KOROTKOV VADIM (DE)
SIEBER STEPHAN (DE)
WO2009106211A1 | 2009-09-03 | |||
WO2013083724A1 | 2013-06-13 |
US20140163094A1 | 2014-06-12 |
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Claims 1. A compound having general formula I wherein X is s l cted from Ri is selected from R2 is selected from H and R3 is selected from R4 is selected from or a pharmaceutically acceptable salt thereof for use in the treatment of bacterial infections. 2. to claim 1, wherein Ri is selected from 3. The compound for use according to claim 1 or 2, selected from 341 343 350 356-2 357 363 376-2 4. The compound for use according to any one of claims 1 to 3, wherein the bacterial infections treated are infections with Staphylococcus aureus, preferably multi-resistant Staphylococcus aureus (MRSA). 5. The compound for use according to any one of claims 1 to 4, in combination with further agent(s) or drug(s), such as (an)other antibiotic(s). 6. The compound as defined in any one of claims 1 to 3 for use in the treatment of leukemia. 7. The compound for use according to claim 6, in combination with further agent(s) or drug(s), such as cytostatic compound(s). 8. In vitro use of a compound as defined in any one of claims 1 to 3 as inhibitor of chaperone ClpX. 9. In vitro use of a compound as defined in any one of claims 1 to 3 as antibiotic. 10. A method of treatment of a bacterial infection, comprising the step of administering to a subject a therapeutically effective amount of at least one compound according to any of claims 1 to 3 or a pharmaceutical composition comprising at least one compound according to any of claims 1 to 3, wherein the bacterial infection is preferably selected from infections with Staphylococcus aureus, more preferably multi-resistant Staphylococcus aureus (MRSA). 11. A method of treatment of leukemia, comprising the step of administering to a subject a therapeutically effective amount of at least one compound according to any of claims 1 to 3 or a pharmaceutical composition comprising at least one compound according to any of claims 1 to 3. 12. The method of claim 10 or 11, comprising administering to a subject a therapeutically effective amount of at least one compound according to any of claims 1 to 3 or a pharmaceutical composition comprising at least one compound according to any of claims 1 to 3, in combination with further agent(s) or drag(s), such as (an)other antibiotic(s) or cytostatic compound(s). |
The present invention relates to antibiotic compounds and their use as ClpX inhibitors and in the treatment of bacterial infections, such as infections with multi-resistant Staphylococcus aureas. The present invention further relates to respective methods of treatment.
BACKGROUND OF THE INVENTION
Staphylococcus aureus is a gram-positive pathogen responsible for devastating infections of lung, skin and bones (Lowy 1998). A diverse arsenal of virulence factors propagates these infections by infiltrating host cells (proteases), eliminating immune response (leukotoxin) and causing cytotoxicity (hemolysins) (Zecconi and Scali, 2013). Severe acute disease pattern such as sepsis and toxic shock syndrome are direct consequences of these molecular mechanisms. With the onset of multiresistant S. aureus (MRS A) their treatment and prevention becomes challenging with classical antibiotics. Thus, alternative strategies that focus on virulence rather than viability have been reported (Clatworthy et al, 2007; Liu et al, 2008; Rasko and Sperandio, 2010). S. aureus virulence is controlled by a complex network involving quorum sensing mediated by the accessory gene regulator (agr) system (George et al, 2007). Agr expresses autoinducing peptides that sense bacterial growth and initiate the production of toxins by binding to receptors of two component systems upon a threshold density (Cheung et al, 2004; Novick 2003). Thus, interference with agr signaling represents an intriguing possibility to silence virulence (George et al, 2008; Tal-Gan et al, 2013; Murray et al, 2014).
Caseinolytic protease (ClpP) was shown to play a crucial role in agr dependent toxin expression (Frees et al, 2005; Michel et al, 2006) and genetic deletions revealed a dramatic reduction in hemolysin alpha (hla) production (Frees et al, 2003). ClpP associates with chaperones such as ClpX that recognize and unfold substrate proteins (Sauer and Baker 2011; Maglica et al, 2009). Accordingly, also ClpX represents a crucial target for anti-virulence strategies (Frees et al , 2003), however, only little is known about selective inhibitors (Cheng et al, 2007; McGillivray et al, 2012). Contrary, several specific ClpP inhibitors have been reported foremost beta-lactones attenuating S. aureus virulence in vitro and in vivo (Bottcher and Sieber, 2008; Weinandy et al, 2014). However, their facile ester bond limited pharmacological applications and initiated the search for alternative inhibitor scaffolds. In fact, a recent high-throughput screen (HTS) of 140.000 compounds against ClpP revealed oxazoles as novel inhibitor scaffolds with enhanced stability and potency (Pahl et al, 2015). Unfortunately these reversible binders were rapidly ejected from the ClpP active site upon chaperone binding via conformational selection.
Thus, there is a need in the art for improved means and methods for treating bacterial infections.
SUMMARY OF THE INVENTION
According to the present invention this object is solved by a compound having general formula I
(I)
wherein
X is selected from
NH
Ri is selected from
R 2 is selected from H and O
R3 is selected from
R4 is selected from or a pharmaceutically acceptable salt thereof for use in the treatment of bacterial infections / as antibiotic.
According to the present invention this object is solved by a compound having said general formula I for use in the treatment of leukemia.
According to the present invention this object is solved by in vitro using the compound(s) of the present invention as inhibitor(s) of chaperone ClpX.
According to the present invention this object is solved by in vitro using the compound(s) of the present invention as antibiotic. According to the present invention this object is solved by a method of treatment of a bacterial infection, comprising the steps of
administering to a subject a therapeutically effective amount of at least one compound of the present invention or a pharmaceutical composition comprising at least one compound of the present invention.
According to the present invention this object is solved by a method of treatment of leukemia, comprising the steps of
administering to a subject a therapeutically effective amount of at least one compound of the present invention or a pharmaceutical composition comprising at least one compound of the present invention.
DESCRIPTION OF THE PREFERRED EMBODIMENTS OF THE INVENTION
Before the present invention is described in more detail below, it is to be understood that this invention is not limited to the particular methodology, protocols and reagents described herein as these may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention which will be limited only by the appended claims. Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art. For the purpose of the present invention, all references cited herein are incorporated by reference in their entireties.
Concentrations, amounts, and other numerical data may be expressed or presented herein in a range format. It is to be understood that such a range format is used merely for convenience and brevity and thus should be interpreted flexibly to include not only the numerical values explicitly recited as the limits of the range, but also to include all the individual numerical values or sub-ranges encompassed within that range as if each numerical value and sub-range is explicitly recited. Furthermore, such an interpretation should apply regardless of the breadth of the range or the characteristics being described.
ClpX inhibitory compounds As discussed above, the present invention provides a compound having general formula I
wherein
X is selected from
\ \ \
H o
Ri is selected from
R 2 is selected from H and \ 5
R4 is selected from or a pharmaceutically acceptable salt thereof.
The compounds of the present invention are provided as antibiotic(s).
The compounds of the present invention are provided for use in the treatment of bacterial infections.
In a preferred embodiment, a compound of the present invention is a compound having general formula I wherein Ri is selected from
a preferred embodiment, a compound of the present invention is a compound selected from
334
341
356-2 357
363
375 376-1
376-2
In a preferred embodiment, the compound is compound 334.
Preferably, the bacterial infections treated with the compound(s) of the present invention are infections with Staphylococcus aureus, preferably multi-resistant Staphylococcus aureus
(MRSA).
In one embodiment, the compound(s) of the present invention are used in combination with further agent(s) or drug(s), such as (an)other antibiotic(s).
The compounds of the present invention are provided for use in the treatment of leukemia.
In one embodiment, the compound(s) of the present invention are used in combination with further agent(s) or drug(s), such as cytostatic compound(s).
The administration of the compounds according to this invention may be performed in any of the generally accepted modes of administration available in the art. Illustrative examples of suitable modes of administration include intravenous, oral, nasal, inhalative, parenteral, topical, transdermal and rectal delivery. Oral and intravenous deliveries are preferred.
As discussed above, the present invention provides the compound(s) of the present invention as inhibitor(s) of chaperone ClpX. In particular, for an in vitro use of the compound(s) of the present invention as inhibitor(s) of chaperone ClpX.
As discussed above, the present invention provides the compound(s) of the present invention as antibiotic.
In particular, for an in vitro use of the compound(s) of the present invention as an antibiotic. Treatment methods
As discussed above, the present invention provides a method of treatment of a bacterial infection.
Said method comprises the step of
administering to a subject a therapeutically effective amount of at least one compound according to the present invention or a pharmaceutical composition comprising at least one compound according to the present invention,
Preferably, the bacterial infection is selected from infections with Staphylococcus aureus, preferably multi-resistant Staphylococcus aureus (MRSA).
In one embodiment, the method comprises a combination with further agent(s) or drug(s), such as (an)other antibiotic(s).
As discussed above, the present invention provides a method of treatment of leukemia.
Said method comprises the step of
administering to a subject a therapeutically effective amount of at least one compound according to the present invention or a pharmaceutical composition comprising at least one compound according to the present invention.
In one embodiment, the method comprises a combination with further agent(s) or drug(s), such as cytostatic compound(s).
A "therapeutically effective amount" of a compound according to the invention preferably refers to the amount necessary to achieve the therapeutic outcome. As discussed above, the administration of the compounds according to this invention and pharmaceutical compositions according to the invention may be performed in any of the generally accepted modes of administration available in the art. Illustrative examples of suitable modes of administration include intravenous, oral, nasal, inhalative, parenteral, topical, transdermal and rectal delivery. Oral and intravenous deliveries are preferred.
Further description of preferred embodiments
The inventors performed a high-throughput screen (HTS) against the whole ClpXP complex and identified one potent hit molecule that inhibits proteolysis by disruption of ClpX and globally reduces virulence of S. aureus and MRSA.
For ClpXP inhibitor screens we utilized an established assay based on the degradation of green fluorescent protein (GFP) equipped with an SsrA peptide-tag (Kim et al, 2000). This native peptide sequence, usually appended to ribosome stalled proteins, is recognized by ClpX which unfolds the tagged GFP under ATP consumption. Subsequently, the linear peptide chain is digested within the proteolytic ClpP barrel resulting in a loss of fluorescence signal. Multiple turnover is achieved by ATP regeneration with creatin kinase. Putative inhibitors of proteolysis thus either address ClpP, ClpX, the interaction between the two or the kinase requiring careful validation of hits in secondary assays. We adapted the fluorescence assay for the needs of HTS and performed a pilot run against 3000 compounds. Subsequently, a full screen with about 40.000 compounds from the FMP library was performed (Figure 1 A). 332 molecules were identified as primary hits reducing activity by >50 %. To select the most potent compounds and exclude inhibitors of the ATP regenerating system IC50 values were determined and molecules assayed against creatin kinase in secondary screens (Figure 4A). 158 molecules were identified as sole ClpXP inhibitors with a potency < 5 μΜ and 8 compounds with IC5 0 values ranging between 0.6 and 3.1 μΜ were selected for a closer inspection of their mode of action (Figure IB).
To unravel the mode of action we performed several assays focusing on either ClpP, ClpX or the ClpXP complex. Interestingly, none of the hit compounds inhibited ClpP peptidase activity suggesting that these molecules exhibit a novel mechanism (Figure 4B). Compounds 334 and 336, with a similar structural core motif, blocked ClpX ATPase activity with an IC 50 of 1.6 and 2.4 μΜ, respectively, while all other compounds were largely inactive (Figure 1C). Inhibition of chaperone activity is an intriguing finding as no specific ClpX inhibitor has been reported so far and previous molecules addressing ClpC, a related chaperone, either displayed no effect on ATPase activity or even stimulated it (Schmitt et at, 2011; Gavrish et at, 2014). Both hits did not alter ClpCP proteolysis demonstrating selectivity for solely ClpX (Figure 4C).
We further focused on compound 334 as the most potent ClpX inhibitor and analyzed its mechanism of action. First, the 334 structure does not exhibit any obvious reactive electrophilic moieties and accordingly no covalent modification of ClpX was obtained by intact mass spectrometry (MS, Figure 5A). Second, ClpX hexamer stability was evaluated in presence and absence of 334 via analytical size-exclusion chromatography (Figure ID). Importantly, a dramatic disruption of the oligomeric state to dimeric/trimeric species was observed upon compound addition. Third, even the whole ClpXP proteolytic complex collapsed in response to 334 binding (Figure ID). Fourth, 334 induced deoligomerization of ClpX was associated with a decrease in the melting temperature of 2.8 K as obtained in thermal shift assays indicating destabilization of the complex (Figure 5B).
To dissect the molecular prerequisites of 334 inhibition we prepared several derivatives varying in their aromatic ring substitutions. Introduction of a methoxy substituent at the upper benzene ring almost completely abolished inhibition of ClpXP suggesting that this site is less suited for structural modifications (Figure 2A). An exchange of the lower phenol ring by thiophene (336) retained potency while a replacement by pyridine (352) resulted in a significantly decreased IC 50 value. Interestingly, the phenol ring turned out to be amenable for the introduction of additional hydroxy- (347) or methoxy- (344) groups. However, positioning of the phenolic hydroxy-group in meta (parent 334) or para (343) was crucial for activity while ortho (351) resulted in a significant drop of potency. Other substituents at meta- and para-position including fluorine (348) and acetoxy (353) were tolerated while benzoic acid (349) completely abolished inhibition. The left benzene ring showed flexibility for larger substituents. For example, the introduction of a photocrosslinker (376), enabling covalent crosslinking, retained potency in the ClpXP protease. Moreover, we realized that some compounds, although they exhibited comparable IC 5 0 values, largely varied in the extent of maximum inhibition ranging from 100 % (e.g. 334) to 35 % (e.g. 353) (Figure 2A). ClpX is an essential protein for bacterial pathogenesis and as a confirmed intracellular target of our new inhibitor series we analyzed virulence factor expression upon 334 treatment. One predominant trait of S. aureus virulence is lysis of red blood cells by secretion of hemolysins. We thus treated S. aureus with various concentrations of 334 overnight and applied supernatants to agar plates substituted with sheep erythrocytes. Satisfyingly, we obtained a concentration dependent reduction of hemolysis with an IC 5 0 of 3 μΜ (Figure 3 A). Moreover, overall proteolysis, an additional hallmark of host cell invasion, was significantly reduced (Figure 5C). For a more comprehensive analysis of 334 anti-virulence effects we performed a whole secretome analysis of 334 treated S. aureus and monitored the expression of virulence proteins. Cells were incubated with 334 or DMSO overnight. The supernatant was collected, tryptically digested and peptides modified by dimethyl labeling for quantitative analysis. Importantly, the visualization of 334/DMSO protein ratios shows a dramatic and global down- regulation of major virulence factors including toxins (hemolysin gamma) and diverse proteases (serine proteases A-F, staphopain) upon compound treatment (Figure 3 B). The observed IC50 values are in the low μΜ range comparable with common antibiotics. This molecule class is highly beneficial in the treatment of infectious diseases either alone or in combination with antibiotics.
It should be mentioned here that this compound class is also very effective in inhibition of human ClpXP (Figure 6). Previous studies have shown that human ClpP is essential for leukemia cell survival (Cole et al, 2015). Interestingly, our previously disclosed beta-lactone inhibitors (see WO 2009/106211, WO 2013/083724 Al and US 2014/163094 Al) were used in a study showing a significant reduction of leukemia in mice. However, the authors of this study state that compounds with increased pharmacological stability are desperately needed (Cole et al, 2015). Plasma stability tests of 334 showed a half live of >240 min (Figure 7). Cell viability assays in three leukemia cell lines (K562, S-Jurkat and CCRF-CEM) show growth inhibition after treatment with compounds 334 and 336 with IC50 values in the low μΜ range (Figure 8). Thus our new compounds exhibit a huge potential for the treatment of bacterial virulence as well as leukemia. Furthermore, MTT assays in the A549 cell line revealed low toxicity for some compounds (etc. 355 and 367), enabling prospective species selective use as an antiinfective compound. The following examples and drawings illustrate the present invention without, however, limiting the same thereto.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1. Evaluation of novel ClpXP inhibitors.
(A) A high-throughput-screen of 40,000 compounds at a concentration of 10 μΜ revealed potent inhibitors against the ClpXP protease complex. (B) Structures of the six most potent hits all showing IC 0 values below enzyme concentration. (C) Hits 334 and 336 disrupt ClpXP activity through inhibition of ClpX in the same ICso range. (D) Analytical SEC showing the disruption of ClpX hexamer and ClpXP complex after incubation with 334.
Figure 2. Structure-activity relationship studies
of several derivatives of the 334 structure class (A and B). Inhibition of ClpXP protease activity and relative activity difference between the active and inhibited species (C and D).
Figure 3. Several virulence factors of S. aureus NCTC 8325 are down-regulated upon treatment with 334.
(A) Left: Supernatants of S. aureus cultures show a dose-dependent reduction of hemolysis after 20 h treatment with 334. Right: Colonies grown in the presence of 334 show reduced hemolytic activity. Cultures were grown on small paper filter slides on Columbia agar containing 5 % sheep blood.
(B) Several extracellular proteins are down-regulated in a concentration dependent manner. Serine protease A-F, gamma hemolysin component C and Staphopain A show a reduction with a mean EC50 of ca. 3 μΜ 334. Secretome analysis was performed using supernatants of 334 and DMSO treated S. aureus NCTC 8325 cultures and analyzed via label- free quantification.
Figure 4.
(A) All validated primary active hits were counter-screened against inhibition of the creatine kinase which is needed for ATP regeneration.
(B) None of the six most potent identified ClpXP inhibitors alters ClpP peptidase activity in a fluorescent assay. (C) ClpCP activity is not inhibited by the six compounds at 100 μΜ concentration. Phenylester 170 was used as a positive control.
Figure 5.
(A) No covalent modifications of ClpX can be observed upon treatment with 334 at 100 μΜ.
(B) Thermal-shift assay performed with 10 μΜ ClpX and 100 μΜ 334 reveal a destabilization of ca. 2.8 compared to DMSO treated ClpX.
(C) Inhibition of extracellular proteolysis activity after treatment of S. aureus NCTC 8325 with different amounts of 334. Cells (3 μΕ) and different concentrations of 334 (3 iL) were pipetted on small paper filters on LB agar plates containing skimmed milk.
Figure 6.
Compound 334 and 336 show inhibition of the human ClpXP protease complex. Figure 7.
Compound 334 (25 μΜ) is stable in mouse plasma even after 240 min. Procain (100 μΜ) is used as a positive control, procainamide (100 μΜ) as a negative control.
Figure 8. Cell viability assay in leukemia cells.
Cells of the three different leukemia cell lines K562 (A), S-Jurkat (B) and CCRF-CEM (C) were stimulated with compounds 334 or 336 in the indicated concentrations for 72h. Cell viability was determined using Cell Titer Blue reagent. n=l, normalization to untreated control.
Figure 9. Cell viability assay in lung carcinoma cells.
A549 cells were incubated with different concentrations of compounds 334, 343, 344, 347, 355, 358, 359 or 367 for 24 h. The residual metabolic activity of the cells was determined using the MTT assay.
EXAMPLE
Example 1
1.1 Materials and Methods High-throughput screening:
A library consisting of 40,480 small molecule compounds was screened using the ClpXP GFP degradation assay. On every plate 352 compounds, as well as 16 wells containing negative controls and 16 wells containing positive controls were measured. 25 μί, of ClpXP-master mix (25 mM Hepes, pH 7.6, 200 mM KC1, 5 mM MgCl 2 , 1 mM DTT, 10% glycerol, 4 mM ATP, 16 mM creatine phosphate, 20 U/mL creatine phosphokinase (Roche), 0.3 μΜ ClpPu, 0.6 μΜ ClpX 6 ; final assay concentrations) or negative control (same composition as above, except without ClpXP) were dispensed in a black 384-well plate (Corning, #3573) and 0.3 μΐ, of compound (in DMSO) or DMSO was added to each well (10 μΜ final compound concentration). The plate was shaken for 15 s at 2000 rpm, centrifuged to collect sample at bottom and incubated at r.t. for 15 min. The GFP-ssrA substrate (1.5 μΜ in H 2 0) was added and the plate was again shaken and centrifuged. Kinetic fluorescence measurement was performed in a Tecan Safire 2 microplate reader (2 min interval, 10 cycles, λ βχ = 465 nm, em = 535 nm, 20 nm bandwidth). The overall Z' factor was calculated to be 0.69 ± 0.087.
The 332 best inhibiting (z-score > 3) and 20 best activating (z-score < -4) compounds were cherry picked and subsequently analyzed for dose dependent inhibition (or activation). This was done by measurement of a twofold dilution series of 9 concentrations, starting at 50 μΜ.
In vitro ClpXP/ClpCP protease assay:
The ClpXP protease activity was monitored using a fluorescent GFP substrate, which was tagged with a ssrA degradation tag. Assays were performed in PZ buffer (25 mM Hepes, pH 7.6, 200 mM KC1, 5 mM MgCl 2 , 1 mM DTT, 10% glycerol) with 60 μΐ, reaction volume at 30 °C. GFP fluorescence was monitored in white, flat-bottom well plates (Brand) using an Infinite M200 Pro (Tecan; λ βχ = 465 nm, λ β ιη = 535 nm). Degradation reactions contained 0.6 μΜ ClpX 6 , 0.3 μΜ ClpP 14 , 0.25 μΜ GFP-ssrA and an ATP regeneration system (4 mM ATP, 16 mM creatine phosphate, 20 U/mL creatine phosphokinase). In case of ClpCP protease assays 1.0 μΜ ClpC 6 , 0.3 μΜ ClpP 14 and 2.5 μΜ MecA were used. 0.6 μί ^ of inhibitor (in DMSO) were added to the wells followed by all other reaction partners (in 50 μΐϊ) except the substrate. After pre-incubation for 15 min at 30 °C the substrate (10 μΐ,) was added and fluorescence was monitored. Unless stated otherwise all data were collected in duplicates and in three independent experiments. The slope of the curves in the linear region was determined via linear regression using GraphPad Prism. DMSO treated samples were normalized to 100% activity and samples without ClpX were used as a negative control. Analytical size exclusion chromatography:
Analytical size exclusion chromatography experiments were performed using a Superdex 200 10/300 GL (GE) or a Superose 6 Increase 10/300 GL (GE) column at 4 °C. For most experiments PZ buffer (with 0.5 mM ATP) was used. Samples (200 μΤ) were mixed, incubated 10 min at 37 °C and loaded into a 500 μΕ loop. Elution was monitored at 280 nm. Runs were referenced against the salt peak of the conductivity trace and normalized to the highest peak for easier comparison.
Extracellular proteolytic and hemolytic activity:
Extracellular proteolytic activity was tested on LB agar plates containing 1% skimmed milk. Hemolytic activity was tested on Columbia Sheep Blood Agar plates (PB5039A, Thermo Scientific). Sterile filter paper disks (5 mm diameter) were placed on agar plates and 3 μΤ DMSO/compound stock and 3 xL of stationary S. aureus diluted with B-Medium (1 :100) were added. The agar plates were incubated for 20 h at 37 °C and photos were taken using LAS4000 (GE Healthcare).
For the analysis of hemolytic activity of S. aureus supernatants, the DMSO/compound treated cells were centrifuged (5 min, 6000 g) and the sterile filtered supernatant (0.22 μιη) directly added on blood agar plates (20-50 μΕ) following incubation at 37 °C.
In vitro creatine kinase assay:
To account for inhibitors of the ATP regeneration system a counter-screen against creatine kinase was performed using Kinase-Glo assay (Promega). Assays were performed in PZ buffer in a white flat-bottom 96-well plate (Brand) with 50 μΤ reaction volume at r.t. 0.5 μΕ of compound stock (lOOx, 50 μΜ final concentration) or DMSO were mixed with 47 μΕ of master mix (creatine kinase in PZ buffer, 52 μg/mL final concentration). After incubation for 10 min at r.t. 3 μΤ of substrate mix (20 μΜ ADP, 20 μΜ creatine phosphate; final concentrations) were added and incubated for another 10 min at r.t. 50 μΐ ^ of Kinase-Glo reagent were added and luminescence was recorded after 10 min incubation at r.t. using an Infinite M200 Pro (Tecan). DMSO treated samples were normalized to 100% activity and samples without creatine kinase were used as a negative control.
In vitro ClpP peptidase assay: Activity of SaClpP was measured using a fluorogenic peptidic substrate (Suc-Leu-Tyr-AMC, Bachem). 1 μΐ, of DMSO or compound stock was pipetted to a black flat-bottom 96-well plate (Greiner) and 88 μΐ, of assay-buffer (100 mM Hepes, pH 7.0, 100 mM NaCl) containing 1 μΜ ClpP (final concentration) were added. After incubation for 15 min at 32 °C the reaction was started by addition of 10 μί ^ substrate (0.2 mM final concentration) and fluorescence was recorded for 60 min in an infinite M200 pro plate reader (Tecan; λ εχ = 380 nm, X em = 440 nm). Data was analyzed by calculating the initial slope (GraphPad Prism) and normalizing DMSO treated samples to 100% activity.
In vitro ClpXATPase assay:
Activity of ClpX was measured by detection of free phosphate, which is formed by hydrolysis of ATP using the malachite green assay. The dye solution (0.045% malachite green in H 2 0:4.2% ammonium molybdate in 4 N HC1: 1% Triton X-100 in H 2 0 / 36: 12: 1) was always prepared freshly and filtered (0.45 μηι) after incubation for 1 h at r.t. In a reaction tube 4 μΜ ClpX in PZ buffer (200 )xL reaction volume) were incubated with 2 μΐ, of DMSO/compound stock for 10 min at r.t. and then ATP (2 mM final concentration) was added to start the reaction. After defined time points (e.g. 2, 6 and 14 min) 50 μΐ ^ of the sample were pipetted into a plastic cuvette filled with 800 μΤ dye solution and 50 μΐ, PZ buffer. The cuvette was sealed and inverted. After 60 s 100 μΤ of 35% citric acid (in H 2 0) were added to the cuvette and absorbance at 650 nm was measured in triplicates after inverting the mixture again. Data was analyzed by calculating the slope (GraphPad Prism) via linear regression and normalizing DMSO treated samples to 100% activity. Samples without ClpX were used as a negative control to account for potential autohydrolysis of ATP.
In vitro hClpXP assay:
Per each reaction 25 μΐ, 4x PZ' buffer (100 mM Hepes, pH 7.6, 30 mM MgCl 2 , 4 mM DTT, 800 mM KC1, 40% glycerol) were filled up to 80 μΤ with 3.0 μΜ hClpP (or hClpP SI 53 A), 2.8 μΜ hClpX, 10 μΤ ATP regeneration mix (40 mM ATP, 160 mM creatine phosphate, 200 U/mL creatine kinase in 100 mM Hepes, pH 7.0) and water. The protein mix was transferred to a black flat-bottom 96 well plate well including 1 Τ of DMSO dissolved compound (lOOx final concentration). After a preincubation time of 15 min at 37 °C, 20μΤ substrate (10 mM unlabeled casein, 2 mM FITC-labeled casein) mix was added. Fluorescence was recorded using an Infinite M200 Pro (Tecan; λ εχ = 494 nm, em = 525 nm). Intact protein mass spectrometry:
A solution of 3 μΜ ClpX wt or ClpX E183Q in PZ-Buffer (containing 0.5 mM ATP) was incubated (60 min at 30 °C) with up to 100 μΜ of inhibitor (1% DMSO final concentration). Measurements were performed on a Dionex Ultimate 3000 HPLC system coupled to a Thermo LTQ-FT Ultra mass spectrometer with electrospray ionization source (spray voltage 4.2 kV, tube lens 110 V, capillary voltage 48 V, sheath gas 60 arb, aux gas 10 arb). 5 of reaction mixture were on-line desalted using a Massprep desalting cartridge (Waters). The mass spectrometer was operated in positive mode collecting full scans at high resolution (R = 200,000) from m/z = 600 to m/z = 2000. Collected spectra were deconvoluted using Thermo Xcalibur Xtract algorithm.
Thermal-shift assay:
To each well of a white 96-well PCR plate 50 i of a 10 μΜ ClpX solution in PZ-buffer containing Sypro Orange (1:2000, Sigma- Aldrich) were added. To this solution 0.5 \iL of DMSO or lOOx compound stock was added and fluorescence intensity was measured while heating from 20 °C to 89.6 °C (0.3 K steps) in a CFX96 Real-Time System (BioRad). Data was analyzed using Bio-Rad CFX Manager 3.0.
Plasma stability:
Prior to measurements mouse plasma (biowest) was diluted 1 :2 with potassium phosphate buffer (0.1 M, pH 7.4) and pre-incubated at 37 °C for at least 30 min. Test compounds and controls (positive control: procaine; negative control: procainamide) were added at 100 μΜ and incubated at 37 °C at 600 rpm in a shaker. 25 μΤ aliquots were taken after several time points and pipetted into 2>0 \L acetonitrile. The mixture was vortexed, cooled down (-20 °C) and centrifuged (10 min, 17,000 g). The supernatant was transferred into vials and analyzed using a Dionex Ultimate 3000 HPLC system (XBridge™ BEH130 CI 8 5 μΜ, 4.6 χ 100 mm) coupled to a Thermo LTQ-FT Ultra mass spectrometer with an APCI ionization source (vaporizer temperature 400 °C, sheath gas 60 arb, aux gas 20 arb, source voltage 6 kV, capillary voltage 4 V, tube lens 45 V). Data was collected in positive mode using full scans (R = 12,500) from m/z = 100 to m/z = 500 and SIM scans (R = 12,500) for each compound. Xcalibur 2.2 Quan Browser was used for calculation of peak areas (SIM scans) and quantification.
Secretome analysis: 5 n L of B-Mediuni was inoculated 1:100 from an overnight culture of S. aureus NCTC8325 (NRS77) or S. aureus USA300 (BAA-1556) and incubated (37 °C, 200 rpm) until OD 60 o = 0.3-0.5. The culture was then diluted to 3><10 4 CFU/mL with fresh B-Medium and split into 1.5 mL aliquots in 14 mL PP plastic tubes (17x100 mm, VWR). 15 μΐ of DMSO or compound stocks were added to the aliquots following incubation for 19-20 h at 37 °C and 200 rpm. On the next day the cultures were transferred to 2 mL Eppendorf tubes and centrifuged (10 min, 6000 g). The supernatant was removed and sterile filtered (0.22 μπι) into a 50 mL Falcon tube and 12 mL cold acetone (-80 °C) were added. Proteins were allowed to precipitate overnight at -80 °C. The precipitate was centrifuged (15 min, 18,000 g) and washed twice with 1 mL cold methanol (-80 °C) with resuspension (5 s ultrasonic bath) and centrifugation steps (10 min, 18,000 g) in between. The washed pellet was dissolved in 200 L X-buffer (20 mM Hepes, pH 7.5, 7 M urea, 2 M thiourea) and transferred in low-bind Eppendorf tubes for further analysis. Proteins were reduced by addition of 0.2 iL dithiothreitol (DTT, 1 M) and incubation for 45 min at r.t. and 450 rpm). Alkylation was performed by adding 2 μΤ iodoacetamide (IAA, 550 mM) and incubation for 30 min at r.t. in the dark with subsequent quenching of the reaction with 0.5 μΕ DTT (1 M) for 30 min. The samples were pre-digested by addition of 1 μΤ LysC (0.5 mg/mL) and incubation at r.t. for 4 h. After diluting with 600 μΐ, triethylammonium bicarbonate buffer (TEAB, 50 mM) 1.5 trypsin (0.5 mg/mL in 50 mM acetic acid) was added followed by overnight incubation at 37 °C. Digestion was stopped by addition of 8 iL formic acid (FA). Desalting of the samples was conducted on 50 mg SepPak CI 8 columns (Waters). The columns were equilibrated with 1 mL acetonitrile (ACN), 1 mL elution buffer (80% ACN, 0.5% FA) and 3 mL aqueous 0.5% FA solution. The acidified samples were loaded by gravity flow, washed five times with 1 mL 0.5%) FA and then labeled with five times 1 mL of the respective dimethyl labeling agents (light (L): 30 mM NaBH 3 CN, 0.2 % CH 2 0, 45 mM sodium phosphate buffer, pH 7.5; medium (M): 30 mM NaBH 3 CN, 0.2 % CD 2 0, 45 mM sodium phosphate buffer, pH 7.5; heavy (H): 30 mM NaBD 3 CN, 0.2 % 13 CD 2 0, 45 mM sodium phosphate buffer, pH 7.5). Column bound peptides were washed two more times with 1 mL 0.5%> FA and then eluted with two times 250 μΕ elution buffer. 900 μΕ of each sample were combined in a 15 mL tube, frozen in liquid nitrogen and lyophilized. Samples, which were analyzed by label-free quantification (titration experiment) were eluted directly after the washing step and lyophilized individually. Prior to LC-MS/MS measurement the samples were dissolved in 40 ]iL 1% FA and filtered with 0.22 μιη ultrafree centrifugal filters (Merck) equilibrated with 300 μΐ 1% FA. The filtrate was transferred into MS vials and queued for LC-MS/MS measurement.
Prior to LC-MS/MS measurements all peptide samples were reconstituted in 40 μΐ ^ ddH 2 0/l % FA and filtered using centrifugal filters (0.22 μηι, Merck).
Nanoflow LC-MS/MS analysis was performed with an UltiMate 3000 Nano HPLC system (Thermo Scientific) coupled to an Orbitrap Fusion (Thermo Scientific). Peptides were loaded on a trap column (Acclaim CI 8 PepMaplOO 75 μηι ID x 2 cm) and washed for 10 min with 0.1 % FA, then transferred to an analytical column (Acclaim CI 8 PepMap RSLC, 75 μΜ ID x 15 cm) and separated using a 125 min gradient from 3 % to 40 % (120 min from 3 % to 25 % and 5 min to 40 %) MeCN in 0.1 % FA and 5 % DMSO at a flow rate of 200 nL/min. Peptides were ionised using a nanospray source at 1.9 kV and a capillary temperature of 275 °C. Orbitrap Fusion was operated in a top speed data dependent mode with a cycle time of 3 s. Full scan acquisition (scan range of 300 - 1700 m/z) was performed in the orbitrap at a resolution of 120000 (at m/z 200) and with an automatic gain control ion target value of 4e5. Monoisotopic precursor selection as well as dynamic exclusion of 60 s were enabled. Internal calibration was performed using the ion signal of fluoranthene cations (EASY-ETD/IC source). Most intense precursors with charge states of 2 - 7 and intensities greater than 5e3 were selected for fragmentation. Isolation was performed in the quadrupole using a window of 1.6 m/z. Ions were collected to a target of le2 for a maximum injection time of 250 ms with "inject ions for all available parallelizable time" enabled ("Universal" method, Eliuk et al, Thermo Scientific Poster Note PN40914). Fragments were generated using higher-energy collisional dissociation (HCD) and detected in the ion trap at a rapid scan rate.
Raw files were analysed using MaxQuant software (version 1.5.3.8) with the Andromeda search engine. The search included carbamidomethylation of cysteines as a fixed modification and oxidation of methionines and acetylation of protein N-termini as variable modifications. Trypsin was specified as the proteolytic enzyme with N-terminal cleavage to proline and two missed cleavages allowed. Precursor mass tolerance was set to 4.5 ppm (main search) and fragment mass tolerance to 0.5 Da. Searches were performed against the Uniprot database for Staphylococcus aureus NCTC 8325 (taxon identifier: 93061, including isoforms). The second peptide identification option was enabled. False discovery rate determination was carried out using a decoy database and thresholds were set to 1 % FDR both at peptide- spectrum match and at protein levels. "I— L", "requantification" and "match between runs" (0.7 min match and 20 min alignment time windows) options were enabled. For quantification the label free quantification option MaxLFQ was enabled. Statistical analysis was performed with Perseus software (version 1.5.2.6). Putative contaminants, reverse sequences and only identified by site hits were filtered out. Missing LFQ values were replaced by imputation from a normal distribution. The replicate values for each group was averaged and the ratio regarding to the DMSO control was calculated.
Cell viability assay in leukemia cell lines
Cells of the three different leukemia cell lines K562, S-Jurkat and CCRF-CEM were seeded in 96-well plates in 0,1*10 6 cells/ml (CCRF-CEM: 0,2*10 6 cells/ml) and stimulated with 334 and 336 in the indicated concentrations for 72h. Cell viability was determined using Cell Titer Blue reagent. n=l, normalization to untreated control.
MTT assay in A549 cell line
This assay was performed in flat bottom 96 well plates. A549 cells were grown to 30-40 % confluence. The medium was removed and 100 μΤ medium/well containing 1 μΐ, DMSO compound stock were added to the cells and incubated for 24 h. All concentrations as well as a DMSO control were done in triplicates. 20 μΐ, Thiazolyl Blue Tetrazolium bromide (5 mg/mL in PBS) were added to the cells and incubated for 2-4 h until complete consumption was observed. After removal of the medium, the resulting formazan was dissolved in 200 μΕ DMSO. Optical density was measured at 570 nm (562 nm) and background subtracted at 630 nm (620 nm) by a TECAN M200 Pro.
1.2 Organic Synthesis
General remarks
All reactions were carried out under argon in oven-dried glassware unless noted otherwise. All chemicals were of reagent grade or better and used without further purification. Chemicals and solvents were purchased from Sigma Aldrich. Solvents for chromatography and workup purposes were generally of reagent grade. In all reactions, temperatures were measured externally. H NMR and C spectra of small molecules were recorded on Bruker instruments (250MHz, 360 MHz or 500 MHz) and referenced to the residual proton signal of the deuterated solvent. Carbon samples were reference externally against the residual 13 C signal of CDC1 3 . HR-MS-ESI spectra were recorded with a Thermo Scientific LTQ FT.
3-Hydroxy-5-methoxybenzaldehyde was synthesized from methyl 3,5-dihydroxybenzoate via methylation with Mel (Shioe et al., 2011) followed by the reduction with LiAlH 4 (Seidel et al., 1990) and oxidation with PCC. 2- Aniino-5-metlioxythioplienol was synthesized according to (Mathis et al., 2003).
3- (tert-Butoxycarbonylamino)-benzyl alcohol was synthesized according to (Harmrolfs et al., 2010).
3-Hydroxy-2-(methoxycarbonyl)-l-oxo-lH-indene-6-carboxylic acid was obtained according to (Matsumoto et al., 2012).
2-(3-(But-3-inyl)-3H-diazirin-3-yl)ethylamine was synthesized according to (Li et al, 2013).
5-Eth nyl-indan-l,3-dione
5-Ethynyl-indan-l,3-dione was synthesized according to (Buckle et al., 1973). 4- Ethynylphthalic anhydride (1.26 g, 7.3 mmol) was added to Ac 2 0 (4 mL, 4.32 g, 42 mmol), Et 3 N (2 mL) was added to this suspension and the obtained mixture was treated with tert- butyl acetoacetate (1.2 g, 7.6 mmol). The reaction mixture was stirred at r. t. for 18 h, then ice (4 g) and concentrated HCl (4 mL, 37%) were added. Then one more portion of HCl was added (10 mL, 5 M) and the dark solution was stirred at 75-80 °C for 1 h. The reaction mixture was allowed to cool down to r. t. and extracted with CH 2 C1 2 (3 times). The combined organic phases were dried over Na 2 S0 4 , filtered and concentrated under reduced pressure yielding 1.2 g (97%) the product that was used for the next step without further purification. - 1H NMR (400 MHz, DMSO-d 6 ): 8.03 - 7.88 (m, 3 H), 4.67 (s, 1 H), 3.38 (s, 2 H). - 13 C NMR (100 MHz, DMSO-d 6 ): 197.75, 143.62, 142.89, 138.88, 129.05, 125.86, 123.49, 86.05, 82.49, 45.86.
5,6-Dichloroindan-l,3-dione
5,6-Dichloroindan-l,3-dione was synthesized according to (Buckle et al., 1973). 4,5- Dichlorophthalic anhydride (2.0 g, 9.2 mmol) was added to Ac 2 0 (4.7 mL, 5.0 g, 50 mmol), Et 3 N (2.6 mL) was added to this suspension and the obtained mixture was treated with tert- butyl acetoacetate (1.5 mL, 1.5 g, 9.2 mmol). The reaction mixture was stirred at r. t. for 18 h, then ice (3 g) and concentrated HCl (3 mL, 37%) were added. Then one more portion of HCl was added (13 mL, 5 M) and the dark solution was stirred at 75-80 °C for 1 h. The reaction mixture was allowed to cool down to r. t. and extracted with CH 2 CI 2 (3 times). The combined organic phases were dried over Na 2 S0 4 , filtered and concentrated under reduced pressure. Recrystallization from hexane : EtOAc 1 : 1 yielded 1.1 g (56%) of the product. - 1H NMR (400 MHz, DMSO-d 6 ): 8.17 (s, 2 H), 3.39 (s, 2 H). - 13 C NMR (100 MHz, DMSO-d 6 ): 196.43, 142.85, 139.07, 125.21, 45.77.
3-(tert-Buty ldimeth lsilyloxymeth l)b enzyl alcohol
To a solution of 1,3-benzenedimethanol (2.3 g, 16.7 mmol) in CH 2 C1 2 (80 niL) was added imidazole (1.14 g, 16.7 mmol) and TBSC1 (2.52 g, 16.7 mmol). The reaction mixture was stirred for 16 h at r. t, washed with H 2 0, aq. NaHC0 3 (10%), and finally H 2 0. The organic phase was dried over Na 2 S0 4 , filtered and concentrated under reduced pressure. Column chromatography (hexane : EtOAc = 10 : 1 to 4 : 1) yielded 1.1 g (24%) of the desired product. - 1H NMR (400 MHz, CDCl 3 ): 7.39 - 7.25 (m, 4 H), 4.77 (s, 2 H), 4.72 (d, J= 6.0 Hz, 2H), 0.97 (s, 9 H), 0.13 (s, 6 H). - 13 C NMR (100 MHz, CDCI3): 141.86, 140.83, 128.51, 125.57, 125.42, 124.67, 65.48, 64.88, 25.98, 18.46, -5.24.
3-Bromo-5-hydroxybenzaldehyde
A solution of 3,5-dibromophenol (2.5 g, 10 mmol) in THF (25 mL) was cooled to -78 0 C using the dry ice-acetone bath and a solution of BuLi (9 mL, 22.5 mmol, 2.5 M solution in hexane) was added dropwise. The reaction mixture was stirred for 30 min at this temperature and DMF (15.3 mL) was added. The solution was stirred for 30 min at -78 °C and for 2 h at r. t. The reaction was quenched with sat. NH 4 C1 and extracted with EtOAc (3 times). The combined organic layers were dried over Na 2 S0 4 , filtered and concentrated under reduced pressure. The residue was separated by column chromatography (hexane : EtOAc = 20 : 1 to 4 : 1) yielding 1.0 g (50%) of a desired product. - 1H NMR (300 MHz, DMSO-d 6 ) 10.55 - 10.39 (m, 1 H), 9.89 (s, 1 H), 7.53 - 7.50 (m, 1 H), 7.29 - 7.24 (m, 2 H). Spectral data are identical to those published in the literature (Yang et al., 2016). 3-Azido-5-hydroxybenzaldehyde
3-Azido-5-hydroxybenzaldehyde was synthesized according to (Andersen et al., 2005). 3- Bromo-5-hydroxybenzaldehyde (1.8 g, 9 mmol), NaN 3 (1.2 g, 18 mmol), sodium ascorbate (89 mg, 0.45 mmol), Cul (171 mg, 0.9 mmol) were placed into a Schlenk flask and it was evacuated. EtOH (13 mL), H 2 0 (5 mL), DMF (0.3 mL) and rara-N,N'-dimethylcyclohexane- 1,2-diamine
(192 mg, 1.35 mmol) were added and the suspension was degassed for 15 min by bubbling Ar through it. Then the reaction mixture was refluxed under Ar for 3 h and finally allowed to cool down to r.t. H 2 0 was added and the mixture was extracted with EtOAc (3 times). The combined organic phases were dried over Na 2 S0 4 , filtered and concentrated under reduced pressure. Column chromatography (hexane: EtOAc = 20 : 1 to 4 : 1) yielded 400 mg (27%) of a desired compound. - 1H NMR (500 MHz, DMSO-d 6 ): 10.34 (s, 1 H), 9.90 (s, 1 H), 7.11 - 7.07 (m, 2 H), 6.79 - 6.75 (m, 1 H). - 13 C NMR (125 MHz, DMSO-d 6 ): 193.00, 159.61, 142.10, 139.18, 112.81, 112.03, 111.32.
3-(fert-Butoxycarbonylamino)benzaldehyde
To a solution of 3-(tert-butoxycarbonylamino)-benzyl alcohol (2.0 g, 9.0 mmol) in CH 2 CI 2 (50 mL) was added activated Mn0 2 (7.8 g, 90 mmol). The resulting suspension was stirred for 16 h at r. . The reaction mixture was filtered through a pad of Celite and a filtrated was concentrated under reduced pressure yielding 1.7 g (85%) of the desired product. - 1H NMR (400 MHz, CDCh): 9.97 (s, 1 H), 7.92 (t, J = 1.8 Hz, 1 H), 7.63 (dd, J = 8.0, 1.1 Hz, 1 LI), 7.55 (ddd, J= 7.6, 1.4, 1.1 Hz, 1 H), 7.44 (t, J= 7.6 Hz, 1 H), 1.53 (s, 9 H).
3-(teri-ButyldimethyIsilyloxymethyl)benzaldehyde To a solution of 3-(tert-butyldimethylsilyloxymethyl)-benzyl alcohol (1.0 g, 4.0 mmol) in CH 2 C1 2 (30 mL) was added activated Mn0 2 (3.5 g, 40 mmol). The resulting suspension was stirred for 16 h at r. . The reaction mixture was filtered through a pad of Celite and a filtrated was concentrated under reduced pressure yielding 950 mg (90%) of the desired product. - 1H NMR (400 MHz, CDCl 3 ): 10.02 (s, 1 H), 7.83 (s, 1 H), 7.77 (d, J= 7.5 Hz, 1 H), 7.61 (d, J= 7.5 Hz, 1 H), 7.50 (t, J = 7.5 Hz, 1 H), 4.81 (s, 2 H), 0.95 (s, 9 H), 0.12 (s, 6 H). - 13 C NMR (100 MHz, CDCh): 192.42, 142.65, 136.45, 132.06, 128.93, 128.36, 127.17, 64.32, 25.93, 18.42, -5.27.
3 -(Hex-5-y ny 1) oxy-5-hy droxyb enzald ehy d e
To a solution of 3,5-dihydroxybenzaldehyde (552 mg, 4 mmol) in DMF (5 mL) at 0 °C under argon was added NaH (164 mg, 4.1 mmol, 60% in mineral oil). The mixture was stirred for 30 min at r. t. and a solution of hex-5-ynyl iodide (390 mg, 1.9 mmol) in DMF (5 mL) was added. The reaction mixture was stirred for 18 h at r. t. The reaction was quenched with 1 M HCl, extracted with EtOAc (3 times). The combined organic phases were washed with H 2 0 (2 times), dried over Na 2 S0 4 , concentrated under reduced pressure and the residue was separated by column chromatography (hexane: EtOAc = 10 : 1 to 1 : 1) yielding the desired product (240 mg, 58%) as a colorless solid. - 1H NMR (500 MHz, CD 3 OD): 9.84 (s, 1 H), 6.96 - 6.93 (m, 1 H), 6.92 - 6.90 (m, 1 H), 6.65 (t, J= 2.3 Hz, 1 H), 4.03 (t, J= 6.3 Hz, 2 H), 2.32 - 2.21 (m, 3 H), 1.95 - 1.87 (m, 2 H), 1.75 - 1.67 (m, 2 H). - 13 C NMR (125 MHz, DMSO-d 6 ): 192.63, 160.86, 159.14, 138.64, 108.46, 107.85, 105.98, 83.28, 68.47, 67.36, 27.94, 24.88, 17.40.
3- (?/Y-Butyldimethylsilyloxy-5-methoxybenzaldehyde
To a solution of 3-hydroxy-5-methoxybenzaldehyde (380 mg, 2.5 mmol) in DMF (5 mL) was added diisopropylethylamine (1 mL) at r.t, followed by the solution of tert-butyldimethylsilyl chloride (453 mg, 3.0 mmol) in DMF (5 mL). The reaction mixture was stirred for 18 h at r. t. and H 2 0 was added. The mixture was extracted with EtOAc (3 times). The combined organic phases were dried over Na 2 S0 4 , concentrated under reduced pressure and separated by column chromatography (hexane : EtOAc = 1 : 0 to 20 : 1) yielding 500 mg (75%) of a desired product. - 1H NM (400 MHz, CDC¾): 9.90 (s, 1 H), 7.05 (dd, J= 2.3, 1.3 Hz, 1 H), 6.96 (dd, J= 2.3, 1.3 Hz, 1 H), 6.67 (t, J= 2.3 Hz, 1 H), 3.86 (s, 3 H), 1.01 (s, 9 H), 0.25 (s, 6 H). - 13 C NMR (100 MHz, DMSO-d 6 ): 191.88, 161.20, 157.36, 138.39, 114.54, 113.08, 106.59, 55.59, 25.61, 18.19, -4.44.
3-Bromo-5-(ter/-butyldimethylsilyloxy)benzaldehyde
To a solution of 3-bromo-5-hydroxybenzaldehyde (1.0 g, 5 mmol) in CH 2 C1 2 (40 mL) was added imidazole (0.41 g, 6 mmol) at r.t., followed by fert-butyldimethylsilyl chloride (0.91 g, 6 mmol). The reaction mixture was stirred for 18 h at r. t. The mixture was concentrated under reduced pressure and separated by column chromatography (hexane : EtOAc = 1 : 0 to 20 : 1) yielding 1.1 g (70%) of a desired product. - 1H NMR (400 MHz, CD0 3 ): 9.88 (s, 1 H), 7.60 (dd, J = 1.7, 1.4 Hz, 1 H), 7.25 - 7.23 (m, 1 H), 7.24 (dd, J= 1.4, 0.6 Hz, 1 H), 0.99 (s, 9 H), 0.24 (s, 6 H). - 13 C NMR (100 MHz, CDCl 3 ): 190.50, 157.20, 130.08, 129.32, 126.08, 123.33, 118.99, 25.54, 18.18, - 4.47.
3-Azido-5-(teri-butyldimethylsilyloxy)benzaldehyde
To a solution of 3-azido-5-hydroxybenzaldehyde (400 mg, 2.5 mmol) in CH 2 C1 2 (20 mL) was added imidazole (400 mg, 6 mmol) at r.t., followed by the solution of tert-butyldimethylsilyl chloride (906 mg, 6.0 mmol). The reaction mixture was stirred for 18 h at r. t, concentrated under reduced pressure and separated by column chromatography (hexane : EtOAc = 1 : 0 to 20 : 1) yielding 550 mg (79%) of a desired product. - 1H NMR (400 MHz, CDC¾): 9.90 (s, 1 H), 7.17 (dd, J= 2.2, 1.3 Hz, 1 H), 7.09 (dd, J= 2.3, 1.3 Hz, 1 H), 6.72 (t, J= 2.3 Hz, 1 H), 0.99 (s, 9 H), 0.24 (s, 6 H). - 13 C NMR (100 MHz, CZX¾): 191.00, 157.64, 138.75 (2C), 117.38, 116.68, 112.87, 25.56, 18.20, -4.42. - IR (film) v = 2956 cm "1 , 2930, 2859, 2111, 1704, 1588, 1458, 1330, 1248, 1148, 833, 782. 3-te , i-Butyldimethylsilyloxy-5-(hex-5-ynyloxy)benzaldehyde
To a solution of 3-(hex~5-ynyl)oxy-5~hydroxybenzaldehyde (240 mg, 1.1 mmol) in DMF (5 mL) was added diisopropylethylamine (1 mL) at r.t, followed by the solution of tert- butyldimethylsilyl chloride (241 mg, 1.6 mmol) in DMF (5 mL). The reaction mixture was stirred for 18 h at r. t. and ¾0 was added. The mixture was extracted with EtOAc (3 times). The combined organic phases were dried over Na 2 S0 4 , concentrated under reduced pressure and separated by column chromatography (hexane : EtOAc = 20 : 1 to 10 : 1) yielding 196 mg (54%) of a desired product. R { = 0.42 (hexane / EtOAc = 10 : 1). - 1H NMR (400 MHz, CDCh): 9.89 (s, 1 H), 7.03 (s, 1 H), 6.94 (s, 1 H), 6.66 (s, 1 H), 4.03 (t, J= 6.2 Hz, 2 H), 2.30 (td, J= 7.0, 2.5 Hz, 2 H), 2.00 (td, J= 2.5, 0.9 Hz, 1 H), 1.97 - 1.90 (m, 2 H), 1.74 (p, J= 7.2 Hz, 2 H), 1.01 (s, 9 H), 0.25 (s, 6 H). - 13 C NMR (100 MHz, CDCh): 191.90, 160.59, 157.34, 138.35, 114.35, 113.48, 107.33, 83.94, 68.75, 67.70, 28.14, 25.61, 24.98, 18.19, 18.14, -4.42.
General Procedure 1 GP1) for the Synthesis of 2-(Arylmethylene)indan-l,3-diones
2-(Arylmethylene)indan-l,3-diones were obtained according to the modified procedure of (Sarvesh and Nizamuddin, 2008). A solution of indan-l,3-dione (1.46 g, 10 mmol), corresponding arylaldehyde (10 mmol) and NaOAc (0.9 g, 11 mmol) in acetic acid (20 mL) was stirred at reflux (oil bath temperature 150 °C) for 3 h. The reaction mixture was cooled to r. t. and poured onto ice. The formed precipitate was filtered off and recrystallized from aq. EtOH.
2-Benzylideneindan-l,3-dione
2-Benzylideneindan-l,3-dione was obtained according to GPl from indan-l,3-dione (1.46 g, 10 mmol), benzaldehyde (1.1 g, 10 mmol) and NaOAc (0.9 g, 11 mmol) in acetic acid (20 mL). Yield: 1.2 g (51%). Spectral data are identical to those published in the literature.
2-(3-Methox benzylidene)indan-l,3-dione
2-(3-Methoxybenzylidene)indan-l,3-dione was obtained according to GPl from indan-1,3- dione (1.46 g, 10 mmol), 3-methoxybenzaldehyde (1.4 g, 10 mmol) and NaOAc (0.9 g, 11 mmol) in acetic acid (20 mL). Yield: 0.8 g (30%). Spectral data are identical to those published in the literature.
2-(4-Hydroxybenzylidene)indan-l,3-dione
2-(4-Hydroxybenzylidene)indan-l,3-dione was obtained according to GPl from indan-1,3- dione (1.2 g, 8.2 mmol), 4-hydroxybenzaldehyde (1.0 g, 8.2 mmol) and NaOAc (0.9 g, 11 mmol) in acetic acid (20 mL). Yield: 1.2 g (59%). Spectral data are identical to those published in the literature.
2-(4-Fluorobenz lidene)indan-l,3-dione
2-(4-Fluorobenzylidene)indan-l,3-dione was obtained according to GPl from indan-l,3-dione (1.46 g, 10 mmol), 4-fluorobenzaldehyde (1.24 g, 8.2 mmol) and NaOAc (0.9 g, 11 mmol) in acetic acid (20 mL). Yield: 1.5 g (73%). Spectral data are identical to those published in the literature. General Procedure 2 (GP2) for the Synthesis of 2-(Arylmeth lene)indan-l,3-diones
2-(Arylmethylene)indan-l,3-diones were obtained according to the modified procedure of (Lee et al., 2014). To a solution of indan-l,3-dione (1.46 g, 10 mmol) in MeOH (20 mL) was added (Z)-proline (0.35g, 3 mmol) followed by the corresponding arylaldehyde (11 mmol). This mixture was stirred for 16 h at r. t, the formed precipitate was filtered off, washed with MeOH and dried in vacuo.
2-(3-Hydrox benzylidene)indan-l,3-dione
2-(3-Hydroxybenzylidene)indan-l,3-dione was obtained according to GP1 from indan-1,3- dione (1.46 g, 10 mmol), 3-hydroxybenzaldehyde (1.2 g, 10 mmol) and NaOAc (0.9 g, 11 mmol) in acetic acid (20 mL). Yield: 0.6 g (24%).
Alternatively 2-(3-hydroxybenzylidene)indan-l,3-dione was obtained according to GP2 from indan-l,3-dione (5.6 g, 37 mmol), 3-hydroxybenzaldehyde (5.0 g, 41 mmol) and (L)-proline (1.4 g, 12mmol) in MeOH (80 mL). Yield: 7.1 g (77%).
Spectral data are identical to those published in the literature.
2-(3-Fluorobenz lidene)indan-l,3-dione
2-(3-Fluorobenzylidene)indan-l,3-dione was obtained according to GP2 from indan-l,3-dione (1.46 g, 10 mmol), 3-fluorobenzaldehyde (1.36 g, 11 mmol) and (J)-proline (0.35g, 3 mmol) in MeOH (20 mL). Yield: 1.80 g (71%). Spectral data are identical to those published in the literature.
2-(3,5-DihydroxybenzyIidene)indan-l,3-dione
2-(3,5-Dihydroxybenzylidene)indan-l,3-dione was obtained according to GP2 from indan- 1,3-dione (0.95 g, 6.5 mmol), 3,5-dihydroxybenzaldehyde (1.00 g, 7.2 mmol) and (L)-proline (0.20 g, 2.0 mmol) in MeOH (15 mL). Yield: 1.25 g (72%). - 1H NMR (400 MHz, DMSO- d 6 ): 9.71-9.62 (br m, 2 H), 8.00-7.88 (m, 4 H), 7.61 (s, 1 H), 7.41 (d, J = 2.0 Hz, 2 H), 6.52 (t, J = 2.0 Hz, 1 H). - 13 C NMR (100 MHz, DMSO-d 6 ): 189.97, 188.86, 158.86, 146.86, 142.44, 139.81, 136.37, 136.19, 134.65, 129.25, 123.50, 123.48, 112.46, 108.55.
2-(4-Carbox benzylidene)indan-l,3-dione
2-(4-Carboxybenzylidene)indan-l,3-dione was obtained according to GP2 from indan-1,3- dione (1.46 g, 10.0 mmol), 4-carboxybenzaldehyde (1.65 g, 11.0 mmol) and (L)-proline (0.35 g, 3.0 mmol) in MeOH (20 mL). Yield: 2.2 g (79%). Spectral data are identical to those published in the literature.
2-(2-Hydrox benzylidene)indan-l,3-dione
2-(2-Hydroxybenzylidene)indan-l,3-dione was obtained according to GP2 from indan-1,3- dione (1.46 g, 10.0 mmol), 2-hydroxybenzaldehyde (1.34 g, 11.0 mmol) and (Z)-proline (0.35 g, 3.0 mmol) in MeOH (20 mL). Yield: 1.7 g (68%). Spectral data are identical to those published in the literature.
5,6-Dichloro-2-(3-hydroxybenzylidene)indan-l,3-dione
5,6-Dichloro-2-(3-hydroxybenzylidene)indan-l,3-dione was obtained according to GP2 from 5,6-dichloroindan-l,3-dione (600 mg, 2.8 mmol), 3-hydroxybenzaldehyde (375 mg, 3.1 mmol) and (I)-proline (97 mg, 0.8 mmol) in MeOH (10 mL). Yield: 590 mg (67%). - 1H NMR (400 MHz, DMSO-d 6 ): 10.18 - 9.56 (br. m, 1 H), 8.21 (s, 1 H), 8.18 (s, 1 H), 8.02 (s, 1 H), 7.83 (d, J= 8.0 Hz, 1 H), 7.76 (s, 1 H), 7.37 (t, J = 8.0 Hz, 1 H), 7.06 (ddd, J = 8.0, 2.4, 0.8 Hz, 1 H). - 13 C MR (100 MHz, DMSO-d 6 ): 187.96, 186.86, 157.93, 147.69, 141.74, 139.30, 139.21, 139.11, 134.22, 130.28, 128.86, 126.27, 125.54, 125.51, 121.68, 120.20.
(E)~ and (Z)-5-Eth nyl-2-(3-hydroxybenzylidene)-indan-l,3-dione
A mixture of (E)- and (Z)-5-ethynyl-2-(3-hydroxybenzylidene)-indan-l,3-dione was obtained according to GP2 from 5-ethynylindane-l,3-dione (510 mg, 3.0 mmol), 3- hydroxybenzaldehyde (400 mg, 3.31 mmol) and (L)-proline (103 mg, 0.9 mmol) in MeOH (20 mL). Column chromatography (hexane: EtOAc = 4 : 1 to 1 : 1) yielded 320 mg (39%) of the desired product. - ! H NMR (400 MHz, DMSO-d 6 ): 9.88 - 9.85 (m, 1 H), 8.07 - 8.04 (m, 1 H), 8.00 - 7.98 (m, 3 H), 7.87 - 7.83 (m, 1 H), 7.77 (s, 1 H), 7.35 - 7.34 (m, 1 H), 7.08 - 7.04 (m, 1 H), 4.70 (d, J= 4.2 Hz, 1 H).
2-(3-Bromo-5-te , /-butyldimethylsilyloxybenzyIidene)indan-l,3-dione
2-(3-Bromo-5-tert-butyldimethylsilyloxybenzylidene)indan-l 5 3-dione was obtained according to GP2 from indan-l,3-dione (440 mg, 3.0 mmol), 3-bromo-5-(tert- butyldimethylsilyloxy)benzaldehyde (1.1 g, 3.3 mmol) and (Z)-proline (120 mg, 1.0 mmol) in MeOH (10 mL). Column chromatography (hexane : EtOAc = 20 : 1 to 10 : 1) yielded 530 mg (40%) of the desired product. - 1H NMR (400 MHz, DMSO-d 6 ): 8.34 (t, J = 1.5 Hz, 1 H), 8.17 - 8.15 (m, 1 H), 8.08 - 7.94 (m, 4 H), 7.81 (s, 1 H), 7.29 (t, J= 2.0 Hz, 1 H), 0.99 (s, 9 H), 0.29 (s, 6 H). - 13 C NMR (100 MHz, DMSO-d 6 ): 189.52, 189.05, 156.52, 143.78, 142.57, 140.07, 136.64, 136.55, 136.13, 131.24, 129.87, 127.48, 123.86, 123.72, 123.68, 122.37, 25.97, 18.44, -4.23.
2-(3-(/e/*i-Butoxycarbon lamino)benzyIidene)indan-l,3-dione
2-(fert-Butoxycarbonylaminobenzylidene)indan-l,3-dione was obtained according to GP2 from indan-l,3-dione (1.0 g, 7.0 mmol), 3-(ter^butoxycarbonylamino)benzaldehyde (1.7 g, 7.7 mmol) and (Z)-proline (230 mg, 2.0 mmol) in MeOH (20 mL). Yield: 1.5 g (61%). - 1H NMR (400 MHz, DMSO-d 6 ): 9.58 (s, 1 H), 8.34 - 8.31 (m, 1 H), 8.22 (d, J = 7.8 Hz, 1 H), 8.02 - 7.93 (m, 4 H), 7.71 (s, 1 H), 7.66 (dd, J= 8.0, 2.1 Hz, 1 H), 7.45 (t, J = 8.0 Hz, 1 H), 1.50 (s, 9 H). - 13 C NMR (100 MHz, DMSO-d 6 ): 189.80, 188.76, 153.27, 145.92, 142.44, 140.37, 139.95, 136.50, 136.33, 133.49, 129.81, 129.50, 127.73, 123.92, 123.61, 123.58, 79.80, 28.59.
2-(3-(ieri-Butyldimeth lsilyloxymethyl)benzylidene)indan-l,3-dione
2-(3-(tert-Butyldimethylsilyloxymethyl)benzylidene)indan- l,3-dione was obtained according to GP2 from indan-l,3-dione (526 mg, 3.6 mmol), 3-(tert- butyldimethylsilyloxymethyl)benzaldehyde (900 mg, 3.6 mmol) and (Z)-proline (115 mg, 1.0 mmol) in MeOH (10 mL). The crude product was purified by column chromatography (hexane : EtOAc = 20 : 1 to 10 : 1). Yield: 320g (24%). - 1H NMR (400 MHz, DMSO-d 6 ): 8.46 (s, 1 H), 8.41 - 8.37 (m, 1 H), 8.04 - 7.94 (m, 4 H), 7.85 (s, 1 H), 7.60 - 7.53 (m, 2 H), 0.93 (s, 9 H), 0.13 (s, 6 H). - 13 C NMR (100 MHz, DMSO-d 6 ): 189.89, 188.96, 145.90, 142.46, 142.32, 139.98, 136.53, 136.36, 133.14, 133.02, 131.80, 131.46, 129.83, 129.19, 123.61, 123.59, 64.48, 26.33, 18.52, -4.79. (£)- and (Z)-2-(3-A2ido-5-te */-butyldimethylsilyloxybenzylidene)-5-ethylideneindan-l,3- dione
A mixture of (£)- and (Z)-2-(3-azido-5-¾rt-butyldimethylsilyloxybenzylidene)-5- ethylideneindan-l,3-diones was obtained according to GP2 from 5-ethynyl-indan-l,3-dione (306 mg, 1.8 mmol), 3-azido-5-(ter/-butyldimethylsilyloxy)benzaldehyde (540 mg, 1.95 mmol) and (Z)-proline (57 mg, 0.5 mmol) in MeOH (15 mL). Volatiles were removed under reduced pressure and the residue was purified by column chromatography (hexane: EtOAc = 20 : 1 to 4 : 1) yielding 508 mg (66%). - 1H NMR (500 MHz, DMSO-d 6 ): 8.11 - 7.81 (m, 6 H), 6.79 (s, 1 H), 4.74 (s, 1 H), 0.99 (s, 9 H), 0.29 (s, 6 H). IR (film) v = 2930 cm , 2857, 2108, 1729, 1687, 1608, 1576, 1314, 1235, 835.
2-(3- ^-ButyIdimethylsil Ioxy-5-methoxybenzylidene)indan-l,3-dione
2-(3 -½rt-Butyldimethylsilyloxy-5-methoxybenzylidene)indan- 1 ,3 -dione was obtained according to GP2 from indan- 1,3 -dione (0.25 g, 1.7 mmol), 3-fert-butyldimemylsilyloxy-5- methoxybenzaldehyde (0.50 g, 1.9 mmol) and (i)-proline (58 mg, 0.5 mmol) in MeOH (10 mL). Yield: 540 mg (81%). - 1H NMR (400 MHz, DMSO-d 6 ): 8.06 - 7.92 (m, 4 H), 7.85 - 7.74 (m, 3 H), 6.68 - 6.61 (m, 1 H), 3.84 (s, 3 H), 0.99 (s, 9 H), 0.28 (s, 6 H). - 13 C NMR (100 MHz, DMSO-d ): 189.84, 189.13, 160.74, 156.66, 146.01 (2C), 139.90, 136.50, 136.37, 134.96, 130.07, 123.74, 123.57, 1 17.66, 1 13.04, 1 11.85, 55.94, 26.04, 18.44, -4.11.
2-(3-/£? i-Butyldimethylsilyloxy-5-(hex-5-ynyloxy)benzylidene)indan-l ,3-dione
2-(3-ter^-Butyldimethylsilyloxy-5-(hex-5-ynyloxy)benzylidene )indan-l ,3-dione was obtained according to GP2 from indan-l,3-dione (219 mg, 1.5 mmol), 3-(fer -butyldimethylsilyloxy)-5- (hex-5-ynyloxy)benzaldehyde (350 mg, 1.1 mmol) and (L) -proline (35 mg, 0.3 mmol) in MeOH (5 mL). The reaction mixture was concentrated under reduced pressure (due to the possible instability of the product the removal of the solvent should be done at r. t.) and the residue was separated by column chromatography (hexane : EtOAc = 20 : 1 to 10 : 1). Yield: 200 mg (40%). - 1H NMR (500 MHz, DMSO-d 6 ): 8.06 - 7.94 (m, 4 H), 7.82 (s, 1 H), 7.80 - 7.77 (m, 2 H), 6.66 (s, 1 H), 4.07 (t, J = 6.4 Hz, 2 H), 2.81 (t, J= 2.7 Hz, 1 H), 2.26 (td, J = 7.1, 2.6 Hz, 2 H), 1.88 - 1.80 (m, 2 H), 1.67 - 1.58 (m, 2 H), 0.99 (s, 9 H), 0.28 (s, 6 H). - 13 C NMR (125 MHz, DMSO-d 6 ): 189.91, 189.20, 160.07, 156.66, 146.07, 142.54, 139.91, 136.52, 136.39, 134.95, 130.01, 123.78, 123.59, 117.60, 113.66, 112.29, 84.77, 71.96, 67.78, 28.17, 26.04, 25.11, 18.45, 17.92, -4.10. £)- and (Z)-5-Carboxy-2-(3-methoxybenzylidene)-indan-l,3-dione
A suspension of 3-hydroxy-2-(methoxycarbonyl)-l-oxo-lH-indene-6-carboxylic acid (1.1 g, 4.4 mmol) and 3-methoxybenzaldehyde (0.68 g, 5.0 mmol) in acetic acid (15 mL) was stirred at 100 °C for 2 h. The obtained mixture was poured onto the ice. The obtained precipitate was filtered off and purified by column chromatography (CH 2 C1 2 : MeOH = 1 : 0 to 97 : 1). Yield: 450 mg (33%) of the ca. \ : \ mixture of (E)- and (Z)-5-carboxy-2-(3-methoxybenzylidene)- indan-l,3-diones. - 1H NMR (500 MHz, DMSO-d 6 ) 13.92 - 13.59 (br s, 1 H), 8.45 - 8.39 (m, 1 H), 8.39 - 8.30 (m, 2 H), 8.12 - 8.06 (m, 1 H), 8.05 - 7.96 (m, 1 H), 7.91 - 7.84 (m, 1 H), 7.53 - 7.46 (m, 1 H), 7.26 - 7.19 (m, 1 H), 3.88 (s, 1.5 H), 3.87 (s, 1.5 H). - 13 C NMR (125 MHz, DMSO-d 6 ): 189.43, 189.25, 188.58, 188.43, 166.40, 166.38, 159.67, 159.66, 146.92, 146.90, 145.05, 142.61, 142.56, 140.08, 137.65, 137.62, 137.52, 136.75, 136.59, 134.43, 130.40, 130.35, 129.94, 129.92, 127.62, 127.58, 124.15, 124.03, 123.93, 123.83, 120.63, 120.54, 118.32, 118.13, 55.81, 55.79. General Procedure 3 (GP3) for the Synthesis of ll-Aryl-5,ll-dihydro-12H- benzo[b]indeno[l,2-e][l,4]thiazepin-12-ones
1 l-Aryl-5,1 l-dihydro-12H-benzo[b]indeno[l,2-e][l,4]thiazepin-12-ones were obtained according to the modified procedure of (Krysin et al, 2003). To a suspension of a corresponding 2-(arylmethylene)indan-l,3-dione (2.0 mmol) in the mixture of z ' PrOH (15 mL) and HOAc (10 mL) was added 2-aminothiophenol (256 μ∑, 300 mg, 2.4 mmol). The reaction mixture was stirred for 18 h at r. t. The reaction mixture was neutralized by sat. NaHC0 3 , z ' PrOH was removed under reduced pressure and the residue was extracted with EtOAc (3x). The combined organic phases were dried over Na 2 S0 4 , filtered and concentrated under reduced pressure. The residue was separated by column chromatography (hexane : EtOAc) to yield the desired product as a red solid.
General Procedure 4 (GP4) for the Synthesis of ll-Aryl-5,ll-dihydro-12H- benzo [b] indeno [1 ,2-e] [1 ,4] thiazepin-12-ones
To a suspension of a corresponding 2-(arylmethylene)indan-l,3-dione (2.0 mmol) in EtOH (20 mL) was added 2-aminothiophenol (256 μΕ, 300 mg, 2.4 mmol). The reaction mixture was stirred for 18 h at r. t. The reaction mixture was concentrated under reduced pressure. The residue was separated by column chromatography (hexane : EtOAc) to yield the desired product as a red solid.
5,ll-Dihydro-ll-phenyl-12H-benzo[b]indeno[l,2-e] [l,4]thiazepin-12-one (340)
To a solution of 2-benzylideneindan-l,3-dione (468 mg, 2.0 mmol) in DMF (20 mL) was added 2-aminothiophenol (256 μΤ, 300 mg, 2.4 mmol). The reaction mixture was stirred for 18 h at r. t. Water was added and the obtained mixture was extracted with EtOAc (3x).The combined organic phases were dried over Na 2 S0 4 , filtered and concentrated under reduced pressure. The residue was separated by column chromatography (hexane : EtOAc 10 : 1 to 2 : 1) to yield 120 mg (18%) of the desired product as a red solid. R f = 0.54 (hexane / EtOAc = 2
: 1). - 1H NMR (500 MHz, DMSO-d 6 ): 10.06 (s, 1 H), 8.12 (d, J = 7.3 Hz, 1 H), 7.61 - 7.52 (m, 2 H), 7.44 - 7.36 (m, 2 H), 7.32 (td, J = 7.3, 1.6 Hz, 1 H), 7.10 - 7.00 (m, 5 H), 6.98 - 6.94 (m, 1 H), 6.92 - 6.87 (m, 1 H), 5.52 (s, 1 H). - 13 C NMR (125 MHz, DMSO-d 6 ): 189.78, 156.99, 143.86, 142.69, 139.79, 136.52, 133.19, 132.12, 130.49, 129.47, 128.21, 127.66, 126.83, 125.10, 124.67, 128.85, 120.91, 119.61, 109.55, 46.19. -HRMS (ESI) calcd. for C 22 H 16 NOS [M+H] + 342.0953, found 342.0947.
5,1 l-Dihydro-ll-(3-methoxyphenyl)-12H-benzo [b] indeno [1 ,2-e] [1,4] thiazepin-12-one
To a solution of 2-(3-methoxybenzylidene)indan-l,3-dione (528 mg, 2.0 mmol) in DMF (10 mL) was added 2-aminothiophenol (256 μΕ, 300 mg, 2.4 mmol). The reaction mixture was stirred for 18 h at r. t. Water was added and the obtained mixture was extracted with EtOAc (3x).The combined organic phases were dried over Na 2 S0 4 , filtered and concentrated under reduced pressure. The residue was separated by column chromatography (hexane : EtOAc 10 : 1 to 2 : 1) to yield 520 mg (70%) of the desired product as a red solid. R f = 0.46 (hexane /
EtOAc = 2 : 1). - 1H NMR (500 MHz, DMSO-d 6 ): 10.05 (s, 1 H), 8.11 (d, J = 7.3 Hz, 1 H), 7.58 (d, J= 8.1 Hz, 1 H), 7.54 (t, J= 7.3 Hz, 1 H), 7.43 - 7.36 (m, 2 H), 7.32 (td, J= 7.3, 1.5 Hz, 1 H), 7.02 - 6.90 (m, 3 H), 6.62 - 6.56 (m, 2 H), 6.55 (s, 1 H), 5.47 (s, 1 H), 3.54 (s, 3 H). - 13 C NMR (125 MHz, DMSO-d 6 ): 189.79, 159.19, 156.85, 144.32, 143.82, 139.77, 136.57, 133.15, 132.12, 130.49, 129.47, 129.17, 125.19, 124.74, 123.83, 120.91, 120.10, 119.64, 113.27, 112.48, 109.60, 55.26, 46.14. -HRMS (ESI) calcd. for C 23 H 18 N0 2 S [M+H] + 372.1058, found 372.1054.
■ Dihydro-ll-(4-hydroxyphenyl)-12H-benzo[b]indeno[l,2-e] [l,4]thiazepin-12-
5,11-Dihydro-l l -(4-hydroxyphenyl)-12H-benzo[b]indeno[l,2-e][l,4]thiazepin-1 2-one was obtained according to GP3 from 2-(4-hydroxybenzylidene)indan-l,3-dione (150 mg, 0.6 mmol) and 2-aminothiophenol (106 μΐ,, 125 mg, 1.0 mmol) in z ' PrOH (15 mL) and HOAc (10 mL) at r. t. Column chromatography (hexane : EtOAc 10 : 1 to 1 : 1) yielded 116 mg (54%) of the desired product as a red solid. R f = 0.20 (hexane / EtOAc = 2 : 1). - ! H NMR (400 MHz, DMSO-d 6 ): 9.98 (s, 1 H), 9.17 (s, 1 H), 8.10 (d, J= 7.4 Hz, 1 H), 7.60 - 7.49 (m, 2 H), 7.44 - 7.28 (m, 3 H), 7.00 (dd, J= 7.7, 1.6 Hz, 1 H), 6.92 (td, J= 7.7, 1.3 Hz, 1 H), 6.82 (d, J = 8.4 Hz, 2 H), 6.45 (d, J = 8.4 Hz, 2 H), 5.42 (s, 1 H). - 13 C NMR (100 MHz, DMSO-d 6 ): 189.67, 156.77, 156.13, 143.88, 139.85, 136.63, 133.25, 132.98, 132.02, 130.38, 129.28, 128.76, 125.08, 125.03, 123.76, 120.83, 119.49, 114.98, 110.40, 45.96. -HRMS (ESI) calcd. for C 22 Hi 6 N0 2 S [M+H] + 358.0902, found 358.0895.
5,ll-Dihydro-ll-(3-hydroxyphenyl)-12H-benzo[b]indeno[l,2- e] [l,4]thiazepin-12-one
5,11-Dihydro-l l-(3-hydroxyphenyl)-12H-benzo[b]indeno[l,2-e][l,4]thiazepin- 12-one was obtained according to GP4 from 2-(3-hydroxybenzylidene)indan-l,3-dione (500 mg, 2.0 mmol) and 2-aminothiophenol (256 μΕ, 300 mg, 2.4 mmol) in EtOH (20 mL) at r. t. Column chromatography (hexane : EtOAc 10 : 1 to 1 : 1) yielded 200 mg (28%) of the desired product as a red solid. R f = 0.22 (hexane / EtOAc = 2 : 1). - 1H NMR (500 MHz, DMSO-d 6 ): 10.02 (s, 1 H), 9.14 (br s, 1 H), 8.12 (d, J = 7.3 Hz, 1 H), 7.59 - 7.52 (m, 2 H), 7.44 - 7.36 (m, 2 H), 7.33 (td, J= 7.7, 1.5 Hz, 1 H), 7.03 (dd, J= 7.7, 1.5 Hz, 1 H), 6.93 (td, J = 7.5, 1.2 Hz, 1 H), 6.86 (t, J= 7.8 Hz, 1 H), 6.49 (d, J= 7.7 Hz, 1 H), 6.46 (s, 1 H), 6.41 (dd, J= 7.8, 1.8 Hz, 1 H), 5.40 (s, 1 H). - 13 C NMR (125 MHz, DMSO-d 6 ): 189.78, 157.23, 156.81, 143.95, 143.79, 139.81, 136.47, 133.20, 132.11, 130.47, 129.39, 129.11, 125.08, 124.75, 123.76, 120.90, 119.55, 118.62, 114.87, 113.82, 109.84, 46.06. -HRMS (ESI) calcd. for C 22 H 16 N0 2 S [M+H] + 358.0902, found 358.0896.
5,ll-Dihydro-ll-(2-hydroxyphenyl)-12H-benzo[b]indeno[l,2- e][l,4]thiazepin-12-one
5,1 1-Dihydro-l l-(2-hydroxyphenyl)-12H-benzo[b]indeno[l,2-e][l,4]t iazepin-12-one was obtained according to GP3 from 2-(2-hydroxybenzylidene)indan-l,3-dione (500 mg, 2.0 mmol) and 2-aminothiophenol (256 iL, 300 mg, 2.4 mmol) in z ' PrOH (15 niL) and HOAc (10 mL) at r. t. Column chromatography (hexane : EtOAc 3 : 1 to 1 : 1 to 0 : 1) yielded 319 mg (45%) of the desired product as a dark red solid. - 1H NMR (400 MHz, DMSO-d 6 ): 10.00 (s, 1 H), 9.73 (s, 1 H), 8.11 (d, J= 7.4 Hz, 1 H), 7.60 (d, J= 8.0 Hz, 1 H), 7.53 (d, J= 7.4 Hz, 1 H), 7.39 (d, J= 7.4 Hz, 1 H), 7.37 - 7.29 (m, 2 H), 6.97 - 6.81 (m, 3 H), 6.75 (d, J = 8.0 Hz, 1 H), 6.28 - 6.18 (m, 2 H), 5.64 (s, 1 H). - 13 C NMR (100 MHz, DMSO-d 6 ): 189.66, 157.25, 154.48, 143.81, 139.94, 136.58, 133.22, 132.02, 130.35, 129.26, 129.04, 128.05, 126.97, 125.08, 124.96, 123.64, 120.81, 119.44, 118.09, 115.32, 110.21, 49.09. -HRMS (ESI) calcd. for C 22 Hi 6 N0 2 S [M+H] + 358.0902, found 358.0897. ll-(4-Fluorophenyl)-5,ll-dihyd -12H-benzo[b]indeno[l,2-e][l,4]thiazepin-12-one
1 l-(4-Fluorophenyl)-5,l l-dihydro-12H-benzo[b]indeno[l,2-e][l,4]thiazepin-12-one was obtained according to GP3 from 2-(4-fluorobenzylidene)indan-l,3-dione (500 mg, 2.0 mmol) and 2-aminothiophenol (256 pL, 300 mg, 2.4 mmol) in z ' PrOH (15 mL) and HOAc (10 mL) at r. t. Column chromatography (hexane : EtOAc 10 : 1 to 1 : 1) yielded 550 mg (77%) of the desired product as a red solid. R f = 0.40 (hexane / EtOAc = 2 : 1). - 1H NMR (400 MHz, DMSO-d 6 ): 10.06 (s, 1 H), 8.11 (d, J = 7.2 Hz, 1 H), 7.64 - 7.48 (m, 2 H), 7.46 - 7.25 (m, 3 H), 7.10 - 7.00 (m, 2 H), 7.00 - 6.80 (m, 4 H), 5.54 (s, 1 H). - 13 C NMR (100 MHz, DMSO- d 6 ): 189.70, 160.94 (d, J CF = 243 Hz), 157.01, 143.88, 139.73, 138.97 (d, J CF = 3 Hz), 136.57, 133.15, 132.12, 130.50, 129.56, 129.48, 125.24, 124.56, 123.96, 120.91, 119.64, 114.87 (d, JCF = 22 Hz), 109.44, 45.49. -HRMS (ESI) calcd. for C 22 H 15 FNOS [M+H] + 360.0858, found 360.0853.
1 l-(3-Fluorophenyl)-5,l 1-dihy dro-^/f-benzo [b] indeno [1 ,2-e] [1 ,4] thiazepin-12-one
1 l-(3-Fluorophenyl)-5,l l-dihydro-12H-benzo[b]indeno[l,2-e][l,4]thiazepin-12-one was obtained according to GP4 from 2-(3-fluorobenzylidene)indan-l,3-dione (500 mg, 2.0 mmol) and 2-aminothiophenol (256 pL, 300 mg, 2.4 mmol) in EtOH (20 mL) at r. t. Column chromatography (hexane : EtOAc 10 : 1 to 1 : 1) yielded 180 mg (25%) of the desired product as a red solid. R f = 0.24 (hexane / EtOAc = 2 : 1). - 1H NMR (400 MHz, DMSO-d 6 ): 10.09 (s, 1 H), 8.12 (d, J = 7.3 Hz, 1 H), 7.62 - 7.51 (m, 2 H), 7.46 - 7.30 (m, 3 H), 7.13 - 7.05 (m, 1 H), 7.02 - 6.90 (m, 2 H), 6.88 - 6.79 (m, 3 H), 5.56 (s, 1 H). - 13 C NMR (100 MHz, DMSO- d 6 ): 189.75, 162.20 (d, J CF = 243 Hz), 157.13, 145.72 (d, J CF = 7 Hz), 143.82, 139.72, 136.49, 133.14, 132.13, 130.54, 129.98 (d, J CF = 8 Hz), 129.63, 125.27, 124.43, 123.97, 123.73 (d, J CF = 2 Hz), 120.95, 119.73, 114.43 (d, J CF = 22 Hz), 113.68 (d, J CF = 21 Hz), 108.90, 45.73. - HRMS (ESI) calcd. for C 22 H 15 FNOS [M+H] + 360.0858, found 360.0854. ll-(4-CarboxyphenyI)-5,H-dihydro-12H-benzo[b]indeno[l,2-e] [l,4]thiazepin-12-one
1 l-(4-Carboxyphenyl)-5,l l-dihydro-12H-benzo[b]indeno[l,2-e][l,4]thiazepin-12-one was obtained according to GP4 from 2-(4-carboxybenzylidene)indan-l,3-dione (556 mg, 2.0 mmol) and 2-aminothiophenol (256 iL, 300 mg, 2.4 mmol) in EtOH (20 mL) at r. t. Column chromatography (hexane : EtOAc 10 : 1 to 1 : 1) yielded 330 mg (43%) of the desired product as a red solid. - 1H NMR (500 MHz, DMSO-d 6 ): 13.22 - 12.26 (br s, 1 H), 10.11 (s, 1 H), 8.13 (d, J = 7.3 Hz, 1 H), 7.65 (d, J= 8.2 Hz, 2 H), 7.61 - 7.52 (m, 2 H), 7.44 - 7.37 (m, 2 H), 7.34 (td, J= 7.6, 1.6 Hz, 1 H), 7.14 (d, J= 8.2 Hz, 2 H), 6.99 - 6.87 (m, 2 H), 5.60 (s, 1 H). - 13 C MR (125 MHz, DMSO-d 6 ): 189.78, 167.46, 157.18, 147.69, 143.83, 139.72, 136.49, 133.14, 132.17, 130.58, 129.67, 129.35, 129.15, 127.81, 125.25, 124.34, 123.97, 120.98, 119.73, 108.77, 45.96. -HRMS (ESI) calcd. for C 23 H 16 N0 3 S [M+H] + 386.0851, found 386.0846.
5,ll-Dihydro-ll-(3,5-dihydroxyphenyl)-12H-benzo[b]indeno[ l,2-e] [l,4]thiazepin-12-one
5,11 -Dihydro-11 -(3 ,5-dihydroxyphenyl)- 12H-benzo [b]indeno[ 1 ,2-e] [1 ,4]thiazepin- 12-one was obtained according to GP4 from 2-(3,5-dihydroxybenzylidene)indan-l,3-dione (532 mg, 2.0 mmol) and 2-aminothiophenol (256 μΐ,, 300 mg, 2.4 mmol) in EtOH (20 mL) at r. t. Column chromatography (hexane : EtOAc 10 : 1 to 1 : 1) yielded 290 mg (39%) of the desired product as a red solid. - 1H NMR (400 MHz, DMSO-d 6 ) 9.95 (s, 1 H), 8.95 (br s, 2 H), 8.12 (d, J= 7.2 Hz, 1 H), 7.59 - 7.49 (m, 2 H), 7.44 - 7.29 (m, 3 H), 7.10 (dd, J= 7.6, 1.1 Hz, 1 H), 6.97 (t, J= 7.6 Hz, 1 H), 5.97 (s, 2 H), 5.87 (s, 1 H), 5.27 (s, 1 H). - 13 C NMR (100 MHz, DMSO-d 6 ): 189.80, 158.17, 156.64, 144.40, 143.74, 139.84, 136.41, 133.23, 132.07, 130.43, 129.29, 125.02, 124.82, 123.66, 120.88, 119.46, 110.16, 106.38, 101.32, 46.07. - HRMS (ESI) calcd. for C 22 H 16 N0 3 S [M+H] + 374.0851, found 374.0847. 5,ll-Dihydro-ll-(3-hydroxyphenyl)-8-methoxy-12H-benzo[b]inde no[l,2- e] [l,4]thiazepin-12-one
5,11 -Dihydro- 11 -(3 -hydroxyphenyl)— 8 -methoxy- 12H-benzo [b]indeno [ 1 ,2-e] [1 ,4]thiazepin- 12-one was obtained according to GP4 from 2-(3-hydroxybenzylidene)indan-l,3-dione (375 mg, 1.5 mmol) and 2-amino-5-methoxy-thiophenol (230 mg, 1.5 mmol) in EtOH (20 mL) at r. t. Column chromatography (hexane : EtOAc 10 : 1 to 1 : 1) yielded 150 mg (26%) of the desired product as a red solid. - 1H NMR (500 MHz, DMSO-d 6 ): 9.98 (s, 1 H), 9.15 (s, 1 H), 8.08 (d, J= 7.4 Hz, 1 H), 7.53 (td, J= 7.4, 1.3 Hz, 1 H), 7.49 (d, J= 9.0 Hz, 1 H), 7.43 - 7.35 (m, 2 H), 6.94 (dd, J = 9.0, 3.0 Hz, 1 H), 6.89 (t, J= 7.8 Hz, 1 H), 6.56 (d, J= 3.0 Hz, 1 H), 6.53 (d, J= 7.8 Hz, 1 H), 6.49 (s, 1 H), 6.43 (dd, J= 7.8, 1.8 Hz, 1 H), 5.39 (s, 1 H), 3.62 (s, 3 H). - 13 C NMR (125 MHz, DMSO-d 6 ): 189.35, 157.20, 156.84, 156.09, 143.82, 139.63, 136.71, 133.55, 131.86, 130.41, 129.09, 126.24, 124.98, 120.70, 120.41, 119.40, 118.75, 115.29, 114.97, 113.79, 108.79, 55.78, 46.08. -HRMS (ESI) calcd. for C 23 Hi 8 N0 3 S [M+H] + 388.1007, found 388.1003. ll-([l,l'-biphenyl]-4-yl)-5H-benzo[b]indeno[l,2-e] [l,4]thiazepin-12(llH)-one
1 l-([l, -biphenyl]-4-yl)-5H-benzo[b]indeno[l,2-e][l,4]thiazepin-12(H H)-one was obtained according to GP3 from 2-([l,l'-biphenyl]-4-ylmethylene)-lH-indene-l,3(2H)-dione (500 mg, 1.6 mmol) and 2-aminothiophenol (207 xL, 242 mg, 1.9 mmol) in the mixture of z ' PrOH (12 mL) and HO Ac (8 mL) at r. t. Column chromatography (hexane : EtOAc 10 : 1 to 1 : 1) yielded 350 mg (52%) of the desired product as a red solid. - *H NMR (500 MHz, DMSO-d 6 ): 10.06 (s, 1H), 8.14 (d, J= 5.0 Hz, 1H), 7.61-7.29 (m, 12H), 7.12-6.91 (m, 4H), 5.55 (s, 1H). - 1J C NMR (100 MHz, DMSO-d 6 ) 189.78, 156.89, 143.88, 142.01, 139.89, 139.80, 138.41, 136.54, 133.15, 132.12, 130.50, 129.53, 129.30, 128.27, 127.77, 126.83, 126.41, 125.13, 124.62, 123.89, 120.91, 119.63, 109.56, 45.94. -HRMS (ESI) c ded, for C 28 H 19 NOS [M+H] + 418.1265, found 418.1258. ll-(4-(trifluoromethyl)phenyl)-5H-benzo[b]indeno[l,2-e][l,4] thiazepin-12(llH)-one
1 l-(4-(trifluoromethyl)phenyl)-5H-benzo[b]indeno[l,2-e][l,4]t hiazepin-12(l lH)-one was obtained according to GP3 from 2-(4-(trifluoromethyl)benzylidene)-lH-indene-l,3(2H)-dione (500 mg, 1.6 mmol) and 2-aminothiophenol (212 xL, 248 mg, 1.9 mmol) in the mixture of z ' PrOH (12 mL) and HO Ac (8 mL) at r. t. Column chromatography (hexane : EtOAc 10 : 1 to 1 : 1) yielded 500 mg (74%) of the desired product as a red solid. - 1H NMR (500 MHz, DMSO-d 6 ): 10.12 (s, 1H), 8.14 (d, J = 10.0 Hz, 1H), 7.60-7.24 (m, 9H), 6.99-6.91 (m, 2H), 5.64 (s, 1H). - 13 C NMR (100 MHz, DMSO-d 6 ): 189.77, 157.21, 147.47, 143.87, 139.71, 136.45, 133.10, 132.16, 130.57, 129.75, 128.39, 127.34 (q, J CF = 30 Hz), 125.95, 125.10 (q, JCF = 4 Hz), 124.17, 124.02, 123.25, 120.97, 119.75, 108.52, 45.75. -HRMS (ESI) calcd. for C 23 Hi4F 3 NOS [M+H] + 410.0826, found 410.0819. ll-(4-fluorophenyI)-5H-benzo[b]indeno[l,2-e][l,4]thiazepin-1 2(llH)-one 10,10-dioxide
To a solution of l l-(4-fluorophenyl)-5H-benzo[b]indeno[l,2-e][l,4]thiazepin-12 (l lH)-one (50 mg, 0.13 mmol) in THF (1.4 mL) was added mCPBA(79 mg, 0.45 mmol), and the mixture was stirred for 5 hours. The mixture was quenched with water and extracted by ethyl acetate. The organic layer was dried over sodium sulfate, and filtered. The filtrate was purified by column chromatography (hexane : EtOAc 10 : 1 to 1 : 1) to afford the desired product (38 mg, 70%) as a yellow solid. 1H NMR (500 MHz, DMSO-d 6 ): 10.53 (s, 1H), 8.20 (d, J = 10.0 Hz, 1H), 7.86-7.45 (m, 6H), 7.22-6.97 (m, 5H), 5.77 (s, 1H). - 13 C NMR (100 MHz, DMSO-d 6 ): 191.04, 162.44 (d, J CF = 145 Hz), 156.15, 139.42, 136.79, 135.35, 132.82, 132.59, 131.77 (d, J CF = 8 Hz), 131.04, 129.47, 127.42, 124.77, 123.90, 121.50, 120.57, 115.58, 115.36, 100.35, 66.00. -HRMS (ESI) calcd. for C 22 Hi 4 FN0 3 S [M+H] + 392.0756, found 392.0753. ll-(3-hydroxyphenyl)-5H-benzo[b]indeno[l,2-e][l,4]thiazepin- 12(HH)-one 10,10- dioxide
To a solution of l l-(3-hydroxyphenyl)-5H-benzo[b]indeno[l,2-e][l,4]thiazepin-1 2(HH)-one (40 mg, 0.11 mmol) in THF (1.2 mL) was added mCPBA(63 mg, 0.36 mmol), and the mixture was stirred for 5 hours. The mixture was quenched with water and extracted by ethyl acetate. The organic layer was dried over sodium sulfate, and filtered. The filtrate was purified by column chromatography (hexane : EtOAc 10 : 1 to 1 : 1) to afford the desired product (27 mg, 62%) as a orange solid. 1H NMR (500 MHz, 10.49 (s, 1H), 9.36 (s, 1H), 8.20 (d, J = 5.0 Hz, 1H), 7.85-7.47 (m, 6H), 7.23-7.19 (m, 1H), 6.93-6.90 (m, 1H), 6.55-6.48 (m, 3H), 5.58 (s, 1H). - 13 C NMR (100 MHz, DMSO-d 6 ): 191.07, 157.44, 155.83, 139.40, 136.77, 135.62, 135.19, 132.80, 132.60, 131.00, 129.46, 129.38, 127.65, 124.63, 123.69, 121.48, 120.44, 120.13, 116.62, 115.93, 100.84, 66.71. -HRMS (ESI) calcd. for C 22 H 15 N0 4 S [M+H] + 390.0800, found 390.0795. ll-(3-methoxyphenyl)-5H-benzo[b]indeno[l,2-e][l,4]thiazepin- 12(llH)-one 10,10- dioxide
To a solution of l l-(3-methoxyphenyl)-5H-benzo[b]indeno[l,2-e][l,4]thiazepin-1 2(l lH)-one (70 mg, 0.18 mmol) in THF (1.8 niL) was added mCPBA(107 mg, 0.62 mmol), and the mixture was stirred for 5 hours. The mixture was quenched with water and extracted by ethyl acetate. The organic layer was dried over sodium sulfate, and filtered. The filtrate was purified by column chromatography (hexane : EtOAc 10 : 1 to 1 : 1) to afford the desired product (36 mg, 47%) as a yellow solid. 1H NMR (500 MHz, CDCl 3 ): 8.83 (s, 1H), 7.63 (d, J = 5.0 Hz, 1H), 7.52 (d, J = 5.0 Hz, 1H), 7.28-6.98 (m, 6H), 6.83-6.80 (m, 1H), 6.69-6.65 (m, 3H), 5.94 (s, 1H), 3.62 (s, 3H). - 13 C NMR (100 MHz, CDCl 3 ): 191.05, 159.32, 154.24, 138.28, 135.46, 134.70, 134.36, 131.98, 131.90, 130.31, 129.29, 129.16, 126.45, 123.65, 122.46, 121.81, 121.60, 177.94, 155.20, 114.50, 100.64, 66.85, 55.16. -HRMS (ESI) calcd. for C 23 H 17 N0 4 S [M+H] + 404.0956, found 404.0954. ll-([l,r-biphenyl]-4-yl)-5H-benzo[b]indeno[l,2-e][l,4]thiaze pin-12(HH)-one 10,10- dioxide
To a solution of l l-([l,l'-biphenyl]-4-yl)-5H-benzo[b]indeno[l,2-e][l,4]thiaze pin-12(HH)- one (70 mg, 0.16 mmol) in THF (1.7 mL) was added mCPBA(95 mg, 0.55 mmol), and the mixture was stirred for 5 hours. The mixture was quenched with water and extracted by ethyl acetate. The organic layer was dried over sodium sulfate, and filtered. The filtrate was purified by column chromatography (hexane : EtOAc 10 : 1 to 1 : 1) to afford the desired product (55 mg, 73%) as a yellow solid. 1H NMR (500 MHz, DMSO-d 6 ): 10.55 (s, 1H), 8.22 (d, J = 5.0 Hz, 1H), 7.88 (d, J = 5.0 Hz, 1H), 7.75-7.64 (m, 2H), 7.56-7.31 (m, 10H), 7.21- 7.18 (m, 3H), 5.79 (s, 1H). - 13 C NMR (100 MHz, DMSO-de): 191.11, 156.02, 140.51, 139.46, 139.43, 136.88, 135.36, 133.68, 132.84, 132.60, 131.05, 130.25, 129.58, 129.37, 128.19, 127.64, 127.02, 126.75, 124.73, 123.87, 121.52, 120.57, 100.59, 66.60. -HRMS (ESI) calcd. for C 23 Hi 7 N0 4 S [M+H] + 450.1164, found 450.1156. ll-(4-(trifluoromethyl)phenyl)-5H-benzo[b]indeno[l,2-e][l,4] thiazepm-12(llH)-one 10,10-dioxide
To a solution of l l-(4-(trifluoromethyl)phenyl)-5H-benzo[b]indeno[l,2-e][l,4]t hiazepin- 12(l lH)-one (70 mg, 0.17 mmol) in THF (1.7 mL) was added mCPBA(97 mg, 0.56 mmol), and the mixture was stirred for 5 hours. The mixture was quenched with water and extracted by ethyl acetate. The organic layer was dried over sodium sulfate, and filtered. The filtrate was purified by column chromatography (hexane : EtOAc 10 : 1 to 1 : 1) to afford the desired product (50 mg, 66%) as a yellow solid. 1H NMR (500 MHz, DMSO-d 6 ): 10.59 (s, 1H), 8.22 (d, J= 10.0 Hz, 1H), 7.87 (d, J- 5.0 Hz, 1H), 7.76-7.64 (m, 2H), 7.54-7.38 (m, 7H), 7.20 (t, J = 5.0 Hz, 1H) 5.94 (s, 1H). - 13 C NMR (100 MHz, DMSO-d 6 ): 191.00, 156.41, 139.39, 139.19, 136.86, 135.58, 132.87, 132.55, 131.13, 130.56, 129.50, 129.34 (q, J CF = 30 Hz), 127.32, 125.43 (q, J CF = 4 Hz), 124.85, 124.03, 122.96, 121.56, 120.65, 99.80, 66.46. - HRMS (ESI) calcd. for C 23 H 17 N0 4 S [M+H] + 442.0724, found 442.0717.
2,3-Dichloro-5,ll-dihydro-ll-(3-hydroxyphenyl)-12H-benzo[ b]indeno[l,2- e] [l,4]thiazepin-12-one
2,3 -Dichloro-5, 11 -dihydro- 11 -(3 -hydroxyphenyl)- 12H-benzo [b] indeno [ 1 ,2-e] [ 1 ,4]thiazepin- 12-one was obtained according to GP4 from 5,6-dichloro-2-(3-hydroxybenzylidene)indan- 1,3-dione (300 mg, 0.96 mmol) and 2-aminothiophenol (143 xL, 123 mg, 1.2 mmol) in EtOH (10 mL) at r. t. Column chromatography (hexane : EtOAc 10 : 1 to 1 : 1) yielded 130 mg (31%) of the desired product as a red solid. - 1H NMR (500 MHz, DMSO-d 6 ): 10.18 (s, 1 H), 9.16 (s, 1 H), 8.41 (s, 1 H), 7.52 (s, 2 H), 7.33 (t, J= 7.5 Hz, 1 H), 7.02 (dd, J= 7.7, 1.3 Hz, 1 H), 6.94 (t, J = 7.3 Hz, 1 H), 6.85 (t, J= 7.7 Hz, 1 H), 6.49 - 6.37 (m, 3 H), 5.40 (s, 1 H). - 13 C NMR (125 MHz, DMSO-d 6 ): 186.62, 157.28, 155.93, 143.81, 143.63, 139.91, 136.43, 134.10, 133.91, 132.77, 129.51, 129.08, 125.30, 124.90, 123.97, 122.51, 121.92, 118.52, 114.88, 113.93, 111.38, 46.12. -HRMS (ESI) calcd. for C 22 H 14 C1 2 N0 2 S [M+H] + 426.0122, found 426.0117.
Mixture of 2-Ethynyl-5,ll-dihydro-ll-(3-hydroxyphenyl)-12H-benzo[b]inde no[l,2- e][l,4]thiazepin-12-one and 3-EthynyI-5,ll-dihydro-ll-(3-hydroxyphenyI)-12/f- -e] [1 ,4] thiazepin-12-one
An inseparable mixture of 2-ethynyl-5,l l-dib.ydro-l l-(3-hydroxyphenyl)-12H- benzo[b]indeno[l,2-e][l,4]thiazepin-12-one and 3-ethynyl-5,l 1-dihydro-l l-(3- hydroxyphenyl)-12H-benzo[b]indeno[l ,2-e][l,4]thiazepin-12-one was obtained according to GP4 from the mixture of (E)- and (Z)-5-ethynyl-2-(3-hydroxybenzylidene)-indan-l,3-dione (320 mg, 1.2 mmol) and 2-aminothiophenol (150 μΐ ^ , 175 mg, 1.4 mmol) in EtOH (10 mL) at r. t. Column chromatography (hexane : EtOAc 10 : 1 to 1 : 1) yielded 79 mg (18%) of the desired product as a red solid.- 1H NMR (500 MHz, CD 3 OD) S.04 (s, 1 H), 7.53 (dd, J= 7.7, 1.0 Hz, 1 H), 7.46 (dd, J = 8.0, 1.0 Hz, 1 H), 7.40 (d, J = 7.4 Hz, 1 H), 7.28 (dt, J = 7.7, 1.5 Hz, 1 H), 7.06 (dd, J= 7.7, 1.4 Hz, 1 H), 6.93 (td, J= 7.4, 1.1 Hz, 1 H), 6.87 (t, J= 7.7 Hz, 1 H), 6.55 - 6.50 (m, 2 H), 6.43 (dd, J = 7.9, 2.2 Hz, 1 H), 5.40 (s, 1 H), 3.78 (s, 1 H). - 13 C NMR (125 MHz, CD 3 OD): 189.71, 157.23, 156.60, 143.27, 143.12, 139.59, 136.25, 133.73, 133.31, 128.65, 128.31, 125.76, 125.17, 124.72, 122.69, 121.30, 120.29, 118.57, 114.19, 113.03, 110.94, 82.65, 79.79, 45.88. -HRMS (ESI) calcd. for C 24 H 16 N0 2 S [M+H] + 382.0902, found 382.0895.
5,ll-Dihydro-ll-(3-(/eri-butyloxycarbonylamino)phenyI)-12 H-benzo[b]indeno[l,2- e][l,4]thiazepin~12-one
5,11 -Dihydro- 11 - (3 -(tert-butyloxycarbonylamino)phenyl)- 12H-benzo [b] indeno [1,2- e][l,4]thiazepin-12-one was obtained according to GP4 from 2-(3-(tert- butyloxycarbonyl)benzylidene)indan-l,3-dione (800 mg, 2.3 mmol) and 2-aminothiophenol (534 μί, 625 mg, 5.0 mmol) in EtOH (40 mL) at r. t.. Column chromatography (hexane : EtOAc 20 : 1 to 1 : 1) yielded 470 mg (45%) of the desired product as a red solid. - 1H NMR (500 MHz, DMSO-d 6 ): 10.00 (s, 1 H), 9.11 (br s, 1 H), 8.12 (d, J= 7.4 Hz, 1 H), 7.58 - 7.51 (m, 2 H), 7.45 - 7.28 (m, 3 H), 7.22 - 7.15 (m, 2 H), 7.10 (dd, J = 7.7, 1.6 Hz, 1 H), 6.95 - 6.89 (m, 2 H), 6.59 (d, J= 7.7 Hz, 1 H), 5.40 (s, 1 H), 1.42 (s, 9 H).-HRMS (ESI) calcd. for C2 7 H 25 N 2 0 3 S [M+H] + 457.1586, found 457.1582.
5,11-Dihydro-l 1 -(3-aminophenyl)-12H-benzo [b] indeno [1 ,2-e] [1 ,4] thiazepin-12-one
5,11 -Dihydro- 11 -(3 -(tert-butyloxycarbonylamino)phenyl)- 12H-benzo [bjindeno [1,2- e][l,4]thiazepin-12-on (200 mg, 0.44 mmol) was dissolved in CH 2 C1 2 (4 mL) and cooled down to 0 °C. Trifluoroacetic acid (1 mL) was added and the reaction mixture was stirred for 3 h at 0 °C. The volatiles were removed under reduced pressure and the residue was purified by column chromatography (hexane : EtOAc = 4 : 1 to 1 : 3) to yield the desired product as a red solid. Yield: 76 mg (49%). - 1H NMR (500 MHz, CD 3 OD): 7.91 (d, J = 7.3 Hz, 1 H), 7.52 - 7.46 (m, 2 H), 7.43 - 7.37 (m, 2 H), 7.28 (td, J= 7.3, 1.6 Hz, 1 H), 7.11 (t, J = 7.7 Hz, 1 H), 7.04 (dd, J= 7.7, 1.5 Hz, 1 H), 7.00 - 6.97 (m, 2 H), 6.93 - 6.84 (m, 2 H), 5.49 (s, 1 H). - 13 C NMR (125 MHz, CD 3 OD): 191.00, 158.21, 144.44, 143.30, 139.20, 136.16, 135.04, 133.28, 131.60, 129.98, 128.91 (2C), 124.92, 124.77, 124.76, 122.91, 120.46, 119.74, 118.63, 118.26, 108.73, 45.67. -HRMS (ESI) calcd. for C 22 H 17 N 2 OS [M+H] + 357.1062, found 357.1059.
S j ll-Dihydro-ll-iS-fefi-butyldimethylsilyloxy-S-^ex-S-yn yloxyJ-pheny^-llH- benzo [b] indeno [1,2-e] [1 ,4] thiazepin-12-one
5,11 -Dihy dro- 11 - (3 -ter^butyldimethylsilyloxy- 5 -(hex- 5 -ynyloxy) -phenyl)- 12H- benzo[b]indeno[l,2-e][l,4]thiazepin-12-one was obtained according to GP4 from 2-(3-(tert- butyldimethylsilyloxy)-5-(hex-5-ynyloxy)-benzylidene)indan-l ,3-dione (200 mg, 0.4 mmol) and 2-aminothiophenol (230 μΐ ^ , 269 mg, 2.2 mmol) in EtOH (20 niL) at r. t. Column chromatography (hexane : EtOAc 10 : 1 to 1 : 1) yielded 110 mg (49%) of the desired product as a red solid. - 1H NMR (400 MHz, CD 3 OD ): 7.89 (d, J = 7.3 Hz, 1 H), 7.53 - 7.37 (m, 3 H), 7.33 - 7.28 (m, 1 H), 7.13 - 7.09 (m, 2 H), 6.95 (td, J = 7.5, 1.3 Hz, 1 H), 6.31 (s, 1 H), 6.14 (s, 1 H), 6.05 (t, J = 2.2 Hz, 1 H), 5.38 (s, 1 H), 3.86 - 3.69 (m, 2 H), 2.26 - 2.15 (m, 3 H), 1.82 - 1.72 (m, 2 H), 1.67 - 1.55 (m, 2 H), 0.89 (s, 9 H), 0.03 (s, 6 H). -HRMS (ESI) calcd. for C 34 H 38 N0 3 SSi [M+H] + 568.2342, found 568.2338.
5,ll-Dihydro-ll-(3-^/"i-butyIdimethylsilyloxy-5-methoxyph enyl)-12H- benzo [b] indeno [1 ,2-e] [1 ,4] thiazepin-12-one
5,11 -Dihy dro- 11 -(3 -/er^-butyldimethylsilyloxy-5-methoxyphenyl)- 12H-benzo [bjindeno [ 1 ,2- e][l,4]thiazepin-12-one was obtained according to GP4 from 2-(3-(tert- butyldimethylsilyloxy)-5-methoxybenzylidene)indan-l,3-dione (540 mg, 1.4 mmol) and 2- aminothiophenol (160 μΕ, 190 mg, 1.5 mmol) in EtOH (20 mL) at r. t. Column chromatography (hexane : EtOAc 10 : 1 to 3 : 1) yielded 370 mg (54%) of the desired product as a red solid. - 1H NMR (400 MHz, DMSO-d 6 ): 10.02 (s, 1 H), 8.11 (d, J = 7.4 Hz, 1 H), 7.59 - 7.51 (m, 2 H), 7.45 - 7.30 (m, 3 H), 7.04 (dd, J = 7.7, 1.6 Hz, 1 H), 6.95 (td, J = 7.7, 1.2 Hz, 1 H), 6.30 (s, 1 H), 6.06 - 6.02 (m, 2 H), 5.40 (s, 1 H), 3.55 (s, 3 H), 0.82 (s, 9 H), - 0.02 (s, 6 H). - 13 C NMR (100 MHz, DMSO-d 6 ): 189.72, 160.22, 156.74, 155.94, 144.99, 143.75, 139.72, 136.45, 133.10, 132.14, 130.49, 129.47, 125.14, 124.61, 123.76, 120.90, 119.56, 111.82, 109.57, 107.15, 104.48, 55.41, 45.98, 25.98, 18.31, -4.18. -HRMS (ESI) calcd. for C 2 9H3 2 N0 3 SSi [M+H] + 502.1872, found 502.1870.
5,ll-Dihydro-ll-(3-bromo-5-fer/-butyIdimethylsilyloxyphen yl)-12H-benzo[b]indeno[l,2- e] [l,4]thiazepin-12-one
5,11 -Di ydro- 11 -(3 -bromo-5-tert-butyldimethylsilyloxyphenyl)- 12H-benzo [b]indeno [ 1 ,2- e][l,4]thiazepin-12-one was obtained according to GP4 from 2-(3-bromo-5-tert- butyldimethylsilyloxybenzylidene)indan-l,3-dione (528 mg, 1.2 mmol) and 2- aminothiophenol (150 μί, 175 mg, 1.4 mmol) in EtOH (10 mL) at r. t.. Column chromatography (hexane : EtOAc 10 : 1 to 3 : 1) yielded 357 mg (54%) of the desired product as a red solid. - 1H NMR (500 MHz, DMSO-d 6 ): 10.11 (s, 1 H), 8.12 (d, J = 7.4 Hz, 1 H), 7.62 - 7.54 (m, 1 H), 7.43 (t, J = 7.4 Hz, 1 H), 7.41 - 7.34 (m, 1 H), 7.12 - 6.95 (m, 2 H), 6.93 (s, 1 H), 6.73 (dd, J = 8.1, 0.9 Hz, 1 H), 6.70 (t, J = 1.8 Hz, 1 H), 6.43 (td, J = 7.8, 1.2 Hz, 1 H), 6.39 (s, 1 H), 5.51 (s, 1 H), 0.82 (s, 9 H), -0.02 (s, 6 H). - 13 C NMR (125 MHz, 157.05, 155.90, 150.21, 146.50, 143.75, 139.58, 136.43, 135.89, 132.98, 132.25, 131.60, 130.65, 129.77, 125.38, 124.25, 124.17, 123.93, 121.64, 121.24, 121.02, 119.73, 118.20, 116.90, 116.50, 115.25, 108.61, 45.34, 31.44, 25.88, 18.30, -4.26, -4.30. - HRMS (ESI) calcd. for C 2 gH 29 BrN0 2 SSi [M+H] + 550.0872/552.0851, found 550.0872/552.0851.
Mixture of 2-Ethynyl-5,ll-dihydro-ll-(3-azido-5-( eri-butyldimethylsiIyIoxy)phenyl)- 12H-benzo[b]indeno[l,2-e][l,4]thiazepin-12-one and 3-Ethynyl-5,ll-dihydro-ll-(3- azido-5- (/e */-butyldimethylsilyloxy)phenyl)-12H-benzo[b]indeno[l,2-e] [l,4]thiazepin-
12-one
An unseparable mixture of 2-ethynyl-5,l l-dihydro-l l-(3-azido-5-(fert- butyldimethylsilyloxy)phenyl)- 12H-benzo [bjindeno [ 1 ,2-e] [ 1 ,4]thiazepin- 12-one and 3 - ethynyl-5 , 11 -dihydro- 1 1 -(3 -azido-5 - (fert-butyldimethylsilyloxy)phenyl)- 12H- benzo[b]indeno[l,2-e][l,4]thiazepin-12-one was obtained according to GP4 from (E)- and (Z)-2-(3-azido-5-tert-butyldimethylsilyloxybenzylidene)-5-et hylideneindan-l,3-dione
(460 mg, 1.1 mmol) and 2-aminothiophenol (182 μί, 212 mg, 1.7 mmol) in EtOH (20 mL) at r. t. Column chromatography (hexane : EtOAc 20 : 1 to 4 : 1) yielded 380 mg (64%) of the desired product as a red solid. - 1H NMR (500 MHz, DMSO-d 6 ): 10.36 - 9.97 (br. s, 1 H), 8.17 (s, 1 H), 7.59 - 7.46 (m, 2 H), 7.38 - 7.26 (m, 2 H), 7.10 - 6.88 (m, 2 H), 6.27 (s, 1 H), 6.20 (s, 1 H), 6.12 (s, 1 H), 5.40 (s, 1 H), 4.42 (s, 1 H), 0.84 (s, 9 H), -0.04 (s, 6 H). . IR (film) v = 3293- cm "1 , 2927, 2857, 2108, 1589, 1558, 1456, 1251, 837.-HRMS (ESI) calcd. for C 3 oH 2 9N 4 0 2 SSi [M+H] + 537.1781, found 537.1775.
S j ll-Dihydro-ll-iS-^e 'i-butyldimethylsilyloxymethy^pheny^-llH-benzoIblindenoJl,!- e][l,4]thiazepin-12-one
5,11 -Dihydro- 11 -(3 -(ter^-butyldimethylsilyloxymethy^phenyl)- 12H-benzo [b] indeno [ 1 ,2- e][l,4]thiazepin-l 2-one was obtained according to GP4 from 2-(3 -(tert- butyldimethylsilyloxymethyl)benzylidene)indan-l,3-dione (320 mg, 0.9 mmol) and 2- aminothiophenol (110 μί, 125 mg, 1.0 mmol) in EtOH (10 mL) at r. t. Column chromatography (hexane : EtOAc 10 : 1 to 4 : 1) yielded 130 mg (30%) of the desired product as a red solid. - 1H NMR (500 MHz, DMSO-d 6 ): 10.04 (s, 1 H), 8.11 (s, 1 H), 7.63 - 6.35 (m, 11 H), 5.48 (s, 1 H), 4.46 (s, 2 H), 0.77 (s, 9 H), -0.07 (s, 6 H). - 13 C NMR (125 MHz, DMSO-d 6 ): 189.68, 157.10, 150.23, 144.04, 142.59, 140.99, 139.94, 136.42, 135.90, 133.31, 131.99, 131.61, 130.38, 129.33, 128.03, 126.58, 123.92, 120.79, 119.53, 116.90, 115.24, 64.19, 46.19, 26.13, 18.27, -4.97. -HRMS (ESI) calcd. for C 29 H 32 N0 2 SSi [M+H] + 486.1923, found 486.1918.
Mixture of 2-carboxy-5,ll-dihydro-ll-(3-methoxyphenyl)-12H-benzo[b]inde no[l,2- e] [l,4]thiazepin-12-one and 3-carboxy-5,ll-dihydro-ll-(3-methoxyphenyl)-12H- -e] [1 ,4] thiazepin-12-one
A mixture of (£)- and (Z)-5-carboxy-2-(3-methoxybenzylidene)-indan-l,3-diones (450 mg, 1.46 mmol) was suspended in EtOH (30 mL) and DMF (20 mL). 2-Aminothiophenol (312 μΐ,, 365 mg, 2.92 mmol) was added and the reaction mixture was stirred for 6 h at r. t. A new portion of 2-aminothiophenol (156 μΕ, 182mg, 1.46 mmol) was added and the reaction mixture was stirred for 18 h at r. t. EtOH was evaporated under reduced pressure and H 2 0 was added to the residue. The obtained mixture was extracted with EtOAc (3 times), the combined organic phases were dried over Na 2 S0 4 , concentrated under reduced pressure and purified by column chromatography (CH 2 C1 2 : MeOH = 1 : 0 to 9 : 1) yielding 600 mg (99%) of an inseparable mixture 2 : 1 of 2-carboxy-5,l l-dihydro-l l-(3-methoxyphenyl)-12H- benzo[b] indeno [l,2-e][l,4]thiazepin-12-one and 3-carboxy-5,l l-dihydro-l l-(3- methoxyphenyl)-12H-benzo[b]indeno[l,2-e][l,4]thiazepin-12-on e. - 1H NMR (400 MHz, DMSO-d 6 ): 13.38 - 13.16 (br s, 1 H), 10.35 (s, 0.66 H), 10.20 (s, 0.33 H), 8.79 (s, 0.66 H), 8.26 (d, J= 7.8 Hz, 0.33 H), 8.19 (dd, J= 7.8, 1.5 Hz, 0.33 H), 8.04 (dd, J= 7.4, 1.2 Hz, 0.66 H), 7.80 - 7.78 (m, 0.33 H), 7.66 - 7.57 (m, 1 H), 7.47 (d, J = 7.4 Hz, 0.66 H), 7.39 - 7.32 (m, 1 H), 7.03 - 6.92 (m, 3 H), 6.62 - 6.52 (m, 3 H), 5.50 (s, 0.33 H), 5.49 (s, 0.66 H), 3.55 (s, 1 H), 3.54 (s, 2 H). - HRMS (ESI) calcd. for C 24 H 18 N0 4 S [M+H] + 416.0957, found 416.0949.
Mixture of 2-(Propargylaminocarbonyl)-5,ll-dihydro-ll-(3-methoxyphenyl) -12H r - benzo[b]indeno[l,2-e] [l,4]thiazepin-12-one and 3-propargylaminocarbonyl-5,ll- dihydro-ll-(3-methoxyphenyl)-12H-benzo[b]indeno[l,2-e][l,4]t hiazepin-12-one
A mixture of 2-carboxy-5,l l-dihydro-l l-(3-methoxyphenyl)-12H-benzo[b]indeno[l,2- e][l,4]thiazepin-12-one and 3-carboxy-5,l 1 -dihydro- 11 -(3 -methoxyphenyl)-12H- benzo[b]indeno[l,2-e][l,4]thiazepin-12-one (606 mg, 1.46 mmol) was dissolved in 0¾0 2 (22 mL). Diisopropylethylamine (330 μϋ, 245 mg, 1.9 mmol), propargylamine (122 ΐ ^ , 105 mg, 1.9 mmol), HOBt*l¾0 (257 mg, 1.9 mmol) were added to this mixture. Finally O-(6- chlorberizotriazol-l-y^-NNN'^'-tetramethyluronium-hexafluoro phosphat (HCTU) (787 mg, 1.9 mmol) was added and the reaction mixture was stirred for 2 h at r. t. Volatiles were removed under reduced pressure and the residue was separated by column chromatography (hexane : EtOAc - 10 : 1 to 1 : 1) yielding 508 mg (77%) of the inseparable ca. 2 : 1 mixture of 2-(propargylaminocarbonyl)-5 , 11 -dihydro- 11 -(3 -methoxyphenyl)- 12H- benzo[b]indeno[l,2-e][l,4]thiazepin-12-one and 3 -propargylaminocarbonyl-5, 11 -dihydro- 11- (3-methoxyphenyl)-12H-benzo[b]indeno[l,2-e][l,4]thiazepin-12 -one. - 1H NMR (500 MHz, DMSO-di): 9.15 - 9.07 (m, 1 H), 8.65 (s, 0.67 H), 7.95 (s, 0.33 H), 7.89 - 7.85 (m, 1 H), 7.64 (d, J= 7.4 Hz, 0.67 H), 7.59 (d, J= 7.4 Hz, 0.33 H), 7.45 (d, J= 7.4 Hz, 0.67 H), 7.38 - 7.32 (m, 1 H), 7.03 - 6.92 (m, 3.33 H), 6.62 - 6.52 (m, 3 H), 5.50 (s, 0.67 H), 5.48 (s, 0.33 H), 4.12 (dd, J= 5.5, 2.5 Hz, 1.34 H), 4.08 (dd, J = 5.5, 2.5 Hz, 0.66 H), 3.54 (s, 1 H), 3.53 (s, 2 H), 3.18 (t, J = 2.5 Hz, 0.67 H), 3.15 (t, J = 2.5 Hz, 0.33 H). -HRMS (ESI) calcd. for C 27 H 2 oN 2 0 3 S [M+H] + 453.1273, found 453.1264.
2-(Propargylaminocarbonyl)-5,ll-dihydro-ll-(3-hydroxyphen yI)-12H- benzo[b]indeno[l,2-e][l,4]thiazepin-12-one and 3-propargylaminocarbonyl-5,ll- dihydro-ll-(3-hydroxyphenyl)-12H r -benzo[b]indeno[l,2-e][l,4]thiazepin-12-one
To a solution of a mixture of 2-(propargylaminocarbonyl)-5,l l-dihydro-l l-(3- methoxyphenyl) - 12H-benzo [b] indeno [ 1 ,2-e] [ 1 ,4]thiazepin- 12-one and 3- propargylaminocarbonyl-5, 11 -dihydro- 11 -(3 -methoxyphenyl)- 12H-benzo [bjindeno [ 1 ,2- e][l,4]thiazepin-12-one (212 mg, 0.47 mmol) in CH 2 C1 2 ( 5 mL) was added BBr 3 (2.35 mL, 2.35 mmol, 1 M in CH 2 C1 2 ) dropwise at r. t. The reaction was stirred for 3 h, quenched with H 2 0. The obtained mixture was extracted with EtOAc (3 times). Combined organic phases were dried over Na 2 S0 4 and concentrated under reduced pressure. The residue was separated by column chromatography (hexane: EtOAc = 2 : 1 to 1 : 1) yielding 2- (propargylaminocarbonyl)-5 , 11 -dihydro- 11 -(3 -hydroxyphenyl)- 12H-benzo [bjindeno [ 1 ,2- e][l,4]thiazepin-12-one and 3-propargylaminocarbonyl-5,l 1 -dihydro- 1 1 -(3 -hydroxyphenyl)- 12H-benzo [b] indeno [ 1 ,2-e] [ 1 ,4] thiazepin- 12-one .
2-(PropargylaminocarbonyI)-5,ll-dihydro-ll-(3-hydroxyphen yl)-12H- benzo [b] indeno [1,2-e] [1 ,4] thiazepin-12-one
Yield: 45.3 mg (22%). i¾=0.25 (hexane: EtOAc = 1 : 1). - 1H NMR (500 MHz, CD 3 OD): 8.34 (s, 1 H), 7.81 (d, J= 7.3 Hz, 1 H), 7.50 - 7.40 (m, 2 H), 7.29 - 7.22 (m, 1 H), 7.05 (d, J = 7.5 Hz, 1 H), 6.91 (t, J= 7.2 Hz, 1 H), 6.85 (t, J= 7.9 Hz, 1 H), 6.55 - 6.50 (m, 2 H), 6.43 (d, J = 7.9 Hz, 1 H), 5.41 (s, 1 H), 4.22 (s, 2 H), 3.34 - 3.30 (m, 1 H). - 13 C NMR (125 MHz, CD 3 OD): 189.01, 167.80, 157.89, 157.00, 143.42, 143.26, 139.76, 137.19, 136.67, 136.18, 129.08, 128.60, 128.29, 125.36, 124.72, 123.16, 119.94, 118.41, 117.41, 114.43, 113.21, 111.17, 78.78, 70.76, 46.00, 28.74. HRMS (ESI) calcd. for CaeH^OaS [M+H] + 439.1116, found 439.1108.
3-(Propargylaminocarbonyl)-5,l l-dihydro-ll-(3-hydroxyphenyl)-12H r - benzo [b] indeno [1 ,2-e] [1 ,4] thiazepin-12-one
Yield: 22.5 mg (11%). i^0.18 (hexane: EtOAc = 1 : 1). - 1H NMR (500 MHz, CD 3 OD): 8.02-7.94 (m, 2 H), 7.80 (s, 1 H), 7.49 - 7.40 (m, 1 H), 7.26 (t, J= 7.2 Hz, 1 H), 7.05 (d, J= 7.2 Hz, 1 H), 6.91 (t, J = 7.5 Hz, 1 H), 6.85 (t, J= 7.5 Hz, 1 H), 6.55 - 6.49 (m, 2 H), 6.42 (d, J= 7.5 Hz, 1 H), 5.43 (s, 1 H), 4.18 (s, 2 H), 3.37 (s, 1 H). - 13 C NMR (125 MHz, CD 3 OD): 188.85, 167.25, 157.61, 157.38, 143.68, 143.23, 142.79, 136.20, 135.34, 134.27, 131.12, 128.59, 128.25, 125.37, 124.62, 123.12, 118.54, 118.20, 118.04, 114.60, 113.34, 111.54, 78.82, 70.57, 46.08, 28.64. HRMS (ESI) calcd. for C 26 H 19 N 2 0 3 S [M+H] + 439.1116, found 439.1108.
Mixture of 2-(2-(3-(But-3-inyl)-3H-diazirin-3-yl)ethylaminocarbonyl)-5, ll-dihydro-ll- (3-methoxyphenyl)-12H-benzo[b]indeno[l,2-e] [l,4]thiazepin-12-one and 3-(2-(3-(But-3- inyl)-3H-diazirin-3-yl)ethylaminocarbonyl)-5 1-dihydro-ll-(3-methoxyphenyl)-12H- -e][l,4]thiazepin-12-one (374 + 375)
A mixture of 2-carboxy-5,l l-dihydro-l l-(3-metrioxyplienyl)-12H-benzo[b]indeno[l,2- e][l,4]thiazepin-12-one and 3-carboxy-5,l 1-dihydro-l l-(3-methoxyphenyl)-12H- benzo[b]indeno[l,2-e][l,4]thiazepin-12-one (90 mg, 0.22 mmol) was dissolved in CH 2 C1 2 (5 mL). Diisopropylethylamine (87 \iL, 64 mg, 0.5 mmol), 2-(3-(but-3-inyl)-3H-diazirin-3- yl)ethylamine (41 mg, 0.3 mmol), HOBt*H 2 0 (41 mg, 0.3 mmol) were added to this mixture. Finally 0-(6-chlorbenzotriazol- 1 -yl)-N,N,N',N'-tetramethyluronium-hexafluorophosphat (HCTU) (124 mg, 0.3 mmol) was added and the reaction mixture was stirred for 1 h at r. t. Volatiles were removed under reduced pressure and the residue was separated by column chromatography (hexane : EtOAc = 10 : 1 to 1 : 1) yielding 27 mg (23%) of 2-(2-(3-(but-3- inyl)-3H-diazirin-3 -yl)ethylaminocarbonyl)-5 , 11 -dihydro- 11 -(3 -methoxyphenyl)- 12H- benzo[b]indeno[l,2-e][l,4]thiazepin-12-one and 19 mg (16%) of 3-(2-(3-(but-3-inyl)-3H- diazirin-3 -yl)ethylaminocarbonyl)-5, 11 -dihydro- 1 1 -(3 -methoxyphenyl)- 12H- benzo [b] indeno [ 1 ,2-e] [ 1 ,4]thiazepin- 12-one.
2-(2-(3~(But-3-inyl)-3H-diazirm-3-yl)ethyI aminocarbonyl)-5,ll-dihydro-ll-(3- hydro he -lZ f-benzoIbJindenoIljZ-eJtl^jthi ze in-l -one (374)
Yield: 27 mg (23%).- 1H NMR (500 MHz, DMSO-d 6 ): 10.32 (s, 1 H), 8.66 - 8.62 (m, 2 H), 7.86 (dd, J= 7.5, 1.2 Hz, 1 H), 7.64 (dd, J= 8.2, 1.0 Hz, 1 H), 7.47 (d, J= 7.4 Hz, 1 H), 7.37 - 7.32 (m, 1 H), 7.03 - 6.92 (m, 3 H), 6.62 - 6.57 (m, 2 H), 6.55 - 6.53 (m, 1 H), 5.49 (s, 1 H), 3.54 (s, 3 H), 3.23 (q, J= 7.0 Hz, 2 H), 2.87 (t, J= 2.6 Hz, 1 H), 2.04 (td, J= 7.3, 2.6 Hz, 2 H), 1.71 (t, J = 7.0 Hz, 2 H), 1.66 (t, J = 7.3 Hz, 2 H). - 13 C NMR (125 MHz, DMSO-d 6 ) 188.77, 166.55, 159.19, 156.70, 144.14, 143.76, 139.92, 138.45, 136.53, 135.49, 133.05, 129.52, 129.21, 125.38, 124.76, 123.95, 120.49, 120.11, 119.46, 113.20, 112.59, 110.74, 83.65, 72.35, 55.27, 46.08, 38.72, 35.07, 32.30, 31.74, 13.21. HRMS (ESI) calcd. for C 3 iH 27 N 4 0 3 S [M+H] + 535.1804, found 535.1794.
3-(2-(3-(But-3-inyl)-3H-diazirin-3-yl)ethylaminocarbonyl) -5,ll-dihydro-ll-(3- hydroxyphenyl)-12H-benzo[b]indeno[l,2-e][l,4]thiazepin-12-on e (375)
Yield: 19 mg (16%). - 1H NMR (500 MHz, DMSO-d 6 ): 10.18 (s, 1 H), 8.66 (t, J = 5.5 Hz, 1 H), 8.21 (d, J= 7.7 Hz, 1 H), 8.08 (dd, J= 7.7, 1.6 Hz, 1 H), 7.86 (d, J= 1.6 Hz, 1 H), 7.66 -
7.54 (m, 1 H), 7.39 - 7.34 (m, 1 H), 7.04 - 6.93 (m, 3 H), 6.63 - 6.53 (m, 3 H), 5.51 (s, 1 Ή),
3.55 (s, 3 H), 3.19 (q, J= 7.0 Hz, 2 H ), 2.85 (t, J= 2.6 Hz, 1 H), 2.02 (td, J= 7.4, 2.7 Hz, 2 H), 1.69 (t, J = 7.1 Hz, 2 H), 1.64 (t, J = 7.4 Hz, 2 H). - 13 C NMR (125 MHz, DMSO-d 6 ) 188.89, 165.47, 159.21, 156.34, 144.14, 142.38, 136.60, 136.18, 132.00, 131.92, 131.90, 129.56, 129.29, 129.20, 125.36, 124.76, 123.89, 120.08, 119.61, 113.27, 112.57, 111.08, 83.63, 72.31, 55.28, 46.16, 38.72, 32.25, 31.74, 27.81, 13.19. HRMS (ESI) calcd. for C 31 H 27 N 4 0 3 S [M+H] + 535.1804, found 535.1793.
Mixture of 2-carboxy-5,ll-dihydro-ll-(3-hydroxyphenyl)-12H-benzo[b]inde no[l,2- e] [l,4]thiazepin-12-one and 3-carboxy-5,ll-dihydro-ll-(3-hydroxyphenyl)-12H- ,2-e] [1 ,4]thiazepin-12-one
To a solution of a mixture of 2-carboxy-5,l 1-dihydro-l 1 -(3 -methoxyphenyl)-12H- benzo[b]indeno[l,2-e][l,4]thiazepin-12-one and 3-carboxy-5,l 1-dihydro-l l-(3- methoxyphenyl)-12H-beriZo[b]indeno[l,2-e][l,4]thiazepin-12-o ne (64.0 mg, 0.15 mmol) in CH 2 C1 2 (4 mL) was added BBr 3 (0.77 mL, 0.77 mmol, 1 M in C¾C1 2 ) dropwise at r. t. The reaction was stirred for 3 h, quenched with H 2 0. The obtained mixture was extracted with EtOAc (3 times). Combined organic phases were dried over Na 2 S0 4 and concentrated under reduced pressure. The residue was separated by column chromatography (hexane: EtOAc — 2 : 1 to 1 : 1) yielding a mixture of 2-carboxy-5,l 1-dihydro-l l-(3-hydroxyphenyl)-12H- benzo[b]indeno[l,2-e][l,4]thiazepin-12-one and 3 -carboxy-5,11-dihydro-l 1 -(3- hydroxyphenyl)- 12H-benzo [bjindeno [ 1 ,2-e] [ 1 ,4]thiazepin- 12-one.
Mixture of 2-(2-(3-(but-3-inyI)-3H-diazirin-3-yl)ethylaminocarbonyI)-5, ll-dihydro-ll- (3-hydroxyphenyl)-12H-benzo[b]indeno[l,2-e] [l,4]thiazepin-12-one and 3-(2-(3-(but-3- inyl)-3H r -diazirin-3-yl)ethyIaminocarbonyI)-5,ll-dihydro-ll-(3- hydroxyphenyl)-12H- benzo[b]indeno[l,2-e][l,4]thiazepin-12-one (376-1 and 376-2)
A mixture of 2-carboxy-5, 11-dihydro-l 1 -(3 -hydroxyphenyl)-12H-benzo[b]indeno[l, 2- e][l,4]thiazepin-12-one and 3-carboxy-5,l 1-dihydro-l l-(3-hydroxyphenyl)-12H- benzo[b]indeno[l,2-e][l,4]thiazepin-l 2-one obtained from 2-carboxy-5,l 1-dihydro-l 1 -(3- methoxyphenyl)-12H-benzo[b]indeno[l,2-e][l,4]thiazepin-12-on e and 3-carboxy-5,l 1- dihydro- 11 -(3 -methoxyphenyl)- 12H-benzo [bjindeno [ 1 ,2-e] [ 1 ,4]thiazepin- 12-one (64.0 mg, 0.15 mmol) was dissolved in 0¾0 2 (4 mL) and DMF (0.2 mL). Diisopropylethylamine (87 μΐ ^ , 64 mg, 0.5 mmol), the solution of 2-(3-(but-3-inyl)-3H-diazirin-3-yl)ethylamine (14 mg, 0.1 mmol) in CH 2 Ci2 (4 mL) were added to this mixture. Finally O-(6-chlorbenzotriazol- l-y^-N.NjN'jiV'-tetramethyluronium-hexafluorophosphat (HCTU) (42 mg, 0.1 mmol) was added and the reaction mixture was stirred for 48 h at r. t. Volatiles were removed under reduced pressure and the residue was separated by column chromatography (hexane : EtOAc = 10 : 1 to 1 : 1) yielding 5.5 mg (7% over two steps) of a mixture of 2-(2-(3-(but-3-inyl)-3H- diazirin-3-yl)ethylaminocarbonyl)-5,l l-dihydro-l l-(3-hydroxyphenyl)-12H- benzo[b]indeno[l,2-e][l,4]thiazepin-12-one and 3-(2-(3-(but-3-inyl)-3H-diazirin-3- yl)ethylaminocarbonyl)-5, 11 -dihydro-11 -(3-hydroxyphenyl)-12H-benzo[b]indeno[l ,2- e][l,4]thiazepin-12-one. - 1H NMR (400 MHz, CD 3 OD): 8.42 (dd, J = 1.7, 0.7 Hz, 0.5 H), 8.34 (dd, J = 1.4, 0.6 Hz, 0.5 H), 8.06 (dd, J= 8.1, 1.7 FIz, 0.5 H), 7.89 (dd, J = 8.1, 0.7 FIz, 0.5 H), 7.84 (dd, J= 7.4, 1.4 Hz, 0.5 H), 7.64 (d, J= 8.4, 0.5 H), 7.57 (dd, J= 8.1, 1.1 Hz, 0.5 H), 7.52 - 7.48 (m, 2 H), 7.37 - 7.32 (m, 0.5 H), 7.32 - 7.27 (m, 0.5 H), 7.24 (td, J= 7.7, 1.4 Hz, 0.5 H), 7.07 (dd, J = 7.8, 1.4 Hz, 0.5 H), 6.97 - 6.92 (m, 0.5 H), 6.89 - 6.84 (m, 0.5 H), 6.84 - 6.80 (m, 0.5 H), 6.56 - 6.51 (m, 1 H), 6.49 - 6.42 (m, 1 H), 5.42 (s, 1 H), 3.40 - 3.31 (m, 2 H), 2.31 (t, J = 2.6 Hz, 0.5 H), 2.30 (t, J= 2.7 Hz, 0.5 H), 2.12 - 2.04 (m, 2 H), 1.85 - 1.67 (m, 4 H). HRMS (ESI) calcd. for C 30 H 25 N4O3S [M+H] + 521.1647, found 521.1637. -Deprotection
To a solution of substituted 5,1 1 -dihydro-11 -(3 -feri-butyldimethylsilyloxyphenyl)- 12H- benzo[b]indeno[l,2-e][l,4]thiazepin-l 2-one (0.7 mmol) in THF (10 mL) at r. t. was added H 2 0 (4 drops) followed by Bu 4 NF*3H 2 0 (283 mg, 0.9 mmol) and the reaction mixture was stirred for 2 h at r. t. The volatiles were removed under reduced pressure and the residue was purified by column chromatography (hexane : EtOAc = 10 : 1 to 1 : 1) yielding the desired product. 5,ll-Dihydro-ll-(3-bromo-5-hydroxyphenyI)-12H-benzo[b]indeno [l,2-e][l,4]thiazepin- -one
5,11 -Dihydro- 11 -(3 -bromo-5-hydroxyphenyl)- 12H-benzo [b]indeno[ 1 ,2-e] [ 1 ,4]thiazepin- 12- one was obtained according to GP5 from 5,1 1 -dihydro- 1 l-(3-bromo-5-te? - butyldimethylsilyloxyphenyl)-12H-benzo[b]indeno[l,2-e][l,4]t hiazepin-12-one (320 mg, 0.58 mmol) and tetrabutylammonium fluoride trihydrate (283 mg, 0.9 mmol) in THF (10 mL) and H 2 0 (4 drops) at r. t. Column chromatography (hexane : EtOAc 10 : 1 to 1 : 1) yielded 160 mg (63%) of the desired product as a red solid. - 1H NMR (500 MHz, DMSO-d 6 ): 10.07 (s, 1 H), 9.64 (s, 1 H), 8.12 (d, J= 7.4 Hz, 1 H), 7.61 - 7.53 (m, 2 H), 7.46 - 7.34 (m, 3 H), 7.08 - 7.03 (m, 1 H), 7.02 - 6.97 (m, 1 H), 6.68 (s, 1 H), 6.59 (s, 1 H), 6.40 (s, 1 H), 5.43 (s, 1 H). - 13 C NMR (125 MHz, DMSO-d 6 ): 189.72, 158.29, 156.99, 146.21, 143.72, 139.64, 136.47, 133.06, 132.20, 130.62, 129.65, 125.37, 124.42, 123.90, 121.63, 121.40, 121.00, 119.71, 116.60, 114.08, 108.91, 45.52. -HRMS (ESI) calcd. for C 22 H 15 BrN0 2 S [M+H] + 436.0007/437.9986, found 436.0002/437.9981.
Mixture of 2-Ethynyl-5,ll-dihydro-l l-(3-azido-5-hydroxyphenyl)-12H- benzo [b] indeno [1 ,2-e] [1 ,4] thiazepin-12-one and 3-Ethynyl-5,l 1-dihydro-l l-(3-azido-5- hydroxyphenyl)-12H-benzo[b]indeno[l,2-e][l,4]thiazepin-12-on e
An inseparable mixture of 2-ethynyl-5,l 1-dihydro-l l-(3-azido-5-hydroxyphenyl)-12H- benzo[b]indeno[l,2-e][l,4]thiazepin-12-one and 3-ethynyl-5,l 1-dihydro-l l-(3-azido-5- hydroxyphenyl)-12H-benzo[b]indeno[l,2-e][l,4]thiazepin-12-on e was obtained according to GP5 from the mixture of 2-ethynyl-5,l l-dihydro-l l-(3-azido-5-(tert- butyldimethylsilyloxy)phenyl)- 12H-benzo [bjindeno [ 1 ,2-e] [ 1 ,4]thiazepin- 12-one and 3 - ethynyl-5,11-dihydro-l l-(3-azido-5- (tert-butyldimethylsilyloxy)phenyl)- 12H- benzo[b]indeno[l,2-e][l,4]thiazepin-12-one (380 mg, 0.71 mmol), tetrabutylammonium fluoride trihydrate (252 mg, 0.8 mmol) in THF (10 mL) and H 2 0 (4 drops). Yield: 30 mg (10%)
- 1H NMR (500 MHz, DMSO-d 6 ): 10.23 - 10.04 (br s, 1 H), 9.56 (s, 1 H), 8.29 (s, 1 H), 7.62 - 7.51 (m, 2 H), 7.42 - 7.32 (m, 2 H), 7.11 - 6.95 (m, 2 H), 6.31 - 6.09 (m, 3 H), 5.42 (s, 1 H), 4.50 (s, 1 H). - 13 C NMR (125 MHz, DMSO-d 6 ): 188.60, 158.48, 156.25, 145.68, 143.57, 140.09, 140.00, 136.50, 134.26, 133.12, 129.68, 125.47, 125.21, 124.48, 123.81, 122.53, 121.11, 112.13, 110.42, 109.23, 104.49, 83.68, 83.65, 45.69. IR (film) v= 3290 cm " 2108, 1668, 1589, 1539, 1475, 1374. - HRMS (ESI) calcd. for C 24 H 15 N 4 0 2 S [M+H] + 423.0916, found 423.0909.
5,ll-Dihydro-ll-(3-hydroxy-5-methoxyphenyl)-12H-benzo[b]i ndeno[l,2- e] [l,4]thiazepin-12-one
5,11 -Dihydro- 11 - (3 -hydroxy- 5 -methoxyphenyl)- 12H-benzo [b] indeno [ 1 ,2-e] [ 1 ,4]thiazepin- 12-one was obtained according to GP5 from 5,11-dihydro-l l-(3-tert-butyldimethylsilyloxy-5- methoxyphenyl)-12H-benzo[b]indeno[l,2-e][l,4]thiazepin-12-on e (350 mg, 0.7 mmol) and tetrabutylammonium fluoride trihydrate (283 mg, 0.9 mmol) in THF (10 mL) and H 2 0 (4 drops) at r. t. Column chromatography (hexane : EtOAc 10 : 1 to 1 : 1) yielded 175 mg (65%) of the desired product as a red solid. R f = 0.20 (hexane / EtOAc = 2 : 1). - 1H NMR (400 MHz, CD 3 OD): 7.84 (d, J= 7.3 Hz, 1 H), 7.48 - 7.32 (m, 4 H), 7.26 (td, J= 7.7, 1.5 Hz, 1 H), 7.09 (dd, J= 7.7, 1.5 Hz, 1 H), 6.93 (td, J= 7.5, 1.2 Hz, 1 H), 6.18 (s, 1 H), 6.12 (s, 1 H), 6.03 (t, J = 2.2 Hz, 1 H), 5.35 (s, 1 H), 3.53 (s, 3 H). - 13 C NMR (100 MHz, CD 3 OD): 191.08, 160.37, 157.86, 157.55, 144.34, 143.17, 139.24, 136.30, 133.36, 131.46, 129.84, 128.64, 125.22, 124.67, 122.62, 120.41, 118.00, 109.84, 107.31, 104.38, 99.42, 54.15, 46.06. - HRMS (ESI) calcd. for C 23 H 18 N0 3 S [M+H] + 388.1007, found 388.1007. 5,ll-Dihydro-ll-(3-hydroxy-5-(hex-5-ynyloxy)-phenyl)-12H-ben zo[b]indeno[l,2- e] [l,4]thiazepin-12-one
5,11 -Dihydro- 11 -(3 -hydroxy-5 -(hex-5 -ynyloxy)-phenyl)- 12H-benzo [bjindeno [ 1 ,2- e][l,4]thiazepin-12-one was obtained according to GP5 from 5,11 -dihydro- 11 -(3 -iert- butyldimethylsilyloxy-5-(hex-5-ynyloxy)-phenyl)-12H-benzo[b] indeno[l,2-e][l,4]thiazepin- 12-one (110 mg, 0.19 mmol), tetrabutylammonium fluoride trihydrate (80 mg, 0.25 mmol) and ¾0 (4 drops) in THF (3 mL) at r. t. Column chromatography (hexane : EtOAc 10 : 1 to 1 : 1) yielded 55 mg (64%) of the desired product as a red solid. - 1H NMR (500 MHz, CD 2 OD): 7.89 (d, J= 7.3 Hz, 1 H), 7.52 - 7.46 (m, 2 H), 7.44 - 7.38 (m, 2 H), 7.34 - 7.29 (m, 1 H), 7.11 (dd, J = 7.7, 1.4 Hz, 1 H), 6.97 (td, J = 7.5, 1.3 Hz, 1 H), 6.18 (s, 1 H), 6.07 (s, 1 H), 6.02 (t, J= 1.2 Hz, 1 H), 5.34 (s, 1 H), 3.79 - 3.73 (m, 1 H), 3.71 - 3.65 (m, 1 H), 2.24 - 2.17 (m, 3 H), 1.78 - 1.71 (m, 2 H), 1.63 - 1.65 (m, 2 H). - 13 C NMR (125 MHz, DMSO-d 6 ): 191.01, 159.63, 157.85, 157.55, 144.26, 143.24, 139.30, 136.33, 133.39, 131.48, 129.84, 128.65, 125.27, 124.68, 122.62, 120.39, 118.02, 109.82, 107.27, 104.84, 100.03, 83.34, 68.39, 66.83, 46.00, 27.87, 24.85, 17.35. -HRMS (ESI) calcd. for C 2 8H 24 N0 3 S [M+H] + 454.1477, found 454.1471.
5,ll-Dihydro-ll-(3-(hydroxymethyl)phenyl)-12H-benzo[b]ind eno[l,2-e][l,4]thiazepin- -one
5,11 -Dihydro- 11 -(3 -(hydroxymethyl)phenyl)- 12H-benzo [bjindeno [ 1 ,2-e] [ 1 ,4]thiazepin- 12- one was obtained according to GP5 from 5,11 -dihydro- 11 -(3- (tert- butyldimethylsilyloxymethyl)phenyl)- 12i -benzo [bjindeno [ 1 ,2-e] [ 1 ,4jthiazepin- 12-one (130 mg, 0.27 mmol), tetrabutylammonium fluoride trihydrate (95 mg, 0.3 mmol) and H 2 0 (4 drops) in THF (10 mL) at r. t. Column chromatography (hexane : EtOAc 10 : 1 to 1 : 1) yielded 30 mg (30%) of the desired product as a red solid. - 1H NMR (500 MHz, DMSO-d 6 ): 10.04 (s, 1 H), 8.12 (d, J = 7.4 Hz, 1 H), 7.61 - 7.53 (m, 2 H), 7.45 - 7.30 (m, 3 H), 7.05 - 6.88 (m, 5 H), 6.82 (d, J = 7.4 Hz, 1 H), 5.49 (s, 1 H), 5.08 (t, J = 5.7 Hz, 1 H), 4.29 (d, J = 5.7 Hz, 2 H). - I3 C NMR (125 MHz, DMSO-d 6 ): 189.77, 156.88, 143.86, 142.52, 142.49, 139.81, 136.51, 133.19, 132.12, 130.48, 129.44, 127.85, 125.91, 125.85, 125.12, 124.94, 124.70, 123.81, 120.90, 119.61, 109.72, 63.08, 46.27. -HRMS (ESI) calcd. for C 23 Hi 8 N0 2 S [M+H] + 372.1058, found 372.1053.
The features disclosed in the foregoing description, in the claims and/or in the accompanying drawings may, both separately and in any combination thereof, be material for realizing the invention in diverse forms thereof.
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