Synthesis and evaluation of pseudosaccharin amine derivatives as potential elastase inhibitions [Elektronische Ressource] / vorgelegt von  Rode Haridas Baburado

Synthesis and evaluation of pseudosaccharin amine derivatives as potential elastase inhibitions [Elektronische Ressource] / vorgelegt von Rode Haridas Baburado

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Synthesis and evaluation of pseudosaccharin amine derivatives as potential elastase inhibitors INAUGURALDISSERTATION zur Erlangung des akademischen Grades Doctor rerum naturalium (Dr. rer. nat.) an der Mathematisch-Naturwissenschaftlichen Fakultät der Ernst-Moritz-Arndt-Universität Greifswald Vorgelegt von Rode Haridas Baburao geb. am 03.12.1974 Greifswald, Dezember 2005 in Aambi (Indien) Dekan: Prof. Dr. J. -P. Hildebrandt 1. Gutachter: Prof. Dr. H. -H. Otto 2. Gutachter: Prof. Dr. T. Schirmeister Tag der Promotion: 01.02.2006 Dedicated to my parents “In chemistry, one’s ideas, however beautiful, logical, elegant, imaginative that they may be in their own right, are simply without value unless they are actually applicable to the one physical environment we have- in short, they are only good if they work!” R. B. Woodward Publications 1) Rode, H. B.; Sprang, T.; Besch, A.; Loose, J.; Otto, H.-H. Pseudosaccharin amine derivatives: synthesis and elastase Inhibitory activity. Die Pharmazie 2005, 60(10), 723-731. 2) Rode, H.; Koerbe, S.; Besch, A.; Methling, K.; Loose, J.; Otto, H.-H.

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Synthesis and evaluation of pseudosaccharin amine
derivatives as potential elastase inhibitors






INAUGURALDISSERTATION


zur
Erlangung des akademischen Grades
Doctor rerum naturalium (Dr. rer. nat.)
an der
Mathematisch-Naturwissenschaftlichen Fakultät
der
Ernst-Moritz-Arndt-Universität Greifswald














Vorgelegt von
Rode Haridas Baburao
geb. am 03.12.1974
Greifswald, Dezember 2005 in Aambi (Indien)
















































Dekan: Prof. Dr. J. -P. Hildebrandt
1. Gutachter: Prof. Dr. H. -H. Otto
2. Gutachter: Prof. Dr. T. Schirmeister
Tag der Promotion: 01.02.2006


















Dedicated to my parents












































“In chemistry, one’s ideas, however beautiful, logical, elegant, imaginative that
they may be in their own right, are simply without value unless they are
actually applicable to the one physical environment we have- in short, they
are only good if they work!”

R. B. Woodward Publications

1) Rode, H. B.; Sprang, T.; Besch, A.; Loose, J.; Otto, H.-H. Pseudosaccharin amine
derivatives: synthesis and elastase Inhibitory activity. Die Pharmazie 2005, 60(10),
723-731.

2) Rode, H.; Koerbe, S.; Besch, A.; Methling, K.; Loose, J.; Otto, H.-H. Synthesis and
in vitro evaluation of pseudosaccharin amine derivatives as potential elastase
inhibitors. Bioorg. Med. Chem. 2005, in press.

Presentation

1) Rode, H.; Besch, A.; Otto, H.-H. Studies on pseudosaccharin derivatives. A poster
presented at the annual meeting of the Medicinal Chemistry “Frontiers in Medicinal
Chemistry” Erlangen, 15-17 March 2004.
































List of abbrevations
aq. Aqueous
BTAC Benzyl triethyl ammonium chloride
BOC tert-Butoxycarbonyl
δ Chemical shift in ppm
CC Column chromatography
J Coupling constant in Hz
dec. Decomposition
DSC Differential scanning calorimetry
CDCl Deuterochloroform 3
°C Degree Celsius
DBU 1,8-Diazabicyclo[5.4.0]undec-7-en
DCM Dichloromethane
[D]DMSO Deuterodimethylsulfoxide 6
DMF Dimethylformamide
DMSO Dimethylsulfoxide
EtOH Ethanol
AcOEt Ethyl acetate
g Gram
HDTMAB Hexadecyltrimethylammonium bromide
HPLC High performance liquid chromatography
HLE Human Leukocyte Elastase
K Inhibitor constant i
LG Leaving group
IBCF Isobutyl chloroformate
IR Infrared
M.p. Melting point
MeOH Methanol
µM Micromolar
mg Milligram
mM Millimolar
pNA 4-Nitroanilide
NMM N-Methyl morpholine
NMR Nuclear magnetic resonance
PPE Porcine Pancreatic Elastase
KI Potassium iodide
R Ratio of front f
T Retention time RList of abbrevations
RP-HPLC Reverse phase high performance liquid chromatography
rt Room temperature
THF Tetrahydrofuran
TMS Tetramethylsilane
TLC Thin layer chromatography
TEA Triethylamine
UV Ultraviolet light


Amino acids
Ala Alanine
Asp Aspartic acid
Glu Glutamic
Phe Phenylalanine
Gly Glycine
His Histidine
Ile Isoleucine
Leu Leucine
Met Methionine
α-Me-Ala α-Methyl alanine
Pro Proline
Tyr Tyrosine
Ser Serine
Val Valine






Table of contents
Table of contents
1. Introduction and theoretical background .............................................................1
1.1. Human Leukocyte Elastase (HLE) ....................................................................1
1.2. Porcine Pancreatic Elastase (PPE)...................................................................2
1.3. Leukocyte Elastase-related diseases................................................................2
1.3.1. ARDS and Lung injury...............................................................................2
1.3.2. Cystic fibrosis (CF) ....................................................................................2
1.3.3. Pulmonary emphysema.............................................................................3
1.3.4. Smoking-related chronic bronchitis ...........................................................3
1.3.5. Ischaemic-reperfusion injury......................................................................4
1.3.6. Rheumatoid arthritis (RA)..........................................................................4
1.3.7. Gastric mucosal injury .........4
1.4. Mechanism of peptide hydrolysis by HLE .........................................................4
1.5. Elastase Inhibitors .............................................................................................8
1.5.1. Peptide based inhibitors ............................................................................8
1.5.2. Heterocyclic Inhibitors .............................................................................12
1.5.2.1. Enzyme-activated inhibitors.............................................................12
1.5.2.2. Heterocyclic acylating agents ..........................................................13
1.5.3. Nonheterocyclic alkylating/acylating agents ............................................14
2. Aim of the work .....................................................................................................17
3. Results and Discussion........................................................................................19
3.1. Pseudosaccharinamines from (1,1-dioxobenzo[d]isothiazol-3-
ylsulfanyl)acetonitrile .......................................................................................19
3.2. Pseudosaccharinamine synthesis from thiosaccharinates..............................24
3.3. Pseudosaccharinamine synthesis from 3-ethoxybenzo[d]isothiazole 1,1-
dioxide .............................................................................................................26
3.4. Pseudosaccharinamine derivatives from 3-chlorobenzo[d]isothiazole 1,1-
dioxide and their further modifications.............................................................32
3.4.1. Pseudosaccharinamines .........................................................................32
3.4.2. Amide and methyl ester derivatives.........................................................33
3.4.3. Ester hydrolysis .......................................................................................35
3.4.4. Different ester derivatives containing pseudosaccharinamines...............37
3.4.5. Alcohol derivative: (2S,3S)-2-(1,1-dioxobenzo[d]isothiazol-3-ylamino)-
3-methylpentan-1-ol.................................................................................40
3.4.6. Pseudosaccharin amine containing thiazole or thiophene ring
analogues................................................................................................41 Table of contents
3.4.7. Nitration of 3-(1,1-dioxobenzo[d]isothiazol-3-ylamino)thiophene-2-
carboxylic acid methyl ester ....................................................................47
3.5. Peptide synthesis ............................................................................................50
3.6. Enzyme assay .................................................................................................58
3.6.1. Reversible inhibition ................................................................................58
3.6.2. Inhibitory activity of the compounds ........................................................61
3.7. Molecular mechanics and docking studies......................................................69
3.7.1. Molecular Mechanics...............................................................................69
3.7.2. Docking....................................................................................................69
4. Conclusions...........................................................................................................78
5. Experimental..........................................................................................................80
5.1. General Information.........................................................................................80
5.2. Synthesis of the compounds ...........................................................................82
5.3. X-ray crystallography of 3-(1,1-dioxobenzo[d]isothiazol-3-ylamino)-5-nitro-
thiophene-2-carboxylic acid methyl ester ......................................................120
5.4. Elastase inhibition studies .............................................................................121
5.4.1. Buffers ...................................................................................................121
5.4.2. Determination of percent inhibition........................................................121
5.4.2.1. Percent inhibition of PPE...............................................................121
5.4.2.2. Percent inhibition of HLE121
5.4.3. Determination of K ................................................................................122 i
5.4.4. Stability of cyanomethyl (2S,3S)-2-(1,1-dioxobenzo[d]isothiazol-3-
ylamino)-3-methylpentanoate during the enzyme assay .......................123
5.5. Docking Studies ............................................................................................124
6. References...........................................................................................................126
7. Appendix..............................................................................................................132

1. Introduction and theoretical background 1
1. Introduction and theoretical background
Life depends on a well-orchestrated series of chemical reactions. Many of these
reactions, however, proceed too slowly to sustain life on their own. Hence, nature
has designed catalysts, which we now refer to as enzymes, to greatly accelerate the
[1]rates of these chemical reactions . As the biocatalysts that regulate the rates at
which all physiologic processes take place, enzymes occupy central roles in health
and disease. While in health all physiologic processes occur in an ordered, regulated
manner and homeostasis is maintained, homeostasis can be profoundly disturbed in
[2]pathologic states . One of the most exiting fields of modern Enzymology is the
application of enzyme inhibitors as drugs in human and veterinary medicine. Many of
the drugs that are commonly used today function by inhibiting specific enzymes that
[1]are associated with the disease process . Elastases are possibly the most
destructive enzymes in the body, having the ability to degrade virtually all connective
tissue components. Uncontrolled proteolytic degradation by elastase has been
[3]implicated in a number of pathological conditions .
1.1. Human Leukocyte Elastase (HLE)
HLE (EC 3.4.21.37) is often called as neutrophil elastase. It is one of the several
hydrolytic enzymes contained in azurophil granules of human neutrophils and also
found in thrombocytes, macrophages, in the spleen, aorta and skin, in snake venoms
and in some microorganisms. Elastases are endopeptidases (serine proteases)
[4, 5]which by definition are able to solubilize elastin by proteolytic cleavage . Besides
elastin, collagen type III, proteoglycans, azocasein, haemoglobin, fibrinogen and
histones are degraded, among other proteins. HLE is responsible (at least in part) for
[4]inflammatory and arthritis condition . HLE consists of a single basic polypeptide
chain of 218 amino acid residues, joined together by four disulfide bonds. It contains
two asparagine linked carbohydrate side chains, and is in fact synthesized as a
series of isoenzymes each containing different amounts of carbohydrate. The
backbone architect of HLE and PPE is conserved, and the structures of the active
[6, 7]sites near the cleavage site are very similar . The isoforms do obviously not differ
[8, 9]in catalytic activity and are immunologically identical . Comparison of the
sequence to other serine proteinases indicates only moderate homology with porcine
[6]pancreatic elastase (43.0%) or neutrophil cathepsin G (37.2%) . The N-terminal
amino acid sequence is strongly homologous with that of the porcine pancreatic
[8]elastase .