Natural Product Derived Antiprotozoal Agents ... - ACS Publications

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Natural Product Derived Antiprotozoal Agents: Synthesis, Biological Evaluation, and Structure−Activity Relationships of Novel Chromene and Chromane Derivatives Dipak Harel,† Dirk Schepmann,† Helge Prinz,† Reto Brun,‡ Thomas J. Schmidt,§ and Bernhard Wünsch*,† †

Institut für Pharmazeutische und Medizinische Chemie der Westfälischen Wilhelms-Universität Münster, Corrensstraße 48, D-48149 Münster, Germany ‡ Swiss Tropical and Public Health Institute (Swiss TPH), Socinstraße 57, CH-4002 Basel, Switzerland § Institut für Pharmazeutische Biologie und Phytochemie der Westfälischen Wilhelms-Universität Münster, Corrensstraße 48, D-48149 Münster, Germany S Supporting Information *

ABSTRACT: Various natural products with the chromane and chromene scaffold exhibit high antiprotozoal activity. The natural product encecalin (7) served as key intermediate for the synthesis of different ethers 9, amides 11, and amines 12. The chromane analogues 14 and the phenols 15 were obtained by reductive amination of ketones 13 and 6, respectively. Angelate 3, ethers 9, and amides 11 did not show considerable antiprotozoal activity. However, the chromene and chromane derived amines 12, 14, and 15 revealed promising antiprotozoal activity and represent novel lead compounds. Whereas benzylamine 12a and α-methylbenzylamine 12g were active against P. falciparum with IC50 values in the range of chloroquine, the analogous phenols 15a and 15b were unexpectedly 10- to 25-fold more potent than chloroquine with selectivity indexes of 6760 and 1818, respectively. The phenylbutylamine 14d based on the chromane scaffold has promising activity against T. brucei rhodesiense and L. donovani.

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INTRODUCTION Neglected tropical diseases caused by protozoan parasites are among the most serious public health problems in developing countries. Some of the neglected diseases also affect the people living in Europe and U.S. According to the World Health Organization (WHO) World Global Burden of Disease analysis report in 2008, around 17% of deaths worldwide are caused by neglected tropical diseases.1 However, only very few drugs are available for the treatment of these lethal infections. Malaria, leishmaniasis, Chagas disease, and human African trypanosomiasis (HAT or sleeping sickness) are among the most serious neglected tropical diseases. Plasmodium falciparum is one of the protozoan agents leading to malaria after a bite of an infected female Anopheles mosquito. Malaria causes almost 800 000 deaths per year, mostly in children.2 Visceral leishmaniasis (Kala Azar) is caused by Leishmania donovani (India and Near East) and Leishmania infantum (around the Mediterranean Sea), and an estimated 1.3 million new cases and 20 000 to 30 000 deaths occur annually.3,4 There are about 10 million people worldwide suffering from Chagas disease (American trypanosomiasis), which is caused by Trypanosoma cruzi.5 Human African trypanosomiasis is caused by Trypanosoma brucei rhodesiense and gambiense (East and West African forms, respectively), which are transmitted by the tsetse fly © XXXX American Chemical Society

(Glossina spp.). It is estimated that about 20 000 people are currently suffering from human African trypanosomiasis.6 The absence of vaccines and the availability of only few chemotherapeutics, some with reduced efficacy and considerable adverse effects, impede the efficient treatment of these diseases. Therefore, the discovery and development of novel effective, safe, and affordable antiprotozoal agents remain an urgent need. Natural products can play an important role as potential lead structures toward such novel pharmacologically active compounds.7,8 Chromanes and chromenes represent a widely distributed class of natural products. The chromenodihydrochalcone 1 isolated from Crotalaria ramosissima (a medicinal plant endemic to India) inhibited in an in vitro assay the growth of L. donovani. The corresponding chalcone with a double bond in the side chain between the carbonyl moiety and the phenyl residue was almost as active as the natural product 1.9 The synthetic tricyclic chromene 2 with the exocyclic benzylidene moiety also showed strong antileishmanial acitivity.10 The medicinal plant Ageratum conyzoides L. (Asteraceae) is used in folk medicine against various diseases including protozoan Received: July 8, 2013

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dx.doi.org/10.1021/jm401007p | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

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infections.11 Recently, it was discovered that the dichloromethane extract of A. conyzoides L. displays considerable activity against T. b. rhodesiense.12 In a search for the active constituents, the chromene encecalol angelate (3)13 was identified in the extract but showed only low antitrypanosomal activity when tested after total synthesis.14 The stability of 3 was found to be very low so that the weak bioactivity may be related to its rapid degradation when tested as a pure compound. The instability of 3 originates from facile dissociation of the angelate anion, since the remaining benzylic cation is stabilized by two Osubstituents. Thus, a rapid formation of the corresponding methyl ether was observed when storing the angelate 3 in methanolic solution14 (Figure 1).

Herein the synthesis, antiprotozoal activity, and relationships between the structure and the antiprotozoal activity of chromenes and chromanes related to the lead compounds 1− 3 are reported.

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RESULTS AND DISCUSSION

Chemistry. The natural product encecalin (7)15 was the central intermediate for the synthesis of various ethers 9, amides 11, and amines 12 (Scheme 1). In contrast to the first reported synthesis of 715 we followed our previously reported more efficient synthetic strategy,14 which started with resorcinol (4). Formic acid catalyzed annulation of 4 with 2-methylbut-3en-2-ol provided a chromanol derivative, which was acetylated to give acetylchromanol 5. Dehydrogenation of 5 afforded chromenol 6, which upon methylation yielded the methyl ether encecalin (7).14 For the synthesis of ethers 9, encecalin (7) was reduced with NaBH4 to afford encecalol (8). In order to obtain compounds structurally related to the lead compounds 1−3, the alcohol 8 was deprotonated with NaH and subsequently treated with different arylalkyl halides to form the ethers 9a−c in good yields. The arylalkyl substructure of the ethers 9 should imitate the arylalkyl residues of the lead compounds 1 and 2 as well as the alkenoyl substructure of angelate 3. In contrast to 3 the ethers 9 are very stable against hydrolytic degradation. Reaction of the ketone 7 with ammonium acetate and NaBH3CN16 led to the primary amine 10 in 65% yield. The primary amine 10 was converted into amides 11 by acylation with various acids in the presence of carbonyl diimidazole (CDI). The acyl residues were selected because of their similarity to the lead compounds 1−3. Reductive amination of ketone 7 with benzylamine in the presence of NaBH(OAc)3 failed to give the secondary amine 12a. However, microwave assisted condensation of ketone 7

Figure 1. Chromenes with antiprotozoal activity.

In order to study the antiprotozoal properties of chromene derivatives related to 1−3, we planned to synthesize more stable analogues of the angelate 3. In particular the hydrolytically very labile ester group of 3 should be replaced by ether (9), amide (11), and amine (12, 14, 15) moieties, taking the arylalkyl side chains of the chromenes 1 and 2 into account. Scheme 1a

a Reagents and conditions: (a) HCO2H, 100 °C; (b) CH3COCl, AlCl3, CH2Cl2, 0 °C; (c) DDQ, toluene, 110 °C; (d) CH3I, K2CO3, DMF, rt; (e) NaBH4, THF, rt; (f) R-Br, NaH, DMF, rt, residues R are defined in Table 2; (g) NH4OAc, NaBH3CN, MeOH, rt; (h) RCO2H, CDI, THF, rt, residues R are defined in Table 2; (i) (1) R2NH, MeOH, microwave irradiation, (2) NaBH4, rt, the substituents of the amino moiety NR2 are defined in Table 3. (j) oOnly for synthesis of 12o: (1) PhCH2CH2CH2NH2, MeOH, microwave irradiation, NaBH4, rt; (2) HCHO, NaBH(OAc)3, THF, rt.

B



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against nonpathogenic lizard-infecting L. tarentolae (strain P10, Jena Biosciences) promastigotes, which can be cultured costefficiently with hemin-supplemented BHI medium and which rapidly grow in vitro. This relatively simple L. tarentolae system has recently been propagated as a model for the in vitro screening of compounds for antileishmanial activity.18−20 In Table 1 the activity of reference compounds (positive controls)

with various primary amines led to formation of imines, which were reduced subsequently with NaBH4 to give the secondary amines 12a−l in 52−75% yields. In order to synthesize the cyclic amines 12m and 12n an intermediate iminium ion was formed upon microwave irradiation, which was reduced with NaBH4. Methylation of the secondary amine 12c with formaldehyde and NaBH(OAc)3 resulted in the tertiary amine 12o. In order to broaden the structure−activity relationships in this compound class, chromanamines 14 without a double bond in the heterocycle were taken into consideration. After methylation of the phenol 5 without the endocyclic double bond, the resulting ketone 13 was reacted with primary amines assisted by microwave irradiation. Subsequent reduction with NaBH4 led to the secondary amines 14a−e in 56−68% yields (Scheme 2). The selection of the amines for the synthesis of

Table 1. In Vitro Antiprotozoal Activity of Reference Compounds (Positive Controls) and Cytotoxicity of Podophyllotoxin (IC50 [μM])

Scheme 2a

parasite

reference drug

IC50 [μM]

P. falciparum T. brucei rhodesiense T. cruzi L. donovani L. tarentolae L6 cells (cytotoxicity)

chloroquine melarsoprol benznidazole miltefosine pentamidine podophyllotoxin

0.263 0.005 1.573 0.361 0.030 0.010

toward the different protozoan parasites is summarized. Moreover, the cytotoxic effect of podophyllotoxin (used as a positive control) toward L6 rat skeletal myoblasts is included. The ester 3, ethers 9a−c, and amides 11a−c generally show only low activity against the protozoan parasites. Only the benzyl ether 9a reveals an IC50 value against Pfc below 10 μM. The compounds also display low selectivity indices (SI < 5) indicating a small gap between the desired antiprotozoal activity (anti-Pfc, anti-Tbr) and the undesired cytotoxicity. In contrast to the human pathogens, the nonpathogenic L. tarentolae promastigotes were much more sensitive to these compounds, with IC50 values generally lower than 10 μM. The cinnamyl ether 9c (IC50 = 0.86 μM) and the phenylethyl ether 9b (IC50 = 1.17 μM) represent the most potent anti-Ltr compounds of this series, although their activity is still about 30-fold lower than the activity of the reference compound pentamidine (IC50 = 0.030 μM; see Table 1). In contrast to the ester, ether, and amide derivatives 3, 9, and 11, the chromen- and chromanamines 12, 14, and 15 show much better antiprotozoal activity. Some of the amines also reveal activity against Tbr. As summarized in Table 3, the antiprotozoal activity in the chromenamine series 12 decreases in the order anti-Pfc > anti-Tbr > anti-Tcr > anti-Ldo. In contrast to the chromenamines 12, the chromanamines 14 display promising anti-Ldo activity. The phenylalkylamines 12a, 12c, 12e, 12f, and 12g display IC50 values against Pfc below 1 μM and can hence be classified as potent antiplasmodial agents with activity in the same range as the positive control chloroquine (Table 3). Phenylalkylamines are generally more active against Pfc than aliphatic amines (e.g., 12i, 12j). With the exception of 12c, the phenylalkylamines 12a, 12e, 12f, and 12g also show relatively low cytotoxicity indicating a big difference between the antiplasmodial effect and potential side effects so that these compounds deserve further attention as potent and safe antimalarials. Replacement of the methoxy group of 12 with a phenolic hydroxy moiety led to 15 with surprisingly high activity against P. falciparum. The benzylamine 15a and the phenylpropylamine 15b show IC50 values of 0.02 and 0.01 μM, respectively. This activity is about 10- to 25-fold higher than the activity of the standard compound chloroquine (IC50 = 0.263 μM). Moreover, the cytotoxicity of both phenols 15a and 15b is very low

a

Reagents and conditions: (a) CH3I, K2CO3, DMF, rt; (b) (1) RNH2, MeOH, microwave irradiation, NaBH4, rt.

chromenamines 12 and chromanamines 14 was stimulated by the corresponding substituents in the lead compounds 1−3 and, moreover, by the biological activity obtained during the synthetic work. Finally the chromene derived phenols 15a and 15b were synthesized (Scheme 3). For this purpose a two-step reductive Scheme 3a

Reagents and conditions: (a) RNH2, Ti(OEt)4,THF, 65 °C, then NaBH4, rt.

a

amination of ketone 6 (see Scheme 1) with benzylamine and 3phenylpropan-1-amine was performed. At first the ketone 6 was reacted with the primary amines in the presence of Ti(OEt)4 to form imine intermediates, which were subsequently reduced with NaBH417 to afford the secondary amines 15a and 15b. Antiprotozoal Activity. In a search of compounds with improved stability and antiprotozoal activity in comparison with the natural product 3, derivatives with ether (9), amide (11), and amino (12, 14, 15) groups instead of the labile ester moiety of 3 were synthesized. The activities of the compounds 3, 9, 11, 12, 14, and 15 were evaluated in vitro against four pathogenic protozoan parasites: P. falciparum (Pfc, erythrocytic stage, K1 strain, which is chloroquine and pyrimethamine resistant), T. b. rhodesiense (Tbr, trypomastigote stage, STIB 900 strain), T. cruzi (Tcr, intracellular amastigote stage, Tulahuen C4 strain), and L. donovani (Ldo, axenic amastigote stage, MHOM-ET-67/L82) (Table 2). A preliminary antileishmanial assay was performed C



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Table 2. Activity of Ester 3, Ethers 9a−c, and Amides 11a−c against the Protozoan Parasites P. falciparum (Pfc), T. brucei rhodesiense (Tbr), T. cruzi (Tcr), L. donovani (Ldo), and L. tarentolae (Ltr), Cytotoxicity against L6 Rat Skeletal Myoblasts, and Selectivity Indices (SI) for Pfc and Tbra

antiprotozoal activity IC50 [μM] compd 312 9a 9b 9c 11a 11b 11c a

R

Pfc

Tbr

Tcr

Ldo

Ltr

cytotoxicity IC50 [μM], L6 cells

SI (Pfc)

SI (Tbr)

SI (LDo)

PhCH2 PhCH2CH2 PhCHCHCH2 Ph PhCH2 PhCHCH

18.9 9.48 59.9 nd 11.5 31.0 nd

157 76.1 180 94.7 23.4 103 35.9

61.4 45.0 56.7 73.0 36.8 44.7 23.1

46.2 21.2 63.5 23.0 26.3 53.8 26.3

21.3 1.53 1.17 0.86 8.61 7.69 1.78

139 43.9 123 64.4 34.8 86.5 107

7.35 4.64 2.08 nd 3.01 2.79 nd

0.89 0.58 0.69 0.68 1.45 0.83 2.99

3.0 2.1 1.9 2.8 1.3 1.6 4.1

nd: not determined.

Table 3. Antiprotozoal Activity of Amino Substituted Chromenes 12a−o, Chromanes 14a−e, and Phenols 15a,b: Activity against the Parasites P. falciparum, T. brucei rhodesiense, T. cruzi, L. donovani, and L. tarentolae, Cytotoxicity against L6 Rat Skeletal Myoblasts, and Selectivity Indices (SI) for Pfc and Tbra

antiprotozoal activity IC50 [μM] compd

R2N

Pfc

Tbr

Tcr

Ldo

Ltr

cytotoxicity IC50 [μM], L6 cells

SI (Pfc)

SI (Tbr)

SI (Ldo)

12a 12b 12c 12d 12e 12f 12g 12h 12i 12j 12k 12l 12m 12n 12o 14a 14b 14c 14d 14e 15a 15b

PhCH2NH PhCH2CH2NH PhCH2CH2CH2NH PhCH2CH2CH2CH2NH p-MeOPhCH2NH (R)-PhCH(CH3)NH (S)-PhCH(CH3)NH PhCHCHCH2NH CH3CH2NH CH3CH2CH2CH2NH Et2N(CH2)3CH(CH3)NH C6H11CH2NH O(CH2CH2)2N (CH2CH2)2N PhCH2CH2CH2NCH3 PhCH2NH PhCH2CH2NH PhCH2CH2CH2NH PhCH2CH2CH2CH2NH CH3CH2CH2CH2NH PhCH2NH PhCH2CH2CH2NH

0.64 2.42 0.85 1.41 0.93 0.72 0.37 4.41 8.12 4.18 2.39 2.46 9.92 16.4 1.24 1.51 2.49 2.13 1.34 3.22 0.02 0.01

2.38 2.22 1.03 3.15 2.42 2.02 5.10 5.84 22.8 8.19 1.86 2.16 96.2 41.1 3.17 4.98 4.15 2.27 1.84 7.82 6.18 5.31

33.9 9.10 13.6 4.19 33.1 nd nd 8.13 91.5 25.2 35.5 12.9 110 68.2 9.17 13.3 12.1 5.35 5.03 38.4 nd nd

159 127 101 71.7 225 nd nd 62.4 365 nd nd 139 148 270 84.3 3.70 1.73 nd 0.57 10.8 nd nd

2.40 1.04 0.22 0.77 9.80 8.61 9.01 3.26 8.76 6.25 3.24 0.65 12.4 18.6 9.01 270 126 129 90.6 nd 12.4 6.20

21.0 15.4 1.08 4.73 14.9 15.9 16.5 14.6 176 52.5 41.9 14.5 157 177 16.3 15.3 14.5 2.63 5.28 59.0 109 27.0

32.8 6.34 1.27 3.35 16.0 22.1 44.4 3.32 22.0 12.6 17.5 5.88 15.9 10.8 13.5 10.1 5.80 1.23 3.94 18.3 6760 1818

8.83 6.93 1.05 1.50 6.13 7.85 3.23 2.50 7.71 6.41 22.5 6.69 1.63 4.31 5.12 3.08 3.48 1.15 2.87 7.54 17.7 5.08

0.13 0.12 0.11 0.07 0.07

a

0.23 0.48

0.10 1.06 0.66 0.19 4.1 8.4 9.3 5.5

nd: not determined.

With respect to antitrypanosomal potency, the homologous phenylalkylamines 12a−d show increasing activity with increasing number of methylene moieties in the side chain from the benzylamine 12a to the phenylpropylamine 12c, which represents the most active anti-Tbr compound with an IC50 value of 1.03 μM. The phenylbutylamine 12d is 3-fold less

leading to selectivity indices of 6760 and 1818, respectively. This extraordinarily high anti-Pfc activity was completely unexpected, since the starting point of this project was the optimization of the antitrypanosomal activity, which is rather low for both phenols 15. D



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chromene derivative 12d. The SI for Ldo of 14d is about 9.3, which makes this phenylbutylamine an interesting starting compound for the development of novel anti-Ldo compounds. The phenylpropylamine 12c, the phenylbutylamine 12d, and the cyclohexylmethylamine 12l show submicromolar activity against the nonpathogenic parasite L. tarentolae. However, the activities of these compounds are still 10- to 30-fold lower than the activity of the reference compound pentamidine (IC50 = 0.03 μM). The high anti-Ldo activity of the chrmomane derivatives 14a−d was not observed for L. tarentolae, indicating a different sensitivity of the different Leishmania species. The data in Table 3 do not reveal an obvious correlation between the activity against the Ldo axenic amastigotes and Ltr promastigotes. Different activities of structurally related compounds against different L. ssp. are well-known and may be in general due to species differences.21 Furthermore, data from pro- and amastigote stages of Leishmania cannot easily be compared, not even among the same L. ssp. L. donovani axenic amastigotes express significant levels of amastigote-specific proteins and enzymes,22 whereas the expression of several promastigote form enzymes is diminished.23,24 The L. donovani axenic amastigote model is widely used as an initial test for new agents against visceral leishmaniasis, whereas L. tarentolae promastigotes neither represent a relevant parasite stage nor a human pathogen. The obvious lack of correlation between the activity data obtained with the two models indicates that the results obtained with the L. tarentolae promastigote model have to be treated with caution, when new agents for the treatment of the human disease are to be developed.

active than 12c against Tbr but about 3 times more active against Tcr, indicating different optimal chain lengths for the two related Trypanosoma species. Quite interestingly, the activity against Pfc shows an opposite behavior with respect to the chain length in the phenylalkyl series than observed for the antitrypanosomal potency. Here, activity decreases with increasing number of carbon atoms in the side chain (exception 12b). Thus, benzylamine 12a has the highest antimalarial activity with an IC50 value of 0.64 μM, which is only 2-fold higher than the IC50 value of the reference compound chloroquine (IC50 = 0.26 μM). The Pfc selectivity index of 12a with a value of 32.8 also appears quite promising and indicates a specific activity. The p-methoxybenzylamine 12e (IC50 = 0.93 μM) shows a slightly reduced antimalarial activity compared to the unsubstituted benzylamine 12a. Introduction of a methyl group in α-position of the benzyl group led to the αmethylbenzylamines 12f and 12g which display comparable or slightly increased antiplasmodial activity compared with benzylamine 12a. The (S)-configured α-methylbenzylamine 12g (IC50 = 0.37 μM) represents the most potent and safe (SI = 44) compound of the series of methyl ethers. It is noteworthy that the (R)-enantiomer 12f is about 2 times less active than the (S)-configured enantiomer 12g. The influence of the stereochemistry on the antiplasmodial activity of these compounds indicates the interaction with a particular but yet unknown target in the parasite Pfc. Within the series of alkylamines, 12k with a chloroquine-like diamine side chain displays higher anti-Pfc and anti-Tbr activity (IC50 = 2.4 and 1.9 μM, respectively) compared to other alkylamines like ethylamine 12i and butylamine 12j. In order to compare the antiparasitic effect of secondary and tertiary amines, some tertiary amines 12m−o were prepared. The morpholine 12m and the pyrrolidine derivative 12n display very low antiparasitic activity but also very low cytotoxicity. N-Methylation of the secondary amine 12c resulted in 12o with slightly decreased antiprotozoal activity against the parasites (exception anti-Tcr activity). It can be concluded that secondary amines are generally more potent antiprotozoal agents than tertiary amines. With exception of the anti-Ldo activity, the antiprotozoal activity of the chromane derivatives 14a−e is in most cases lower than the activity of the corresponding chromene derivatives 12a−d and 12j. Obviously, the Δ3,4-double bond of the chromene ring increases the antiprotozoal activity. As discussed for the chromene derivatives 12a−d, the anti-Tbr activity of the phenylalkylamines 14a−d correlates nicely with the number of methylene moieties in the side chain. The phenylbutylamine 14d displays the highest anti-Pfc and anti-Tbr activity but also reveals higher cytotoxicity than the benzylamine 14a and phenylethylamine 14b. The benzylamine 14a has the highest selectivity index of 10.1 for Pfc in this series of compounds. The aliphatic butylamine 14e possesses an even higher SI of 18.3, but its antiplasmodial activity is rather low. The anti-Ldo activity of the chromanamines 14a−d with a phenylalkyl residue at the N-atom is remarkable. An increased number of methylene moieties between the N-atom and the phenyl ring leads to increased anti-Ldo activity. The phenylbutylamine 14d represents the most potent anti-Ldo compound of the whole series of test compounds. The IC50 value of 0.57 μM is in the same range as the IC50 value of the reference compound miltefosine (IC50 = 0.36 μM; see Table 1) and more than 100-fold lower than the IC50 value of the corresponding

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CONCLUSION This study unraveled a new class of potent antiprotozoal compounds, starting from the unstable natural product 3 found in an extract of Ageratum conyzoides L. The high activity of the crude extract from this plant against T. brucei rhodesiense prompted us to search primarily for stable analogues of 3 with increased antritrypanosomal activity. This goal was achieved with some of the phenylalkylamine substituted chromene and chromane derivatives; i.e., the phenylpropylamine 12c and the phenylbutylamine 14d show the highest anti-Tbr activity of this series of compounds with IC50 values of 1.03 and 1.84 μM, respectively. In addition to antitryoanosomal activity, the chromanamines 14 exhibit high anti-Ldo activity. The phenylbutylamine 14d represents the most potent compound of this class with an IC50 value of 0.57 μM, which is close to the IC50 value of the reference compound miltefosine. The systematic variation of the natural products 3 and 7 led to unexpected high in vitro antiplasmodial activity and selectivity indices against P. falciparum. Whereas the benzylamine 12a with the methoxy moiety possesses an anti-Pfc activity (IC50 = 0.64 μM) in the range of chloroquine, the corresponding phenol 15a (IC50 = 0.02 μM) is >10-fold more potent than chloroquine. The selectivity index of 6760 indicates a high safety of 15a. Altogether, the amino substituted ethers 12 and phenols 15 represent the first members of a new promising class of potent and safe natural product derived antimalarials.

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EXPERIMENTAL PART

General, Chemistry. For flash chromatography, silica gel 60, 40− 64 μm (Merck) was used. Results in parentheses include diameter of the column, length of column, fraction size, eluent, and Rf value. E



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N-[1-(7-Methoxy-2,2-dimethylchroman-6-yl)ethyl]-4-phenylbutan-1-amine (14d). According to general procedure C, ketone 13 (80 mg, 0.34 mmol) was reacted with phenylbutylamine (63 mg, 0.51 mmol) and the product was purified by flash chromatography (dichloromethane/MeOH = 95/5, ⌀ = 2.0 cm, h = 15 cm, Rf = 0.26). Colorless oil, yield 59 mg (51%). 1H NMR (CDCl3): δ (ppm) = 1.21 (d, J = 6.7 Hz, 3H, CH3), 1.26 (s, 6H, CH3), 1.39−1.60 (m, 5H, PhCH2CH2CH2CH2NH), 1.70 (t, J = 6.7 Hz, 2H, 3-CH2), 2.34−2.49 (m, 2H, PhCH2CH2CH2CH2NH), 2.51 (t, J = 6.7 Hz, 2H, PhCH2CH2CH2CH2NH), 2.61 (t, J = 6.7 Hz, 2H, 4-CH2), 3.66 (s, 3H, OCH3), 3.89 (q, J = 6.7 Hz, 1H, CH3CHNH), 6.24 (s, 1H, 8CH), 6.85 (s, 1H, 5-CH), 7.05−7.20 (m, 5H, Ph). 6-[1-(Benzylamino)ethyl]-2,2-dimethyl-2H-chromen-7-ol (15a). Under N2, phenol 6 (100 mg, 0.46 mmol) was dissolved in dry THF (10 mL). Benzylamine (59 mg, 0.55 mmol) and Ti(OEt)4 (126 mg, 0.55 mmol) were added, and the reaction mixture was heated to reflux for 12 h. The reaction mixture was cooled to 0 °C. NaBH4 (26 mg, 0.69 mmol) was added, and the mixture was stirred for 3 h at room temperature. Water was added. The reaction mixture was extracted with EtOAc (3 × 100 mL). The organic layer was dried (Na2SO4) and concentrated in vacuum, and the residue was purified by flash chromatography (petroleum ether/EtOAc = 70/30, ⌀ = 2.0 cm, h = 12 cm, Rf = 0.27). Colorless oil, yield 85 mg (60%). 1H NMR (CDCl3): δ (ppm) = 1.34 (s, 6H, CH3), 1.36 (d, J = 6.7 Hz, 3H, HNCHCH3), 3.58 (d, J = 13.1 Hz, 1H, HNCH2Ph), 3.77 (d, J = 13.1 Hz, 1H, HNCH2Ph), 3.82 (q, J = 6.7 Hz, HNCHCH3), 5.36 (d, J = 9.8 Hz, 1H, 3-CH), 6.16 (d, J = 9.8 Hz, 1H, 4-CH), 6.24 (s, 1H, 8-CH), 6.49 (s, 1H, 5-CH), 7.14−7.34 (m, 5H, Ph). 2,2-Dimethyl-6-{1-[(3-phenylpropyl)amino]ethyl}-2H-chromen-7-ol (15b). Under N2, phenol 6 (100 mg, 0.46 mmol) was dissolved in dry THF (10 mL). 3-Phenylpropylamine (74 mg, 0.55 mmol) and Ti(OEt)4 (126 mg, 0.55 mmol) were added, and the reaction mixture was heated to reflux for 12 h. The reaction mixture was cooled to 0 °C. NaBH4 (26 mg, 0.69 mmol) was added, and the mixture was stirred for 3 h at room temperature. Water was added. The reaction mixture was extracted with EtOAc (3 × 100 mL). The organic layer was dried (Na2SO4) and concentrated in vacuum, and the residue was purified by flash chromatography (petroleum ether/ EtOAc = 70/30, ⌀ = 2.0 cm, h = 12 cm, Rf = 0.29). Colorless oil, yield 75 mg (48%). 1H NMR (CDCl3): δ (ppm) = 1.32 (s, 6H, CH3), 1.34 (d, J = 6.7 Hz, 3H, HNCHCH 3 ), 1.65−1.88 (m, 2H, HNCH2CH2CH2Ph), 2.42−2.69 (m, 4H, HNCH2CH2CH2Ph), 3.73 (q, J = 6.7 Hz, HNCHCH3), 5.35 (d, J = 9.8 Hz, 1H, 3-CH), 6.13 (d, J = 9.8 Hz, 1H, 4-CH), 6.20 (s, 1H, 8-CH), 6.46 (s, 1H, 5-CH), 6.90− 7.16 (m, 3H, Ph), 7.18−7.26 (m, 2H, Ph). Investigation of the Antiprotozoal Activity. In vitro assays for activity against Trypanosoma brucei rhodesiense (bloodstream trypomastigote stage, STIB 900 strain), T. cruzi (intracellular amastigote stage, Tulahuen C4 strain), Leishmania donovani (axenic amastigote stage, MHOM-ET-67/L82), and Plasmodium falciparum (erythrocytic stage, K1 strain) as well as cytotoxicity determinations against L6 rat skeletal myoblasts were carried out at the Swiss Tropical and Public Health Insitute according to established standard protocols described earlier.24 Compounds used as positive controls were of commercial origin, with the exception of melarsoprol, which was a gift from WHO. Their purity (generally >95%) was specified by the manufacturers. Investigation of Anti Leishmania tarentolae Activity. The assay is described in detail in the Supporting Information.

Melting point apparatus SMP 3 (Stuart Scientific) was used, and melting points were uncorrected. For 1H NMR (400 MHz) and 13C NMR (100 MHz), a Mercury Plus 400 spectrometer (Varian) was used. δ in ppm is referenced to tetramethylsilane. Coupling constants are given with 0.5 Hz resolution. Where necessary, the assignment of the signals in the 1H NMR and 13C NMR spectra was performed using 1 H−1H and 1H−13C COSY NMR spectra. The purity of all test compounds was determined by HPLC analysis (purity of >95%). General Procedure A for the Synthesis of Ethers 9. Under N2 alcohol 8 (80 mg, 0.34 mmol) was dissolved in dry DMF (2.5 mL). At 0 °C NaH (19 mg, 0.40 mmol) and then the respective halogen alkane (0.38 mmol) were added dropwise, and the reaction mixture was stirred at room temperature for 4 h. Then water was added and the solution was extracted with ethyl acetate (3 × 10 mL). The organic layer was dried (Na2SO4) and concentrated in vacuum, and the residue was purified by fc. General Procedure B for the Synthesis of Amides 11. Under N2 commercially available carboxylic acid (1 equiv.) was dissolved in dry THF (5 mL). CDI (67 mg, 0.41 mmol) was added slowly and the reaction mixture was stirred for 10 min at rt. A solution of primary amine 10 (80 mg, 0.33 mmol) in dry THF was added and the reaction mixture was stirred at rt for 4 h. Then water was added and the solution was extracted with ethyl acetate (3 x10 mL). The organic layer was dried (Na2SO4), concentrated in vacuum and the residue was purified by flash chromatography. General Procedure C for the Synthesis of Amines 12 and 14. Under N2, ketone 7 (100 mg, 0.43 mmol) or ketone 13 (80 mg, 0.34 mmol) was dissolved in dry MeOH (2 mL) in a microwave vial. Amine (0.65 mmol) was added, and the reaction mixture was treated under microwave irradiation at 80 °C for 10 min. The reaction mixture was cooled to 0 °C. NaBH4 (30 mg, 0.78 mmol) was added slowly, and the mixture was stirred at room temperature for 2 h. Then it was concentrated in vacuum. The residue was dissolved in water, and the solution was extracted with ethyl acetate (3 × 10 mL). The organic layer was dried (Na2SO4) and concentrated in vacuum, and the residue was purified by flash chromatography. Synthetic Procedures. 6-[1-(Benzyloxy)ethyl]-7-methoxy2,2-dimethyl-2H-chromene (9a). According to general procedure A, alcohol 8 (80 mg, 0.34 mmol) was reacted with benzyl bromide (65 mg, 0.38 mmol) and the product was purified by flash chromatography (petroleum ether/EtOAc = 80/20, ⌀ = 1.5 cm, h = 15 cm, Rf = 0.29). Colorless oil, yield 62 mg, (56%). 1H NMR (CDCl3): δ (ppm) = 1.40 (d, J = 6.4 Hz, 3H, CHCH3), 1.44 (s, 6H, CH3), 3.77 (s, 3H, OCH3), 4.32 (d, J = 12.0 Hz, 1H, OCH2Ph), 4.47 (d, J = 11.9 Hz, 1H, OCH2Ph), 4.86 (q, J = 6.4 Hz, 1H, OCHCH3), 5.47 (d, J = 9.8 Hz, 1H, 3-CH), 6.30 (d, J = 9.8 Hz, 1H, 4-CH), 6.36 (s, 1H, 8-CH), 7.08 (s, 1H, 5-CH), 7.29−7.23 (m, 2H, Ph), 7.30−7.37 (m, 3H, Ph). N-[1-(7-Methoxy-2,2-dimethyl-2H-chromen-6-yl)ethyl]benzamide (11a). According to general procedure B, primary amine 10 (80 mg, 0.33 mmol) was reacted with benzoic acid (40 mg, 0.33 mmol) and the product was purified by flash chromatography (petroleum ether/EtOAc = 60/40, ⌀ = 2 cm, h = 12 cm, Rf = 0.26). Colorless solid, mp 91 °C, yield 105 mg (94%). 1H NMR (CDCl3): δ (ppm) = 1.41 (s, 3H, CH3), 1.42 (s, 3H, CH3), 1.53 (d, J = 6.9 Hz, 3H, CHCH3), 3.87 (s, 3H, OCH3), 5.34 (dq, J = 8.7/6.9 Hz, 1H, CH3CHNH-CO), 5.47 (d, J = 9.8 Hz, 1H, 3-CH), 6.25 (d, J = 9.8 Hz, 1H, 4-CH), 6.40 (s, 1H, 8-CH), 6.88 (s, 1H, 5-CH), 7.07 (d, J = 8.7 Hz, 1H, NHCO), 7.50−7.38 (m, 3H, Ph), 7.77−7.72 (m, 2H, Ph). N-Benzyl-1-(7-methoxy-2,2-dimethyl-2H-chromen-6-yl)ethanamine (12a). According to general procedure C, ketone 7 (100 mg, 0.43 mmol) was reacted with benzylamine (69 mg, 0.65 mmol) and the product was purified by flash chromatography (dichloromethane/MeOH = 95/5, ⌀ = 1.5 cm, h = 15 cm, Rf = 0.26). Colorless oil, yield 85 mg (61%). 1H NMR (CDCl3): δ (ppm) = 1.34 (d, J = 6.7 Hz, 3H, CHCH3), 1.43 (s, 6H, CH3), 1.73 (bs, 1H, NH), 3.60 (d, J = 12.9 Hz, 1H, PhCH2NH), 3.66 (d, J = 12.9 Hz, 1H, PhCH2NH), 3.77 (s, 3H, OCH3), 4.05 (q, J = 6.7 Hz, 1H, CH3CH-NH), 5.46 (d, J = 9.8 Hz, 1H, 3-CH), 6.29 (d, J = 9.8 Hz, 1H, 4-CH), 6.37 (s, 1H, 8-CH), 6.97 (s, 1H, 5-CH), 7.36−7.19 (m, 5H, Ph).

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ASSOCIATED CONTENT

S Supporting Information *

Physical and spectroscopic data of all new compounds, purity data, general chemistry methods, and description of the anti L. tarentolae assay. This material is available free of charge via the Internet at http://pubs.acs.org. F



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Article

(11) Kamboj, A.; Saluja, A. K. Ageratum conyzoides L.: a review on its phytochemical and pharmacological profile. Int. J. Green Pharm. 2008, 2, 59−68. (12) Nour, A. M. M.; Khalid, S. A.; Kaiser, M.; Brun, R.; Abdalla, W. E.; Schmidt, T. J. The antiprotozoal activity of methylated flavonoids from Ageratum conyzoides L. J. Ethnopharmacol. 2010, 129, 127−130. (13) Bohlmann, F.; Tsankova, E.; Jakupovic, J.; King, R. M.; Robinson, H. Dimeric chromenes and mixed dimers of a chromene from Encelia canescens. Phytochemistry 1983, 22, 557−560. (14) Harel, D.; Khalid, S. A.; Kaiser, M.; Brun, R.; Wünsch, B.; Schmidt, T. J. Encecalol angelate, an unstable chromene from Ageratum conyzoides L.: total synthesis and investigation of its antiprotozoal activity. J. Ethnopharmacol. 2011, 137, 620−625. (15) Steelink, C.; Marshall, G. P. Structures, syntheses, and chemotaxonomic significance of some new acetophenone derivatives from Encelia farinosa Gray. J. Org. Chem. 1979, 44, 1429−1433. (16) Borch, R. F.; Hassid, A. I. New method for the methylation of amines. J. Org. Chem. 1972, 37, 1673−1674. (17) Bhattacharyya, S. A high throughput synthesis of N,N-dimethyl tertiary amines. Synth. Commun. 2000, 30, 2001−2008. (18) Morgenthaler, J. B.; Peters, S. J.; Cedeño, D. L.; Constantino, M. H.; Edwards, K. A.; Kamowski, E. M.; Passini, J. C.; Butkus, B. E.; Young, A. M.; Lash, T. D.; Jones, M. A. Carbaporphyrin ketals as potential agents for a new photodynamic therapy treatment of leishmaniasis. Bioorg. Med. Chem. 2008, 16, 7033−7038. (19) Taylor, V. M.; Muñoz, D. L.; Cedeño, D. L.; Vélez, I. D.; Jones, M. A.; Robledo, S. M. Leishmania tarentolae: utility as an in vitro model for screening of antileishmanial agents. Exp. Parasitol. 2010, 126, 471− 475. (20) St. George, S.; Bishop, J. V.; Titus, R. G.; Selitrennikoff, C. P. Novel compounds active against Leishmania major. Antimicrob. Agents Chemother. 2006, 50, 474−479. (21) Debrabant, A.; Joshi, M. B.; Pimenta, P. F. P.; Dwyer, D. M. Generation of Leishmania donovani axenic amastigotes: their growth and biological characteristics. Int. J. Parasitol. 2004, 34, 205−217. (22) Zilberstein, D.; Blumenfeld, N.; Liveanu, V.; Gepstein, A.; Jaffe, C. L. Growth at acidic pH induces an amastigote stage-specific protein in Leishmania promastigotes. Mol. Biochem. Parasitol. 1991, 45, 175− 178. (23) Chavali, A. K.; Whittemore, J. D.; Eddy, J. A.; Williams, K. T.; Papin, J. A. Systems analysis of metabolism in the pathogenic trypanosomatid Leishmania major. Mol. Syst. Biol. 2008, 4, 177. (24) Nour, A. M. M.; Khalid, S. A.; Brun, R.; Kaiser, M.; Schmidt, T. J. The antiprotozoal activity of sixteen Asteraceae species native to Sudan and bioactivity-guided isolation of xanthanolides from Xanthium brasilicum. Planta Med. 2009, 75, 1363−1368.

AUTHOR INFORMATION

Corresponding Author

*Phone: +49-251-8333311. Fax: +49-251-8332144. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was done within the framework of the NRW International Graduate School of Chemistry, Mü nster, Germany, which is funded by the Government of the State Nordrhein-Westfalen and Westfälische Wilhelms-Universität Münster. The financial support is deeply acknowledged. The biological part of this study is an activity of the Research Network Natural Products against Neglected Diseases (ResNet NPND, http://www.uni-muenster.de/ResNetNPND).

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ABBREVIATIONS USED WHO, World Health Organization; HAT, human African trypanosomiasis; Pfc, Plasmodium falciparum; Tbr, Trypanosoma brucei rhodesiense; Tcr, Trypanosoma cruzi; Ldo, Leishmania donovani; Ltr, Leishmania tarentolae; SI, selectivity index

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REFERENCES

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