Final NH Report

Chapter-1INTRODUCTION1.1 Gastric Cancer- Symptoms, Causes and epidemiology

Gastric cancer develops from the lining of the stomach. The stomach wall is made up of three layers of tissues, namely themucosal layer (innermost), muscularis (the middle layer), and theserosallayer (outermost).Gastric cancerbegins in thecellslining the mucosal layer and spreads through the outer layers as it grows [1]. Gastric cancer is often asymptomatic or it may show symptoms which are not specific to cancer. The symptoms may include upper abdominal pain, loss of appetite, nausea, heartburn, weight loss, difficulty in swallowing, blood in stool. By the time symptoms occur, the cancer has often reached an advanced stage and may have also metastasized, which is one of the main reasons for its relatively poor prognosis [2]. Globally gastric cancer is the fifth leading cause of cancer and third leading cause of cancer death [3] which renders it as a matter of great concern. The factors that enhance the risk of stomach cancer include chronic stomach inflammation, environmental factors, smoking [4], diet high in salt, preservatives and frozen food, obesity, Helicobacter pylori infection. Ethnicity is also proven to be one of the factors responsible for the risk of gastric cancer. It is less common in the United States and other Western countries than in countries in Asia and South America. It has a high prevalence in developing world. Out of all above mentioned factors, H. pylori infection is the primary cause of stomach cancer [5].

1.2 H. pylori: MorphologyH. pylori is a gram negative, spiral shaped bacterium that infest the stomach or duodenum of the intestines. It is 2 to 4 m in long and 0.5 to 1 m in diameter. It has 2 to 6 unipolar, sheathed flagella approximately 3 m long which often carry a distinctive bulb at the end [6].This flagella acts as locomotive tool and assists the rapid movement of the bacterium in mucus layer overlying gastric epithelial cells [6]. Attributed to the similarity of the DNA base composition, spiral shape and growth requirements of the bacterium with Campylobacter species, it was initially called as Campylobacter pyloridis but further studies showed that its ribosomal RNA, fatty acid composition and ultrastructural appearance differs largely from other Campylobacter species, it was renamed Helicobacter pylori in 1989[7],[8] . It is found only on gastric epithelium where it clusters around the junction between cells. It is not found in blood, and with rare exceptions, has not been found in other parts of the body [9]. H. pylori grows optimally at a neutral pH and mitigates acid exposure as long as urea is available and maintains its cytoplasm at 7.4[10].It is not a true microaerophile as shown by a study that under high cell density or under high partial pressure of CO2 H. pylori can grow in the presence of atmospheric oxygen[11],[12].

1.3 HistoryThe presence of bacteria in human stomach has been known for almost a century. However, the bacteria present in the stomach were thought to be contaminants from digested food rather than true colonizers until the discovery of H. pylori and its role in peptic ulcer disease [13] by Barry & Marshall for which they received Nobel Prize in 2005[14]. It busted the myth that no bacteria can survive the strong acid environment of the stomach. Self- ingestion experiments by Marshall and later studies revealed that these bacteria can colonize and induce inflammation of gastric mucosa[15 ],[16].These initial reports led to further research which showed that gastric colonization with H. pylori can lead to several upper gastrointestinal disorders such as peptic ulcers, chronic gastritis, gastric mucosa associated lymphoid tissue (MALT) lymphoma[17].

1.4 Pathogenesis:Acid Resistance H. pylori bacteria have a unique way to adapt themselves to survive in the extremely acidic environment of the human stomach. Upon encountering the gastric mucosa, it drills inside the gastric mucus propelled by its polar flagella and adheres to the gastric epithelial cells via its adhesins interacting with host cell receptors. Once H. pylori is safely entrenched in the mucus, it is able to fight the stomach acid that does reach it with an enzyme it possesses called urease. Urease converts urea, of which there is an abundant supply in the stomach (from saliva and gastric juices), into bicarbonate and ammonia, which are strong bases. This creates a cloud of acid neutralizing chemicals around the H. pylori, protecting it from the acid in the stomach as shown by the following urea hydrolysis reaction:

Urease enzyme also provides a nitrogen source for protein synthesis [18], [19]. The host bodys immune system responds to H. pylori infection, by sending white blood cells and cytotoxic-T cells. However, these potential H. pylori eradicators cannot reach the bacteria, because they cannot easily get through stomach lining. They do not go away either and the immune response grows and grows. Polymorphs die, and spill their destructive compounds (superoxide radicals) on stomach lining cells. Extra nutrients are sent to strengthen the white cells, and the H. pylori can feed on this [20].

1.5 Diagnosis of H. pylori infectionInvasive and non-invasive are two types of tests available for the detection of H. pylori infection. The choice of test depends upon issues such as cost, availability, clinical situation, population prevalence of infection, pretest probability of infection, and factors such as the use of proton pump inhibitors and antibiotics that may influence certain test results[21],[22,[23]1. Carbon-Isotope Urea Breath test: This test is one of the most important non-invasive test to detect H. pylori infection. It can also be used to check whether the infection is fully treated or not. The person being diagnosed is asked to exhale with the help of straw into two tubes and drink a small amount of water in which 13C urea tablet is dissolved. The exhaling procedure is repeated after some time. If present, H. pylori convert urea into 13CO2, leading to increased concentration of labeled carbon dioxide later measured using an Isotope Ratio Mass Spectrometer (IRMS) [24]. Other than being expensive, it sometimes gives false results due to infection with coccoid form of H. pylori which does not produce much urease[25] .2. Serology test: Serology tests are appropriate where the prevalence of H. pylori infection is greater than 30%.It is based on the quantization of immunoglobulin G antibodies against H. pyloriby the means of an enzyme-linked immunosorbent assay [26]. A negative H. pylori serology test confirms the absence of infection in the majority of cases. It is inexpensive but not a very reliable method as it cannot prove ongoing infection due to immunological memory [27] .

3. Stool Antigen Test: It includes fecal antigen testing by enzyme immunoassay or immunochromatography is one of the simplest and least expensive methods available. It is as simple and noninvasive as serology, can be used regardless of prior testing or treatment, and detects active infection as effectively as urea breath testing with less potential for false negative results while taking acid suppression or bismuth medications[28],[29].The sensitivity and specificity of fecal antigen testing exceed 90%

4. Antibiogram: In geographic areas with a high resistance rate against metronidazole and clarithromycin, culture for antibiotic susceptibility testing seems to be useful[30],[31]

5. Invasive Tests: Histology, culture biopsy and rapid urease tests are considered as gold standard in routine diagnostics. Rapid urease test is Cost-effective .While histology gives provides histologicaldata on inflammation and atrophy, culture biopsy Allows for testing of antimicrobial sensitivity.[27,[25]

1.6 Treatments and their limitationsThere are several therapies which have been used for H. pylori infection mainly a combinatorial therapy of proton pump inhibitor (PPI) and antibiotic. Dual Therapy consists of Clarithromyacin and Omeprazole; triple therapy consists of Clarithromycin, amoxicillin and Omeprezole and quadruple theory which is a combination of bismuth salt, antibiotics and PPI. These therapies have been restricted due to side effects and growing drug resistance. Though multiple regimens have been developed to treat H. pylori infection, any optimal therapeutic regimen hasnt been developed yet. Hence, there is a need to find new drug targets for H. pylori infection.

1.7 IMPDH as a potential drug targetH. pylori cannot synthesize purine nucleotides via de novo pathway hence depends only on the salvage pathway for purine nucleotide biosynthesis[32],[33]George Weber recognized the association of neoplastic progression with a set of pacemaker enzymes and postulated that inhibition of these enzymes can be a promising strategy for cancer chemotherapy[34]. Subsequently, he discovered that Inosine 5-monophosphate dehydrogenase (IMPDH) is amplified in tumors and rapidly proliferating tissues and hence can be addressed as a target for drug design[35]. Guanine nucleotide is a precursor for DNA & RNA, therefore it is essential for the maintenance of cell function and growth. IMPDH catalyses the most significant step of guanosine-5 monophosphate (GMP) biosynthesis and thus regulates the guanine nucleotide pool hence, governing cell proliferation [36]. The IMPDH-catalyzed conversion of IMP to XMP is the first committed and rate-limiting step in guanine nucleotide biosynthesis. XMP is subsequently converted to GMP by the action of GMP synthase (GMPS). IMPDH catalyses two very different chemical transformations: i) a dehydrogenase reaction to form NADH and the covalent intermediate E-XMP* and ii) a hydrolysis reaction which converts E-XMP* to XMP:

Fig. 1

During the dehydrogenase reaction, the catalytic Cys319 attacks C2 of IMP and hydride is transferred to NAD+, producing the covalent intermediate E-XMP*. In this step IMPDH exists in open conformation (that allows NAD to bind). After NADH departs, a mobile flap folds into NAD site forming a closed conformation bringing the catalytic Arg418 to the active site where hydrolysis of the covalent intermediate E-XMP* takes place by conserved Arg418 residue (acting as a general base for the production of XMP). The affinity of NAD+ and the flap are precisely balanced- if the flap binds too tightly, NAD+ will not be able to compete effectively and the dehydrogenase reaction cannot occur. If NAD+ binds too tightly, then the flap will not close and EXMP* cannot hydrolyze[36] .Thus, the flap competes with inhibitors that bind in the NAD site, and this competition is an important determinant of inhibitor potency [37]. The following figure shows the two conformations of IMPDH: an open conformation for redox reaction and closed conformation for hydrolysis of E-XMP*[38] :

Fig. 2Hedstrom et al. reported that the presence of potassium ions accelerates the conformational change in CpIMPDH. This study revealed that in the presence of K+, rate of flap closure increases by 65 fold [39] as K+ mobilizes residues on the Cys319 loop by providing alternative interactions for the main chain carbonyl oxygens. Active Site:The IMP binding site is conserved in all IMPDHs but NAD binding site and the flap are highly divergent. Hence, selective inhibitors can be designed that specifically interact with NAD binding site. [40] Micophenolic acid (MPA) and Mizoribine monophosphate (MZP) have been reported to inhibit C. albicans IMPDH[41]. Micophenolic acid traps E-XMP* by competing with the flap for NDH site[42] while Mizoribine monophosphate binds to the IMP site and induces the flap to close[43]. Thus, the equilibrium between open and closed conformations of IMPDH controls drug sensitivity. Other IMPDH inhibitors such as merimepodib, tiazofurin, and ribavirin are also used in immunosuppressive, cancer, and antiviral therapy. Several reports have shown that Cryptosporidium parvum (a protozoa that causes cryptosporidiosis and several other gastrointestinal disorders) has derived its IMPDH gene from epsilon-proteobacterium (H. pylori) via lateral transfer and adenosine from host for the purine synthesis.[44]Also, C. parvum IMPDH has a 60 % similarity with H. pylori IMPDH and CpIMPDH inhibitors have shown inhibitory activity for H. pylori IMPDH. Based on C. parvum IMPDH inhibitors, H. pylori inhibitor molecules were designed[45],[46].

1.8 In-silico approach: Molecular Docking of potent inhibitors#

Series of benzoxazole and benzimidazole derivatives (based on C91) were designed and docking score is as follows:

Molecule Docking Score

NH- 02 -5.5

NH- 01 -5.1

NH- 03 -4.7

NH- 04 -4.6

NH- 05 -4.2

The Docking images of designed inhibitors showing the interaction between inhibitor molecule and NAD binding site: 2D and 3D docking images of compound NH-02:

Chapter - 2EXPERIMENTAL SECTION

2.1 Scheme 1: Synthesis of N-(3,4-dimethoxyphenyl)-2-(2-(thiazol-4-yl)-1H-benzo[d]imidazol-1-yl)acetamide (compound-5)

The mechanism involves nucleophilic addition-elimination reaction between amine 1 and acyl halide 2 in the first step to yield amide 3. The second step involves nucleophilic substitution of bromine by tiabendazole 4 aided by K2CO3 to yield acetamide derivative 5.

2.1.1 Synthesis of 2-bromo-N-(3,4-dimethoxyphenyl)acetamide (compound-3)

To a solution of compound 1 (1.62g, 0.01 mol) in DCM (5 mL), bromoacetyl chloride 2 was added drop wise at 5o C under N2 atmosphere. The reaction mixture was degassed for 5- 10 minutes. After degassing, the reaction mixture was stirred at room temperature for half an hour. After stirring, DCM was evaporated under reduced pressure from the reaction mixture followed by dilution with water. Pale grey solid precipitate was filtered, washed with water and dried.MS (ESI) : Exact mass of C10H12BrNO3 (compound 3) : 273 Found (M+1) peak : m/z 274

2.1.2 Synthesis of N-(3,4-dimethoxyphenyl)-2-(2-(thiazol-4-yl)-1H-benzo[d]imidazol-1-yl)acetamide (compound-5)

To a solution of tiabendazole 4 (0.2g, 0.001 mol) in DMF (4 mL) stirred under N2 atmosphere, added K2CO3 (0.4 g, 0.003 mol) and allowed to stir at room temperature for five minutes. Compound 3 (0.27g, 0.001 mol) was added I portions and allowed to stir for 6-7 hours under inert atmosphere. After the completion of reaction, crushed ice was added to the resulting mixture and then extracted twice with ethyl acetate. The combined organic layers were washed with brine followed by drying over anhydrous Na2SO4. The solvent was evaporated under reduced pressure. The residue was purified using flash column chromatography (1% MeOH: DCM) to yield compound 5 as pale brown solid.MS (ESI): Exact mass of C20H18N4O5S (compound 5) : 394.11 Found (M+1): m/z 395.021H- NMR: 1H NMR (500 MHz, DMSO-d6) 10.32 (d, J = 4.4 Hz, 1H), 9.36 9.27 (m, 1H), 8.56 (dd, J = 4.5, 2.1 Hz, 1H), 7.73 7.60 (m, 2H), 7.29 (tt, J = 7.0, 3.6 Hz, 3H), 7.02 (ddd, J = 9.1, 4.6, 2.3 Hz, 1H), 6.88 (dd, J = 8.8, 4.3 Hz, 1H), 5.68 (d, J = 4.3 Hz, 2H), 3.69 (dd, J = 14.5, 4.3 Hz, 6H).13C NMR (126 MHz, DMSO) 165.77, 156.04, 155.73, 149.03, 147.56, 147.51, 147.39, 145.33, 142.77,136.94, 132.91, 123.25, 123.07, 122.79, 122.67, 122.24, 119.87, 119.39, 119.22, 112.59, 112.24, 111.32,111.12, 104.59, 79.62, 56.20, 55.76, 48.28.

2.2 Scheme 2: Synthesis of N-(pyridin-3-yl)-2-(2-(thiazol-4-yl)-1H-benzo[d]imidazol-1-yl)acetamide

The mechanism involves nucleophilic addition-elimination reaction between amine 6 and acyl halide 7 in the first step to yield amide 8. The second step involves nucleophilic substitution of bromine by tiabendazole 9 aided by K2CO3 to yield acetamide derivative 10.

2.2.1 Synthesis of 2-bromo-N-(pyridin-3-yl)acetamide compound 8)

To a solution of compound 6 (1g, 0.001 mol) in DCM (3 mL), bromoacetyl chloride 7 was added drop wise at 5o C under N2 atmosphere. The reaction mixture was degassed for 5- 10 minutes. After degassing, the reaction mixture was stirred at room temperature for half an hour. After stirring, DCM was evaporated under reduced pressure from the reaction mixture followed by dilution with water. Extraction was done using ethyl acetate. The combined organic layers were dried over sodium sulfate and the solvent was evaporated using reduced pressure. The residue was purified using flash column chromatography and compound 8 was obtained.MS (ESI): Exact mass of C7H2BrN2O (compound 8): 213.97 Found (M+1) peak: 214.92

2.2.2 Synthesis of N-(pyridin-3-yl)-2-(2-(thiazol-4-yl)-1H-benzo[d]imidazol-1-yl)acetamide(compound 10)

To a solution of tiabendazole 9 (0.1g, 0.0005 mol) in DMF (3 mL) stirred under N2 atmosphere, added K2CO3 (0.4 g, 0.003 mol) and allowed to stir at room temperature for five minutes. Compound 8 (0.13g, 0.00059 mol) was added in portions and allowed to stir for 6-7 hours under inert atmosphere. After the completion of reaction, crushed ice was added to the resulting mixture and then extraction was done twice with ethyl acetate. The combined organic layers were washed with brine followed by drying over anhydrous Na2SO4. The solvent was evaporated under reduced pressure. The residue was purified using flash column chromatography (4%MeOH: DCM) to yield compound 10.MS (ESI): Exact Mass of C17H13N5OS (compound 10) = 335.08 Found (M+1) peak: 336.041H-NMR: 1H NMR (500 MHz, DMSO-d6) 10.69 (s, 1H), 9.31 (d, J = 2.0 Hz, 1H), 8.73 (d, J = 2.5 Hz, 1H), 8.57 (d, J = 2.1 Hz, 1H), 8.27 (d, J = 4.6 Hz, 1H), 7.99 (d, J = 8.2 Hz, 1H), 7.77 7.63 (m, 2H), 7.32 (dt, J = 23.5, 5.1 Hz, 3H), 5.74 (s, 2H).13C NMR (126 MHz, DMSO) 165.61, 146.32, 142.58, 138.35, 134.93, 133.41, 131.06, 129.64, 129.62,129.47, 129.02, 128.34, 128.02, 127.96, 127.65, 127.58, 125.91, 106.91, 76.29, 76.18, 75.36, 73.75, 70.59,70.42, 67.66, 58.23.

2.3 Scheme 3: Synthesis of 2-(naphthalen-2-yloxy)-N-(2-(pyridin-4-yl)benzo[d]oxazol-5-yl)acetamide

2.3.1 Synthesis of 5-nitro-2-(pyridin-4-yl)benzo[d]oxazole compound 13)

In a sealed round bottomed flask, polyphosphoric acid (5.7 mL, 0.12 mol) was added followed by aminoalcohol 11 (0.5 g, 0.003 mol) and stirred for 10 minutes and temperature was maintained 110o C. When the mixture became stirrable (due to high density of PPA), isonicotinic acid 12 (0.395 g, 0.003 mol) was added to it and the reaction mixture was allowed to stir for 8 hours at 110o C. The reaction mixture was allowed to cool and neutralized with sodium bicarbonate followed by extraction with ethyl acetate. The combined extracted organic layers were dried over sodium sulfate. The solvent was evaporated to give the residue, which was purified by flash column chromatography (90% Ethyl Acetate: to afford orange colored compound 13.MS(ESI) : Exact mass of C12H7N3O3 (compound 13) :241.05 Found (M+1) peak: 242.011H NMR : (500 MHz, Chloroform-d) 8.89 (s, 2H), 8.74 (d, J = 2.4 Hz, 1H), 8.41 (dd, J = 9.0, 2.4 Hz, 1H), 8.12 (d, J = 4.8 Hz, 2H), 7.76 (d, J = 9.0 Hz, 1H).

2.3.2 Synthesis of 2-(pyridin-4-yl)benzo[d]oxazol-5-amine (compound 14)

In a round bottom flask 5 mL of ethanol was taken, added benzoxazole 13 (0.09 g, 0.0003 mol) and SnCl2 (0.417g, 0.0018 mol). The reaction mixture was allowed to stir for 1 hour and 30 minutes at room temperature under N2 atmosphere. After the completion of reaction, ethanol was evaporated and the residue was washed with water to remove tin salts. Purification was done by column chromatography and the compound 14 was obtained at 3% MeOH: DCM. MS(ESI) : Exact mass of C12H9N3O (compound 14) : 211.07 Found (M+1) peak: 212.091H-NMR: 1H NMR (500 MHz, Chloroform-d) 8.80 (d, J = 5.1 Hz, 2H), 8.04 (d, J = 5.1 Hz, 2H), 7.40 (d, J = 8.6 Hz, 1H), 7.07 (s, 1H), 6.79 (d, J = 8.6 Hz, 1H), 3.91 3.66 (m, 2H).

2.3.3 Synthesis of 2-bromo-N-(2-(pyridin-4-yl)benzo[d]oxazol-5-yl)acetamide (compound 16)

To a stirred solution of compound 14 (0.038 g, 0.00018 mol) in DCM (1 mL), added K2CO3 (0.074 g, 0.00054) and stirred for 10 minutes at room temperature under N2 atmosphere. After degassing, bromoacetyl chloride 15 (0.0425 g, 0.00027 mol) was added drop wise to the reaction mixture at 5oC. The reaction mixture was allowed to stir for 30 minutes at room temperature. After the completion of reaction, DCM was evaporated under reduced pressure from the reaction mixture followed by dilution with water. The residue was filtered, thoroughly washed with water and dried to afford compound 16.MS (ESI): Exact mass of C14H10BrN3O2 compound 16 = 331.0 Found (M+2) peak: 331.9

2.3.4 Synthesis of 2-(naphthalen-2-yloxy)-N-(2-(pyridin-4-yl)benzo[d]oxazol-5-yl)acetamide (compound 18)

In a round bottom flask, 2 mL DMF, K2CO3 (0.066 g, 0.00048 mol) and 2- naphthol 17 (0.023g, 0.00016 mol) were stirred for 10 minutes under N2 atmosphere at room temperature. Compound 16 (0.053g, 0.00016mol) was then added and the reaction mixture was allowed to stir for 8 hours. Crushed ice was added to reaction mixture followed by extraction in ethyl acetate. The compound was purified using flash column chromatography to afford compound 18 at 50% Ethyl acetate: Hexane.MS (ESI) : Exact mass of the compound 18 : 395.13 Found (M+1) peak: 396.11

2.4 Scheme 4: Synthesis of 2-(2-(pyridin-2-yl)-1H-benzo[d]imidazol-1-yl)-N-(pyridin-3-yl)acetamide (compound 20)

2.4.1 Synthesis of 2-(2-(pyridin-2-yl)-1H-benzo[d]imidazol-1-yl)-N-(pyridin-3-yl)acetamide (compound 20)

To a solution of compound 9 (0.050g, 0.00025 mol) in DMF (1 mL) stirred under N2 atmosphere, added K2CO3 (0.1 g, 0.00075 mol) and allowed to stir at room temperature for five minutes. Compound 8 (0.055g, 0.00025 mol) was added in portions and allowed to stir for 6-7 hours under inert atmosphere. After the completion of reaction, crushed ice was added to the resulting mixture and then extraction was done twice with ethyl acetate. The combined organic layers were washed with brine followed by drying over anhydrous Na2SO4. The solvent was evaporated under reduced pressure. The residue was purified using flash column chromatography (4%MeOH: DCM) to yield compound 20.MS (ESI): Exact mass of C19H15N5O (compound 20) = 329.13 Found (M+1) peak: 330.081H-NMR: 1H NMR (500 MHz, Chloroform-d) 10.68 (s, 1H), 8.71 (d, J = 2.5 Hz, 1H), 8.64 8.59 (m, 1H), 8.43 8.37 (m, 1H), 8.26 (dd, J = 4.7, 1.5 Hz, 1H), 8.04 7.93 (m, 2H), 7.78 7.75 (m, 1H), 7.72 (s, 1H), 7.50 7.45 (m, 1H), 7.37 7.28 (m, 3H).13C-NMR (126 MHz, DMSO) : 167.38, 150.28, 149.83, 149.00, 144.78, 142.38, 141.19, 137.96, 137.89, 136.04, 126.57, 124.73, 124.21, 124.19, 123.82, 122.96, 119.95, 111.31, 49.21.

Results and discussion:Designed potent inhibitors molecules benzoxazole and benzimidazole derivatives NH-01, NH-02,NH-03,NH-05 were synthesized and purified using Flash Column Chromatography. The compounds were characterized using High Resolution Mass Spectroscopy, 1H-NMR and 13C- NMR spectroscopy. The designed molecules were docked to HpIMPDH using Maestro Glide. These potent inhibitors of H. pylori IMPDH will further be investigated under biological screening and inhibition activity. Also, they will be used for crystallization studies of IMPDH inhibitor binding.

SUPPORTING INFORMATION:

1. Mass Spectra of compound 3


3. 1H- NMR of compound 5

s




7. Mass spectra of compound 13

242.01242.01



10. 1H-NMR of compound 14






16. 13C spectra of compound 5



ACKNOWLEDGEMENTS

I would like to express my deep gratitude to my project supervisor Dr. Sivapriya Kirubakaran for her guidance and motivation throughout the course of training. I would also like to thank Dr. Kapil Juvale and Mr. Althaf Shaik for helping me learn the synthetic skills and Dr. Vijay Singh for making me familiar with the basics of molecular modeling and performing docking studies for the inhibitor molecules. I would like to thank all other members of the research group who have been of great help in some or the other way.

REFERENCES:

1. "Gastric Cancer Treatment." National Cancer Institute. N.p., n.d. Web. 27 Apr. 2015.2. "Statistics and Outlook for Stomach Cancer." Statistics and Outlook for Stomach Cancer. N.p., n.d. Web. 27 Apr. 2015.3. "Chapter 1.1".World Cancer Report 2014. World Health Organization. 2014.4. Kurata, J. H., & Nogawa, A. N. (1997). Meta-analysis of risk factors for peptic ulcer: nonsteroidal antiinflammatory drugs, Helicobacter pylori, and smoking.Journal of clinical gastroenterology,24(1), 2-17.5. Graham, D. Y. (1999). Helicobacter pylori infection is the primary cause of gastric cancer.Journal of gastroenterology,35, 90-97.6. Paul, W. O., Lane, M. C., & Porwollik, S. (2000). Helicobacter pylori motility.Microbes and infection,2(10), 1207-1214.7. GOODWIN, C. S., ARMSTRONG, J. A., CHILVERS, T., PETERS, M., COLLINS, M. D., SLY, L & HARPER, W. E. (1989). Transfer of Campylobacterpylori and Campylobactermustelae to Helicobacter gen. nov. as Helicobacter pylori comb. nov. and Helicobacter mustelae comb. nov., respectively.International Journal of Systematic Bacteriology,39(4), 397-405.8. Cover, T. L., & Blaser, M. J. (1992). Helicobacter pylori and gastroduodenal disease.Annual review of medicine,43(1), 135-145.9. Peterson, W. L. (1991). Helicobacter pylori and peptic ulcer disease.New England journal of medicine,324(15), 1043-104810. Stingl, K., & De Reuse, H. (2005). Staying alive overdosed: how does Helicobacter pylori control urease activity?.International journal of medical microbiology,295(5), 307-315.11. BuryMon, S., Kaakoush, N. O., Asencio, C., Mgraud, F., Thibonnier, M., De Reuse, H., & Mendz, G. L. (2006). Is Helicobacter pylori a true microaerophile?.Helicobacter,11(4), 296-303.12. Park, S. A., Ko, A., & Lee, N. G. (2011). Stimulation of growth of the human gastric pathogen Helicobacter pylori by atmospheric level of oxygen under high carbon dioxide tension.BMC microbiology,11(1), 96.13. Warren JR, Marshall BJ. Unidentified curved bacilli on gastric epithelium in active chronic gastritis. Lancet. 1983;1:1273-127514. Hellstrm, P. M. (2006). This years Nobel Prize to gastroenterology: Robin Warren and Barry Marshall awarded for their discovery of Helicobacter pylori as pathogen in the gastrointestinal tract.World journal of gastroenterology: WJG,12(19), 312615. Marshall, B. J., Armstrong, J. A., McGechie, D. B., & Glancy, R. J. (1985). Attempt to fulfil Koch's postulates for pyloric Campylobacter.The Medical Journal of Australia,142(8), 436-439.16. Morris, A. J., Ali, M. R., Nicholson, G. I., Perez-Perez, G. I., & Blaser, M. J. (1991). Long-term follow-up of voluntary ingestion of Helicobacter pylori.Annals of internal medicine,114(8), 662-663. 17. Ernst, P. B., & Gold, B. D. (2000). The disease spectrum of Helicobacter pylori: the immunopathogenesis of gastroduodenal ulcer and gastric cancer.Annual Reviews in Microbiology,54(1), 615-640.18. Peterson, W. L. (1991). Helicobacter pylori and peptic ulcer disease.New England journal of medicine,324(15), 1043-1048.19. McGee, D. J., & Mobley, H. L. T. (1999). Mechanisms of Helicobacter pylori infection: bacterial factors. InGastroduodenal Disease and Helicobacter pylori(pp. 155-180). Springer Berlin Heidelberg.20. Mendz, G. L., & Hazell, S. L. (1995). Aminoacid utilization by Helicobacter pylori.The international journal of biochemistry & cell biology,27(10), 1085-1093.21. Mons, J., Gisbert, J. P., Borda, F., Domnguez-Muoz, E., & Grupo Conferencia Espaola de Consenso sobre Helicobacter pylori. (2005). Indications, diagnostic tests and Helicobacter pylori eradication therapy. Recommendations by the 2nd Spanish Consensus Conference.Rev Esp Enferm Dig,97, 348-7422. Cutler, A. F., Havstad, S., Ma, C. K., Blaser, M. J., Perez-Perez, G. I., & Schubert, T. T. (1995). Accuracy of invasive and noninvasive tests to diagnose Helicobacter pylori infection.Gastroenterology,109(1), 136-141.23. Vaira, D., Malfertheiner, P., Mgraud, F., Axon, A. T., Deltenre, M., Hirschl, A. M., ... & HpSA European Study Group. (1999). Diagnosis of Helicobacter pylori infection with a new non-in vasive antigen-based assay.The Lancet,354(9172), 30-3324. Savarino, V., Vigneri, S., & Celle, G. (1999). The 13C urea breath test in the diagnosis ofHelicobacter pylori infection.Gut,45(suppl 1), I18-I22.25. Bazzoli F, et. al. Urea breath tests for the detection of Helicobacter pylori infection. Helicobacter 1997; 2 Suppl: S34-7.26. She, R. C., Wilson, A. R., & Litwin, C. M. (2009). Evaluation of Helicobacter pylori immunoglobulin G (IgG), IgA, and IgM serologic testing compared to stool antigen testing.Clinical and Vaccine Immunology,16(8), 1253-125527. Kusters, J. G., van Vliet, A. H., & Kuipers, E. J. (2006). Pathogenesis of Helicobacter pylori infection.Clinical microbiology reviews,19(3), 449-490.28. Ni, Y. H., Lin, J. T., Huang, S. F., Yang, J. C., & Chang, M. H. (2000). Accurate diagnosis Helicobacter pylori infection by stool antigen test and 6 other currently available tests in children.The Journal of pediatrics,136(6), 823-827.29. Horiki N, Omata F, Uemura M, et al. Annual change of primary resistance to clarithromycin among Helicobacter pylori isolates from 1996 through 2008 in Japan.Helicobacter. Oct 2009;14(5):86-9030. Fallone CA. Epidemiology of the antibiotic resistance of Helicobacter pylori in Canada.Can J Gastroenterol. Nov 2000;14(10):879-8231. Cutler, A. F., Havstad, S., Ma, C. K., Blaser, M. J., Perez-Perez, G. I., & Schubert, T. T. (1995). Accuracy of invasive and noninvasive tests to diagnose Helicobacter pylori infection.Gastroenterology,109(1), 136-141.32. Liechti, G., & Goldberg, J. B. (2012). Helicobacter pylori relies primarily on the purine salvage pathway for purine nucleotide biosynthesis.Journal of bacteriology,194(4), 839-85433. Mendz, G. L., A. J. Shepley, S. L. Hazell, and M. A. Smith. 1997. Purine metabolism and the microaerophily of Helicobacter pylori. Archives of Microbiology 168:448-456.34. Hedstrom, Lizbeth. IMP Dehydrogenase: Structure, Mechanism and Inhibition. Chemical Reviews 109, no. 7 (July 2009): 290328. doi:10.1021/cr900021w.35. Jackson, R. C., Weber, G., & MORRIS, H. P. (1975). IMP dehydrogenase, an enzyme linked with proliferation and malignancy.36. Hedstrom, L., G. Liechti, J. B. Goldberg, and D. R. Gollapalli. The Antibiotic Potential of Prokaryotic IMPDehydrogenase Inhibitors. Current Medicinal Chemistry 18, no. 13 (2011): 19091837. Gollapalli, D. R., MacPherson, I. S., Liechti, G., Gorla, S. K., Goldberg, J. B., & Hedstrom, L. (2010). Structural determinants of inhibitor selectivity in prokaryotic IMP dehydrogenases.Chemistry & biology,17(10), 1084-1091.38. Hedstrom, L., & Gan, L. (2006). IMP dehydrogenase: structural schizophrenia and an unusual base.Current opinion in chemical biology,10(5), 520-525.39. Riera, T. V., Zheng, L., Josephine, H. R., Min, D., Yang, W., & Hedstrom, L. (2011). Allosteric activation via kinetic control: potassium accelerates a conformational change in IMP dehydrogenase.Biochemistry,50(39), 8508-851840. Khler, G. A., Gong, X., Bentink, S., Theiss, S., Pagani, G. M., Agabian, N., & Hedstrom, L. (2005). The functional basis of mycophenolic acid resistance in Candida albicans IMP dehydrogenase.Journal of Biological Chemistry,280(12), 11295-11302.41. Khler, G. A., Gong, X., Bentink, S., Theiss, S., Pagani, G. M., Agabian, N., & Hedstrom, L. (2005). The functional basis of mycophenolic acid resistance in Candida albicans IMP dehydrogenase.Journal of Biological Chemistry,280(12), 11295-11302.42. Link, J. O., & Straub, K. (1996). Trapping of an IMP dehydrogenase-substrate covalent intermediate by mycophenolic acid.Journal of the American Chemical Society,118(8), 2091-2092.43. Gan, L., Seyedsayamdost, M. R., Shuto, S., Matsuda, A., Petsko, G. A., & Hedstrom, L. (2003). The immunosuppressive agent mizoribine monophosphate forms a transition state analogue complex with inosine monophosphate dehydrogenase.Biochemistry, 42(4), 857-863.44. Umejiego, N.; N. et al. Cryptosporidium parvum IMP Dehydrogenase. J. Biol. Chem. 2004, 279, 40320 40327. 45. Kirubakaran, S. et al. The Structural Basis of Cryptosporidium-Specific IMP Dehydrogenase Inhibitor Selectivity. J. Am. Chem. Soc. 2010, 132, 1230 1231.46. Kirubakaran, S. et al. Structure-activity relationship study of selective benzimidazolebased inhibitors of Cryptosporidium parvum IMPDH. Bioorg Med Chem Lett. 2012, 22(5), 1985 198847. # Unpublished Results

1

41

9 8 7 6 5 4 3 2 1 0 ppm

3.80

4

6.78

16.

796

7.07

27.

391

7.40

7

8.04

7

8.79

9

1.80

1.00

1.01

1.15

2.07

2.06

PC 1.00GB 0LB 0.30 HzSSB 0WDW EMSF 500.0900143 MHzSI 65536F2 Processing parameters

PLW1 17.00000000 WP1 12.15 usecNUC1 1HSFO1 500.0930883 MHz======== CHANNEL f1 ========

TD0 1D1 1.00000000 secTE 297.2 KDE 6.50 usecDW 50.000 usecRG 200.08AQ 3.2767999 secFIDRES 0.152588 HzSWH 10000.000 HzDS 2NS 32SOLVENT CDCl3TD 65536PULPROG zg30PROBHD 5 mm PABBO BB/INSTRUM spectTime 12.21Date_ 20150409F2 Acquisition Parameters

PROCNO 1EXPNO 1100NAME nishaCurrent Data Parameters

benzox redn

-100102030405060708090100110120130140150160170180190200210f1 (ppm)

0

500

1000

1500

2000

2500

3000

3500

4000

4500

nisha.1094.fiddimethoxy tbz

55.7

656.2

0

79.6

2

104.

5911

1.12

111.

3211

2.59

119.

3911

9.871

22.6

712

2.79

123.

25

132.

9113

6.94

142.

7714

5.33

147.5

614

9.0315

5.73

165.

77

Parameter Value

1 Data File Name G:/ Work/ NMR data/nisha/ 1094/ fid

2 Title nisha.1094.fid3 Comment dimethoxy tbz4 Origin Bruker BioSpin GmbH5 Owner IITG_Chemistry6 Site

7 Spectrometer spect8 Author

9 Solvent DMSO10 Temperature 299.011 Pulse Sequence zgpg3012 Experiment 1D13 Probe 5 mm PABBO BB/ 19F-1H/

D Z-GRD Z119467/ 000514 Number of Scans 800015 Receiver Gain 11316 Relaxation Delay 2.000017 Pulse Width 8.900018 Presaturation

Frequency19 Acquisition Time 1.101020 Acquisition Date 2015-04-09T09:04:0021 Modification Date 2015-04-09T09:34:3922 Class

23 SpectrometerFrequency

125.76

24 Spectral Width 29761.925 Lowest

Frequency-2307.3

26 Nucleus 13C27 Acquired Size 3276828 Spectral Size 65536

-100102030405060708090100110120130140150160170180190200210f1 (ppm)

-1000

0

1000

2000

3000

4000

5000

6000

7000

8000

9000

10000

11000

12000

13000

14000

15000

16000

17000nisha.111.fidNH 1 58

.23

67.6

670

.42

70.5

973

.75

75.3

676

.18

76.2

9

106.

91

125.

9112

7.58

127.6

512

7.96

128.

0212

8.34

129.

0212

9.47

129.

6212

9.64

131.

0613

3.41

134.

9313

8.35

142.

5814

6.32

165.

61

13C NMR (126 MHz, DMSO) 165.61, 146.32, 142.58, 138.35, 134.93, 133.41, 131.06, 129.64, 129.62,129.47, 129.02, 128.34, 128.02, 127.96, 127.65, 127.58, 125.91, 106.91, 76.29, 76.18, 75.36, 73.75, 70.59,70.42, 67.66, 58.23.

-100102030405060708090100110120130140150160170180190200210f1 (ppm)

0

500

1000

1500

2000

2500

3000

3500

4000

nisha.121.fidp pyridine

49.2

1111.

31119

.95

122.

9612

3.82

124.

2112

4.73

136.

0413

7.89

137.9

614

1.19

142.

3814

4.78

149.

0014

9.83

150.

28

167.3

8

13C NMR (126 MHz, DMSO) 167.38, 150.28, 149.83, 149.00, 144.78, 142.38, 141.19, 137.96, 137.89,136.04, 126.57, 124.73, 124.21, 124.19, 123.82, 122.96, 119.95, 111.31, 49.21.

-100102030405060708090100110120130140150160170180190200210f1 (ppm)

0

500

1000

1500

2000

2500

3000

3500

4000

nisha.121.fidp pyridine

49.2

1111.

31119

.95

122.

9612

3.82

124.

2112

4.73

136.

0413

7.89

137.9

614

1.19

142.

3814

4.78

149.

0014

9.83

150.

28

167.3

8

13C NMR (126 MHz, DMSO) 167.38, 150.28, 149.83, 149.00, 144.78, 142.38, 141.19, 137.96, 137.89,136.04, 126.57, 124.73, 124.21, 124.19, 123.82, 122.96, 119.95, 111.31, 49.21.

Documents

Final NH Report