Thiazolyl-thiadiazines as Beta Site Amyloid Precursor Protein Cleaving Enzyme-1 (BACE-1) Inhibitors and Anti-inflammatory Agents: Multitarget-Directed Ligands for the Efficient Management of Alzheimer's Disease

Article abstract of DOI:10.1021/acschemneuro.8b00063

Alzheimer's disease (AD) is associated with multiple neuropathological events including β-site amyloid precursor protein cleaving enzyme-1 (BACE-1) inhibition and neuronal inflammation, ensuing degeneracy, and death to neuronal cells. Targeting such a complex disease via a single target directed treatment was found to be inefficacious. Hence, with an intention to incorporate multiple therapeutic effects within a single molecule, multitarget-directed ligands (MTDLs) have been evolved. Herein, for the first time, we report the discovery of novel thiazolyl-thiadiazines that can serve as MTDLs as evident from the in vitro and in vivo studies. These MTDLs exhibited BACE-1 inhibition down to micromolar range, and results from the in vivo studies demonstrated efficient anti-inflammatory activity with inherent gastrointestinal safety. Moreover, compound 6d unveiled noteworthy antioxidant, antiamyloid, neuroprotective, and antiamnesic properties. Overall, results of the present study manifest the potential outcome of thiazolyl-thiadiazines for AD treatment.

Full text of DOI:10.1021/acschemneuro.8b00063

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Thiazolyl-thiadiazines as BACE-1 Inhibitors and Anti-
inflammatory Agents: Multitarget-Directed Ligands
for the Efficient Management of Alzheimer’s Disease
Sneha Sagar, Devendra Pratap Singh, Nirupa B Panchal, Rajesh D Das, Dhaivat
H Pandya, Vasudevan Sudarsanam, Manish Nivsarkar, and Kamala K. Vasu
ACS Chem. Neurosci., Just Accepted Manuscript • DOI: 10.1021/acschemneuro.8b00063 • Publication Date (Web): 26 Apr 2018
Downloaded from http://pubs.acs.org on April 27, 2018
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Thiazolylꢀthiadiazines as BACEꢀ1 Inhibitors and Antiꢀinflammatory Agents: Multitargetꢀ
*
*
Directed Ligands for the Efficient Management of Alzheimer’s Disease
a,c
b
a,c
a
Sneha R. Sagar , Devendra Pratap Singh , Nirupa B. Panchal , Rajesh D. Das , Dhaivat H.
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Pandya , Vasudevan Sudarsanam , Manish Nivsarkar , Kamala K. Vasu
a
Department of Medicinal Chemistry, B. V. Patel Pharmaceutical Education and Research
Development (PERD) Centre, S. G. Highway, Thaltej, Ahmedabadꢀ380054, Gujarat, India
b
Department of Pharmacology and Toxicology, B. V. Patel Pharmaceutical Education and
Research Development (PERD) Centre, S. G. Highway, Thaltej, Ahmedabadꢀ380054, Gujarat,
India
c
Institute of Pharmacy, NIRMA University, S. G. Highway, Chandlodia, Gota, Ahmedabadꢀ
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*
82481, Gujarat, India
Corresponding author. Tel.: + 91 79 27439375; Fax: + 91 79 27450449
Eꢀmail: kamkva@gmail.com, kamalav@perdcentre.com
#
Communication Ref. No.: PERD95012018
Abstract:
Alzheimer’s disease (AD) is associated with multiple neuropathological events including BACEꢀ
1
inhibition and neuronal inflammation, ensuing degeneracy and death to neuronal cells.
Targeting such a complex disease via a single target directed treatment was found to be
inefficacious. Hence, with an intention to incorporate multiple therapeutic effects within a single
molecule; multitargetꢀdirected ligands (MTDLs) have been evolved. Herein, for the first time we
report the discovery of novel thiazolylꢀthiadiazines which can serve as MTDLs as evident from
the in vitro and in vivo studies. These MTDLs exhibited BACEꢀ1 inhibition down to micromolar
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range and results from the in vivo studies demonstrated efficient antiꢀinflammatory activity with
inherent gastroꢀintestinal safety. Moreover, compound 6d unveiled noteworthy antiꢀoxidant, antiꢀ
amyloid, neuroprotective, and antiꢀamnesic properties. Overall, results of the present study
manifest the potential outcome of thiazolylꢀthiadiazines for AD treatment.
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Keywords: Drug discovery, BACEꢀ1, Inflammation, Multitargetꢀdirected ligands
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Introduction:
Alzheimer’s disease (AD) is a progressive degenerative neurological disorder of the brain. The
two widely accepted hallmarks of AD pathogenesis are amyloid plaques and neurofibrillary
tangles. Certain other factors that affect the pathophysiology of this disorder are inflammation,
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neurotransmitter imbalance, oxidative stress and rising cholesterol levels. Currently, the
available drugs treat only behavioral and cognitive symptoms related to this disease but none of
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them are applicable for disease modifying treatment of AD. As a result, there is an unmet need
to develop antiꢀamyloid drugs in order to combat complex AD pathogenesis. In recent years,
BACEꢀ1 (Beta site APP cleaving enzymeꢀ1) has emerged as one of the main targets for AD
intervention. BACEꢀ1 is the key enzyme of amyloidogenic cascade that acts on amyloid
precursor protein (APP) and eventually helps in formation of amyloid plaques in the extracellular
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site. Despite being the prime target of AD, preclinical BACEꢀ1 inhibitors have failed so far to
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show promising efficacy during clinical studies. Therefore, it can be presumed that AD
treatment interferes with more than one neuropathological mechanism simultaneously. In this
context, multitargetꢀdirected ligands (MTDLs) have emerged as an effective approach for disease
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modifying AD treatment and proven to be advantageous over single target antiꢀamyloid therapy.
Amyloid plaques induces the release of various inflammatory cytokines like TNFꢀα, ILꢀ1β and
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cyclooxygenases (COXs). Therefore, neuronal inflammation is considered to be the downstream
factor for AD pathogenesis. The collaborative influence of BACEꢀ1 and neuronal inflammation
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lead to neuronal cell degeneration and cell death.
Broadly speaking, the MTDLs are compounds which endow ability to interact with multiple
biological targets simultaneously. Besides, they reduce the pharmacokinetic and drugꢀdrug
interaction problems linked to multipleꢀmedication therapy and generally show a synergistic
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effect. The design and development of new MTDLs seems to be an attractive and widely adopted
approach in AD drug discovery, as attested by numerous researchers around the globe. Certain
examples of MTDLs are: dual BACEꢀ1 and acetylcholinesterase (AChE) inhibitors, dual BACEꢀ
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and glycogen synthase kinase (GSK)ꢀ3β inhibitors, microtubule stabilizing agents with COX
and LOX (5ꢀlipoxygenase) inhibitors, dual binding site cholinesterase inhibitors with βꢀamyloid
antiꢀaggregation properties, hybrid AChE and butyrylcholinesterase (BuChE) with antiꢀoxidant
and neuroprotective properties, dual acting compounds targeting histaminergic system, hybrid
AChE inhibitors with cannabinoid receptor antagonism, multifunctional chelators, hybrid
BACEꢀ1 inhibitors and metal chelators and dual acting γꢀsecreatse modulators with peroxisome
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, 8ꢀ10
proliferator activated receptor (PPAR)ꢀγ.
Our study focuses on MTDLs acting on BACEꢀ1 and inflammation concurrently. We have
designed MTDLs comprising of two scaffolds in a single molecule to generate potential
prototype ligand for complex AD treatment.
Design of molecules
BACEꢀ1 is an aspartic protease enzyme developed as a one of the major targets for AD
intervention. Based on the crystal structures of BACEꢀ1 with ligands, catalytic machinery of the
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enzyme is well reported. The aspartic dyad formed by Asp32 and Asp228 are located at the
center of the binding site. BACEꢀ1 catalyzes hydrolysis of peptide bonds by acidꢀbase catalysis
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of water molecule present between aspartic acid dyad and forms tight hydrogen bond. We
aimed at identifying a potential moiety with ability to bind the enzymes catalytic aspartic dyad.
Based on the reported literature we found, various thiadiazines are emerging as a BACEꢀ1
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0,13ꢀ14
inhibitor scaffold.
Iminothiadiazinane dioxide inhibitor was developed to exhibit selectivity
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of BACEꢀ1 over BACEꢀ 2 by forming Hꢀbond with ASP32 and Asp228. Verubecestat (MKꢀ
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931ꢀ1) is a clinical candidate developed by Merck as a BACEꢀ1 inhibitor contains
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iminothiadiazinane dioxide core scaffold with 2.2 nM K for BACEꢀ1.
5‑Arylꢀ1ꢀiminoꢀ1ꢀ
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oxoꢀ[1,2,4]thiadiazines (2) exhibited BACEꢀ1 inhibition at IC of 0.009 nM. (S)ꢀ3ꢀaminoꢀ5ꢀ(3ꢀ
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chloroꢀ4ꢀ(5ꢀ(propꢀ1ꢀynꢀ1ꢀyl)pyridinꢀ3ꢀyl)thiophenꢀ2ꢀyl)ꢀ2,5ꢀdimethylꢀ5,6ꢀdihydroꢀ2Hꢀ1,2,4ꢀ
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thiadiazine 1,1ꢀdioxide (3) showed BACEꢀ1 inhibitory activity with the IC value of 2.4 nM.
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These molecules signify the therapeutic importance of thiadiazines as BACEꢀ1 inhibitors.
Neuronal inflammation is consider to be the downstream factor for the AD generation. The
COXꢀ1 and COXꢀ2 expression are beneficial for neurodegeneration in AD. COXꢀ1 is expressed
in the glia and neurons of the AD brain. Little role of COXꢀ1 is reported for the inflammatory
mechanism through its glia expression. While the COXꢀ2 expression is preferentially elevated in
neurons with amyloid plaques and neurofibrillary tangles. Therefore, selective COXꢀ2 inhibitors
might influence the AD progression and nonꢀselective COXꢀ1 and COXꢀ2 inhibitors mediate the
antiꢀAlzheimer’s effect by acting on glia and neurons. Thus, the design strategy for COX
inhibitors with CNS penetration ability can be an effective solution for AD threat. Our lab has
been exploring various small heterocycles as antiꢀinflammatory agents. Thiazole moiety has been
established as a privileged structure to confer the drug likeliness properties. Also, the Hꢀbond
donor as well as Hꢀbond acceptor features of thiazole are advantageous for Hꢀbonding to the
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receptor, which impart the therapeutic activity.
Methyl 3ꢀ(4ꢀ(dimethylamino)ꢀ2ꢀ
(methylamino)thiazolꢀ5ꢀyl)ꢀ2ꢀ(methoxyimino)ꢀ3ꢀoxopropanoate (4) is reported as an antiꢀ
inflammatory agent with 83% inhibition of rat paw edema in acute inflammation and 66%
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inhibition of rat paw edema in chronic inflammation. 2ꢀ(4ꢀChlorophenyl)ꢀ3ꢀ(4ꢀmethylꢀ2ꢀ
(phenylamino)thiazolꢀ5ꢀyl)naphthalenꢀ1(2H)ꢀone (5) showed 72% inhibition of edema in acute
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inflammation rat model. Based on the reported literature, design of multitargetꢀdirected ligands
for BACEꢀ1 enzyme and inflammation using thiadiazines and thiazoles is depicted in Figure 1.
INSERT FIGURE 1 HERE
Figure 1. Design strategy of MTDLs for AD
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Results & Discussion:
Docking studies
To obtain substantial or rather conclusive theoretical evidence of therapeutic activity, the
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thiazolylꢀthiadiazines were virtually screened by docking studies using Schrödinger software.
In the process, a library of thiazolylꢀthiadiazines was prepared as shown in Figure 2 and docked
against BACEꢀ1, BACEꢀ2, Cathepsin D, COXꢀ1 and COXꢀ2 enzymes. BACEꢀ2 enzyme is a
homolog of BACEꢀ1 enzyme with nearly 64% of structural similarity and it can efficiently
cleave APP but cannot form βꢀamyloid plaques. An important criterion to fulfill is selectivity of
BACEꢀ1enzyme over BACEꢀ2 and Cathepsin D. Cathepsin D is a lysosomal aspartic
endopeptidase associated with apoptosis regulation, metabolic degradation of intracellular
proteins, stimulation and downgrading of polypeptide growth factors and hydrolysis of lowꢀ
density lipoprotein (LDL). Nonꢀinhibition of Cathepsin D is essential for the cell viability.
Therefore, compounds with the best docking score values against BACEꢀ1 and least docking
scores against BACEꢀ2 and Cathepsin D were selected for synthesis.
βꢀamyloid plaques and the damaged neurons induce inflammatory response as a natural
phenomenon due to cell damage. The elevated levels of prostaglandins can be attributed to COXꢀ
1 and COXꢀ2 enzymes. Also, certain proꢀinflammatory substances are released from the
activated microglia viz. reactive oxygen species (ROS), interlukinꢀ1, interlukinꢀ6 and tumor
necrosis factor alleviate the AD progression. Thus, this increased oxidative stress causes the
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neuronal cell degradation and amyloidꢀβ neurotoxicity. To check the antiꢀinflammatory potential
of designed molecules docking studies were performed using COXꢀ1 and COXꢀ2 enzymes.
Noteworthy interactions between the molecules and catalytic pocket of BACEꢀ1, COXꢀ1 and
COXꢀ2 enzymes were observed during this scrutiny. Compounds with single target efficacy were
forbidden from further studies as they deviate from our approach to target multiple enzymes.
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Results of top ranked compounds are displayed in Table 1 (For specific substitutions R , R and
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R refer Scheme 3 &4). Compounds showing least docking scores are mentioned in “Supporting
Information”.
INSERT FIGURE 2 HERE
Figure 2. General structure for docking library
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R = R = H, Ph, 4ꢀOCH ꢀPh, 4ꢀCH ꢀPh, 4ꢀFꢀPh, 4ꢀClꢀPh, 3,4ꢀdiꢀClꢀPh, 4ꢀNO ꢀPh, 3,4ꢀ(CH ) ꢀPh, 4ꢀCF ꢀPh
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R = H, Ph, CH , N(CH ) , CH ꢀPh
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Table 1. Results of docking studies
Code
Docking Score
BACEꢀ2 Cathepsin D
BACEꢀ1
COXꢀ1
COXꢀ2
ꢀ9.0
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a
ꢀ7.3
ꢀ7.4
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ꢀ4.1
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ꢀ5.2
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ꢀ8.1
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6c
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d
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AZD3839
Diclofenac
Celecoxib
Hydroxyethylenamine
inhibitor
ꢀ5.2
Docking results were further extended as given in Figure 3A & 3B. The crystal structures of
BACEꢀ1 (PDB ID: 4B05) and COXꢀ2 (PDB ID: 3LN1) enzymes were employed to conduct
docking studies. Binding interactions and docking scores were considered for binding affinity of
compounds with the respective enzymes. A side by side overlay of most potent compound 6d
with clinical candidate (AZD839) on BACEꢀ1 enzyme binding site is demonstrated in Figure 3A.
Figure 3B represents the overlay of standard drug Celecoxib and compound 6d for COXꢀ2
binding site influence. Binding interactions with the catalytic amino acid residues are found to be
similar to that of reference drugs.
INSERT FIGURE 3A & 3B HERE
Figure 3. Docking study results A) Overlay of compound 6d (green) with AZD3839 (orange) docked into the
catalytic region of BACEꢀ1. Compound 6d showed Hꢀbonding interactions (yellow dotted line) with aspartic acid
dyad Asp32, Asp228 and πꢀπ staking interaction (blue dotted line) with Tyr71. Docking score value was ꢀ7.5.
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AZD3839 showed Hꢀbonding interactions with Asp32, Asp228, Trp76 and πꢀπ stacking interactions with Tyr71 and
Phe108 with docking score of ꢀ8.8. B) Overlay of compound 6d (green) with Celecoxib (orange) docked into COXꢀ
2 binding site. Compound 6d showed Hꢀbonding interaction with Tyr341, πꢀπ stacking interactions with Tyr341,
Trp373, Tyr371 and πꢀcation interaction (green dotted line) with Arg106. Docking score value was ꢀ9.6. Celecoxib
showed Hꢀbonding interactions with Tyr341, Ser339, Arg499, Phe504 and πꢀcation interaction with Arg106 with
docking score of ꢀ12.2.
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From the library of our designed molecules, those with the best docking scores against BACEꢀ1
and inflammatory pathway enzymes were selected for synthesis. A complete synthetic route for
compounds 6aꢀe & 8aꢀc is portrayed in Scheme 1 to 4.
Prediction of physicochemical properties
An examination of physicochemical properties of central nervous system (CNS) targeting
molecules is necessary from the view point of developing drug like molecules. Many active
molecules fail to emerge out as drug candidates at the later stage of drug discovery due to lower
blood brain barrier permeability. Hence, prediction of physicochemical properties of the
1
8
molecules were carried out using Qikprop module of Schrödinger (Table 2) . The selected
compounds were found to obey the Lipinski’s rule of five and Jorgensen’s rule of three. Some
other physicochemical parameters like QPlogBB, QPlogPo/w, QPPCaco, %HOA and
QPlogHERG were also predicted. Compounds showed satisfactory drug likeliness properties
with higher BBB permeation capacity and lower toxicity by HERG inhibition.
Table 2. Physicochemical properties prediction
a
b
c
d
e
Code
QPlogBB
QPlogPo/w
QPPCaco
%HOA
QPlogHERG
6
a
ꢀ0.11
6.40
3459
100
ꢀ6.43
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4.40
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92
ꢀ6.40
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ꢀ7.70
ꢀ6.40
ꢀ7.86
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ꢀ7.96
ꢀ7.83
ꢀ7.98
ꢀ6.42
ꢀ2.95
ꢀ5.78
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b
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AZD3839
Diclofenac
Celecoxib
500
398
358
a
b
QPlogBB: Predicted brain/blood partition coefficient (ꢀ3.0 to 1.2), QPlogPo/w: Predicted octanol/water partition
c
d
coefficient (ꢀ2.0 to 6.5), QPPCaco: Predicted Cacoꢀ2 cell permeability (<25 poor, >500 great), %HOA: Predicted
e
human oral absorption (>80% is high, <25% is poor), QPlogHERG: Predicted IC value for blockage of HERG K+
50
channels (below ꢀ5)
Chemistry
The designed compounds were synthesized as per the Schemes 1ꢀ4. Scheme1 demonstrates the
synthesis of intermediates ꢀ substituted Nꢀphenylhydrazinecarbothioamide 3aꢀe. Reaction of
substituted anilines in presence of TEA and carbon disulfide in THF at 0ꢀ5 °C yielded
dithiocarbamate salt. This salt was further treated with TEA and ethyl chloroformate in
chloroform at RT to get substituted isothiocyanatobenzenes 2aꢀf. Synthesis of substituted Nꢀ
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phenylhydrazinecarbothioamides 3aꢀe were carried out using substituted isothiocyanatobenzenes
in presence of hydrazine hydrate in THF at RT.
INSERT SCHEME 1 HERE
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Scheme 1. Synthesis of substituted Nꢀphenylhydrazinecarbothioamides
Reagents and Conditions: (a) TEA (3.2 eq), CS (1.0 eq), 0ꢀ5 °C, THF, 6ꢀ7 hr; (b) TEA (1.0 eq), ethyl chloroformate
2
(1.1 eq), 0 °C to RT, chloroform, 2 hr; (c) hydrazine hydrate, RT, THF, 2 hr
2
ꢀChloroꢀ1ꢀ(4ꢀphenylꢀ2ꢀ(phenylamino)thiazolꢀ5ꢀyl)ethanone derivatives 5aꢀd were synthesized
as depicted in Scheme 2. Isothiocyanatobenzene reacted with N,Nꢀdiethylbenzimidamide in ethyl
acetate at RT to yield N,NꢀdiethylꢀN'ꢀ(phenylcarbamothioyl)benzimidamide 4aꢀd. Substituted 2ꢀ
chloroꢀ1ꢀ(4ꢀphenylꢀ2ꢀ(phenylamino)thiazolꢀ5ꢀyl)ethanones were synthesized from 4aꢀd using
1
,3ꢀDichloropropanꢀ2ꢀone in acetonitrile at RT as described in Scheme 2.
INSERT SCHEME 2 HERE
Scheme 2. Synthesis of substituted 2ꢀChloroꢀ1ꢀ(4ꢀphenylꢀ2ꢀ(phenylamino)thiazolꢀ5ꢀyl)ethanones
Reagents and Conditions: (d) N,Nꢀdiethylbenzimidamide (1.0 eq), RT, ethyl acetate, 3ꢀ4 hr; (e) 1,3ꢀDichloropropanꢀ
ꢀone (1.5 eq), RT, acetonitrile, 3ꢀ4 hr
2
Scheme 3 depicts the reaction of final compounds 6aꢀg. Reactions of the intermediates 3aꢀe and
aꢀd were carried out by heating in THF at 70 °C for 6ꢀ7 hr to afford substituted Nꢀphenylꢀ5ꢀ(4ꢀ
5
phenylꢀ2ꢀ(phenylamino)thiazolꢀ5ꢀyl)ꢀ6Hꢀ1,3,4ꢀthiadiazinꢀ2ꢀamines (final compounds).
INSERT SCHEME 3 HERE
Scheme 3. Synthesis of substituted Nꢀphenylꢀ5ꢀ(4ꢀphenylꢀ2ꢀ(phenylamino)thiazolꢀ5ꢀyl)ꢀ6Hꢀ1,3,4ꢀthiadiazinꢀ2ꢀ
amines (Final compounds)
Reagents and Conditions: (f) 70 °C, THF, 6ꢀ7 hr
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Final compounds 8aꢀc were synthesized as per the Scheme 4. Hydrazinecarbothioamide was
reacted with intermediate 5bꢀd in THF at 70 °C for 6ꢀ7 hr to get the final compounds substituted
5ꢀ(4ꢀphenylꢀ2ꢀ(pꢀtolylamino)thiazolꢀ5ꢀyl)ꢀ6Hꢀ1,3,4ꢀthiadiazinꢀ2ꢀamine 8aꢀc.
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INSERT SCHEME 4 HERE
Scheme 4. Synthesis of substituted 5ꢀ(4ꢀphenylꢀ2ꢀ(pꢀtolylamino)thiazolꢀ5ꢀyl)ꢀ6Hꢀ1,3,4ꢀthiadiazinꢀ2ꢀamines (Final
compounds)
Reagents and Conditions: (f) 70 °C, THF, 6ꢀ7 hr
Biological Evaluation
In vitro BACEꢀ1 inhibition assay
The molecules (6aꢀg and 8aꢀc) were investigated by in vitro BACEꢀ1 inhibition assay (Table 3).
2
0
IC values were obtained and compared with the reference molecule AZD3839. Compounds
5
0
nd
6
aꢀg have aromaticꢀNH substitutions and 8aꢀc have NH substitutions at 2 position of
2
1
thiadiazine. Compounds bearing trifluromethyl substitution at R position were found to be the
most active BACEꢀ1 inhibitors. Compounds 6b and 6d showed BACEꢀ1 inhibition with IC of
5
0
9
µM. As shown in Figure 3A, 6d had binding interactions with the catalytic aspartic dyad of the
enzyme Asp32 and Asp228 along with πꢀπ staking interaction with Tyr71. Docking score value of
6b and 6d were found as ꢀ7.4 & ꢀ7.5, respectively. Compounds 6b and 6d have similarity in
2
1
structure except the R position with methyl group in 6b. Compound 6a has methoxy group at R
nd
position which showed BACEꢀ1 inhibition with IC of 11 µM. NH group at 2 position of
5
0
2
thiadiazine moderately inhibited BACEꢀ1 with IC of 50, 29 and 20 µM in compound 8a, 8b
50
2
and 8c, respectively. Fluoro and chloro substitutions at R position favored BACEꢀ1 inhibitory
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activity in 8b and 8c. Compounds 6f and 8a showed BACEꢀ1 enzyme inhibition with IC of 50
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0
µM. Whereas, 6e and 6g were found to be less potent with IC greater than 100 µM.
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0
Table 3. In vitro inhibitory potencies (IC ) of compounds against BACEꢀ1 and in vivo % edema
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inhibition against acute inflammation rat model
Compound BACEꢀ1 [IC (µM) % inhibition of edema against
5
0
a
b
±
SEM ]
acute inflammation [Mean ±
SEM]
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a
11 ± 0.08
9 ± 0.35
40 ± 0.2
9 ± 0.09
112 ± 0.08
50 ± 0.09
114 ± 0.5
50 ± 0.09
29 ± 0.7
20 ± 0.5
NT
48 ± 0.45
63 ± 0.6
48 ± 0.75
70 ± 0.4
56 ± 0.5
70 ± 0.55
29 ± 0.9
67 ± 0.7
60 ± 1.0
29 ± 0.95
72 ± 0.65
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c
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6g
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a
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8c
Diclofenac
a
IC50 values are reported as concentration of each compound resulting in 50% inhibition. Data represented as
Mean ± SEM (n=3)
b
Data shown are refer to the % change in edema size relative to the disease control group. Statistical analysis
was performed by Oneꢀway ANOVA followed by Tukey’s multiple comparison test. Mean ± SEM (n=6
animals/group) *p<0.05
NT Not Tested
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In vivo carrageenan induced acute inflammation study
Another crucial aspect in understanding the efficacy of these developed molecules against AD
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1
was estimated by carrying out in vivo acute inflammation study in rats. We observed
satisfactory results for compounds (6aꢀg and 8aꢀc), as compared with reference drug; Diclofenac
sodium. % edema inhibition in case of compounds 6d, 6f and 8c was found to be 70%, 70% and
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7 %, respectively. In case of Diclofenac, 72 % inhibition was noticed, as mentioned in Table 4.
Compounds 6b, 6e, 8a and 8b showed antiꢀinflammatory activity with 63%, 56%, 67% and 60%
of rat paw edema. Importantly, these developed molecules fit into the catalytic pocket of COXꢀ1
and COXꢀ2 enzyme nonꢀselectively and subsequently elicit antiꢀinflammatory activity as
evidenced from the present study.
In vivo formalin induced chronic inflammation study
As illustrated above, compounds 6b, 6d and 6f were found as promising agents with BACEꢀ1
inhibition and acute antiꢀinflammatory properties. In spite of this, the results obtained were
rather insufficient to identify these MTDLs as candidate for treatment of AD because chronic
inflammation has been characterized to cause major AD mechanism reinforced by release of
2
2
cytokines and chemokines from activated microglia and astrocytes. Therefore, the developed
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1
MTDLs were further evaluated against the chronic inflammation model developed in rats. It
was very captivating to note that the % edema inhibition for compounds 6b, 6d, 6f were 49%,
th
5
8% and 47%, respectively on the 5 day of chronic model. Celecoxib was taken as a reference
th
drug and showed 68% edema inhibition on the 5 day as shown in Table 4.
Table 4. % edema inhibition against chronic inflammation rat model
a
Compound
% inhibition of edema in chronic inflammation [Mean ± SEM]
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Day 1
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45 ± 0.7
54 ± 1.7
49 ± 1.5
67 ± 0.9
52 ± 1.1
55 ± 0.4
43 ± 1.2
74 ± 0.6
53 ± 2.1
59 ± 1.0
39 ± 1.3
70 ± 2.2
54 ± 1.4
60 ± 2.0
43 ± 0.9
70 ± 1.5
49 ± 1.6
58 ± 0.6
47 ± 0.8
68 ± 0.5
6d
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Celecoxib
a
Data shown are refer to the % change in edema size relative to the disease control group. Statistical analysis was
performed by Oneꢀway ANOVA followed by Tukey’s multiple comparison test. Mean ± SEM (n=6 animals/group)
p<0.05
*
AlCl induced Alzheimer’s disease model
3
Taking into account the potency of compound 6d against BACEꢀ1, acute and chronic
23
inflammation, it was further examined for its effect on AlCl induced rat model of AD. During
3
the course of this examination the behavioral parameters were assessed by using pole climbing
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25
26
apparatus , elevated plus maze and Yꢀmaze. We wanted to ascertain the memory enhancing
effects of compound 6d in the developed model of AD. In this purview, elevated plus maze test
(
EPM) was used for testing the spatial working memory of compound 6d treated rats. Transfer
latency (TL) i.e. the time taken by an animal to move from an open arm to enclosed arm was
calculated. It was measured as a parameter of animal’s ability to recall the location of enclosed
arms since animals feel unsafe in open and high spaced maze. Interestingly, rats treated with
compound 6d showed significant improvement from the AlCl induced memory impairment and
3
significantly decreased TL as shown in Figure 4A. Further, based on the rodent’s innate tendency
to explore new environment, Yꢀmaze test was carried out to test immediate working memory of
6
d treated rats. In this test, they showed preference towards novel unexplored arm over the
previously explored one. Also, % alteration was measured and animals treated with compound
6
d showed elevation in the % alteration which indicated an increase in their cognitive function as
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shown in Figure 4B. Consequently, compound 6d when tested in the rat model of conditioned
avoidance response (CAR), it was found to reverse the amnesic effect of AlCl in comparison to
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,
the disease group as displayed in Figure 4C, Compound 6d decreased the CAR and showed
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INSERT FIGURE 4A, 4B, 4C AND FIGURE 4 DATA LABEL HERE
Figure 4. Effect of compound 6d on behavioral parameters of AD. NC= Normal control group animals treated with
normal saline, DC = Disease control group animals treated with AlCl (4.2 mg/kg i.p.), PC = Positive control group
3
,
animals treated with AlCl (4.2 mg/kg i.p) and Meloxicam (5.0 mg/kg, per oral), 6d = MTDL group animals treated
3
with AlCl (4.2 mg/kg i.p) and compound 6d (50.0 mg/kg, per oral) A) transfer latency (Sec) was calculated using
3
EPM test. Data expressed as mean TL (Sec) ± SEM. B) % alteration assessed by Yꢀmaze apparatus expressed as
mean % alteration ± SEM. C) inhibition of CAR by compound 6d on AlCl induced amnesia using pole climbing
3
apparatus. Results represent the mean of CAR (Sec) ± SEM. Asterisk denotes statistical significance (*p<0.05)
#
when compared to NC and ( p<0.05) when compared to DC, using twoꢀway ANOVA followed by Tukey’s multiple
comparison test.
27
Oxidative stress is an important marker of AD. We studied the free radical scavenging effect of
compound 6d on AlCl treated animals. Antiꢀoxidative benefits of compound 6d were observed
3
in the rat brain homogenates when tested by lipid peroxidation (LPO) levels and superoxide
dismutase (SOD) activity. High level of malonaldehyde (MDA), a marker for LPO indicates
28,29
higher oxidative stress.
AlCl treated rats showed significant elevation in MDA levels and
3
interestingly, MDA level was noticed to have significantly reduced in animals treated with
#
compound 6d ( p<0.05) in comparison to DC group (Figure 5A). Additionally, as expected the
28
antiꢀoxidant enzyme SOD activity was significantly decreased in AD induced animals.
Notably, SOD activity was found to be significantly increased in compound 6d treated animals
#
as compared to the animals in the DC group ( p<0.05) as shown in Figure 5B. These results
signify the antiꢀoxidative properties of compound 6d.
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INSERT FIGURE 5A & 5B HERE
Figure 5. Antiꢀoxidative effect of compound 6d: A) Lipid peroxidation (LPO) expressed as MDA level in rat brain
homogenates B) Super oxide dismutase (SOD) activity in rat brain homogenates. Data shown as Mean ± SEM (n=6
#
animals/group) *p<0.05 when compared to NC and p<0.05 when compared to DC group using oneꢀway ANOVA
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followed by Tukey’s multiple comparison test.
Haematological parameters estimation:
Haemoglobin and haematocrit level were estimated for all the groups of animals. Results
indicated that there was a significant decrease in the haemoglobin (HGB) and haematocrit (HCT)
level in PC group animals treated with 5 mg/kg of Meloxicam. Whereas, DC and compound 6d
treated animals showed HGB and HCT level same as that of NC animals (Figure 6A & 6B).
These studies indicated that Meloxicam causes loss of blood due to the gastric injury. Which was
further confirmed by results of gastroꢀintestinal (GI) safety studies.
INSERT FIGURE 6A & 6B HERE
Figure 6. Estimation of haematological parameters: A) Estimation of HGB level in all the groups. PC animals
showed significant decrease in the HGB level compared to all the groups. Here, data presented as Mean ± SEM
(*p<0.05). B) HCT level estimation in all the groups indicated that there was a significant decrease in the HCT level
in PC group when compared to all the groups. Data expressed as Mean ± SEM (*p<0.05) by using oneꢀway
ANOVA followed by Tukey’s multiple comparison test.
Estimation of Serum biochemical parameters:
From the study of serum biochemical parameters, it was observed that there was no significant
difference in all the groups’ serum albumin level (Figure 7A). Whereas, PC and DC animals
showed significant difference in the total protein when compared with NC and compound 6d
treated animals (Figure 7B).
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INSERT FIGURE 7A & 7B HERE
Figure 7: Serum albumin & Total protein estimation: A) Graph represent estimation of serum albumin level in all
the groups. There was no significant difference in the serum albumin level in all the groups. B) Total protein level
was significantly different in DC and PC group. Decrease in the total protein level was observed in DC and PC
group when compared to NC and compound 6d treated animals. Data expressed as Mean ± SEM (*p<0.05) by using
oneꢀway ANOVA followed by Tukey’s multiple comparison test.
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AlCl by causing oxidative damage to the neuronal cells forms aggregates of amyloid peptides
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which leads to the degeneration of neuronal cells. To observe the effect of compound 6d on
cellular morphology of hippocampal neuronal cells, histopathological studies by hematoxylinꢀ
eosin (HE) stain was employed. Results of HE stain in rat brain indicated the curative effect of
compound 6d against the neuronal degeneration. Figure 8A represents the normal and intact
hippocampal neurons (NC rats). In Figure 8B, it can be observed that neurons of DC animals
have degenerated. While, in PC group (Figure 8C) and compound 6d treated group (Figure 8D)
healthy and intact hippocampus neuronal region similar to that of NC animals was observed.
These results proved that in compound 6d treated animals, there was no sign of neuronal cell
degeneration as compound 6d protected the neurons from the detrimental effect of AlCl3.
Eventually it signifies the protective effect of compound 6d against oxidativeꢀstress and amyloid
plaques aggregations induced cell damage. In addition to this, the effect of compound 6d on
amyloid plaques formation was checked by Congo red staining and compound 6d was found to
act as an excellent antiꢀamyloid agent (Figure 9). The serial sections were stained with Congo
red to detect the amyloid plaques in the cortex region of the brain. Animals treated with AlCl3
showed formation of amyloid plaques in the cortex region of the brain. Whereas, compound 6d
treated group did not show plaques in the cortex region of the brain.
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INSERT FIGURE 8 HERE
Figure 8. Typical microscopic HE stained sections (under 100X magnification) of brain in all the groups. A) NC rat
brain showed healthy and intact neurons B) DC rat brain showed neuronal loss with shrunken and degenerated
hippocampal region C) PC rat brain showed intact neurons but thin hippocampal region and D) Compound 6d
treated rat brain showed intact, healthy neurons with dense hippocampal region
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INSERT FIGURE 9 HERE
Figure 9: Typical microscopic Congo red sections (under 100X magnification) of brain in all the groups: A) NC rat
brain without any amyloid plaques B) DC rat brain with amyloid plaques in the cortex region C) PC rat brain
showed no amyloid plaque region in the cortex D) compound 6d treated rat brain had no plaque in the cortex.
Gastroꢀintestinal (GI) safety studies of compound 6d
Macroscopic evaluation: PC animals treated with Meloxicam showed gastric and intestinal
damage. Lesion indices of PC treated animals in stomach and intestine were found to be 12.19 ±
0.7 mm and 29.15 ± 1.3 mm, respectively. Whereas, compound 6d treated rats did not show any
haemorrhagic damage in the stomach and intestine as shown in Figure 10.
INSERT FIGURE 10 HERE
Figure 10: Lesion index. Data expressed as Mean ± SEM (*p<0.05) by using twoꢀway ANOVA followed by
Tukey’s multiple comparison test.
Histopathological studies of stomach and intestine: Microscopic evaluation of stomach and
intestinal HE stained sections revealed focal erosions and perforations in the stomach of PC rats.
Moreover, epithelium erosions and stratifications were observed in intestines of PC treated
animals. Whereas, DC group and compound 6d treated animals did not show any focal erosions
and damage in the stomach and intestinal region as shown in Figure 11 & 12. These studies
proved the gastric safety of compound 6d when compared to the standard drug Meloxicam.
INSERT FIGURE 11 HERE
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Figure 11: Typical microscopic HE stained sections (under 100X magnification) of stomach in all the groups. A)
NC group without any gastric damage B) DC rats did not show erosions C) PC treated rats showed focal erosions
and perforations indicated by an arrow D) Compound 6d treated animals had no erosions or perforations in stomach
INSERT FIGURE 12 HERE
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Figure 12: Typical microscopic HE stained sections (under 100X magnification) of intestine in all the groups. A)
NC rats without epithelium damage B) DC rats did not show any erosions C) PC rats showed epithelium erosions
and stratification indicated by an arrow D) Compound 6d treated animals with intact intestinal epithelium
Although, Meloxicam is a potent nonꢀsteroidal antiꢀinflammatory drug (NSAIDs) being used in
AD, it is not gastrointestinal (GI) safe. NSAIDsꢀinduced gastroenteropathy is a serious risk
against the usage of such NSAIDs. It is essential to note that, in present study, while GI adverse
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events were observed in all the NSAIDs (Diclofenac, Celecoxib and Meloxicam)
, compound
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d despite having proportional antiꢀinflammatory activity demonstrated excellent GI safety in
comparison to standard drug Meloxicam.
Conclusion
th
AD has become a major burden worldwide and it is the 6 leading cause of death in US
population. The currently available drugs do not possess disease modifying potential, which is
the major drawback for AD treatment. Even though drugs targeting βꢀamyloid plaques have
recently emerged to be a useful tactic to get the disease modifying benefits, the reported antiꢀ
amyloid moieties have failed during clinical trials. Hence, to target the multiꢀfactorial nature of
AD, and to overcome the drawbacks of single targeted drugs; multitarget drug design approach
seems to be an appropriate strategy in quest for new antiꢀAlzheimer’s agents. Inflammation is
another major hallmark for AD pathogenesis. Antiꢀinflammatory activity may hold the greater
potential in AD treatment by reducing the oxidative damage and plaque formation. Thus to
achieve the disease modifying benefits, BACEꢀ1 inhibitory and antiꢀinflammatory activities are
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conjoined in one single molecule. We designed thiazolylꢀthiadiazines as MTDLs for the first
time, acting on BACEꢀ1 and inflammation simultaneously. In silico docking studies suggested
the multitargeted profile of these molecules. They were found to interact with enzyme’s catalytic
domain of BACEꢀ1, COXꢀ1 and COXꢀ2. These MTDLs showed effective results in in vitro
BACEꢀ1 inhibition assay and in vivo acute and chronic antiꢀinflammatory studies. One of the
molecules; compound 6d showed promising result in behavioral studies conducted in rats.
Compound 6d was found to have excellent antiꢀoxidant properties. It was demonstrated to have
antiꢀamyloid potency and neuroprotective effect which is an advantageous feature of these
studies. The results are encouraging and may lead to the development of disease modifying
treatment for AD. Furthermore, the GI safety profile of the molecule offers excellent advantage
in relation to the oral toxicity over NSAIDs.
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Our future endeavor is to bring this multifaceted category of molecules to the forefront of
therapeutic agents for the management of AD and many other diseases as well and emerge as a
forerunner in this field. Translation of this knowledge is eagerly awaited.
Materials & methods
Reagents and solvents were purchased from commercial suppliers and were used without further
purification. Melting points were determined using scientific melting point apparatus (Veego;
Model VMPꢀDS) and are uncorrected. FTꢀIR spectra were obtained using IR Affinity 1 by
Shimadzu. Mass spectra were recorded using LCꢀESIꢀMS (Perkin Elmer mass spectrometer).
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HRMS spectral analysis was done by waters QꢀTOF micromass (ESIꢀMS). H NMR and C
NMR samples analysis were carried out using Bruker Avance II 400 MHz NMR
spectrophotometer with chemical shift values represented in parts per million. Signals are
described as s (singlet), d (doublet), t (triplet), q (quartet), m (multiplet) and dd (doublet of
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doublets). Reaction monitoring was done by thin layer chromatography (TLC, Merck, Preꢀ
coated). TLC spot visualization was done under UV light using UV chamber. Purity of
compounds was determined by using SHIMADZUꢀLCꢀ2010 HPLC. Schrödinger software was
used for in silico docking and physical properties prediction. Fluorimetric BACEꢀ1 inhibition
assay readings were taken from Varioskan® Flash multimode plate reader. Paw volumes were
recorded using manual mercury plethysmometer. Polytron homogenizer (Kinematica,
Switzerland) was used for homogenization. Centrifugation was carried out using Sorvall Legend
X1R centrifuge (Thermo Scientific). Samples were vortexed in Vortex Mixer (Eltek, VM301).
UVꢀVisible absorbance of the samples were taken using UV/Visible Spectrophotometer
Shimadzu UVꢀ1800. Serum albumin and total protein were determined using fully automated
random access clinical chemistry analyser Transasia EM 360. Haemoglobin and haematocrit
level estimation were obtained using VetScan HMꢀ5. Histopathological slides were observed
under optical microscope ProgRes C3 OLYMPUS, UꢀTV1 X, Japan.
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Experimental procedures
1
) Docking studies
Docking studies were carried out using Maestro 11.1.012 software of Schrödinger, LLC,
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New York, NY, 2017, USA.
Ligand preparation: Library of molecules was generated and all the structures were drawn
and cleaned up in the Maestro software 2D sketcher. Ligprep module was used for the energy
minimization of all the structures using OPLS3 force field and possible states were obtained
by using Epik (pH 7.0). Results of the Ligprep studies were taken further for docking
process. The general structure for library of thiazolylꢀthiadiazines is given in Figure S1.
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Different R and R substituents were taken for docking process.
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Protein preparation
BACEꢀ1 protein preparation: Docking studies were performed using BACEꢀ1 crystal
structure from protein data bank. PDB ID: 4B05 was retrieved from protein data bank which
contains complex structure of BACEꢀ1 with the bound ligand AZD3839 (clinical
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candidate). Protein structure was prepared using protein preparation wizard of Schrödinger
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1.1.012 software. Bond orders were assigned and hydrogens were added to the structure.
Zero order bonds to metals and disulfide bonds were added. Missing side chains were added
using prime and water molecules within 5 Å of het groups were removed. All the ions were
deleted and protein structures were optimized using OPLS3 force field and minimized for
receptor grid generation. Grid was obtained at the centroid of the ligand (AZD3839) of
BACEꢀ1 receptor.
BACEꢀ2 protein preparation: Crystal structure of human BACEꢀ2 in complex with a
hydroxyethylenamine transitionꢀstate inhibitor was retrieved from the protein data bank
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(
PDB ID: 2EWY). Protein preparation was carried out using the same protocol as described
in BACEꢀ1 protein preparation. Chain A was used for the docking study.
Cathepsin D protein preparation: Crystal structure of Cathepsin D (PDB ID: 1LYW) was
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retrieved from protein data bank. Protein was prepared using protein preparation wizard by
adding bond orders, hydrogens and removing water within 5 Å of het groups. Protein
structure was optimized and minimized. Binding site generation was carried out by using
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SiteMap module of Schrödinger. Top ranked potential binding sites were identified by
using SiteScore. Highest ranked binding site was selected for receptor grid generation. Grid
was generated at the binding site pocket of the enzyme and used for the docking process.
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COXꢀ1 protein preparation: Crystal structure of COXꢀ1 in complex with Celecoxib (PDB
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ID: 3KK6) was used for the docking process. All the steps for the protein preparation were
followed as described in BACEꢀ1 protein preparation protocol.
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COXꢀ2 protein preparation: Structure of COXꢀ2 with coꢀcrystallized Celecoxib ligand
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(
PDB ID: 3LN1) was used for the docking process. All the steps for the COXꢀ2 protein
preparation were followed as per the given protocol of BACEꢀ1 protein preparation using
chain A of the enzyme.
Docking study: Ligand docking was performed using Glide module of Schrödinger suite.
Extra precision (XP) mode was used for the docking of all the molecules with proteins.
Docking score and interactions of ligands with protein were taken into consideration for
predicting activity of the molecules. Validation of docking process was done by re docking
of coꢀcrystallized ligand with the proteins.
2) Physicochemical properties prediction
Prediction of physicochemical properties of the compounds was done using QikProp
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application of Schrödinger. Properties like brain/blood partition coefficient (QPlogBB),
octanol/water partition coefficient (QPlogPo/w), Cacoꢀ2 cell permeability (QPPCaco), %
human oral absorption (HOA), HERG inhibition (QPlogHERG) were taken into
consideration for the brain permeability and toxicity prediction of the molecules.
3
) Chemistry
General procedure A: Synthesis of substituted hydrazinecarbothioamides (3aꢀe)
To the solution of substituted amines 1aꢀe (1.0 eq) in THF, addition of triethylamine (3.2 eq)
and carbon disulfide was done at 0ꢀ5 °C. Reaction was allowed to stir for 6ꢀ7 hr. Formation
of dithiocarbamate salt was confirmed by TLC (Methanol: Hexane: Ethyl acetate =
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.5:3:1.5). It was filtered off and dried. Salt was dissolved in chloroform and addition of
triethylamine (1.0 eq) and ethyl chloroformate (1.1 eq) was done at 0ꢀ5 °C and continued to
stir at room temperature (RT) for 2 hr. Reaction was monitored by TLC (Hexane: Ethyl
acetate = 1:4). After completion of reaction, 3M HCl was added to the reaction mixture.
Chloroform layer was separated and washed with water (3×50 ml). Chloroform was
evaporated under vacuo and dried over sodium sulfate to yield 2aꢀe. Purification of 2aꢀe was
done by column chromatography using 100% hexane as a mobile phase.
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After that, 2aꢀe (1.0 eq) was reacted with hydrazine hydrate in THF at RT for 2 hr. Reaction
was monitored by TLC (Hexane: Ethyl acetate = 3:2). THF was evaporated under vacuo and
crystallized using methanol to get pure white product of 3aꢀe.
Nꢀ(4ꢀmethoxyphenyl)hydrazinecarbothioamide (3a)
Synthesis of intermediate 2a was done using starting material 4ꢀmethoxyaniline 1a. 3a was
synthesized using 1ꢀisothiocyanatoꢀ4ꢀmethoxybenzene 2a and hydrazine hydrate as described
in general procedure A. Colorless solid; Yield: 81%; Melting point: 162ꢀ164 °C; R : 0.30
f
+
(Hexane: Ethyl acetate = 3:2); LCꢀESIꢀMS (m/z): 198.1 [M+H]
Nꢀ(4ꢀ(trifluoromethyl)phenyl)hydrazinecarbothioamide (3b)
Synthesis of intermediate 2b was done using starting material 4ꢀ(trifluoromethyl)aniline 1b.
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b was synthesized using 1ꢀisothiocyanatoꢀ4ꢀ(trifluoromethyl)benzene 2b and hydrazine
hydrate as described in general procedure A. Colorless solid; Yield: 83%; Melting point:
+
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59ꢀ161 °C; R : 0.32 (Hexane: Ethyl acetate = 3:2); LCꢀESIꢀMS (m/z): 236.0 [M+H]
f
Nꢀ(pꢀtolyl)hydrazinecarbothioamide (3c)
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Synthesis of intermediate 2c was done using starting material pꢀtoluidine 1c. 3c was
synthesized using 1ꢀisothiocyanatoꢀ4ꢀmethylbenzene 2c and hydrazine hydrate as described
in general procedure A. Colorless solid; Yield: 79%; Melting point: 165ꢀ167 °C; R : 0.28
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Nꢀphenylhydrazinecarbothioamide (3d)
Synthesis of intermediate 2d was done using starting material aniline 1d. 3d was synthesized
using isothiocyanatobenzene 2d and hydrazine hydrate as described in general procedure A.
Colorless solid; Yield: 72%; Melting point: 173ꢀ175 °C; R : 0.26 (Hexane: Ethyl acetate =
f
+
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:2); LCꢀESIꢀMS (m/z): 168.2 [M+H]
Nꢀ(4ꢀfluorophenyl)hydrazinecarbothioamide (3e)
Synthesis of intermediate 2e was done using starting material 4ꢀfluoroaniline 1e. 3e was
synthesized using 1ꢀfluoroꢀ4ꢀisothiocyanatobenzene 2e and hydrazine hydrate as described in
general procedure A. Colorless solid; Yield: 71%; Melting point: 184ꢀ186 °C; R : 0.31
f
+
(Hexane: Ethyl acetate = 3:2); LCꢀESIꢀMS (m/z): 186.2 [M+H]
General procedure B: Synthesis of substituted 1ꢀ(2ꢀaminoꢀ4ꢀphenylthiazolꢀ5ꢀyl)ꢀ2ꢀ
chloroethanones (5aꢀd)
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To a solution of N,Nꢀdiethylbenzimidamide (1.0 eq) in ethyl acetate, isothiocyanatobenzene
(1.0 eq) 2cꢀe was added and stirred at RT for 3ꢀ4 hr. Progress of reaction was monitored by
TLC (Hexane: Ethyl acetate = 7:3). Yellowish solid was filtered off, washed with hexane and
dried to yield pure substituted N,NꢀdiethylꢀN'ꢀ(phenylcarbamothioyl)benzimidamide 4aꢀd.
After that, 1,3ꢀDichloropropanꢀ2ꢀone (1.5 eq) in acetonitrile was taken into round bottom
flask and 4aꢀd (1.0 eq) was added slowly. Reaction was allowed to stir for 3ꢀ4 hr at RT.
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Progress of reaction was checked by TLC. (Hexane: Ethyl acetate = 7:3) and upon
completion of reaction, reaction mixture was poured into ice cold water to get yellow
precipitates of 5aꢀd. Pure 5aꢀd were obtained by crystallization using methanol.
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ꢀChloroꢀ1ꢀ(4ꢀphenylꢀ2ꢀ(pꢀtolylamino)thiazolꢀ5ꢀyl)ethanone (5a)
Intermediate 4a was synthesized using starting material 1ꢀIsothiocyanatoꢀ4ꢀmethylbenzene
c. Synthesis of 5a was done using N,NꢀdiethylꢀN'ꢀ(pꢀtolylcarbamothioyl)benzimidamide 4a
and 1,3ꢀDichloropropanꢀ2ꢀone as described in general procedure B. Brown solid; Yield:
9%; Melting point: 171ꢀ173 °C; R : 0.50 (Hexane: Ethyl acetate = 3:2); LCꢀESIꢀMS (m/z):
2
6
f
+
+
342.5 [M] , 344.5 [M+2]
2
ꢀChloroꢀ1ꢀ(4ꢀphenylꢀ2ꢀ(phenylamino)thiazolꢀ5ꢀyl)ethanone (5b)
Intermediate 4b was synthesized using starting material isothiocyanatobenzene 2d. Synthesis
of 5b was done using N,NꢀdiethylꢀN'ꢀ(phenylcarbamothioyl)benzimidamide 4b and 1,3ꢀ
Dichloropropanꢀ2ꢀone as described in general procedure B. Brown solid; Yield: 75%;
Melting point: 167ꢀ169 °C; R : 0.48 (Hexane: Ethyl acetate = 3:2); LCꢀESIꢀMS (m/z): 328.5
f
+
+
[
M] , 330.5 [M+2]
2
ꢀChloroꢀ1ꢀ(2ꢀ((4ꢀfluorophenyl)amino)ꢀ4ꢀphenylthiazolꢀ5ꢀyl)ethanone (5c)
Intermediate 4c was synthesized using starting material 1ꢀFluoroꢀ4ꢀisothiocyanatobenzene
e. Synthesis of 5c was done using N,NꢀdiethylꢀN'ꢀ((4ꢀ
2
fluorophenyl)carbamothioyl)benzimidamide 4c and 1,3ꢀDichloropropanꢀ2ꢀone as described
in general procedure B. Brown solid; Yield: 75%; Melting point: 177ꢀ179 °C; R : 0.52
f
+
+
(
Hexane: Ethyl acetate = 3:2); LCꢀESIꢀMS (m/z): 346.5 [M] , 348.5 [M+2]
2ꢀChloroꢀ1ꢀ(2ꢀ((4ꢀchlorophenyl)amino)ꢀ4ꢀphenylthiazolꢀ5ꢀyl)ethanone (5d)
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Intermediate 4d was synthesized using starting material 1ꢀChloroꢀ4ꢀisothiocyanatobenzene
2
f. Synthesis of 5d was done using N'ꢀ((4ꢀchlorophenyl)carbamothioyl)ꢀN,Nꢀ
diethylbenzimidamide 4d and 1,3ꢀDichloropropanꢀ2ꢀone as described in general procedure
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B. Brown solid; Yield: 65%; Melting point: 183ꢀ185 °C; R : 0.53 (Hexane: Ethyl acetate =
f
+
+
+
3
:2); LCꢀESIꢀMS (m/z): 346.5 [M] , 348.5 [M+2] , 350.5 [M+4]
General procedure C for final compounds: Synthesis of substituted 5ꢀ(2ꢀaminoꢀ4ꢀ
phenylthiazolꢀ5ꢀyl)ꢀ6Hꢀ1,3,4ꢀthiadiazinꢀ2ꢀamines (6aꢀg & 8aꢀc)
To a solution of substituted hydrazinecarbothioamides 3aꢀe and hydrazinecarbothioamide 7a
(1.2 eq) in THF, addition of substituted 1ꢀ(2ꢀaminoꢀ4ꢀphenylthiazolꢀ5ꢀyl)ꢀ2ꢀchloroethanones
5aꢀd (1.0 eq) was done. Reaction mixture was allowed to stir for 6ꢀ7 hr at 70 °C. Reaction
was monitored by TLC (Hexane: Ethyl acetate = 7:3). Reaction mixture was poured into cold
water to get precipitates of 6aꢀg & 8aꢀc. Compounds were purified by column
chromatography to yield pure compounds. HPLC purity was determined using SHIMADZUꢀ
LCꢀ2010 [Mobile phase (10 mM): KH PO buffer: Acetonitrile (50:50); Column: Reverse
2
4
phase; pH: 7]
Nꢀ(4ꢀmethoxyphenyl)ꢀ5ꢀ(4ꢀphenylꢀ2ꢀ(pꢀtolylamino)thiazolꢀ5ꢀyl)ꢀ6Hꢀ1,3,4ꢀthiadiazinꢀ2ꢀ
amine (6a)
Final
compound
6a
was
synthesized
3a
using
and
starting
material
Nꢀ(4ꢀ
methoxyphenyl)hydrazinecarbothioamide
2ꢀChloroꢀ1ꢀ(4ꢀphenylꢀ2ꢀ(pꢀ
tolylamino)thiazolꢀ5ꢀyl)ethanone 5a as described in general procedure C. Pale yellow solid;
Yield: 81%; Purity: 99.8%; Melting point: 177ꢀ179 °C; Molecular formula: C H N OS ;
26
23
5
2
+
+
LCꢀESIꢀMS (m/z): 486.1 [M+H] ; HRMS (TOF) m/z calcd for C H N OS [M+H]
2
6
23
5
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ꢀ1
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4
86.1344, found: 486.1142; IR (KBr, cm ): 3319.49, 2908.65, 1608.63, 769.60; H NMR
(
400MHz, DMSOꢀd ) δ: 2.254 (s, 3H, CH ), 3.224 (s, 2H, CH ), 3.718 (s, 3H, CH O), 6.865
6 3 2 3
(d, J = 8.8 Hz, 2H, meta to CH O), 7.131 (d, J = 8.4 Hz, 2H, ortho to CH O), 7.447ꢀ7.495
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(
m, 4H, CH ꢀC H ), 7.532 (d, J = 8.8 Hz, 2H, C H ), 7.568ꢀ7.591 (m, 3H, C H ), 10.439 (s,
3 6 4 6 5 6 5
D O exchangeable, 1H, NH linked to thiazole), 10.452 (s, D O exchangeable, 1H, NH linked
2
2
13
to thiadiazine); C NMR (400 MHz, DMSOꢀd ) δ: 20.42 (CH ), 29.56 (CH ), 52.51 (CH ),
6
3
2
3
1
1
16.40 (2×CH, CH OꢀC H ), 117.47 (2×CH, CH OꢀC H ), 118.35 (2×CH, CH ꢀC H ),
3 6 4 3 6 4 3 6 4
22.03 (2×CH, C H ), 124.78 (2×CH, CH ꢀC H ), 128.12 (CH, C H ), 128.50 (CH, CH ꢀ
6
5
3
6
4
6
5
3
C H ), 128.96 (CH, CH ꢀC H ), 129.24 (C, C H ), 129.56 (C, CH OꢀC H ), 129.87 (C,
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3
6
4
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5
3
6
4
thiazole), 134.81 (C, C H ), 140.11 (C, CH OꢀC H ), 141.36 (C, CH OꢀC H ), 151.93 (C,
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6
4
3
6
4
thiazole), 157.45 (C, thiadiazine), 160.82 (C, thiazole), 170.71 (C, thiadiazine)
ꢀ(4ꢀPhenylꢀ2ꢀ(pꢀtolylamino)thiazolꢀ5ꢀyl)ꢀNꢀ(4ꢀ(trifluoromethyl)phenyl)ꢀ6Hꢀ1,3,4ꢀ
thiadiazinꢀ2ꢀamine (6b)
Final compound 6b
trifluoromethyl)phenyl)hydrazinecarbothioamide 3b and 2ꢀChloroꢀ1ꢀ(4ꢀphenylꢀ2ꢀ(pꢀ
5
was
synthesized
using
starting
material
Nꢀ(4ꢀ
(
tolylamino)thiazolꢀ5ꢀyl)ethanone 5a as described in general procedure C. Pale yellow solid;
°
Yield: 64%; Purity: 99.9%; Melting point: 165ꢀ167 C; Molecular formula: C H F N S ;
26 20
3
5 2
+
+
LCꢀESIꢀMS (m/z): 524.1 [M+H] ; HRMS (TOF) m/z calcd for C H F N S [M+H]
2
6
20
3
5 2
ꢀ1
1
5
24.1112, found: 524.1035; IR (KBr, cm ): 3159.40, 2916.37, 1508.33, 769.60; H NMR
(
400MHz, DMSOꢀd ) δ: 2.259 (s, 3H, CH ), 3.274 (s, 2H, CH ), 7.148 (d, J = 8.4 Hz, 2H,
6
3
2
meta to CF ), 7.469ꢀ7.479 (m, 3H, CH ꢀC H & C H ), 7.538 (d, J = 8.4 Hz, 2H, ortho to
3
3
6
4
6
5
CH ), 7.583ꢀ7.593 (m, 2H, CF ), 7.643 (d, J = 8.4 Hz, 4H, C H ), 9.789 (s, D O
3
3
6
5
2
exchangeable, 1H, NH linked to thiazole), 10.488 (s, D O exchangeable, 1H, NH linked to
2
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