Pyrazolo[4,3-d]pyrimidine Derivatives as a Novel Hypoxia-Inducible Factor Prolyl Hydroxylase Domain Inhibitor for the Treatment of Anemia

Abstract

Inhibition of hypoxia-inducible factor prolyl hydroxylase domain (HIF-PHD) promotes erythropoietin (EPO) production by stabilizing the HIFα subunit. Thieno[2,3-d]pyrimidine 8 identified based on X-ray crystal structure analysis was optimized to lead to the discovery of pyrazolo[4,3-d]pyrimidine 13 as the lead compound of orally bioavailable HIF-PHD inhibitors. Conversion of the benzyl moiety in 13 gave pyrazolopyrimidine 19 with high solubility and bioavailability, which increased hemoglobin levels in anemic model rats after repeated oral administration. It was shown that pyrazolo[4,3-d]pyrimidine derivatives are promising therapeutic agents for renal anemia through the inhibition of HIF-PHD.

Renal anemia refers to a condition where low erythrocyte and hemoglobin levels are known to be caused by a decline of the production of erythropoietin (EPO), which plays an important role in the promotion of erythropoiesis, due to decreased renal function such as chronic renal failure.¹ Erythropoiesis stimulating agents (ESAs) such as epoetin alfa and darbepoetin alfa have greatly contributed to the treatment of renal anemia.² However, ESAs have several unsolved issues; for example, exacerbation of patient prognosis occurs in high dose therapy and results in a reduction of reactivity to EPO. In addition, ESAs are expensive, and the treatment is inconvenient because subcutaneous or intravenous injection is required for drug delivery. Therefore, therapeutic agents that improve the current status of ESAs are greatly desired.

Hypoxia-inducible factor-prolyl hydroxylase domain (HIF-PHD) regulates HIF levels by hydroxylation of proline under normoxia (normal level of oxygen). Conversely, the degradation of HIF is suppressed under hypoxia (deficiency of oxygen), and activation of EPO gene transcription and hematopoiesis occur.³ HIF-PHD inhibitors should be able to effectively improve anemia by not only enhancing EPO production by stabilizing the HIFα subunit but also multifaceted abilities, such as improving iron metabolism,⁴ protecting kidney tissue,⁵ and elevating the expression of EPO receptors.⁶ HIF-PHD inhibitors are expected to be categorized in a new type of therapeutic agents for renal anemia without the problems traditionally associated with ESAs, which are in late phase clinical trials (Figure 1).⁷⁻¹⁶

Figure 1.

Representative HIF-PHD inhibitors.

Herein, we report the identification as a HIF-PHD inhibitor of pyrazolo[4,3-d]pyrimidine derivatives, which have novel scaffold and iron coordination moiety, and the optimization to discover pyrazolopyrimidine 19 that showed potent improvement in blood hemoglobin level in anemic rats.

In our initial exploration of a new scaffold to improve the biological activities and pharmacokinetic properties, a thienopyrimidine compound, which is frequently used in drug discovery,^17,18 was designed on the basis of the cocrystal structure of 7 and HIF-PHD2 registered in the Protein Data Bank (PDB, 2HBU), which has the minimal structure required to express inhibitory activity, that is, the coordination site of iron and the interaction sites with Tyr310 and Arg383. A docking study using the structure from PDB as a template confirmed that 8 had the same interactions as 7 as shown in Figure 2. In fact, 8 showed moderate inhibitory activity (IC₅₀ 290 nM).

Figure 2.

Docking study of 8 into the binding pocket using 7. Compounds 7 and 8 are shown as a ball–stick model in light green and yellow green, respectively.

Next, the transformation of the 3-methyl group in 8 was performed to improve the inhibitory activity. Compound 9 with a cyclohexylmethyl group showed a 2-fold increase in activity, and 10 with a benzyl group showed a 4-fold increase in activity. Compound 11, in which a bulky biphenyl group was introduced in anticipation of further improved activity, showed potent HIF-PHD2 inhibitory activity (Table 1). Activity improvement by scaffold conversion was tried for 12 and 13. As a result, while the activity of the pyrrolopyrimidine 12 was reduced, the activity of the pyrazolopyrimidine 13 was further improved more than that of thienopyrimidine 11. The regioisomer 14 of 13 also exhibited potent inhibitory activity. Moreover, oral administration of 13, which had the strongest HIF-PHD2 inhibitory activity among the series, at 10 and 30 mg/kg to ICR mice induced very high levels of EPO in the serum, approximately 63000 and 89000 pg/mL, respectively, versus 125 pg/mL with vehicle treatment (Figure 3). On the other hand, 14, which induced weak levels of EPO in the serum, approximately 450 pg/mL versus 146 pg/mL with vehicle treatment at 100 mg/kg in ICR mice, was not effective (Figure 3).

Table 1. HIF-PHD2 Inhibitory Activity of Bicyclic Pyrimidine Derivatives.

Figure 3.

Serum EPO concentration at 6 h after single oral dosing in ICR mice: (A) 13 at 10 and 30 mg/kg, (B) 14 at 100 mg/kg. Data are expressed as the mean ± SEM (N = 5), and statistical significance was assessed by Dunnett’s test. **P < 0.01 vs vehicle.

Although 13 showed good in vivo efficacy, its bioavailability in rats was only 16% (Table 3). This was thought to be due to its low solubility in pH 6.5 buffer (9.5 μg/mL). Therefore, the optimization of lead 13 for solubility was carried out. We adopted the strategy of reducing the planarity of molecules without decreasing lipophilicity to prevent waning in vivo efficacy due to low membrane permeability. The designed pyrazolopyrimidine derivatives and their profiles are shown in Table 2. For 15, we expected that the introduction of fluorine groups onto the biphenyl moiety would increase the dihedral angle. The resulting solubility of 15 in pH 6.5 buffer increased to 49 μg/mL.^19,20 However, since the improvement was not sufficient, the solubility improvement was tried in 16 with the single phenyl ring side chain. As a result, the solubility of 16 was twice as high as that of 15. Next, a methyl group was introduced at the benzyl position of 17 to reduce the crystal packing ability.²¹ However, the solubility of 17, which showed more potent HIF-PHD2 inhibitory activity than enantiomer 18, was not improved (75 μg/mL). The solubility of 19 was dramatically improved (>1000 μg/mL), which was attributed to the packing reduction effects, which are the torsion of phenyl ring by steric hindrance between the ortho-chloro group and the methyl group. As a result of further biological studies of 19, strong EPO production (Figure 4) and good pharmacokinetic profiles (Table 3) were observed.

Table 3. Pharmacokinetic Parameters of 13 and 19 in Male SD Rats.

	13	19
po dose (mg/kg)	10	1
Cmax (ng/mL)	2708	1839
AUC0–∞ (ng·h/mL)	17813	23463
F (%)	16	77
iv dose (mg/kg)	0.5	1
Cmax (ng/mL)	4908	12357
AUC0–∞ (ng·h/mL)	5558	30430
CLtot (mL·h–1·kg–1)	91	34
Vdss (mL/kg)	197	249

Table 2. HIF-PHD2 Inhibitory Activity of Pyrazolo[4,3-d]pyrimidine Derivatives with Various Benzyl Groups and Their Solubility.

^aSolubility in pH 6.5 buffer.

Figure 4.

Serum EPO concentration at 8 h after single oral dose of 19 at 10 mg/kg in SD rats. Data are expressed as the mean ± SEM (N = 4), and statistical significance was assessed by Student’s t test. **P < 0.05 vs vehicle.

Moreover, rats artificially induced to develop renal anemia by excising five-sixths of their kidneys were used in this study. Three doses of 19 (0.5, 1, and 2 mg/kg) were orally administered once daily for 4 weeks. Improvement in blood hemoglobin levels were observed starting at weeks 2 and 1 in the groups receiving 1 and 2 (mg/kg)/day, respectively (Figure 5).

Figure 5.

Effects of 19 on hemoglobin levels in anemic rats after repeated oral administration. Data are expressed as the mean ± SEM (N = 9–10), and statistical significance was assessed. **P < 0.001 sham vs control (Student’s t test); *P < 0.025 vs control (Williams’ test).

Finally, we summarize the synthesis of our compounds using the coupling reaction that was reported in the literature (Scheme 1, 2).²²

Scheme 1. Synthesis of Thieno[2,3-d]pyrimidine 8.

Reagents and conditions: (a) NaOMe, THF, MeOH, 0 °C–rt, 96%; (b) ethyl 4-pyrazolecarboxylate, Pd₂(dba)₃, Me₄^tBuXPhos, K₃PO₄, ^tBuOH, 90 °C, 80%; (c) TMSI, CH₃CN, 50 °C; (d) aqueous NaOH, THF, EtOH, 40 °C, 12% (2 steps).

Scheme 2. Synthesis of Pyrazolo[4,3-d]pyrimidine Derivatives 13–19.

Reagents and conditions: (a) TFA, 60 °C, 80%; (b) RBr, K₂CO₃, CH₃CN, 80 °C; (c) ROH, Mitsunobu reagent, PPh₃, rt; (d) aqueous NaOH, THF, EtOH, 60 °C; (e) 3-fluoro-4-bromobenzyl alcohol, DIAD, PPh₃, rt, 55%; (f) 4-fluorophenyl boronic acid, PdCl₂(amphos)₂, K₃PO₄, dioxane, 100 °C, 86%; (g) aqueous NaOH, THF, EtOH, 60 °C, 99%.

Thieno[2,3-d]pyrimidine 8 was synthesized as shown in Scheme 1. Compound 20 was converted to 21 by substitution of the chloro group to the methoxy group, and the pyrazole moiety was introduced to afford 22 under Pd-coupling conditions.²² Demethylation with TMSI was performed, followed by hydrolysis of the ethyl ester to obtain thieno[2,3-d]pyrimidine 8.

The synthesis of pyrazolo[4,3-d]pyrimidine derivatives 13–19 was conducted as shown in Scheme 2. Acidic removal of the p-methoxy benzyl group of known pyrimidine 23(22) was employed. The side chains were introduced with benzyl bromide and potassium carbonate or under Mitsunobu conditions, followed by a basic hydrolysis of the ethyl ester and the methoxy group to afford 13, 14, and 16–19. Compound 25 was synthesized in the same manner and transformed to biphenyl-containing 26 using a Suzuki–Miyaura coupling reaction, which was then hydrolyzed to give 15.

In summary, we have developed pyrazolo[4,3-d]pyrimidine derivatives as novel orally bioavailable HIF-PHD inhibitors for the treatment of renal anemia. Starting from novel pyrazole–carboxylic acid type inhibitor 8, which was designed from the X-ray crystal structure of HIF-PHD2 protein complex with 7, we found that the lead compound 13 improved HIF-PHD2 inhibitory activity by the conversion of the methyl group and thienopyrimidine scaffold in 8. Next, the side chains were optimized to improve the solubility of the HIF-PHD inhibitors. Consequently, we discovered 19, which possessed strong HIF-PHD2 inhibitory activity and a good pharmacokinetic profile and was effective in a pathological model. Further research is currently in progress to diversify the structure of HIF-PHD inhibitor class.

Glossary

Abbreviations

EPO

erythropoietin

ESA

erythropoiesis stimulating agent

HIF-PHD

hypoxia-inducible factor prolyl hydroxylase domain

ICR mice

Institute of Cancer Research mice

SD rat

Sprague–Dawley rat

PK

pharmacokinetics

HGB

hemoglobin

DIAD

diisopropyl azodicarboxylate

TMSI

trimethylsilyl iodide

dba

dibenzylideneacetone

Xphos

2-dicyclohexylphosphino-2′,4′,6′-triisopropylbiphenyl

amphos

(4-dimethylaminophenyl)di-tert-butylphosphine

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsmedchemlett.0c00108.

• Biological assays, synthetic procedures, and analytical data of compounds (PDF)

Author Contributions

The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.

This research was supported by Mitsubishi Tanabe Pharma Corporation, JSPS KAKENHI Grant Number 15H05836 (K.F.) in Middle Molecular Strategy, and JSPS KAKENHI Grant Number 16H01885 (K.F.).

The authors declare no competing financial interest.