Progress in the Studies on Rutaecarpine

Rutaecarpine is an indolopyridoquinazolinone alkaloid isolated from Evodia rutaecarpa and related herbs, which has shown a variety of intriguing biological properties such as anti-thrombotic, anticancer, anti-inflammatory and analgesic, anti-obesity and thermoregulatory, vasorelaxing activity, as well as effects on the cardiovascular and endocrine systems. Recent progress in the studies on the isolation, synthesis, structure-activity relationship studies, biological activities and metabolism of rutaecarpine are reviewed.


Introduction
Herbs have been widely employed as important remedies all over the world [1]. Progress in science and technology in recent decades has made possible not only to isolate and characterize the biologically active constituents of herbs, but also to evaluate their biological activities. Rutaceous plants, especially Evodia rutaecarpa (whose dried fruit is named 'Wu-Chu-Yu' in China), have long been used for the treatment of gastrointestinal disorders, headache, amenorrhea, and postpartum hemorrhage in traditional oriental medicine [2]. Rutaecarpine (8,13-dihydroindolo-[2',3':3,4]pyrido[2,1-b]quinazolin-5(7H)-one, 1a), first isolated by Asahina and Kashiwaki from acetone extracts of Evodia rutaecarpa after basic treatment [3] in 1915 and later from 'Wu-Chu-Yu' [4] is one of the intriguing indolopyridoquinazoline alkaloids of the Rutaceae plants.
The structure of rutaecarpine was determined by degradation methods [3]. Hydrolysis of rutaecarpine with KOH in amyl alcohol afforded anthranilic acid, while fusion with KOH yielded aniline, CO 2 , NH 3 and indole-2-carboxylic acid [3]. Later infrared [5], ultraviolet spectroscopy [6], mass spectrometry [7], 1 H-NMR [8] and 13 C-NMR [8] spectroscopy were employed to confirm the structure. Finally, the molecular structure was confirmed by X-ray analysis by Fujii, Kobayashi and Hirayama [9], who reported that rutaecarpine (C 18  noted that rutaecarpine exists in two forms. The X-ray crystallographic structure of rutaecarpine is an essentially planar structure in which rings A and B as well as rings D and E are coplanar and ring C has a half-chair conformation, which can undergo a ring flip, thus having dihedral angles between the two coplanar rings (co-plane A-B vs D-E) of 6.20° and 6.45°, for forms A and B, respectively ( Figure  1).

Figure 1.
Structure and two forms of rutaecarpine molecules with numbering [9]. A search if the SCIFinder database (provided by the American Chemical Society) afforded 320 references, including 22 patents, covered isolation, biological activity, synthesis, metabolism, toxicology, etc, since the first report in 1915 ( Table 2). Examination of these references provided three noticeable trends: references covering pharmacological properties, the annual numbers of references and number of references originating from China, are increasing (data not shown) (Table 1). Among the 10 review papers written so far, five were focused on synthesis [10], three on pharmacology [11], one on the modulation of cytochrome P450 [12], and one on detection methods [13]. A review written in 1983 by Bergman [10e] is a well organized one covering nomenclature, structure, synthesis and pharmacological properties of rutaecarpine, as well as related quinazolinone alkaloids. In addition, a review [11c], written in 1999 by Sheu, covered in vitro as well as vivo pharmacology of rutaecarpine. Although a recent review [10a], written in Chinese in 2006, provided good coverage of the syntheses of rutaecarpine based on the construction patterns for the five-ring system, since the present work will emphasize the progress in the isolation, synthesis, studies on additional pharmacological properties and the metabolism of rutaecarpine, some overlap between parts of this review and prior publications is inevitable.

1) 2,3,4,9-Tetrahydro-1H-pyrido[3,4-b]indol-1-one -Derived Synthesis.
Since the first synthesis [57], several attempts to synthesize rutaecarpine and its derivatives used 5 as a starting material [59]; some of them employed structurally modified derivatives of 5 which required consturction of the D-ring at the final stage. It should be noted that compound 5, a degradation product of rutaecarpine and related quinazolinone alkaloids, was isolated from the extract of E. rutaecarpa [60] as an alkaloid.
When the corresponding O-methyllactim 7a, prepared from 5 by reacting with trialkyloxonium ion, was employed, the yield was improved up to 66% [61]. Recently, methylthiolactim (7b) was replaced for 7a, which somewhat improved the yield (89%) [62]. The starting 7b could be readily prepared in two steps from compound 5 [63] (Scheme 2).  The imino chloride 9, generated from the HCl salt of 5 and POCl 3 , was condensed with methyl anthranilate to afford rutaecarpine in excellent yield [65a]. In addition, a one-pot synthesis of rutaecarpine from 5, anthranilic acid (6a), and SOCl 2 in refluxing pyridine has also been established [65b] (Scheme 4). Although the procedures described above have advantages for introducing substituent(s) on the 4(3H)-quinazolinone skeleton, the relative unavailability of 5 and its derivatives limits the general utility of this procedure.

2) Tryptamine-Derived Synthesis
Tryptamine-derive syntheses of rutaecarpine are basically one step or stepwise constructions of the C-and D-rings of rutaecarpine (Scheme 5). A decade after its discovery, Schöf and Steuer reported a synthesis of rutaecarpine under so-called physiological conditions by condensing anthranilic acid with 5,6-dihydro-4-carboline [66], which suggested the possible utility of tryptamine (10). They supported their biochemical studies by a chemical synthesis of rutaecarpine from 10 via 5 [67]. The carbon added to the N of tryptamine that would be the C2 of the quinazolinone ring, is generally afforded by formic acid, alkyl formate, triethyl orthformate, alkyloxycarbonyl halide, etc. Tryptamine (10) and/or its equivalents (i.e. 11) can thus be readily available starting materials for the synthesis of rutaecarpine. First, N-formyltryptamine (11) was used as a starting material, which was reacted with sulfonamide anhydride 12 or iminoketene 13, generated from anthranilic acid and SOCl 2 , to afford N-[2-(indol-2-yl)ethyl]-4(3H)-quinazolinone, which was then cyclized by HCl in HOAc to afford rutaecarpine in 45% yield [68] (Scheme 6). The harsh reaction temperature (110 o C) and long time (166 h) as well as the relatively low yield may be the drawbacks to be overcome. Condensation of tryptamine with isatoic anhydride (15) led to N-(2-aminobenzoyl)tryptamine (16). Although Horvath-Dora and Clauder claimed that reaction of 16 with triethyl orthoformate afforded 13b, 14-dihydrorutaecarpine (17) [69b], the real structure of the product was corrected by Bergman and Bergman [8b,70] to be 14a (Scheme 7). The structure of 17 was further confirmed by isolation from Zanthoxylum flavum [71] and Evodia rutaecarpa [72], as well as chemical synthesis [72].  15 16

14a
Bergman and Bergman additionally introduced a modified procedure for the preparation of 14a by reacting N-(2-aminobenzoyl)tryptamine (16) with triethyl orthoformate in 89% yield [8b]. Attempts to convert 17 directly to rutaecarpine (1a) using oxidative coupling reagents such as Hg(OAc) 2 , FeCl 3 , and Pb(OAc) 2 were rather indiscriminate and resulted complex reaction mixtures (Scheme 8). A rationale to improve the conversion of 14a to rutaecarpine could be to introduce a good leaving group at C2 of either the 4(3H)-quinazolinone or the indole ring. The introduction of a good leaving group such as CF 3 at C2 of quinazolinone ring would not only facilitate the acid-catalyzed ring closure of 14b to the pentacyclic intermediate 19, but would also act as a leaving group in the final step [70]. Reaction may proceed by protonation of the 4(3H)-quinazolinone moiety of 14b, followed by electrophilic attack on the indole ring leads to 19, which has an angular CF 3 group in the 13b-position. The reaction time required for the three steps is less than 1 h, the reaction temperature is approximately 150 o C, and the yield for each step is over 95%. This procedure has been applied for the introduction of various substituent(s) on ring A to pursue structure-activity relationship [73]. Prerequisite compound 18 could be readily prepared by reacting isatoic anhydride (15) with trifluoroacetic anhydride [70]. It should be noted that the reactions attempted with a relatively poor leaving Cl moiety at C2 of the quinazolinone ring afforded a mixture of products [74]. The distributions of the products 1a, 22, 23, and 24 were highly dependent on the reaction conditions (Table 6). Such information and additional experimentation gave enough information to explore the mechanism of this-type of reaction as shown below (Scheme 10).  Tryptamine was further condensed with methyl N-(4-chloro-5H-1,2,3-dithiazol-5ylidene)anthranilate, prepared from methyl anthranilate and 4,5-dichloro-1,2,3-dithiazolium chloride (Appel's salt, 25), to give a cyano compound 26, which then underwent cyclization on heating with TFAA/HCl(g) to afford rutaecarpine in 37% overall yields [75] (Scheme 11). Recently a radical reaction was employed for the preparation of rutaecarpine. Bowman et al. used indol-2-yl radical cyclization onto the quinazolinone motif. Previously reported 27 [76] was treated with (Me 3 Sn) 2 to afford an indole radical 29 which would undergo 6-exo cyclization to lead to rutaecarpine in 55% yield [77] (Scheme 13).

12/13
A four-step synthesis of rutaecarpine from 32 was additionally reported. Although the keto group at C5 and dehydrogenation at C15a and N16 position onto 33 could be accomplished with MnO 2 , overdehydrogenation of 1a led 7,8-dehydrorutaecarpine (2a) in 11% yield [80]. This method has, however, somewhat limited general applicability due to the unavailability of the staring carboline and/or its precursor.

5) Miscellaneous
Use of 2,3-polymethylene-4(3H)-quinazolinone was further pursued by Kökösi et al. by employing deoxyvasicinone (38) as a starting material [87]. The Vilsmeier-Haack reaction on 38 yielded corresponding N,N-dimethylmethylidene compound 39, which was treated with aniline in refluxing EtOH to give hydrazone 40 in 87% yield. Fischer indole synthesis was then applied to 40 to yield rutaecarpine in 40% over three-step yields via spiro compound 41 (Scheme 17) [74]. Although a lot of synthetic procedures have been reported for the synthesis of rutaecarpine, many of them were limited to for the introduction of substituent(s) only on a part of the 5 rings in the molecules. For the further pursuing the structure-activity relationship study on rutaecarpine derivatives, it definitely needs either to develop more practical methods to introduce substituents all over the 5 rings or to design (a) new procedure(s) combining methods reported so far.

Biological Properties
A comprehensive review written in 1999 by Sheu [11c], covered very well the in vitro as well as the in vivo pharmacology of rutaecarpine, whose pharmacological actions were reviewed under several subheadings: (1) cardiovascular effects, (2) antithrombotic activity, (3) anticancer effects, (4) antiinflammatory and analgesic effects, (5) effects on endocrine system, (6) antiobesity and thermoregulatory effects, (7) effects on smooth muscle except cardiovascular, and (8) others. Herein we cover only studies on the cytotoxicity and inhibitory activity on COX. Studies on the cytotoxicity of rutaecarpine and its derivatives are summarized in Table 7. In general, a substitution on the ring resulted in selectivity towards specific cell lines. 11-Methoxyrutaecarpine showed selective cytotoxicity against the lung and renal cancer subpanel at 1.38 and 0.31 μM level, respectively, while the 10,11-methylenedioxy analogue showed selective cytotoxicity for the ovarian cancer subpanel [73a]. 10-Bromo-and 10-methylthiorutaecarpine showed high selectivity on a renal carcinoma cell line and strong cytotoxicity at 0.3 and 0.08 μM level, respectively [73b, 88] while 12fluororutaecarpine showed strong cytotoxicity with selectivity on the HT-29 human cell line [89]. Introduction of substitutions on ring E affected the cytotoxicity more significantly. 2-Chlororutaecarpine showed strong inhibitory activity against topoisomerase I and II, with potency comparable to camptothecin [89]. It should be noted that the inhibitory activities on topo I and II seemed to be affected by a substitution on the E-ring but not by substitutions on ring A and C [55,89]. In addition, rutaecarpine inhibited tumor cell migration approximately 30-40% level compared to control at 100 μg/mL [90], which would be a new vista of the further studies on rutaecarpine. Strong and selective inhibitory activity on COX-2 has been claimed as the origin of the antiinflammatory activity of rutaecarpine [91]. A series of substituted rutaecarpines were therefore prepared by employing Fischer indole synthesis as the key step and their inhibitory activities on COX-1 and 2 as well as selectivity on COX-2 were evaluated. The compounds 10-methanesulfonylrutaecarpine and 10-bromorutaecarpine showed promising inhibitory activity (IC 50 = 0.27 and 0.35 μM, respectively) with selectivity (39 and 62, respectively) [92].
It should be noted that more compounds with various substituents on each ring as well as both Aand E-ring together are definitely required to lead better and more decisive conclusion for the structure-activity relationship and to pursue further studies in the process of drug development for specified biological activity.

Metabolism
Understanding of metabolic profiles of biologically active compounds in human body would be a critical step in the process of developing new candidates of drugs. In this regard, systematic studies on the metabolic biotransformations of a plant-derived biologically active rutaecarpine have been pursued. We have summarized herein the in vitro (rat and human liver microsomes) and in vivo (rat) studies done to identify the phase I and phase II metabolites of rutaecarpine.

1) Phase I Studies
In vitro phase I metabolites of rutaecarpine were determined by collision-induced dissociation (CID) fragmentation spectra of MS 2 , MS 3 and retention times by LC/ESI-MS [93]. Incubation of rutaecarpine with rat liver microsomes in the presence of NADPH afforded five mono-hydroxylated rutaecarpines (M1-M5), of which three isomers were hydroxylated on ring E, and one on ring C, and one on ring A, as well as 4 di-hydroxylated rutaecarpines (Figure 2). Ueng et al. confirmed the structures of four mono-hydroxylated metabolites as 3-, 10-, 11-, and 12-hydroxyrutaecarpines, by synthesis and comparison of their spectral data such as mass, UV absorbance and 1 H-NMR spectra with those of authentic synthetic standards [94]. A later in vitro study with human liver microsomes afforded six mono-hydroxylated rutaecarpines of which an additional mono-hydroxylated one (M6) was found to be 9-hydroxyrutaecarpine [95]. In addition, four dihydroxylated rutaecarpines could be divided to three di-hydroxylated metabolites on the A-and C-rings and one on C-and D-rings [95]. By using combinations of chemical inhibition, immunoinhibition and metabolism by cDNA expressed cytochrome P450 (CYP) enzymes, CYP isozyme responsible for each rutaecarpine metabolite was specified as shown ( Figure 1) [94,95,96].  [97].

In vitro
In vivo  In vivo phase I studies on male Sprague-Dawley rats were also pursued to give four monohydroxylated metabolites such as 3-hydroxyrutaecarpine (M5, 10.1%), 9-hydroxyrutaecarpine (M4, 7.2%), 10-hydroxyrutaecarpine (M2, 24.6%) and 11-hydroxyrutaecarpine (M3, 58.1%) and 4 isobaric di-hydroxylated metabolites (M7-M10) (Figure 1) in urine [97], which were identical to the in vitro metabolites except one (M1) that was hydroxylated in the aliphatic moiety. On the other hand, in faeces, the distribution of monohydroxylated metabolites were somewhat different from those in urine. Additionally, M9 was not detected in faeces. It should be noted that the formation of M1 in the rat liver microsomes accounted for 47% the total metabolites, which was not detected in the in vivo system. Such result could be explained by the relative amount of CYP2B enzymes in control liver vs in rat in which amount of CYP2B is usually very low or does not exist [98].

2) Phase II Studies
From the urine of male Sprague-Dawley rats, pretreated intravenously with rutaecarpine, 16 different phase I and II metabolites were identified including four sulfate and four glucuronide conjugates (Figure 3) [97]. Phase I metabolites of rutaecarpine were identified as four monohydroxylated rutaecarpines and four isobaric di-hydroxylated rutaecarpines as metabolites (vide ante). In addition, eight phase II metabolites were identified as conjugated with 4-sulfate (S1-S4) and 4glucuronide (G1-G4). On the other hand, in faeces, 11 different metabolites were identified, in which the di-hydroxylated metabolite M9 and 4 glucuronides G1-G4 were not detected.

Conclusions
Herbal medicines have been employed as the main source for the development of new agents for treating diseases as well as disorders. Although many of them are still used as mixtures and/or extracts, without identification of active ingredient(s), or with unknown biological action mechanisms, many drugs of the currently used in clinics as a single identity are originally derived from herbs and other plants. Rutaecarpine is one of the important alkaloids isolated from the Rutaceae, exhibits a variety of interesting biological properties, thus continuously attract the scientists in academy as well as industry. Recent years have witnessed the steady progress in the chemistry and biology of rutaecarpine. In the present review, we focused only at the area of natural sources, derivatives, synthesis, biological activities and metabolism of rutaecarpine.
We anticipate that recent progress in science and technology will permit the isolation and identification of new minor rutaecarpine-related components, as well as their biological activities. In addition, more efficient and/or practical methods for the synthesis of rutaecarpine derivatives are still required to pursue structure-activity relationship study and to find more potent compounds for drug development.