University of Groningen
Development of novel anticancer agents for protein targets
Estrada Ortiz, Natalia
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CHAPTER
4
A
RTIFICIAL MACROCYCLES AS POTENT P
53-
MDM
2
INHIBITORS
Natalia Estrada-Ortiz,
a, *Constantinos G. Neochoritis,
a, *Aleksandra
Twarda-Clapa,
b, cBogdan Musielak,
dTad A. Holak,
b, dAlexander Dömling
aa
Department of Drug Design, University of Groningen, A. Deusinglaan 1, Groningen 9700AV, The Netherlands. b
Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Gronostajowa 7, 30-387 Krakow, Poland.
c
Malopolska Centre of Biotechnology, Jagiellonian University, Gronostajowa 7a, 30-387 Krakow, Poland. d
Department of Chemistry, Jagiellonian University, Ingardena 3, 30-060 Krakow, Poland. * Shared first authors
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Abstract: ABSTRACT: Based on a combination of an Ugi four component reaction and a ring closing metathesis, a library of novel artificial macrocyclic inhibitors of the p53-MDM2 interaction was designed and synthesized. These macrocycles, alternatively to stapled peptides, target for the first time the large hydrophobic surface area formed by Tyr67, Gln72, His73 Val93 and Lys94 yielding derivatives with affinity to MDM2 in the nanomolar range. Their binding affinity with MDM2 was evaluated using fluorescence polarization (FP) assay and 1H-15N 2D HSQC NMR experiments.
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In nature, macrocycles are not uncommon and often exhibit interesting biological activities.1 Often
macrocyclic compounds have distinct advantages over their open chain analogous including higher affinity and selectivity,2 preferable entropic signature, better membrane permeation and oral bioavailability or higher stability.1,2 Thus, macrocycles can increase affinity and selectivity for a specific
target.1 In spite of their potential, macrocycles pose considerable synthesis problems and also the
accurate prediction of their conformation makes it difficult to predict activity.1,2
The tumor suppressor protein p53 is involved in controlling pathways of cell cycle, apoptosis, angiogenesis metabolism, senescence and autophagia.3,4 TP53 gene is one of the most frequently
mutated gene in a multitude of human cancer.5 Additionally, in multiple cases where TP53 is intact,
p53’s function is impaired by its negative regulators: MDM2 and MDMX, due to amplification or enhanced expression of their coding genes.3-5 Multiple potent and selective compound classes to inhibit the p53-MDM2 interaction have been discovered, described and evaluated in early clinical trials.6 However, the pharmacokinetic and pharmacodynamics properties of the studied scaffolds
could still be optimized to minimize the side effects. Thus, the discovery of new p53-MDM2 inhibitors with diverse structures to improve their properties is still of importance.6-8 The three finger
pharmacophore model for p53-MDM2 is recognized as responsible for the binding of small molecules and peptides to the MDM2.9,10 We already described several series of potent p53-MDM2 antagonists,
proposing an extended four finger model; the intrinsically disordered MDM2 N-terminus is ordered by certain small molecules, which can be obtained by multicomponent reaction chemistry,11,12 as shown by co-crystallization.13,14 Recently, several macrocyclic stapled peptides have been described with great affinity towards MDM2 and MDMX.6 ALRN-6924 (Aileron Therapeutics) is currently undergoing
phase I and II clinical trials in patients suffering of solid tumors, lymphoma and myeloid leukemias (ClinicalTrials.gov ID: NCT02264613 and NCT02909972).
Here we propose a novel series of non peptidic artificial macrocyclic compounds that inhibit the p53-MDM2 interaction, which might have a different activity profile from the currently available scaffolds. Our synthesis strategy is shown in Figure 1. Based on the Ugi scaffold, we introduced two terminal ene-functionalities, via the carboxylic acid and the isocyanide component and we were able to cyclize the compounds by ring closing metathesis (RCM, Figure 1).15, 16
Figure 1. Macrocyclization strategy to inhibit the p53-MDM2 interaction.
Our previously introduced three and four-point phar-macophore models were the basis of the discovery and development of the current inhibitors. Thus, we used as the starting point our formerly ĚĞƐĐƌŝďĞĚɲ-aminoacylamide (YH300, shown in cyan sticks, Figure 2) with a Ki of 600 nM and its crystal structure in complex with MDM2 recep-tor (PDB ID 4MDN).13 Accordingly, the 6-chloroindole-2-carboxylic acid was used as an ‘anchor’ in order to mimic the Trp23 amino acid and constrain the position of other substituents and three additional binding sites were defined, Phe19, Leu26 and the induced Leu26 subpocket
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Figure 2. Four and three-point pharmacophore modelling of macrocyclic compounds; Left: YH300 -PDB ID: 4MDN- (cyan lines), Tyr67, Gln72, His73 Val93 and Lys94 (blue sticks), proposed macrocycle to explore the four-pharmacophore point (pink sticks). Right: SAH-p53-8 stapled-peptide -PDB ID: 3V3B- (cyan lines), Tyr67, Gln72, His73, Val93 and Lys94 (blue sticks), proposed macrocycle to explore the three-pharmacophore point (pink sticks).
KƵƌ ĚĞƐŝŐŶ ŽĨ ƚŚĞ ŵĂĐƌŽĐLJĐůŝĐ ůŝŶŬĞƌ ĂŝŵƐ ƚŽ ďŝŶĚ ŽŶ ƚŽƉ ŽĨ ƚŚĞ ůŽŽƉ ůŝŶŬŝŶŐ ɲϮ͛ ĂŶĚ ɲϭ͛ ŚĞůŝĐĞƐ͕ covering a large hydrophobic surface area formed by Tyr67, Gln72, His73, Val93 and Lys94 (Figure 2) and potentially increasing the affinity to the receptor. Moreover, we reasoned that the addition of a methyl group on the ring could mimic the tert-butyl group of YH300,13 likely leading to increased
affinity.
The retrosynthetic plan of the designed macrocycle 1 foresees (Scheme 1), a ring closing metathesis reaction (RCM), followed by a classical Ugi four-component (U-4CR). The Ugi adduct is formed of the anchoring 6-chloro-3-carboxaldehyde 6, suitably substituted benzylamines 5 and the long-chained aliphatic carboxylic acids 8 along with the isocyanides 7 incorporating terminal double bonds.
Aldehyde 6 was synthesized from the 6-chloro-indole derivative using the Vilsmeier-Haack formylation reaction.17,18 For the preliminary SAR analysis of the Leu26 and the induced pockets, the benzylamines
5 were used as commercially available (5b-h) or obtained via Williamson ether synthesis (5a). Probing both the Phe19 pocket and the larger hydrophobic surface area formed by Tyr67, Gln72, His73, Val93 and Lys94, the isocyanides 7a,b were synthesized from the corresponding formamides, whereas the carboxylic acids are commercially available. In particular, the amine 5a with the oxygen linker that designed to probe the induced pocket was synthesized through a Williamson ether synthesis of the protected 4-hydroxybenzylamine with the 3,4-benzyl chloride under basic conditions (Supporting information, Scheme 1). The isocyanides 7a,b were synthesized from the formamides via the revised Leuckart-Wallach reaction of the corresponding carbonyl compounds (SI, Scheme 2).19
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Scheme 1. Retrosynthetic plan based on a U-4CR and a ring clousing metathesis
N N H O O R1 X n m N H CO2H Cl N N H O O R1 X n N H CO2Et Cl CO2H R1 NC N H CHO CO2Et Cl + NH2 X m n N N H O O R1 X n N H CO2Et Cl disconnection point U-4CR m m 1 3 4 5 6 7 8 RCM
Next, we proceeded in the Ugi four-component reaction. Equimolar mixture of the substituted benzylamine 5, aldehyde 6, isocyanide 7 and carboxylic acid 8 in TFE was irradiated at 120 oC for 1 h in a microwave oven yielding compounds 4 (Scheme 2). Afterwards, we successfully performed the ring closing metathesis with the 2nd generation of Grubbs catalystTM affording compounds 3 as mixture of isomers (E and Z). Due to the fact that the existence of the double bond gives rise to two possible isomers and most importantly reduces the flexibility of the macrocycle, we decided to subject the mixture to hydrogenation on Pd/C isolating compounds 2. Last step was the ester hydrolysis obtaining the final screening compounds 1 (Scheme 2). Performing a preliminary SAR, we built a small library of macrocycles of various ring sizes (12, 13, 18, 19 and 24 number of atoms) targeting the hydrophobic region around Tyr67, Gln72, His73 Val93 and Lys94. We maintained the anchoring indole group for the Trp23 and the phenyl group for Leu26 and further explored the induced pocket with the extended dichlorobenzyloxy moiety.
Scheme 2. Synthesis of the macrocycle library based on the U-4CR/RCM strategy
CO2H R1 NC N H CHO CO2Et Cl + N N H O O R1 X n NH2 X m n N H CO2Et Cl N N H O O R1 X n N H CO2Et Cl N N H O O R1 X n N H CO2Et Cl Grubbs catalyst, 2ndgeneration (8-10%) DCM, reflux,48 h H2,Pd/C, DCM, 1 h MW, 120oC, 1h TFE N N H O O R1 X n N H CO2H Cl m m m m EtOH-H2O 1:1, rt, 3 d LiOH 5a-g 6 7a,b 8a-c 4a-k (15-60%) 3a-k (10-96%) 2a-k (70-99%) 1a-k (11-70%)
5a:X= 4-OCH2-3,4-di-Cl-C6H3
5b:X= 4-F 5c:X=4-Cl 5d:X= 2,4-di-Cl 5e:X= 4-OMe 5f:X= 3-OMe 5g: X= 3,4-di-Cl 5h:X= 3,4,5-tri-F 7a:R1= Me, m= 1 7b:R1= H, m= 7 8a: n=1 8b: n=2 8c: n=7
«ÖãÙψ N N H O O N H CO2H Cl O Cl Cl 1a (d.r. 1:1) N N H O O N H Cl CO2H O Cl Cl N NH O O F N H Cl CO2H 1d N NH O O Cl N H Cl CO2H N NH O O O N H Cl CO2H 1g N NH O O Cl N H Cl CO2H 1i N NH O O N H Cl CO2H O N H N O O N H Cl CO2H F F F 1j N N H O O O N H Cl CO2H Cl Cl 1k Cl N NH O O Cl N H Cl CO2H 1f Cl 1b (d.r. 1:1) 1c (d.r 1:1) 1e 1h N NH O O O N H Cl CO2H Cl Cl N H N O O N H Cl CO2H F F F 1ja N H N O O N H Cl CO2H F F F 1jb N H N O O N H Cl CO2H F F F 3j N N H O O O N H Cl CO2H Cl Cl 3k N N H O O N H CO2Et Cl O Cl Cl 2a N N H O O O N H Cl CO2Et Cl Cl 2k HN N OO N H Cl CO2H F F F 9 N NH O O N H Cl 10 N NH O O Cl Cl 11
Two complementary assays based on independent physicochemical principles, fluorescence polarization (FP) and 1H-15N 2D HSQC NMR were used to measure affinity and to exclude false positive hits. Fluorescence polarization (FP) assay was employed to determine the inhibitory affini-ties (Ki) of the macrocycles against MDM2 as previously described20 and the results are presented in Table 1.
Examining both the three and four-finger pharmaco-phore model, it seems that most of the obtained macrocy-cles are active towards MDM2, many of them demonstrat-ing an affinity below 1 μM. Although it is a preliminary SAR study, we can conclude that the ideal ring size for the four-finger model, seems to be around 18 (entry 3). The affinity improves while increasing the size from 12 (1a, entry 1) to 13 (1b, entry 2) and eventually 18 (1c, entry 3) atom ring size. Moreover, compound 1c has ĂŶĂĨĨŝŶŝƚLJŽĨϭϬϬŶɀĂƐĂŵŝdžƚƵƌĞŽĨĚŝĂƐƚĞƌĞŽŵĞƌƐ͕ǁŚĞƌĞĂƐƚŚĞůĂƌŐĞƌϮϰ-membered macrocycle 1k ;ĞŶƚƌLJϭϭͿƐŚŽǁƐĚĞĐƌĞĂƐĞĚĂĐƚŝǀŝƚLJ;ϭ͘ϵϭʅM).
In addition, we focused on the three-finger-pharmacophore model, which characterizes the vast ma-jority of the currently available small-molecule MDM2 inhibitors.6 We synthesized various macrocycles with a different substitution pattern. The position of halogens on the phenyl group seems to play a significant role since the para fluoro (1d, entry 4) or chloro substituted (1f, entry 6) derivatives are only slightly or not active at all. On the contrary, the addition of a second chlorine in o- or m-position influenced the binding mode; the 2,4-dichloro derivative (1e) showed activity of 5.25 ʅɀ;ĞŶƚƌLJϱͿĂŶĚƚŚĞĐŽƌƌĞƐƉŽŶĚŝŶŐϯ͕ϰ-dichloro compound 1h (entry 8) displayed an activity of 80 nM. Whereas placing the donor group -OMe in compound 1g (entry 7), improved the affin-ity with ϭ͘Ϯϲʅɀ͘EŽŶĞƚŚĞůĞƐƐ͕ŵ-OMe substitution keeping the same ring size of 18 (1i, entry 9), did not show ĂŶLJƐŝŐŶŝĨŝĐĂŶƚĚŝĨĨĞƌĞŶĐĞ͕ĞdžŚŝďŝƚŝŶŐĂŶĂĐƚŝǀŝƚLJŽĨϭ͘ϳϴʅɀ͘ƵĞƚŽŽƵƌƉƌĞǀŝŽƵƐƌĞƐƵůƚƐ͕ϮϭǁŚĞƌĞǁĞ were able to synthe-size fluorinated phenyls as compound 9 with Ki up to 100 nM, we employed the 3,4,5-trifluorobenzylamine and we synthesized compound 1j (entry 10) which exhibited an interesting affinity as a racemic mixture of 140 nM. Enan-tiomeric separation of the racemic mixture via chiral SFC
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provided the enantiomers (+)-1ja and (-)-ϭũďǁŝƚŚĂĨĨŝŶŝƚŝĞƐŽĨϵϬŶDĂŶĚϳϬϬŶɀ͕ƌĞƐƉĞĐƚŝǀĞůLJ͘Ɛŝƚ was expected, the separated enantiomers showed a significant increase of the activity compared to the racemic mixture.
The existence of the double bond in the macrocycles 3j and 3k, as anticipated, reduced significantly ƚŚĞĂĨĨŝŶŝƚLJƚŽϯϰϬŶDĂŶĚϮ͘ϱϯʅɀƌĞƐƉĞĐƚŝǀĞůLJ;ĞŶƚƌŝĞƐϭϮ͕ϭϯͿ͕ĐŽŵ-pared with the corresponding ŚLJĚƌŽŐĞŶĂƚĞĚĐŽŵƉŽƵŶĚƐϭũ;<ŝсϭϰϬŶDͿĂŶĚϭŬ;<ŝсϭ͘ϵϭʅɀͿ͘dŚĞĐŽƌƌesponding esters, 2a and 2k (entries 14, 15), are orders of magnitude less reactive or inactive comparing with the acids in ac-cordance with our previous experience (entries 1, 11).11,12,22-25 Interestingly, the acyclic Ugi-adduct 4j was also proven to be practically inactive both in the ester and acid forms (SI, Table S1, entry 15). Moreover, changing the anchor to a non-substituted indole moiety (compound 10) or to the 3- or 4-phenyl moiety (compound 11), resulted in nearly no activity. The expected ligand-induced perturbations in 1H-15N 2D HSQC NMR spectra were indeed observed (Figure 3). The 15N-labeled MDM2 was titrated with increasing concentration of the compound. Since all cross peaks in the MDM2 spectrum were assigned to particular amino acid residues,26 it was possible to analyze the
interaction within the MDM2/1j complex. Particularly, Val93 is clearly involved in the interaction, as its cross peak shifted between titration steps for MDM2:1j molar ratios equal to 2:1 and 1:1. After 1:1 step the peak remained in the same position. NMR titration also confirmed the tight binding of 1j, as e.g. for Arg29 NMR signal splitting was observed (Figure 3), which indicated strong interaction with MDM2 at Kd ďĞůŽǁϭʅD;ĂŶĚĂƐůŽǁĐŚĞŵŝĐĂůĞdžĐŚĂŶŐĞͿ͘
Table 1. Synthesized MDM2 inhibitors Entry Compound Ring size Ki(μM)
1 1a 12 0.35 2 1b 13 0.32 3 1c 18 0.10 4 1d 18 N/A 5 1e 18 5.25 6 1f 18 N/A 7 1g 18 1.26 8 1h 18 0.08 9 1i 18 1.78 10 1j 19 0.14 (rac-1j) 0.09 {(+)-1ja} 0.70 {(-)-1jb} 11 1k 24 1.91 12 3j 19 0.34 13 3k 24 2.53 14 2a 12 2.98 15 2k 24 N/A
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Figure 3. Spectrum of the 15N-labeled MDM2 (blue) superimposed with spectrum after addition of 1j in MDM2/1j molar ratio equal to 2:1 (red) and 1:1 (green). The close-up view shows selected peaks assigned to Val93 and Arg29. For Arg29, NMR signal splitting indicates strong interaction at Kd ďĞůŽǁϭʅD͘
Three of the macrocyclic compounds (1c, 1h and 1j) obtained, demonstrated improved binding affinities (Ki ~100 nM) over the lead acyclic molecule, YH300 (Ki = 600 nM). In order to rationalize the
tight receptor ligand interaction, we exploit modelling studies using MOLOC27 based on the HSQC binding data having as template a known co-crystal structure (PDB ID: 3TU1)21 and the small network
analysis using Scorpion software (Figure 4).28 It revealed the existence of van der Waals interactions of
the aliphatic handle with Tyr67 and His73, the expected alignment of the 6-chloro-indole moiety of the designed compounds with the p53Trp23 pocket, whereas the 3,4,5-trifluorophenyl ring occupied the p53>ĞƵϮϲ ŚLJĚƌŽƉŚŽďŝĐ ƉŽĐŬĞƚ͘ DŽƌĞŽǀĞƌ͕ ƚŚĞ ʋ-ʋ ŝŶƚĞƌĂĐƚŝŽŶ ŽĨ ,ŝƐϵϲ ǁŝƚŚ ƚŚĞ ϯ͕ϰ͕ϱ-trifluorophenyl fragment and several van der Waals interactions with Leu54, Ile61, Phe86, Phe91, Val93, His96 and Tyr100 are depicted. These findings support our initial hypothesis of the divergent hydrophobic handle position compared to staple peptides shown before,29-34 suggesting a new approach to improve and diversify the extensive collection of MDM2/X inhibitors.
To analyze and compare the physicochemical properties of our newly synthesized macrocycles with the orally available macrocycle drugs,35 we plotted molecular weight, clogP, TPSA, number of HBDs
and HBAs as well as the number of rotatable bonds (Figure 5). Interestingly, the values of the properties in most of our macrocycles are set in the appropriate range, demonstrating the significance of this new strategy to develop potentially oral bioavailable macrocycles targeting p53-MDM2 interaction. In the case of 1c and 1k, clogP, goes off the limits as expected since we were targeting a very lipophilic surface. However, this will be overcome in the future with a strategy to incorporate heteroatoms (oxygens) on both the acid and isocyanide linker. After this initial SAR study, we will in the future synthesize libraries of novel macrocycles as potent p53-MDM2 inhibitors with higher diversity and complexity.
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Figure 4. Small network analysis of 1j ;ǁŚŝƚĞƐƚŝĐŬƐͿŵŽĚĞůůĞĚŝŶƚŽƚŚĞDDϮƌĞĐĞƉƚŽƌ;W/͗ϯdhϭ͕ĐLJĂŶƐƚŝĐŬƐͿ͗ʋ-ʋ and van der Waals interactions are shown in orange and yellow dotted lines, respectively.
Figure 5. Physicochemical properties of the synthesized macrocycles, compared with the oral macrocycle marketed drugs on a hexagon radar graph. The dark gray area contains the low limits of the oral macrocycle drugs, whereas the light gray the highest limits.
S
UPPLEMENTARY DATAExperimental 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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1. R
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L.; Camacho, C.; Holak, T. A.; Dömling, A. 1,4-Thienodiazepine-2,5-Diones via MCR (I): Synthesis, Virtual Space and p53-Mdm2 Activity. Chem. Biol. Drug Des. 2010, 76 (2), 116–129. (25) Srivastava, S.; Beck, B.; Wang, W.;
Czarna, A.; Holak, T. A.; Dömling, A. Rapid and Efficient Hydrophilicity Tuning of p53/mdm2 Antagonists. J. Comb. Chem. 2009, 11 (4), 631–639. (26) Rehm, T.; Huber, R.; Holak, T. A.
Application of NMR in Structural Proteomics: Screening for Proteins Amenable to Structural Analysis. Structure 2002, 10 (12), 1613–1618. (27) Gerber, P. R.; Müller, K. MAB, a
Generally Applicable Molecular Force Field for Structure Modelling in Medicinal Chemistry. J. Comput. Aided Mol. Des. 1995, 9 (3), 251–268.
(28) Kuhn, B.; Fuchs, J. E.; Reutlinger, M.; Stahl, M.; Taylor, N. R. Rationalizing Tight Ligand Binding through Cooperative Interaction Networks. J. Chem. Inf. Model. 2011, 51 (12), 3180– 3198.
(29) Phan, J.; Li, Z.; Kasprzak, A.; Li, B.; Sebti, S.; Guida, W.; Schönbrunn, E.; Chen, J. Structure-Based Design of High Affinity Peptides Inhibiting the Interaction of p53 with MDM2 and MDMX. J. Biol. Chem. 2010, 285 (3), 2174–2183.
(30) Baek, S.; Kutchukian, P. S.; Verdine, G. L.; Huber, R.; Holak, T. A.; Lee, K. W.; Popowicz, G. M. Structure of the Stapled p53 Peptide Bound to Mdm2. J. Am. Chem. Soc. 2012, 134 (1), 103– 106.
(31) 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.
(32) Zhan, C.; Zhao, L.; Wei, X.; Wu, X.; Chen, X.; Yuan, W.; Lu, W.-Y.; Pazgier, M.; Lu, W. An Ultrahigh Affinity D-Peptide Antagonist Of MDM2. J. Med. Chem. 2012, 55 (13), 6237–6241. (33) Liu, M.; Li, C.; Pazgier, M.; Li, C.; Mao,
Y.; Lv, Y.; Gu, B.; Wei, G.; Yuan, W.; Zhan, C.; Lu, W.-Y.; Lu, W. D-Peptide Inhibitors of the p53-MDM2 Interaction for Targeted Molecular Therapy of Malignant Neoplasms. Proc. Natl. Acad. Sci. U. S. A. 2010, 107 (32), 14321–14326.
(34) Chee, S. M. Q.; Wongsantichon, J.; Soo Tng, Q.; Robinson, R.; Joseph, T. L.; Verma, C.; Lane, D. P.; Brown, C. J.; Ghadessy, F. J. Structure of a Stapled Peptide Antagonist Bound to Nutlin-Resistant Mdm2. PLoS ONE 2014, 9 (8), e104914.
(35) Villar, E. A.; Beglov, D.;
Chennamadhavuni, S.; Jr, J. A. P.; Kozakov, D.; Vajda, S.; Whitty, A. How Proteins Bind Macrocycles. Nat. Chem. Biol. 2014, 10 (9), 723–731.
«ÖãÙψ
SUPPORTING
INFORMATION
1.
E
XPERIMENTALM
ATERIALS ANDM
ETHODS1.1. SYNTHESIS AND ANALYSIS
All the reagents and solvents were purchased from Sigma-Aldrich, AK Scientific, Fluorochem, Abcr GmbH, Acros and were used without further purification. All microwave irradiation reactions were carried out in a Biotage Initiator™ Microwave Synthesizer. Thin layer chromatography was performed on Millipore precoated silica gel plates (0.20 mm thick, particle size ϮϱʅŵͿ͘EƵĐůĞĂƌŵĂŐŶĞƚŝĐƌĞƐŽŶĂŶĐĞƐƉĞĐƚƌĂǁĞƌĞƌĞĐŽƌĚĞĚŽŶĂƌƵŬĞƌǀĂŶĐĞ 500 or 600 spectrometers {1, EDZ ;ϱϬϬ D,nj͖ ϲϬϬ D,njͿ͕13 EDZ ;ϭϮϲ D,nj͖ ϭϱϭ D,njͿ͘
Chemical shifts for 1H NMR were reported as ɷ values and coupling constants were in hertz
;,njͿ͘ dŚe following abbreviations were used for spin multiplicity: s = singlet, br s = broad singlet, d = doublet, t = triplet, q = quartet, quin = quintet, dd = double of doublets, ddd = double doublet of doublets, m = multiplet. Chemical shifts for 13C NMR were reported in ppm
relative to the solvent peak. Flash chromatography was performed on a Reveleris® X2 Flash ŚƌŽŵĂƚŽŐƌĂƉŚLJ͕ƵƐŝŶŐ'ƌĂĐĞΠZĞǀĞůĞƌŝƐ^ŝůŝĐĂĨůĂƐŚĐĂƌƚƌŝĚŐĞƐ;ϭϮŐƌĂŵƐͿ͘DĂƐƐƐƉĞĐƚƌĂǁĞƌĞ measured on a Waters Investigator Supercritical Fluid Chromatograph with a 3100 MS ĞƚĞĐƚŽƌ; ^/ͿƵƐŝŶŐĂƐŽůǀĞŶƚƐLJƐƚĞŵŽĨŵĞƚŚĂŶŽůĂŶĚK2 on a Viridis silica gel column (4.6 x
ϮϱϬŵŵ͕ϱђŵƉĂƌƚŝĐůĞƐŝnjĞͿŽƌsŝƌŝĚŝƐϮ-ethyl pyridine column (4.6 x 250 mm, 5 μm particle ƐŝnjĞͿ͘ŶĂůLJƚŝĐĂůĐŚŝƌĂů^&ǁĂƐƉĞƌĨŽƌŵĞĚ on a Reprosil Chiral-IC column (4.6 x 250 mm, 5 μm ƉĂƌƚŝĐůĞ ƐŝnjĞͿ ĂŶĚ ƐĞŵŝ-preparative SFC was performed with stacked injector (250 μL ŝŶũĞĐƚŝŽŶƐͿ ŽŶ Ă ZĞƉƌŽƐŝů ŚŝƌĂů-/ ĐŽůƵŵŶ ;ϭϬ dž ϮϱϬ ŵŵ͕ ϱ ђŵ ƉĂƌƚŝĐůĞ ƐŝnjĞͿ ǁŝƚŚ Ϯϱй MeOH/CO2 as mobile phase. High resolution mass spectra were recorded using a
LTQ-Orbitrap-y>;dŚĞƌŵŽͿĂƚĂƌĞƐŽůƵƚŝŽŶŽĨϲϬϬϬϬΛŵͬnjϰϬϬ͘KƉƚŝĐĂůƌŽƚĂƚŝŽŶƐǁĞƌĞŵĞĂƐƵƌĞĚŝŶ DĞK,ŽŶĂ^ĐŚŵŝĚƚн,ĂĞŶƐĐŚƉŽůĂƌŝŵĞƚĞƌ;WŽůĂƌƚƌŽŶŝĐD,ϴͿǁŝƚŚĂϭϬĐŵĐĞůů;ĐŐŝǀĞŶŝŶ ŐͬϭϬϬŵ>Ϳ͘
1.2. PROTEIN EXPRESSION AND PURIFICATION
Fragment of the N-terminal domain of human MDM2 (residues 1-ϭϭϴͿ ǁĂƐ ĐůŽŶĞĚ ŝŶƚŽ ƚŚĞ pET-ϮϬ ;EŽǀĂŐĞŶͿ ĂŶĚ ĞdžƉƌĞƐƐĞĚ ŝŶ ͘ ĐŽůŝ ƐƚƌĂŝŶ >Ϯϭ-ŽĚŽŶWůƵƐ; ϯͿ-RIL as described previously.1 In brief, cells were cultured at 37 °C. Protein expression was induced with 1 mM IPTG at OD600 of 0.8 and cultured for additional 5 h at 37 °C. Cells were collected by centrifugation and lysed by sonication. Inclusion bodies were collected by centrifugation, ǁĂƐŚĞĚǁŝƚŚW^ĐŽŶƚĂŝŶŝŶŐϬ͘ϬϱйdƌŝƚŽŶ-X100 and subsequently solubilized in 6 M guanidine hydrochloride in 100 mM Tris-HCl, pH 8.0, containing 1 mM EDTA and 10 mM ɴ-mercaptoethanol. The protein was dialyzed against 4 M guanidine hydrochloride pH 3.5 supplemented with 10 mM ɴ-mercaptoethanol. Following, the protein was refolded by dropwise addition into 10 mM Tris-HCl, pH 7.0, containing 1 mM EDTA and 10 mM ɴ-mercaptoethanol and slow mixing overnight at 4 °C. Ammonium sulfate was added to the
Ù㮥®®½ÃÙÊù½ÝÝÖÊãÄãÖωχͲDDφ®Ä«®®ãÊÙÝ
4
5
6
7
8
9
final concentration of 1.5 M and the refolded protein was recovered on Butyl Sepharose 4 &ĂƐƚ&ůŽǁ;' ,ĞĂůƚŚĐĂƌĞͿ͘dŚĞƉƌŽƚĞŝŶǁĂƐĞůƵƚĞĚƵƐŝŶŐϭϬϬŵDdƌŝƐ-HCl pH 7.2 containing 5 ŵDɴ-mercaptoethanol and further purified by gel filtration on HiLoad 16/600 Superdex75 ;' ,ĞĂůƚŚĐĂƌĞͿŝŶϱϬŵDƉŚŽƐƉŚĂƚĞďƵffer pH 7.4 containing 150 mM NaCl and 5 mM DTT ;&WͬEDZďƵĨĨĞƌͿ͘
1.3. FLUORESCENCE POLARIZATION ASSAY
All FP measurements were performed using Tecan Infinite® 200 PRO plate reader. The assay
was conducted in 50 mM NaCl, 10 mM Tris pH 8.0, 1 mM EDTA contaiŶŝŶŐ ϱй D^K͘ dŽ determine the optimal concentration of the protein for the competition binding assay, the effective concentration of MDM2 (1-ϭϭϴͿ ǁĂƐ ĞĂĐŚ ƚŝŵĞ ĂƐĐĞƌƚĂŝŶĞĚ ďLJ ĚĞƚĞƌŵŝŶŝŶŐ ƚŚĞ apparent Kd towards 5’FAM->d& ,ztY>d^;WϮ͕ϭϬŶDͿ͘ŽŵƉĞƚŝƚŝŽŶassay was performed
by contacting serial dilutions of tested compounds with 10 nM P2 at protein concentration yielding f0 = 0.8. Fluorescence polarization was determined at 485 nm excitation and 535 nm
emission 15 min after mixing all assay components. All tests were performed using Corning black 96-well NBS assay plates at room temperature.
1.4. NMR EXPERIMENTS
All NMR spectra were acquired at 300 K using a Bruker Avance 600 MHz spectrometer. Uniform 15N isotope labelling was achieved by expression of the protein in the M9 minimal medium containing 15NH
4Cl as the sole nitrogen source. MDM2(1-ϭϭϴͿ ǁĂƐ ƉƌĞƉĂƌĞĚ ŝŶ ϱϬ
ŵDƉŚŽƐƉŚĂƚĞďƵĨĨĞƌƉ,ϳ͘ϰĐŽŶƚĂŝŶŝŶŐϭϱϬŵDEĂůĂŶĚϱŵDdd͘ϭϬй;ǀͬǀͿŽĨ2O was
added to the samples to provide lock signal. Water suppression was carried out using the WATERGATE sequence.2 Stock solutions of inhibitors used for titration were prepared in d6-DMSO. Several titrĂƚŝŽŶ ƐƚĞƉƐ ;ĐŽŵƉŽƵŶĚͬDDϮ͗ ϭ͗ϱ͕ ϭ͗ϰ͕ ϭ͗ϯ͕ ϭ͗Ϯ͕ ϭ͗ϭ͕ Ϯ͗ϭ͕ ϱ͗ϭͿ ǁĞƌĞ performed in order to assess the binding. The spectra were processed with TopSpin 3.2 software. 1H-15N heteronuclear correlations were obtained using the fast HSQC pulse sequence.3 Assignment of the amide groups of MDM2 was obtained as previously reported.4
2.
S
YNTHETIC PROCEDURES AND ANALYTICAL DATA2.1. PROCEDURE AND ANALYTICAL DATA FOR AMINE
5
ANHBoc NH2 OH O 3,4-benzylchloride NH2 OH (Boc)2O, NaHCO3 MeOH Cl Cl 9 5a K2CO3,MeCN Scheme 1.
«ÖãÙψ
2.1.1. Tert-butyl 4-ŚLJĚƌŽdžLJďĞŶnjLJůĐĂƌďĂŵĂƚĞ;ϵͿ
4-ŚLJĚƌŽdžLJďĞŶnjLJůĂŵŝŶĞ;ϭ͘ϬĞƋƵŝǀ͘Ϳ͕;ŽĐͿ2K ;ϭ͘ϭĞƋƵŝǀ͘ͿĂŶĚ
NaHCO3 ;Ϯ͘ϭĞƋƵŝǀ͘ͿǁĞƌĞƌĞĨůƵdžĞĚŝŶŵĞƚŚĂŶŽůŽǀĞƌŶŝŐŚƚ;ϭϲ
ŚͿ͘ĨƚĞƌǁĂƌĚƐ͕ƚŚĞƌĞĂĐƚŝŽŶǁĂƐĐŽŽůĞĚƚŽƌƚ and methanol was evaporated. To the resulting slurry, water and dichloromethane were added and the aqueous phase was extracted with dichloromethane. Organic layers were collected, washed with water, dried over anhydrous MgSO4 and evaporated, giving crude product as brown oil. The crude product ǁĂƐ ƉƌĞĐŝƉŝƚĂƚĞĚ ĂŶĚ ǁĂƐŚĞĚ ǁŝƚŚ ĚŝĞƚŚLJů ĞƚŚĞƌͬƉĞƚƌŽůĞƵŵ ĞƚŚĞƌ ;ĂƉƉƌŽdž͘ ϭ͗ϭͿ ŐŝǀŝŶŐ compound 9͖>ŝŐŚƚďƌŽǁŶƐŽůŝĚ͕ϵϵйLJŝĞůĚ͖1H NMR (600 MHz, CDCl3Ϳ͗ɷ [ppm] 7.10 (d, J = 7.3
,nj͕ Ϯ,Ϳ͕ ϲ͘ϳϳ;Ěƚ͕ J = ϴ͘ϲ͕ Ϯ͘Ϭ ,nj͕ Ϯ,Ϳ͕ ϰ͘ϴϲ ;Ɛ͕ ϭ,Ϳ͕ ϰ͘ϮϮ;Ɛ͕ Ϯ,Ϳ͕ ϭ͘ϰϲ;Ɛ͕ ϵ,Ϳ͖13C NMR (151
MHz, CDCl3Ϳ͗ɷ [ppm] 156.3, 155.5, 130.5, 129.0, 115.6, 79.9, 44.3, 28.6. LC-D^;ͬ ^/Ϳ͗ƚR =
5.31 min, Calcd for C12H17NO3 ;ŵͬnjͿ͗D-H]- 222.26, found: [M-H]- 222.10.
2.1.2. (4-((3,4-ĚŝĐŚůŽƌŽďĞŶnjLJůͿŽdžLJͿƉŚĞŶLJůͿŵĞƚŚĂŶĂŵŝŶĞ;ϱĂͿ
Tert-butyl 4-hydroxybenzylcarbamate 9 ;ϭ͘ϬĞƋƵŝǀ͘Ϳ͕ϯ͕ϰ-ďĞŶnjLJů ĐŚůŽƌŝĚĞ ;ϭ͘ϱ ĞƋƵŝǀ͘Ϳ͕ <2CO3 ;Ϯ͘Ϯ ĞƋƵŝǀ͘Ϳ ǁĞƌĞ
ƌĞĨůƵdžĞĚŝŶĂĐĞƚŽŶŝƚƌŝůĞŽǀĞƌŶŝŐŚƚ;ϭϲŚͿ͘ĨƚĞƌǁĂƌĚƐ͕ƚŚĞ reaction was cooled to rt and the solvent was evaporated. To the resulting solid, water and dichloromethane were added and the aqueous phase was extracted with dichloromethane. Organic layers were collected washed with water, dried over anhydrous MgSO4 and
evaporated, giving crude product as white-yellowish powder. Afterwards the amine was ĚĞƉƌŽƚĞĐƚĞĚ ŝŶƉƌĞƐĞŶĐĞ ŽĨ ƚƌŝĨƵŽƌŽĂĐĞƚŝĐĂĐŝĚ ;ϮϬ ĞƋƵŝǀ͘Ϳ ĂŶĚ DĂƐƐŽůǀĞŶƚ͕ ƚŚĞ ŵŝdžƚƵƌĞ was stirred at rt for 1.5 h. Water was added to stop the reaction and stirred for 5 min more, followed by washing with 2 times with DCM and once with water, to obtain the free amine in the aqueous phase. Further addition of NaOH 1 N and DCM to extract the amine, the organic layer were collected, washed with water, dried over anhydrous MgSO4 and evaporated, giving
the final product 5a͖tŚŝƚĞƉŽǁĚĞƌ͕ϲϳйLJŝĞůĚ͘1H NMR (500 MHz, CDCl
3Ϳɷ [ppm] 7.54 (d, J = Ϯ͘Ϭ,nj͕ϭ,Ϳ͕ϳ͘ϰϱ;Ě͕J = ϴ͘Ϯ,nj͕ϭ,Ϳ͕ϳ͘Ϯϵ– 7.22 (m, 3,Ϳ͕ϲ͘ϵϱ– ϲ͘ϴϴ;ŵ͕Ϯ,Ϳ͕ϱ͘Ϭϭ;Ɛ͕Ϯ,Ϳ͕ϯ͘ϴϭ ;Ɛ͕ Ϯ,Ϳ͖13C NMR (126 MHz, CDCl 3Ϳɷ [ppm] 137.5, 136.4, 130.7, 129.3, 128.5, 126.6, 115.0, 68.7, 46.0.
2.2. PROCEDURE AND ANALYTICAL DATA FOR ETHYL
6-CHLORO-3-FORMYL-1H-INDOLE-2-CARBOXYLATE (6
)
The 6-chloro-1H-indole-2-ĐĂƌďŽdžLJůŝĐĂĐŝĚĞƚŚLJůĞƐƚĞƌ;ϭ͘ϬĞƋƵŝǀ͘Ϳ ĂŶĚD&;ϮϬŵůͿǁĞƌĞƉůĂĐĞĚŝŶƌŽƵŶĚ-bottom flask equipped with CaCl2 tube. Then, POCl3 ;ϭ͘Ϯ ĞƋƵŝǀ͘Ϳ ǁĂƐĂĚĚĞĚĚƌŽƉǁŝƐĞ
anĚƚŚĞƌĞĂĐƚŝŽŶŵŝdžƚƵƌĞǁĂƐŚĞĂƚĞĚŽǀĞƌŶŝŐŚƚ;ϭϲŚͿĂƚϱϬǑ͘ N H O O HO O Cl NH2 Cl N H CO2Et CHO Cl
Ù㮥®®½ÃÙÊù½ÝÝÖÊãÄãÖωχͲDDφ®Ä«®®ãÊÙÝ
4
5
6
7
8
9
Afterwards, the reaction was cooled to rt, quenched with saturated NaHCO3 solution and
extracted with ethyl acetate. Organic layer was collected, washed with water and brine, dried over anhydrous MgSO4 and evaporated. The crude product was washed with diethyl ether giving compound 6;5 >ŝŐŚƚ LJĞůůŽǁ ƐŽůŝĚ͕ ϵϮй LJŝĞůĚ͖1H NMR (600 MHz, DMSO-d6Ϳ͗ ɷ [ppm] ϭϮ͘ϵϬ;Ɛ͕ϭ,Ϳ͕ϭϬ͘ϱϴ;Ɛ͕ϭ,Ϳ͕ϴ͘ϮϮ;Ě͕J = ϴ͘ϲ,nj͕ϭ,Ϳ͕ϳ͘ϱϲ;Ě͕J = ϭ͘ϱ,nj͕ϭ,Ϳ͕ϳ͘ϯϮ;ĚĚ͕J = 8.6, ϭ͘ϵ,nj͕ϭ,Ϳ͕ϰ.46 (q, J = ϳ͘ϭ,nj͕Ϯ,Ϳ͕ϭ͘ϰϬ;ƚ͕J = ϳ͘ϭ,nj͕ϯ,Ϳ͖13C (151 MHz, DMSO-d6Ϳ͗ɷ [ppm]
187.5, 159.9, 136.1, 133.5, 130.4, 124.0, 123.9, 123.4, 118.2, 112.6, 62.0, 14.1; LC-MS ;ͬ ^/Ϳ͗ƚR = 2.97 min, Calcd for C12H10ClNO3 ;ŵͬnjͿ͗D-H]- 250.03, [M+2-H]- 250.02, found:
[M-H]- 250.05, [M+2-H]- 252.05.
2.3. PROCEDURE AND ANALYTICAL DATA FOR ISOCYANIDES
7
O m NH2CHO R1 R1= H, Me m = 1,7 NHCHO m R1 HCO2H POCl3, Et3N DCM m NC R1 10a,b 7a,b Scheme 2.
2.3.1. General procedure of the modified Leuckart Wallach formamide synthesis
The 10-undecenal or 5-hexen-2-ŽŶĞ ;ϭ͘Ϭ ĞƋƵŝǀ͘Ϳ͕ ĨŽƌŵĂŵŝĚĞ ;ϱϰ͘Ϭ ĞƋƵŝǀ͘Ϳ ĂŶĚ ĨŽƌŵŝĐ ĂĐŝĚ ;ϭϭ͘ϱĞƋƵŝǀ͘ͿǁĞƌĞƌĞĨůƵdžĞĚĂƚϭϴϬΣĨŽƌϭŚ͘ĨƚĞƌǁĂƌĚƐ͕ƚŚĞƌĞĂĐƚŝŽŶǁĂƐĐŽŽůĞĚƚŽƌƚĂŶĚ extracted with DCM, organic layer was collected, washed with water, dried over anhydrous MgSO4 and evaporated. The products were obtained without further purification.
N-(hex-5-en-2-LJůͿĨŽƌŵĂŵŝĚĞ;ϭϬĂͿ
ƌŽǁŶŽŝů͕ϵϬйLJŝĞůĚ͖mixture of rotamers is observed, major rotamer is given; 1H NMR (500 MHz, CDCl3Ϳɷ ƉƉŵϴ͘ϭϮ ;Ɛ͕ ϭ,Ϳ͕ϲ͘ϭϯ;ďƌƐ͕ ϭ,Ϳ͕ϱ͘ϴϲ– ϱ͘ϳϮ;ŵ͕ϭ,Ϳ͕ϱ͘ϭϮ– ϰ͘ϵϬ;ŵ͕Ϯ,Ϳ͕ϰ͘Ϭϴ;Ěƚ͕J = 14.0, 6.8 ,nj͕ϭ,Ϳ͕Ϯ͘Ϯϭ– Ϯ͘Ϭϯ;ŵ͕Ϯ,Ϳ͕ϭ͘ϲϲ– ϭ͘ϰϴ;ŵ͕Ϯ,Ϳ͕ϭ͘ϭϳ;Ě͕J = ϲ͘ϲ,nj͕ϯ,Ϳ͖13C NMR (126 MHz, CDCl3Ϳɷ [ppm] 160.7, 137.7, 115.0, 43.7, 35.8, 30.0, 20.8. N-(undec-10-en-1-LJůͿĨŽƌŵĂŵŝĚĞ;ϭϬďͿ zĞůůŽǁ Žŝů͕ ϵϮй LJŝĞůĚ͖ mixture of rotamers is observed, major rotamer is given; 1H NMR (500 MHz, CDCl3Ϳɷ ƉƉŵϴ͘ϭϲ;Ɛ͕ϭ,Ϳ͕ϱ͘ϴϰ– ϱ͘ϳϳ;ŵ͕ϭ,Ϳ͕ϱ͘ϬϬ– ϰ͘ϵϮ;ŵ͕Ϯ,Ϳ͕ϯ͘Ϯϵ;Ƌ͕J = 6.6 Hz, ϭ,Ϳ͕Ϯ͘Ϭϰ;Ƌ͕J = ϲ͘ϵ,nj͕Ϯ,Ϳ͕ϭ͘ϱϮ;Ƌ͕J = ϲ͘ϴ,nj͕Ϯ,Ϳ͕ϭ͘ϯϯ;Ě͕J = ϰϲ͘Ϭ,nj͕ϭϯ,Ϳ͖13C NMR (126 MHz, CDCl3Ϳ ɷ [ppm] 161.4, 139.1, 114.1, 41.9, 38.2, 33.8, 29.44, 29.36, 29.2, 29.05, 28.9, 26.83. NHCHO NHCHO
«ÖãÙψ
2.3.2. General procedure of the isocyanide synthesis
The corresponding formamides 10a and 10b ;ϭ͘ϬĞƋƵŝǀ͘Ϳ͕ƚƌŝĞƚŚLJůĂŵŝŶĞ;ϰ͘ϬĞƋƵŝǀ͘ͿĂŶĚD were put together in a round-bottom flask and cooled to 0 °C. Then POCl3 ;ϭ͘ϭ ĞƋƵŝǀ͘ͿǁĂƐ
added dropwise and the reaction was stirred at rt for 3-4 h. After the reaction was completed, the products were poured into ice cold solution of NaHCO3 and left to reach rt. The
precipitated solid was filtered off and washed with DCM. The remaining water phase was extracted with DCM. Organic layers were collected, washed with water, dried over anhydrous MgSO4 and evaporated. The resulting oils were purified on silica pad with copious amount of
DCM, which was then collected and evaporated, giving the pure isocyanides 7a,b.
5-isocyanohex-1-ĞŶĞ;ϳĂͿ
ƌŽǁŶ Žŝů͕ ϴϳй LJŝĞůĚ͖ mixture of rotamers is observed, major rotamer is given; 1H NMR (500 MHz, CDCl 3Ϳɷ [ppm] 5.77 (ddt, J = 17.0, 10.2, 6.7 Hz, ϭ,Ϳ͕ϱ͘ϭϯ– ϰ͘ϵϳ;ŵ͕Ϯ,Ϳ͕ϯ͘ϲϰ;ĚĚĚ͕J = ϴ͘ϲ͕ϲ͘ϳ͕ϰ͘ϵ,nj͕ϭ,Ϳ͕Ϯ͘Ϯϯ;ĚĚƋ͕J = ϯϳ͘ϰ͕ ϭϰ͘ϵ͕ ϳ͘ϰ͕ ϲ͘ϳ ,nj͕ Ϯ,Ϳ͕ ϭ͘ϳϵ – ϭ͘ϱϲ ;ŵ͕ Ϯ,Ϳ͕ ϭ͘ϰϭ – ϭ͘ϯϮ ;ŵ͕ ϯ,Ϳ͖13C NMR (126 MHz, CDCl3Ϳɷ [ppm] 154.6, 136.4, 116.1, 49.6, 46.0, 35.8, 29.8, 21.6, 8.6. 11-isocyanoundec-1-ĞŶĞ;ϳďͿ
ƌŽǁŶ Žŝů͕ ϳϱй LJŝĞůĚ͖ mixture of rotamers is observed, major rotamer is given; 1H NMR (500 MHz, CDCl
3Ϳ ɷ
[ppm] 5.90 – ϱ͘ϳϬ;ŵ͕ϭ,Ϳ͕ϱ͘Ϭϱ– 4.89 (m, 2HͿ͕ϯ͘ϯϴ;ĚĚƚ͕J = ϲ͘ϳ͕ϯ͘ϳ͕ϭ͘ϴ,nj͕ϭ,Ϳ͕Ϯ͘Ϭϰ;Ƌ͕J = ϲ͘ϵ ,nj͕ Ϯ,Ϳ͕ϭ͘ϲϴ ;ĚĚĚ͕J = ϭϬ͘Ϭ͕ ϰ͘ϲ͕Ϯ͘ϭ ,nj͕ϭ,Ϳ͕ϭ͘ϰϴ– ϭ͘ϭϯ;ŵ͕ ϭϰ,Ϳ͖13C NMR (126 MHz, CDCl3Ϳɷ [ppm] 154.4, 136.5, 116.1, 49.6, 45.9, 35.8, 31.6, 29.9, 21.6, 8.6.
2.4. GENERAL PROCEDURE OF THE UGI FOUR-COMPONENT REACTION (UT-4CR)
The corresponding amine 5 ;ϭ͘ϬĞƋƵŝǀ͘Ϳ͕ĂůĚĞŚLJĚĞ6 ;ϭ͘ϬĞƋƵŝǀ͘Ϳ͕ŝƐŽĐLJĂŶŝĚĞ7 ;ϭ͘ϬĞƋƵŝǀ͘ͿĂŶĚ acid 8 ;ϭ͘ϬĞƋƵŝǀ͘ͿǁĞƌĞĚŝƐƐŽůǀĞĚŝŶϮ͕Ϯ͕Ϯ-ƚƌŝĨůƵŽƌŽĞƚŚĂŶŽů;Ϯŵ>ͿĂŶĚƉůĂĐĞĚŝŶƚŽĂŵŝĐƌŽǁĂǀĞ vial which irradiated at 12ϬǑĨŽƌϲϬŵŝŶ͘ĨƚĞƌǁĂƌĚƐ͕ƚŚĞƐŽůǀĞŶƚǁĂƐĞǀĂƉŽƌĂƚĞĚĂŶĚƚŚĞ crude mixture was purified by flash chromatography (hexane-ĞƚŚLJů ĂĐĞƚĂƚĞͿ ŐŝǀŝŶŐ ƚŚĞ corresponding compounds 4 (yields 15-ϲϬйͿĂƐLJĞůůŽǁŽŝůƐ͘
NC
Ù㮥®®½ÃÙÊù½ÝÝÖÊãÄãÖωχͲDDφ®Ä«®®ãÊÙÝ
4
5
6
7
8
9
Ethyl 6-chloro-3-(1-(N -(4-((3,4-ĚŝĐŚůŽƌŽďĞŶnjLJůͿŽdžLJͿďĞŶnjLJůͿƉĞŶƚ-4-ĞŶĂŵŝĚŽͿ-2-(hex-5-en-2-LJůĂŵŝŶŽͿ-2-ŽdžŽĞƚŚLJůͿ-1H-indole-2-ĐĂƌďŽdžLJůĂƚĞ;ϰĂͿ
ϭϱй LJŝĞůĚ͖ mixture of rotamers and diastereomers observed; 1H
NMR (500 MHz, CDCl3Ϳɷ ƉƉŵϵ͘ϯϬ;Ɛ͕ϭ,Ϳ͕ϳ͘ϴϯ;Ě͕J = ϴ͘ϴ,nj͕ϭ,Ϳ͕ 7.48 – ϳ͘ϰϬ;ŵ͕Ϯ,Ϳ͕ϳ͘Ϯϵ– ϳ͘ϭϰ;ŵ͕ϰ,Ϳ͕ϳ͘Ϭϴ;ĚĚ͕J = 14.5, 8.6 Hz, ϭ,Ϳ͕ϲ͘ϵϳ– ϲ͘ϴϰ;ŵ͕Ϯ,Ϳ͕ϲ͘ϱϯ;Ěƚ͕J = ϮϬ͘ϰ͕ϵ͘ϲ,nj͕ϰ,Ϳ͕ϱ͘ϴϮ;ĚĚƚ͕J = ϭϲ͘ϳ͕ϭϮ͘ϱ͕ϲ͘ϭ,nj͕ ϭ,Ϳ͕ ϱ͘ϱϴ;ĚĚ͕J = ϭϯ͘ϰ͕ ϳ͘ϱ,nj͕ϭ,Ϳ͕ϱ͘Ϭϵ– 4.90 ;ŵ͕ϱ,Ϳ͕ϰ͘ϵϭ– ϰ͘ϲϭ;ŵ͕ϰ,Ϳ͕ϰ͘ϰϴ– ϰ͘Ϯϲ;ŵ͕ϰ,Ϳ͕ϰ͘Ϭϱ;ĚĚ͕J = 13.5, ϲ͘ϴ,nj͕ϭ,Ϳ͕ϯ͘ϰϳ;Ɛ͕ϭ,Ϳ͕Ϯ͘ϱϳ;ĚĚ͕J = ϭϯ͘Ϯ͕ϳ͘ϱ,nj͕ϭ,Ϳ͕Ϯ͘ϰϰ;ĚĚƚ͕J = Ϯϱ͘Ϯ͕ϭϯ͘ϵ͕ϲ͘ϴ,nj͕ϰ,Ϳ͕Ϯ͘ϯϬ;ƚ͕J = ϳ͘ϰ ,nj͕ ϭ,Ϳ͕Ϯ͘Ϭϳ;Ě͕J = 7.3 Hz, ϭ,Ϳ͕ϭ.99 – ϭ͘ϴϵ;ŵ͕ϭ,Ϳ͕ϭ͘ϴϬ;Ƌ͕J = ϳ͘Ϭ,nj͕ϭ,Ϳ͕ϭ͘ϰϯ;Ěƚ͕J = 12.5, ϳ͘Ϯ,nj͕ϭ,Ϳ͕ϭ͘ϯϲ;Ƌ͕J = ϵ͘Ϭ͕ϴ͘Ϭ,nj͕ϯ,Ϳ͕ϭ͘ϯϬ– ϭ͘Ϯϭ;ŵ͕ϭ,Ϳ͕ϭ͘ϭϭ;Ě͕J = ϲ͘ϱ,nj͕ϯ,Ϳ͖13C NMR (126 MHz, CDCl3Ϳɷ [ppm] 173.5, 172.2, 169.3, 162.8, 160.9, 157.6, 156.8, 137.8, 137.5, 137.0, 135.8, 132.6, 131.8, 131.6, 131.2, 130.8, 130.5, 130.3, 130.0, 129.2, 129.2, 129.0, 128.0, 127.2, 126.5, 126.4, 126.3, 125.7, 122.9, 122.4, 115.7, 115.3, 115.0, 114.7, 114.3, 111.8, 100.0, 77.3, 77.1, 76.8, 68.5, 68.4, 61.6, 54.4, 50.2, 49.1, 45.5, 43.9, 43.0, 35.8, 35.5, 33.1, 30.2, 29.9, 29.6, 29.4, 20.5, 14.4; LC-D^ ;ͬ ^/Ϳ͗ ƚR = 3.22 min, Calcd for C38H40Cl3N3O5
;ŵͬnjͿ͗D-H]- 722.20, found: [M-H]- 722.13.
Ethyl 6-chloro-3-(1-(N -(4-((3,4-ĚŝĐŚůŽƌŽďĞŶnjLJůͿŽdžLJͿďĞŶnjLJůͿŚĞdž-5-ĞŶĂŵŝĚŽͿ-2-(hex-5-en-2-ylamiŶŽͿ-2-ŽdžŽĞƚŚLJůͿ-1H-indole-2-ĐĂƌďŽdžLJůĂƚĞ;ϰďͿ
ϮϭйLJŝĞůĚ͖mixture of rotamers and diastereomers observed; 1H NMR
(500 MHz, CDCl3Ϳɷ ƉƉŵϵ͘ϯϬ;Ɛ͕ϭ,Ϳ͕ϳ͘ϴϱ;ĚĚ͕J = ϴ͘ϳ͕ϰ͘ϵ,nj͕ϭ,Ϳ͕ 7.58 – ϳ͘ϱϬ;ŵ͕Ϯ,Ϳ͕ϳ͘ϱϬ– ϳ͘ϰϭ;ŵ͕ϯ,Ϳ͕ϳ͘ϯϭ– ϳ͘ϭϱ;ŵ͕ϱ,Ϳ͕ϳ͘ϭϬ;Ě͕ J = 8.6 ,nj͕ϭ,Ϳ͕ϲ͘ϵϯ– ϲ͘ϴϲ;ŵ͕Ϯ,Ϳ͕ϲ͘ϲϮ– ϲ͘ϰϰ;ŵ͕ϯ,Ϳ͕ϱ͘ϳϲ;ĚĚƚĚ͕J = ϭϴ͘Ϭ͕ϭϰ͘ϲ͕ϭϬ͘ϯ͕ϳ͘ϯ ,nj͕ϯ,Ϳ͕ϱ͘ϭϰ– ϰ͘ϴϵ;ŵ͕ϴ,Ϳ͕ϰ͘ϵϬ– 4.63 (m, ϰ,Ϳ͕ ϰ͘ϱϭ– ϰ͘ϯϰ;ŵ͕ ϰ,Ϳ͕ϰ͘ϯϮ– ϰ͘ϮϮ ;ŵ͕ Ϯ,Ϳ͕ ϰ͘ϭϮ– ϯ͘ϵϴ;ŵ͕ ϭ,Ϳ͕ 2.37 – Ϯ͘Ϯϰ;ŵ͕ Ϯ,Ϳ͕ Ϯ͘Ϯϰ– Ϯ͘ϭϱ ;ŵ͕ Ϯ,Ϳ͕Ϯ͘Ϭϴ ;ƚƚ͕J = 14.3, 7.3 Hz, ϰ,Ϳ͕ ϭ͘ϴϲ– ϭ͘ϲϱ;ŵ͕ϰ,Ϳ͕ ϭ͘ϱϵ– ϭ͘ϯϵ ;ŵ͕ ϯ,Ϳ͕ϭ͘ϰϭ– ϭ͘Ϯϳ;ŵ͕ ϰ,Ϳ͕ 1.28 – ϭ͘ϭϱ;ŵ͕ϭ,Ϳ͕ϭ͘ϭϭ;ĚĚ͕J = ϭϱ͘ϱ͕ϲ͘ϱ,nj͕Ϯ,Ϳ͕Ϭ͘ϵϱ;Ě͕J = 6.5 Hz, ϭ,Ϳ͖13C NMR (126 MHz, CDCl 3Ϳ ɷ [ppm] 190.8, 177.3, 174.1, 172.8, 169.3, 161.0, 157.7, 156.8, 138.1, 138.0, 137.8, 137.7, 137.3, 137.2, 135.9, 132.7, 132.6, 132.1, 131.9, 131.8, 131.7, 131.2, 130.9, 130.7, 130.6, 130.5, 129.3, 129.2, 129.0, 127.1, 126.5, 126.4, 126.3, 125.8, 124.7, 123.0, 122.8, 122.4, 115.4, 115.4, 115.1, 115.0, 114.9, 114.7, 114.2, 112.2, 111.8, 77.3, 77.1, 76.8, 68.7, 68.5, 68.4, 61.7, 54.4, 49.1, 45.5, 45.4, 43.9, 43.0, 35.9, 35.4, 33.3, 33.2, 33.1, 33.0, 33.0, 30.2, 29.9, 24.8, 24.5, 24.0, 20.6, 20.5, 14.4; LC-D^;ͬ ^/Ϳ͗ƚR =
3.21 min, Calcd for C39H42Cl3N3O5 ;ŵͬnjͿ͗D-H]- 736.22, found: [M-H]- 736.15.
N O HN Cl O HN O EtO2C Cl Cl N O HN Cl O HN O EtO2C Cl Cl
«ÖãÙψ
Ethyl 6-chloro-3-(1-(N -(4-((3,4-ĚŝĐŚůŽƌŽďĞŶnjLJůͿŽdžLJͿďĞŶnjLJůͿƵŶĚĞĐ-10-ĞŶĂŵŝĚŽͿ-2-(hex-5-en-2-LJůĂŵŝŶŽͿ-2-ŽdžŽĞƚŚLJůͿ-1H-indole-2-ĐĂƌďŽdžLJůĂƚĞ;ϰĐͿ
ϭϴй LJŝĞůĚ͖ mixture of rotamers and diastereomers observed; 1H
NMR (500 MHz, CDCl3Ϳ ɷ [ppm] 9.64 (d, J = ϵ͘ϰ ,nj͕ ϭ,Ϳ͕ ϳ͘ϵϮ – ϳ͘ϴϭ;ŵ͕ϭ,Ϳ͕ϳ͘ϱϳ– ϳ͘ϯϴ;ŵ͕ϰ,Ϳ͕ϳ͘ϯϳ– ϳ͘Ϭϲ;ŵ͕ϱ,Ϳ͕ϲ͘ϵϵ;Ě͕J = ϱ͘ϵ ,nj͕ϭ,Ϳ͕ϲ͘ϴϳ;ƚ͕J = ϳ͘ϱ ,nj͕ ϭ,Ϳ͕ ϲ͘ϲϲ– ϲ͘ϰϰ ;ŵ͕ ϯ,Ϳ͕ ϱ͘ϴϲ– ϱ͘ϲϵ;ŵ͕ϯ,Ϳ͕ ϱ͘Ϯϴ;Ɛ͕ϭ,Ϳ͕ϱ͘ϭϰ– ϰ͘ϴϴ;ŵ͕ ϲ,Ϳ͕ϰ͘ϴϰ ;Ě͕J = 15.4 ,nj͕Ϯ,Ϳ͕ϰ͘ϴϬ– ϰ͘ϲϳ;ŵ͕ϭ,Ϳ͕ϰ͘ϰϱ;ĚĚ͕J = ϭϳ͘ϱ͕ϴ͘Ϭ,nj͕ϭ,Ϳ͕ϰ͘ϯϲ (t, J = ϳ͘ϴ,nj͕ϭ,Ϳ͕ϰ͘ϯϭ– ϰ͘Ϯϭ;ŵ͕ϭ,Ϳ͕ϰ͘ϭϭ– ϰ͘ϬϬ;ŵ͕ϭ,Ϳ͕Ϯ͘ϱϮ– 2.39 (m, ϭ,Ϳ͕Ϯ͘ϯϭ;Ěƚ͕J = ϭϭ͘ϳ͕ϲ͘ϴ,nj͕ϭ,Ϳ͕Ϯ͘ϮϬ;ƚ͕J = ϳ͘ϲ,nj͕ϭ,Ϳ͕ 2.02 (dq, J = ϭϰ͘ϱ͕ϳ͘ϳ͕ϳ͘ϭ,nj͕ϰ,Ϳ͕ϭ͘ϳϵ;Ƌ͕J = ϳ͘ϭ,nj͕ϭ,Ϳ͕ϭ͘ϳϱ– ϭ͘ϱϮ;ŵ͕ϯ,Ϳ͕ϭ͘ϱϮ– ϭ͘ϭϲ;ŵ͕Ϯϭ,Ϳ͕ϭ͘ϭϳ– ϭ͘ϬϮ;ŵ͕ϯ,Ϳ͕Ϭ͘ϵϱ;Ě͕J = ϲ͘ϱ,nj͕ϭ,Ϳ͖͘13C NMR (126 MHz, CDCl3Ϳ ɷ [ppm] 177.5, 174.4, 174.4, 173.3, 169.5, 169.4, 161.1, 160.2, 157.6, 156.8, 139.2, 139.2, 139.1, 138.0, 137.8, 137.3, 137.2, 136.0, 132.7, 132.6, 131.9, 131.8, 131.5, 131.3, 130.9, 130.5, 130.5, 129.2, 129.1, 129.0, 128.1, 127.7, 127.2, 126.5, 126.4, 126.3, 125.7, 122.9, 122.8, 122.3, 121.7, 115.3, 115.1, 115.0, 115.0, 114.9, 114.7, 114.6, 114.2, 114.2, 114.2, 114.1, 113.9, 112.3, 111.9, 77.4, 77.1, 76.9, 68.5, 68.4, 61.6, 61.3, 57.2, 56.9, 54.5, 54.3, 53.5, 49.2, 49.1, 46.3, 45.7, 45.5, 45.4, 43.0, 36.8, 35.9, 35.4, 34.1, 33.8, 30.2, 29.9, 29.6, 29.4, 29.4, 29.3, 29.3, 29.2, 29.2, 29.1, 28.9, 25.8, 25.4, 24.9, 20.6, 20.5, 14.3; LC-D^ ;ͬ ^/Ϳ͗ ƚR = 3.14 min, Calcd for C44H52Cl3N3O5 ;ŵͬnjͿ͗ D-H]- 806.30, found: [M-H]
-806.32.
Ethyl 6-chloro-3-(1-(N -(4-ĨůƵŽƌŽďĞŶnjLJůͿƉĞŶƚ-4-ĞŶĂŵŝĚŽͿ-2-oxo-2-(undec-10-en-1-LJůĂŵŝŶŽͿĞƚŚLJůͿ-1H-indole-2-ĐĂƌďŽdžLJůĂƚĞ;ϰĚͿ
Ϯϯй LJŝĞůĚ͖ mixture of rotamers observed; 1H NMR (500 MHz, CDCl 3Ϳ ɷ [ppm] 7.50 (d, J = 8.5 ,nj͕ϭ,Ϳ͕ϳ͘ϭϯ;ƚ͕J = ϵ͘ϱ,nj͕Ϯ,Ϳ͕ϲ͘ϯϱ;Ɛ͕ϯ,Ϳ͕ϲ͘ϭϯ;Ɛ͕ ϭ,Ϳ͕ϱ͘ϳϬ;Ɛ͕ϯ,Ϳ͕ϱ͘ϭϰ;Ě͕J = ϭϲ͘ϲ,nj͕ϯ,Ϳ͕ϰ͘ϳϱ;Ě͕J = ϭϳ͘ϴ,nj͕Ϯ,Ϳ͕ϰ͘ϰϲ (d, J = ϭϳ͘ϳ,nj͕Ϯ,Ϳ͕ϳ͘ϴϮ;Ě͕J = ϴ͘ϴ,nj͕ϯ,Ϳ͕ϳ͘Ϭϭ;Ě͕J = ϭϳ͘Ϭ,nj͕ϭ,Ϳ͕ϲ͘ϲϮ (dt, J = Ϯϭ͘ϲ͕ϴ͘ϭ,nj͕ϰ,Ϳ͕ϲ͘ϱϯ;Ɛ͕ϭ,Ϳ͕ϱ͘ϴϮ;ƚĚĚ͕J = ϭϲ͘ϳ͕ϭϬ͘ϯ͕ϰ͘ϯ,nj͕ϯ,Ϳ͕ 5.08 – ϰ͘ϵϴ;ŵ͕ϯ,Ϳ͕ϰ͘ϵϰ;ĚĚ͕J = ϭϲ͘ϰ͕ϭϬ͘ϲ,nj͕ϰ,Ϳ͕ϰ͘ϯϴ;Ěƚ͕J = 14.3, 7.2 ,nj͕ϭ,Ϳ͕ϰ͘ϯϮ;Ěƚ͕J = ϭϰ͘ϰ͕ϳ͘ϭ,nj͕Ϯ,Ϳ͕ϯ͘ϴϮ;Ě͕J = ϭϱ͘Ϯ,nj͕ϭ,Ϳ͕ϯ͘Ϯϵ;Ěƚ͕J = ϭϯ͘ϭ͕ϲ͘ϱ,nj͕ϭ,Ϳ͕ϯ.21 (dt, J = ϭϯ͘ϵ͕ϲ͘ϳ,nj͕ϭ,Ϳ͕Ϯ͘ϱϳ;ĚƋ͕J = 17.0, 10.8, ϵ͘Ϯ,nj͕Ϯ,Ϳ͕Ϯ͘ϰϳ;Ěƚ͕J = ϭϯ͘ϲ͕ϳ͘ϭ,nj͕ϯ,Ϳ͕Ϯ͘ϯϲ;ƚƚ͕J = ϭϯ͘ϱ͕ϴ͘Ϭ,nj͕Ϯ,Ϳ͕ 2.02 (q, J = ϲ͘ϲ,nj͕ϯ,Ϳ͕ϭ͘ϰϴ;Ě͕J = Ϯϰ͘ϭ,nj͕ϭ,Ϳ͕ϭ͘ϯϱ;ĚƋ͕J = ϭϱ͘ϲ͕ϴ͘ϰ͕ϳ͘ϳ,nj͕ϭϭ,Ϳ͕ϭ͘ϭϳ;Ě͕J = ϭϴ͘ϲ,nj͕ϵ,Ϳ͖13C NMR (126 MHz, CDCl 3Ϳɷ [ppm] 173.4, 169.8, 160.8, 160.4, 139.2, 137.4, 135.8, 133.6, 131.7, 127.6, 127.2, 126.5, 126.5, 125.7, 122.6, 121.6, 115.5, 115.3, 114.7, 114.6, 114.1, 113.9, 112.3, 111.9, 77.3, 77.1, 76.8, 61.7, 61.4, 56.4, 54.1, 49.1, 46.8, 40.0, 33.8, 33.1, 32.5, 29.4, 29.4, 29.2, 29.1, 28.9, 26.9, 14.4; LC-D^;ͬ ^/Ϳ͗ƚR = 2.82 min, Calcd for C36H45ClFN3O4 ;ŵͬnjͿ͗D-H]- 636.31, found: [M-H]- 636.31. N O N H Cl O HN O CO2Et Cl Cl N HN Cl O HN O EtO2C F
Ù㮥®®½ÃÙÊù½ÝÝÖÊãÄãÖωχͲDDφ®Ä«®®ãÊÙÝ
4
5
6
7
8
9
Ethyl 6-chloro-3-(1-(N -(2,4-ĚŝĐŚůŽƌŽďĞŶnjLJůͿƉĞŶƚ-4-ĞŶĂŵŝĚŽͿ-2-oxo-2-(undec-10-en-1-LJůĂŵŝŶŽͿĞƚŚLJůͿ-1H-indole-2-ĐĂƌďŽdžLJůĂƚĞ;ϰĞͿ
ϮϲйLJŝĞůĚ͖ mixture of rotamers observed; 1H NMR (500 MHz, CDCl
3Ϳɷ ƉƉŵϵ͘ϰϬ;Ɛ͕Ϯ,Ϳ͕ϵ͘Ϯϱ;Ɛ͕ϭ,Ϳ͕ϳ͘ϴϮ;Ě͕J = ϴ͘ϴ,nj͕ϭ,Ϳ͕ϳ͘ϱϮ;Ě͕J = 8.6 ,nj͕ϭ,Ϳ͕ϳ͘ϯϭ– ϳ͘Ϯϲ;ŵ͕Ϯ,Ϳ͕ϳ͘ϭϰ– ϳ͘Ϭϳ;ŵ͕Ϯ,Ϳ͕ϳ͘ϬϬ;Ě͕J = 12.4 Hz, Ϯ,Ϳ͕ϲ͘ϲϰ;Ɛ͕ϭ,Ϳ͕ϱ͘ϴϮ;ƚĚƚ͕J = ϭϲ͘ϴ͕ϵ͘ϵ͕ϲ͘ϰ,nj͕ϯ,Ϳ͕ϱ͘ϲϲ;ƚ͕J = 5.5 Hz, ϯ,Ϳ͕ϱ͘ϭϭ– ϱ͘Ϭϯ;ŵ͕ϭ,Ϳ͕5.02 – ϰ͘ϴϵ;ŵ͕ϱ,Ϳ͕ϰ͘ϲϴ;Ɛ͕Ϯ,Ϳ͕ϰ͘ϰϰ– 4.32 ;ŵ͕ϯ,Ϳ͕ϯ͘Ϯϵ;ƚƚ͕J = ϭϮ͘ϰ͕ϲ͘Ϯ,nj͕ϭ,Ϳ͕ϯ͘Ϯϭ;ĚƋ͕J = ϭϮ͘ϴ͕ϲ͘ϵ,nj͕ϭ,Ϳ͕ 3.10 – Ϯ͘ϵϵ;ŵ͕ϭ,Ϳ͕Ϯ͘ϴϵ– Ϯ͘ϴϭ;ŵ͕ϭ,Ϳ͕ Ϯ͘ϱϯ– Ϯ͘ϯϱ ;ŵ͕ϱ,Ϳ͕Ϯ͘Ϯϲ– Ϯ͘ϭϲ;ŵ͕ϭ,Ϳ͕Ϯ͘ϬϮ;Ƌ͕J = ϳ͘Ϭ,nj͕ϯ,Ϳ͕ϭ͘ϯϴ;Ěƚƚ͕J = 22.7, 15.1, 7.4 Hz, ϵ,Ϳ͕ ϭ͘ϭϱ ;ƚ͕ J = ϭϮ͘ϰ ,nj͕ ϳ,Ϳ͖13C NMR (126 MHz, CDCl 3Ϳ ɷ [ppm] 173.5, 173.5, 169.4, 169.4, 160.9, 160.9, 139.2, 139.2, 137.2, 137.2, 137.1, 137.1, 136.8, 135.8, 135.8, 133.8, 133.8, 132.8, 132.8, 132.3, 132.3, 132.0, 132.0, 131.1, 131.1, 129.3, 129.3, 128.8, 128.8, 128.0, 128.0, 127.5, 127.5, 127.3, 127.3, 126.9, 126.9, 126.4, 126.4, 125.8, 125.8, 125.5, 125.5, 122.8, 122.8, 121.8, 121.8, 115.8, 115.8, 115.5, 115.5, 115.3, 115.3, 115.2, 115.2, 114.3, 114.3, 114.1, 114.1, 112.1, 112.1, 111.9, 111.9, 77.3, 77.3, 77.0, 77.0, 76.8, 76.8, 61.9, 61.9, 61.6, 61.6, 56.3, 54.2, 54.2, 47.3, 47.3, 45.3, 40.9, 40.9, 40.3, 40.3, 40.1, 40.1, 35.7, 35.7, 33.8, 33.8, 32.8, 32.8, 32.4, 32.4, 29.5, 29.5, 29.4, 29.4, 29.4, 29.4, 29.2, 29.2, 29.2, 29.2, 29.1, 29.1, 28.9, 28.9, 27.0, 27.0, 26.9, 26.9, 23.4, 23.4, 14.5, 14.5, 14.3, 14.3; LC-D^;ͬ ^/Ϳ͗ƚR = 2.86 min, Calcd for C36H44Cl3N3O4 ;ŵͬnjͿ͗D-H]- 686.24, found: [M-H]- 686.23.
Ethyl 6-chloro-3-(1-(N -(4-ĐŚůŽƌŽďĞŶnjLJůͿƉĞŶƚ-4-ĞŶĂŵŝĚŽͿ-2-oxo-2-(undec-10-en-1-LJůĂŵŝŶŽͿĞƚŚLJůͿ-1H-indole-2-ĐĂƌďŽdžLJůĂƚĞ;ϰĨͿ
Ϯϱй LJŝĞůĚ͖ mixture of rotamers observed; 1H NMR (500 MHz, CDCl 3Ϳ ɷ ƉƉŵϵ͘ϰϵ;Ɛ͕ϭ,Ϳ͕ϵ͘ϯϳ;Ɛ͕ϭ,Ϳ͕ϳ͘ϴϬ(d, J = ϴ͘ϴ,nj͕ϭ,Ϳ͕ϳ͘ϱϬ;Ě͕J = 8.6 Hz, ϭ,Ϳ͕ϳ͘ϯϭ– ϳ͘ϮϬ;ŵ͕ϭ,Ϳ͕ϳ͘ϭϭ;Ě͕J = ϴ͘ϵ,nj͕ϭ,Ϳ͕ϳ͘ϬϬ;Ɛ͕ϭ,Ϳ͕ϲ͘ϵϯ;Ě͕J = ϴ͘ϭ,nj͕ϭ,Ϳ͕ϲ͘ϴϬ;Ě͕J = ϳ͘ϴ,nj͕ϭ,Ϳ͕ϲ͘ϱϵ;Ě͕J = ϴ͘Ϭ,nj͕ϭ,Ϳ͕ϲ͘ϯϭ;Ě͕J = 7.7 ,nj͕ϭ,Ϳ͕ϲ͘ϭϱ;Ɛ͕ϭ,Ϳ͕ϱ͘ϳϵ;ƚĚĚ͕J = Ϯϰ͘ϰ͕ϭϮ͘ϲ͕ϲ͘ϳ,nj͕Ϯ,Ϳ͕ϱ͘ϭϰ;Ě͕J = 17.4 ,nj͕ϭ,Ϳ͕ϰ͘ϵϵ;ĚĚ͕J = ϭϳ͘ϱ͕ϭϭ͘Ϯ,nj͕Ϯ,Ϳ͕ϰ͘ϵϮ;Ě͕J = ϭϭ͘ϯ,nj͕ϭ,Ϳ͕ϰ͘ϳϰ;Ě͕J = ϭϴ͘Ϭ,nj͕ϭ,Ϳ͕ ϰ͘ϰϲ;Ě͕J = ϭϴ͘ϭ,nj͕ϭ,Ϳ͕ϰ͘ϯϰ;ĚƚĚĚ͕J = 23.8, 19.8, 11.5, ϳ͘Ϭ ,nj͕ Ϯ,Ϳ͕ ϯ͘ϳϵ ;Ě͕ J = ϭϱ͘ϲ ,nj͕ ϭ,Ϳ͕ ϯ͘ϰϮ – 3.34 (m͕ ϭ,Ϳ͕ ϯ͘ϯϬ ;ĚƋ͕ J = ϭϯ͘ϭ͕ϲ͘ϳ,nj͕ϭ,Ϳ͕ϯ͘ϮϬ;ĚƋ͕J = ϭϯ͘Ϭ͕ϲ͘ϴ,nj͕ϭ,Ϳ͕Ϯ͘ϱϲ;Ěƚ͕J = 14.4, 6.5 Hz, ϭ,Ϳ͕Ϯ͘ϱϭ– Ϯ͘ϰϮ;ŵ͕ϭ,Ϳ͕Ϯ͘ϰϬ;ĚĚ͕J = ϭϬ͘ϯ͕ϱ͘ϵ,nj͕ϭ,Ϳ͕Ϯ͘ϯϴ– Ϯ͘Ϯϵ;ŵ͕ϭ,Ϳ͕Ϯ͘Ϭϯ;Ɖ͕J = 7.5, ϲ͘ϲ,nj͕Ϯ,Ϳ͕ϭ͘ϰϵ;Ɛ͕ϭ,Ϳ͕ϭ͘ϰϰ– ϭ͘ϯϮ;ŵ͕ϲ,Ϳ͕ϭ͘Ϯϯ;Ě͕J = ϯϱ͘ϰ,nj͕ϳ,Ϳ͕ϭ͘ϭϱ;Ɛ͕ϰ,Ϳ͖13C NMR (126 MHz, CDCl3Ϳɷ [ppm] 173.9, 173.4, 169.9, 160.8, 139.2, 137.6, 137.3, 136.5, 135.9, 132.2, 131.7, 129.0, 127.9, 127.4, 127.4, 127.2, 126.4, 125.6, 124.9, 122.6, 122.5, 121.5, 115.4, 115.1, 114.4, 114.1, 112.4, 112.0, 77.3, 77.1, 76.8, 61.7, 61.4, 56.4, 54.2, 51.6, 49.1, 46.9, 40.2, 40.0, 33.8, 33.1, 32.5, 29.4, 29.4, 29.2, 29.1, 28.9, 27.1, 27.0, 26.9, 23.6, 14.4; LC-MS ;ͬ ^/Ϳ͗ƚR = 2.89 min, Calcd for C36H45Cl2N3O4 ;ŵͬnjͿ͗D-H]- 652.28, found: [M-H]- 652.31.
N HN Cl O HN O EtO2C Cl Cl N HN Cl O HN O EtO2C Cl
«ÖãÙψ
Ethyl 6-chloro-3-(1-(N -(4-ŵĞƚŚŽdžLJďĞŶnjLJůͿƉĞŶƚ-4-ĞŶĂŵŝĚŽͿ-2-oxo-2-(undec-10-en-1-LJůĂŵŝŶŽͿĞƚŚLJůͿ-1H-indole-2-ĐĂƌďŽdžLJůĂƚĞ;ϰŐͿ
Ϯϱй LJŝĞůĚ͖ mixture of rotamers observed; 1H NMR (500 MHz, CDCl 3Ϳ ɷ ƉƉŵϵ͘ϭϳ;Ɛ͕ϭ,Ϳ͕ϳ͘ϴϮ;Ě͕J = ϴ͘ϳ,nj͕ϭ,Ϳ͕ϳ͘Ϯϲ;Ě͕J = ϲ͘Ϭ,nj͕ϭ,Ϳ͕ϳ͘ϭϭ (d, J = ϴ͘ϱ,nj͕ϭ,Ϳ͕ϲ͘ϵϴ;Ɛ͕ϭ,Ϳ͕ϲ͘ϱϲ;Ě͕J = ϴ͘ϯ,nj͕ϭ,Ϳ͕ϲ͘ϱϬ;Ě͕J = 8.3 ,nj͕Ϯ,Ϳ͕ϲ͘ϰϯ;Ě͕J = ϭϭ͘ϱ,nj͕ϭ,Ϳ͕ϱ͘ϴϵ– ϱ͘ϳϰ;ŵ͕Ϯ,Ϳ͕ϱ͘ϲϵ;Ɛ͕ϭ,Ϳ͕ϱ͘Ϭϱ– ϰ͘ϵϴ;ŵ͕Ϯ,Ϳ͕ϰ͘ϵϴ– ϰ͘ϴϵ;ŵ͕Ϯ,Ϳ͕ϰ͘ϳϮ;Ě͕J = ϭϳ͘ϲ,nj͕ϭ,Ϳ͕ϰ͘ϰϮ– 4.35 ;ŵ͕ϭ,Ϳ͕ϰ͘ϯϮ;Ěƚ͕J = ϭϬ͘ϳ͕ϳ͘Ϭ,nj͕ϭ,Ϳ͕ϯ͘ϲϲ;Ɛ͕ϯ,Ϳ͕ϯ͘ϯϬ;Ěƚ͕J = 13.2, 6.7 ,nj͕ϭ,Ϳ͕ϯ͘Ϯϱ– 3.ϭϵ;ŵ͕ϭ,Ϳ͕Ϯ͘ϱϴ;ĚĚ͕J = ϭϯ͘ϯ͕ϳ͘ϱ,nj͕ϭ,Ϳ͕Ϯ͘ϱϯ– 2.43 ;ŵ͕Ϯ,Ϳ͕Ϯ͘ϯϵ ;Ěƚ͕J = ϭϭ͘Ϯ͕ ϲ͘ϯ,nj͕ Ϯ,Ϳ͕Ϯ͘ϬϮ ;Ƌ͕J = ϲ͘ϱ ,nj͕ Ϯ,Ϳ͕ ϭ͘ϰϵ– 1.31 (m, J = ϴ͘Ϭ͕ϳ͘ϰ,nj͕ϳ,Ϳ͕ϭ͘Ϯϯ;Ě͕J = ϯϯ͘ϳ,nj͕ϲ,Ϳ͕ϭ͘ϭϲ;Ɛ͕ϱ,Ϳ͖13C NMR (126 MHz, CDCl3Ϳɷ [ppm] 169.9, 139.2, 139.2, 137.5, 137.5, 135.8, 135.8, 131.6, 131.6, 129.9, 129.2, 129.2, 126.2, 126.2, 122.8, 122.8, 122.5, 122.5, 115.5, 115.2, 115.2, 114.1, 114.1, 114.1, 114.1, 113.3, 113.3, 111.8, 111.8, 77.3, 77.3, 77.0, 77.0, 76.8, 76.8, 61.7, 61.7, 55.2, 55.2, 54.3, 54.3, 49.1, 49.1, 43.1, 40.0, 40.0, 33.8, 33.8, 33.1, 33.1, 29.4, 29.4, 29.4, 29.4, 29.2, 29.2, 29.1, 29.1, 28.9, 28.9, 26.9, 26.9, 14.4, 14.4; LC-MS ;ͬ ^/Ϳ͗ƚR = 3.03 min, Calcd for C37H48ClN3O5 ;ŵͬnjͿ͗D-H]- 648.33, found: [M-H]- 648.28.
Ethyl 6-chloro-3-(1-(N -(3,4-ĚŝĐŚůŽƌŽďĞŶnjLJůͿƉĞŶƚ-4-ĞŶĂŵŝĚŽͿ-2-oxo-2-(undec-10-en-1-LJůĂŵŝŶŽͿĞƚŚLJůͿ-1H-indole-2-ĐĂƌďŽdžLJůĂƚĞ;ϰŚͿ
Ϯϱй LJŝĞůĚ͖ mixture of rotamers observed; 1H NMR (500 MHz, CDCl
3Ϳɷ ƉƉŵϵ͘ϲϲ;Ɛ͕ϭ,Ϳ͕ϵ͘ϰϲ;Ɛ͕ϭ,Ϳ͕ϳ͘ϳϵ;Ě͕J = ϴ͘ϴ,nj͕ϭ,Ϳ͕ϳ͘ϯϱ– 7.27 (m, ϭ,Ϳ͕ ϳ͘ϭϰ ;ĚĚ͕ J = ϭϳ͘ϲ͕ ϴ͘ϲ ,nj͕ ϭ,Ϳ͕ ϳ͘Ϭϴ – ϲ͘ϵϳ ;ŵ͕ ϭ,Ϳ͕ ϲ͘ϳϱ ;Ɛ͕ ϭ,Ϳ͕ 6.44 (d, J = ϴ͘ϭ,nj͕ϭ,Ϳ͕ϲ͘Ϯϭ;Ě͕J = ϴ͘ϭ,nj͕ϭ,Ϳ͕ϱ͘ϵϰ;ĚĚ͕J = 16.7, 10.2 ,nj͕ϭ,Ϳ͕ϱ͘ϴϵ– ϱ͘ϳϰ;ŵ͕ϯ,Ϳ͕ϱ͘Ϯϰ;Ě͕J = ϭϱ͘ϳ,nj͕ϭ,Ϳ͕ϱ͘ϭϰ;Ě͕J = 17.4 ,nj͕ϭ,Ϳ͕ϱ͘Ϭϳ– ϱ͘ϬϮ;ŵ͕ϭ,Ϳ͕ϱ͘ϬϬ;Ɛ͕ϭ,Ϳ͕ϰ͘ϵϵ– ϰ͘ϵϱ;ŵ͕ϭ,Ϳ͕ϰ͘ϵϮ;Ě͕J = ϭϬ͘Ϭ,nj͕ϭ,Ϳ͕ϰ͘ϳϰ;Ě͕J = ϭϴ͘ϭ,nj͕ϭ,Ϳ͕ϰ͘ϰϮ– ϰ͘ϯϳ;ŵ͕ϭ,Ϳ͕ϰ͘ϯϮ;ĚƋ͕J = ϭϮ͘ϱ͕ϲ͘ϰ͕ϱ͘ϲ,nj͕ϭ,Ϳ͕ϯ͘ϲϵ;Ě͕J = ϭϱ͘ϲ,nj͕ϭ,Ϳ͕ϯ͘ϯϵ;Ɛ͕ϭ,Ϳ͕ϯ͘ϯϬ;ĚƋ͕J = ϭϯ͘ϯ͕ϲ͘ϵ,nj͕ϭ,Ϳ͕ϯ͘ϭϵ;ĚƋ͕J = Ϯϭ͘ϱ͕ϳ͘ϴ͕ϳ͘Ϯ,nj͕ϭ,Ϳ͕Ϯ͘ϵϵ;ĚĚ͕J = 12.8, ϲ͘ϲ,nj͕Ϯ,Ϳ͕Ϯ͘ϵϰ– Ϯ͘ϴϰ;ŵ͕ϭ,Ϳ͕Ϯ͘ϰϮ;ĚƚĚĚ͕J = 58.9, 36.3, 16.5, 9.6 Hz, ϲ,Ϳ͕Ϯ͘Ϭϵ– ϭ͘ϵϴ;ŵ͕ϯ,Ϳ͕ϭ͘ϲϭ;Ě͕J = ϭϬ͘ϯ,nj͕ϭ,Ϳ͕ϭ͘ϱϭ;Ɛ͕Ϯ,Ϳ͕ϭ͘ϯϴ;ĚĚƋ͕J = 23.2, 16.5, 9.4, ϳ͘ϲ,nj͕ϴ,Ϳ͕ϭ͘ϯϭ– ϭ͘ϭϴ;ŵ͕ϭϬ,Ϳ͕ϭ͘ϭϴ– ϭ͘Ϭϰ;ŵ͕ϰ,Ϳ͕Ϭ͘ϵϯ– Ϭ͘ϴϭ;ŵ͕ϭ,Ϳ͖ 13C NMR (126 MHz, CDCl3Ϳ ɷ [ppm] 173.4, 169.9, 160.8, 139.2, 139.2, 138.5, 137.2, 137.2, 131.8, 131.8, 129.7, 129.7, 129.1, 129.1, 127.8, 127.8, 127.0, 127.0, 125.4, 125.4, 124.4, 124.4, 122.7, 122.7, 122.2, 122.2, 115.6, 115.6, 115.3, 115.3, 114.1, 114.1, 112.5, 112.5, 112.2, 112.2, 77.3, 77.3, 77.1, 77.1, 76.8, 76.8, 61.9, 61.9, 61.6, 61.6, 56.1, 54.1, 54.1, 48.8, 48.8, 46.7, 40.1, 40.1, 33.8, 33.8, 33.0, 33.0, 32.4, 32.4, 29.4, 29.4, 29.1, 29.1, 28.9, 28.9, 26.9, 26.9, 14.3; LC-MS ;ͬ ^/Ϳ͗ƚR = 2.94 min, Calcd for C36H44Cl3N3O4 ;ŵͬnjͿ͗D-H]- 686.24, found: [M-H]-686.23.
N HN Cl O HN O EtO2C O N HN Cl O HN O EtO2C Cl Cl
Ù㮥®®½ÃÙÊù½ÝÝÖÊãÄãÖωχͲDDφ®Ä«®®ãÊÙÝ
4
5
6
7
8
9
Ethyl 6-chloro-3-(1-(N -(3-ŵĞƚŚŽdžLJďĞŶnjLJůͿƉĞŶƚ-4-ĞŶĂŵŝĚŽͿ-2-oxo-2-(undec-10-en-1-LJůĂŵŝŶŽͿĞƚŚLJůͿ-1H-indole-2-ĐĂƌďŽdžLJůĂƚĞ;ϰŝͿ
ϯϭй LJŝĞůĚ͖ mixture of rotamers observed; 1H NMR (500 MHz, CDCl 3Ϳ ɷ ƉƉŵϵ͘ϴϬ;Ɛ͕ϭ,Ϳ͕ϳ͘ϴϭ;Ě͕J = ϴ͘ϳ,nj͕ϭ,Ϳ͕ϳ͘ϰϲ;Ě͕J = ϴ͘ϱ,nj͕ϭ,Ϳ͕ϳ͘Ϯϳ;Ɛ͕ ϭ,Ϳ͕ϳ͘ϭϬ;Ě͕J = ϴ͘ϴ,nj͕ϭ,Ϳ͕ϳ͘Ϭϰ;Ɛ͕ϭ,Ϳ͕ϲ͘ϴϲ;Ěƚ͕J = ϭϰ͘ϳ͕ ϳ͘ϵ,nj͕ϭ,Ϳ͕ 6.51 – ϲ͘ϰϬ;ŵ͕ϭ,Ϳ͕ϲ͘Ϯϳ;Ě͕J = ϳ͘ϰ,nj͕ϭ,Ϳ͕ϲ͘ϭϬ;Ɛ͕ϭ,Ϳ͕ϱ͘ϵϱ;ƚ͕J = 12.4 ,nj͕ϭ,Ϳ͕ϱ͘ϴϮ;ĚĚƋ͕J = Ϯϱ͘ϱ͕ ϭϲ͘ϭ͕ϵ͘Ϭ͕ ϴ͘Ϭ ,nj͕ Ϯ,Ϳ͕ ϱ͘Ϯϱ– ϱ͘ϭϭ;ŵ͕ ϭ,Ϳ͕ ϱ͘Ϭϭ;Ɛ͕ϭ,Ϳ͕ϰ͘ϵϳ;Ě͕J = ϰ͘ϲ,nj͕ϭ,Ϳ͕ ϰ͘ϵϯ;ƚ͕J = ϴ͘ϳ,nj͕ϭ,Ϳ͕ϰ͘ϳϱ;Ě͕J = 17.ϴ,nj͕ϭ,Ϳ͕ϰ͘ϱϰ– ϰ͘ϰϭ;ŵ͕Ϯ,Ϳ͕ϰ͘ϯϯ;Ěƚ͕J = ϭϰ͘ϰ͕ϳ͘Ϯ,nj͕ϭ,Ϳ͕ϰ͘Ϯϰ;Ěƚ͕J = ϭϳ͘ϱ͕ϲ͘ϵ,nj͕ϭ,Ϳ͕ϰ͘ϭϭ;Ƌ͕J = ϳ͘ϭ,nj͕ϭ,Ϳ͕ϯ͘ϳϳ;Ě͕J = ϭϮ͘ϲ,nj͕ϭ,Ϳ͕ϯ͘ϰϲ ;Ɛ͕Ϯ,Ϳ͕ϯ͘Ϯϵ;Ěƚ͕J = ϭϮ͘ϴ͕ϲ͘ϱ,nj͕ϭ,Ϳ͕ϯ͘Ϯϭ;Ěƚ͕J = ϭϯ͘Ϭ͕ϲ͘ϯ,nj͕ϭ,Ϳ͕Ϯ͘ϱϱ (ddd, J = 22.3, 16.2͕ϱ͘ϳ,nj͕ϭ,Ϳ͕Ϯ͘ϰϰ;Ěƚ͕J = ϭϮ͘ϳ͕ϳ͘ϲ,nj͕Ϯ,Ϳ͕Ϯ͘ϰϭ– Ϯ͘ϯϬ;ŵ͕ϭ,Ϳ͕Ϯ͘Ϭϯ;Ƌ͕J = ϳ͘ϭ,nj͕Ϯ,Ϳ͕ϭ͘ϰϵ;Ɛ͕ϭ,Ϳ͕ϭ͘ϯϱ;Ɖ͕J = ϭϭ͘ϲ͕ϭϬ͘Ϯ,nj͕ϲ,Ϳ͕ϭ͘Ϯϰ;ĚĚ͕J = ϭϯ͘ϴ͕ϲ͘ϱ,nj͕ϲ,Ϳ͕ϭ͘ϭϱ;Ɛ͕ ϰ,Ϳ͖13 C NMR (126 MHz, CDCl3Ϳ ɷ [ppm] 173.7, 170.1, 161.1, 159.3, 139.7, 139.2, 137.4, 136.8, 136.1, 131.5, 128.9, 127.4, 125.9, 122.5, 122.4, 117.4, 115.4, 115.3, 114.3, 114.1, 112.5, 112.1, 110.3, 77.4, 77.1, 76.9, 61.7, 56.8, 55.2, 54.8, 54.3, 49.7, 47.4, 40.0, 35.8, 33.8, 33.1, 29.4, 29.4, 29.3, 29.2, 29.1, 28.9, 28.8, 26.9, 21.0, 14.2. LC-D^;ͬ ^/Ϳ͗ƚR = 4.55 min,
Calcd for C37H48ClN3O5 ;ŵͬnjͿ͗D-H]- 648.33, found: [M-H]- 648.39.
Ethyl 6-chloro-3-(2-oxo-1-(N -(3,4,5-ƚƌŝĨůƵŽƌŽďĞŶnjLJůͿŚĞdž-5-ĞŶĂŵŝĚŽͿ-2-(undec-10-en-1-LJůĂŵŝŶŽͿĞƚŚLJůͿ-1H-indole-2-ĐĂƌďŽdžLJůĂƚĞ;ϰũͿ
ϰϱй LJŝĞůĚ͖ mixture of rotamers observed; 1H NMR (500 MHz, CDCl 3Ϳ ɷ ƉƉŵϵ͘ϬϮ;Ɛ͕ϭ,Ϳ͕ϴ͘ϴϰ;Ě͕J = ϭϯ͘ϴ,nj͕ϭ,Ϳ͕ϳ͘ϴϮ;Ě͕J = ϴ͘ϳ,nj͕ϭ,Ϳ͕ϳ͘ϱϴ (d, J = ϴ͘ϰ ,nj͕ϭ,Ϳ͕ϳ͘ϯϬ;Ě͕J = ϯϱ͘ϲ ,nj͕ϯ,Ϳ͕ϳ͘ϭϳ;ĚĚ͕J = 21.2, 8.4 Hz, Ϯ,Ϳ͕ϲ͘ϵϴ;Ɛ͕ϭ,Ϳ͕ϲ͘ϯϮ;Ɛ͕ϭ,Ϳ͕ϱ͘ϵϵ;Ɛ͕ϭ,Ϳ͕ϱ͘ϴϱ– ϱ͘ϳϭ;ŵ͕ϰ,Ϳ͕ϱ͘ϱϳ;Ɛ͕ ϭ,Ϳ͕ϱ͘Ϯϴ;Ě͕J = ϭϲ͘ϰ,nj͕ϭ,Ϳ͕ϰ͘ϵϵ;ƚĚ͕J = Ϯϳ͘ϰ͕Ϯϲ͘ϳ͕ϭϰ͘ϯ,nj͕ϴ,Ϳ͕ϰ͘ϳϳ – ϰ͘ϲϳ;ŵ͕ϭ,Ϳ͕ϰ͘ϰϮ;ĚĚ͕J = Ϯϵ͘ϴ͕ϭϭ͘ϭ,nj͕ϱ,Ϳ͕ϰ͘Ϭϳ;Ɛ͕ϭ,Ϳ͕ϯ͘ϲϴ;Ě͕J = ϭϲ͘Ϭ,nj͕ϭ,Ϳ͕ϯ͘ϯϯ– ϯ͘Ϯϲ;ŵ͕ϭ,Ϳ͕ϯ͘Ϯϯ;Ě͕J = ϱ͘ϱ ,nj͕ϭ,Ϳ͕Ϯ͘ϰϲ– 2.34 ;ŵ͕Ϯ,Ϳ͕Ϯ͘ϮϬ;Ɛ͕ϯ,Ϳ͕Ϯ͘ϭϭ;ĚƋ͕J = ϭϮ͘ϵ͕ϲ͘ϴ,nj͕ϰ,Ϳ͕Ϯ͘Ϭϱ– Ϯ͘ϬϬ;ŵ͕ϲ,Ϳ͕ 1.95 – ϭ͘ϱϳ;ŵ͕ ϱ,Ϳ͕ϭ͘ϱϱ– Ϭ͘ϵϳ;ŵ͕ϱ,Ϳ͖13C NMR (126 MHz, CDCl 3Ϳɷ [ppm] 174.4, 173.8, 169.7, 169.5, 160.7, 139.2, 138.2, 137.9, 135.8, 132.1, 127.1, 125.5, 124.8, 123.0, 123.0, 122.3, 121.4, 115.4, 115.3, 115.1, 114.1, 112.3, 112.0, 109.8, 109.6, 109.1, 109.0, 77.3, 77.0, 76.8, 62.0, 61.7, 55.9, 54.0, 48.7, 46.8, 40.2, 40.1, 33.8, 33.3, 33.0, 33.0, 32.8, 32.6, 32.2, 29.5, 29.4, 29.4, 29.2, 29.1, 28.9, 26.9, 26.8, 24.2, 23.9, 23.8, 14.3; LC-D^;ͬ ^/Ϳ͗ƚR = 3.47 min, Calcd for C37H45ClF3N3O4 ;ŵͬnjͿ͗D-H]
-686.31, found: [M-H]- 686.10. N HN Cl O HN O EtO2C O N HN Cl O HN O EtO2C F F F
«ÖãÙψ
Ethyl 6-chloro-3-(1-(N -(4-((3,4-ĚŝĐŚůŽƌŽďĞŶnjLJůͿŽdžLJͿďĞŶnjLJůͿƵŶĚĞĐ-10-ĞŶĂŵŝĚŽͿ-2-oxo-2-(undec-10-en-1-LJůĂŵŝŶŽͿĞƚŚLJůͿ-1H-indole-2-ĐĂƌďŽdžLJůĂƚĞ;ϰŬͿ
ϭϳй LJŝĞůĚ͖ mixture of rotamers observed; 1H NMR (500 MHz,
CDCl3Ϳɷ ƉƉŵϴ͘ϵϵ ;Ɛ͕ ϭ,Ϳ͕ ϳ͘ϴϮ(d, J = ϴ͘ϴ,nj͕ ϭ,Ϳ͕ ϳ͘ϱϬ– 7.40 ;ŵ͕Ϯ,Ϳ͕ϳ͘Ϯϰ– ϳ͘ϭϲ;ŵ͕Ϯ,Ϳ͕ϳ͘ϭϬ;Ě͕J = ϴ͘ϳ,nj͕ϭ,Ϳ͕ϲ͘ϵϲ;Ɛ͕Ϯ,Ϳ͕ 6.53 (d, J = ϵ͘ϱ,nj͕ϯ,Ϳ͕ϲ͘ϰϵ– ϲ͘Ϯϵ;ŵ͕ϭ,Ϳ͕ ϱ͘ϴϬ;ĚĚĚ͕J = 14.7, ϭϬ͘ϰ͕ϰ͘Ϯ,nj͕ Ϯ,Ϳ͕ ϱ͘ϲϮ;Ɛ͕ϭ,Ϳ͕ ϱ͘Ϭϰ– ϰ͘ϴϵ;ŵ͕ ϱ,Ϳ͕ϰ͘ϴϳ ;Ɛ͕Ϯ,Ϳ͕ 4.71 (d, J = ϭϳ͘ϱ,nj͕ϭ,Ϳ͕ϰ͘ϰϳ– ϰ͘ϯϲ;ŵ͕Ϯ,Ϳ͕ϰ͘ϯϱ– ϰ͘Ϯϲ;ŵ͕ϭ,Ϳ͕ 3.25 (dt, J = ϯϳ͘ϰ͕ϲ͘ϵ,nj͕ϰ,Ϳ͕Ϯ͘ϰϲ;Ě͕J = ϴ͘ϳ,nj͕ϭ,Ϳ͕Ϯ͘ϯϭ;ĚĚ͕J = ϭϱ͘ϲ͕ ϳ͘ϳ,nj͕ ϭ,Ϳ͕ Ϯ͘ϭϮ– ϭ͘ϵϲ;ŵ͕ϱ,Ϳ͕ ϭ͘ϴϳ– ϭ͘ϲϲ;ŵ͕ Ϯ,Ϳ͕ϭ͘ϯϳ (dq, J = ϭϰ͘ϵ͕ϴ͘ϯ͕ϱ͘ϴ,nj͕ϭϯ,Ϳ͕ϭ͘ϯϮ– ϭ͘ϮϬ;ŵ͕ϭϱ,Ϳ͕ϭ͘ϮϬ – 1.04 ;ŵ͕ϳ,Ϳ͖13C NMR (126 MHz, CDCl3Ϳɷ [ppm] 174.2, 169.9, 160.9, 156.7, 139.2, 139.2, 137.3, 135.8, 132.7, 131.9, 131.7, 130.9, 130.6, 129.3, 129.0, 127.6, 127.1, 126.4, 126.3, 125.8, 122.9, 122.5, 121.8, 115.1, 115.0, 114.9, 114.2, 114.1, 113.9, 112.1, 111.7, 77.3, 77.0, 76.8, 68.6, 68.4, 61.7, 61.3, 56.7, 54.1, 49.1, 46.6, 43.0, 40.0, 33.8, 33.8, 33.3, 29.4, 29.4, 29.3, 29.2, 29.1, 28.9, 26.9, 25.4, 24.9, 24.8, 14.4, 14.2; LC-D^ ;ͬ ^/Ϳ͗ ƚR = 3.41 min, Calcd for
C49H62Cl3N3O5 ;ŵͬnjͿ͗D-H]- 876.38, found: [M-H]- 876.27.
2.5. GENERAL PROCEDURE FOR THE RING-CLOSURE METATHESIS (RCM) REACTION
The corresponding UT-4CR compound 4 ;ϭ͘ϬĞƋƵŝǀ͘ͿǁĂƐĚŝƐƐŽůǀĞĚŝŶĚƌLJDĂŶĚƚŚĞƐĞĐŽŶĚ ŐĞŶĞƌĂƚŝŽŶ 'ƌƵďďƐ ĐĂƚĂůLJƐƚ ;Ϭ͘Ϭϴ ĞƋƵŝǀ͘Ϳ ǁĂƐ ĂĚĚĞĚ ƵŶĚĞƌ E2 atmosphere. The reaction
mixture was refluxed for 3 d. The solvent was evaporated and the crude mixture was purified by flash chromatography (hexane-ĞƚŚLJů ĂĐĞƚĂƚĞͿ ŐŝǀŝŶŐ ƚŚĞ ĐŽƌƌĞƐƉŽŶĚŝŶŐ ĐŽŵƉŽƵŶĚƐ 3 (yields 10-ϵϲйͿĂƐŐƌĞĞŶ-brown powders.
Ethyl 6-chloro-3-(1-(4-((3,4-ĚŝĐŚůŽƌŽďĞŶnjLJůͿŽdžLJͿďĞŶnjLJůͿ-5-methyl-3,12-dioxo-1,4-diazacyclododec-8-en-2-LJůͿ-1H-indole-2-ĐĂƌďŽdžLJůĂƚĞ;ϯĂͿ
ϴϵй LJŝĞůĚ͖ mixture of rotamers and diastereomers observed;
1H NMR (500 MHz, CDCl 3Ϳ ɷ [ppm] 7.71 (d, J = ϳ͘Ϯ ,nj͕ ϭ,Ϳ͕ 7.57 – ϳ͘ϰϴ;ŵ͕ϭ,Ϳ͕ϳ͘ϰϴ– ϳ͘ϰϬ;ŵ͕Ϯ,Ϳ͕ϳ͘ϰϬ– ϳ͘ϯϭ;ŵ͕ϭ,Ϳ͕ 7.29 – ϳ͘ϮϬ;ŵ͕ϭ,Ϳ͕ϳ͘ϮϬ– ϳ͘ϭϮ;ŵ͕ϭ,Ϳ͕ϳ͘ϭϮ– ϳ͘Ϭϱ;ŵ͕ϭ,Ϳ͕ 7.04 – ϲ͘ϵϳ;ŵ͕ϭ,Ϳ͕ϲ͘ϳϮ;Ě͕J = ϴ͘ϭ,nj͕ϭ,Ϳ͕ϲ͘ϲϳ;Ě͕J = 8.6 Hz, ϭ,Ϳ͕ ϲ͘ϰϮ;Ě͕J = ϴ͘Ϯ,nj͕ϭ,Ϳ͕ϲ͘ϯϱ ;ĚĚ͕J = ϭϮ͘ϱ͕ ϴ͘ϲ ,nj͕ ϭ,Ϳ͕ ϱ͘ϴϱ;Ɛ͕ϭ,Ϳ͕ϰ͘ϵϰ;Ě͕J = ϭϮ͘Ϯ,nj͕ϭ,Ϳ͕ϰ͘ϴϭ;ƚ͕J = ϭϬ͘ϲ,nj͕Ϯ,Ϳ͕ϰ͘ϲϴ;ĚĚ͕J = ϭϳ͘ϭ͕ϳ͘ϯ,nj͕ϭ,Ϳ͕ 4.35 – ϰ͘ϭϴ;ŵ͕ϯ,Ϳ͕Ϯ͘ϳϮ– Ϯ͘ϲϱ;ŵ͕ϭ,Ϳ͕Ϯ͘ϱϵ;ĚĚ͕J = ϭϮ͘ϴ͕ϲ͘Ϭ,nj͕ϭ,Ϳ͕Ϯ͘ϱϱ– Ϯ͘ϯϵ;ŵ͕ϭ,Ϳ͕ Ϯ͘ϯϯ;Ɛ͕Ϯ,Ϳ͕Ϯ͘ϬϮ;ĚƋ͕J = ϰϮ͘ϲ͕ϭϯ͘ϱ͕ϭϮ͘ϵ,nj͕Ϯ,Ϳ͕ϭ͘ϳϳ– ϭ͘ϲϮ;ŵ͕ϭ,Ϳ͕ϭ͘ϰϱ;Ě͕J = ϲ͘ϲ,nj͕ϭ,Ϳ͕ N N H O O N H CO2Et Cl O Cl Cl N HN Cl O HN O EtO2C O Cl Cl
Ù㮥®®½ÃÙÊù½ÝÝÖÊãÄãÖωχͲDDφ®Ä«®®ãÊÙÝ
4
5
6
7
8
9
1.42 – ϭ͘ϯϬ;ŵ͕ϰ,Ϳ͕ϭ͘Ϯϳ;Ě͕J = ϭϯ͘ϲ,nj͕ϰ,Ϳ͕ϭ͘Ϭϲ;Ě͕J = ϲ͘ϭ,nj͕ϭ,Ϳ͕Ϭ͘ϴϳ;Ƌ͕J = 10.6, 8.6 Hz, ϭ,Ϳ͖13C NMR (126 MHz, CDCl 3Ϳ ɷ [ppm] 176.4, 173.6, 169.5, 168.9, 160.3, 160.1, 157.2, 156.9, 139.0, 137.3, 137.1, 135.9, 135.1, 132.6, 132.5, 131.9, 131.1, 130.6, 130.5, 130.3, 129.5, 129.1, 129.1, 127.7, 127.0, 126.9, 126.5, 125.8, 125.7, 124.9, 124.5, 124.0, 121.5, 121.4, 121.1, 115.6, 114.4, 114.2, 114.1, 113.6, 112.7, 112.3, 77.3, 77.1, 76.8, 68.4, 61.3, 61.2, 58.8, 54.6, 48.2, 46.8, 46.2, 45.7, 34.5, 33.6, 33.1, 32.8, 31.9, 31.4, 31.3, 30.2, 29.8, 29.7, 29.5, 29.4, 27.1, 26.7, 22.7, 21.8, 20.5, 18.2, 14.3, 14.2; LC-D^;ͬ ^/Ϳ͗ƚR = 5.52 min,Calcd for C36H36Cl3N3O5 ;ŵͬnjͿ͗D-H]- 694.17, found: [M-H]- 694.15.
Ethyl 6-chloro-3-(1-(4-((3,4-ĚŝĐŚůŽƌŽďĞŶnjLJůͿŽdžLJͿďĞŶnjLJůͿ-5-methyl 3,13-dioxo-1,4-diazacyclododec-8-en-2-LJůͿ-1H-indole-2-ĐĂƌďŽdžLJůĂƚĞ;ϯďͿ
ϮϵйLJŝĞůĚ͖ mixture of rotamers and diastereomers observed; 1H
NMR (500 MHz, CDCl3Ϳ ɷ ƉƉŵ ϳ͘ϱϮ ;Ɛ͕ ϭ,Ϳ͕ ϳ͘ϱϬ – 7.42 (m, ϭ,Ϳ͕ϳ͘ϯϱ– ϳ͘ϮϮ;ŵ͕Ϯ,Ϳ͕ϳ͘Ϭϲ;Ƌ͕J = ϲ͘Ϯ͕ϱ͘ϭ,nj͕ϭ,Ϳ͕ϲ͘ϴϴ;ĚĚ͕J = ϭϮ͘Ϯ͕ϴ͘ϴ,nj͕ϭ,Ϳ͕ϲ͘ϳϭ(d, J = ϴ͘Ϭ,nj͕Ϯ,Ϳ͕ϲ͘ϱϯ;Ɛ͕ϭ,Ϳ͕ϲ͘ϮϮ;Ě͕ J = ϲ͘ϳ ,nj͕ ϭ,Ϳ͕ϱ͘ϴϳ– ϱ͘ϲϳ;ŵ͕ ϭ,Ϳ͕ ϱ͘ϯϭ ;Ě͕J = ϭϯ͘Ϭ,nj͕ ϭ,Ϳ͕ 4.99 (q, J = ϭϬ͘ϴ͕ϭϬ͘Ϭ,nj͕Ϯ,Ϳ͕ϰ͘ϯϳ;Ě͕J = ϱ͘ϲ,nj͕ϭ,Ϳ͕ϰ͘Ϯϴ;ĚĚƚ͕ J = ϭϴ͘Ϭ͕ϭϬ͘ϳ͕ϱ͘ϰ,nj͕ϭ,Ϳ͕ϰ͘ϭϮ;Ƌ͕J = ϳ͘ϭ,nj͕ϯ,Ϳ͕Ϯ͘ϵϮ;Ě͕J = 16.8 Hz, ϭ,Ϳ͕Ϯ͘ϰϯ;ĚĚĚ͕J = 34.3, Ϯϱ͘ϳ͕ϭϯ͘ϭ,nj͕ϭ,Ϳ͕Ϯ͘ϯϰ– Ϯ͘ϭϱ;ŵ͕Ϯ,Ϳ͕Ϯ͘Ϭϱ;Ɛ͕ϱ,Ϳ͕ϭ͘ϳϲ;Ěƚ͕J = ϭϰ͘ϴ͕ϳ͘ϰ,nj͕ϭ,Ϳ͕ϭ͘ϳϭ– 1.56 ;ŵ͕ϭ,Ϳ͕ϭ͘ϯϯ;Ƌ͕J = ϴ͘Ϭ͕ϳ͘ϱ,nj͕Ϯ,Ϳ͕ϭ͘Ϯϲ;ƚ͕J = ϳ͘ϭ,nj͕ϰ,Ϳ͕ϭ͘ϭϮ;Ě͕J = ϲ͘ϯ,nj͕Ϯ,Ϳ͖13C NMR (126 MHz, CDCl3Ϳɷ [ppm] 174.1, 171.2, 169.7, 160.0, 157.3, 137.3, 135.9, 132.5, 131.9, 131.4, 130.6, 130.5, 129.7, 129.3, 129.2, 129.1, 126.7, 126.5, 124.9, 121.9, 121.5, 115.8, 115.3, 115.0, 114.5, 112.6, 77.3, 77.0, 76.8, 68.6, 68.4, 61.2, 60.4, 57.8, 48.6, 45.0, 43.0, 35.9, 34.8, 33.1, 30.8, 30.2, 30.0, 24.7, 22.5, 22.2, 21.1, 14.3, 14.2; LC-D^;ͬ ^/Ϳ͗ƚR = 3.50 min, Calcd for C37H38Cl3N3O5 ;ŵͬnjͿ͗D-H]- 708.19, found: [M-H]- 708.30. Ethyl 6-chloro-3-(1-(4-((3,4-ĚŝĐŚůŽƌŽďĞŶnjLJůͿŽdžLJͿďĞŶnjLJůͿ-5-methyl-3,18-dioxo-1,4-diazacyclooctadec-8-en-2-LJůͿ-1H-indole-2-ĐĂƌďŽdžLJůĂƚĞ;ϯĐͿ
ϮϲйLJŝĞůĚ͖ mixture of rotamers and diastereomers observed; 1H NMR (500 MHz, CDCl3Ϳɷ [ppm] 7.98 (d, J = ϴ͘ϳ,nj͕ ϭ,Ϳ͕ ϳ͘ϳϳ– ϳ͘ϲϳ;ŵ͕ϭ,Ϳ͕ϳ͘ϲϬ– ϳ͘ϰϬ;ŵ͕ϰ,Ϳ͕ϳ͘ϯϵ– ϳ͘ϮϮ;ŵ͕Ϯ,Ϳ͕ϳ͘ϭϳ;ĚĚ͕J = ϭϲ͘ϵ͕ ϳ͘ϳ ,nj͕ Ϯ,Ϳ͕ϳ͘Ϭϳ ;ĚĚ͕ J = ϭϵ͘ϰ͕ ϴ͘ϰ ,nj͕ ϭ,Ϳ͕ϲ͘ϱϲ ;Ě͕J = ϭϲ͘ϴ,nj͕ϰ,Ϳ͕ϱ͘ϰϱ– ϱ͘Ϯϱ;ŵ͕Ϯ,Ϳ͕ϰ͘ϵϰ– ϰ͘ϴϴ;ŵ͕ϭ,Ϳ͕ϰ͘ϴϯ;Ě͕J = ϵ͘ϭ,nj͕Ϯ,Ϳ͕ϰ͘ϰϲ– 4.34 (m, 1HͿ͕ϰ͘ϯϯ– ϰ͘ϭϴ;ŵ͕Ϯ,Ϳ͕ϰ͘ϭϭ;Ěƚ͕J = ϭϯ͘ϯ͕ϲ͘ϵ,nj͕ϭ,Ϳ͕Ϯ͘ϭϱ– 1.97 ;ŵ͕ϯ,Ϳ͕ϭ͘ϵϰ;Ɛ͕ϭ,Ϳ͕ϭ͘ϴϲ;Ɛ͕ϭ,Ϳ͕ϭ͘ϲϬ– ϭ͘ϭϭ;ŵ͕Ϯϯ,Ϳ͕ϭ͘Ϭϵ– ϭ͘Ϭϯ;ŵ͕ϭ,Ϳ͕Ϭ͘ϵϭ;ĚƋ͕J = 22.1, ϴ͘ϲ͕ϳ͘Ϭ,nj͕ϱ,Ϳ͖13C NMR (126 MHz, CDCl 3Ϳɷ [ppm] 174.4, 169.6, 161.1, 157.1, 156.7, 137.3, 135.9, 132.6, 131.8, 131.6, 131.5, 130.8, 130.5, 130.2, 129.4, 129.1, 129.0, 128.6, 127.2, 126.4, 126.4, 125.9, 123.3, 122.3, 122.0, 114.9, 114.2, 113.6, 111.8, 77.3, 77.1, 76.8, 68.4, 61.6, 61.3, 54.5, 49.2, 45.8, 45.2, 44.1, 38.0, 36.9, 33.6, 33.2, 31.8, 30.9, 30.2, 29.7, 29.4, 28.8, 28.5, 28.4, 28.1, 27.5, 27.2, 26.9, 26.5, 25.8, 25.6, 24.5, 20.7, 20.6, 19.5, 14.4; LC-MS ;ͬ ^/Ϳ͗ƚR = 3.53 min, Calcd for C42H48Cl3N3O5 ;ŵͬnjͿ͗D-H]- 778.27, found: [M-H]- 781.52.
N N H O O N H Cl CO2Et O Cl Cl N NH O O O N H Cl CO2Et Cl Cl
