SAR:s for the Antiparasitic Plant Metabolite Pulchrol. 1. The Benzyl Alcohol Functionality

Pulchrol (1) is a natural benzochromene isolated from the roots of Bourreria pulchra, shown to possess potent antiparasitic activity towards both Leishmania and Trypanozoma species. As it is not understood which molecular features of 1 are important for the antiparasitic activity, several analogues were synthesized and assayed. The ultimate goal is to understand the structure–activity relationships (SAR:s) and create a QSAR model that can be used for the development of clinically useful antiparasitic agents. In this study, we have synthesized 25 2-methoxy-6,6-dimethyl-6H-benzo[c]chromen analogues of 1 and its co-metabolite pulchral (5a), by semi-synthetic procedures starting from the natural product pulchrol (1) itself. All 27 compounds, including the two natural products 1 and 5a, were subsequently assayed in vitro for antiparasitic activity against Trypanozoma cruzi, Leishmania brasiliensis and Leishmania amazoniensis. In addition, the cytotoxicity in RAW cells was assayed, and a selectivity index (SI) for each compound and each parasite was calculated. Several compounds are more potent or equi-potent compared with the positive controls Benznidazole (Trypanozoma) and Miltefosine (Leishmania). The compounds with the highest potencies as well as SI-values are esters of 1 with various carboxylic acids.


Introduction
Parasites belonging to the Trypanozoma and Leishmania genera affect a large proportion of the world's inhabitants, approximately 12 and 8 million people, respectively, especially in the tropic regions. Surprisingly little is known about these organisms, and there are few efficient drugs against them on the market. This is partly due to the fact that the affected regions in general are poor and cannot pay for advanced medications, and these parasitic diseases are consequently labelled as neglected [1,2]. Benzochromenes are polycyclic aromatic compounds formed by the fusion of a benzene ring with a chromene moiety [3]. The benzochromene structure is found in many natural products isolated from plants [4][5][6][7][8], lichens and fungi [9], and a wide range of biological activities such as antibacterial [5,10], antioxidant and anticancer activities have been reported for natural benzochromenes [9,11]. They have been reported to be able to intercalate with DNA [11,12], and to bind selectively to the estrogen receptor ERβ [13]. Several cannabinoids are benzochromenes and bind to the cannabinoid receptors CB1 and CB2 [5,14].
(1), the synthetic procedure of 1 was further developed in order to improve the yields and increase the throughput. In this study, we present the synthesis of 25 pulchrol analogues systematically varied in the benzyl alcohol region while keeping the 2-methoxy-6,6-dimethyl-6H-benzo[c]chromen structure intact, and report their in vitro antiparasitic activity against T. cruzi epimastigotes and L. amazoniensis, as well as L. brasiliensis promastigotes. In addition, their cytotoxicity in a murine macrophage RAW cell line was investigated, in order to get a general overview of the selectivity of the assayed compounds. The SAR:s that this and future studies suggest will be used for the development of a QSAR model to enable the design of more potent and selective antiparasitic drug candidates. In addition, improved understanding of the molecular targets of the parasites could be obtained, facilitating the developing novel antiparasitic agents. (1) Several synthetic strategies to prepare benzo[c]chromenes have been reported in the literature [18,19]. However, as the focus in this study is on the benzyl alcohol functionality of 1, we essentially relied on the synthetic manipulation of pulchrol (1) itself. An important intermediate in the synthesis of 1 is the biaryl intermediate 2 (see Scheme 1), which can be transformed into 1 by intramolecular cyclization. 2 can be obtained by a metal catalyzed Suzuki-Miyaura cross coupling reaction, using phenyl boronic acid and a o-halo-benzoic acid [3,13,14,20,21]. Other synthetic routes to 2 are by a dicarbonyl cycloaddition to chromenes [20], or by the direct intramolecular biaryl formation from phenylbenzyl ethers, as the subsequent cyclization can be performed by metal catalysis [20,22,23] or by a radical reaction promoted by KtBuO [20,24]. A synthetic route to pulchrol was reported in 2014, in which the biaryl formation was achieved with a Suzuki-Miyaura coupling and the final cyclization was acid catalyzed [18]. The procedure for preparing 1 in this study was based on this strategy, although we introduced conditions that are milder, increased the yields, and shortened the reaction times. As in the reported synthesis [18], commercially available 3-iodo-4-(methoxycarbonyl)benzoic acid (also known as 1-methyl-2-iodoterephthalate) was used as the starting material. This was

Results and Discussion
2.1. Improvements of the Synthetic Route to Pulchrol (1) Several synthetic strategies to prepare benzo[c]chromenes have been reported in the literature [18,19]. However, as the focus in this study is on the benzyl alcohol functionality of 1, we essentially relied on the synthetic manipulation of pulchrol (1) itself. An important intermediate in the synthesis of 1 is the biaryl intermediate 2 (see Scheme 1), which can be transformed into 1 by intramolecular cyclization. 2 can be obtained by a metal catalyzed Suzuki-Miyaura cross coupling reaction, using phenyl boronic acid and a o-halo-benzoic acid [3,13,14,20,21]. Other synthetic routes to 2 are by a dicarbonyl cycloaddition to chromenes [20], or by the direct intramolecular biaryl formation from phenylbenzyl ethers, as the subsequent cyclization can be performed by metal catalysis [20,22,23] or by a radical reaction promoted by KtBuO [20,24]. A synthetic route to pulchrol was reported in 2014, in which the biaryl formation was achieved with a Suzuki-Miyaura coupling and the final cyclization was acid catalyzed [18]. The procedure for preparing 1 in this study was based on this strategy, although we introduced conditions that are milder, increased the yields, and shortened the reaction times.
As in the reported synthesis [18], commercially available 3-iodo-4-(methoxycarbonyl)benzoic acid (also known as 1-methyl-2-iodoterephthalate) was used as the starting material. This was reduced to the corresponding benzyl alcohol (methyl 4-(hydroxymethyl)-2-iodobenzoate) using a borane-tetrahydrofuran complex in THF as solvent, at 0 • C (step a). The benzylic hydroxyl group was protected with tert-butyldiphenylchlorosilane (TBDPSCl) using pyridine as solvent (step b), and this intermediate was then coupled with 2,5-dimethoxyphenyl boronic acid using palladiumtetrakis(triphenylphosphine) (Pd(PPh 3 ) 4 ) as catalyst and K 2 CO 3 as base, in dimethoxyethane (DME)/H 2 O 4:1, to yield the biaryl precursor of 2 (step c). This coupling was reported to work successfully at 120 • C for a period of 14 h in 83% yield [18]. However, microwave-assisted Suzuki couplings have been shown to reduce the reaction time and increase the yields [25], and we obtained 85-90% yield at 100 • C in 30 min using a microwave reactor. Instead of MeMgBr in tetrahydrofuran (THF) at 40 • C for 18 h, the methyl ester group was transformed to the tertiary alcohol 2 by two equivalents of methyllithium (MeLi) in THF at 0 • C for 8 h in similar yields, and the milder conditions produced a cleaner product that was considerably easier to purify (step d). By using a larger excess of hydroiodic acid (HI) (10 equiv) for the cyclization of 2 to 1 (step e) we completely avoided the formation of cannabidiol-type biaryl by-products and obtained the deprotected product directly.
Molecules 2020, 25, x FOR PEER REVIEW 3 of 16 reduced to the corresponding benzyl alcohol (methyl 4-(hydroxymethyl)-2-iodobenzoate) using a borane-tetrahydrofuran complex in THF as solvent, at 0 °C (step a). The benzylic hydroxyl group was protected with tert-butyldiphenylchlorosilane (TBDPSCl) using pyridine as solvent (step b), and this intermediate was then coupled with 2,5-dimethoxyphenyl boronic acid using palladiumtetrakis(triphenylphosphine) (Pd(PPh3)4) as catalyst and K2CO3 as base, in dimethoxyethane (DME)/H2O 4:1, to yield the biaryl precursor of 2 (step c). This coupling was reported to work successfully at 120 °C for a period of 14 h in 83% yield [18]. However, microwave-assisted Suzuki couplings have been shown to reduce the reaction time and increase the yields [25], and we obtained 85-90% yield at 100 °C in 30 minutes using a microwave reactor. Instead of MeMgBr in tetrahydrofuran (THF) at 40 °C for 18 h, the methyl ester group was transformed to the tertiary alcohol 2 by two equivalents of methyllithium (MeLi) in THF at 0 °C for 8 h in similar yields, and the milder conditions produced a cleaner product that was considerably easier to purify (step d). By using a larger excess of hydroiodic acid (HI) (10 equiv) for the cyclization of 2 to 1 (step e) we completely avoided the formation of cannabidiol-type biaryl by-products and obtained the deprotected product directly.  Figure 2 summarizes the structure types of the analogues prepared from 1. See the experimental part for details about how each individual analogue was prepared. The biological activities are given in Table 1. Analogues prepared from 1. 3a R = H; 3b R = Cl; 3c R = methoxy; 3d R = isopropyloxy; 3e R = 4-methylpentyloxy; 3f R = isopropylamino; 3g R = isobutylamino; 3h R = isopentylamino; 4a R = Me; 4b R = isopropyl; 4c R = tert-butyl; 4d R = propyl; 4e R = isobutyl; 4f R = neopentyl; 4g R = pentyl; 4h R = 2-cyclopentylethyl; 4i R = cyclohexyl; 4j R = vinyl; 4k R = 2-furanyl; 4l R = phenyl; 5a R = H; 5b R = Me; 5c R = OH; 5d R = methoxy; 5e R = NH2. See Experimental for synthetic details.  Figure 2 summarizes the structure types of the analogues prepared from 1. See the experimental part for details about how each individual analogue was prepared. The biological activities are given in Table 1. reduced to the corresponding benzyl alcohol (methyl 4-(hydroxymethyl)-2-iodobenzoate) using a borane-tetrahydrofuran complex in THF as solvent, at 0 °C (step a). The benzylic hydroxyl group was protected with tert-butyldiphenylchlorosilane (TBDPSCl) using pyridine as solvent (step b), and this intermediate was then coupled with 2,5-dimethoxyphenyl boronic acid using palladiumtetrakis(triphenylphosphine) (Pd(PPh3)4) as catalyst and K2CO3 as base, in dimethoxyethane (DME)/H2O 4:1, to yield the biaryl precursor of 2 (step c). This coupling was reported to work successfully at 120 °C for a period of 14 h in 83% yield [18]. However, microwave-assisted Suzuki couplings have been shown to reduce the reaction time and increase the yields [25], and we obtained 85-90% yield at 100 °C in 30 minutes using a microwave reactor. Instead of MeMgBr in tetrahydrofuran (THF) at 40 °C for 18 h, the methyl ester group was transformed to the tertiary alcohol 2 by two equivalents of methyllithium (MeLi) in THF at 0 °C for 8 h in similar yields, and the milder conditions produced a cleaner product that was considerably easier to purify (step d). By using a larger excess of hydroiodic acid (HI) (10 equiv) for the cyclization of 2 to 1 (step e) we completely avoided the formation of cannabidiol-type biaryl by-products and obtained the deprotected product directly.  Figure 2 summarizes the structure types of the analogues prepared from 1. See the experimental part for details about how each individual analogue was prepared. The biological activities are given in Table 1. Analogues prepared from 1. 3a R = H; 3b R = Cl; 3c R = methoxy; 3d R = isopropyloxy; 3e R = 4-methylpentyloxy; 3f R = isopropylamino; 3g R = isobutylamino; 3h R = isopentylamino; 4a R = Me; 4b R = isopropyl; 4c R = tert-butyl; 4d R = propyl; 4e R = isobutyl; 4f R = neopentyl; 4g R = pentyl; 4h R = 2-cyclopentylethyl; 4i R = cyclohexyl; 4j R = vinyl; 4k R = 2-furanyl; 4l R = phenyl; 5a R = H; 5b R = Me; 5c R = OH; 5d R = methoxy; 5e R = NH2. See Experimental for synthetic details.

Transformations of the Benzylic Alcohol Functionality
4h R = 2-cyclopentylethyl; 4i R = cyclohexyl; 4j R = vinyl; 4k R = 2-furanyl; 4l R = phenyl; 5a R = H; 5b R = Me; 5c R = OH; 5d R = methoxy; 5e R = NH 2 . See Experimental for synthetic details.       Tables 2 and 3 give the 1D 1 H and 13 C-NMR shifts of the assayed compounds. In general, 1-H is a doublet (d) with coupling constant (J) close to 2 Hz, 3-H is a doublet of doublet (dd) with J = 9 and 2 Hz, while 4-H is a d with J close to 9 Hz. 10-H is a d with J close to 2 Hz, 8-H is a dd with J = 8 and 2 Hz, while 7-H is a d with J close to 8 Hz.
Molecules 2020, 25, x FOR PEER REVIEW 4 of 16  Tables 2 and 3 give the 1D 1 H and 13 C-NMR shifts of the assayed compounds. In general, 1-H is a doublet (d) with coupling constant (J) close to 2 Hz, 3-H is a doublet of doublet (dd) with J = 9 and 2 Hz, while 4-H is a d with J close to 9 Hz. 10-H is a d with J close to 2 Hz, 8-H is a dd with J = 8 and 2 Hz, while 7-H is a d with J close to 8 Hz. 15 Tables 2 and 3 give the 1D 1 H and 13 C-NMR shifts of the assayed compounds. In general, 1-H is a doublet (d) with coupling constant (J) close to 2 Hz, 3-H is a doublet of doublet (dd) with J = 9 and 2 Hz, while 4-H is a d with J close to 9 Hz. 10-H is a d with J close to 2 Hz, 8-H is a dd with J = 8 and 2 Hz, while 7-H is a d with J close to 8 Hz.   Tables 2 and 3 give the 1D 1 H and 13 C-NMR shifts of the assayed compounds. In general, 1-H is a doublet (d) with coupling constant (J) close to 2 Hz, 3-H is a doublet of doublet (dd) with J = 9 and 2 Hz, while 4-H is a d with J close to 9 Hz. 10-H is a d with J close to 2 Hz, 8-H is a dd with J = 8 and 2 Hz, while 7-H is a d with J close to 8 Hz.   Tables 2 and 3 give the 1D 1 H and 13 C-NMR shifts of the assayed compounds. In general, 1-H is a doublet (d) with coupling constant (J) close to 2 Hz, 3-H is a doublet of doublet (dd) with J = 9 and 2 Hz, while 4-H is a d with J close to 9 Hz. 10-H is a d with J close to 2 Hz, 8-H is a dd with J = 8 and 2 Hz, while 7-H is a d with J close to 8 Hz. 24 Tables 2 and 3 give the 1D 1 H and 13 C-NMR shifts of the assayed compounds. In general, 1-H is a doublet (d) with coupling constant (J) close to 2 Hz, 3-H is a doublet of doublet (dd) with J = 9 and 2 Hz, while 4-H is a d with J close to 9 Hz. 10-H is a d with J close to 2 Hz, 8-H is a dd with J = 8 and 2 Hz, while 7-H is a d with J close to 8 Hz. 21 Tables 2 and 3 give the 1D 1 H and 13 C-NMR shifts of the assayed compounds. In general, 1-H is a doublet (d) with coupling constant (J) close to 2 Hz, 3-H is a doublet of doublet (dd) with J = 9 and 2 Hz, while 4-H is a d with J close to 9 Hz. 10-H is a d with J close to 2 Hz, 8-H is a dd with J = 8 and 2 Hz, while 7-H is a d with J close to 8 Hz.

Antiparasitic Activity of Pulchrol (1), the Starting Point
Pulchrol (1) and pulchral (5a) have previously been shown to possess antiparasitic activity, 1 towards T.cruzi epimastigotes and three strains of Leishmania promastigotes (L. mexicana, L. brasiliensis and L. amazoniensis), and 5a towards L. brasiliensis and L. amazoniensis [4]. The IC 50 value of 1 against T.cruzi in this investigation was 18.5 µM (see Table 3), and it is thereby equipotent with the positive control benznidazole (19.2 µM). Benznidazole is currently on the market for the treatment of Chagas disease, caused by T. cruzi, under the trade names Rochagan and Radanil. The potency of pulchrol against the Leishmania parasites, with the IC 50 values 59.2 µM for L. brasiliensis and 77.7 µM for L. amazoniensis, is more moderate, although promastigotes of L. mexicana (not part of this investigation) are more sensitive with an IC 50 value of 17 µM [4]. As the chemical structure of pulchrol (1) does not raise any red flags, it contains no functionalities that are associated with reactivity or unspecific biological activity, we were motivated to synthesize and assay analogues of 1. This study focuses on the importance of the benzyl alcohol functionality for the biological activity, and the natural products (1 and 5a) together with 25 analogues were prepared as discussed above, and assayed. The assays against T. cruzi epimastigotes and L. amazoniensis as well as L. brasiliensis promastigotes essentially follows the protocol used in previous investigations, but the cytotoxicity in a mammalian murine macrophage RAW cell line was also assayed in order to get an impression of the compounds selectivity for the parasites over a mammalian cell line. The biological results are presented in Table 1.

Antiparasitic Activities towards Trypanozoma cruzi Epimastigotes
To determine the importance of the hydroxyl group for pulchrol's activity, the 9-methyl analogue (3a) was prepared and found to be considerably less active (IC 50 = 51.1 µM) compared to 1. With the intention of mimicking the Van der Waals interactions around the benzylic carbon, the hydroxyl group was replaced by a chlorine (3b), but this analogue was also less potent than 1 (IC 50 = 38.1 µM). It would appear to be beneficial to have an oxygen in the benzylic position, although 3a and 3b are by no means inactive. To evaluate if the hydroxyl group acts as a hydrogen bond donor, the methyl ether 3c was prepared and assayed. It is slightly less potent compared to 1 (IC 50 = 24.6 µM), indicating that the hydroxyl group is more a hydrogen bond acceptor than donor. However, the two bulkier ethers 3d and 3e were actually more potent than 1 (IC 50 = 12.9 and 9.0 µM, respectively), suggesting that there also is a lipophilic pocket close to the binding site of the benzylic moiety in a target protein. In addition, 3d and 3e show an improved SI compared to 1 and 3c. Somewhat surprisingly, the isopropylamino analogue 3f is considerably less potent and selective compared to the isopropyl ether 3d, while the isobutyl and isopentyl analogues 3g and 3h (IC 50 = 15.4 and 5.9 µM, respectively) are as potent as the bulkier ethers but less selective.
Moving to the pulchrol esters 4a-4l, it is clear that this group of analogues is interesting as most of them are more potent and selective compared to 1. For the saturated esters 4a-4i it is especially those with branched alkyl groups that are good, with the 3-methylbutanoic acid ester 4e standing out with IC 50 = 4.2 µM and SI = 6.7. All the unsaturated esters 4j-4l prepared and assayed are potent and selective, indicating that a π-π interaction with the binding pocket is favourable. The furan-2-carboxylic acid ester 4k is actually the most potent (IC 50 = 3.8 µM) and the most selective (SI = 7.9) towards T. cruzi of all analogues prepared in this investigation, and has considerably better antiparasitic activity towards T. cruzi epimastigotes compared to the positive control Benznidazol (see Table 1). Also the selectivity is noteworthy, as it is twice that of the positive control.
Among the 1'-carbonyl analogues (5a-5e) included in this study, the aldehyde 5a and the methyl ketone 5b can be compared to 1, both with respect to potency and selectivity. However, the carboxylic acid 5c, the methyl ester 5d and especially the amide 5e are less potent, although the carboxylic acid 5c is considerably less cytotoxic than the others. The N-hydroxy-9-carboximidamide 6 was obtained as a by-product and assayed; it did not show any interesting activities, although it was more potent than the 9-carboxamide 5e. If anything, analogue 6 underlines the importance of a lipophilic component at the benzylic moiety.

Antiparasitic Activities towards Leishmania brasiliensis Promastigotes
As for T.cruzi, transforming the benzylic alcohol moiety to a methyl group is not beneficial, and 3a was found to be slightly less potent than 1. On the contrary, the benzyl chloride (3b) showed both an interesting potency (IC 50 = 17.1 µM) and selectivity (SI = 3.5), suggesting that the presence of an oxygen in the benzylic position is less important. The differences in antiparasitic effects of 3b in T. cruzi epimastigotes and L. brasiliensis promastigotes indicate that the molecular targets in the two species are different. Although the two ethers 3c and 3d are slightly more potent than 1, the 4-methylpentyl ether 3e is considerably less potent and the positive effect of bulky ethers observed for T. cruzi is not seen with L. brasiliensis. However, for the secondary amines 3f-3h the trend is identical, and the isopentylamino analogue 3h (IC 50 = 15.9 µM) is one of the most potent against L. brasiliensis. Most of the esters 4a-4l, except for 4h, are more potent compared to 1, and among the saturated esters there is again a tendency that branched alkyl groups are better than straight. The aromatic esters 4k and 4l are potent towards L. brasiliensis as well, 4k (IC 50 = 12.8 µM) is as potent as the positive control, although the selectivity observed towards T. cruzi is less prominent. The vinyl ester 4j is the most potent and selective towards L. brasiliensis, with IC 50 = 5.7 µM and SI = 7.0, overshadowing the positive control. For the 1'-carbonyl analogues, the aldehyde 5a, the methyl ketone 5b and especially the methyl ester 5d are more potent, while the carboxylic acid 5c and the N-hydroxy-9-carboximidamide (6) are comparable to 1. The carboxamide 5e is considerably less potent than 1.

Antiparasitic activities towards Leishmania amazoniensis promastigotes
For L. amazoniensis, too, the replacement of the benzylic alcohol moiety for a methyl group (3a) does not improve the antiparasitic activity, and as for L. brasiliensis, a chlorine substituent in this position (3b) increases the potency more than two-fold. Ethers of pulchrol (1) are more potent; the methyl and isopropyl ethers (3c and 3d) only slightly, but the 4-methylpentyl ether 3e more clearly. This is in contrast to the poor potency of 3e towards L. brasiliensis. For the secondary amines the sensitivity of L. amazoniensis follows that observed already for T. cruzi and L. brasiliensis, lower potency for the isopropylamino analogue 3f and higher for the isobutyl-and isopentylamino analogues 3g and 3h. For the esters 4a-4l, the results follow those obtained with L. brasiliensis (vide supra) closely.
There is only one exception, the 3-cyclopentylpropanic acid ester 4h, which displays a more expected potency towards L. amazoniensis than towards L. brasiliensis (vide supra). Again, the vinyl ester 4j is the most potent towards L. amazoniensis among the esters, and actually among all compounds assayed here, and as the SI value is 5.8 it is also by far the most selective compound. Among the 9-carbonyl analogues, the aldehyde 5a and the methyl ketone 5b are more potent, while the methyl ester 5d and the N-hydroxy-9-carboximidamide (6) are only slightly more potent. The carboxylic acid 5c and the carboxamide 5e are both considerably less potent compared to 1.

General
1 H-NMR spectra (400 MHz) and 13 C-NMR spectra (100 MHz) were recorded with a Bruker Avance II (Bruker Biospin AG, Industriestrasse 26, 8117 Fällanden, Switzerland) in CDCl 3 . The individual 1D signals were assigned using 2D NMR experiments (COSY, HSQC, HMBC). The chemical shifts are given in ppm with the solvent signal as reference (7.27 ppm for 1 H and 77.0 for 13 C). Infrared spectra were recorded with a Bruker Alpha-P FT/IR instrument (Bruker Biospin AG, Industriestrasse 26, 8117 Fällanden, Switzerland) with a Diamond ATR sensor as films, and the intensities are given as vw (very weak), w (weak), m (medium), s (strong) and vs (very strong). High-resolution mass spectra (HRMS) were recorded with a Waters XEVO-G2 QTOF equipment (Waters Corp, Milford, Worcester County, Massachusetts, United States), with electrospray ionization (ESI). Synthetic reactions were monitored by TLC using alumina plates coated with silica gel and visualized using either UV light and/or spraying/heating with vanillin/H 2 SO 4 . Flash chromatography was performed with silica gel (35-70 µm, 60 Å). THF was distilled from sodium, acetonitrile was distilled from CaH 2 and other reaction solvents were dried with Al 2 O 3 . Commercially available compounds were obtained from Aldrich.
Methyl 5-(((tert-butyldiphenylsilyl)oxy)methyl)-2',5'-dimethoxy-[1,1'-biphenyl]-2-carboxylate (intermediate in the synthesis of 1), 2,5-dimethoxyphenylboronic acid (155 mg, 0.85 mmol), K 2 CO 3 (394 mg, 2.85 mmol) and tetrakis(triphenylphosphine)palladium(0) (115 mg, 0.1 mmol), were added to a stirred solution of methyl 4-(((tert-butyldiphenylsilyl)oxy)methyl)-2-iodobenzoate (prepared as described above, 300 mg, 0.57 mmol) dissolved in 4:1 DME/water (15 mL). The mixture (contained in a microtube) was degasified under vacuum/N 2 at −78 • C five times. The microwave reaction conditions were 100 • C, high pressure, and 10 s of pre-stirring. After 30 min in the microwave reactor, the mixture was filtered through a plug of celite and washed with ethyl acetate (250 mL) before drying (Na 2 SO 4 ) and removal of solvent under This presumed active site on which pulchrol and its analogues may be acting, is probably somewhat different in the different organisms. In L. brasiliensis there appear to be limited for branched chains of around five carbons, while T. cruzi and L. amazoniensis seem to have some flat hydrophobic regions that enhance the activity of aromatic and planar substituents. The selectivity as the SI is below or around 1 for most of the compounds, but is considerably higher in some examples. This is an important property to learn to understand, if compounds developed from this model ever should move forward into clinic trials. The best compound developed in this investigation is the vinyl ester 4j. It shows potencies well below those of the positive controls and selectivity indexes of more than 5 for all three organisms. Another name for 4j is an acrylic acid ester, and it is known that the acrylic acid derivatives may be weakly reactive as Michael acceptors. However, this is only relevant when an acrylic acid ester is bound to a pocket of a protein and presented to a highly reactive nucleophile (e.g., a thiol group of a cysteine, acting as an irreversible 'covalent inhibitor'). If this is the explanation for the potency and selectivity of 4j in this investigation, this type of analogues can eventually be used for the fishing out of the molecular targets of the parasites, and enable studies of such.