Guanylate cyclase

In vitro activity: BAY 41-2272 causes the human and rabbit cavernosum to relax in response to concentration, with EC50 values of 489.1 nM and 406.3 nM in vitro, respectively.

---

Background: The most important receptor for nitric oxide is the soluble guanylate cyclase (sGC), a heme containing heterodimer. Recently, a pyrazolopyridine derivative BAY 41-2272, structurally related to YC-1, was identified stimulating soluble guanylate cyclase in an NO-independent manner, which results in vasodilatation and antiplatelet activity. The study described here addresses the identification of the NO-independent site on soluble guanylate cyclase. Results: We developed a photoaffinity label (3H-meta-PAL) for the direct and NO-independent soluble guanylate cyclase (sGC) stimulator BAY 41-2272 by introducing an azido-group into the tritium labeled compound. The synthesized photoaffinitylabel directly stimulates the purified sGC and shows in combination with NO a synergistic effect on sGC activity. Irradiation with UV light of 3H-meta-PAL together with the highly purified sGC leads to a covalent binding to the alpha1-subunit of the enzyme. This binding is blocked by unlabeled meta-PAL, YC-1 and BAY 41-2272. For further identification of the NO-independent regulatory site the 3H-meta-PAL labeled sGC was fragmented by CNBr digest. The 3H-meta-PAL binds to a CNBr fragment, consisting of the amino acids 236-290 of the alpha1-subunit. Determination of radioactivity of the single PTH-cycles from the sequencing of this CNBr fragment detected the cysteines 238 and 243 as binding residues of the 3H-meta-PAL.
Conclusions: Our data demonstrate that the region surrounding the cysteines 238 and 243 in the alpha1-subunit of the sGC could play an important role in regulation of sGC activity and could be the target of this new type of sGC stimulators. [1]

---

Through its interaction with this binding site in sGC, BAY 41-2272 possesses an unique pharmacological in vitro and in vivo profile. In phenylephrine-preconstricted (3 × 10-8g ml-1) rabbit aortic rings, BAY 41-2272 elicited a concentration-dependent relaxation with a half-maximal inhibitory concentration (IC50) of 304 ± 63 nM (n = 6), whereas glycerol trinitrate and YC-1 used as controls had an IC50 of 1,300 ± 385 nM (n = 6) and 10,035 ± 632 nM (n = 26).
We and others have shown that YC-1 activates purified sGC and sensitizes the enzyme for NO in vitro, in human platelets, on a sGC-overexpressing cell line and in smooth muscle cells. Compared with BAY 41-2272, about 100-fold higher concentrations of YC-1 would be needed to give similar stimulation at sGC. The structural similarity between BAY 41-2272 and YC-1 might suggest that both compounds act by the same mechanism. In contrast to YC-1 (ref. 21), however, BAY 41-2272, up to 10-5 M, is devoid of any PDE-5 inhibitory activity. [2]

---

BAY41-2272 resulted in concentration dependent relaxation of human and rabbit cavernosum (mean EC50 +/- SEM 489.1 +/- 22.5 and 406.3 +/- 21.5 nM., respectively). The compound was 32 times more potent than YC-1 and twice as potent as spermine-NONOate. ODQ decreased the potency of BAY41-2272, such that in the presence of 30 microM. ODQ the EC50 of BAY41-2272 induced relaxation was 1,407.3 +/- 158.0 and 1,902.7 +/- 11.0 nM. in human and rabbit tissues, respectively. L-NAME also inhibited relaxations elicited by BAY41-2272 in rabbit tissue. In the presence of 500 microM. L-NAME the EC50 of BAY41-2272 induced responses was 836.7 +/- 46.7 nM. BAY41-2272 at subthreshold concentrations of 30 to 50 nM. potentiated nitrergic responses. Moreover, the inhibition of nitrergic responses by L-NAME was reversed by 0.3 to 3 microM. BAY41-2272. Conclusions: We report that a nonNO based soluble guanylate cyclase activator relaxes human and rabbit corpus cavernosum, and potentiates nitrergic responses. [3]

---

In previous study, researchers have found that 5-cyclopropyl-2-[1-(2-fluoro-benzyl)-1H-pyrazolo[3,4-b]pyridine-3-yl]-pyrimidin-4-ylamine (BAY 41-2272), a guanylate cyclase agonist, activates human monocytes and the THP-1 cell line to produce the superoxide anion, increasing in vitro microbicidal activity, suggesting that this drug can be used to modulate immune functioning in primary immunodeficiency patients [4].

BAY 41-2272 (10 mg/kg, p.o.) exhibits antiplatelet effect, significantly lowers blood pressure, and improves survival in female spontaneously hypertensive rats. [2] In addition to improving macrophage functions, BAY 41-2272 (10 mg/kg, i.p.) significantly increases macrophage-dependent cell influx to the peritoneum in C. albicans-infected mice while also lowering the death rate. [4] BAY 41-2272 ameliorates impaired corpus cavernosum (CC) relaxation in db/db-/- type II diabetic and obese mice. [5]

---

In the present work, we investigated the potential of the in vivo administration of BAY 41-2272 for the treatment of Candida albicans and Staphylococcus aureus infections introduced via intraperitoneal and subcutaneous inoculation. We found that intraperitoneal treatment with BAY 41-2272 markedly increased macrophage-dependent cell influx to the peritoneum in addition to macrophage functions, such as spreading, zymosan particle phagocytosis and nitric oxide and phorbol myristate acetate-stimulated hydrogen peroxide production. Treatment with BAY 41-2272 was highly effective in reducing the death rate due to intraperitoneal inoculation of C. albicans, but not S. aureus. However, we found that in vitro stimulation of peritoneal macrophages with BAY 41-2272 markedly increased microbicidal activities against both pathogens. Our results show that the prevention of death by the treatment of C. albicans-infected mice with BAY 41-2272 might occur primarily by the modulation of the host immune response through macrophage activation. [4]

---

Peritoneal cell influx and cell recruitment to lymphoid organs - The mice were treated (or not) with BAY 41-2272 (0.3-10 mg/kg IP) for 48 h, after which the peritoneal cavity was harvested and the spleen, BM and LN were collected (Fig. 1A). The cellular distribution showed that treatment with BAY 41-2272 induced a significant increase in the total number of cells in the peritoneum compared with the control group (Fig. 1B). This cell population was composed primarily of macrophages (Fig. 1C), but the percentage of polymorphonuclear leukocytes (PMNs) was also elevated in the group treated with this drug (Fig. 1D). All vehicles used (transcutol, Cremophor-EL and water solution and DMSO) had no effect on this or the other assays performed in this study (data not shown). [4]

There were no differences in the numbers of cells in the other lymphoid organs, such as the spleen, BM or mesenteric LN s, in the animals treated with BAY 41-2272 compared with the untreated animals (Fig. 1E-G). There was a trend of an increase in cell numbers only in the mesenteric LN s, indicating the recruitment of cells to this draining organ.[4]

Carrageenan-induced footpad oedema - To evaluate the effect of BAY 41-2272 on the inflammatory process we used a carrageenan-induced mouse paw oedema model. Mice were treated (or not) with BAY 41-2272 (0.3-10 mg/kg, IP) for 48 h, after which carrageenan (300 μg/paw) was injected into the footpad to measure oedema formation every hour for 4 h (Fig. 2A). Intraperitoneal pre-treatment with BAY 41-2272 significantly increased paw oedema, which was observed at 180 and 240 min after carrageenan injection (Fig. 2B). Similar data were observed for Con A. These results confirm the pro-inflammatory potential of BAY 41-2272.[4]

Ex vivo macrophage activation induced by BAY 41-2272 - spreading and phagocytosis - With regard to the pro-inflammatory activity generated by BAY 41-2272, spreading and phagocytosis were assessed as markers of peritoneal macrophage activation. The peritoneal cavity of mice that were treated (or not) with BAY 41-2272 (0.3-10 mg/kg IP) for 48 h was harvested and peritoneal cells were incubated on glass slides to measure spreading or were incubated with zymosan to evaluate phagocytosis (Fig. 3A). The macrophages obtained from the BAY 41-2272-treated mice showed an increase in spreading compared with the untreated animals (Fig. 3B), which is consistent with their increased phagocytic activities (Fig. 3C).[4]

NO and H 2 O 2 production - Mice were treated (or not) with BAY 41-2272 (0.3-10 mg/kg IP) for 48 h, after which the peritoneal cavity was harvested and peritoneal cells were incubated for 1 h with or without PMA (30 nM) to evaluate H2O2 release or were incubated for 48 h to evaluate NO production (Fig. 4A). It is known that phagocytosis and ROS release are related and are responsible for many antimicrobial responses. However, in this study, despite an increase in phagocytic activity, we did not observe alterations in spontaneous H2O2 release (Fig. 4C). However, the addition of PMA to the macrophage cultures from the BAY 41-2272-treated mice significantly increased the level of this metabolite (Fig. 4C).[4]

Although pre-treatment did not induce the spontaneous release of H2O2, BAY 41-2272 significantly increased the spontaneous production of NO compared with the macrophages from the control group (Fig. 4B).[4]

BAY 41-2272 increases survival of mice infected with fungi - The increases in phagocytosis and microbicidal activity suggest that BAY 41-2272 has potential for the treatment for infections. Therefore, C3H/HePas mice were challenged with C. albicans and S. aureus and the survival rates of these animals were evaluated. Mice were inoculated with C. albicans or S. aureus IP and after 48 h, they were treated (or not) with either BAY 41-2272 (0.3-10 mg/kg IP) or itraconazole (20 mg/kg), penicillin G (5 KU/kg) and tetracycline (1 mg/kg) for three days. The survival rates of the animals were evaluated for 20 days (Fig. 5A). [4]

The results showed that intraperitoneal treatment with BAY 41-2272 at 48 h after infection significantly increased the survival rate of the C. albicans-infected mice (Fig. 5B), but had no effects on that of the S. aureus-infected mice (Fig. 5C). In addition, as expected, itraconazole was completely effective in controlling Candida infection, maintaining the mouse survival rate at 100%. [4]

BAY 41-2272 increases mouse response against local C. albicans, but not S. aureus infection - According to the observation that BAY 41-2272 increased the survival rate of C. albicans-infected mice, a model of infection in the animal footpad paw with the same pathogens was used (Fig. 6A). This protocol allowed for the evaluation of the direct effects of BAY 41-2272 on the site of infection (intralesional drug injection) and systemically (intraperitoneal drug administration). [4]

Intralesional injection of BAY 41-2272 significantly reduced footpad swelling as induced by C. albicans, whereas the intraperitoneal treatment had no significant effect (Fig. 6B, C). The footpad swelling produced by S. aureus was not significantly altered by the subcutaneous or intraperitoneal BAY 41-2272 treatment (Fig. 6D, E). [4]

BAY 41-2272 increases in vitro and ex vivo microbicidal activities against C. albicans and S. aureus - For the in vivo models of infection, BAY 41-2272 generated a better response to C. albicans than to S. aureus. Thus, the effect of the in vitro or ex vivo treatment of peritoneal macrophages with BAY 41-2272 was investigated by assessing its microbicidal activity in relation to both of these pathogens. Mice were treated (or not) with BAY 41-2272 (0.3-10 mg/kg IP) for 48 h, after which the peritoneal cavity was harvested and peritoneal cells were incubated with C. albicans or S. aureus for 2 h to assess microbicidal activity (Fig. 7A).[4]

---

Type 2 diabetes mellitus (DM2) and obesity are major risk factors for erectile dysfunction (ED). In diabetes, increased oxidative stress leads to decreased nitric oxide (NO) bioavailability, and diabetic patients appear to be less responsive to conventional therapy with phosphodiesterase type 5 inhibitors. We investigated whether the soluble guanylyl cyclase stimulator BAY 41-2272 (5-cyclopropyl-2-[1-(2-fluoro-benzyl)-1H-pyrazolo[3,4-b]pyridine-3-yl]pyrimidin-4ylamine) is effective in improving impaired corpus cavernosum (CC) relaxation in obese DM2 mice by reducing oxidative stress. Adult db/db(-/-) mice or their lean db(/+) littermates were used to assess vascular function, cGMP levels, antioxidant status, NADPH oxidase expression, and superoxide formation in the absence or presence of BAY 41-2272. Results showed that BAY 41-2272 (10(-8) to 10(-5) M) potently relaxed CC from db(/+) or db/db(-/-) mice in a similar manner. BAY 41-2272 significantly enhanced both endothelium-dependent and nitrergic relaxation induced by electrical field stimulation (EFS), and improved the impaired relaxation to acetylcholine and EFS in the diabetic animals in a concentration-dependent manner (10(-8) to 10(-7) M). BAY 41-2272 increased cGMP levels and potentiated relaxation responses to exogenous NO in CC. Total antioxidant status was reduced in plasma and urine whereas expression of vascular NADPH oxidase subunits (gp91phox, p22phox, and p47phox) was increased in the CC of db/db(-/-) mice, suggesting a state of oxidative stress. These effects were prevented by BAY 41-2272 in a concentration-dependent manner. These results suggest that BAY 41-2272 improves CC relaxation in db/db(-/-) mice by increasing cGMP and augmenting antioxidant status, making this drug is a potential novel candidate to treat ED [5].

BAY 41-2272 is an activator of guanylyl cyclase sensitive to nitric oxide (NO-sensitive GC), with EC50 values of 3 μmol/L and 0.3 μmol/L in the presence and absence of 100 nmol/L DEA-NO, respectively.

---

Purification of soluble guanylate cyclase (sGC) and determination of sGC activity [1]
sGC was highly purified from a baculovirus / Sf9 expression system and enzyme activity was measured by formation of [32P]-cGMP from [α-32P]-GTP modified according to Gerzer in the presence of Mg2+ as the divalent metal cation as described. Incubations were performed in the presence and absence of 1 mM DTT. All measurements were performed in duplicate and were repeated three times unless otherwise indicated. The specific activity of sGC was calculated as nmol cGMP formed per mg protein per min incubation time. For characterisation of the different sGC stimulators the specific activity of sGC was expressed as x-fold stimulation vs. specific basal activity. The highest DMSO concentration in the test was 1% (v/v) and did not elicit any effect per se on cGMP production.

---

sGC assay [2]
We purified sGC by using a baculovirus/Sf9 expression system and measured enzyme activity in the presence of Mg2+ as described.

---

We investigated the effect of BAY 41-2272 on the tone and nitrergic relaxation responses of human and rabbit cavernous strips in the absence and presence of the soluble guanylate cyclase inhibitor ODQ (1H-[1,2,4]oxadiazolo[4-3a]quinoxalin-1-one) or the NO synthase inhibitor L-NAME (N-nitro-L-arginine-methyl ester HCl). The potency of BAY 41-2272 was compared to that of another soluble guanylate cyclase activator YC-1, and the NO releasing compound spermine NONOate (N-2-aminoethyl-N-2-hydroxy-2-nitrosohydroazino-1,2-ethylenediamine).[3]

BAY 41-2272 may have a sensitizing effect on NO-sensitive GC in platelets when GSNO at 3 μmol/L (a submaximally effective concentration) is evaluated. Applying NO at this concentration without BAY 41-2272 produced a cGMP response that was merely marginal. A quick rise in cGMP up to 1000 pmol/109 platelets was observed following treatment with GSNO at 3 μmol/L in the presence of BAY 41-2272 at 100 μmol/L.

---

Platelet aggregation [2]
We prepared washed human platelets as described14, and measured platelet aggregation by the turbidometric method. The platelet suspension was pre-incubated with the test compound at 37 °C for 10 min, and collagen (0.1–2 µg ml-1) was added to induce platelet aggregation.

---

Spreading assay [4]
A spreading assay was performed according to Rabinovitch et al. (1977). Peritoneal cell suspensions containing 2 × 106 cells were centrifuged and suspended in 1 mL of 5 mM glucose in PBS. Fifty microlitres of cell suspension were layered on glass coverslips and incubated for 1 h at 37ºC. The coverslips were gently rinsed in PBS and the glass-adherent cells were fixed in 2.5% glutaraldehyde and examined with a phase contrast microscope at a 400X magnification. Two hundred macrophages were counted and scored as round or spread. An index of macrophage spreading (SI) was then calculated as follows: SI = number of spreading macrophages × 100)/200, i.e., SI = % of spreading macrophages.

---

Zymosan phagocytosis assay [4]
A phagocytosis assay was performed according to Pinello et al. (2006). Peritoneal cell suspensions containing 2 × 106 cells were centrifuged and suspended in 1 mL of RPMI medium. The cells were dispensed over round glass coverslips (20 mm) in six-well flat-bottomed microtest plates and the cultures were incubated at 37ºC for 20 min. After incubation, culture supernatants were aspirated and the non-adherent cells were removed. Adherent monolayers were rinsed with PBS. Subsequently, 1 mL of RPMI-1640 medium containing 5% heat-inactivated foetal bovine serum was added to the cultures. The cultures were maintained at 37ºC for 1 h in the presence of 1 mg/L S. cerevisiae zymosan (Sigma). The cultures were then washed with cold PBS to remove non-internalised particles. The cells were then fixed with 0.5% glutaraldehyde. An average of 200 macrophages were counted using phase contrast microscopy to determine the phagocytic percentage. The phagocytosis index (PI) was calculated as follows: PI = the number of macrophages with phagocytic activity × 100)/200 adherent cells counted, i.e., PI = % of macrophages with at least two phagocytised zymosan particles.

---

H 2 O 2 release and nitric oxide (NO) production [4]
H2O2 release and NO production were determined in a single macrophage sample using a previously described method (Cruz et al. 2007). To evaluate H2O2 release, a HRP-dependent phenol red oxidation microassay was used (Pick & Mizel 1981 ). For this assay, 2.0 x 106 peritoneal cells were suspended in 1 mL of freshly prepared phenol red solution [ice-cold PBS containing 5.5 mM dextrose, 0.56 mM phenol red and 8.5 U/mL HRP type II]. One hundred microlitres of the cell suspension were added to each well and incubated with or without PMA (30 nM) for 1 h at 37ºC in a 5% CO2 humid atmosphere. Plates were centrifuged once at 150 g for 3 min and the supernatants were transferred to another plate. The reaction was stopped with 10 μL sodium hydroxide. Absorbance was measured at 620 nm with a microplate reader. The conversion of absorbance to μM of H2O2 was performed by comparison with a standard curve obtained with known concentrations of H2O2 (5-40 μM) diluted in RMPI medium (Pick & Keisari 1980).

Thereafter, the plates containing the cells were washed three times with PBS and the remaining adherent macrophages were cultured in 100 μL of RPMI-1640 medium (supplemented with 10 mM HEPES, 11 mM sodium bicarbonate, 100 U/mL penicillin, 100 μg/mL streptomycin, 2 mM L-glutamine, 23 mM L-asparagine, 1 mM folic acid, 0.1 mM pyruvic acid and 5% foetal calf serum) for 48 h at 37ºC in a 5% CO2 humid atmosphere. After the incubation, 50 μL of supernatants were collected and incubated with an equal volume of Griess reagent (1% sulfanilamide/0.1% naphthalene diamine dihydrochloride/2.5% phosphoric acid) for 10 min at room temperature (RT) to quantify the accumulation of nitrite (Ding et al. 1988). Absorbance was determined at 550 nm. The conversion of absorbance to μM of NO was performed by comparison to a standard curve obtained with known concentrations (5-60 μM) of sodium nitrite diluted in RPMI medium.

---

Ex vivo and in vitro peritoneal macrophage microbicidal activity [4]
To assess microbicidal activity, an MTT oxidation microassay was used after the incubation of the cells with bacteria or fungi. For this assay, two protocols were used to treat the peritoneal macrophages as follows: (i) the resident peritoneal cells were stimulated outside of the untreated animals (in vitro) and (ii) the cells were harvested from the animals treated as described previously in this protocol (ex vivo). After preparation, 2.5 × 105 cells were suspended in 200 µL of RPMI-1640 (without supplements) and distributed in a 96-well plate. The pathogens were then added at a 10:1 (pathogens:macrophages) ratio for S. aureus and a 2:1 ratio for C. albicans. Co-cultures were incubated for 2 h at 37ºC with 5% CO2. After incubation, the plate was centrifuged and the supernatants were collected and stored at -80ºC for the subsequent cytokine dosage assay. The cell pellets were washed twice with PBS to remove non-phagocytosed pathogens. After the washings, Triton X-100 (1.5%) was added for 10 min at RT to lyse the macrophages and release the pathogens. The cells were then washed twice with PBS to remove the Triton X-100, 100 µL of MTT (0.5 mg/mL) was added and they were incubated for another 2 h at RT away from light. After this incubation, 100 µL of DMSO was added and another 30-min incubation was performed to release formazan precipitate into the supernatant. After incubation, the plates were centrifuged (300 g for 3 min) and the supernatants were transferred to a new plate. Absorbance was determined at λ = 570 nm with a microplate reader. The conversion from absorbance to percentage of cell death was achieved with the following equation: 1 - (OD of sample - OD of 90% killing)/(OD of 0% killing - OD of 90% killing) × 100. This calculation was performed based on the concentrations of pathogens representing 100-10% of the total number of pathogens incubated with the cells.

Animal treatments [4]
For the in vivo experiments, BAY 41-2272 was diluted in a transcutol, Cremophor-EL and water solution (10/20/70 ratio, vol/vol/vol) to a final concentration of 1 mg/mL, as previously described (Bischoff et al. 2003). The animals were then weighed and the drug doses were adjusted to 0.3, 1.0, 3.0 and 10.0 mg/kg. For in vitro stimulation, BAY 41-2272 diluted in a 0.7% DMSO solution was used at concentrations of 1.0 and 3.0 µM, according to Bischoff et al. (2003). Treatment with BAY 41-2272 was administered intraperitoneally (IP) for 48 h. A negative control group was injected with a saline solution and a positive control group was treated with 4% thioglycolate or Con A (0.5 mg/kg) (Sigma). An additional control group was treated with a dilution solution only.
The ex vivo experiments were performed using resident macrophages or macrophages obtained from mice treated with BAY 41-2272. Treatments with penicillin G (5 kU/kg) and tetracycline (1 mg/kg) or itraconazole (20 mg/kg) were also administered in the infection assays. To evaluate hydrogen peroxide (H2O2) production, an additional in vitro treatment with PMA (30 nM) was performed. Other reagents, treatments and models are described in the following specific methodologies.

---

Footpad oedema induction [4]
Animals were anaesthetised and injected subcutaneously (SC) with carrageenan (300 μg/paw in saline) into the right paw. Differences in the sizes of the injected vs. un-injected paws were used as an indicator of inflammation (paw oedema) (Winter et al. 1962). The properties of BAY 41-2272 were assessed by injecting various doses of this drug (0.01-1.0 mg kg-1) IP at 48 h before the administration of carrageenan. Control mice were injected with same volume of a solvent (0.5 mL olive oil). Con A (100 mg kg-1) served as a positive control. Inflammation was assessed at 60-min intervals during a 4-h period.

---

Resistance of mice to C. albicans and S. aureus infections [4]
To assess the resistance of the BAY 41-2272-treated animals to C. albicans (ATCC 90028) and S. aureus (ATCC 25923), two models of infection were used as follows: (i) the inoculation of pathogens in the peritoneal cavity followed by survival rate evaluation and (ii) the inoculation of pathogens SC into the footpad of the animals. For the first model, the animals were inoculated IP with 0.5 × 106 C. albicans blastospores or 5 × 106 colony-forming units of S. aureus. Forty-eight hours from inoculation to the establishment of infection, the animals were also treated daily IP with BAY 41-2272 (1 or 3 mg/kg) or itraconazole (20 mg/kg) or penicillin G (5 KU/kg) and tetracycline (1 mg/kg) for three days. The survival rate of the animals was evaluated for 20 days from the first day of inoculation. We attempted to perform survival experiments in mice with less than 12 days of infection, but the results were reliable only for those infected for 20 days. For the second model, the animals were inoculated SC with the same concentrations of pathogens into the footpad of the left paw and the right paw served as the control. At 48 h from inoculation to the establishment of infection, the animals were treated daily IP or intralesionally (inoculated paw) with BAY 41-2272 (1 or 3 mg/kg) or itraconazole (20 mg/kg) or penicillin G (5 KU/kg) and tetracycline (1 mg/eg) for three days. Paw thickness was then measured after seven days from inoculation to assess the development of the lesion or infection.

---

Dissolved in 10/20/70 (v/v/v) Transcutol/Cremophor EL/water; 1 mg/kg; p.o.

Female spontaneously hypertensive rats

[1]. BMC Pharmacol . 2001:1:13.

[2]. Nature . 2001 Mar 8;410(6825):212-5.

[3]. J Urol . 2003 Feb;169(2):761-6.

[4]. Mem Inst Oswaldo Cruz . 2015 Feb;110(1):75-85.

[5]. J Pharmacol Exp Ther . 2015 May;353(2):330-9.

BAY 41-2272 is a pyrazolopyridine that is 1H-pyrazolo[3,4-b]pyridine which is substituted by a 2-fluorobenzyl group at position 1 and by a 4-amino-5-cyclopropylpyrimidin-2-yl group at position 3. It is an activator of soluble guanylate cyclase. It has a role as a soluble guanylate cyclase activator, a platelet aggregation inhibitor, a vasodilator agent and an antihypertensive agent. It is a pyrazolopyridine, a member of monofluorobenzenes, an aminopyrimidine and a member of cyclopropanes.

---

In summary, using photoaffinity labelling, we identified the region of the cysteines 238 and 243 in the α1 subunit of sGC as the target for NO-dependent sGC stimulators. However, the relevance of the identified region as a regulatory unit remains to be confirmed by mutational analysis and co-crystallization studies.[1]

---

In summary, the effects of BAY 41-2272on sGC and the photoaffinity labelling studies suggest the existence of a new NO-independent regulatory site on sGC in the Cys 238 and Cys 243 region of the α1-subunit that modulates the catalytic rate and the responsiveness towards the haem ligand. Our data offer both an approach to understanding the regulation of sGC and a potent new stimulator of sGC, BAY 41-2272, which induces vasodilation without developing nitrate tolerance, antiplatelet activity, and finally reduces mortality. [2]

---

Nitric oxide (NO) is a widespread, potent, biological mediator that has many physiological and pathophysiological roles. Research in the field of NO appears to have followed a straightforward path, and the findings have been progressive: NO and cyclic GMP are involved in vasodilatation; glycerol trinitrate relaxes vascular smooth muscles by bioconversion to NO; mammalian cells synthesize NO; and last, NO mediates vasodilatation by stimulating the soluble guanylate cyclase (sGC), a heterodimeric (alpha/beta) haem protein that converts GTP to cGMP2-4. Here we report the discovery of a regulatory site on sGC. Using photoaffinity labelling, we have identified the cysteine 238 and cysteine 243 region in the alpha1-subunit of sGC as the target for a new type of sGC stimulator. Moreover, we present a pyrazolopyridine, BAY 41-2272, that potently stimulates sGC through this site by a mechanism that is independent of NO. This results in antiplatelet activity, a strong decrease in blood pressure and an increase in survival in a low-NO rat model of hypertension, and as such may offer an approach for treating cardiovascular diseases. [2]

---

Purpose: In cavernous smooth muscle nitric oxide (NO) activates soluble guanylate cyclase, which catalyzes the synthesis of cyclic guanosine 3',5'-monophosphate, leading to smooth muscle relaxation, increased blood flow and penile erection. The pyrazolopyridine derivative BAY 41-2272 (5-cyclopropyl-2-[1-(2-fluoro-benzyl)-1H-pyrazolo[3,4-b]pyridine-3-yl]pyrimidin-4ylamine) was identified and found to stimulate soluble guanylate cyclase in a NO independent manner. We investigated the effect of BAY41-2272 on human and rabbit corpus cavernosum. [3]

---

In the in vivo models of infection, BAY 41-2272 was more effective in responding to the fungi compared to the bacteria. Thus, we investigated the in vitro and ex vivo microbicidal activities of peritoneal macrophages against the same pathogens. Our results showed that the in vitro treatment enhanced the microbicidal activities of the peritoneal macrophages against C. albicans and S. aureus and these increases were even more significant ex vivo. These results confirm the potential of BAY 41-2272 for treating fungal infections, specifically C. albicans. We also show that this treatment is effective in promoting S. aureus killing. These data support our hypothesis that the apparent non-resolution of S. aureus infection in vivo involves the maintenance of inflammation generated by the pathogen and potentiated by BAY 41-2272.

This increase in microbicidal activity is probably related to the oxidative burst, reactive nitrogen production and phagocytosis. However, we cannot exclude the possible involvement of other processes, such as phagosome pH acidification and lysosomal/granular enzyme release (Sokolovska et al. 2012), in addition to the participation of other cells. Importantly, the extensiveness of the ex vivo response indicates the relevance of chemical mediators and cells present in the physiological environment to the activation and modulation of phagocyte responses. It is likely that the action of BAY 41-2272 on other immune cells creates an environment with significantly more stimuli for macrophage activation. These data, considering a complex physiological system, provide new evidence in support of the notion that BAY 41-2272, or its pathway (sGC-cGMP), can be used as a treatment for some infections, especially in immunocompromised patients. It is important to emphasise that the cardiovascular effects of BAY 41-2272 (Thorsen et al. 2010, Joshi et al. 2011) did not limit its in vivo application.

We conclude that BAY 41-2272 causes a pro-inflammatory effect, activating mononuclear phagocytes (peritoneal macrophages). Moreover, treatment with BAY 41-2272 significantly increases mouse responses to C. albicans (in vivo and in vitro) and S. aureus (in vitro), improving peritoneal macrophage microbicidal activities against these pathogens. Our group is actively investigating the pharmacological aspects of BAY 41-2272, aiming to clarify its signalling pathways and elucidate its effects on mononuclear phagocytes. With this information, we intend to develop novel treatments to increase the quality of life of patients susceptible to infections, especially those with PID. [4]

---

BAY 41-2272 (5-cyclopropyl-2-[1-(2-fluoro-benzyl)-1H-pyrazolo[3,4-b]pyridine-3-yl]pyrimidin-4ylamine) is an sGC stimulator that has been shown to produce antiproliferative and vasodilatory effects (Evgenov et al., 2006), as well as to potentiate erectile responses (Bischoff et al., 2003) and relax the CC of humans and animals (Baracat et al., 2003; Kalsi et al., 2003; Claudino et al., 2011). This compound was suggested to have a high potency and no PDE inhibitory activity (Stasch et al., 2001). In a NO-deficient rat model, long-term oral treatment with BAY 41-2272 improved the impaired cavernosal relaxation (Claudino et al., 2011). In a previous investigation of the effects of BAY 41-2272 in mice CC, our group showed that this compound reverses the increased NADPH oxidase-dependent superoxide generation by decreasing protein expression of its subunits gp91phox and p22phox (Teixeira et al., 2007).

BAY 41-2272, but not a PDE-5 inhibitor, enhances the nitrergic relaxation response in anococcygeus and retractor penile muscle (Kalsi et al., 2004) (ideal tissues to study nitrergic neurotransmission), which are impaired in streptozotocin-induced diabetic rats (Cheah et al., 2002). These data suggest that endogenous NO from nitrergic nerves is decreased in diabetes and show that sGC stimulators are more effective than PDE-5 inhibitors in the treatment of diabetes-induced ED.

To the best of our knowledge, there are no previous studies investigating the action of BAY 41-2272 on diabetic CC. Additionally, few studies have been performed using db/db−/− mice to investigate ED, even though these animals have shown altered vasoreactivity consistent with impaired cavernosal relaxation and penile veno-occlusive disorder. The db/db−/− mice lack leptin receptors, and this deficiency contributes to the development of both diabetes and obesity. Therefore, these mice are widely considered an appropriate model for DM2, which has been used for the study of DM2-associated ED (Luttrell et al., 2008). In addition, db/db−/− mice develop hyperglycemia and hyperinsulinemia, the latter of which raises resting sympathetic output and contributes to impaired cavernosal relaxation (Anderson et al., 1991).

In this study, we examine the effect of BAY 41-2272 on relaxation of the CC from db/db−/− obese DM2 mice and their lean db/+ counterparts in response to vasodilatory agonists and the effects of the drug on markers of oxidative stress in these animals.

Our data showed that in diabetic, obese (db/db−/−) mice BAY 41-2272 ameliorated impaired endothelial and nitrergic cavernosal relaxation by elevating the intracellular cGMP concentration, preventing elevated expression of NADPH oxidase enzyme subunits, and decreasing superoxide formation. Although the pathogenesis of ED in diabetes is multifactorial, vascular dysfunction is a major contributor to the high incidence of ED in men with diabetes (Chu and Edelman, 2002). [5]

C20H17FN6

360.39

360.149

C, 66.65; H, 4.75; F, 5.27; N, 23.32

256376-24-6

9798973

White to off-white solid powder

1.5±0.1 g/cm3

496.1±45.0 °C at 760 mmHg

253.8±28.7 °C

0.0±1.3 mmHg at 25°C

1.767

1.99

1

6

4

27

517

0

FC1=C([H])C([H])=C([H])C([H])=C1C([H])([H])N1C2=C(C([H])=C([H])C([H])=N2)C(C2=NC([H])=C(C(N([H])[H])=N2)C2([H])C([H])([H])C2([H])[H])=N1

ATOAHNRJAXSBOR-UHFFFAOYSA-N

InChI=1S/C20H17FN6/c21-16-6-2-1-4-13(16)11-27-20-14(5-3-9-23-20)17(26-27)19-24-10-15(12-7-8-12)18(22)25-19/h1-6,9-10,12H,7-8,11H2,(H2,22,24,25)

5-cyclopropyl-2-[1-[(2-fluorophenyl)methyl]pyrazolo[3,4-b]pyridin-3-yl]pyrimidin-4-amine

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Solubility in Formulation 1: ≥ 1.75 mg/mL (4.86 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 17.5 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

---

Solubility in Formulation 2: ≥ 1.75 mg/mL (4.86 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 17.5 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

---

View More

Solubility in Formulation 3: ≥ 1.75 mg/mL (4.86 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 17.5 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

---

 (Please use freshly prepared in vivo formulations for optimal results.)