Discovery and Optimization of 4-(Imidazo[1,2-a]pyrimidin-3-yl)thiazol-2-amine Derivatives as Novel Phosphodiesterase 4 Inhibitors

doi:10.21203/rs.3.rs-4852333/v1

Phosphodiesterases (PDEs) are important intracellular enzymes that hydrolyze the second messengers cAMP and/or cGMP. Now several studies have shown that PDE4 received particular attention due to it represents the most prominent cAMP-metabolising enzyme involved in many diseases. In this study, the hit compound (PTC-209) with moderate PDE4 inhibitory activity (IC₅₀ of 4.8±0.2 μM) was discovered by screening of our internal compound library. And a series of 4-(imidazo[1,2-a]pyrimidin-3-yl)thiazol-2-amine derivatives as novel PDE4 inhibitors starting from PTC-209 were successfully designed and synthesized using a structure-based discovery strategy. L19, the most potent inhibitor, exhibited good inhibitory activity (IC₅₀ of 0.51±0.02 μM) and remarkable metabolic stability in rat liver microsomes. The results emphasized the successful application of screening and discovery of new scaffolds that can be helpful for optimization of novel PDE4 inhibitors.

Phosphodiesterase

Phosphodiesterase 4

Inhibitors

Structural optimization

Metabolic stability

Phosphodiesterases (PDEs) are important intracellular enzymes that hydrolyze the second messengers cAMP and/or cGMP to their inactive forms through the cleavage of a phosphodiester bond [1–3]. There are 11 gene families have been identified in the PDEs superfamily, each with its unique substrate properties and tissue distribution characteristics and play a central role in modulating the signal transduction relayed by cyclic nucleotide-based secondary messengers inside the cell [4].

PDE4, distributed in various tissues and organs in the body, is the predominant cAMP-specific PDE and which consists of 4 subtypes (PDE4A, PDE4B, PDE4C and PDE4D) sharing a highly conserved catalytic domain [2]. Now several studies have shown that PDE4 received particular attention due to it represents the most prominent cAMP-metabolising enzyme in immune cells involved in the pathophysiology of lung inflammation underlying asthma and chronic obstructive pulmonary diseases (COPD)[2, 5]. Other studies show that its overexpression can also promote the development of Alzheimer's disease, glioma, stroke, depression and other neurological diseases [6].

In fact, PDE4 inhibitors have shown extensive therapeutic potential and have been used in the treatment of asthma, COPD, rheumatoid arthritis, psoriasis, inflammatory bowel disease, depression, and cognitive dysfunction [7–9]. In addition, some studies have reported that natural product PDE4 inhibitors have great potential in the treatment of anti-vascular dementia (VaD) [10].However, the clinical application of most PDE4 inhibitors are limited by their unexpected and severe side effects such as nausea, vomiting, and diarrhea. Therefore, the discovery of novel PDE4 inhibitors with new scaffolds continues to attract much attention in both academics and industry.

2.1 Structure‑based strategies applied for screening PDE4 inhibitors

In order to screen novel PDE4 inhibitors, a dataset of 2,530 PDE4 inhibitors was collected from the literature report. By using the Partial Least Squares (PLS) algorithm, we developed a molecular predictive model for the activity prediction of anti-PDE4. As shown in Fig. 1A, the optimal predictive model achieved R² values of 0.812 for the training set and 0.731 for the test set, suggesting robust predictive performance. We then applied this model to predict the activity of 50 compounds procured by our laboratory, finding that the hit compound PTC-209, N-(2,6-dibromo-4-methoxyphenyl)-4-(2-methylimidazo[1,2-a]pyrimidin-3-yl)thiazol-2-amine (Fig. 1B), had the highest predicted pIC₅₀ of 9.38. PTC-209 was initially developed as a selective anti-tumor agent mediated by Bmi-1 [11, 12]. To our knowledge, this is the first report of PTC-209 exhibiting PDE4 inhibitory activity. Subsequent bioassays confirmed that PTC-209 exhibited considerable inhibitory affinity against PDE4, with an IC₅₀ of 4.8 ± 0.2 µM.

2.2 Chemistry

In order to understand its structure–activity relationship (SAR) around the novel scaffold and improve the inhibitory activities, four series of 4-(imidazo[1,2-a]pyrimidin-3-yl)thiazol-2-amine derivatives were designed and synthesized (Schemes 1–4). As shown in Scheme 1, -(2-methylimidazo[1,2-a]pyrimidin-3-yl)ethan-1-one (3) was synthesized by the reaction of pyrimidin-2-amine (1), 1,1-dimethoxy-N,N-dimethylethylamine (2), and chloracetone in toluene [13]. Then, compound 3 was brominated with Br₂ in acetic acid [14] to afford the α-bromo ketones (4) and followed by the cyclization with substituted thioureas to get the product L1-L3 [15], respectively. Meanwhile, the α-bromo ketones 4 reacted with the N-Boc thiourea to get the N-Boc-4-(imidazo[1,2-a]pyrimidin-3-yl)thiazol-2-amine (5). And the Boc group of 5 was deprotected by TFA in tetrahydrofuran to afford 4-(imidazo[1,2-a]pyrimidin-3-yl)thiazol-2-amine (L4) [16], which was treated with acetic anhydride in pyridine or treated with carboxylic acids and HATU in the presence of DIPEA to obtain the product L5-L9 [17–19], respectively.

Finally, the same procedures as Schemes 1–2 were used to yield L10-L22, which was firstly synthesized the N-aryl thioureas by the reaction of amines and ammonium thiocyanate in acetic acid [20] and followed by the cyclization with 4 to get the product L10-L22 [21, 22], respectively (Schemes 3–4).

Reagents and conditions: (a) (i) PhMe, 100℃, 3 h; (ii) Chloracetone, rt, 12 h; (b) (i) AcOH, Br₂, 110℃, 3 h; (ii) rt, 12 h; (c) Thioureas, EtOH, reflux, 12 h.

Reagents and conditions: (a) N-Boc thiourea, EtOH, Et₃N, reflux, 12 h; (b) TFA, THF, rt, 1 h; (c) Ac₂O, pyridine, 100℃, 1 h; or RCOOH, HATU, DIPEA, DMF, 100℃, MW, 10 min.

Reagents and conditions: (a) NH₄SCN, AcOH, 73℃, 12 h; (b) 5, EtOH, reflux, 12 h.

2.3 Structure–activity relationships (SARs)

Herein, to understand the structure–activity relationships (SARs) and improve the potency around the 4-(imidazo[1,2-a]pyrimidin-3-yl)thiazol-2-amine scaffold, 22 derivatives were synthesized. Their inhibitory activities against PDE4 are shown in Table 1 and the inhibitory curves of compounds 1, L19, L20, and Rolipram against the PDE4D are shown in Fig. 2, protocols for the expression and enzyme assay of PDE isoforms were similar to our previous works [23–25]. And for the measurement of IC₅₀, eight concentrations at least were used and IC₅₀ values were calculated by the nonlinear regression. In this study, our initial investigations of the SARs of 4-(imidazo[1,2-a]pyrimidin-3-yl)thiazol-2-amines were carried out by substituting the amino position with alkyl or acyl group. However, all the compounds (L1-L9) exhibited weaker PDE4 inhibitory activities than compound 1, indicating that alkyl or acyl substitution at this position is unfavorable for the formation interactions and this subpocket is more favorable for holding an aromatic ring. Subsequently, we focus on change the aryl groups of the amino position. As a result, most of the derivatives (L10-L22) show considerable inhibitory potencies. Specifically, compounds L19 and L20 have a different substitution, in which the introduction of 3,5-dichloropyridin-4-yl/3,5-difluoropyridin-4-yl groups (L19 and L20, IC₅₀ = 0.51 ± 0.02 and 2.1 ± 0.2 µM, respectively) resulted in tighter binding than that with other groups. Finally, compound L19 bearing a 3,5-difluoropyridin-4-yl group has the best inhibitory activity with an IC₅₀ of 0.51 ± 0.02 nM, suggesting that a 3,5-difluoropyridin-4-yl group at this position was favorable for the formation of interactions in the binding pocket.

Table 1. Inhibitory Activities of 4-(imidazo[1,2-a]pyrimidin-3-yl)thiazol-2-amines towards PDE4.

2.4 Computational Study

In order to better understand the structural basis of PDE4 inhibition by the new inhibitors, we have conducted a computational study to identify the binding mode allowing to explain the experimental SARs observed within the 4-(imidazo[1,2-a]pyrimidin-3-yl)thiazol-2-amines (Fig. 3). We performed the hit compound (1) and the most potent compounds L19-L20 docking into the catalytic domain of PDE4D, and the docking pose with the best docking score. The results show that the imidazo[1,2-a]pyrimidine scaffold of L1 (Fig. 3A), L19 (Fig. 3B) or L20 (Fig. 3C) formed H-bonds with the residue Gln369 and the aromatic ring formed π − π stacking interactions against residue Phe372, which are two characteristic interactions of PDE4 inhibitors.

2.5 Rat Liver Microsomal Stability

Metabolic stability is an important parameter to be optimized during the process of design and synthesis of new active compounds [26], and the metabolic stability in human/rat liver microsomes are extensively used in the pharmaceutical industry. Herein, we examined the 4-(imidazo[1,2-a]pyrimidin-3-yl)thiazol-2-amines (1, L11, and L17-L19) using a standard microsomal stability assay with comparison to the testosterone control compound. The results shows that all tested compounds have moderate metabolic stability except L11. Among them, L19 has the best metabolic stability with a t_1/2 of 50.2 min and metabolic bioavailability (MF) of 52.7%, which is significantly better than those for the positive control (t_1/2 of 2.5 min, and MF of 5.3%).

Table 2

Metabolic Stability of Compounds in Rat Liver Microsomes.
Compounds	k	t_1/2 (min)	CL_int (mL·min^− 1·mg^− 1)	CL_h (mL·min^− 1·kg^− 1)	MF (%)
testosterone^a	0.2765	2.5	995.4	52.3	5.3
1	0.0272	25.5	97.9	35.3	36.1
L11	0.0529	13.1	190.4	42.8	22.5
L17	0.0357	19.4	128.5	38.6	30.1
L18	0.0337	20.6	121.3	37.9	31.3
L19	0.0138	50.2	49.7	26.1	52.7

^aTestosterone was the positive control; CL_int: intrinsic clearance; CL_h: hepatic clearance; MF: metabolic bioavailability.

In summary, the hit compound (PTC-209) with moderate PDE4 inhibitory activity (IC₅₀ of 4.8±0.2 μM) was discovered by screening of our internal compound library. And a series of 4-(imidazo[1,2-a]pyrimidin-3-yl)thiazol-2-amine derivatives as novel PDE4 inhibitors starting from PTC-209 were successfully designed and synthesized using a structure-based discovery strategy. In total, 22 derivatives of 4-(imidazo[1,2-a]pyrimidin-3-yl)thiazol-2-amines were discovered, resulting 13 compounds (L10-L22) show considerable inhibitory potencies. L19, the most potent inhibitor, exhibited good inhibitory activity (IC₅₀ of 0.51±0.02 μM) and remarkable metabolic stability in rat liver microsomes. The results emphasized the successful application of screening and discovery of new scaffolds that can be helpful for optimization of novel PDE4 inhibitors.

Supporting Information Available

Chemistry, ¹H NMR, ¹³C NMR, and High-resolution mass spectra (HRMS) data for tested compounds.

Author information

Corresponding Author

*To whom correspondence should be addressed. Phone: +86-898-66254967. Fax: +86-898-66254967.

E-mail: [email protected] (D. Wu), [email protected] (Y.-Y. Huang)

¹These authors made equal contributions to this work.

ORCID

Hai-Bin Luo: 0000-0002-2163-0509

Deyan Wu: 0009-0001-4640-6781

Notes

The authors declare no competing financial interest.

Acknowledgments

This work was supported by Natural Science Foundation of China (82373732, 22307031), Hainan Provincial Natural Science Foundation of China (222RC556, 823CXTD375, 324MS018) Fundamental Research Funds for Hainan University (KYQD(ZR)-21126, KYQD(ZR)-23003).

Silverberg JI, French LE, Warren RB, Strober B, Kjøller K, Sommer MOA, Andres P, Felding J, Weiss A, Tutkunkardas D, Skak‐Nielsen T, Guttman E (2023) Pharmacology of orismilast, a potent and selective PDE4 inhibitor. J Eur Acad Dermatol 37:721-729. https://doi: 10.1111/jdv.18818
Carzaniga L, Amari G, Rizzi A, Capaldi C, De Fanti R, Ghidini E, Villetti G, Carnini C, Moretto N, Facchinetti F, Caruso P, Marchini G, Battipaglia L, Patacchini R, Cenacchi V, Volta R, Amadei F, Pappani A, Capacchi S, Bagnacani V, Delcanale M, Puccini P, Catinella S, Civelli M, Armani E (2017) Discovery and Optimization of Thiazolidinyl and Pyrrolidinyl Derivatives as Inhaled PDE4 Inhibitors for Respiratory Diseases. J Med Chem 60:10026-10046. https://doi: 10.1021/acs.jmedchem.7b01044
Beavo JA (1995) Cyclic nucleotide phosphodiesterases: functional implications of multiple isoforms. Physiol Rev 75:725-48. https://doi: 10.1152/physrev.1995.75.4.725
Newton AC, Bootman MD, Scott JD (2016) Second Messengers. Csh Perspect Biol 8: a005926. https://doi: 10.1101/cshperspect.a005926
Hatzelmann A, Morcillo EJ, Lungarella G, Adnot S, Sanjar S, Beume R, Schudt C, Tenor H (2010) The preclinical pharmacology of roflumilast – A selective, oral phosphodiesterase 4 inhibitor in development for chronic obstructive pulmonary disease. Pulm Pharmacol Ther 23:235-256. https://doi: 10.1016/j.pupt.2010.03.011
Li H, Zuo J, Tang W (2018) Phosphodiesterase-4 Inhibitors for the Treatment of Inflammatory Diseases. Front Pharmacol 9:1048. https://doi: 10.3389/fphar.2018.01048
Xu Y, Li Z, Wu S, Guo L, Jiang X (2023) Oral small-molecule tyrosine kinase 2 and phosphodiesterase 4 inhibitors in plaque psoriasis: a network meta-analysis. Front Immunol 14: 1180170. https://doi: 10.3389/fimmu.2023.1180170
Song Z, Huang Y-Y, Hou K-Q, Liu L, Zhou F, Huang Y, Wan G, Luo H-B, Xiong X-F (2022) Discovery and Structural Optimization of Toddacoumalone Derivatives as Novel PDE4 Inhibitors for the Topical Treatment of Psoriasis. J Med Chem 65:4238-4254. https://doi: 10.1021/acs.jmedchem.1c02058
Martinez JM, Shen A, Xu B, Jovanovic A, de Chabot J, Zhang J, Xiang YK (2023) Arrestin-dependent nuclear export of phosphodiesterase 4D promotes GPCR-induced nuclear cAMP signaling required for learning and memory. Sci Signal 16:eade3380. https://doi: 10.1126/scisignal.ade3380
Liang J, Huang Y-Y, Zhou Q, Gao Y, Li Z, Wu D, Yu S, Guo L, Chen Z, Huang L, Liang SH, He X, Wu R, Luo H-B (2020) Discovery and Optimization of α-Mangostin Derivatives as Novel PDE4 Inhibitors for the Treatment of Vascular Dementia. J Med Chem 63:3370-3380. https://doi: 10.1021/acs.jmedchem.0c00060
Xu J, Zhang Y, Xu J, Wang M, Liu G, Wang J, Zhao X, Qi Y, Shi J, Cheng K, Li Y, Qi S, Nie G (2019) Reversing tumor stemness via orally targeted nanoparticles achieves efficient colon cancer treatment. Biomaterials 216:119247. https://doi: 10.1016/j.biomaterials.2019.119247
Kreso A, van Galen P, Pedley NM, Lima-Fernandes E, Frelin C, Davis T, Cao L, Baiazitov R, Du W, Sydorenko N, Moon YC, Gibson L, Wang Y, Leung C, Iscove NN, Arrowsmith CH, Szentgyorgyi E, Gallinger S, Dick JE, O'Brien CA (2014) Self-renewal as a therapeutic target in human colorectal cancer. Nat Med 20:29-36. https://doi: 10.1038/nm.3418
Bartucci M, Hussein MS, Huselid E, Flaherty K, Patrizii M, Laddha SV, Kui C, Bigos RA, Gilleran JA, El Ansary MMS, Awad MAM, Kimball SD, Augeri DJ, Sabaawy HE (2017) Synthesis and Characterization of Novel BMI1 Inhibitors Targeting Cellular Self-Renewal in Hepatocellular Carcinoma. Target Oncol 12:449-462. https://doi: 10.1007/s11523-017-0501-x
Sachse F, Gebauer K, Schneider C (2020) Continuous Flow Synthesis of 2H‐Thiopyrans via thia‐Diels–Alder Reactions of Photochemically Generated Thioaldehydes. Eur J Org Chem 2021:64-71. https://doi: 10.1002/ejoc.202001343
Kabalka GW, Mereddy AR (2006) Microwave promoted synthesis of functionalized 2-aminothiazoles. Tetrahedron Lett 47:5171-5172. https://doi: 10.1016/j.tetlet.2006.05.053
Yadav L, Tiwari MK, Shyamlal BRK, Chaudhary S (2020) Organocatalyst in Direct C(sp2)–H Arylation of Unactivated Arenes: [1-(2-Hydroxyethyl)-piperazine]-Catalyzed Inter-/Intra-molecular C–H Bond Activation. J Org Chem 85:8121-8141. https://doi: 10.1021/acs.joc.0c01019
Thutewohl M, Waldmann H (2003) Solid-phase synthesis of a pepticinnamin E Library. Bioorgan Med Chem 11:2591-2615. https://doi: 10.1016/s0968-0896(03)00159-7
Dai Z, Ye G, Pittman CU, Li T (2012) Solution‐phase synthesis and evaluation of tetraproline chiral stationary phases. Chirality 24:329-338. https://doi: 10.1002/chir.22001
Song E, Kim HY, Oh K (2017) Palladium-Catalyzed Aerobic Oxidative Hydroamination of Vinylarenes Using Anilines: A Wacker-Type Amination Pathway. Org Lett 19:5264-5267. https://doi: 10.1021/acs.orglett.7b02532
Ahdenov R, Mohammadi AA, Makarem S, Taheri S, Mollabagher H (2022) Eelectrosynthesis of benzothiazole derivatives via C–H thiolation. Heterocycl Commun 28:67-74. https://doi: 10.1515/hc-2022-0008
Kidwai M, Bhatnagar D, Mothsra P, Singh AK, Dey S (2009) Molecular iodine as a versatile reagent for Hantzsch synthesis of 2-aminothiazole derivatives. J Sulfur Chem 30:29-36. https://doi: 10.1080/17415990802422365
Mishra A, Srivastava M, Rai P, Yadav S, Tripathi BP, Singh J, Singh J (2016) Visible light triggered, catalyst free approach for the synthesis of thiazoles and imidazo[2,1-b]thiazoles in EtOH : H₂O green medium. RSC Adv 6:49164-49172. https://doi: 10.1039/c6ra05385h
Huang Y, Liu X, Wu D, Tang G, Lai Z, Zheng X, Yin S, Luo H-B (2017) The discovery, complex crystal structure, and recognition mechanism of a novel natural PDE4 inhibitor from Selaginella pulvinata. Biochem Pharmacol 130:51-59. https://doi: 10.1016/j.bcp.2017.01.016
Huang Y-Y, Yu Y-F, Zhang C, Chen Y, Zhou Q, Li Z, Zhou S, Li Z, Guo L, Wu D, Wu Y, Luo H-B (2019) Validation of Phosphodiesterase-10 as a Novel Target for Pulmonary Arterial Hypertension via Highly Selective and Subnanomolar Inhibitors. J Med Chem 62:3707-3721. https://doi: 10.1021/acs.jmedchem.9b00224
Zhang T, Lai Z, Yuan S, Huang Y-Y, Dong G, Sheng C, Ke H, Luo H-B (2020) Discovery of Evodiamine Derivatives as Highly Selective PDE5 Inhibitors Targeting a Unique Allosteric Pocket. J Med Chem 63:9828-9837. https://doi: 10.1021/acs.jmedchem.0c00983
Podlewska S, Kafel R (2018) MetStabOn—Online Platform for Metabolic Stability Predictions. Int J Mol Sci 19: 1040. https://doi: 10.3390/ijms19041040

Table 1 is available in the Supplementary Files section

Schemes 1-4 are available in the Supplementary Files section.

No competing interests reported.

SupplementaryMaterial.docx
GraphicalAbstract.png
Table1.docx
Scheme1.tif
Scheme 1. Syntheses of N-alkyl-4-(imidazo[1,2-a]pyrimidin-3-yl)thiazol-2-amines L1-3. Reagents and conditions: (a) (i) PhMe, 100℃, 3 h; (ii) Chloracetone, rt, 12 h; (b) (i) AcOH, Br₂, 110℃, 3 h; (ii) rt, 12 h; (c) Thioureas, EtOH, reflux, 12 h.
Scheme2.tif
Scheme 2. Syntheses of N- acyl-4-(imidazo[1,2-a]pyrimidin-3-yl)thiazol-2-amines L4-9. Reagents and conditions: (a) N-Boc thiourea, EtOH, Et₃N, reflux, 12 h; (b) TFA, THF, rt, 1 h; (c) Ac₂O, pyridine, 100℃, 1 h; or RCOOH, HATU, DIPEA, DMF, 100℃, MW, 10 min.
Scheme3.tif
Scheme 3. Syntheses of N-aryl-4-(imidazo[1,2-a]pyrimidin-3-yl)thiazol-2-aminesL10-18. Reagents and conditions: (a) NH₄SCN, AcOH, 73℃, 12 h; (b) 5, EtOH, reflux, 12 h.
Scheme4.tif
Scheme 4. Syntheses of N- pyridyl-4-(imidazo[1,2-a]pyrimidin-3-yl)thiazol-2-aminesL19-22. Reagents and conditions: (a) NH₄SCN, AcOH, 73℃, 12 h; (b) 5, EtOH, reflux, 12 h.

Discovery and Optimization of 4-(Imidazo[1,2-a]pyrimidin-3-yl)thiazol-2-amine Derivatives as Novel Phosphodiesterase 4 Inhibitors

Status:

Journal Publication

Version 1

Abstract

Figures

1. Introduction

2. Results and discussion

2.1 Structure‑based strategies applied for screening PDE4 inhibitors

2.2 Chemistry

2.3 Structure–activity relationships (SARs)

2.4 Computational Study

2.5 Rat Liver Microsomal Stability

3. Conclusion

Declarations

References

Table

Schemes

Additional Declarations

Supplementary Files

Status:

Journal Publication

Version 1