2, com-
pound 6c was converted into the saturated ester and
corresponding cyclopentene- and cyclopentane-linked
carboxylic and hydroxamic acids (compounds 7c-11c).
However, unlike previous examples, the carboxylic and
hydroxamic acids for the cyclopentene and cyclopentane-
based linkers were isolated from the hydroxamic acid
synthesis. Once formed, these compounds were easily
separated utilizing reversed-phase HPLC. The com-
pounds isolated from this scheme possessed the (1R, 3R)
cyclopentene and (1S, 3S) cyclopentane configurations.
by the numbering in Figure 3. Similarly, the cyclopen-
tane analogues possess the (1S, 3S) configuration. As
this study focused not only on the rigidity of the tether,
but also on the stereochemistry of the substituents
within the tether, preparation of all possible stereoiso-
mers was of interest. Therefore, the next goal was to
synthesize the enantiomer of compound 6a and all
relevant derivatives. As shown in Scheme 3, this goal
was realized beginning with the previously described
intermediate 2. Initial protection of the allylic alcohol
as a trityl ether followed by cleavage of the silyl ether
with TBAF gave the allylic alcohol, 12. The allylic
alcohol was converted to the corresponding allyl chlo-
ride, 13, on treatment with mesyl chloride, with stereo-
chemical inversion. Compound 13 was then used to
alkylate dimethylmalonate with inversion of the ster-
eochemistry at the chloride center. The resulting com-
pound 14 was decarboxylated on treatment with lithium
iodide and the trityl ether was subsequently cleaved
under acidic conditions giving the allylic alcohol 15.
Finally, utilizing Mitsunobu conditions, compound 15
and adenine were coupled with stereochemical inversion
at the hydroxyl center giving compound 6b.
Utilizing the chemistry described in Scheme 2, com-
pound 6b was converted into the saturated ester and
corresponding cyclopentene- and cyclopentane-linked
carboxylic and hydroxamic acids (compounds 7b-1b).
In this series, the (1S, 3R) cyclopentene and (1R, 3R)
cyclopentane configurations were obtained.
Having prepared two series of derivatives bearing
enantiomeric linkers, attention was directed toward the
synthesis of the series containing the corresponding
diastereomeric linkers. As illustrated in Scheme 4, the
key step in the preparation of the first set of diastereo-
In pursuit of the final family of diastereomers,
compound 12 (Scheme 3) was utilized as illustrated in
Scheme 5. Applying the Mitsunobu coupling of com-
pound 12 with p-nitrobenzoic acid followed by cleavage
of the p-nitrobenzoate with sodium methoxide, the
stereochemistry of the hydroxyl group was inverted
giving compound 20. Conversion of 20 to the corre-
sponding allyl chloride 21 was achieved on treatment
with mesyl chloride accompanied by stereochemical
inversion. Compound 21 was then used to alkylate
dimethylmalonate with inversion of the stereochemistry

2180 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 11
Levy et al.
Sch em e 6
Sch em e 7
Sch em e 8
at the chloride center. The resulting compound 22 was
decarboxylated on treatment with lithium iodide and
the trityl ether was subsequently cleaved under acidic
conditions giving the allylic alcohol, 23. Finally, utilizing
Mitsunobu conditions, compound 23 and adenine were
coupled with stereochemical inversion at the hydroxide
center giving compound 6d .
Using the previously described chemistry, compound
6d was converted to its saturated ester and correspond-
ing cyclopentene- and cyclopentane-linked carboxylic
and hydroxamic acids (compounds 7d -11d ). The (1S,
3S) cyclopentene and (1R, 3S) cyclopentane configura-
tions were obtained using this scheme.
Having completed the cyclopentene/cyclopentane ace-
tic acid derived group, attention was focused on the
preparation of compounds with a direct linkage between
the cyclopentene/cyclopentane ring and the metal che-
lator. To accomplish this goal, commercially available
(1R,4S)-4-amino-cyclopent-2-ene carboxylic acid, 24a ,
was used. As shown in Scheme 6, compound 24a was
converted to its methyl ester, 25a , in acidic methanol.
The amine was then coupled with 4,6-dichloro-5-nitro-
pyrimidine.20 Following reduction of the nitro group
with tin chloride,21 cyclization to the chloropurine, 26a ,
was achieved on treatment with trimethylorthoformate
and catalytic methanesulfonic acid.22 Subsequent con-
version to the azidopurine, 27a , was achieved on treat-
ment with sodium azide.
Continuing as illustrated in Scheme 7, the azidopu-
rine, 27a , was converted to its corresponding substituted
adenine, 28a , utilizing tin chloride. Alternately, the
azide and the olefin were simultaneously reduced via
catalytic hydrogenation giving compound 29a . On treat-
ment with hydroxylamine and potassium hydroxide,
compound 29a was converted to the corresponding
hydroxamic acid, 30a .
all compounds prepared in Schemes 6-8 (compounds
25b-33b) were prepared under identical conditions
beginning with commercially available (1S,4R)-4-amino-
cyclopent-2-ene carboxylic acid, 24b.
SAR An a lysis. The compounds of the present study
were assayed using recombinant human type V AC
expressed in HEK293 cells. Membranes isolated from
these cells demonstrated a 40-50-fold stimulation by
recombinant activated GsR when compared to empty
vector (pcDNA3) control populations. Stimulation with
activated GsR demonstrates that 90-98% of the cAMP
generated in the human AC V populations is due to
expression of the recombinant adenylyl cyclase.
In agreement with our previously reported data,10,11
all ester analogues, regardless of stereochemistry, failed
to demonstrate any measurable inhibitory activity
against type V AC (data not shown). With respect to
the carboxylic acids prepared in this study (Table 1),
some of the acids prepared did show moderate to weak
inhibitory activity (compounds 8b, 11b, and 8d ). This
activity was highly dependent upon the stereochemical
configuration around the cyclopentene/cyclopentane ring
and directly correlated with the activity of the most
potent hydroxamic acids (Table 2) in this series (com-
pounds 7b, 10b, and 7d ). With respect to the cyclopen-
Completion of this family of compounds proceeded as
shown in Scheme 8. As illustrated, compound 28a was
treated with hydroxylamine and potassium hydroxide
forming a mixture of diastereomeric hydroxamic acids
31a and 32a , in a ratio of approximately 2:1 favoring
compound 31a . Following separation of these isomers
by preparative reversed-phase HPLC, compound 32a
was subjected to catalytic hydrogenation giving com-
pound 33a . It should be noted that the enantiomers of

Metal Coordination-Based Inhibitors of AC
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 11 2181
Ta ble 1. Cyclopentene/Cyclopentane Acetic Acids
compds
stereochemistry
IC50 (µM)
8a
11a
8b
11b
8c
11c
8d
1R, 3S
1S, 3S
1S, 3R
1R, 3R
1R, 3R
1S, 3R
1S, 3S
1R, 3S
>200
>100
8.2 ( 5.2 (n ) 3)
28.8 ( 13.4 (n ) 2)
>200
>200
73.2 (n ) 1)
>200
11d
Ta ble 2. Cyclopentene/Cyclopentane Acetic Acid
Hydroxamates
F igu r e 5. Evolution of potent hydroxamic acid-based AC
inhibitors.
compds
stereochemistry
IC50 (µM)
7a
10a
7b
10b
7c
10c
7d
1R, 3S
1S, 3S
1S, 3R
1R, 3R
1R, 3R
1S, 3R
1S, 3S
1R, 3S
4.3 ( 1.8 (n ) 5)
9.4 ( 3.1 (n ) 5)
0.4 ( 0.1 (n ) 3)
0.6 ( 0.2 (n ) 3)
16.3 ( 1.4 (n ) 2)
55.0 ( 4.6 (n ) 2)
2.6 ( 0.9 (n ) 3)
25.1 ( 27.6 (n ) 3)
contrast, the activity of compound 33a appeared to be
dependent upon both the stereochemistry and the
absence of an endocyclic double bond in the cyclic tether.
The stereochemical relationship between the adenine
and the hydroxamate were identical among all three
compounds.
10d
Con clu sion s
Ta ble 3. Cyclopentene/Cyclopentane Hydroxamic Acids
Rationally designed inhibitors of type V AC were
prepared and tested in this study. Three hydroxamic
acid-based inhibitors with cyclic tethers showed signifi-
cantly enhanced activity over the corresponding acyclic
analogues previously prepared. In addition, the level of
activity was highly dependent upon the stereochemistry
within the cyclic tether and, in one case, upon the
absence of the endocyclic double bond within the tether.
While the most potent analogues possessed strongly
chelating hydroxamic acid functionality, some carboxylic
acids showed activity presumably due to favorable
positioning of the adenine and the weaker chelator by
the rigid linker. These results validate our hypothesis
that more potent inhibitors could be obtained by intro-
ducing conformational restriction into the tether of the
designed adenine-linked hydroxamic acids.
compds
stereochemistry
IC50 (µM)
30a
30b
31a
31b
32a
32b
33a
33b
1R, 3S
1S, 3R
1S, 3R
1R, 3S
1S, 3S
1R, 3R
1R, 3R
1S, 3S
4.6 ( 2.2 (n ) 4)
> 70
188 ( 68 (n ) 2)
> 200
4.9 ( 1.5 (n ) 2)
100 ( 82 (n ) 2)
0.2 ( 0.2 (n ) 3)
78 ( 68 (n ) 2)
tene/cyclopentane carboxylic acid-derived series (Table
3), compound 33a was found to have exceptional inhibi-
tory activity. As noted in the previous series, this
activity was highly dependent upon stereochemistry and
was only noted for the cyclopentane-containing ana-
logue. The most potent hydroxamic acids prepared in
this study, illustrated in Figure 5, inhibited type V AC
with 13-89-fold increases in activity over the predeces-
sor acyclic analogues. While the activity of compounds
7b and 10b did not appear to be dependent upon the
presence of an endocyclic double bond in the linker, it
was highly dependent upon the stereochemistry around
the cyclic tether with a preferred trans relationship
between the adenine and hydroxamate groups. By
While the introduction of conformational restriction
into the linker did enhance the potency of the inhibitors
as measured against type V AC, it is currently unclear
if these restrictions altered the expected mode of binding
illustrated in Figure 3. However, the illustrated active
site amino acid residues Arg1029, Ser1032, Ser1028,
Thr401, and Asn1025, shown to interact with 2-deoxy-
3′-AMP in crystallographic studies,10,12,13 are not ex-
pected to interact with the reported synthetic inhibitors
due to the absence of hydrogen bond donors and accep-
tors within the tether region. Although it is anticipated
that the model reflects the probable binding mode, its
relevance to type V AC is limited because of the
inclusion of the C2 domain from type II AC. Neverthe-

2182 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 11
Levy et al.
Gen er a l P r oced u r e F - TBDMS P r otection . An alcohol
(72.24 mmol) was dissolved in THF (200 mL) and imidazole
(108.36 mmol) was added followed by TBDMS-Cl (90.30 mmol).
The reaction was stirred at room temperature for 24 h after
which the solids were removed by filtration and the filtrate
was concentrated to dryness. The residue was dissolved in
EtOAc (300 mL) and washed with HCl (1 N, 3 × 50 mL),
saturated NaHCO3 (3 × 50 mL), and brine (50 mL). The
organic phase was dried over anhydrous MgSO4, filtered, and
concentrated, and the residue was used with no further
purification.
Gen er a l P r oced u r e G - TBDMS Clea va ge (AcOH/THF /
Wa ter ). A TBDMS ether (4.52 mmol) was combined with THF
(1 mL), H2O (1 mL), and acetic acid (3 mL). The reaction was
stirred at room temperature for 6 h after which it was
azeotroped with benzene (3 × 15 mL). The residue was dried
under vacuum and purified on silica gel (25% EtOAc/hexane).
Gen er a l P r oced u r e H - p-Nitr oben zoic Acid Mitsu n o-
bu . An allylic alcohol (60.70 mmol), p-nitrobenzoic acid (242.81
mmol), and triphenylphospine (242.81 mmol) were combined
with THF (200 mL) and cooled to 0 °C under argon. Diethyl-
azodicarboxylate (242.81 mmol) was added dropwise and the
reaction was stirred at room temperature for 15 h and 40 °C
for an additional 3 h. After the sample was cooled to room
temperature, the reaction was concentrated to dryness and the
residue was diluted with EtOAc (200 mL). The resulting
solution was washed with HCl (1 N, 3 × 50 mL), brine (50
mL), saturated NaHOC3 (3 × 50 mL), and brine (50 mL). After
drying of the sample over anhydrous MgSO4, the organics were
filtered, concentrated, and stirred with Et2O (150 mL) for 18
h. The resulting solids were removed by filtration and the
filtrate was concentrated to dryness. The isolated residue was
used without further purification.
less, crystallographic studies placing the inhibitors of
this report into the active site of the type V/type II AC
heterodimer are currently underway.
Exp er im en ta l Section
Gen er al P r ocedu r e. 1H Nuclear magnetic resonance (NMR)
spectra were recorded on a Varian Unity +400 spectrometer.
Low-resolution mass spectra were recorded with a HP 1100-
MSD LC-MS spectrometer. High-resolution mass spectra
(HRMS) were recorded with a VG ZAB2-EQ high-resolution
mass spectrometer and performed at the University of Cali-
fornia, Berkeley. Final compounds were purified by reversed
phase high-performance liquid chromatography (HPLC) using
a Waters 4000Prep, Waters 490E multiwavelength detector
and Vydac 218TP1022 column (10 µM, C18, 22 × 250 mm).
Purity of the compounds was confirmed by two diverse HPLC
systems using Waters 600 controller, Waters 996 photodiode
array detector, and a Keystone Beta Basic column (C18, 4.6 ×
50 mm). HPLC method A (isocratic) - 2% CH3CN/10 min
HPLC method B (gradient) - 5-85% CH3CN/9 min. The new
compounds synthesized were characterized by mass spectrom-
etry (MS) and NMR, and purity was determined by HRMS
and two HPLC systems. We did not obtain melting points,
elemental analyses, or optical rotations of these compounds
as most of the derivatives were amorphous, hygroscopic, and
prepared in small quantities. Commercially available reagents
and starting materials were obtained from Aldrich and Chi-
rotech and were used with no additional purification. Chro-
matography generally refers to flash silica gel chromatography
(J . T. Baker, 40 µM, flash chromatography silica gel). The
typical experimental procedures used in this study are de-
scribed as follows.
Gen er a l P r oced u r e A - Hyd r oxa m ic Acid s. KOH (3.8
M in MeOH, 0.45 mL) was added to HONH2‚HCl (1.6 M in
MeOH, 0.67 mL) and cooled to 0 °C for 2 h. A methyl or ethyl
ester (0.15 mmols) was dissolved in MeOH (0.31 mL) and the
HONH2 solution was added by filtration. After the sample was
stirred for 45 min at room temperature, the reaction was
concentrated to dryness and the residue was purified by
reverse phase preparative HPLC (0-10% CH3CN/30 min). The
isolated product was desalted with MP-carbonate resin (Ar-
gonaut) in MeOH, filtered, and concentrated to dryness giving
the desired hydroxamic acid.
Gen er a l P r oced u r e B - Ca r boxylic Acid s. A methyl or
ethyl ester (0.53 mmols) was dissolved in MeOH (2.40 mL)
and NaOH (2.00 M in H2O, 1.60 mmols) was added. The
reaction was stirred at room temperature for 2.5 h after which
it was acidified to pH ) 2 with DOWEX acid resin (50WX2-
100, MeOH washed). The reaction was filtered and concen-
trated to dryness giving the desired carboxylic acid.
Gen er a l P r oced u r e I - TBDMS Clea va ge (TBAF ). A
TBDMS ether (69.58 mmol) was dissolved in THF (500 mL)
and tetrabutylammonium fluoride (1 M in THF, 104 mL) was
added. The reaction was stirred at room temperature for 2 h
and concentrated to dryness. The residue was filtered through
silica gel (EtOAc) and again concentrated to dryness. Final
purification was achieved on silica gel (10% then 25% then
50% EtOAc/hexane).
Gen er a l P r oced u r e J - Allyl Ch lor id e. An allylic alcohol
(46.17 mmol) was dissolved in CH2Cl2 and diisopropylethyl-
amine (69.25 mmol) was added. The resulting solution was
cooled to 0 °C under argon and methanesulfonyl chloride (57.71
mmol) was added. After stirring at 0 °C for 3 h, the reaction
was diluted with EtOAc (600 mL). The mixture was then
washed with HCl (1 N, 3 × 50 mL), saturated NaHCO3 (3 ×
50 mL), and brine (50 mL). The organics were dried over
anhydrous MgSO4, filtered, and concentrated, and the residue
was purified on silica gel (5% EtOAc/hexane).
Gen er al P r ocedu r e C - Acetate/p-Nitr oben zoate Cleav-
a ge (Na OMe). An acetate or p-nitrobenzoate (5.18 mmol) was
dissolved in anhydrous MeOH (15 mL), and catalytic NaOMe
(solution in MeOH) was added. The reaction was stirred at
room temperature for 24 h after which it was quenched with
H2O (1.0 mL) and concentrated to dryness. The product was
purified on silica gel (50% EtOAc/hexane).
Gen er a l P r oced u r e D - Ad en in e Mitsu n obu . An allylic
alcohol (10.93 mmol), triphenylphosphine (10.93 mmol), and
adenine (10.93 mmol) were dissolved in THF (40 mL) and
cooled to 0 °C. Diethyl azodicarboxylate (10.93 mmol) was
added dropwise and the reaction was stirred at room temper-
ature for 18 h. After heating the reaction to 40 °C for an
additional 4 h, the mixture was cooled to room temperature
and the solids were removed by filtration. The filtrate was
concentrated to dryness and the residue was purified on silica
gel (EtOAc then 5% MeOH/CHCl3).
Gen er a l P r oced u r e K - Ma lon a te Cou p lin g. NaH (60%,
149.03 mmol) was suspended in anhydrous THF (400 mL) and
cooled to 0 °C under argon. Dimethylmalonate (149.03 mmol)
was added dropwise over 30 min and the reaction was allowed
to warm to room temperature. An allyl chloride (29.81 mmols)
was dissolved in anhydrous THF (100 mL) and added to the
malonate solution via cannula. After heating of the sample to
75 °C for 19 h, the reaction was cooled, concentrated to a
volume of 150 mL, and diluted with 50% EtOAc/hexane (300
mL). The resulting solution was washed with saturated NH4-
Cl (3 × 50 mL) and brine (2 × 50 mL). Following concentration,
the organics were partitioned between hexane (150 mL) and
H2O (150 mL). The hexane layer was further washed with H2O
(2 × 50 mL), dried over anhydrous MgSO4, filtered, and
concentrated. The residue was purified on silica gel (5% EtOAc/
hexane).
Gen er a l P r oced u r e
L - Deca r boxyla tion (LiI). A
Gen er a l P r oced u r e E - Olefin Hyd r ogen a tion (Also
Azid e Red u ction ). An olefin (100 mg) and 10% Pd/C (25 mg)
were placed under argon and MeOH (10 mL) was added. The
mixture was degassed under vacuum and stirred under H2 (1
atm) for 20 h. The reaction was filtered and concentrated
giving the desired product.
substituted malonate (31.93 mmol) was combined with LiI
(191.58 mmol) and dissolved in DMF (260 mL). The mixture
was degassed under vacuum, placed under argon, and heated
to 130 °C for 17 h. After the sample was cooled to room
temperature, the reaction was diluted with 25% EtOAc/hexane
(1500 mL) and washed with H2O (3 × 300 mL) and brine (100

Metal Coordination-Based Inhibitors of AC
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 11 2183
mL). The organic phase was dried over anhydrous MgSO4,
filtered, and concentrated. The resulting residue was purified
on silica gel (5% EtOAc/hexane).
(1R,3S)-1-Hyd r oxy-3-(ter t-Bu tyl-d im eth ylsiloxy)-4-cy-
clop en ten e (2). (1R,3S)-1-Acetoxy-3-hydroxy-4-cyclopentene,
1, was subjected to general procedure F. Yield ) 100%. TLC:
Rf ) 0.48 (10% EtOAc/hexane). Subsequent subjection of the
product to general procedure C gave compound 2. Yield ) 90%.
TLC: Rf ) 0.43 (25% EtOAc/hexane).
(1R,3R)-1-(ter t-Bu tyl-d im eth ylsiloxy)-3-h yd r oxy-4-cy-
clop en ten e (16). Compound 2 was subjected to general
procedure H. Yield ) 81%. TLC: Rf ) 0.50 (10% EtOAc/
hexane). Subsequent subjection of the product to general
procedure C gave compound 16. Yield ) 85%. TLC: Rf ) 0.33
(25% EtOAc/hexane).
(1S,3R)-1-H yd r oxy-3-t r ip h en ylm et h oxy-4-cyclop en -
ten e (12). Compound 2 was subjected to general procedure
M. TLC: Rf ) 0.27 (hexane). Subsequent subjection of the
product to general procedure I gave compound 12. Yield ) 87%
(2 steps). TLC: Rf ) 0.32 (25% EtOAc/hexane).
(1R ,3R )-1-Ch lor o-3-t r ip h e n ylm e t h oxy-4-cyclop e n -
ten e (13). Compound 13 was prepared by subjecting com-
pound 12 to general procedure J . Yield ) 84%. TLC: Rf ) 0.61
(10% EtOAc/hexane).
(1S,3R)-1-(2-Dim eth ylm a lon yl)-3-tr ip h en ylm eth oxy-4-
cyclop en ten e (14). Compound 14 was prepared by subjecting
compound 13 to general procedure K. Yield ) 72%. TLC: Rf
) 0.27 (10% EtOAc/hexane).
(1R,3R)-1-Hyd r oxy-3-(m eth yl-ca r boxym eth yl)-4-cyclo-
p en ten e (15). Compound 14 was subjected to general proce-
dure L. Yield ) 80%. TLC: Rf ) 0.45 (10% EtOAc/hexane).
Subsequent subjection of the product to general procedure N
gave compound 15. Yield ) 39%. TLC: Rf ) 0.38 (50% EtOAc/
hexane).
(1S,3R)-1-(9-Ad en en yl)-3-(m et h yl-ca r b oxym et h yl)-4-
cyclop en ten e (6b). Compound 6b was prepared by subjecting
compound 15 to general procedure D. Yield ) 9%. TLC: Rf )
0.22 (5% MeOH/CHCl3). Purity: >85% (HPLC method B).
(1S,3R)-1-(9-Aden en yl)-3-(N-h ydr oxycar bam oylm eth yl)-
4-cyclop en ten e (7b). Compound 7b was prepared by subject-
ing compound 6b to general procedure A. Yield ) 37%. TLC:
Rf ) 0.40 (CHCl3/MeOH/H2O 150/45/5). Purity: >97% (HPLC
method A); >97% (HPLC method B).
Gen er a l P r oced u r e M - Tr ityla tion . An allylic alcohol
(9.39 mmol), trityl chloride (46.96 mmol), and DMAP (56.36
mmol) were combined and dissolved in DMF (30 mL). After
heating of the sample to 100 °C for 20 h, the reaction was
cooled to room temperature and diluted with H2O (200 mL).
The aqueous mixture was washed with 50% EtOAc/hexane
(200 mL), and the organics were sequentially washed with HCl
(1 N, 3 × 25 mL), saturated NaHCO3 (3 × 25 mL), and brine
(25 mL). The organics were dried over MgSO4, filtered,
concentrated to dryness, and used without further purification.
Gen er a l P r oced u r e N - Tr ityl Clea va ge (TsOH). A
trityl ether (15.75 mmol) was dissolved in MeOH (100 mL)
and p-toluenesulfonic acid (0.79 mmol) was added. After
stirring of the sample at room temperature for 1.25 h, the
reaction was quenched with saturated NaHCO3 (100 mL). The
resulting mixture was washed with EtOAc (3 × 100 mL), and
the combined organic extracts were washed with brine (50
mL). After drying of the sample over anhydrous MgSO4, the
product was purified on silica gel (25% then 50% EtOAc/
hexane).
Gen er a l P r oced u r e
O - Meth yl Ester F or m a tion
(MeOH, Ac-Cl). Acetyl chloride (9.00 mmol) was slowly
added to MeOH (35.00 mL) and cooled to 0 °C. A carboxylic
acid (7.87 mmol) was added and the resulting mixture was
stirred at room temperature for 4 h. Concentration of the
reaction mixture provided the desired product requiring no
further purification.
Gen er a l P r oced u r e P - Cou p lin g w ith P yr im id in e. An
amine hydrochloride (7.97 mmol) was combined with dichlo-
ronitropyrimidine (11.95 mmol) and EtOH (80 mL). Triethyl-
amine (23.90 mmol) was added and the reaction was stirred
at room temperature for 3.5 h. Following dilution with EtOAc
(320 mL), the mixture was sequentially washed with HCl (1
N, 3 × 50 mL), saturated NaHCO3 (3 × 30 mL), and brine (30
mL). The organics were dried over anhydrous MgSO4, filtered,
and concentrated. The isolated residue was used with no
further purification.
(1S,3R)-1-(9-Ad en en yl)-3-ca r b oxym et h yl-4-cyclop en -
ten e (8b). Compound 8b was prepared by subjecting com-
pound 6b to general procedure B. Yield ) 97%. TLC: Rf )
0.42 (CHCl3/MeOH/H2O 150/45/5). Purity: >86% (HPLC
method B).
(1R,3R)-1-(9-Ad en en yl)-3-(m et h yl-ca r b oxym et h yl)cy-
clop en ta n e (9b). Compound 9b was prepared by subjecting
compound 6b to general procedure E. Yield ) 95%. TLC: Rf
) 0.21 (CHCl3/MeOH/H2O 150/45/5). Purity: >86% (HPLC
method B).
(1R,3R)-1-(9-Ad en en yl)-3-(N-h yd r oxyca r ba m oylm eth -
yl)cyclop en ta n e (10b). Compound 10b was prepared by
subjecting compound 9b to general procedure A. Yield ) 39%.
TLC: Rf ) 0.34 (CHCl3/MeOH/H2O 150/45/5). Purity: >97%
(HPLC method A); >99% (HPLC method B).
(1R ,3R )-1-(9-Ad e n e n yl)-3-ca r b oxym e t h ylcyclop e n -
ta n e (11b). Compound 11b was prepared by subjecting
compound 9b to general procedure B. Yield ) 83%. TLC: Rf
) 0.38 (CHCl3/MeOH/H2O 150/45/5). Purity: >80% (HPLC
method B).
(1S,3S)-1-Ch lor o-3-(ter t-Bu tyl-d im eth ylsiloxy)-4-cyclo-
p en ten e (3). Compound 3 was prepared by subjecting com-
pound 2 to general procedure J . Yield ) 79%. TLC: Rf ) 0.80
(10% EtOAc/hexane).
(1R,3S)-1-(2-Dim eth ylm a lon yl)-3-(ter t-Bu tyl-d im eth yl-
siloxy)-4-cyclop en ten e (4). Compound 4 was prepared by
subjecting compound 3 to general procedure K. Yield ) 74%.
TLC: Rf ) 0.32 (10% EtOAc/hexane).
(1S,3S)-1-Hyd r oxy-3-(m eth yl-ca r boxym eth yl)-4-cyclo-
p en ten e (5). Compound 4 was subjected to general procedure
L. Yield ) 75%. TLC: Rf ) 0.57 (10% EtOAc/hexane).
Subsequent subjection of the product to general procedure G
gave compound 5. Yield ) 61%. TLC: Rf ) 0.39 (50% EtOAc/
hexane).
Gen er al P r ocedu r e Q - Nitr o Gr ou p Redu ction (Sn Cl2).
A nitropyrimidine (9.36 mmol) was dissolved in EtOH (75 mL),
and SnCl2 (28.09 mmol) was added. The reaction was heated
to reflux for 50 min and cooled to room temperature. Following
quenching with saturated NaHCO3 (300 mL), the reaction was
washed with EtOAc (3 × 75 mL). The organic extracts were
washed with brine (2 × 75 mL), dried over anhydrous MgSO4,
filtered, and concentrated. No further purification was re-
quired.
Gen er a l P r oced u r e R - P u r in e F or m a tion (Or th ofor -
m a te, Ms-OH). A diaminopyrimidine (9.36 mmol) was dis-
solved in trimethylorthoformate (25 mL) and methanesulfonic
acid (0.22 mL) was added. The reaction was stirred at room
temperature for 4.5 h and diluted with EtOAc (150 mL). The
resulting mixture was washed with saturated NaHCO3 (3 ×
25 mL) and brine (25 mL). The organic phase was dried over
anhydrous MgSO4, filtered, and concentrated. The product was
purified on silica gel (50% EtOAc/hexane).
Gen er a l P r oced u r e S - Azid op u r in e. A chloropurine
(3.02 mmol), sodium azide (9.06 mmol), EtOH (13 mL), and
H2O (6.5 mL) were combined and heated to 50 °C for 20 h.
After stirring of the sample for an additional 17 h at room
temperature, the reaction was concentrated to dryness. The
residue was diluted with H2O (20 mL) and the resulting solids
were filtered, washed with H2O, and dried in a desiccator. No
further purification was required.
Gen er a l P r oced u r e T - Azid e Red u ction (Sn Cl2). An
azidopurine (0.42 mmol) was dissolved in EtOH (3.25 mL) and
SnCl2 (1.27 mmol) was added. The reaction was heated to
reflux for 20 min and cooled to room temperature. Following
quenching of the sample with saturated NaHCO3 (15 mL), the
reaction was washed with EtOAc (3 × 15 mL). The organic
extracts were dried over anhydrous MgSO4, filtered, and
concentrated. No further purification was required.

2184 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 11
Levy et al.
(1R,3S)-1-(9-Ad en en yl)-3-(m et h yl-ca r b oxym et h yl)-4-
cyclop en ten e (6a ). Compound 6a was prepared by subjecting
compound 5 to general procedure D. Yield ) 10%. TLC: Rf )
0.14 (5% MeOH/CHCl3). Purity: >85% (HPLC method B).
(1R,3S)-1-(9-Aden en yl)-3-(N-h ydr oxycar bam oylm eth yl)-
4-cyclop en ten e (7a ). Compound 7a was prepared by subject-
ing compound 6a to general procedure A. Yield ) 63%. TLC:
Rf ) 0.40 (CHCl3/MeOH/H2O 150/45/5). Purity: >84% (HPLC
method A); >90% (HPLC method B).
(1R,3R)-1-H yd r oxy-3-t r ip h en ylm et h oxy-4-cyclop en -
ten e (20). Compound 12 was subjected to general procedure
H. TLC: Rf ) 0.36 (10% EtOAc/hexane). Subsequent subjection
of the product to general procedure C gave compound 20. Yield
) 77% (two steps). TLC: Rf ) 0.32 (25% EtOAc/hexane).
(1S ,3R )-1-C h lo r o -3-t r ip h e n y lm e t h o x y -4-c y c lo p e n -
ten e (21). Compound 21 was prepared by subjecting com-
pound 20 to general procedure J . Yield ) 93%. TLC: Rf ) 0.58
(10% EtOAc/hexane).
(1R,3R)-1-(2-Dim eth ylm a lon yl)-3-tr ip h en ylm eth oxy-4-
cyclop en ten e (22). Compound 22 was prepared by subjecting
compound 21 to general procedure K. Yield ) 74%. TLC: Rf
) 0.22 (10% EtOAc/hexane).
(1R,3S)-1-Hyd r oxy-3-(m eth yl-ca r boxym eth yl)-4-cyclo-
p en ten e (23). Compound 22 was subjected to general proce-
dure L. Yield ) 63%. TLC: Rf ) 0.45 (10% EtOAc/hexane).
Subsequent subjection of the product to general procedure N
gave compound 23. Yield ) 54%. TLC: Rf ) 0.35 (50% EtOAc/
hexane).
(1R,3S)-1-(9-Ad en en yl)-3-ca r b oxym et h yl-4-cyclop en -
ten e (8a ). Compound 8a was prepared by subjecting com-
pound 6a to general procedure B. Yield ) 100%. TLC: Rf )
0.31 (CHCl3/MeOH/H2O 150/45/5). Purity: >85% (HPLC
method B).
(1S,3S)-1-(9-Ad en en yl)-3-(m et h yl-ca r b oxym et h yl)cy-
clop en ta n e (9a ). Compound 9a was prepared by subjecting
compound 6a to general procedure E. Yield ) 93%. TLC: Rf
) 0.22 (CHCl3/MeOH/H2O 150/45/5). Purity: >93% (HPLC
method B).
(1S,3S)-1-(9-Ad en en yl)-3-(m eth yl-ca r boxym eth yl)-4-cy-
clop en ten e (6d ). Compound 6d was prepared by subjecting
compound 23 to general procedure D. Yield ) 16%. TLC: Rf
) 0.17 (5% MeOH/CHCl3). Purity: >87% (HPLC method B).
(1S,3S)-1-(9-Aden en yl)-3-(N-h ydr oxycar bam oylm eth yl)-
4-cyclop en ten e (7d ). Compound 7d was prepared by subject-
ing compound 6d to general procedure A. Yield ) 49%. TLC:
Rf ) 0.36 (CHCl3/MeOH/H2O 150/45/5). Purity: >88% (HPLC
method A); >89% (HPLC method B).
(1S,3S)-1-(9-Ad en en yl)-3-ca r b oxym et h yl-4-cyclop en -
ten e (8d ). Compound 8d was prepared by subjecting com-
pound 6d to general procedure B. Yield ) 75%. TLC: Rf )
0.34 (CHCl3/MeOH/H2O 150/45/5). Purity: >84% (HPLC
method B).
(1S,3S)-1-(9-Ad en en yl)-3-(N-h yd r oxyca r b a m oylm et h -
yl)cyclop en ta n e (10a ). Compound 10a was prepared by
subjecting compound 9a to general procedure A. Yield ) 34%.
TLC: Rf ) 0.39 (CHCl3/MeOH/H2O 150/45/5). Purity: >98%
(HPLC method A); >85% (HPLC method B).
(1S ,3S )-1-(9-Ad e n e n yl)-3-c a r b oxym e t h ylc yc lop e n -
ta n e (11a ). Compound 11a was prepared by subjecting
compound 9a to general procedure B. Yield ) 83%. TLC: Rf
) 0.33 (CHCl3/MeOH/H2O 150/45/5). Purity: >80% (HPLC
method B).
(1R,3S)-1-Ch lor o-3-(ter t-Bu tyl-d im eth ylsiloxy)-4-cyclo-
p en ten e (17). Compound 17 was prepared by subjecting
compound 16 to general procedure J . Yield ) 99%. TLC: Rf )
0.86 (10% EtOAc/hexane).
(1R,3S)-1-(9-Ad en en yl)-3-(m et h yl-ca r b oxym et h yl)cy-
clop en ta n e (9d ). Compound 9d was prepared by subjecting
compound 6d to general procedure E. Yield ) 92%. TLC: Rf
) 0.27 (5% MeOH/CHCl3). Purity: >87% (HPLC method B).
(1S,3S)-1-(2-Dim eth ylm a lon yl)-3-(ter t-Bu tyl-d im eth yl-
siloxy)-4-cyclop en ten e (18). Compound 18 was prepared by
subjecting compound 17 to general procedure K. Yield ) 75%.
TLC: Rf ) 0.36 (10% EtOAc/hexane).
(1R,3S)-1-(9-Ad en en yl)-3-(N-h yd r oxyca r ba m oylm eth -
yl)cyclop en ta n e (10d ). Compound 10d was prepared by
subjecting compound 9d to general procedure A. Yield ) 56%.
TLC: Rf ) 0.42 (CHCl3/MeOH/H2O 150/45/5). Purity: >96%
(HPLC method A); >94% (HPLC method B).
(1R ,3S )-1-(9-Ad e n e n yl)-3-ca r b oxym e t h ylcyclop e n -
ta n e (11d ). Compound 11d was prepared by subjecting
compound 9d to general procedure B. Yield ) 99%. TLC: Rf
) 0.42 (CHCl3/MeOH/H2O 150/45/5). Purity: >82% (HPLC
method B).
(1S,3R)-1-Hyd r oxy-3-(m eth yl-ca r boxym eth yl)-4-cyclo-
p en ten e (19). Compound 18 was subjected to general proce-
dure L. Yield ) 74%. TLC: Rf ) 0.58 (10% EtOAc/hexane).
Subsequent subjection of the product to general procedure I
gave compound 19. Yield ) 59%. TLC: Rf ) 0.40 (50% EtOAc/
hexane).
(1R,3R)-1-(9-Ad en en yl)-3-(m et h yl-ca r b oxym et h yl)-4-
cyclop en ten e (6c). Compound 6c was prepared by subjecting
compound 19 to general procedure D. Yield ) 4.3%. TLC: Rf
) 0.17 (5% MeOH/CHCl3). Purity: >81% (HPLC method B).
(1S ,3R )-Me t h yl-1-a m in ocyclop e n t -4-e n e -3-ca r b oxy-
la te h yd r och lor id e (25a ). Compound 25a was prepared by
subjecting (1R,4S)-4-amino-cyclopent-2-ene carboxylic acid to
general procedure O. Yield ) 100%.
(1R ,3S )-Me t h yl-1-a m in ocyclop e n t -4-e n e -3-ca r b oxy-
la te h yd r och lor id e (25b). Compound 25b was prepared by
subjecting (1S,4R)-4-amino-cyclopent-2-ene carboxylic acid to
general procedure O. Yield ) 100%.
(1S,3R)-1-[9-(1-Ch lor oa d en en yl)]-3-m et h ylca r b oxy-4-
cyclop en ten e (26a ). Compound 25a was subjected to general
procedure P. Subsequent subjection of the crude product to
general procedure Q yielded the desired crude aminopyrimi-
dine. Without purification, the crude aminopyrimidine was
subjected to general procedure R giving compound 26a . Yield
) 40% (3 steps). TLC: Rf ) 0.50% (EtOAc/hexane). Purity:
>90% (HPLC method B).
(1R,3S)-1-[9-(1-Ch lor oa d en en yl)]-3-m et h ylca r b oxy-4-
cyclop en ten e (26b). Compound 25b was subjected to general
procedure P. Subsequent subjection of the crude product to
general procedure Q yielded the desired crude aminopyrimi-
dine. Without purification, the crude aminopyrimidine was
subjected to general procedure R giving compound 26b. Yield
) 38% (three steps). TLC: Rf ) 0.50% (EtOAc/Hexane).
Purity: >90% (HPLC method B).
(1R,3R)-1-(9-Aden en yl)-3-(N-h ydr oxycar bam oylm eth yl)-
4-cyclop en ten e (7c) a n d (1R,3R)-1-(9-Ad en en yl)-3-ca r -
boxym eth yl-4-cyclop en ten e (8c). Compound 6c was sub-
jected to general procedure A, and the products were separated
by preparative TLC (CHCl3/MeOH/H2O 150/45/5). Hydroxamic
acid (7c): Yield ) 21%. TLC: Rf ) 0.29 (CHCl3/MeOH/H2O
150/45/5). Purity: >89% (HPLC method A); >90% (HPLC
method B). HRMS: calcd (C12H14N6O2) m/z 275.1257 (M+
+
1), found 275.1257 (M+ + 1). Carboxylic acid (8c): Yield )
31%. TLC: Rf ) 0.25 (CHCl3/MeOH/H2O 150/45/5). Purity:
>99% (HPLC method B).
(1S,3R)-1-(9-Ad en en yl)-3-(m et h yl-ca r b oxym et h yl)cy-
clop en ta n e (9c). Compound 9c was prepared by subjecting
compound 6c to general procedure E. Yield ) 85%. TLC: Rf
) 0.27 (5% MeOH/CHCl3).
(1S,3R)-1-(9-Ad en en yl)-3-(N-h yd r oxyca r ba m oylm eth -
yl)cyclop en ta n e (10c) a n d (1S,3R)-1-(9-Ad en en yl)-3-ca r -
boxym eth ylcyclop en ta n e (11c). Compound 9c was sub-
jected to general procedure A, and the products were separated
by preparative HPLC and isolated as TFA salts. Hydroxamic
acid (10c): Yield ) 32%. TLC: Rf ) 0.33 (CHCl3/MeOH/H2O
150/45/5). Purity: >95% (HPLC method A); >90% (HPLC
method B). HRMS: calcd (C12H16N6O2) m/z 277.1413 (M+
+
1), found 277.1421 (M+ + 1). Carboxylic acid (11c): Yield )
11%. TLC: Rf ) 0.33 (CHCl3/MeOH/H2O 150/45/5). Purity:
>95% (HPLC method B).
(1S,3R)-1-[9-(1-Azid oa d en en yl)]-3-m eth ylca r boxy-4-cy-
clop en ten e (27a ). Compound 27a was prepared by subjecting

Metal Coordination-Based Inhibitors of AC
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 11 2185
compound 26a to general procedure S. Yield ) 52%. Purity:
>99% (HPLC method B).
(1R,3S)-1-[9-(1-Azid oa d en en yl)]-3-m eth ylca r boxy-4-cy-
clop en ten e (27b). Compound 27b was prepared by subjecting
compound 26b to general procedure S. Yield ) 60%. Purity:
>99% (HPLC method B).
(1R ,3S )-1-(9-Ad e n e n yl)-3-m e t h ylca r b oxycyclop e n -
ta n e (29a ). Compound 29a was prepared by subjecting
compound 27a to general procedure E. Yield ) 99%. Purity:
>99% (HPLC method B).
(1S ,3R )-1-(9-Ad e n e n yl)-3-m e t h ylca r b oxycyclop e n -
ta n e (29b). Compound 29b was prepared by subjecting
compound 27b to general procedure E. Yield ) 96%. Purity:
>99% (HPLC method B).
(1R,3S)-1-(9-Ad en en yl)-3-(N-h yd r oxyca r ba m oyl)cyclo-
p en ta n e (30a ). Compound 30a was prepared by subjecting
compound 29a to general procedure A. Yield ) 53%. Purity:
>91% (HPLC method A); >95% (HPLC method B).
(1S,3R)-1-(9-Ad en en yl)-3-(N-h yd r oxyca r ba m oyl)cyclo-
p en ta n e (30b). Compound 30b was prepared by subjecting
compound 29b to general procedure A. Yield ) 53%. Purity:
>98% (HPLC method A); >95% (HPLC method B).
(1S,3R)-1-(9-Ad en en yl)-3-m et h ylca r b oxy-4-cyclop en -
ten e (28a ). Compound 28a was prepared by subjecting
compound 27a to general procedure T. Yield ) 98%. Purity:
>99% (HPLC method B).
flasks for 3 days at 37 °C under 5% CO2. Cells were washed
with sterile phosphate buffered saline (PBS), trypsinized,
concentrated, and seeded in 500 mL Bell-Co spinner flasks at
a density of 1 × 106/mL and grown at 37 °C at medium speed
on a Cellgro stirrer. After 5 days, cells were collected in 500
mL centrifuge bottles and pelleted at 3000 rpm for 10 min.
Pellets were resuspended in tissue culture PBS and harvested
at 1200 rpm for 10 min. Supernatants were discarded and
pellets were frozen at -20 °C.
Adenylyl cyclase membranes were prepared after thawing
cell pellets on ice. Thawed pellets were washed with 15 mL
PBS and centrifugation at 3000 rpm for 10 min at 4 °C in a
Beckman J 2-21 centrifuge. Washed cells were resuspended
in 20 mL of 20 mM Tris-HCl, 5 mM EDTA buffer freshly
supplemented with 2 mM DTT and 1:25 dilution of complete
protease inhibitor and dounced in a tissue grinder 10 times to
homogenization. The suspension was sonicated for 18 cycles
and centrifuged at 12000 rpm for 30 min in a J A-17 rotor.
Pellets were resuspended in 300 mL of 25 mM Tris-HCl, 20
mM MgCl2, and 10% sucrose freshly supplemented with DTT
and Complete as described above to a final concentration of
10 mg/mL protein. Supernatants were then triturated seven
1
times through a 22 /2 gauge needle on a 5 mL syringe. Final
protein concentration was determined and protein was ali-
quoted and stored at -80 °C.
Typ e V AC Assa y. Type V AC activity was evaluated with
or without added inhibitors. Membranes from HEK293 cells
expressing recombinant human type V AC (1.4 µg/mL) were
used in the presence of 60 mM HEPES, pH 8.0, 0.6 mM EDTA,
0.01% (w/v) Bovine serum albumin, 25 nM activated recom-
binant GsR, 1 mM ATP, 2 mM isobutyl methyl xanthine and
2 mM MgCl2. Compounds were added to the mixture and the
reaction was run for 30 min at 30 °C. Terminated reactions
were evaluated for the enzymatic product, cAMP using a
commercially available New England Nuclear flash plate
system. Degree of inhibition was determined by comparing to
control reactions which did not contain compound.
(1R,3S)-1-(9-Ad en en yl)-3-m et h ylca r b oxy-4-cyclop en -
ten e (28b). Compound 28b was prepared by subjecting
compound 27b to general procedure T. Yield ) 97%. Purity:
>99% (HPLC method B).
(1S,3R)-1-(9-Ad en en yl)-3-(N-h yd r oxyca r ba m oyl)-4-cy-
clop en t en e (31a ) a n d (1S,3S)-1-(9-Ad en en yl)-3-(N-h y-
d r oxyca r ba m oyl)-4-cyclop en ten e (32a ). Compound 28a
was subjected to general procedure A, and the products were
separated by preparative HPLC as described. The isolated TFA
salts were converted to free bases utilizing MP-carbonate resin
(Argonaut) in MeOH. Compound 31a : Yield ) 44%. TLC: Rf
) 0.34 (CHCl3/MeOH/H2O 150/45/5). Purity: >96% (HPLC
method A); >99% (HPLC method B). Compound 32a : Yield
) 26%. TLC: Rf ) 0.29 (CHCl3/MeOH/H2O 150/45/5). Purity:
>99% (HPLC method A); >99% (HPLC method B).
(1R,3S)-1-(9-Ad en en yl)-3-(N-h yd r oxyca r ba m oyl)-4-cy-
clop en ten e (31b) a n d (1R,3R)-1-(9-Ad en en yl)-3-(N-h y-
d r oxyca r ba m oyl)-4-cyclop en ten e (32b). Compound 28b
was subjected to general procedure A and the products were
separated by preparative HPLC as described. The isolated TFA
salts were converted to free bases utilizing MP-carbonate resin
(Argonaut) in MeOH. Compound 31b: Yield ) 44%. TLC: Rf
) 0.32 (CHCl3/MeOH/H2O 150/45/5). Purity: >99% (HPLC
method A); >99% (HPLC method B). Compound 32b: Yield
) 24%. TLC: Rf ) 0.26 (CHCl3/MeOH/H2O 150/45/5). Purity:
>99% (HPLC method A); >99% (HPLC method B).
(1R,3R)-1-(9-Ad en en yl)-3-(N-h yd r oxyca r ba m oyl)cyclo-
p en ta n e (33a ). Compound 33a was prepared by subjecting
compound 32a to general procedure E where 10% Pd/C was
replaced with 20% Pd(OH)2/C. Yield ) 99%. TLC: Rf ) 0.27
(CHCl3/MeOH/H2O 150/45/5). Purity: >99% (HPLC method
A); >97% (HPLC method B).
(1S,3S)-1-(9-Ad en en yl)-3-(N-h yd r oxyca r ba m oyl)cyclo-
p en ta n e (33b). Compound 33b was prepared by subjecting
compound 32b to general procedure E where 10% Pd/C was
replaced with 20% Pd(OH)2/C. Yield ) 95%. TLC: Rf ) 0.27
(CHCl3/MeOH/H2O 150/45/5). Purity: >98% (HPLC method
A); >97% (HPLC method B).
HEK293 Mem br a n e P r ep a r a tion s. Cell membranes en-
riched with human adenylyl cyclase isozyme type-5 (hAC5)
were generated by splicing hAC5 into pcDNA3 plasmid DNA
and expressed in human embrionic kidney cells (HEK293).
Cells were grown under nonadherent conditions in F-12
nutrient mixture (HAM) supplemented with 10% fetal bovine
serum (Gemini Bio-Products), 450 µg/mL Geneticin (Gobco
BRL), 1% penicillin-streptomycin, 2 mM final l-glutamine,
0.1% pluronic F-68, 10 units/mL heparin, and buffered with
10 mM HEPES. Cells were grown in T-150 tissue culture
Su p p or tin g In for m a tion Ava ila ble: All 1H NMR and
HRMS data. This material is available free of charge via the
Internet at http://pubs.acs.org.
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