Microwave Heated Flow Synthesis of Spiro-oxindole

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Microwave heated flow synthesis of spiro-oxindol dihydroquinazolinone based IRAP inhibitors Karin Engen, Jonas Savmarker, Ulrika Rosenström, Johan Wannberg, Thomas Lundbäck, Annika Jenmalm-Jensen, and Mats Larhed Org. Process Res. Dev., Just Accepted Manuscript • DOI: 10.1021/op500237k • Publication Date (Web): 09 Oct 2014 Downloaded from http://pubs.acs.org on October 11, 2014

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Organic Process Research & Development

Microwave heated flow synthesis of spiro-oxindole dihydroquinazolinone based IRAP inhibitors. Karin Engen†, Jonas Sävmarker#, Ulrika Rosenström†, Johan Wannberg†, Thomas Lundbäck±, Annika Jenmalm-Jensen±, Mats Larhed§* †

Department of Medicinal Chemistry, Organic Pharmaceutical Chemistry, Uppsala University

#

Department of Medicinal Chemistry, Beijer Laboratory, Uppsala University

±

Chemical Biology Consortium Sweden, Science for Life Laboratory, Department of Medical

Biochemistry and Biophysics, Karolinska Institutet, Tomtebodavägen 23A, SE-171 65 Solna, Sweden

§

Department of Medicinal Chemistry, Science for Life Laboratory, BMC, Uppsala University

P.O. Box 574, SE-751 23 Uppsala, Sweden Tel.: +46 18 471 4667 Fax: +46 18 471 44 74 E-mail: [email protected] *Corresponding author Keywords: Microwave, flow synthesis, IRAP, Insulin-regulated aminopeptidase, silicon carbide, spiro-oxindol dihydroquinazolinone

ACS Paragon Plus Environment

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ABSTRACT: A fast and convenient synthetic route towards spiro-oxindole dihydroquinazolinones as novel and drug-like insulin-regulated aminopeptidase (IRAP) inhibitors is reported. The synthesis is performed using a MW heated continuous flow system employing 200 mm x 3 mm Øi MW absorbing silicon carbide (SiC) or MW transparent borosilicate tubular reactors. A three-component MW-flow reaction to build up the spiro compounds (9 examples, 40-87% yield), using the SiC reactor, as well as a Suzuki-Miyaura cross-coupling reaction (71%), employing the borosilicate reactor, are presented with residence times down to 168 s. The continuous MW-flow routes provide a smooth and scalable synthetic methodology towards this class of IRAP inhibitors. INTRODUCTION Almost 30 years ago, microwave-assisted organic synthesis was introduced as a new way of promoting organic reactions.1–4 This technique has since then gone through rapid development and is nowadays an established heating-method both in academia and industry for quick smallscale synthesis of a wide range of compounds5, including drug-like compounds6, peptides7 and polymers8. The general advantages with microwave (MW) heating over conventional heating are rapid and uniform in situ heating, reduced reaction times and in many cases higher reaction yields.9 During the last two decades, lab-scale continuous flow (CF) synthesis has enjoyed considerable progress as well.10,11 In this field, a small reaction volume is heated in a continuous manner instead of a large volume as in batch chemistry. Thus, this organic synthesis methodology is very beneficial from a safety perspective for processing of explosive reagents, toxic materials and gas releasing reactions.10,11 Most commonly, the heating method for CF has been conductive heating.


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The problems featured with this heating methodology are time requirements both regarding heating as well as cooling. In order to facilitate CF synthesis in a standard hood and to provide on-the-fly temperature control, we and others have developed purpose-built small foot-print MW-flow instrumentation for organic synthesis.4,12–27 One of the key features with our design is the tubular, easy access, 200 mm reactor consisting of disposable borosilicate glass, or wear and chemically resistant SiC.24,27–29 Inhibition of insulin-regulated aminopeptidase (IRAP), a single-spanning transmembrane zincmetallopeptidase, might become a future pharmacotherapy against cognitive disorders.30–35 Previously, our division has reported promising constrained macrocycles derived from Ang IV as potent IRAP inhibitors.36,37 However, these inhibitors are peptidic in character and thus not likely to be able to enter the brain. To reveal a new starting point for development of IRAP inhibitors, a small molecule based library of 10 500 compounds were screened for their inhibitory activity against the enzymatic activity of IRAP. After hit confirmation, the spiro-oxindole dihydroquinazolinone 1a (Figure 1) with an IC50 value of 1.5 µM was identified as a representative member of a new class of IRAP inhibitors. Figure 1. Structure of the hit compound 1a.


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Two and three-component syntheses of structurally similar compounds to 1a have been published, involving both Brønstedt and Lewis acid catalyzed reactions.38–42 Commonly, the reactions involve reflux for several hours. We aimed to improve the synthetic methodology for use in a drug discovery project towards the development of novel drug-like IRAP inhibitors. Herein, we present a fast three-component MW-heated CF synthesis of a series of spiro-oxindole dihydroquinazolinone analogs of 1a. RESULTS & DISCUSSION We have previously developed a three-component batch synthesis of IRAP inhibitor 1a from 5bromo-1-methylisatin 2a, isatoic anhydride 3 and p-toluidine 4a using a 50:50 toluene:acetic acid solvent system. Thus, 5-bromoisatin was first N-methylated to afford the starting material 2a using a standard batch procedure. After a few test reactions, pure acetic acid was selected as the solvent as it facilitated the yield and the purification since 1a precipitated out upon cooling to room temperature (72% yield). We next decided to change from conventional reflux heating of 2 h to MW-heating and sealed vessels to reduce the reaction time. When heated to 150 °C with a dedicated MW batch instrument, the reaction was completed in 10 min, resulting in 75% yield of 1a. Scheme 1. MW synthesis of compound 1a.


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The first MW-flow attempt was thereafter to synthesize 1a using a MW transparent 200 mm x 3 mm Øi tubular borosilicate glass reactor in our purpose-built MW-flow applicator.24 We started by employing similar parameters as previously developed for the MW batch synthesis. Hence, 2a, 3 and 4a were used in a 1:1:1 relationship using acetic acid as solvent with a concentration of 0.05 M (Scheme 1). A flow rate of 1 mL/min was selected with a planned process temperature range between 140 and 220 °C. This initial temperature screen indicated that a temperature of 200 °C gave the best result and collection for 5 min (1 mL/min) was performed providing an isolated yield of 40% of the desired compound 1a (entry 1, Table 1). However, it was noted that the conversion was not complete and that the starting material 2a still remained in the processed reaction mixture together with uncharacterized byproducts. In parallel with this work, Konda et al. developed MW heated CF synthesis using a different kind of reactor, a MW absorbing 200 mm x 3 mm Øi silicon carbide (SiC) tubular reactor, with very promising results.28 Thus, we decided to investigate this reactor for the multicomponent reaction. First of all, the reactants were used with the same stoichiometry and concentration as with the borosilicate reactor. Temperatures ranging from 100-240 ºC were investigated, using 20 ºC stepwise increasing increments. The collected reaction mixtures were analyzed by LC-MS, which showed that a reaction temperature of 120-160 °C was most beneficial. Lower


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temperatures resulted in poor product formation while temperature above 160 °C afforded product degradation. Product mixture was collected at 120 °C affording 55% isolated yield (entry 4, Table 1). A flow rate of 0.5 mL/min was used since lower flow (0.25 mL/min) furnished plugging of the system, and higher flow rate (1 mL/min) resulted in less product formation. In order to provide a more direct comparison between the two reactor materials, a reaction using the same conditions as with SiC was performed using the corresponding borosilicate reactor (entry 3, Table 1), furnishing poor conversion and only 8% isolated yield. Furthermore, to investigate a lower flow rate and a higher temperature and to evaluate whether the reported advantages noted in other studies regarding more gentle heating using MWs could be observed, a reaction at 200 °C with a flow rate of 0.5 mL/min was performed (entry 2, Table 1). The reaction furnished 43% yield, an outcome comparable with the 40% yield obtained at 1 mL/min (entry 1, Table 1). Noticeable is that the commonly described advantages4,5,9 with in situ MWs vs wall heating regarding higher temperatures, providing shorter reaction times and cleaner reactions were not observed in this case. In contrast, employing the SiC reactor enabled lower reaction temperatures, less complex reaction mixtures and hence, easier purification. We will continue to investigate plausible explanations for the unexpected results observed by the different mode of heating, but it is obvious to us that flow profiles and heat gradients in the reaction mixture will differ between the two reactors (SiC and borosilicate). The positive outcome with the SiC reactor is in line with the conclusions by Kappe, e.g. the positive effects of MW processing should be considered as an effect of rapid heat exchange, something that could be reproduced in small scale CF chemistry by wall heating using reactor materials such as SiC.29 Both in the previously performed batch reactions and in the flow synthesis of 1a, it was noted that remaining 5-bromo-N-methylisatin (2a) was observed in the mixture after the reaction was


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performed. To investigate whether this could be avoided, the nucleophilic p-toluidine (4a) was used in excess affording a reaction yield of 65% with the SiC reactor (entry 5, Table 1). Next, using both 3 and 4a in excess (1.2 equiv) but with otherwise identical conditions (120 °C, 0.5 mL/min) furnished 74% isolated yield of 1a (entry 6, Table 1). Surprisingly, using 1.5 equivalents of 3 and 4a respectively, resulted in similar yield (entry 7, Table 1) however; increasing the temperature to 160 ºC provided 1a in 82% yield (entry 8, Table 1). Hence, using 1.5 equivalents of 3 and 4a respectively at 160 °C with a flow rate of 0.5 mL/min through the SiC reactor provided full conversion of 2a and the highest isolated yield of 1a. The total residence time for the three-component synthesis of 1a was only 168 s (Table 1, entry 8). Table 1. Three-component scaffold formation of spiro-oxindole dihydroquinazolinone 1a.

Entry 1

2a 3 4a Flow Temp Reactor Yield (equiv) (equiv) (equiv) (mL/min) (ºC) 1 1 1 1 200 Borosilicate 40%

2

1

1

1

0.5

200

Borosilicate

43%

3

1

1

1

0.5

120

Borosilicate

8%

4

1

1

1

0.5

120

SiC

55%

5

1

1

1.5

0.5

120

SiC

65%

6

1

1.2

1.2

0.5

120

SiC

74%

7

1

1.5

1.5

0.5

120

SiC

76%

8

1

1.5

1.5

0.5

160

SiC

82%

A series of different analogues to the hit IRAP inhibitor 1a was then synthesized utilizing the developed MW-SiC-CF conditions from entry 8 in Table 1. A variety of anilines and isatins were used to afford compounds 1b-i in 40-87% yield (Table 2). We also intended to use aliphatic amines, however this did not work under these conditions probably due to protonation. Likewise


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to the preparation of 1a from 2a, the additional isatins 2b,c were prepared by N-methylation in batch mode before participating in the multicomponent reaction. Table 2. Synthesis of spiro-oxindole dihydroquinazolinones. Entry

Isatin

Aniline

Product

1

Yield

85% 2a

4b 1b

2

40% 2a

4c 1c

3

78% 2a

4d

1d

4

87% 2a

4e 1e


8

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N O

5

61% 2a

H2N

4f 1f

6

75% 2b

4a 1g

7

74% 4a 2c 1h

8

78% 2d

4a 1i

Biological testing of the spiro-oxindole dihydroquinazolinones 1a-i indicated that the bromine is preferred in the 5-position, since removal of it (1g) decreased the activity more than 10-fold, and switching of the bromine from the 5 to the 7-position (1h) gave an inactive compound. In an attempt to further look into the structure-activity-relationship (SAR), the ability to perform a Suzuki-Miyaura cross-coupling reaction on 1a using MW-CF was investigated.43,44 The reaction conditions were first optimized using MW batch chemistry with the aim to reduce


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dehalogenation and hydrolysis of the aryl bromide 1a. Different Pd-catalyst (Pd(PPh3)4, Pd(tBu3)2, PdCl2(dppf)), bases (DBU, NaOH, KOH), solvent systems (MeCN, DMF, DMA and methyl ethyl ketone with adducts of H2O) , temperatures (160 °C, 180 °C, 200 °C) and 4tolylboronic acid in excess (1.5–3 equiv) were evaluated.45 From these investigations it was observed that minimization of water content and slight excess of base were necessary to avoid hydrolysis of the aminal part of 1a. Dehalogenation was not highly dependent on concentration, temperature or Pd-source. However, it was affected by solvent and base. The following reaction parameters were found to be highly suitable for the Suzuki-Miyaura cross coupling; 1a (0.1 mmol, 1 equiv), boronic acid (2 equiv), DBU (1.5 equiv), PdCl2(dppf) (2 mol%) and MeCN with 2% H2O (5 mL). These MW-batch conditions, using p-tolylboronic acid (5) as arylating agent and 180 °C for 1 min provided an isolated yield of 93% of 1j. Next, we transferred the cross-coupling to the flow system going back to the 3 mm Øi borosilicate reactor and aiming to produce a library of biphenyl compounds in a CF process. A temperature screen was performed ranging from 160 to 220 °C with 20 °C increasing increments at a flow of 0.5 mL/min, which showed that the highest temperature gave full conversion of 1a. The flow rate was thereafter investigated and the productivities at 0.75, 1.0, 1.5 and 2.0 mL/min were analyzed, respectively. However, when increasing the flow rate, full conversion of 1a could not be obtained. Product mixture was thus collected for 5 min with a flow rate of 0.5 mL/min at 220 °C, which furnished a residence time of 168 s and a 71% isolated yield of 1j (Scheme 2). Compared to the related work by Konda et al. no issues with clogging or reactor rupture were observed during these experiments.28 Scheme 2. MW-heated continuous flow Suzuki-Miyaura cross coupling reaction.


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Disappointingly, elongated biaryl compound 1j furnished no IRAP inhibiting activity. This results indicated that substitution of the bromine in the 5-position is not permitted and the idea of producing Suzuki-Miyaura based IRAP-inhibitor library by MW-CF methodology was ultimately abandoned. In the near future, other parts of the molecule will be investigated to gain further knowledge about the SAR for this class of IRAP inhibitors. CONCLUSION As a result of a screen of 10 500 compound a new type of spiro-oxindole dihydroquinazolinone based IRAP inhibitors (1a) was identified. Hence, a fast, productive and scalable synthetic method for this class of structures was needed. Thus, we developed a synthetic route employing MW-heated CF synthesis with a MW-absorbent 200 mm x 3 mm Øi SiC reactor to smoothly produce 1a in 82% isolated yield. This method was compatible with different kinds of anilines and isatines, delivering 9 test compounds (1a-i) in moderate to high yields. Further, we also showed that derivatization of 1a by Suzuki-Miyaura cross-coupling was possible using the MWflow set-up. This time a MW-transparent borosilicate reactor was used providing an isolated yield of 71% of the cross-coupling product 1j. While it may be debatable if the synthetic procedures described within this report is a better route to the target compounds 1a-j, or not, we


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wish to inform the reader of our work, which allows an alternative approach to be considered. The utilized MW-CF system is limited to lab-scale, but the technique as such is scalable. We will continue to investigate the flow and temperature profiles of the reaction mixture in the CF tube reactors, as well as various technical modifications/additions in order to disrupt a laminar flow profile. EXPERIMENTAL SECTION General Information. The MW generator, reactor, applicator and cavity are part of the ArrheniusOne™ system by WaveCraft AB. This system operates in a nonresonant mode and features a straight tube borosilicate glass or Si/C reactor. The pump used was a Shimadzu LC10AD HPLC pump. WaveCraft adjustable back-pressure regulator ranging from 10-150 bars was used to regulate the pressure. The temperature was determined using a calibrated external IR sensor. An Optris CT sensor with a LT22 sensing head was used for the SiC tube reactor, while the sensor used for the borosilicate glass tube reactor was an Optris CSmicro 3M sensor. For a schematic picture of the MW-flow system, see Supporting Information. Commercially available starting materials and solvents were used without further purification. 1H and 13C NMR spectra were recorded at 25 °C and at 400 and 100 MHz, respectively. Chemical shift are reported in ppm with the solvent residual peak as internal standard (CDCl3 δH 7.26, CDCl3 δC 77.16; CD3OD δH 3.31, CD3OD δC 49.0; (CD3)2SO δH 2.50, (CD3)2SO δC 39.52). When using a mixture of CD3OD and CDCl3 as solvent for NMR, the reference peak was set to CD3OD. The batch microwave experiments were conducted using a Biotage Smith Synthesizer™ single mode cavity and sealed 2-5 mL process vials. Analytical thin-layer chromatography was performed on silica gel 60 F-254 plates and visualized with UV-light. Flash column chromatography was performed on silica gel 60 (40-63 µM). Analytical HPLC-UV/MS was performed on a Dionex


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Ultimate 3000 HPLC system with a Bruker Amazon SL ion trap mass spectrometer and detection by UV (DAD) and MS (ESI+), using a Phenomenex Kinetex C18 column (50x3.0 mm, 2.6 µM particle size, 100 Å pore size) and a flow rate of 1.5 mL/min. A gradient of H2O/MeCN/0.05% HCOOH was used. High-resolution MS (HRMS) data was produced using an ES-TOF instrument. General procedure for N-methylation of isatins (2a-c). To a suspension of the appropriate isatin (1 equiv) and Cs2CO3 (2 equiv) in dry MeCN (0.05 M isatin) was added MeI (1.1 equiv). The reaction mixture was stirred for 7 h at ambient temperature. Subsequently the reaction mixture was concentrated under reduced pressure, dissolved in EtOAc and washed with water and brine, dried over MgSO4, filtered and concentrated. The product was used in the next step without further purification. 5-Bromo-1-methylindolone-2,3-dione (2a). The compound was synthesized according to the General procedure from 5-bromo-isatin (1.00 g, 4.42 mmol), Cs2CO3 (2.90 g, 8.90 mmol) and MeI (0.31 mL, 4.98 mmol) to give 2a as a red solid (1.05 g, 98%).46 1H NMR (400 MHz, DMSO-d6) δ 7.85 (dd, J=8.4, 2.1 Hz, 1H), 7.69 (d, J=2.1 Hz, 1H), 7.12 (d, J=8.4 Hz, 1H), 3.12 (s, 3H). 13C NMR (101 MHz, DMSO-d6) δ 182.6, 158.2, 150.7, 140.2, 126.9, 119.6, 115.3, 113.1, 26.5. 1-Methylindoline-2,3-dione (2b). The compound was synthesized according to the General procedure from isatin (2.00 g, 13.59 mmol), Cs2CO3 (8.90 g, 27.32 mmol) and MeI (0.95 mL, 15.26 mmol) to give 2b as a red solid (1.99 g, 91%).46 1H NMR (400 MHz, CDCl3) δ 7.58 (td, J=7.8, 1.3 Hz, 1H), 7.53 (ddd, J=7.4, 1.4, 0.7 Hz, 1H), 7.09 (td, J=7.5, 0.8 Hz, 1H), 6.88 (dt,


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J=7.9, 0.7 Hz, 1H), 3.21 (s, 3H). 13C NMR (101 MHz, CDCl3) δ 183.4, 158.3, 151.5, 138.5, 125.2, 123.9, 117.4, 110.1, 26.3. 7-Bromo-1-methylindoline-2,3-dione (2c). The compound was synthesized according to the General procedure from 7-bromo-isatin (1.00 g, 4.24 mmol), Cs2CO3 (2.90 g, 8.90 mmol) and MeI (0.31 mL, 4.98 mmol) to give 2c as a red solid (973 mg, 92%).47 1H NMR (400 MHz, CDCl3) δ 7.70 (dd, J=1.3, 8.1 Hz, 1H), 7.56 (dd, J=1.3, 7.3 Hz, 1H), 6.99 (ddd, J=8.1, 7.3, 0.6 Hz, 1H), 3.65 (s, 3H). 13C NMR (101 MHz, CDCl3) δ 182.6, 158.7, 148.4, 143.9, 125.2, 124.6, 120.6, 104.5, 29.9. General procedure for the optimization or the three-component formation of 1a. A 0.05 M solution of 2a (1 equiv), p-toluidine (1-1.5 equiv) and isatoic anhydride (1-1.5 equiv) in AcOH was stirred at ambient temperature for approximately 30 min to afford all reactants in solution, and the reaction mixture was subsequently filtered through a 25 mm syringe filter with 0.45 µM PTFE membrane to removed remaining particles. The reaction mixture was then pumped through the 200 mm x 3 mm Øi borosilicate or SiC reactor at a flow rate of 0.25, 0.5 and 1 mL/min and at temperatures of 100-240 ºC with intervals of 20 ºC. The wash-out volume was 3 mL. General procedure for three-componentformation of 1a-h. A 0.05 M solution of the appropriate N-methylated isatin 2a-c (1 equiv), the appropriate aniline (1.5 equiv) and isatoic anhydride (1.5 equiv) in AcOH was stirred at ambient temperature for 30 min to afford all reactants in solution, and the reaction mixture was subsequently filtered through a 25 mm syringe filter with 0.45 µM PTFE membrane to removed remaining particles. The reaction mixture was pumped through the 200 mm x 3 mm Øi SiC reactor at a flow of 0.5 mL/min and at


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a temperature of 160 °C. The obtained product was either purified by suspension in hot EtOH, cooling to ambient temperature and filtration, or by flash column chromatography. 5-Bromo-1-methyl-3’-(p-tolyl)-1’H-spiro[indoline-3,2’-quinazoline]-2,4’(3’H)-dione (1a). The product was synthesized according to the general procedure and a reaction mixture volume of 21.0 mL was collected. The reaction mixture was left for 14 h for the product to precipitate out. The crude product was filtered and subsequently suspended in hot EtOH, cooled to ambient temperature and filtered, and 1a was obtained as a beige solid (286 mg, 82%). 1H NMR (400 MHz, DMSO-d6) δ 7.82 (d, J=2.0 Hz, 1H), 7.68 (dd, J=7.7, 1.5 Hz, 1H), 7.62 (s, 1H), 7.46 (dd, J=8.4, 2.1 Hz, 1H), 7.32 (ddd, J=8.0, 7.3, 1.6 Hz, 1H), 7.07–6.99 (m, 2H), 6.90–6.82 (m, 3H), 6.79 (td, J=7.6, 1.1 Hz, 1H), 6.69 (dd, J=8.1, 1.0 Hz, 1H), 3.01 (s, 3H), 2.19 (s, 3H). 13C NMR (101 MHz, DMSO-d6) δ 174.0, 163.8, 146.2, 142.9, 137.6, 135.5, 134.2, 134.1, 129.7, 129.4, 129.3, 127.9, 118.4, 115.0, 114.96, 114.94, 114.6, 111.5, 76.5, 26.5, 21.0. HRMS (ES) m/z calcd. for C23H18BrN3O2: [M+H]+ 448.0661, found: 448.0662. 5-Bromo-1-methyl-3’-phenyl-1’H-spiro[indoline-3,2’-quinazoline]-2,4’(3’H)-dione (1b). The product was synthesized according to the general procedure and a reaction mixture volume of 21.0 mL was collected. The reaction mixture was left for 14 h for the product to precipitate out. The crude product was filtered and subsequently suspended in hot EtOH, cooled to ambient temperature and filtered, and 1b was obtained as a yellow solid (389 mg, 85%). 1H NMR (400 MHz, CD3OD) δ 7.85 (dd, J=7.8, 1.5 Hz, 1H), 7.64 (d, J=2.0 Hz, 1H), 7.37 (dd, J=8.3, 2.0 Hz, 1H), 7.32 (ddd, J=8.1, 7.3, 1.6 Hz, 1H), 7.24–7.14 (m, 3H), 6.98–6.91 (m, 2H), 6.85 (td, J=7.6, 1.0 Hz, 1H), 6.70–6.65 (m, 1H), 6.57 (d, J=8.3 Hz, 1H), 2.99 (s, 3H). 13C NMR (101 MHz, CD3OD) δ 174.0, 165.4, 145.9, 142.4, 137.5, 134.9, 134.4, 129.6, 129.5, 129.1, 128.9, 128.7,


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119.7, 116.2, 115.1, 114.9, 110.9, 77.3, 26.3. HRMS (ES) m/z calcd. for C22H16BrN3O2: [M+H]+ 434.0493, found: 434.0504. 5-Bromo-1-methyl-3’-(4-(trifluoromethyl)phenyl)-1’H-spiro[indoline-3,2’-quinazoline]2,4’(3’H)-dione (1c). The product was synthesized according to the general procedure and a reaction mixture volume of 21.0 mL was collected. The crude product was purified by flash column chromatography using 20% EtOAc in toluene followed by a second column using 40% EtOAc in pentane as eluent to afford 1c as a light orange solid (211 mg, 40%). 1H NMR (400 MHz, CD3OD) δ 7.85 (dd, J=7.8, 1.6 Hz, 1H), 7.65 (d, J=2.0 Hz, 1H), 7.48 (d, J=8.3 Hz, 2H), 7.41 (dd, J=8.4, 2.0 Hz, 1H), 7.37-7.30 (m, 1H), 7.11 (d, J=8.2 Hz, 2H), 6.86 (td, J=7.6, 1.1 Hz, 1H), 6.69 (dd, J=8.1, 1.0 Hz, 1H), 6.62 (d, J=8.4 Hz, 1H), 3.01 (s, 3H). 13C NMR (101 MHz, CD3OD) δ 173.6, 165.3, 145.8, 142.3, 141.1, 135.1, 134.8, 130.3, 129.2, 128.9, 128.7, 126.5, 119.9, 116.4, 115.0, 114.9, 111.1, 110.6, 78.0, 26.4. HRMS (ES) m/z calcd. for C23H15BrF3N3O2: [M+H]+ 502.0365, found: 502.0378. 5-Bromo-3’-(4-(tert-butyl)phenyl)-1-methyl-1’H-spiro[indoline-3,2’-quinazoline]-2,4’(3’H)dione (1d). The product was synthesized according to the general procedure and a reaction mixture volume of 21.0 mL was collected. The crude product was purified by flash column chromatography using 20% EtOAc in toluene followed by a second column using 40% EtOAc in pentane as eluent to afford 1d as a light pink solid (404 mg, 78%). 1H NMR (400 MHz, CDCl3) δ 8.00 (ddd, J=7.8, 1.5, 0.5 Hz, 1H), 7.58 (dd, J=2.0, 0.4 Hz, 1H), 7.39 (ddd, J=8.4, 2.0, 0.5 Hz, 1H), 7.37-7.33 (m, 1H), 7.23-7.16 (m, 2H), 7.02-6.95 (m, 1H), 6.84 (d, J=8.1 Hz, 1H), 6.65 (ddd, J=8.0, 1.0, 0.5 Hz, 1H), 6.53 (d, J=8.3 Hz, 1H), 2.97 (s, 3H), 1.21 (s, 9H). 13C NMR (101 MHz, CDCl3) δ 172.6, 164.1, 151.5, 144.0, 141.8, 134.29, 134.27, 134.1, 129.6, 129.2, 128.6, 128.4,


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126.1, 120.0, 116.7, 116.1, 115.4, 110.4, 76.6, 34.7, 31.3, 26.4. HRMS (ES) m/z calcd. for C26H24BrN3O2: [M+H]+ 490.1135, found: 490.1130. 5-Bromo-3’-(4-iodophenyl)-1-methyl-1’H-spiro[indoline-3,2’-quinazoline]-2,4’(3’H)-dione (1e). The product was synthesized according to the general procedure and a reaction mixture volume of 8.0 mL was collected. The crude product was purified by flash column chromatography using 20% EtOAc in toluene as eluent to afford 1e as an off-white solid (195 mg, 87%). 1H NMR (400 MHz, DMSO-d6) δ 7.85 (d, J=2.0 Hz, 1H), 7.71–7.65 (m, 2H), 7.63– 7.58 (m, 2H), 7.50 (dd, J=8.4, 2.1 Hz, 1H), 7.34 (ddd, J=8.0, 7.3, 1.6 Hz, 1H), 6.89 (d, J=8.4 Hz, 1H), 6.79 (ddd, J=7.5, 5.8, 1.2 Hz, 3H), 6.69 (dd, J=8.2, 1.0 Hz, 1H), 3.03 (s, 3H). 13C NMR (101 MHz, DMSO-d6) δ 173.3, 163.3, 145.8, 142.5, 137.8, 137.6, 134.0, 133.9, 131.5, 129.1, 128.5, 127.5, 118.1, 114.7, 114.3, 111.3, 94.3, 75.9, 26.2. HRMS (ES) m/z calcd. for C22H15BrIN3O2: [M+H]+ 559.9459, found: 559.9471. 5-Bromo-1-methyl-3’-(3-(oxazol-5-yl)phenyl)-1’H-spiro[indoline-3,2’-quinazoline]2,4’(3’H)-dione (1f). The product was synthesized according to the general procedure and a reaction mixture volume of 15.0 mL was collected. The reaction mixture was left for 14 h for the product to precipitate out. The crude product was filtered and subsequently suspended in hot EtOH, cooled to ambient temperature and filtered, and 1f was obtained as a brown solid (230 mg, 61%). 1H NMR (400 MHz, DMSO-d6) δ 8.43 (s, 1H), 7.93 (d, J=2.1 Hz, 1H), 7.74–7.69 (m, 2H), 7.62 (s, 1H), 7.53 (dt, J=7.9, 1.2 Hz, 1H), 7.44 (dd, J=8.4, 2.1 Hz, 1H), 7.40–7.31 (m, 3H), 6.96 (d, J=7.7 Hz, 1H), 6.84 (d, J=8.4 Hz, 1H), 6.80 (dd, J=7.6, 1.0 Hz, 1H), 6.72 (dd, J=8.2, 1.0 Hz, 1H), 3.02 (s, 3H). 13C NMR (101 MHz, DMSO-d6) δ 173.5, 163.3, 152.1, 149.5, 145.8, 142.5, 138.4, 134.0, 133.8, 129.8, 129.3, 128.4, 128.0, 127.6, 123.5, 122.4, 118.2, 114.6, 114.4,


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114.3, 111.1, 76.1, 26.1. HRMS (ES) m/z calcd. for C25H17BrN4O3: [M+H]+ 501.0544, found: 501.0562. 1-Methyl-3’-(p-tolyl)-1’H-spiro[indoline-3,2’-quinazoline]-2,4’(3’H)-dione (1g). The product was synthesized according to the general procedure and a reaction mixture volume of 21.0 mL was collected. The reaction mixture was left for 14 h for the product to precipitate out. The crude product was filtered and subsequently suspended in hot EtOH, cooled to ambient temperature and filtered, and 1g was obtained as a yellow solid (289 mg, 75%). 1H NMR (400 MHz, CD3OD) δ 7.84 (dd, J=7.8, 1.5 Hz, 1H), 7.51 (ddd, J=7.5, 1.3, 0.6 Hz, 1H), 7.30 (ddd, J=8.1, 7.3, 1.6 Hz, 1H), 7.25 (td, J=7.8, 1.3 Hz, 1H), 7.02 (td, J=7.6, 1.0 Hz, 1H), 6.97–6.91 (m, 2H), 6.87–6.75 (m, 3H), 6.70–6.63 (m, 2H), 3.02 (s, 3H), 2.19 (s, 3H). 13C NMR (101 MHz, CD3OD) δ 174.5, 165.8, 146.1, 143.2, 138.5, 135.0, 134.6, 131.6, 129.9, 128.6, 127.7, 126.0, 123.9, 119.5, 115.4, 114.8, 109.3, 77.4, 26.3, 21.1. HRMS (ES) m/z calcd. for C23H19N3O2: [M+H]+ 370.1554, found: 370.1556. 7-Bromo-1-methyl-3’-(p-tolyl)-1’H-spiro[indoline-3,2’-quinazoline]-2,4’(3’H)-dione (1h). The product was synthesized according to the general procedure and a reaction mixture volume of 13.0 mL was collected. The reaction mixture was left for 14 h for the product to precipitate out. The crude product was filtered and subsequently suspended in hot EtOH, cooled to ambient temperature and filtered, and 1h was obtained as a yellow solid (215 mg, 74%). 1H NMR (400 MHz, DMSO-d6) δ 7.71–7.66 (m, 2H), 7.61 (s, 1H), 7.44 (dd, J=8.2, 1.2 Hz, 1H), 7.33 (dddd, J=8.1, 7.3, 1.6, 0.8 Hz, 1H), 7.06–7.00 (m, 2H), 6.96 (dd, J=8.2, 7.3 Hz, 1H), 6.84–6.75 (m, 3H), 6.68 (ddd, J=8.2, 1.1, 0.5 Hz, 1H), 2.19 (s, 3H). 13C NMR (101 MHz, DMSO-d6) δ 174.6, 163.5, 145.8, 145.7, 140.3, 137.2, 136.2, 135.1, 135.1, 133.9, 130.2, 129.4, 127.6, 126.1, 124.7, 118.1,


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114.5, 114.2, 101.7, 75.6, 29.5, 20.6. HRMS (ES) m/z calcd. for C23H18BrN3O2: [M+H]+ 448.0667, found: 448.0661. 5-Bromo-3’-(p-tolyl)-1’H-spiro[indoline-3,2’-quinazoline]-2,4’(3’H)-dione (1i). The product was synthesized according to the general procedure and a reaction mixture volume of 21.0 mL was collected. The crude product was purified by flash column chromatography using 40% EtOAc in hexane to afford 1i as a grey solid (354 mg, 78%). 1H NMR (400 MHz, CD3OD) δ 7.85 (dd, J=7.8, 1.5 Hz, 1H), 7.54 (d, J=2.0 Hz, 1H), 7.29 (ddd, J=8.1, 7.3, 1.6 Hz, 1H), 7.25 (dd, J=8.3, 2.0 Hz, 1H), 6.97 (dt, J=7.3, 0.9 Hz, 2H), 6.90–6.81 (m, 3H), 6.63 (dd, J=8.1, 0.9 Hz, 1H), 6.51 (d, J=8.3 Hz, 1H), 2.19 (s, 3H). 13C NMR (101 MHz, CD3OD) δ 175.4, 164.9, 145.0, 140.0, 138.3, 134.5, 134.3, 133.9, 131.8, 130.0, 129.8, 128.9, 128.5, 119.6, 115.3, 115.3, 114.6, 112.2, 77.5, 21.0. HRMS (ES) m/z calcd. for C22H16BrN3O2: [M+H]+ 434.0509, found: 434.0504. 1-Methyl-3’,5-di-p-tolyl-1’H-spiro[indoline-3,2’-quinazoline]-2,4’(3’H)-dione (1j). A 0.02 M solution of 1a (90 mg, 0.2 mmol), p-tolylboronic acid (54 mg, 0.4 mmol), DBU (60 µL, 0.4 mmol) and PdCl2(dppf) (2 mol%) in 2% H2O in MeCN was mixed and filtered through a 25 mm syringe filter with 0.45 µM PTFE membrane to remove remaining particles. The reaction mixture was pumped through the 200 mm x 3 mm Øi borosilicate reactor at a flow of 0.5 mL/min and a temperature of 220 ºC. The crude product was concentrated and purified by flash column chromatography using 0.5-2.5% MeOH in DCM as eluent to afford 1j as a white solid (92 mg, 71%). 1H NMR (400 MHz, DMSO-d6) δ 7.91 (dd, J=2.0, 0.5 Hz, 1H), 7.69 (dd, J=7.8, 1.6 Hz, 1H), 7.63 (s, 1H), 7.58–7.49 (m, 3H), 7.31 (ddd, J=8.1, 7.3, 1.6 Hz, 1H), 7.27–7.22 (m, 2H), 7.01–6.96 (m, 2H), 6.92 (m, 3H), 6.77 (ddd, J=7.8, 7.3, 1.1 Hz, 1H), 6.70 (ddd, J=8.1, 1.1, 0.5 Hz, 1H), 3.06 (s, 3H), 2.33 (s, 3H), 2.15 (s, 3H). 13C NMR (101 MHz, DMSO-d6) δ 174.1, 163.6,


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146.0, 142.4, 136.9, 136.46, 136.47, 135.4, 134.9, 133.6, 129.5, 129.2, 128.8, 127.5, 127.4, 126.1, 124.2, 117.8, 114.7, 114.1, 109.4, 76.2, 26.1, 20.6, 20.5. HRMS (ES) m/z calcd. for C30H25N3O2: [M+H]+ 460.2003, found: 460.2025. Enzymatic assay. An enzymatic assay measuring IRAP activity was applied for screening purposes as well as follow-up dose-response characterization. Membrane preparations from Chinese Hamster Ovary (CHO) cells are used as a source of enzyme activity. The extent of enzymatic activity in each sample was quantified in a microtiter based screening assay using the peptide like substrate L-leucine-p-nitroanilide (L-Leu-pNA), which upon IRAP mediated cleavage produces p-nitroanilide which absorbs at 405 nm. The screen (reported elsewhere) was conducted in a 384-well format using transparent microplates, whereas follow-up dose-response experiments were conducted in the equivalent transparent 96-well plate (Nunc, Product number: 26962). In this format the assay volume was 200 µL and the buffer was 50 mM Tris-HCl, 150 mM NaCl, and 0.1 mM phenylmethanesulfonylfluoride (PMSF) at pH 7.4. The assays were conducted in the presence of a final concentration of 1 mM L-Leu-pNA and CHO cell membranes from a total of 50.000-200.000 cells per well. The CHO membrane preparation was performed as described in Demaegt et al., 2004, with the lysis of washed cells being done by means of ultrasonication followed by multiple strokes (>20) with a Dounze homogenizer and an ultracentrifugation procedure for pelleting and washing the membranes.48 The protocol for running the dose-response assay started with a serial dilution of the compound stock solutions at 10 mM in pure DMSO by a factor of 1/3 in columns 1 through to 11 of the U-formed 96-well plates. Column 12 was reserved for controls meaning the equivalent amount of DMSO was added to the wells A12-D12, whereas a 10 mM stock solution of 6 (Figure 2) was placed in wells E12-H12 serving as a control representing 100% inhibition of the enzymatic activity. The DMSO


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solutions were then diluted with assay buffer and transferred to the transparent assay plates in triplicates (50 µL to each well both for samples and controls), followed by addition of diluted homogenized CHO cell membranes (50 µL) and substrate (100 µL) to initiate the enzymatic reactions. The plates were covered with lids and then incubated at room temperature until a statistically significant difference (6-12 hours) was observed between the controls (Z’ values above 0.8). Care was taken to ensure the readings were taken at a time point when the absorbance signal still increased linearly with time. Raw data were then imported into Microsoft Excel for a conversion to % inhibition data based on the controls on each plate. Following averaging of data from the triplicate samples the curves were fitted to a four parameter dose response model within XLfit (model 205) to obtain best-fit values for the IC50 value, Hill slope and the upper and lower limits of the dose-response curve. Cl N N N N H

H N

S Br

S O O

6

Figure 2. Structure of inhibitor used as control for enzymatic activity. AUTHOR INFORMATION Corresponding Author *Mats Larhed, Department of Medicinal Chemistry, Science for Life Laboratory, Uppsala University, P. O. Box 574, SE-751 23 Uppsala, Sweden Tel.: +46 18 471 4667 Fax: +46 18 471 4474 Email: [email protected] Notes


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The authors declare no competing financial interest. ACKNOWLEDGMENT Funding for the work at Chemical Biology Consortium Sweden was provided by the Swedish Research Council and Karolinska Institutet (A. J. J & T. L). We thank the Knut and Alice Wallenberg Foundation for financial support. We also want to express our gratitude to Magnus Fagrell and Wavecraft AB for their help with the fabrication of the SiC reactors. SUPPORTING INFORMATION. Copies of spectra and chromatograms. This material is available free of charge via the Internet at http://pubs.acs.org/. ABBREVIATIONS MW, microwave; CF, continuous flow; SiC, silicon carbide; IRAP, Insulin-Regulated Aminopeptidase; Ang IV, Angiotensin IV.

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Microwave Heated Flow Synthesis of Spiro-oxindole

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