Synthesis of trypanocidal tetrahydrofuran lignans

Several tetra-substituted 2,5-dihydrofuran lignans have been prepared using a sequential Michael addition-carbocyclization with palladium as the catalyst. The synthetic compounds were evaluated against trypomastigote forms of Trypanossoma cruzi and the higher activity for diastereoisomeric compounds could be correlated to the trans configuration of the aromatic rings. The highest activity was observed for compound 14b in which IC 50 was 1.5µM


Introduction
Chagas disease is an important tropical disease caused by the protozoan Trypanosoma cruzi which has the hematophagous reduviid bug Rhodnius prolixus (Hemiptera) as major parasite vector.2][3] Approximately 30-40% of the patients are affected by irreversible heart and gastrointestinal tract lesions. 4,5Thus, such disease has caused approximately 3 million disabilities each year.Therefore, it represents a very serious public health and economic problem in these countries.
The search for solutions has been attempted in several different directions.2][3] To date there have been only two trypanocidal drugs, Nifurtimox (4-[(5-nitrofurfurylidene)amino]-3-(methylthio)morpholine 1,1-dioxide) and benznidazole (N-benzyl-2-nitro-1-imidazoleacetamide).The daily administration of benznidazole to the early chronic patients for two months has diminished the risk of developing cardiomyopathy. 1][8] Due to the increasing influx of people into urban area and the transfusion of contaminated blood, the disease characteristics has changed from endemic to epidemic. 1,9 etween 1960 and 1989, the prevalence of infected blood in blood banks in selected cities of South America ranged from 1.7 % in São Paulo, Brazil to 53.0 % in Santa Cruz, Bolivia, a percentage far higher than that observed for hepatitis or HIV infection.In this regard, an approach to eliminate the parasite in blood involve the chemoprophylactic agent, crystal violet (N-{4-bis[ [4-(dimethylamino)phenyl]methylene]-2,5-cyclohexadien-1-ylidene}-N-methylammonium chloride), a dye discovered to be effective many years ago. 10Nevertheless, such treatment has not been well accepted by patients due to the bluish colour it confers to the blood.
A number of investigations dealing with effective trypanocidals described the isolation of diterpene, 11 naphtoquinones, 12 and lignans [13][14][15] as active compounds.Our research group started with the investigation of natural products as a potential source of trypanocidal drugs since 1998 13 when the lignans (-)-grandisin ( 1) and (+)-veraguensin (2) were discovered as the most active natural products against trypomatigote forms of Trypanosoma cruzi.These structurally simple lignans were formerly isolated from twigs of Virola surinamensis, a common woody species from Amazon Forest.Since both compounds showed potency about forty times higher than crystal violet (Figure 1), we envisaged further investigations of synthetic analogs in order to better understand the structure-activity relationship.

Figure 1
A drug candidate with simple structure would be desirable, because it would allow the access of a large number of derivatives and then studies involving structure-activity relationship would be carried out.
Such considerations prompted us to synthesize several derivatives of 1 and 2, via a short and simple convergent route.Our general strategy was based on the construction of a tetrahydrofuran (THF) ring by using sequential Michael addition-carbocyclization reaction with palladium catalyst. 16We report herein, the synthesis of several THF lignan derivatives and their trypanocidal activity.

Results and Discussion
In the strategic analysis to synthesize THF lignans and its derivatives, methylenetetrahydrofuran 3 was chosen as a viable intermediate for the easy introduction of several functional groups at the THF ring (Scheme 1).The key step in the proposed sequence, a Michael additioncarbocyclization, would provide 3 from 1.5 equivalent (eq) of propargyl alcohol 4 as a donor and arylidenemalonate 5 with vicinal electron withdrawing groups as a acceptor (EWG) by using substoichiometric amounts of palladium reagent and base. 16Moreover, this reaction could be carried out in hindered substrates at the aromatic moieties such as 4 and 5.

Scheme 1
The first objective in the synthesis was the development of workable synthesis of propargyl alcohol 7 and arylidenemalonate 10 (Scheme 2).The 3,4,5-trimethoxybenzaldehyde (6) was converted to 7 via addition of ethynylmagnesium bromide in high yield.On the other hand, preparation of 10 started with syringaldehyde (8), which was converted to the corresponding benzyl ether 9 using K 2 CO 3 in dimethylformamide (DMF).In this step, the typical reaction conditions using NaH in THF or DMF, and K 2 CO 3 in acetone, were conducted to the formation of a complex mixture.Knoevenagel reaction with diethyl malonate and 9 by using catalytic amount of pyrrolidinium acetate gave rise to 10 in high yield but without decarboxylation. 17,18

Scheme 2
The formation of the methylenetetrahydrofuran 11 from 7 and 10 was accomplished through a sequential Michael addition-carbocyclization protocol using improved Balme's palladiummediated approach (Figure 2). 16Treatment of 7 with catalytic amount of n-butyl lithium (n-BuLi) generated the corresponding alkoxide in few minutes.After sequential addition of catalytic amount of Pd(AcOH) 2 , triphenylphosphine (TPP), and 10, the substrates were smoothly converted to a diastereomeric mixture 11a and 11b after 12 h in excellent yield (entry 2).The low diastereoselectivity was interesting in this case since many types of compounds could be generated.This 'one pot reaction' to couple tertiary alcohol and arylidenemalonate in high yield could not be promoted by adding over 0.1 eq of n-BuLi, as shown in the original reference (entry 3 and 4) 16 but using a reaction time of 12 hr (entry 1).These results paved a way to the possibility of coupling between several hindered Michael acceptor and donor substituted at the aromatic moiety.Since the mixture of diastereomeric compounds was quite difficult to separate by silica gel column, the following reaction was then performed with the diastereomeric mixture.
At the next step, although several conditions mentioned in Krapcho's report were tried, simply mono-decarboethoxylated THF compound from 11 could be obtained only in low yield. 24or example, the treatment of KCN in dimethylsulfoxide (DMSO) under 150 o C was obtained to corresponding mono-decarboethoxylated furan in 26% yield.The desired compound could be one of the precursors to synthesize THF lignan derivatives, but due to the low yield, the optimization of the next reaction step was further investigated.Treatment of 11 with an alkaline aqueous solution followed by acidification provided monocarboxylic acid 12 via sequential reactions of hydrolysis of ester, decarboxylation, and isomerization of olefin in high yield (Scheme 3).Since it was difficult to separate the mixture of each diastereomer by silica gel chromatography, the mixture 12a/12b was treated with excess of LiAlH 4.However, complete transformation to the corresponding allyl alcohol 14 did not occur.Accordingly, 12a/12b was treated with MeI and 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) to yield methyl ester 13a/13b 25 , which was then converted to 14a/14b by using LiAlH 4 in high yield without 1,4-reduction products.The allyl alcohols 14a and 14b were easily separated using silica gel column chromatography.

Figure 2
Allyl alcohol 14b, which was the major diastereoisomer of 14, was transformed in a twosteps sequence using tosylation with tosyl chloride (TsCl) and 4-(dimethylamino)pyridine (DMAP), followed by reduction with excess of LiAlH 4 to yield 7,7′-dihydrofuran 16b (Scheme 4.).Catalytic hydrogenation of 16b with Pd(OH) 2 /C gave the phenol 17b by hydrogenolysis.In this step, we attempted to transform 16b to the desired THF lignan 18b directly, but this conversion was not possible under normal hydrogenation conditions due to the low reactivity at the hindered tetrasubstituted olefin.For example, the reaction with 16b by using Pd(OH) 2 on carbon at room temperature (rt) for 48 h led to the formation of complex mixture as indicated by TLC.

Scheme 3
In short steps, the allyl alcohol 25a and 25b was obtained in high yield via the same steps (Scheme 6.).Although it was difficult to separate most of isomers of intermediates by using silica gel column chromatography, 25a and 25b exhibited excellent resolution without the application of preparative TLC.

Scheme 6
The first synthesis of 7,7′-dehydrofuran 17b was accomplished by using a convergent strategy via nine steps in 9% total yield from syringaldehyde (8).Then, from aldehyde 6, allyl alcohol 25a and 25b were obtained in 41% total yield.The advantage of the reaction sequence can be summarized by two points.One is the facile induction of several moieties at THF ring.The second is the possibility to scale up this synthesis at inexpensive cost using a catalytic amount and easily available reagents.
The evaluation of trypanocidal activity was carried out according to the procedure described in the literature (Table 1) 21 .In comparison with grandisin (1) and veraguensin (2) which exhibited total tripomastigote lysis at the concentration of 3.7 and 2.3 µM, all synthetic lignans showed lower activities, except for the lignan 14b in which a IC 50 of 1.5µM was determined.The trans configuration between aromatic ring seems to be an important requirement for activity since 11b and 14b were more active than the corresponding 11a and 14a, although for veraguensin (cis) and grandisin (trans) no significant difference could be observed.
Since the tetrahydrofuran lignans have showed promising trypanocidal activity [13][14][15] , further systematic investigations are required for a better evaluation of this potential.

Experimental Section
General Procedures.EI-MS were measured at 70 eV on a HP 5990/5988 A spectrometer. 1 H and 13 C NMR spectra were measured on a Bruker DPX-300 (300 and 75 MHz) while 2D experiments (HMQC and HMBC) were recorded in a Bruker DRX-500 (500 and 125 MHz) using CDCl 3 (Aldrich) as solvent and TMS as int.standard.Chemical shifts were reported in units (ppm) and coupling constants (J) in Hz.ESI analysis were performed on a triple quadrupole (Quattro-LC, Micromass, UK).Dilute standards (10mg ml-1) were prepared daily in 80% v/v (methanol/water) with 0.25mg ml-1 as the final concentration.Routine TLC analysis were performed Silica gel (Merck, 70-230 mesh) was used for CC and silica-gel 60 PF 254 Merck (0.50 mm and 1 mm) for anal.and prep.TLC.Spots on chromatograms were detected under UV light (254 and 365 nm) and by spraying H 2 SO 4 60% and ceric sulphate solutions followed by heating.