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Design, Synthesis, and Structure–Activity Relationship Studies of Novel 3-Alkylindole Derivatives as Selective and Highly Potent Myeloperoxidase Inhibitors Jalal Soubhye, Iyas Aldib, Betina Elfving, Michel Gelbcke, Paul Georg Furtmüller, Manuel Podrecca, Raphael Conotte, Jean-Marie Colet, Alexandre Rousseau, Florence Reye, Ahmad Sarakbi, Michel Vanhaeverbeek, Jean Michel Kauffmann, Christian Obinger, Jean Nève, Martine Prévost, Karim Zouaoui Boudjeltia, Francois M U Dufrasne, and Pierre G Van Antwerpen J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/jm4001538 • Publication Date (Web): 14 Apr 2013 Downloaded from http://pubs.acs.org on April 16, 2013

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Journal of Medicinal Chemistry

Design, Synthesis, and Structure–Activity Relationship Studies of Novel 3-Alkylindole Derivatives as Selective and Highly Potent Myeloperoxidase Inhibitors Jalal Soubhyea, Iyas Aldiba, Betina Elfving b, Michel Gelbckea, Paul G. Furtmüllerc, Manuel Podreccad, Raphaël Conotted, Jean-Marie Coletd, Alexandre Rousseaue, Florence Reyea, Ahmad Sarakbif, Michel Vanhaeverbeeke, Jean-Michel Kauffmannf, Christian Obingerd, Jean Nèvea, Martine Prévostg, Karim Zouaoui Boudjeltiae, Francois Dufrasnea† and Pierre Van Antwerpena,h†* a

Laboratoire de Chimie Pharmaceutique Organique, Faculté de Pharmacie, Université Libre de

Bruxelles (ULB), Brussels, Belgium. b c

Centre for Psychiatric Research, Aarhus University Hospital Risskov, 8240 Risskov, Denmark.

Department of Chemistry, Division of Biochemistry at the Vienna Institute of BioTechnology,

BOKU—University of Natural Resources and Life Sciences, Muthgasse 18, A-1190 Vienna d

Department of Human Biology and Toxicology, Faculty of Medicine and Pharmacy, University of

Mons, Mons, Belgium. e

Laboratory of Experimentral Medicine, CHU Charleroi, A. Vesale Hospital, Université Libre de

Bruxelles (ULB), Montigny-le-Tilleul, Belgium. f

Laboratory Instrumental Analysis and Bioelectrochemistry, Faculté de Pharmacie, Université Libre

de Bruxelles (ULB), Brussels, Belgium. g

Laboratoire de Structure et Fonction des Membranes Biologiques, Université Libre de Bruxelles

(ULB), Brussels, Belgium. h

Analytical Platform of the Faculty of Pharmacy, Université Libre de Bruxelles (ULB), Brussels,

Belgium

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Abstract 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Due to its production of potent antimicrobial oxidants including hypochlorous acid, human myeloperoxidase (MPO) plays a critical role in innate immunity and inflammatory diseases. Thus MPO is an attractive target in drug design. Aminoalkyl-fluoroindole derivatives were detected to be very potent MPO inhibitors; however, they also promote inhibition of the serotonin reuptake transporter (SERT) at the same concentration range. Using structure-based drug design, a new series of MPO inhibitors derived from 3-alkylindole were synthesized and their effects were assessed on the MPO-mediated taurine chlorination and LDL oxidation as well as on inhibition of SERT. The fluoroindole compound with 3 carbons in the side chain and one amide group exhibited a selectivity index of 35 (Ki/IC50) with a high inhibition of MPO activity (IC50= 18 nM) whereas its effect on SERT was in the micromolar range. Structure-function relationships, mechanism of action and safety of the molecule were discussed in the manuscript.


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Introduction 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Myeloperoxidase (MPO, EC 1.11.2.2) is a heme-containing enzyme stored in large amount in azurophilic granules of neutrophils antiviral system

1-3

. It plays a fundamental role in the human antimicrobial and

4, 5

. Upon phagocytosis of pathogens, azurophilic granules discharge their content

including MPO into phagolysosomes 6. The microbicidal activity of MPO is due to its ability to oxidize halide ions (Cl-, Br- and I-) and the pseudohalide (SCN-) by H2O2 thereby producing the respective hypohalous acids (HOX) 7. However, MPO can be released outside the phagocytes, and contribute to tissue damage by oxidizing biomolecules (lipids, proteins, DNA, etc.) 3. Active MPO was detected in atheromatous plaques, brain tissue showing Alzheimer-type alterations or Parkinson’s disease, multiple sclerosis lesions, synovial fluid of patients with rheumatoid arthritis, glomerular basement membrane at membranous glomerulonephritis and in polymorphonuclear neutrophils-mediated liver diseases 8. Moreover, enhanced plasma levels of MPO are regarded as a risk factor for coronary artery disease 9. These findings demonstrate that MPO is implicated in many chronic inflammatory diseases. Lipoproteins have a central role in the pathogenesis of plaques. Data support a strong association between plasma lipoprotein levels and the risk of cardiovascular diseases 10. The role of lipoproteins, especially low-density lipoproteins (LDL), in the formation and evolution of atheromatous plaques has been largely documented showing that the oxidative modification of LDL is an important step in this process 11, 12. It has been demonstrated that oxidized LDL can induce the formation of foam cells and of a number of potentially pro-atherogenic metabolites such as proinflammatory cytokines and chemokines in monocytes, endothelial cells and smooth muscle cells. As a consequence, oxidized LDLs accumulate in the vascular cell wall and promote a local inflammatory process

13

. The key role of MPO in this process has been documented: the enzyme

selectively binds to apolipoprotein B-100 producing oxidative modifications of the protein moiety of LDLs in the intima and at the surface of endothelial cells

13, 14

. Furthermore, high-density

lipoproteins (HDLs), which are the major carriers of lipid hydroperoxides in circulating plasma, can be a target of oxidative species produced by MPO. It has been reported that MPO binds to (major) ACS Paragon Plus Environment

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apolipoprotein A-I and produces oxidative modifications leading to an impairment of the cholesterol 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

efflux 15. MPO therefore appears as a crucial pharmacological target. Many authors investigated its inhibition focusing either on the scavenging of the oxidative species produced by MPO or on a direct inhibition of the enzyme 16-27. Recently, we demonstrated that 3-(aminoalkyl)-5-fluoroindole derivatives are efficient inhibitors of MPO. These compounds with an electron-withdrawing halogen atom on the indole ring can act as substrates. They are easily oxidized by MPO Compound I promoting accumulation of Compound II which is inactive in the halogenation activity (Compound II)

27

. However, these

inhibitors are structurally similar to serotonin (5-hydroxytryptamine, 5HT) (Figure 1), and, unfortunately, also inhibit the 5HT reuptake transporter (SERT) at nanomolar range (data not shown). Using structure-based drug design, we designed a novel series of alkylindole analogs featuring a potent affinity for the active site of MPO but not for SERT. Four strategies were followed: (1) varying the nature and the position of the electron-withdrawing group on the aromatic ring; (2) modulating the substitution on the nitrogen of the side-chain; (3) inserting an electron-donating sulfur atom in the side chain promoting oxidation of the compound by MPO and thus conversion of Compound I to (inactive) Compound II; (4) replacing the basic amino group of the side chain by other groups. The last modification was undertaken to reduce the affinity of the inhibitors for SERT since a basic amino group is mandatory for the inhibition of this transporter 28. The designed analogs were then synthesized and their inhibition potency on recombinant human MPO was measured by the taurine chloramine assay. The effect of these new compounds was also determined on the MPOdependent LDL oxidation and mechanism was proposed for the inhibition of the most relevant compounds. The best MPO inhibitors were tested on SERT. Finally, the toxicological effects of these selected compounds were investigated in mice.


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Results 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Docking experiments – preliminary comparison of SHA-MPO crystal structure. A former docking study of fluorotryptamine derivatives

27

was used to design new analogs. The latter were

docked into MPO active site to get insight into their binding modes and explore virtually their interactions with the residues in the heme cavity and substrate access channel. To validate the procedure, docking of salicylhydroxamic acid (SHA) was performed and compared with the SHA position in the crystal structure of the MPO complex (Figure 2)

18

. The best affinity score pose

obtained by docking was very similar to its crystal position reflecting the capacity of the docking procedure in evaluating other compounds. Docking of 5-fluorotryptamine to the active site of MPO showed that its best affinity position features stacking (as in the MPO-SHA crystal structure) of the indole 6-membered ring onto the pyrrole ring D of the heme (Figure 2)27. Moreover, the amine group forms two salt bridges respectively with Glu102 and with one propionate group of the heme 27. Other poses were found which still include stacking of the indole ring and one salt bridge of the amine with Glu102 but exhibits a different hydrogen bond with Thr100 (Figure 2)27. Other docked positions show a shifted stacking and one H-bond with the propionate group of the heme. Based on these binding modes, different strategies were suggested to conceive new specific analogs. Fluorine at position 7 of the indole. Having a fluorine atom at position 7 on the indole ring brings no significant change in the predicted affinity relative to compounds with fluorine at position 5. The docked poses show that the orientation of the indole ring differs and that a fluorine atom in position 7 does not point towards the heme iron as does fluorine in position 5 (see supporting information). Compounds 26 and 27 feature a shifted stacking and two salt bridges with Glu102 and the heme propionate (Figure 3A and supporting information). One H-bond is formed with the other heme propionate and one with Arg239. Compound 29 is characterized by stacking, one salt bridge with Glu102 and one H-bond with the backbone of Thr100. Its fluorine shows a H-bond to Gln91 (see supporting information). Replacement of fluorine by a carboxylic group. Compounds that bear a carboxylic acid group at position 5 show a binding mode with a stacking arrangement. The carboxylic acid group fits into ACS Paragon Plus Environment

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the pocket located on top of the heme. It points to the heme iron and makes one salt bridge with 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Arg239 and one H-bond with Gln91. The amino group of compound 28 and 30 forms one salt bridge with Glu102 (Figure 3A and supporting information). The nitrile group of compound 53 makes hydrogen bonds with the backbone NH of Glu102. Compound 30 makes an additional H-bond to the backbone of Thr100 (see supporting information). Modulating the substitution on the side-chain nitrogen. A phenyl piperazine and a benzyl group were added to increase the volume around the nitrogen (compounds 31 and 32). Compound 31 features a stacking arrangement of the fluoroindole moiety with pyrrole ring D. The fluorine atom accommodates the pocket between the heme iron and the guanidinium group of Arg239. A salt bridge of the piperazine N1 with Glu102 is observed (Figure 3B). The docked positions of compound 32, which has one benzyl substituent on the amine of the side chain, show a stacking of the fluoroindole with the heme group and formation of one salt bridge with Glu102 and one hydrogen bond with one of the propionate groups of the heme (see supporting information). Compound 32 is predicted to have a better affinity relative to compound 31. Substitution by a hydroxyl-ethyl group (compounds 33 and 34) was also examined. The best affinity pose of compound 33 shows a shifted stacking with the heme, one salt bridge with Glu102, one H-bond of the hydroxyl group in the side chain with Glu102 and an additional H-bond of the indole NH with the heme propionate. In contrast the binding modes of compound 34 feature no stacking with the heme. Instead its hydroxyl group points towards the iron of the heme and forms a H-bond with the distal histidine (His95). Interestingly, this pose (that is not typical for tryptamine derivatives) bears some resemblance with the experimental position of the hydroxyl group in SHA (see supporting information). Using the induced fit protocol rather than performing the docking with a rigid receptor (see Material and Methods section) compound 34 features poses similar to the other fluorotryptamine derivatives, i.e. a stacking with the heme and two salt bridges between the amino group and Glu102 and the heme propionate. The hydroxyl group makes two hydrogen bonds with the backbone of Arg424 and Phe147 backbone. The indole NH forms also one H-bond with the heme propionate group (Figure 3B and supporting information). ACS Paragon Plus Environment

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Introduction of a sulfur atom in the side chain. Compounds 35-38 demonstrate a stacking 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

arrangement with pyrrole ring D of the heme. Compounds 36 and 38 could only achieve a stacking with the induced-fit protocol. The best poses of compound 35 and compound 36 reveal a salt bridge with Glu102. Compound 36 also forms an additional salt bridge with propionate of pyrrole ring D. The indole nitrogen is also involved in a H-bond with the propionate A (Figure 3C). Compounds 37 and 38, which have the fluorine atom in position 7, feature one salt bridge with Glu102 and one with propionate heme group (ring D). In addition the indole NH group of compound 38 forms a hydrogen bond with the heme propionate on ring C (see supporting information). Replacement of the amino group of the side chain by different groups. The lack of a polar group at the extremity of the side chain was examined by removing the amino group. A shifted stacking is observed in the binding modes of compounds 39 - 41 and only one H-bond is formed between the indole NH group and one propionate (see supporting information). The lack of specific interactions (H bonds, salt bridges etc.) is illustrated by a relatively low affinity (< - 5.5 kcal/mol). A second series of compounds was designed with the replacement of the amino group by different polar groups on the alkyl side chain of 5-fluorotryptamine. The idea was to maintain a potential interaction with Glu102 and/or the propionic heme group or even to reach further residues, such as Glu116, Asp214, or the C-terminus of Ala104. The binding modes of compounds containing one sulfonamide group at the side chain extremity (compounds 42 and 43) exhibit a shifted stacking and one H-bond between the sulfonyl and either Arg424, for compound 42 or Phe147 backbone for compound 43. The indole NH group hydrogen binds to the propionate group (see supporting information). Adding one hydroxyl structure to form hydroxylamine group (compound 44) produces a binding mode featuring no stacking but with the hydroxylamine pointing toward the iron of the heme and forming two hydrogen bonds with the distal histidine (His95) and with Gln91 (see supporting information). This mode is similar to the binding of SHA in the crystal structure except that no stacking is observed

18

. The binding modes obtained using an induced fit protocol feature

unusual concomitant interactions: a stacking with the heme and three hydrogen bonds, two of the hydroxyl with His95 and Gln91 and one of the amine with Asp94. This mode is very similar to the MPO-SHA complex structure. The fluorine atom hydrogen bonds with Gln91 (see supporting ACS Paragon Plus Environment

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information). Compound 45 with a thiocyanate group at the side chain extremity features two 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

different types of position. One shows a shifted stacking and one hydrogen bond between the NH of the indole and the propionate of the heme. Interestingly, the other depicts the thiocyanate group at the same position as the cyanide in the crystal MPO structure (code 1DNW) and forms a hydrogen bond with Gln91 but no stacking (See supporting information). Furthermore, two amino groups were introduced in order to increase the polarity at the extremity of the side chain (Compound 46). The two diastereoisomers were docked in two different protonated forms either on the N1 or N4. The predicted affinity differs between all forms by at most 1.3 kcal/mol. Both feature either a stacking or a shifted stacking and one or two salt bridges and hydrogen bonds with Glu102 and one heme propionate. The fluorine atom oriented towards the heme iron makes one hydrogen bond with Gln91 (see supporting information). Interestingly, compound 50 features an interaction between the nitrile group of the side chain and the heme iron -

but exhibits no stacking of the indole ring. The position of the nitrile almost fits the CN position in the crystal structure of 1DNW. Using an induced fit protocol a similar binding mode is observed as well as two additional differing positions with either a shifted stacking with the cyanide oriented towards the heme iron or stacking of the indole moiety and the cyanide forming a hydrogen bond with Phe147 (see supporting information). By contrast, in compound 53 the carboxylic group rather than the nitrile group occupies the pocket on top of the heme iron (see supporting information). Compound 57 that contains an amide group at the side chain extremity is predicted to bind in the active site through a stacking arrangement with the heme pyrrole ring D and two H-bonds between the amide group and Glu102 and Arg333. A hydrogen bond is also observed between the fluorine atom and Gln91 residue (Figure 3D and see supporting information). With the exception of compounds 44-46 and 53, all compounds in which the amino group was replaced by various polar moieties (sulfonamide, hydroxylamine, thiocyanate, nitrile and amide) showed decreased predicted affinities.


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Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

A series of 3-(hydroxyalkyl) indole derivatives 1, 2, 3, 4, 5, 6, 8, 9, and 10 was obtained following a general procedure based on Fischer indole synthesis

29

, starting from commercially

available phenylhydrazine hydrochloride derivatives and either cyclic enol ether (compounds 1-6) or 6-hydroxyhexanal (7), which was obtained by the reduction of ε-caprolactone using DIBAL-H as indicating in scheme 1 (compounds 8-10)27,

30

. The hydroxyl groups in these compounds were

transformed first into mesylate in presence of methanesulfonyl chloride and amino-basic compound to give compounds 11-19 (scheme 2) 31. Mesylate groups in compounds 11, 12, 13, 14, 17, and 19 were then converted into azidoalkane by adding NaN3 in DMSO at 100 °C to give compounds 20, 21, 22, 23, 24, and 25 in good yields

31

. These azidoalkane compounds were hydrogenated using

LiAlH4 (or C/Pd for the compounds 22 and 25) to create compounds 26, 27, 28, 29, 30, and 46 (scheme 2) 32. Starting from mesylate compounds 11, 12, 15, and 16, the compounds from 31 to 45 were obtained using the same synthetic method but in different conditions (scheme 3) 33. Compounds with one amide group at the side chain extremity 54-61, were synthesized starting from mesylate derivatives 11, 12, 15, 16, 17, 18, and 19. The mesylate groups were converted to nitrile to give the compounds 47-53 34 which were further hydrolyzed by KOH in t-BuOH to produce the carboxamide 54-59 (scheme 4)

35

. The N-methyl and N-ethyl derivatives of amide 57 (compounds 60 and 61

respectively) were obtained by nucleophilic substitution using the suitable bromoalkane. Inhibition of the chlorination activity of myeloperoxidase A microplate reader was used to monitor the inhibition of MPO-mediated taurine chlorination in a high-throughput screening mode26. Table 1 lists the IC50 values of the 3-alkylindole analogs. These data clearly demonstrate that the fluorotryptamine compounds with 4 and 5 atoms between the indole ring and the amino group are among the most active compounds. However, compound 38 with 6 atoms between indole ring and amino group is inactive. Compound 37 with a fluorine atom on position 7 and containing 5 atoms between indole ring and amino group, shows the best activity. ACS Paragon Plus Environment

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Compounds with fluorine at position 7 (compounds 26, 27, 29 & 37) and those with fluorine at 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

position 5 (compounds A1-3 & 35)

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almost have the same activity. In contrast, all compounds

substituted with one carboxyl group at position 5 on the indole ring have no effect on the chlorination activity of MPO (compound 28, 30, 53). Introduction of bulky and rather apolar substituents on the nitrogen of the side chain (Compounds 31 and 32) resulted in a slightly increased activity as compared to compounds with the same number of carbon atoms between the indole ring and the amino group. The IC50 of 3-(3-aminopropyl)-5-fluoro-1H-indole, compound A2, is 50 ± 8 nM, and that of N-methyl-5-fluoro-3-[3-(1-piperazinyl)propyl]-1H-indole is 350 ± 60 nM 27. Substitution with one hydroxyl ethyl group (compounds 33 and 34) significantly improves the inhibitory potency as compared to compounds with the same number of carbon atoms between the indole ring and the amino group (compounds A1-2

27

). Introduction of one sulfur atom at the third

position of the alkyl side chain (compounds 35 and 37) slightly increases the activity as compared to the compound with the same length of the alkyl chain (compound A4) while its insertion at the fourth position (compounds 36 & 38) has various effects depending on the position of the fluorine atom on the indole ring (Table 1). In compounds featuring 3 to 5 atoms between the indole ring and the functional group, the IC50 values also show that the presence of a polar group at the chain extremity is crucial to achieve a high MPO inhibition. Indeed, amine, sulfonamide, hydroxylamine, thiocyanate or amide group, which can form H-bonds and/or salt bridges, brings additional interactions in particular with Glu102 and one proprionate of the heme. However, addition of a second primary amine group on the side chain Cβ (compound 46) decreases the activity as compared to compound A3. Besides the effect of these newly designed compounds on the chlorination of taurine, their interaction with the MPO-mediated oxidation of LDL was determined by an ELISA which is based on a mouse monoclonal antibody (Mab AG9) that specifically recognizes MPO-oxidized APO B100 on LDL 13. The IC50 values for the inhibition of MPO-dependent LDL oxidation by the designed compounds are summarized in Table 2. Most of these compounds inhibit LDL oxidation at nanomolar concentrations. The IC50 values measured with this method are correlated with the taurine ACS Paragon Plus Environment

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chlorination inhibition assay. Compounds 35 and 37 exhibit the best LDL oxidation inhibitory 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

activities. As seen in the taurine chlorination assay, compounds containing an alkyl side chain with no polar group (compounds 39, 40, and 41) are bad inhibitors with IC50 values in the micromolar range. No inhibiting activity was measured for compounds containing one substituted carboxyl group at position 5 on the indole ring. Interaction with the redox intermediates of myeloperoxidase In order to determine the mechanism of inhibition of MPO by the newly synthesized compounds, a transient kinetic study was performed using multi-mixing stopped-flow spectroscopy. Native ferric MPO [Fe(III)…Por] is oxidized (k1) by H2O2 thereby producing water and Compound I {oxoiron(IV) combined with a porphyrin cation radical: [Fe(IV)=O…Por•+]} (Figure 4). In the halogenation cycle Compound I is directly reduced back to the resting state (k2) by chloride [or other (pseudo-)halides] thereby releasing hypochlorous acid. Alternatively, in the presence of one-electron donors the peroxidase pathway is followed, including Compound I reduction to Compound II [Fe(IV)-OH…Por] (k3) and Compound II reduction to the ferric state (k4) (Figure 4) 36. In the present study, Compound I reduction was performed and probed for reaction with some selected molecules including active and non-active compounds (28, 40, 42, 37, and 57). With all tested compounds, there was a direct and fast transition of Compound I to Compound II (Soret maximum at 456 nm) with clear isosbestic points (Figure 5A, representative example: compound 37). The reactions were monophasic (Figure 5B) and from the slope of the linear plot of kobs-values versus inhibitor concentration, the apparent bimolecular rate constant (k3) of Compound I to Compound II reduction was calculated (Figure 5C). Table 3 summarizes the k3 values of the selected molecules ranging from 1.0 × 106 M-1 s-1 to 3.7× 107 M-1 s-1. It clearly indicates that all the selected molecules behaved as very good one-electron donors of Compound I. By contrast, the reaction rates of these molecules with Compound II shows a broad variety. The compounds which have no effect on the chlorination activity of MPO (compare with Tables 1 & 2) showed k4 values > 103 M-1 s-1 suggesting that they are effectively able to close the peroxidase cycle (Figure 4). Those compounds that have an inhibitory effect on MPO are bad electron donors of Compound II and promote the ACS Paragon Plus Environment

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accumulation of the enzyme in this redox state. This is clear by examining the initial phase of the 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

constant absorbance at 456 nm (see Figure 5E for compound 37). The length of this phase is strictly correlated with the amount of H2O2 present in the system. After complete hydrogen peroxide consumption, Compound II was converted to the resting state (Figures 5D & 5E) in a concentrationdependent manner (Figure 5F). Comparison of IC50-values (Tables 1 & 2) with the respective k3/k4 ratios unequivocally demonstrates that the inhibitory activity increases with increasing k3/k4 ratios. Accumulation of Compound II shifts the enzyme from the halogenation to the peroxidase cycle thereby decreasing the release of HOCl. To further understand the mechanism of inhibition, the redox properties of the synthesized compounds were determined. The corresponding values of the relevant redox couples of MPO were published. The standard reduction potential (E°’) of the couples Compound I/Compound II and Compound I/ferric MPO are 1.35 V and 0.97 V, respectively 36. The one-electron reduction potential of the inhibitors was determined by cyclic-voltammetry (Ag/AgCl 3 M NaCl) in phosphate buffer, pH 7.4, and is referenced to the normal hydrogen electrode (NHE). E’ values vary between 808 and 923 mV (Table 1). Introduction of a sulfur atom has almost no effect to the redox properties of the respective compound. A fluorine atom or a carboxyl group on the indole moiety decreases the E’ value as compared to that of tryptamine (1.02 V) whereas it significantly increases its redox potential value compared to that of serotonin (0.65 V). Like Tyr and 5HT, all our active or non-active compounds have E’ values dramatically smaller than that of Compound I and even below that of Compound II. Serotonin reuptake inhibition The structure of 3-(alkylamine)-5-fluoroindole is very similar to that of serotonin. As a matter of fact, the homotryptamine derivatives are excellent SERT inhibitors

37

. Consequently, the

activity of the synthesized molecules was tested on this transporter. The Ki’s are listed in Table 2. The most striking observations are the following: Changing the position of the fluorine atom cannot significantly decrease the affinity for SERT compared to previous compounds (2-47 nM for compounds A1-4, data not published). A carboxylic acid group at position 5 of the indole moiety ACS Paragon Plus Environment

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abolishes the inhibition of SERT as well as of MPO (compounds 28, 30, & 53). In general, 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

fluoroindole compounds bearing an amino group or a substituted amino group (compounds 26, 27, 29, 31-37) have the same activity on SERT than previous compounds and the Ki values are in a similar range than the IC50 for MPO (the specificity index Ki/IC50 ranging from 1 to 8, table 2). Compound 38 has a reduced activity on SERT but is a bad inhibitor of MPO. Compounds with an alkyl group on the side chain extremity (compounds 39-41) show no interaction with SERT but are bad MPO inhibitors. Compounds with functional groups (amide, cyanide, thiocyanate and sulfonamide) at the end of the side chain (compounds 42, 45, 50, & 57) exhibit a low affinity for SERT but act as MPO inhibitors. Compound 57 with one amide group has an IC50 of 18 nM on MPO, while its Ki on SERT is 631 nM (Ki/IC50= 35). Compound 45 with a thiocyanate is also very active on MPO (IC50 = 5 nM) and has a weak activity on SERT (Ki = 212 nM). Compound 42 with one sulfonamide group features an IC50 on MPO of 14 nM and a Ki value on SERT of 174 nM. Two further compounds draw attention. Firstly, compound 43 albeit being a homolog of compound 42 shows a significantly better affinity for SERT. Secondly, compound 36 is the most active one on SERT (Ki = 0.9 nM), whereas its isomer 38 with fluorine in position 7 shows a dramatic decrease in inhibition (Ki = 539 nM). Optimization of 3-amidoalkylindole derivatives Based on inhibition of taurine chlorination and LDL oxidation as well as on the interaction with SERT, compound 57 with one amide group at the side chain extremity appeared as a potentially good drug candidate. It was therefore chosen to study the structure-activity relationships in more details. The series of 3-(amidoalkyl)indole (compounds 54-61) was designed based on the docking of compound 57. The effect(s) of a variation in the length of the side chain, a substitution of fluorine atom in position 5 or 7 of the indole ring and an introduction of alkyl groups on the nitrogen of the amide group were probed by docking. Table 4 shows that all these designed compounds have almost the same predicted affinity (∆G ranging from -5.1 to -6.1 kcal/mol). Compounds 55 and 57 with 3 carbons in the side chain and without substitutions on the amide show stacking of the indole moiety with one heme propionate. All other compounds feature a shifted stacking. The fluorine atom is ACS Paragon Plus Environment

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oriented towards the heme iron only for compound 57. All the compounds with 3 and 4 carbons in 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

the side chain (compounds 55 and 58-61) make two hydrogen bonds between the amido group and Glu102 as well as the heme propionate. These 3-amidoalkylindole compounds were assayed using the taurine chloramine assay to determine the respective IC50 values (Table 4). The most active molecules are compounds 55 and 57 that contain 3 carbons between indole ring and the amido group. Compounds 58 and 59 with 4 carbon atoms have a slightly reduced activity. Compounds with the fluorine atom in position 5 are more active as the side chain contains 2 or 3 carbons with the amido group. Introduction of alkyl groups such as methyl (compound 60) and ethyl on the amido group (compound 61) decreases the activity. Thus, the most potent compound contains 3 carbon atoms between the indole ring and the amido group (compound 57). Apart from compound 61 with a substituted ethyl group on the amide nitrogen, all other designed molecules were able to inhibit the oxidation of LDL at about 10-8 M (Table 4). The LDL oxidation inhibition test also demonstrates that compounds with 2 carbons between the indole ring and the amide (compounds 54 and 56) have a higher activity on LDL oxidation than on MPO inhibition. Compounds 55 and 57 are the most potent 3-amidoalkylindole inhibitors of the MPO-mediated oxidation of LDL. Toxicity Test Two compounds were tested in vivo in order to study their toxicity and adverse effects. Compound 37, which had the best activity on MPO among all the molecules studied here, was administered by intraperitoneal injection to Wistar Han male rats (mean B.W. ~ 285 g) after dissolution in physiological solution [alcohol/water 1:3 (v/v)]. The rats were divided into 2 groups; each group consisted of 3 rats. The first group (R1, R2 and R3) received a single dose 10 mg/kg of compound 37 while the second one (R4, R5 and R6) received a single dose of 100 mg/kg. No clinical observation was reported for the first group, whereas in the second group piloerection was observed in all individuals of this group 48 h after administration. Slight paralysis of hind limbs was also reported in these rats (R4, R5 and R6). However, a complete recovery of clinical parameters was observed within 48 h. No mortality was observed in the two groups 4 days after administration. ACS Paragon Plus Environment

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The rats were then killed by exsanguination and the organs were taken at necropsy. Gross 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

examination revealed liver lesions in the individuals receiving the high dose (100 mg/kg). The second selected molecule tested for in vivo risk assessment is compound 57, which - in addition to high activity on MPO- had the highest selectivity on MPO. Three groups of Wistar Han male rats (mean body of 285 g) received a single dose of either 1, 10, or 100 mg/kg of compound 57 by intraperitoneal injection. Each group consisted of three rats (R’1, R’2, and R’3) for 1 mg/kg, (R’4, R’5, and R’6) for 10 mg/kg and (R’7, R’8, and R’9) for 100 mg/kg. No clinical features were reported for the first and second group, with 1 and 10 mg/kg, respectively. In the third group, piloerection and prostrated postures were observed during the first 24 h after exposure to 100 mg/kg of compound 57. However, no significant clinical observation was found in this group 24 h after the administration and the recovery, was complete in terms of clinical signs. No mortality was observed in all groups receiving compound 57. After 4 days, the rats were killed by exsanguination and blood and organs (liver, kidney, heart, and muscle) were collected. No macroscopic lesions were reported at necropsy in all treated groups. The in v ivo toxicological study demonstrates that both candidate inhibitors cause no significant side effects after exposure to a single intraperitoneal dose at low and medium concentrations (1 and 10 mg/kg). In such conditions, no mortality was reported after exposure to compounds 37 and 57, even at the highest dose of 100 mg/kg. At necropsy, only the rats treated with the high dose of compound 37 (100 mg/kg) demonstrated some signs of macroscopic liver lesions. As the plasma concentration of MPO in patients suffering from coronary artery diseases is 100 – 300 ng/ml (0.7 – 2 nM), the dose of 10 mg/kg seems to be higher than the required clinical dose which could confer a wide therapeutic margin to our compounds38.


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Discussion 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

The design of novel 3-alkylindole derivatives aimed at obtaining potent MPO inhibitors that do not block SERT. It was based on structure-based docking of the 5-fluorotryptamine compounds to the active site of MPO

27

. After validation of the docking procedure with cristallographic data of

SHA, all designed derivatives can be docked into the heme pocket which provides information on their virtual interactions with the target. The respective docking energy binding values range from 4.0 to -10.7 kcal/mol. Given the difficulty to evaluate the affinity using the scoring functions developed for the docking programs it is always perilous to rank the ligands based on this property. Some compounds predicted to have a high affinity are inactive (i.e. compounds 28 & 30). Instead, we rationally examined the various interactions formed by the ligands. The best poses of all compounds feature a stacking between the indole and the pyrrole moieties though not ideal in all cases. All compounds with a charged or polar group at the side chain extremity form salt bridges and/or hydrogen bonds with Glu102 and one heme propionate. These interactions were already identified in docking calculations as being important for the affinity of a series of 3-(aminoalkyl)-5fluoroindole derivatives 27. Moreover, in a virtual high-throughput screening study carried out on a database of 700.000 compounds 7 out of the 8 compounds that exhibited a potent inhibition, featured also stacking and an interaction with Glu102 in their docked positions 36. The presence of a hydroxyl group on the side chain extremity (compounds 33, 34 and 44) induces positions with no stacking but with the side chain extremity on the top of the heme iron similar to the position of hydroxylamine in the SHA-MPO complex structure. As far as the specificity of inhibitors is concerned, several rules can be derived from our data. They help to understand how the activity of good inhibiting candidates on MPO can be decoupled from their interaction with SERT. As for the substituents on the indole ring, the shift of fluorine from position 5 to 7 modulates the predicted poses in the catalytic pocket of MPO without affecting the activity of such compounds. Addition of a carboxylic group on the indole ring generates compounds with no activity on both MPO and SERT. Introduction of this voluminous anionic substituent seems to be deleterious for the activities of these compounds. ACS Paragon Plus Environment

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As for the side chain containing an amino group at its extremity, neither the length of the chain 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

nor the substitution on the nitrogen seem to decouple MPO from SERT inhibition. It is noteworthy that previous experiments show that substitution on the nitrogen is in favor of a SERT inhibition 37. Addition of a sulfur atom on the side chain was designed in order to increase the reaction with Compound I of MPO through modification of the redox potential. However, these molecules exhibit activities similar to the non-sulfur counterparts. Moreover, their interaction with SERT remains the same. Thus, the key element seems to be the amino group and its interactions as predicted by the docking calculations. This is supported by the fact that in its absence, with or without sulfur atom (compounds 39, 40, and 41); the compounds are inactive due to loss of salt bridges and hydrogen bonds between the amino group and Glu102 and/or one heme propionate. Only one hydrogen bond between the NH of indole and one heme propionate is formed. Likewise, this modulation of the side chain extremity gives inactive compounds on SERT as well. It is generally admitted that selective serotonin reuptake inhibitors (SSRI) have to bear a positively charged group such as an amine to feature a potent activity. Addition of a supplementary amino function on the side chain (compound 46) decreases the inhibiting activity on MPO but increases the affinity for SERT. Modifying the amino group on the side chain into other polar groups such as sulfonamide, thiocyanate, hydroxylamine, and nitrile generates compounds that maintain the inhibition potency on MPO (IC50: 11-63 nM) whereas the effect on SERT varies (Ki: 12-212 nM). The loss of the amino group decreases in all cases the activity on SERT (compounds 42, 45, 50 and 57). Only compound 43 with a sulfonamide group at the side chain extremity and a 3 carbon side chain length keeps a good inhibition potency. Substitution of the amino group by an amide leads to a dramatic decrease in the affinity for SERT with a specificity index of 35 (Ki/IC50) showing a more potent activity towards MPO. A study of the structure-activity relationship on a relatively small number of derivatives demonstrates that the initial amide compound with 3 carbons between the indole ring and the amido group and the fluorine atom in position 5 has the highest activity on MPO (compound 57).


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There are numerous pieces of evidence that MPO-dependent LDL oxidation plays important 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

roles in atherogenesis. Furthermore, it is also known that the complex of MPO with ApoB-100 of LDL mainly forms through electrostatic interactions and modifies the chemical and steric requirements for efficient penetration of substrates and/or inhibitors into the binding site 13. In this context, the inhibiting potency of the new compounds on the LDL oxidation carried out by the MPO/H2O2/Cl- system has been assessed. Most of those that are active on isolated MPO conserve their inhibitory potency in the MPO complex suggesting that they bound to LDL and could enter the catalytic pocket of MPO. The kinetic study of the reaction of several designed molecules with Compound I and Compound II of MPO show that these molecules are electron donors for both redox intermediates. Compounds 37, 42, and 57, which have a high inhibitory potency, quickly react with Compound I whereas their reaction with Compound II is very slow. This is illustrated by high k3/k4 ratios (see Table 4) that demonstrates these inhibitors accumulate Compound II and shift MPO from the chlorination cycle to the peroxidase cycle (Figure 4). By contrast, compounds that are inactive (e.g. compound 28 that is predicted to bind to MPO with a good affinity) are effectively able to close the peroxidase cycle by reduction of Compound II (k4 > 103 M-1 s-1) and thus enable MPO to act in the halogenation cycle. This is underlined by low k3/k4 ratios together with high values of IC50. In order to explain the correlation between the inhibition potency and the reactivity with MPOCompound II, the following hypothesis was proposed by Jantschko et al. (2005): MPO inhibitors must achieve the redox potential of 0.97 V ≪ E°′ (A• /AH) < 1.35 V where 0.97 and 1.35 V are the redox potentials of the native MPO/Compound II and of the Compound II/Compound I, respectively. Thus, molecules fulfilling this criterion can easily react with Compound I but not with Compound II 36

. However, our compounds (including active and non-active) have reduction potentials lower or

close to 0.97 V suggesting that other factors than pure thermodynamic ones must play a key role in the inhibiting activity. A critical step in the inhibition of MPO by alkylindole derivatives is the dissociation of the reaction product (A●) from the heme cavity and MPO in its Compound II and ferric state (Figure 4). It could be hypothesized that the oxidized form of good inhibitors dissociates ACS Paragon Plus Environment

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slower from the active site compared to less active compounds. Functional groups on the side chain 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

extremity such as one amine or one polar group (H-bond acceptor and/or donor group such as amide, sulfonamide and nitrile) apparently favor this process. This observation is also supported by other studies that reported that the MPO-inhibitor disassociation rate plays a critical role in the case of quercetin 23 derivatives and salicylhydroxamic acid 20.

Conclusion Structure-based drug design led to the synthesis of novel 3-alkylindole derivatives that were pharmacologically evaluated for their interaction with MPO and SERT. For most compounds the modes for binding to MPO predicted by docking experiments feature a stacking of the indole moiety with the pyrrol D of the heme and several salt bridges and/or hydrogen bonds with Glu102 and one propionic heme group. Among these new fluorotryptamine derivatives, compound 57, in which the amino group is replaced by an amido group on the side chain extremity, showed a promising specificity towards MPO. It acts as reversible MPO inhibitor and efficiently prevents MPOdependent LDL oxidation. According to preliminary toxicological tests, it seems well tolerated in acute exposure. These results prompt us to investigate the in vivo impact of this new compound in a suitable mouse model.


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Experimental section 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Synthesis 1

H- and

13

C-NMR spectra were taken on a Bruker Avance 300M Hz spectrometer

(Wissemburg, France) at 293 K. Chemical shifts (δ) are given in ppm relative to TMS and the coupling constants are expressed in Hz. Infrared spectroscopic analysis was performed with a Shimadzu (Kyoto, Japan) IRAffinity-1spectrophotometer and the peaks data are given in cm-1. Mass spectrometric data were obtained on a QTOF 6520 (Agilent, Palo Alto, CA, USA), positive mode, ESI, mode TOF, by diffusion of 0.5 mL/min, by mobile phase 0.1 M HCOOH: CH3OH (50:50), (VCAP 3500, Source T: 350 °C, fragmentation: 110 v, Shimer: 65 v). All reactions were followed by TLC carried out on Fluka (Bornem, Belgium) PET-foils silica gel 60 ®, and compounds were visualized by UV and by spraying Van Urk reagent (0.125 g of p-dimethylaminobenzaldehyde dissolved in 100 mL of 65% sulfuric acid with addition of 0.1 mL of 5% ferric chloride). Column chromatographies were performed with EchoChrom MP silica 63-200 from MP Biomedicals (Santa anna, USA). Organic solutions were dried over Na2SO4 and concentrated with a Buchi rotatory evaporator

(Flawil,

Switzerland).

Starting

material

4-phenylhydrazine

hydrochloride,

7-

phenylhydrazine hydrochloride, 4-hydrazinobenzoic acid, ε-caprolactone, dihydrofuran, and dihydropyran were available from Sigma-Aldrich (Bornem, Belgium). Microwave-promoted reactions were performed with a Start S microwave oven from Milestone (Sorisole, Italy). Purity was determined with LC-DAD (Waters, Milfors, USA) using a 150 mm × 4.6 mm Symmetry C18 column at a mobile phase flow rate of 1 mL/min. The mobile phase was a mixture of methanol (350 mL) and a KH2PO4 solution (0.07 M in water, 650 mL) adjusted to pH 3.0 with a 34 wt % H3PO4 solution. The chromatograms were extracted at maximum absorption wavelengths by using the Max Plot extraction mode. The purity was ≥ 95 % for all the compounds.27 Synthesis of 3-(hydroxyalkyl) indole derivatives (compounds 1-6 and 8-10). A solution of 4-fluorophenylhydrazine

hydrochloride,

7-fluorophenylhydrazine

hydrochloride

or

4-

hydrazinobezoic acid (6.9 mmol) in 4 wt % aqueous H2SO4 (10 mL) (in case of 4-hydrazinobezoic acid H2SO4:20 wt %) and DMA (10 mL) was heated at 100 °C under microwave. Dropwise addition ACS Paragon Plus Environment

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of the corresponding enol lactone (for compounds 1-6), or hydroxyaldehyde (for compounds 8-10) 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

(6.9 mmol) over 2 min. Reaction was heated at 175 °C (temperature ramp from room temperature to 100 °C over 8 min, followed by addition of the reagent, heating to 175 °C over 8 min and maintaining this temperature for 30 min (1000 W). The reaction mixture was cooled to room temperature and extracted two times with EtOAc (25 mL), and the organic layer was washed with water and evaporated. The crude material was purified by column chromatography (eluent CH2Cl2/EtOAc 4:1). Synthesis of Compound 7. A 1 M DIBAL-H solution in CH2Cl2 (7.8 mL, 7.38 mmol) was added slowly to a solution of the lactone (6.6 mmol) in dry CH2Cl2 at −78 °C. After stirring at the same temperature for 1 h, methanol (8.9 mL) was added dropwise and the mixture poured into 0.5 M HCl (200 mL). After stirring for additional 1 h at room temperature, the organic layer was separated. The aqueous layer was extracted with CH2Cl2 (3 × 20 mL), and the combined organic layers were dried with Na2SO4 and evaporated. The residue purified by flash chromatography (first CH2Cl2, then CH2Cl2:EtOAc (60:40)). The second fraction was evaporated to give the desired product. Synthesis of mesylate derivatives (compounds 11-19). A solution of alcohol derivative (5.5 mmol) in CH2Cl2 (25 mL) was cooled with an ice bath. To this solution TEA (0.44 mL, 5.8 mmol) and methanesulfonyl chloride (0.77 mL, 5.5 mmol) were added (the carboxylic alcohol derivatives were dissolved in pyridine and no TEA was added). The solution was stirred at room temperature for 2 h. Then it was washed with a saturated solution of NaHCO3 and H2O and then dried over Na2SO4 and filtered. The solvent was evaporated under reduced pressure. The crude material was purified by flash chromatography (CH2Cl2/EtOAc 4:1). Synthesis of azide derivatives (compounds 20-25). To a solution of sulfonate (3.6 mmol) in DMSO (10 mL), NaN3 (3.6 mmol) was added, and the solution was stirred at room temperature for 1 h at 100 °C. Then H2O (30 mL) and CH2Cl2 (or EtOAc for compounds 15 and 29) (30 mL) were added. After decantation, the organic layer was washed with water, dried with Na2SO4, filtered, and evaporated under reduced pressure. The residue was purified by column chromatography (CH2Cl2).


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Synthesis of amine derivatives (compounds 26-30 and 46). Palladium on charcoal 10% (50 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

mg) was added to a solution of azide derivative (4.6 mmol) in EtOH (20 mL). The suspension was stirred under H2 (4.1 bar) overnight. After filtration on Celite, the solvent was evaporated under reduced pressure. For compounds 28 and 30, after filtration H2O (30 mL) and EtOAc (30 mL) were added, the aqueous layer was collected and evaporated to give the pure compounds. The residue was dissolved in ether, extracted with 1 M HCl, and the resulting solution was washed with ether. A solution of 1 M NaOH was added (pH ≈ 10), and the mixture was extracted with diethylether. The organic layer was washed with water, dried over Na2SO4, and evaporated under reduced pressure. A saturated solution of anhydrous oxalic acid in ether was added to the ethereal solution and the resulting solid was filtered, washed with ether, and dried under vacuum. Synthesis of compounds 31−45. A solution of mesylate 11, 12, 15 or 16 (3.6 mmol) in dioxane (5 mL) was added slowly through an addition funnel to a refluxing solution of the amine or thiol (0.26 mol) in dioxane (15 mL) at 100 °C (some compounds have special conditions; see the supporting information for details). After the addition was completed, the reaction medium was stirred at this temperature for 4 h. After cooling, the mixture was treated with water (20 mL) and extracted with EtOAc (30 mL). The organic layer was dried over Na2SO4 and evaporated to dryness to afford a crude product. The residue was dissolved in 0.1 M HCl. This solution was washed with diethylether and rendered alkaline (pH ≈ 10) with a solution of 1 M NaOH. The mixture was extracted with ether. The organic layer was washed with water, dried over Na2SO4, and evaporated to afford the pure compounds. Some compounds were transformed into oxalic salts, by adding saturated solution of anhydrous oxalic acid in ether to the ethereal solution of compounds, and then the resulting solid was filtered, washed with ether, and dried under vacuum. Synthesis of nitrile derivatives (compounds 47 - 53). To a solution of NaCN (2 g, 41 mmol) in H2O (10 mL) and DMA (10 mL) mesylate derivative (11, 12, 15-19) (7.4 mmol) in DMA (10 mL) was added dropwise and heated at 100 °C. The reaction was stirred at this temperature for 2 h and then cooled to room temperature and extracted with EtOAc. The organic layer was washed with


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water, dried on Na2SO4, and evaporated. The residue was purified by flash chromatography 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

(CH2Cl2/EtOAc 4:1). Synthesis of amide derivatives (compounds 54-61). To a solution of nitrile (47 - 53) (11 mmol) in t-BuOH (40 mL) was added finely powdered KOH (5 g, 89 mmol). The solvent was refluxed for 2 h with stirring. Then a saturated solution of NaCl (50 mL) (or alkyl bromide for the substituted amides) was added and stirred 1 hour. The resulting mixture was extracted three times with CH2Cl2. Flash chromatography was performed (CH2Cl2/EtOAc 4:1 then CH2Cl2/methanol 1:1). The fractions obtained with the second solvent mixture were evaporated. Docking protocols The X-ray structure of human myeloperoxidase complexed to cyanide and thiocyanate (PDB code: 1DNW) was used as the target structure to endeavor the docking studies 39. The X-ray water and other ligand molecules were removed from the active site. The ligand input files were prepared according to the following procedure. The initial 3D structures of the ligands were generated using the Ligprep module from Schrodinger OPLS force-field ligands

40, 41

and the ligand partial charges were ascribed using the

42

. The Epik program was used to predict the different protonation states of all

43

. Docking was performed using the Glide program (version 5.6) which approximates a

systematic search of positions, orientations, and conformations of the ligand in the receptor binding site using a series of hierarchical filters (www.schrodinger.com). The Glide XP docking protocol and scoring function were used. The remaining parameters were set to their default values. Docking experiments including protein flexibility (induced-fit protocol from Schrodinger Inc.) in the form of flexible side chain residues were also carried out for a few molecules (see Table 1) when the Glide docking protocol performed with a rigid receptor did not produce any positions of ligands featuring a stacking interaction of the indole moiety of the compounds. Enzymatic activity assays Taurine chlorination assay. The assay is based on the production of taurine chloramine produced by the MPO/H2O2/Cl− system in the presence of a selected inhibitor at defined ACS Paragon Plus Environment

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concentration. The reaction mixture contained the following reagents in a final volume of 200 µL: 10 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

mM phosphate buffer (pH 7.4, 300 mM NaCl), 15 mM taurine, the compound to be tested (up to 20 µM), and the fixed amount of the recombinant MPO (6.6 µL of MPO batch solution diluted 2.5 times, 40 nM). When necessary, the volume was adjusted with water. This mixture was incubated at 37 °C and the reaction initiated with 10.0 µL of H2O2 (100 µM). After 5 min, the reaction was stopped by the addition of 10 µL of catalase (8 U/µL). To determine the amount of taurine chloramines produced, 50 µL of 1.35 mM solution of thionitrobenzoic acid were added and the volume adjusted to 300 µL with water. Then, the absorbance of the solutions was measured at 412 nm with a microplate reader and the curve of the absorbance as a function of the inhibitor concentration was plotted. IC50 values were then determined using standard procedures taking into account the absence of hydrogen peroxide as 100% of inhibition and the absence of inhibitors as 0% of inhibition. LDL Oxidation-mediated by MPO. Recombinant MPO was prepared as previously described 44. Each batch solution is characterized by its protein concentration (mg/mL), its activity (U/mL), and its specific activity (U/mg). The chlorination activity was determined according to Hewson and Hager

45

. Human plasma served for the isolation of LDL by ultracentrifugation

according to Havel et al

44

. Before oxidation, the LDL fraction (1.019 < d < 1.067 g/mL) was

desalted by two consecutive passages through PD10 gel-filtration columns (Amersham Biosciences, The Netherlands) using PBS buffer. The different steps were carried out in the dark, and the protein concentration was measured by the Lowry assay for both MPO and LDL. LDL oxidation was carried out at 37 °C in a final volume of 500 µL. The reaction mixture contained the following reagents at the final concentrations indicated between brackets: pH 7.2, PBS buffer, MPO (1 µg/mL), LDL (1000 µg/mL), 2 µL 1 M HCl (4 mM), one of the compounds at different concentrations, and H2O2 (100 µM). The reaction was stopped after 5 min by cooling the tubes in ice. The assay was performed as described by Moguilevsky et al. in a NUNC maxisorp plate (VWR, Zaventem, Belgium): 200 ng/well of LDL was coated overnight at 4 °C in a sodium bicarbonate pH 9.8 buffer (100 µL) 46 Afterward, the plate was washed with TBS 80 buffer and then ACS Paragon Plus Environment

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saturated during 1 h at 37 °C with the PBS buffer containing 1% BSA (150 µL/well). After washing 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

the wells twice with the TBS 80 buffer, the monoclonal antibody Mab AG9 (200 ng/well) obtained according to a standard protocol and as previously described was added as a diluted solution in PBS buffer with 0.5% BSA and 0.1% of Polysorbate 20. After incubation for 1 h at 37 °C, the plate was washed four times with the TBS 80 buffer and a 3000 times diluted solution of IgG antimouse alkaline phosphatase (Promega, Leiden, The Netherlands) in the same buffer was added (100 µL/well). The wells were washed again four times and a revelation solution (150 µL/well) containing 5 mg of para-nitrophenyl phosphate in 5 mL of diethanolamine buffer was added for 30 min at room temperature. The reaction was stopped with 60 µL/well of 3 N NaOH solution. The measurement of the absorbance was performed at 405 nm with a background correction at 655 nm with a Bio-Rad photometer for a 96-well plate (Bio-Rad laboratories, CA, USA). Results were expressed as IC50 taking the LDL oxidation in absence of candidate inhibitors as the 100% and the native LDL as the 0% 13. Transient State Kinetics Highly purified myeloperoxidase of a purity index (A430/A280) of a least 0.86 was purchased from Planta Natural Products (http://www.planta.at). Its concentration was calculated using ε430 = 91 mM−1 cm−1. Hydrogen peroxide, obtained from a 30% solution was diluted and the concentration determined by absorbance measurement at 240 nm where the extinction coefficient is 39.4 M−1 cm−1. Alkylindole derivative stock solutions were prepared in dimethylsulfoxide (DMSO) and stored in dark flasks. Dilution was performed with 200 mM phosphate buffer, pH 7.4, to a final DMF concentration of 2% (v/v) in all assays. The multimixing stopped-flow measurements were performed with the Applied Photophysics (UK) instrument SX-18MV. When 100 µL were shot into a flow cell having a 1 cm light path, the fastest time for mixing two solutions and recording the first data point was 1.3 ms. Kinetics were followed both at single wavelength and by using a diode-array detector. At least three determinations (2000 data points) of pseudo-first-order rate constants (kobs) were performed for each substrate concentration (pH 7.4, 25 °C) and the mean value was used in the calculation of the second-order ACS Paragon Plus Environment

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rate constants, which were calculated from the slope of the line defined by a plot of kobs versus 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

substrate concentration. To allow calculation of pseudo-first-order rates, the concentrations of substrates were at least 5 times in excess of the enzyme. Conditions of MPO Compound I formation were described recently 47. Typically, 8 µM MPO were premixed with 80 µM H2O2, and after a delay time of 20 ms, Compound I was allowed to react with varying concentrations of alkylindole derivative in 200 mM phosphate buffer, pH 7.4. The reactions were followed at the Soret maximum of Compound II (456 nm). Compound II formation and reduction could be followed in one measurement. The resulting biphasic curves at 456 nm showed the initial formation of Compound II and its subsequent reduction to native MPO by the alkylindole derivatives (decrease in absorbance at 456 nm). Assessment of 5-HT reuptake inhibition. Inhibition assessment of 5-HT reuptake was performed as follows. The mammalian expression plasmid pcDNA3.1 containing human 5-HT transporter (hSERT) cDNA has been described previously by Kristensen et al

48

. HEK-293 MSR cells (Invitrogen) were cultured as

monolayers in Dulbecco´s modified Eagle´s medium (BioWhitaker) supplemented with 10% fetal calf serum (Invitrogen), 100 units/mL penicillin, 100 µg/mL streptomycin (BioWhitaker) and 6 µg/mL Geneticin (Invitrogen) at 95% humidity, 5% p(CO2) and 37ºC. Cells were detached from the culture flasks by Versene (Invitrogen) and trypsin/EDTA (BioWhitaker) treatment for subculturing or seeding into white TC-microtiter plates (Nunc). Transfection and measurement of [3H]5-HT (PerkinElmer Life Sciences) uptake was performed as described by Larsen et al. (2004) except that HEK-293 MSR cells (Invitrogen) were used instead of COS-1 cells 49. Toxicological tests Wistar Han male rats (Janvier Elevage, Le Genest-Saint-Isle/France) of 7 weeks old at start of the study and with a body weight between 250 and 300 g were used. The rats were individually housed with free access to water and received 30 to 35 g of pelleted food (Rat/Mouse 20% maintenance, RN-01-20K12; Carfil Quality) daily. During the whole duration of the experiment, rats ACS Paragon Plus Environment

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were exposed to a temperature of 22 ± 1 ° C and a circadian cycle of 12 hours. All animal 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

experiments were approved by the local Ethical Committee for animal care of the Institution (Faculty of Medicine & Pharmacy/UMons) Animals were divided into groups of three rats which received a single i.p. injection of either compound 37 or compound 57 dissolved in 2 ml of the vehicle (alcohol/water 1:3/V:V), or the vehicle alone. Compound 43 was tested at the doses of 10 and 100 mg/kg, while compound 57 was tested at 1, 10, and 100 mg/kg. Physical and clinical signs were recorded daily on each individual during the course of the study. After 4 days, the animals were killed by exsanguination and a gross examination of the organs was performed at necropsy. Determination of Redox potentials The experiments were achieved in a conventional three-electrode cell at 25±2 °C, calculated by a potentionstate Epsilon (BASinc. West Lafayette. USA). The working electrode was a glassy carbon disk polished with 0.05 lm alumina (Metkron) before each run, for linear cyclic voltammetry measurements. The auxiliary electrode was a platinum wire. The reference electrode was Ag/AgCl (3 M NaCl). Alkylindole derivatives were dissolved in 0.1 M phosphate buffer, pH= 7.4. The solutions were diluted by the same buffer until alkylindole concentration of 1×10-4 M. Cyclic voltammograms were obtained by a single cycle performed at a scan rate of 100 mV s-1. For the scan rate studies, the scan rate was varied from -200 to 1000 mV s-1

50

. E°’ values were obtained by

converting redox potentials from Ag/AgCl, 3 M NaCl to normal hydrogen electrode NHE by adding +198 mV

51

. Because the oxidation of our compounds is irreversible oxidation, it is difficult to

determine their E°’ values. However in our case E°’ values are slightly less than E’.


27


Page 28 of 50

SUPPORTING INFORMATION 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Docking figures of all compounds, synthetic procedures not described in the Experimental Section, spectral analyses of the compounds, cyclic voltammetry diagrams and purity data of the final compounds. This material is available free of charge via the Internet at http://pubs.acs.org. THE PDB CODE OF MPO IS 1DNW. CORRESPONDING AUTHOR INFORMATION: Dr Pierre Van Antwerpen; Phone: +3226505263, FAX: +3226505249, [email protected] †

these authors equally contributed to the manuscript

ACKNOWLEDGMENTS This study was supported by grants from the Belgian Fund for Scientific Research (FRSFNRS), no. 34553.08, a grant from the FER 2007 (ULB) and a grant from the Department of International Relationship (BRIC 2007). Martine Prévost is a Senior Research Associate at the FRSFNRS (Belgium). We thank Prof. M. Luhmer and Dr. R. D’Orazio from CIREM (ULB) for NMR spectra recording and the technical assistance supplied by Pia Høgh Plougmann for [3H]5-HT uptake is gratefully acknowledged. ABBREVIATIONS USED MPO, myeloperoxidase; 5-HT, serotonin; LDL, low density lipoprotein; APO B-100, apolipoprotein; SERT, serotonin transporter; SSRI, Selective serotonin reuptake inhibitor; SSRI, selective

serotonin

reuptake

inhibitor;

DIBAL-H,

diisobutylaluminium

hydride;

DMA,

dimethylacetamide; DMSO, dimethylsulfoxide; EtOAc, ethyl acetate; EtOH, ethanol; TEA, triethylamine; E°′, standard reduction potential; E′, reduction potential.


28

Page 29 of 50


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1. Agner, K. Acta.Chem.Scand. 1941, 2, (Suppl. 8) 1–62 2. 2. Harrison, J. E.; and Schultz, J. J.Biol.Chem. 1976, 251, 1371–1374. 3. Klebanoff, S. J. Myeloperoxidase: Friend and foe. J.Leukocyte.Biol. 2005, 77, 598–562. 4. Schultz, J.; and Kaminker, K. Arch.Biochem Biophys. 1962, 96, 465−467. 5. Salmon, S. E.; Cline, M. J.; Schultz, J.; and Lehrer, R. I. N.Engl.J.Med. 1970, 282, 250−253. 6. Segal, A.W. Annu.Rev.Immunol. 2005, 23, 197–223 7. Fiedler, T.J.; Davey, C.; Fenna, R. X-ray crystal structure and characterization of halidebinding

sites

of

human

myeloperoxidase

at

1.8

A

resolution.

J.Biol.Chem.

2000,275(16):11964-11971. 8. Arnhold, J.; Flemmig J. Human myeloperoxidase in innate and acquired immunity. Arch.Biochem.Biophys. 2010, 500(1):92-106. 9. Baldus, S.; Heeschen, C.; Meinertz, T.; Zeiher, A.M.; Eiserich, T. Münzel, J.P.; Simoons, M.L.; Hamm, C.W. Myeloperoxidase serum levels predict risk in patients with acute coronary syndromes. Circulation. 2003, 108, 1440–1445. 10. Libby, P.; Radker, P.; Hansson, G. Progress and Challenges in translating the Biology of Atherosclerosis. Nature. 2011, 473, 317-325. 11. Barter, P. The inflammation: lipoprotein cycle. Atheroscler. Suppl. 2005, 6, 15–20. 12. Chisolm, G.; Steinberg, D. The oxidative modification hypothesis of atherogenesis: an overview. Free Radic.Biol.Med.2000, 28, 1815–1826. 13. Van Antwerpen, P.; Boudjeltia, K.; Babar, S.; Legssyer, I.; Moreau, P.; Moguilevsky, N.; Vanhaeverbeek, M.; Ducobu, J.; Nève, J. Thiol-containing molecules interact with the ACS Paragon Plus Environment

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myeloperoxidase/H2O2/chloride 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

system

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inhibit

LDL

oxidation.

Biochem.Biophys.Res.Com. 2005,337, 82-88. 14. Carr, A.C.; Decker, E.A.; Park, Y.; Frei, B. Comparison of low-density lipoprotein modification by myeloperoxidase-derived hypochlorous and hypobromous acids. Free.Radic.Biol.Med. 2001, 31, 62–72. 15. Nicholls,

S.J.;

Hazen

S.L.

Myeloperoxidase

and

cardiovascular

disease.

Arterioscler.Thromb.Vasc.Biol. 2005,25(6):1102-1111. 16. Nève, J.; Parij, N.; Moguilevsky, N. Inhibition of the myeloperoxidase chlorinating activity by non-steroidal anti-inflammatory drugs investigated with human recombinant enzyme. Eur.J.Pharm. 2001;417:37-43. 17. Van Antwerpen, P.; Dufrasne, F.; Lequeux, M.; Zouaoui-Boudjeltia, K.; Lessgyer, I.; Babar, S.; Moreau, P.; Moguilevsky, N.; Vanhaeverbeek, M.; Ducobu, J.; Neve, J. Inhibition of the myeloperoxidase chlorinating activity by nonsteroidal anti-inflammatory drug flufenamic acid and its 5-chloro-derivative directly interact with a recombinant human myeloperoxidase to inhibit the synthesis of hypochlorous acid. Eur. J. Pharmacol. 2007, 570, 235– 243. 18. Van Antwerpen, P.; Prévost, M.; Zouaoui-Boudjeltia, K.; Babar, S.; Legssyer, I.; Moreau, P.; Moguilevsky, N.; Vanhaeverbeek, M.; Ducobu, J.; Neve, J.; Dufrasne, F.Conception of myeloperoxidase inhibitors derived from flufenamic acid by computational docking and structure modification.Med.Chem. 2008, 16, 1702– 1720. 19. Kettle, A.; Gedye, C.; Hampton, MB.; Winterbourn, CC. Inhibition of myeloperoxidase by benzoic acid hydrazides. Biochem.J. 1995;308 ; Pt 2:559-563. 20. Ikeda-Saito, M.; Shelley, D.; Lu, L.; Booth, K.; Caughey, W.; Kimura, S. Salicylhydroxamic acid inhibits myeloperoxidase activity. J.Biol.Chem. 1991;266(6):36116. ACS Paragon Plus Environment

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21. Hanson, S.; Nordvall, G.; Tiden, A.K. Preparation of xanthinethione derivatives as 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

myeloperoxidase inhibitors. PCT Int. Appl. (2003), WO 2003089430 A1 20031030. 22. Regasinia, L.; Vellosab, L.; Silvaa, D.; Furlana, M.; Oliveirab, O.; Khalilc, N.; Brunettic, I.; Youngd, M.; Barreiroe, E.; Bolzania, V. Flavonols from Pterogyne nitens and their evaluation as myeloperoxidase inhibitors. Phytochemistry. 2008, 69(8):1739-1744. 23. Shiba, Y.; Kinoshita, T.; Chuman, H.; Taketani, Y.; Takeda, E.; Kato, Y.; Naito, M.; Kawabata, K.; Ishisaka, A.; Terao, J.; Kawai, Y. Flavonoids as substrates and inhibitors of myeloperoxidase: molecular actions of aglycone and metabolites. Chem.Res.Toxicol. 2008, 21(8):1600-1609. 24. Galijasevic, S.; Abdulhamid, I.; Abu-Soud, HM. Melatonin is a potent inhibitor for myeloperoxidase. Biochemistry. 2008, 47(8):2668-2677. 25. Ximenes, VF.; Paino, IMM.; Faria-Oliveira, OMMD.; Fonseca, LMD.; Brunetti, IL. Indole ring oxidation by activated leukocytes prevents the production of hypochlorous acid. Braz.J.Med.Biol.Res. 2005;38(11):1575-1583. 26. Van Antwerpen, P.; Moreau, P.; Zouaoui-Boudjeltia, K.; Babar, S.; Dufrasne, F.; Moguilevsky, N.; Vanhaeverbeek, M.; Ducobu, J.; Neve, J.Development and validation of screening procedure for the assessment of inhibition using a recombinant enzyme. Talanta. 2008, 75, 503– 510. 27. Soubhye, J.; Prévost, M.; Van Antwerpen, P.; Boudjeltia, K.; Rousseau, A.; Furtmuller, P.; Obinger, C.; Vanhaeverbeek, M.; Ducobu, J.; Neve, J.; Gelbcke, M.; Dufrasne, F. Structure-based design, synthesis, and pharmacological evaluation of 3-(aminoalkyl)-5fluoroindoles as myeloperoxidase inhibitors. J.Med.Chem. 2010;53(24):8747-8759. 28. Rupp, A.; Kovar, K.-A.; Beuerle, G.; Ruf, C.; Folkers, G. A new pharmacophoric model for 5-HT reuptake-inhibitors: differentiation of amphetamine analogs. Pharmac. Acta Helv. 1994; 68: 235-244. ACS Paragon Plus Environment

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29. Campos, K.; Woo, J.; Lee, S.; Tillyer, R. A general synthesis of substituted indoles from 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

cyclic enol ethers and enol lactones. Org.Lett. 2004, 6, 79– 82. 30. Deo, M. Di.; Marcantoni, E.; Torregiani, E.; Bartoli, G.; Bellucci, M. C.; Bosco M.; Sambri, v. Simple, efficient, and general method for the conversion of alcohols into alkyl iodides by a CeCl3•7H2O/NaI system in acetonitrile. J. Org. Chem. 2000, 65, 2830-2833. 31. Dufrasne, F.; Nève, J. Synthesis of 1,2-diamino-1-phenylpropane diastereoisomers from uN-trifluoroacetyl-2-amino-1-phenylpropan-1-ol. Monatsh. Chem. 2005, 163, 739–746. 32. Mewshaw, R. E.; Zhou, D.; Zhou, P.; Shi, X.; Hornby, G.; Spangler, T.; Scerni, R.; Smith, D.; Schechter, L. E.; Andree, T. H.Studies toward the Discovery of the Next Generation of Antidepressants. 3. Dual 5-HT1A and Serotonin Transporter Affinity within a Class of NAryloxyethylindoleylalkylamines. J. Med. Chem. 2004, 47, 3823– 3842. 33. Stevenson, R.; Sant Milid, P.; Haider, R.; Hilmi, A.; Al Farham, E. Method for making substituted indole. PCT Int. Appl. 1999. WO9959970. 34. Hatzenbuhler, N. T.; Evrard, D. A.; Mewshaw, R. E.; Zhou, D.; Shah, U. S.; Inghrim, J. A.; Lenicek, S. E.; Baudy, R. B.; Butera, J. A.; Sabb, A. L.; Failli, A. A.; Ramamoorthy, P. S. A preparation of 3-aminochroman and 2-aminotetralin derivatives, useful in the treatment of serotonin-mediated disorders. PCT Int. Appl. 2005. WO2005012291. 35. Hall, H.; Gisler, M. A Simple Method for Converting Nitriles to Amides. Hydrolysis with potassium hydroxide in tert-butyl alcohol. J. Org. Chem. 1976, 41, 3769– 3770. 36. Jantschko, W.; Furtmuller, G. P.; Zederbauer, M.; Neugschwandtner, K.; Lehner, I.; Jakopitsch, C.; Arnhold, J.; Obinger, C. Exploitation of the unusual thermodynamic properties

of

human

myeloperoxidase

in

inhibitor

design.

Biochem.

Pharmacol. 2005, 69, 1149– 1157.


32

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37. Schmitz, W.; William, D.; Brenner, J.; Ditta, A.; Mattson, R.; Mattson, G.; Molski T.; 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Macor, J. Homotryptamines as potent and selective serotonin reuptake inhibitors (SSRIs). Bioorg.Med. Chem. Lett. 2005, 15, 1619-1621. 38. Franck, T.; Kohnen, S.; Boudjeltia, KZ.; Van Antwerpen, P.; Bosseloir, A.; Niesten, A.; Gach, O.; Nys, M.; Deby-Dupont, G.; Serteyn, D. A new easy method for specific measurement of active myeloperoxidase in human biological fluids and tissue extracts. Talanta, 2009, 80, 723-729. 39. Blair-Johnson, M.; Fiedler, T.; Fenna, R.Human myeloperoxidase: structure of a cyanide complex and its interaction with bromide and thiocyanate substrates at 1.9 Å resolution. Biochemistry. 2001, 40, 13990– 1399. 40. Sadowski, J.; Gasteiger, J.From atoms and bonds to three-dimensional atomic coordinates: automatic model builders. Chem. Rev. 1993, 93, 2567– 2581. 41. Aldib, I.; Soubhye, J.; Zouaoui Boudjeltia, K.; Vanhaeverbeek, M.; Rousseau, A.; Furtmüller, P.; Obinger, C.; Dufrasne, F.; Nève, J.; Van Antwerpen P.; Prévost, M. Evaluation of New Scaffolds of Myeloperoxidase Inhibitors by Rational Design Combined with High-Throughput Virtual Screening. J. Med. Chem. , 2012. 55(16):7208-7218. 42. Friesner, R. A.; Banks, J. L.; Murphy, R. B.; Halgren, T. A.; Klicic, J. J.; Mainz, D. T.; Repasky, M. P.; Knoll, E. H.; Shelley, M.; Perry, J. K.Glide: a new approach for rapid, accurate docking and scoring. 1. Method and assessment of docking accuracy J. Med. Chem. 2004, 47, 1739– 1794. 43. Shelley, J. C.; Cholleti, A.; Frye, L. L.; Greenwood, J. R.; Timlin, M. R.; Uchimaya, M.Epik: a software program for pK(a) prediction and protonation state generation for druglike molecules J. Comput.-Aided Mol. Des. 2007, 21, 681–691.


33


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44. Havel, R. J.; Eder, H. A.; Bragdon, J. H.Distribution and chemical composition of 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ultracentrifugally separated lipoproteins in human serum. J. Clin. Invest. 1955, 34, 1345– 1353. 45. Hewson, W. D.; Hager, L. P.Mechanism of the chlorination reaction catalyzed by horseradish peroxidase with chlorite J. Biol. Chem. 1979, 254, 3175– 3181. 46. Moguilevsky, N.; Zouaoui-Boudjeltia, Z.; Babar, S.; Delre e, P.; Legssyer, I.; Carpentier, Y.; Vanhaeverbeek, M.; Ducobu, J.Monoclonal antibodies against LDL progressively oxidized by myeloperoxidase react with ApoB-100 protein moiety and human atherosclerotic lesions. Biochem. Biophys. Res. Commun. 2004,323, 1223– 1228. 47. Furtmuller, P. G.; Burner, U.; Obinger, C. Reaction of human myeloperoxidase Compound I with chloride, bromide, iodide, and thiocyanate; Biochemistry. 1998, 37, 17923–17930. 48. Kristensen, A.; Larsen, M.; Johnsen, L.; Wiborg, O. Mutational scanning of the human serotonin transporter reveals fast translocating serotonin transporter mutants. Eur. J. Neurosc. 2004, 19, 1513-1523. 49. Larsen, M.; Elfving, B.; Wiborg, O. The Chicken Serotonin Transporter Discriminates between Serotonin-selective Reuptake Inhibitors: A Species-scanning mutagenesis study. J. Biol. Chem. 2004 , 279, 42147-42156. 50. Estevãoa, M.; Carvalhoa, L.; Ferreiraa, L.; Fernandesb, E.; Marques, M. Analysis of the antioxidant activity of an indole library: cyclic voltammetry. Tetrahedron Letters, 2010, 52, 101–106. 51. Garrels, R.; Christ, C. Minerals, Solutions, and Equilibria., USA: Jones and Bartlett, 1990.


34

Page 35 of 50


TABLES 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Table 1. Synthesized compounds and IC50 values for the inhibition of taurine chlorination by myeloperoxidase (mean ± SD, n = 4) and the reduction potentials of the synthesized compounds. The free energies of binding ∆G predicted from docking experiments are also listed. R2

n

R3

N H R1

Compound

n

R1

R2

R3

A1 [27]

2

H

F

-NH2

200±30

-6.5

-

A2 [27]

3

H

F

-NH2

50±8

-6.4

-

A3 [27]

4

H

F

-NH2

15±4

-6.6

-

A4 [27]

5

H

F

-NH2

8±2

-6.4

-

26

2

F

H

-NH2

29±9

-6.4

923±13

27

3

F

H

-NH2

24±2

-7.4

862±22

28

3

H

COOH

-NH2

Not active

-9.4

908±14

29

4

F

H

-NH2

6±1

-6.4

895±24

30

4

H

COOH

-NH2

Not active

-10.7

889±12

31

3

H

F

N

22±6

-4.0

916±20

32

3

H

F

13±2

-7.6

817±14

33

2

H

F

H N

10±2

-7.4

850±14

34

3

H

F

H N

10±1

-7.1/-9.6&

849±13

35

2

H

F

S

NH2

4±0.2

-6.0

890±13

36

3

H

F

S

NH2

35±4

-8.1$

843±12

37

2

F

H

S

NH2

3±1

-7.6

805±13

38

3

F

H

S

NH2

>100

-8.9$

920±14

39

2

H

F

>100

-5.2

806±15

N

IC50 (nM)

H N

OH

∆G (kcal/mol)

E’ (mV)

OH

S


35


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 36 of 50

40

3

H

F

S

>100

-5.4

805±18

41

2

H

F

-CH3

>100

-4.8

870±14

42

2

H

F

14±2

-5.4

881±6

37±3

-5.5

849±23

C2H5 N O C2H5

43

3

H

F

N O

&

† $

S

S

O

O

44

3

H

F

-NHOH

11±2

-7.2/-8.2&

869±12

45

3

H

F

-S-CN

5±0.6

-5.0/-5.2†

869±8

46

2

H

F

60±9

-6.9 to -8.2*

869±11

50

3

H

F

-CN

63±4

-5.7

869±13

53

3

H

COOH

-CN

Not active

-8.1

893±12

57

3

H

F

-CONH2

18±2

-5.7

879±11

NH2 NH2

Values from docking with a rigid receptor and with induced fit protocol are given. Values obtained from two different types of positions (see text). Values obtained from docking with induced fit protocol.

* Four molecules were docked accounting for the different protonation states of the two amines and the asymmetry of the β−carbon of the side chain. For the R form ∆G= -7.2 kcal/mol with α amine bearing a positive charge and -8.2 kcal/mol with β amine bearing a positive charge. For S form, ∆G=-6.9 kcal/mol with α amine bearing a positive charge and -8.0 kcal/mol with β amine bearing a positive charge.


36

Page 37 of 50


Table 2. IC50-values for the inhibition of oxidation of LDL mediated by the MPO/Cl−/H2O2 system. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

the Ki-values of SERT inhibition are given (mean ± SD, n = 3) and the specificity index SERT Ki/ MPO IC50 values.

R2

n

R3

N H R1

Compound

n

R1

R2

R3

LDL oxid. IC50 (nM)

SERT Ki (nM)

SERT Ki(nM)/ MPO IC50(nM)

26

2

F

H

-NH2

28±4

29±7

1

27

3

F

H

-NH2

26±2

24±6

1

28

3

H

COOH

-NH2

Not active

Not active

-

29

4

F

H

-NH2

21±6

8±2

1.3

30

4

H

COOH

-NH2

Not active

Not active

-

31

3

H

F

N

60±12

36±9

1.6

32

3

H

F

H N

30±7

53±17

4.1

33

2

H

F

H N

OH

31±6

10±5

1

34

3

H

F

H N

OH

43±8

62±23

6.2

35

2

H

F

S

NH2

8±2

2.3±0.2

0.6

36

3

H

F

S

NH2

21±8

0.9±0.4

0.02

37

2

F

H

S

NH2

6±1

24±11

8

38

3

F

H

S

NH2

>100

539±158

-

39

2

H

F

S

>100

Not active

-

40

3

H

F

S

>100

Not active

-

41

2

H

F

>100

Not active

-

42

2

H

F

N

27±10

174±63

12

43

3

H

F

N

27±6

12±2

0.3

N

-CH3 C2H5 S O O C2H5 O

44

3

H

45

3

H

S

O

F

-NHOH

21±6

20±11

1.8

F

-S-CN

23±6

212±46

42


37


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

46

2

H

F

50

3

H

F

53

3

H

57

3

H

NH2

Page 38 of 50

26±8

12±2

0.2

-CN

72±8

184±12

2.9

COOH

-CN

Not active

Not active

-

F

-CONH2

18±3

631±118

35

NH2


38

Page 39 of 50


Table 3. Apparent bimolecular rate constants for the reactions between the synthesized 31 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

alkylindole derivatives with Compounds I (k3) and II (k4) of human myeloperoxidase. For comparison the IC50-values as well as the reduction potential (E’) of the compounds are listed.

Molecule

k3 (M-1 s-1)

k4 (M-1 s-1)

k3/k4

IC50 (nM)

E’ (mV)

Serotonin 36

1.7 × 107

1.4 × 106

12

Not active

650

Tyrosine 36

7.7 × 106

1.6 × 104

48

Not active

930

Tryptamine 36

8.6 × 106

5.2 × 102

16538

770

1015

28

1.0 × 106

2.3 × 103

435

Not active

908

37

3.0 × 107

8

3750000

3±1

805

40

4.6 × 106

3.1 × 104

148

>100

805

42

1.2 × 107

28

428571

14 ± 2

881

57

1.6 × 107

92

173913

18 ± 2

879


39


Page 40 of 50

Table 4. IC50-values for the inhibition of taurine chlorination and for the inhibition of LDL 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

oxidation by myeloperoxidase (moxLDL, mean ± SD, n = 3). The free energies of binding ∆G predicted from docking experiments are given.

R2

n

NHR3

O N H R1

IC50 nM

∆G (kcal/mol)

moxLDL nM

271±33

-5.9

64±24

H

26±0.4

-5.5

24±14

F

H

132±35

-5.7

34±12

H

F

H

18±2

-5.7

18±3

4

H

F

H

39±4.5

-5.5

43±9

59

4

F

H

H

34±9.9

-5.1

44±11

60

3

H

F

CH3

40±8

-5.8

95±15

61

3

H

F

C2H5

182±34

-6.1

243±42

Compound

n

R1

R2

R3

54

2

F

H

H

55

3

F

H

56

2

H

57

3

58


40

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FIGURE CAPTIONS 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 1. General structure of 5-hydroxy tryptamine (5HT, serotonin), 3-(aminoalkyl)-5-fluroindole derivatives and 3-alkylindole derivatives. Figure 2. Comparison of the best-scored docking poses of SHA (green) and of 5-fluorotryptamine (light brown) into the catalytic site of MPO, showing stacking pose of both compounds with ring D of heme. Ionic interactions are with residues Glu102, Thr100 shown in yellow-black lines. The small blue sphere indicates the location of the ferric ion at the center of the heme. Figure 3. (A) Comparison of the best-scored docking poses of compounds 27 and 28. Shift stacking pose with heme for compound 27 while compound 28 shows stacking pose with orientation of carboxyl group toward ferric atom of heme (B) Best-scored docking pose of compound 31 and 34 that have fluorine on position 5 and the same alkyl chain. Compound 31 (in orange) features a stacking with fluorine atom away from heminic iron and compound 34 (in green) shows a stacking with fluorine position toward the heme iron. (C) Best-scored docking pose of compound 36 onto the active site of MPO. It shows stacking on the heme, its fluorine atom (in green) is not oriented toward the heminic iron. Hydrogen bond is formed between the indole nitrogen and propionate, and three salt bridges were observed between the amine of the side chain and Glu102 and propionate. (D) Best-scored docking pose of compound 57 with amide group features a stacking. Oxygen of amide contributes to binding by formation hydrogen bond with Arg333 and the NH2 group hydrogen bonds to Glu102. Fluorine also forms hydrogen bond with Gln91. Figure 4. Scheme of chlorination and peroxidase cycle of myeloperoxidase (MPO). Compound I [oxoiron(IV) plus porphyrin cation radical] is the key intermediate. Depending on the substrate availability (one- versus two-electron donors) and bimolecular rate constants (k2 versus k3) it is reduced directly to the ferric state (halogenation cycle: release of HOCl) or it is converted to Compound II [oxoiron(IV)-species] thereby following the peroxidase cycle. A good reversible inhibitor has a high k3/k4 ratio, i.e. it promotes accumulation of Compound II.


41


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Figure 5. Reaction of MPO Compounds I and II with compound 37 (pH 7.4 and 25 °C). (A) Spectral 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

changes upon mixing 2.0 µM MPO Compound I with 5 µM compound 37. (B) Typical time trace at 456 nm and fit for the reaction between 1 µM Compound I and 7.5 µM compound 37. (C) Pseudofirst-order rate constants for Compound I reduction plotted against compound 37 concentration. (D) Spectral changes upon 2 µM Compound II reduction by 500 µM compound 37 (pH 7.4 and 25 °C). (E) Typical time trace and fit of the reaction between MPO Compound II (1 µM) and 1.5 mM compound 37 followed at 456 nm. (F) Pseudo-first-order rate constants for MPO Compound II reduction plotted against compound 37 concentration.


42

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Figure 1. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

NH2

HO

F

n

N

R1

R1

n

R3

R2 N H

N H R1,R2= H; n= R1,R2= H; n= R1,R2= H; n= R1,R2= H; n=

N H 2 A1 3 A2 4 A3 5 A4

R2


43


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Figure 2. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

OH H N SHA

OH

O

NH2

F N H 5-Fluorotryptamine


44

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Figure 3 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

NH2

A N H

27

F

O NH2

HO N H

28

F

B

N N N H

31 F

OH

N H N H

34

C

F

NH 2

S N H

36

D

NH 2

F O N H

57


45


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Figure 4. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 ACS Paragon Plus Environment

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Figure 5. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 ACS Paragon Plus Environment

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SCHEMES 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Scheme 1. Synthesis of 3-(hydroxyalkyl) indole. (i) H2SO4 4% or 20%, DMA; (ii) DIBAL-H, CH2Cl2, -78 °C; (iii) H2SO4 4%, DMA.

n= 1, 2 R3= H, CH2OH

O

OH

R2

n

n

(i)

R3

R3

R1= R1= R1= R1= R1= R1=

N H R1

F, R2= H , R3= H, n= 1 F, R2= H , R3= H, n= 2 H, R2= COOH , R3= H, n= 2 H, R2= F , R3= CH2OH, n= 2 H, R2= F , R3= H, n= 1 H, R2= F , R3= H, n= 2

R1

R2

NHNH2.HCl

R1= H, F R2= F, H, COOH

O

OH

R2 O HO

(ii)

O

7

R1= F, R2= H 8 R1= H, R2= F 9 R1= H, R2= COOH 10

N H

(iii)

R1

Scheme 2. Synthetic path of 3- (alkylamino) indole compounds. (i) CH2Cl2, CH3SO2Cl, TEA or pyridine and CH3SO2Cl; (ii) DMSO, NaN3 100 °C; (iii) Pd/C, EtOH or AlLiH4, dioxane.

OH

R2

n

OMs

R2 R3

N H R1 R1= F, R2= H , R3= H, n= 1 1 R1= F, R2= H , R3= H, n= 2 2 R1= H, R2= COOH ,R3= H, n= 2 3 R1= H, R2= F, R3= CH2OH, n= 2 4 R1= H, R2= F , R3= H, n= 1 5 R1= H, R2= F , R3= H, n= 2 6 R1= F, R2=H , R3= H, n= 3 8 R1= H, R2= F , R3= H, n= 3 9 R1= H, R2= COOH, R3= H, n= 3 10

(i)

R4

R2

n

n

(ii)

R3

R3 N H

N H

R1

R1 R1= F, R2= H , R3= H, n= 1 11 R1= F, R2= H , R3= H, n= 2 12 R1= H, R2= COOH , R3= H, n= 2 13 R1= H, R2= F, R3= CH2OMs, n= 2 14 R1= H, R2= F , R3= H, n= 1 15 R1= H, R2= F , R3= H, n= 2 16 R1= F, R2= H , R3= H, n= 3 17 R1= H, R2= F , R3= H, n= 3 18 R1= H, R2= COOH ,R3= H, n= 3 19

R4=

R1= F, R2= H , R3= H, n= 1 R1= F, R2= H , R3= H, n= 2 N3 R1= H, R2= COOH , R3= H, n= 2 R1= H, R2= F, R3= CH2N3, n= 2 R1= F, R2= H , R3= H, n= 3 R1= H, R2= COOH ,R3= H, n= 3

20 21 22 23 24 25

(iii)

R4= NH2

R1= F, R2= H , R3= H, n= 1 26 R1= F, R2= H , R3= H, n= 2 27 R1= H, R2= COOH , R3= H, n= 2 28 R1= H, R2= F, R3= CH2NH2, n= 2 46 R1= F, R2= H , R3= H, n= 3 29 R1= H, R2= COOH ,R3= H, n= 3 30


48

1 2 3 4 5 6

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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Scheme 3. Synthesis of compounds 31-45. (i) Dioxan, 100 °C, for R1, R2 and R3 see supporting information. R2

n

R2

OMs

+

N H

n

(i) HR3

R3

N H R1

R1

Scheme 4. Synthesis of 3- (alkylamido) indole compounds. (i) H2O, DMA, KCN; (ii) t-ButOH, KOH, NaCl/H2O; (iii) CH3Br; (iv) C2H5Br. R2

n

R2

OMs

n

CN

(i) N H R1= F, R2= H , n= 2 R1= F, R2= H , n= 3 R1= H, R2= F , n= 2 R1= H, R2= F , n= 3 R1= H, R2= F , n= 4 R1= F, R2= H , n= 4 R1= H, R2=COOH , n= 3

R1= F, R2= H R1= F, R2= H R1= H, R2= F R1= H, R2= F R1= H, R2= F R1= F, R2= H R1= H, R2=COOH

(ii) O

R1

R1 11 12 15 16 17 18 19

n

N H

N H

R1

NHR3

R2

, n= 2 , n= 3 , n= 2 , n= 3 , n= 4 , n= 4 , n= 3

47 48 49 50 51 52 53

R1= F, R2= H R1= F, R2= H R1= H, R2= F R1= H, R2= F R1= H, R2= F R1= F, R2= H R1= H, R2= F R1= H, R2= F

, R3= H, n= 2 , R3= H, n= 3 , R3= H, n= 2 , R3= H, n= 3 , R3= H, n= 4 , R3= H, n= 4 , R3= CH3, n= 3 ,R3= C2H5, n= 3

54 55 56 57 58 59 60 61

(iii)

(iv)


49


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TABLE OF CONTENTS GRAPHIC 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 ACS Paragon Plus Environment

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Design, Synthesis, and StructureâActivity ... - ACS Publications

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