Synthesis and evaluation of a phosphonate analogue of the soluble guanylate cyclase activator YC-1

Article abstract of DOI:10.1016/j.bmcl.2007.06.039

Soluble guanylate cyclase (sGC) is activated by the known benzylindazole derivative YC-1 [1-benzyl-3-(5′-hydroxymethyl-2′-furyl)-indazole]. YC-1 also acts synergistically with CO, activating sGC to a level comparable to that achieved upon binding of nitric oxide, the endogenous activator of sGC. We here describe the synthesis of a YC-1 phosphonate analogue with improved aqueous solubility as well as its effects on sGC.

Full text of DOI:10.1016/j.bmcl.2007.06.039

Bioorganic & Medicinal Chemistry Letters 17 (2007) 4938–4941
Synthesis and evaluation of a phosphonate analogue of the
soluble guanylate cyclase activator YC-1
Nathaniel I. Martin,a Emily R. Derbyshireb and Michael A. Marlettaa,b,c,
*
aDepartment of Chemistry, University of California, Berkeley, CA 94720, USA
bDepartment of Molecular and Cell Biology, University of California, Berkeley, CA 94720, USA
cDivision of Physical Biosciences, Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USA
Received 26 April 2007; revised 6 June 2007; accepted 7 June 2007
Available online 14 June 2007
Abstract—Soluble guanylate cyclase (sGC) is activated by the known benzylindazole derivative YC-1 [1-benzyl-3-(50-hydroxy-
methyl-20-furyl)-indazole]. YC-1 also acts synergistically with CO, activating sGC to a level comparable to that achieved upon bind-
ing of nitric oxide, the endogenous activator of sGC. We here describe the synthesis of a YC-1 phosphonate analogue with improved
aqueous solubility as well as its effects on sGC.
Ó 2007 Elsevier Ltd. All rights reserved.
Soluble guanylate cyclase (sGC)1 is a heterodimeric
O
HO
O
hemoprotein consisting of an a1 and b1 subunit. sGC
is the primary nitric oxide (NO) receptor in mammals
and is activated several hundred-fold over the basal
activity upon the binding of NO, catalyzing the conver-
sion of GTP to cGMP and pyrophosphate. cGMP is re-
quired for vascular smooth muscle relaxation, neuronal
signaling, and platelet aggregation.1–4 Compounds capa-
ble of activating sGC present an approach to treating
diseases involving the NO-cGMP pathway.
P
O
O
HO
N
OH
N
N
N
1a
1b
Figure 1. (a) YC-1 and (b) YC-1 phosphate analogue initially pursued.
results of these studies leave YC-1’s precise mode of ac-
tion an open question.
In addition to NO activation, sGC can also be activated
3- to 5-fold upon binding of carbon monoxide (CO) to
the heme, or 8- to 12-fold in the presence of the benzy-
lindazole derivative YC-1 [3-(50-hydroxymethyl-20-fur-
yl)-1-benzylindazole] (Fig. 1a), a known activator of
sGC.5 Furthermore, YC-1 and CO together act synergis-
tically to activate the enzyme to a level similar to that
To further explore the effects of YC-1 on sGC, a phos-
phate analogue (Fig. 1b) was initially pursued. The ratio-
nale behind the phosphate analogue was twofold: (1)
while YC-1 has been shown to be efficacious in vivo, its
aqueous solubility is extremely low (<200 lM). Phos-
phorylation of the YC-1 hydroxy group would be
expected to improve solubility. (2) Also, the analogue
could be used to test the theory that YC-1 is a nucleotide
mimic capable of activating sGC by binding to a
‘pseudosymmetric’ site.15,17 If YC-1 binds to a substrate
binding site, phosphorylation of YC-1 may provide a
better mimic thus increasing the potency of the activator.
Phosphorylation of the YC-1 hydroxy group was there-
fore attempted to address both of these factors.
observed with NO.6,7
A variety of experimental
approaches, aimed at locating the YC-1 binding site
within sGC, have been inconclusive.8–14 YC-1 binding
in the heme pocket on the b1 subunit,9,10 to a predicted
PAS domain on the a1 subunit,12 and to a regulatory
GTP binding site on the catalytic domains (a1 and b1
subunits)15,16 have all been proposed. The conflicting
Keywords: YC-1; Soluble guanylate cyclase; NO signaling.
*
Initial attempts at the phosphorylation of YC-1 em-
ployed standard approaches. Treatment with phospho-
Corresponding author. Tel.: +1 510 643 9325; fax: +1 510 643
9388; e-mail: marletta@berkeley.edu
0960-894X/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.06.039

N. I. Martin et al. / Bioorg. Med. Chem. Lett. 17 (2007) 4938–4941
4939
While compounds 1 and 2 are stable and observable by
NMR it seems that upon oxidation to the phosphate a
rapid elimination/addition process occurs. The elimina-
tion of the phosphate is likely aided by the nitrogen
lone-pair present in the extended conjugated system.
This elimination product is then quenched by an
available nucleophile, in this case residual tetrazole
(Scheme 3).
N
O
P
O
a
YC-1
N
O
N
CN
1
O
O
Based upon these findings it seems unlikely that phos-
phorylation of the YC-1 hydroxy group is possible.
The insights gained into the inherent reactivity of the
molecule itself however suggested that a phosphonate
analogue might be accessible by a modified Michaelis–
Arbuzov approach. It was previously shown by the
group of Marder18 that very electron-rich, benzylic-type,
phosphonates can be prepared by treatment of the cor-
responding alcohol in triethyl phosphite with one equiv-
alent of iodine in a general one-pot process (Scheme 4).
P
O
N
b
O
YC-1
N
2
Scheme 1. Conversion of YC-1 to phosphoramidite intermediate 1 and
phospho-triester intermediate 2. Reagents: (a) 2-cyanoethyl N,N-
diisopropylchlorophosphoramidite, DIPEA, MeCN; (b) diallyl N,N-
diisopropylphosphoramidite, tetrazole, CH2Cl2.
This approach to a YC-1 phosphonate analogue seemed
appealing with the exception of the use of triethyl phos-
phite. Diethyl phosphonates typically require harsh (Le-
wis or protic acid) hydrolysis conditions to yield the
deprotected phosphonate.19 To circumvent this issue
we chose to investigate the use of tribenzyl phosphite
in the iodine-promoted preparation of a dibenzyl phos-
phonate analogue of YC-1. The resulting dibenzyl phos-
phonate could then be deprotected using milder
hydrogenation conditions. Tribenzyl phosphite20 is not
commercially available and was readily prepared by
the treatment of phosphorus trichloride with benzyl
alcohol (Scheme 5).
rus oxychloride followed by aqueous work-up failed to
yield the desired product. Other attempts involving the
low temperature deprotonation followed by treatment
with tetrabenzyl pyrophosphate were also unsuccessful.
As a third option phosphoramidite chemistry was then
explored. While not successful in yielding the desired
phosphorylated YC-1 species, the phosphoramidite
approach revealed a unique pattern of reactivity within
the molecule. Treatment of YC-1 with 2-cyanoethyl
N,N-diisopropylchlorophosphoramidite
or
diallyl
N,N-diisopropylphosphoramidite successfully yielded
the expected phosphoramidite 1 or phosphite-triester 2
intermediates (Scheme 1).
In the attempted oxidative conversion of both 1 and 2 to
their respective phosphate derivatives however, the
unexpected product 3 was obtained (Scheme 2).
O
OH
P
OC2H5
OC2H5
triethyl phosphite
1.0 eq. I2
N
(reference 18)
1
N
a, b, c
N
N
N
N
Ar
Ar
Ar
Ar
O
N
N
Scheme 4. One-pot preparation of electron-rich phosphonates from
the corresponding alcohol.
d
3
2
3 BnOH
PCl3
P(OBn)3
NEt3, Et2O
81%
Scheme 2. Conversion of compounds 1 and 2 to unexpected adduct 3.
Reagents: (a) tetrazole, MeCN/H2O; (b) I2, THF/Pyr/H2O; (c) DBU;
(d) mCPBA.
Scheme 5. Preparation of tribenzyl phosphite.
N
N
O
N
NH
RO
P
O
OX
O
O
1 or 2
3
OR
N
N
N
N
Scheme 3. Mechanistic rationale for conversion of compounds 1 and 2 to tetrazole adduct 3.

4940
N. I. Martin et al. / Bioorg. Med. Chem. Lett. 17 (2007) 4938–4941
O
P
O
P
HO
BnO
BnO
HO
a
b
O
O
O
HO
N
N
N
N
N
N
4
5
Scheme 6. Preparation of the phosphonate analogue of YC-1 5 via dibenzyl phosphonate 4. Reagents: (a) P(OBn)3, 1.0 equiv I2; (b) H2, Pd/C,
MeOH.
When treated with an excess of tribenzyl phosphite and
one equivalent of iodine at room temperature, YC-1 is
slowly converted to the desired dibenzyl phosphonate
4 (conversion accelerated upon heating to 40 °C). As ex-
pected, hydrogenation of 4 successfully provided the
deprotected YC-1 phosphonate analogue 5 (Scheme 6).
a lower Kd of binding for the GTP binding site in the
Fe–NO form of the enzyme. It is well established that
the Km for sGC with its endogenous substrate (GTP)
is dependent on the ligation state of the enzyme.9
In conclusion, while the preparation of a phosphory-
lated YC-1 species was not possible, the information
gained from our attempts has shed light onto an inher-
ent reactivity pattern for this important sGC-activating
molecule. From the insights gained, a phosphonate ana-
logue of YC-1 was successfully prepared via a modified
Michaelis–Arbuzov approach. This analogue shows im-
proved aqueous solubility and a comparable ability to
activate sGC at concentrations typically used in the
YC-1 activation of sGC. With improved solubility prop-
erties the phosphonate analogue was also tested at high-
er concentrations showing a somewhat unexpected
inhibitory effect. Future work will be aimed at establish-
ing the nature of this apparent inhibition.
Effect of YC-1 and phosphonate analogue 5 on sGC
activity. In agreement with our prediction, the YC-1
phosphonate analogue 5 shows improved aqueous solu-
bility (>800 lM) compared with YC-1 (aqueous solubil-
ity <200 lM). The ability of the phosphonate analogue
5 to activate sGC was next investigated. Activity assays
of YC-1 (at 150 lM) and analogue 5 (at 150 and
800 lM) with sGC gave the results shown in Table 1.
At 150 lM, YC-1 and compound 5 both activate sGC
(6.4- and 5.3-fold, respectively). In the presence of CO,
the same concentrations of YC-1 and compound 5 act
synergistically, activating 87-fold and 16-fold, respec-
tively. However, NO induced activity decreased from
77- to 38-fold in the presence of compound 5. This inhi-
bition is unlike the synergism that is observed with YC-1
and NO.
Supplementary data
Supplementary data associated with this article can be
found in the online version. Supplementary data associ-
ated with this article can be found, in the online version,
at doi:10.1016/j.bmcl.2007.06.039.
Somewhat surprisingly, the phosphonate analogue 5
shows much different effects with sGC at the higher con-
centration tested (800 lM). In all cases this concentra-
tion of compound 5 suppressed sGC activity relative
to the fold activation observed at a concentration of
150 lM (Table 1). A possible explanation for this effect
may be that compound 5 is also able to bind at the cat-
alytic substrate (GTP) binding site, acting as a compet-
itive inhibitor at the higher concentration tested. Also of
interest are the differing effects observed for compound 5
with the Fe–NO and Fe–CO complexes of sGC. A pos-
sible explanation for this could be that compound 5 has
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