Chinese Chemical Letters  2016, Vol. 27 Issue (10): 1626-1629   PDF    
Design and synthesis of 5-cyclopropyl substituted cyclic acylguanidine compounds as BACE1 inhibitors
Liu Jia-Kuo, Gu Wei, Cheng Xiao-Rui, Cheng Jun-Ping, Nie Ai-Hua, Zhou Wen-Xia     
Beijing Institute of Pharmacology and Toxicology, Beijing, 100850, China
Abstract: By taking compound 1 as a lead, a series of 5-cyclopropyl substituted cyclic acylguanidine compounds were designed and synthesized as BACE1 inhibitors, compound 4d exhibited 84-fold improved inhibition efficiency than lead compound 1. The diphenyl fragment at the P3 position and the substituents at the second phenyl ring were essential for the compounds to achieve improved inhibition efficiency. This SAR studies provides new insights into the design and synthesis of more promising BACE1 inhibitors for the potential treatment of AD.
Key words: Alzheimer's disease     BACE1 inhibitor     β-Secretase     Drug discovery     Cyclic acylguanidine    
1. Introduction

Alzheimer's disease (AD) is a chronic neurodegenerative disorder. According to the amyloid cascade hypothesis [1], abnormal accumulation of amyloid peptides (Aβ) in the brain resulting in neuronal toxicityis the maincause ofAD [2]. Although the amyloid cascade hypothesis remains polemic as it has not been fully validated [3], it still represents a widely supported theory, substantiated by genetic evidence from the various mutations of amyloid precursor protein (APP) [4]. BACE1 (β-secretase) is a rate- limiting enzyme that hydrolyzes b-amyloid precursor protein (β- APP) to produce amyloid peptides (Aβ) while most amino-acid mutations close to the cleavage site of APP result in faster proteolysis and increased rate of disease progression [5]. In currently, BACE1 is considered as an attractive therapeutic target for the treatment of AD [6]. To date, different structural classes BACE1 inhibitors have been designed and developed [7], but it is still a challenge to discover brain penetration BACE1 inhibitor with low molecule weight, potent activity and high selectivity. A cyclic acylguanidine compound, compound 1, which was discovered each other independently by Schering-Plough [8], Wyeth [9] and Pfizer [10], is a weak BACE1 inhibitor (IC50 = 7.1 μmol/L) [8] with brain penetration. This finding is a milesto ne to the development of BACE1 inhibitor based on the amyloid cascade hypothesis. Many excellent BACE1 inhibitors of this type that were effective in vivo have been discovered, some of which have entered clinical trial [11, 12].

In our primary research, by taking compound 1 asa lead, we had design and synthesized a series of new cyclic acylguanidine compounds as BACE1 inhibitors and compound 2 exhibited submicromolar activity in vitro [13]. In this report, based on the SAR studies revealed in our previous paper [13], we then designed another type of compounds (3 and 4) (Fig. 1). The cyclopropyl fragment was introduced to make the molecule more flexible and lower the molecular weight. It is hoped that this could help the molecule to adjust its conformation to better fill the BACE1 active binding site to form the interactions essential for its biological activity.

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Figure 1. Structures of lead and the designed compounds

2. Experimental

The synthetic route of target compounds 3 was shown in Scheme 1. To the mixture of cyclopropyl acetylene and 3- bromophenol in THF was added TBAF/PdCl2(PPh3)2, the resulting mixture was heated to 80 ℃ for 8hto get acetylene intermediate 6 (yield 92%). Then 6 was dissolved in a co-solvent of acetone and H2O to which MgSO4/Na2CO3/KMnO4 was added. After reacting at r.t. for 2 h, dione intermediate 7 was obtained in a yield of 63%. A mixture of 7, 1-methylguanidine hydrochloride and Na2CO3 in EtOH/H2O was refluxed for 2h to give compound 8 (yield 42%). The phenol group then reacted with ethyl methyl-carbamic chloride to give one of the final product 3a (yield 21%). The synthetic route of the other target compound 3b also started from cyclopropyl acetylene (Scheme 1), which reacted with 1, 3-dibromobenzene to give intermediate 9 (yield 70%). One of the acetylene fragments of 9 was the n oxidized into dione intermediate 10 by MgSO4/Na2CO3/ KMnO4 in acetone/H2O (yield 20%). Cyclization and rearrangement of 10 with 1-methylguanidine hydrochloride at the presence of Na2CO3 gives final product 3b (yield 24%).

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Scheme. 1. Synthetic route of the designed compounds 3a, 3b. Reagents and conditions: a) 3-bromophenol, TBAF, PdCl2(PPh3)2, reflux; b) KMnO4, MgSO4, Na2CO3; c) 1- methylguanidine hydrochloride, Na2CO3, EtOH/H2O; d) DIPEA/THF, reflux; e) 1, 3-dibromobenzene, TBAF, PdCl2(PPh3)2, reflux; f) KMnO4, MgSO4, Na2CO3; g) 1- methylguanidine hydrochloride, Na2CO3, EtOH/H2O

The general synthetic route of compounds 4 (Scheme 2) also started from cyclopropyl acetylene, which reacted with 1, 3- dibromobenzene at the presence of TBAF/PdCl2(PPh3)2 in THF to get acetylene intermediate 11 (yield 72%). Oxidization of 11 with MgSO4/Na2CO3/KMnO4 gives dione intermediate 12 (yield 65%). K2CO3 and PdCl2(dppf) were added to a solution of 12 and 3- hydroxyphenyl boronic acid in 1, 4-dioxane at room temperature, then H2O was added until a clear solution was made, the resulting mixture was then heated to 90 ℃ for 12 h to afford 13 (yield 95%). The dione fragment then cyclized with 1-methylguanidine hydrochloride in EtOH/H2O under reflux to give cyclic acylguani- dine intermediate 14 (yield 62%). Then 14 was dissolved in acetone to which K2CO3 and the corresponding carbamic chloride or 3- bromopropyne was added. After reacting at r.t. for 36h, the mixture was filtered and the filtrate was directly separated on a silica column to give final products 4a-4i (yield 19%-34%). Target compound 4j was synthesized from 12. K2CO3/PdCl2(dppf) catalyzed Suzuki-coupling of 12 with 3-aminobenzeneboronic acid in 1, 4-dioxane and H2O gives intermediate 17. Then 17 was cyclized with 1-methylguanidine hydrochloride to get the acylguanidine fragment. The amino group then reacted with 3- bromopropyne in acetone at r.t. for 36 h to give target compound 4j. The bromobenzene group of 12 reacted with 3-bromobenze- neboronic acid to give the diphenyl fragment, the product was then mixed with cyclopropyl acetylene and TBAF/PdCl2(PPh3)2 in THF. After refluxing for 24 h, intermediate 15 was obta ined in a yield of 50%. The dione group of 15 cyclized with 1-methylguanidine hydrochloride to give target compound 4k (yield 10%). Suzuki coupling of 12 and phenylboronic acid gives intermediate 16, which reacted with 1-methylguanidine hydrochloride to give target compound 4l.

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Scheme. 2. Synthetic route of the designed compounds 4a-41. Reagents and conditions: a) TBAF, PdCl2(PPh3)2, reflux; b) KMnO4, MgSO4, Na2CO3; c) 3-hydroxyphenyl boronic acid, K2CO3 and PdCb(dppf); d) 1-methylguanidine hydrochloride, Na2CO3, EtOH/H2O; e) acetone/K2CO3; f) 3-aminobenzeneboronic acid, K2CO3 and PdCl2(dppf); g) i: 1-methylguanidine hydrochloride, Na2CO3, EtOH/H2O, ii: 3-bromopropyne, acetone/K2CO3; h) i: 3-bromobenzeneboronic acid, ii: TBAF, PdCl2(PPh3)2, reflux; i) 1-methylguanidine hydrochloride, Na2CO3, EtOH/H2O; j) phenylboronic acid, K2CO3 and PdCl2(dppf); k) 1-methylguanidine hydrochloride, Na2CO3, EtOH/H2O

TruPointTM Beta-Secretase Assay Kit is used for the BACE1 inhibition activity evaluation. A 15 μL of human recombinant BACE1 solution (0.67 mU/μL in reaction buffer) was mixed with 15 μL reaction buffer on a 384-well optiplate. To this mixture was added 2 μL of the tested compounds in DMSO (three concentrations for each compound 2.1×10-5, 2.1×10-6 and 2.1×10-7 mol/L). After a pre-incubation of 30 min at r.t., BACE1 substrate Eu-CEVNLDAEFK-Qsy 7 (400 nmol/L in reaction buffer) 15 μL was added. This incubation lasts 6hat r.t., and then 10 μL stop solution was added. The fluorescence of th e product was measured on an Envision Multilabel Plate Reader with an excitation wavelength of 340 nm and an emission wavelength of 615 nm. Blank control and negative control experiments were also conducted for the calculation of inhibition rates. The compounds that exhibited obviously improved inhibition efficiency were further tested to determine their IC50 value (five concentrations for each compound 2.1×10-5, 2.1×10-6, 2.1×10-7, 2.1×10-8 and 2.1×10-9mol/L to make the IC50 accurate).

The structure of the new compounds was characterized by 1H NMR and MS. Detailed experimental procedures, characterization data and1H NMR and MS spectra of target compounds are available in supporting information.

3. Results and discussion

In the previous work [13], attaching the rivastigmine pharmacophore phenyl ester group to the lead compound 1 to interact with the unoccupied S3 pocket, we have successfully discovered compound 2 with much improved inhibition efficiency. Encouraged by this result, we then designed and synthesized compounds 3 and 4. The cyclopropyl was introduced to replace one phenyl ring of the lead compound 1 to obtain compounds 3a and 3b. The phenyl ester 3a and cyclopropyl acetylene 3b fragments were applied to interact with the unoccupied S3 pocket or S3 subpocket. It is hoped that this additional interactions would improve the inhibition efficiency and druggability. However, BACE1 inhibition tests showed that these compounds did not exhibit much improved inhibition efficiency as expected (Table 1). As can be seen, compound 3a showed decreased inhibitory activity than lead compound 1, while compound 3b showed an IC50 of 0.807 mmol/L. It seems that when the cyclopropyl was introduced, steric rigidified-G was preferred to give compounds with relatively higher inhibition efficiency. This is in accordance with the SAR we have previously concluded. Docking results of 3b with BACE1 active binding site (Fig. 2) revealed that only part of the terminal cyclopropyl fragment was suited in the S3 sub-pocket to form hydrophobic interactions. This is believed to be an important cause to prevent the enzymatic inhibitory activity of 3b from further improving.

Table 1
Inhibitory activity of compounds 3 and 4 against BACE1 at the concentration of 1 μmol/L.a, b

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Figure 2. Docking result of compound 3b (left) and 4k (right) with BACE1 active binding site (PDB: 4DJY)

To further improving the inhibitory activity, we then designed and synthesized compounds 4. In compounds 4 the diphenyl fragment was applied to form enhanced interactions as the second phenyl ring has been proved to be a better substitute to bind the S3 pocket. The-Y fragment was attached at the meta-position of this phenyl ring to interact with the S3 sub-pocket. Compounds 4a-k were synthesized and tested their enzymatic inhibition efficiency against BACE1 (Table 1). As can be seen, these compounds showed generally much improved inhibitory activity than lead compound 1 except 4a and 4b, which means the phenyl estergroup is not well suited in the S3 sub-pocket as they are expected. While the propyne (4c, 4j) or linear paraffin (4d-4i) or cyclopropyl acetylene (4k) helps improving the bioactivity of the compounds. Linker atom of -Y plays the role of a certain degree of influencing the bioactivity, as 4j showed relatively higher IC50 value (IC5O = 1.15 mmol/L) when the basic N atom was used. Linear paraffin -Y is important for the compound to maintain its bioactivity. Ethyl seems to be the most suitable (4e IC50 = 0.303 mmol/L), when the chain gets longer (4g, 4h), the bioactivity decreases sharply. Introduction of the F atom at the terminal of ethyl of 4e gives the most potent compound 4d with an IC50 of 0.0847 mmol/L. This is believed to be due to the polarization effect induced by the strong electron-attracting ability of the F atom. Compound 4i also showed higher inhibition efficiency than the corresponding 4g. To better understand the binding mode of this series of compounds with BACE1, compound 4k was docked into BACE1 active binding site (Fig. 2) and this confirmed our hypothesis that the second phenyl ring interact well with the S3 pocket while the linear cyclopropyl acetylene fragment nicely fitted the S3 sub-pocket. This also explained why compound 4a and 4b showed sharply reduced inhibitory activity. Efforts to discovery compounds with further improved inhibition efficiency are ongoing.

4. Conclusion

In conclusion, based on the work we previously reported, we have design and synthesized a series of new compounds as BACE1 inhibitors. By introducing the cyclopropyl to lower the molecule weight and make the molecule more flexible, compounds with sub-micromolar activity were obtained, the most potent 4d exhibited 84-fold improved inhibition efficiency than lead compound 1. SAR studies revealed that it is important for the molecule to maintain the rigid conformation at the P3 position exemplified by the diphenyl fragment. The -Y fragment was attached at the meta-position of the second phenyl ring and this was important to guide this branch into the S3 sub-pocket to form further enhanced interactions. Introduction of F atom to the ethyl of 4e gives the most potent 4d, this illustrates that short polarized- Y is essential for the molecule to maintain the bioactivit y. This SAR studies provide new insights into the design and synthesis of more promising BACE1 inhibitors for the potential treatment of AD.

Acknowledgment

This work was supported by grants from the National Natural Science Foundation of China (No. 81172924) and Beijing Municipal Natural Science Foundation (No. 7112106).

Appendix A. Supplementary data

Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/jxclet.2016.05. 002.

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