2-Aminothiazole derivatives as antimycobacterial agents: Synthesis, characterization, in vitro and in silico studies.

Accepted Manuscript 2-Aminothiazole Derivatives as antimycobacterial agents: Synthesis, characterization, in vitro and in silico studies Parameshwar Makam, Tharanikkarasu Kannan PII:

S0223-5234(14)00914-3

DOI:

10.1016/j.ejmech.2014.09.086

Reference:

EJMECH 7394

To appear in:

European Journal of Medicinal Chemistry

Received Date: 21 February 2014 Revised Date:

21 June 2014

Accepted Date: 9 September 2014

Please cite this article as: P. Makam, T. Kannan, 2-Aminothiazole Derivatives as antimycobacterial agents: Synthesis, characterization, in vitro and in silico studies, European Journal of Medicinal Chemistry (2014), doi: 10.1016/j.ejmech.2014.09.086. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT

Graphical abstract 2-Aminothiazole derivatives as antimycobacterial agents: Synthesis, characterization, in

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vitro and in silico studies

A library of 2-aminothiazole derivatives were designed, synthesized and evaluated against

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Mycobacterium tuberculosis, H37Rv.

ACCEPTED MANUSCRIPT 2-Aminothiazole derivatives as antimycobacterial agents: Synthesis, characterization, in

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vitro and in silico studies

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Parameshwar Makam, Tharanikkarasu Kannan*

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Department of Chemistry, Pondicherry University, Puducherry-605 014, India.

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*

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E -mail address: [email protected]; [email protected]

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Abstract: A series of 2-aminothiazole derivatives with a wide range of substitutions at 2-, 4-

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and 5- positions were designed and synthesized using Hantzsch thiazole synthesis. These

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compounds were evaluated for their inhibitory potential against Mycobacterium tuberculosis

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(Mtb), H37Rv. The compound, 7n showed high antimycobacterial activity with MIC value of

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6.25 µM and the succeeding compounds, 7b, 7e and 7f also exhibited antimycobacterial

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activity with MIC value of 12.50 µM. Docking studies of these molecules with β-Ketoacyl-

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ACP Synthase (KasA) protein of Mtb have been carried out to understand the mechanism of

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antimycobacterial action. The compound, 7n showed good interaction with KasA protein

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with the Ki value of 0.44 µM.

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Key words: Mycobacterium tuberculosis; tuberculosis; 2-aminothiazole; drug like molecule;

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β-Ketoacyl-ACP Synthase; docking studies

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Corresponding author. Tel: +91-413-265 4708; Fax: +91-413-265 6740.

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1. Introduction Tuberculosis (TB) is one of the world’s leading airborne contagious diseases that is mainly caused by Mycobacterium tuberculosis (Mtb), a species of genus Mycobacterium.

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According to the eighteenth global TB report of the World Health Organization (WHO), it

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was estimated that 1.3 million people died out of 8.6 million new infections in 2012 [1].

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Although the death rate declined 45 % between 1990 and 2012, TB is next only to HIV/AIDS

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in killing the highest numbers of people through single infectious disease. Whilst TB is an

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ancient disease, its pathogen, Mtb was discovered in 1882 and the chemotherapy for this

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disease was started in 1944 using streptomycin [2]. In the following years, the first line drugs

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such as isoniazid, ethambutol, rifampicin, etc., and the second line drugs such as

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ethionamide, p-amino salicylic acid, cycloserine, kanamycin, etc., have been used to treat TB

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either independently or in combination with other drugs [3]. But, after 1950s, the rate in

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developing novel and effective drugs was slow [4-6]. As a result, there is no sufficient

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number of anti-tuberculosis drugs in the market. Due to this, TB patients still depend on the

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drugs discovered in 1950s [7]. In addition, the constant emergence of multi, extensively and

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totally drug – resistant strains of Mtb, and close synergy with HIV/AIDS [8] have made the

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goal of wiping out TB from this world difficult. Poverty, complexity of the disease,

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prolonged and poor administration of drugs are added reasons for the resurgence of TB as the

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major global health burden [9-12], particularly in high TB burden countries [13].

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To find out a solution to this alarming increase of TB infections, many attempts have been made to understand the reasons behind the evolution and existence of resistant strains of

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Mtb [14-16]. On the other hand, the generation and high-throughput screening of large

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chemical entities with a wide spectrum of known and novel scaffolds have also been carried

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out to identify lead anti-TB molecules with high efficacy, unique and novel mode of action.

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[7, 17-20]. Among the diverse and medicinally relevant scaffolds, 2-aminothiazole

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derivatives have exhibited good activity against Mtb, H37Rv [17, 21, 22]. Interestingly, 2-

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aminothiazole derivatives are found to be structurally similar to thiolactomycin (TLM) [23] ,

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a β-Ketoacyl-ACP Synthase (KasA) protein inhibitor [24]. TLM inhibits the biosynthesis of

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mycolic acid, an essential cell wall component of Mtb, by inhibiting KasA protein [25].

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Hence, it is expected that 2-aminothiazole derivatives may also inhibit growth of Mtb by

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inhibiting biosynthesis of mycolic acid. 2-Aminothiazole derivatives have been validated

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recently against UDP-galactopyranose mutase thwart to inhibit Mtb and found to have

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ACCEPTED MANUSCRIPT improved activities more than their parent thiazolidinone derivatives [26]. Nitazoxanide [27,

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28], 2-aminothiazole-4-carboxylates and 2-amino-4-(2-pyridyl) thiazoles are other molecules

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of this class now in clinical evaluations [19]. In addition to these, 2-aminothiazole scaffold is

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attracting medicinal chemists continuously by being an integral part of drugs which are in

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clinical practice for the treatment against tumor and cancer [29, 30], hypertension [31],

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inflammation [32], analgesics [33], hypnotics [34], schizophrenia [35], HIV/AIDS [36],

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malaria [37, 38] and microbial infections [39], to mention a few.

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Limited exploration of 2-aminothiazole derivatives against Mtb and the possibility of generating novel and potent Mtb inhibitors from this class encouraged us. In our previous

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studies, we have synthesized novel 2-(2-hydrazinyl) thiazole derivatives comprising 2-

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aminothiazole scaffold and evaluated for their inhibitory potentials against Mtb, H37Rv [40]

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and Plasmodium falciparum, NF54 [41]. The good results obtained in these studies

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encouraged us to go on to synthesize novel 2-aminothiazole derivatives. Hence, in the present

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study, novel 2-aminothiazole derivatives are designed with a wide range of substitutions at

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2-, 4- and 5- positions and evaluated their inhibitory potentials against Mtb by in vitro assays.

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In addition, with the help of ligand – protein interaction studies through docking studies, an

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insight into the mechanism of action has also been carried out against KasA protein.

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2. Results and Discussion

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2.1. Rational design

The physico-chemical properties play a vital role in defining drug like molecule

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(DLM) character and pharmacokinetics properties such as absorption, distribution,

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metabolism, excretion and toxicity (ADMET) of a molecule [42, 43]. Certainly, the necessary

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physico - chemical properties and their importance in the early drug discovery are best

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explicated by the works of Lipinski [44] and others [43, 45, 46]. These rules state that the

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molecule with a molecular mass less than 500, Log P value less than 5, hydrogen bond

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donors less than 5, hydrogen bond acceptors less than 10, polar surface area less than 140 and

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the number of rotatable bonds less than 10 could be a molecule with DLM character.

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In our previous investigation, 2-(2-hydrazinyl) thiazole derivatives have been designed without violating rule of five and synthesized. The synthesized compounds have

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been evaluated for their inhibitory potential against Mtb, H37Rv and found that some of these

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compounds show inhibitory potential against Mtb, H37Rv. As most of the anti-TB and

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antibacterial compounds are exceptions to the rule of five [20, 45], in the present

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investigation, we intentionally increased the lipophilicity of 2-aminothiazole derivatives by

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designing compounds with the Log P values beyond 5. While designing, the structural

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features of reported anti-TB molecules have been considered as shown in Figure 1. An

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expectation during the designing is that the molecules with a defined optimal range of the

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lipophilicity could influence in identifying molecules with high therapeutic success by

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monitoring the ADMET properties of the molecules [47-49]. Bedaquiline, the only anti-TB

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drug that has been approved by FDA in the last 40 years for drug-resistant tuberculosis

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treatment is also a lipophilic drug [20]. Hence, by considering the cell wall permeability and

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the solubility, during designing of 2- aminothiazole derivatives, core 2-aminothiazole

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scaffold has been retained and additionally, substituted phenyl, naphthalene and positional

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isomeric pyridyl systems have been incorporated on either side of 2-aminothiazole, i.e., at

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both 2- and 4- positions of the thiazole ring to induce lipophilicity and hydrophilicity

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correspondingly. In addition to this, the H at 5- position has been replaced with other

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functional groups such as CH3 and Br to understand the effect of this position on

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antimycobacterial activity.

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The physico – chemical properties of the designed 2-aminothiazole derivatives have

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been determined and the results are summarized in Table 1. The data reveal that all the

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compounds follow the Lipinski rule of five except Log P values. As mentioned earlier, Log P

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values of N-phenyl thiazol-2-amine derivatives (7a-7e, 7g, 7i-j and 7q-7s) and N-2-pyridyl

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thiazol-2-amine derivatives (8c-j) have been intentionally maintained higher than 5. In

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addition to these, the other parameters such as polar surface area and number of rotatable

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bonds are found to be in the range of 24.919 - 83.63 and 3 - 6 respectively that are in the

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range of recommended values.

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2.2. Synthesis of 2-aminothiazole derivatives

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The designed 2-aminothiazole derivatives have been synthesized according to the synthetic pathways described in Scheme 1. First, arylthioureas were synthesized by refluxing 4

ACCEPTED MANUSCRIPT corresponding anilines with ammonium thiocyanate in the presence dil. HCl [50] as shown in

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Scheme 1 (i). But, pyridyl thioureas could not be achieved with the same procedure and

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hence, an alternative procedure was used [51]. In this procedure, first, 2-pyridylamine,

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ammonium thiocyanate and benzoyl chloride were reacted in acetone to yield N-(pyridin-2-

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ylcarbamothioyl) benzamide. This was further hydrolyzed with 10% NaOH solution to yield

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the corresponding pyridyl thioureas [51] as shown in Scheme 1 (ii). The phenyl and the

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pyridyl thioureas have been characterized by the spectral methods and the data were found to

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be in good agreement with the literature data [50-52].

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In the next step, pyridyl α-bromo ketones have been synthesized from corresponding acetyl pyridines [51] as shown in Scheme 1 (iii). Finally, the aryl or pyridyl thioureas have

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been heterocyclized to thiazole by reacting with the aryl or pyridyl α-halo ketones in the

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presence of ethanol using the classical Hantzsch thiazole procedure [51] as shown in Scheme

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1 (iv). All the synthesized compounds have been characterized using nuclear magnetic

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resonance (NMR), infra-red (IR) and mass spectroscopic techniques. The aromatic protons

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present in the pyridyl and phenyl systems are observed at 6.5 – 9 ppm. The appearance of the

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characteristic peak of thiazole ring H at around 6.50 ppm and the disappearance of the

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characteristic peaks of reactants in the 1H NMR spectra confirm the formation of the thiazole

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ring. The characteristic peak of the NH at the 2nd position of the thiazole ring resonates at

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around the 10.5 to 12 ppm. Similarly, the precise matching of the experimental mass and

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CHNS data with the calculated data further confirms the formation of analytically pure

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desired products. All the synthesized 2-aminothiazole derivatives have been classified into

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four groups based on the structural similarities of the substitutions at the 2nd and 4th positions

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on the 2-aminothiazole scaffold as shown in Table 2. In the first class of compounds,

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substituted phenyl ring systems are attached on both 2nd and 4th positions on the 2-

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aminothiazole scaffold. The spectral data of the compounds, 7a [53], 7b [53], 7f [54] 7g [55,

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56] and 7h [55] are in agreement with the literature reports. In the second class, the

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substituted 2-pyridyl and phenyl systems are incorporated on the 2nd and 4th positions of the

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2-aminothiazole scaffold respectively. Among this class of compounds, the compounds, 7l

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[51], 7o [51, 55] and 7p [55] are reported and the data is in agreement with the reported

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literature. Similarly, in the third class of compounds, substituted pyridyl and phenyl systems

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are incorporated on the 4th and 2nd positions of the 2-aminothiazole scaffold respectively.

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The fourth class of compounds includes substituted pyridyl systems on both 2nd and 4th

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positions of the 2-aminothiazole scaffold. The spectral data of the reported compounds, 8l

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[57] and 8n [58] are in agreement with the literature.

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2.3. Single crystal and crystal packing studies The spatial arrangement of the drug molecules is an important factor and its

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understanding is more helpful in designing and identifying the better hit and lead drug

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molecules with enhanced biological activity [51, 59, 60]. The spatial arrangement of the

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molecules in its stable conformation can be identified by the single crystal X-ray studies. The

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present 2-aminothiazole derivatives contain commonly three aromatic cyclic systems in their

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structures viz. substituted phenyl, thiazole and pyridyl systems. Hence, based on the structural

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diversity of the molecules, 7g and 8l have been selected as the representative molecules of 2-

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aminothiazole derivatives. The molecule 7g contains both phenyl rings attached to 2nd and 4th

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positions of 2-aminothiazole ring and the molecule 8l contains both pyridyl rings attached to

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the 2nd and 4th positions of the 2-aminothiazole scaffold. The single crystal structures and

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crystal packing diagrams of 7g and 8l have been solved and the results are summarized in

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Table 3.

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The single crystal structure of 7g is represented in the ORTEP diagram with 40% ellipsoid probability and its crystal packing is depicted in Figure 2. The single crystal

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structure reveals that 7g is stabilized by its monohydrate form i.e., solvation by one water

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molecule. The alignment of the three planar rings of the molecule, the 4-methoxy phenyl ring

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systems attached to 4th positions of thiazole ring and the 2-aminothiazole ring are oriented

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almost in the same plane. Whereas, the 4-hydroxy phenyl system attached to NH of the 2-

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aminothiazole ring is perpendicular to the 2-aminothiazole ring (Figure 2a). The single

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crystals are oriented in the opposite directions i.e., head to tail dimer fashion and are

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stabilised by inter molecular hydrogen bonds. There are two inter molecular hydrogen

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bonding (2.284 Å, 159.98°) exist between -NH group present in the 2-aminothiazole ring of

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one molecule and the N present in the thiazole ring of other molecule and vice versa. In

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addition, π – π interactions also exist between two 4-hydroxy phenyl rings (2.844 Å, 2.915 Å)

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present in 7g. The solvent, water molecule is sandwiched between the two 7g molecules and

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it is stabilising the packing of the crystals by forming four inter molecular hydrogen bond

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interactions (2.05 Å, 126.51°; 2.04 Å, 126.41°) with two 7g molecules. The interaction

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between Cl atom present in 7g and H2O (2.844 Å, 139.05°) also exits. All the hydrogen

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bonding interactions and the orientation of the molecules in the crystal lattice are shown in

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Figure 2b and 2c along the c- and a-axis respectively. The single helix arrangement can also

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be visualised from the a-axis is shown in Figure 2c.

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The single crystal structure of 8l reveals that the three thiazole ring and the pyridyl

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ring systems attached to 2nd and 4th positions are aligned in same plane. Hence, the molecule

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is planar as shown in the single crystal structure in ORTEP diagram with 40% ellipsoid

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probability (Figure 3a). The crystal packing reveals that the two molecules of 8l are

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stabilised by the two intermolecular hydrogen bonds (2.101 Å, 167.91°) between the NH

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group present in the 2nd position of the thiazole ring and the N present in the 3-pydidyl ring

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and vice versa. The crystal packing of the molecules with two intermolecular hydrogen bonds

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along a- and b-axis is shown in Figure 3 b and c respectively. The alignment of 8l molecules

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in the layered and single helical fashion can be viewed along the a-axis as shown in Figure

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3b.

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2.4. Antimycobacterial activity and structure activity relationship (SAR) studies

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After successful completion of design, synthesis and structural confirmation, these

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four classes of 2-aminothiazole derivatives have been evaluated for their antimycobacterial

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activities by Resazurin microtiter assay (REMA), an in vitro assay against Mtb, H37Rv [40]. It

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is interesting to note that anti-TB activity results are varied based on the substitutions at 2nd,

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4th and 5th positions of thiazole ring. In the first category of compounds, firstly, 4-fluoro

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phenyl attached in the 2nd positions of the 2-aminothiazole scaffold kept constant and the

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substitutions at 4th position and 5th positions have been varied. The variations at 4th position

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i.e., 4-chloro phenyl system (7a), exhibits good activity with MIC value 25 µM. When the

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chlorine is replaced with bromine (7b), the potency increases to one fold with MIC value of

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12.5 µM. Interestingly, when the H at 5th position of the 7b is replaced with the methyl group

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(7c) and bromine (7d), the potentency drops down to the MIC values of 25 µM and 100 µM

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respectively. This clearly indicates the importance of the 5th position and its scope in

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identifying the successful candidates. Similarly, when the 4-bromo phenyl of 7b is replaced

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with 4-methoxy phenyl (7f) and naphthalene, the potential is retained with 12.5 µM. To

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understand the effect of phenyl derivatives attached on the 2nd positions of the 2-

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aminothiazole scaffold, 4-hydroxy and 2-nitro- 4-chloro phenyl derivatives have been

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prepared. When the fluorine present in the compounds 7a and 7f is replaced with 4-hydroxy

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group, the compounds 7g and 7h loses their potentials and exhibit MIC values 100 µM and

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200 µM respectively. Similarly, the compounds 7i and 7j, the 2-nitro- 4-chloro phenyl

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replaced derivatives of 7f and 7e show no activity with MIC value of >200 µM and this result

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shows the importance of fluorine at 4th position. In the second category of compounds, the substituted phenyl systems at 2nd position

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are same and phenyl systems at 4th position are replaced with pyridyl systems. Keeping the 3-

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pyridyl system intact at 4th position and varying the 2nd position with 4-flouro (7k), 4-bromo

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(7m), 4-hydroxy (7o) and 2-nitro- 4-chloro (7r) phenyl systems lead to formation of the

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compounds with no antimycobacterial activity (>200 µM). Interestingly, when 3-pyridyl

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system is replaced with 4-pyridyl system, the activity enhances in the case of 4-flouro (7l)

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and 4-bromo (7n) with MIC values 25 µM and 6.25 µM respectively. But, the compounds

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with 4-hydroxy (7p) and 2-nitro- 4-chloro (7s) phenyl systems show no activity. The 2-

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pyridyl systems with 2-nitro- 4-chloro phenyl (7q) do not make any difference in the activity.

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In the third class of compounds, the 2-pyridyl group is introduced at the 2nd position of the 2-aminothiazole. In addition to this, substituted phenyl systems at 4th position and

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methyl or bromine at 5th position have also been introduced to investigate the effect of 2-

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pyridyl system. Unfortunately, all these compounds have exhibited no inhibition with MIC

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value >200 µM against the Mtb, H37Rv. Similarly, to understand the effect pyridine systems

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on both sides of 2-aminothiazole scaffold, fourth category of compounds have been

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synthesized by keeping 3- or 4-pyridyl system at 4th position and 2-pyridyl derivative of NH

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at 2nd position. In this category, 8m and 8n inhibit Mtb with MIC value 100 µM and rest of

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the compounds are inactive with MIC value >200 µM.

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Overall, 4-flourophenyl system at 2nd position and H at 5th position of 2-aminothiazole

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are enhancing the activity in the first class of compounds. Similarly, in the second class of

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compounds, 4-flouro and 4-bromophenyl system on the 2nd position and 4-pyridyl systems on

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the 4th position are enhancing the activity. The structural changes in the third and fourth class

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of compounds did not influence much on enhancing the antimycobacterial activity. The

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structure activity relationship studies are summarised in the Figure 4. The effect of Log P on

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inhibition of Mtb also analysed for all the 34 compounds and the results are summarised in

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Figure 5. When the Log P value is increased from 3.08 to 3.46 (8n, 8f) there is no difference

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in antimycobacterial activity, but, when it increases to 3.71 (7l) the activity also increased.

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When the log P value is further increased to 4.33 (7g) the antimycobacterial activity

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decreased significantly. Further increase of Log P value up to 4.77 (7m, 7f) increases the 8


activity enormously. After this, the activity reduced when log P value reaches 5.09 (7e).

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From 5.54 to 6.35 (7d, 7a, 7i and 7b) the antimycobacterial activity is very good. Further

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increase from 6.35 to 6.64 (7c) decreases the activity. Overall, the antimycobacterial activity

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of 2-aminothiazole was found to be good when the Log P value is from 4.25 to 4.75 and from

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5.5 to 6.5.

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2.5. Docking studies with KasA protein

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The positive in vitro results encouraged us to study their possible mode of action through in silico methods using docking studies. As 2-aminothiazole derivatives are

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structurally similar to TLM and TLM is potential inhibitor of the KasA protein of Mtb, in the

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present study, KasA protein has been chosen as receptor target. KasA protein plays a vital

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role in the biosynthesis of mycolic acid, long chain fatty acids and the essential components

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of the Mtb cell wall [24]. Hence among all the 2-aminothiazole derivatives, the compounds,

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7b, 7n, 8a and 8n have been chosen as the representative compounds based on their in vitro

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activities and structural variations.

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As anticipated, the selected compounds have exhibited good interactions and inhibition constant (Ki) against KasA protein. The compounds, 7b, 7n, 8a and 8n have shown

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the Ki values of 1.28 µM, 0.44 µM, 30.20 µM and 30.19 µM with a binding energy of -8.04

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Kcal/mol, -8.66 Kcal/mol, -11.66 Kcal/mol and -11.81 Kcal/mol respectively. The inhibition

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of the protein by 7n is attained through hydrogen bonding interactions of N present in the 4-

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pyridyl ring present at the 4th position of the 2-aminothiazole ring with COOH of the

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phenylalanine (PHE) – 402. In addition to this, the NH present at the 2nd position of the 2-

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aminothiazole ring has shown the hydrogen bond interactions with threonine (THR) – 315

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and THR – 313 residues as shown in Figure 6. Hence, from the correlation of the results

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obtained by in vitro and in silico analyses, it may be concluded that the present molecules

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could be targeting the KasA protein and in turn disturbing the cell wall biosynthesis by

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obstructing mycolic acid synthesis.

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3. Conclusion

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The designed 2-aminothiazole derivatives with variations in the Log P values have

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been designed and synthesized. All the synthesized compounds have been evaluated for their

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inhibitory potentials against Mtb, H37Rv and their SAR studies have been carried out. Among 9

ACCEPTED MANUSCRIPT all the compounds, 4-flourophenyl group at 2nd position of the 2-aminothiazole ring system

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has been proven to be responsible for contributing the activity. The compound 7n has shown

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the highest inhibition with the MIC values of 6.25 µM and 0.44 µM by in vitro and in silico

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analyses respectively. The continuation of the efforts in identifying the new molecules with

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special focus on the present lead compounds to achieve improved antimycobacterial activities

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are under progress in our laboratory.

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4.

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4.1. Materials and methods

Experimental

The substituted anilines, 2-, 3- or 4- acetyl pyridines, benzoyl chloride and aryl α –

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haloketones were purchased either from Sigma–Aldrich, USA or Himedia Biosciences or

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Spectrochem Pvt. Ltd (Mumbai, India). The chemicals procured were used without further

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purification. The spectral data obtained by 1H NMR and 13C NMR were recorded with 400

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MHz and 101 MHz Bruker Avance-II NMR instrument. Thermo Nicolet 6700 FT-IR

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spectrometer was used to record the IR spectra and only major peaks are reported in cm-1.

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Similarly, Elementar Vario EL-II CHNS analyzer was used for the Elemental analysis of all

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the compounds. Chemical ionization (CI) or negative-ion electrospray ionization (ESI)

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method was used to record the mass spectra (MS) by Thermo Scientific High Resolution

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Magnetic Sector MS DFS. For the analysis of single crystals, Oxford diffractometer was used

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and CrysAlis PRO, Oxford Diffraction Ltd., Version 1.171.34.44 (release 25-10-2010

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CrysAlis171. NET) software has been used for the data collection, cell refinement and data

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reduction purposes. The olex2.solve (compiled Oct 25 2010, 18:11:34) and SHELXL have

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been used for solving the structure and to refine single crystal respectively. ChemBioDraw

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Ultra has been used to deduce all the IUPAC names for the synthesized compounds.

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4.2. General procedure for the synthesis of phenylthioureas (1a-d)

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anilines (0.1 mol) were added and warmed to get a clear solution. Ammonium thiocyanate

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(0.11 mol) dissolved in water (15 mL) was gradually added to this solution. The reaction

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mixture was refluxed till the completion of the reaction and then more than the half of the

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volume of the water present in the reaction mixture was distilled off. The phenyl thiourea was

To the dilute hydrochloric acid (15 mL) solution, the corresponding substituted

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obtained on gradual cooling of the reaction mixture, and it was filtered, washed with water

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and recrystallized from ethanol.

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4.3. General procedure for the synthesis of pyridylthioureas (3a-c)

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chloride (65 mmol) was added dropwise by maintaining temperature between 20 – 25 °C.

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Then the temperature of reaction mixture was raised to reflux temperature and stirred for 15

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to 30 min. Then, the reaction mixture was cooled to 25 °C and appropriate pyridyl amine (64

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mmol) was added gradually. The mixture was stirred at reflux temperature for a further 30

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min, cooled to 20 °C, and poured on to ice. The mixture was stirred for another 30 min., and

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then the precipitate was filtered, washed with water (15 mL) and dried. The solid was added

328

to a solution of NaOH (10% w/v, 100 mL) at 80 °C and continued stirring for 30 min, then

329

cooled to 20 °C and poured onto ice/HCl (6 M, 50 mL) solution. The pH of the mixture was

330

adjusted to 10 using conc. NH3 solution and stirred for 30 min. The precipitate was filtered

331

and washed with water (20 mL) and dried. The precipitate was purified by recrystallization

332

from ethanolic solution to afford the desired product in 85-95% yield.

M AN U

SC

RI PT

In dry acetone (50 mL), NH4SCN (69 mmol) was taken and to this solution, benzoyl

333

335 336

4.4. General procedure for the synthesis of (bromoacetyl)pyridines (5a-c)

TE D

334

Corresponding acetylpyridine (80 mmol) was taken in 30 % HBr/AcOH (100 mL) and to this stirred solution, Br2 (80 mmol) was added dropwise at 15 °C. The mixture was stirred

338

at 40 °C for 1 h and then at 75 °C for 1 h. Then, the reaction mixture was cooled to 20 °C.

339

The cooled reaction mixture was diluted with Et2O (400 mL), and continued to stir for 30

340

min. The precipitate obtained was filtered, washed with Et2O (25 mL), and dried under

341

vacuum to give the (bromoacetyl)pyridines as the hydrobromide salt [51].

AC C

342

EP

337

343 344 345

4.5. General procedure for the synthesis of titled 2-aminothiazole analogues (7a-8o)

346

solution, corresponding α-bromoacetylpyridine (1 mmol) or α-haloacetophenone (1 mmol)

347

derivative was added at 25- 30 °C. The reaction mixture was refluxed for 3-5 h or until the

348

reaction was complete as evidenced using TLC. Then, the reaction mixture was gradually

349

poured into ice-water (20 mL) and stirred for another 30 min. To this, aqueous 1N Na2CO3

350

solution was added to neutralise and to maintain pH ∼ 8. The precipitated aminothiazole

The appropriate thiourea (1 mmol) was dissolved in ethanol (∼10 mL) and to this

11


derivatives were filtered and collected. The crude 2-aminothiazoles were purified by

352

recrystallization from ethanol.

353 354

4.5.1. 4-(4-Chlorophenyl)-N-(4-fluorophenyl)thiazol-2-amine (7a) White solid from EtOH (88% yield). m.p. 170.3-171.5 °C; 1H NMR (400 MHz,

356

CDCl3): δ 7.75 – 7.73 (m, 1H), 7.48 – 7.36 (m, 2H), 7.13 (dd, J = 9.1, 8.1 Hz, 1H), 6.70 (s,

357

1H), 3.40 (s, 1H);

358

132.46, 129.58, 127.58, 123.68, 122.73, 122.64, 117.06, 117.06, 116.83, 116.83, 104.05; IR

359

(KBr): 3255, 3066, 1607, 1505, 1404, 1238, 1091, 841, 769 cm-1. Anal. Calcd. for

360

C15H10ClFN2S: C, 59.11; H, 3.31; N, 9.19; S, 10.52; found: C, 59.13; H, 3.37; N, 9.22; S,

361

10.53; HR-MS (ESI+): m/z [M + H]

362

02330.

C NMR (101 MHz, DMSO-d6): δ 167.43, 159.22, 145.12, 134.49,

364

Calcd. for C15H10ClFN2S: 304.02326; found: 304.

M AN U

363

+

SC

13

RI PT

355

4.5.2. 4-(4-Bromophenyl)-N-(4-fluorophenyl)thiazol-2-amine (7b) White solid from EtOH (86% yield). m.p. 179.5 – 180.5 °C; 1H NMR (400 MHz,

366

CDCl3): δ 7.67 (d, J = 8.7 Hz, 1H), 7.56 (d, J = 8.7 Hz, 1H), 7.40 (dd, J = 9.1, 4.5 Hz, 1H),

367

7.12 (dd, J = 9.1, 8.1 Hz, 1H), 6.72 (s, 1H). 13C NMR (101 MHz, CDCl3): δ 167.43, 159.22,

368

145.12, 134.49, 132.46, 129.58, 127.58, 123.68, 122.73, 122.64, 117.06, 116.83, 100.55; IR

369


370

C15H10BrFN2S: C, 51.59; H, 2.89; N, 8.02; S, 9.18; found: C, 51.57; H, 2.92; N, 8.04; S, 9.21;

371

HR-MS (ESI+): m/z [M + H] + Calcd. for C15H10BrFN2S: 347.97320; found: 347.97322.

TE D

365

373

4.5.3

EP

372

4-(4-Bromophenyl)-N-(4-fluorophenyl)-5-methylthiazol-2-amine (7c) White solid from EtOH (88% yield). m.p. 252.6-254°C; 1H NMR (400 MHz, DMSO-

375

d6): δ 7.67 (d, J = 8.7 Hz, 1H), 7.56 (d, J = 8.7 Hz, 1H), 7.40 (dd, J = 9.1, 4.5 Hz, 1H), 7.12

376

(dd, J = 9.1, 8.1 Hz, 1H), 6.72 (s, 1H), 2.45(s, 3H);

377

160.96, 158.81, 156.40, 141.84, 137.03, 132.91, 131.42, 130.25, 120.83, 119.88, 117.12,

378

115.65, 11.94; IR (KBr): 3448, 3074, 1621, 1594, 1506, 1230, 1002, 826, 645 cm-1. Anal.

379

Calcd. for C16H12BrFN2S: C, 52.90; H, 3.33; N, 7.71; S, 8.83; found: C, 52.92; H, 3.34; N,

380

7.70; S, 8.81; HR-MS (ESI+): m/z [M + H]

381

361.98890.

AC C

374

+

13

C NMR (101 MHz, DMSO-d6): δ

Calcd. for C16H12BrFN2S: 361.98886; found:

382 383

4.5.4. 5-Bromo-4-(4-bromophenyl)-N-(4-fluorophenyl)thiazol-2-amine (7d)

12


White solid from EtOH (92% yield). m.p. 258.7-259.5°C; 1H NMR (400 MHz,

385

DMSO-d6): δ 10.32 (s, 1H), 8.88 (d, J = 8.7 Hz, 2H), 8.42 (d, J = 8.7 Hz, 2H), 7.53 (d, J =

386

8.1Hz, 2H), 6.80 (d, J = 8.1 Hz, 2H);

387

152.22, 138.30, 136.91, 134.14, 128.84, 123.01, 118.54, 116.33, 94.05; IR (KBr): 3438,

388

3068, 1625, 1598, 1511, 1236, 1001, 828, 644 cm-1. Anal. Calcd. for C15H9Br2FN2S: C,

389

42.08; H, 2.12; N, 6.54; S, 7.49; found: C, 42.10; H, 2.13; N, 6.57; S, 7.43; HR-MS (ESI+):

390

m/z [M + H] + Calcd. for C15H9Br2FN2S: 427.88168; found: 427.88168.

C NMR (101 MHz, DMSO-d6): δ 165.58, 158.06,

RI PT

13

391 392

4.5.5. N-(4-Fluorophenyl)-4-(naphthalen-2-yl)thiazol-2-amine (7e)

White solid from EtOH (91% yield). m.p. 171.5-175.3 °C; 1H NMR (400 MHz,

394

CDCl3): δ 8.35 (d, J = 1.3 Hz, 1H), 7.96 (dd, J = 6.1, 3.4 Hz, 1H), 7.89 (d, J = 8.6 Hz, 1H),

395

7.82 (dd, J = 6.0, 3.4 Hz, 1H), 7.75 (dd, J = 8.6, 1.9 Hz, 1H), 7.55 – 7.50 (m, 2H), 7.38 (dd, J

396

= 9.0, 4.5 Hz, 2H), 7.15 (dd, J = 8.9, 8.1 Hz, 2H), 6.78 (s, 1H), 5.27 (s, 1H); 13C NMR (101

397

MHz, CDCl3): δ 168.41, 133.84, 133.29, 129.48, 129.04, 127.75, 125.99 123.59, 122.66,

398

117.38, 117.15, 99.32; IR (KBr): 3251, 3049, 1625, 1503, 1231, 1158, 818, 750 cm-1. Anal.

399

Calcd. for C19H13FN2S: C, 71.23; H, 4.09; N, 8.74; S, 10.01; found: C, 71.24; H, 4.11; N,

400

8.75; S, 10.03. HR-MS (ESI+): m/z [M + H]

401

320.07839.

+

Calcd. for C19H13FN2S: 320.07835; found:

TE D

402 403

M AN U

SC

393

4.5.6. N-(4-Fluorophenyl)-4-(4-methoxyphenyl)thiazol-2-amine (7f) White solid from EtOH (87% yield). m.p. 231.5-234.3 °C; 1H NMR (400 MHz,

405

CDCl3): δ 11.54 (s, 1H), 7.72 (d, J = 8.9 Hz, 2H), 7.44 – 7.34 (m, 2H), 7.18 (dd, J = 9.1, 8.0

406

Hz, 2H), 7.01 (d, J = 8.9 Hz, 2H), 6.49 (s, 1H), 3.85 (s, 3H); 13C NMR (101 MHz, CDCl3): δ

407

164.08, 144.85, 142.28, 136.60, 129.41, 122.08, 121.99, 121.62, 120.49, 116.64, 116.41,

408

113.23, 17.46, 14.55; IR (KBr): 3212, 3015, 1619, 1505, 1292, 1255, 1022, 883 cm-1. Anal.

409

Calcd. for C16H13FN2OS: C, 63.98; H, 4.36; N, 9.39; S, 10.68; found: C, 63.99; H, 4.39; N,

410

9.37; S, 10.71. HR-MS (ESI+): m/z [M + H] + Calcd. for C16H13FN2OS: 300.07326; found:

411

300.07331.

AC C

EP

404

412 413

4.5.7. 4-(4-(4-Chlorophenyl)thiazol-2-ylamino)phenol (7g)

414


415

DMSO-d6): δ 9.93 (s, 1H), 9.15 (s, 1H), 7.90 (d, J = 8.7 Hz, 1H), 7.47 (d, J = 1.2 Hz, 1H),

416

7.45 (d, J = 1.4 Hz, 1H), 6.75 (d, J = 8.9 Hz, 1H); 13C NMR (101 MHz, DMSO-d6): δ 164.43,

417

152.42, 148.83, 133.54, 133.22, 131.85, 128.64, 127.36, 119.35, 115.52, 102.70; IR (KBr): 13


3562, 3293, 3067, 1573, 1511, 1224, 672 cm-1. Anal. Calcd. for C15H11ClN2OS: C, 59.50; H,

419

3.66; N, 9.25; S, 10.59; found: C, 59.52; H, 3.68; N, 9.28; S, 10.62. HR-MS (ESI+): m/z [M

420

+ H] + Calcd. for C15H11ClN2OS: 302.02806; found: 302.02809.

421 422

4.5.8. 4-((4-(4-Methoxyphenyl)thiazol-2-yl)amino)phenol (7h) White solid from EtOH (88% yield). m.p. 126.3-128.3 °C; 1H NMR (400 MHz,

424


425

7.45 (d, J = 1.4 Hz, 1H), 6.75 (d, J = 8.9 Hz, 1H), 3.85 (s, 3H );

426

DMSO-d6): δ 161.13, 151.39, 147.49, 132.45, 128.01, 125.22, 121.34, 117.15, 111.85,

427

104.90, 55.63; IR (KBr): 3562, 3293, 3067, 1573, 1511, 1224, 672 cm-1. Anal. Calcd. for

428

C15H11ClN2OS: C, 59.50; H, 3.66; N, 9.25; S, 10.59; found: C, 59.52; H, 3.68; N, 9.28; S,

429

10.62. HR-MS (ESI+): m/z [M + H]

430

302.02809.

RI PT

423

SC

C NMR (101 MHz,

Calcd. for C15H11ClN2OS: 302.02806; found:

M AN U

431 432

+

13

4.5.9. N-(4-Chloro-2-nitrophenyl)-4-(4-methoxyphenyl)thiazol-2-amine (7i) Yellow solid from EtOH (89% yield). m.p. 150.2-151.5 °C; 1H NMR (400 MHz,

434

DMSO-d6): δ 10.39 (s, 1H), 9.02 (d, J = 9.3 Hz, 1H), 8.37 (d, J = 2.6 Hz, 1H), 8.33 (dd, J =

435

9.3, 2.6 Hz, 1H), 7.93 (d, J = 8.7 Hz, 1H), 7.49 (s, 1H), 7.04 (d, J = 8.8 Hz, 1H), 3.85 (s, 1H);

436

13

437

124.15, 119.91, 117.73, 114.07, 105.16, 55.17; IR (KBr): 3390, 3102, 1590, 1503, 1335,

438

1280, 1176, 835, 739 cm-1. Anal. Calcd. for C16H12ClN3O3S: C, 53.11; H, 3.34; N, 11.61; S,

439

8.86; found: C, 53.14; H, 3.36; N, 11.68; S, 8.89. HR-MS (ESI+): m/z [M + H] + Calcd. for

440

C16H12ClN3O3S: 361.02879; found: 361.02883.

442 443

EP

C NMR (101 MHz, DMSO-d6): δ 161.41, 159.03, 149.71, 143.41, 140.19, 127.17, 125.09,

AC C

441

TE D

433

4.5.10. N-(4-Chloro-2-nitrophenyl)-4-(naphthalen-2-yl)thiazol-2-amine (7j) Yellow solid from EtOH (92% yield). m.p. 223.2-224.4 °C; 1H NMR (400 MHz,

444

DMSO-d6): δ 10.40 (s, 1H), 9.08 (d, J = 9.3 Hz, 1H), 8.48 (s, 1H), 8.34 (dd, J = 9.3, 2.4 Hz,

445

1H), 8.30 (d, J = 2.4 Hz, 1H), 8.07 (d, J = 8.6 Hz, 1H), 8.03 (d, J = 7.7 Hz, 1H), 7.95 (d, J =

446

8.6 Hz, 1H), 7.91 (d, J = 7.7 Hz, 1H), 7.73 (s, 1H), 7.56 – 7.46 (m, 1H); 13C NMR (101 MHz,

447

DMSO-d6): δ 161.58, 149.71, 143.28, 140.19, 133.14, 132.50, 128.25, 128.15, 127.51,

448

126.35, 126.05, 124.97, 124.41, 124.19, 123.90, 107. 86; IR (KBr): 3377, 3108, 1589, 1544,

449

1496, 1324, 1281, 1121, 892, 746 cm-1. Anal. Calcd. for C19H12ClN3O2S: C, 59.76; H, 3.17;

450

N, 11.00; S, 8.40; found: C, 59.79; H, 3.19; N, 11.04; S, 8.43. HR-MS (ESI+): m/z [M + H] +

451

Calcd. for C19H12ClN3O2S: 381.03388; found: 381.03393. 14

ACCEPTED MANUSCRIPT 452 453

4.5.11. N-(4-Fluorophenyl)-4-(pyridin-3-yl)thiazol-2-amine (7k)

454

Antique white solid from EtOH (91% yield). m.p. 181.5-188.1 °C; 1H NMR (400

455

MHz, CDCl3 + DMSO-d6): δ 10.19 (s, 1H), 9.60 (s, 1H), 9.10 (d, J = 1.6 Hz, 1H), 8.46 (dd, J

456

= 4.7, 1.3 Hz, 1H), 8.30 – 8.09 (m, 1H), 7.72 (dd, J = 9.1, 4.8 Hz, 2H), 7.36 (dd, J = 7.9, 4.8

457

Hz, 1H), 7.05 (t, J = 8.8 Hz, 2H);

458

147.85, 147.07, 137.35, 132.67, 130.08, 123.28, 118.26, 115.15, 114.93, 103.69; IR (KBr):

459

3443, 3114, 1619, 1576, 1508, 1402, 1225, 1021, 819, 710 cm-1. Anal. Calcd. for

460

C14H10FN3S: C, 61.98; H, 3.72; N, 15.49; S, 11.82; found: C, 61.99; H, 3.75; N, 15.54; S,

461

11.85. HR-MS (ESI+): m/z [M + H] + Calcd. for C14H10FN3S: 271.05795; found: 271.05797.

C NMR (101 MHz, CDCl3 + DMSO-d6): δ 163.58,

SC

RI PT

13

462 463

4.5.12. N-(4-Fluorophenyl)-4-(pyridin-4-yl)thiazol-2-amine (7l)

Yellow solid from EtOH (88% yield). m.p. 283.5-284.3 °C; 1H NMR (400 MHz,

465

DMSO-d6): δ 10.72 (s, 1H), 8.91 (d, J = 6.7 Hz, 2H), 8.45 (d, J = 6.7 Hz, 2H), 8.33 (s, 1H),

466

7.79 (dd, J = 8.9, 4.8 Hz, 2H), 7.18 (t, J = 8.8 Hz, 2H); 13C NMR (101 MHz, DMSO-d6): δ

467

163.93, 156.00, 148.73, 145.25, 142.12, 137.13, 122.31, 118.82, 115.72, 115.50; IR (KBr):

468

3238, 3204, 3048, 1630, 1545, 1501, 1410, 1214, 826, 770 cm-1. Anal. Calcd. for

469

C14H10FN3S, C, 61.98; H, 3.72; N, 15.49; S, 11.82; found: C, 61.99; H, 3.75; N, 15.53; S,

470

11.85. HR-MS (ESI+): m/z [M + H] + Calcd. for C14H10FN3S: 271.05795; found: 271.05798.

471

4.5.13. N-(4-Bromophenyl)-4-(pyridin-3-yl)thiazol-2-amine (7m)

TE D

M AN U

464


473

DMSO-d6): δ 10.67 (s, 1H), 9.36 (d, J = 7.2 Hz, 1H), 9.06 – 8.88 (m, 1H), 8.86 – 8.74 (m,

474

1H), 8.06 (s, 1H), 7.91 (d, J = 10.4 Hz, 1H), 7.82 – 7.75 (m, 1H), 7.39 (d, J = 8.8 Hz, 1H);

475

13

476

109.09. IR (KBr): 3227, 3174, 3059, 1600, 1559, 1486, 1246, 820, 678 cm-1. Anal. Calcd. for

477

C14H10BrN3S: C, 50.61; H, 3.03; N, 12.65; S, 9.65; found: C, 50.64; H, 3.04; N, 12.68; S,

478

9.64. HR-MS (ESI+): m/z [M + H] + Calcd. for C14H10BrN3S, 330.97788; found: 330.97792.

AC C

EP

472

C NMR (101 MHz, DMSO-d6): δ 163.62, 140.86, 139.63, 132.82, 128.77, 126.97, 118.60,

479 480

4.5.14. N-(4-Bromophenyl)-4-(pyridin-4-yl)thiazol-2-amine (7n)

481

Yellow solid from EtOH (94% yield). m.p. 281-282.5 °C; 1H NMR (400 MHz,

482


483

7.80 (d, J = 9.0 Hz, 2H), 7.39 (d, J = 8.7 Hz, 2H); 13C NMR (101 MHz, DMSO-d6): δ 163.57,

15


142.23, 128.88, 122.30, 118.71, 116.08; IR (KBr): 3221, 3173, 3043, 2717, 1631, 1544,

485

1488, 1310, 820, 667 cm-1. Anal. Calcd. for C14H10BrN3S: C, 50.61; H, 3.03; N, 12.65; S,

486


487

C14H10BrN3S: 330.97788; found: 330.97793.

488

4.5.15. 4-(4-(Pyridin-3-yl)thiazol-2-ylamino)phenol (7o)

RI PT

489

Brown solid from EtOH (95% yield). m.p. 256.2-257.8 °C; 1H NMR (400 MHz,

491

CDCl3 + DMSO-d6): δ 9.66 (s, 1H), 9.05 (s, 1H), 8.84 (s, 1H), 8.43 (s, 1H), 8.13 (d, J = 7.7

492

Hz, 1H), 7.41 (d, J = 8.0 Hz, 2H), 7.33 (d, J = 4.3 Hz, 1H), 6.99 (s, 1H), 6.71 (d, J = 8.0 Hz,

493

2H); 13C NMR (101 MHz, CDCl3 + DMSO-d6): δ 164.92, 152.27, 147.39, 147.04, 146.52,

494

132.87, 119.29, 115.21, 102.35; IR (KBr): 3229, 3125, 1606, 1568, 1410, 1241, 831, 722 cm-

495

1

496

4.14; N, 15.63; S, 11.94. HR-MS (ESI+): m/z [M + H] + Calcd. for C14H11N3OS: 269.06228;

497

found: 269.06231.

SC

490

M AN U

. Anal. Calcd. for C14H11N3OS: C, 62.43; H, 4.12; N, 15.60; S, 11.91; found: C, 62.45; H,

498 499

4.5.16. 4-(4-(Pyridin-4-yl)thiazol-2-ylamino)phenol (7p)

Dark red solid from EtOH (91% yield). m.p. 267.2-268.0 °C; 1H NMR (400 MHz,

501

DMSO-d6): δ 10.32 (s, 3H), 9.07 (m, 2H), 8.87 (d, J = 6.7 Hz, 6H), 8.41 (d, J = 6.8 Hz, 6H),

502

8.22 (s, 3H), 7.52 (d, J = 8.8 Hz, 6H), 6.79 (d, J = 8.8 Hz, 6H); 13C NMR (101 MHz, DMSO-

503

d6): δ 164.85, 152.80, 148.96, 145.36, 142.00, 132.71, 122.30, 119.61, 115.63, 115.02; IR

504


505

C14H11N3OS, C, 62.43; H, 4.12; N, 15.60; S, 11.91; found: C, 62.45; H, 4.16; N, 15.63; S,

506

11.94. HR-MS (ESI+): m/z [M + H] + Calcd. for C14H11N3OS: 269.06228; found: 269.0623.

EP

TE D

500

508 509

AC C

507

4.5.17. N-(4-Chloro-2-nitrophenyl)-4-(pyridin-2-yl)thiazol-2-amine (7q) Yellow solid from EtOH (92% yield). m.p. 311.6-312.5 °C; 1H NMR (400 MHz,

510

DMSO-d6): δ 11.91 (s, 1H), 8.60 (d, J = 7.8, 1H), 8.22 (d, J = 7.8, 1H), 8.05 (d, J = 7.8, 1H),

511

7.86 (t, J = 7.3, 1H), 7. 63 (d, J = 7.8, 1H), 7.42 – 7.37 (t, J = 7.3, 1H), 7.19 (s, 1H), 6.83 (d, J

512

= 7.7, 1H);

513

137.47, 135.24, 132.45, 127.45, 122.72, 121.60, 117.99, 105.18; IR (KBr): 3205, 3085, 1597,

514

1544, 1494, 1335, 1275, 756 cm-1. Anal. Calcd. for C14H9ClN4O2S: C, 50.53; H, 2.73; N,

515

16.84; S, 9.64; found: C, 50.55; H, 2.74; N, 16.86; S, 9.65. HR-MS (ESI+): m/z [M + H]

516

Calcd. for C14H9ClN4O2S: 332.01347; found: 332.01349.

13

C NMR (101 MHz, DMSO-d6): δ 161.41, 154.44, 149.98, 143.31, 141.64,

517 16

+


4.5.18. N-(4-Chloro-2-nitrophenyl)-4-(pyridin-3-yl)thiazol-2-amine (7r)

519

Yellow solid from EtOH (88% yield). m.p.342.3-343.7 °C; 1H NMR (400 MHz,

520


521

8.85 (d, J = 5.1 Hz, 1H), 8.28 (d, J = 2.2 Hz, 1H), 8.22 (dd, J = 9.3, 2.3 Hz, 1H), 8.14 (s, 1H),

522

8.08 (dd, J = 7.9, 5.8 Hz, 1H);

523

140.88, 140.31, 139.57, 132.33, 126.82, 124.75, 123.80, 120.11, 118.22, 112.47; IR (KBr):

524

3212, 3060, 1589, 1549, 1498, 1326, 1282, 1121, 816, 678 cm-1. Anal. Calcd. for

525

C14H9ClN4O2S: C, 50.53; H, 2.73; N, 16.84; S, 9.64; found: C, 50.56; H, 2.72; N, 16.85; S,

526

9.67. HR-MS (ESI+): m/z [M + H]

527

332.01352.

13

Calcd. for C14H9ClN4O2S: 332.01347; found:

SC

+

RI PT

C NMR (101 MHz, DMSO-d6): δ 162.45, 143.42, 142.62,

528 529

4.5.19. N-(4-Chloro-2-nitrophenyl)-4-(pyridin-4-yl)thiazol-2-amine (7s)

Yellow solid from EtOH (89% yield). m.p. 332.8 – 333.4 °C; 1H NMR (400 MHz,

531


532

8.49 (d, J = 5.5 Hz, 5H), 8.36 (s, 3H), 8.28 (d, J = 8.9 Hz, 2H); 13C NMR (101 MHz, DMSO-

533

d6): δ 162.82, 147.85, 144.93, 142.77, 140.82, 129.29, 125.12, 123.98, 122.26, 120.60,

534

118.56; IR (KBr): 3231, 3046, 1632, 1602, 1548, 1502, 1324, 1120, 825, 738 cm-1. Anal.

535

Calcd. for C14H9ClN4O2S: C, 50.53; H, 2.73; N, 16.84; S, 9.64; found: C, 50.54; H, 2.75; N,

536

16.86; S, 9.65. HR-MS (ESI+): m/z [M + H] + Calcd. for C14H9ClN4O2S: 332.01347; found:

537

332.01353.

TE D

M AN U

530

538

4.5.20. 4-(4-Methoxyphenyl)-N-(pyridin-2-yl)thiazol-2-amine (8a)

EP

539

Light yellow solid from EtOH (87% yield). m.p. 261.5-262.3 °C; 1H NMR (400 MHz,

541

DMSO-d6): δ 11.91 (s, 1H), 8.60 (d, J = 7.8 Hz, 1H), 8.05 (d, J = 7.8 Hz, 1H), 7.86 (t, J = 7.8

542

Hz, 1H), 7.65 (s, 1H) 7.52 (d, J = 8.2 Hz, 2H), 7.40 (t, J = 7.8 Hz, 1H), 7.07 (d, J = 8.2 Hz,

543

2H), 2.98 (s, 3H);

544

138.30, 128.01, 124.39, 117.42, 114.36, 108.25, 103.23, 55.92; IR (KBr): 3231, 3077, 1618,

545

1559, 1474, 1429, 1344, 1238, 771, 671 cm-1. Anal. Calcd. for C15H13N3OS: C, 63.58; H,

546

4.62; N, 14.83; S, 11.32; found: C, 63.55; H, 4.65; N, 14.86; S, 11.36. HR-MS (ESI+): m/z

547

[M + H] + Calcd. for C15H13N3OS: 283.07793; found: 283.07795.

AC C

540

13

C NMR (101 MHz, DMSO-d6): δ 162.24, 154.73, 150.56, 147.77,

548 549

4.5.21. 4-(4-Bromophenyl)-N-(pyridin-2-yl)thiazol-2-amine (8b)

550

Light yellow solid from EtOH (92% yield). m.p. 281.5-282.1 °C; 1H NMR (400 MHz,

551

DMSO-d6): δ 12.49 (s, 1H), 8.44 (s, 1H), 7.96 (d, J = 3.8 Hz, 1H), 7.80 (d, J = 7.8 Hz, 2H), 17


7.51 (d, J = 6.6 Hz, 2H), 7.42 (s, 1H), 7.36 (s, 1H), 7.14 (s, 1H);

553

DMSO-d6): δ 161.41, 155.84, 149.98, 148.04, 138.30, 132.73, 131.35, 128.01, 123.27,

554

117.99, 107.69, 104.61, 40.15, 39.94, 39.74, 39.53, 39.32, 39.11, 38.90; IR (KBr): 3231,

555

3077, 1618, 1559, 1474, 1429, 1344, 1238, 771, 671 cm-1. Anal. Calcd. for C14H10BrN3S: C,

556

50.61; H, 3.03; N, 12.65; S, 9.65; found: C, 50.64; H, 3.05; N, 12.67; S, 9.68 HR-MS (ESI+):

557

m/z [M + H] + Calcd. for C14H10BrN3S: 330.97788; found: 330.97788.

558 559

C NMR (101 MHz,

RI PT

552

4.5.22. 4-(4-Bromophenyl)-5-methyl-N-(pyridin-2-yl)thiazol-2-amine (8c)


561

DMSO-d6): δ 11.85 (s, 1H), 8.35 (dd, J = 5.4, 1.0 Hz, 1H), 7.90 – 7.85 (m, 1H), 7.64 (d, J =

562

2.9 Hz, 2H), 7.21 (d, J = 8.5 Hz, 1H), 7.09 – 7.04 (m, 1H), 2.45 (s, 1H); 13C NMR (101 MHz,

563

DMSO-d6): δ 161.52, 156.39, 150.39, 144.03, 141.70, 133.22, 131.37, 130.22, 120.72,

564

120.46, 116.65, 112.07, 11.82; IR (KBr): 3231, 3077, 1618, 1559, 1474, 1429, 1344, 1238,

565

771, 671 cm-1. Anal. Calcd. for C15H12BrN3S: C, 52.03; H, 3.49; N, 12.14; S, 9.26; found: C,

566

52.06; H, 3.51; N, 12.17; S, 9.28. HR-MS (ESI+): m/z [M + H] + Calcd. for C15H12BrN3S:

567

344.99353; found: 344.99356.

568 569

M AN U

SC

560

4.5.23. 5-Bromo-4-(4-bromophenyl)-N-(pyridin-2-yl)thiazol-2-amine (8d) White solid from EtOH (86% yield). m.p. 230.1-232 °C; 1H NMR (400 MHz, DMSO-

571

d6): δ 11.89 (s, 1H), 8.38 (d, J = 4.8 Hz, 1H), 7.90 (d, J = 8.3 Hz, 2H), 7.62 (s, 1H), 7.60 (d, J

572

= 2.7 Hz, 1H), 7.23 (d, J = 8.4 Hz, 1H), 7.09 – 7.04 (m, 1H); 13C NMR (101 MHz, DMSO-

573

d6): δ 159.96, 150.63, 147.58, 144.18, 140.05, 133.38, 131.61, 127.88, 120.83, 116.54,

574

112.03, 107.50; IR (KBr): 3474, 3070, 1649, 1615, 1525, 1452, 1198, 832, 767, 668 cm-1.

575

Anal. Calcd. for C14H9Br2N3S: C, 40.90; H, 2.21; N, 10.22; S, 7.80; found: C, 40.93; H, 2.23;

576

N, 10.25; S, 7.84. HR-MS (ESI+): m/z [M + H] + Calcd. for C14H9Br2N3S: 408.88839; found:

577

408.88842.

579

EP

AC C

578

TE D

570

4.5.24. 4-(3,4-Dichlorophenyl)-N-(3-methylpyridin-2-yl)thiazol-2-amine (8e)

580


581

DMSO-d6): δ 11.21 (s, 1H), 8.06 (d, J = 8.9 Hz, 1H), 7.95 (s, 1H), 7.86 (d, J = 5.2 Hz, 1H),

582

7.62 (s, 1H), 7.49 (d, J = 7.5 Hz, 1H), 7.05 (t, J = 7.5 Hz, 1H), 6.91 (d, J = 7.8 Hz, 1H), 2.46

583

(s, 3H); 13C NMR (101 MHz, DMSO-d6): δ 163.08, 160.02, 151.66, 146.10, 138.30, 134.40,

584

131.90, 131.84, 129.39, 129.11, 125.78, 117.71, 113.52, 106.02; IR (KBr): 3421, 3083, 1637,

585

1583, 1489, 1258, 1191, 783, 738 cm-1. Anal. Calcd. for C15H11Cl2N3S: C, 53.58; H, 3.30; N, 18


12.50; S, 9.54; found: C, 53.62; H, 3.32; N, 12.55; S, 9.56. HR-MS (ESI+): m/z [M + H]

587

Calcd. for C15H11Cl2N3S: 335.00507; found: 335.00509.

+

588 589

4.5.25. 4-(4-Bromophenyl)-5-methyl-N-(3-methylpyridin-2-yl)thiazol-2-amine (8f) Light Yellow solid from EtOH (94% yield). m.p. 243.1-244.5 °C; 1H NMR (400

591

MHz, DMSO-d6): δ 11.73 (s, 1H), 7.77 (t, J = 7.3 Hz, 1H), 7.69 (dd, J = 16.6, 8.5 Hz, 1H),

592

7.02 (d, J = 8.1 Hz, 1H), 6.95 (d, J = 7.2 Hz, 1H), 2.55 (s, 1H); 13C NMR (101 MHz, DMSO-

593

d6): δ 162.80, 157.78, 147.77, 145.25, 136.64, 133.29, 131.62, 128.56, 123.82, 117.42,

594

115.76, 113.52; IR (KBr): 3444, 3073, 1653, 1586, 1455, 1202, 1167, 997, 806, 667 cm-1.

595

Anal. Calcd. for C16H14BrN3S: C, 53.34; H, 3.92; N, 11.66; S, 8.90; found: C, 53.36; H, 3.95;

596

N, 11.69; S, 8.94. HR-MS (ESI+): m/z [M + H] + Calcd. for C16H14BrN3S: 359.00918; found:

597

359.00921.

SC

RI PT

590

599

M AN U

598

4.5.26. 5-Bromo-4-(4-bromophenyl)-N-(3-methylpyridin-2-yl)thiazol-2-amine (8g) White solid from EtOH (87% yield). m.p. 286.9-288.1 °C; 1H NMR (400 MHz,

601

DMSO-d6): δ 11.34 (s, 1H), 8.01 (d, J = 8.9 Hz, 1H), 7.76 (d, J = 8.6 Hz, 2H), 7.56 (d, J =

602

8.6 Hz, 2H), 7.26 (d, J = 10.9 Hz, 1H), 6.62 (t, J = 8.9 Hz, 1H), 2.30 (s, 1H); 13C NMR (101

603

MHz, DMSO-d6) δ 164.05, 161.00, 152.38, 144.08, 136.59, 132.15, 132.03, 128.93, 123.85,

604

118.76, 112.83, 93.34, 16.79; IR (KBr): 3439, 3072, 1653, 1588, 1453, 1211, 1165, 985, 811,

605

655 cm-1. Anal. Calcd. for C15H11Br2N3S: C, 42.38; H, 2.61; N, 9.88; S, 7.54; found: C,

606

42.39; H, 2.66; N, 9.91; S, 7.56. HR-MS (ESI+): m/z [M + H]

607

424.90200; found: 424.9023.

608

610

Calcd. for C15H11Br2N3S:

4.5.27. 4-(3,4-Dichlorophenyl)-N-(6-methylpyridin-2-yl)thiazol-2-amine (8h) White solid from EtOH (94% yield). m.p. 281.3-282 °C; 1H NMR (400 MHz, DMSO-

AC C

609

+

EP

TE D

600

611

d6): δ 11.91 (s, 1H), 8.60 (d, J = 3.8 Hz, 1H), 8.05 (d, J = 7.8 Hz, 1H), 7.86 (t, J = 7.3 Hz,

612

1H), 7.57 (s, 1H), 7.46 – 7.32 (m, 1H), 7.14 (d, J = 7.1 Hz, 1H), 6.96 (d, J = 2.7 Hz, 1H),

613

2.52 (S, 1H);

614

133.74, 132.05, 129.50, 127.52, 126.67, 120.18, 107.19, 104.92, 21.58; IR (KBr): 3385,

615

3075, 2857, 1631, 1585, 1452,1392, 1314, 1260, 1188,1071, 1003, 910, 832, 784 cm-1. Anal.

616

Calcd. for C15H11Cl2N3S: C, 53.58; H, 3.30; N, 12.50; S, 9.54; found: C, 53.60; H, 3.34; N,

617

12.53; S, 9.55. HR-MS (ESI+): m/z [M + H] + Calcd. for C15H11Cl2N3S: 335.00507; found:

618

335.00510.

13

C NMR (101 MHz, DMSO-d6) δ 160.86, 155.20, 153.22, 150.11, 138.26,

619 19


4.5.28. 4-(4-Bromophenyl)-5-methyl-N-(6-methylpyridin-2-yl)thiazol-2-amine (8i)

621


622

DMSO-d6): δ 11.91 (s, 1H), 8.60 (d, J = 3.8 Hz, 1H), 7.87 (d, J = 7.3 Hz, 1H), 7.40 (t, J =

623

7.3, 1H), 6.92 (d, J = 6.3 Hz, 1H), 6.59 (d, J = 8.4 Hz, 1H), 2.50 (s, 4H), 2.40 (s, 3H);

624

NMR (101 MHz, DMSO-d6) δ 162.26, 158.31, 153.22, 148.99, 137.41, 132.31, 131.19,

625

128.38, 123.01, 119.89, 117.63, 103.52, 24.41, 13.97; IR (KBr): 3519, 3074, 1658, 1584,

626

1216, 1168, 987, 821, 643 cm-1. Anal. Calcd. for C16H14BrN3S: C, 53.34; H, 3.92; N, 11.66;

627

S, 8.90; found: C, 53.36; H, 3.89; N, 11.69; S, 8.92. HR-MS (ESI+): m/z [M + H] + Calcd. for

628

C16H14BrN3S: 359.00918; found: 359.00920.

RI PT

C

SC

629 630

13

4.5.29. 4-(4-Methoxyphenyl)-N-(6-methylpyridin-2-yl)thiazol-2-amine (8j) Antique white solid from EtOH (89% yield). m.p. 221.5-222.5 °C; 1H NMR (400

632

MHz, CDCl3 + DMSO-d6): δ 7.89 (s, 1H), 7.68 (d, J = 8.7 Hz, 2H), 7.29 (d, J = 7.1 Hz, 1H),

633

7.18 (s, 1H), 7.05 (d, J = 7.3 Hz, 1H), 6.92 (d, J = 8.8 Hz, 2H), 4.39 (s, 1H), 3.77 (s, 4H),

634

2.64 (s, 3H);

635

136.96, 129.23, 126.17, 116.70, 113.28, 109.67, 103.86, 54.20, 22.47; IR (KBr): 3518, 3068,

636

1644, 1580, 1454, 1256, 1172, 1029, 799 cm-1. Anal. Calcd. for C16H15N3OS, C, 64.62; H,

637

5.08; N, 14.13; S, 10.78; found: C, 64.65; H, 5.10; N, 14.17; S, 10.81. HR-MS (ESI+): m/z

638

[M + H] + Calcd. for C16H15N3OS: 297.09358; found: 297.09361.

640

13

C NMR (101 MHz, CDCl3 + DMSO-d6): δ 163.65, 159.06, 157.67, 150.31,

TE D

639

M AN U

631

4.5.30. N-(6-Methylpyridin-2-yl)-4-(naphthalen-1-yl)thiazol-2-amine (8k) White Solid from EtOH (94% yield). m.p. 227.5-228.4 °C; 1H NMR (400 MHz,

642

DMSO-d6): δ 11.68 (s, 1H), 8.42 (s, 1H), 8.04 (dd, J = 8.6, 1.7 Hz, 1H), 7.99 – 7.86 (m, 2H),

643

7.69 – 7.45 (m, 2H), 7.02 – 6.77 (m, 1H), 2.47 (s, 1H).

644

158.36, 155.05, 150.37, 145.25, 138.56, 128.23, 127.73, 127.55, 127.07, 126.52, 125.63,

645

115.48, 107.56, 94.97, 23.16; IR (KBr): 3057, 3028, 2703, 1649, 1590, 1457, 1170, 885, 784,

646

746 cm-1. Anal. Calcd. for C19H15N3S: C, 71.90; H, 4.76; N, 13.24; S, 10.1; found: 1 C,

647

71.92; H, 4.79; N, 13.28; S, 10.12. HR-MS (ESI+): m/z [M + H]

648

317.09867; found: 317.09872.

13

C NMR (101 MHz, DMSO-d6): δ

AC C

EP

641

+

Calcd. for C19H15N3S:

649 650

4.5.31. N-(Pyridin-2-yl)-4-(pyridin-3-yl)thiazol-2-amine (8l)

651

Antique white solid from EtOH (94% yield). m.p. 235.6-237.6 °C; 1H NMR (400

652

MHz, DMSO-d6): δ 11.48 (s, 1H), 9.14 (s, 1H), 8.51 (d, J = 3.9 Hz, 1H), 8.32 (dd, J = 5.0,

653

1.0 Hz, 1H), 8.28 – 8.21 (m, 1H), 7.72 (ddd, J = 8.8, 7.3, 1.8 Hz, 1H), 7.61 (s, 1H), 7.11 (d, J 20


C NMR (101 MHz, DMSO-d6): δ 160.01,

654

= 8.3 Hz, 1H), 6.94 (dd, J = 6.4, 5.2 Hz, 1H);

655

151.70, 148.19, 146.78, 146.43, 137.97, 132.79, 123.79, 116.12, 110.86, 107.53; IR (KBr):

656

3241, 3097, 2913, 1621, 1547, 1483, 1404, 1311, 765, 715 cm-1. Anal. Calcd. for C13H10N4S:

657

C, 61.40; H, 3.96; N, 22.03; S, 12.61; found: C, 61.43; H, 3.99; N, 22.07; S, 12.62. HR-MS

658

(ESI+): m/z [M + H] + Calcd. for C13H10N4S: 254.06262; found: 254.06266.

660

RI PT

659

4.5.32. N-(3-Methylpyridin-2-yl)-4-(pyridin-3-yl)thiazol-2-amine (8m)

661


662

DMSO-d6): δ 10.54 (s, 1H), 9.16 (d, J = 1.7 Hz, 1H), 8.49 (dd, J = 4.7, 1.5 Hz, 1H), 8.28 –

663

8.23 (m, 1H), 8.18 (dd, J = 4.9, 1.1 Hz, 1H), 7.60 (s, 1H), 7.57 – 7.52 (m, 1H), 7.43 (ddd, J =

664

8.0, 4.8, 0.7 Hz, 1H), 6.90 (dd, J = 7.2, 5.0 Hz, 1H), 2.35 (s, 3H);

665

CDCl3): δ 160.56, 149.64, 148.56, 147.57, 146.41, 144.29, 138.31, 133.19, 130.79, 123.55,

666

117.84, 116.66, 107.51, 16.67; IR (KBr): 3219, 3102, 2921, 1591, 1463, 1189, 811, 781, 731

667

cm-1.. Anal. Calcd. for C14H12N4S C, 62.66; H, 4.51; N, 20.88; S, 11.9; found: 5 C, 62.68; H,

668

4.53; N, 20.94; S, 11.98. HR-MS (ESI+): m/z [M + H]

669

found: 268.07831.

SC

C NMR (101 MHz,

M AN U

670 671

13

+

Calcd. for C14H12N4S: 268.07827;

4.5.33. N-(6-Methylpyridin-2-yl)-4-(pyridin-4-yl)thiazol-2-amine (8n) Yellow solid from EtOH (94% yield). m.p. 294.5-295.4 °C; 1H NMR (400 MHz,

673


674

7.67 – 7.61 (m, 1H), 6.93 (d, J = 8.2 Hz, 1H), 6.84 (d, J = 7.3 Hz, 1H), 2.48 (s, 1H);

675

NMR (101 MHz, DMSO-d6): δ 166.66, 160.71, 155.13, 150.52, 143.66, 142.25, 138.58,

676

122.01, 118.20, 115.59, 107.68, 23.24; IR (KBr): 3211, 3187, 3093, 2729, 1629, 1604, 1541,

677

1463, 1323, 1199, 828, 777 cm-1. Anal. Calcd. for C14H12N4S: C, 62.66; H, 4.51; N, 20.88; S,

678


679

C14H12N4S: 268.07827; found: 268.07829.

681

13

EP

C

AC C

680

TE D

672

4.5.34. N-(3-Methylpyridin-2-yl)-4-(pyridin-4-yl)thiazol-2-amine (8o)

682


683


684

8.28 (d, J = 6.7 Hz, 1H), 7.73 (d, J = 6.7 Hz, 1H), 7.10 – 7.01 (t, J = 6.7 Hz, 1H); 13C NMR

685

(101 MHz, DMSO-d6) δ 161.98, 157.74, 148.99, 145.60, 140.78, 137.69, 136.56, 123.85,

686

116.80, 113.12, 108.88, 40.15, 39.94, 39.74, 39.53, 39.32, 39.11, 38.90, 16.22; IR (KBr):

687

3472, 3390, 3079, 1635, 1581, 1530, 1499, 1458, 1373, 1256, 1190, 827, 790, 670 cm-1. 21


Anal. Calcd. for C14H12N4S: C, 62.66; H, 4.51; N, 20.88; S, 11.95; found: C, 62.67; H, 4.55;

689

N, 20.94; S, 11.98. HR-MS (ESI+): m/z [M + H] + Calcd. for C14H12N4S: 268.07827; found:

690

268.07829.

691

4.6.

In vitro assay for evaluation of antimycobacterial activity

RI PT

692

The in vitro assay method previously reported by our group has been used for

694

evaluation of antimycobacterial assay. DMSO was used for the preparation of the test

695

compound stocks as well as for dilutions. The test compounds MICs against Mtb were

696

analysed in 7H9 broth using a standard micro dilution method [61]. The test compounds

697

which were prepared with 1µl of serial twofold dilutions in DMSO were added in to the 384

698

well plates with the final concentrations ranging from 100 mM - 0.19 mM. Two sets of

699

control wells were prepared: a media control with only medium (Middlebrook 7H9 medium

700

supplemented with 0.2% glycerol, 0.05% Tween 80 (Sigma), and 10% albumin dextrose

701

catalase (Difco Laboratories, Detroit, Mich.)) and a culture control with a bacterial

702

suspension but no compound. 40µl (3-7 x 105 CFU/ml) of the bacterial culture was added to

703

all the wells except the media control wells. The plates were packed in gas permeable

704

polythene bags and incubated at 37°C for 5 days. Following this incubation period, 8 µl of a

705

freshly prepared 1:1 mixture of Resazurin (0.02% in water) and 10% Tween 80 was added to

706

all the wells. The plates were re-incubated for an additional 24 h at 37°C and the colour

707

conversion of all wells recorded. A blue colour in the well indicates no growth and a pink

708

colour indicates growth. Absorbance at 575 nm and 610 nm was monitored and the ratio

709

(A575/A610) calculated. The least concentration which yielded 80% inhibition was considered

710

as MIC; the media control is considered 100% inhibition and the culture control as 0%

711

inhibition. Isoniazid was used as a reference drug for the assay.

712

4. 7.

M AN U

TE D

EP

AC C

713

SC

693

Molecular docking studies

The docking simulations were performed using Auto Dock 4.2 [62]. The recently

714

solved X-ray crystal structure of KasA of Mtb (PDB code: 2WGD) in complex with TLM has

715

been retrieved from the RCSB Protein Data Bank and all heteroatoms were removed. The

716

2WGD was setup for a standard protocol for docking. Lamarckian Genetic Algorithm was

717

used to carry out the simulation. Columbic electrostatic potential, Vander Waals interaction

718

represented as a Lennard-Jones12–6 dispersion/repulsion term and hydrogen bonding 22

ACCEPTED MANUSCRIPT represented as a directional 12–10 term were the three parameters taken into account for

720

evaluating binding energy in the docking step. The most favourable free energy of binding

721

were attained by considering the docking orientations lying within the range of 2.0 Å in the

722

root-mean square deviation (rmsd) tolerance and clustering the each other to get the result.

723

The post-docking energy minimization on Discovery Studio 2.5 was performed on top-posed

724

docking conformations obtained.

725

Acknowledgements

RI PT

719

We are grateful to AstraZeneca India Pvt. Ltd., Bangalore, India for generation of the

726

MIC data against Mtb. The authors thank Council of Scientific and Industrial Research

728

(CSIR), New Delhi, India for financial support through a sponsored project (01 (2262)

729

/08/EMR-II). Mr. Parameshwar Makam thanks Pondicherry University for awarding

730

University Research Fellowship (URF).

731

Appendix A. Supplementary information

M AN U

SC

727

Supplementary information related to this article can be found at…

732 733

References

734

[1]

TE D

Tuberculosis: WHO global tuberculosis report 2013, in, World Health Organization, World Health Organization, Geneva, 2013.

735

[2]

H. Herzog, History of tuberculosis, Respiration 65 (1998) 5-15.

737

[3]

C. Lienhardt, S.V. Cook, M. Burgos, V.Y. Edwards, L. Rigouts, G. Anyo, K.S. Jae,

EP

736

A. Jindani, D.A. Enarson, A.J. Nunn, Efficacy and safety of a 4-drug fixed-dose

739

combination regimen compared with separate drugs for treatment of pulmonary

740

tuberculosis: The study c randomized controlled trial, JAMA 305 (2011) 1415-1423.

741

[4]

359 (2002) 2188-2194.

743

[5]

[6]

749

R. Osborne, First novel anti-tuberculosis drug in 40 years, Nat. Biotech. 31 (2013) 8991.

747 748

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871

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904

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905

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AC C

907

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913

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914

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919

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920

receptor flexibility, J. Comput. Chem. 30 (2009) 2785-2791.

921

AC C

EP

TE D

M AN U

SC

RI PT

922

29


Figure and Scheme Legends

924

Figure 1. Rational design of the 2-aminothiazole derivatives. Log P is considered as the

926

decisive factor.

927

Figure 2. Single crystal X-ray diffraction results of 7g. (a) The ORTEP diagram with 40%

928

ellipsoid probability (b) The crystal packing diagram along the c-axis and (c) The single helix

929

packing arrangement along the a-axis.

930

Figure 3. Single crystal X-ray diffraction results of 8l. (a) The ORTEP diagram with 40%

931

ellipsoid probability (b) The crystal packing diagram along the a-axis and (c) The single helix

932

packing arrangement along the b-axis.

933

Figure 4. The summary of structure activity relationship studies.

934

Figure 5. The summary of correlation between Log P and in vitro antimycobacterial activity.

935

Figure 6. Hydrogen bond interactions of compound 7n with Mtb KasA protein.

936

Scheme 1. Synthesis of (i) arylthioureas (ii) pyridyl thioureas (iii) (bromoacetyl)pyridines

937

and (iv) 2-aminothiazole derivatives.

AC C

EP

TE D

M AN U

SC

RI PT

925

30

ACCEPTED MANUSCRIPT Table 1. Physico-chemical properties a and antimycobacterial activity of thiazol-2-amine derivatives against Mtb, H37Rv. S. No. Compound Log P H Freely MIC Mol. H Lipinski Polar Weight rule Acceptors Donors Rotatable Surface (µM) violations Area Bonds 1 5.70 349.228 2 1 1 3 24.919 12.5 7a 7b

6.35

363.255

2

1

1

3

7c

6.64

428.124

2

1

1

4

7d

5.54

304.777

2

1

1

5

7e

5.09

302.786

3

2

0

6

7f

4.77

300.358

3

1

7

7g

4.33

298.367

4

2

8

7h

6.48

361.803

5

1

9

7i

5.92

320.392

2

10

7j

7.62

381.835

11

7k

5.80

332.764

12

7l

3.71

271.32

13

7m

4.34

332.226

14

7n

3.27

15

7o

5.42

16

7p

3.71

17

7q

4.34

18

7r

19

7s

20

8a

21

3

24.919

RI PT

2

25

24.919

100

3

24.919

25

3

45.147

100

0

4

34.153

12.5

0

4

54.381

200

1

5

79.97

>200

1

1

3

24.919

12.5

4

1

1

4

70.74

>200

5

1

1

4

83.63

>200

3

1

0

3

37.811

25

3

1

0

3

37.811

6.25

269.329

4

2

0

3

58.039

>200

332.764

5

1

1

4

83.63

>200

271.32

3

1

0

3

37.811

>200

332.226

3

1

0

3

37.811

>200

M AN U

TE D

AC C

EP

SC

3

3.27

269.329

4

2

0

3

58.039

>200

5.42

332.764

5

1

1

4

83.63

>200

4.01

283.356

4

1

0

4

47.045

>200

8b

4.93

332.226

3

1

0

3

37.811

>200

22

8c

5.58

346.253

3

1

0

3

37.811

>200

23

8d

5.87

411.122

3

1

1

3

37.811

>200

24

8e

2.95

254.318

4

1

0

3

50.703

>200

25

8f

3.46

268.345

4

1

0

3

50.703

100

26

8g

3.46

268.345

4

1

0

3

50.703

>200

27

8h

5.89

336.247

3

1

1

3

37.811

>200

28

8i

6.38

425.149

3

1

1

3

37.811

>200

29

8j

6.09

360.28

3

1

1

3

37.811

>200

30

8k

5.71

360.28

3

1

0

3

37.811

>200

31

8l

4.14

297.383

4

1

0

4

47.045

>200

32

8m

5.51

336.247

3

1

1

3

37.811

>200

33

8n

3.08

268.345

4

1

0

3

50.703

100

34

8o

5.29

317.417

3

1

0

3

37.811

>200

EP

TE D

RI PT

M AN U

SC

Calculated from online server http://www.molinspiration.com/cgi-bin/properties

AC C

a

ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT Table 2. Library of N-phenyl thiazol-2-amine and N-2-pyridyl thiazol-2-amine derivatives. Category I: N,4-Diphenylthiazol-2-amine derivatives

8a

8f

RI PT

SC

M AN U

7p

7d

7g 7h 7i Category II: N-Phenyl-4-(pyridinyl)thiazol-2-amine derivatives

7l

7m

7n

8b

7j

7o

8g

8c

8d

8e

8h

8i

8j

8k Category IV: N,4-Di(pyridinyl)thiazol-2-amine derivatives

8l

7e

7q 7r 7s Category III: 4-Phenyl-N-(pyridinyl)thiazol-2-amine derivatives

TE D

7k

7c

EP

7f

7b

AC C

7a

8m

8n

8o

ACCEPTED MANUSCRIPT Table 3. Crystal and measurement details of 7g and 8l molecules. Crystal Parameter

7g

8l

C15H12.5ClN2OS

C13H10N4S

CCDC Number

903724

903727

Formula weight

624.58

254.32

Crystal system

Monoclinic

Monoclinic

Crystal morphology

Colourless block

Pale Yellow needle

Crystal size (mm)

0.45 × 0.37 × 0.25

0.42 × 0.14 × 0.14

Space group

C2/c

P21/C

Temperature/K

293

293

Radiation, λ

Mo Kα , 0.71073 Å

Mo Kα , 0.71073 Å

a (Å)

31.9952 (16)

14.612 (3)

b (Å)

11.3729 (6)

c (Å)

7.8990 (4)

14.715 (3)

α (Å)

-

90°

β (Å)

93.304 (4)°

104.79 (2)°

γ (Å)

-

90°

Volume (Å3)

2869.5 (3)

SC 5.5299 (11)

M AN U

TE D

Z

RI PT

Formula

1149.6 (4)

4

4

1292

528.0

Density (Mg m−3)

1.446

1.469

µ (1/mm)

0.41

0.27

θ (min, max)

3.2, 25.0°

2.9, 25.0°

No. Unique refln

2538

2032

No. of parameters

194

163

Rint, wR(F2)

0.022, 0.109

0.075, 0.128

∆ρmin(eÅ-3), ∆ρmax(eÅ-3)

−0.47, 0.39

−0.34, 0.33

GooF

1.02

0.96

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Figure 5

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Figure 6

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Research Highlights Library of 2-aminothiazole derivatives were synthesized and characterized.

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All the compounds were evaluated against M. tuberculosis, H37Rv, by in vitro assay.

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4-Halophenyl at 2nd position of 2-aminothiazole derivatives enhances activity.

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2-Pyridyl at 2nd position of 2-aminothiazole derivatives decreases activity.

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The MIC and Ki values of compound 7n are 6.5 µM and 0.44 µM respectively.

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