Some compounds added to urea or urea-containing fertilizers can reduce the rate of the first “hydrolysis” step, and slow the rate of ammonia production. Under certain conditions, this can help reduce ammonia loss to the atmosphere Urea is the most widely used form of N fertilizer, and can be formulated as dry granules, prills, or as a fluid alone or mixed with ammonium nitrate (UAN). Urea is also present in animal manures. All these forms of urea have the disadvantage of undergoing considerable losses as ammonia gas if not incorporated into soil soon after application. Once dissolved in water, urea is converted to ammonium bicarbonate within a few days following application by the naturally occurring enzyme, urease. Urease is produced by many soil microorganisms and plants, and is present in nearly all soils. When urea is hydrolyzed by urease, much of the resulting ammonium is held on soil cation exchange sites. During the conversion, the pH temporarily rises and ammonia gas is produced. The loss of ammonia, termed volatilization, can be from nil to over 50% The factors conducive to N loss as ammonia from urea are: surface application, less than 10 mm (0.4 in.) of rainfall and/or irrigation in the first few days after application, presence of crop residues, open crop canopies, high temperatures, high soil pH and low cation exchange capacity soils. Moving the applied urea below the soil surface with tillage or through rainfall and irrigation also effectively minimizes ammonia loss from urea. Reducing Urease Activity Urease inhibitors are used to temporarily reduce the activity of the enzyme and slow the rate at which urea is hydrolyzed. There are many compounds that can inhibit urease, but only a few that are non-toxic, effective at low concentrations, chemically stable and able to be mixed with or coated onto urea-containing fertilizers. The most widely used urease inhibitor is N-(n-Butyl) triphosphoric triamide (NBTPT), which converts to active NBPT (N-(n-Butyl) phosphoric triamide). Other widely studied urease inhibitors include phenylphosphorodiamidate (PPD/PPDA) and hydroquinone. Ammonium thiosulfate and some metals can also inhibit urea hydrolysis. There are many other organic compounds, especially structural analogues of urea, capable of inhibiting urease. Management Practices Urease inhibitors are potentially useful tools for controlling or reducing gaseous losses of ammonia following fertilization with urea. They can restrict urea hydrolysis for up to 7 to 14 days, after which rain, irrigation, or soil mixing would be required to further restrict ammonia losses. Because the magnitude of ammonia loss varies with soil type, climate and crop cover, the reduction due to the use of a urease inhibitor can also be variable. Research suggests NBTPT-treated urea use can reduce ammonia loss by 50% to 90% when compared to untreated urea.

1. 1
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2. Title:
Synthesis of novel N-phenyl-2-((5-(((2,4,6-trioxo tetrahydro pyrimidine-
5(2H)-ylidine)methyl)amino-1,3,4-thiadiazol-2-yl)thio)acetamid derivatives
as novel urease inhibitors
Supervisors:
Dr. Massoud Amanlou
Dr. Mahmoud Biglar
By:
Amin Shirini
2
Sep 2023 3/14/2024

3. Introduction
3
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4. etiologic agent
 H. pylori is a gram-negative bacillus .
 It lives in gastric mucus.
 its distribution is never systemic.
 Its spiral shape and flagella render H. pylori motile in the mucus environment.
 The organism has several acid-resistance mechanisms, most notably a highly expressed
urease that catalyzes urea hydrolysis to produce buffering ammonia.
 H. pylori is microaerophilic (requiring low levels of oxygen), is slow-growing.
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5. epidemiology
 The prevalence of H. pylori among adults is ∼30% in the United States and other developed
countries.
 In developing nations, where the majority of children are infected before the age of 10, the
prevalence in adults peaks at more than 80 percent before age 50.
 Humans are the only important reservoir of H. pylori.
 The risk of acquiring H. pylori infection is related to socioeconomic status and living
conditions early in life. Factors such as density of housing, overcrowding, number of
siblings, sharing a bed, and lack of running water have all been linked to a higher
acquisition rate of H. pylori infection.
 Children may acquire the organism from their parents(more often from the mother) or
from other children.
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6. Diagnosis
Tests for the presence of H. pylori can be divided into two groups:
 Invasive tests, which require EGD and are based on the analysis of gastric biopsy
specimens
 noninvasive tests .
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7.  Invasive tests:
 The presence of H. pylori urease leads to a pH alteration and therefore to a color change,
which often occurs within minutes but can require up to 24 h.
 Histologic examination of biopsy specimens for H. pylori also is accurate, provided that a
special stain (e.g., a modified Giemsa or silver stain) permitting optimal visualization of
the organism is used.
 Microbiologic culture is most specific but may be insensitive because of difficulty with H.
pylori isolation.
 Non-Invasive tests:
 Urea breath test (the most consistently accurate test ):
 The patient drinks a solution of urea labeled with the nonradioactive isotope 13C and
then blows into a tube. If H. pylori urease is present, the urea is hydrolyzed and labeled
carbon dioxide is detected in breath samples.
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8. pathophysiology of h.pylori
 severe inflammation and tissue damage in the stomach.
 Cag A also activates signaling pathways in host cells, leading to the release of pro-inflammatory cytokines
and chemokines, further enhancing the inflammatory response.
 The VacA toxin secreted by Helicobacter pylori enhances the ability of the bacteria to colonize the stomach
and contributes to the pathogenesis of gastric adenocarcinoma and peptic ulcer disease.
 The combination of Vac A and Cag A contributes to the development of peptic ulcers and gastric cancer.
 H. pylori also has the ability to modulate the host immune response by suppressing certain immune cells,
such as T cells, and promoting the expansion of regulatory T cells, which can dampen the immune response.
 Urease enzymes in colonization of the host by neutralizing gastric acid and providing Nitrogen for bacterial
protein synthesis.
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9. 9
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10. 10
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11. Treatment protocols:
 PPI based triple therapy including a PPI + Clarithromycin + Amoxicillin / Metronidazole
 Bismuth based quadruple therapy including a PPI + Bismuth + Tetracycline +
Metronidazole
 Sequential therapy
 Levofloxacin based triple therapy including Levofloxacin + Amoxicillin + PPI
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12. Drug resistance and new treatment methods
 Targeting bacteria's most important tools for survival:
 flagellum
 binding proteins
 Enzymes such as urease: as the most important enzyme in bacterial survival and its
targeting
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13. 13 Urease inhibitors:
mechanism of action
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14. Urease active site and
mechanism of action
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15. Urease enzyme inhibitor classification
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16. Reasearch classsification: Urease inhibitors
1,2,3‐triazole–(thio)barbituric acid and barbituric-phenoxy-N-phenylacetamide hybrids and 5-Aminomethylene Barbituric derivatives
1. Asgari MS, Azizian H, Nazari Montazer M, Mohammadi‐Khanaposhtani M,
Asadi M, Sepehri S, Ranjbar PR, Rahimi R, Biglar M, Larijani B, Amanlou M.
Archiv der Pharmazie. 2020 Sep;353(9):2000023.
2. Sedaghati S, Azizian H, Montazer MN, Mohammadi-Khanaposhtani M, Asadi M,
Moradkhani F, Ardestani MS, Asgari MS, Yahya-Meymandi A, Biglar M, Larijani
B. Structural Chemistry. 2020 Aug 22:1-2.
3. Asadi M, Mahdavi M, Mahernia S, Rezaei Z, Safavi M, Saeedi M, Amanlou M.
Letters in Drug Design & Discovery. 2018 Apr 1;15(4):428-36.
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17. Design:
Hybrid Structure
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18. Experimental
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19. Synthesis:
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20. Step 1:
 Alfa chloroacetamide derivatives Synthesis
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21. Step 2:
 Step II: 2-amino-5-mercaptoalkyl thiadiazol derivatives
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22. Step 3:
 Ethoxy methylene barbiturate Synthesis
22
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23. Step 4:
 Synthesis of final products
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24. Step 1 mechanism:
24
Amidation
reaction
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25. Step 2 Mechanism
25
SN2
reaction
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26. Step 3 Mechanism:
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27. Step 4 Mechanism:
27
SN2
reaction
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28. Yields of product synthesis:
28
Entry
Compound R M.p./°C Yield/%a
1 9a Phenyl 194-196 74
2 9b 2-methoxyphenyl 201-203 70
3 9c 4-methoxyphenyl 208 – 210 74
4 9d 3,4-dimethoxyphenyl 210 – 212 75
5 9e 3-chlorophenyl 222-224 68
6 9f 4-chlorophenyl 216 – 218 74
7 9g 4-Chloro benzyl 203 – 205 75
8 9h 2,5-dichlorophenyl 191 – 193 60
9 9i 4-bromophenyl 218-220 59
10 9j 2-fluoro-4-nitrophenyl 181 – 183 70
11 9k 5-chloropyridin-2-yl 182-184 75
12 9l 5-methylpyridin-2-yl 191-193 68
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29. Result & discussion
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30. 30
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IR
Spectroscopy:
NH
CH-
Aromatic
CH-
Aliphatic
C=O
amide

31. 31
1H-NMR- compound 9g
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32. 32
13C-NMR- compound 9g
8(C-Aromatic)
3(C=O)
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33. Mass Spectroscopy
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MW= 452 m/z

34. In-Silico Study: Compound 9g Docking
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35. Pharmacologic Test
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36. 36
Urease inhibitory
result
Compound
Number
R IC50
[a](ΜM)
9a Phenyl 14.8±1.18
9b 2-methoxyphenyl 23.75±0.91
9c 4-methoxyphenyl 36.6±1.50
9d 3,4-dimethoxyphenyl 51.79±1.40
9e 3-chlorophenyl 10.02±1.10
9f 4-chlorophenyl 2.89±1.07
9g 4-Chloro benzyl 1.3±1.10
9h 2,5-dichlorophenyl 18.3±1.20
9i 4-bromophenyl 15.31±1.12
9j 2-fluoro-4-nitrophenyl 4.6±0.08
9k 5-chloropyridin-2-yl 3.89±1.07
9l 5-methylpyridin-2-yl 6.23 ±1.16
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37. 37
‫نتایج‬
‫فارماکولوژیک‬ ‫تست‬
IC50 = 14.8±1.18 IC50 = 23.75±0.91 IC50 = 36.6±1.50
IC50 =
10.02±1.10
IC50 = 51.79±1.40 IC50 = 2.89±1.07
IC50 = 1.3±1.10 IC50 = 18.3±1.20 IC50 = 15.31±1.12
9a 9b 9c
9d 9e 9f
9g 9h 9i
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38. 38
‫نتایج‬
‫فارماکولوژیک‬ ‫تست‬
IC50 = 4.6±0.08
IC50 = 3.89±1.07
IC50 = 6.23 ±1.16 IC50 = 4.15±0.58
9j 9k
9l 9m
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39. Suggestion
 Further investigation in cell culture in terms of toxicity
 Check against the desired bacteria
 Review in Animal model
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40. 40
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