Synthesis and inhibitory effect of piperine derivates on monoamine oxidase

Bioorganic & Medicinal Chemistry Letters 22 (2012) 3343–3348

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Synthesis and inhibitory effect of piperine derivates on monoamine oxidase Li-Hua Mu a, Bo Wang a, , Hao-Yang Ren a, , Ping Liu a,⇑, Dai-Hong Guo a, Fu-Meng Wang a, Lin Bai a, Yan-Shen Guo b a b

Department of Clinical Pharmacology, General Hospital of PLA, Beijing 100853, China Institute of Materia Medica, Chinese Academy of Medical Sciences & Peking Union Medical College, Xian Nong Tan Street, Beijing 100050, China

a r t i c l e

i n f o

Article history: Received 28 September 2011 Revised 15 February 2012 Accepted 27 February 2012 Available online 8 March 2012 Keywords: Monoamine oxidase Inhibitors Piperine derivates Structure–activity relationship Molecular docking

a b s t r a c t A series of piperine derivates (1–19) have been designed, synthesized and evaluated in vitro for their monoamine oxidase (MAO) A and B inhibitory activity and selectivity. It is worth noting that most of the small amine moieties substituted on the piperidine ring proved to be potent and selective inhibitors of MAO-B rather than of MAO-A. 5-(3,4-methylenedioxyphenyl)-2E,4E-pentadienoic acid n-propyl amide (3) showed the greatest MAO-B inhibitory activity (IC50(MAO-B) = 0.045 lM) and good selectivity (IC50(MAO-A) = 3.66 lM). The conjugated double bond and carbonyl group of piperine are proved to be an essential feature for piperine and related alkylamides to exhibit MAO-inhibitory activity. Binding mode of the titled compounds was predicted using FlexX algorithm. The design and optimization of novel small molecule monoamine oxidase inhibitors will be guided by the results of this report. Ó 2012 Elsevier Ltd. All rights reserved.

Monoamine oxidase (MAO) catalyzes the oxidative deamination of monoamine neurotransmitters such as serotonin, dopamine, and norepinephrine, and appears to play important roles in several psychiatric and neurological disorders.1,2 MAO has been divided into two subtypes, MAO-A and MAO-B, on the basis of their aminoacid sequence, substrate and inhibitor selectivity, and tissue distribution.3,4 Selective MAO-A inhibitors are useful in the therapy of mental disorders, mainly as antidepressants and anxiety, whereas MAO-B inhibitors are expected to be useful in the therapy of Parkinson’s and Alzheimer’s disease.5–7 To date, a number of MAO inhibitors such as coumarins, xanthones, and isoquinoline alkaloids have been isolated from natural products or synthesized.8–11 The high-resolution structures of MAO-A and MAO-B has been available and these can be exploited in the design of selective MAO inhibitors with minimum side effects.12–14 Piperine was the first amide isolated from Piper species and was reported to display central nervous system depression, antipyretic, and anti-inflammatory activity.15,16 Moreover, previous studies have demonstrated that piperine and its derivatives present sedative-hypnotic, tranquilizing, and muscle-relaxing actions and can intensify the depressive action of other depressants.17 Amidst the vast spectrum of piperine’s activities, it was particularly intriguing to notice the consistent outcome from several independent studies, suggesting significant antidepressant-like activity of this compound. For example, piperine and its analog, antiepilepsirine exhibited antidepressant-like activity in classical behavioral ⇑ Corresponding author. Tel.: +86 10 66936676; fax: +86 10 66936678.  

E-mail address: [email protected] (P. Liu). These authors contributed equally to this work.

0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.bmcl.2012.02.090

Figure 1. The piperine structure divided into three key regions.

models such as forced swimming test and tail suspension tests in rodents.18–20 Piperine treatment also led to increased level of biogenic amines such as noradrenaline and serotonin in some regions of mouse brain, which indirectly suggests its ability to interfere with the metabolism of these amines.19,20 Lastly, piperine and its analog—methylpiperate have been directly shown to inhibit the relevant enzymes that is, monoamine oxidases (MAOs; EC 1.4.3.4) in vitro.21–23 Previously, piperine was found to be more MAO-A selective for rat21 while it was more MAO-B selective for mouse.22 So, with a view to having some preliminary idea, we design a series of derivatives which were from different parts of piperine. MAO inhibitory activity and structure–activity relationship of these synthesized derivatives were studied. To give structural insights regarding the binding mode of these inhibitors, we carried out docking simulations of the most potent and selective MAO-B inhibitor 3. In our present work, several piperine derivatives were prepared, using a simple coupling method foramide synthesis, and tested for monoamine oxidase inhibitory activity. The piperine structure (Fig. 1) can be split into three fragments namely methylenedioxyphenyl (MDP) ring (compartment A), side chain with conjugated

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Scheme 1. Reagents and conditions: (a) KOH (b) H2SO4/MeOH, EtOH reflux 6 h (c) SOCl2 in CH2Cl2 reflux 1 h (d) R1R2NH, rt 30 min (e) 2-octanol/180 °C 72 h.

Scheme 2. Reagents and conditions: (a) NaBH4/I2, THF, 0 °C nitrogen atmosphere, 48 h (b) BBr3 in CH2Cl2 rt 48 h (c) Ac2O/pyridine.

double bonds (compartment B) and a basic piperidine moiety attached through a carbonylamide linkage to side chain (compartment C). Piperinic acid 2 was obtained by alkaline hydrolysis of piperine 1 (Scheme 1). The amides (3–6) were readily obtained

from the carboxylic acids through acyl chloride formation with appropriate amines (Scheme 1).24 The amide functional groups of piperine were also replaced by a range of ester groups, yielding the corresponding analogues 10–11 (Scheme 1).25 In. order to

Scheme 3. Reagents NR1R2 = piperidine (13), n-propyl amide (14), n-butyl amide(15), N,N-diethyl amide (16), N-methyl piperazine amide (17).

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L.-H. Mu et al. / Bioorg. Med. Chem. Lett. 22 (2012) 3343–3348 Table 1 The inhibition of rat main MAO isoforms by the newly synthesized piperine derivativesa. No.

IC50 for MAO-Ab (lM)

IC50 for MAO-Bb (lM)

SIc

MAO-inhibitory selectivity

N

0.404

0.26

1.55

Selective for MAO-B

OH

18.34

13.59

1.35

Selective for MAO-B

N

3.66

0.045

81.5

Selective for MAO-B

N

>100

0.33

—

Selective for MAO-B

N

3.82

0.078

49.0

Selective for MAO-B

2.04

0.96

2.12

Selective for MAO-B

0.80

1.57

0.51

Selective for MAO-A

1.28

0.75

1.71

Selective for MAO-B

>100

>100

—

—

O

2.74

0.23

11.9

Selective for MAO-B

O

7.55

0.33

22.8

Selective for MAO-B

>100

>100

—

—

>100

1.97

—

Selective for MAO-B

Compound

O 1

O O O

2

O O O

3

O O O

4

O O O

5

O O O

6

O

N N

O O 7

HO

N

HO O

O

O 8

N

O O O

N

9

O O 10

O O O

11

O O

O

N

O

N 12

O O

O O O

13

O

N

O (continued on next page)

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L.-H. Mu et al. / Bioorg. Med. Chem. Lett. 22 (2012) 3343–3348

Table 1 (continued) No.

IC50 for MAO-Ab (lM)

IC50 for MAO-Bb (lM)

SIc

MAO-inhibitory selectivity

N

>100

>100

—

—

N

16.08

3.50

4.59

Selective for MAO-B

N

5.21

1.75

2.98

Selective for MAO-B

>100

>100

—

—

O

3.17

1.80

1.76

Selective for MAO-B

O

5.57

1.23

4.53

Selective for MAO-B

Compound

O 14

O O O

15

O O O

16

O O O

17

O

N N

O O 18

O O O

19

O O

20 21

Selegiline Clorgyline

>100 0.42  10

3

13.91  10 0.26

3

— 1.62  10

3

Selective for MAO-B Selective for MAO-A

a Selegiline or clorgyline were added at 1 lM to determine the isoenzymes A and B. Newly synthesized compounds were preincubated with the homogenates for 10 min at 37 °C. b IC50 values were determined from plots of residual activity percentage, calculated in relation to a sample of the enzyme treated under the same conditions without inhibitor. c SI = IC50 of MAO-A/IC50 of MAO-B.

Figure 2. Lineweaver–Burk plots of the inhibition of MAO-A and MAO-B by compound 3. The reciprocals of MAO-A and MAO-B activities were plotted against the reciprocals of substrate concentration. Compound 3 concentrations: MAO-A 0 lM (rhombus); 50 lM (rectangle);100 lM (triangle), MAO-B 0 lM (rhombus); 3.5 lM (rectangle);7 lM (triangle).

achieve the diacetylphenyl containing analogue 8, Piperine 1 was sequentially treated with BBr3 and Ac2O (Scheme 2). 5-(3,4-methylenedioxyphenyl)-penta-2E,4E-dienyl piperidine 9 (Scheme 2) was isolated during an attempt to reduce a single double bond of piperine according to the method of Das et al.26 Compounds 1, 3–6, 10 and 11 were subjected to hydrogenation(Scheme 3) and saturated molecules 13–19 thus obtained were bioevaluated.25–30 MAO-A and MAO-B inhibitory activities of the synthesized piperine derivates were determined by a fluorometric assay, using kynuramine as a substrate, in the presence of their specific inhibitors (selegiline 1 lM for MAO-A and clorgyline 1 lM for MAO-B).31 Rat brain mitochondria were isolated from Sprague–Dawley rats

according to the method of Clark and Nicklas.32 Protein concentration was determined according to the method of Bradford33 in which bovine serum albumin was used as standard. The MAO inhibitory activities, expressed as IC50 values are summarized in Table 1. Some of the synthesized compounds showed potent MAO-B inhibitory activity with the IC50 value at low micromolar. Among all the compounds, only compound 7 is selective MAO-A inhibitors (IC50 (MAO-A) = 0.80 and IC50(MAO-B) = 1.57). Compounds 3 and 5 showed the good MAO-B inhibitory activity (IC50(MAO-B) = 0.045 and 0.078 lM, respectively) and good selectivity (IC50(MAOA) = 3.66 and 3.82 lM, respectively). Compounds 2, 9, 12, 14 and

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Figure 3. FlexX-modeled binding mode of compound 3 (carbon atoms colored green) with MAO-A(a) and MAO-B(b). H-bonding interactions are presented with red line.

17 showed no inhibitory activity to both MAO-A and MAO-B isoforms. The inhibitory of mouse and rat brain-derived MAO-B by piperine was competitive in previous studies, the mode of its inhibitory action of rodent brain-derived MAO-A was found to be either competitive or mixed type for mouse and rat MAO-A.21,22 The Lineweaver–Burk plots of the inhibition of MAO-A and MAO-B by compound 3 showed the inhibitory actions are both competitive (Fig. 2).34 Structure–activity relationships (SARs) were inferred from the data of enzymatic experiments reported in Table 1.Compounds 3–6, 10 and 11 has a propyl, butyl, diethyl, N-methyl phneyl, methoxy and ethoxyl group in place of the piperidine moiety of piperine respectively and they have been shown to inhibit both MAO-A and MAO-B with more selectivity for the latter. Two compounds (3, 5) with propyl and diethyl substituent showed the better MAO-B inhibitory activity and good selectivity comparing with piperine. So, the small molecule amines substitute the piperidine moiety may improve the MAO-B inhibitory activity. But all the compounds losing piperidine moiety showed poor MAO-A inhibitory activity comparing to piperine. Thus, the piperidine moiety (compartment C, Fig. 1) of piperine seems to aid in achieving better MAO-A selectivity while its omission barely affects the MAO-B selectivity. But the previous docking of piperine and methyl piperate onto MAO enzymes by Rahman and Rahmatullah given the contrast conclusion.35 The reasons for such discrepancy are not clear but species difference is likely to be among plausible explanations. In order to determine the effect of the compartment B(Fig. 1) of piperine that can act as an effective inhibitor of MAOs, compounds 12, 13–19 and compound 9 were synthesised. Compounds 13–19 were obtained from compounds 3–6, 10 and 11 by catalytic hydrogenation but they showed no inhibition or very poor inhibition to both MAO-A and MAO-B. Compound 9 was synthesised by removing the carbonyl of piperine and it lost the inhibition to both MAO-A and MAO-B. Compound 12 (dimer of piperine) also has no MAO-A and MAO-B inhibition. This result indicated that the conjugated double bond and carbonyl group (compartment B, Fig. 1) are likely to be an essential feature for piperine and related alkylamides to exhibit MAO-inhibitory activity. Compounds 7, 8 screened in our study had the changed MDP ring (compartment A, Fig. 1) and compounds changing such moiety seem to offer poor inhibitory to monoamine oxidase. So, the MDP ring of piperine is likely to impart MAO-B inhibitory activity. This result is analogy with the previous study of Rahman and Rahmatullah.35 Interestingly com-

pound 7 is the only compound showing MAO-A inhibition, this result indicated that the phenolic hydroxyl is likely to increase the MAO-A inhibitory selectivity. In order to get some insight for further structure-based modification of MAO inhibitors, the binding mode was investigated using FlexX algorithm implemented in SYBYL 7.2.36 The coordinates of X-ray co-crystal structure of MAO-A&B (pdb code: 2Z5X & 3PO7) was employed for docking the synthesized compounds.37,38 The docking calculations add to the understanding of the structural requirements for binding to the MAO enzyme. In FlexX calculations, hydrogen bonding interactions, van der Waals interactions and steric as well as electrostatic interactions were evaluated. The topology of the active site for both the proteins MAO-A and MAO-B are comparable in Figure 3 which shows the binding modes of compound 3 (a) into the MAO-A binding cavity and (b) into the MAO-B binding cavity. Visual inspection of the pose of compound 3 into the MAO-A binding site revealed that the pyridine ring of 3 is placed in the ‘aromatic cage’ framed by Tyr197, Tyr407, Tyr444, and the aromatic ring is oriented to establish p–p stacking interactions with Tyr407. Conjugated double bonds are embedded in a large hydrophobic pocket formed by Ile180, Phe208, Asn181, Gln215, and Ile335. Moreover, one hydrogen bond is observable for 3 between the NH of 3 and the carbonyl oxygen of Phe208. Visual inspection of the pose of compound 3 into the MAO-B binding site revealed that compound 3 is inserted into the ‘aromatic cage’ framed by Tyr60, Tyr326, Tyr398, Tyr435, and the aromatic ring. Moreover the carbonyl oxygen of compound 3 formed hydrogen bonds with NH on the side chain of SER59 and TYR60. Compound 3 forms more hydrogen bond interactions with the MAO-B active site compared to the MAO-A active site which may indicate that 3 interacts more tightly with MAO-B. Overall, a series of piperine derivatives (1–19) were designed and evaluated in vitro for their MAO-A and MAO-B inhibitory activity. Most of the synthesized compounds proved to be potent, and selective inhibitors of MAO-B rather than of MAO-A. It is worth noting that the small molecule amines moieties substituted on the piperidine ring may improve the MAO-B inhibitory activity. Among those derivatives, compound 3 showed the greatest MAOB inhibitory activity (IC50(MAO-A) = 3.66 lM) and good selectivity (IC50(MAO-B) = 0.045 lM). The conjugated double bond and carbonyl group of piperine are likely to be an essential feature for piperine and related alkylamides to exhibit MAO-inhibitory activity.

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Molecular dockings of compound 3 into MAO-A and MAO-B were performed. Compound 3 forms more hydrogen bond interactions with the MAO-B active site compared to the MAO-A active site which explains its MAO-B selectivity. Acknowledgments We are grateful to Mr Li-Ping Kang for the NMR and HR-EI-MS measurement in the Instrumentation Center of the Academy of Military Medical Sciences. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.bmcl.2012.02.090. References and notes 1. Shih, J. C.; Chen, K.; Ridd, M. J. Annu. Rev. Neurosci. 1999, 22, 197. 2. Bach, A. W.; Lan, N. C.; Johnson, D. L.; Abell, C. W.; Bembenek, M. E.; Kwan, S. W.; Seeburg, P. H.; Shih, J. C. Proc. Natl. Acad. Sci. U.S.A. 1988, 85, 4934. 3. Abell, C. W.; Kwan, S. W. Prog. Nucl. Acids Res. Mol. Biol. 2001, 65, 129. 4. Kalgutkar, A. S.; Castagnoli, N., Jr Med. Res. Rev. 1995, 15, 325. 5. Jegham, S.; George, P. Expert. Opin. Ther. Patents 1998, 8, 1143. 6. Youdim, M. B.; Edmondson, D.; Tipton, K. F. Nat. Rev. Neurosci. 2006, 7, 295. 7. Chimenti, F.; Cottiglia, F.; Bonsignore, L.; Casu, L.; Casu, M.; Floris, C.; Secci, D.; Bolasco, A.; Chimenti, P.; Granese, A.; Befani, O.; Turini, P.; Alcaro, S.; Ortuso, F.; Trombetta, G.; Loizzo, A.; Guarino, I. J. Nat. Prod. 2006, 69, 945. 8. Gnerre, C.; Catto, M.; Leonetti, F.; Weber, P.; Carrupt, P. A.; Altomare, C.; Carotti, A.; Test, B. J. Med. Chem. 2000, 43, 4747. 9. Suzuki, O.; Katsumata, Y.; Oya, M.; Chari, V. M.; Vermes, B.; Wagner, H.; Hostettmann, K. Planta Med. 1981, 42, 17. 10. Ro, J. S.; Lee, S. S.; Lee, K. S.; Lee, M. K. Life Sci. 2001, 70, 639. 11. Kong, L. D.; Cheng, C. H.; Tan, R. X. Planta Med. 2001, 67, 74. 12. Edmondson, D. E.; Binda, C.; Mattevi, A. Arch. Biochem. Biophys. 2007, 464, 269.

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