University of Groningen Development of novel anticancer agents for protein targets Estrada Ortiz, Natalia

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University of Groningen

Development of novel anticancer agents for protein targets

Estrada Ortiz, Natalia

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2017

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2,3'-B

IS

(1'H-

INDOLE

)

H

ETEROCYCLES

:

N

EW

P

53/MDM2/MDMX

A

NTAGONISTS

Constantinos G. Neochoritis,

a

Kan Wang,

b

Natalia Estrada-Ortiz,

a

Eberhardt

Herdtweck,

c

Katarzyna Kubica,

d

Aleksandra Twarda,

e, f

Krzysztof M. Zak,

e

Tad A.

Holak,

d, e

and Alexander Dömlinga,

b*

a

Department of Drug Design, University of Groningen, A. Deusinglaan 1, 9713 AV Groningen, The Netherlands.

b

Department of Chemistry, University of Pittsburgh, 3501 Fifth Avenue, Pittsburgh, PA 15261, USA.

c_{Department Chemie, Technische Universität München, Lichtenbergstrasse 4, D-85747 Garching bei München,}

Germany.

d

Department of Organic Chemistry, Jagellonian University, Ingardena 3, 30-060 Krakow, Poland.

e

Malopolska Centre of Biotechnology, Jagiellonian University, Gronostajowa 7a, 30-387 Krakow, Poland

f

Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Gronostajowa 7, 30-387 Krakow, Poland.

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Abstract: The protein-protein interaction of p53 and MDM2/X is a promising non genotoxic anticancer target. A rapid and efficient methodology was developed to synthesize the 2,3'-bis(1'H-indole) heterocyclic scaffold 2 as ester, acid and amide derivatives. Their binding affinity with MDM2 was evaluated using both fluorescence polarization (FP) assay and HSQC experiments, indicating good inhibition and a perfect starting point for further optimizations.

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The transcription factor and tumor suppressor p53 has multiple roles in stabilizing the genome by preventing mutations.1,2_{However p53, our genome guardian, is the most mutated protein in human}

cancer with >50% of cancers not showing functional p53.3 _{The remainder group of cancers also exhibit}

reduced p53 pathway activity due to the negative regulation through the protein-protein interaction (PPI) with inhibitors as MDM2/X or viral proteins. MDM2 is a key regulator of p53 activity via a complex regulatory feedback system that involves all levels of expression control including transcription, mRNA translation and protein degradation. MDM2 inhibits the N-terminal trans-activation domain (TAD) of p53, and promotes p53 degradation through the ubiquitin-proteasome system (E3 ligase activity).4,5_{Similarly to MDM2, MDMX (90% homology with MDM2) its negative}

regulation of p53 is via inhibition of the TAD domain and forming heterodimers with MDM2 can increase the rates of ubiquitinylation of p53 by MDM2.6,7_{Therefore, blocking the interaction between}

wild-type p53 and its negative regulators MDM2 and MDMX has become an important target in oncology to restore the anti-tumor activity of p53.8–10

The discovery of novel p53-MDM2/X inhibitors was one of the highlights in anti-tumor agents.11 Since the disclosure of Nutlin-3,12_{many scaffolds have been prepared and evaluated, including}

indo-imidazole, imidazoline, benzodiazepinedione and spirooxindole scaffolds.13 Thus, antagonizing the p53-DDϮ ŽƌͬĂŶĚ DDɍ ŝŶƚĞƌĂĐƚŝŽŶ ŝƐ Ă ƉƌŽŵŝƐŝŶŐ ĂŶƚŝĐĂŶĐĞƌ Ɛtrategy where several compounds presently undergo early clinical evaluations.14–17_{However, the discovery of new p53/MDM2/MDMX}

scaffolds is still of high interest due to low single agent activity currently seen in clinical trials and insufficient PKPD properties.

A three finger pharmacophore model is now widely accepted to be responsible for the binding of small molecules to the MDM2 and recently an extended four finger model was experimentally shown by co-crystallization.18,19 The corresponding amino acid residues in p53 are tryptophan (Trp23), leucine (Leu26) and phenylalanine (Phe19). Initially, indole fragment was taken as starting point to mimic tryptophan residue, where an important hydrogen bond was observed. Studies by Garcia-Echeverria et al. on a p53-derived linear octapeptide showed that a Trp23 to (6-Cl) Trp substitution gave rise to a 63-fold increase in affinity for MDM2.20 _{The starting point for our antagonist discoveries was the}

anchoring side chain of tryptophan embedded in a deep hydrophobic pocket formed by the residues Leu57, Phe86 and Ile99 using our pharmacophore based virtual screening platform ANCHOR.QUERY.10,18,21–25_{This had led to several scaffolds potently antagonizing p53-MDM2. Amongst}

them we could also solve representative co-crystal structures of the imidazoloindole derivative 1 binding to MDM2 (PDB ID: 3LBK) and the close relative MDMX (PDB ID: 3LBJ) confirming the initial binding hypothesis.21,22_{Based on these findings, a new class of 1,2,3-trisubstituted bis(indoles)}

heterocycles (Figure 1) was designed. In this paper, we report the novel synthesis and preliminary biophysical evaluation of this first of its kind antagonists bisindoles 2. It should be noted that the class of p53-MDM2 antagonistic imidazoloindole has also discovered independently by another group.21

Figure 1. Protein-ƉƌŽƚĞŝŶ ŝŶƚĞƌĂĐƚŝŽŶ ŽĨƉϱϯ ƉĞƉƚŝĚĞ ;ŽƌĂŶŐĞ ɲ-helix with sticks) with MDM2 (grey surface) (PDB ID 1YCR). Key amino acids Leu22 (marine sticks), Phe19 (green sticks), Trp23 (blue sticks) and Leu26 (red sticks) are ŵŽƵŶƚĞĚŽŶƚŽƚŚĞŽƌĂŶŐĞƉϱϯɲ-helix. Leu26 is embedded into the hydrophobic receptor amino acids Ile99, Ile103,

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Leu54 and Val93. The Trp23 pocket is formed by Leu57, Phe86 and Ile99. The Phe19 pocket consists of Val93, Gln72, Val75, Tyr67, Met62, Il261 and Gly58. Val93 undergoes hydrophobic interactions with p53

Leu22.

Our hypothesis is that the additional phenyl annulated ring in scaffold 2 will make additional hydrophobic interactions with the mdm2_Val93,13_mimickingp53_{Leu22 (Figure 2). To test this speculation}

we developed a short synthesis towards scaffold 2.

N N NH Cl Y N NH Y Cl Leu22 1 2

Figure 2. Conversion of central imidazole scaffold 1 into indole scaffold 2 to address p53Leu22.

Our retrosynthetic plan of the designed derivatives 2 is based on the indole acetyl derivative 3 and hydrazine 4 which can react in a Fischer indole synthesis (Scheme 1).

Scheme 1. Retrosynthesis of scaffold 2

3 4 N H Y Cl O N NH2 + N N H Y Cl 2 Fischer indole

After some optimization, derivative 3a was easily synthesized by selective acylation at the 3-position of the 6-chloroindole derivative 5a in high yields in the presence of Lewis acids, such as SnCl4 (Scheme

2).26–28 Compound 4a was derived by the direct alkylation of the secondary amine of the phenylhydrazine 7a, with no need of protecting groups.29,30 Finally, refluxing derivatives 3a and 4a in acetic acid, gave the targeted bisindole 2a in good to very good yields in a just 3-step sequence.

Scheme 2. 3-step sequence for preparing 2,3'-bis(1'H-indole) heterocycles.

N NH O OEt Cl NNH2 N H NH2+ Br Et3N, toluene N H OEt O Cl N H OEt O Cl Ph Cl O O Ph SnCl4,CH2Cl2/MeNO2 5a 6a 3a 7a 8a 4a 2a AcOH

The synthesis of bisindoles 2, offers many possibilities for SARs since both of the main components (3 and 4) could easily be modified (Figure 3). Different halogens as R1, R2 and R3 and different sizes in the linkers of the two aromatic groups were tested. Moreover, the importance of carboxylate moiety was taken into account, as found in previous MDM2 binders.18,19,21,22_{Using the ethyl esters, the}

corresponding acids and various amides could be synthesized in order to optimize some physicochemical properties (Figure 3).

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N NH Y Cl 2 R3 R1 R2

Figure 3. SAR potential of bisindoles.

The 3-alkylation of the 6-chloroindole derivatives proceeded smoothly affording the corresponding compounds in excellent yields as shown in table 1.28_{The crystal structure obtained of the indole}

derivative 3c, is described in supporting information (CCDC 981828).

Table 1. 3-Acylation of 6-chloroindole derivatives

N H Cl N H Y Cl X Cl O O X SnCl4, CH2Cl2/MeNO2 5 6 3a-e Y Entry Y X Compound (yield %) 1 -CO2Et Ph 3a (99) 2 -CO2Et 4-Cl-C6H4 3b (97) 3 -CO2Et 4-F-C6H4 3c (95) 4 -CO2Et 4-Cl-C6H4CH2 3d (95) 5 H Ph 3e (99)

The hydrazine derivatives 4a-c were synthesized by the regioselective alkylation of the secondary amine, thus different hydrazines and benzyl halides were utilized in order to initially explore the aforementioned interactions in the MDM2 pocket (Table 2).

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Table 2. Alkylation of the secondary amine of hydrazines 7. NNH2 N H NH2+ Br 7 8 4a-c toluene Et3N R1 R2 R1 R2 N NH2 4d

Entry R1 _R2 _{Compound (yield %)}

1 H H 4a (40)

2 H Cl 4b (60) 3 Cl Cl 4c (42)

Next, the synthesis of the desired bisindoles 2a-h was performed. Combining the two moieties, the indole derivatives 3 and hydrazines 4 into a Fischer indole synthesis produced a series of active compounds (Scheme 3). All the combinations and compounds that were synthesized are shown below.

Scheme 3. Library of the indole derivatives 2

N X R 2 N H Y Cl 3 4 R1 N H Y Cl O X NNH2 R2 R1 + 2 AcOH reflux N H Cl N Ph O OEt Cl 2a (64%) N H Cl N Ph O OEt Ph 2b (65%) N H Cl N Ph Cl 2c (70%) N H Cl N O OEt Cl Cl 2d (61%) N H Cl N O OEt Cl F 2e (66%) N H Cl N Ph O OEt Cl Cl 2f (45%) mixture of isomers 1:1 N H Cl N Ph O OEt Cl 2g (50%) N H Cl N Ph O OEt F 2h (56%)

The structures of the above compounds were unambiguously characterized and confirmed by the crystal structures of the compounds 2a and 2h (SI, CCDCs 981829 and 981827 respectively). Due to the fact that ester groups are readily cleaved in cells by esterases and are sometimes metabolically unstable, we hydrolyzed, under basic conditions specific derivatives 2 into the corresponding acids 9 in

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5

6

7

8

9

order to compare and evaluate the activity. Additionally we know from our previous studies on scaffold with the indole anchor, that the free –COOH is always 1-2 orders of magnitude more potent towards MDM2 protein.10,18–20,25,31

Scheme 4. Hydrolysis of derivatives 2 to the corresponding acids 9.

N X R 2 N H CO2Et Cl R1 2 N X R 2 N H CO2H Cl R1 LiOH EtOH/H2O 9 N H Cl N Ph O OH Cl N H Cl N Ph O OH N H Cl N O OH Cl Cl 9a (61%) 9b (45%) 9c (52%) N H Cl N O OH Cl F 9d (55%) N H Cl N Ph O OH Cl 9e (58%)

Apart from poor absorption-distribution-metabolism-excretion-toxicology (ADMET) properties, insufficiently water-soluble compounds often lead to poor reproducibility and unreliable results or even false positive hits during in vitro screening. In order to potentially improve the properties of our compounds, we converted the ester group of the derivatives 2 into the corresponding better water soluble amides 10 with a one-step TBD-catalyzed amidation procedure (Scheme 5).32 In our previous studies, amidation of the indole-2-carboxylate gave often improved water solubility.31

Scheme 5. TBD-catalyzed amidation of the bisindoles 2

N X R 2 N H CO2Et Cl R1 2 NHR4_R5 TBD, THF N X R 2 N H Cl R1 O NR4_R5 10 N H Cl N Ph O N Cl H N O 10a (45%) N H Cl N O N Cl H N O Cl 10b (46%) N H Cl N Ph O N Cl H OH 10c (50%) N H Cl N Ph O N Cl H N O 10d (70%) N H Cl N Ph O HN Cl O OH 10e (35%) N H Cl N Ph O N Cl O 10f (46%)

Binding affinities were determined by the fluorescence polarization method (FP) towards MDM2 and MDMX (SI).25_{Representatives from each class compounds, esters, acids and an amide, were evaluated}

as inhibitors of the PPI. The results are summarized in the table 3. Noticeably, the acids 9a and 9d comparing with the corresponding esters 2a and 2e were found highly active towards both MDM2 and MDMX as previously reported by the indole anchor.10,18–20,25,31_{In addition, compounds 9a,d provided}

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fail to combine.17_{Moreover comparing the binding affinities of scaffold 1 (WK23)}22_{ǁŚŝĐŚǁĂƐϬ͘ϵʅɀ}

ĂŶĚϯϲʅDĨŽƌDDϮĂŶĚDDyƌĞƐƉĞĐƚŝǀĞůǇ͕ƚŚĞŶŽǀĞůƐĐĂĨĨŽůĚ9, is more potent towards MDMX without significant loss of activity in MDM2 (9a) or even slight improvements (9d).

Table 3. Results of the evaluation of inhibitory activity of compounds 2,3'-bis(1'H-indole) Heterocycles towards MDM2/MDMX as determined by FP. Ki (μM) Compound MDM2 MDMX 2a 17 NA 2c 10 NA 2d >22 NA 2e 6.7 5.0 2f NA NA 9a 1.8 0.2 9d 0.7 1.5 10d NA 6,09 WK23 (1) 0.92 36

NA: not active, Ki > 60 μM.

FP-based screening of protein-protein interactions often gives a high fraction of false positives especially with hydrophobic molecules and therefore it is advisable to run a second orthogonal biophysical assay. As a second, orthogonal screening system we performed the well-known heteronuclear single quantum coherence (HSQC) experiment where compound the 15_{N labeled MDM2}

is titrated with compound 9d.The expected ligand-induced perturbations in NMR chemical shifts are indeed observed (Figure 4).33_{Since all cross peaks in the MDM2 spectrum were assigned to particular}

amino acid residues before 34–36_{it is possible to analyze the way of interaction in the MDM2/9d}

complex. For example, the interaction of 9d with the MDM2 tryptophan subpocket is expressed by the movement of the peak assigned to Mdm2Val93 (Figure 4). Chemical shift changes of T101 and M62 indicate interaction with the leucine and phenylalanine subpocket, respectively. Both the FP assay and HSQC test indicate that the acid derivatives are the active species as p53-MDM2/X inhibitors.

N N NH Cl CO2H Cl WK23

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Figure 4. Superposition of NMR HSQC spectra of the 15N-labeled MDM2 titrated against 9d. The spectrum of free

MDM2 is shown in blue. The spectrum of 9d-MDM2 (ratio 1:1, respectively) is shown in red.

Our modelling (Figure 5) based on the HSQC binding data and known co-crystal structures and using MOLOC software37 revealed the nice alignment of the 6-chloro-indole moiety of compound 9d with the anchoring p53_{Trp23, whereas the two phenyl rings occupy the hydrophobic pockets mimicking} p53_{Phe19 and}p53_{Leu26. Moreover, the additionally introduced phenyl-annulated ring of the second}

indole moiety is predicted to cover mdm2Val93 through hydrophobic interactions. Then we rationalized the tight receptor ligand interaction using a small world network approach using Scorpion software (Figure 5).31 His96 is forming a pi-pi interaction with the p-Cl phenyl fragment and a hydrogen bond is formed between the Leu57 backbone carbonyl and the indole NH. Multiple van der Waals interactions can be used to rationalize the tight interactions. Amongst the major contributors of the interaction, shown as red balls, are F37-Tyr67, F27-Met62, C20-Met62, N7-Leu57. Cl10 contributes to an extended network including Phe86, Ile99, Ile103 and Leu57. Less strong contributors are C13-Val93, C3-, C1-, C6- of the buried indole moiety and C31-Leu57 and Cl36-Ile99. Interestingly small world network analysis of WK23 (PDB ID 3LBK) reveals no direct contribution of the scaffold imidazole to the tight interaction, whereas in 9d C13 interacts with Val93, supporting our hypothesis.

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Figure 5. above: Modeled binding pose of the most potent compound 9d (green sticks) in the MDM2 pocket (grey surface presentation, PDB ID 1YCR) and alignment with the hot-spot triade F19W23L26 (blue sticks). L57 and V93 are shown as grey sticks; below: Small network analysis of 9d in the MDM2 receptor. ʋ-ʋ, hydrogen bonding and van der Waals interactions are shown in red, blue and magenta dotted lines, respectively. Each ligand non-H atom is shown as colored ball according to its importance of contribution to the network (descending importance: red, purple, grey). Atom numbering of 9d is given in the right corner.

In conclusion, the designed 2,3'-bis(1'H-indole) scaffold 9 is active as dual action inhibitor of the p53-MDM2/X interactions with initial sub-μM affinities. Our hypothesis, that the extra phenyl ring in scaffold 2 makes additional hydrophobic interactions with the Mdm2_{Val93 comparing with the}

derivatives 1, is suggested by 2D NMR and modeling studies. Further studies are ongoing to introduce more ‘drug-like’ properties into this scaffold and to investigate cellular mechanism-based anti-cancer behaviors.

S

UPPLEMENTARY DATA

Experimental procedures for the synthesis of compounds, characterization of compounds, crystal data, as well FP assay and NMR HSQC are provided in the supporting information.

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9 SUPPORTING

INFORMATION

G

ENERAL METHODS

All reactions were performed under air atmosphere. All other reagents and solvents are purchased without further purification. Analytical thin-layer chromatography (TLC) was performed on SiO2 plates on Alumina available from Whatman. Visualization was

accomplished by UV irradiation at 254 nm, or by staining with any one of the following reagents: iodine, ninhydrin (0.3% w/v in glacial acetic acid/n-butyl alcohol 3:97), Vaughn’s reagent (4.8 g of (NH4)6Mo7O24.4H2O and 0.2 g of Ce(SO4)2.4H2O in 10 mL of conc. H2SO4 and

90 mL of H2O). Flash column chromatography was performed using SiO2 60 (particle size

0.040-0.055 mm, 230-400 mesh, EM science distributed by Bioman). Preparative TLC was conducted using preparative silica gel TLC plates ;ϭϬϬϬʅŵ͕ϮϬĐŵпϮϬĐŵͿ͘WƌŽƚŽŶĂŶĚĐĂƌďŽŶ NMR spectra were obtained on Bruker Avance™ 600 MHz NMR spectrometer. Chemical shifts ĂƌĞƌĞƉŽƌƚĞĚĂƐɷǀĂůƵĞƐŝŶƉĂƌƚƐƉĞƌŵŝůůŝŽŶ;ƉƉŵͿĂƐƌĞĨĞƌĞŶĐĞĚƚŽƌĞƐŝĚƵĂůƐŽůǀĞŶƚ͘1_{H NMR}

spectra are tabulated as follows: chemical shift, multiplicity (s = singlet, br s = broad singlet, d = doublet, t = triplet, q = quartet, quin = quintet, dd = double of doublets, ddd = double of doublet of doublets, m = multiplet), coupling constant(s) and number of protons. High Resolution Mass spectra were obtained at the University of Pittsburgh Mass Spectrometry facility. LC-MS analysis was performed on an SHIMADZU instrument, using an analytical C18 ĐŽůƵŵŶ;ŝŽŶĞǆĐĐůĂŝŵϭϮϬ͕Ϯ͘ϭпϱϬŵŵ͕ϯ͘Ϭʅŵ͕Ϭ͘Ϯŵ>ͬŵŝŶͿ͘

P

ROCEDURE AND ANALYTICAL DATA OF INDOLE DERIVATIVES

3

To a stirred solution of the corresponding indole derivatives (5.0 mmol) in DCM (10 mL), SnCl4

(1.0 M in DCM, 7.5 mmol) was added. The reaction mixture stirred for 30 min at rt and then, the corresponding phenyl acetyl chloride (6.0 mmol) was added slowly following the addition of MeNO2 (10 mL). After stirring overnight at rt, the reaction was quenched with ice/water,

extracted with ethyl acetate, washed by 1.0 M NaOH solution three times and one time by brine. The solvent was removed and the product was generated and used in the next step without further purification.

ethyl 6-chloro-3-(2-phenylacetyl)-1H-indole-2-carboxylate(3a) yellow solid, 99% yield; 1H NMR (CDCl3, 600 MHz): ɷ 9.49 (s, 1H),

7.78 (d, J = 8.4 Hz, 1H), 7.36 (s, 1H), 7.28 (d, J = 7.2 Hz, 2H), 7.20-7.27 (m, 3H), 7.15 (d, J = 8.4 Hz, 1H), 4.48 (q, J = 6.6 Hz, 2H), 4.45 (s, 2H), N H OEt O Cl O Ph

(15)

1.43 (t, J = 6.6 Hz, 3H) ppm; 13_{C NMR (CDCl}

3, 150 MHz): ɷ 198.3, 160.5, 135.2, 134.7, 133.2,

129.6, 128.5, 126.8, 126.4, 125.4, 123.6, 123.5, 121.5, 111.6, 62.2, 50.5, 14.3 ppm. ESL-TOF for C19H16ClNO3 (M+) found: m/z: 341.0818; Calc. Mass: 341.0819.

ethyl 6-chloro-3-(2-(4-chlorophenyl)acetyl)-1H-indole-2-carboxylate (3b)

yellow solid, 97% yield; 1_{H NMR (DMSO-d6, 600 MHz): ɷ 12.5 (br s,}

1H), 7.79 (d, J = 8.4 Hz, 1H), 7.53 (s, 1H), 7.35 (d, J = 7.8 Hz, 2H), 7.25 (d, J = 7.8 Hz, 2H), 7.21 (d, J = 8.4 Hz, 1H), 4.44 (q, J = 6.6 Hz, 2H), 4.37 (s, 2H), 1.36 (t, J = 6.6 Hz, 3H) ppm; 13C NMR (DMSO-d6, 150 MHz): ɷ 197.1, 161.1, 136.2, 134.8, 132.0, 131.7, 130.4, 128.8, 128.6, 125.1, 123.7, 123.2, 120.1, 112.7, 62.3, 48.9, 14.5 ppm. ESL-TOF for C19H16ClNO3Na (M+) found: m/z:

398.0329; Calc. Mass: 398.0327.

ethyl 6-chloro-3-(2-(4-fluorophenyl)acetyl)-1H-indole-2-carboxylate (3c)

yellow solid, 95% yield; 1_{H NMR (600 MHz, CDCl}

3): ɷ 9.20 (s, 1H), 7.81

(d, 1H, J = 8.0 Hz), 7.40 (s, 1H), 7.18-7.23 (m, 3H), 6.98-7.01 (m, 2H), 4.50 (q, 2H, J = 7.2 Hz), 4.43 (s, 2H), 1.46 (t, 3H, J = 7.2 Hz); 13_{C NMR}

(150 MHz, CDCl3): ɷ 14.3, 49.4, 62.3, 111.5, 115.3, 115.4, 121.5,

123.7, 123.9, 125.5, 126.4, 131.2, 132.4, 135.1, 160.3, 161.1, 162.7, 197.9 ppm. ESL-TOF for C19H15ClFNO3 (M+) found: m/z: 359.0729; Calc. Mass: 359.0725.

ethyl 6-chloro-3-(3-(4-chlorophenyl)propanoyl)-1H -indole-2-carboxylate (3d)

yellow solid, 95% yield; 1_{H NMR (DMSO-d6, 600 MHz): ɷ}

7.90 (d, J = 8.4 Hz, 1H), 7.58 (d, J = 2.4 Hz, 1H), 7.37 (d, J = 7.8 Hz, 2H), 7.33 (d, J = 7.8 Hz, 2H), 7.27 (dd, J = 7.8, 2.4 Hz, 1H), 4.43 (q, J = 7.2 Hz, 2H), 3.38 (t, J = 7.2 Hz, 2H), 3.00 (t, J = 7.2 Hz, 2H), 1.37 (t, J = 7.2 Hz, 3H) ppm; 13_{C NMR (DMSO-d6, 150 MHz): ɷ 198.0, 160.8, 140.3, 136.0, 130.4, 130.2, 129.6,}

128.1, 124.6, 123.3, 122.6, 119.3, 112.3, 61.7, 43.9, 29.3, 13.9 ppm; ESL-TOF for C20H17Cl2NO3

(M+) found: m/z: 269.0610; Calc. Mass: 269.0607.

1-(6-chloro-1H-indol-3-yl)-2-phenylethanone(3e) yellow solid, 99% yield; 1_{H NMR (CDCl}

3, 600 MHz): ɷ 12.06 (s, 1H), 8.44 (s, 1H), 8.09 (d, J = 7.8 Hz, 1H), 7.52 (s, 1H), 7.25-7.29 (m, 4H), 7.14-7.19 (m, 2H), 4.10 (s, 2H) ppm; 13C NMR (CDCl3, 150 MHz): ɷ 193.7, 137.6, 136.4, 135.9, 129.7, 128.8, 128.1, 126.9, 124.6, 123.0, 122.7, N H Cl OEt O O F N H Cl OEt O O Cl N H Cl OEt O O Cl N H Cl O Ph

(16)

5

6

7

8

9

116.3, 112.4, 46.1 ppm. ESL-TOF for C16H12ClNO (M+) found: m/z: 269.0612; Calc. Mass:

269.0607.

P

ROCEDURE AND ANALYTICAL DATA OF HYDRAZINE DERIVATIVES

4

To a stirred solution of the corresponding benzyl chloride (10.0 mmol) in toluene (10 mL), the corresponding phenyl hydrazine (10.0 mmol) and triethylamine (10.0 mmol) were added and the reaction mixture was refluxed overnight. Afterwards, the solvent was evaporated and the crude product purified with flash chromatography on silica gel, eluted with hexane-ethyl acetate (1:1).

1-benzyl-1-phenylhydrazine (4a)1

yellow oil, 40% yield; 1_{H NMR (CDCl}

3, 400 MHz,): ɷ 7.39-7.27 (m, 7H),

7.11-7.15 (m, 2H), 6.87-6.82 (m, 1H), 4.62 (s, 2H), 3.58 (s, 2H) ppm; 13C NMR (CDCl3, 100 MHz): ɷ 148.1, 139.4, 129.2, 128.6, 127.5, 127.2, 117.5, 112.8,

48.3 ppm.

1-(4-chlorobenzyl)-1-phenylhydrazine (4b)2

yellow oil, 60% yield; 1_{H NMR (CDCl}

3, 600 MHz): ɷ 7.32-7.22 (m, 6H),

7.05 (d, J = 7.8 Hz, 2H), 6.83 (t, J = 7.2 Hz, 1H), 4.56 (s, 2H), 3.58 (s, 2H) ppm; 13C NMR (CDCl3, 150 MHz): ɷ 151.5, 136.2, 133.1, 129.17,

129.15, 128.8, 118.8, 113.5, 59.8 ppm. ESL-TOF for C13H13ClN2 (M+)

found: m/z: 232.0769; Calc. Mass: 232.0767.

1-(4-chlorobenzyl)-1-(3-chlorophenyl)hydrazine (4c) yellow oil, 42% yield; 1_{H NMR (CDCl}

3, 600 MHz): ɷ 7.33 (d, J = 7.2

Hz, 2H), 7.22 (d, J = 7.2 Hz, 2H), 7.17 (t, J = 7.2 Hz, 1H), 7.11 (s, 1H), 6.90 (d, J = 7.8 Hz, 1H), 6.79 (d, J = 7.2 Hz, 1H), 4.58 (s, 2H), 3.59 (s, 2H) ppm; 13_{C NMR (CDCl}

3, 150 MHz): ɷ 152.5, 135.5, 135.1, 133.4,

130.1, 129.1, 129.0, 118.4, 111.4, 111.3, 59.2 ppm. ESL-TOF for C13H12Cl2N2 (M+) found: m/z:

266.0376; Calc. Mass: 266.0378.

P

ROCEDURE AND ANALYTICAL DATA OF BIS

-

INDOLE DERIVATIVES

2

To a stirred solution of the corresponding compounds 3a-e (4.0 mmol) in acetic acid (2 mL), compounds 4a-d (4.0 mmol) were added and the reaction mixture refluxed for 30 min. The

N NH2 Cl N NH2 Cl Cl N NH2

(17)

solvent was removed and the crude product purified with flash chromatography on silica gel eluted with hexane-ethyl acetate (4:1).

ethyl 6'-chloro-1-(4-chlorobenzyl)-3-phenyl-1H,1'H -[2,3'-biindole]-2'-carboxylate (2a)

yellow solid, yield 64%; 1H NMR (CDCl3, 600 MHz): ɷ 9.03 (s,

1H), 7.87 (d, J = 7.8 Hz, 1H), 7.42 (d, J = 1.8 Hz, 1H), 7.31 (d, J = 8.4 Hz, 1H), 7.28-7.15 (m, 7H), 7.10 (t, J= 7.2 Hz, 1H), 7.05 (d, J = 8.4 Hz, 2H), 7.05 (d, J = 9.0 Hz, 1H), 6.77 (d, J = 8.4 Hz, 2H), 5.23 (d, J = 16.8 Hz, 1H), 5.07 (d, J = 16.8 Hz, 1H), 4.05 (qd, J = 7.2, 2.4 Hz, 2H), 1.03 (t, J = 7.2 Hz, 3H) ppm; 13_{C NMR (CDCl} 3, 150 MHz): ɷ 160.9, 137.0, 136.3, 135.7, 135.1, 132.9, 132.0, 128.8, 128.5, 128.4, 128.1, 127.8, 127.5, 127.3, 126.9, 125.7, 122.8, 122.5, 122.4, 120.2, 119.9, 118.2, 112.5, 111.8, 110.1, 61.2, 47.0, 14.0 ppm. ESL-TOF for C32H24Cl2N2O2 (M+) found: m/z:

538.1215; Calc. Mass: 538.1215.

ethyl 1-benzyl-6'-chloro-3-phenyl-1H,1'H -[2,3'-biindole]-2'-carboxylate(2b)

yellow solid, yield 65%; 1_{H NMR (CDCl}

3, 600 MHz): ɷ 8.95 (s, 1H), 7.78 (d, J = 1.8 Hz, 1H), 7.35 (s, 1H), 7.28 (d, J = 7.8 Hz, 1H), 7.21-7.15 (m, 4H), 7.14-7.08 (m, 3H), 7.04-7.00 (m, 4H), 6.97 (d, J = 9.0 Hz, 1H), 6.80-6.75 (m, 2H), 5.20 (d, J = 16.2 Hz, 1H), 5.02 (d, J = 16.2 Hz, 1H), 3.95 (t, J = 7.8 Hz, 2H), 0.93 (t, J = 7.2 Hz, 3H) ppm; 13_{C NMR (CDCl} 3, 150 MHz): ɷ 161.0, 137.8, 137.1, 135.7, 135.3, 131.9, 128.8, 128.7, 128.3, 128.1, 127.6, 127.2, 127.1, 127.0, 126.5, 125.6, 122.7, 122.6, 122.2, 120.1, 119.8, 118.0, 112.6, 111.8, 110.3, 61.1, 47.7, 14.0 ppm. ESL-TOF for C32H25ClN2O2 (M+) found: m/z: 504.1607; Calc. Mass: 504.1605.

6'-chloro-1-(4-chlorobenzyl)-3-phenyl-1H,1'H-2,3'-biindole (2c)

white solid, yield 70%; 1_{H NMR (CDCl}

3, 600 MHz): ɷ 8.22 (s, 1H), 7.87 (d, J = 7.8 Hz, 1H), 7.36 (s, 2H), 7.35 (s, 1H), 7.28-7.10 (m, 9H), 6.98 (dd, J = 7.8, 1.8 Hz, 2H), 6.85 (d, J = 7.8 Hz, 2H), 5.24 (s, 2H) ppm; 13C NMR (CDCl3, 150 MHz): ɷ 137.0, 136.8, 136.1, 135.3, 132.9, 130.4, 129.3, 128.8, 128.6, 128.2, 127.5, 126.2, 126.0, 125.6, 122.3, 121.4, 120.8, 120.4, 119.7, 117.2, 111.3, 110.2, 107.4, 46.9 ppm. ESL-TOF for C29H20Cl2N2 (M+) found: m/z: 466.0998; Calc. Mass: 466.1004.

ethyl 6'-chloro-1-(4-chlorobenzyl)-3-(4-chloro phenyl)-1H,1'H-[2,3'-biindole]-2'-carboxylate(2d) N H Cl N Ph O OEt Cl N H Cl N Ph O OEt Ph N H Cl N Ph Cl Cl Cl

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5

6

7

8

9

3, 600 MHz): ɷ 9.10 (s, 1H), 7.81 (d, J = 7.8 Hz, 1H), 7.44

(s, 1H), 7.32 (d, J = 7.8 Hz, 1H), 7.29-7.10 (m, 7H), 7.03-7.09 (m, 3H), 6.76 (d, J = 8.4 Hz, 2H), 5.22 (d, J = 16.8 Hz, 1H), 5.05 (d, J = 16.8 Hz, 1H), 4.07 (q, J = 6.6 Hz, 2H), 1.04 (t, J = 6.6 Hz, 3H) ppm; 13C NMR (CDCl3, 150 MHz): ɷ 137.0, 136.1, 135.7, 133.7, 133.0, 132.2, 131.4, 130.0,

128.7, 128.5, 128.4, 127.8, 127.3, 127.0, 126.9, 122.9, 122.6, 122.3, 120.5, 119.6, 117.0, 112.2, 112.0, 110.2, 61.3, 47.1, 14.0 ppm. ESL-TOF for C32H23Cl3N2O2 (M+) found: m/z:

572.0823; Calc. Mass: 572.0825.

ethyl 6'-chloro-1-(4-chlorobenzyl)-3-(4-fluoro phenyl)-1H,1'H-[2,3'-biindole]-2'-carboxylate(2e)

yellow solid, yield 66%; 1_{H NMR (DMSO/CDCl}

3, 600 MHz): ɷ 12.20 (s, 1H), 7.69 (d, J = 7.8 Hz, 1H), 7.46 (s, 1H), 7.35 (d, J = 7.8 Hz, 1H), 7.08-7.22 (m, 5H), 7.04 (d, J = 7.8 Hz, 2H), 6.94-6.86 (m, 3H), 6.79 (d, J = 6.6 Hz, 2H), 5.23 (d, J = 16.2 Hz, 1H), 5.10 (d, J = 16.2 Hz, 1H), 4.11-3.99 (m, 2H), 1.04 (t, J = 7.2 Hz, 3H) ppm; 13C NMR (DMSO/CDCl3, 150 MHz): ɷ 160.7 (t, J = 243 Hz), 160.7, 136.9 (d, J = 9 Hz), 136.8, 132.3, 131.6, 130.5, 130.3 (d, J = 8 Hz), 129.7, 128.4, 128.3, 127.6, 127.0, 126.8, 122.4, 122.1, 122.0, 120.4, 119.1, 116.0, 115.3, 115.2, 112.8, 111.2, 110.7, 60.8, 46.8, 14.3 ppm. ESL-TOF for C32H23Cl2FN2O2 (M+) found: m/z: 556.1200; Calc. Mass: 556.1121.

ethyl 6,6'-dichloro-1-(4-chlorobenzyl)-3-phenyl-1H,1'H-[2,3'-biindole]-2'-carboxylate, ethyl 4,6'-dichloro-1-(4-chlorobenzyl)-3-phenyl-1H,1'H-[2,3'-biindole]-2'-carboxylate(2f)

N H Cl N Ph O OEt Cl Cl N H Cl N Ph O OEt Cl Cl

Crude proton NMR indicated a mixture of two isomers in a 1:1 ratio

Yellow solid, yield 45%, Isomer 1: 1_{H NMR (CDCl}

3, 600 MHz): ɷ 8.97 (s, 1H), 7.35 (d, J = 1.8 Hz, 1H), 7.19-7.23 (m, 4H), 7.14 (s, 1H), 7.14 (d, J = 7.2 Hz, 1H), 7.12-7.05 (m, 5H), 7.04 (dd, J = 8.4, 1.8 Hz, 1H), 6.80 (d, J = 8.4 Hz, 2H), 5.18 (d, J = 16.8 Hz, 1H), 5.06 (d, J = 16.8 Hz, 1H), 4.10-4.18 (m, 2H), 1.11 (t, J = 7.2 Hz, 3H) ppm; Isomer 2: 1H NMR (CDCl3, 600 MHz): ɷ 9.06 (s, 1H), 7.75 (d, J = 9.0 Hz, 1H), 7.66 (s, 1H), 7.65 (s, 1H), 7.59 (d, J = 8.4 Hz, 2H), 7.42 (d, J = 1.2 Hz, 1H), 7.35 (d, J = 8.4 Hz, 2H), 7.29 (d, J = 1.8 Hz, 1H), 7.22-6.88 (m, 3H), 6.83 (d, J = 7.8 Hz, 1H), 6.75 (d, J = 8.4 Hz, 2H), 5.16 (d, J = 16.2 Hz, 1H), 5.03 (d, J = 16.2 Hz, 1H), 4.02-4.09 (m, 2H), 1.04 (t, J = 7.2 Hz, 3H) ppm. ESL-TOF for C32H23Cl3N2O2 (M+) found: m/z: 572.0823; Calc.

Mass: 572.0825. 13C NMR (CDCl3, 150 MHz): ɷ 160.6, 137.8, 135.8, 135.5, 134.6, 133.1, 131.9, 131.3, 130.8, 128.6, 127.8, 127.5, 127.1, 126.8, 126.6, 126.2, 124.2, 122.7, 122.5, 122.2, N H Cl N O OEt Cl F

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121.3, 118.5, 111.9, 111.8, 108.8, 61.3, 47.4, 14.1 ppm; ESL-TOF for C32H23Cl3N2O2 (M+) found:

m/z: 572.0823; Calc. Mass: 572.0825.

ethyl 6'-chloro-3-(4-chlorobenzyl)-1-phenyl-1H,1'H -2,3'-biindole-2'-carboxylate (2g) yellow solid, 50%; 1H NMR (CDCl3, 600 MHz): ɷ 8.93 (s, 1H), 7.50 (d, J = 7.8 Hz, 1H), 7.40 (s, 1H), 7.40-7.37 (m, 2H), 7.36 (d, J = 8.4 Hz, 1H), 7.24-7.28 (m, 3H), 7.20 (t, J = 7.2 Hz, 3H), 7.17-7.04 (m, 2H), 6.98 (d, J = 8.4 Hz, 2H), 6.93 (t, J = 7.2 Hz, 1H), 4.13 (dq, J = 11.4, 7.2 Hz, 1H), 4.06 (d, J = 16.2 Hz, 1H), 4.04 (dq, J = 11.4, 7.2 Hz, 1H), 3.88 (d, J = 16.2 Hz, 1H), 1.05 (t, J = 7.2 Hz, 3H) ppm; 13_{C NMR (CDCl} 3, 150 MHz): ɷ 160.7, 139.7, 138.1, 137.8, 135.6, 131.8, 131.2, 129.6, 129.5, 128.7, 128.0, 127.9, 127.5, 126.9, 126.7, 122.6, 122.4, 120.0, 119.3, 115.1, 112.9, 111.8, 110.5, 61.0, 30.4, 14.1 ppm. ESL-TOF for C32H24Cl2N2O2 (M+) found: m/z: 5638.1210; Calc. Mass: 538.1215.

ethyl 6'-chloro-3-(4-fluorophenyl)-1-phenyl-1H,1'H -2,3'-biindole-2'-carboxylate (2h) yellow solid, 56%; 1_{H NMR (CDCl} 3, 600 MHz): ɷ 8.87 (s, 1H), 7.84 (d, J = 8.4 Hz, 1H), 7.37 (d, J = 7.8 Hz, 1H), 7.33 (s, 1H), 7.30 (t, J = 7.8 Hz, 2H), 7.27-7.18 (m, 6H), 7.12 (s, 2H), 6.99 (d, J = 8.4 Hz, 1H), 6.91 (t, J = 8.4 Hz, 2H), 4.20-4.04 (m, 2H), 1.06 (t, J = 7.8 Hz, 3H) ppm; 13_{C NMR (CDCl} 3, 150 MHz): ɷ 161.0, 140.1, 139.1, 138.2, 136.3, 130.4, 130.2, 129.4, 129.3, 129.1, 128.0, 127.7, 126.5, 125.9, 125.7, 122.4, 121.4, 120.0, 119.3, 114.8, 113.9, 112.8, 110.5, 30.2, 14.5. ESL-TOF for C31H22ClFN2O2 (M+)

found: m/z: 508.1360; Calc. Mass: 508.1354;

P

ROCEDURE AND ANALYTICAL DATA OF THE BIS

-

INDOLE DERIVATIVES

9

To a stirred solution of the corresponding compounds 2 (1.0 mmol) in EtOH-water (1:1), LiOH (10.0 mmol) was added and the reaction mixture refluxed overnight. Then, pH was adjusted to approximately 6 with the addition of 1 N HCl and the reaction mixture extracted with DCM (3x20 mL). The organic layer was separated, washed with water, dried with magnesium sulfate, filtered and concentrated in vacuo, affording the product as solid.

6'-chloro-1-(4-chlorobenzyl)-3-phenyl-1H,1'H -[2,3'-biindole]-2'-carboxylic acid (9a)

white solid, yield 61%; 1_{H NMR (MeOH-d4, 600 MHz): ɷ 7.78}

(d, J = 7.8 Hz, 1H), 7.45 (d, J = 1.8 Hz, 1H), 7.39 (d, J = 8.4 Hz, 1H), 7.31 (d, J = 7.8 Hz, 2H), 7.21 (t, J = 7.2 Hz, 1H), 7.17 (t, J = N H Cl N Ph O OH Cl N H Cl N Ph O OEt Cl N H Cl N Ph O OEt F

(20)

5

6

7

8

9

8.4 Hz, 1H), 6.87 (dd, J = 8.4, 1.8 Hz, 1H), 6.81 (d, J = 8.4 Hz, 2H), 5.24 (s, 2H) ppm; 13_{C NMR} (MeOH-d4, 150 MHz): ɷ 162.3, 137.2, 136.9, 136.4, 135.5, 132.3, 130.7, 129.3, 128.7, 127.85, 127.84, 127.6, 127.2, 126.9, 125.2, 121.8, 121.7, 121.3, 119.6, 118.9, 117.3, 112.2, 111.6, 109.8, 46.4 ppm. ESL-TOF for C30H20Cl2N2O2 (M+) found: m/z: 510.0904; Calc. Mass: 510.0902.

1-benzyl-6'-chloro-3-phenyl-1H,1'H-[2,3'-biindole]-2'-carboxylic acid(9b)

yellow solid, yield 45%; 1H NMR (MeOH-Ěϰ͕ ϲϬϬ D,ǌͿ͗ ɷ ϳ͘ϲϰ (d, J = 7.8 Hz, 1H), 7.30 (d, J = 1.8 Hz, 1H), 7.21 (d, J = 7.8 Hz, 1H), 7.18 (d, J = 7.2 Hz, 2H), 7.05-6.97 (m, 4H), 6.91 (t, J = 7.8 Hz, 1H), 6.89-6.86 (m, 4H), 6.87-6.91 (m, 3H), 5.10 (s, 2H) ppm;

13_{C NMR (MeOH-d4, 150 MHz): ɷ ϭϲϮ͘ϰ͕ ϭϯϴ͘ϭ͕ ϭϯϳ͘Ϯ͕ ϭϯϲ͘ϱ͕ ϭϯϱ.6, 130.6, 129.5, 128.7,}

127.82, 127.80, 127.6, 127.2, 127.0, 126.5, 126.2, 125.1, 122.0, 121.6, 121.1, 119.5, 118.8, 117.1, 112.5, 111.6, 110.1, 47.1 ppm. HRMS ESL-TOF for C30H21ClN2O2 (M+) found: m/z:

476.1295; Calc. Mass 476.1292.

6'-chloro-1-(4-chlorobenzyl)-3-(4-chlorophenyl)-1H,1'H -[2,3'-biindole]-2'-carboxylic acid(9c)

white solid, yield 52%; 1H NMR (MeOH-Ěϰ͕ϲϬϬD,ǌͿ͗ɷϳ͘ϲϰ (d, J = 7.8 Hz, 1H), 7.35 (s, 1H), 7.28 (d, J = 7.8 Hz, 1H), 7.15 (d, J = 8.4 Hz, 2H), 7.10 (t, J = 7.2 Hz, 1H), 7.05 (d, J = 7.8 Hz, 3H), 6.90 (d, J = 7.8 Hz, 2H), 6.89 (d, J = 8.4 Hz, 1H), 6.79 (d, J = 9.0 Hz, 1H), 6.78 (d, J = 8.4 Hz, 2H), 5.12 (s, 2H) ppm; 13_{C NMR (MeOH-d4, 150 MHz): ɷ} 163.0, 138.6, 138.2, 137.9, 135.7, 133.7, 132.3, 132.2, 131.4, 131.1, 129.27, 129.25, 129.2, 128.3, 128.2, 123.3, 123.1, 122.8, 121.3, 120.1, 117.5, 113.1, 111.4, 47.8 ppm. HRMS ESL-TOF for C30H19Cl3N2O2 (M+) found: m/z: XX; Calc. Mass 544.0512.

6'-chloro-1-(4-chlorobenzyl)-3-(4-fluorophenyl)-1H,1'H -[2,3'-biindole]-2'-carboxylic acid(9d)

yellow solid, yield 55%; 1H NMR (DMSO-Ěϲ͕ ϲϬϬ D,ǌͿ͗ ɷ 8.12 (s, 1H), 7.70 (d, J = 7.8 Hz, 1H), 7.46 (s, 1H), 7.35 (d, J = 8.4 Hz, 1H), 7.26 (dd, J = 8.4, 6.0 Hz, 2H), 7.17 (t, J = 7.8 Hz, 1H), 7.13 (t, J = 7.2 Hz, 1H), 7.07 (d, J = 7.8 Hz, 2H), 7.04 (d, J = 8.4 Hz, 1H), 6.94 (t, J = 8.4 Hz, 2H), 6.87 (t, J = 6.6 Hz, 3H), 5.21 (s, 2H) ppm; 13C NMR (DMSO-d6, 150 MHz): ɷϭϲϳ͘Ϭ͕ϭϲϱ͘ϱ;Ě͕J = 243 Hz), 141.7, 141.4 (d, J = 15 Hz), 136.9, 136.5 (d, J = 3 Hz), 135.2 (d, J = 8 Hz), 134.8 (d, J = 5 Hz), 133.5, 133.2 (d, J = 8 Hz), 131.8, 131.6, 127.0, 126.8, 126.3, 125.1, 123.8 (d, J = 12 Hz), 120.5, 120.1, 119.9, 117.4, 115.6, 115.6, 47.7 ppm. ESL-TOF for C30H19Cl2FN2O2 (M+) found: m/z: 528.0810; Calc. Mass:

528.0808. N H Cl N O OH Cl F N H Cl N Ph O OH N H Cl N O OH Cl Cl

(21)

6'-Chloro-3-(4-chlorobenzyl)-1-phenyl-1H,1'H-2,3'-biindole-2'-carboxylic acid (9e)

yellow solid, yield 58%; 1H NMR (MeOH-Ěϰ͕ϲϬϬD,ǌͿ͗ɷϵ͘ϬϬ;Ɛ͕ϭ,Ϳ͕ 7.50 (d, J = 7.8 Hz, 1H), 7.40 (s, 1H), 7.37 (t, J = 8.4 Hz, 2H), 7.31 (t, J = 7.2 Hz, 1H), 7.25-7.10 (m, 7H), 7.07 (d, J = 8.4 Hz, 2H), 7.01 (d, J = 8.4 Hz, 2H), 4.08 (d, J = 16.2 Hz, 1H), 3.94 (d, J = 16.2 Hz, 1H) ppm; 13_C

NMR (MeOH-d4, 150 MHz): ɷϭϯϵ͘ϰ͕ϭϯϴ͘ϭ͕ϭϯϴ͘Ϭ͕ϭϯϱ͘ϵ͕ϭϯϮ͘ϲ͕ϭϯϭ͘ϯ͕ 129.7, 129.5, 129.0, 128.8, 128.1, 127.9, 127.4, 127.2, 126.9, 122.9, 122.59, 122.65, 120.2, 119.5, 115.4, 115.0, 111.9, 110.6, 30.4 ppm. ESL-TOF for C30H20Cl2N2O2 (M+) found: m/z:

510.0910; Calc. Mass: 510.0902.

P

ROCEDURE AND ANALYTICAL DATA OF THE INDOLE AMIDE DERIVATIVES

10

To a stirred solution of the corresponding compounds 2 (1.0 mmol) in THF, triazabicyclodecene (TBD, 1.0 mmol) and the corresponding amine (10.0 mmol) were added and the reaction mixture refluxed for 12 h. Afterwards, the solvent was evaporated and the crude product purified with flash chromatography on silica gel eluted with ethyl acetate with 1% triethylamine.

6'-chloro-1-(4-chlorobenzyl)-N-(3-morpholino propyl)-3-phenyl-1H,1'H-[2,3'-biindole]-2'-carboxamide(10a) white solid, yield 45%; 1_{H NMR (DMSO-d6, 600 MHz): ɷ}

9.88 (s, 1H), 7.91 (d, J = 8.4 Hz, 1H), 7.50 (d, J = 1.2 Hz, 1H), 7.46 (d, J = 8.4 Hz, 1H), 7.35 (t, J = 7.8 Hz, 1H), 7.24-7.30 (H), 7.22 (t, J = 7.8 Hz, 2H), 7.17 (t, J = 5.4 Hz, 1H), 7.11 (dd, J = 8.4, 1.8 Hz, 1H), 7.08 (d, J = 8.4 Hz, 2H), 6.73 (d, J = 8.4 Hz, 2H), 6.25 (t, J = 6.0 Hz, 1H), 5.20 (t, J = 15.6 Hz, 1H), 4.99 (d, J = 15.6 Hz, 1H), 3.51-3.60 (m, 4H), 3.05-3.10 (m, 2H), 1.97-2.07 (m, 4H), 1.82-1.91 (m, 2H), 1.27-1.40 (m, 2H) ppm; 13_{C NMR (DMSO-d6, 150 MHz): ɷ 160.2, 137.6, 135.6, 135.3, 133.6, 133.4, 131.1, 130.1,} 128.73, 128.72, 128.6, 128.1, 127.8, 126.9, 126.7, 123.5, 122.6, 121.6, 121.1, 120.3, 118.7, 112.3, 110.5, 106.0, 66.9, 55.4, 53.3, 47.1, 37.1, 26.0 ppm. HRMS ESL-TOF for C37H34Cl2N4O2

(M+_{) found: m/z: 636.2072; Calc. Mass 636.2059.}

6'-chloro-1-(4-chlorobenzyl)-3-(4-chlorophenyl)-N -(3-morpholino propyl)-1H,1'H-[2,3'-biindole]-2'-carboxamide (10b)

white solid, yield 46%; 1H NMR (CDCl3, 600 MHz): ɷ 10.50

(br s, 1H), 7.86 (d, J = 8.4 Hz, 1H), 7.54 (s, 1H), 7.47 (d, J = N H Cl N Ph O N Cl H N O N H Cl N O N Cl H N O Cl N H Cl N Ph O OH Cl

(22)

5

6

7

8

9

8.4 Hz, 1H), 7.36 (t, J = 7.2 Hz, 1H), 7.28 (t, J = 7.8 Hz, 1H), 7.18-7.22 (m, 5H), 7.10 (d, J = 8.4 Hz, 1H), 7.07 (d, J = 8.4 Hz, 2H), 6.73 (d, J = 7.8 Hz, 2H), 6.22 (t, J = 5.4 Hz, 1H), 5.20 (d, J = 16.2 Hz, 1H), 4.99 (d, J = 16.2 Hz, 1H), 3.50-3.60 (m, 4H), 3.11 (q, J = 6.0 Hz, 2H), 1.97-2.10 (m, 4H), 1.83-1.92 (m, 2H), 1.30-1.42 (m, 2H) ppm; 13C NMR (CDCl3, 150 MHz): ɷ 160.3, 137.6, 135.6, 135.5, 133.5, 132.4, 132.2, 131.1, 130.1, 129.8, 129.0, 128.7, 128.2, 128.1, 127.6, 126.6, 123.7, 122.7, 121.4, 121.3, 120.0, 117.4, 112.6, 110.7, 105.6, 66.9, 55.4, 53.3, 47.1, 37.2, 26.0 ppm. HRMS ESL-TOF for C37H33Cl3N4O2 (M+) found: m/z: 670.1660; Calc. Mass 670.1669.

6'-chloro-1-(4-chlorobenzyl)-N -(2-hydroxyethyl)-3-phenyl-1H,1'H-[2,3'-biindole]-2'-carboxamide(10c)

3, 600 MHz): ɷ 10.11 (s, 1H), 7.91 (d, J = 7.8 Hz, 1H), 7.49 (d, J = 1.2 Hz, 1H), 7.44 (d, J = 7.8 Hz, 1H), 7.35 (t, J = 7.2 Hz, 1H), 7.26-7.30 (m, H), 7.23 (t, J = 7.8 Hz, 2H), 7.17 (t, J = 7.8 Hz, 1H), 7.12 (dd, J = 9.0, 1.8 Hz, 1H), 7.07 (d, J = 8.4 Hz, 2H), 6.71 (d, J = 8.4 Hz, 2H), 6.56 (t, J = 5.4 Hz, 1H), 3.40-3.46 (m, 1H), 3.35-3.40 (m, 1H), 3.19-3.25 (m, 1H), 3.08-3.15 (m, 1H), 1.75 (s, 1H) ppm; 13_{C NMR (CDCl} 3, 150 MHz): ɷ 161.2, 137.7, 135.8, 135.5, 133.7, 133.4, 131.2, 129.5, 128.72, 128.67, 128.6, 127.9, 127.8, 126.9, 126.7, 123.5, 122.7, 121.6, 121.0, 120.3, 118.9, 112.4, 110.5, 106.5, 61.6, 47.1, 42.2 ppm. HRMS ESL-TOF for C32H25Cl2N3O2 (M+) found:

m/z: 553.1321; Calc. Mass 553.1324.

6'-chloro-1-(4-chlorobenzyl)-N-(2-morpholino ethyl)-3-phenyl-1H,1'H-[2,3'-biindole]-2'-carboxamide(10d)

1H), 7.90 (d, J = 7.8 Hz, 1H), 7.47 (d, J = 1.8 Hz, 1H), 7.45 (d, J = 8.4 Hz, 1H), 7.34 (t, J = 7.2 Hz, 1H), 7.28 (d, J = 7.2 Hz, 2H), 7.20 (t, J = 7.8 Hz, 2H), 7.15 (t, J = 3.6 Hz, 1H), 7.14 (d, J = 8.4 Hz, 1H), 7.05 (d, J = 8.4 Hz, 2H), 7.04 (dd, J = 8.4, 1.8 Hz, 1H), 6.78 (d, J = 8.4 Hz, 2H), 6.63 (d, J = 4.2 Hz, 1H), 5.18 (d, J = 16.2 Hz, 1H), 5.04 (d, J = 16.2 Hz, 1H), 3.18-3.31 (m, 2H), 2.88-3.10 (m, 4H), 2.22 (ddd, J = 12.0, 7.6, 4.8 Hz, 1H), 2.02-2.12 (m, 3H), 1.88-1.93 (m, 2H) ppm; 13_{C NMR (CDCl} 3, 150 MHz): ɷ 160.6, 137.6, 135.7, 135.5, 134.0, 130.2, 128.7, 128.6, 128.5, 128.3, 127.6, 127.5, 127.2, 126.5, 123.5, 122.4, 121.6, 121.0, 120.3, 119.0, 112.3, 110.4, 106.5, 66.1, 56.7, 53.2, 47.1, 35.9 ppm. HRMS ESL-TOF for C36H32Cl2N4O2 (M+) found:

m/z: 622.1899; Calc. Mass 622.1902.

6'-chloro-1-(4-chlorobenzyl)-N -(2-(2-hydroxyethoxy)ethyl)-3-phenyl-1H,1'H -[2,3'-biindole]-2'-carboxamide(10e)

white solid, yield 35%; 1H NMR (CDCl3, 600 MHz): ɷ 10.40

(s, 1H), 7.92 (s, 1H), 7.48 (s, 1H), 7.44 (d, J = 7.2 Hz, 1H), 7.35 (t, J = 7.8 Hz, 1H), 7.25-7.30 (m, 4H), 7.22 (t, J = 7.2 N H Cl N Ph O N Cl H OH N H Cl N Ph O N Cl H N O N H Cl N Ph O HN Cl O OH

(23)

Hz, 2H), 7.10 (d, J = 7.2 Hz, 1H), 7.02-7.12 (m, 3H), 6.75 (t, J = 7.2 Hz, 2H), 6.57 (s, 1H), 5.22 (d, J = 16.2 Hz, 1H), 5.04 (d, J = 16.2 Hz, 1H), 3.35 (s, 3H), 3.26 (s, 2ȸ), 3.14 (s, 1H), 3.06-3.10 (m, 3H) ppm; 13_{C NMR (CDCl} 3, 150 MHz): ɷ 160.4, 137.6, 135.6, 135.5, 133.7, 133.3, 131.0, 129.8, 128.7, 128.62, 128.59, 128.0, 127.8, 127.7, 127.1, 126.6, 123.4, 122.5, 121.5, 120.9, 120.1, 118.9, 112.4, 110.6, 106.3, 72.1, 69.0, 61.3, 47.0, 39.1 ppm. HRMS ESL-TOF for C34H29Cl2N3O3

(M+_{) found: m/z: 597.1581; Calc. Mass 597.1586.}

(6'-chloro-1-(4-chlorobenzyl)-3-phenyl-1H,1'H -[2,3'-biindol]-2'-yl)(morpholino)methanone(10f)

1H), 7.90 (d, J = 7.2 Hz, 1H), 7.40 (s, 1H), 7.39 9d, J = 9.0 Hz, 1H), 7.31 (t, J = 7.2 Hz, 1H), 7.22-7.28 (m, H), 7.19 (t, J = 8.4 Hz, 2H), 7.07 (d, J = 8.4 Hz, 2H), 7.02 (d, J = 7.8 Hz, 1H), 6.79 (d, J = 6.6 Hz, 2H), 5.28 (d, J = 16.2 Hz, 1H), 5.23 (d, J = 16.2 Hz, 1H),3.70 (s, 2H), 2.85-3.60 (m, 4H), 2.89 (s, 2H) ppm; 13C NMR (CDCl3, 150 MHz): ɷ 162.4, 137.5, 136.3, 135.7, 134.4, 133.3, 130.6, 129.9, 128.7, 128.63, 128.58, 128.55, 128.2, 127.2, 126.5, 126.2, 123.1, 122.3, 122.0, 120.8, 120.2, 117.8, 112.0, 110.5, 107.2, 68.2, 66.3, 47.1, 46.5 ppm. HRMS ESL-TOF for C34H27Cl2N3O2 (M+) found: m/z: 579.1476; Calc. Mass 579.1480.

S

INGLE CRYSTAL X

-

RAY STRUCTURE DETERMINATION

G

ENERAL

:

Data were collected on an X-ray single crystal diffractometer equipped with a CCD detector (Bruker APEX II,

N

CCD), a rotating anode (Bruker AXS, FR591) with MoKD radiation (

O

= 0.71073 Å), and a graphite monochromator by using the SMART software package (compounds 2h, 3c). Data were collected on an X-ray single crystal diffractometer equipped with a CCD detector (APEX II,

N

CCD) at the window of a fine-focused sealed tube with MoKɲ

radiation (

O

= 0.71073 Å) and a graphite monochromator by using the SMART software package (compound 2a).3 The measurements were performed on single crystals coated with perfluorinated ether. Each crystal was fixed on the top of a glass fiber and transferred to the diffractometer. The crystals were frozen under a stream of cold nitrogen. A matrix scan was used to determine the initial lattice parameters. Reflections were merged and corrected for Lorenz and polarization effects, scan speed, and background using SAINT.4_Absorption

corrections, including odd and even ordered spherical harmonics were performed using SADABS.4_{Space group assignments were based upon systematic absences, E statistics, and}

successful refinement of the structures. Structures were solved by direct methods with the aid of successive difference Fourier maps, and were refined against all data using WinGX5

based on SIR-92.6 If not mentioned otherwise, non-hydrogen atoms were refined with anisotropic displacement parameters. Compound 2a: All hydrogen atom positions were found in the difference map calculated from the model containing all non-hydrogen atoms. The

N H Cl N Ph O N Cl O

(24)

5

6

7

8

9

hydrogen positions were refined with individual isotropic displacement parameters. Compound 2h: In the difference map(s) calculated from the model containing all non-hydrogen atoms, not all of the non-hydrogen positions could be determined from the highest peaks. For this reason, the hydrogen atoms were placed in calculated positions (dC-H = 95, 98,

99 pm; dN-H = 88 pm). Isotropic displacement parameters were calculated from the parent

carbon atom (UH = 1.2/1.5 UC; UH = 1.2 UN). The hydrogen atoms were included in the

structure factor calculations but not refined. Compound 3c: The hydrogen atom bound to the nitrogen atom was found in the difference Fourier. The hydrogen position was refined with an individual isotropic displacement parameter. All other hydrogen atoms were placed in calculated positions (dC-H = 95, 98, 99 pm). Isotropic displacement parameters were calculated

from the parent carbon atom (UH = 1.2/1.5 UC). The hydrogen atoms were included in the

structure factor calculations but not refined. Methyl hydrogen atoms were refined as part of rigid rotating groups. Full-matrix least-squares refinements were carried out by minimizing

6w (Fo2-Fc2)2 with SHELXL-977 weighting scheme. Neutral atom scattering factors for all atoms

and anomalous dispersion corrections for the non-hydrogen atoms were taken from

International Tables for Crystallography.8 _{Images of the crystal structures were generated by}

PLATON.9 CCDC 981829 (2a), CCDC 981827 (2h), and CCDC 981828 (3c) contains the supplementary crystallographic data for this compound. This data can be obtained free of charge from The Cambridge Crystallographic Data Centre via

www.ccdc.cam.ac.uk/data_request/cif or via

https://www.ccdc.cam.ac.uk/services/structure_deposit/ Special:

2a: Small extinction effects were corrected with the SHELXL-977 _procedure.

H

_{refined to}

H

= 0.021(2)

2h: Problems with unresolvable solvent molecules were cured by using the calc squeeze procedure.8

(25)

Figure 1. Ortep drawing of compound 2a with 50% ellipsoids. 8

Operator: *** Herdtweck *** Molecular Formula: C32 H24 Cl2 N2 O2

Crystal Color / Shape Pale yellow fragment

Crystal Size Approximate size of crystal fragment used for data

collection: 0.36 u 0.51 u 0.76 mm

Molecular Weight: 539.43 a.m.u.

F000: 560

Systematic Absences: none

Space Group: Triclinic P 1 (I.T.-No.: 2)

Cell Constants: Least-squares refinement of 9841 reflections with the programs "APEX suite" and "SAINT" [1,2]; theta range 1.74° < T < 25.48°; Mo(K

D

); l = 71.073 pm a = 1031.81(5) pm a = 115.551(2)° b = 1196.13(5) pm b = 90.435(2)° c = 1327.37(6) pm g = 113.632(2)° V = 1321.37(11)· 106_pm3_{; Z = 2; D} calc = 1.356 g cm-3; Mos. = 0.95

Diffractometer: Kappa APEX II (Area Diffraction System; BRUKER AXS); sealed tube; graphite monochromator; 50 kV; 30 mA;

O = 71.073 pm; Mo(K

D

)

Temperature: (20±1) °C; (293±1) K

Measurement Range: 1.74° < T < 25.48°; h: -12/12, k: -14/14, l: -16/16 Measurement Time: 2 u 7.5 s per film

(26)

5

6

7

8

9

j- and w-movement; Increment: Dj/Dw = 1.00°;

dx = 40.0 mm LP - Correction: Yes [2]

Intensity Correction No/Yes; during scaling4

Absorption Correction: Multi-scan; during scaling; m = 0.279 mm-1 4

Correction Factors: Tmin = 0.6931

Tmax = 0.7452

Reflection Data: 35612 reflections were integrated and scaled 35612 reflections to be merged

4868 independent reflections

0.020 Rint: (basis Fo2)

4868 independent reflections (all) were used in

refinements

4139 independent reflections with Io > 2s(Io)

99.0 % completeness of the data set 440 parameter full-matrix refinement 11.1 reflections per parameter Solution: Direct Methods [3]; Difference Fourier syntheses Refinement Parameters: In the asymmetric unit:

38 Non-hydrogen atoms with anisotropic

displacement parameters

24 Hydrogen atoms with isotropic displacement

parameters

Hydrogen Atoms: All hydrogen atom positions were found in the difference map calculated from the model containing all non-hydrogen atoms. The hydrogen positions were refined with individual isotropic displacement parameters.

Atomic Form Factors: For neutral atoms and anomalous dispersion [4]

Extinction Correction: Fc (korr) = kFc[1+ 0.001 · e · Fc2· l3/sin(2Q)]-1/4 SHELXL-97 [5]; e

refined to e = 0.021(2) Weighting Scheme: w-1_{= s}2_(F

o2)+(a*P)2+b*P

with a: 0.0427; b: 0.6518; P: [Maximum(0 or Fo2)+2*Fc2]/3

Shift/Err: Less than 0.001 in the last cycle of refinement: Resid. Electron Density: +0.37 e₀-_/Å3_{; -0.43 e}

0 -_/Å3 R1: S(||Fo|-|Fc||)/S|Fo| [Fo > 4s(Fo); N=4139]: = 0.0386 [all reflctns; N=4868]: = 0.0480 wR2: [Sw(Fo2-Fc2)2/Sw(Fo2)2]1/2 [Fo > 4s(Fo); N=4139]: = 0.0947 [all reflctns; N=4868]: = 0.1049

Goodness of fit: [Sw(Fo2-Fc2)2/(NO-NV)]1/2 = 1.060

(27)

Compound 2h

Figure 2. Ortep drawing of compound 2h with 50% ellipsoids.8 Operator: *** Herdtweck ***

Molecular Formula: C31 H22 Cl F N2 O2

Crystal Color / Shape Colorless fragment

F000: 528

D

); l = 71.073 pm a = 1104.86(6) pm a = 114.043(2)° b = 1269.09(7) pm b = 97.343(2)° c = 1318.92(6) pm g = 102.677(3)° V = 1598.01(15)· 106_pm3_{; Z = 2; D} calc = 1.058 g cm-3; Mos. = 0.60

Diffractometer: Kappa APEX II (Area Diffraction System; BRUKER AXS); rotating anode; graphite monochromator; 50 kV; 40 mA; O = 71.073 pm; Mo(K

D

)

(28)

5

6

7

8

9

Measurement Time: 2 u 10 s per film

Measurement Mode: measured: 8 runs; 3052 films / scaled: 8 runs; 3052 films

Intensity Correction No/Yes; during scaling [2]

Absorption Correction: Multi-scan; during scaling; m = 0.151 mm-1_[2]

Tmax = 0.7452

Reflection Data: 49951 reflections were integrated and scaled 49951 reflections to be merged

refinements

4768 independent reflections with Io > 2s(Io)

displacement parameters

Hydrogen Atoms: In the difference map(s) calculated from the model containing all non-hydrogen atoms, not all of the hydrogen positions could be determined from the highest peaks. For this reason, the hydrogen atoms were placed in calculated positions (dC-H = 95,

98, 99 pm; dN-H = 88 pm). Isotropic displacement parameters

were calculated from the parent carbon atom (UH_{= 1.2/1.5 U}C;

UH_{= 1.2 U}N). The hydrogen atoms were included in the structure

factor calculations but not refined.

Atomic Form Factors: For neutral atoms and anomalous dispersion [4] Extinction Correction: no

Weighting Scheme: w-1 = s2(Fo2)+(a*P)2+b*P

Shift/Err: Less than 0.001 in the last cycle of refinement: Resid. Electron Density: +0.63 e₀-_/Å3_{; -0.37 e}

0 -_/Å3 R1: S(||Fo|-|Fc||)/S|Fo| [Fo > 4s(Fo); N=4768]: = 0.0477 [all reflctns; N=5722]: = 0.0559 wR2: [Sw(Fo2-Fc2)2/Sw(Fo2)2]1/2 [Fo > 4s(Fo); N=4768]: = 0.1354 [all reflctns; N=5722]: = 0.1399

(29)

Remarks: Refinement expression Sw(Fo2-Fc2)2

Compound 3c

Figure 3. Ortep drawing of compound 3c with 10% ellipsoids.8

Operator: *** Herdtweck *** Molecular Formula: C20 H17 Cl2 N O3

Crystal Color / Shape Colorless fragment

F000: 404

D

); l = 71.073 pm a = 544.89(2) pm a = 85.0829(13)° b = 1268.09(4) pm b = 78.8518(13)° c = 1366.51(4) pm g = 86.7201(15)° V = 922.19(5)· 106 pm3; Z = 2; Dcalc = 1.405 g cm-3; Mos. = 0.70

Diffractometer: Kappa APEX II (Area Diffraction System; BRUKER AXS); rotating anode; graphite monochromator; 50 kV; 40 mA;

O = 71.073 pm; Mo(K

D

)

(30)

5

6

7

8

9

Measurement Time: 2 u 5 s per film

Measurement Mode: measured: 11 runs; 4279 films / scaled: 5 runs; 2150 films

Intensity Correction No/Yes; during scaling [2]

Absorption Correction: Multi-scan; during scaling; m = 0.372 mm-1 [2]

Tmax = 0.7452

Reflection Data: 14074 reflections were integrated and scaled 1 obvious wrong intensity and rejected 14073 reflections to be merged

refinements

3159 independent reflections with Io > 2(Io)

Displacement parameters

1 Hydrogen atoms with isotropic displacement

parameters

Hydrogen Atoms: The hydrogen atom bound to the nitrogen atom was found in the difference Fourier. The hydrogen positions was refined with an individual isotropic displacement parameter.

Hydrogen Atoms: All other hydrogen atoms were placed in calculated positions (d C-H = 95, 98, 99 pm). Isotropic displacement parameters were

calculated from the parent carbon atom (UH_{= 1.2/1.5 U}C). The

hydrogen atoms were included in the structure factor calculations but not refined.

Atomic Form Factors: For neutral atoms and anomalous dispersion [4] Extinction Correction: no

Weighting Scheme: w-1 = s2(Fo2)+(a*P)2+b*P

Shift/Err: Less than 0.001 in the last cycle of refinement: Resid. Electron Density: +0.50 e₀-_/Å3

; -0.47 e₀-_/Å3

R1: S(||Fo|-|Fc||)/S|Fo|

[Fo > 4s(Fo); N=3159]: = 0.0359

[all reflctns; N=3352]: = 0.0379

(31)

[Fo > 4s(Fo); N=3159]: = 0.0905

[all reflctns; N=3352]: = 0.0925

Goodness of fit: [Sw(Fo2-Fc2)2/(NO-NV)]1/2 = 1.037

Remarks: Refinement expression Sw(Fo2-Fc2)2

F

LUORESCENCE POLARIZATION BINDING ASSAYS

All fluorescence experiments were performed as described by Czarna et al.10_{Briefly, the}

fluorescence polarization experiments were read on an Ultra Evolution 384-well plate reader (Tecan) with the 485 nm excitation and 535 nm emission filters. The fluorescence intensities parallel (Intparallel) and perpendicular (Intperpedicular) to the plane of excitation were measured in black 384-well NBS assay plates (Corning) at room temperature (~20°C). The background fluorescence intensities of blank samples containing the references buffer were subtracted and steady-state fluorescence polarization was calculated using the equation: P = (Intparallel – GIntperpedicular)/ (Intparallel + GIntperpedicular), and the correction factor G (G = 0.998 determined empirically) was introduced to eliminate differences in the transmission of vertically and horizontally polarized light. All fluorescence polarization values were expressed in millipolarization units (mP). The binding affinities of the fluorescent p53-derived peptide of Hu at al.11_{(the P4 peptide in Czarna et al.) towards MDM2 and MDMX}

proteins were determined in the buffer which contained 50 mM NaCl, 10 mM Tris pH 8.0, 1 mM EDTA, 10% DMSO. Each sample contained 10 nM of the fluorescent P4 peptide and MDM2 (the MDM2 concentration used, from 0 to 1 ʅM and MDMX, from 0 to 10 ʅM) in a final volume of 50 ʅl. Competition binding assays were performed using the 10 nM fluorescent P4 peptide, 15 nM MDM2 or 120 nM MDMX. Plates were read at 30 min after mixing all assay components. Binding constant and inhibition curves were fitted using the SigmaPlot (SPSS Science Software). Fluorescein labeled p53 mimicking peptide TSFAEYWNLLSP described previously was used.12

Entry Compound Plot (MDM2/MDMX) Ki (uM)

MDM2 MDMX

1 2a 17 ND

2 2c 10 ND

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5

6

7

8

9

4 2e 6.7 5.0 5 2f ND ND 6 9a 0.01 0.1 1 10 0.2 0.4 0.6 0.8 1.0 nor m al iz ed v al ue of FP compound [P0@ 1.5 0.18 7 9d 0.7 1.5 8 10d 0.1 1 10 100 0.2 0.4 0.6 0.8 1.0 1.2 nor m al iz ed val ue of F P compound [P0@ ND 6.09

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2D-NMR

MEASUREMENTS

Uniform 15_{N isotope labeling was achieved by expression of the protein in the M9 minimal}

media containing 15_NH

4Cl as the sole nitrogen source. Final step of purification of MDM2 for

NMR consisted of gel filtration into the NMR buffer (50 mM phosphate buffer pH 7.4 containing 150 mM NaCl, 5 mM DTT). 10% (v/v) of D2O was added to samples to provide lock

signal. All the spectra were recorded at 300 K using a Bruker Avance 600 MHz spectrometer.

1_H-15_{N heteronuclear correlations were obtained using the fast HSQC pulse sequence.}13

Assignment of the amide groups of MDM2 was obtained after Stoll et al.14

ADMET

PROPERTIES OF SELECTED COMPOUNDS

Compound logP PSA (pH = 7.4) Solubility (logS, pH = 7.4) 2a 7.82 47.02 -10.75 2c 7.61 20.72 -10.05 2d 8.38 47.02 -11.40 2e 7.98 47.02 -10.98 2f 8.38 47.02 -11.40 9a 7.22 60.85 -7.87 9d 7.38 60.85 -8.11 10d 6.4 62.29 -10.00

R

EFERENCES

(1) Zhu, M.; Zheng, N. Photoinduced Cleavage of N-N Bonds of Aromatic Hydrazines and Hydrazides by Visible Light. Synthesis 2011, 2011 (14), 2223– 2236.

(2) Nara, S.; Sakamoto, T.; Miyazawa, E.; Kikugawa, Y. A Convenient Synthesis of 1Alkyl1Phenylhydrazines from N -Aminophthalimide. Synth. Commun. 2003, 33 (1), 87–98.

(3) APEX Suite of Crystallographic Software. APEX 2 Version 2008.4. Bruker AXS Inc., Madison, Wisconsin, USA (2008).

(4) SAINT, Version 7.56a and SADABS Version 2008/1. Bruker AXS Inc., Madison, Wisconsin, USA (2008). (5) Farrugia, L. J. WinGX Suite for

Small-Molecule Single-Crystal

Crystallography. J. Appl. Crystallogr. 1999, 32 (4), 837–838.

(6) Altomare, A.; Cascarano, G.;

C.; Polidori, G.; Camalli, M. SIR92 – a Program for Automatic Solution of Crystal Structures by Direct Methods. J.

Appl. Crystallogr. 1994, 27 (3), 435–

435.

(7) Sheldrick, G. M. “SHELXL-97”, University of Göttingen, Göttingen, Germany, (1998).

(8) International Tables for

Crystallography, Vol. C, Tables 6.1.1.4 (Pp. 500-502), 4.2.6.8 (Pp. 219-222), and 4.2.4.2 (Pp. 193-199), Wilson, A. J. C., Ed., Kluwer Academic Publishers, Dordrecht, The Netherlands, 1992. (9) A. L. Spek, “PLATON”, A Multipurpose

Crystallographic Tool, Utrecht University, Utrecht, The Netherlands, (2010).

(10) Czarna, A.; Popowicz, G. M.; Pecak, A.; Wolf, S.; Dubin, G.; Holak, T. A. High Affinity Interaction of the P53 Peptide-Analogue with Human Mdm2 and

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5

6

7

8

9

Mdmx. Cell Cycle Georget. Tex 2009, 8

(8), 1176–1184.

(11) Hu, B.; Gilkes, D. M.; Chen, J. Efficient P53 Activation and Apoptosis by Simultaneous Disruption of Binding to MDM2 and MDMX. Cancer Res. 2007,

67 (18), 8810–8817.

(12) Mori, S.; Abeygunawardana, C.; Johnson, M. O.; Vanzijl, P. C. M. Improved Sensitivity of HSQC Spectra of Exchanging Protons at Short Interscan Delays Using a New Fast HSQC (FHSQC) Detection Scheme That Avoids Water Saturation. J. Magn.

Reson. B 1995, 108 (1), 94–98.

(13) Pazgier, M.; Liu, M.; Zou, G.; Yuan, W.; Li, C.; Li, C.; Li, J.; Monbo, J.; Zella, D.; Tarasov, S. G.; Lu, W. Structural Basis for High-Affinity Peptide Inhibition of P53 Interactions with MDM2 and MDMX. Proc. Natl. Acad. Sci. 2009, 106 (12), 4665–4670.

(14) Stoll, R.; Renner, C.; Hansen, S.; Palme, S.; Klein, C.; Belling, A.; Zeslawski, W.; Kamionka, M.; Rehm, T.; Mühlhahn, P.; Schumacher, R.; Hesse, F.; Kaluza, B.; Voelter, W.; Engh, R. A.; Holak, T. A. Chalcone Derivatives Antagonize Interactions between the Human Oncoprotein MDM2 and P53.