2-Arylpaullones are selective antitrypanosomal agents

Accepted Manuscript 2-Arylpaullones are Selective Antitrypanosomal Agents Jasmin Ryczak, Ma’ayan Papini, Annette Lader, Abedelmajeed Nasereddin, Dmitry Kopelyanskiy, Lutz Preu, Charles L. Jaffe, Conrad Kunick PII:

S0223-5234(13)00225-0

DOI:

10.1016/j.ejmech.2013.03.065

Reference:

EJMECH 6103

To appear in:

European Journal of Medicinal Chemistry

Received Date: 18 January 2013 Revised Date:

27 March 2013

Accepted Date: 28 March 2013

Please cite this article as: J. Ryczak, M.’a. Papini, A. Lader, A. Nasereddin, D. Kopelyanskiy, L. Preu, C.L. Jaffe, C. Kunick, 2-Arylpaullones are Selective Antitrypanosomal Agents, European Journal of Medicinal Chemistry (2013), doi: 10.1016/j.ejmech.2013.03.065. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT

2-Arylpaullones are Selective Antitrypanosomal Agents

Jasmin Ryczak, Ma’ayan Papini, Annette Lader, Abedelmajeed Nasereddin, Dmitry

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Kopelyanskiy, Lutz Preu, Charles L. Jaffe, Conrad Kunick

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Graphical abstract

ACCEPTED MANUSCRIPT 1 2-Arylpaullones are Selective Antitrypanosomal Agents

Jasmin Ryczak,a Ma’ayan Papini,b Annette Lader,a Abedelmajeed Nasereddin,b

a

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Dmitry Kopelyanskiy,b Lutz Preu,a Charles L. Jaffe,b* Conrad Kunicka, c*

Technische Universität Braunschweig, Institut für Medizinische und

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Pharmazeutische Chemie, Beethovenstraße 55, D-38106 Braunschweig, Germany

Department Microbiology and Molecular Genetics, Kuvin Centre for Study of

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b

Infectious and Tropical Diseases, IMRIC, P.O. Box 12272, Hebrew UniversityHadassah Medical School, Jerusalem 91120, Israel c

Science Center of Pharmaceutical Engineering (SCOPE), Beethovenstraße 55,

Abstract

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D-38106 Braunschweig, Germany

Antileishmanial paullone-chalcone hybrid molecules display antiparasitic activity

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against Trypanosoma brucei rhodesiense blood stream forms, albeit with low selectivity against human THP-1 cells. In order to develop less toxic analogues,

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paullones with acrylamide or aryl substituents in 2-position were synthesized, of which the latter exhibited potent antiparasitic activity with excellent selectivity profiles. The most potent compound identified in this study was 9-tert-butyl-2-(4morpholinophenyl)paullone (3i) which inhibited the parasites at submicromolar concentrations (GI50 = 510 nM) with a selectivity index of 157.

Keywords: Neglected tropical diseases, Paullones, Suzuki reaction, Trypanosoma brucei rhodesiense, Trypanosomiasis.

ACCEPTED MANUSCRIPT 2

* Corresponding authors: C. L. J.: Phone: +972-2-6757435; Fax. +972-2-6757425; e-mail: [email protected].

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C.K.: Phone: +49-531-391-2751; Fax: +49-531-391-2799; e-mail: c.kunick@tu-

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braunschweig.de.

ACCEPTED MANUSCRIPT 3 1. Introduction

Sleeping sickness or human African trypanosomiasis (HAT) is a neglected disease

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threatening millions of people in sub-Sarahan Africa. This disease is caused by two unicellular parasites belonging to the genus Trypanosoma, Trypanosoma brucei gambiense and T. b. rhodesiense, which are transmitted by tsetse flies. HAT is

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characterized by two stages: first a haemolymphatic stage, where the extracellular parasites multiply in the blood, lymph and subcutaneous tissues; and second a

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neurological phase, where the parasites cross the blood-brain barrier and the infection spreads to the central nervous system. HAT is fatal if untreated. Medicinal options for treatment are rather limited. Of the four registered drugs against sleeping sickness none is orally available, and all show significant side effects. Pentamidine and suramin only work against first stage disease, melarsoprol and eflornithine are

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used against the second or late stage of disease. Melarsoprol exhibits severe toxicity, and is fatal in 3 – 10% of the cases. Eflornithine treatment is laborious and not effective against T. b. rhodesiense infections. Reports of treatment failures resulting

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from increasing parasite drug resistance are alarming and underscore the urgent need for safe, orally administered drugs against HAT. Coadministration of eflornithine

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and nifurtimox, a drug used against Chagas disease, is the only new development in this field. The eflornithine/nifurtimox combination simplifies administration, and has a better safety and efficacy profile than eflornithine alone. This combination therapy has recently been recommended by the WHO for use in treating second stage HAT caused by T. b. gambiense.[1-3]

In a recent paper, the development of paullone-chalcone hybrid molecules 1 as antileishmanial agents was reported. [4] These structures can be regarded as hybrids

ACCEPTED MANUSCRIPT 4 of chalcones (1,3-diarylpropenones) [5-9] and paullones (7,12-dihydroindolo[3,2d][1]benzazepin-6(5H)-ones) [10, 11], which are both well known for various

Since Leishmania and Trypanosoma parasites

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biological activities.

both belong to the order

Trypanosomatida, and share many unique metabolic and biochemical pathways it

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was interesting to see whether these compounds are also active against extracellular African trypanosomes. An initial screen of representatives 1a-1k for activity at 5 µM

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against T. b. rhodesiense bloodstream trypomastigotes was performed. Indeed, most of the congeners exhibited considerable antitrypanosomal activity with inhibition rates between 35 and 100% at this concentration (data not shown). Subsequent evaluation of the compounds at different concentrations revealed GI50 values in the µM range

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without obvious structure-activity relationships (Table 1).

1 contain in position 2 of the paullone ring system an electrophilic alpha,betaunsaturated ketone partial structure which can form Michael type adducts with

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biomolecules. As long as the mechanism by which the paullone-chalcone hybrids 1 display their antiparasitical activity is unknown, the role of this enone element and of

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its reactivity remains obscure. The electrophilic property of alpha,beta-unsaturated carbonyl compounds has been linked to toxicity and mutagenicity [12-14] and therefore is generally undesirable in drug molecules. Indeed, all structures 1 listed in Table 1 showed considerable toxicity against the human macrophage cell line THP-1. Therefore, we modified the Michael acceptor substructure in 1 aiming for congeners with reduced toxicity against host cells. For this purpose, we designed four groups of structural analogs:

ACCEPTED MANUSCRIPT 5 (a) The acrylamides 2 in which a nitrogen atom is inserted next to the carbonyl group. Because alpha,beta-unsaturated

amides

2

are less

electrophilic

than the

corresponding ketones, they are supposed to be less prone to unwanted toxicity

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against human cells. In line with this consideration, several alpha,beta-unsaturated amides are currently undergoing clinical trials, namely as irreversible kinase inhibitors for cancer treatment (e.g. neratinib, canertinib, pelitinib, BIBW-2992). In these drugs

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the electrophilicity of the acrylamide partial structure is obviously well balanced so that the drug molecules reach the specific nucleophilic target structure without

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previous unspecific reaction with ubiquitous other biologic nucleophiles. [15-17] (b) The 2-aryl-derivatives 3 which lack the alpha,beta unsaturated partial structure and thus are not reactive as electrophiles. As a structural similarity to 1, 3 also display a sequential arrangement of three sp2-hybridized carbon atoms attached to position 2 of the paullone scaffold.

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(c) The paullone-chalcone hybrid structure 4 which constitutes an isomer of 1a with “inverted” propenone structure. 4 was designed to study whether a defined focus of electrophilicity in the compounds is necessary for antitrypanosomal activity.

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(d) The dihydro analog 5 of the paullone-chalcone hybrid 1a was designed to evaluate a compound with overall similar structure but without the Michael acceptor

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element.

<>

2. Chemistry

For the synthesis of the alpha,beta-unsaturated amides 2, the 2-iodopaullone 6 [4] was reacted with suitable acrylamides by means of a Heck reaction in the presence

ACCEPTED MANUSCRIPT 6 of palladium acetate in DMF. The 2-arylpaullones 3 were obtained from the same starting material 6 by a microwave assisted Suzuki synthesis employing aromatic boronic acids as reagents (Scheme 1). The derivative 4 with the inverted alpha,beta-

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unsaturated ketone moiety was prepared in a four-step synthesis. Initially, 6 was reacted with trimethylsilylacetylene by means of a Sonogashira reaction. The resulting 2-[(trimethylsilyl)ethinyl]paullone was deprotected by TBAF to give the

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terminal alkyne 7, which was subsequently hydrolyzed by a Kucherov reaction. [18] Finally, the obtained 2-acetylpaullone 8 was reacted with benzaldehyde in the

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presence of potassium hydroxide to yield the desired paullone-chalcone hybrid 4 together with a number of unidentified side products, from which it was separated by column chromatography. The dihydrochalcone 5 was prepared by reduction of the paullone-chalcone hybrid 1a with zink in acetic acid. Also in this case a number of unidentified side products had to be removed by chromatography, leaving pure 5 in

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rather poor yields (Scheme 2).

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Table 1: Antitrypanosomal Activity, Toxicity and Selectivity of 2-Substituted Paullones a

b

R

Activity (GI50) against T.b. rhodesiense [µM ± se]

(95% CI)

Toxicity (GI50) against THP-1 macrophages [µM ± se]

(95% CI)

1a

Ph

4.0

(1.3 - 12.6)

17.6

1b

Pyridin-3-yl

9.0

(6.0 - 13.5)

47.9

1c

2,4-di-MeO-Ph

5.4

(3.8 - 7.4)

31.4

1d

2,5-di-MeO-Ph

1.2

(0.8 - 1.7)

2.2

(15.6 19.8) (33.2 69.0) (26.5 37.1) (2.2 - 2.2)

SI

c

4.4 8.7 5.8 1.8

ACCEPTED MANUSCRIPT 7 1e

Thiophen-2-yl

1.1

(0.9 - 1.4)

3.7

(3.2 - 4.3)

3.4

1f

4-HO-Ph

7.4

(5.0 - 10.8)

72.6

9.8

1g

4-MeO-Ph

12.2

(6.9 - 21.4)

32.6

1h

4-Cl-Ph

18.3

(11.8 -

40.8

(57.9 91.5) (22.4 48.0) (27.9 59.6)

1i

3,4-di-MeO-Ph

4.2

(3.1 - 5.8)

9.6

1j

3,4,5-tri-MeO-Ph

2.6

(1.8 - 3.8)

14.7

1k

Furan-2-yl

0.57

(0.54 -

1.3

Morpholin-4-yl

3.9

(2.6 - 5.7)

2b

Piperidin-1-yl

4.7

(3.3 - 6.7)

2c

Diethylamino

1.1

2d

Benzylamino

3.5

2e

4-Methylpiperazin-

24.4

1-yl

13.7 6.2

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2a

(4.4 21.0) (11.2 19.2) (0.2 - 7.3)

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0.60)

2.2

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28.3)

2.7

2.3

5.6

2.3

(10.5 17.9) (5.3 - 7.3)

3.5 1.3

(0.7 - 1.6)

4.1

(2.6 - 6.5)

3.7

(2.3 - 5.3)

0.93

<1.0

(20.9 -

17.8

(0.84 1.0) (14.6 21.7)

<1.0

28.8)

Anilino

1.5

(1.0 - 2.2)

0.34

2g

3-Cl-Anilino

1.6

(1.1 - 2.2)

0.85

2h

4-Cl-Anilino

2.5

(1.8 - 3.6)

0.47

2i

4-Me-Anilino

1.7

(1.3 - 2.2)

1.3

2j

4-MeO-Anilino

1.5

(0.8 - 2.7)

0.36

3a

Ph

1.8

(1.1 - 2.9)

>100

3b

3-[4-(2-Methoxy-

1.7

(1.1 - 2.6)

12.9

2.6

(1.6 - 4.1)

55.1

(31.5 96.3)

21.2

3.0

(1.9 - 4.7)

>100

(166.0 257.3)

>33.3

0.72

(0.60 -

>100

(111.2 135.5)

>138.9

>100

(115.1 130.5)

>62.5

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2f

(0.08 1.4) (0.65 1.1) (0.41 0.54) (1.2 - 1.5)

<1.0

(0.30 0.45) (67.7 241.4) (9.8 17.0)

<1.0

<1.0 <1.0 <1.0

>55.6 7.6

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ethyl)phenoxymethyl]phenyl

3c

4-Trifluor-

methylphenyl

3d

4-Trifluormethoxyphenyl

3e

4-Methoxyphenyl

0.86) 3f

3-Methoxyphenyl

1.6

(0.8 - 3.4)

ACCEPTED MANUSCRIPT 8 3g

3-(Dimethyl-

2.3

(1.7 - 3.1)

93.9

(71.5 123.4)

40.8

0.76

(0.70 -

>100

(130.0 197.3)

>131.6

80.0

(48.7 131.2)

156.9

(183.8 307.2)

>163.9

(171.5 243.4)

>88.8

amino)phenyl 1,3-Benzodioxol5-yl 3i

0.83)

4-(Morpholin-4-yl)-

0.51

phenyl 4

0.59)

Refer to Scheme

0.61

2

(0.48 -

>100

0.77)

Refer to Scheme

2.3

(1.2 - 4.6)

>100

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5

(0.45 -

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3h

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2

a GI50 ± se – concentration that inhibits 50% growth ± standard error. DFMO (6.5 µM) was included on each plate as a trypanocidal positive control reagent, and routinely gave >95% inhibition of growth. 95% CI - 95% confidence b c interval in µM of GI50; Toxicity (GI50, µM ± se) determined against THP 1 macrophages;. Selectivity Index (SI) = GI50 THP-1/GI50 T. b. rhodesiense.

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3. Biological evaluation and discussion

Assays for activity against blood stage trypomastigotes of the new compounds revealed that most of the alpha,beta unsaturated amides 2 were equipotent or

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superior to the paullone-chalcone hybrids 1, displaying single digit micromolar GI50 values against T. b. rhodesiense bloodstream forms (Table 1). However, similar to

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the members of compound class 1, all tested alpha,beta-unsaturated amides 2 exhibited strong toxicity in the test system. In this regard, our intention to minimize the toxicity by switching from alpha,beta-unsaturated ketone structures to acrylamides was not realized. In contrast, if the Michael acceptor structure of the paullone-chalcone hybrids 1 was inverted (ref. to structure 4) or partially reduced (ref. to structure 5), the resulting compounds retained antiparasitic activity and display decreased toxicity. Therefore, we concluded on one hand that the Michael acceptor motif present in the paullone-chalcone hybrids 1 and in the acrylamides 2 is not

ACCEPTED MANUSCRIPT 9 essential for the antiparasitic activity, and on the other hand may cause unspecific toxicity. Consistent with this observation, the 2-aryl paullones 3 showed potent antitrypanosomal activity with considerably decreased toxicity for the tested human

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THP-1 cells. With the exception of 3b, all other representatives exhibited selectivity indices above 20. The best compounds of the series (3e, 3h and 3i) exhibited submicromolar antitrypanosomal activities and selectivity indices >100. For further

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development, the 2-aryl substituted paullones 3 appear to be particular promising, since they are easy to prepare, show high activities against T.b.rhodesiense

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bloodstream forms and high selectivity indices. The elucidation of the hitherto unknown mechanism of action by identification of the biological target will greatly facilitate a rational drug development in the compound class. Investigations are underway to identify such target structures from parasite lysates by pull-down assays with immobilized paullone structures. In follow-up studies, selected congeners of

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4. Conclusion

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series 3 will be evaluated for in vivo efficacy in animal models of trypanosomiasis.

Taken together, the data presented in this work demonstrate that the enone partial of

paullone-chalcone

hybrid

molecules

1

is

not

necessary

for

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structure

antitrypanosomal activity. Moreover, the results with both the chalcone derivatives 1 and the related acrylamide analogues 2 show that the Michael acceptor structure displayed by the two series may cause considerable toxicity against THP-1 cells. In contrast, substitution of the propenone partial structure in 1 or 2 for aryl rings led to derivatives 3 which inhibited the parasites with considerably improved toxicity profiles. The most potent compound identified in this study was 9-tert-butyl-2-(4morpholinophenyl)paullone (3i), which exhibited a GI50 value towards T. b.

ACCEPTED MANUSCRIPT 10 rhodesiense of 510 nM and a selectivity index of 157 versus human THP-1 macrophages. These findings warrant a further design and synthesis campaign of 2arylpaullones 3 as selective antitrypanosomal agents and further evaluation of the

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compounds in animal studies.

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5. Experimental protocols

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5.1. Synthetic Chemistry

5.1.1. General

Melting points (mp) were determined on an electric variable heater (Barnstead Electrothermal IA 9100) and were not corrected. IR-spectra were recorded as KBr discs on a Thermo Nicolet FT-IR 200. 1H NMR-spectra and

13

C NMR-spectra were

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recorded on the following instruments: Bruker Avance DRX-400 and Bruker Avance II-600; solvent DMSO-d6 if not stated otherwise; internal standard trimethylsilane; signals in ppm (δ scale). Elemental analyses were determined on a CE Instruments

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FlashEA® 1112 Elemental Analyser (Thermo Quest). Mass spectra were recorded on a double-focused sector field mass spectrometer Finnigan-MAT 90. Accurate

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measurements where conducted according to the peakmatch method using perfluorokerosene (PFK) as an internal mass reference; (EI)-MS: ionisation energy 70 eV. TLC: Polygram® Sil G/UV254, Macherey-Nagel, 40 x 80 mm, visualization by UV-illumination (254 nm). Column chromatography: silica gel 60 (Merck), column width: 2 cm, column height 10 cm unless stated otherwise. Purity was determined by HPLC using the following devices and settings: [19] Elite LaChrom (Merck/Hitachi), Pump L-2130, autosampler L-2200, Diode Array Detector L-2450, organizer box L2000; column: Merck LiChroCART 125-4, LiChrosphere 100, RP 18, 5 µm; flow rate

ACCEPTED MANUSCRIPT 11 1.000 mL/min, isocratic, volume of injection: 10 µL; detection (DAD) at 254 and 280 nm; AUC-%-method.; time of detection 15 min, net retention time (tN), dead time (tm) related to DMSO. Preparation of H2O + (Et3NH)2SO4-buffer (pH 2.5) for HPLC:

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triethylamine (20.0 mL) and sodium hydroxide (242 mg) are dissolved in water (980 mL). The solution is adjusted to pH 2.5 by addition of sulfuric acid. Preparation of H2O/TFA mixture pH 1.5 for HPLC: water is adjusted to pH 1.5 by addition of TFA.

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With the exception of 4 (purity > 88%), all compounds employed in biological tests were used in > 95% purity. The paullone-chalcone hybrids 1a-1k and the starting

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material 6 were synthesized according to the method published previously. [4] The acrylamides used as starting materials for the synthesis of the 2-substituted paullones 2a-2j were either purchased from commercial suppliers or prepared according to literature methods [20-22]. Commercially available benzaldehyde was purified by washing with saturated Na2CO3 solution prior to use. Other starting

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materials were purchased from commercial suppliers (Acros Organics, Geel, Belgium), Sigma Aldrich (St. Louis, MO, USA), Merck KGaA (Darmstadt, Germany) and were used without further purification. [4] Synthesis procedures and

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characterization of 2a-2b, 2d-2j, 3a-e, 3g-3i, 4, 5, 7, and 8 are described in detail in

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the supporting information.

5.1.2. General Procedure for the Synthesis of 2a-2j: 9-tert-Butyl-2-iodo-7,12-dihydroindolo[3,2-d][1]benzazepin-6(5H)-one (6; 215 mg, 0.500 mmol), the indicated amount of a suitable acryl amide, palladium(II)acetate (11 mg, 0.049 mmol) and triethylamine (2 mL) are stirred under nitrogen at 150 °C in DMF (5 mL). The reaction is monitored by TLC. As soon as all starting material 6 is consumed, the mixture is filtered. After addition of silical gel (1.5 g) the filtrate is

ACCEPTED MANUSCRIPT 12 evaporated to dryness. The residue is eluated over a short silica gel column (3.5 cm) using mild vacuum and the indicated eluent. The solution is evaporated and the solid residue is purified by crystallization from the indicated solvent.

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5.1.2.1. (2E)-3-(9-tert-Butyl-6-oxo-5,6,7,12-tetrahydroindolo[3,2-d][1]benzazepin-2yl)-N,N-diethylacrylamide (2c)

Prepared from 6 and N,N-diethylacrylamide (63 mg, 0.50 mmol). Reaction time 50

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min, eluated with ethyl acetate (400 mL). Crystallization from EtOH yielded 71 mg (33%) of light brown powder, mp: 286-288 °C (dec); IR (KBr): 3200 cm-1 (NH), 1667

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cm-1 (C=O); 1H NMR (DMSO-d6, 600.1 MHz): δ (ppm) = 1.09 (t, 3H, J = 7.0 Hz, CH3), 1.19 (t, 3H, J = 7.0 Hz, CH3), 1.37 (s, 9H, CH3), 3.40 (q, 2H, J = 7.0 Hz, CH3), 3.54 (q, 2H, J = 7.0 Hz, CH3), 3.55 (s, 2H, azepine CH2), 7.14 (d, 1H, J = 15.3 Hz, vinyl-H), 7.26 (d, 1H, J = 8.5 Hz, ArH), 7.29 (dd, 1H, J = 8.7/1.9 Hz, ArH), 7.40 (d, 1H, J = 8.5 Hz, ArH), 7.56 (d, 1H, J = 15.3 Hz, Vinyl-H), 7.61 (d, 1H, J =1.7 Hz, ArH), 7.74

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(dd, 1H, J = 8.5/1.9 Hz, ArH), 7.99 (d, 1H, J = 1.9 Hz, ArH), 10.25 (s, 1H, NH), 11.47 (s, 1H, NH); 13C NMR (DMSO-d6, 150.9 MHz): δ (ppm) = 13.2 (CH3), 15.2 (CH3), 31.5 (azepine CH2), 31.7 (3x CH3), 40.1 (CH2), 41.4 (CH2); 111.0, 113.5, 118.0, 120.5,

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122.4, 126.7, 127.0, 140.3 (tert. C); 34.3, 107.6, 122.9, 126.2, 130.5, 132.05, 135.6, 136.0, 141.5, 164.5, 171.3 (quat. C); C27H31N3O2 (429.55); MS (EI): m/z (%) = 429

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[M]+· (100); HRMS (EI): m/z [M]+·calcd 429.24108; found 429.24124; HPLC: 97.8% at 254 nm and 98.1% at 280 nm, tN = 7.03 min, tM = 1.02 min (ACN/H2O 50:50), λmax: 305 nm.

5.1.3. General Procedure for the Synthesis of 3a-3i: A mixture of 9-tert-butyl-2-iodo-7,12-dihydroindolo[3,2-d][1]benzazepin-6(5H)-one (6; 112 mg, 0.260 mmol), the indicated amount of a suitable boronic acid (0.54 mmol), tetrakis(triphenylphosphin)palladium(0) (10 mg, 0.0087 mmol), 2 M aqeous sodium carbonate solution (0.80 mL, 1.6 mmol), and propan-1-ol (4 mL) are stirred in a

ACCEPTED MANUSCRIPT 13 sealed microwave vessel in a synthetic monomode microwave device at 150 °C for 20 min (200 W, ramp time 5 min, maximum pressure 250 psi). After cooling, the suspension is filtered. The residue is washed successively with water and ethyl

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acetate (50 mL each). The combined filtrates are separated by means of a separation funnel. The aqueous layer is extracted with ethyl acetate (20 mL each) three times. After drying by means of anhydrous sodium sulfate, silica gel (1.5 g) is added to the

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organic layers and the solvent is evaporated. The remaining solid residue is charged onto a silica gel pad in a glass frit and eluted with ethyl acetate (200 mL) applying

crystallization from ethanol 96%.

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mild vacuum. After evaporation of the filtrate, the remaining solid is purified by

5.1.3.1. 9-tert-Butyl-2-(3-methoxyphenyl)-7,12-dihydroindolo-[3,2-d][1]benzazepin6(5H)-one (3f)

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Preparation from 6 and 4-methoxyphenylboronic acid yielded a light green powder (58%), mp: 316-317 °C (dec); IR (KBr): 3260 cm 1 (N H), 1647 cm 1 (C=O); 1H NMR (DMSO-d6, 400.4 MHz): δ (ppm) = 1.37 (s, 9H, CH3), 3.55 (s, 2H, azepine CH2), 3.82

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(s, 3H, OCH3), 7.04-7.11 (m, 2H, ArH), 7.25-7.31 (m, 2H, ArH), 7.38 (d, 1H, J = 8.6 Hz, ArH), 7.58-7.63 (m, 2H, ArH), 7.70-7.76 (m, 2H, ArH), 7.97 (d, 1H, J = 2.0 Hz,

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ArH), 10.13 (s, 1H, NH), 11.53 (s, 1H, NH); 13C NMR (DMSO d6, 100.7 MHz): δ (ppm) = 31.6 (azepine CH2), 31.8 (3x CH3), 55.2 (OCH3); 111.0, 113.5, 114.4 (2C), 120.5, 122.7, 124.3, 125.6, 127.7 (2C) (tert. C); 34.4, 107.6, 123.2, 126.3, 131.8, 132.6 134.1, 135.2, 135.6, 141.6, 158.9, 171.5 (quat. C); C27H26N2O2 (410.51); MS (EI): m/z (%) = 410.2 [M]+· (100); HRMS (EI): m/z [M]+·calcd 410.19888; found 410.19871; HPLC: 97.7% at 254 nm and 98.5% at 280 nm, tN = 6.42 min, tM = 1.07 min (ACN/H2O 65:35), λmax: 283 nm and 388 nm.

ACCEPTED MANUSCRIPT 14 5.2. Biological assays

5.2.1. Drug screening on trypomastigotes.

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Drug testing on T. b. rhodesiense strain STIB900 was performed by an alamarBlue viability assay essentially as previously described. [23, 24] Test compounds were incubated with parasites for 70 h at 37 °C. Initial screening for antitrypanosomal

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activity was carried out at 5 µM, after which GI50 values were determined by measuring growth inhibition in serial dilutions of drug. Details are described in the

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Supporting Information. All assays were carried out in triplicate. Sigmoidal doseresponse linear regression curve fitting comparing a fixed and variable slope by GraphPad Prism version 4.0b was used to calculate the GI50.

5.2.2. Toxicity assay.

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Toxicity of the compounds for mammalian cells was measured on THP-1 cells by an alamarBlue assay as previously described [4] For the assay, cells were incubated with test compounds at 37 °C for 48 hours. Details are described in the Supporting

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Information. All assays were carried out in triplicate, and the percentage inhibition of

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cell growth ± SEM (± 3.0 - 5.) calculated using Graphpad Prism version 4.0b.

Acknowledgements

Funding of the project by the Deutsche Forschungsgemeinschaft (to J. R. and C. K.) is gratefully acknowledged. This joint research project was, in part, financially supported by the State of Lower-Saxony, Hannover, Germany (to M. P., A. L., C. L. J. and C. K). C. L. J. holds the Michael and Penny Feiwel Chair in Dermatology.

ACCEPTED MANUSCRIPT 15 Appendix. Supplementary material. Supplementary data associated with this article (Synthesis procedures and characterization of 2a-2b, 2d-2j, 3a-e, 3g-3i, 4, 5, 7 and 8) can be found, in the

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online version, at doi: XXX.

[1]

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References

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Collaborative actions in anti-trypanosomatid chemotherapy with partners from disease endemic areas, Trends Parasitol. 26 (2010) 395-403. [2]

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S.R. Wilkinson, J.M. Kelly, Trypanocidal drugs: mechanisms, resistance and

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new targets, Expert Rev. Mol. Med. 11 (2009) e31.

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antileishmanial chemotype, J. Med. Chem. 51 (2008) 659-665. P. Boeck, C. Bandeira Falcao, P. Leal, R. Yunes, V. Filho, E. Torres-Santos,

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B. Rossi-Bergmann, Synthesis of chalcone analogues with increased antileishmanial activity, Bioorg. Med. Chem. 14 (2006) 1538-1545.

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M. Liu, P. Wilairat, S. Croft, A.L.-C. Tan, M.-L. Go, Structure-activity relationships of antileishmanial and antimalarial chalcones, Bioorg. Med. Chem. 11 (2003) 2729-2738. S. Nielsen, S. Christensen, G. Cruciani, A. Kharazmi, T. Liljefors,

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Heck reaction modification using ketone Mannich bases as enone precursors: parallel synthesis of anti-leishmanial chalcones, Bioorg. Med. Chem. Lett. 18 (2008) 1985-1989. [10]

C. Schultz, A. Link, M. Leost, D.W. Zaharevitz, R. Gussio, E.A. Sausville, L. Meijer, C. Kunick, Paullones, a series of cyclin-dependent kinase inhibitors:

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synthesis, evaluation of CDK1/ cyclin B inhibition, and in vitro antitumor activity, J. Med. Chem. 42 (1999) 2909-2919. [11]

X. Xie, T. Lemcke, R. Gussio, D.W. Zaharevitz, M. Leost, L. Meijer, C. Kunick,

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Epoxide-containing side chains enhance antiproliferative activity of paullones, Eur. J. Med. Chem. 40 (2005) 655-661. Y.K. Koleva, J.C. Madden, M.T. Cronin, Formation of categories from

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structure-activity relationships to allow read-across for risk assessment: toxicity of alpha,beta-unsaturated carbonyl compounds, Chem. Res. Toxicol. 21 (2008) 2300-2312.

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A. Pérez-Garrido, A.M. Helguera, G.C. López, M.N. Cordeiro, A.G. Escudero, A topological substructural molecular design approach for predicting mutagenesis end-points of alpha, beta-unsaturated carbonyl compounds, Toxicology 268 (2010) 64-77.

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resistance to gefitinib, Proc. Natl. Acad. Sci. U. S. A. 102 (2005) 7665-7670. G.P. Sagitullina, M.A. Vorontsova, A.K. Garkushenko, N.V. Poendaev, R.S. Sagitullin, Nitropyridines: X. Palladium-catalyzed cross-coupling of 2-bromo-5-

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A.-M. Egert-Schmidt, J. Dreher, U. Dunkel, S. Kohfeld, L. Preu, H. Weber, J.E.

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nitropyridine with terminal acetylenes, Russ. J. Org. Chem. 46 (2010) 1830-

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diseases: the DNDi model, Drug Des. Devel. Ther. 5 (2011) 175-181. B. Räz, M. Iten, Y. Grether-Bühler, R. Kaminsky, R. Brun, The Alamar Blue

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assay to determine drug sensitivity of African trypanosomes (T.b. rhodesiense

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and T.b. gambiense) in vitro, Acta Trop. 68 (1997) 139-147.

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Highlights

> 2-Arylpaullones and analogues were synthesized by palladium catalyzed reactions.

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> 2-Arylpaullones inhibited Trypanosoma brucei rhodesiense bloodstream forms in

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vitro. > 2-Arylpaullones exhibited good selectivity against the human THP-1 cell line.

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Chart 1: 2-Substituted Paullones

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Scheme 1. Synthesis of 2-Substituted Paullones 2 and 3.a

a

Reagents and conditions: (a) Pd(OAc)2, DMF, Et3N, 150 °C, N 2; (b) Pd(PPh3)4,

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Na2CO3, propan-1-ol, µW, 150 °C, 200 W.

Scheme 2. Synthesis of 2-Substituted Paullones 4 and 5.a

a

Reagents and conditions: (a) 1. Trimethylsilyacetylene, CuI, PdCl2(PPh3)2, Et3N,

DMF, 50 °C, 60 min., 2. TBAF x 3 H 2O, THF, N2, RT, 20 min (b) Hg(OAc)2, H2SO4,

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dust, HOAc, RT, 4 h.

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acetone, H2O, reflux, 50 min (c) benzaldehyde, KOH, EtOH, 0 °C → RT, 8 d (d) Zn

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ACCEPTED MANUSCRIPT

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Chart 1

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Scheme 1

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Scheme 2

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Supporting Information

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2-Arylpaullones are Selective Antitrypanosomal Agents

Lutz Preu,a Charles L. Jaffe,b* Conrad Kunicka*

a

Technische Universität Braunschweig, Institut für Medizinische und Pharmazeutische Chemie,

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Beethovenstraße 55, D-38106 Braunschweig, Germany b

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Jasmin Ryczak,a Ma’ayan Papini,b Annette Lader,a Abedelmajeed Nasereddin,b Dmitry Kopelyanskiy,b

Department Microbiology and Molecular Genetics, Kuvin Centre for Study of Infectious and Tropical Diseases, IMRIC, P.O. Box 12272, Hebrew University-Hadassah Medical School,

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Jerusalem 91120, Israel

Supporting Information Contents Biological assays:

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Drug screening on trypomastigotes…………………………………………………………………SI2 Toxicity assay on THP1 cells……………………………………………………………………….SI3

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Synthetic Chemistry:

General………………………………………………………………………………………………SI4 Synthesis of paullones 2a-2b, 2d-2j, 3a-e, 3g-3i, 4 and 5…………………………………….…….SI5 References………………………………………………………………………………………......SI27

SI1  

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Drug screening on trypomastigotes Trypanosoma brucei rhodesiense (STIB 900 (S704) Tanzania) blood stage trypomastigotes, a gift from Dr. Reto Brun, Swiss Tropical Institute, Basel, Switzerland, were grown in HMI-11 supplemented with 10% heat inactivated horse serum at 37 °C and 5% CO2 in 24-well flat

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bottom plates. The parasites were grown up to 1-2×106 per mL and then passaged at 1:1000 dilution in a 2 mL volume.

Drug testing was performed essentially as previously described. 1, 2 In brief, actively dividing

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blood stage trypomastigotes were diluted to 4×103 parasites/mL in HMI-11 medium. Parasites were aliquoted in triplicate (200 trypomastigotes, 50 μl/well) into 96-well flat bottom plates

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containing the drugs to be tested diluted in medium (50 μl/well). Serial drug dilutions ranging from 400 μM to 0.1 μM depending on the compound were tested in order to obtain accurate inhibition curves for calculation of the GI50’s. In order to prevent artifacts due to medium evaporation, all of the outer wells were filled with double distilled water (DDW, 100 μL/well). Negative controls, parasites in medium or medium containing 0.15% DMSO, and a positive control, medium containing 6.5 μM DFMO (DL-α-Difluoromethylornithine hydrochloride

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hydrate) final concentration, were included on each plate. Plates were incubated at 37 °C and 5% CO2 for 70 hours followed by addition of 10% alamarBlue (10 μL/well). Plates were incubated for an additional 4 hours, the fluorescence measured as described above, and the

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results analyzed using Prism 4.0b (GraphPad software).

SI2  

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Toxicity assay on THP1 cells Toxicity of the compounds for mammalian cells was measured on THP-1 cells, as previously described  3. Prior to assaying drug activity proliferation was blocked by passaging the cells into fresh THP-1 culture medium, 3 supplemented with 1 μM retinoic acid for an additional 72

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hours. Cells were harvested by centrifugation (250 × g, 10', 4 °C), washed thrice with medium, and diluted to 8×105 cells/mL. The cells were aliquoted in triplicate (125 μL/well) into 96-well flat bottom plates containing drugs diluted in complete medium (125 μL/well, containing 0.15% DMSO). Toxicity, over a range of concentrations from 400 to 0.1 μM, was

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tested in order to calculate the GI50’s. THP-1 cells in wells containing medium or medium plus 0.15% DMSO served as negative controls, while KuRei 317 (compound 12i in reference

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3) at the same final concentrations as the drugs tested, served as positive control. Cells were incubated at 37 °C and 5% CO2 for 48 hours prior to the addition of 10% alamarBlue (25 μL/well). After an additional 4 hours the fluorescence was read (λex=544 nm, λem= 590 nm) using a microplate reader (Fluoroskan Ascent FL). Results were analyzed using Prism 4

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(GraphPad software), and toxicity was determined.

SI3  

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Synthetic Chemistry: General Melting points (mp) were determined on an electric variable heater (Barnstead Electrothermal FT-IR 200. 1H NMR-spectra and

13

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IA 9100) and were not corrected. IR-spectra were recorded as KBr discs on a Thermo Nicolet C NMR-spectra were recorded on the following

instruments: Bruker Avance DRX-400 and Bruker Avance II-600; solvent DMSO-d6 if not stated otherwise; internal standard trimethylsilane; signals in ppm ( scale). Elemental analyses were determined on a CE Instruments FlashEA® 1112 Elemental Analyser (Thermo

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Quest). Mass spectra were recorded on a double-focused sector field mass spectrometer Finnigan-MAT 90. Accurate measurements where conducted according to the peakmatch

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method using perfluorokerosene (PFK) as an internal mass reference; (EI)-MS: ionisation energy 70 eV. TLC: Polygram® Sil G/UV254, Macherey-Nagel, 40 x 80 mm, visualization by UV-illumination (254 nm). Column chromatography: silica gel 60 (Merck), column width: 2 cm, column height 10 cm unless stated otherwise. Purity was determined by HPLC using the following devices and settings: Elite LaChrom (Merck/Hitachi), Pump L-2130, autosampler L-2200, Diode Array Detector L-2450, organizer box L-2000; column: Merck

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LiChroCART 125-4, LiChrosphere 100, RP 18, 5 µm; flow rate 1.000 mL/min, isocratic, volume of injection: 10 µL; detection (DAD) at 254 and 280 nm; AUC-%-method.; time of detection 15 min, net retention time (tN), dead time (tm) related to DMSO. Preparation of H2O

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+ (Et3NH)2SO4-buffer (pH 2.5) for HPLC: triethylamine (20.0 mL) and sodium hydroxide (242 mg) are dissolved in water (980 mL). The solution is adjusted to pH 2.5 by addition of sulfuric acid. Preparation of H2O/TFA mixture pH 1.5 for HPLC: water is adjusted to pH 1.5

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by addition of TFA. With the exception of 4 (purity > 88%), all compounds employed in biological tests were used in > 95% purity. The chalcone-paullone hybrids 1a-1k and the starting material 6 were synthesized according to the method published previously.  3 The acrylamides used as starting materials for the synthesis of the 2-substituted paullones 2a-2j were either purchased from commercial suppliers or prepared according to literature methods 4-6

. Commercially available benzaldehyde was purified by washing with saturated Na2CO3

solution prior to use. Other starting materials were purchased from commercial suppliers (Acros Organics, Geel, Belgium), Sigma Aldrich (St. Louis, MO, USA), Merck KGaA (Darmstadt, Germany) and were used without further purification.

SI4  

ACCEPTED MANUSCRIPT 9-tert-Butyl-2-[(1E)-3-(morpholin-4-yl)-3-oxoprop-1-en-1-yl]-7,12-dihydroindolo[3,2-

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d][1]benzazepin-6(5H)-one (2a)

9-tert-Butyl-2-iodo-7,12-dihydroindolo[3,2-d][1]benzazepin-6(5H)-one (6; 215 mg, 0.500

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mmol), 4-acryloylmorpholine (630 µL, 5.00 mmol), palladium(II)acetate (11 mg, 0.049 mmol) and triethylamine (2 mL) were suspended in DMF (5 mL). After stirring for 30 min under N2, the mixture was filtered hot. Silica gel (1.5 g) was added, and the mixture was evaporated. The residue was applied on a short silica gel column (3.5 cm) and eluted with ethyl acetate (500 mL) by mild vacuum. Evaporation of the filtrate produced a residue which was crystallized twice from EtOH to yield 89 mg (40%) of a light yellowish powder, mp 293-

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294 °C (dec); IR (KBr): 3434 cm-1 (NH), 2963 cm-1 (CH aliph.), 1669 cm-1 (C=O); 1H NMR (DMSO-d6, 600.1 MHz): δ (ppm) = 1.37 (s, 9H, CH3), 3.56 (s, 2H, azepine CH2), 3.56 - 3.75 (m, 8H, CH2), 7.25 (d, 1H, J = 8.5 Hz, ArH), 7.29 (dd, 1H, J = 8.7/1.9 Hz, ArH), 7.29 (d, 1H, J = 15.2 Hz, vinyl-H), 7.39 (d, 1H, J = 8.7 Hz, ArH), 7.58 (d, 1H, J = 15.3 Hz, vinyl-H), 7.61

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(d, 1H, J = 1.7 Hz, ArH), 7.74 (dd, 1H, J = 8.5/2.1 Hz, ArH), 8.04 (d, 1H, J = 1.9 Hz, ArH), 10.26 (s, 1H, NH), 11.46 (s, 1H, NH);

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C NMR (DMSO-d6, 150.9 MHz): δ (ppm) = 31.51

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(azepine CH2), 31.7 (3x CH3), 42.0 (CH2), 45.5 (CH2), 66.1 (CH2), 66.3 (CH2); 110.9, 113.5, 117.0, 120.5, 122.3, 126.6, 127.2, 141.0 (tert. C); 34.3, 107.6, 122.9, 126.1, 130.3, 132.0, 135.6, 136.1, 141.5, 164.4, 171.3 (quat. C); C27H29N3O3 (443.54); MS (EI): m/z (%) = 443 [M]+· (100); HRMS (EI): m/z [M]+· calcd 443.22034; found 443.22011; HPLC: 98.3% at 254 nm and 98.5% at 280 nm, tN = 5.76 min, tM = 1.02 min (ACN/H2O 45:55), λmax: 306 nm.

SI5  

ACCEPTED MANUSCRIPT 9-tert-Butyl-2-[(1E)-3-oxo-3-(piperidin-1-yl)prop-1-en-1-yl]-7,12-dihydroindolo[3,2-d]-

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[1]benzazepin-6(5H)-one (2b)

9-tert-Butyl-2-iodo-7,12-dihydroindolo[3,2-d][1]benzazepin-6(5H)-one (6; 108 mg, 0.251

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mmol), 1-acryloylpiperidine (40a; 174 mg, 1.25 mmol), palladium(II)acetate (11.0 mg, 0.0490 mmol) and triethylamine (2 mL) were stirred under N2 in DMF (5 mL) at 150 °C for 45 min. The hot mixture was filtrated and evaporated. Crystallization from EtOH yielded 27 mg (24%) of a light yellow powder, mp 277-278 °C (dec); IR (KBr): 3429 cm-1 (NH), 3194 cm-1 (NH), 2949 cm-1 (CH aliph.), 1668 cm-1 (C=O); 1H-NMR (DMSO-d6, 600.1 MHz): δ (ppm) = 1.37 (s, 9H, CH3), 1.49 - 1.55 (m, 4H, CH2), 1.61 - 1.65 (m, 2H, CH2) 3.55 (s, 2H,

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azepine CH2), 3.55 - 3.68 (m, 4H, CH2), 7.25 (d, 1H, J = 8.5 Hz, ArH), 7.29 (dd, 1H, J = 6.8/1.7 Hz, ArH), 7.30 (d, 1H, J = 15.3 Hz, vinyl-H), 7.39 (d, 1H, J = 9.0 Hz, ArH), 7.53 (d, 1H, J = 15.2 Hz, vinyl-H), 7.61 (d, 1H, J = 1.9 Hz, ArH), 7.72 (dd, 1H, J = 8.5/1.9 Hz, ArH), 8.03 (d, 1H, ArH), 10.25 (s, 1H, NH), 11.46 (s, 1H, NH); 13C-NMR (DMSO-d6, 150.9 MHz):

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δ (ppm) = 24.1 (CH2), 25.4 (CH2), 26.5 (CH2), 31.5 (azepine CH2), 31.7 (3x CH3), 42.5 (CH2), 46.0 (CH2); 110.9, 113.4, 117.7, 120.5, 122.3, 126.4, 127.2, 140.4 (tert. C); 34.3,

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107.5, 122.9, 126.1, 130.5, 132.0, 135.6, 135.9, 141.5, 164.0, 171.3 (quat. C); C28H31N3O2 (441.57); MS (EI): m/z (%) = 441 [M]+· (100) HRMS (EI): m/z [M]+· calcd 441.24108; found 441.24040; HPLC: 97.9% at 254 nm and 98.2% at 280 nm, tN = 6.04 min, tM = 1.02 min (ACN/H2O 50:50), λmax: 306 nm.

SI6  

ACCEPTED MANUSCRIPT (2E)-N-Benzyl-3-(9-tert-butyl-6-oxo-5,6,7,12-tetrahydroindolo[3,2-d][1]benzazepin-2-

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yl)acrylamide (2d)

A mixture of 9-tert-butyl-2-iodo-7,12-dihydroindolo[3,2-d][1]benzazepin-6(5H)-one (6; 215

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mg, 0.500 mmol), N-benzylacrylamide (161 mg, 1.00 mmol), palladium(II)acetate (11 mg, 0.049 mmol) and triethylamine (2 mL) was stirred in DMF (5 mL) at 150 °C under N2 for 170 min. The mixture was filtered hot. After addition of silica gel (1.5 g), the filtrate was evaporated. The residue was applied to a short column of silica gel (3.5 cm) and eluted with ethyl acetate (600 mL). After evaporation of the eluate, silica gel (1.5 g) was added and the solvent was evaporated. The residue was applied to a silica gel column (15 cm) and eluted

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with a toluene/ethyl acetate mixture. Evaporation of selected fractions yielded 62 mg (27%) of a light beige powder, mp 270-271 °C (dec); IR (KBr): 3416 cm-1 (NH), 2952 cm-1 (CH aliph.), 1666 cm-1 (C=O); 1H-NMR (DMSO-d6, 600.1 MHz): δ (ppm) = 1.37 (s, 9H, CH3), 3.55 (s, 2H, azepine CH2), 4.43 (d, 2H, J = 6.0 Hz, NCH2), 6.75 (d, 1H, J = 15.6 Hz, vinyl-H),

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7.25 - 7.31 (m, 5H, ArH), 7.34 - 7.38 (m, 3H, ArH), 7.53 (d, 1H, J = 15.8 Hz, vinyl-H), 7.57 (dd, 1H, J = 8.7/2.0 Hz, ArH), 7.61 (d, 1H, J = 1.9 Hz, ArH), 7.94 (d, 1H, J = 1.9 Hz, ArH),

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8.69 (t, 1H, J = 6.0 Hz, NH), 10.27 (s, 1H, NH), 11.52 (s, 1H, NH);

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C-NMR (DMSO-d6,

150.9 MHz): δ (ppm) = 31.5 (azepine CH2), 31.7 (3x CH3), 42.2 (CH2); 110.9, 113.5, 120.6, 121.4, 122.5, 126.2, 126.3, 126.8, 127.3 (2C), 128.3 (2C), 138.1 (tert. C); 34.3, 107.7, 123.1, 126.2, 130.1, 132.0, 135.7, 136.0, 139.3, 141.6, 164.9, 171.4 (quat. C); C30H29N3O2 (463.57); MS (EI): m/z (%) = 463 [M]+· (100); HRMS (EI): m/z [M]+· calcd 463.22543; found 463.22563; HPLC: 98.3% at 254 nm and 94.4% at 280 nm, tN = 3.43 min, tM = 1.03 min (ACN/H2O 60:40), λmax: 306 nm.

SI7  

ACCEPTED MANUSCRIPT 9-tert-Butyl-2-[(1E)-3-(4-methylpiperazin-1-yl)-3-oxopropen-1-yl]-7,12-dihydroindolo[3,2-

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d][1]benzazepin-6(5H)-one (2e)

A mixture of 9-tert-Butyl-2-iodo-7,12-dihydroindolo[3,2-d][1]benzazepin-6(5H)-one (6; 215

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mg, 0.500 mmol), 1-acryloyl-4-methylpiperazine (77 mg, 0.50 mmol), palladium(II)acetate (11 mg, 0.049 mmol) and triethylamine (2 mL) was stirred in DMF (5 mL) at 150 °C under N2 for 1 h. The mixture was filtered hot. After cooling to room temperature, the filtrate was poured into aqueous sodium hydroxide solution (4M, 20 mL). A precipitate formed, which was filtered off by suction and washed with water. Crystallization from EtOH yielded 72 mg (31%) of yellowish powder; mp 276-277 °C; IR (KBr): 3429 cm-1 (NH), 2951 cm-1 (CH

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aliph.), 1649 cm-1 (C=O); 1H-NMR (DMSO-d6, 600.1 MHz): δ (ppm) = 1.37 (s, 9H, CH3), 2.21 (s, 3H, NCH3), 2.31 - 2.36 (m, 4H, CH2), 3.55 (s, 2H, azepine CH2), 3.58 - 3.73 (m, 4H, CH2), 7.25 (d, 1H, J = 8.5 Hz, ArH), 7.29 (dd, 1H, J = 8.7/1.7, ArH) 7.30 (d, 1H, J = 15.3 Hz, vinyl-H), 7.39 (d, 1H, J = 8.5 Hz, ArH), 7.55 (d, 1H, J = 15.2 Hz, vinyl-H), 7.61 (d, 1H, J =

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1.7 Hz, ArH), 7.73 (dd, 1H, J = 8.5/1.9 Hz, ArH), 8.03 (d, 1H, J = 1.9 Hz, ArH), 10.25 (s, 1H, NH), 11.45 (s, 1H, NH); 13C-NMR (DMSO-d6, 150.9 MHz): δ (ppm) = 31.5 (azepine CH2),

AC C

31.7 (3x CH3), 41.5 (CH2), 44.9 (CH2), 45.5 (CH3), 54.3 (CH2), 55.1 (CH2); 110.9, 113.5, 117.4, 120.5, 122.3, 126.5, 127.2, 140.8 (tert. C); 34.3, 107.6, 122.9, 126.2, 130.4, 132.1, 135.6, 136.0, 141.5, 164.3, 171.3 (quat. C); C28H32N4O2 (456.58); MS (EI): m/z (%) = 456 [M]+· (70); HRMS (EI): m/z [M]+· calcd 456.25198; found 456.25164; HPLC: 99.3% at 254 nm and 99.1% at 280 nm, tN = 3.44 min, tM = 1.02 min (ACN/buffer (pH = 2.7) 35:65), λmax: 308 nm.

SI8  

ACCEPTED MANUSCRIPT (2E)-3-(9-tert-Butyl-6-oxo-5,6,7,12-tetrahydroindolo[3,2-d][1]benzazepin-2-yl)-N-

Prepared

according

to

the

General

SC

RI PT

phenylacrylamide (2f)

Procedure (6;

215

mg,

M AN U

dihydroindolo[3,2-d][1]benzazepin-6(5H)-one

from

9-tert-butyl-2-iodo-7,12-

0.500

mmol)

and

N-

phenylacrylamide (74 mg, 0.50 mmol), reaction time 55 min, elution with ethyl acetate (400 mL). Crystallization from EtOH yielded 122 mg (54%) yellow powder; mp 295-296 °C (dec); IR (KBr): 3262 cm-1 (NH), 2957 cm-1 (CH aliph.), 1668 cm-1 (C=O); 1H-NMR (DMSO-d6, 600.1 MHz): δ (ppm) = 1.37 (s, 9H, CH3), 3.57 (s, 2H, azepine CH2), 6.90 (d, 1H, J = 15.6 Hz, vinyl-H), 7.06 - 7.09 (m, 1H, ArH), 7.30 (d, 2H, J = 8.5 Hz, ArH), 7.33 - 7.36 (m, 2H,

TE D

ArH), 7.39 (d, 2H, J = 8.7 Hz, ArH), 7.62 - 7.66 (m, 3H, vinyl-H, ArH), 7.73 (d, 1H, J = 7.5 Hz, ArH), 8.01 (d, 1H, J = 1.7 Hz), 10.29 (s, 1H, NH), 10.33 (s, 1H, NH), 11.55 (s, 1H, NH); 13

C-NMR (DMSO-d6, 150.9 MHz): δ (ppm) = 31.6 (azepine CH2), 31.7 (3x CH3); 111.0,

113.5, 119.1 (2C), 120.6, 121.5, 122.6, 123.3, 126.4, 127.0, 128.7 (2C), 139.3 (tert. C); 34.3,

EP

107.8, 123.1, 126.2, 129.9, 132.0, 135.7, 136.3, 139.3, 141.6, 163.5, 171.4 (quat. C); C29H27N3O2 (449.54); MS (EI): m/z (%) = 449 [M]+· (100); HRMS (EI): m/z [M]+· calcd

AC C

449.20978; found 449.20952; HPLC: 97.6% at 254 nm and 98.1% at 280 nm, tN = 4.67 min, tM = 1.03 min (ACN/H2O 55:45), λmax: 310 nm.

SI9  

ACCEPTED MANUSCRIPT (2E)-3-(9-tert-Butyl-6-oxo-5,6,7,12-tetrahydroindolo[3,2-d][1]benzazepin-2-yl)-N-(3-

Prepared

according

to

the

SC

RI PT

chlorophenyl)acrylamide (2g)

General

Procedure

from

9-tert-butyl-2-iodo-7,12-

M AN U

dihydroindolo[3,2-d][1]benzazepin-6(5H)-one (6; 215 mg, 0.500 mmol) and N-(3chlorophenyl)acrylamide (91 mg, 0.50 mmol), reaction time 75 min, elution with ethyl acetate (400 mL). The residue was cystallized twice from EtOH to yield 19 mg (8%) of a light yellow powder; mp 283-284 °C (dec); IR (KBr): 3423 cm-1 (NH), 2955 cm-1 (CH aliph.), 1666 cm-1 (C=O); 1H-NMR (DMSO-d6, 600.1 MHz): δ (ppm) = 1.37 (s, 9H, CH3), 3.58 (s, 2H, azepine CH2), 6.86 (d, 1H, J = 15.6 Hz, vinyl-H), 7.13 - 7.15 (m, 1H, ArH), 7.29 - 7.31 (m, 2H, ArH),

TE D

7.36 - 7.39 (m, 2H, ArH), 7.54 - 7.56 (m, 1H, ArH), 7.62 (d, 1H, J = 1.7 Hz, ArH), 7.64 (dd, 1H, J = 8.5/1.9 Hz, ArH), 7.67 (d, 1H, J = 15.6 Hz, vinyl-H), 7.98 (t, 1H, J = 1.9 Hz, ArH), 8.01 (d, 1H, J = 1.9 Hz, ArH), 10.33 (s, 1H, NH), 10.48 (s, 1H, NH), 11.55 (s, 1H, NH); 13CNMR (DMSO-d6, 150.9 MHz): δ (ppm) = 31.6 (azepine CH2), 31.7 (3x CH3); 110.9, 113.5,

EP

117.5, 118.5, 120.6, 121.0, 122.6, 123.0, 126.5, 127.0, 130.5, 140.0 (tert. C); 34.3, 107.8, 123.1, 126.2, 129.7, 132.0, 133.1, 135.8, 136.4, 140.7, 141.6, 163.8, 171.4 (quat. C);

AC C

C29H26ClN3O2 (483.99); MS (EI): m/z (%) = 483 [M]+· (100); HRMS (EI): m/z [M]+· calcd 483.17081; found 483.17104; HPLC: 97.8% at 254 nm and 97.6% at 280 nm, tN = 5.21 min, tM = 1.03 min (ACN/H2O 60:40), λmax: 314 nm.

SI10  

ACCEPTED MANUSCRIPT (2E)-3-(9-tert-Butyl-6-oxo-5,6,7,12-tetrahydroindolo[3,2-d][1]benzazepin-2-yl)-N-(4-

Prepared

according

to

the

SC

RI PT

chlorophenyl)acrylamide (2h)

General

Procedure

from

9-tert-butyl-2-iodo-7,12-

M AN U

dihydroindolo[3,2-d][1]benzazepin-6(5H)-one (6; 215 mg, 0.500 mmol) and N-(4chlorophenyl)acrylamide (91 mg, 0.50 mmol), reaction time 50 min, elution with ethyl acetate (400 mL). Crystallization from EtOH yielded 142 mg (59%) light yellow powder; mp 316317 °C (dec); IR (KBr): 3368 cm-1 (NH), 2952 cm-1 (CH aliph.), 1657 cm-1 (C=O); 1H-NMR (DMSO-d6, 600.1 MHz): δ (ppm) = 1.37 (s, 9H, CH3), 3.57 (s, 2H, azepine CH2), 6.87 (d, 1H, J = 15.6 Hz, vinyl-H), 7.29 - 7.30 (m, 2H, ArH), 7.39 (d, 1H, J = 8.7 Hz, ArH), 7.40 - 7.42

TE D

(m, 2H, ArH), 7.62 - 7.64 (m, 2H, ArH), 7.66 (d, 1H, J = 15.6 Hz, vinyl-H), 7.75 - 7.77 (m, 2H, ArH), 8.01 (d, 1H, J = 1.9 Hz, ArH), 10.33 (s, 1H, NH), 10.43 (s, 1H, NH), 11.54 (s, 1H, NH);

13

C-NMR (DMSO-d6, 150.9 MHz): δ (ppm) = 31.6 (azepine CH2), 31.7 (3x CH3);

110.9, 113.5, 120.6 (3C), 121.2, 122.6, 126.5, 127.0, 128.7 (2C), 139.7 (tert. C); 34.3, 107.8,

EP

123.1, 126.2, 126.8, 129.7, 132.0, 135.7, 136.3, 138.2, 141.6, 163.6, 171.4 (quat. C); C29H26ClN3O2 (483.99); MS (EI): m/z (%) = 483 [M]+· (100); HRMS (EI): m/z [M]+· calcd

AC C

483.17081; found 483.17084; HPLC: 95.8% at 254 nm and 96.7% at 280 nm, tN = 4.41 min, tM = 1.03 min (ACN/H2O 60:40), λmax: 311 nm.

SI11  

ACCEPTED MANUSCRIPT (2E)-3-(9-tert-Butyl-6-oxo-5,6,7,12-tetrahydroindolo[3,2-d][1]benzazepin-2-yl)-N-(4-

Prepared

according

to

the

SC

RI PT

methylphenyl)acrylamide (2i)

General

Procedure

from

9-tert-butyl-2-iodo-7,12-

M AN U

dihydroindolo[3,2-d][1]benzazepin-6(5H)-one (6; 215 mg, 0.500 mmol) and N-(4methylphenyl)acrylamide (81 mg, 0.50 mmol), reaction time 90 min, elution with ethyl acetate (400 mL). Crystallization from EtOH yielded 134 mg (58%) light yellow powder; mp 336-337 °C (dec); IR (KBr): 3304 cm-1 (NH), 2957 cm-1 (CH aliph.), 1670 cm-1 (C=O); 1HNMR (DMSO-d6, 600.1 MHz): δ (ppm) = 1.37 (s, 9H, CH3), 2.27 (s, 3H, CH3), 3.57 (s, 2H, azepine CH2), 6.88 (d, 1H, J = 15.6 Hz, vinyl-H), 7.15 (d, 2H, ArH), 7.29 - 7.30 (m, 2H,

TE D

ArH), 7.39 (d, 1H, J = 8.5 Hz, ArH), 7.61 - 7.64 (m, 5H, vinyl-H, ArH), 7.99 (d, 1H, J = 1.9 Hz, ArH), 10.20 (s, 1H, NH), 10.31 (s, 1H, NH) 11.54 (s, 1H, NH);

13

C-NMR (DMSO-d6,

150.9 MHz): δ (ppm) = 20.4 (CH3), 31.6 (azepine CH2), 31.7 (3x CH3); 110.9, 113.5, 119.1 (2C), 120.6, 121.7, 122.6, 126.4, 126.9, 129.1 (2C), 139.0 (tert. C); 34.3, 107.8, 123.1, 120.6,

EP

129.9, 132.0, 132.2, 135.7, 136.2, 136.6, 141.6, 163.3, 171.4 (quat. C); C30H29N3O2 (463.57); MS (EI): m/z (%) = 463 [M]+· (100); HRMS (EI): m/z [M]+· calcd 463.22543; found

AC C

463.22517; HPLC: 97.1% at 254 nm and 97.8% at 280 nm, tN = 4.39 min, tM = 1.03 min (ACN/H2O 55:45), λmax: 311 nm.

SI12  

ACCEPTED MANUSCRIPT (2E)-3-(9-tert-Butyl-6-oxo-5,6,7,12-tetrahydroindolo[3,2-d][1]benzazepin-2-yl)-N-(4-

Prepared

according

to

the

SC

RI PT

methoxyphenyl)acrylamide (2j)

General

Procedure

from

9-tert-butyl-2-iodo-7,12-

M AN U

dihydroindolo[3,2-d][1]benzazepin-6(5H)-one (6; 215 mg, 0.500 mmol) and N-(4methoxyphenyl)acrylamide (89 mg, 0.50 mmol), reaction time 150 min, elution with ethyl acetate (400 mL). Crystallization from EtOH yielded 109 mg (46%) light beige powder; mp 307-308 °C (dec); IR (KBr): 3195 cm-1 (NH), 2956 cm-1 (CH aliph.), 1656 cm-1 (C=O); 1HNMR (DMSO-d6, 600.1 MHz): δ (ppm) = 1.37 (s, 9H, CH3), 3.57 (s, 2H, azepine CH2), 3.74 (s, 3H, OCH3), 6.86 (d, 1H, J = 15.6 Hz, vinyl-H), 6.91 - 6.94 (m, 2H, ArH), 7.28 - 7.30 (m,

TE D

2H, ArH), 7.39 (d, 1H, J = 8.5 Hz, ArH), 7.60 - 7.62 (m, 3H, vinyl-H, ArH), 7.63 - 7.66 (m, 2H, ArH), 7.99 (d, 1H, J = 1.9 Hz, ArH), 10.16 (s, 1H, NH), 10.31 (s, 1H, NH), 11.54 (s, 1H, NH); 13C-NMR (DMSO-d6, 150.9 MHz): δ (ppm) = 31.6 (azepine CH2), 31.7 (3x CH3), 55.1 (OCH3); 110.9, 113.5, 113.9 (2C), 120.5 (2C), 120.6, 121.7, 122.6, 126.3, 126.9, 138.8 (tert.

EP

C); 34.3, 107.8, 123.1, 126.2, 130.0, 132.0, 132.4, 135.7, 136.2, 141.6, 155.2, 163.0, 171.4 (quat. C); C30H29N3O3 (479.57); MS (EI): m/z (%) = 479 [M]+· (100); HRMS (EI): m/z [M]+·

AC C

calcd 479.22034; found 479.22034; HPLC: 98.3% at 254 nm and 99.2% at 280 nm, tN = 4.95 min, tM = 1.03 min (ACN/H2O 50:50), λmax: 312 nm.

SI13  

ACCEPTED MANUSCRIPT 9-tert-butyl-2-phenyl-7,12-dihydroindolo[3,2-d][1]benzazepin-6(5H)-one (3a)

HN

O

CH3

RI PT

H N

CH3 CH3

Preparation according to the general procedure from 6 and phenyl boronic acid yielded 42 mg

SC

(43%) white powder, mp: 320 °C (dec); IR (KBr): 3423 cm-1 (NH), 3254 cm-1 (NH), 1648 cm-1 (C=O); 1H NMR (DMSO-d6, 600.1 MHz): δ (ppm) = 1.37 (s, 9H, CH3), 3.56 (s, 2H,

M AN U

azepine CH2), 7.29 (dd, 1H, J = 8.6/1.7 Hz, ArH), 7.32 (d, 1H, J = 8.4 Hz, ArH), 7.39 (m, 2H, ArH), 7.48-7.53 (m, 2H, ArH), 7.62 (d, 1H, J = 0.7 Hz, ArH), 7.67 (dd, 1H, J = 8.4/2.0 Hz, ArH), 7.78-7.81 (m, 2H, ArH), 8.03 (d, 1H, J = 1.9 Hz, ArH), 10.20 (s, 1H, NH), 11.56 (s, 1H, NH); 13C NMR (DMSO-d6, 150.9 MHz): δ (ppm) = 31.7 (azepine CH2), 31.9 (3x CH3); 111.0, 113.6, 120.6, 122.8, 125.0, 126.1, 126.6 (2C), 127.4, 129.0 (2C) (tert. C); 34.4, 107.7, 123.3, 126.4, 132.6, 134.7, 135.5, 135.7, 139.5, 141.6, 171.6 (quat. C); C26H24N2O (380.48); MS

TE D

(EI): m/z (%) = 380.2 [M]+· (98); HRMS (EI): m/z [M]+·calcd 380.18831; found 380.18841; HPLC: 98.0% at 254 nm and 98.4% at 280 nm, tN = 4.09 min, tM = 1.09 min (ACN/H2O

AC C

EP

70:30), λmax: 250 nm, 276 nm and 317 nm.

SI14  

ACCEPTED MANUSCRIPT 9-tert-butyl-2-(3-{[4-(2-methoxyethyl)phenoxy]methyl}phenyl)-7,12-dihydroindolo[3,2d][1]benzazepin-6(5H)-one (3b)

O

according

to

the

general

CH3

SC

HN

RI PT

H3CO

Preparation

O

H N

procedure

from

6

CH3 CH3

and

3-{[4-(2-

M AN U

methoxyethyl)phenoxy]methyl}phenylboronic acid yielded 99 mg (69%) of off-white powder, mp: 228 °C; IR (KBr): 3419 cm-1 (NH), 3300 cm-1 (NH), 1671 cm-1 (C=O); 1H NMR (DMSO-d6, 600.1 MHz): δ (ppm) = 1.37 (s, 9H, CH3), 2.72 (t, 2H, J = 6.9 Hz, CH2), 3.22 (s, 3H, OCH3), 3.47 (t, 2H, J = 6.9 Hz, CH2), 3.56 (s, 2H, azepine CH2), 5.16 (s, 2H, OCH2), 6.94-6.98 (m, 2H, ArH), 7.13-7.16 (m, 2H, ArH), 7.28 (dd, 1H, J = 8.6/1.9 Hz, ArH), 7.37-

TE D

7.40 (m, 1H, ArH), 7.46 (d, 1H, J = 7.7 Hz, ArH), 7.53 (t, 1H, J = 7.6 Hz, ArH), 7.61 (d, 1H, J = 1.8 Hz, ArH), 7.67 (dd, 1H, J = 8.4/2.2 Hz, ArH), 7.73-7.77 (m, 1H, ArH), 7.85-7.87 (m, 1H, ArH), 8.04 (d, 1H, J = 2.2 Hz, ArH), 10.21 (s, 1H, NH), 11.56 (s, 1H, NH);

13

C NMR

(DMSO-d6, 150.9 MHz): δ (ppm) = 31.7 (azepine CH2), 31.9 (3x CH3), 34.5 (OCH2CH2Ar),

EP

57.8 (OCH3), 69.2 (OCH2), 73.1 (OCH2); 111.0, 113.6, 114.6 (2C), 120.6, 122.8, 125.0, 126.0, 126.1(2C), 126.9, 129.2, 129.8 (2C) (tert. C); 34.4, 107.8, 123.3, 126.4, 131.3, 132.5, 134.8, 135.3, 135.7, 138.0, 139.6, 141.6, 156.7, 171.6 (quat. C); C36H36N2O3 (544.68); MS (EI): m/z

AC C

(%) = 544.2 [M]+· (100); HRMS (EI): m/z [M]+·calcd 544.27204; found 544.27214; HPLC: 96.4% at 254 nm and 97.0% at 280 nm, tN = 7.38 min, tM = 1.08 min (ACN/H2O 70:30), λmax: 278 nm and 317 nm.

SI15  

ACCEPTED MANUSCRIPT 9-tert-Butyl-2-[4-(trifluoromethyl)phenyl]-7,12-dihydroindolo[3,2-d][1]benzazepin-6(5H)one (3c)

F

F HN

O

RI PT

H N

CH3

CH3 CH3

SC

F

Preparation according to the general procedure from 6 and 4-(trifluoromethyl)phenylboronic

M AN U

acid yielded 31 mg (27%) of off-white powder, mp: 343 °C (dec); IR (KBr): 3272 cm-1 (NH), 3082 cm-1 und 3052 cm-1 (CH, arom.), 2957 cm-1 (CH, aliph.), 1648 cm-1 (C=O); 1H NMR (DMSO-d6, 400.4 MHz): δ (ppm) = 1.37 (s, 9H, CH3), 3.58 (s, 2H, azepine CH2), 7.29 (dd, 1H, J = 8.6/1.9 Hz, ArH), 7.36 (d, 1H, J = 8.5 Hz, ArH), 7.40 (dd, 1H, J = 8.6/0.5 Hz, ArH), 7.62 (d, 1H, J = 1.8 Hz, ArH), 7.75 (dd, 1H, J = 8.5/2.2 Hz, ArH), 7.84-7.90 (m, 2H, ArH), 13

TE D

8.01-8.06 (m, 2H, ArH), 8.11 (d, 1H, J = 2.2 Hz, ArH), 10.26 (s, 1H, NH), 11.57 (s, 1H, NH); C NMR (DMSO-d6, 100.7 MHz): δ (ppm) = 31.7 (azepine CH2), 31.8 (3x CH3); 111.0,

113.6, 120.7, 122.9, 125.4, 125.8, 125.9, 126.3, 127.3 (2C) (tert. C); 34.4, 107.8, 123.1, 123.3, 127.9, 132.3, 133.7, 135.5, 135.7, 141.7, 143.4, 171.5 (2C) (quat. C); C27H23N2O (448.48);

EP

MS (EI): m/z (%) = 448.2 [M]+· (86); HRMS (EI): m/z [M]+·calcd 448.17570; found 448.17574; HPLC: 97.4% at 254 nm and 99.0% at 280 nm, tN = 4.12 min, tM = 1.11 min

AC C

(ACN/H2O 75:25), λmax: 283 nm.

SI16  

ACCEPTED MANUSCRIPT 9-tert-Butyl-2-[4-(trifluoromethoxy)phenyl]-7,12-dihydroindolo[3,2-d][1]benzazepin-6(5H)one (3d)

F F

F HN

O

O

RI PT

H N

CH3

SC

CH3 CH3

Preparation according to the general procedure from 6 and 4-(trifluoromethoxy)phenylboronic

M AN U

acid yielded 75 mg (70%) of off-white powder, mp: 321-327 °C (dec); IR (KBr): 3276 cm-1 (NH), 1649 cm-1 (C=O); 1H NMR (DMSO-d6, 400.4 MHz): δ (ppm) = 1.37 (s, 9H, CH3), 3.57 (s, 2H, azepine CH2), 7.29 (dd, 1H, J = 8.6/1.8 Hz, ArH), 7.33 (d, 1H, J = 8.5 Hz, ArH), 7.38 (dd, 1H, J = 8.6/0.5 Hz, ArH), 7.48-7.53 (m, 2H, ArH), 7.62 (d, 1H, J = 1.8 Hz, ArH), 7.69 (dd, 1H, J = 8.4/2.2 Hz, ArH), 7.88-7.94 (m, 2H, ArH), 8.04 (d, 1H, J = 2.2 Hz, ArH), 10.22

TE D

(s, 1H, NH), 11.54 (s, 1H, NH); 13C NMR (DMSO-d6, 100.7 MHz): δ (ppm) = 31.6 (azepine CH2), 31.8 (3x CH3); 111.0, 113.5, 120.6, 121.5 (2C), 122.8, 125.2, 126.1, 128.4 (2C) (tert. C); 34.4, 107.7, 121.4, 123.3, 126.3, 132.4, 134.0, 135.0, 135.7, 138.8, 141.6, 147.8, 171.5 (quat. C); C27H23F3N2O2 (464.48); MS (EI): m/z (%) = 464.2 [M]+· (88); HRMS (EI): m/z

EP

[M]+·calcd 464.17061; found 464.17018; HPLC: 98.5% at 254 nm and 98.6% at 280 nm, tN =

AC C

6.38 min, tM = 1.08 min (ACN/H2O 70:30), λmax: 277 nm, 318 nm and 387 nm.

SI17  

ACCEPTED MANUSCRIPT 9-tert-Butyl-2-(4-methoxyphenyl)-7,12-dihydroindolo[3,2-d][1]-benzazepin-6(5H)-one (3e)

H3C

HN

O

O

RI PT

H N

CH3

SC

CH3 CH3

Preparation according to the general procedure from 6 and 4-methoxyphenylboronic acid yielded a light green powder (58%), mp: 316-317 °C (dec); IR (KBr): 3260 cm-1 (NH), 1647

M AN U

cm-1 (C=O); 1H NMR (DMSO-d6, 400.4 MHz): δ (ppm) = 1.37 (s, 9H, CH3), 3.55 (s, 2H, azepine CH2), 3.82 (s, 3H, OCH3), 7.04-7.11 (m, 2H, ArH), 7.25-7.31 (m, 2H, ArH), 7.38 (d, 1H, J = 8.6 Hz, ArH), 7.58-7.63 (m, 2H, ArH), 7.70-7.76 (m, 2H, ArH), 7.97 (d, 1H, J = 2.0 Hz, ArH), 10.13 (s, 1H, NH), 11.53 (s, 1H, NH); 13C NMR (DMSO-d6, 100.7 MHz): δ (ppm) = 31.6 (azepine CH2), 31.8 (3x CH3), 55.2 (OCH3); 111.0, 113.5, 114.4 (2C), 120.5, 122.7,

TE D

124.3, 125.6, 127.7 (2C) (tert. C); 34.4, 107.6, 123.2, 126.3, 131.8, 132.6 134.1, 135.2, 135.6, 141.6, 158.9, 171.5 (quat. C); C27H26N2O2 (410.51); MS (EI): m/z (%) = 410.2 [M]+· (100); HRMS (EI): m/z [M]+·calcd 410.19888; found 410.19871; HPLC: 97.7% at 254 nm and

AC C

EP

98.5% at 280 nm, tN = 6.42 min, tM = 1.07 min (ACN/H2O 65:35), λmax: 283 nm and 388 nm.

SI18  

ACCEPTED MANUSCRIPT 9-tert-Butyl-2-[3-(dimethylamino)phenyl]-7,12-dihydroindolo[3,2-d][1]benzazepin-6(5H)one (3g)

H N

O

N H3C HN

RI PT

CH3

CH3

SC

CH3 CH3

Preparation according to the general procedure from 6 and 3-(dimethylamino)phenylboronic

M AN U

acid yielded 77 mg (69%) of a yellow powder, mp: 310 °C (dec); IR (KBr): 3264 cm-1 (NH), 1652 cm-1 (C=O); 1H NMR (DMSO-d6, 400.4 MHz): δ (ppm) = 1.37 (s, 9H, CH3), 2.98 (s, 6H, N(CH3)2), 3.55 (s, 2H, azepine CH2), 6.73-6.78 (m, 1H, ArH), 7.02-7.07 (m, 2H, ArH), 7.26-7.32 (m, 3H, ArH), 7.39 (dd, 1H, J = 8.6/0.5 Hz, ArH), 7.61 (d, 1H, J = 1.7 Hz, ArH), 7.64 (dd, 1H, J = 8.4/2.2 Hz, ArH), 7.98 (d, 1H, J = 2.1 Hz, ArH), 10.15 (s, 1H, NH), 11.53 (s,

TE D

1H, NH); 13C NMR (DMSO-d6, 100.7 MHz): δ (ppm) = 31.6 (azepine CH2), 31.8 (3x CH3), 40.3 (2C, N(CH3)2); 110.6, 111.0, 111.6, 113.5, 114.8, 120.5, 122.6, 124.9, 126.3, 139.4 (tert. C); 34.4, 107.7, 123.2, 126.4, 132.6, 134.5, 135.7, 136.6, 140.3, 141.6, 151.0, 171.5 (quat. C); C28H29N3O (423.23); MS (EI): m/z (%) = 423.2 [M]+· (100); HRMS (EI): m/z [M]+·calcd =

1.12

min

(ACN/H2O

75:25),

AC C

tM

EP

423.23105; found 423.23117; HPLC: 97.2% at 254 nm and 97.4% at 280 nm, tN = 6.77 min,

SI19  

λmax:

268

nm

and

318

nm.

ACCEPTED MANUSCRIPT

2-(1,3-Benzodioxol-5-yl)-9-tert-butyl-7,12-dihydroindolo[3,2-d][1]benzazepin-6(5H)-one (3h)

O HN

O

O

RI PT

H N

CH3

SC

CH3 CH3

M AN U

Preparation according to the general procedure from 6 and 1,3-benzodioxol-5-ylboronic acid yielded 69 mg (62%) of off-white powder, mp: 307 °C (dec); IR (KBr): 3428 and 3259 cm-1 (NH), 1650 cm-1 (C=O); 1H NMR (DMSO-d6, 400.4 MHz): δ (ppm) = 1.37 (s, 9H, CH3), 3.55 (s, 2H, azepine CH2), 6.08 (s, 2H, dioxole CH2), 7.05 (d, 1H, J = 8.1 Hz, ArH), 7.26 (d, 1H, J = 1.7 Hz, ArH), 7.27-7.30 (m, 2H, ArH), 7.38 (dd, 1H, J = 8.6/0.5 Hz, ArH), 7.40 (d, 1H, J = 1.7 Hz, ArH), 7.58-7.62 (m, 2H, ArH), 7.96 (d, 1H, J = 2.2 Hz, ArH), 10.14 (s, 1H, NH), 13

C NMR (DMSO-d6, 100.7 MHz): δ (ppm) = 31.6 (azepine CH2), 31.8

TE D

11.51 (s, 1H, NH);

(3x CH3), 101.2 (dioxole CH2); 107.0, 108.7, 110.9, 113.5, 120.1, 120.5, 122.7, 124.5, 125.8, (tert. C); 34.4, 107.6, 123.2, 126.3, 132.6, 133.7, 134.3, 135.2, 135.6, 141.6, 146.8, 148.1, 171.4 (quat. C); C27H24N2O3 (424.50); MS (EI): m/z (%) = 424.2 [M]+· (100); HRMS (EI):

EP

m/z [M]+·calcd 424.17868; found 424.17878; HPLC: 97.8% at 254 nm and 98.9% at 280 nm,

AC C

tN = 6.23 min, tM = 1.09 min (ACN/H2O 65:35), λmax: 283 nm, 307 nm and 383 nm.

SI20  

ACCEPTED MANUSCRIPT 9-tert-Butyl-2-[4-(4-morpholinyl)phenyl]-7,12-dihydroindolo[3,2-d][1]benzazepin-6(5H)-one (3i)

HN

N

O

RI PT

H N

CH3

CH3 CH3

SC

O

Preparation according to the general procedure from 6 and 4-(4-morpholinyl)phenylboronic

M AN U

acid yielded 23 mg (19%) of yellow powder, mp: 312 °C (dec); IR (KBr): 3423 and 3318 cm-1 (NH), 1655 cm-1 (C=O); 1H NMR (DMSO-d6, 600.1 MHz): δ (ppm) = 1.37 (s, 9H, CH3), 3.15-3.21 (m, 4H, CH2 morpholine), 3.54 (s, 2H, azepine CH2), 3.74-3.79 (m, 4H, CH2 morpholine), 7.05-7.09 (m, 2H, ArH), 7.27 (d, 1H, J = 8.5 Hz, ArH), 7.29 (d, 1H, J = 1.8 Hz, ArH), 7.38 (d, 1H, J = 8.7 Hz, ArH), 7.59-7.62 (m, 2H, ArH), 7.65-7.71 (m, 2H, ArH), 7.96

TE D

(d, 1H, J = 2.1 Hz, ArH), 10.11 (s, 1H, NH), 11.53 (s, 1H, NH); 13C NMR (DMSO-d6, 150.9 MHz): δ (ppm) = 31.5 (azepine CH2), 31.7 (3x CH3), 48.1 (2x NCH2CH2O), 66.0 (2x NCH2CH2O); 110.9, 113.4, 115.2 (2C), 120.4, 122.6, 123.8, 125.2, 127.0 (2C) (tert. C); 34.3, 107.5, 123.2, 126.2, 129.8, 132.6, 133.7, 135.3, 135.5, 141.5, 150.4, 171.4 (quat. C);

EP

C30H31N3O2 (465.59); MS (EI): m/z (%) = 465.3 [M]+· (100); HRMS (EI): m/z [M]+·calcd 465.24164; found 465.24178; HPLC: 95.0% at 254 nm and 97.3% at 280 nm, tN = 6.18 min,

AC C

tM = 1.08 min (ACN/H2O 65:35), λmax: 308 nm.

SI21  

ACCEPTED MANUSCRIPT

RI PT

9-tert-Butyl-2-[(trimethylsilyl)ethynyl]-7,12-dihydroindolo[3,2-d][1]benzazepin-6(5H)-one

SC

9-tert-Butyl-2-iodo-7,12-dihydroindolo[3,2-d][1]benzazepin-6(5H)-one (6; 430 mg, 1.00 mmol), bis(triphenylphosphin)palladium(II)dichloride (14 mg, 0.020 mmol), copper(I)iodide

M AN U

(7.6 mg, 0.040 mmol), and triethylamine (5 mL) were stirred in DMF (10 mL) at 50 °C under N2. Trimethylsilylacetylene (555 µL, 4.00 mmol) was added drop wise by means of a dropping funnel, which was afterwards rinsed with DMF (1 mL). After stirring for 1 h at 50°C under N2, acetone (50 mL) was added and the mixture was filtered. After addition of silica gel (1.5 g), the filtrate was evaporated. The residue was applied to a silica gel column (15 cm) and the product was eluted by a mixture of toluene/cyclohexane (1:1). Evaporation of selected

TE D

fractions yielded raw material, which was crystallized from EtOH to yield 221 mg (55%) light brown crystals, which were used for the following steps without further purification. Mp 297298 °C; IR (KBr): 3436 cm-1 (NH), 2957 cm-1 (CH aliph.), 2155 cm-1 (-C≡C-), 1671 cm-1 (C=O); 1H-NMR (DMSO-d6, 400.4 MHz): δ (ppm) = 0.25 (s, 9H, CH3 trimethylsilyl), 1.36 (s,

EP

9H, CH3), 3.54 (s, 2H, azepine CH2), 7.22 (d, 1H, J = 8.5 Hz, ArH), 7.28 (dd, 1H, J = 8.8/2.0 Hz, ArH), 7.35 (dd, 1H, J = 8.5/0.5 Hz, ArH), 7.43 (dd, 1H, J = 8.3/1.8 Hz, ArH), 7.60 (d, 13

AC C

1H, J = 1.5 Hz, ArH), 7.84 (d, 1H, J = 2.0 Hz, ArH), 10.27 (s, 1H, NH), 11.51 (s, 1H, NH); C-NMR (DMSO-d6, 100.7 MHz): δ (ppm) = 0.1 (3x CH3 trimethylsilyl), 31.6 (azepine

CH2), 31.8 (3x CH3); 111.1, 113.5, 120.7, 122.4, 130.0, 130.6 (tert. C); 34.4, 93.8, 104.8, 107.9, 117.1, 123.0, 126.2, 131.4, 135.6, 135.8, 141.7, 171.4 (quat. C); C25H28N2OSi (400.59); HPLC: 99.0% at 254 nm and 99.1% at 280 nm, tN = 4.16 min, tM = 1.03 min (ACN/H2O 75:25), λmax: 279 nm, 232 nm and 318 nm.

SI22  

ACCEPTED MANUSCRIPT

RI PT

9-tert-Butyl-2-ethynyl-7,12-dihydroindolo[3,2-d][1]benzazepin-6(5H)-one (7)

added

dropwise

to

a

solution

of

SC

A solution of tetrabutylammonium fluoride trihydrate (1.27 g, 4.03 mmol) in THF (5 mL) was 9-tert-butyl-2-[(trimethylsilyl)ethinyl]-7,12-

M AN U

dihydroindolo[3,2-d][1]benzazepin-6(5H)-one (338 mg, 0.844 mmol) in THF (5 mL) and stirred for 20 min under N2 at room temperature. After addition of saturated sodium chloride solution (30 mL) the mixture was extracted three times with ethyl acetate (30 mL each). The combined organic layers were dried over sodium sulfate and evaporated to yield 257 mg (93%) of a beige powder, mp > 360 °C (dec), 1H-NMR (DMSO-d6, 400.0 MHz): δ (ppm) = 1.36 (s, 9H, CH3), 3.54 (s, 2H, azepine CH2), 4.21 (s, 1H, C≡CH), 7.23 (d, 1H, J = 8.6 Hz,

TE D

ArH), 7.28 (dd, 1H, J = 8.6/1.8 Hz, ArH), 7.36 (d, 1H, J = 8.6 Hz, ArH), 7.45 (dd, 1H, J = 8.3/1.8 Hz, ArH), 7.60 (d, 1H, J = 1.5 Hz, ArH), 7.86 (d, 1H, J = 1.8 Hz, ArH), 10.25 (s, 1H, NH), 11.50 (s, 1H, NH); 13C-NMR (DMSO-d6, 100.6 MHz): δ (ppm) = 31.6 (azepine CH2), 31.7 (3x CH3); 80.4 (C ethinyl), 83.1 (C ethinyl); 111.1, 113.5, 120.7, 122.4, 130.1, 130.7

EP

(tert. C); 34.3, 108.0, 116.7, 123.1, 126.2, 131.4, 135.5, 135.8, 141.7, 171.4 (quat. C). The

AC C

material was used for the following reaction steps without further purification.

SI23  

ACCEPTED MANUSCRIPT

RI PT

2-Acetyl-9-tert-butyl-7,12-dihydroindolo[3,2-d][1]benzazepin-6(5H)-one (8)

SC

9-tert-Butyl-2-ethynyl-7,12-dihydroindolo[3,2-d][1]benzazepin-6(5H)-one (7; 181 mg, 0.551 mmol) was dissolved in 70% aqueous acetone (20 mL). Concentrated H2SO4 (0.0588 mL,

M AN U

1.10 mmol) and Hg(II)acetate (176 mg, 0.552 mmol) were added and the mixture was refluxed until the starting material was no longer detectable by TLC (50 min). After cooling to room temperature, the mixture was neutralized by addition of saturated sodium carbonate solution. After addition of water (30 mL), the mixture was extracted three times with ethyl acetate (30 mL each). The combined organic layers were dried (Na2SO4). After addition of silica gel (1.5 mg), the mixture was evaporated. The residue was applied to a short silica gel

TE D

column (3.5 cm) and eluted with ethyl acetate (400 mL) by mild vacuum. Evaporation of the eluate yielded 109 mg (57%) of a beige powder; Mp > 360 °C (dec);1H-NMR (DMSO-d6, 400.0 MHz): δ (ppm) = 1.37 (s, 9H, CH3), 2.65 (s, 3H, CH3), 3.58 (s, 2H, azepine CH2), 7.30 (dd, 1H, J = 8.6/1.8 Hz, ArH), 7.34 (d, 1H, J = 8.6 Hz, ArH), 7.40 (d, 1H, J = 8.5 Hz, ArH),

EP

7.62 (d, 1H, J = 1.8 Hz, ArH), 7.92 (dd, 1H, J = 8.3/2.3 Hz, ArH), 8.36 (d, 1H, J = 2.0 Hz, ArH) 10.42 (s, 1H, NH), 11.62 (s, 1H, NH);

13

C-NMR (DMSO-d6, 150.9 MHz): δ (ppm) =

AC C

26.6, 31.6 (azepine CH2), 31.7 (3x CH3); 111.0, 113.5, 126.7, 122.0, 127.3, 127.5, (tert. C); 34.3, 107.8, 122.4, 126.1, 131.7, 131.9, 135.8, 138.9, 141.6, 171.9, 196.6 (quat. C). The material was used for the following reaction step without further purification.

SI24  

ACCEPTED MANUSCRIPT 9-tert-Butyl-2-[(2E)-3-phenylprop-2-enoyl]-7,12-dihydroindolo[3,2-d][1]benzazepin-6(5H)-

SC

RI PT

one (4)

A mixture of benzaldehyde (440 µL, 4.35 mmol) and 2-acetyl-9-tert-butyl-7,12-dihydro-

M AN U

indolo[3,2-d][1]benzazepin-6(5H)-one (8; 377 mg, 1.09 mmol) in EtOH (8 mL) was stirred at 0 °C. The mixture was adjusted to pH 9 by addition of a 5.5% aqueous KOH. Stirring was continued for 15 min at 0 °C and for 8 d at room temperature. After neutralizing by means of 10% hydrochloric acid,water (50 mL) was added and the mixture was extracted four times with ethyl acetate (60 mL each). Evaporation of the combined organic layers yielded 414 mg (87.5%) raw product, which was contaminated by several side products (TLC). Addition of

TE D

silica gel (1.5 g) before evaporation and application of the evaporation residue to the head of a silica gel column (25 cm) yielded 59 mg (12%) of the product by column chromatography (eluent toluene/ethyl acetate 1:1). An analytical sample was prepared by crystallization of this material from EtOH, yielding 11 mg (2%) of a yellow powder; mp 346-347 °C (dec); IR

EP

(KBr): 3274 cm-1 (NH), 2954 cm-1 (CH aliph.), 1655 cm-1 (C=O); 1H-NMR (DMSO-d6, 600.1 MHz): δ (ppm) =: 1.38 (s, 9H, CH3), 3.62 (s, 2H, azepine CH2), 7.31 (dd, 1H, J = 8.7/1.9 Hz,

AC C

ArH), 7.40 (d, 1H, J = 8.5 Hz, ArH), 7.42 (d, 1H, J = 8.5 Hz, ArH), 7.48 - 7.52 (m, 3H, ArH), 7.64 (d, 1H, J = 1.9 Hz, ArH), 7.83 (d, 1H, J = 15.6 Hz, vinyl-H), 7.93 (dd, 2H, J =8.1/2.0 Hz, ArH), 8.06 (d, 1H, J = 15.6, vinyl-H), 8.15 (dd, 1H, J = 8.7/2.1 Hz, ArH), 8.53 (d, 1H, J = 2.0 Hz, ArH), 10.48 (s, 1H, NH), 11.65 (s, 1H, NH); 13C-NMR (DMSO-d6, 150.9 MHz): δ (ppm) = 31.6 (azepine CH2), 31.7 (3x CH3); 111.0, 113.5, 120.7. 121.9, 122.1, 127.7, 127.8, 128.8 (2C), 128.9 (2C), 130.6, 143.7 (tert. C); 34.3, 107.8, 122.6, 126.2, 131.7, 132.7, 134.6, 135.8, 139.0, 141.6, 171.4, 187.6 (quat. C); C29H26N2O2 (434.53); MS (EI): m/z (%) = 434 [M]+· (92); HRMS (EI): m/z [M]+· calcd 434.19888; found 434.19902; HPLC: 89.7 % at 254 nm and 88.5% at 280 nm, tN = 3.35 min, tM = 1.03 min (ACN/H2O 70:30), λmax: 314 nm and 225 nm. SI25  

ACCEPTED MANUSCRIPT

9-tert-Butyl-2-(3-oxo-3-phenylpropyl)-7,12-dihydroindolo[3,2-d][1]benzazepin-6(5H)-one

SC

RI PT

(5)

M AN U

9-tert-Butyl-2-[(1E)-3-oxo-3-phenylprop-1-enyl]-7,12-dihydroindolo[3,2-d][1]benzaze-pin6(5H)-one (1a; 485 mg, 1.12 mmol) was stirred with zinc dust (742 mg, 11.2 mmol)in acetic acid (15 mL) at room temperature. The reaction progress was monitored by TLC. After complete consumption of the starting material (4 h) the mixture was filtered. The residue was washed three times with ethyl acetate (20 mL each). The combined organic layers were evaporated and the residue was purified twice by flash chromatography (first eluent:

TE D

toluene/ethyl acetate 1:1; second eluent toluene/ethyl acetate 2:1) to yield 67 mg (14%) white powder, mp 270-271 °C; IR (KBr): 3284 cm-1 (NH), 2958 cm-1 (CH aliph.),

1691 cm-1

(C=O), 1646 cm-1 (C=O); 1H-NMR (DMSO-d6, 600.1 MHz): δ (ppm) = 1.36 (s, 9H, CH3), 3.01 (t, 2H, J = 7.53 Hz, CH2), 3.47 (t, 2H, J = 7.53 Hz, CH2), 3.48 (s, 2H, azepine CH2), 7.15

EP

(d, 1H, J = 8.28 Hz, ArH), 7.26 (dd, 1H, J = 8.66/1.89 Hz, ArH), 7.29 (dd, 1H, J = 8.28/2.07 Hz, ArH), 7.37 (dd, 1H, J = 8.66/0.56 Hz, ArH), 7.52 - 7.55 (m, 2H, ArH), 7.59 (dd, 1H, J =

AC C

1.69 Hz, ArH), 7.63 - 7.66 (m, 2H, ArH), 8.01 - 8.03 (m, 2H, ArH), 10.01 (s, 1H, NH), 11.40 (s, 1H, NH); 13C-NMR (DMSO-d6, 150.9 MHz): δ (ppm) = 28.9 (CH2), 31.5 (azepine CH2), 31.7 (3x CH3), 39.4 (CH2); 110.9, 113.3, 120.3, 122.1, 126.4, 127.9 (2C), 128.0, 128.7 (2C), 133.1 (tert. C); 34.3, 107.4, 122.8, 126.2, 132.6, 133.3, 135.5, 136.5, 136.5, 141.4, 171.4, 199.0 (quat. C); C29H28N2O2 (436.55); calcd C 79.79, H 6.46, N 6.42; found C 79.77, H 6.40 N 6.14; MS (EI): m/z (%) = 436 [M]+· (100); HRMS (EI): m/z [M]+· calcd 436.21453; found 436.21493; HPLC: 97.4% at 254 nm and 94.6% at 280 nm, tN = 5.93 min, tM = 1.03 min (ACN/H2O 50:50), λmax: 234 nm , 317 nm and 395 nm.

SI26  

ACCEPTED MANUSCRIPT

References

(4) (5)

AC C

EP

TE D

(6)

RI PT

(3)

SC

(2)

Chatelain, E., Ioset, J.-R. Drug discovery and development for neglected diseases: the DNDi model. Drug Des. Devel. Ther. 2011, 5, 175-181. Räz, B., Iten, M., Grether-Bühler, Y., Kaminsky, R., Brun, R. The Alamar Blue assay to determine drug sensitivity of African trypanosomes (T.b. rhodesiense and T.b. gambiense) in vitro. Acta Trop. 1997, 68, 139-147. Reichwald, C., Shimony, O., Dunkel, U., Sacerdoti-Sierra, N., Jaffe, C. L., Kunick, C. 2-(3Aryl-3-oxopropen-1-yl)-9-tert-butyl-paullones: a new antileishmanial chemotype. J. Med. Chem. 2008, 51, 659-665. Eriksson, J., Åberg, O., Långström, B. Synthesis of [11C]/[13C]acrylamides by palladiummediated carbonylation. Eur. J. Org. Chem. 2007, 455-461. Le Sann, C., Huddleston, J., Mann, J. Synthesis and preliminary evaluation of novel analogues of quindolines as potential stabilisers of telomeric G-quadruplex DNA. Tetrahedron 2007, 63, 12903-12911. Kuhnert, N., Le-Gresley, A. Synthesis and capsule formation of upper rim substituted tetraacrylamido calix[4]arenes. Org. Biomol. Chem. 2005, 3, 2175-2182.

M AN U

(1)

SI27