Hydroxamate based inhibitors of adenylyl cyclase. Part 2: The effect of cyclic linkers on P-site binding

Article abstract of DOI:10.1016/S0960-894X(02)00654-6

The adenylyl cyclases (ACs) are a family of enzymes that are key elements of 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 purine bindin

Full text of DOI:10.1016/S0960-894X(02)00654-6

Bioorganic & Medicinal Chemistry Letters 12 (2002) 3089–3092
Hydroxamate Based Inhibitors of Adenylyl Cyclase.
Part 2: The Effect of Cyclic Linkers on P-Site Binding
Daniel E. Levy,* Ming Bao, James E. Tomlinson and Robert M. Scarborough
Millennium Pharmaceuticals, Inc., 256 East Grand Avenue, South San Francisco, CA 94080, USA
Received 20 May 2002; accepted 1 August 2002
Abstract—The adenylyl cyclases (ACs) are a family of enzymes that are key elements of 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 purine
binding site (P-site) followed by metal mediated cyclization with loss of pyrophosphate. Previous work in our group identified novel
inhibitors which possess an adenine ring joined to a metal-coordinating hydroxamic acid through flexible linkers. Considering the
spatial positioning of the metals with respect to the adenine binding site coupled with potentially favorable entropic factors, con-
formational restriction of the tether through a stereochemistry based SAR employing a rigid cyclic scaffold was explored.
# 2002 Elsevier Science Ltd. All rights reserved.
b-Adrenergic signaling is a key process in CV,1 CNS2
and metabolic regulation.3 Unfortunately, the prolonged
use of b-agonists/antagonists is plagued by poor tissue
selectivity, sensitization/desensitization following therapy,
and dynamic changes to the b-adrenergic receptors that
are inconsistent among disease states.4 Since the use of
b-blockers in clinical settings is intended to regulate
cAMP production, direct regulation of AC activity may
afford a novel pharmacological approach to b-adrenergic
signaling.
required to order flexible linkers within the active site
has potentially negative effects on inhibitory activity,
inhibitors bearing conformationally restricted linkers
were proposed. As shown in Figure 3, linker rigidity was
designed utilizing cyclopentene and cyclopentane rings.
Because this modification results in the introduction of
two stereogenic centers, all stereoisomeric hydroxamic
acids were prepared and compared to their corresponding
carboxylic acid and ester analogues.
As shown in Scheme 1, the commercially available
(1R,3S)-cyclopentene, 1, was alkylated with ethyl diazo-
acetate utilizing rhodium catalysis.8 Subsequent cleavage
of the acetate group, with simultaneous conversion of
the ethyl ester to a methyl ester, gave the alcohol, 2.
Alkylation of adenine at N9 with alcohol, 2, was
achieved via Mitsunobu methodology9,10 and gave the
desired compound, 3a, with inversion.
Previous studies in our labs demonstrated that AC P-site
antagonists may be obtained by linking adenine to a
metal chelating hydroxamic acid via flexible linkers.
Figure 1 illustrates several representative inhibitors
derived from this approach.5 As shown in our initial
study, inhibitory activity was dependent on both the
length and heteroatom content of the linkers.
Previously reported crystallographic studies6,7 of adenylyl
cyclase/Gsa complexes with bound P-site substrates con-
firmed our hypothesis regarding the importance of
metal chelating groups in enzyme catalysis. Figure 2,
based on these studies, illustrates expected binding
interactions between one of our designed inhibitors and
the AC active site residues. Realizing the energy
Conversion of 3a 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
hydrolyzed to its carboxylic acid, 4a, on treatment with
sodium hydroxide in aqueous methanol. Alternately,
the ester group was converted to its hydroxamic acid,
5a, on treatment with hydroxylamine and potassium
hydroxide in methanol.11 Following hydrogenation of
*Corresponding author. Tel.: +1-650-246-7073; fax:+1-650-244-
9287; e-mail: dan.levy@mpi.com
0960-894X/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(02)00654-6

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the cyclopentene ester, 3a, to its corresponding cyclo-
pentane derivative, 6a, the analogous carboxylic acid
and hydroxamic acid analogues (compounds 7a and 8a,
respectively) were prepared.
(Scheme 1) and all relevant derivatives. As shown in
Scheme 3, this goal was realized beginning with the same
cyclopentene starting material, 1. Initial protection of the
alcohol as its TBDMS ether followed by cleavage of the
acetate group and alkylation of the resulting hydroxyl
group with ethyl diazoacetate gave compound 9. Cleavage
of the silyl group followed by coupling with adenine under
Mitsunobu conditions gave the desired enantiomer, 3b.
The cyclopentene products prepared according to
Scheme 1 possess the (1S, 3S) configuration as defined
by the numbering in Figure 3. Similarly, the cyclo-
pentane analogues possess the (1R, 3R) configuration.
As this study focused not only on the rigidity of the
tether, but also on the stereochemistry of the sub-
stituents around the tether, preparation of all possible
stereoisomers was of interest. Therefore, the next goal
was to synthesize the enantiomer of compound 3a
Utilizing the chemistry described in Scheme 2, compound
3b was converted to its saturated ester and corresponding
cyclopentene and cyclopentane linked carboxylic and
hydroxamic acids (compounds 4b–8b). In this series, the
(1R, 3R) cyclopentene and (1S, 3S) cyclopentane con-
figurations were realized.
Having prepared two series bearing enantiomeric linkers,
attention was directed towards the synthesis of the series
containing the corresponding diastereomeric linkers. As
illustrated in Scheme 4, protecting group manipulation
gave the intermediate alcohol, 10. Subsequent inversion
Scheme 1. Reagents and conditions: (a) ethyl diazoacetate,
[Rh(OAc)2]2, CH2Cl2; (b) NaOMe, MeOH, rt; (c) adenine, DEAD,
PPh3, THF.
Figure 1. Representative P-site inhibitors with flexible linkers.
Scheme 2. Reagents and conditions: (a) NaOH, MeOH, H2O, rt; (b)
HONH2.HCl, KOH, MeOH, rt; (c) H2, 10% Pd/C, MeOH.
Figure 2. Proposed interactions between ATP binding site and flexible
P-site inhibitor.
Scheme 3. Reagents and conditions: (a) TBDMS-Cl, imidazole, THF,
rt; (b) NaOMe, MeOH, rt; (c) ethyl diazoacetate, [Rh(OAc)2]2,
CH2Cl2; (d) AcOH, THF, H2O, rt; (e) adenine, DEAD, PPh3, THF.
Figure 3. Design of cyclic tethers from acyclic analogues.

D. E. Levy et al. / Bioorg. Med. Chem. Lett. 12 (2002) 3089–3092
3091
In agreement with our previously reported data,5 all
ester analogues, regardless of stereochemistry, failed to
demonstrate any measure of inhibitory activity against type
V AC (data not shown). However, unlike the carboxylic
acids presented in previous reports,5 some analogues
presented herein did exhibit a weak level of inhibitory
activity (Table 1). Due to the low level of inhibition, it is
difficult to assess whether this activity relies on the ste-
reochemistry around the cyclic tether. Perhaps the most
striking results noted relate to the hydroxamic acid
analogues (Table 2). While all of these compounds
demonstrated some level of inhibitory activity, two
compounds stood out, analogues 5b and 8b. These
compounds (Fig. 4), were found to inhibit type V AC
with approximately an eight-fold increase in activity
over the predecessor acyclic analogue. While the activity
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.
Scheme 4. Reagents and conditions: (a) TBDMS-Cl, imidazole, THF,
rt; (b) NaOMe, MeOH, rt; (c) p-nitrobenzoic acid, DEAD, PPh3,
THF; (d) ethyl diazoacetate, [Rh(OAc)2]2, CH2Cl2; (e) TBAF, THF,
rt; (f) adenine, DEAD, PPh3, THF.
of the hydroxyl stereochemistry was achieved utilizing the
Mitsunobu reaction with p-nitrobenzoic acid followed by
cleavage of the resulting p-nitrobenzoate. The resulting
alcohol, 11, was alkylated with ethyl diazoacetate and
the silyl group was cleaved. Treatment of compound 12
with adenine under Mitsunobu conditions gave the
ester, 3c, bearing the desired diastereomeric linker.
Table 1. IC50 values of carboxylic acids against type V AC
Applying the chemistry described in Scheme 2, com-
pound 3c was converted into its saturated ester and
corresponding cyclopentene and cyclopentane linked
carboxylic and hydroxamic acids (compounds 4c–8c).
The (1R, 3S) cyclopentene and (1S, 3R) cyclopentane
configurations were realized in this scheme.
In pursuit of the final family of diastereomers, com-
pound 9 (Scheme 3) was utilized as illustrated in Scheme
5. As shown, initial cleavage of the silyl group followed
by inversion of the hydroxyl stereochemistry under
Mitsunobu conditions gave the desired p-nitrobenzoate,
13. Subsequent cleavage of the p-nitrobenzoate and
coupling with adenine under Mitsunobu conditions
gave the desired ester, 3d.
Compd
Stereochemistry
IC50 (mM)
4a
7a
4b
7b
4c
7c
4d
7d
1S, 3S
1R, 3R
1R, 3R
1S, 3S
1R, 3S
1S, 3R
1S, 3R
1R, 3S
98Æ61 (n=2)
>200
78Æ33 (n=4)
79Æ48 (n=3)
72.0( n=1)
>200
>200
>200
Using previously described chemistry, compound 3d
was converted its saturated ester and corresponding
cyclopentene and cyclopentane linked carboxylic and
hydroxamic acids (compounds 4d–8d). The (1S, 3R)
cyclopentene and (1R, 3S) cyclopentane configurations
were realized using this scheme.
Table 2. IC50 values of hydroxamic acids against type V AC
Following isolation, all adenine based esters, carboxylic
and hydroxamic acids were evaluated against type V
AC expressed in HEK 293 cells.12
Compd
Stereochemistry
IC50 (mM)
8b
5b
5d
5a
8d
8a
5c
8c
1S, 3S
1R, 3R
1S, 3R
1S, 3S
1R, 3S
1R, 3R
1R, 3S
1S, 3R
10.8Æ3.6 (n=13)
13.6Æ4.0( n=12)
96Æ78 (n=2)
102Æ13 (n=2)
109Æ46 (n=3)
110Æ12 (n=2)
116Æ35 (n=3)
>200
Scheme 5. Reagents and conditions: (a) AcOH, THF, H2O, rt; (b)
p-nitrobenzoic acid, DEAD, PPh3, THF; (c) NaOMe, MeOH, rt; (d)
Adenine, DEAD, PPh3, THF.

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References and Notes
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4. Braunwald, E.; Zipes, D. P.; Libby, P. In Heart Disease,
6th Ed., W. B. Saunders Company, Philadelphia, 1997.
5. Levy, D. E.; Marlowe, C.; Kane-Maguire, K.; Bao, M.;
Cherbavaz, D.; Tomlinson, J.; Sedlock, D.; Scarborough, R.
M. Bioorg. Med. Chem. Lett. 2002, 12, 3085.
6. Dessauer, C. W.; Tesmer, J. J. G.; Sprang, S. R.; Gilman,
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7. Tesmer, J. J. G.; Sunahara, R. K.; Johnson, R. A.; Gosselin,
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Teyessie, Ph. Tetrahedron Lett. 1973, 14, 2233.
Figure 4. Most potent cyclic analogues derived from acyclic lead.
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11. Levy, D. E.; Lapierre, F.; Liang, W.; Ye, W.; Lange,
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12. Measurement of AC activities was achieved utilizing
human type V recombinant AC expressed in HEK293 cells.
Isolated membranes (140ng/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 recombinant Gsa, 1 mM
ATP, 2 mM isobutyl methyl xanthine and 2 mM MgCl2.
Compounds were added to the mixture and the reaction was
run for 30min at 30 ꢀC. Terminated reactions were evaluated
for the enzymatic product, cAMP, using a commercially
available New England Nuclear flash plate system. The degree
of inhibition was determined by comparing the measured
cAMP concentrations to those measured in control reactions
containing no compound.
In conclusion, rationally designed inhibitors of type V
AC were prepared and tested. Two hydroxamic acid
based inhibitors with cyclic tethers showed significantly
enhanced activity over the corresponding acyclic analogue.
In addition, the level of activity was highly dependent upon
the stereochemistry within the cyclic tether. While the
most potent analogues possessed strongly chelating hydr-
oxamic acid groups, the weaker chelating carboxylic acids
showed some activity presumably due to favorable
positioning of the adenine and chelator by the rigid linker.
These results validate our hypothesis that more potent
inhibitors could be obtained by introducing conforma-
tional restriction into the tether of the designed adenine-
linked hydroxamic acids. Further attempts to improve
the activity of these type V AC inhibitors will be the
subject of future communications from this laboratory.