Cinnamide Derivatives as Mammalian Arginase Inhibitors - MDPI

5 downloads 287 Views 3MB Size Report
Sep 29, 2016 - Caldwell, R.B.; Toque, H.A.; Narayanan, S.P.; Caldwell, R.W. Arginase: An old enzyme with new tricks. Trends Pharmacol. Sci. 2015, 36 ...
International Journal of

Molecular Sciences Article

Cinnamide Derivatives as Mammalian Arginase Inhibitors: Synthesis, Biological Evaluation and Molecular Docking Thanh-Nhat Pham 1 , Simon Bordage 1,2 , Marc Pudlo 1 , Céline Demougeot 1 , Khac-Minh Thai 3 and Corine Girard-Thernier 1, * 1

2 3

*

PEPITE EA4267, University Bourgogne Franche-Comté, F-25000 Besançon, France; [email protected] (T.-N.P.); [email protected] (S.B.); [email protected] (M.P.); [email protected] (C.D.) University Lille, EA 7394-ICV-Institut Charles Viollette, F-59000 Lille, France Department of Medicinal Chemistry, Faculty of Pharmacy, University of Medicine and Pharmacy at Ho Chi Minh City, 41 Dinh Tien Hoang, Dist 1, Ho Chi Minh City 700000, Vietnam; [email protected] Correspondence: [email protected]; Tel.: +33-3-8166-5559

Academic Editor: Alejandro Cifuentes Received: 22 July 2016; Accepted: 23 September 2016; Published: 29 September 2016

Abstract: Arginases are enzymes that are involved in many human diseases and have been targeted for new treatments. Here a series of cinnamides was designed, synthesized and evaluated in vitro and in silico for their inhibitory activity against mammalian arginase. Using a microassay on purified liver bovine arginase (b-ARG I), (E)-N-(2-phenylethyl)-3,4-dihydroxycinnamide, also named caffeic acid phenylamide (CAPA), was shown to be slightly more active than our natural reference inhibitor, chlorogenic acid (IC50 = 6.9 ± 1.3 and 10.6 ± 1.6 µM, respectively) but it remained less active that the synthetic reference inhibitor Nω -hydroxy-nor-L-arginine nor-NOHA (IC50 = 1.7 ± 0.2 µM). Enzyme kinetic studies showed that CAPA was a competitive inhibitor of arginase with Ki = 5.5 ± 1 µM. Whereas the activity of nor-NOHA was retained (IC50 = 5.7 ± 0.6 µM) using a human recombinant arginase I (h-ARG I), CAPA showed poorer activity (IC50 = 60.3 ± 7.8 µM). However, our study revealed that the cinnamoyl moiety and catechol function were important for inhibitory activity. Docking results on h-ARG I demonstrated that the caffeoyl moiety could penetrate into the active-site pocket of the enzyme, and the catechol function might interact with the cofactor Mn2+ and several crucial amino acid residues involved in the hydrolysis mechanism of arginase. The results of this study suggest that 3,4-dihydroxycinnamides are worth being considered as potential mammalian arginase inhibitors, and could be useful for further research on the development of new arginase inhibitors. Keywords: arginase inhibitor; cinnamide; docking; screening; structure-activity relationships

1. Introduction L -Arginine ( L -Arg) is an amino acid involved in distinct metabolic routes for the synthesis of many different compounds including proteins, urea, polyamines, proline or nitric oxide (NO), and is consequently the substrate of various enzymes. Therefore, many studies focused on the search for bioactive compounds that are able to regulate L-arginine metabolism [1]. Nitric oxide synthase (NOS) hydrolyses L-Arg to produce L-citrulline and NO, a crucial vasorelaxant factor. Arginase (amidinohydrolase, EC 3.5.3.1), by hydrolysing L-arginine into L-ornithine and urea, plays an important role in the ammonia detoxification in mammals [2]. Interestingly, it has been shown these last few years that this enzyme also plays a crucial role in the bioavailability of L-arginine for nitric oxide synthase (NOS) by substrate competition [3]. Therefore, an increased activity of

Int. J. Mol. Sci. 2016, 17, 1656; doi:10.3390/ijms17101656

www.mdpi.com/journal/ijms

Int. J. Mol. Sci. 2016, 17, 1656

2 of 17

arginase is associated with various diseases by reducing the supply of L-arginine needed by NOS to produce NO, and by raising production of L-ornithine resulting in vascular structural problems [4]. In fact, L-ornithine is converted into either polyamines (putrescine, spermidine and spermine) or into proline, and these downstream products can promote cell proliferation and collagen production [4,5]. Over the last few decades, interest has grown concerning the use of arginase for therapeutic uses. Indeed, the use of arginase inhibitors has proven to be beneficial in various pathophysiological states such as hypertension [6], erectile dysfunction [7], pulmonary hypertension [8], atherosclerosis [9], Int. J. Mol. Sci. 2016, 17, 1656 2 of 16 diabetic renal injury [10], asthma and allergic rhinitis [11], myocardial ischemia-reperfusion injury [12], wound healing [13], and cancer [14].byThe first generation arginase inhibitors associated with various diseases reducing the supply of of L-arginine needed by NOS comprised to produce analogs NO, andLby raising production of the L-ornithine resulting in structural problems [4]. In L fact, of Nω -hydroxy-arginine (NOHA), intermediate in vascular the production of NO from -arginine by L-ornithine is converted into either polyamines (putrescine, spermidine and spermine) or into NOS [15,16]. These inhibitors are characterized by N-hydroxy-guanidium side chains. Among them, proline, and these downstream products can promote cell proliferation and collagen production Nω -hydroxy-nor-arginine most potent arginase inhibitor to date [17]. [4,5]. OverLthe last few (nor-NOHA) decades, interest is hasthe grown concerning the use of arginase for known therapeutic Boronic acid analogs of L -arginine formed the second generation of arginase inhibitors, among uses. Indeed, the use of arginase inhibitors has proven to be beneficial in various pathophysiological states such as hypertension [6], erectile dysfunction [7], pulmonary hypertension [8], atherosclerosis which S-(2-boronoethyl)-L-cysteine (BEC) and 2-(S)-amino-6-boronohexanoic acid (ABH) are the main [9], diabetic renal injury [10], asthma and allergic rhinitis [11], myocardial ischemia-reperfusion representative compounds [18,19]. Some of these synthetic arginase inhibitors, such as nor-NOHA, BEC injury [12], wound healing [13], and cancer [14]. The first generation of arginase inhibitors and ABH,comprised are currently available commercially. However, the use of such molecules for therapeutic analogs of Nω-hydroxyL-arginine (NOHA), the intermediate in the production of NO purposes has poor bioavailability and potential toxicity (BEC, and from several L-argininelimitations: by NOS [15,16]. These inhibitors are characterized by N-hydroxy-guanidium sideABH) [20], Among them, Nω-hydroxy-nor(nor-NOHA) the most potent that arginase very shortchains. half-life (nor-NOHA) [21], andL-arginine also high cost [22].is Considering theinhibitor development of known to date [17]. Boronic acid analogs of L-arginine formed the second generation of arginase arginase inhibitors is of great therapeutic relevance in various human diseases, investigations have inhibitors, among which S-(2-boronoethyl)-L-cysteine (BEC) and 2-(S)-amino-6-boronohexanoic acid been undertaken in order develop prodrugs NOHA [23] optimized ABH by substituting the (ABH) are the mainto representative compoundsof[18,19]. Some of or these synthetic arginase inhibitors, Cα-aminosuch acidasfunction [12,24–26] andare bycurrently replacing the boronic acidHowever, functionthe[27–31]. Plants have nor-NOHA, BEC and ABH, available commercially. use of such molecules therapeutic purposes has several inhibitors limitations: [32]. poor bioavailability andhemisynthesis potential been shown to be aforsource of promising arginase Nevertheless, has toxicity (BEC, and ABH) [20], very short half-life (nor-NOHA) [21], and also high cost [22]. never been used for the design of new arginase inhibitors [32]. Among the natural compounds Considering that the development of arginase inhibitors is of great therapeutic relevance in various tested as potential arginase inhibitors, webeen previously that chlorogenic (CGA) human diseases, investigations have undertakenreported in order to develop prodrugs acid of NOHA [23] displayed an interesting activityABH on bovine liver arginase I (b-ARG I) [33,34]. We therefore considered it as a or optimized by substituting the Cα-amino acid function [12,24–26] and by replacing the boronic acid function [27–31]. Plants been shown to be a acids. source We of promising potential lead compound. CGA is an esterhave of caffeic and quinic showed arginase that the caffeoyl inhibitors [32]. Nevertheless, hemisynthesis has never been used for the design of new arginase (i.e., dihydroxy cinnamoyl) part is probably more involved in the arginase inhibition than the quinoyl inhibitors [32]. Among the natural compounds tested as potential arginase inhibitors, we previously moiety [33,34]. we replaced latteranmoiety, containing in reportedTherefore, that chlorogenic acid (CGA)this displayed interesting activity on four bovineasymmetric liver arginasecarbons, I order to improve inhibitory activity and ittoassimplify Considering (b-ARG I)the [33,34]. We therefore considered a potentialthe leadchemical compound.structure. CGA is an ester of caffeic that the quinic acids. is We showed that the caffeoyl (i.e., dihydroxy cinnamoyl) part is probably stability ofand a compound improved when its ester bond is replaced by an amide bondmore [35], and that involved in the arginase inhibition than the quinoyl moiety [33,34]. Therefore, we replaced this latter the amidification with a range of phenethylamines has already been noted for their inhibitory activity moiety, containing four asymmetric carbons, in order to improve the inhibitory activity and to on tyrosinase (which is another binuclear metalloenzyme) we decided to evaluate this kind of simplify the chemical structure. Considering that the stability[36], of a compound is improved when its amides forester their arginase inhibition. Finally, given polyphenolic products have bond is replaced by an amide bond [35],that and several that the natural amidification with a range of has already for their inhibitoryhas activity onmainly tyrosinase (which ison another significantphenethylamines effects on arginase [34],been thenoted functionalization been focused methoxy and binuclear metalloenzyme) [36], we decided to evaluate this kind of amides for their arginase hydroxyl groups which are common in such compounds. The optimal size of compounds and the inhibition. Finally, given that several natural polyphenolic products have significant effects on contribution of each been explored to identify portions of the molecule that groups are essential for arginase [34],part the have functionalization has been mainly focused on methoxy and hydroxyl the expected biological activity. which are common in such compounds. The optimal size of compounds and the contribution of each part havework been explored to identify portions of thenew molecule that are essential the expected The present first aimed at synthesizing or already known for cinnamide derivatives biological activity. in order to test their inhibitory property on a purified bovine liver arginase (b-ARG I) (Figure 1). The present work first aimed at synthesizing new or already known cinnamide derivatives in Then, the order mosttoactive compound of thisonseries wasbovine evaluated on a (b-ARG recombinant test their inhibitory property a purified liver arginase I) (Figurehuman 1). Then,arginase I (h-ARG I).the Additionally, enzyme-kinetic studies were alsoI (h-ARG performed on the most active compound of this seriesand was molecular evaluated on docking a recombinant human arginase I). enzyme-kinetic and molecular studies were alsotheir performed on the mechanism most active and the most activeAdditionally, compounds and compared to CGAdocking in order to analyze inhibition compounds and compared to CGA in order to analyze their inhibition mechanism and the protein-ligand interactions, respectively. protein-ligand interactions, respectively.

Figure 1. Design of cinnamide derivatives from chlorogenic acid.

Figure 1. Design of cinnamide derivatives from chlorogenic acid.

Int. J. Mol. Sci. 2016, 17, 1656

3 of 17

2. Results and Discussion 2.1. Chemistry Int. J. Mol. Sci. 2016, 17, 1656

3 of 16

Cinnamide derivatives were synthesized using phosphonium salts as condensing reagents 2. Results adapted and Discussion by a procedure from Okombi et al. [37]. The synthesis protocol was modified by replacing 2.1. benzotriazol-1-yloxytris(dimethylamino)-phosphonium hexafluorophosphate (BOP) by Chemistry benzotriazol-1-yloxytri(pyrrolidino)-phosphonium hexafluorophosphate (PyBOP) [38] to avoid the Cinnamide derivatives were synthesized using phosphonium salts as condensing reagents formationbyofaInt.the side-product acidwas triamide [39]. J. Mol.carcinogenic Sci. 2016,adapted 17, 1656 3 of 16 (HMPA) procedure from Okombi et hexamethylphosphoric al. [37]. The synthesis protocol modified by The amidereplacing linkagebenzotriazol-1-yloxytris(dimethylamino)-phosphonium was generated by mixing the free acid with a hexafluorophosphate variety of amines (BOP) in theby presence of 2. Results and Discussion benzotriazol-1-yloxytri(pyrrolidino)-phosphonium (PyBOP) [38] to avoid the PyBOP, triethylamine (Et3 N) in dimethylformamidehexafluorophosphate (DMF) and CH2 Cl 2 (Scheme 1). The by-product formation of the carcinogenic side-product hexamethylphosphoric acid triamide (HMPA) [39]. The 2.1. Chemistry tripyrrolidinophosphoric acid triamide (TPPA) derived from PyBOP was more viscous and less polar amide linkage was generated by mixing the free acid with a variety of amines in the presence of Cinnamide derivatives were synthesized using phosphonium salts as condensing reagents than HMPA. Furthermore, its3N) boiling point was higher, making separation of the cinnamide PyBOP, triethylamine (Et in dimethylformamide (DMF) and CH2Cl2the (Scheme 1). The by-product by a procedure adapted from Okombi et al. [37]. The synthesis protocol was modified by derivativetripyrrolidinophosphoric and TPPA more difficult and leading to low yields [39]. Cinnamide derivatives acid triamide (TPPA) derived from PyBOP was more viscous and less (Table 1) replacing benzotriazol-1-yloxytris(dimethylamino)-phosphonium hexafluorophosphate (BOP) by polarbenzotriazol-1-yloxytri(pyrrolidino)-phosphonium than yields HMPA. ranging Furthermore, boiling point was higher, making the hexafluorophosphate (PyBOP) [38]separation to avoid theof the were obtained with fromits19% to 98%. cinnamide derivative and TPPAside-product more difficult and leading to lowacid yields [39]. Cinnamide derivatives formation of the carcinogenic hexamethylphosphoric triamide (HMPA) [39]. The (E)-N-(2-phenylethyl)-3,4-dihydroxycinnamide (compound 1, CAPA) was chosen as the basic (Table 1) were obtained with yields ranging 98%. amide linkage was generated by mixing thefrom free 19% acid to with a variety of amines in the presence of structure (Table 1). Subsequently, compounds 2–15 were synthesized to explore the PyBOP, triethylamine (Et3N) in dimethylformamide (DMF) and CH1, 2ClCAPA) 2 (Scheme 1). The by-product (E)-N-(2-phenylethyl)-3,4-dihydroxycinnamide (compound was chosen as the basic effect on tripyrrolidinophosphoric acid triamide (TPPA) derived from PyBOP was more viscous and lessrings structure (Table 1). Subsequently, 2–15positions were synthesized explore the effect on and also arginase inhibition of hydroxyl groupscompounds at different on thetotwo aromatic polarinhibition than HMPA. Furthermore, its atboiling point was higher, making the separation of also the their arginase of hydroxyl groups different positions on the two aromatic rings and their substitutions by methyl groups. Compounds 16 and 17 were synthesized to help explain cinnamide derivative and TPPA more difficult and leading to low yields [39]. Cinnamide derivatives substitutions by methyl groups. Compounds 16 and 17 were synthesized to help explain contribution of the1) double bond caffeoyl and (Table were obtained withof yields ranging moiety from 19% to 98%.of the single bond of the phenethylamine contribution of the double bond of caffeoyl moiety and of the single bond of the phenethylamine (E)-N-(2-phenylethyl)-3,4-dihydroxycinnamide (compound 1, CAPA) was chosen as the basic group on the moiety. Compound 18 was18synthesized to explore of aa methylcarboxylate methylcarboxylate moiety. Compound was synthesized to explorethe the effect effect of group on structure (Table 1). Subsequently, compounds 2–15 were synthesized to explore the effect on the arginase phenethylamine moiety. Finally, to evaluate the importance of the aromatic ring on the phenethylamine moiety. Finally, to evaluate the importance of the aromatic ring on the phenethylamine inhibition of hydroxyl groups at different positions on the two aromatic rings and also their it was replaced by a(compound sulfhydryl group 19). The of structures of by groups. Compounds 16 and 17 were synthesized to help explain moiety, it phenethylamine wassubstitutions replaced moiety, by methyl a sulfhydryl group 19).(compound The structures these compounds thesecontribution compounds were confirmed NMR,moiety HRMS (ESI), IRsingle spectrum, comparison with of the double bond ofby caffeoyl and of the bond ofand the by phenethylamine were confirmed by NMR, HRMS (ESI), IR spectrum, and by comparison with previous published data. moiety. Compound 18 was synthesized to explore the effect of a methylcarboxylate group on previous published data.

the phenethylamine moiety. Finally, to evaluate the importance of the aromatic ring on the phenethylamine moiety, it was replaced by a sulfhydryl group (compound 19). The structures of these compounds were confirmed by NMR, HRMS (ESI), IR spectrum, and by comparison with previous published data.

Scheme 1. General procedure for the synthesis of cinnamide derivatives. Reagents and conditions:

Scheme 1.PyBOP, General procedure the(0.5 synthesis of cinnamide derivatives. Reagents and conditions: DMF, Et3N, CH2Clfor 2, 0 °C h) then room temperature (overnight). ◦ PyBOP, DMF, Et3 N, CH2 Cl2 , 0 C (0.5 h) then room temperature (overnight). Scheme 1. Generalofprocedure for the synthesis of cinnamide conditions: Table 1. Structures the synthesized compounds and theirderivatives. inhibitory Reagents potenciesand toward b-ARG I. PyBOP, DMF, Et3N, CH2Cl2, 0 °C (0.5 h) then room temperature (overnight).

Table 1. Structures of the synthesized compounds and their inhibitory potencies toward b-ARG I. Table 1. Structures of the synthesized compounds and their inhibitory potencies toward b-ARG I. b-ARG I

Compound

Inhibition a (%) (100 μM)

CompoundCompound

R1 R2 R3 1 (CAPA) OH OH H 2 OH H3 OCH R13 R2 R RH 3 13(CAPA)R1 HOH R2 OH OH 2 OH H OCH3 1 (CAPA) 4 OH OCH3 OHOCH3 OCH H 3 OH H 5 3 OCH HH OH H 2 HH 3 3 OCH3 4 OCH 3 OCH 6 OH OH H 3 H HH 5 H OH H 7 H OCH3 4 OCHOH 6 OCH3 OH H3 3OH OCH 8 OH 7 H 5 H H HH OCH3 H OH 9 8 OCH 3 OCH H3 OH OH3 OCH 6 OH HH 10 9 H 3 H 3 OCH H OCH 7 OCH3 OCHOH H 3 11 10 OHH OH H 8 H OH H HH 12 11 3 OH H OCH OH OH 9 OCH3 OCH3 OCH3 OH OCH3 13 12 H OH H

10 11 12 13 14 15

13

H OH OCH3 H OCH3 H

H OH HH OH H OH H OH H OCH3 OCH3 H H

H

R4 R5 H H RH 4 R5H HHR4 HH HH H H H HH HH H H H OH H H H H OH H OHH H OH H OHH H OH HH OH OH OH HH OH OH OH OH OH OH H OH OH OH OH OH OH OH OH OH OHOHOH

OH OH OH OH OH

IC50 b (µM)

b-ARG I b-ARG I b IC50 (µM) IC b Inhibition a (%) a (%) 50 Inhibition (100 64 μM) ±2 6.9 ± 1.3

(100 µM)

37 ± 4

R5 64 31 ± 2± 4 37 ± 4

35 ± 2 H 31 34 ±4 H 35 ± 2 ± 2 58 H 34 ± 2 ± 2 ±2 H 58 24 ±2 ±2 ±2 H 24 40 30 ±2 40 ± 2 H 34 ± 3 H 30 ± 2 34 62 ± 3± 3 H 62 61 ± 3± 3 H 61 54 ± 3± 1 H 54 ± 1 OH OH OH OH OH

(µM)

n.d c

6.9 ± 1.3n.d c ± 2 n.d n.d 6.9 ± 1.3 n.d n.d ± 4 n.d n.d c 22.1 ± 1.6 n.d ± 4 n.d ± 22.1 2 ± 1.6n.d n.d ± 2 n.d n.d n.d ± 2 n.d n.d 22.1 ± 1.6 n.d ± 2 n.d n.d 114.9 ± 1.3 n.d ±2 n.d 114.9198.7 ± 1.3 ± 1.4 ±198.7 2 n.d ± 1.4 ± 1.7 170.4 ±170.4 3 ± 1.7 n.d

64 37 31 35 34 58 24 40 30 34 62 ± 3 61 ± 3 54 ± 1 53 ± 1 68 ± 3

114.9 ± 1.3 198.7 ± 1.4 170.4 ± 1.7 193.6 ± 1.4 39.3 ± 1.4

previous published data.

Int. J. Mol. Sci. 2016, 17, 1656

4 of 17

Scheme 1. General procedure for the synthesis of cinnamide derivatives. Reagents and conditions: PyBOP, DMF, Et3N, CH2Cl2, 0 °C (0.5 h) then room temperature (overnight).

Table 1. Cont.

Table 1. Structures of the synthesized compounds and their inhibitory potencies toward b-ARG I.

Compound Compound Int. Int. J. J. Mol. Mol. Sci. Sci. 2016, 2016, 17, 17, 1656 1656 Int. J. Mol. Sci. 2016, 17, 1656 Int. J. Mol. Sci. 2016, 17, 1656 Int. J. Mol. Sci. 2016, 17, 1656 Int. J. Mol. Sci. 2016, 17, 1656 Int. J. Mol. Sci. 2016, 14 17, 1656

14 1 (CAPA)R1 14 14 15 14 15 2 14 15 14 15 3 15 15 15

16 16 16 16 16 16 16 16 d 17 17 dd 17 d17 d 17 17 dd 17 17 d

4 5 6 7 8 9 10 11 12 13

b-ARG I b-ARG I b IC50 (µM) Inhibition a (%) Inhibition a (%) (100 μM) R1 OH R2 OCH3 H OCH3 H OH OCH3 H OCH3 H OH OCH3 H

R2 OH OH OH OCH3 H OH OH OH OCH3 H OH OH OH

R3

RH 3 H H OCH3 H H H H OCH3 H H H H

R4 R5 H R4 H H H H H H H H H OH H OH H OH H OH H OH H OH OH OH OH OH OH

R5 64 ± 2 37 ± 4 31 ± 4 35 ± 2 34 ± 2 58 ± 2 24 ± 2 40 ± 2 30 ± 2 34 ± 3 62 ± 3 61 ± 3 54 ± 1

(100 µM) 53 ±1 53 53 ±±± 1131 6.9 ± 1.3 53 68 53 68 ±± 3113 n.d c 53 68 53 ± 31 68 n.d 68 ± 3 68 68 ±± 33 n.d 59 ±± 11± 1 n.d 59 ±59 59 1 22.1 ± 1.6 59 ± 59 ±± 111 59 n.d 59 ± 1

IC50

b

(µM)

193.6 1.4 193.6 ±±± 1.4 1.4 193.6 193.6 ± 1.4 39.3 ± 1.4 193.6 ± 1.4 39.3 ± 1.4 193.6 ± 1.4 39.3 ±±1.4 193.6 1.4 39.3 1.4 39.3 ±±± 1.4 1.4 39.3 39.3 ± 1.4

44 of of 16 16 4 of 16 44 of 16 of 16 4 of 16 4 of 16

35.6 ± 1.3 35.6 ± 1.3 35.6 1.3 35.6 ±±± 1.3 35.6 1.3 35.6 ±± 1.3 1.3 35.6 35.6 ± 1.3

n.d n.d n.d 52 ±± 22±114.9 175.3 ± 1.5 52 ±52 175.3 1.5 2 ± 1.3 175.3 175.3 ± 1.5 52 2 ±±± 1.5 52 ±± 22 198.7 ± 1.4 175.3 1.5 52 175.3 ± 1.5 52 ± 2 175.3 ± 1.5 52 ± 2 170.4 ± 1.7 175.3 ± 1.5

18 18 18 18 18 18 18 18

62 ±± 11 62 ±62 62 62 ±± 111± 1 62 62 62 ±± 11

41.9 ± 1.3 41.9 1.3 41.9 ±±± 1.3 41.9 ± 1.3 41.9 1.3 41.9 ± 1.3 1.3 41.9 ± 41.9 ± 1.3

19 19 19 19 19 19 19 19

67 67 ±±± 111 67 67 ±67 67 ± 11± 1 67 67 ±± 11

37.0 1.3 37.0 ±±± 1.3 1.3 37.0 37.0 ±± 1.3 37.0 ± 1.3 37.0 1.3 37.0 ± 1.3 37.0 ± 1.3

Caffeic acid Caffeic acid acid Caffeic Caffeic acid Caffeic acid Caffeic acidacid Caffeic Caffeic acid

61 61 ±±± 444 61 61 ±61 61 ± 44± 4 61 61 ±± 44

86.7 8.0 86.7 ±±± 8.0 8.0 86.7 86.7 ± 8.0 86.7 ± 8.0 86.7 ± 8.0 86.7 ± 8.0 86.7 ± 8.0

CGA CGA CGA CGA CGA CGA CGA CGA

71 71 ±±± 111 71 71 ±± 11 71 71 71 ±71 ± 11± 1

10.6 1.6 10.6 ±±± 1.6 1.6 10.6 10.6 ±± 1.6 10.6 1.6 10.6 ±± 1.6 10.6 ± 1.6 10.6 1.6

e 99 1.7 0.2 nor-NOHA 99 ±±± 111 1.7 ±±± 0.2 0.2 nor-NOHA ee 99 1.7 nor-NOHA e 99 ± 1 1.7 ± 0.2 nor-NOHA e 99 ± 1 1.7 ± 0.2 nor-NOHA e 99 ± 1 1.7 ± 0.2 nor-NOHA e e 99 99 ± 1± 1 1.7 nor-NOHA nor-NOHA 1.7± 0.2 ± 0.2 a The values were obtained from three separate in-duplicate experiments; b IC50: Half maximal inhibitory a The values were obtained from three separate in-duplicate experiments; b IC50: Half maximal inhibitory a The b were obtained from three separate in-duplicate experiments; 50: Half maximal inhibitory a The values b IC c n.d: were obtained from three separate in-duplicate experiments; : Half maximal inhibitory a The values b IC not determined; concentration was obtained from three separate in-triplicate experiments; c n.d: values were were obtained from three separate in-duplicate experiments; IC50 50: Half maximal inhibitory aconcentration b IC not determined; determined; wasobtained obtained from three separate in-triplicate experiments; values from three separate in-duplicate experiments; :: cHalf maximal inhibitory a The b IC50 n.d: not was obtained from three separate in-triplicate experiments; c The values were obtained from three separate in-duplicate experiments; 50 Half maximal inhibitory a Theconcentration b d e ω n.d: not determined; concentration was obtained from three separate in-triplicate experiments; c Half Compound 17 was prepared from 3,4-dihydroxybenzoic acid; N -hydroxy-norL -arginine were obtained from three separate in-duplicate experiments; IC : maximal inhibitory d values e ω 50 n.d: not determined; concentration was obtained from three separate in-triplicate experiments; c-hydroxy-norCompound 17 was prepared from 3,4-dihydroxybenzoic acid; N L -arginine d e ω n.d: not determined; concentration was obtained from three separate in-triplicate experiments; c n.d: notd determined; Compound 17 was prepared from 3,4-dihydroxybenzoic acid; N -hydroxy-norL-arginine c d e ω concentration was obtained from three separate in-triplicate experiments; 17 was prepared from 3,4-dihydroxybenzoic acid; L -arginine Compound 17 concentration was obtained from three separate in-triplicate experiments; n.d: not determined; d Compound e N ω-hydroxy-nor(nor-NOHA): used as reference inhibitor. Compound 17 was prepared from 3,4-dihydroxybenzoic acid; N -hydroxy-norL -arginine d e Nω-hydroxy-nor-L-arginine (nor-NOHA): used as reference inhibitor. Compound 17 was prepared from 3,4-dihydroxybenzoic acid; e N ω -hydroxy-nordprepared e Nω-hydroxy-norused as reference inhibitor. was (nor-NOHA): from L -arginine (nor-NOHA): used as Compound 173,4-dihydroxybenzoic was prepared fromacid; 3,4-dihydroxybenzoic acid; L-arginine (nor-NOHA): used as reference inhibitor. (nor-NOHA): used as reference inhibitor. (nor-NOHA): reference reference inhibitor. used (nor-NOHA): used as as reference inhibitor. inhibitor. 2.2. Arginase Inhibitory Activity

2.2. Arginase Arginase Inhibitory Inhibitory Activity Activity 2.2. 2.2. Arginase Inhibitory Activity 2.2. Arginase Inhibitory Activity 2.2. Inhibitory Activity Arginase inhibitory activity was first evaluated using an in vitro assay optimizing the protocol 2.2. Arginase Arginase Inhibitory Activity Arginase inhibitory activity was was first first evaluated evaluated using using an an in in vitro vitro assay assay optimizing optimizing the the protocol protocol 2.2. Arginase Inhibitory Activity Arginase inhibitory activity Arginase inhibitory activity was first evaluated using an in vitro assay optimizing the protocol of Corraliza et al. (1994) [40] by miniaturization and by using commercially-available purified Arginase inhibitory activity was first evaluated using an in vitro assay optimizing the protocol of Corraliza et al. (1994) [40] by miniaturization and by using commercially-available purified Arginase inhibitory activity was first evaluated using an in vitro assay optimizing the protocol of Corraliza et al. (1994) [40] by miniaturization and by using commercially-available purified Arginase inhibitory activity was first evaluated using an invitro vitro assay optimizing the protocol of Corraliza et al. (1994) [40] by miniaturization and by using commercially-available purified Arginase inhibitory activity was first evaluated using an in assay optimizing the protocol bovine arginase (b-ARG I) instead of animal tissue [34]. The most active compound was then of Corraliza et al. (1994) [40] by miniaturization and by using commercially-available purified bovine arginase (b-ARG I) instead of animal tissue [34]. The most active compound was then of of Corraliza et al. (1994) [40] by miniaturization and by using commercially-available purified bovine arginase (b-ARG instead of animal tissue [34]. most active compound was then of Corraliza et al. (1994)I) [40] by miniaturization and byNThe using commercially-available purified ω bovine arginase (b-ARG I) instead of animal tissue [34]. The most active compound was then evaluated on a recombinant human arginase (h-ARG I). -hydroxy-norL -arginine (nor-NOHA) Corraliza et al. (1994) [40] by miniaturization and by using commercially-available purified bovine ω bovine arginase (b-ARG I) instead of animal tissue [34]. The most active compound was then evaluated on aa recombinant recombinant human of arginase (h-ARG I). N NωThe -hydroxy-norL-arginine (nor-NOHA) bovine arginase (b-ARG I) animal tissue most compound was evaluated on human arginase I). -hydroxy-norL-arginine (nor-NOHA) bovine arginase (b-ARG I) instead instead of animal(h-ARG tissue [34]. [34]. The most active active compound was then then ω evaluated on a recombinant human arginase (h-ARG I). N L -arginine (nor-NOHA) ω-hydroxy-norand CGA served as reference inhibitors. evaluated on a recombinant human arginase (h-ARG I). N -hydroxy-norL -arginine (nor-NOHA) arginase I) instead of animal tissue [34]. The mostI).active compound -arginine was then(nor-NOHA) evaluated on a ω-hydroxy-norand(b-ARG CGA served served as reference reference inhibitors. evaluated on recombinant human arginase (h-ARG ω-hydroxy-nor-L and CGA as inhibitors. evaluated on aa in recombinant humaninhibition arginase (h-ARG I). N L-arginine (nor-NOHA) and CGA served as reference inhibitors. ω -hydroxy-norAs shown 1, arginase on b-ARG IINwas higher than 50% for eleven target and CGA CGA servedarginase asTable reference inhibitors. As shown in Table 1, arginase inhibition on b-ARG was higher than 50% for eleven target as recombinant human (h-ARG I). N L -arginine (nor-NOHA) and CGA served and served as reference inhibitors. As shown in 1, arginase inhibition on b-ARG II was higher than 50% for eleven target and CGA served asTable reference inhibitors. As shown in Table 1, arginase inhibition on b-ARG was higher than 50% for eleven target compounds at 100 μM. IC 50 values of these eleven compounds were determined to better estimate As shown shown in Table Table 1,50arginase arginase inhibition on b-ARG b-ARG I was waswere higher than 50% 50% to forbetter eleven target compounds at 100 μM. IC values of these eleven compounds determined estimate As in 1, inhibition on I higher than for eleven target reference inhibitors. compounds at 100 μM. IC values of these eleven compounds determined better estimate As shown in Table 1,50 inhibition on b-ARG I waswere higher than 50%to for eleven target compounds at 100 μM. IC 50arginase values of these eleven compounds were determined to better estimate their potencies and discuss structure–activity It is worth noting that all eleven of compounds at 100 100 μM. IC50 50 the values of these these eleven elevenrelationships. compounds were were determined to better estimate their potencies andμM. discuss the structure–activity relationships. Ithigher is worth worth noting that alleleven eleven of compounds at IC values of compounds determined to better estimate As shown in Table 1, arginase inhibition on b-ARG I was than 50% for target their potencies and discuss the structure–activity relationships. It is noting that all eleven of compounds at 100 μM. IC 50 values of these eleven compounds were determined to better estimate their potencies and discuss the structure–activity relationships. It is worth noting that all eleven of these compounds contain a catechol group, on the cinnamoyl (compounds 1, 6, 16 and 19), benzoyl their potencies andcontain discussa the the structure–activity relationships. It is is worth worth noting noting that all eleven of these compounds catechol group, on the cinnamoyl (compounds 1, 6, 16 and 19), benzoyl their potencies and discuss structure–activity relationships. It that all eleven of these compounds contain aa catechol group, on the cinnamoyl (compounds 1, 6, 16 and 19), benzoyl compounds at 100 µM. IC of(compounds these eleven compounds were determined to better estimate their potencies and discuss the structure–activity relationships. It isparts worth noting that all eleven of 50 values these compounds contain catechol group, on the cinnamoyl (compounds 1, 6, 16 and 19), benzoyl (compound 17) or phenethyl parts 12–15), or on both (compounds and 18). In these compounds contain catechol group, on on the the cinnamoyl (compounds 1, 6, 6, 16 16 and and11 19), benzoyl (compound 17) or or contain phenethyl parts (compounds (compounds 12–15), or on on both both parts (compounds (compounds 1119), andbenzoyl 18). In In these compounds aaa catechol group, cinnamoyl (compounds 1, (compound 17) phenethyl parts 12–15), or parts 11 and 18). these compounds contain catechol group, on the cinnamoyl (compounds 1, 6, 16 and 19), benzoyl their potencies and discuss the structure–activity relationships. It is worth noting that all eleven (compound 17) or phenethyl parts (compounds 12–15), or on both parts (compounds 11 and 18). In fact, suppression methylation one or two hydroxyl groups the catechol function to aa of (compound 17) or oror phenethyl partsof (compounds 12–15), or on on both of parts (compounds 11 and andled 18). In fact, suppression suppression or methylation of(compounds one or or two two 12–15), hydroxyl groups of the(compounds catechol function function led toIn (compound 17) phenethyl parts or both parts 11 18). fact, or methylation of one hydroxyl groups of the catechol led to a (compound 17) or phenethyl parts (compounds 12–15), or on both parts (compounds 11 and 18). In these compounds contain a catechol group, thehydroxyl cinnamoyl 1, 6, 16 and 19), benzoyl fact, suppression or methylation of one or two groups of the catechol function led to aa severe decrease in inhibition (compound 1(compounds vs. Our main result is that fact, suppression or arginase methylation of one activity orontwo two hydroxyl groups of2–5). the catechol catechol function led to severe decrease in in arginase inhibition activity (compound vs.of 2–5). Our main main resultled is that that fact, or methylation of or hydroxyl groups the function to severe decrease inhibition activity (compound 111 vs. Our result is fact, suppression suppression or arginase methylation of one one or two 12–15), hydroxyl groups of2–5). the catechol function led to aa 18). severe decrease in arginase inhibition activity (compound vs. 2–5). Our main result is that (compound 17) or phenethyl parts (compounds or on both parts (compounds 11 and structural modifications led to an improvement of the activity for compound 1, caffeic acid severe decrease in arginase arginase inhibition activity (compound (compound vs. for 2–5).compound Our main main1,result result is acid that structural modifications led inhibition to an an improvement improvement of the the activity activity caffeic severe in activity 11 vs. 2–5). Our is that structural modifications led to of for compound 1, caffeic severe decrease decrease in arginase inhibition activity (compound vs.μM, 2–5). Our 3A) main result is acid that structural modifications led to an improvement of the activity for compound 1, caffeic acid phenylamide (CAPA), which aaor lower 50 value ±±1 1.3 Figure compared our In fact,structural suppression or methylation twoIC hydroxyl groups of catechol function led structural modifications led displayed to of an one improvement of the the(6.9 activity forthe compound 1, caffeicto acid phenylamide (CAPA), which displayed lower IC 50 of value (6.9 1.3 μM, Figure 3A) compared to ourto a modifications led to an improvement activity for compound 1, caffeic acid phenylamide (CAPA), which displayed a lower IC 50 value (6.9 ± 1.3 μM, Figure 3A) compared to our structural modifications led to an improvement of the activity for compound 1, caffeic acid phenylamide (CAPA), which displayed a lower IC 50 value (6.9 ± 1.3 μM, Figure 3A) compared to our natural reference compound CGA (IC 50 = 10.6 ± 1.6 µM). Nevertheless, this value still remains higher phenylamide (CAPA), which displayed a lower IC 50 value (6.9 ± 1.3 μM, Figure 3A) compared to our severenatural decrease in arginase inhibition activity (compound 1 vs. 2–5). Our main result is that structural natural reference compound CGA (IC 50 = 10.6 ± 1.6 µM). Nevertheless, this value still remains higher phenylamide (CAPA), which displayed lower IC 50 value (6.9 ±± 1.3 Figure 3A) compared to reference compound CGA (IC 50 =a 10.6 ±± 1.6 µM). Nevertheless, this value still remains higher phenylamide (CAPA), which displayed lower ICIn 50 value (6.9 1.3 μM, μM, Figure 3A) compared to our our natural reference compound CGA (IC 50 =a10.6 1.6 µM). Nevertheless, this value still remains higher than that of the reference compound terms of structure–activity natural reference compound CGA (IC (ICnor-NOHA. 50 = 10.6 ± 1.6 µM). Nevertheless, this value valuerelationships still remains remains(SARs), higher than that that of the the reference reference compound nor-NOHA. InµM). terms of structure–activity structure–activity relationships (SARs), natural reference compound CGA 50 = 10.6 ± 1.6 Nevertheless, this still higher than of compound nor-NOHA. In terms of relationships (SARs), natural reference compound CGA (ICnor-NOHA. 50 = 10.6 ± 1.6 µM). Nevertheless, this value still remains higher than that of the reference compound In terms of structure–activity relationships (SARs), functionalization of the phenethyl of by one (compound 6) or two (compound 11) hydroxyl than that of of the the reference reference compoundpart nor-NOHA. In terms terms of structure–activity structure–activity relationships (SARs), functionalization of the the phenethyl phenethyl part of 111 by by one one (compound 6) or or two two (compound (compound 11) hydroxyl hydroxyl than that compound nor-NOHA. In of relationships (SARs), functionalization of part of (compound 6) 11) than that of the reference compound nor-NOHA. In terms of structure–activity relationships (SARs), functionalization of the phenethyl part of 1 by one (compound 6) or two (compound 11) hydroxyl groups decreased However, the cinnamoyl part remained unsubstituted, functionalization ofthe theactivity. phenethyl part of ofif by one one (compound 6) or or two (compound (compound 11)inhibition hydroxyl groups decreased decreasedof the activity. However, if11 the the cinnamoyl part remained remained unsubstituted, inhibition functionalization the phenethyl part by (compound 6) 11) hydroxyl groups the activity. However, cinnamoyl part functionalization of the phenethyl part ofif 1 the by one (compound 6) or two two unsubstituted, (compound 11)inhibition hydroxyl groups decreased the activity. However, if cinnamoyl part remained unsubstituted, inhibition groups decreased the activity. However, if the cinnamoyl part remained unsubstituted, inhibition groups groups decreased decreased the the activity. activity. However, However, if if the the cinnamoyl cinnamoyl part part remained remained unsubstituted, unsubstituted, inhibition inhibition

Int. J. Mol. Sci. 2016, 17, 1656

5 of 17

modifications led to an improvement of the activity for compound 1, caffeic acid phenylamide (CAPA), which displayed a lower IC50 value (6.9 ± 1.3 µM, Figure 3A) compared to our natural reference compound CGA (IC50 = 10.6 ± 1.6 µM). Nevertheless, this value still remains higher than that of the reference compound nor-NOHA. In terms of structure–activity relationships (SARs), functionalization Int. J. Mol. Sci. 2016, 17, 1656 5 of 16 of the phenethyl part of 1 by one (compound 6) or two (compound 11) hydroxyl groups decreased the activity. However, if thebycinnamoyl partof remained unsubstituted, was partially restored by was partially restored the presence a catechol group on theinhibition phenethyl part (compound 15 vs. the presence of a catechol group on the phenethyl part (compound 15 vs. 11). This result confirmed 11). This result confirmed the important role played by the catechol function for the arginase the important roleof played by the function for the inhibitory activity of this seriesa inhibitory activity this series of catechol compounds. Reducing thearginase length of the molecule by suppressing of compounds. Reducing the length of the molecule by suppressing a bond in either bond in either the caffeoyl side or the phenethyl side also decreased activity (compound the 6 vs.caffeoyl 16, 17). side or the phenethyl side also decreased activity (compound 6 vs. 16, 17). However, retaining However, retaining most or all of the cinnamoyl part seemed to be of greater importance than that of most or all of the cinnamoyl part seemed to be of greater importance than that of the phenethyl the phenethyl part. Concerning the single bond of the phenylethylamine moiety, a preliminary part. Concerning the single of the the phenylethylamine moiety, preliminary investigation has investigation has been madebond involving substitution of the ethyla linker by a methylcarboxylate been made involving the substitution of the ethyl linker by a methylcarboxylate group (18) and by group (18) and by replacing the aromatic ring by a sulfhydryl group (19). The methylcarboxylate replacing the aromatic ring by a sulfhydrylmoiety group improved (19). The methylcarboxylate group at position of group at position 1 of the phenethylamine the inhibitory activity (compound 181vs. the phenethylamine moiety improved the inhibitory activity (compound 18 vs. 11), and replacing the 11), and replacing the aromatic ring of the phenethylamine moiety by the sulfhydryl group had little aromatic the phenethylamine moiety by the group hadthat littleno effect on theinteraction inhibitory effect onring the ofinhibitory activity (compound 18 sulfhydryl vs. 19), suggesting specific activity (compound 18 vs. 19), suggesting that no specific interaction occurred between this part of the occurred between this part of the molecule and the mouth of the active site (Figure 2). Therefore, molecule and the mouth of the active site (Figure 2). Therefore, pharmacomodulations in this position pharmacomodulations in this position could be expected to increase compounds affinity toward the could expected to increase compounds affinity toward the active site. active be site.

Figure 2. Structure–activity relationships for cinnamide derivatives on b-ARG I. Figure 2. Structure–activity relationships for cinnamide derivatives on b-ARG I.

Considering its effect on b-ARG I, CAPA (1) was evaluated on a recombinant h-ARG I. The Considering effect on was b-ARG I, CAPA (1) was on evaluated on and a recombinant value ofI. reference inhibitorits nor-NOHA previously evaluated this model gave an IC50h-ARG The reference inhibitor nor-NOHA was previously evaluated on this model and gave an IC value of that 5.7± 0.6 µM. The IC50 value of CAPA was found to be 60.3 ± 7.8 µM. This value is higher50than 5.7 ± 0.6 µM.on The IC50 value CAPA found to results be 60.3illustrated ± 7.8 µM. the Thisfact value higher than that determined b-ARG I (IC50of = 6.9 ± 1.3was µM). These thatisthe evaluation on determined on b-ARG I (IC = 6.9 ± 1.3 µM). These results illustrated the fact that the evaluation 50 b-ARG I as an easy-to-handle and cheap source of mammalian arginase could be used inona b-ARG I as anstudy easy-to-handle cheapnew source of mammalian arginase could be usedan in evaluation a preliminary preliminary in order toand search potential active compounds. However, of study in order to search new potential active compounds. However, an evaluation of the most active the most active compounds on h-ARG I is required before further in vivo studies. compounds on h-ARG I is required before further in vivo studies. 2.3. Enzyme Kinetic Studies for CAPA (1) 2.3. Enzyme Kinetic Studies for CAPA (1) The mechanism of inhibition and the inhibition constant (Ki) of compound 1 (CAPA) was The mechanism of inhibition and the inhibition constant (Ki ) of compound 1 (CAPA) was assessed assessed by enzyme-kinetic studies. The kinetic data were used to generate the Lineweaver–Burk, by enzyme-kinetic studies. The kinetic data were used to generate the Lineweaver–Burk, Dixon and Dixon and Cornish–Bowden plots (Figure 3B–D), showing that CAPA is a competitive inhibitor of Cornish–Bowden plots (Figure 3B–D), showing that CAPA is a competitive inhibitor of arginase [41]. arginase [41]. The Ki values for CAPA, CGA and nor-NOHA could be calculated on the basis of the The Ki values for CAPA, CGA and nor-NOHA could be calculated on the basis of the Cheng–Prusoff equation [Ki = IC50 /(1 + [S]/Km )] [42], with [S] = 14.3 mM of L-arginine and the Cheng–Prusoff equation [Ki = IC50/(1 + [S]/Km)] [42], with [S] = 14.3 mM of L-arginine and the Michaelis–Menten constant of b-ARG I Km = 55.5 ± 10.5 µM [34] (Table 2). Michaelis–Menten constant of b-ARG I Km = 55.5 ± 10.5 µM [34] (Table 2). The in vitro affinity of CAPA was better than that of CGA (Ki 5.5 ± 1.0 vs. 8.4 ± 1.2 µM, respectively). In the last few years, several chemical groups that target the bimanganese cluster have been identified. Here, the in vitro affinity of CAPA was similar to that described for amino imidazole derivative [28], and better than thiosemicarbazide [43], sulfamide [27], nitro [29] and aldehyde [30] derivatives, which displayed Ki greater than 50 µM. Cinnamides could be added to this list and may

Int. J. Mol. Sci. 2016, 17, 1656

6 of 17

The in vitro affinity of CAPA was better than that of CGA (Ki 5.5 ± 1.0 vs. 8.4 ± 1.2 µM, respectively). In the last few years, several chemical groups that target the bimanganese cluster have been identified. Here, the in vitro affinity of CAPA was similar to that described for amino imidazole derivative [28], and better than thiosemicarbazide [43], sulfamide [27], nitro [29] and aldehyde [30] derivatives, which displayed Ki greater than 50 µM. Cinnamides could be added to this list and may help design future inhibitors. Int. J. to Mol. Sci. 2016, 17, 1656 6 of 16

Figure3.3.Kinetics Kineticsofofarginase arginase inhibition 1 (CAPA). The was IC50 determined was determined a sigmoidal Figure inhibition by by 1 (CAPA). The IC from afrom sigmoidal curve 50 curve (A). The competitive inhibition mechanism was determined by analysis of the: Lineweaver– (A). The competitive inhibition mechanism was determined by analysis of the: Lineweaver–Burk (B); Burk (B); (C); and Cornish–Bowden (D) plots. Eachrepresents data pointthe represents mean of three Dixon (C); Dixon and Cornish–Bowden (D) plots. Each data point mean of the three in-triplicate in-triplicate independent experiments. independent experiments. Table2.2.Comparison Comparisonof ofKKi values values for for arginase arginase inhibition inhibition by by CAPA, CAPA,CGA CGAand andnor-NOHA. nor-NOHA. Table i

Inhibitor Inhibitor CAPA (1) CGA CAPA (1) nor-NOHACGA

nor-NOHA

Ki (µM) 5.5 ± 1.0 5.5 ± 1.0± 1.2 8.4 8.4 ± 1.2 1.3 ± 0.1

Type of Inhibition competitive competitive competitive competitive competitive

Ki (µM)

Type of Inhibition

1.3 ± 0.1

competitive

2.4. Molecular Docking Studies 2.4. Molecular Docking Studies In order to gain further insight into the inhibitory mechanism, the binding modes of CAPA (1) In order to gain further insight into the inhibitoryon mechanism, the binding modes CAPA (1) and CGA were determined by docking simulations h-ARG I (pdb id: 3kv2). The of structure of and CGAI is were docking h-ARG I (pdb id:homology 3kv2). Thewith structure of b-ARG b-ARG notdetermined available inby the Proteinsimulations Data Bankon but present 100% h-ARG I in the Iactive is notsite. available in the Protein Data Bank present 100% homology with h-ARG I instand-alone the active In addition, the alignment of thebut b-ARG I and h-ARG I sequences using the site. In addition, the alignment of the b-ARG I and h-ARG I sequences using the stand-alone Java Web Start application, accessible from the PDB server (http://www.rcsb.org), revealed a Java high Web application, accessible the PDB server high level of levelStart of similarity between the from two sequences (91%(http://www.rcsb.org), and 95%, respectively).revealed A few aresidues were similarity between the two sequences (91% and 95%, respectively). A few residues were different, but different, but not involved in the active site of the two arginases, whose residues are strictly conserved. The most active compound 1 (CAPA), CGA and nor-NOHA were successfully docked into the active pocket of h-ARG I by using a FlexX docking program implemented in LeadIt 2.0.2 software [44]. The results obtained with CGA confirmed that the quinoyl moiety interact with residues of the mouth of the pocket formed by the catalytic site, helping the caffeoyl moiety of chlorogenic acid to

Int. J. Mol. Sci. 2016, 17, 1656

7 of 17

not involved in the active site of the two arginases, whose residues are strictly conserved. The most active compound 1 (CAPA), CGA and nor-NOHA were successfully docked into the active pocket of h-ARG I by using a FlexX docking program implemented in LeadIt 2.0.2 software [44]. The results obtained with CGA confirmed that the quinoyl moiety interact with residues of the mouth of the pocket formed by the catalytic site, helping the caffeoyl moiety of chlorogenic acid to approach the bottom of the active site and interact with Asp124, Asp232, Asp234 by H-interactions and His141 via cation–π interaction (Figure 4). Interestingly, the catechol group could chelate the Mn2+ ions in the of arginase. Int. J. Mol. Sci.active 2016, 17,site 1656 7 of 16

Figure ofof CGA inside thethe active sitesite of h-ARG I. For the the 3D Figure 4. 4. 3D 3D(left); (left);and and2D 2D(right) (right)binding bindingmodes modes CGA inside active of h-ARG I. For binding mode, CGA is represented as a “ball and stick” form: C (grey), O (red), and H (white); 3D binding mode, CGA is represented as a “ball and stick” form: C (grey), O (red), and H (white); manganese manganese ions ions are are represented represented as as cyan cyan spheres; spheres; and and the the crucial crucial residues residues of of the the binding binding site site are are represented by lines. H-bonds are represented as pink dotted lines, while the metal coordination represented by lines. H-bonds are represented as pink dotted lines, while the metal coordination bonds bonds are are represented represented by by cyan cyan dotted dotted lines. lines. Docking Docking simulations simulations were were performed performed by by FlexX FlexX program program implemented in LeadIt 2.0.2 software, and the pictures of docking solution were created implemented in LeadIt 2.0.2 software, and the pictures of docking solution were created by by MOE MOE 2008.10 2008.10 software. software.

Since CAPA was identified as a potential lead compound of the series, interactions of this Since CAPA was identified as a potential lead compound of the series, interactions of this compound with the residues of the active site of the enzyme were examined more closely. We noted compound with the residues of the active site of the enzyme were examined more closely. We noted with interest that the caffeoyl moiety of CAPA is able to interact with the2+Mn2+ cofactor and can with interest that the caffeoyl moiety of CAPA is able to interact with the Mn cofactor and can make make hydrogen interactions with Asp234 and Thr246 as well as π–π interactions with His141 and hydrogen interactions with Asp234 and Thr246 as well as π–π interactions with His141 and His126 of His126 of h-ARG I active site. The phenethyl moiety could have hydrophobic interactions residues at h-ARG I active site. The phenethyl moiety could have hydrophobic interactions residues at the mouth the mouth of active pocket (Figure 5). of active pocket (Figure 5). In addition, it is worth noting that compound 15 took a position that was opposite to that of CAPA, the cinnamoyl moiety staying at the mouth whereas the phenethylamine with a catechol group approached the active pocket of b-ARG I (data not shown). These opposite binding modes confirmed the hypothesis of the crucial role played by the catechol group in arginase inhibition, and clearly explained why the activity was recovered by compound 15 despite the fact the cinnamoyl moiety was unfunctionalized.

Figure 5. 3D (left); and 2D (right) binding modes of CAPA (1) inside the active site of h-ARG I. For

Since CAPA was identified as a potential lead compound of the series, interactions of this compound with the residues of the active site of the enzyme were examined more closely. We noted with interest that the caffeoyl moiety of CAPA is able to interact with the Mn2+ cofactor and can make hydrogen interactions with Asp234 and Thr246 as well as π–π interactions with His141 and of h-ARG I active site. The phenethyl moiety could have hydrophobic interactions residues Int.His126 J. Mol. Sci. 2016, 17, 1656 8 of 17 at the mouth of active pocket (Figure 5).

Figure 3D(left); (left);and and 2D 2D (right) (right) binding binding modes h-ARG I. For Figure 5. 5.3D modes of of CAPA CAPA (1) (1)inside insidethe theactive activesite siteofof h-ARG I. the 3D binding mode, CAPA is represented as a “ball and stick” form: C (grey), O (red), and For the 3D binding mode, CAPA is represented as a “ball and stick” form: C (grey), O (red), and H H (white); manganese ions represented cyan spheres; and crucial residues binding site (white); manganese ions areare represented as as cyan spheres; and thethe crucial residues of of thethe binding site are represented by lines. H-bonds are represented as pink dotted lines, while the metal coordination are represented by lines. H-bonds are represented as pink dotted lines, while the metal coordination bonds represented cyan dotted lines. Docking simulations were performed FlexX program bonds areare represented byby cyan dotted lines. Docking simulations were performed byby FlexX program implemented in LeadIt 2.0.2 software, and the pictures of docking solution were created MOE implemented in LeadIt 2.0.2 software, and the pictures of docking solution were created byby MOE 2008.10 software. 2008.10 software.

3. Materials and Methods 3.1. Chemistry All reagents were purchased from Sigma-Aldrich and used without further purification, except for the purified liver bovine arginase I (b-ARG I) which was from MP Biomedicals (One unit (1 U) of b-ARG I is defined by this manufacturer as the amount of enzyme that converted 1 µmole of L -arginine to urea and L -ornithine per minute at pH 9.5 and 37 ◦ C), and for the recombinant human arginase I (h-ARG I) (BXC572/n P0408M10) which was from Interchim (Number of units is not defined by the manufacturer and thus a preliminary study (data not shown) allowed us to choose the concentration of enzyme leading to an absorbance of about 1). Solvents (methanol, ethyl acetate, cyclohexane and dichloromethane) were supplied by Carlo Erba Reagents and VWR Chemicals companies. Reactions were monitored by thin-layer chromatography (TLC) on pre-coated silica gel aluminum plates (Macherey-Nagel) and visualized under UV light (254 and 365 nm). Flash column chromatography was carried out using EasyVarioFlash® DL cartridges (Merck, Fontenay-sous-Bois, France) for dry loading, eluting with CH2 Cl2 and MeOH. 1 H-NMR at 300 MHz and 13 C-NMR at 75 MHz (only for compounds lacking analytical data from literature) were acquired using a Bruker AC300 spectrometer (Bruker BioSpin, Wissembourg, France). Chemical shifts (δ) were reported in parts per million (ppm) relative to the residual solvent signals. Coupling constants (J) were reported in hertz (Hz). Data were presented as follows: chemical shift (δ, ppm), multiplicity (s, singlet; bs, broad singlet; d, doublet; dd, doublet of doublet; t, triplet; q, quadruplet; m, multiplet), coupling constant (J, Hz), and integration. High-resolution electrospray ionisation mass spectra (HRMS-ESI) were analyzed by using the SCA Illkirch QToF instrument. 3.2. General Procedure for the Synthesis of Amide Derivatives 1−19 A solution of substituted cinnamic acid or 3,4-dihydroxybenzoic acid (1.66 mmol) and trimethylamine (Et3 N) (0.35 mL, 2.49 mmol, 1.5 equivalent (eq)) in dimethylformamide (DMF) (3.5 mL)

Int. J. Mol. Sci. 2016, 17, 1656

9 of 17

was cooled in an ice bath. A corresponding amine (1.66 mmol, 1 eq) was added and followed by addition of a solution of benzotriazolyloxy-tris(pyrrolidino)-phosphonium hexafluorophosphate (PyBOP) (864 mg, 1.66 mmol, 1 eq) in dichloromethane (CH2 Cl2 ) (3.5 mL). The mixture was stirred for 30 min at 0 ◦ C and left overnight at room temperature before CH2 Cl2 was evaporated under vacuum. Then, 30 mL of water was added to the remaining solution and the resulting mixture was extracted with ethyl acetate (EtOAc) (3 × 75 mL). The organic phase was successively washed with 100 mL of 1 M HCl solution, 100 mL of water, 100 mL of 1 M NaHCO3 solution and 100 mL of brine, then dried over Na2 SO4 , filtered and evaporated under vacuum. The crude product was purified by silica gel flash chromatography (eluent: CH2 Cl2 /MeOH 98:2 to 80:20) to afford the desired compound [37]. (E)-N-(2-Phenylethyl)-3,4-dihydroxycinnamide (1). General procedure using 3,4-dihydroxycinnamic acid (300 mg, 1.66 mmol) and 2-phenylethylamine (209 µL, 1.66 mmol) afforded compound 1 (213 mg, 45%) as a white solid. Rf = 0.2 (CH2 Cl2 /MeOH 95:5); IR (ATR) γ cm−1 : 3259, 3087, 3061, 3027, 2969, 2935, 2870, 1702, 1652, 1590, 1513; 1 H-NMR (Acetone-d6 , 300 MHz): δ 8.23 (s, 2H), 7.41 (d, J = 15.6 Hz, 1H), 7.31–7.17 (m, 6H), 7.06 (s, 1H), 6.93 (d, J = 8.0 Hz, 1H), 6.83 (d, J = 8.0 Hz, 1H), 6.43 (d, J = 15.6 Hz, 1H), 3.57–3.50 (m, 2H), 2.90–2.82 (m, 2H); HRMS (ESI) m/z calcd for C17 H18 NO3 [M + H]+ : 284.1208, found: 284.1288. The analytical data were in line with previously described data [45]. (E)-N-(2-Phenylethyl)-3-methoxy-4-hydroxycinnamide (2). General procedure using 3-methoxy-4-hydroxy cinnamic acid (322 mg, 1.66 mmol) and 2-phenylethylamine (209 µL, 1.66 mmol) afforded compound 2 (388 mg, 79%) as a white solid. Rf = 0.33 (Cyclohexane/EtOAc 4:6); IR (ATR) γ cm−1 : 3270, 3063, 3027, 2935, 1652, 1585, 1510; 1 H-NMR (MeOD-d4 , 300 MHz): δ 7.44 (d, J = 15.7 Hz, 1H), 7.32–7.17 (m, 5H), 7.12 (s, 1H), 7.03 (d, J = 8.4 Hz, 1H), 6.79 (d, J = 8.4 Hz, 1H), 6.40 (d, J = 15.7 Hz, 1H), 3.88 (s, 3H), 3.52 (t, J = 7.2 Hz, 2H), 2.86 (t, J = 7.2 Hz, 2H); HRMS (ESI) m/z calcd for C18 H20 NO3 [M + H]+ : 298.1365, found: 298.1442. The analytical data were in line with previously described data [46]. (E)-N-(2-Phenylethyl)-4-hydroxycinnamide (3). General procedure using 4-hydroxycinnamic acid (272 mg, 1.66 mmol) and 2-phenylethylamine (209 µL, 1.66 mmol) afforded compound 3 (288 mg, 65%) as a colorless syrup. Rf = 0.56 (CH2 Cl2 /MeOH 95:5); IR (ATR) γ cm−1 : 3400, 3263, 3085, 3064, 3026, 2934, 2813, 2750, 2687, 2608, 1651, 1600, 1579, 1511; 1 H-NMR (MeOD-d4 , 300 MHz): δ 7.47–7.38 (m, 3H), 7.31–7.17 (m, 5H), 6.78 (d, J = 8.4 Hz, 2H), 6.38 (d, J = 15.7 Hz, 1H), 3.51 (t, J = 7.2 Hz, 2H), 2.85 (t, J = 7.2 Hz, 2H); HRMS (ESI) m/z calcd for C17 H18 NO2 [M + H]+ : 268.1259, found: 268.1336. The analytical data were in line with previously described data [47]. (E)-N-(2-Phenylethyl)-3,4,5-trimethoxycinnamide (4). General procedure using 3,4,5-trimethoxycinnamic acid (395 mg, 1.66 mmol) and 2-phenylethylamine (209 µL, 1.66 mmol) afforded compound 4 (511 mg, 90%) as a white solid. Rf = 0.56 (CH2 Cl2 /MeOH 95:5); IR (ATR) γ cm−1 : 3305, 3087, 3062, 3030, 3005, 2967, 2933, 2862, 2835, 1653, 1615, 1581, 1536, 1506; 1 H-NMR (CDCl3 , 300 MHz): δ 7.53 (d, J = 15.5 Hz, 1H), 7.36–7.22 (m, 5H), 6.71 (s, 2H), 6.22 (d, J = 15.5 Hz, 1H), 5.55 (bs, 1H), 3.88 (s, 6H), 3.87 (s, 3H), 3.71–3.65 (m, 2H), 2.89 (t, J = 6.8 Hz, 2H); 13 C-NMR (CDCl3 , 75 MHz): δ 165.9, 153.5, 141.1, 139.6, 138.9, 130.5, 128.9, 128.8, 126.7, 120.0, 104.9, 61.1, 56.2, 40.9, 35.7; HRMS (ESI) m/z calcd for C20 H24 NO4 [M + H]+ : 342.1627, found: 342.1709. (E)-N-(2-Phenylethyl)cinnamide (5). General procedure using cinnamic acid (246 mg, 1.66 mmol) and 2-phenylethylamine (209 µL, 1.66 mmol) afforded compound 5 (330 mg, 79%) as a white solid. Rf = 0.7 (CH2 Cl2 /MeOH 95:5); IR (ATR) γ cm−1 : 3265, 3062, 3030, 2971, 2944, 2862, 1959, 1895, 1819, 1660, 1651, 1603, 1579, 1544; 1 H-NMR (CDCl3 , 300 MHz): δ 7.62 (d, J = 15.5 Hz, 1H), 7.50–7.47 (m, 2H), 7.40–7.31 (m, 5H), 7.25–7.22 (m, 3H), 6.32 (d, J = 15.5 Hz, 1H), 5.58 (bs, 1H), 3.71–3.65 (m, 2H), 2.90 (t, J = 6.6, 2H); HRMS (ESI) m/z calcd for C17 H18 NO [M + H]+ : 252.1310, found : 252.1386. The analytical data were in line with previously described data [48]. (E)-N-(2-(4-Hydroxyphenyl)ethyl)-3,4-dihydroxycinnamide (6). General procedure using 3,4-dihydroxy cinnamic acid (300 mg, 1.66 mmol) and 2-(4-hydroxyphenyl)ethylamine (228 mg, 1.66 mmol) afforded compound 6 (358 mg, 72%) as a slight yellow solid. Rf = 0.64 (Cyclohexane/EtOAc 1:9); IR (ATR)

Int. J. Mol. Sci. 2016, 17, 1656

10 of 17

γ cm−1 : 3349, 3167, 3030, 2960, 2930, 2877, 1727, 1645, 1602, 1578, 1535, 1514; 1 H-NMR (DMSO-d6 , 300 MHz): δ 9.23 (bs, 3H), 8.02 (bs, 1H), 7.21 (d, J = 15.6 Hz, 1H), 7.0 (d, J = 8.2 Hz, 2H), 6.93 (s, 1H), 6.82 (d, J = 8.0 Hz, 1H), 6.73 (d, J = 8.0 Hz, 1H), 6.67 (d, J = 8.2 Hz, 2H), 6.31 (d, J = 15.6 Hz, 1H), 3.34–3.28 (m, 2H), 2.63 (t, J = 7.3 Hz, 2H); HRMS (ESI) m/z calcd for C17 H18 NO4 [M + H]+ : 300.1236, found: 300.1235. The analytical data were in line with previously described data [49]. (E)-N-(4-Hydroxyphenethyl)-3-methoxy-4-hydroxycinnamide (7). General procedure using 3-methoxy-4hydroxycinnamic acid (322 mg, 1.66 mmol) and 4-hydroxyphenethylamine (228 mg, 1.66 mmol) afforded compound 7 (298 mg, 57%) as a white solid. Rf = 0.22 (Cyclohexane/EtOAc 5:5); IR (ATR) γ cm−1 : 3287, 3015, 2936, 1651, 1586, 1510; 1 H-NMR (MeOD-d4 , 300 MHz): δ 7.43 (d, J = 16.0 Hz, 1H), 7.12 (s, 1H), 7.07–7.01 (m, 3H), 6.79 (d, J = 8.1 Hz, 1H), 6.71 (d, J = 8.0 Hz, 2H), 6.40 (d, J = 16.0 Hz, 1H), 3.88 (s, 3H), 3.47 (t, J = 6.9 Hz, 2H), 2.76 (t, J = 6.9 Hz, 2H); HRMS (ESI) m/z calcd for C18 H20 NO4 [M + H]+ : 314.1314, found: 314.1396. The analytical data were in line with previously described data [50]. (E)-N-(4-Hydroxyphenethyl)-4-hydroxycinnamide (8). General procedure using 4-hydroxycinnamic acid (272 mg, 1.66 mmol) and 4-hydroxyphenethylamine (228 mg, 1.66 mmol) afforded compound 8 (233 mg, 50%) as a white solid. Rf = 0.30 (Cyclohexane/EtOAc 5:5); IR (ATR) γ cm−1 : 3431, 3171, 3024, 2942, 1895, 1660, 1622, 1602, 1590, 1530, 1510; 1 H-NMR (MeOD-d4 , 300 MHz): δ 7.46–7.38 (m, 3H), 7.05 (d, J = 8.1 Hz, 2H), 6.78 (d, J = 8.3 Hz, 2H), 6.71 (d, J = 8.1 Hz, 2H), 6.38 (d, J = 15.6 Hz, 1H), 3.45 (t, J = 7.3 Hz, 2H), 2.75 (t, J = 7.3 Hz, 2H); HRMS (ESI) m/z calcd for C17 H18 NO3 [M + H]+ : 284.1208, found: 284.1285. The analytical data were in line with previously described data [51]. (E)-N-(4-Hydroxyphenethyl)-3,4,5-trimethoxycinnamide (9). General procedure using 3,4,5-trimethoxy cinnamic acid (395 mg, 1.66 mmol) and 4-hydroxyphenethylamine (228 mg, 1.66 mmol) afforded compound 9 (586 mg, 98%) as a white solid. Rf = 0.26 (Cyclohexane/EtOAc 5:5); IR (ATR) γ cm−1 : 3323, 3012, 2963, 2940, 2909, 2888, 2847, 2824, 2605, 1994, 1652, 1605, 1580, 1544, 1516, 1504; 1 H-NMR (MeOD-d4 , 300 MHz): δ 7.44 (d, J = 15.7 Hz, 1H), 7.06 (d, J = 8.1 Hz, 2H), 6.86 (s, 2H), 6.72 (d, J = 8.1 Hz, 2H), 6.51 (d, J = 15.7 Hz, 1H), 3.87 (s, 6H), 3.78 (s, 3H), 3.48 (t, J = 7.2 Hz, 2H), 2.76 (t, J = 7.2 Hz, 2H); 13 C-NMR (MeOD-d4 + 10% CDCl3 , 75 MHz): δ 168.5, 156.8, 154.5, 141.6, 140.4, 132.1, 131.1, 130.7, 121.2, 116.2, 106.1, 61.3, 56.7, 42.5, 35.7; HRMS (ESI) m/z calcd for C20 H24 NO5 [M + H]+ : 358.1576, found: 358.1657. (E)-N-(4-Hydroxyphenethyl)-cinnamide (10). General procedure using cinnamic acid (246 mg, 1.66 mmol) and 4-hydroxyphenethylamine (228 mg, 1.66 mmol) afforded compound 10 (359 mg, 81%) as a white solid. Rf = 0.44 (Cyclohexane/EtOAc 5:5); IR (ATR) γ cm−1 : 3429, 3268, 3059, 3022, 2941, 2922, 2858, 1889, 1665, 1624, 1611, 1592, 1532, 1512; 1 H-NMR (MeOD-d4 , 300 MHz): δ 7.56–7.49 (m, 3H), 7.42–7.33 (m, 3H), 7.06 (d, J = 8.2 Hz, 2H), 6.72 (d, J = 8.2 Hz, 2H), 6.57 (d, J = 16.0 Hz, 1H), 3.47 (t, J = 6.9 Hz, 2H), 2.76 (t, J = 6.9 Hz, 2H); HRMS (ESI) m/z calcd for C17 H18 NO2 [M + H]+ : 268.1259, found: 268.1337. The analytical data were in line with previously described data [52]. (E)-N-(3,4-Dihydroxyphenethyl)-3,4-dihydroxycinnamide (11). General procedure using 3,4-dihydroxy cinnamic acid (300 mg, 1.66 mmol) and 3,4-dihydroxyphenethylamine hydrochloride (315 mg, 1.66 mmol) afforded compound 11 (229 mg, 44%) as a slight yellow solid. Rf = 0.45 (CH2 Cl2 /MeOH 85:15); IR (ATR) γ cm−1 : 3214, 2938, 2723, 1695, 1650, 1586, 1514; 1 H-NMR (Acetone-d6 , 300 MHz): δ 8.15 (bs, 4H), 7.43 (s, 1H), 7.42 (d, J = 15.6 Hz, 1H), 7.06 (s, 1H), 6.89 (d, J = 8.1 Hz, 1H), 6.8 (d, J = 8.2 Hz, 1H), 6.72 (s, 1H), 6.69 (d, J = 8.2 Hz, 1H), 6.52 (d, J = 8.1 Hz, 1H), 6.44 (d, J = 15.6 Hz, 1H), 3.47 (m, 2H), 2.67 (t, J = 6.9 Hz, 2H); HRMS (ESI) m/z calcd for C17 H18 NO5 [M + H]+ : 316.1185; found: 316.1181. The analytical data were in line with previously described data [53]. (E)-N-(3,4-Dihydroxyphenethyl)-3-methoxy-4-hydroxycinnamide (12). General procedure using 3-methoxy4-hydroxycinnamic acid (322 mg, 1.66 mmol) and 3,4-dihydroxyphenethylamine hydrochloride (315 mg, 1.66 mmol) afforded compound 12 (164 mg, 30%) as a white solid. Rf = 0.14 (CH2 Cl2 /MeOH 95:5); IR (ATR) γ cm−1 : 3321, 2969, 2937, 2841, 2720, 1651, 1585, 1510; 1 H-NMR (MeOD-d4 , 300 MHz):

Int. J. Mol. Sci. 2016, 17, 1656

11 of 17

δ 7.43 (d, J = 16.0 Hz, 1H), 7.12 (s, 1H), 7.02 (d, J = 8.0 Hz, 1H), 6.79 (d, J = 8.3 Hz, 1H), 6.71–6.67 (m, 2H), 6.55 (d, J = 8.0 Hz, 1H), 6.40 (d, J = 16.0 Hz, 1H), 3.88 (s, 3H), 3.46 (t, J = 7.0 Hz, 2H), 2.70 (t, J = 7.0 Hz, 2H). 13 C-NMR (MeOD-d4 , 75 MHz): δ 169.3, 149.9, 149.4, 146.4, 144.9, 142.2, 132.2, 128.4, 123.4, 121.2, 118.9, 117.0, 116.6, 116.5, 111.6, 56.5, 42.7, 36.2; HRMS (ESI) m/z calcd for C18 H20 NO5 [M + H]+ : 330.1263; found: 330.1342. (E)-N-(3,4-Dihydroxyphenethyl)-4-hydroxycinnamide (13). General procedure using 4-hydroxycinnamic acid (272 mg, 1.66 mmol) and 3,4-dihydroxyphenethylamine hydrochloride (315 mg, 1.66 mmol) afforded compound 13 (204 mg, 41%) as a white solid. Rf = 0.15 (CH2 Cl2 /MeOH 95:5); IR (ATR) γ cm−1 : 3270, 3025, 2938, 2817, 2701, 1696, 1648, 1577, 1511; 1 H-NMR (MeOD-d4 , 300 MHz): δ 7.44 (d, J = 16.0 Hz, 1H), 7.40 (d, J = 8.4 Hz, 2H), 6.79 (d, J = 8.4 Hz, 2H), 6.70–6.67 (m, 2H), 6.55 (d, J = 8.0 Hz, 1H), 6.38 (d, J = 16.0 Hz, 1H), 3.45 (t, J = 7.0 Hz, 2H), 2.70 (t, J = 7.0 Hz, 2H); HRMS (ESI) m/z calcd for C17 H18 NO4 [M + H]+ : 300.1158; found: 300.1233. The analytical data were in line with previously described data [54]. (E)-N-(3,4-Dihydroxyphenethyl)-3,4,5-trimethoxycinnamide (14). General procedure using 3,4,5-trimethoxy cinnamic acid (395 mg, 1.66 mmol) and 3,4-dihydroxyphenethylamine hydrochloride (315 mg, 1.66 mmol) afforded compound 14 (436 mg, 70%) as a white solid. Rf = 0.26 (CH2 Cl2 /MeOH 95:5); IR (ATR) γ cm−1 : 3337, 3105, 3040, 2963, 2944, 2843, 2728, 1706, 1658, 1613, 1605, 1585, 1544, 1504; 1 H-NMR (MeOD-d , 300 MHz): δ 7.44 (d, J = 15.7 Hz, 1H), 6.87 (s, 2H), 6.71–6.67 (m, 2H), 6.57–6.48 4 (m, 2H), 3.87 (s, 6H), 3.78 (s, 3H), 3.47 (t, J = 7.2 Hz, 2H), 2.71 (t, J = 7.2 Hz, 2H); HRMS (ESI) m/z calcd for C20 H24 NO6 [M + H]+ : 374.1525; found: 374.1605. The analytical data were in line with previously described data [55]. (E)-N-(3,4-Dihydroxyphenethyl)cinnamide (15). General procedure using cinnamic acid (246 mg, 1.66 mmol) and 3,4-dihydroxyphenethylamine hydrochloride (315 mg, 1.66 mmol) afforded compound 15 (293 mg, 62%) as a colorless syrup. Rf = 0.29 (CH2 Cl2 /MeOH 95:5); IR (ATR) γ cm−1 : 3265, 2927, 1702, 1655, 1599, 1519; 1 H-NMR (MeOD-d4 , 300 MHz): δ 7.56–7.49 (m, 3H), 7.42–7.35 (m, 3H), 6.71–6.67 (m, 2H), 6.60–6.54 (m, 2H), 3.47 (t, J = 7.2 Hz, 2H), 2.71 (t, J = 7.2 Hz, 2H); HRMS (ESI) m/z calcd for C17 H18 NO3 [M + H]+ : 284.1208; found: 284.1283. The analytical data were in line with previously described data [54]. (E)-N-(4-Hydroxyphenyl)-3,4-dihydroxycinnamide (16). General procedure using 3,4-dihydroxycinnamic acid (300 mg, 1.66 mmol) and 4-hydroxyphenylamine (181 mg, 1.66 mmol) afforded compound 16 (84 mg, 19%) as a brown solid. Rf = 0.65 (Cyclohexane/EtOAc 1:9); IR (ATR) γ cm−1 : 3210, 1696, 1648, 1588, 1535, 1508; 1 H-NMR (DMSO-d6 , 300 MHz): δ 9.83 (s, 1H), 9.46 (bs, 1H), 9.21 (bs, 2H), 7.46 (d, J = 8.6 Hz, 2H), 7.33 (d, J = 15.6 Hz, 1H), 6.98 (s, 1H), 6.88 (d, J = 8.3 Hz, 1H), 6.76 (d, J = 8.3 Hz, 1H), 6.70 (d, J = 8.6 Hz, 2H), 6.49 (d, J = 15.6 Hz, 1H); HRMS (ESI) m/z calcd for C15 H14 NO4 [M + H]+ : 272.0923, found: 272.0918. The analytical data were in line with previously described data [56]. N-(4-Hydoxyphenethyl)-3,4-dihydroxybenzamide (17). General procedure using 3,4-dihydroxybenzoic acid (256 mg, 1.66 mmol) and 4-hydroxyphenethylamine (228 mg, 1.66 mmol) afforded compound 19 (53 mg, 12%) as a colorless syrup. Rf = 0.19 (CH2 Cl2 /MeOH 95:5); IR (ATR) γ cm−1 : 3177, 2970, 2948, 2869, 1698, 1632, 1589, 1553, 1510; 1 H-NMR (MeOD-d4 , 300 MHz): δ 7.24 (d, J = 2.0 Hz, 1H), 7.15 (dd, J1 = 8.2 Hz, J2 = 2.0 Hz, 2H), 7.06 (d, J = 8.3 Hz, 2H), 6.78 (d, J = 8.2 Hz, 1H), 6.71 (d, J = 8.3 Hz, 2H), 3.49 (t, J = 7.3 Hz, 2H), 2.78 (t, J = 7.3 Hz, 2H). HRMS (ESI) m/z calcd for C15 H16 NO4 [M + H]+ : 274.1001, found: 274.1072. The analytical data were in line with previously described data [57]. 3-(3,4-Dihydroxyphenyl)-2-[3-(3,4-dihydroxyphenyl)-acryloylamino]-propionic acid methyl ester (18). General procedure using 3,4-dihydroxycinnamic acid (300 mg, 1.66 mmol) and L-3,4-dihydroxyphenylalanine methyl ester (315 mg, 1.66 mmol, previously prepared following [58]) afforded compound 18 (332 mg, 54%) as a yellow solid. Rf = 0.5 (CH2 Cl2 /MeOH 85:15); IR (ATR) γ cm−1 : 3308, 2953, 2838, 2725, 1722, 1650, 1594, 1514; 1 H-NMR (DMSO-d6 , 300 MHz): δ 8.35 (d, J = 6.9 Hz, 1H), 7.20 (d, J = 15.6 Hz, 1H), 6.93 (s, 1H), 6.83 (d, J = 8.1 Hz, 1H), 6.73 (d, J = 8.1 Hz, 1H), 6.61–6.59 (m, 2H), 6.45 (d, J = 8.1 Hz,

Int. J. Mol. Sci. 2016, 17, 1656

12 of 17

1H), 6.38 (d, J = 15.7 Hz, 1H), 4.46 (m, 1H), 3.60 (s, 3H), 2.89–2.72 (m, 2H); HRMS (ESI) m/z calcd for C19 H20 NO7 [M + H]+ : 374.1240; found: 374.1240. The analytical data were in line with previously described data [59]. (E)-N-(3,4-Dihydroxycinnamoyl)-L-cysteine methyl ester (19). General procedure using 3,4-dihydroxy cinnamic acid (300 mg, 1.66 mmol) and L-cysteine methyl ester hydrochloride (285 mg, 1.66 mmol) afforded compound 19 ( 50.8 mg, 10%) as a white solid. Rf = 0.19 (CH2 Cl2 /MeOH 95:5); IR (ATR) γ cm−1 : 3472, 3400, 3318, 3163, 2952, 2551, 1730, 1654, 1592, 1524; 1 H-NMR (Acetone-d6 , 300 MHz): δ 8.31 (bs, 2H), 7.52 (d, J = 7.6 Hz, 1H), 7.44 (d, J = 15.6 Hz, 1H), 7.09 (d, J = 1.8 Hz, 1H), 6.97 (dd, J1 = 1.8 Hz, J2 = 8.2 Hz, 1H), 6.84 (d, J = 8.2 Hz, 1H), 6.62 (d, J = 15.6 Hz, 1H), 4.87–4.80 (m, 1H), 3.73 (s, 3H), 3.05–2.93 (m, 2H), 1.96 (t, J = 8.5 Hz, 1H); HRMS (ESI) m/z calcd for C13 H16 NO5 S [M + H]+ : 298.0671; found: 298.0750. The analytical data were in line with previously described data [53]. 3.3. Biological Evaluation 3.3.1. In Vitro Enzymatic Assay Arginase catalyzes the hydrolysis of L-arginine into L-ornithine and urea. Urea amount produced by this reaction can be detected using a colorimetric assay. We adapted the Corraliza et al. (1994) method [40] by miniaturizing this colorimetric assay and by using a purified bovine liver arginase or a recombinant human arginase I instead of cell lysates [34], as described below. In each well of a microplate, the following solutions were added in this order: (1) 10 µL of a buffer containing and Tris-HCl (50 mM, pH 7.5) 0.1% of Bovine Serum Albumin (TBSA buffer), with or without (control) arginase (0.025 U/µL for b-ARGI or 3.125 µg/mL for h-ARGI); (2) 30 µL of Tris-HCl solution (50 mM, pH 7.5) containing MnCl2 10 mM as a co-factor; (3) 10 µL of a solution containing an inhibitor or its solvent (as a control—see Section 3.3.2); and (4) 20 µL of L-arginine (pH 9.7, 0.05 M). The microplate was covered with a plastic sealing film and incubated for 60 min in a 37 ◦ C water bath. In the case of recombinant h-ARGI the enzyme was activated for 10 min at 55 ◦ C prior to Step (2). The reaction was stopped by adding 120 µL of H2 SO4 /H3 PO4 /H2 O (1:3:7) after placing the microplate on ice. Thereafter, 10 µL of alpha-isonitrosopropiophenone (5% in absolute ethanol) was added and the microplate was covered with an aluminum sealing film and heated in an oven at a temperature of 100 ◦ C for 45 min. The microplate was kept in the dark until reading. After 5 min centrifugation and cooling for another 10 min, the microplate was shaken for 2 min and the absorbance was read at 550 nm and 25 ◦ C using a spectrophotometer (Synergy HT BioTeck, Winooski, VT, USA). The level of arginase activity was expressed as relative to the “100% arginase activity” (see Section 3.3.2). Each experimental condition (e.g., various inhibitor concentrations) was repeated three times per microplate. 3.3.2. Determination of IC50 Values and Percentages of Arginase Inhibition For each tested compound, a stock solution (70 mM) was prepared in DMSO and stored at −26 ◦ C. Just before the assay, these stock solutions were successively diluted in ultra-pure water to get the following concentrations: 7000, 2100, 700, 210, 70, 21, 7, 2.1 and 0.7 µM (i.e., final concentrations in the wells of 1000, 300, 100, 30, 10, 3, 1, 0.3, and 0.1 µM, respectively). For a first screening, compounds were only tested at 100 µM (final concentration). Each dilution was incubated with arginase for one hour, as described above. The resulting absorbance was converted into percentage of arginase inhibition, i.e., relative to the absorbance of controls with solvent (“100% arginase activity”), and plotted on a semi-logarithmic scale. The IC50 values were estimated with Prism® (GraphPad Software, version 5.0.3, La Jolla, CA, USA) by non-linear sigmoidal curve-fitting. 3.3.3. Enzyme Kinetic Study and Determination of Ki Values The type of inhibition was determined with the same experimental approach with three concentrations of CAPA (10, 20 and 30 µM) and a control under increasing L-arginine concentrations (2.86, 7.15, 14.30 mM). The kinetics data were analyzed using Lineweaver–Burk plot (i.e., 1/velocity vs.

Int. J. Mol. Sci. 2016, 17, 1656

13 of 17

1/[L-arginine] (Figure 2B), Dixon plot (i.e., 1/velocity vs. [inhibitor]) (Figure 2C) and Cornish–Bowden plot (i.e., [L-arginine]/velocity vs. [inhibitor]) (Figure 2D). The type of inhibition was determined following the graphical method described by Cornish–Bowden [41]. The Ki values of competitive inhibitors were calculated based on the Cheng–Prusoff equation [Ki = IC50 /(1 + [S]/Km )] [42]. 3.4. Molecular Docking Study 3.4.1. Preparation of Target Protein The 3D crystal structure of h-ARG I complexed with co-ligand nor-NOHA (pdb id: 3kv2) was retrieved from Protein Data Bank (http://www.rcsb.org). The receptor was prepared following the standard procedure of LeadIt 2.0.2 software. The binding site was defined as all the amino acid residues enclosed within 16.0 Å radius sphere centered by the co-ligand, nor-NOHA. All water molecules were removed. 3.4.2. Preparation of Ligands Ligands were built and prepared by using Sybyl-X 2.0 software [60]. Hydrogen were added to the 3D structure, which was optimized by energy minimization using Conj Grad Method with convergence criterion set at 0.0001 kcal/(Åmol), max iterations (10,000) and with Gasteiger Hükel charge assigned in Tripos force field; other parameter values were kept at default. Then, the structures were subjected to a simulated annealing run and further optimized to obtain the lowest-energy conformers, which were stored in the database for the docking simulation. 3.4.3. Docking Simulation The docking simulations were performed using the FlexX docking program [44,61] implemented in LeadIt 2.0.2 software with number of poses set at 10 for analyzing, maximum number of solutions per iteration set at 1000 and maximum number of solutions per fragmentation set at 200, other docking parameters were kept at default setting. The molecular docking technique used in this study was a flexible one in which the ligand binding process was an induced-fit process. The 3D and 2D binding modes of docking solutions were generated by using MOE 2008.10 software [62]. 4. Conclusions In summary, a series of nineteen cinnamide derivatives were synthesized and first evaluated for their arginase inhibitory capacities on mammalian arginase I (b-ARG I), together with the two natural caffeic and chlorogenic acids. Among them, chlorogenic acid and six of the synthesized compounds showed an IC50 lower than 50 µM. Caffeic acid phenethylamide (CAPA, compound 1) was the most active of this series, with an IC50 value of 6.9 µM. Whereas the activity of nor-NOHA was retained (IC50 = 5.7 ± 0.6 µM) on an assay using a human recombinant arginase (h-ARG I), CAPA showed poorer activity (IC50 = 60.3 ± 7.8 µM) on h-ARG I compared to b-ARG-I. Therefore, although preliminary studies on b-ARG I, which is much cheaper than h-ARG I, could be useful to identify new mammalian arginase inhibitors, the most active compounds should also be tested on h-ARG I before going further with in vivo experiments. Enzyme kinetic studies, performed on b-ARG I, identified CAPA as a competitive inhibitor with Ki = 5.5 ± 1.0 µM. SARs and docking studies indicated the crucial role of caffeoyl moiety, which could penetrate into the active site of ARG-I in order to chelate the Mn2+ cofactor and interact with the important preserved residues (Asp124, His126, His141, Asp232, Asp234, and Thr246) involved in the catalytic site of arginase, thus disturbing the enzymatic activity. Overall, our results identified for the first time the important role of the caffeoyl moiety in binding the arginase active site. Considering the high IC50 value obtained with CAPA against h-ARG I, cinnamide derivatives could constitute potential new lead compounds for the development of arginase inhibitors with potential therapeutic applications. For instance, the phenethylamine moiety could be replaced by

Int. J. Mol. Sci. 2016, 17, 1656

14 of 17

a part displaying specific interactions on the top of the arginase active site in order to design a new generation of cinnamides. Acknowledgments: The authors gratefully acknowledge the Ministère Français de l’Enseignement supérieur et de la Recherche for awarding a PhD fellowship to Thanh-Nhat Pham and the Université de Bourgogne Franche-Comté for giving a travel grant to have research collaborations in Vietnam. Thanks are also due to Maude Nappey and Andy Zedet for technical assistance and Stéphanie Boullanger for NMR measurements. The molecular modeling work was supported by the Vietnam's National Foundation for Science and Technology Development-NAFOSTED (Grant # 106-YS.05-2015.31 to Khac-Minh Thai). Author Contributions: Ideas and experiments design: Corine Girard-Thernier and Khac-Minh Thai. Computational development: Thanh-Nhat Pham and Khac-Minh Thai. Chemistry and biology: Thanh-Nhat Pham, Simon Bordage, Marc Pudlo. Analysis and data interpretation: Thanh-Nhat Pham, Marc Pudlo, Céline Demougeot and Corine Girard-Thernier. Writing and review of the paper: all authors. Study supervision: Corine Girard-Thernier and Khac-Minh Thai. Conflicts of Interest: The authors declare no conflict of interest.

References 1. 2. 3. 4. 5. 6.

7.

8.

9.

10. 11. 12.

13.

Maccallini, C.; Fantacuzzi, M.; Amoroso, R. Amidine-based bioactive compounds for the regulation of arginine metabolism. Mini Rev. Med. Chem. 2013, 13, 1305–1310. [CrossRef] [PubMed] Dimski, D.S. Ammonia metabolism and the urea cycle: Function and clinical implications. J. Vet. Intern. Med. Am. Coll. Vet. Int. Med. 1994, 8, 73–78. [CrossRef] Morris, S.M. Regulation of enzymes of the urea cycle and arginine metabolism. Annu. Rev. Nutr. 2002, 22, 87–105. [CrossRef] [PubMed] Caldwell, R.B.; Toque, H.A.; Narayanan, S.P.; Caldwell, R.W. Arginase: An old enzyme with new tricks. Trends Pharmacol. Sci. 2015, 36, 395–405. [CrossRef] [PubMed] Jenkinson, C.P.; Grody, W.W.; Cederbaum, S.D. Comparative properties of arginases. Comp. Biochem. Physiol. B Biochem. Mol. Biol. 1996, 114, 107–132. [CrossRef] Bagnost, T.; Ma, L.; da Silva, R.F.; Rezakhaniha, R.; Houdayer, C.; Stergiopulos, N.; André, C.; Guillaume, Y.; Berthelot, A.; Demougeot, C. Cardiovascular effects of arginase inhibition in spontaneously hypertensive rats with fully developed hypertension. Cardiovasc. Res. 2010, 87, 569–577. [CrossRef] [PubMed] Segal, R.; Hannan, J.L.; Liu, X.; Kutlu, O.; Burnett, A.L.; Champion, H.C.; Kim, J.H.; Steppan, J.; Berkowitz, D.E.; Bivalacqua, T.J. Chronic oral administration of the arginase inhibitor 2(S)-amino-6boronohexanoic acid (ABH) improves erectile function in aged rats. J. Androl. 2012, 33, 1169–1175. [CrossRef] [PubMed] Grasemann, H.; Dhaliwal, R.; Ivanovska, J.; Kantores, C.; McNamara, P.J.; Scott, J.A.; Belik, J.; Jankov, R.P. Arginase inhibition prevents bleomycin-induced pulmonary hypertension, vascular remodeling, and collagen deposition in neonatal rat lungs. Am. J. Physiol. Lung Cell. Mol. Physiol. 2015, 308, L503–L510. [CrossRef] [PubMed] Olivon, V.C.; Fraga-Silva, R.A.; Segers, D.; Demougeot, C.; de Oliveira, A.M.; Savergnini, S.S.; Berthelot, A.; de Crom, R.; Krams, R.; Stergiopulos, N.; et al. Arginase inhibition prevents the low shear stress-induced development of vulnerable atherosclerotic plaques in ApoE−/− mice. Atherosclerosis 2013, 227, 236–243. [CrossRef] [PubMed] Morris, S.M.; Gao, T.; Cooper, T.K.; Kepka-Lenhart, D.; Awad, A.S. Arginase-2 mediates diabetic renal injury. Diabetes 2011, 60, 3015–3022. [CrossRef] [PubMed] Meurs, H.; Zaagsma, J.; Maarsingh, H.; van Duin, M. Use of Arginase Inhibitors in the Treatment of Asthma and Allergic Rhinitis. US 20150164930 A1, 4 March 2010. Van Zandt, M.C.; Whitehouse, D.L.; Golebiowski, A.; Ji, M.K.; Zhang, M.; Beckett, R.P.; Jagdmann, G.E.; Ryder, T.R.; Sheeler, R.; Andreoli, M.; et al. Discovery of (R)-2-Amino-6-borono-2-(2-(piperidin-1-yl) ethyl)hexanoic acid and congeners as highly potent inhibitors of human arginases I and II for treatment of myocardial reperfusion injury. J. Med. Chem. 2013, 56, 2568–2580. [CrossRef] [PubMed] Kavalukas, S.L.; Uzgare, A.R.; Bivalacqua, T.J.; Barbul, A. Arginase inhibition promotes wound healing in mice. Surgery 2012, 151, 287–295. [CrossRef] [PubMed]

Int. J. Mol. Sci. 2016, 17, 1656

14.

15.

16.

17.

18.

19.

20. 21.

22. 23.

24.

25.

26.

27. 28.

29.

30. 31.

32.

15 of 17

Singh, R.; Pervin, S.; Karimi, A.; Cederbaum, S.; Chaudhuri, G. Arginase Activity in human breast cancer cell lines: Nω -Hydroxy-L-arginine selectively inhibits cell proliferation and induces apoptosis in MDA-MB-468 cells. Cancer Res. 2000, 60, 3305–3312. [PubMed] Boucher, J.L.; Custot, J.; Vadon, S.; Delaforge, M.; Lepoivre, M.; Tenu, J.P.; Yapo, A.; Mansuy, D. Nω-Hydroxy-L-arginine, an intermediate in the L-arginine to nitric oxide pathway, is a strong inhibitor of liver and macrophage arginase. Biochem. Biophys. Res. Commun. 1994, 203, 1614–1621. [CrossRef] [PubMed] Custot, J.; Boucher, J.-L.; Vadon, S.; Guedes, C.; Dijols, S.; Delaforge, M.; Mansuy, D. Nω -Hydroxyaminoα-amino acids as a new class of very strong inhibitors of arginases. JBIC J. Biol. Inorg. Chem. 1996, 1, 73–82. [CrossRef] Custot, J.; Moali, C.; Brollo, M.; Boucher, J.L.; Delaforge, M.; Mansuy, D.; Tenu, J.P.; Zimmermann, J.L. The new α-amino acid Nω -hydroxy-nor-L-arginine: A high-affinity inhibitor of arginase well adapted to bind to its manganese cluster. J. Am. Chem. Soc. 1997, 119, 4086–4087. [CrossRef] Kim, N.N.; Cox, J.D.; Baggio, R.F.; Emig, F.A.; Mistry, S.K.; Harper, S.L.; Speicher, D.W.; Morris, S.M.; Ash, D.E.; Traish, A.; et al. Probing erectile function: S-(2-Boronoethyl)-L-cysteine binds to arginase as a transition state analogue and enhances smooth muscle relaxation in human penile corpus cavernosum. Biochemistry 2001, 40, 2678–2688. [CrossRef] [PubMed] Baggio, R.; Emig, F.A.; Christianson, D.W.; Ash, D.E.; Chakder, S.; Rattan, S. Biochemical and functional profile of a newly developed potent and isozyme-selective arginase inhibitor. J. Pharmacol. Exp. Ther. 1999, 290, 1409–1416. [PubMed] Ivanenkov, Y.A.; Chufarova, N. V. Small-molecule arginase inhibitors. Pharm. Pat. Anal. 2013, 3, 65–85. [CrossRef] [PubMed] Havlinova, Z.; Babicova, A.; Hroch, M.; Chladek, J. Comparative pharmacokinetics of Nω -hydroxy-norL -arginine, an arginase inhibitor, after single-dose intravenous, intraperitoneal and intratracheal administration to brown Norway rats. Xenobiotica 2013, 43, 886–894. [CrossRef] [PubMed] Morris, S.M., Jr. Recent advances in arginine metabolism: Roles and regulation of the arginases. Br. J. Pharmacol. 2009, 157, 922–930. [CrossRef] [PubMed] Schade, D.; Kotthaus, J.; Klein, N.; Kotthaus, J.; Clement, B. Prodrug design for the potent cardiovascular agent Nω -hydroxy-L-arginine (NOHA): Synthetic approaches and physicochemical characterization. Org. Biomol. Chem. 2011, 9, 5249–5259. [CrossRef] [PubMed] Ilies, M.; di Costanzo, L.; Dowling, D.P.; Thorn, K.J.; Christianson, D.W. Binding of α,α-disubstituted amino acids to arginase suggests new avenues for inhibitor design. J. Med. Chem. 2011, 54, 5432–5443. [CrossRef] [PubMed] Golebiowski, A.; Beckett, R.P.; van Zandt, M.; Ji, M.K.; Whitehouse, D.; Ryder, T.R.; Jagdmann, E.; Andreoli, M.; Mazur, A.; Padmanilayam, M.; et al. 2-Substituted-2-amino-6-boronohexanoic acids as arginase inhibitors. Bioorg. Med. Chem. Lett. 2013, 23, 2027–2030. [CrossRef] [PubMed] Golebiowski, A.; Whitehouse, D.; Beckett, R.P.; van Zandt, M.; Ji, M.K.; Ryder, T.R.; Jagdmann, E.; Andreoli, M.; Lee, Y.; Sheeler, R.; et al. Synthesis of quaternary α-amino acid-based arginase inhibitors via the Ugi reaction. Bioorg. Med. Chem. Lett. 2013, 23, 4837–4841. [CrossRef] [PubMed] Cama, E.; Shin, H.; Christianson, D.W. Design of amino acid sulfonamides as transition-state analogue inhibitors of arginase. J. Am. Chem. Soc. 2003, 125, 13052–13057. [CrossRef] [PubMed] Ilies, M.; di Costanzo, L.; North, M.L.; Scott, J.A.; Christianson, D.W. 2-Aminoimidazole amino acids as inhibitors of the binuclear manganese metalloenzyme human arginase I. J. Med. Chem. 2010, 53, 4266–4276. [CrossRef] [PubMed] Zakharian, T.Y.; di Costanzo, L.; Christianson, D.W. S-2-Amino-6-nitrohexanoic acid binds to human arginase I through multiple nitro−metal coordination interactions in the binuclear manganese cluster. J. Am. Chem. Soc. 2008, 130, 17254–17255. [CrossRef] [PubMed] Shin, H.; Cama, E.; Christianson, D.W. Design of amino acid aldehydes as transition-state analogue inhibitors of arginase. J. Am. Chem. Soc. 2004, 126, 10278–10284. [CrossRef] [PubMed] Zakharian, T.Y.; di Costanzo, L.; Christianson, D.W. Synthesis of (2S)-2-amino-7,8-epoxyoctanoic acid and structure of its metal-bridging complex with human arginase I. Org. Biomol. Chem. 2008, 6, 3240–3243. [CrossRef] [PubMed] Girard-Thernier, C.; Pham, T.-N.; Demougeot, C. The promise of plant-derived substances as inhibitors of arginase. Mini Rev. Med. Chem. 2015, 15, 798–808. [CrossRef] [PubMed]

Int. J. Mol. Sci. 2016, 17, 1656

33.

34.

35.

36. 37.

38. 39.

40. 41.

42.

43.

44. 45.

46.

47.

48. 49.

50. 51. 52.

16 of 17

Pham, T.; Guglielmetti, A.; Fimbel, S.; Demougeot, C.; Girard-Thernier, C. Arginase inhibitory activity of several natural polyphenols using a novel in vitro test on purified bovine arginase. Planta Med. 2014, 80, P1L9. [CrossRef] Bordage, S.; Pham, T.-N.; Zedet, A.; Gugglielmetti, A.-S.; Nappey, M.; Demougeot, C.; Girard-Thernier, C. Investigation of mammal arginase inhibitory properties of natural ubiquitous polyphenols by using an optimized colorimetric microplate assay. Planta Med. 2016. accepted. Wang, L.-N.; Wang, W.; Hattori, M.; Daneshtalab, M.; Ma, C.-M. Synthesis, Anti-HCV, antioxidant and reduction of intracellular reactive oxygen species generation of a chlorogenic acid analogue with an amide bond replacing the ester bond. Molecules 2016, 21, 737. [CrossRef] [PubMed] Le Mellay-Hamon, V.; Criton, M. Phenylethylamide and phenylmethylamide derivatives as new tyrosinase inhibitors. Biol. Pharm. Bull. 2009, 32, 301–303. [CrossRef] [PubMed] Okombi, S.; Rival, D.; Boumendjel, A.; Mariotte, A.-M.; Perrier, E. Para-Coumaric Acid or Para-Hydroxycinnamic Acid Derivatives and Their Use in Cosmetic or Dermatological Compositions. US 8481593 B2, 9 August 2007. Coste, J.; Le-Nguyen, D.; Castro, B. PyBOP: A new peptide coupling reagent devoid of toxic by-product. Tetrahedron Lett. 1990, 31, 205–208. [CrossRef] Berndt, M.; Hölemann, A.; Niermann, A.; Bentz, C.; Zimmer, R.; Reissig, H.-U. Replacement of HMPA in Samarium Diiodide Promoted Cyclizations and Reactions of Organolithium Compounds. Eur. J. Org. Chem. 2012, 2012, 1299–1302. [CrossRef] Corraliza, I.M.; Campo, M.L.; Soler, G.; Modolell, M. Determination of arginase activity in macrophages: A micromethod. J. Immunol. Methods 1994, 174, 231–235. [CrossRef] Cornish-Bowden, A. A simple graphical method for determining the inhibition constants of mixed, uncompetitive and non-competitive inhibitors (Short Communication). Biochem. J. 1974, 137, 143–144. [CrossRef] [PubMed] Yung-Chi, C.; Prusoff, W.H. Relationship between the inhibition constant (KI) and the concentration of inhibitor which causes 50 per cent inhibition (I50) of an enzymatic reaction. Biochem. Pharmacol. 1973, 22, 3099–3108. [CrossRef] Di Costanzo, L.; Pique, M.E.; Christianson, D.W. Crystal structure of human arginase I complexed with thiosemicarbazide reveals an unusual thiocarbonyl µ-sulfide ligand in the binuclear manganese cluster. J. Am. Chem. Soc. 2007, 129, 6388–6389. [CrossRef] [PubMed] BioSolveIT–GmbH. FlexX (Protein–Ligand Docker) user & technical reference as part of LeadIT 2.0. Germany, 2012; p. 15. Available online: https://www.biosolveit.de/FlexX (accessed on 18 March 2016). Weng, Y.-C.; Chuang, C.-F.; Chuang, S.-T.; Chiu, H.-L.; Kuo, Y.-H.; Su, M.-J. KS370G, a synthetic caffeamide derivative, improves left ventricular hypertrophy and function in pressure-overload mice heart. Eur. J. Pharmacol. 2012, 684, 108–115. [CrossRef] [PubMed] Shi, Z.-H.; Li, N.-G.; Shi, Q.-P.; Hao-Tang, B.S.P.; Tang, Y.-P.; Wei-Li, B.S.P.; Lian-Yin, B.S.P.; Yang, J.-P.; Duan, J.-A. Design, synthesis and biological evaluation of ferulic acid amides as selective matrix metalloproteinase inhibitors. Med. Chem. 2013, 9, 947–954. [CrossRef] [PubMed] Yamazaki, Y.; Kawano, Y.; Uebayasi, M. Induction of adiponectin by natural and synthetic phenolamides in mouse and human preadipocytes and its enhancement by docosahexaenoic acid. Life Sci. 2008, 82, 290–300. [CrossRef] [PubMed] Yao, H.; Tang, Y.; Yamamoto, K. Metal-free oxidative amide formation with N-hydroxysuccinimide and hypervalent iodine reagents. Tetrahedron Lett. 2012, 53, 5094–5098. [CrossRef] Hong, S.S.; Jeong, W.; Kwon, J.G.; Choi, Y.-H.; Ahn, E.-K.; Ko, H.-J.; Seo, D.-W.; Oh, J.S. Phenolic Amides from the Fruits of Tribulus terrestris and Their Inhibitory Effects on the Production of Nitric Oxide. Bull. Korean Chem. Soc. 2013, 34, 3105–3108. [CrossRef] Kan, S.; Chen, G.; Han, C.; Chen, Z.; Song, X.; Ren, M.; Jiang, H. Chemical constituents from the roots of Xanthium sibiricum. Nat. Prod. Res. 2011, 25, 1243–1249. [CrossRef] [PubMed] Lin, C.-F.; Lay, H.-L.; Ni, C.-L.; Chen, C.-C. Phenolic Components of Dendrobium antennatum. Chem. Nat. Compd. 2013, 49, 520–522. [CrossRef] Tamiz, A.P.; Cai, S.X.; Zhou, Z.-L.; Yuen, P.-W.; Schelkun, R.M.; Whittemore, E.R.; Weber, E.; Woodward, R.M.; Keana, J.F.W. Structure−Activity relationship of N-(Phenylalkyl)cinnamides as novel NR2B subtype-selective NMDA receptor antagonists. J. Med. Chem. 1999, 42, 3412–3420. [CrossRef] [PubMed]

Int. J. Mol. Sci. 2016, 17, 1656

53. 54.

55.

56.

57. 58.

59.

60. 61. 62.

17 of 17

Son, S.; Lewis, B.A. Free radical scavenging and antioxidative activity of caffeic acid amide and ester analogues: Structure−activity relationship. J. Agric. Food Chem. 2001, 50, 468–472. [CrossRef] Wu, Z.; Zheng, L.; Li, Y.; Su, F.; Yue, X.; Tang, W.; Ma, X.; Nie, J.; Li, H. Synthesis and structure–activity relationships and effects of phenylpropanoid amides of octopamine and dopamine on tyrosinase inhibition and antioxidation. Food Chem. 2012, 134, 1128–1131. [CrossRef] [PubMed] Michalet, S.; Cartier, G.; David, B.; Mariotte, A.-M.; Dijoux-franca, M.-G.; Kaatz, G. W.; Stavri, M.; Gibbons, S. N-Caffeoylphenalkylamide derivatives as bacterial efflux pump inhibitors. Bioorg. Med. Chem. Lett. 2007, 17, 1755–1758. [CrossRef] [PubMed] Shi, Z.-H.; Li, N.-G.; Shi, Q.-P.; Tang, H.; Tang, Y.-P.; Li, W.; Yin, L.; Yang, J.-P.; Duan, J.-A. Design, synthesis and biological evaluation of caffeic acid amides as selective MMP-2 and MMP-9 inhibitors. Drug Dev. Res. 2012, 73, 343–351. [CrossRef] Chou, S.-C.; Su, C.-R.; Ku, Y.-C.; Wu, T.-S. The constituents and their bioactivities of Houttuynia cordata. Chem. Pharm. Bull. 2009, 57, 1227–1230. [CrossRef] [PubMed] Larsson, R.; Blanco, N.; Johansson, M.; Sterner, O. Synthesis of C-1 indol-3-yl substituted tetrahydroisoquinoline derivatives via a Pictet–Spengler approach. Tetrahedron Lett. 2012, 53, 4966–4970. [CrossRef] Lee, S.U.; Shin, C.-G.; Lee, C.-K.; Lee, Y.S. Caffeoylglycolic and caffeoylamino acid derivatives, halfmers of L-chicoric acid, as new HIV-1 integrase inhibitors. Eur. J. Med. Chem. 2007, 42, 1309–1315. [CrossRef] [PubMed] SYBYL-X, Tripos International. Available online: https://www.certara.com (accessed on 18 March 2016). Rarey, M.; Kramer, B.; Lengauer, T.; Klebe, G. A fast flexible docking method using an incremental construction algorithm. J. Mol. Biol. 1996, 261, 470–489. [CrossRef] [PubMed] Chemical Computing Group Inc. Molecular Operating Environment (MOE). Available online: https://www. chemcomp.com (accessed on 18 March 2016). © 2016 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC-BY) license (http://creativecommons.org/licenses/by/4.0/).