Indirect Iodination on the Vinyl Double Bond of Andrographolide

Andrographolide is a bicyclic diterpenoid constituent of Andrographis paniculata which is used extensively in the traditional medicine in Indonesia to treat inflammations. The structure of andrographolide contains an a-alkylidene r-butyrolactone moiety, two olefin bonds at C-8(C-17) and C-12(C-13), and three hydroxyls at C-3, C-19 and C-14. Andrographolide's structure lacks of aromatic ring, hence the iodination reaction of this compound is quite an interesting challenge to be investigated. Iodine atom was incorporated at vinyl position by indirect reaction. The reaction was divided into two steps, which was started by a bromination in non polar medium, then followed by iodination. It is shown that the principal reaction is the addition of bromine atom to an sp2 carbon atom of the vinyl group via electrophilic substitution. Bromination was carried out in chloroform solvent at room temperature to produce bromo-andrographolide. The iodination was applied further by using palladium triphenylphosphine catalyst. Positive charge iodine species was produced in situ with the addition of chloramine-T, an oxidizing agent, at 40 oC. 1H-NMR study shows that iodine attacked the C-12(C-13) vinyl bond which was confirmed by (1) the disappearance of proton chemical shift of C-12 at SH 6.86 ppm and (2) the change of proton chemical shifts of C-11 which shifted downfield at SH 2.74 ppm and 2.63 ppm, due to the deshielding effect of iodine.


Introduction
The main reactions of halogenation with iodine belong either to nucleophilic or electrophilic substitutions.Direct iodination (replacement of hydrogen atom by a iodine atom) represents an exception almost for electrophilic substitution in arenes.Electrophilic iodination is a process in which formally a positively charged iodine attacks a system with high electron density such as an aromatic ring or a double bond.As a result a covalent carbon-iodine bond is formed with loss of a positively charged leaving group.The leaving group (the electrofuge) must necessarily depart without its electron pair.The most important leaving groups are those that can best exist without the pair of electrons necessary to fill the outer shell, i.e. the weakest Lewis acids.The most common leaving group is the proton (Eersels et al., 2005).When bromine is used as a leaving group for iodination, the advantage of such an iodine-for-bromine exchange (non-isotopic exchange) reaction is that a very high specific activity can be obtained, provided the radioiodinated compound is efficiently separated from its brominated precursor (Coenen et al., 2006).
Generally, halogenation is preferably carried out to aromatic moieties, but sometimes it has to be applied on a double bond.In recent years, Verbeek and his co-workers committed that they were the first to apply iodination reaction successfully on vinyl double bond of tiagabine molecule.In principal, tiagabine might be labelled in one of the thiophene moieties.However, to circumvent Z,E formation they decided to label the vinylic part (Figure 1).On the first step Verbeek directly brominated tiagabine in carbon tetrachloride medium to the vinyl double bond, to produce bromotiagabine in 70% yield.This reaction was followed by iodination to substitute the bromine by using Na-I 123 to form iodotiagabine by using the Cu(I) mediated non-isotopical exchange reaction generating the Cu(I) in situ with gentisic acid as the catalyst.The overall radiochemical yield after preparative HPLC (Rt 2 = 42 min.,Rt 3 = 48 min.)was 50%, with a radiochemical purity of > 99%.(Verbeek et al., 2007).
Andrographolide (Figure 2) has an α-alkylidene γ-butyrolactone, two olefin bonds at C-8 and C-12, and three hydroxyls at C-3, C-19 and C-14 (Nanduri et al., 2004).Its molecular formula is C 20 H 30 O 5 .Andrographolide is soluble in methanol, ethanol, pyridine, acetic acid and acetone, but it is slightly dissolved in ether and water.Its melting point is 228 -230 ℃ and its ultraviolet λmax in ethanol is 223 nm.Andrographolide's structure lacks of aromatic ring, hence the iodination reaction of this compound is quite an interesting challenge to be investigated.Iodine atom was incorporated at vinyl position by indirect reaction via electrophilic substitution.Electrophilic substitution was chosen due to the instability of andrographolide to high temperature as often needed in nucleophilic reaction.The reaction was divided into two steps, which was started by a bromination in non polar medium, and then followed by iodination.It is shown that the principal reaction is the addition of bromine atom to an sp 2 carbon atom of the vinyl group via electrophilic substitution.Bromination was carried out in chloroform solvent at room temperature to produce bromo-andrographolide.The iodination was applied further by using palladium triphenylphosphine catalyst.

Chemicals
Andrographolide 98% 500 mg CAS 5508-58-7 for R & D use and palladium triphenylphosphine analytical grade were purchased from Aldrich.Prior to use the compound was dissolved in methanol and chloroform, respectively.Bromine liquid and chloroform analytical grade were purchased from E.Merck.

Iodination
Accurately weighed +35 mg andrographolide (0.1 mmol) was added by 0.15 ml of bromine liquid in CHCl 3 (1:1 v/v).The mixture was vortex-mixed (Maximix plus-Thermolyne) for 5 minutes at room temperature.The excess of bromine was evaporated, and added by 0.3 ml of palladium triphenylphosphine in chloroform (1 µmol).The solution was vortex-mixed for one minute and followed by the addition of 20 µl of sodium iodide solution and 0.25 ml of chloramine-T (1 mg/ml in sterilized distilled water).The solution was vortex-mixed for one minute and heated at 40 ℃ for 5 minutes.Oxidation was stopped by the addition of 0.25 ml of sodium metabisulfite solution (1 mg/ml in sterilized distilled water).

Computational study
Computational study was carried out to predict (1) the position of halogenations, and (2) the chemical shifts of protons in andrographolide and iodo-andrographolide structures.Softwares used in this step were ChemOffice 2004, ArgusLab (Mark A. Thompson Planaria Software LLC, Seattle, WA, http://www.arguslab.com),and HyperChem Professional 7.

Computational study
The main reactions of halogenation with iodine belong either to nucleophilic or electrophilic substitutions.Electrophilic iodination is a process in which positive charged iodine attacks a system with high electron density such as an aromatic ring or a double bond.ArgusLab software calculates and predicts the position of iodination based on the electron density (Figure 3).HyperChem Professional 7.0 calculates that iodination on andrographolide increases its lypophylicity as showed by the increasing of the cLog P value (Table 1).
The addition of bromine molecule which was carried out in chloroform as a non polar medium, produced bromo-andrographolide, and then iodo-andrographolide was yielded from further iodination reaction catalyzed by palladium triphenylphosphine (Figure 4).
The structure of andrographolide and iodo-andrographolide were compared by the 1 H-NMR spectra which showed that iodination was occurred at C-12 (Figure 5 and Table 2).
The two olefin bonds at C-12 and C-17 gave their chemical shifts at δ H 4.5 -7.0 ppm which were caused by the increasing of electronegativity of sp 2 Carbon and anisotropy of C=C bond hence resulted in higher frequency (lower magnetic field) of the peaks.Proton at C-12 double bond, which located near a carbonyl, was strengthened by both the anisotropic effect of the carbonyl and conjugation, hence it occured in higher frequency at δ H 6.86 ppm.While proton at C-17 double bond occurred at δ H 4.89 ppm and 4.67 ppm.Proton position of these two double bonds is very important to identify the position of halogenation. 1 H-NMR study shows that iodine attacked the C-12(C-13) vinyl bond which was confirmed by the disappearance of proton chemical shift of C-12 at δ H 6.86 ppm.The iodination position was further confirmed by studying the chemical shift of H-11 which are in the neighbouring position with H-12.The peaks of H-11 show a downfield shift at δ H 2.74 ppm and 2.63 ppm, probably due to the deshielding effect of iodine.Iodination was positively confirmed by these data.The result of 1 H-NMR analysis was compared with computational study which was calculated by using ChemOffice software as showed in Figure 6.ChemOffice calculation showed estimation of proton chemical shift of andrographolide and 12-bromo-andrographolide.The chemical shift of H-12 (δ H 6.86 ppm) which was available in andrographolide molecule (Figure 6 left) was not detected in bromo-andrographolide molecule (Figure 6 right).Bromine ion (Br + ) preferred to attack C-12 double bond instead of C-17 because C-12 which was located near a carbonyl had a higher electronegativity than C-17.

Conclusion
Indirect iodination on the vinyl bond of andrographolide via electrophilic substitution had successfully produced iodo-andrographolide. Iodine was incorporated at C-12 double bond as proven by 1 H-NMR which showed the disappearance of proton chemical shift of C-12 at δ H 6.86 ppm and the change of proton chemical shifts of C-11 which shifted downfield at δ H 2.74 ppm and 2.63 ppm, due to the deshielding effect of iodine.

Table 2 .
Chemical shift and proton position of andrographolide and iodo-andrographolide