INVESTIGATION OF IRON MEDIATED MOLECULAR TRANSFORMATIONS AND SYNTHESIS OF 2-AMINO-Î±-CARBOLINE AND ITS ANALOGUES RELEVANT FOR DNA ADDUCT FORMATION

The discovery of new synthetic methods and routes for making new bonds is one of the highest incentives to an organic chemist. Innovative, efficient, effective, economical and exceptionally environmentally friendly synthetic methods are being continuously discovered. Transition metal catalyzed C-H activation has been unearthed as one of these methods, especially iron catalyzed direct C-H activation. Fundamental to making more discoveries is understanding the mechanism behind a specific reaction. To this end, the mechanism behind iron catalyzed C-H activation was investigated. Results indicate the intermediary transition state for the iron species involves an Fe(I) species but the attempt to synthesize this Fe(I) species failed. We also confirmed that Fe(II) is definitely not the active species for this reaction. The nitrogen-based ligand might not have afforded us the Fe(I) complexes but there was evidence to show the reduction of the Fe(acac)3 due to the formation of the biphenyl product In the same vein of discovery, we identified a mild, one-pot, FeCl2 mediated procedure to produce 3-substituted allylic alcohols from α,β-unsaturated ketones. The addition of an organolithium nucleophile produced tertiary allylic alcohol as an intermediate, which underwent a 1,3-OH migration assisted by FeCl2. The proposed mechanism indicates that a syn-facial migration occurs for the significant product and we obtained yield as high as 98% from this one-pot reaction. New synthetic methods are also very beneficial to the world outside chemistry. The study of the carcinogen 2-amino--carboline (AC) and its interaction with DNA involved the synthesis of the AC-DNA adduct. We report the synthesis of AC and 2-nitro--carboline (AC-NO2) which also facilitate the synthesis of DNA adducts for biophysical studies. An attempt was made to synthesize the fluorinated 2-nitro--carboline which will enable the use of F-NMR for monitoring of these studies. Cyclization of the coupled product for the fluorinated analogue could not be achieved due to the electron deficiency in the ring systems. AC was obtained in good yields of 88%, and the AC-NO2 was obtained in 60% yield.


LIST OF TABLES
Understanding how iron works is important because it has been established as a very versatile metal, able to be used in various chemical processes. A chemical process of interest is tertiary allylic alcohol rearrangement, which is an applied method where certain molecules, especially drugs, have been synthesized. 3 Iron, an inexpensive first row transition metal has not been used for tertiary allylic alcohol rearrangement, though other rare and first row transitions metals like chromium 4 and rhenium 5 have been used. This prompted the investigation in using iron for this important rearrangement.
Finding new synthetic routes is also beneficial to the biological world. The effect of carcinogens on human cells have been studied extensively and these studies have focused on the interactions of the carcinogen with DNA. To facilitate these studies, there is the need for the synthesis of DNA adducts of these carcinogen --a research frontier identified through previous studies . 6 It was found to be cost effective to synthesize the carcinogen of interest to be used for synthesis of the DNA adducts used for these biophysical studies hence the need to find quick, efficient and effective methods to synthesize these carcinogens.

WHY C-H ACTIVATION?
Synthetically the direct functionalization of a C-H bond into either a C-C or C-heteroatom will be of great advantage to the synthesis world, especially the pharmaceutical and fine chemical industry. Over a long period of time, organic chemists have explored ways to make molecular transformations. The use of nucleophiles, such as Grignard reagents 7-12 , organocuprates 13 In recent times, the innovative methods being explored involving direct functionalization of the C-H bond 1 . The need for direct C-H activation arises primarily to eliminate the need for prefunctionalization of the C-H bond into halides or other leaving groups. By exploring the direct C-H functionalization, the organic chemist increases the sturdy and reliable methods already available.
In the discussion of green and sustainable chemistry, direct C-H activation also eliminates a number of problems related to waste. Direct C-H activation also saves time, money and efforts and this is a key advantageous component for industries. 1,2 Iron Catalyzed C-H Activation Until recently the most widely used transition metals for direct C-H activation had been limited to the 4d transition metals like palladium 47,48 , rhodium 49 , iridium 50 and ruthenium. 51 Most recent interest has been in iron 52 , cobalt 1 and nickel 53 mainly due to availability and relatively low cost of these metals as compared to the other precious metals. Iron compared to nickel and cobalt is also very inexpensive and non-toxic. The advantages in the use of iron speared off an intense investigation into its versatility. It is interesting to note that these iron catalyzed reactions are sometimes of better yields than their palladium counterparts 54 as shown in scheme 1. The mechanism for iron cross-coupled reactions has been studied extensively compared to direct C-H activation. 11,[55][56][57][58] The multifaceted nature of the electronic structure of iron makes the study of its intermediate in reaction cycles quite an exasperating task to achieve. Even though, there has been a number of physical methods that can be used to study or monitor these iron catalyzed C-H bonds transformation, the intrinsic mechanism of the iron species in the cycle has not been identified yet. There has been a couple of theories in working now but without a direct probing of the iron species generated in situ, there are restrictions on how these theories can be applied.
So far, two general mechanisms of how the iron activates the substrate have been proposed, 1. a low valent iron complex undergoes an oxidative addition into a C-H bond of the substrate to form the organoiron complex; 2. the iron complex deprotonates the substrates before the formation of the Fe-C bond, also known as direct deprotonation metalation. 59 The pioneering works done by Nakamura et al 54 In the study to extend the scope of their reaction conditions to form biaryl heterocycles since these heterocycles form the basis for most of biological and pharmaceutical molecules, the iron catalyzed ortho directed arylation of the heterocycles such as pyridines, furans and thiophenes was investigated with good yields as shown in scheme 1.4. The reaction gave a complete conversion of starting material in 15 minutes and yields as high as 88%. Investigations led to findings that questioned the mechanism proposed by Nakamura.
We observed that the Fe(II) species proposed by Nakamura et al was not present because the use of Fe(acac)2 did not yield any product. A reduced imine as a byproduct was detected, this implied a hydride was being formed during the reaction which can be only be introduced into the reaction if there were an Fe(I)H being formed. This led an investigation into finding the intermediary species for this reaction. This will be discussed further in manuscript one. Scheme 1.4: Iron catalyzed direct arylation of heterocycles 54

1,3-TERTIARY ALLYLIC ALCOHOL REARRANGEMENT
Iron as a metal is very versatile 56 . It has been shown to be comparable to palladium and in some cases, a better metal catalyst. 68 Iron complexes have been found to be better Lewis acids in the Mukaiyama aldol condensation reaction of silyl ketene acetals and aldehydes. The iron complexes had better turnovers and stability to moisture and air and they were able to give good enantioselective aldol additions. 69 Iron catalyzed Negishi reactions were also found to be robust, highly reproducible and made studies for reaction mechanism manageable. 41 In view of these trends can iron outperform other known systems?
Allylic rearrangements involve the movement of a double bond in an allylic system usually facilitated by the addition of a nucleophile or an oxidant. The most practically known ones are the Cope and Claisen rearrangements, which are shown in scheme 1.5. 70-73 3',3-disubstituted allylic alcohol is often times a difficult system to synthesize though these systems are crucial in the synthesis of a lot of natural products.

Scheme 1.5: Cope and Claisen rearrangement
The most common methods of synthesizing these disubstituted molecules involve nucleophilic additions using organometallic reagents to propargyl alcohols and conjugated addition of nucleophiles to ynones. [74][75][76][77][78] . Findings have shown that migration of the hydroxy group in tertiary allylic alcohol can help synthesize some of these complex and difficult systems but, until recent, not much attention had been given to this use of the tertiary alcohol 1,3-rearrangement.
Uses of Tertiary Allylic Alcohol Iron has been involved in a few substitution reactions via -allylic system 99-102 but the use of iron for 1,3-rearrangement has not been explored yet. We explored iron for this allylic rearrangement considering the fact that transition metals have been used for over long periods of time for this same type of rearrangement. It was interesting to discover that iron can break and functionalize strong C-O bonds, a unique synthetic characteristic that most conventional transition metals cannot do.
We reported a one pot iron mediated 1,3-rearrangement of tertiary allylic alcohols This involved the addition of organolithium reagents to α,β-unsaturated ketones, which then undergo a 1,3rearrangement in the presence of FeCl2 (scheme 1.8) This mechanism we believe for this novel reaction involves a cleavage of the C-O bond of the tertiary allylic alcohol and the intermediacy of an allylic cation. This is discussed further in manuscript 2.

FOR DNA ADDUCT BIOPHYSICAL STUDIES
A greater number of HAAs have been found to be more carcinogenic, higher mutagenicity than the usual carcinogens like nitrosamines, aflatoxins B1 and benzo[α]pyrene. 108 Studies have also shown that these HAAs are principal causes of most cancers like lung, breast, colon, stomach and prostate. [109][110][111][112] Alpha carboline, a subgroup of HAAs has been found in the core structure for some natural products 113,114 as shown in figure 1.2. They have a similar structure, relative to indoles and carbazoles and have served as a platform as a building block structure in medicinal chemistry 115 and optoelectronic materials. 116 They were isolated and identified several years ago and were found to be mutagenic against Salmonella typhimurium TA 98 (TA 98). They were different from its isomers, β-carboline and γ-carboline 117 in structure and activity. The latter carbolines had been identified to exhibit anticancer, antimalarial and antidopamine 118 effects, thus interest to find the therapeutic or mutagenicity of -carboline intensified.  show a direct correlation between cancerous cells developed and the consumption of cooked meat. 105 AαC has also shown antitumor activity towards Glioblastoma multiforme but unfortunately, the understanding of exactly how the HAA interact s with DNA leading to mutations is understudied.
It has already been reported on how HAAs are metabolized in vivo. 104 The reactive intermediate is the N-acetoxyamine, which is easily transferred to DNA by the N-acetyltransferases found in vivo. Synthetically the best way is to convert an amine to N-acetoxyamine is by first converting the amine to nitro group, then partially reduce the nitro group to hydroxylamine, before acetylating the hydroxylamine. There has been a report though, of the use of a polyaniline nanofiber supported FeCl3 to acetylate an alcohol or amine to an acetoxy group. 122 To study the biophysical interaction between AαC and DNA and how this leads to mutation, we looked into the synthesis of AαC and then 2-nitro-α-carboline (AαC-NO2).

Synthesis of Alpha-carboline
There have been various methods report for the synthesis of AαC (scheme 1.9). These include a modified Graebe-Ullmann reaction, intramolecular Diels Alder, annulation of substituted benzene and pyridines, photocyclization and transition metal catalyzed cross coupling.

a. Graebe-Ullmann reaction
This coupling reaction was discovered in the early 1900s but it was modified to aid the

Total synthesis
Total synthesis involves reacting the most reactive form of the AC with guanine, one of the nucleic acid base, before it is reacted with a sugar, before the addition of a phosphate group. This resultant nucleotide then undergoes an automated oligopeptide synthesis. The advantage with this route is the selectivity in terms of building a specific nucleotide sequence. It eliminates the worry of having to find exactly where the AC bounded to the DNA. The disadvantage with this method is that it is expensive, time-consuming and involves a lot of steps.

Biomimetic
Biomimetic involves the reaction of the reactive form of the AC with a nucleotide sequence. The advantage with this route that it is quick, fewer resources needed and fewer steps needed. The disadvantage however is knowing the exact location of the adduct. Should there be more than one guanine in the sequence the AC will react with all of them making detection and monitoring quite cumbersome and difficult.
The best method to suit our aim was the Graebe-Ullmann modified method since it has fewer steps and the starting materials are readily available and this is discussed further in manuscript three.

REFERENCES
(    The use of iron for catalysis dates as far back as 1940 2 and 1970 3 and these iron-catalyzed reaction are ideal because iron is economically sustainable due to its abundance, 4 biologically nontoxic 5 nature and its inexpensiveness. The reactions have also been proven to be efficient and easily scalable. Iron catalyzed reactions have also been found to be robust, highly reproducible and are known to be 'green' considering that it is easily processed and its environmentally benign waste 6 is appealing to the pharmaceutical and fine chemical industries Even though this discovery of iron's reactivity and it use for the cross-coupled reactions was made before the use of palladium [7][8][9] and nickel based catalysis, 10 Nakamura and co-workers were the first to report a direct C-H bond functionalization at low temperature (0 o C) which they discovered during a cross coupling reaction between diphenylzinc and 2-bromopyridine. 16 Optimization of this reaction conditions resulted in arylation of the ortho hydrogen on phenylpyridines, phenylpyrazoles, phenylpyrimidines and benzoquinoline. These ortho hydrogens were susceptible to the transformation regardless of the electronic effect of the substituents. 17 Other groups then researched into the mechanism for iron catalyzed C-H activation. [18][19][20] It should also be noted that iron has been found to activate not just C(sp 2 )-H, but also C(sp)-H and C(sp 3 )-H bonds using Fe complexes to form Fe-C bonds in the presence or absence of directing groups. 21,22 The proposed mechanism for this iron catalyzed ortho directed arylation as shown in scheme 2.2 Scheme 2.2: Nakamura's proposed mechanism for iron catalyzed ortho directed arylation As the interest in iron catalyzed direct C-H activation increased, the inquiry into the mechanism also began. In respect of this, two general mechanisms of how the iron activates the activation have been proposed, a low valent iron complex undergoing an oxidative addition into a C-H bond to form the organoiron complex, or, the iron complex deprotonates the substrates before forming the Fe-C bond, also known as direct deprotonation metalation. 23 These proposed mechanisms involves either a double electron transfer or a single electron transfer between the substrate and the iron. Unfortunately, the active Fe species involved in these reactions is dependent on many conditions, such as ligand, type of Grignard (nucleophile) used and but not limited to the presence or absence of a β hydride on the nucleophile. 24 The key step of interest was the reduction of Fe(acac)3 by a Grignard reagent. The active Fe species is dependent on the type of Grignard reagent used. Nakamura used an aryl Grignard reagent which has been shown to form clusters of anionic Ph3Fe(II) or Ph4Fe(III) in THF. 25 This implies the presence of both Fe(II) and Fe(III) species in solution. Computational studies also confirmed the Fe(II)/Fe(III) system but added the additional information of the presence of an Fe(I) species after the C-C bond formation through reductive elimination. 26 Nakamura also proposed that the main intermediary species to be Fe(II)/Fe(III) system for a similar C-H activation using organoborons. 27 Though the Fe(II)/Fe(III) system seems to explain a lot of these iron reactions, the fast rates of reactions and side products had led others to propose other mechanisms which include an Fe(I)/Fe(III) system, 28,29 Fe oxo species 30,31 and Fe -1 species 32 for other Fe-catalyzed processes.
Our group's previous work 33 expanded Nakamura's ortho directed arylation of arenes containing directing groups to include the azoles and thiophenes heterocyclic substrates (scheme 2.3). The investigation led to the conclusion that Fe (III) catalysts, preferably Fe(acac)3, were required for the reaction. Importantly, The Fe(II) catalyst precursor, Fe(acac)2, did not catalyze the reaction.
The by-product obtained was the reduced imine (7) implying a hydride was being produced in the catalytic cycle. The use of radical scavenger, TEMPO, did not affect the reaction implying the catalysis did not proceed through radicals, eliminating the single electron transfer method of activation. High amounts of Grignard reagents had to be used to account for the high amounts of biphenyl produced even though the additive KF was added to reduce the homo-coupling as shown in scheme 2.3. Scheme 2.3: Iron catalyzed direct ortho arylation of arylamine.

RESULT AND DISCUSSION
This led us to propose a mechanism that involves an Fe(III)/Fe(I) system (scheme 2.4a) or an The deprotonation of hydrogen happens as the Fe binds to the C to form species (13), which then undergoes reductive elimination to give product (14). The ligand dtbpy is added to maintain the Fe(I) species (15)      The imine((E)-N,1-diphenylethan-1-imine and arylamine product ((E)-N-([1,1'-biphenyl]-2-yl)-1phenylethan-1-imine) were synthesized using the methods from previous work 31

Synthesis of Imine
To an oven dried 50 mL RBF with a stir bar was added 20 g of 3 Å molecular sieves and 30 mL of toluene. The aniline (60 mmol) and acetophenone (50 mmol to yield an orange-red color.

Synthesis of Fe (I) species (4)
The Fe(II) complex (0.02 mmol) was dissolved in 1.5 mL of toluene(anhydrous) and cooled to -40 degrees, then the Grignard, tolyl MgBr (0.06 mmol) was added dropwise and the mixture was stirred for 20 minutes, then the mixture was allowed to warm to room temperature for 40 minutes under nitrogen. The solvent was removed to 2/3 its volume then the mixture was cooled to -20 degrees in order to obtain red crystals.

Investigation of Iron Catalysts:
For GC/MS analysis An oven dried Schleck vial was evacuated using vacuum three times, the tube was filled with nitrogen intermittently. Iron catalyst (0.055 mmol) and 1,2-bis(diphenylphosphino)benzene (dppbz, 0.118 mmol) were added to the Schleck vial. Then PhMgBr Grignard (0.168 mmol) was added over 15mins. After 15 mins, 0.1 mL of the reaction mixture was taken into a GC vial and 990 mL of EtOAc was added for GC/MS analysis.

Investigation of Iron Complexes for Direct C-H ortho Arylation
The imine (0.55 mmol), chlorobenzene (2 mL), Iron complex (0.055 mmol) and the dppbz (0.118 mmol) were added to an oven dried Schleck vial sequentially. The vial was evacuated using vacuum three times, the tube was filled with nitrogen intermittently before the addition was done. The mixture was then cooled to -78 o C for 15 minutes. The Grignard (0.168 mmol) was added over 15mins. The reaction was then allowed to warm to room temperature. 0.1 mL of the reaction mixture was taken into a GC vial containing 990 mL of EtOAc and used for GC/MS analysis .   than their mono-substituted counterparts. Though methods such as the conjugate addition of nucleophiles to ynones and the addition of organometallic reagents to propargylic alcohols have been described, [9][10][11][12][13] one method that has received little attention is the 1,3-migration of the hydroxy group in tertiary allylic alcohols. This transformation has been primarily catalyzed by oxo catalysts, 14-16 though oxidative palladium catalysis has also been employed to perform the migration and oxidize the allylic alcohol to a β-disubstituted enone. 17 This form of rearrangement can be done by enzymes to form enones. 2 Rhenium assisted rearrangement has also been used in the synthesis of semisquarates. 18 Trifluoroacetic acid has also been used to isomerize allylic alcohols, and this method has been applied to the synthesis of valerenic acid, which binds to both the GABAA and 5-HT5A receptors, and is used as a treatment of insomnia. 19 Acid assisted allylic alcohol rearrangement was also used in the synthesis of two quinolone natural products isolated from Pseudonocardia sp. 20 Additionally one can envision that this rearrangement could be used to create artemisinin-like antimalarial drugs via Singh's synthetic (Scheme 3.1). 21 Iron has recently been studied as a catalyst for a number of coupling reactions. [12][13][14][15][16][17][18][19][20][21][22][23][24][25][26][27][28] The most widely used of these processes are alternatives to traditional palladium-catalyzed transformations, such as the Kumada coupling and C-H arylation and alkylation reactions. 22,29,30 Even a few examples of substitution reactions via π-allyl iron intermediates have been reported. [31][32][33][34][35] Scheme 3.1: Application of allylic 1,3-migrations

REFERENCES
Iron is preferable to late transition metal catalysts due to its low expense and toxicity. Additionally, iron catalysts are often capable of performing synthetic steps that conventional late transition metal catalysts cannot perform, such as breaking and functionalizing strong C-O bonds. Herein, we describe a one-pot addition of an organolithium reagent to an ,-unsaturated ketone, followed by an iron-mediated 1,3-rearrangement reaction. We propose that the novel reaction proceeds via the formal cleavage of a C-O bond and the intermediacy of an allylic cation.

RESULTS AND DISCUSSION
During the course of our studies on iron-mediated reactions involving organolithium and organomagnesium nucleophiles, 30,36 we discovered that the addition of an organolithium reagent to an α,β-unsaturated ketone in the presence of an iron salt, does not result in the expected 1,4addition products, but rather an isomeric allylic alcohol is formed (2).
Cyclohex-2-enone, (1) was chosen as a reliable and simple substrate for reaction optimization (Table 3.1). A number of both iron(II) and iron(III) salts were selected, out of which FeCl2 was determined as the most efficient reagent. In general, iron(III) salts were inferior to iron(II) salts.
Diethyl ether (with or without BHT stabilization) was the most desirable solvent for the rearrangement and the other common ethers solvents, THF and Me-THF, produced low or no yields of 2.
Colder temperatures (entry 10) allowed for the formation of the 1,2-addition product, 3, but hindered the formation of the desired rearranged product 2. While the reaction proceeded in both inhibited and purified Et2O, the addition of one equiv of BHT further hindered the reaction (entry 11), indicating that the BHT preservative was not involved in the reaction and that commercial, stabilized ether was a suitable solvent for the addition/migration. Reduction of the loading of FeCl2 from one equiv to 10 mol% resulted in an 8% yield (entry 13), indicating that the reaction required a stoichiometric amount of the iron reagent. The scope and limitations of the addition-rearrangement with respect to both linear and cyclic α,β unsaturated ketone substrates were also investigated ( Table 2). Cycloalkenones were found to be the best substrates, with cyclohex-2-enone (1) giving the highest yield. Linear α,β-unsaturated ketones (entries 3-5) produced only the 1,2 addition product.   a Aryllithium was synthesized via lithium-halogen exchange from the arylbromide and butyllithium.
We then investigated the scope and limitation of organolithium and Grignard nucleophiles ( Table   3). Alkyllithium reagents did not yield any results, presumably due to their basicity. The naphthyllithium reagent produced only the 1,2-addition product 15, indicating that the 1,3 migration may be inhibited due to steric hindrance. Only trace amounts of the product were obtained from a Grignard reagent (entry 6). We investigated the feasibility of this method using heterocyclic aryllithiums, and we obtained only a trace amount of products 20 and 22.
To probe the mechanism of the reaction, the biphenyl lithium reagent was added to 1 and stirred for 3 hrs. Purification by flash chromatography gave 3. FeCl2 (1 equiv) was then added to a solution of 3 (1 equiv) and the reaction was stirred at room temperature for 3 hrs, providing 2 after column chromatography (Scheme 3.2). This confirmed that the tertiary allylic alcohol (3)  alcohol. This indicates that the 1,3-migration proceed primarily via a syn-facial pathway due to less energy needed for the iron-oxo species to approach from the same face of the allylic cation than to approach from the opposite face as the methyl group to give the syn product.
Based on these data and the previous work by McCubbin, 37 we propose the mechanism shown in Scheme 3.4. The organolithium reagent reacts with the α,β-unsaturated ketone to give the tertiary alkoxide (1,2-addition). The FeCl2 coordinates to the alkoxide (28) and LiCl is formed. The iron-oxo species (29) cleaves the C-O bond, forming an allylic carbocation (30), and the iron-oxo species then attacks the 3-position of the allylic cation, forming a new C-O bond. The major product of this process arises from the syn migration of the iron-oxo species. We hypothesize that the intimate ionic pair (30) could explain the formation of both diastereomers (26 and 27) and the preference for the trans isomer (26). DFT calculations indicate that both the trans (26) and cis (27) rearranged products have similar ground-state energies, so the observed 2:1 selectivity likely arises from kinetic control. When aryllithium nucleophiles are used, the final allylic alcohol is conjugated which we believe is the overall driving force for the reaction.

Scheme 3.3: Diastereoselective OH-migration
Finally, the extent of the OH-migration was investigated (scheme 3.5). Phenyllithium was added to a solution of the conjugated dieneone, 32, then after an hour, FeCl2 was added. The mixture was then stirred at room temperature for 24 hours, producing 33, a 1,2-addition product, and 34, the product of a 1,5-OH migration. The 1,3-migration product (35) was not observed, likely because it was less conjugated than 33 or 34. This result corroborates the proposed OH-migration mechanism and confirms the overall driving force for the reaction is the creation of an extended conjugated system.

CONCLUSION
In summary, we have developed a novel iron-mediated process that isomerizes allylic alcohols.
The system can be used to effect the transformation of cyclic -unsaturated enones to 3,3'-disubstituted allylic alcohols. Future work in this field could involve the enhancement of the diastereoselectivity of the process and its application to the synthesis of medicinal compounds.

EXPERIMENTAL SECTION
All reactions were carried out in oven-dried glassware under nitrogen atmosphere unless stated otherwise. Yields refer to chromatographically and spectroscopically pure compounds unless stated otherwise. 1   (1.0 equiv) was added. The reaction was allowed to run at room temperature overnight. Silica was then added to the reaction. Purification by flash column chromatograph using hexane and ethyl acetate provided the corresponding desired 1,3 rearranged allylic alcohols.       Studies have shown the presence of AαC in the urine of smokers 4 and that even its concentration in tobacco smoke is higher than the known carcinogen 4-aminobiphenyl, (4-ABP) (which is a wellknown human bladder carcinogen). 5 AαC has also been found to cause cancer of lungs in mice 6 and also mutagenic towards Salmonella typhimurium. 7 It is interesting to note that the isomer of -carboline, -carboline has been found to have anti-cancer, 8 anti-malaria and anti-dopaminergic activity. -Carboline though, was not known for these properties until a recent study showed αcarboline as an antitumor agent against Glioblastoma multiforme. 9 Figure 4.1: Natural products with AαC as a backbone structure 2-Amino-α-carboline (AαC), isolated and identified from soybean several decades ago, though has been found to be the second most consumed HAA with a daily dietary intake of 5 ng/Kg/day, 10 does not have enough biophysical data on its interaction with DNA. Though there has not been a definitive correlation between AC and tumors in humans, studies in aminals 6 found a direct correlation between consumption of cooked meat and cancers. 11 The understanding of how AC interacts with DNA and its resultant mutation needs to be unfolded. This makes looking into the biophysical study of the mechanism through which AC cause these mutations, crucial.
A factor that needed to be considered in synthesizing fluorinated AC was a means to detect the DNA adduct formed after synthesis since, for small or minor DNA conformers, 1 H NMR is mostly not useful. 19 FNMR is considered a powerful tool to help detect 23,24 and track the molecule of our interest; hence fluorinated analogues of AαC were also synthesized.
The conventional means to form an N-acetoxyamine from an arylamine is to partially reduce a nitro group to hydroxylamine before acylation thus nitro-α-carboline (AαC-NO2) had to be synthesized to initiate the synthesis of the DNA adduct 25 (oxone) and acetone in the presence of a base (NaHCO3) 10 (scheme 4.7) and the use of Na2WO4 with H2O2 in MeOH though the latter had lower yields than the former and purification was difficult.  This method involves concerted metalation deprotonation, CMD or a double C-H activation.
Though this proposed scheme has many advantages, inexpensive starting materials, and fewer steps, many challenges were faced during the experimental. The Goldberg coupling gave low yields of the desired product 12, due to the formation of other side products. Cyclization was also not achieved due to the absence of electron donating groups on the rings. In order to increase the efficiency of this pathway, the modification as shown in scheme 4.9 can be done.

DNA ADDUCT REACTION
Our biomimetic synthesis for DNA adduct formation imitates the pathway for the metabolism of HAA as explained above. The AC-NO2 is partially reduced to hydroxylamine. The hydroxylamine will be acetylated using acetic acid or trifluoroacetic acid to form the very reactive acetamide as shown in scheme 4.10.
Scheme 4.10: Biomimetic pathway for synthesis of DNA adduct In order to obtain the reactive N-acetoxy--carboline, the AC-NO2 will be partially reduced using Pd/C and hydrazine to obtain the hydroxylamine, which is then acetylated to obtain the Nacetoxy--carboline using Acyl chloride as shown in scheme 4.11. Scheme 4.11: Synthetic pathway to form N-acetoxy-α-carboline

CONCLUSION
In summary, we have synthesized 2-amino--carboline (AC) and 2-nitro--carboline (AC-NO2) with high yields. Future works would involve synthesizing the fluorinated analogue and DNA adduct for studies into the thermodynamic stability and other biophysical parameters of the adduct to ascertain how the carcinogen AC interaction with DNA leads to mutation.

EXPERIMENTAL SECTION
All reactions were carried out in oven-dried glassware under a nitrogen atmosphere unless stated otherwise. Yields refer to chromatographically and spectroscopically pure compounds unless stated otherwise. 1