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Article

Synthesis and Evaluation of Novel 1,2,6-Thiadiazinone Kinase Inhibitors as Potent Inhibitors of Solid Tumors

by
Andreas S. Kalogirou
1,2,*,
Michael P. East
3,
Tuomo Laitinen
4,
Chad D. Torrice
5,
Kaitlyn A. Maffuid
5,
David H. Drewry
6,7,
Panayiotis A. Koutentis
2,
Gary L. Johnson
3,7,
Daniel J. Crona
5,7 and
Christopher R. M. Asquith
3,*
1
Department of Life Sciences, School of Sciences, European University Cyprus, 6 Diogenis Str., Engomi, P.O. Box 22006, Nicosia 1516, Cyprus
2
Department of Chemistry, University of Cyprus, P.O. Box 20537, Nicosia 1678, Cyprus
3
Department of Pharmacology, School of Medicine, University of North Carolina, Chapel Hill, NC 27599, USA
4
School of Pharmacy, Faculty of Health Sciences, University of Eastern Finland, 70211 Kuopio, Finland
5
Division of Pharmacotherapy and Experimental Therapeutics, Eshelman School of Pharmacy, University of North Carolina, Chapel Hill, NC 27599, USA
6
Structural Genomics Consortium, Eshelman School of Pharmacy, University of North Carolina, Chapel Hill, NC 27599, USA
7
Lineberger Comprehensive Cancer Center, School of Medicine, University of North Carolina, Chapel Hill, NC 27599, USA
*
Authors to whom correspondence should be addressed.
Molecules 2021, 26(19), 5911; https://0-doi-org.brum.beds.ac.uk/10.3390/molecules26195911
Submission received: 6 September 2021 / Revised: 22 September 2021 / Accepted: 24 September 2021 / Published: 29 September 2021
(This article belongs to the Special Issue Polysulfur- and Sulfur-Nitrogen Heterocycles)
Browse Figures
Versions Notes

Abstract

:
A focused series of substituted 4H-1,2,6-thiadiazin-4-ones was designed and synthesized to probe the anti-cancer properties of this scaffold. Insights from previous kinase inhibitor programs were used to carefully select several different substitution patterns. Compounds were tested on bladder, prostate, pancreatic, breast, chordoma, and lung cancer cell lines with an additional skin fibroblast cell line as a toxicity control. This resulted in the identification of several low single digit micro molar compounds with promising therapeutic windows, particularly for bladder and prostate cancer. A number of key structural features of the 4H-1,2,6-thiadiazin-4-one scaffold are discussed that show promising scope for future improvement.

1. Introduction

Heteroatom-rich scaffolds are underexplored in medicinal chemistry, potentially leading to missed opportunities [1,2,3,4,5,6]. One such ring system is the non S-oxidized 4H-1,2,6-thiadiazinone, selected examples of which have applications as plant protectants [7,8,9,10,11], organic photovoltaics (OPVs) [12,13], liquid crystals [14], and potential anti-cancer agents [15]. The precursor to this 1,2,6-thiadiazinone scaffold, is the 3,4,4,5-tetrachloro-4H-1,2,6-thiadiazine (1) which was first reported in 1973 [16]. Subsequently, the chemistry was later developed to allow broad diversification [17]. Synthetic interest in thiadiazones/thiadiazines is currently focused around two scaffolds: 3,5-dichloro-4H-1,2,6-thiadiazin-4-one (2) and its precursor, 3,4,4,5-tetrachloro-4H-1,2,6-thiadiazine (1), which can be derived from dichloromalononitrile (Scheme 1) [18]. The substitution chemistry of both chlorines of 3,5-dichloro-4H-1,2,6-thiadiazin-4-one (2) has been explored in detail and affords a versatile platform to prepare suitable 3,5-substituted derivatives.
3-Anilino-1,2,6-thiadiazinones occupy a similar chemical space to more commonly used medicinal chemistry scaffolds, including the mono- and di-anilinopyrimidines. The anilinopyrimidines are a common chemotype found in ~10% of the clinically approved kinase inhibitor drugs, including palbociclib and ceritinib (Figure 1) [19]. Furthermore, anilinopyrimidines are found in a number of tool compounds for kinase interrogation, including GNF-2 for ABL [20], GSK854 for TNNi3K [21,22], and THZ-P1-2 for the PI5P4K isoforms [23]. The broad activity profile of the dianilinopyrimidine chemotype can also be seen within several kinase inhibitor screening sets, including PKIS, PKIS2, and KCGS, with more than 35 examples showing activity on >400 different protein kinases (excluding mutants) in either enzyme inhibition or affinity capture assays [24,25,26].
The chemistry of 3,5-dichloro-4H-1,2,6-thiadiazin-4-one (2) is dominated by the substitution of the two chlorides. In particular, substitution can occur with N, O, and S nucleophiles as well as with organometallic reagents (carbon nucleophiles). Interestingly, while the synthesis of symmetrical 3,5-diarylthiadiazines is easy via Suzuki and Stille reactions [27], the synthesis of unsymmetrical analogues is more difficult as selective reaction of one of the two sites is not possible [28]. However, with amine or alkoxide nucleophiles, a stepwise reaction is possible as the first displacement is considerably more facile that the second one [17,18]. This allows for the preparation of unsymmetrical 3,5-diaminothiadiazines or even 3-amino-5-aryl derivatives via a combination of nucleophilic attack by an amine followed by a Suzuki reaction in the remaining C-5 position [15]. Moreover, a palladium-catalyzed C-N coupling protocol was developed for the more difficult amino-displacement of the second chloride of thiadiazinone 4 to afford disubstituted derivatives 5 that avoids the use of elevated temperatures and excess amine (Scheme 2) [29].

2. Results

2.1. Synthesis

An in-depth analysis of the kinome-wide profiling of the dianilinopyrimidines in PKIS, PKIS2, and KCGS, and within the literature, led us to select a series of R1 and R2 substituent patterns (Figure 2, Table 1) that showed the broadest range of activity on human kinases. These compounds were designed to capture as much of the kinome as possible and, while unoptimized, they should provide attractive starting points for future work. In addition, several powerful hinge binding motifs were selected to enhance the scaffolds potential to capture extra kinases [30,31,32].
The corresponding 1,2,6-thiadiazin-4-ones (14–19, 21–25) were synthesized in two-steps starting from 3,5-dichloro-4H-1,2,6-thiadiazin-4-one (2) [18]. The first chloride of thiadiazinone 2 can be readily displaced by anilines and alkylamines by reaction with stoichiometric amounts of the amine (1 equiv.) and the base 2,6-lutidine (1 equiv.) in EtOH, at ca. 0–20 °C. The required 5-amino-3-chloro-4H-1,2,6-thiadiazin-4-ones 6–13 were isolated in 67–99% yields with a chromatography-free work-up (Scheme 3).
Subsequently, scaffolds 6–12 were subjected to a Suzuki coupling with (1H-pyrrolo [2,3-b]pyridin-4-yl)boronic acid. The seven desired products 14–19 were obtained in moderate to good yields (36–83%) (Scheme 4).
A similar Suzuki coupling reaction was used to prepare 3-(4-methylpiperazin-1-yl)-5-(1H-pyrrolo[2,3-b]pyridin-4-yl)-4H-1,2,6-thiadiazin-4-one (20) starting from 3-chloro-5-(4-methylpiperazin-1-yl)-4H-1,2,6-thiadiazin-4-one (12) and (1H-pyrrolo[2,3-b]pyridin-4-yl)boronic acid (Scheme 5). Moreover, the two 3,5-diamino-substituted thiadiazines 21 and 22 were prepared via our previously developed C-N coupling methodology [15] from 5-(4-methylpiperazin-1-yl)thiadiazin-4-one 12. The products were isolated in 64 and 92% yields, respectively (Scheme 5).
Finally, a modified Suzuki coupling procedure was required for the synthesis of the 3-[(1H-indazol-5-yl)amino]-4H-1,2,6-thiadiazin-4-ones. A brief optimization showed that the combination of the catalyst Pd(dppf)Cl2 (10 mol%) and the base Na2CO3 (2 equiv.), in dioxane/H2O 90:10, in a sealed tube at ca. 140 °C was required to drive the reactions to completion. The four desired products 23–26 were obtained in medium to good yields of 68–92% (Scheme 6).
It is important to note that the stability of dianilinothiadiazinones in biological systems has previously been assessed [15]. 3,5-Bis(phenylamino)-4H-1,2,6-thiadiazin-4-one [18] is stable to neutral, acidic, or slightly basic aqueous conditions, the presence of amine or thiol nucleophiles, and oxidizing and reducing conditions. We similarly tested thiadiazinone 16 from our current study and found the same results.

2.2. Cancer Cell Screening

Compounds 14–26 were screened on an array of cancer cell lines to explore the structural requirements for anti-cancer activity within the 1,2,6-thiadiazinone scaffold (Table 1). This included pancreatic, bladder, prostate, breast, chordoma, and lung cancer cell lines while a skin fibroblast cell line was used as a toxicity control [33,34,35,36]. These cancer cell lines present different drug resistance profiles and when combined in a panel, each represents a distinct therapeutic challenge to overcome [37].
The first analogue 3-[(3-acetylphenyl)amino]-5-(1H-pyrrolo[2,3-b]pyridin-4-yl)-4H-1,2,6-thiadiazin-4-one (14) demonstrated limited inhibition across the panel of cell lines with the most inhibition shown on the bladder cancer cell line (IC50 = 15 μM). Switching from the acetyl substitution 14 to the methoxy 15 showed no improvement across the panel. Removal of the methyl on the methoxy 15, to afford a hydroxy functionality 16, led to a 10-fold increase in potency on the bladder cancer cell line (IC50 = 1.6 μM) with no toxicity in the WS-1 cell line (IC50 = >100 μM). The introduction of a 4-methyl substituent 17 removed weak inhibition on PANC1 and UCH-1 but did not show any improvement on the other cell lines in the panel. Interestingly, the introduction of a 2-methyl substituent 18 removed nearly all anti-cancer activity. Switching to the aliphatic a morpholine substituent again changed the preference toward pancreatic (IC50 = 11 μM) and prostate (IC50 = 14 μM) cancers with little activity towards bladder and the other cell lines.
We then sought to look at changing the hinge binding motif using the methyl piperazine as the fixed substituent. This tertiary amine substitution affords compounds with favorable properties [38,39,40,41,42]. However, in the case of our three powerful hinge binders [30], we observed very limited activity across both azaindoles, 4-postion 20 and 6-position 21, along with the indazole 22. The indazole 22 showed a small amount of toxicity in the WS-1 cell line (IC50 = 24 μM).
Switching to the indazole scaffold afforded a different potential hinge binding orientation. 5-(3-Hydroxyphenyl)-3-[(1H-indazol-5-yl)amino]-4H-1,2,6-thiadiazin-4-one (23) had only weak activity in the panel of cell lines but also showed no toxicity on the WS-1 cell line (IC50 = >100 μM). The introduction of a 2-methyl group to compound 23 to afford 24 improved the overall anti-cancer profile of the scaffold, with the most potency observed on breast cancer cell line (IC50 = 13 μM) and no toxicity on WS-1. The introduction of the methoxy group at the 3-position of thiadiazinone 25 changed the anti-cancer profile significantly with increased potency against bladder cancer (IC50 = 13 μM). Finally, we tested an isosteric replacement for the 3-methoxy the 3-fluoro and introduced a 4-pyridyl substitution pattern to afford compound 26; this substituent orientation is observed in a number of kinase tool compounds [43,44]. 5-(2-Fluoropyridin-4-yl)-3-[(1H-indazol-5-yl)amino]-4H-1,2,6-thiadiazin-4-one (26) demonstrated potent activity against both bladder (IC50 = 8.4 μM) and prostate (IC50 = 5.7 μM) cancer with no toxicity on the WS-1 cell line (IC50 = >100 μM).

2.3. Kinome Profiling of Thiadiazinones 16, 17, and 26

We then assessed the kinome profile of thiadiazinones 16, 17, and 26 all at 1 μM using a multiplexed kinase inhibitor bead set and quantitative mass spectrometry for detection of kinase peptides (MIB-MS) [36,45,46]. The MIB-MS proteomics was able to detect between 350 and 400 kinases in the cell lysates and competitive displacement of specific kinases from the beads was used to determine the kinome profile of the thiadiazinone kinase inhibitors. Compound 16 showed a narrow spectrum kinome profile with weak interactions on MAP4K5, MAP4K3, PRKCD, and PKN1 just below the threshold (Figure 3). Compound 17 also had a narrow spectrum kinome profile, with AKT2, MAP2K4, MAP2K1, and STK35 being just below the threshold (Figure 4). Compound 26 had a narrow spectrum kinome profile but was a weak inhibitor, showing affinity for ACVR1B, ACVR2A, and STK35 (Figure 5).

2.4. Modelling of Hits from Kinome Profiling on Thiadiazinones 16, 17, and 26

The key hits on thiadiazinones 16, 17, and 26 from the MIBS profiling, were then modelled to understand their interactions in the respective ATP binding site (Figure 6, Figure 7 and Figure 8). First, compound 16 was analyzed against MAP4K5 [47], MAP4K3 [48], PRKCD [49], and PKN1 [50]. Docking of the compounds was performed using Schrödinger Maestro [51]. Before docking into the ATP-binding site, prepared from the PDB structure as needed, the compounds were minimized using LigPrep. The docking pose that placed the azo-indole in a position to make a hydrogen bond from the backbone NH of the hinge region of the respective kinases was the most favorable (Figure 6A–D). The 1,2,6-thiadiazinone is relatively passive, acting as a linker to the solvent region where the phenolic alcohol can form a series of different hydrogen bonding interactions. In the case of MAP4K5, MAP4K3, and PRKCD (Figure 6A–C), this is both accepting and donating, forming a rigid fixture at the mouth of the ATP binding site. PKN1 is not able to accommodate this accepting interaction, so there is only a much weaker hydrogen bonding interaction with D764 and no interaction with K748.
In the case of compound 17 (Figure 7), the azo-indole was similarly positioned at the hinge in both AKT2 [52] and STK35 [47,53]. However, the rest of the molecule was not orientated differently in both cases. This enabled additional interactions in both cases where the 1,2,6-thiadiazinone is actively involved, forming a hydrogen bond with the residue T292 in AKT2 and D323 in STK35. This is in addition to a hydrogen bond interaction by the phenolic alcohol with N280 and N365 in AKT2 and STK35, respectively.
Thiadiazinone 26 (Figure 8), similarly to compounds 16 and 17, formed a strong hydrogen bond in the hinge region of ACVR1B [54] and STK35 [53]. In AVCR1B, the 1,2,6-thiadiazinone was passive with no strong interactions. The pyridine nitrogen did, however, form a hydrogen bond with a solvent-exposed lysine residue K337. In the case of STK35, the 1,2,6-thiadiazinone was actively involved, forming an interaction with a solvent-exposed lysine residue K231. This was in addition to another hydrogen bond interaction with the bringing amine between the indazole and 1,2,6-thiadiazinone. The ATP was too shallow to accommodate the pyridine interaction as seen in AVCR1B and interactions involved with the phenolic alcohol of compound 17.

3. Discussion

Previously, we demonstrated that the 4H-1,2,6-thiadiazin-4-one chemotype can function as an ATP-competitive kinase inhibitor, acting as a novel hinge binding motif. This was also the first report of a protein co-crystallization with this rare heterocycle [15]. We have now expanded the repertoire of the 4H-1,2,6-thiadiazin-4-one as a spacer unit within a kinase inhibitor affording unique chemical properties. The electronics of the 4H-1,2,6-thiadiazin-4-one core allows for negative charge transfer from the sulfur atom towards the conjugated system [55]. This electronic property, exploited in solar cell applications, can partly explain the general lack of kinome promiscuity compared to the dianilinopyrimidine [15]. The modular synthesis and relative narrow kinome spectrum make the 4H-1,2,6-thiadiazin-4-one an attractive chemotype for further development.
The chemical tractability of the protein kinases makes them an attractive target for drug development. Over 90 drugs have been approved that target the ATP binding site, predominantly in the field of oncology [19,56]. However, with emerging indications beyond cancer including the treatment of chronic diseases, such as inflammation and neurodegeneration, along with the emergence of resistance, development of compounds with improved potency and selectivity profiles are essential to remain at the cutting edge [57,58]. A number of indirect assays exist to assess the inhibitor affinity, and binding assays are some of the most accurate and robust methods to measure the potency and selectivity of ATP-competitive kinase inhibitors [59,60,61,62,63]. Ligand binding displacement assays are particularly important to provide an accepted form of direct measurement of kinase inhibition in drug optimization of ATP-binding site inhibitors for neglected kinases, where there are currently no robust and validated enzyme activity assays [59,63].
Our results afford new data on a series of interesting starting points for further optimization towards new kinases targets including MAP4K3, MAP4K5, PRKCD, PKN1, AKT2, STK35, and ACVR1B, all of which have some indication in proliferation. The early kinome data, combined with modelling and the accompanying phenotype on bladder cancer, afford exciting prospects for this scaffold. This work firmly puts 1,2,6-thiadiazin-4-one into the medicinal chemistry toolbox and provides another example of the application of sulfur heterocycles in drug design.

4. Materials and Methods

4.1. Cancer Cell Line Screening Panel

U-CH1 and U-CH2 cell lines were cultured in 80:20 IMDM/RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS), 1% penicillin/streptomycin in gel-coated flasks. A-431, PANC-1, and WS1 cell lines were cultured in DMEM medium supplemented with 10% FBS and 1% penicillin/streptomycin. DU145 and MCF-7 cells were cultured in MEM medium supplemented with 10% FBS and 1% penicillin/streptomycin. 5637 cells were cultured in RPMI-1640 medium supplemented with 10% FBS and 1% penicillin/streptomycin. Cells were seeded in 384-well plates and were treated with test compound in quadruplicate 24 h after plating. Cell viability was assessed at 72 h using alamarBlue (ThermoFisher, Waltham, MA, USA). Fluorescence was measured using Tecan Infinite 200 PRO (Tecan, Männedorf, Switzerland) plate reader with an excitation at 535 nM and emission at 590 nM. IC50 values were determined by nonlinear regression using GraphPad PrismTM software (GraphPad Software Inc., San Diego, CA, US). (Positive controls: MCF7, lapatinib IC50 = 6.3 μM [64]; 5637, pazopanib IC50 = 15 μM [65]; PANC1, panobinostat IC50 = 1.0 μM [66]; DU145, docetaxel IC50 = 5 nM [67]; UCH1, gefitinib IC50 = 1.4 μM [36]; UCH2, gefitinib IC50 = 23 μM [36]; A431, lapatinib IC50 = 100 nM [68]; WS-1, lapatinib IC50 = 13 μM [36]).

4.2. MIBS Profiling Methods

Previously described [45]. Briefly, multiplexed inhibitor beads (MIBS) affinity chromatography/MS analysis: SUM159 cells were cultured in a 50:50 mixture of DMEM and Nutrient Mixture F-12 medium (Gibco) supplemented with 5% fetal bovine serum, 1% anti/anti, 5 mg mL−1 insulin, and 1 mg·mL−1 hydrocortisone. Cells were maintained at 37 °C in a humidified 5% CO2 atmosphere. SUM159 cells were grown to 80% confluency, washed twice with PBS, and harvested by scraping cells in lysis buffer [50 mM HEPES, pH 7.5, 150 mM NaCl, 0.5% Triton X-100, 1 mM EDTA, 1 mM EGTA, 10 mM NaF, 2.5 mM Na3VO4, complete protease inhibitor cocktail (Roche, Basel, Switzerland), phosphatase inhibitor cocktail 2, and 3 (Sigma, St. Louis, MO, USA)]. Lysates were sonicated then clarified by centrifugation at 14000 V g for 15 min at 4 °C. Lysate was then filtered through a 0.2 mm syringe filter and frozen at −80 °C until use. Protein concentration was quantified using a Bradford assay the day of the experiment. DMSO or the indicated concentration of 16, 17, and 26 were added to lysate containing 4 mg of total protein. Lysates were vortexed briefly then incubated for 30 min on ice. Kinases were affinity purified as previously described [46]. Briefly, lysates were diluted to 1.33 mg mL−1 with lysis buffer then NaCl concentration brought to 1 M.
Diluted lysates were passed over a mixture of 25 mL of settled beads of each of the following inhibitors conjugated to ECH Sepharose beads: Purvalanol B, PP58, VI-16832, UNC21474A, UNC8088A, and 37.5 mL of settled beads conjugated to CTx-0294885 and VI-16832 [69]. The kinase inhibitor–bead conjugates were previously equilibrated in high-salt buffer (50 mM HEPES pH 7.5, 1M NaCl, 0.5% Triton X-100, 1 mM EDTA, and 1 mM EGTA). MIBs columns were sequentially washed with high-salt buffer, low-salt buffer (50 mm HEPES pH 7.5, 150 mM NaCl, 0.5% Triton X-100, 1 mM EDTA, and 1 mM EGTA), and SDS buffer (50 mm HEPES pH 7.5, 150 mm NaCl, 0.5% Triton X-100, 1 mm EDTA, 1 mm EGTA, and 0.1% SDS). Proteins were eluted by boiling samples in elution buffer (100 mM Tris·HCl pH 6.8, 0.5% SDS, and 1% β-mercaptoethanol) for 15 min twice. Dithiothreitol (DTT) was added to a final concentration of 5 mm and samples were incubated at 60 °C for 25 min. Samples were then cooled to room temperature on ice and alkylated by adding iodoacetamide to a final concentration of 20 mm for 30 min in the dark at room temperature. Samples were then concentrated in 10K Amicon Ultra centrifugal concentrators (Millipore, Burlington, MA, USA) followed by methanol and chloroform precipitation of proteins. The final protein pellets were re-suspended in 50 mM HEPES pH 8.0 and incubated with trypsin at 37 °C overnight. Residual detergent was removed by three sequential ethyl acetate extractions then desalted using Pierce C18 spin columns (Thermo Scientific, Waltham, MA, USA) according to the manufacturer’s protocol.

4.3. Molecular Modelling Methods

4.3.1. Ligand Preparation

Structures of small molecules were parametrized and minimized using the LigPrep module of Schrodinger 2020-4 suite employing OPLS3e force field [51].

4.3.2. Protein Preparation

The kinase a crystal structures co-crystallized with substrate analogues or inhibitors were downloaded from RCSB-databank: MAP4K3-(PDB:5J5T) [70]; PKN1-(PDB:4OTI) [71]; AKT2-(PDB:2X39) [72]. The structures were H-bond optimized and minimized using the standard protein preparation procedure of Schrödinger suite.

4.3.3. Development of Homology-Based Model

Three-dimensional models of ACVR1B, STK35, and MAP4K5 were developed using Biovia Discovery Studio 2019 program (Dassault Systèmes, San Diego, CA, USA). The human forms of target sequences were downloaded from uniport, and psi-blast searched against the PDB-template database. Pre-aligned template structures (ACVR1B-(PDB:5E8X) [73], STK35-(PDB:3MA6), and MAP4K5-(PDB:5J5T) [70]) were downloaded to Discovery Studio and Alignments to corresponding kinase domains. The sequence of corresponding unknown kinase was then aligned to template structures. The models were built using standard settings of the modeler: optimization level = high, Number of models = 20, Refine loops = false, original ligands were copied from templates. Models were then ranked according to the PDF score and highest ranked models were uploaded to Maestro for protein preparation protocol. Models of AVCR1B and MAP4K5 were also successfully generated using protein structure prediction functionality available at GalaxyWEB-modelling site (http://galaxy.seoklab.org/ Accessed on: 6 September 2021), and cross checked with in-house generated models. During the final stage of this study, several AlphaFold structures available including ACVR1B, STK35, and MAPK5 were downloaded [74], prepared, and aligned to compare the positions of key residues with previously generated in-house models, which were consistent with our findings (data not shown).

4.3.4. Molecular Docking

The ligand docking was performed using an induced fit protocol using the standard settings of Schrodinger suite 2020-4 (up to 20 models were generated, dockings were done using SP-level of IFD-setting during Glide docking and redocking steps). The centers of glide grids were set to the centroid of co-crystallized ligands or template-derived ligands. The highest ranked docking poses were visually examined to conclude favorable hinge binding forms.

4.4. Chemistry Experimental Section

4.4.1. General Methods and Materials

All chemicals were commercially available except those whose synthesis is described. Anhydrous Na2SO4 was used for drying organic extracts and all volatiles were removed under reduced pressure. 1,4-Dioxane was dried by refluxing over CaH2. All reaction mixtures and column eluents were monitored by TLC using commercial glass-backed thin layer chromatography (TLC) plates (Merck Kieselgel 60 F254, Darmstadt, Germany) [75]. The plates were observed under UV light at 254 and 365 nm. The technique of dry flash chromatography was used throughout for all non-TLC scale chromatographic separations using Merck Silica Gel 60 (less than 0.063 mm). Melting points were determined using a PolyTherm-A, Wagner & Munz, Koefler-Hotstage Microscope apparatus (Wagner & Munz, Munich, Germany). Solvents used for recrystallization are indicated after the melting point. UV spectra were obtained using a Perkin-Elmer Lambda-25 UV/vis spectrophotometer (Perkin-Elmer, Waltham, MA, USA) and inflections are identified by the abbreviation “inf”. IR spectra were recorded on a Shimadzu FTIR-NIR Prestige-21 spectrometer (Shimadzu, Kyoto, Japan) with Pike Miracle Ge ATR accessory (Pike Miracle, Madison, WI, USA) and strong, medium, and weak peaks are represented by s, m, and w, respectively. 1H and 13C NMR spectra were recorded on a Bruker Avance 300 (at 300 and 75 MHz, respectively), or a 500 machine (at 500 and 125 MHz, respectively, Bruker, Billerica, MA, USA, 1H and 13C NMR spectra of new compounds could be found from Supplementary Materials). Deuterated solvents were used for homonuclear lock and the signals are referenced to the deuterated solvent peaks. Attached proton test (APT) NMR studies were used for the assignment of the 13C peaks as CH3, CH2, CH, and Cq (quaternary). The Matrix-Assisted Laser Desorption/Ionization-Time of Flight (MALDI-TOF) mass spectra (+ve mode) were recorded on a Bruker Autoflex III Smartbeam instrument (Bruker, Leipzig, Germany), while ESI-APCI+ mass spectra were recorded on a Model 6110 Quadrupole MSD, Agilent Technologies (Agilent Technologies, Santa Clara, CA, USA). High resolution mass spectra were recorded in a ThermoFisher Q Exactive HF-X (ThermoFisher, Bremen, Germany) mass spectrometer coupled with a Waters Acquity H-class liquid chromatograph system. 3,5-Dichloro-4H-1,2,6-thiadiazin-4-one (2) [18], 3-chloro-5-[(3-hydroxy-4-methylphenyl)amino]-4H-1,2,6-thiadiazin-4-one (9) [15] and 3-chloro-5-morpholino-4H-1,2,6-thiadiazin-4-one (11) [29] were prepared according to the reported procedures.

4.4.2. Preparation of 3-Aminosubstituted-4H-1,2,6-thiadiazines

3-[(3-Acetylphenyl)amino]-5-chloro-4H-1,2,6-thiadiazin-4-one (6) (General procedure). To a stirred solution of 3,5-dichloro-4H-1,2,6-thiadiazin-4-one (2) (366 mg, 2 mmol) in EtOH (4 mL), at ca. 20 °C was added 1-(3-aminophenyl)ethan-1-one (135 mg, 1 mmol) in one portion and the mixture stirred for 30 min at this temperature. Then, 2,6-lutidine (233 μL, 4 mmol) was added and the mixture was stirred at this temperature until complete consumption of the starting material (TLC, 2 h). The yellow solid formed was then filtered under vacuum and washed with EtOH (2 mL), t-BuOMe (5 mL), and n-hexane (5 mL) to give the title compound 6 (190 mg, 67%) as yellow needles, mp 201–202 °C (from EtOH/THF); Rf 0.41 (DCM); (found: C, 46.70; H, 2.97; N, 14.73. C11H8ClN3O2S requires C, 46.90; H, 2.86; N, 14.92%); λmax(DCM)/nm 295 (log ε 4.12), 321 (4.25), 334 inf (4.18), 413 (3.69); vmax/cm−1 3258m (N-H), 1680s, 1632s, 1589m, 1547s, 1518m, 1487w, 1439m, 1431s, 1356m, 1315w, 1312w, 1287m, 1260m, 1240m, 1177m, 1169m, 997m, 908s, 868m, 810m, 795m; δH(300 MHz; DMSO-d6) 10.32 (1H, s, NH), 8.39 (1H, dd, J 1.8, 1.8, Ar H), 8.04 (1H, ddd, J 8.1, 2.2, 1.0, Ar H), 7.72 (1H, ddd, J 7.7, 1.3, 1.1, Ar H), 7.51 (1H, dd, J 8.0, 8.0, Ar H), 2.58 (3H, s, CH3); δC(75 MHz; DMSO-d6) 197.5 (Cq), 157.1 (Cq), 150.2 (Cq), 141.4 (Cq), 138.4 (Cq), 137.2 (Cq), 129.0 (CH), 125.1 (CH), 123.9 (CH), 120.2 (CH), 26.7 (CH3); m/z (MALDI-TOF) 284 (MH++2, 39%), 282 (MH+, 100), 265 (37), 240 (66).
3-Chloro-5-[(3-methoxyphenyl)amino]-4H-1,2,6-thiadiazin-4-one (7). Similar treatment of 3,5-dichloro-4H-1,2,6-thiadiazin-4-one (2) (183 mg, 1 mmol) in EtOH (2 mL), with 3-methoxyaniline (246 mg, 2 mmol) and 2,6-lutidine (116 μL, 2 mmol) for 1 h gave the title compound 7 (494 mg, 92%) as yellow plates, mp 176–177 °C (from PhH/EtOH); Rf 0.54 (DCM); (found: C, 44.45; H, 2.97; N, 15.56. C10H8ClN3O2S requires C, 44.53; H, 2.99; N, 15.58%); λmax(DCM)/nm 324 (log ε 4.61), 419 (4.00); vmax/cm−1 3306m (N-H), 3067w (C-H ar), 2833 (C-H alip.), 1624s, 1613m, 1609m, 1589m, 1566s, 1514w, 1501m, 1489m, 1483m, 1445w, 1435m, 1427m, 1321m, 1294m, 1275m, 1215m, 1206m, 1188w, 1175w, 1148s, 1059s, 1011w, 932m, 874m, 870m, 831s, 772m, 733m; δH(500 MHz; CDCl3) 8.66 (1H, br. s, NH), 7.31 (1H, dd, J 2.2, 2.2, Ar H), 7.29 (1H, d, J 8.2, Ar H), 7.14 (1H, dd, J 8.0, 1.8, Ar H), 6.74 (1H, dd, J 8.3, 2.4, Ar H), 3.84 (3H, s, CH3); δC(125 MHz; CDCl3) 160.3 (Cq), 156.6 (Cq), 149.7 (Cq), 142.0 (Cq), 137.9 (Cq), 130.0 (CH), 112.2 (CH), 110.3 (CH), 105.9 (CH), 55.4 (CH3); m/z (MALDI-TOF) 272 (MH++2, 39), 270 (MH+, 79%), 234 (100), 180 (92).
3-Chloro-5-[(3-hydroxyphenyl)amino]-4H-1,2,6-thiadiazin-4-one (8). Similar treatment of 3,5-dichloro-4H-1,2,6-thiadiazin-4-one (2) (183 mg, 1 mmol) in EtOH (2 mL), with 2-aminophenol (109 mg, 1 mmol), and 2,6-lutidine (116 μL, 2 mmol) for 30 min gave the title compound 8 (228 mg, 89%) as yellow needles, mp 204–205 °C (from PhH/THF); Rf 0.25 (DCM); (found: C, 42.11; H, 2.46; N, 16.24. C9H6ClN3O2S requires C, 42.28; H, 2.37; N, 16.44%); λmax(DCM)/nm 259 (log ε 3.78), 288 inf (4.40), 323 (4.23), 417 (4.03); vmax/cm−1 3379br (O-H), 3256 (N-H), 1640m, 1599s, 1560m, 1551s, 1524m, 1466m, 1451m, 1439m, 1321w, 1304m, 1277m, 1227w, 1148m, 1092w, 972m, 870m, 860m, 841m, 783m, 739m, 731m; δH(300 MHz; DMSO-d6) 9.96 (1H, s), 9.52 (1H, br. s), 7.28 (1H, dd, J 2,1, 2.1, Ar H), 7.17 (1H, dd, J 7.7, 1.3, Ar H), 7.12 (1H, dd, J 8.0, 8.0, Ar H), 6.53 (1H, ddd, J 7.7, 2.2, 1.3, Ar H); δC(125 MHz; DMSO-d6) 157.4 (Cq), 157.1 (Cq), 150.0 (Cq), 141.0 (Cq), 138.8 (Cq), 129.2 (CH), 111.7 (CH), 111.2 (CH), 107.7 (CH); m/z (MALDI-TOF) 258 (MH++ 2, 32), 256 (MH+, 100%), 238 (56), 220 (45), 166 (32).
3-Chloro-5-[(5-hydroxy-2-methylphenyl)amino]-4H-1,2,6-thiadiazin-4-one (10). Similar treatment of 3,5-dichloro-4H-1,2,6-thiadiazin-4-one (2) (183 mg, 1 mmol) in EtOH (2 mL), with 3-amino-4-methylphenol (123 mg, 1 mmol) and 2,6-lutidine (116 μL, 2 mmol) for 1 h gave the title compound 10 (262 mg, 97%) as yellow needles, mp 191–192 °C (from PhH/c-hexane); Rf 0.18 (DCM); (found: C, 44.59; H, 2.91; N, 15.66. C10H8ClN3O2S requires C, 44.53; H, 2.99; N, 15.58%); λmax(DCM)/nm 319 (log ε 4.27), 398 (3.73); vmax/cm−1 3420br (O-H), 3364 (N-H), 1620m, 1609m, 1551s, 1518m, 1514m, 1466m, 1462m, 1445m, 1356m, 1267m, 1217m, 1153m, 1105m, 1015m, 1005m, 974m, 864m, 847m, 810m, 737m; δH(500 MHz; DMSO-d6) 9.52 (1H, s), 9.33 (1H, s), 7.04 (1H, d, J 8.3, Ar H), 6.90 (1H, d, J 2.5, Ar H), 6.58 (1H, dd, J 8.2, 2.5, Ar H), 2.09 (3H, s, CH3); δC(125 MHz; DMSO-d6) 157.0 (Cq), 155.5 (Cq), 151.4 (Cq), 140.4 (Cq), 135.9 (Cq), 130.9 (CH), 122.6 (Cq), 113.1 (CH), 111.9 (CH), 16.6 (CH3); m/z (MALDI-TOF) 272 (MH++2, 70%), 270 (MH+, 100), 252 (96), 234 (55), 211 (34), 180 (37).
3-Chloro-5-(4-methylpiperazin-1-yl)-4H-1,2,6-thiadiazin-4-one (12). Similar treatment of 3,5-dichloro-4H-1,2,6-thiadiazin-4-one (2) (183 mg, 1 mmol) in EtOH (2 mL), with N-methylpiperazine (215 μL, 2 mmol) for 30 min gave the title compound 12 (245 mg, 99%) as yellow needles, mp 76–77 °C (from c-hexane); Rf 0.29 (DCM/t-BuOMe, 50:50); (found: C, 38.72; H, 4.66; N, 22.53. C8H11ClN4OS requires C, 38.95; H, 4.49; N, 22.71%); λmax(DCM)/nm 269 (log ε 3.78), 312 (4.15), 320 inf (4.18), 406 (3.78); vmax/cm−1 2965w, 2934w, 2845w and 2804w (C-H), 1624s, 1493m, 1445m, 1433m, 1402m, 1356m, 1335w, 1306m, 1288m, 1271m, 1196m, 1159m, 1144m, 1080m, 1059m, 1005m, 984m, 939m, 864m, 854m, 843m, 791m, 775m, 760m, 727m; δH(500 MHz; CDCl3) 3.92 (4H, br. s, CH2), 2.51 (4H, t, J 5.1, CH2), 2.32 (3H, s, CH3); δC(125 MHz; CDCl3) 158.7 (Cq), 152.9 (Cq), 145.2 (Cq), 54.8 (CH2), 46.6 (CH2), 45.9 (CH3); m/z (ESI+) 249 (MH++2, 37%), 247 (MH+, 100), 130 (82).
5-Chloro-3-[(1H-indazol-5-yl)amino]-4H-1,2,6-thiadiazin-4-one (13). Similar treatment of 3,5-dichloro-4H-1,2,6-thiadiazin-4-one (2) (183 mg, 1 mmol) in EtOH (2 mL), with 1H-indazol-5-amine (133 mg, 1 mmol) and 2,6-lutidine (116 μL, 2 mmol) for 1 h gave the title compound 13 (255 mg, 91%) as orange plates, mp 290–291 °C (from PhMe/THF); Rf 0.53 (DCM/t-BuOMe, 80:20); (found: C, 42.82; H, 2.09; N, 24.91. C10H6ClN5OS requires C, 42.94; H, 2.16; N, 25.04%); λmax(THF)/nm 254 (log ε 4.57), 286 (4.59), 325 (4.40), 338 (4.38), 423 (3.77); vmax/cm−1 3393m (N-H), 3213w and 3086w (C-H), 1626m, 1589m, 1576w, 1562m, 1557m, 1508s, 1504m, 1427m, 1281m, 1198m, 1138m, 961w, 937s, 872s, 841w, 808m, 754m; δH(500 MHz; DMSO-d6) 13.06 (1H, s, NH), 10.18 (1H, s, NH), 8.18 (1H, s, Ar H), 8.07 (1H, s, Ar H), 7.65 (1H, d, J 7.5, Ar H), 7.52 (1H, d, J 7.5, Ar H); δC(125 MHz; DMSO-d6) 157.1 (Cq), 150.5 (Cq), 140.5 (Cq), 137.2 (Cq), 133.5 (CH), 130.6 (Cq), 122.5 (Cq), 122.0 (CH), 111.7 (CH), 110.1 (CH); m/z (MALDI-TOF) 282 (MH++2, 33%), 280 (MH+, 100), 244 (44), 190 (50).

4.4.3. Preparation of 5-Amino-Substituted 3-Arylthiadiazinones

3-[(3-Acetylphenyl)amino]-5-(1H-pyrrolo[2,3-b]pyridin-4-yl)-4H-1,2,6-thiadiazin-4-one (14). Similar treatment of 3-[(3-acetylphenyl)amino]-5-chloro-4H-1,2,6-thiadiazin-4-one (6) (56.3 mg, 0.20 mmol) with (1H-pyrrolo[2,3-b]pyridin-4-yl)boronic acid (40.8 mg, 0.22 mmol) for 18 h gave the title compound 14 (35.5 mg, 49%) as yellow needles, mp 271–273 °C (from EtOH/PhH); Rf 0.46 (DCM/Et2O, 50:50); (found: C, 59.25; H, 3.72; N, 19.06. C18H13N5O2S requires C, 59.49; H, 3.61; N, 19.27%); λmax(THF)/nm 290 (log ε 4.00), 360 (4.40), 415 inf (4.18); vmax/cm−1 3148w, 2916w, 1672 m, 1612m, 1583m, 1451s, 1535s, 1483w, 1443m, 1429w, 1323m, 1287w, 1265s, 1223m, 953m, 908m, 898m, 878m, 851m, 843m, 833m, 825m, 816m, 783m, 768m, 739m, 723m, 704m; δH(500 MHz; DMSO-d6) 11.80 (1H, s), 10.39 (1H, s), 8.47 (1H, dd, J 1.8, 1.8, Ar H), 8.33 (1H, d, J 5.1, Ar H), 8.12 (1H, dd, J 8.0, 1.1, Ar H), 7.75 (1H, d, J 5.0, Ar H), 7.73 (1H, d, J 1.1, Ar H), 7.57–7.52 (2H, m, Ar H), 6.77 (1H, dd, J 3.3, 1.9, Ar H); δC(75 MHz; DMSO-d6) 159.3 (Cq), 152.4 (Cq), 151.9 (Cq), 149.4 (Cq), 141.8 (CH), 138.4 (Cq), 137.2 (Cq), 133.6 (Cq), 129.0 (CH), 126.9 (CH), 125.2 (CH), 123.7 (CH), 120.3 (CH), 116.9 (Cq), 114.8 (CH), 100.8 (CH), 26.7 (CH3); m/z (ESI+) 363 (M+, 56%), 362 (M+-H, 100), 350 (62); HRMS (ESI+) found for MH+ 364.08444, C18H14N5O2S+ requires 364.08682.
3-[(3-Methoxyphenyl)amino]-5-(1H-pyrrolo[2,3-b]pyridin-4-yl)-4H-1,2,6-thiadiazin-4-one (15) (General procedure A). To a mixture of 3-chloro-5-[(3-methoxyphenyl)amino]-4H-1,2,6-thiadiazin-4-one (7) (53.9 mg, 0.20 mmol), (1H-pyrrolo[2,3-b]pyridin-4-yl)boronic acid (35.7 mg, 0.22 mmol), Pd[3,5-(F3C)2C6H3]3 (21.2 mg, 5 mol%), DPEPhos (5.3 mg, 5 mol%) and powdered dry K2CO3 (66.4 mg, 0.48 mmol) was added dioxane (2 mL) and H2O (0.3 mL). The stirred suspension was then deaerated by bubbling of Ar through it for 5 min and then heated at reflux under Ar until complete consumption of the starting thiadiazine (TLC, 20 h). The mixture was cooled to ca. 20 °C, then filtered and washed with H2O (5 mL) and EtOH (5 mL) and dried under vacuum to give the title compound 15 (58.4 mg, 83%) as orange needles, mp 257–258 °C (from c-hexane); Rf 0.59 (DCM/t-BuOMe, 80:20); (found: C, 58.12; H, 3.67; N, 19.85. C17H13N5O2S requires C, 58.11; H, 3.73; N, 19.93%); λmax(THF)/nm 360 (log ε 4.05), 422 inf (3.79); vmax/cm−1 3136w (C-H), 1613m, 1605m, 1587m, 1541m, 1534s, 1531s, 1483m, 1462m, 1327m, 1288m, 1240w, 1223m, 1211m, 1155s, 1092w, 1049m, 966w, 897m, 872w, 792m, 777m, 768m, 760m, 718m; δH(500 MHz; DMSO-d6) 11.79 (1H, br, NH), 10.14 (1H, s, NH), 8.32 (1H, d, J 5.0, Ar H), 7.38 (1H, dd, J 5.0, Ar H), 7.57–7.48 (3H, m, Ar H), 7.28 (1H, dd, J 8.2, 8.2, Ar H), 6.76 (1H, d, J 3.0, 1.7, Ar H), 6.72 (1H, dd, J 8.1, 1.7, Ar H), 3.77 (3H, s, CH3); δC(75 MHz; DMSO-d6) 159.4 (Cq), 159.3 (Cq), 152.3 (Cq), 151.6 (Cq), 149.4 (Cq), 141.8 (CH), 139.1 (Cq), 133.7 (Cq), 129.4 (CH), 129.9 (CH), 116.9 (Cq), 114.8 (CH), 112.9 (CH), 109.3 (CH), 106.6 (CH), 100.9 (CH), 55.0 (CH3); m/z (MALDI-TOF) 352 (MH+, 100%), 351 (M+, 35); HRMS (ESI+) found for MH+ 352.08567, C17H14N5O2S+ requires 352.08682.
3-[(3-Hydroxyphenyl)amino]-5-(1H-pyrrolo[2,3-b]pyridin-4-yl)-4H-1,2,6-thiadiazin-4-one (16). Similar treatment of 3-chloro-5-[(3-hydroxy-4-methylphenyl)amino]-4H-1,2,6-thiadiazin-4-one (8) (53.9 mg, 0.20 mmol) with (1H-pyrrolo[2,3-b]pyridin-4-yl)boronic acid (40.3 mg, 0.22 mmol) for 24 h gave after chromatography (DCM/t-BuOMe, 50:50) the title compound 16 (24.2 mg, 36%) as yellow plates, mp 326–327 °C (from PhH); Rf 0.56 (DCM/t-BuOMe, 50:50); (found: C, 57.14; H, 3.22; N, 20.52. C16H11N5O2S requires C, 56.97; H, 3.29; N, 20.76%); λmax(THF)/nm 366 (log ε 4.33), 423 inf (4.07); vmax/cm−1 3401w and 3331w (N-H), 1612m, 1591s, 1587s, 1543s, 1503m, 1381w, 1332m, 1269w, 1204m, 1167m, 1153m, 989m, 793m, 789m, 770m, 727m, 716m; δH(500 MHz; DMSO-d6) 11.79 (1H, s), 10.03 (1H, s), 9.48 (1H, s), 8.31 (1H, d, J 5.0, Ar H), 7.74 (1H, d, J 5.0, Ar H), 7.55 (1H, dd, J 2.9, 2.9, Ar H), 7.39 (1H, dd, J 1.9, 1.9, Ar H), 7.25 (1H, d, J 8.0, Ar H), 7.15 (1H, dd, J 8.0, 8.0, Ar H), 6.76 (1H, dd, J 3.2, 1.9, Ar H), 6.55 (1H, dd, J 8.0, 1.7, Ar H); δC(75 MHz; DMSO-d6) 159.3 (Cq), 157.5 (Cq), 152.2 (Cq), 151.5 (Cq), 149.4 (Cq), 141.8 (CH), 138.9 (Cq), 133.7 (Cq), 129.2 (CH), 126.9 (CH), 116.9 (Cq), 114.8 (CH), 111.7 (CH), 107.7 (CH), 100.9 (CH); m/z (MALDI-TOF) 338 (MH+, 100%), 337 (M+, 67); HRMS (ESI+) found for MH+ 338.06991, C16H12N5O2S+ requires 338.07117.
3-[(3-Hydroxy-4-methylphenyl)amino]-5-(1H-pyrrolo[2,3-b]pyridin-4-yl)-4H-1,2,6-thiadiazin-4-one (17). Similar treatment of 3-chloro-5-[(3-hydroxy-4-methylphenyl)amino]-4H-1,2,6-thiadiazin-4-one (9) (53.9 mg, 0.20 mmol) with (1H-pyrrolo[2,3-b]pyridin-4-yl)boronic acid (30.0 mg, 0.22 mmol) for 2 h gave after chromatography (DCM/Et2O/THF, 50:40:10) the title compound 17 (51.4 mg, 73%) as yellow needles, mp 292–293 °C (from PhH); Rf 0.61 (DCM/Et2O/THF, 50:40:10); (found: C, 58.12; H, 3.67; N, 19.85. C17H13N5O2S requires C, 58.11; H, 3.73; N, 19.93%); λmax(THF)/nm 295 (log ε 3.75), 366 (4.00), 430 inf (3.68); vmax/cm−1 3298w, 3138w, 2882w, 1614m, 1597m, 1591m, 1535s, 1503m, 1422m, 1277w, 1260w, 1231w, 1219w, 1207w, 1186w, 1159w, 1155w, 1128w, 1111m, 995m, 899m, 856m, 789m, 729m, 718s; δH(300 MHz; DMSO-d6) 11.79 (1H, br s), 9.98 (1H, s), 9.39 (1H, s), 8.31 (1H, d, J 5.0, Ar H), 7.74 (1H, d, J 5.0, Ar H), 7.55 (1H, dd, J 2.8, 2.8, Ar H), 7.42 (1H, d, J 1.9, Ar H), 7.13 (1H, dd, J 8.2, 1.9, Ar H), 7.03 (1H, d, J 8.0, Ar H), 6.76 (1H, dd, J 3.3, 1.9, Ar H), 2.10 (3H. s, CH3); δC(75 MHz; DMSO-d6) 159.3 (Cq), 155.1 (Cq), 152.2 (Cq), 151.2 (Cq), 149.4 (Cq), 141.7 (CH), 136.3 (Cq), 133.8 (Cq), 130.2 (CH), 126.8 (CH), 119.8 (Cq), 116.9 (Cq), 114.8 (CH), 111.8 (CH), 107.3 (CH), 100.9 (CH), 15.5 (CH3); m/z (MALDI-TOF) 352 (MH+, 90%), 351 (M+, 100); HRMS (ESI+) found for MH+ 352.08558, C17H14N5O2S+ requires 352.08682.
3-[(5-Hydroxy-2-methylphenyl)amino]-5-(1H-pyrrolo[2,3-b]pyridin-4-yl)-4H-1,2,6-thiadiazin-4-one (18). Similar treatment of 2-[(5-chloro-4-oxo-4H-1,2,6-thiadiazin-3-yl)amino]-N-methylbenzamide (10) (59.3 mg, 0.20 mmol) with (1H-pyrrolo[2,3-b]pyridin-4-yl)boronic acid (39.2 mg, 0.22 mmol) for 3 h gave after chromatography (DCM/Et2O, 50:50) the title compound 18 (39.1 mg, 56%) as yellow needles, mp 304–305 °C (from PhH); Rf 0.63 (DCM/Et2O, 50:50); (found: C, 58.36; H, 3.69; N, 19.74. C17H13N5O2S requires C, 58.11; H, 3.73; N, 19.93%); λmax(THF)/nm 292 (log ε 3.34), 364 (3.60), 420 inf (3.37); vmax/cm−1 3339w, 3102w, 2880w, 1612m, 1587m, 1541s, 1537s, 1481m, 1477m, 1381w, 1321m, 1287w, 1221w, 1204w, 1155m, 1130w, 1115w, 1007m, 982m, 899m, 866m, 812m, 808m, 797m, 791m, 764m, 731m, 727m, 716m; δH(300 MHz; DMSO-d6) 11.79 (1H, br s), 9.58 (1H, s), 9.34 (1H, s), 8.31 (1H, d, J 5.0, Ar H), 7.80 (1H, d, J 5.1, Ar H), 7.56 (1H, dd, J 3.0, 3.0, Ar H), 7.12 (1H, d, J 2.5, Ar H), 7.07 (1H, d, J 8.3, Ar H), 6.78 (1H, dd, J 3.4, 1.9, Ar H), 6.58 (1H, dd, J 8.3, 2.5, Ar H); δC(75 MHz; DMSO-d6) 159.2 (Cq), 155.6 (Cq), 153.1 (Cq), 150.7 (Cq), 149.4 (Cq), 141.8 (CH), 136.0 (Cq), 133.6 (Cq), 130.8 (CH), 126.9 (CH), 121.7 (Cq), 116.9 (Cq), 114.9 (CH), 112.6 (CH), 111.0 (CH), 100.8 (CH), 16.6 (CH3); m/z (MALDI-TOF) 352 (MH+, 17%), 351 (M+, 100); HRMS (ESI+) found for MH+ 352.08576, C17H14N5O2S+ requires 352.08682.
3-Morpholino-5-(1H-pyrrolo[2,3-b]pyridin-4-yl)-4H-1,2,6-thiadiazin-4-one (19). Similar treatment of 3-chloro-5-[(3-hydroxy-4-methylphenyl)amino]-4H-1,2,6-thiadiazin-4-one (11) (53.9 mg, 0.20 mmol) with (1H-pyrrolo[2,3-b]pyridin-4-yl)boronic acid (30.2 mg, 0.22 mmol) for 22 h gave after chromatography (DCM/t-BuOMe, 50:50) the title compound 19 (36.7 mg, 58%) as yellow needles, mp 256–257 °C (from EtOH/THF); Rf 0.75 (DCM/t-BuOMe, 50:50); (found: C, 53.08; H, 4.12; N, 22.35. C14H13N5O2S requires C, 53.22; H, 4.16; N, 22.21%); λmax(THF)/nm 291 (log ε 3.63), 298 (3.62), 358 (3.71), 408 inf (3.55); vmax/cm−1 3140w, 3069w, 2978w, 2887w, 2852w, 1603s, 1560w, 1493m, 1483m, 1439m, 1404m, 1327m, 1319m, 1310m, 1283m, 1269m, 1248s, 1217m, 1192m, 1124s, 1065m, 1007m, 959m, 922m, 903m, 878m, 858m, 835m, 810s, 795s, 746m, 727m, 710m; δH(300 MHz; DMSO-d6) 11.76 (1H, br s, NH), 8.27 (1H, d, J 5.0, Ar H), 7.53 (1H, dd, J 3.2, 2.7, Ar H), 7.51 (1H, d, J 5.0, Ar H), 6.60 (1H, dd, J 3.4, 1.8, Ar H), 3.78 (4H, br. s, CH2), 3.72 (4H, br. s, CH2); δC(75 MHz; DMSO-d6) 162.3 (Cq), 155.0 (Cq), 154.3 (Cq), 149.3 (Cq), 141.7 (CH), 134.0 (Cq), 126.8 (CH), 117.0 (Cq), 114.9 (CH), 100.7 (CH), 65.7 (CH2), 46.2 (CH2); m/z (MALDI-TOF) 316 (M+H+, 100%), 315 (M+, 89); HRMS (ESI+) found for MH+ 316.08558, C14H14N5O2S+ requires 316.08682.
3-(4-Methylpiperazin-1-yl)-5-(1H-pyrrolo[2,3-b]pyridin-4-yl)-4H-1,2,6-thiadiazin-4-one (20). Similar treatment of 3-chloro-5-(4-methylpiperazin-1-yl)-4H-1,2,6-thiadiazin-4-one (12) (49.4 mg, 0.20 mmol) with (1H-pyrrolo[2,3-b]pyridin-4-yl)boronic acid (26.0 mg, 0.22 mmol) for 1 h gave after chromatography (DCM/THF, 50:50) the title compound 20 (60.8 mg, 93%) as yellow needles, mp 195–196 °C (from c-hexane/CHCl3); Rf 0.21 (DCM/THF, 50:50); (found: C, 54.59; H, 5.14; N, 25.36. C15H16N6OS requires C, 54.86; H, 4.91; N, 25.59%); λmax(DCM)/nm 259 (log ε 4.08), 358 (4.26), 409 (4.06); vmax/cm−1 3134w, 2938w, 2884w, 2843w, 2787w, 1611m, 1593m, 1564m, 1463m, 1447m, 1410m, 1368w, 1331m, 1281m, 1259m, 1234m, 1219m, 1196m, 1165m, 1148m, 1090m, 1078m, 1001m, 1051m, 895m, 870m, 827s, 789s, 746m, 733m, 708s, 660m, 613m; δH(500 MHz; DMSO-d6) 11.76 (1H, s, NH), 8.27 (1H, d, J 5.0, Ar H), 7.53 (1H, dd, J 3.0, 3.0, Ar H), 7.51 (1H, d, J 5.0, Ar H), 6.60 (1H, dd, J 3.4, 1.9, Ar H), 3.77 (4H, br. s, CH2), 2.45 (4H, t, J 5.0, CH2), 2.22 (3H, s, CH3); δC(125 MHz; DMSO-d6) 162.3 (Cq), 155.0 (Cq), 154.2 (Cq), 149.3 (Cq), 141.8 (CH), 134.1 (Cq), 126.8 (CH), 117.0 (Cq), 114.9 (CH), 100.7 (CH), 54.2 (CH2), 45.54 (CH2), 45.5 (CH3); m/z (APCI+) 329 (MH+, 100%), 309 (34); HRMS (ESI+) found for MH+ 329.11702, C15H17N6OS+ requires 329.11845.
5-(3-Hydroxyphenyl)-3-[(1H-indazol-5-yl)amino]-4H-1,2,6-thiadiazin-4-one (23) (General procedure B). To a mixture of 5-chloro-3-[(1H-indazol-5-yl)amino]-4H-1,2,6-thiadiazin-4-one (13) (55.9 mg, 0.20 mmol), (3-hydroxyphenyl)boronic acid (55.2 mg, 0.40 mmol), Pd(dppf)Cl2 (14.6 mg, 10 mol%) and powdered dry Na2CO3 (42.4 mg, 0.40 mmol), in a sealed tube, was added dioxane (0.9 mL) and H2O (0.1 mL). The stirred suspension was then deaerated by bubbling of Ar through it for 5 min and then heated at ca. 140 °C under Ar until complete consumption of the starting thiadiazine (TLC, 1.5 h). The mixture was cooled to ca. 20 °C, adsorbed onto silica and chromatographed (DCM/t-BuOMe, 50:50) to give the title compound 23 (46.1 mg, 68%) as orange needles, mp 282–283 °C (from MeOH/THF); Rf 0.52 (DCM/t-BuOMe, 50:50); (found: C, 59.79; H, 3.43; N, 20.81. C16H11N5O2S requires C, 56.97; H, 3.29; N, 20.76%); λmax(THF)/nm 326 inf (log ε 3.81), 374 (4.00), 391 inf (3.79), 447 (3.49); vmax/cm−1 3331w, 1605m, 1591m, 1584m, 1580m, 1553s, 1510m, 1503m, 1346m, 1300m, 1229m, 1169w, 1086w, 997w, 968w, 951m, 895w, 866m, 829m, 814m, 800m, 814m, 779m, 754m; δH(500 MHz; DMSO-d6) 13.06 (1H, s), 10.15 (1H, s), 9.60 (1H, s), 8.25 (1H, s, Ar H), 8.08 (1H, s, Ar H), 7.68 (1H, dd, J 8.9, 1.4, Ar H), 7.56–7.51 (3H, m, Ar H), 7.27 (1H, dd, J 7.8, 7.8, Ar H), 6.86 (1H, dd, J 8.3, 2.4, Ar H); δC(125 MHz; DMSO-d6) 159.6 (Cq), 156.4 (Cq), 152.5 (Cq), 151.0 (Cq), 137.2 (Cq), 136.5 (Cq), 133.5 (CH), 130.9 (Cq), 128.9 (CH), 122.6 (Cq), 122.1 (CH), 119.0 (CH), 116.6 (CH), 114.9 (CH), 111.5 (CH), 110.1 (CH); m/z (APCI+) 338 (MH+, 100%), 313 (36); HRMS (ESI+) found for MH+ 338.06985, C16H12N5O2S+ requires 338.07117.
5-(5-Hydroxy-2-methylphenyl)-3-[(1H-indazol-5-yl)amino]-4H-1,2,6-thiadiazin-4-one (24). Similar treatment of 5-chloro-3-[(1H-indazol-5-yl)amino]-4H-1,2,6-thiadiazin-4-one (13) (55.9 mg, 0.20 mmol) with (5-hydroxy-2-methylphenyl)boronic acid (60.8 mg, 0.40 mmol) for 1.5 h gave after chromatography (DCM/t-BuOMe, 75:25) the title compound 24 (48.8 mg, 69%) as yellow needles, mp 268–269 °C (from PhH/THF); Rf 0.35 (DCM/t-BuOMe, 75:25); (found: C, 58.46; H, 3.51; N, 19.69. C17H13N5O2S requires C, 58.11; H, 3.73; N, 19.93%); λmax(THF)/nm 307 (log ε 3.90), 358 (3.93), 426 (3.44); vmax/cm−1 3337w, 3233w, 2689w, 1612m, 1591m, 1576m, 1557s, 1514m, 1504m, 1333m, 1300m, 1267w, 1238w, 1213w, 1167w, 1097w, 966w, 953m, 937w, 883w, 866m, 847m, 802m, 752m; δH(500 MHz; DMSO-d6) 13.06 (1H, s), 10.11 (1H, s), 9.35 (1H, s), 8.26 (1H, d, J 1.2, Ar H), 8.08 (1H, s, Ar H), 7.69 (1H, dd, J 9.0, 1.9, Ar H), 7.53 (1H, d, J 8.9, Ar H), 7.07 (1H, d, J 8.3, Ar H), 6.78 (1H, d, J 2.6, Ar H), 6.75 (1H, dd, J 8.2, 2.6, Ar H), 2.10 (3H, s, CH3); δC(125 MHz; DMSO-d6) 159.0 (Cq), 155.8 (Cq), 154.7 (Cq), 152.1 (Cq), 137.2 (Cq), 136.3 (Cq), 133.5 (CH), 130.9 (CH), 130.8 (Cq), 126.1 (Cq), 122.6 (Cq), 122.1 (CH), 115.9 (CH), 113.8 (CH), 111.5 (CH), 110,1 (CH), 18.6 (CH3); m/z (APCI+) 352 (MH+, 100%), 177 (M+, 67); HRMS (ESI+) found for MH+ 352.08560, C17H14N5O2S+ requires 352.08682.
3-[(1H-Indazol-5-yl)amino]-5-(3-methoxyphenyl)-4H-1,2,6-thiadiazin-4-one (25). Similar treatment of 5-chloro-3-[(1H-indazol-5-yl)amino]-4H-1,2,6-thiadiazin-4-one (13) (55.9 mg, 0.20 mmol) with (3-methoxyphenyl)boronic acid (60.8 mg, 0.40 mmol) for 2 h gave after chromatography (DCM/t-BuOMe, 95:5) the title compound 25 (55.1 mg, 78%) as orange needles, mp 220–221 °C (from EtOH/THF); Rf 0.29 (DCM/t-BuOMe, 95:5); (found: C, 58.20; H, 3.65; N, 19.89. C17H13N5O2S requires C, 58.11; H, 3.73; N, 19.93%); λmax(DCM)/nm 376 (log ε 4.04), 453 (3.53); vmax/cm−1 3335w (N-H), 2943w (C-H), 1605m, 1595m, 1587m, 1547m, 1512m, 1501m, 1491m, 1343m, 1323w, 1287m, 1244m, 1227m, 1215m, 1182w, 1167w, 1038m, 941m, 868m, 829m, 782m, 731m; δH(300 MHz; DMSO-d6) 13.06 (1H, br. s, NH), 10.22 (1H, s, NH), 8.26 (1H, d, J 1.4, Ar H), 8.08 (1H, s, Ar H), 7.72–7.66 (3H, m, Ar H), 7.54 (1H, d, J 9.1, Ar H), 7.41 (1H, dd, J 8.0, 8.0, Ar H), 7.05 (1H, ddd, J 4.8, 2.5, 0.9, Ar H), 3.81 (3H, s, CH3); δC(75 MHz; DMSO-d6) 159.6 (Cq), 158.7 (Cq), 152.5 (Cq), 150.6 (Cq), 137.2 (Cq), 136.5 (Cq), 133.5 (CH), 130.9 (Cq), 129.1 (CH), 122.6 (Cq), 122.0 (CH), 120.4 (CH), 115.2 (CH), 113.4 (CH), 111.5 (CH), 110.1 (CH), 55.1 (CH3); m/z (APCI+) 352 (MH+, 67%), 231 (52), 177 (46), 152 (98), 135 (82), 109 (100); HRMS (ESI+) found for MH+ 352.08557, C17H14N5O2S+ requires 352.08682.
(2-Fluoropyridin-4-yl)-3-[(1H-indazol-5-yl)amino]-4H-1,2,6-thiadiazin-4-one (26). Similar treatment of 5-chloro-3-[(1H-indazol-5-yl)amino]-4H-1,2,6-thiadiazin-4-one (13) (55.9 mg, 0.20 mmol) with (2-fluoropyridin-4-yl)boronic acid (56.4 mg, 0.40 mmol) for 1 h gave after chromatography (DCM/t-BuOMe, 50:50) the title compound 26 (54.3 mg, 92%) as orange needles, mp 280 °C (decomp., from EtOH/CHCl3); Rf 0.79 (DCM/t-BuOMe, 50:50); (found: C, 52.80; H, 2.72; N, 24.42. C15H9FN6OS requires C, 52.94; H, 2.67; N, 24.69%); λmax(DCM)/nm 252 (log ε 4.02), 322 (3.80), 384 (3.99), 453 inf (3.68); vmax/cm−1 3319w and 3221 br (N-H), 1620m, 1612m, 1587m, 1560m, 1557s, 1551s, 1547s, 1503s, 1483m, 1381m, 1352w, 1288m, 1223m, 1202w, 1161m, 1007m, 937m, 837s, 799s; δH(500 MHz; DMSO-d6) 13.10 (1H, s, NH), 10.41 (1H, s, NH), 8.38 (1H, d, J 5.3, Ar H), 7.24 (1H, d, J 1.2, Ar H), 8.09 (1H, s, Ar H), 8.01 (1H, d, J 5.2, Ar H), 7.83 (1H, s, Ar H), 7.70 (1H, dd, J 8.9, 1.8, Ar H), 7.55 (1H, d, J 8.9, Ar H); δC(125 MHz; DMSO-d6) 163.3 (Cq, d, 1JCF 232.5), 159.7 (Cq), 153.1 (Cq), 147.8 (CH, d, 3JCF 15.3), 147.5 (Cq, d, 3JCF 8.7), 146.3 (Cq, d, 4JCF 3.7), 137.3 (Cq), 133.6 (CH), 130.5 (Cq), 122.6 (Cq), 122.2 (CH), 120.0 (CH, d, 4JCF 3.7), 112.2 (CH), 110.2 (CH), 107.3 (CH, d, 2JCF 40.3); m/z (APCI+) 341 (MH+, 100%); HRMS (ESI+) found for MH+ 341.06105, C15H10FN6OS+ requires 341.06208.

4.4.4. Preparation of 3,5-Diaminosubstituted Thiadiazines

5-(4-Methylpiperazin-1-yl)-3-[(1H-pyrrolo[2,3-b]pyridin-4-yl)amino]-4H-1,2,6-thiadiazin-4-one (21) (general procedure). To a mixture of 3-chloro-5-(4-methylpiperazin-1-yl)-4H-1,2,6-thiadiazin-4-one (12) (49.4 mg, 0.20 mmol), Pd[3,5-(F3C)2C6H3]3 (5.3 mg, 1.25 mol%), DPEPhos (5.3 mg, 5 mol%), powdered dry K2CO3 (66.4 mg, 0.48 mmol) and 1H-pyrrolo[2,3-b]pyridin-4-amine (29.3 mg, 0.22 mmol) was added dry dioxane (4 mL). The stirred suspension was then deaerated by bubbling of Ar through it for 5 min and then heated at reflux under Ar until complete consumption of the starting thiadiazine (TLC, 2 h). The mixture was cooled to ca. 20 °C, then filtered and washed with H2O (5 mL) and EtOH (5 mL), and dried under vacuum to give the title compound 21 (43.6 mg, 64%) as yellow plates, mp 286–287 °C (from c-hexane/CHCl3); Rf 0.30 (THF); (found: C, 52.41; H, 4.83; N, 28.47. C15H17N7OS requires C, 52.46; H, 4.99; N, 28.55%); λmax(DCM)/nm 272 (log ε 4.06), 338 (4.71), 351 (4.71), 442 (4.17); vmax/cm−1 3130w, 3022w, 2961w, 1841w, 2792w, 1611m, 1587s, 1549m, 1535m, 1503m, 1485m, 1479m, 1443m, 1439m, 1402m, 1333m, 1306m, 1290m, 1271m, 1252w, 1225m, 1207w, 1161m, 1121m, 951w, 899m, 858w, 818m, 787m, 781m, 773m, 743m, 704m; δH(500 MHz; DMSO-d6) 11.66 (1H, s, NH), 9.28 (1H, s, NH), 8.13 (1H, d, J 5.4, Ar H), 7.73 (1H, d, J 5.4, Ar H), 7.38 (1H, dd, J 2.9, 2.9, Ar H), 6.61 (1H, dd, J 3.3, 1.8, Ar H), 3.64 (4H, br. s, CH2), 2.25 (3H, s, CH3), 4H missing due to overlap with H2O peak (2×CH2); δC(125 MHz; DMSO-d6) 156.7 (Cq), 151.6 (Cq), 149.4 (Cq), 148.6 (Cq), 143.6 (CH), 137.7 (Cq), 124.2 (CH), 110.2 (Cq), 103.4 (CH), 97.0 (CH), 54.0 (CH2), 45.9 (CH2), 45.4 (CH3); m/z (MALDI-TOF) 344 (MH+, 100%), 287 (95); HRMS found for MH+ 344.12803, C15H18N7OS+ requires 344.12935.
3-[(1H-Indazol-5-yl)amino]-5-(4-methylpiperazin-1-yl)-4H-1,2,6-thiadiazin-4-one (22). Similar treatment of 3-chloro-5-(4-methylpiperazin-1-yl)-4H-1,2,6-thiadiazin-4-one (12) (49.4 mg, 0.20 mmol), with 1H-indazol-5-amine (29.3 mg, 0.22 mmol) after 16 h gave after chromatography (DCM/t-BuOMe/Et3N, 50:49:1) the title compound 22 (65.2 mg, 92%) as yellow needles, mp 262–263 °C (from EtOH/THF); Rf 0.16 (DCM/t-BuOMe/Et3N, 50:49:1); (found: C, 52.70; H, 4.78; N, 28.62. C15H17N7OS requires C, 52.46; H, 4.99; N, 28.55%); λmax(THF)/nm 263 (log ε 4.30), 308 (3.69), 469 (3.00); vmax/cm−1 3339w, 1601m, 1585m, 1557m, 1503m, 1493m, 1437w, 1404w, 1306w, 1287w, 1277w, 1246w, 1221m, 1128m, 1078m, 1053s, 978m, 945m, 870m, 799s, 733m; δH(500 MHz; DMSO-d6) 13.01 (1H, br. s, NH), 9.65 (1H, s, NH), 8.21 (1H, s, Ar H), 8.03 (1H, s, Ar H), 7.61 (1H, dd, J 9.0, 1.8, Ar H), 7.49 (1H, d, J 9.0, Ar H), 3.66 (4H, m, CH2), 2.86 (4H, m, CH2), 3H missing due to overlap with DMSO peak (1×CH3); δC(125 MHz; DMSO-d6) 156.8 (Cq), 151.0 (Cq), 149.9 (Cq), 136.9 (Cq), 133.2 (CH), 131.8 (Cq), 122.6 (Cq), 121.7 (CH), 110.2 (CH), 109.9 (CH), 53.1 (CH2), 45.0 (CH2), 44.1 (CH3); m/z (MALDI-TOF) 344 (MH+, 100%), 287 (68); HRMS found for MH+ 344.12806, C15H18N7OS+ requires 344.12935.

Supplementary Materials

The following are available online, mol file, HRMS spectra on the final compounds, 1H and 13C NMR spectra on all new compounds.

Author Contributions

A.S.K., M.P.E., D.H.D., P.A.K., C.R.M.A. conceived and designed the study; A.S.K., M.P.E., T.L., C.D.T., K.A.M., C.R.M.A. performed the experiments; A.S.K., M.P.E., T.L., C.D.T., K.A.M., G.L.J., P.A.K., D.J.C., C.R.M.A. analyzed the data; A.S.K., P.A.K., C.R.M.A. edited the paper; C.R.M.A. wrote the paper and all authors approved the final manuscript. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the Cyprus Research Promotion Foundation, Grant No. NEAYPODOMH/NEKYP/0308/02. This work was partly supported by the NIH Common Fund Illuminating the Druggable Genome (IDG) program (NIH Grant U24DK116204).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Not applicable.

Acknowledgments

The authors thank the following organizations and companies in Cyprus for generous donations of chemicals and glassware: The State General Laboratory; the Agricultural Research Institute; the Ministry of Agriculture; MedoChemie, Ltd.; Medisell, Ltd.; and Biotronics, Ltd. Furthermore, we thank the A. G. Leventis Foundation for helping to establish the NMR facility at the University of Cyprus. We also thank the Biocenter Finland/DDCB for financial support towards the goals of our work and the CSC-IT Center for Science Ltd. (Finland) for the allocation of computational resources. In addition, we are grateful for LC−MS/HRMS support provided by Brandie Ehrmann and Diane E. Wallace in the Mass Spectrometry Core Laboratory at the University of North Carolina at Chapel Hill. The core is supported by the National Science Foundation under grant no. CHE-1726291. We also acknowledge-The SGC is a registered charity (number 1097737) that receives funds from AbbVie, Bayer Pharma AG, Boehringer Ingelheim, Canada Foundation for Innovation, Eshelman Institute for Innovation, Genome Canada, Innovative Medicines Initiative (EU/EFPIA), Janssen, Merck & Co., Novartis Pharma AG, Ontario Ministry of Economic Development and Innovation, Pfizer, São Paulo Research Foundation-FAPESP, Takeda, and Wellcome Trust.

Conflicts of Interest

The authors declare no conflict of interest.

Sample Availability

Samples of the compounds are available from the authors.

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Molecules 26 05911 sch001 550
Scheme 1. Route to access 3,5-dichloro-4H-1,2,6-thiadiazin-4-one (2).
Scheme 1. Route to access 3,5-dichloro-4H-1,2,6-thiadiazin-4-one (2).
Molecules 26 05911 sch001
Molecules 26 05911 g001 550
Figure 1. Representative examples of known anilinopyrimidines (highlighted in red) and general 1,2,6-thiadiazinone core (3).
Figure 1. Representative examples of known anilinopyrimidines (highlighted in red) and general 1,2,6-thiadiazinone core (3).
Molecules 26 05911 g001
Molecules 26 05911 sch002 550
Scheme 2. C-N coupling of 5-subsituted 3-chlorothiadiazinones 4 with (het)arylamines.
Scheme 2. C-N coupling of 5-subsituted 3-chlorothiadiazinones 4 with (het)arylamines.
Molecules 26 05911 sch002
Molecules 26 05911 g002 550
Figure 2. Design rational of novel 1,2,6-thiadiazinone analogues.
Figure 2. Design rational of novel 1,2,6-thiadiazinone analogues.
Molecules 26 05911 g002
Molecules 26 05911 sch003 550
Scheme 3. Synthetic route to access 5-amino-3-chloro-4H-1,2,6-thiadiazin-4-ones 6–13.
Scheme 3. Synthetic route to access 5-amino-3-chloro-4H-1,2,6-thiadiazin-4-ones 6–13.
Molecules 26 05911 sch003
Molecules 26 05911 sch004 550
Scheme 4. Suzuki couplings of thiadiazines 6–12 with (1H-pyrrolo[2,3-b]pyridin-4-yl)boronic acid to afford disubstituted products 14–19.
Scheme 4. Suzuki couplings of thiadiazines 6–12 with (1H-pyrrolo[2,3-b]pyridin-4-yl)boronic acid to afford disubstituted products 14–19.
Molecules 26 05911 sch004
Molecules 26 05911 sch005 550
Scheme 5. Scaffold diversification with tertiary amine substituent to afford 20–22.
Scheme 5. Scaffold diversification with tertiary amine substituent to afford 20–22.
Molecules 26 05911 sch005
Molecules 26 05911 sch006 550
Scheme 6. Suzuki couplings of 5-chloro-3-[(1H-indazol-5-yl)amino]-4H-1,2,6-thiadiazin-4-one (13).
Scheme 6. Suzuki couplings of 5-chloro-3-[(1H-indazol-5-yl)amino]-4H-1,2,6-thiadiazin-4-one (13).
Molecules 26 05911 sch006
Molecules 26 05911 g003 550
Figure 3. MIBS kinome profiling for thiadiazinone 16 (1 μM)/DMSO.
Figure 3. MIBS kinome profiling for thiadiazinone 16 (1 μM)/DMSO.
Molecules 26 05911 g003
Molecules 26 05911 g004 550
Figure 4. MIBS kinome profiling for thiadiazinone 17 (1 μM)/DMSO.
Figure 4. MIBS kinome profiling for thiadiazinone 17 (1 μM)/DMSO.
Molecules 26 05911 g004
Molecules 26 05911 g005 550
Figure 5. MIBS kinome profiling for thiadiazinone 26 (1 μM)/DMSO.
Figure 5. MIBS kinome profiling for thiadiazinone 26 (1 μM)/DMSO.
Molecules 26 05911 g005
Molecules 26 05911 g006 550
Figure 6. Molecular modeling of 16 on MIBS kinome profiling hits: (A) MAP4K5; (B) MAP4K3; (C) PRKCD; and (D) PKN1.
Figure 6. Molecular modeling of 16 on MIBS kinome profiling hits: (A) MAP4K5; (B) MAP4K3; (C) PRKCD; and (D) PKN1.
Molecules 26 05911 g006
Molecules 26 05911 g007 550
Figure 7. Molecular modeling of thiadiazinone 17 on MIBS kinome profiling hits: (A) AKT2 and (B) STK35.
Figure 7. Molecular modeling of thiadiazinone 17 on MIBS kinome profiling hits: (A) AKT2 and (B) STK35.
Molecules 26 05911 g007
Molecules 26 05911 g008 550
Figure 8. Molecular modeling of thiadiazinone 26 on MIBS kinome profiling hits: (A) ACVR1B and (B) STK35.
Figure 8. Molecular modeling of thiadiazinone 26 on MIBS kinome profiling hits: (A) ACVR1B and (B) STK35.
Molecules 26 05911 g008
Table
Table 1. Activity of anilinothiadiazinones on a panel of seven cancer cell lines.
Table 1. Activity of anilinothiadiazinones on a panel of seven cancer cell lines.
Molecules 26 05911 i001
CompoundAr1Ar25637DU145PANC1MCF7UCH1UCH2A431WS-1
IC50 (μM) a
14 Molecules 26 05911 i005 Molecules 26 05911 i00215>100>100>10058>100>10092
15 Molecules 26 05911 i00315>100>100>10067>100>100>100
16 Molecules 26 05911 i0041.641331846>100>100>100
17 Molecules 26 05911 i0062.123>10014>100>100>100>100
18 Molecules 26 05911 i00755>100>100>100>100>100>100>100
19 Molecules 26 05911 i008>100141146>100>100>100>100
20 Molecules 26 05911 i010>100>100>100>100ntntntnt
21 Molecules 26 05911 i009>10051>100>10097>100>100>100
22 Molecules 26 05911 i012>10050>10025>100>100>10024
23 Molecules 26 05911 i011>10084>1009271>100>100>100
24 Molecules 26 05911 i013>10015261367>100>100>100
25 Molecules 26 05911 i01413>100>100385299>10071
26 Molecules 26 05911 i0158.45.7>1005084>100>100>100
a Average of 4 independent experiments, nt = not tested.
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Kalogirou, A.S.; East, M.P.; Laitinen, T.; Torrice, C.D.; Maffuid, K.A.; Drewry, D.H.; Koutentis, P.A.; Johnson, G.L.; Crona, D.J.; Asquith, C.R.M. Synthesis and Evaluation of Novel 1,2,6-Thiadiazinone Kinase Inhibitors as Potent Inhibitors of Solid Tumors. Molecules 2021, 26, 5911. https://0-doi-org.brum.beds.ac.uk/10.3390/molecules26195911

AMA Style

Kalogirou AS, East MP, Laitinen T, Torrice CD, Maffuid KA, Drewry DH, Koutentis PA, Johnson GL, Crona DJ, Asquith CRM. Synthesis and Evaluation of Novel 1,2,6-Thiadiazinone Kinase Inhibitors as Potent Inhibitors of Solid Tumors. Molecules. 2021; 26(19):5911. https://0-doi-org.brum.beds.ac.uk/10.3390/molecules26195911

Chicago/Turabian Style

Kalogirou, Andreas S., Michael P. East, Tuomo Laitinen, Chad D. Torrice, Kaitlyn A. Maffuid, David H. Drewry, Panayiotis A. Koutentis, Gary L. Johnson, Daniel J. Crona, and Christopher R. M. Asquith. 2021. "Synthesis and Evaluation of Novel 1,2,6-Thiadiazinone Kinase Inhibitors as Potent Inhibitors of Solid Tumors" Molecules 26, no. 19: 5911. https://0-doi-org.brum.beds.ac.uk/10.3390/molecules26195911

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