CHAPTER'i SECTION A: Introduction to Tuberculosis and the Drugs Available for Treatment

CHAPTER'I SECTION A: Introduction to Tuberculosis and the drugs available for treatment. Chapter I, Section A

History of Tuberculosis

The truth, tuberculosis (TB) is a communicable disease caused by infection with the tubercle bacillus^ has been established by Robert Koch in the year 1882. Although humankind affliction with TB date backs to at least 5400" years, as evidenced by ancient mummified remains, genetic analysis of the Mycobacterium tuberculosis (Mtb) complex suggests that the common progenitor has infected our hominid ancestors since eons. Unabatedly Mtb has parasitized the human host over ages with its complicated and dynamic series of interaction. Nearly l/S'** of world's population has been infected with Mtb. Globally 9.2 million new cases and 1.7 million deaths occur every year albeit widespread vaccination and chemotherapy. While large chunk of new cases are reported from southeast Asia, the western pacific,^ 95% of all cases are from developing world. The incidence of HIV has refueled the mortality rate among TB cases as it promotes the infection to active clinical disease; While TB accelerates HIV viral replication causing progression to AIDS. Further worsening of the situation was the occurrence of an estimated 49000 new multi-drug resistant tuberculosis (MDR-TB) and extensively drug resistant tuberculosis PCDR-TB) cases every year according to a Global Drug Resistance Surveillance report.''

The Tubercle Bacillus The genus composition of fast growing soil microbes and slow growing pathogens including Mtb, Mycobacterium leprae, Mycobacterium bovis and Mycobacterium marinum constitute the rod shaped mycobacteria. With the exceptions of M microti and live- attenuated vaccine strain M bovis bacille Calmette-Guerin (BCG)' all of the slightly genetical variants of Mtb complex can cause disease in immuno­ competent humans, Mtb being the most important pathogen of Homo sapiens. The complex Mtb cell envelope consists of a plasma membrane, a cell wall, and a capsule-like outer layer (Fig. 1). The cell wall consists of, from innermost to outermost, peptidoglycan (PG), arabinogalactan (AG), mycolic acids (MA), and peripheral lipids. Lipoarabinomannan (LAM) is thought to be anchored in the plasma membrane and is also found in the capsule-like layer anchored in the MAs.* the thick complex Mtb cell wall renders the host ineffective to counter attack on the intra-phagosomal bacteria and

1 IPage Chapter I, Section A

low permeability turns it insensitive to P-lactams and resistant to many other antibiotics. Moreover the retention of carbol ftjchsin stain is due to its unique cell wall mycolic acids. f •}G K ip 4Ah\

>

^

Figure 1: Schematic of Mycobacterial Cell Envelope^ (A) plasma membrane, (B) peptidoglycan, (C) arabinogalactan, (D) mannose-capped lipoarabinomannan, (E) plasma membrane- and cell envelope-associated proteins, (F) mycolic acids and (G) glycolipid surface molecules associated with the mycolic acids.

Pathogenesis of disease When droplets (< 5 |am) housing one or more several Mtb bacteria were inhaled, they get deposited in the alveolar airspace while bigger ones are cleared by the pulmonary mucociliary system.^ Host alveolar macrophages phagocytize these bacteria. Bacterial replication within the membrane-bound phagocytic vesicles eventually overwhelms the macrophages leading to the rupture of the cells and the release of numerous bacilli. Both alveolar and monocyte-derived macrophages then take up these bacteria emigrating from blood stream. The bacterial spread commences approximately after 2 weeks when they begin to spill over from the primary lesion into surrounding tissue and then to regional lymph nodes. The infection remains latent for years or decades after primary exposure^ before reactivation in 5% of cases or takes several years to develop into primary progressive TB

2|Pa -e Chapter I, Section A stage in another 5% cases. But in a healthy individual it can be contained indefinitely or may be completely sterilized over time. In most cases progression to a disease state occurs in infants, the elderly, the malnourished, or those who are immuno-compromised by steroids, genetic predisposition or HIV. The gross cavitation occurring in the lung and necrotic tissue in severe post-primary disease can spill over into airways and the associated cough thereby spreads the bacteria within the lung of an individual and between an individual and his/her contacts. Diagnosis of active TB is based on symptomology, microscopic analysis of sputum stained to reveal acid-fast bacilli, sputum culture, DNA or RNA amplification assays, and/or chest radiograph. Signs and symptoms of TB include: night sweats, productive cough, bloody sputum, weight loss, and consolidated opacities (esp. apical) and/or upper lobe cavitation on lung X-ray. However, it should be noted that with severe immunodeficiencies such as HIV, patients with disseminated Mtb lung infection can display non-typical signs and symptoms mimicking other lung pathologies.

Vaccines The live attenuated bacille Calmette- Guerin (BCG) is the only vaccine used to prevent TB ever since it has been invented by Robert Koch. Even though 90% of vaccinated people infected by Mtb never develop active TB despite lodging the viable tubercle bacilli in their tissue^"'^ lifelong, the apparent protective efficacy of BCG against TB ranged from 80% to nil'^ in large scale, placebo controlled and double-blinded clinical trials. Development of new vaccines to replace BCG demands a thorough understanding of the interaction of Mtb and human immune system. In order to be novel & truly protective they must generate more substantial and enduring immune responses than are seen in the course of natural infection.

Chemotherapy The decade between 1941 and 1952 was a milestone in the history of medicine as it recorded the discovery of trio of drugs by three independent groups that could cure TB. Prior to discovery nearly 1 billion people have yielded to TB in the 2 centuries that spanned. The mortality rate was more than 50%'" due to uncomplicated pulmonar>' TB without antibiotics. In contrast the combination of ^-aminosalicylic acid (PAS) (2)

3 |Page Chapter I, Section A streptomycin (1) and isoniazid (3) could, if administered properly, cure TB completely and nearly universally (Fig. 2). Many effective drugs were identified later and treatment times were shortened.

O

p-Aitiinosalicylic acid (2)

NH,

OH OH

Streptomycin (1) Isoniazid (3) Figure 2

After many trials'^ '^ drug treatment regimen were divided into two, first a two month long treatment with four drugs; either: streptomycin (1), isoniazid (3), rifampin (4) and pyrazinamide (5) or: isoniazid (3), rifampin (4), pyrazinamide (5) and ethambutol (EMB) (6). This is then followed by four months of isoniazid (3) and rifampin (4) (Fig. 3). 0^NH2

N' I^N

Pyrazinamide (5)

OH

HN-

-NH

HO

Rifampin (4) Ethambutol (6) Figure 3

4| Page Chapter /, Section A

While these drugs represent a critical advance in our ability to treat TB, inadequate healthcare infrastructure, financial limitations, the longtime required for full treatment (6- 12 months) and the required number of drug doses, adverse effects, poor patient compliance contributing to appearance of multi-drug resistant (MDR) and extensively drug resistant (XDR) TB strains, the spread of HIV have prevented universal control of the disease. In light of these observations the desirable characteristics of new anti-TB drug include the followings: orally active, long acting, limited toxicity and inexpensive. It should act through novel mechanisms of action such that there is no cross-resistance with current drugs, and it can be active against both drug-sensitive and drug-resistant M. tuberculosis. The drug should preferably be bactericidal, and active against both actively dividing and nonreplicating persistent M. tuberculosis. Ideally, there should be presence of synergistic or additive effects with current drugs, absence of antagonism and no significant interactions with other drugs, in particular the antiretrovirals.'^ Recommendations from a recent survey based on clinical data''"'^^ suggest the use of at least five adequate anti TB drug regimen, the choice of which is driven by the actual or presumed (in view of past failed treatment) resistance characteristic of the strains of M tuberculosis considered. In order of preference they can be chosen from the following, (i) In any case, the first line agents still active on the patient: isoniazid (3), rifampin (4), pyrazinamide (5) and ethambutol (6). (ii) This is followed by the group of injectable drugs: streptomycin (1), kanamycin (7), amikacin (8), capreomycin (9) or viomycin/tuberactinomycin B (10) and the related tuberactinomycins A, N and O (Fig. 4). (iii) One of the many related antibacterial fluoroquinolones such as ciprofloxacin (11), ofloxacin (12a). levofloxacin (12b), or the more recent sparfloxacin (13), gatifloxacin (14), moxifloxacin (15) and sitafloxacin (16) should be included in the regimen. This class of antibiotics has now^'' been proven as indispensable treatment for MDR tuberculosis ' and some of these drugs may leads to shorter antituberculosis regimens.^^' (iv) Second line bacteriostatics, with established clinical efficacy,^' usually have more important side effects,^" they are/^-aminosalicylic acid (2), ethionamide (17a) (the propyl analogue prothionamide (17b) is also used) and cycloserine (18) (Fig. 5).

5|Page Chapter I, Section A

HO

HO,, OH

H2N^V^"'0'S^0^Y"''O H HjN^ ^O OH OH OH NH2 r ^ ^ NH

R = H: kanamycin (7) •^OH R = COCH(OH)CH2CH2NH2: Amikacin (8) O^NH° O V" I OH ^"O H.N-k NH2 O

Viomycin (10) O^NH" O "V

NH2 ^ OH

Capreomycin (9)

O O O O NH2 O O

HN^ F ^

Ciprofloxacin (11) R/S: Ofloxacin (12a) Sparfloxacin (13) S: Levofloxacin (12b)

O O O O o o

HN" VJ 6 u H7N

Gatinoxacin(14) Moxifloxacin(15) Sitafloxacin (16)

Figure 4

6 I Page Chapter I, Section A

(v) Other drugs are also considered. Their use is the subject of debate" and only time and proper observations will provide the necessary data. rio 0~N H

n = 1: Ethionamide (17a) Cycloserine (18) n = 2: Prothionamide (17b)

Figure 5

Mechanisms of action of antituberculosis drugs or antimycobacterials

One of the many fascinating facets of this field is the determination of the biochemical processes targeted by anti-tubercular drugs which is still undergoing and has been reviewed in the recent past.^''^^ The 'deorphaning' of antituberculosis drugs is today of prime importance, as it can lead to the identification of already validated biological targets of M tuberculosis. The recent discovery of Mtb genome and modem development in microbiological methodology afford us unique opportunities for identification of novel bacterial drug targets and rational drug design. Fatty acid biosynthesis inhibitors Mycolic acids are exceptionally long fatty acids covalently linked to arabinogalactam in the thick complex mycobacterial envelope. These are probably part of the keys^'' for the mycobacterial ability to withstand chemical injury from the host. The mycobacteria use two types of fatty acid synthase pathways to produce them. The first type is eukaryotic type of fatty acid synthase (FAS I pathway) which produces CI6 and C24/26 fatty acids in four steps. Second type is prokaryotic type of enzymes (FAS II pathway) which elongate these acids to lengths as long as C56. Amongst the enzymes of the second type are: (i) FabG and FabI which are NADH-dependent enoyl acyl carrier protein reductases, catalysing the reduction steps of fatty acid synthesis (respectively, the P-carbonyl reduction and the a-p-unsaturation reduction), (ii) FabH as well as Kas A or Kas B which are three different P-keto-acyl-carrier protein synthases, catalysing the condensation steps of fatty acid synthesis.

7|Page Chapter I, Section A

Isoniazid Discovered in 1952, this 50-year-old antituberculosis drug targets the mycolic acid synthesis pathway. First it requires an activation by Kat G,^^'^^ an endogenous catalase peroxidase, into an unstable intermediate which reacts with NAD to give compounds such as 19. The resulting adduct then binds the NAD (H) recognition site of Fabl and this leads to the antimycobacterial effect (Fig 6). This orally administered first line drug is associated with adverse reactions like Hepatitis, peripheral neuropathy etc (Table 1).

.NHj KatG

Isoniazid (3)

Figure 6 Pyrazinamide Another first line drug discovered in 1952 gained prominence since 1985 as 3^ most important drug^' in the treatment regimen against TB. Although exact mechanism is still under research, currently suggested^^ is based on the release of pyrazinoic acid (20) in situ. This causes an intake of proton (as protonated pyrazinoic acid) which leads to a complete dysfunction of the pH balance for the group of mycobacteria (which include M tuberculosis) that are lacking an efflux pump capable of dealing with this salt (Fig. 7).

O O

N N

Pyrazinamide (5) Pyrazinoic acid (20) Figure 7

8|Page Chapter I, Section A

This 'death by acidification' mechanism also explains why this drug is efficient on dormant bacilli as, aside from a pyrazinamidase, it does not require much metabolic activity. The side effects include hyperuricemia, gouty arthritis (Table 1).

Ethionamide (17a), prothionamide (17b) and thiacetazone (21). These second line bactereostatics discovered in 1956 are prodrugs oxidized by EthA, a flavoprotein monooxygenase endogenous to Mtb. Oxidation of ethionamide (17a) by the enzyme EthA leads to the sulfmic acid (22) which is likely to be further transformed as amide (23) and alcohol (24) were characterized.'*" In the case of thiacetazone (21), the sulfenic derivative (25) and the carbodiimide (26) were identified.'*''''^ Moreover, it had been known that the sulfinic acid 22 was as active on mycobacterial growth in vitro as ethionamide (Ha).''^"''^ Thus as for isoniazid (3), which is activated by KatG, it has been established that another enzyme endogenous to M tuberculosis, EthA, transforms ethionamide (17a) or thiacetazone (21) into highly reactive species (Fig 8). Even though their clinical efficacy is established the associated Gl toxicity, hepatitis, dizziness have to be considered before these injectable second line drugs are suggested (Table 1).

OH -NH2 S..^NH O^.NH2

17a 22 23 24

O^g.OH S ,NH N-N'-O- N NH2 H

H?N ^O H,N ^O H,N "O

21 25 26

Figure 8 9iPage Chapter I, Section A

PA-824 (27) or OPC-67683 (28). Two related nitroimidazoles PA-824 (27) and OPC-67863 (28) are among the most promising of the present anti-TB leads currently under phase II clinical trials. Both are prodrugs requiring activation by the same F420-dependent enzyme (Rv3547)'* '' and inhibit the growth of Mtb and MDR-TB by inhibiting mycolic acid biosynthesis and protein synthesis. The active species and ultimate targets of each remain unknown. OPC- 67863 (28) inhibits the synthesis of mycolic acid at the stage of methoxy and the keto- mycolic acid syntheses (Fig 9).'^°

O T F3CO ^^ f > F3CO N

\\

H=0 PA-824 (27) OPC-67683 (28) 6'

Figure 9

Arabinogalactam and peptidoglycan biosynthesis inhibitors Arabinogalactam, a polysaccharide made of arabinose and galactose (arabinogalactam), acts as the intermediate binding scaffold between the many types of mycolic acids and the inner peptidoglycan. The structure of peptidoglycan is shared by the Corynebacterium and the Nocardia, it contains D-alanine and differs from other groups of bacteria by the fact that meso-diaminopimelic acid constitutes the di£uninoacid component. Ethambutol This synthetic derivative of ethylene diamine discovered at Lederle laboratories in 1961 is a first line drug. Its effect is focused on arabinan biosynthesis and on many envelope metabolites.'^"'^^ More important, its effect on cell wall improves the efficiency of other antibiotics, for instance clarithromycin (29), which are usually inefficient on M tuberculosis/^'^ Commercially available as myambutol, EMB (6) is associated with side effects like optic neuritis etc (Table 1). out of the 1,00,000 plus diamine analogues of 10|Page Chapter I, Section A

EMB (6) prepared and screened, the remarkable SQ109 (30),which is under phase I clinical trials, was found to be as active as ethambutol (6) with different mechanism of action on cell wall biosynthesis (Fig. 10).^'

SQ109(30)

Clarithromycin (29)

Figure 10

D-CycIoserine(18) D-Cycloserine (18), a second line drug found in 1952 with adverse effects like dizziness, depression, CNS (Table 1), is an oral pill which inhibits either D-alanine racemase or D- alanine D-alanine ligase though exactly not known.

Inhibitors of protein synthesis Ribosome, the biggest and the most ancient enzyme, has not changed over eons as well as the general organization of its 50 plus components assembled, in the case of bacteria, into 30S and 50S subunits. Very schematically, the inhibitors can be classed in three groups. Series that interact with the 30S subunits make the first group and the second is the inhibitors that interact with the 50S subunit. The last group is made of the compounds that inhibit the function of aminoacyl-tRNA synthetases.

Streptomycin (1), kanamycin (7), amikacin (8), capreomycin (9) and viomycin (10). The aminoglycoside streptomycin (1) is the first example of anti-tubercular drug therapy^^ discovered in 1944. Along with the other two aminoglycosides 7, 8 and cyclic peptides 9, 10 it targets the 30s subunit^^ of ribosome inhibiting protein synthesis. All of

11 IPage Chapter I, Section A them were reported with side effects Hke cochlear and vestibular toxicity, nephrotoxicity (Table 1).

Linezolid (31) Among the antibacterials targeting the 50s ribosomal subunit 64-67 linezolid (31) is a fiilly artificial oxazolidinone whose long term use may be plagued with forbidding side effects. ' oxazolidinones with better profile in this regard are ranbezolid/RBx 7644 (32)/'' which has undergone phase I clinical trials in 2004,^' and the thiophene analogue RBx 8700 (33). • Among the inhibitors of amino acyl synthatases 74 are the isoleucyltRNA synthetase inhibitor mupirocin (34) and methionyltRNA synthatase inhibitor^^(35)(Fig. 11).

OoN

A N N—f V—N ^"^ f^ ^O O N—f >-N I H

F HN r Y o X = O: Ranbezolid (32) Linezolid (31) X = S: RBx 8700 (33) o

OH Nc XyJ

O^ OH Mupirocin (34) 35

Figure 11

Inhibitors of DNA-based processes

Rifampin (4) and rifapentin (36). Discovered in 1966, Rifampin (4), a first line drug, and its class of drugs inhibit bacterial RNA synthesis by binding to the P subunit of the DNA-dependent polymerase. Remarkably, rifamycins are the only clinically used antibiotics with this mechanism.

12|Page Chapter I, Section A

Rifapentin (36) and the more recent rifametane, which is undergoing phase II clinical trials, ' are the only exceptional compounds with better pharmacokinetics among those reviewed (Fig 12)7^ Marketed as R-Cinex 600 (in combination with isoniazid) Rifampin (4) has side effects like drug interactions, GI, hepatitis, fever, acute renal failure, haemolytic anaemia etc despite its efficacy (Table 1).

Rifapentin (36)

Figure 12

Fluoroquinolones 11-16 The Fluoroquinolones, most of them discovered in 90's are only a few representatives of the family of at least 25 related antibacterials currently used spanning four generations of analogues/^"^^ They act against M tuberculosis by inhibiting the ATP-dependent DNA gyrase (topoisomerase II). The six quinolones 11,12b-16 depicted earlier, along with clinafloxacin (37), were amongst the best inhibitors according to a classification.^" However all of them are associated with GI toxicity, CNS, tendon rupture (Table 1) and quinolone (37) had to be withdrawn from advanced clinical assessment because of unacceptable side effects (hypoglycaemia; phototoxicity) (Fig 13).^"'*'^

O O

Clinafloxacin (37)

Figur•t3* e 13

13|Page Chapter /, Section A

Inhibitors of dihydrofolate reductase or siderophore biosynthesis

^-Aminosalicylic acid (2). Discovered in 1946 PAS (2) was one among the trio of drugs used to cure to TB then but with the advent of more potent current first line drugs presently this is used as second line drug. Though exact mechanism of action is under research, a suggested mechanism involved an interference with salicylic acid metabolism which would interfere in fine with M.tuberculosis iron intake.*^'^* PAS (2) is associated with adverse effects like Gl toxicity, fever, rash (Table 1) and available under the trade name mesalazine.

Inhibitors of mycobacterial cytochrome P450 monooxygenases The azole class of antifungal, such as econazole (38) or clotrimazole (39) was of interest against mycobacteria, 89-9" 1 as it is likely that their target in mycobacteria is the P450 mono-oxygenase homologue to the eukaryotic 14a-sterol demethylases (CYP51) ' and that the imidazole moiety is binding the iron of these haem-containing enzymes (Fig 14).^^

Clotrimazole (39)

Figure 14

Nucleoside monophosphate kinase inhibitors, pyrimidine or purine nucleoside analogues The seemingly less homology (only 22%) between thymidine kinase of Mtb and of humans' was an advantage for the design of antituberculosis drugs^^ targeting it. The analogues of cytidine,^'''' (40, 41) adenosine^^^^ (42, 43) were known to inhibit their

14|Page Chapter I, Section A respective Mtb kinases where in the latter case 42 was a substrate 43 is an inhibitor (Fig 15).

NH2 ^^/nCioH2i o CX I

o-^J V X oX 'OH \-J. Q ( '0-\ o\ ' -O- H g\ OH OH

40 41 42 43

Figure 15

Signalling kinase inhibitors The thick cell wall^^ is not the only criteria for the survival of M. tuberculosis against the macrophage phagocytosis, but also the disruption of the host-cell defense mechanism against such parasitism'*'" by mycobacterial kinases. Bacterial two component signal transduction system, which involves a histidine kinase, was the focus of research of original antibacterials.*'''"'*'^ Eleven putative eukaryotic-like protein serine-threonine kinases (Pkn A to L) involved in signal transduction were identified in M tuberculosis H37Rv genome.'*'^''°^ The 'generic' kinase inhibitor 44"° as well as other more complex compounds were shown to inhibit the growth of some mycobacteria (Fig 16).

HN

44 Figure 16

Other drug candidates with potential novel mechanism of action Apart from aforesaid compounds many new drug molecules in clinical or preclinical trials are among promising anti TB leads. One of these compounds is the pyrrole LL-3858 (sudoterb) (45), an INH (3) analogue, currently in phase I clinical

15|Page Chapter I, Section A trials, '" is active against drug susceptible and drug resistant M tuberculosis. One more molecule OPC-37306 (46) active against drug susceptible and resistant Mtb strains found in the screening studies of inhibitors of mycolic acid biosynthesis"'' appears to be more potent than RIF (4) in mouse models."'* Another drug molecule FAS20013 (47) with claims that it can eliminate more than 99% of Mtb (including latent bacilli) within 24 h, with no resistant strains observed so far and good pharmacokinetic profile will shortly enter clinical trials (Fig. 17). 115

CF,

xxxx: Q. ,p >r'ex NH ,

LL-3858 (sudoterb) (45) OPC-37306 (46) ^j FAS20013 (47)

Figure 17

Inhibitors of the proton pump FOFIHT ATPase TMC207 (48) Besides the existing, claimed and discussed drug molecules the most potent antituberculosis molecule TMC207 (48)"^ discovered by Andries et al in 2005 is a diarylquinoline (DARQ) compound found potent when a series of in vitro tested"^ DARQs undergone in vivo testing for activity against Mtb. It has unique mechanism of action,"^ as it shows greater potency against mutated drug resistant strains than to fully susceptible isolates. Whilst TMC207-resistant Mtb strains have appeared, they remain fully susceptible to other anti-TB drugs such as RIF (4), INH (3), SM (1) and EMB (6)."^ TMC207 (47) exhibits excellent activity against drug-susceptible, MDR and XDR Mtb strains, with no cross-resistance to current first-line drugs."*'"* In terms of dosage its single use alone stands as effective as a combination of RIF (4), INH (3), PZA (5) and more effective than RIF alone in mouse models."* Bestowed with long half-life,"* a single weekly dosing in mice is sufficiently effective. TMC207 (48) has a potent

16|Page Chapter I, Section A sterilizing ability in guinea pigs, being 100 times more effective than the conventional combination of RIF (4), INH (3) and PZA (5)."^ It has been determined that it can be administered orally in humans. It is likely that TMC207 (48) is incompatible with anti- retrovirals,'^^ as significant decrease in its levels is observed when co-administered with RIF (4) due to CYP3A4 metabolism. TMC207 (48) acts by inhibiting proton pump of Mycobacterium membrane-bound FoFiH^ATP ase. This unique mechanism of action offers great potential as there is little similarity between the mycobacterial and human proteins encoded by the atpE gene that codes for the c subunit of ATP synthase, "^ which has been identified as the specific target of TMC207 (48).'^' A number of mutations (I66M and A63P) have been identified in the c subunit of TMC207-resistant strains "^'^^ near the glutamate residue E61, which is involved in proton transport and is necessary for the synthesis of ATP. ATP synthase has two structural domains (FO and Fl) that act as a biological rotary motor: Fl is located in the cytoplasm, where it generates ATP and FO spans the membrane and is arranged as a disc, containing one a-subunit and between 9 and 12 c-subunits. The flow of protons drives the rotation of this disc. Molecular modeling studies of Mtb ATP synthase have characterized the binding site of TMC207 (48) and suggested its likely mechanism of action. '^^ Normally, the side chain of Arg-186 in the a-subunit adopts an extended conformation that reaches towards Glu-61 in the c-subunit to transfer a proton. This event leads to a conformational change in the c-subunit, making Arg-186 adopt a compact conformation and initiating a 30 ° rotation of the c-subunit. It is believed that the molecular mechanism of action of TMC207 (48) is to mimic the side chain of Arg-186. '^^ TMC207 (48) adopts a folded conformation in solution before binding owing to intramolecular hydrogen bonding, '^'* but this is lost upon entering the binding site, being compensated for by the creation of new hydrogen bonds with Glu-61, as shown in Figure 18. The lack of a cavity large enough to accommodate the bulky dimethyl amino group of TMC207 (48) prevents the necessary rotation required for proton transfer, blocking ATP production. TMC207 (48) synergistic action with current drugs in shortening treatment period is well acknowledged when a combination wdth current first line drugs RIF (4), INH (3) and PZA (5) has resulted in negative culture in mouse models within 2 months."^

17|Page Chapter I, Section A

a-subunit Arg-186 ATP synthase binding pocket

TMC 207 enters ATP synthase and unfolds preventing Arg-186frc transferring a proton to Glu-61

Key: Vanderwaals forces Gtu-6l H-bonding c-subunit Figure 18

Complete eradication of lung Mtb"^ took just 2 months with TMC207 (48) and PZA (5) combination. A combination of TMC207 (47) with the currently recommended MDR-TB regimen (amikacin (8), ethionamide (17a), moxifloxacin (15) emd PZA (5)) has been successful in eradicating lung and spleen infection within two months in drug- sensitive mouse model. '^' Phase Ila clinical trials have demonstrated that TMC207 (48) is well tolerated in patients. The combination of low MIC values, unique mechanism of action, early and late bactericidal activity, reduced drug dosage and treatment period, absence of cross resistance and synergistic action with current drugs and good pharmacokinetic profile makes TMC207 (48) a promising TB drug candidate. The following chart lists promising lead compounds in various stages of development.

PRECLINICAL DEVELOPMENT RS-118641'^^ Linezolid FAS20013 CLINICAL DEVELOPMENT

Phase I Phase il Phase 111 SQ109 PA-824 Gatifloxacin LL-3858 (sudoterb) OPC-67683 Moxifloxacin TMC 207

Chart 1 List of Promising Lead Compounds in Various Stages of Development

18|Page Chapter I, Section A

Table 1. Year of Discovery, Main Characteristics and Most Frequently Reported Adverse Reactions of First and Second Line TB Drugs'^'

First line drugs Year Drug MOA Route Daily dose Major adverse reactions Discovered 5mg/kg Hepatitis, peripheral Cell wall synthesis Isoniazid 1952 POs (max 300 neuropathy, lupus-like inhibitor mg) syndrome, drug interactions Drug interactions, orange lOmg/kg RNA synthesis colour of body fluids, GI, Rifampin 1966 POs (max Inhibitor hepatitis, fever, acute renal 600 mg) failure, haemolytic anaemia Disruption of 15-30 electron transport Hyperuricemia, gouty Pyrazinamide 1952 POs mg/kg across the arthritis, rarely nephritis (max 2 g) membrane • Cell wall synthesis 15-25 Optic neuritis, exfoliative Ethambutol 1961 POs inhibitor mg/kg rash Second line drugs Protein synthesis Cochlear and vestibular Streptomycin 1944 IV/IM 15 mg/kg inhibitor toxicity, nephrotoxicity Protein synthesis 15-30 Cochlear and vestibular Capreomycin 1956 POs inhibitor mg/kg toxicity, nephrotoxicity Protein synthesis 15-30 Cochlear and vestibular Kanamycin 1957 IV/IM inhibitor mg/kg toxicity, nephrotoxicity Protein synthesis 15-30 Cochlear and vestibular Amikacin 1974 IV/IM inhibitor mg/kg toxicity, nephrotoxicity Inhibition of 15-20 GI Ethionamide 1956 mycolic acid IV/IM mg/kg toxicity/hepatitis/dizziness synthesis (cell wall) Inhibition of folic 15-20 PAS 1946 POs/IV GI toxicity, fever, rash acid mg/kg Inhibition of 15-20 Cycloserine 1952 peptidoglycan POs Dizziness, depression, CNS mg/kg Synthesis Inhibition of DM A 750-1550 GI toxicity, CNS, tendon Ciprofloxacin 1986 P Os/IV gyrase mg/d rupture Inhibition of ON A 600-800 GI toxicity, CNS, tendon Ofloxacin 1995* P Os/IV gyrase mg/day rupture Inhibition of ON A GI toxicity, CNS, tendon Levofloxacin 1996* P Os/IV 500 mg/d gyrase rupture Inhibition of ON A GI toxicity, CNS, tendon Moxifloxacin 1999* POs/IV 400 mg/d gyrase rupture Inhibition of DNA GI toxicity, CNS, Gatifloxacin 1999* P Os/IV 400 mg/d gyrase dysglycemia Binding to GI toxicity, cutaneous, mycobacterial 100-300 ocular discoloration/ Clofazimine 1954 P Os/IV DNA mg/d pigmentation QT and mRNA • prolongation, dizziness

• Year of first US patent • proposed

19|Page Chapter I, Section A

The complete eradication of M. tuberculosis in all its forms from the face of mankind would be possible only with a drug regimen which inhibits not only the drug susceptible and resistant forms but also the latent non-replicating and HIV infected forms. Though current drug regimen is successful to a great extent, a perfect regimen which includes all the discussed characteristics is not achieved still. As the recent discoveries are promising a panacea from TB, lot of research has to be undertaken before they get included in the drug regimen. Thus, not only the discovery of the new drugs but the synthesis of the same in substantial amounts is essential to undertake all sorts of research till they reach the public domain efficiently at an affordable cost. In this context, as a highly sought after novel antituberculosis clinical candidate, TMC207 (48) has • ' 128 129 drawn much attention from the synthetic community. '

Previous synthetic approaches

Van Gestel approach. TMC207 (48) is a synthetic molecule reported by Andries et al and the first patented synthetic route was reported from the same research group by Van Gestel, J. F. et al,'^^ which was rather primitive and relied upon stereorandom C-C bond formation between 50 and 51, separation of the diastereomers and optical resolution using chiral HPLC (Scheme 1). Retrosynthesis

:>

50 51

Scheme 1 Results and discussion The synthesis started with Vilsmeier-Haack formylation using POCI3 in DMF on 52 to furnish 54, further nucleophilic displacement of chloro group of 54 with methoxy

20 I Page Chapter I, Section A using NaOMe in refluxing methanol gave 50 (Scheme 2).

POCI3. DMF NaOMe, MeOH 80°C, 12h Reflux, 12h

52 54

Scheme 2

Friedel-Crafts acylation with 3-chloro propionyl chloride in the presence of AICI3 on naphthalene 53 produced 55, further replacement of chloro group of 55 with dimethylamine yielded 51 (Scheme 3).

AICI3, ClCOCH^CHjCl /W''^ MejNH, ACN 80 °C, 2h CH2CI2, 0-5 "C, 2h ^-^^ifi^---^

53 55 51 Scheme 3

Addition of lithiated 50 using LDA on 51 furnished a mixture of possible isomers of 49, the diastereomers were separated through silica gel column chromatography and optical resolution using chiral chromotography gave desired enantiomer TMC 207 (48) (Scheme 4).

LDA, 51 THF, -70 "C

50 49 Scheme 4

21 IPage Chapter I, Section A

Shibasaki approach. Recently Shibasaki et al'^^ have reported the first asymmetric synthesis of TMC 207 (48). Their synthetic strategy was based on the development of two novel catalytic transformations. The retrosynthetic analysis depicted below shows the construction of contiguous tertiary and tetrasubstituted carbon through catalytic enantioselective proton migration from 58 to 57 and diastereoselective allylation on 57 to get 56 and late-stage manipulation of 56 would then lead to TMC 207 (Scheme 5).

1=>

/ N- 48

H Ph

N ^O MOM 57

v

MOM 59

Scheme 5

The synthetic scheme involved the site selective aldol reaction between 59 and 60 followed by dehydration to arrive at 58. After intensive studies, the enantioselective proton migration from 58 to 57 was achieved in 88% ee by using Y(HMDS)3, ligand (63) , MEPO and TBACl at -50 °C in THF in 88% ee. Further the diastereoselective allylation on 57 was performed using CuF-catalyzed allylboration to get 56 in 14/1 dr (Scheme 6).

22 I P a g e Chapter I, Section A

H Ph a) LDA, 60, THF, -78 °C j b) SOCI2, Py, 0 °C MOM MOM 59 58

CuF.3PPh3.EtOH, KO'Bu, ZnClj, Y(HMDS)i,, Ligand 63 PBU4BF4 , 1 ^jj;;v.^BPin THF, rt MEPO, NBU4CI, THF, -50 °C N ^O MOM 57

c) B-Bromobenzodioxaborole, CH2CI2 1—i. ^ d) O3, MeOH/HzO; NaBH4 MOM ^5^ 56 61 Scheme 6

After cleaving the //-methoxymethyl (MOM) group, ozonolysis followed by reduction produced diol 61. Regioselective bromination of 61 with NBS and Selective O- meithylatio n with AgaCOa and Mel ftimished 62. Finally, 0-tosylation followed by substitution with Me2NH afforded TMC 207 (48) (Scheme 7).

i) NBS, NaOAc, DMF 1 ii) Mel, AgjCOj, EtOH, CH3CN

61

ii) TsCl, DMAP, Py

MejNH, DMF, HjO P^ |l.h OH "^^ OH

63 48

Scheme 7

23 I P a g e Chapter I, Section A

References

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26 IP a g e Chapter I, Section A

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27 I P a g e Chapter I, Section A

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94. Johar, M.; Manning, T.; Kunimoto, D. Y.; Kumar, R. Bioorg. Med. Chem. 2005, 13, 6663. 95. Rai, D.; Johar, M.; Manning, T.; Agrawal, B.; Kunimoto, D. Y.; Kumar, R. J. Med. Chem. 2005, 48, 7012. 96. Chen, C. K.; Barrow, E. W.; Allan, P. W.; Bansal, N.; Maddry, J. A.; Suling, W. J.; Barrow, W. W.; Parker, W. B. Microbiology. 2002,148, 289. 97. Long, M. C; Allan, P. W.; Luo, M. Z.; Liu, M. C; Sartorelli, A. C; Parker, W. B. J. Antimicrob. Chemother. 2007,59, 118. 98. Long, M. C; Parker, W. B. Biochem. Pharmacol. 2006,14,1671. 99. Nigou, J.; Gilleron, M.; Rojas, M.; Garcia, L. F.; Thumher, M.; Puzo, G. Microbes Infect. 2002, 4, 945. 100. Koul, A.; Herget, T.; Kleb, B.; Ullrich, A. Nat. Rev. Microbiol. 2004, 2, 189. 101. Hilliard, J. J.; Goldschmidt, R. M.; Licata, L.; Baum, E. Z.; Bush, K. Antimicrob. Agents Chemother. 1998, 43, 1693. 102. Stephenson, K.; Yamaguchi, Y.; Hoch, J. A. J. Biol. Chem. 2000,275, 38900. 103. Weidner-Wells, M. A.; Ohemeng, K. A.; Nguyen, V. N.; Fraga-Spano, S.; Macielag, M. J.; Werblood, H. M.; Foleno, B. D.; Webb, G. C; Barrett, J. F.; Hlasta, D. J. Bioorg Med Chem. Lett. 2001,11,1545. 104. Matsushita, M.; Janda, K. D. Bioorg Med Chem. 2002,10, 855. 105. Stephenson, K.; Hoch, J. A. Curr. Med Chem. 2004,11, 765. 106. Furuta, E.; Yamamoto, K.; Tatebe, D.; Watabe, K.; Kitayama, T.; Utsumi, R. FEBS Lett. 2005,579,2065. 107. Av-Gay, Y.; Everett, M. Trends Microbiol. 2000,8, 238. 108. Greenstein, A. E.; Grundner, C; Echols, N.; Gay, L. M.; Lombana, T. N.; Miecskowski, C. A.; Pullen, K. E.; Sung, P.; Alber, T. J. Mol. Microbiol. Biotechnol. 2005, 9, 167. 109. Peirs, P.; De Wit, L.; Braibant, M.; Huygen, K.; Content, J. Eur. J. Biochem. 1997, 244, 604. 110. Drews, S. J.; Hung, F.: Av-Gay, Y. FEMS Microbiol. Lett. 2001. 205, 369. 111. Davies, J. E.; Waters, B. Patent WO 2002 022138; see Chem. Abstr. 2002, 136, 259921.

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112. Arora, S.K. et al. (2004) Design, synthesis, modelling and activity of novel antitubercular compounds Abstract, 227th. ACS National Meeting, Anaheim, CA, Division of Medicinal Chemistry (Abstract # 63) 113. Arora, S.K. et al. (2004) Pyrrole derivatives as antimycobacterial compounds. International patent WO/2004/026828 114. Matsumoto, M.; Hashizume, H.; Tsubouchi, H.; Sasaki, H.; Itotani, M.; Kuroda, H.; Tomishige, T.; Kawasaki, M.; Komatsu, M. Curr. Top. Med. Chem. 2007, 7,499. 115. FASgen, (2007) TB therapeutics from FASgen ( wwvy.fasgen.com/pipeline-fr.html)

116. Andries, K. et al. A diarylquinoline drug active on the ATP synthase of Mycobacterium tuberculosis. Science. 2005, 307, 223. 117. Van Gestel, J.F.E. et al. Quinoline derivatives and their use as mycobacterial inhibitors. International patent WO/2004/011436. 118. Huitric, E.; Verhasselt, P.; Andries, K.; E. Hoffner, S. E.; Antimicrob. Agents Chemother. 2007, 51,4202. 119. Lenaerts, A.J.; Hoff, D.; Aly, S.; Ehlers, S.; Andries, K.; Cantarero, L.; Orme, I, O.; Basaraba, R. J. Antimicrob. Agents Chemother. 2007, 57, 3338. 120. Open forum 11: Key issues in TB drug development, 12 December 2006, Kaiswemetwork.org. (www.kaisemetwork.org/health_cast/uploaded_files/l 21206_tballiance_welcomea ndpipeline.pdf). 121. Koul, A.; Dendouga, N.; Vergauwen, K.; Molenberghs, B.; Vranckx, L.; Willebrords, R.; Ristic, Z.; Lill, H.; Dorange. I.; Guillemont, J.; Bald, D.; Andries, K. Nat. Chem. Biol. 2007, i, 323. 122. Petrella, S.; Cambau, E.; Chauflfour, A.; Andries. K.; Jarlier, V.; Sougakoff, W. Antimicrob. Agents Chemother. 2006, 50, 2853. 123. de Jonge, M.R.; Luc H.M.; Jerome, K.; Guillemont, E.G.; Koul, A.; Andries, K. Proteins. 2001, 67, 91 \. 124. Gaurrand, S. Desjardins, S.; Meyer, C; Bonnet, P.; ArgouUon, J-M.; Oulyadi, H.; Guillemont, J. Chem. Biol. DrugDes. 2006, 68.11.

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125. Lounis, N.; Veziris, N.; Chauffour, A.; Truffot-Pemot, C; Andries, K.; Jarlier, V. Antimicrob. Agents Chemother. 2006, 50, 3543. 126. Kogal, T; Fukuoka, T.; Doi, N.; Harasaki, T.; Inoue, H.; Hotoda, h.; Kakuta, M.; Muramatsu, Y.; Yamamura, N.; Hoshi, M.; Hirota, T. J. Antimicrob. Chemother. 2004,54, 755. 127. Laurenzi, M.; Ginsberg, A.; Spigelman. M. Infectious Disorders - Drug Targets 2007, 7, 105. 128. Van Gestel, J. F. E.; Guillemont, J. E. G.; Venet, M. G.; Poignet, H. J. J.; Decrane, L. F. B.; Daniel, F. J. V.; Odds, F. C. U.S.A patent US/2005/0148581. 129. Saga, Y.; Motoki, S.; Shimizu, Y.; Kanai, M.; Shibasaki, M. J. Am. Chem. Soc. 2010, J32, 7905.

31 IPage CHAPTER'I SECTION B: Practical synthesis of anti-TB clinical molecule (2^-R207910 and its (IR) isomer. Chapter I, Section B

Our approach towards the synthesis of R207910 The inventorial synthesis by Van Gestel et al' has been rather simple and relied more upon analytical techniques. The first asymmetric synthesis reported by Shibasaki et al during our course of research was appreciative and involved typical reagents and steps which may not be amicable for scale up process. Moreover the overall yield is 5%, to produce substantial amounts of the compound, a more feasible route with commercially viable process is desired.

Synthetic success of a molecule has been relying more upon its retro synthesis since the inception of the latter. The identification of strategic bonds and functional group transformations which go into their construction in arriving at key synthons, play a vital role in sketching a feasible scheme for synthesis, generally containing a pathway of synthetic intermediates connected by possible reaction for the required interconversion. In the present context as is apparent fi-om the structure, the molecule is crowded with three rings around two contiguous tertiary and quaternary carbons with the other active functional groups appended to it. Careful insight into the structure led us to be choosier about starting material, either commercially available or having operationally simple protocol which goes into its preparation or a scalable process to production level would be definitely of more priority.

As the aromatic rings are more accessible to any known electrophilic or nucleophilic reactions, construction of the ftmctionalized quinoline ring starting fi-om either of the two other rings at the final stages of the scheme would be more difficult though not impossible. In view of these limiting factors, construction of functionalized quinoline ring and hence as a starting material was unequivocally opted. In view of the derived conclusions the retrosynthetic analysis is envisioned as shown below (Scheme 1). R207910 (1) could be obtained from 2 in two steps. The stereocenter of the phenyl group could be introduced by regioselctive epoxide opening with PhMgBr and concomitant Payne rearrangement and the stereocenter at the tetrasubstituted carbon could be obtained by Sharpless asymmetric epoxidation.

32 I Page Chapter I, Section B

Retrosynthetic analysis.

\7

COOH

N CI

Scheme 1

Results and Discussion The synthesis commenced with a known intermediate 6-bromo-2-chloroquinoline- 3-carbaldehyde (5), prepared from 4-bromo-acetanilide using modified Vilsmeier reagent.^ The compound thus obtained was in agreement with the literature data. Compound 7 was obtained by condensing 5 with napthyl acetic acid 6 in the presence of acetic anhydride and triethylamine at 100 °C for 4h in 70% yield. The product was confirmed by its 'H NMR spectrum. The absence of peak corresponding to aldehyde proton and presence of 12 aromatic protons indicated the formation of the product. The ESl-MS spectrum has shown a peak at m/z 439 (M+H)^ confirming the product. The next step was to displace chloro atom of 7 with methoxy group. Accordingly a, P-unsaturated acid 7 was treated with NaOMe in refluxing methanol for 12h to give compound 4 in 74% yield (Scheme 2). The conversion was realized by the 'H NMR spectrum analysis. It 33 [Page Chapter I, Section B has shown a peak at 6 4.09 as singlet integrating for 3 protons corresponding to methoxy group along with the rest of 12 aromatic protons. The ESI-MS spectrum of compound 4 has shown a peak at m/z 434 (M+H)* confirming the conversion.

COOH CHO COOH AcjO, EtjN + > 100°C,4h,70%

COOH NaOMe, MeOH Reflux, 12h,74%

Scheme 2

Next the acid group of compound 4 was converted to corresponding methyl ester by treating with diazomethane in diethylether/THF solvent system at 0 "C for half an hour in 80% yield. The PMR spectrum of a, P- unsaturated ester 8 has revealed a peak at 6 3.75 corresponding to methoxy group along with the rest of proton peaks at expected chemical shifts. Further the peak at m/z 448 in the ESI-MS of 8 confirmed the analysis. In order to get the precursor for asymmetric epoxidation the a, p-unsaturated ester 8 was reductively transformed to corresponding allylic alcohol using DIBAl-// at 0 °C in dichloromethane to give 9 in 95% yield (Scheme 3). 'H NMR spectrum of 9 has disclosed the disapj)earance of methoxy singlet at 5 3.75 and shown a broad singlet at 5 4.54 integrating for two protons corresponding to allylic protons attached to primary hydroxyl group and a broad singlet at 6 1.67 for hydroxyl proton, remaining protons resonated at expected chemical shifts. Also the m/z value of 420 for (M+H)* peak in die ESI-MS has added to the conclusion.

As the precursor is ready for the introduction of chirality, the next step was to perform Sharpless asymmetric epoxidation on the allylic double bond of compound 9. Accordingly the 9 was subjected to chiral epoxidation using (+)-DIPT, Ti(0'Pr)4, TBHP at -20 "C in dichloromethane to afford 3 in 80% yield with >90% ee.

34 I Page Chapter I, Section B

COOH COOMe CH2N2, THF:Ether 0°C,0.5h, 80%

D1BA1-//, CH2CI2 »• -Tg^C, 10min,95%

Scheme 3

The product was characterized by its PMR spectrum. The newly formed epoxide proton at the benzylic position was seen at 5 5.01: two protons attached to hydroxyl group resonated as muhiplet at 6 4.31-4.19 and the remaining aromatic, methoxy protons were observed as expected. Additional information in the form of mass peak at m/z 436 in the ESI-MS of 3 has confirmed its structure.

Satisfied with the result the next step was to prepare the precursor for Payne rearrangement. In view of that the primary hydroxyl group of 3 was made as a good leaving group by converting it as its tosylate. Thus treatment with p-toluene sulfonyl chloride, triethylamine in dichloromethane afforded product 10 in 92% yield (Scheme 4). The product was confirmed by its 'H NMR spectrum. It showed the disappearance of hydroxyl proton peak at 5 1.97 and appearance of singlet resonating at 5 2.38 corresponding to methyl group of tosylate. The increment in the proton count to 15 in the aromatic region and the left over protons resonating at respective chemical shifts indicates the transformation. The corresponding mass peak at m/z 591 for (M+H)* in ESl-MS of 10 confirmed the conversion. As the stage is set for Payne rearrangement, the idea behind it was to open the oxirane ring with PhMgBr from the P position of epoxy alcohol and concomitant nucleophilic displacement of tosyl group would result in the terminal epoxide of 2 which would then serve as core structure for further transformations leading to final target 1.

35 I Page Chapter I, Section B

(+)-DlPT, Ti(0'Pr)4 TBHP, CH2CI2, -20 "C 80%

TsCLEtjN OTs CH2CI2, 0 °C-rt 92%

But the expected nucleophilic attack did not occur at the expected site and there was no room for Payne rearrangement. Trails with variations in the reaction protocols only gave unexpected byproducts. Thus fixing the stereocenter of the phenyl ring adjacent to tetrasubstituted carbon was rather difficult (Scheme 5). Hence the scheme was revisited and revised so as to fix the 3° center first followed by chelation controlled introduction of quaternary carbon center.

OTs Nu "TV

Nu: PhLi + Cul PhMgBr + BFj.OEiz

Scheme 5

According to the modified retrosynthetic analysis, 1 could be obtained from 11 in two steps (Scheme 6). The stereocenter at the tetrasubstituted carbon was introduced by chelation controlled Barbier type reaction. The enantiomerically enriched ketone 12 could be obtained by Sharpless asymmetric epoxidation followed by ring opening with PhMgBr.

36 I P a g e Chapter I, Section B

Modified Retrosynthesis

N CI

Scheme 6

Results and discussion Once again the synthesis commenced with the same known intermediate 6-bromo- 2-chloroquinoline-3-carbaldehyde (5), prepared from 4-bromo-acetanilide using modified Vilsmeier reagent.^ The first step was to add on a suitable carbon chain upon which functional group manipulation would provide us a scope to fix the other rings with the required chirality. Accordingly, to convert the aldehyde of 5 to a, p unsaturated ester it was subjected to Homer-Wittig-Emmons olefination using lithium enolate of the phosphonate ((0Et)2 P(0)CH2C02Et] in THF at rt for 2 h to obtain 14 in 89% yield with exclusive E- selectivity. The characterization of the structure was done with the help of its NMR spectra. The typical quartet at 6 4.26 and triplet at 5 1.29 integrating for 2H and 3H indicated the presence of ethyl group. A IH doublet at 6 6.83 for a alkene proton and a set of five aromatic protons including the P alkene proton indicated the transformation. The exclusive (£) geometry was evidenced by the coupling constant of 16.0 Hz between the alkene protons. '^C NMR spectrum has shown a set of 14 carbons peaks, one for ester

37 I Page Chapter I, Section B carbonyl carbon at 5 165.2. IR spectrum revealed an absorption band at 1701 cm'' for carbonyl carbon stretching frequency and finally HRMS value at m/z 339.9739 for (M+H)* peak vindicated the analysis. Next a, P unsaturated ester of 14 was reductively transformed to allylic alcohol with DIBAl-// at 0 °C in dichloromethane to afford 15 in 84% yield (Scheme 7). The 'H NMR spectrum of 15 revealed the absence of characteristic peaks due to ethyl group of ester and shown a broad singlet at 5 4.26 integrating for 2H adjacent to hydroxyl functionality, further two triplet of doublets at 8 6.94 and at 6 6.64 integrating for 1H each are of alkene protons. The remaining protons resonated at their respective chemical shifts. '"C NMR spectrum exhibited a 12 carbon frame work and the presence of OH group was shown by the absorption band at 3285 cm"' in the IR spectrogram. The HRMS value was found to be 297.9616 for (M+H)* peak adding to the confirmation.

^^^^fsT^Cl LiHMDS, THF, 0 "C-rT ^^N^CI 89% 14

DIBAL-// ^'^Nr^'V^^^ ^^^ "OH -^- CH2CI2, 0 "C-rt N CI 84% 15

Scheme 7

Next compound 16 was obtained when chloro functional group of 15 underwent nucleophilic displacement with NaOMe at reflux conditions for 8h in methanol in 92% yield. The nucleophilic displacement was clearly shown by the PMR spectrum of 16 with a peak resonating at 5 4.04 for 3H as a singlet. The remaining set of protons, as expected, resonated at their chemical shifts with slight change. An extra carbon peak due to methoxy functionality was observed at 5 53.5 in the '^C NMR spectrum of 16 along with the earlier 12 carbon network. The HRMS shown a peak at m/z 294.012 for (M+H)^ which concluded the analysis.

38 I Page Chapter I, Section B

Now the comer stone in the strategic scheme was to introduce essential chiraHty, which was achieved through hitherto well explored, commercially viable, Sharpless asymmetric epoxidation. Therefore 16 was subjected to SAE with Ti(0'Pr)4, L-(+)-DIPT, TBHP, 4A° molecular sieves in dichloromethane at -20 °C for 4h to acquire 13 in 86% yield (scheme 8). The 'H NMR spectrogram of 13 showed the absence of alkene protons and presence of newly formed epoxide protons at 5 4.11 as a doublet and at 5 3.21-3.13 as a multiplet integrating for IH proton each respectively whereas the allylic diastereotopic protons were distinguished as multiplets at 5 3.83 and at 5 3.66-3.55 accounting for IH each. As usual the aromatic protons were seen at their respective chemical shifts. Again '^C NMR spectrum has shown similar set of 13 carbon peaks with alkene carbon peaks transposed to epoxy carbon peaks. Further HRMS data disclosed m/z value of 310.0088 for (M+H)* peak and finally chiral HPLC chromatogram gave 95% ee for the asymmetric epoxidation.

^•"^ "'^ ^'^ ^^^^^OH NaOMe, MeOH ^'^'V"*V^'^V^ "^^ ^OH reflux, 8 h 'N -CCI 92% ^ N O 15 16

.xQ Ti(0'Pr)4, L(+)-DIPT ^'^>r^"V^^V^^^^^"'OH TBHP, 4A'' MS, CH,C1-,, -20 "C. 4h Vi^^K,-*^-^ 86% I 13 Scheme 8

Now with 13 in hand the task before us was to introduce phenyl ring at the benzylic position. Having vainly attempted earlier to fix the tertiary center with phenyl substituent after fixing the quaternary center (Scheme 5), this time it was planned to introduce 3° center with phenyl group at the benzylic position. However, many attempts to open oxirane ring with organometallic reagents either directly or catalytically (BF3.0Et2, Cul) have become fruitless. Nevertheless, with surfeit examples banking in the wealthy literature, it was planned to open the epoxide regioselectively at the benzylic position with the help of higher order Gillman reagent. As intended, using phenyl Grignard reagent in the presence of stoichiometric amount of CuCN the oxirane was selectively

39|Page Chapter I, Section B opened from benzylic position. After optimization the protocol involved the treatment of 13 with excess use of phenyl Grignard reagent in the presence of CuCN at -40 "C in THF for 4h to furnish 17 in 86% yield (Scheme 9).'' Compound 17 was characterized by NMR spectroscopy, wherein the PMR spectrogram showed a 5H multiplet at 6 7.29-7.14 for newly formed phenyl ring protons and a 2H broad singlet at 5 4.47 for both benzylic and secondary carbinol attached protons. Rest of the protons resonated as expected. A set of 16 carbon peaks in '^C NMR spectrum and HRMS value of 388.0540 for (M+H/ peak added to confirmation.

BrY^^r^^Y^^^^OH PhMgBr.CuCN ^ ^' KJ^^{^r^ THF,-40 "C

13

Scheme 9

Next the 1,2 diol of 17 was oxidatively cleaved to an unstable aldehyde 18 using Nal04 impregnated over silica in dichloromethane at 0 °C, upon which the 1-naphthyl Grignard reaction"^ was performed immediately to result in an inseparable diastereomeric mixture of 19. Without further characterization compound 19 was subjected to oxidation with Dess-Martin periodinane in dichloromethane at 0 °C for 3h to obtain 12 in 87% yield (Scheme 10). The 'H NMR spectrum of 12 revealed a IH singlet at 5 6.22 for benzylic proton flanked to keto group and plethora of aromatic protons resonating at expected chemical shifts along with the 3H singlet at 5 3.97 for methoxy protons. Additionally '^C NMR spectrogram disclosed an intricate network of 26 carbons while HRMS shown a value of 482.0738 for (M+H)^ peak. The carried over 95% ee substantiated that there was no racemization in the previous reactions.

With the core moiety in hand the next challenge was to create a quaternary center at the keto carbon with a fairly acidic proton adjacent to it. Since generating a quaternary center would mandate the use of nucleophile, the accompanied racemization may not be forestalled unless either milder nucleophile or neutral reaction conditions are engaged.

40 I P a g e Chapter I, Section B

To append the carbon chain and end up introducing required functionalities with feasible functional group transformations, a chiral allylation protocol was envisaged.

NaI04 1-NpMgBr CH2Cl2,0<'C-rt, Ih Ether, 0°C,lh 98% 92%

DMP CH2CI2,0 °C-rt. 3h 87%

Scheme 10

But BINOL catalyzed allylation reactions employing neutral tin reagents didn't elicit any expected reaction. Besides installation of chirality at the keto carbon, the reactive site was hardly prone to any sort of nucleophilic reaction. All attempts using conventional nucleophiles, including lithium enolates, vinyl Grignard reagents, allylmagnesium reagent with/without CeCb, allyl aluminium, allylindium, allylboronate were failed which might be due to the high steric crowding around carbonyl carbon. During this time a solvent free zinc allylation protocol reported'^ has helped us visualize an almost inseparable 2:3 ratio of allylation diastereomers in favor of unwanted isomer when 12 was subjected to neat allylzinc bromide. To improve the ratio towards the required isomer different chelating agents like CuBr, CuCN, Cul, and CuBr.Me2S were added in the reaction, only in the case of CuBr.Me2S the ratio was almost equal rising hopes to further optimization.

The typical protocol involved the treatment of 12 with allylzinc bromide in the presence of catalytic CuBr.Me2S in THF to afford 11a & lib as a diastereomeric mixture in 90% yield (Scheme 11). Preparative HPLC separation of the mixture gave pure 11a. 'H NMR has shown characteristic terminal alkene multiplets at 5 5.17-4.97 and at 5 4.96-

41|Page Chapter I, Section B

4.81 integrating for IH and 2H resj)ectively and the two diastereotopic allylic protons were seen at 6 3.6las a doublet and at 8 2.51-2.37 as a multiplet. At 6 2.66 a IH singlet for quaternary OH along with the remaining protons was seen. '^C NMR spectrogram has shown a set of 22 carbon peaks while IR spectrum showed an absorption peak at 3363 cm' for hydroxyl group. Finally HRMS value at m/z 524.1223 for (M+H)^ peak confirmed the structure.

.. Allylzinc bromide CuBr.Me2S,THF,rt, Ih 90%

11a lib

Scheme 11

Having arrived at a stage, where a few functional group transformations would result in the final compound. Accordingly the terminal double bond of 11a was oxidatively cleaved by using OSO4, NaI04 and 2, 6-lutidine in dioxane-water system at rt to aldehyde of 20a which was subjected to, without purification, NaBH4 reduction in methanol at 0 "C to furnish 21a in 82% over two steps (Scheme 12). The 'H NMR spectrum of 21a revealed the absence of multiplets in the alkene region and presence of newly formed multiplet at 5 3.56-3.42, triplet at 5 3.19, doublet at 6 2.75 and multiplet at 5 2.32-2.17 each for IH, totally accounting for two protons attached to primary hydroxyl group and two diastereotopic protons adjacent to tetrasubstituted carbon. The rest of protons resonated at expected chemical shifts. The 28 carbon framework in the C NMR spectrogram , an absorption band at 3420 cm' in IR spectrum and m/z value 528.1177 for (M+H)^ peak in HRMS data confirmed the analysis. The next objective was to convert the primary hydroxyl functionality of 21a as its mesylate. So compound 21a was treated with methane sulfonyl chloride at 0 °C in the presence of triethylamine in dichloromethane to obtain 22a in 88% yield (Scheme 12). 'H NMR spectrum analysis showed a 3H singlet at 5 2.49 for methyl protons of mesylate. A carbon peak at 6 50.4 due to methyl carbon of mesylate group was seen along with 29

42 I P a g e Chapter I, Section B carbon network in the '^C NMR spectrum of 22a. HRMS value was found to be 606.0955 for (M+H)^ peak.

l)NaI04,Os04, 2,6-lutidine dioxane: water (3:1), rt, 2h

11a

2) NaBH4, MeOH, 0 °C-rt MsCl, EtjN j i »> 82% (over two steps) CH2CI2, 0 °C-rt, 3h 88%

21a

Me,NH THF, 45 °C, 24h 90% ' OMs ' ,N, 22a 1 Scheme 12 At this stage the nucleophilic displacement of mesylate functionality of 22a with dimethylamine group would bestow with the aspired final molecule. Hence 22a was treated with 8 molar solution of dimethylamine in THF in solvent proportions at 45 "C for 24h to furnish TMC 207 (1) in 90% yield (Scheme 12).* The spectral and analytical data of compound were in compliance with reported values. The PMR spectrum disclosed the disappearance of methyl singlet at 6 2.49 and presence of 6H singlet at 5 1.99 integrating for protons of dimethylamine group. The remaining proton set was in good agreement with the reported one. '^C NMR spectnmi has shown a close network of 29 carbon peaks and HRMS data for (M+H)^ peak was came to be 555.1671. In a similar way the other allyl diastereomer lib obtained through HPLC separation was also subjected to the same set of functional group transformations to

43 I P a g e Chapter I, Section B arrive at the diastereomer of R207910 (1') (Scheme 13). All diastereomeric intermediates from that point were in good agreement with spectral and analytical values.

3 Steps

lib

Scheme 13 In a more practical approach the diastereomeric mixture (11a & lib) obtained in the allylation step was carried to the final step with the same set of reaction protocol and the viability of chromatographic separation-at the final stage has been tested. To our satisfaction the products were separable easily by silica gel column chromatography (Ethyl acetate: hexane = 1:6). In fact this approach is operationally simple and amenable for scale up at affordable cost (Scheme 14).

3 Steps Br »~ I +

Scheme 14

Conclusion: In conclusion, we have achieved the synthesis of (2S)-R207910 (1) and (2/?)- R207910 (1') in overall yield of 12 % each in 10 steps. The key steps involved are Sharpless asymmetric epoxidation, regioselective epoxide opening and modified allylzinc bromide addition. Optimization of the allylation reaction would enable the approach amenable for scale up at affordable cost as Sharpless asymmetric epoxidation is a commercially viable process.

44 I P a g e Chapter I, Section B

Experimental 6-bromo-2-chloroquinoline-3-carbaldehyde5

CHO

To a solution of 4-Br-acetanilide (32.1 g, 150 mmol), DMF (231 ml, 300 mmol) in ACN containing a solution of CTAB (30 mL of 0.05 mol) in acetonitrile was added POCI3 (279 ml, 300 mmol) slowly at -5 "C and refluxed for 45 min. The acetonitrile was evaporated and the reaction mixture was dissolved in CH2CI2 and quenched with Na2S203 solution. The organic layer was separated, dried over Na2S04 and evaporated under vacuum. The crude product was silica gel chromatographed using ethylacetate:hexane (3:7) as eluent to get pure 6-bromo-2-chloro-3-formyl quinoline (36.5 g, 90% yield);

'H NMR (CDCI3, 200 MHz) : 6 10.55 (s, IH), 8.63(s, IH), 8.14 (br s, IH), 7.94 (d, J = 1.5H2,2H) LC-MS (ESI) : m/z 270 (M)^ Mp. : 186-188 °C.

(£)-3-(6-bromo-2-chIoroquinolin-3-yI)-2-(naphthaIen-l-yl)acrylicacid 7

Br>^ ^^ ^

To a stirred mixture of 5 (3.0 g, 11 mmol), napthyl acetic acid 6 (2.0 g, 10.8 mmol), acetic anhydride (3.15 mL) was added triethylamine (1.5 mL, 10.8 mmol). The mixture was stirred at 100 "C for 4h. After being cooled the mixture was made alkaline with 10% NaHCOs. The aqueous mixture was warmed to 60 "C and filtered. The filtrate was adjusted to pH 4.5 with 10%HC1, and the resulting precipitate was collected and recrystallized fi-om EtOH to give 7 (3.4 g, 70%).

'H NMR (CDCI3, 200 MHz) : 6 8.31 (s, IH). 7.93-7.77 (m, 3H), 7.74-7.60 (m, 2H), 7.53-7.38 (m, 3H), 7.33-7.26 (m, IH), 7.20 (br s, 2H)

45 I P a g e Chapter I, Section B

LC-MS (ESI) : m/z 439 (M+H)^

(£)-3-(6-bronio-2-methoxyquinolin-3-yI)-2-(naphthaIen-l-yI)acrylic acid 4

XOOH

To a solution of 7 (2.0 g, 4.5 mmol) in MeOH (25 mL), was added NaOMe (1.23 g, 22.7 mmol) and the resulting suspension was refluxed at 80 °C for 8 h. The solvent was evaporated under reduced pressure to give a crude solid which is quenched with water (10 mL) and extracted with ethyl acetate (3 x 25 mL). The combined organic layers were dried over Na2S04 and rotary evaporated. The crude product was silica gel chromatographed (eluent: MeOH/CHCb = 1/8) to furnish 4 (1.46 g, 74%). 'H NMR (CDCI3, 300 MHz) : 6 8.50 (s, IH), 7.92 (d, J- 8.3 Hz, 2H), 7.80 (d, J- 8.3 Hz, IH), 7.54 (s, 2H), 7.52-7.42 (m, 3H), 7.34-7.30, (m, IH), 7.06 (s, IH), 6.98 (s, IH), 4.09 (s, 3H). LC-MS (ESI) : m/z 434 (M+H)^

(£)-methyI3-(6-bromo-2-inethoxyquinoIin-3-yI)-2-(naphthalen-l-yl)acrylate8

XOOMe

To the stirred solution of 4 (1.4 g, 3.2 nunol) in THF (10 mL) was added CH2N2 in diethylether (10 mL) at 0 °C. After 10 min the solution was washed with water (2x10 mL), brine (10 mL) dried over Na2S04 and concentrated under vacuo. The crude product was silicagel chromatographed (eluent: EtOAc/Hexane =1/10) to furnish 8(1.1 g, 80%). 'H NMR (CDCI3, 300 MHz) : 5 8.34 (s, IH). 7.93-7.86 (m, 2H), 7.74 (d, J = 8.3 Hz, IH), 7.53-7.40 (m. 5H). 7.29-7.25 (m, IH), 7.02 (s, IH), 6.91 (s, IH), 4.10 (s, 3H). 3.75 (s, 3H). LC-MS (ESI) : m/z 448 (M+H)^

\ 46 I P a g e Chapter I, Section B

(£)-3-(6-bromo-2-methoxyquinoIin-3-yl)-2-(naphthalen-l-yI)prop-2-en-l-oI9

DIBAL-// (3.5 mL, 4.9 mmol, 1.4 M in hexane) was added slowly to a, P-unsaturated ester 8 (1.1 g, 2.4 mmol) in CH2CI2 (10 mL) at -78 °C, and the resulting mixture was stirred at room temperature for 10 min. The reaction mixture was carefully quenched with saturated aqueous Rochelle's salt (5 mL) at 0 °C and the resulting suspension was stirred vigorously for 4 h. The reaction mixture was filtered and the two layers separated. The aqueous layer was further extracted with CH2CI2 (3 x 10 mL). The combined organic layers were dried over Na2S04 and concentrated under reduced pressure. The crude product was silica gel chromatographed (eluent: AcOEt/hexane = 1/5) to afford 9 (0.97 g, 95%). 'H NMR (CDCI3, 300 MHz) : 5 7.94-7.80 (m, 3H), 7.52-7.37 (m, 5H), 7.33-7.28 (m, IH), 7.22 (s, IH), 7.02 (d, J = 1.5 Hz, IH), 6.94 (s, IH), 4.54 (br s, 2H), 4.10 (s, 3H), 1.67 (br s, IH). LC-MS (ESI) : m/z 420 (M+H)^

((25,35)-3-(6-bromo-2-methoxyquinolin-3-yl)-2-(naphthaIen-l-yl)oxiran-2- yl)methanol 3

Ti(0'Pr)4 (70 \xL, 0.2 mmol) in CH2CI2 (1 mL) was added to a suspension of 4A molecular sieves (0.3 g, 30 % w/w based on substrate) in CH2CI2 (3 mL). The mixture was cooled to -20 °C and L-(+)-diisopropyl tartrate (75 (iL, 0.35 mmol) in CH2CI2 (1 mL) was added and stirred for 30 min followed by 9 (1.0 g, 2.3 mmol) in CH2CI2 (10 mL). The resulting suspension was stirred for 40 min at the same temperature. After this time anhydrous rer/-butylhydrogenperoxide in toluene (0.8 mL, 9.5 mmOl, 4.0 M) was

471 P a g e Chapter I, Section B added drop-wise and stirring continued for 24 h. After warming up to 0 °C, water (1 mL) was added and the mixture was stirred for 1 h. 20% Aqueous NaOH saturated with NaCl (0.5 mL) was added and stirring continued for 1.5 h. The slurry was filtered through a plug of Celite and rinsed thoroughly with CH2CI2. The organic layer was separated and the aqueous layer extracted with CH2CI2 (2x5 mL). The combined organic layer was washed with brine, dried over Na2S04. and rotary evaporated. The crude product was silica gel chromatographed (eluent: AcOEt/hexane = 1/5) to obtain 3 (0.83 g, 80%). 'H NMR (CDCI3, 300 MHz) : 6 7.97 (d, 7 = 8.1 Hz, IH), 7.72 (d, J = 6.8 Hz, IH), 7.65-7.57 (m, 2H), 7.51-7.26 (m, 7H), 5.01 (s, IH), 4.31-4.19 (m, 2H), 4.14 (s, 3H), 1.97 (br s, IH). LC-MS (ESI) : m/z 436 (M+H)^.

((2535)-3-(6-bromo-2-inethoxyquinolin-3-yl)-2-(naphthaIen-l-yI)oxiran-2-yl)methyl 4-methylbenzenesulfonate 10

OTs

To a solution of 3 (0.8 g, 1.8 mmol) in CH2CI2 (6 mL) were added EtsN (77 ^L, 5.5 mmol) and/?-toluene sulfonyl chloride (0.349 g, 1.8 mmol) sequentially at 0 °C. After stirring for 24 h at room temperature, water (2 mL) was added to quench the reaction. The reaction mixture was diluted with CH2CI2 (5 mL). The organic layer was separated and the aqueous layer extracted with CH2CI2 (2x5 mL). The combined organic extracts were washed with brine, dried over Na2S04 and concentrated under reduced pressure. The crude product was silica gel chromatographed (eluent: AcOEt/hexane = 1/8) to afford 10 (0.99 g, 92%). 'H NMR (CDCI3, 300 MHz) : 5 7.82-7.73 (m, IH), 7.73-7.65 (m, IH), 7.63-7.51 (m, 4H), 7.47-7.33 (m, IH), 7.30-7.22 (m, 3H), 7.15-7.08 (m, 2H), 4.86 (s, IH). 4.60-4.50 (m, 2H), 4.12 (s, 3H), 2.38 (s, 3H). LC-MS (ESI) : m/z 590 (M+H)^

48 1 P a g e Chapter I, Section B

(£)-ethyl 3-(6-bromo-2-chloroquinoHn-3-yI)acrylate 14

To a stirred solution of phosphonate (EtO)2P(0)CH2C02Et) (32.2 g, 140 mmol) in THF (200 mL) was added LiHMDS (138.0 mL, 38.0 mmol, 1.0 M) at 0 T slowly for 30 min and stirred further for 30 min at room temperature. The above solution was cannulated to a solution of 5 (30.0 g, 11 mmol) in THF (50 mL) drop-wise and stirred for 2 h. The reaction was quenched with water (100 mL). The organic layer was separated and the aqueous layer extracted with ethyl acetate (2 x 100 mL). The combined organic layers were washed with brine, dried over Na2S04 and concentrated under reduced pressure. The crude product was silica gel chromatographed (eluent: ethyl acetate/hexane = 1:19) to give 14 as a white solid (33.2 g, 89%). 'H NMR (300 MHz, DMSO-Dg): 5 8.98 (s, IH), 8.28 (d, J= 1.5 Hz, IH), 8.03-7.86 (m, 3H), 6.83 (d, J = 16.0 Hz, IH,), 4.26 (q, J = 7.1 Hz, 2H), 1.29(t,J=7.1Hz,3H). '^C NMR (75 MHz, DMS0-D6): 6 165.2, 148.1, 145.6, 137.9, 136.6, 134.7, 130.3, 129.7, 128.0,127.3, 123.4, 120.6,60.5,14.0. IR(KBr) : 1701 Cm'. HRMS (ESI) : calcd for CuHnBrClNOi (M+H)"^ 339.9734. Found 339.9739. M.p. :175''C.

(£)-3-(6-bromo-2-chIoroquinolin-3-yI)prop-2-en-l-oI15

Br^,...... — OH

N CI

DIBAL-// (125.0 mL, 170.0 mmol, 1.4 M in hexane) was added slowly to a,^- unsaturated ester 14 (30.0 g, 88.0 mmol) in CH2CI2 (150 mL) at 0 °C, and the resulting

49 i P a g e Chapter I, Section B

mixture was stirred at room temperature for 2 h. The reaction mixture was carefully quenched with saturated aqueous Rochelle's salt (150 mL) at 0 °C and the resulting suspension was stirred vigorously for 4 h. The reaction mixture was filtered and the two layers separated. The aqueous layer was further extracted with CH2CI2 (3 x 150 mL). The combined organic layers were dried over Na2S04 and concentrated under reduced pressure. The crude product was silica gel chromatographed (eluent: AcOEt/hexane = 1/5) to afford 15 as a pale yellow solid (22.0 g, 84%). 'H NMR (500 MHz, DMSO-De): S 8.62 (s, IH), 8.30-8.24 (m, IH), 7.89-7.83 (m, 2H), 6.94 (td, J= 15.8, 1.9 Hz, IH), 6.64 (td, ./= 15.8, 4.3 Hz, IH), 5.16 (t,y= 5.1 Hz, IH), 4.26 (br s, 2H); '^C NMR (75 MHz, DMSO-De) : 8 149.4, 144.4,137.5, 133.7,133.2, 130.4, 129.8, 129.6, 128.5,122.0,120.2,61.0; IR(KBr) : 3285 Cm'. HRMS (ESI) : calcd for CuHioBrClNO (M+H)^ 297.9629. Found 297.9616. M.p. tno^c.

(£)-3-(6-bromo-2-methoxyquinoUn-3-yl)prop-2-en-l-oI 16

I To a solution of 15 (39.4 g, 130 mmol) in MeOH (150 mL), was added NaOMe (35.6 g, 650 mmol) and the resulting suspension was refluxed at 80 ^^C for 8 h. The solvent was evaporated under reduced pressure to give a crude solid which was quenched with water (100 mL) and extracted with ethyl acetate (3 x 150 mL). The combined organic layers were dried over Na2S04 and rotary evaporated. The crude product was silica gel chromatographed (eluent: AcOEt/hexane = 1/5) to furnish 16 as a pale yellow solid (35.1 g,92%). 'H NMR (300 MHz, DMSO-De): 6 8.32 (s, IH), 8.10 (d, J = 1.7 Hz, IH), 1.15-1 M (m, 2H), 6.80 (d, J= 16.0 Hz, IH), 6.63 (td, 7 = 16.0, 4.5 Hz, IH), 5.04 (br s. IH), 4.21 (br s, 2H), 4.04 (s, 3H);

50 I Page Chapter /, Section B

'^CNMR(75 MHz, DMSO-De): S 159.5, 143.4, 135.4, 133.0, 131.9, 129.3, 128.4, 126.6, 122.7,121.1,116.5,61.4,53.5. IR(KBr) : 3258 Cm'. HRMS (ESI) : calcd for CnHiBBrNOa(M+Hf 294.0124. Found 294.012. M.p. :134°C.

((2535)-3-(6-bromo-2-methoxyquinolin-3-yl)oxiran-2-yl)methanoll3

Ti(0'Pr)4 (2.0-mL, 0.68 mmol) in CH2CI2 (20 mL) was added to a suspension of 4A molecular sieves (7.0 g, 30 % w/w based on substrate) in CH2CI2 (20 mL). The mixture was cooled to -20 °C and L-(+)-diisopropyl tartarate (2.1 mL, 10 mmol) in CH2CI2 (10 mL) was added and stirred for 30 min followed by 16 (20.0 g, 68.0 mmol) in CH2CI2 (200 mL). The resulting suspension was stirred for 40 min at the same temperature. After this time anhydrous /er/-butylhydrogenperoxide in toluene (68.2 mL, 270 mmol, 4.0 M) was added drop-wise and stirring continued for 4 h. After warming up to 0 °C, water (100 mL) was added and the mixture was stirred for 1 h. 20% Aqueous NaOH saturated with NaCl (50 mL) was added and stirring continued for 1.5 h. The slurry was filtered through a plug of Celite and rinsed thoroughly with CH2CI2. The organic layer was separated and the aqueous layer extracted with CH2CI2 (2 x 150 mL). The combined organic layer was washed with brine, dried over Na2S04, and rotary evaporated. The crude product was silica gel chromatographed (eluent: AcOEt/hexane = 1/5) to obtain 13 as a white solid (18.1 g, 86%). 'H NMR (300 MHz, DMSO-De): 5 8.16 (d, J = 1.8 Hz, IH), 7.99 (s, IH), 7.78-7.67 (m, 2H), 5.09 (t, J- 5.8 Hz, IH), 4.11 (d, J- 1.7 Hz , IH), 4.06 (s, 3H), 3.83 (ddd, J = 12.6, 5.4, 2.6 Hz, IH), 3.66-3.55 (m, IH), 3.20-3.13 (m, IH);

51 IPage Chapter I, Section B

'^C NMR(75 MHz, DMSO-De) : d 160.1, 143.7, 133.0, 132.3, 129.6, 128.4, 126.1, 123.3, 116.6,62.8,60.3,53.6,50.3. IR(KBr) :3377,3281cm''. HRMS (ESI) : calcd for CuHnBrNOs (M+H)"^ 310.0073. Found 310.0088. M.p. :153''C. [af'o :(-) 41.8 (c 0.5, CHCI3). HPLC (Chiraipak IC (250 x 4.6min, 5^), IPA/Hexane 1/10, flow 1.0 mL): IR 10.8 min (minor), 15.4 min (major) 95% ee.

(2jR3jl'?)-3-(6-bromo-2-methoxyquinoIin-3-yl)-3-phenyIpropane-l,2-dioI 17

To a suspension of CuCN (23.0 g, 250 mmol) in THF (150 mL) at -40 "C, PhMgBr (258.2 mL, 510 mmol, 2.0 M in Et20) was added. After stirring for 1 h, solution of 13 (16.0 g, 51.0 mmol) in THF (50 mL) was added drop-wise via cannula. After 3 h, the reaction was quenched with a saturated aqueous solution of NH4CI (100 mL), diluted with ethyl acetate (100 mL) and allowed to stir for 3 h. The layers were separated and the aqueous layer extracted with ethyl acetate (3 x 100 mL). The combined organic layers were washed with water, brine, dried over Na2S04, and concentrated under reduced pressure. The crude mixture was silica gel chromatographed (eluent: AcOEt/hexane = 1/4) to afford diol 17 as an off white solid (17.1 g, 86%). 'H NMR ( 300 MHz, CDCI3) : S 8.01 (s, IH), 7.86 (s, IH), 7.67-7.58 (m, 2H), 7.29- 7.14 (m, 5H), 4.47 (br s, 2H), 3.99 (s, 3H), 3.57 (d, J = 10.8 Hz, IH), 3.43 (dd, J- 10.8, 4.0 Hz, IH), 2.82 (br s, lH),2.50(brs, IH). '^C NMR (75 MHz, CDCI3) : S 160.8, 143.8, 139.7, 135.0, 132.1, 129.3, 128.5, 128.4, 126.9, 126.7, 126.4, 117.1, 73.3, 64.8, 53.7,47.4.

52 I Page Chapter I, Section B

IR (KBr) :3374,2924 cm''. HRMS (ESI) : calcd for Ci9H,9BrN03 (M+H)^ 388.0543. Found 388.0540. r i26 :(-) 133.5 (c 0.5, CHCb). [a] D M.p. : 110°C.

(2/?)-2-(6-bromo-2-methoxyquinolin-3-yl)-l-(naphthalen-l-yI)-2-phenylethanol 19

To a solution of 17 (19.0 g, 49 mmol) in CH2CI2 (400 mL) was added NaI04 impregnated over silica (20% w/w on silica, 150 g) and stirred for 1 h. The suspension was filtered and washed with CH2CI2 (100 mL). The filtrate was evaporated under reduced pressure to give crude 18 (17.0 g, 98%) which was used immediately for the next reaction. To a solution of 18 (16.5 g, 46 mmol) in diethyl ether (100 mL) at 0 "C was added freshly prepared napthyl Grignard reagent (230 mL, 0.6 M in Et20, 139.0 mmol) and stirred for 1 h. The reaction was quenched with saturated aqueous solution of NH4CI and the aqueous layer extracted with ether (2 x 150 mL). The combined organic layers were washed with water, brine, dried over Na2S04, and concentrated under reduced pressure. The inseparable mixture of diastereomers were silica gel chromatographed (eluent: ethyl acetate/hexane = 1:10) to give 19 as an off white solid (20.4 g, 92%).

(2/{)-2-(6-bromo-2-methoxyquinolin-3-yl)-l-(naphthalen-l-yl)-2-phenyIethanone 12

To a solution of 19 (20.3 g, 40.0 mmol) in CH2CI2 (150 mL) was added Dess-Martin periodinane (25.4 g, 60.0 mmol). After being stirred at room temperature for 3 h, the

53 I Page Chapter I, Section B reaction mixture was quenched with saturated aqueous Na2S203 (50 mL) and saturated aqueous NaHCOs (50 mL). The organic layer was separated and the aqueous phase was extracted with CH2CI2 (2 x 100 mL). The combined organic extracts were washed with brine (150 mL), dried over Na2S04 and rotary evaporated. The crude product was silica gel chromatographed (eluent: AcOEt/hexane = 1/10) to ftimish 12 as a white solid (17.7 g, 87%). •H NMR (400 MHz., CDCI3) : S 8.55 (d, J - 8.4 Hz, IH), 8.04 (d, J = 7.2 Hz, IH), 7.95 (d, J= 8.3 Hz, IH), 7.85 (d, J- 7.6 Hz, IH), 7.75 (d,J=2.1 Hz, 1H),7.71 (d,J=8.9Hz, IH), 7.64 (dd, J = 8.9, 2.1 Hz, IH), 7.60-7.33 (m, 9H), 6.22 (s, IH), 3.97 (s, 3H). '^C NMR (75 MHz, CDCI3) : d 200.9,160.0,144.3,136.8,136.0,135.4, 133.9, 132.6, 132.3, 130.5, 129.6, 129.5, 129.3, 128.4, 128.3, 127.9, 127.8, 127.6, 126.8, 126.5, 126.4, 125.7, 124.2, 117.1, -57.2, 53.8. IR(KBr) :1684 cm-'. HRMS (ESI) : calcd for C2gH2iBrN02 (M+H)^ 482.0750. Found 482.0738. [a]^'D : (+) 217.9 (c 0.5, CHCI3). M.p. :168°C. HPLC (Chiralpak IC (250 x 4.6mm, 5^), IPA/Hexane 1/10, flow 1.0 mL): XR 5.0 min (major), 6.2 min (minor) 95% ee.

(l/?)-l-(6-Bromo-2-methoxyquinolin-3-yI)-l-(napthaIen-l-yl)-l-phenylpent-4-en-2-oI ll(a&b):

I To a catalytic solution of CuBr.Me2S in THF (50 mL) was added freshly prepared allyl zinc bromide (14.9 g, 80.0 mmol) at room temperature and stirred for 10 min. Compound

54 I P a g e Chapter I, Section B

12 (20.0 g. 40.0 mmol) was then added to it. After 30 min, the reaction was quenched with saturated NH4CI solution (100 mL). The organic layer was separated and the aqueous layer extracted with ethyl acetate (2 x 100 mL). The combined organic extracts were washed with brine, dried over Na2S04 and rotary evaporated. The crude product was silica gel chromatographed (eluent: AcOEt/hexane = 1/19) to give 11 (a & b) as a white solid (19.4 g, 90%) (racemic). IR (KBr, cm"'): 3363; HRMS (ESI) calcd for C3iH27BrN02 [M + H]* 524.1220. Found: 524.1223.

(4/f)-4-(6-Bromo-2-methoxyquinoUn-3-yl)-3-(napthalen-l-yl)-4-phenylbutane-l,3- diol 21 (a & b):

To a solution of 11 (a & b) (14.0 g, 26.0 mmol) in dioxane-water (3:1, 100 mL) were added 2,6-lutidine (6.1 mL, 52.0 mmol), OSO4 (27.2 mL, 0.4 mmol, 0.5% in toluene) and NaI04 (22.8 g, 106.0 mmol). After 2 h, water (20 mL) and CH2CI2 (30 mL) were added. The layers were separated and the aqueous layer was extracted with CH2CI2 (2 x 100 mL). The combined organic extracts were washed with brine, dried over Na2S04 and concentrated under reduced pressure. The crude 20 (a & b) was used for the next reaction without purification. To a stirred solution of 20 (a & b) in MeOH (150 mL) was added NaBH4 (1.2 g, 28.0 mmol) at 0 "C and stirred at room temperature for 2 h. The reaction was quenched with water (50 mL) and methanol was removed under reduced pressure. The aqueous layer was extracted with ethyl acetate (3 x 100 mL). The combined organic layers were washed with brine, dried over Na2S04 and rotary evaporated. The crude product was silica gel chromatographed (eluent: AcOEt/hexane = 1/10) to obtain 21 (a & b) as a white solid (11.2 g, 82% over 2 steps). IR (KBr, cm"'): 3420; HRMS (ESI) calcd for C3oH27BrN03 [M + Hf 528.1169. Found: 528.1177.

55 [Page Chapter I, Section B

(4/f)-4-(6-Bromo-2-methoxyquinolin-3-yI)-3-hydroxyl-3-(napthalen-l-yl)-4- phenyibutylmethane- sulfonate 22 (a & b):

OMs

To a solution of 21 (a & b) (10.6 g, 20.1 mmol) in CH2CI2 (70 mL) were added EtsN (5.4 mL, 40.2 mmol) and methane sulfonyl chloride (2.0 mL, 26.1 mmol) sequentially at 0 °C. After stirring for 3 h at room temperature, water (40 mL) was added to quench the reaction. The reaction mixture was diluted with CH2CI2 (50 mL). The organic layer was separated and the aqueous layer extracted with CH2CI2 (2 x 50 mL). The combined organic extracts were washed with brine, dried over Na2S04 and concentrated under reduced pressure. The crude product was silica gel chromatographed (eluent: AcOEt/hexane - 1/5) to afford 22 (a «& b) as a white solid (10.5 g, 88%). IR (KBr. cm"'): 3432; HRMS (ESI) calcd for C3iH29BrN05S [M + H]^ 606.0944. Found: 606.0955.

(l/?)-l-(6-Bromo-2-methoxyquinolin-3-yI)-4-(dimethyIamino)-2-(napthalen-l-yl)-l- phenylbut- an-2-ol 1 «& 1':

A solution of 22 (a & b) (6.0 g, 10.2 mmol) in Me2NH (200 mL, 8.0 M in THF) was stirred at 45 °C for 24 h. The solution was filtered and the filtrate concentrated under reduced pressure to afford the crude product which on purification by silica gel column chromatography (eluent: ethyl acetate/hexane = 1:5) furnished 1 «& 1' as a white solid (4.8g,90%)(l:l w/w).

56 I P a g e Chapter I, Section B

(l/J,25)-l-(6-bromo-2-methoxyquinolin-3-yl)-4-(dimethylamino)-2-(naphthalen-l- yl)-l-phenylbutan-2-ol 1

'H NMR (300 MHz, CDCI3) 6 8.89 (s, IH), 8.61 (d, J = 8.6 Hz, IH), 7.96 (d, J= 2.0 Hz, IH), 7.92 (d, y = 7.5 Hz, IH), 7.87 (d, J= 8.1 Hz, IH), 7.72 (d, J= 8.8 Hz, IH), 7.68-7.56 (m, 3H), 7.48 (t, J= 7.6 Hz, IH), 7.30 (t, J= 7.7 Hz, IH), 7.17-7.10 (m, 2H), 6.93-6.83 (m, 3H), 5.89 (s, IH), 4.21 (s, 3H), 2.60-2.51 (m, IH), 2.18-2.02 (m, 2H), 1.99 (s, 6H), 1.95-1.85 (m,lH); '^C NMR (75 MHz, CDCI3) : 6 161.3,143.7,141.6,140.5,138.7,134.6, 131.9,129.9, 129.8, 129.7, 128.4, 128.1, 127.8, 127.3, 127.1, 126.8, 125.7, 125.2, 125.1, 125.0, 124.4, 116.9, 82.4, 56.2, 54.1,49.5,44.6,33.4,29.6. IR (KBr) :3441 cm''. HRMS (ESI) : calcd for C32H32BrN202 (M+H)^ 555.1642. Found 555.1671. M.p. : 104 "C. :(-) 165.2 (c 0.8, DMF).

(li?,2/?)-l-(6-bromo-2-methoxyquinoIin-3-yI)-4-(dimethylainino)-2-(naphthalen-l- yI)-l-phenylbutan-2-ol 1'

571 Page Chapter I, Section B

'H NMR (300 MHz, CDCI3) : 5 8.58 (s,lH), 8.48 (d, J = 8.7 Hz, IH), 7.99 (dd, J - 7.4, 0.9 Hz, IH), 7.88 (d, J - 7.3 Hz, 2H), 7.82-7.75 (m, 2H), 7.60-7.50 (m, 2H), 7.47-7.32 (m, 5H), 7.30- 7.21 (m, 2H), 5.73 (s, IH), 3.25 (s, 3H), 2.49 (td, ./- 14.3, 3.0 Hz, lH),2.26(dt,J= 12.2, 3.2 Hz, IH). '^C NMR (75 MHz, CDCI3) : 6 160.4,143.2,141.6,141.1,137.8, 134.6,131.2, 130.0, 129.6, 129.5, 128.1, 128.0, 127.9, 127.6, 127.4, 127.1, 126.5, 126.4, 125.4, 124.9, 124.8, 124.3, 116.2, 81.5, 56.1,52.7,50.9,44.5,34.1,29.6. HRMS (ESI) : calcd for CsaHsaBrNzOz (M+H)* 555.1642. Found 555.1659. M.p. : 145 °C. [af'D : 41.2 (c 0.31, DMF).

(l/?^5)-l-(6-bromo-2-methoxyquinoIin-3-yl)-2-(naphthaIen-l-yl)-l-phenyIpent-4- en-2-oI 11a

The experimental procedure was repeated as of 11 (a <& b) The diastereomers were separated by preparative HPLC (Pheonomenex Luna-C18 (250x10mm, lOjj), ACN/water 9/1, flow 1.5 mL, detection- DAD): tR 10.0 min (syn) and 9.25 min (anti). 'H NMR (300 MHz, CDCI3) : S 8.76-8.60 (m, 2H), 7.97 (d, J = 2.0 Hz, 1H), 7.86 (d, J = 8.1 Hz, IH), 1.15-1.6\ (m, 3H), 7.58 (dd, J= 7.3, 1.1 Hz, IH), 7.49 (t, J - 7.3 Hz, IH), 7.30-7.22 (m,lH), 7.18-7.07 (m, 2H), 6.97-6.86 (m, 3H), 5.97 (br s. IH), 5.17- 4.97 (m, IH), 4.96-4.81 (m, 2H), 4.21 (s, 3H),

58 I Page Chapter I, Section B

3.61 (d, J= 11.5 Hz, IH), 2.66 (s, IH), 2.51-2.37 (m, IH). 13,C NMR (75 MHz, CDCI3) : S 161.0, 143.8, 140.2,139.7, 138.7,134.5, 133.1,132.1, 129.8, 129.7, 128.5, 128.4, 127.5, 126.6, 126.2, 125.5, 124.7,119.7,117.1,54.2,49.6,44.3. IR (KBr) :3363 cm-'. HRMS (ESI) : calcd for C3,H27BrN02 (M+H)"^ 524.1220. Found 524.1223. M.p. : 85 **€. (-) 139.5 (c 0.5, CHCI3).

(35,4/?)-4-(6-bromo-2-methoxyquinolin-3-yl)-3-(naphthalen-l-yI)-4-phenylbutane- 1,3-diol 21a

The experimental procedure was repeated as of 21 (a & b) 'H NMR (300 MHz, CDCI3) : S 8.80 (s, IH), 8.52 (d, J= 7.9 Hz, IH), 7.99 (d, J- 2.0 Hz, IH), 7.88 (d, y= 8.1 Hz, IH), 7.77 (d, J= 7.1 Hz, IH), 7.74-7.56 (m, 4H), 7.54-7.45 (m, IH), 7.31 (t, J- 7.7 Hz, IH), 7.00-7.11 (m, 2H), 6.96-6.86 (m, 3H), 5.89 (br s, IH), 4.48 (br s, IH), 4.18 (s, 3H), 3.56-3.42 (m, IH), 3.19 (t, J = 9.6 Hz, IH), 2.75 (d, J= 14.9 Hz, IH), 2.32-2.17 (m,lH). 13C NMR (75 MHz, CDCI3) : S 161.1, 143.8, 140.2,139.6,138.9, 134.6, 132.1, 131.6, 131.2, 129.9, 128.5, 128.4, 127.4, 126.9, 126.8, 126.7, 126.2, 125.6, 125.2, 125.0, 124.9, 124.7, 117.1, 81.8, 60.7. 54.2, 39.7, 29.7. IR (KBr) 3420 cm

59|Page Chapter I, Section B

HRMS (ESI) : calcd for CaoHayBrNOa (M+H)"^ 528.1169. Found 528.1177. M.p. : 125 ''C. :(-)180(c0.6,CHCl3).

(35,4iR')-4-(6-bromo-2-methoxyquinolin-3-yl)-3-hydroxy-3-(naphthalen-l-yl)-4- phenylbutyl methanesuifonate 22a

OMs The experimental procedure was repeated as of 22 (a i& b) 'H NMR (300 MHz, CDCI3) : 8 8.72 (s, IH), 8.50 (d, J= 7.5 Hz, IH), 8.02 (d, J- 1.7 Hz, IH), 7.92 (d, J= 7.9 Hz, IH), 7.78-7.61 (m, 4H), 7.59-7.49 (m, 2H), 7.32-7.24 (m, IH), 7.01-6.81 (m, 5H), 5.97 (br s, IH), 4.17 (s, 3H), 4.02-3.91 (m, IH), 3.86-3.71 (m, IH), 2.97-2.87 (m, IH), 2.49 (s, 3H), 2.48-2.36 (m,lH). 'X NMR (75 MHz, CDCI3) : 6 160.7,144.0,138.5,138.4,138.0,134.5,132.5,130.0, 129.7, 129.6, 129.1, 128.9, 128.6, 127.7, 126.7, 126.4, 126.2, 125.8, 125.5, 125.2, 124.9, 124.2, 117.4, 79.1, 67.2,54.2, 50.4,38.4,36.6. IR (KBr) : 3432 cm"'. HRMS (ESI) : calcd for CaiHzgBrNOsS (M+H)* 606.0944. Found 606.0955. M.p. : 68 °C. [af'o (-) 155.5 (c 0.5, CHCI3).

60 I P a g e Chapter I, Section B

Diastereomer (l/?^if)-l-(6-bromo-2-methoxyquinolin-3-yl)-2-(naphthaIen-l-yl)-l-phenylpent-4- en-2-olllb

The experimental procedure was repeated as of 11 (a & b) 'H NMR (300 MHz, CDCI3) : 5 8.6-8.46 (m, 2H), 7.82-7.71 (m, 4H), 7.56 (d, J = 7.9 Hz, 2H), 7.49-7.16 (m, 8H), 5.82 (s, IH), 5.17-5.00 (m, IH), 4.98-4.83 (m, 2H), 3.58 (d, 7= 11.5 Hz, IH), 3.36 (s, 3H), 2.68-2.46 (m, 2H). '^C NMR (75 MHz,CDCl3) : 6 160.2,143.3,139.8,139.6,137.5,134.5,133.1, 132.1, 131.6, 131.2, 129.8, 129.7, 129.5, 128.4, 128.2, 128.0, 127.5, 127.0, 126.7, 126.2, 125.1, 124.9, 124.6, 119.7, 116.5,78.5,52.9,51.0,45.4. HRMS (ESI) : calcd for CjiHayBrNOj (M+H)* 524.1220. Found 524.1231. M.p. : 85 °C. [a]% : (-) 27.8 (c 0.5, CHCI3).

(3/?,4/?)-4-(6-bronio-2-methoxyquinolin-3-yl)-3-(naphthaIeii-l-yI)-4-phenylbutane- 13-diol 21b

The experimental procedure was repeated as of 21 (a & b)

61 IPage Chapter I, Section B

'H NMR (300 MHz, CDCI3) : 5 8.49 (s, IH), 8.39 (d, J= 8.3 Hz, IH), 7.91 (d, J= 6.9 Hz, IH), 7.86-7.71 (m, 4H), 7.62-7.48 (m, 2H), 7.48- 7.32 (m, 5H), 7.32-7.19 (m, 2H), 5.71 (s, IH), 4.51 (s, IH), 3.55-3.43 (m, IH), 3.37-3.09 (m, 4H), 2.73 (d, J^ 14.5 Hz, IH), 2.39-2.23 (m, IH), 1.96-1.54 (m, 2H). '^C NMR (300 MHz, CDCI3) : 5 160.2,143.2,140.0,139.9,137.7, 134.4,131.5,131.1, 129.8, 129.5, 128.4, 128.2, 127.9, 126.9, 126.7, 126.6, 126.2, 125.2, 124.9, 124.8, 124.5, 116.4, 80.9, 60.5, 52.8,40.7,29.6. HRMS (ESI) : calcd for CsoHzvBrNOa (M+H)^ 528.1169. Found 528.1153. M.p. :127''C. : 72.0 (c 0.5, CHCI3).

(3/?,4/f)-4-(6-bromo-2-methoxyquinolin-3-yl)-3-hydroxy-3-(naphthalen-l-yI)-4- phenylbutyl methanesulfonate 22b

OMs

The experimental procedure was repeated as of 22 (a & b) "H NMR (300 MHz, CDCI3) : 5 8.46-8.29 (m, 2H), 7.91 (d, J= 5.8 Hz, IH), 7.82-7.64 (m, 4H), 7.63-7.52 (m, 2H), 7.50-7.21 (m, 8H). 5.79 (s, IH), 4.05-3.90 (m, IH), 3.88-3.72 (br s, IH). 3.54-3.29 (m, 3H), 3.20-3.02 (m, IH), 2.88 (s, IH), 2.50 (s, 3H), 2.46-2.32 (m,lH). '^C NMR (75 MHz, CDCI3) : 5 159.9, 143.3, 138.3, 137.7, 137.3, 134.4, 131.8, 130.9, 129.7, 129.6. 129.4, 128.9, 128.7, 128.1. 127.6, 126.1, 126.0, 125.7, 125.0, 124.8, 124.1, 116.7, 78.2, 67.2, 53.1.51.9,40.0,36.6.

62 I P a g e Chapter I, Section B

HRMS (ESI) : calcd for C31 HagBrNOsS (M+H)* 606.0944. Found 606.0954. M.p. : 92 °C. [a]^^D :11.7(c0.5,CHCl3).

63 I P a g e Chapter I, Section B

References

1. Van Gestel, J. F. E.; Guillemont, J. E. G.; Venet, M. G.; Poignet, H. J. J.; Decrane, L. F. B.; Daniel, F. J. V.; Odds, F. C. U.S.A patent US/2005/0148581. 2. Saga, Y.; Motoki, S.; Shimizu, Y.; Kanai, M.; Shibasaki, M. J. Am. Chem. Soc. 2010, 132,7905. 3. (a) Ali, M. M.; Tasneem.; Rajanna, K. C; Sai Prakash, P. K. Synlett, 2001, 2, 251. (b) Meth-Cohn, O.; Narine, B.; Tamowski, B. J. C. S. Perkin Trans 1.1981,1520. 4. Palandoken, H.; Wyatt, J. K.; Hitchcock, S. R.; Olmstead, M. M.; Nantz, M. H. J. Organomet. chem. 1999, 579, 338. 5. Tsuji, R.; Komatsu, K.; Inoue, Y.; Takeuchi, K. J. Org. Chem. 1992,57, 636. 6. Zhang, Y.; Jia, X.; Wang, J.-X.; Eur. J. Org Chem. 2009,2983. 7. Yu, W.; Mei, Y, Kang, Y.; Hua, Z.; Jin, Z.; Org Lett. 2004, 6, 3217. 8. Tam, A.; Raines, R.T. Bioorg Med. Chem. 2009,17,1055.

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