University of Groningen
Discovery of Inhibitors by Combinatorial-Chemistry Approaches
van der Vlag, Ramon
DOI:
10.33612/diss.146091529
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Chapter 4
Optimized Inhibitors of MDM2 via an Attempted
Protein-Templated Reductive Amination
Innovative and efficient hit-identification techniques are required to accelerate drug
discovery. Protein-templated fragment ligations represent a promising strategy in
early drug discovery, enabling the target to assemble and select its binders from a
pool of building blocks. Development of new protein-templated reactions to access
a larger structural diversity and expansion of the variety of targets to demonstrate
the scope of the technique are of prime interest for medicinal chemists. Herein, we
present our attempts to use a protein-templated reductive amination to target
protein-protein interactions (PPIs), a challenging class of drug targets. We address
a flexible pocket, which is difficult to achieve by structure-based drug design. After
careful analysis we did not find one of the possible products in the kinetic
target-guided synthesis (KTGS) approach, however subsequent synthesis and biochemical
evaluation of each library member demonstrated that all the obtained molecules
inhibit MDM2. The most potent library member (K
i= 0.095 μM) identified is almost
as active as Nutlin-3, a potent inhibitor of the p53–MDM2 PPI.
This chapter is adapted from the original publication:
R. van der Vlag*, M.Y. Unver*, T. Felicetti*, A. Twarda-Clapa, F. Kassim, C. Ermis, C.G. Neochoritis, B. Musielak, B. Labuzek, A. Dömling, T.A. Holak, A.K.H. Hirsch, Optimized Inhibitors of MDM2 via an Attempted Protein-Templated Reductive Amination,
ChemMedChem, 2020, 15, 370 * Shared first author
4.1 Introduction
Discovery of fast and efficient techniques to identify bioactive compounds constitutes an important part in today’s drug discovery. A way to accelerate hit-identification is the use of reversible reactions (dynamic combinatorial chemistry, DCC) or irreversible reactions (kinetic target-guided synthesis, KTGS), which are both categories of templated fragment
ligations.1 In these templated fragment ligations, the target selects its own inhibitors by
assembling the corresponding binders from a library of complementary building blocks or by binding and amplifying them from a library of compounds formed in a covalent bond-forming reaction. In KTGS, the biological target accelerates the irreversible reaction
between complementary building blocks upon binding,2,3 whereas in DCC a reversible
reaction between building blocks affords a dynamic combinatorial library (DCL) from which
the biological target selects and amplifies the best binder(s).4,5 Both techniques hold the
potential to accelerate drug discovery and are still relatively underexplored, especially in terms of target scope and availability of biocompatible reactions.
KTGS is a promising hit-identification strategy but only a few reactions with a limited
number of targets have been reported so far.1,2,6–19 The most widely used reaction is the
Huisgen 1,3-dipolar cycloaddition of azides and alkynes and most work in KTGS focuses on acetylcholine esterase (AChE) from various species, although different reactions and targets
have been explored.2,3
Protein-protein interactions (PPIs) are involved in many biological functions, such as intercellular communication and apoptosis. Targeting PPIs using small molecules is considered challenging given the flatness of the interface, a lack of small-molecule starting points for the future design and the difficulties in distinguishing real from artefactual
binding.20
P53 is a tumor-suppressor protein that is activated by cellular stress or damage and leads to cell-cycle arrest, apoptosis and DNA repair. MDM2 is the negative regulator of the
p53 protein and its overexpression leads to loss of p53 function.21,22 MDM2 has a
well-defined and deep pocket, unusual for PPIs, accommodating a hotspot triad consisting of
Trp23, Leu26 and Phe19 from p53.21,23,24 Therefore, design of high-affinity ligands to inhibit
MDM2 should focus on these hotspot amino acids of p53 (three-point pharmacophore
model). As a result, several small-molecule inhibitors have been reported.23–26 Some time
ago, we found the Leu26 pocket to be a flexible pocket, which is enlarged upon ligand
binding (four-point pharmacophore model).27 Very recently, we expanded this work with a
thorough SAR analysis.28 The flexibility of the pocket makes it very difficult to target using
structure-based drug design (SBDD) or other computational techniques such as virtual screening. Therefore, KTGS holds the potential to explore this flexible binding pocket by letting the protein select which combination of building blocks ideally fits.
Use of protein-templated reactions to interrupt PPIs has only been shown for the
Bcl-XL/BAK interaction11,17 and the 14-3-3 protein.7 These targets feature deep cavities in the
binding pocket, which makes them suitable for KTGS just like MDM2. The reactions used for
these two targets are the sulfo-click reaction between thio acids and sulfonyl azides and SN2
thiol ring opening of epoxides, respectively.
The reversible reaction between an aldehyde and an amine to afford imines has been
widely applied in DCC to target various biological targets (Figure 1).29–39 It is generally
building blocks in adjacent pockets, followed by imine formation and subsequent reduction, resulting in an irreversible amine bond. However, since in most cases the reduction agent is present in the mixture during most of the reaction time, it is unknown if this is actually true and difficult, if not impossible, to prove. Additionally, we think that protein-templated reductive amination is an example of KTGS instead of genuine DCC, in which the formed bond remains non-covalent.
Figure 1. Protein-templated reductive amination.
Since the formed imines are unstable in aqueous media, in situ reduction is performed in most cases. After analysis, frequently only the most amplified hits are synthesized and tested for activity. This approach, however, represents several pitfalls, such as: 1) it is assumed that the imines have a similar binding mode as the amines; and 2) the potency of the compound might change upon reduction. Previously, large differences in potency
between imine and amine have been reported.31,39 Furthermore, we noticed that in most
reports the potencies of the starting materials are not reported, although many of the starting materials are used as anchors.
Herein, we present our efforts to use protein-templated reductive amination in the KTGS context for the identification of inhibitors of the MDM2–p53 PPI. Furthermore, it is the first application of KTGS to address a flexible binding pocket (Leu26 pocket). Since we were unable to observe any of the reductive amination products, we evaluated all members of the library.
4.2 Results and Discussion
After the discovery of the extended Leu26 pocket in MDM2,27 we set out to explore this
flexible pocket. Having selected MDM2 as a target, we designed our potential inhibitor scaffold starting from the X-ray crystal structure of inhibitor 1 in complex with MDM2
(Figure 2, PDB ID: 4MDN).27
We designed and optimized a new inhibitor by using the molecular modeling program
SeeSAR.40 Inhibitor 1 occupies the three subpockets of MDM2 that are named according to
the corresponding p53 residues: the 6-chloroindole-2-hydroxamic acid moiety is hosted by the Trp23 pocket, the tert-butyl group occupies the Phe19 pocket and the large 4-clorobenzyl phenyl ether was found to fill the Leu26 and an induced subpocket. In order to occupy this extended subpocket in an optimal way by using the reductive amination reaction, we converted the 4-chlorobenzyl ether moiety into an amine, which can be assembled from the corresponding aldehyde 3 and amine 4 followed by in situ reduction (Scheme 1).
Figure 2. (Top) X-ray crystal structure of MDM2 in complex with docked inhibitor 1 (pink), superimposed with
designed inhibitor 2 (green) (PDB ID: 4MDN). Oxygen, nitrogen and chlorine atoms are shown in red, blue and light green, respectively. (Bottom) Design of inhibitor 2 as a product of reductive amination, based on the previously reported inhibitor 1.
Following the design of the initial inhibitor, we generated a combinatorial library by using aldehyde 3 as a core scaffold and eight different amines (4–11) to explore and fill the extended Leu26 pocket in the best manner and give rise to a focused SAR (Scheme 1).
Scheme 1. Selection of building blocks for the protein-templated reductive amination, which affords eight possible
The use of KTGS to fill flexible pockets is unprecedented. To explore this binding pocket and possibly open-up the Leu26 pocket even further, we used a benzyl- and naphthyl-amine in the library in addition to the aromatic amines to extend the length of the linker between the core scaffold and the amines. A potential extension reaching deeper into the pocket would be an important finding for future drug development.
Having selected the building blocks from commercially available amines, we synthesized the core scaffold (3) as shown in Scheme 2. Literature protocols afforded
compounds 12 and 13, in three and one step(s), respectively.41,42 A four-component Ugi
reaction of aldehyde 12, amine 13, formic acid (14) and tert-butyl-isocyanide (15) furnished the corresponding Ugi product 16 in 63% yield. Oxidation of the alcohol and subsequent hydrolysis of the ester led to the final aldehyde 3 in 86% yield over two steps.
Scheme 2. Synthesis of aldehyde building block 3. Conditions and reagents: i) three-step synthesis41 ii) one-step
synthesis42 iii) MeOH, rt, 6 d, 63%; iv) a) DMP, CH2Cl2, rt, 3 h, b) 1 M LiOH, H2O-EtOH (1:1), rt, 18 h, 86%.
Biochemical evaluation of aldehyde building block 3 showed promising potency
against MDM2 (Ki = 0.55 ± 0.05 μM). We envisioned that additional interactions in the
flexible pocket would enhance compound potency. Therefore, we set up two experiments in parallel using the synthesized aldehyde building block and eight different commercially available amines (4–11), a protein-templated reaction and a blank reaction at pH = 7.4
(100 mM phosphate buffer, 10% DMSO).
In both reactions, we applied standard building block and reducing agent
concentrations of 100 and 200 µM, respectively. One of the stringent requirements for KTGS
is a substantial difference in reaction rate between the blank and the protein-templated reaction. As the imine formation between an aldehyde and an amine is fast, we used dilute
conditions to prevent product formation in the reference reaction for a certain time.43 As a
result, less protein is required, an important consideration especially for precious proteins. By using a reducing agent in the reaction mixture from the beginning, formed imines would be “frozen” by reduction, leading to detectable amounts of products. Therefore, the two
reactions were started by mixing all amines 4–11 (100 µM each), aldehyde 3 (100 µM) and
NaCNBH3 (200 µM). To the protein-templated reaction, we added MDM2 (100 µM). Then,
both reactions were carefully monitored using UPLC-MS analysis (wavelengths: 254 and 305 nm) for two days. After two days, signs of denaturation were observed in all MDM2 containing samples. We did not detect any trace of one of the reductive amination products, while a large amount of the starting materials were observed. Additionally, experiments in which MDM2 was replaced by bovine serum albumin (BSA), experiments without reducing
agent and experiments in which the potent MDM2 inhibitor Nutlin-3 (100 µM, Ki = 36 nM44)
was added, showed identical results (Figure 4). In order to estimate how much of the product(s) should be formed for detection, we assessed the detection limit of the UPLC-MS. The UV-visible spectrum of 3 showed a clear absorption band around 305 nm (Figure S10).
This wavelength has the benefit over 254 nm that there is a lot less background signal. Under identical circumstances as the protein-templated reaction, the detection limit of
aldehyde 3 was estimated to be <2.6 μM and <0.88 μM for 254 and 305 nm, respectively
(Figures S11–13). In case only one reductive amination product is formed and taking into account the protein denaturation step with acetonitrile, this corresponds to as low as 1.8% conversion at 305 nm. Even if all eight amines reacted in a similar fashion, generating all possible products, this would require only 14% conversion of 3. We believe that in order to claim a protein-templated reaction, we should be far above these numbers.
In order to determine the potency of each product we synthesized all possible products (2 and 17–23, Table 1), starting from the core scaffold 3 by using a reductive amination protocol (Scheme 3).
Figure 4. Close-up of the UPLC traces at 305 nm in the area of which all products are expected (after two days
reaction time). From top to bottom: blank (black), reaction without protein (red), MDM2 reaction (green), MDM2 reaction without reducing agent (blue), MDM2 reaction in presence of (±)-Nutlin-3 (yellow) and reaction in presence of BSA instead of MDM2 (purple). * Reduced aldehyde of 3. Note: amines 4, 5, 6, and 11 have limited or no absorption at 305 nm. The retention times of all starting materials and products are shown in Table S1.
Scheme 3. Synthesis of the inhibitors 2 and 17–23. (a) Conditions and reagents: amines 4–11, pyrrolidine, 4Å
molecular sieves, Na(CH3CO2)3BH, dry CH2Cl2, rt, 18 h.
10
Red. 3*
7 8 9 3
We evaluated the inhibitory potency of compounds 2 and 17–23 by using the
fluorescence polarization assay as previously described.45 MDMX is another p53 binding
protein and shows significant homology with MDM2. In order to investigate if the compounds selectively inhibit one of the proteins, we tested all compounds against MDM2 and MDMX (Table 1).
As can be seen from Table 1, the majority of the compounds (2 and 17–21, Ki = 0.40–
0.76 μM) show an activity against MDM2 in the same range as aldehyde 3 (Ki = 0.55 ±
0.05 μM) and lead compound 1 (Ki = 0.60 μM).27 Amine 23 (Ki = 3.18 ± 0.18 μM) is almost
six-fold less potent than aldehyde 3. Interestingly, 18 (Ki = 0.63 ± 0.07 μM) is three times more
potent than its regioisomer in which the CH2–NH is reversed (Ki = 1.9 μM).28 Surprisingly,
compound 22 shows a Ki value of 0.095 ± 0.010 μM, which makes it by far the most potent
inhibitor of this series. In fact, the compound is almost as potent against MDM2 as Nutlin-3
(Ki = 0.036 ± 0.009 μM44).
The inhibitory activities of the synthesized reductive amination products against
MDMX are rather comparable (Ki = 3.73–7.08 μM). Except for compound 17 which has a
slightly higher Ki of 12.2 ± 2.1 μM and 23, which is not active against MDMX under the
applied conditions. Aldehyde 3 (Ki = 11.4 ± 1.6 μM) also has a reduced inhibitory activity
against MDMX in comparison to MDM2. Most compounds have a preference for MDM2 by a factor 7–16. With a selectivity factor of 21, aldehyde 3 is slightly more selective for MDM2 than most of the amines. A remarkable exception is compound 22, which is over four times more selective for MDM2 than inhibitors 2 and 18–21 and two times more selective than
aldehyde 3. Although the activity of 22 against MDM2 is close to the Ki value of Nutlin-3, it
is around six times less selective.
There are several possible explanations as to why we did not find any of the reductive amination products in the protein-templated experiments. Very recently, Van der Veken and co-workers reported their efforts towards the application of the
Table 1. Biochemical evaluation of all reductive amination products. Experiments were performed in duplicate and
values are reported as average ± standard deviation. Note: at physiological pH there is a difference in charge expected between the aniline-type products and benzylamine-type compounds 17 and 23.
Inhibitor Ki (μM) MDM2 Ki (μM) MDMX Selectivity (MDMX/MDM2) 3 Aldehyde 0.55 ± 0.05 11.4 ± 1.6 21 ± 3.5 2 0.40 ± 0.05 4.18 ± 0.27 10 ± 1.5 17 0.76 ± 0.08 12.2 ± 2.1 16 ± 3.2 18 0.63 ± 0.07 4.56 ± 0.49 7.2 ± 1.1 19 0.75 ± 0.08 7.08 ± 0.52 9.4 ± 1.2 20 0.47 ± 0.04 3.73 ± 0.30 7.9 ± 0.9 21 0.49 ± 0.04 4.61 ± 0.39 9.4 ± 1.1 22 0.095 ± 0.010 3.95 ± 0.46 42 ± 6.5 23 3.18 ± 0.18 >15 ≥5 Nutlin-344 0.036 ± 0.009 9.38 ± 0.35 261 ± 66
The imine formation under near-physiological conditions showed to be most challenging. Furthermore, although their final products were also very potent against the protein target, the target itself interfered with the attempted reaction, which could also be the case in our study. Additionally, it could be that the reducing agent does not reach the imine, which is therefore hydrolyzed upon analysis. Last, there are clear differences in rigidity and hydrogen-bond donor/acceptor profiles between the imines and the amines. It can well be that the aldehyde and reductive amination products show activity against MDM2, but that the imines cannot be formed or do not fit in the active site. We performed crystallization studies to confirm the binding mode of the new inhibitors, but due to solubility problems we could not obtain any crystals.
To confirm the inhibitory activity of the compounds further, the uniformly 15N-labelled
MDM2 was titrated with an increasing concentration of compound 17, and 1H-15N HSQC
spectra were recorded after each new portion of the inhibitor was added. Instead of the most potent compound, 22, compound 17 was used due to its better solubility. The method is based on monitoring changes in NMR chemical shifts in protein amide backbone
resonances upon its interaction with a small molecule.47–50 In the course of titration, shifts
or disappearances of cross-peaks assigned to the amino acids of MDM2 affected by binding of 17 were observed, which corroborates the binding event (Figure 5).
Figure 5. (Left) 1H-15N HSQC NMR spectra: blue, reference spectrum of apo-MDM2; red, 1:5 (MDM2: inhibitor 17,
overtitrated). (Right) Chemical shift perturbations plotted onto the structure of the complex of MDM2 (gray) with p53 (green) (PDB ID: 1YCR51); residues which shift or disappear upon titration are labeled on the MDM2 surface
(red).
4.3 Conclusions
In conclusion, herein, we reported our efforts towards the first example of a reductive amination in KTGS applied to a PPI target. By using our target MDM2 in situ, we screened a library of compounds in one-pot, which did not reveal a clear hit over a course of two days. After synthesis of all library members, we found a very potent and rather selective inhibitor
of MDM2 (Ki = 0.095 ± 0.010 μM). Although, the KTGS method can find application in the
early stages of drug discovery, one should be careful in the use of imine-based chemistry. Particularly interesting scenarios for the use of KTGS in drug discovery are flexible protein pockets that are difficult to target by structure-based approaches.
4.4 Experimental Section
4.4.1 Protein purification procedures
4.4.1.1 MDM2
Fragments of human MDM2 (residues 1–118 in pET46) were expressed in the E. coli BL21-CodonPlus (DE3) RIL strain. Cells were cultured in a total volume of 5 L of LB or minimal medium at 37 °C, and induced with 1 mM IPTG at OD600nm of 0.8. Protein was expressed for 5 h at 37 °C. Cells were collected
by centrifugation, re-suspended in 120 mL PBS with protease inhibitor cocktail and lysed by sonication. Inclusion bodies that were collected by centrifugation, were washed twice with 120 mL PBS containing 0.05% Triton-X100 and once with 120 mL PBS and centrifuged after each wash. Purified inclusion bodies were solubilized in 20 mL of 6 M guanidine hydrochloride in 100 mM Tris-HCl, pH 8.0, containing 1 mM EDTA and 10 mM β-mercaptoethanol. The protein was dialyzed against 1 L of 4 M
guanidine hydrochloride, pH 3.5 supplemented with 10 mM β-mercaptoethanol. Following, the protein was refolded by dropwise addition into 1 L of 10 mM Tris-HCl, pH 7.0, containing 1 mM EDTA
and 10 mM β-mercaptoethanol and slow mixing overnight at 4 °C. Ammonium sulphate was added to a final concentration of 1.5 M, mixed for 2 h and centrifuged. The refolded protein was recovered on Butyl Sepharose 4 Fast Flow (GE Healthcare) previously equilibrated with refolding buffer containing 1.5 M (NH4)2SO4. MDM2 was eluted using 100 mM Tris-HCl buffer (pH 7.2), containing 5 mM
β-mercaptoethanol. Fractions containing the protein were pooled, concentrated to <10 mL and further purified by gel filtration on an S75 16/600 column (GE Healthcare) in 50 mM phosphate buffer pH 7.4 containing 150 mM NaCl and 5 mM DTT (FP/NMR buffer).
4.4.1.2 MDMX
Fragments of human MDMX (residues 1–134 in pET46) were expressed in E. coli BL21-CodonPlus (DE3) RIL strain. Cells were cultured in a total volume of 5 L of LB or minimal medium at 37 °C and induced with 0.5 mM IPTG at OD600nm of 0.6. Protein was expressed for 12 h at 20 °C. Cells were collected by
centrifugation, re-suspended in 120 mL 50 mM NaH2PO4 pH 8.0 with 300 mM NaCl, 10 mM imidazole
and protease inhibitor cocktail and lysed by sonication. The protein was purified under native conditions. The lysate was cleared by centrifugation and loaded on Ni-chelating sepharose (GE Healthcare) previously equilibrated with lysis buffer (50 mM NaH2PO4 pH 8.0 with 300 mM NaCl and 10 mM imidazole). MDMX was eluted with 50 mM NaH2PO4 pH 8.0 containing 300 mM NaCl and
300 mM imidazole. Fractions containing the protein were pooled, concentrated and further purified
by gel filtration on S75 16/600 column (GE Healthcare) in the FP/NMR buffer.
4.4.2 Fluorescence polarization assay
A fluorescence polarization (FP) assay was used to monitor interactions between MDM2 and MDMX proteins and their inhibitors.45
For each assay, fresh protein stocks of MDM2 (1–118) and MDMX (1–134) were thawed, and the protein concentrations were determined using Bradford method. The assay buffer contained 50 mM
NaCl, 10 mM Tris pH 8.0, 1 mM EDTA and 5% DMSO. Corning black 96-well NBS assay plates were used. The binding affinity of P2 peptide (sequence: LTFEHYWAQLTS, labelled with carboxyfluorescein) towards MDM2 and MDMX was first determined. For this purpose, 10 nM of the fluorescent P2 peptide was contacted with serial dilutions of tested protein (range from 750 to 0.012 nM for MDM2 and from 3750 to 0.10 nM for MDMX) in a final volume of 100 μL and fluorescence polarization was determined. Kd was determined by fitting the curve described to
FP = FPmin(FP𝑚𝑎𝑥− FP𝑚𝑖𝑛) ∙ c 𝐾d+ c
where FP is the determined value of fluorescence polarization, FPmin is the fluorescence polarization
for ligand only, FPmax is the fluorescence polarization at protein concentration saturating the ligand
and c is the protein concentration.
Competition binding assay was performed using 10 nM fluorescent P2 peptide and optimal protein concentration for the measurement calculated based on determined Kd according to Huang, 2003 (f0
= 0.8).52 Tested compounds were dissolved in DMSO at 50 µM. Serial dilutions (50 μM to 0.05 μM) were
prepared in DMSO.
All the experiments were prepared in duplicate, and plates were read 15 min after mixing of all assay components. Fluorescence polarization was determined using Tecan InfinitePro F200 plate reader with the 485 nm excitation and 535 nm emission filters. Fluorescence polarization values were expressed in millipolarization units (mP).
4.4.3
1H-
15N HSQC NMR experiment
Uniform 15N isotope labelling was obtained by expression of the protein in M9 minimal medium
containing 15NH4Cl as the sole nitrogen source. The 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 the samples to provide lock signal. Stock solutions of
inhibitors of MDM2/MDMX used for titration were prepared in DMSO-d6. The samples were prepared
by adding 50 mM ligand stock solution to the protein solution containing the 15N-labeled MDM2
fragment at a concentration of 0.3 mM. A 2D 1H-15N correlated heteronuclear single quantum
coherence (HSQC) NMR spectrum was recorded at 2–3 different ligand/protein ratios. All the spectra were recorded at 300 K using a Bruker Avance 600 MHz spectrometer. 1H-15N heteronuclear
correlations were obtained using the fast HSQC pulse sequence.53 Assignment of the amide backbone
resonances of MDM2 was obtained according to Stoll et al., 2001.54 The spectra were processed with
the TopSpin 3.2 software.
4.4.4 Modeling and docking
The inhibitor scaffold was modeled starting from the MDM2 X-ray crystal structure (PDB ID: 4MDN).27
Modeling and docking was performed using SeeSAR version 9.2.40 The distances between atoms in
and around the substituted formamide moiety in the X-ray crystal structure are so short, that the BiosolveIT programs show the structure as two connected cyclopropane rings (the authors also incorporated a remark at the beginning of the PDB file). Upon docking of this configuration, many poses were obtained, which are close to the original X-ray crystal structure, indicating the reproducibility by docking. Having restored the original 3D configuration manually, the molecule was docked by generating several poses. Whereas the indole part of the molecule is fully retained in position, the phenoxy moiety shifts a little bit towards the Leu26 pocket. However, the para-chlorine substituted phenyl more or less retains its original position. Subsequently, the pose of inhibitor 1, which resembles most closely the X-ray crystal structure was changed to 2, after which the structure was re-docked by generating several poses. The newly generated poses for 2 showed a high similarity to the poses of 1, which indicates that based on the docking no large shift or difference in binding mode is predicted. Based on these results it is expected that 2 can inhibit MDM2 in a similar way as compound 1, and that our KTGS strategy could potentially work to target the Leu26 pocket. A representative example of the docked poses of 1 and 2 is shown in Figure 2.
4.4.5 Attempted protein-templated reductive amination
To a 2 mL Eppendorf was sequentially added the eight building blocks 4—11 (0.5 μL each, 100 mM in DMSO), aldehyde 3 (0.5 μL, 100 mM in DMSO), DMSO (45.5 μL), phosphate buffer (235 μL, 100 mM, pH 7.4) and MDM2 (214 µL, 0.213 mM in phosphate buffer 100 mM, pH 7.4). After 10 minutes,
NaCNBH3 (1.0 μL, 100 mM in CH3CN) was added. The reaction mixture was allowed to rotate at room
temperature with 10 rpm using a Falc F205 rotary shaker. After one hour and after 1, 2 and 7 days, a 50 μL aliquot of reaction mixture was added to 50 μL of CH3CN, resulting in protein denaturation. The
precipitate was removed by centrifugation and filtration, whereupon the mixture was analyzed by UPLC-MS.
Control reactions
In addition to the reductive amination reaction, the following control reactions were performed: • Blank reaction (negative control) using buffer instead of MDM2
• BSA reaction using BSA (449 μL, 0.111 mM in phosphate buffer 100 mM, pH 7.4) instead of MDM2
• MDM2 reaction in the presence of (±)-Nutlin-3 (2.0 μL, 25 mM in DMSO, solid was obtained from Sanbio B.V., NL)
• MDM2 reaction with the addition of 1 μL CH3CN instead of NaCNBH3 solution
Formation of the individual reductive amination products
Each product of the library was formed by reacting each amine (4–11) with aldehyde 3 in the presence of reducing agent. The amine (2.5 μL, 100 mM in DMSO) and aldehyde 3 (2.5 μL, 100 mM in DMSO) were added to a mixture of MilliQ (450 μL) and DMSO (40 μL). After 10 minutes, NaCNBH3 (5 μL,
100 mM in CH3CN) was added, and the reaction mixtures were followed by UPLC-MS over time (1, 2,
4 and 7 days).
UPLC-MS method
All reactions were repeated twice and analyzed using a Thermo Fischer Scientific Vanquish with LCQ Fleet detector (of which the detection limit was estimated). All analyses were performed using a reversed-phase UPLC column (ACQUITY BEH C8 Column, 130 Å, 1.7 µm, 2.1 mm x 150 mm) on which 1 μL sample was injected. The eluents, acetonitrile and water contained 0.1% of formic acid. The library components were eluted with a gradient from (water): 95% → 10% over 4 min, then at 10% for 11 min, after which the column was re-equilibrated to 95%. The MS (m/z 100-700, ESI+) and the absorbance spectra at 254 and 305 nm were carefully analyzed and compared.
4.4.6 UV-vis absorption spectra of 3, 22 and 24
Compounds 3, 22 and 24 (pre-cursor of 12) were dissolved in MilliQ/ACN 50:50 containing 0.1% formic acid to obtain 0.1 mM solutions. After centrifugation, the solutions were added to a 96-well plate and UV-visible spectra were recorded from 230 – 700 nm using a BioTek Synergy H1 hybrid plate reader to obtain absorption maxima of 0.6 – 1. The blank absorption was subtracted and the absorption spectra were normalized at 305 nm.
4.4.7 Synthesis
General Experimental Details
Starting materials and reagents were purchased from Aldrich or TCI Europe. Yields refer to analytically pure compounds and were not optimized. All solvents were reagent-grade and if necessary, SPS-grade. Column chromatography was performed on silica gel (Silicycle® SiliaSepTM 40–63 μM 60 Å). TLC
was performed with silica gel 60/Kieselguhr F254. Solvents used for the column chromatography were pentane, ethyl acetate, dichloromethane and methanol. 1H and 13C spectra were recorded at 25 °C on
a Varian AMX400 spectrometer (400 MHz for 1H and 101 MHz for 13C, respectively) or a Bruker
Ascend™ 600 MHz (600 MHz for 1H and 151 MHz for 13C, respectively). Chemical shifts (δ) are reported
in ppm relative to the residual solvent peak. Splitting patterns are indicated as (s) singlet, (d) doublet, (t) triplet, (q) quartet, (m) multiplet, (br) broad. The coupling constants (J) are given in Hz. High-resolution mass spectra were recorded with an Orbitrap XL (Thermo Fisher Scientific; Esi pos. mode). UPLC-MS analysis was performed using a Thermo Fischer Scientific Vanquish with LCQ Fleet detector (purity analysis of 20–22) or a Waters Acquity Ultra Performance LC with Acquity TQD detector (purity analysis of the remaining final products). Melting points were measured on a Stuart®SMP11 50 W melting point apparatus.
Final products give a mixture of rotamers in NMR (CD3OD at 25oC). Major peaks were
identified and are reported in 1H-NMR spectra. For the 13C-NMR spectra, all peaks corresponding to
the complete mixture of rotamers are reported. All compounds are ≥95% purity as shown by UPLC-MS analysis (254 nm).
General procedure for compounds 2 and 17–23.
To a solution of compound 3 (1.00 eq), amine (1.05 eq), and pyrrolidine (0.10 eq) in dry CH2Cl2, 4 Å
molecular sieves (100 mg per mmol 3) and Na(CH3CO2)3BH (2.0 eq) were added. The reaction mixture
was stirred at room temperature for 18 h under a nitrogen atmosphere. Then, the mixture was filtered over Celite®, and the filtrate was evaporated under vacuum. The crude product was purified by flash chromatography column, eluting with ammonia-infused (1/1 (v/v) ammonia/CH2Cl2 mixed and CH2Cl2
Ethyl 3-(2-(tert-butylamino)-1-{formyl[4-(hydroxymethyl)benzyl]amino}-2-oxoethyl)-6-chloro-1H-indole-2-carboxylate (16)
In a 50-mL round-bottomed flask charged with MeOH (10 mL), aldehyde
12 (700 mg, 2.78 mmol) was added to amine 13 (381 mg, 2.78 mmol) and
the yellow reaction mixture was stirred for one hour at room temperature. Then, formic acid (110 μL, 2.78 mmol) and tert-butyl isocyanide (312 μL, 2.78 mmol) were added and the reaction mixture was stirred at room temperature for six days. Then, the red reaction mixture was evaporated under vacuum, and the orange crude was purified by flash column chromatography eluting with CH2Cl2/MeOH (99:1 → 95:5). Compound 16
was obtained as a pale yellow solid (871 mg, 1.74 mmol, 63% yield). M.p. = 113–115 °C; HRMS (ESI+) for C26H31ClN3O5 [M + H]+: 500.195 (calc.), 500.195 (found). 1H-NMR (400 MHz, CD3OD, major rotamer,
53%) δ 8.38 (s, 1H), 7.79 (d, 1H, J = 8.8 Hz), 7.39 (d, 1H, J = 1.8 Hz), 7.12 (dd, 1H, J = 8.8 and 1.9 Hz), 7.00 (d, 2H, J = 7.8 Hz), 6.76 (d, 2H, J = 7.9 Hz), 6.19 (s, 1H), 5.11 (d, 1H, J = 15.2 Hz), 4.46 (s, 2H), 4.38 (q, 2H, J = 7.0 Hz), 4.32 – 4.17 (m, 1H), 1.41 (t, 3H, J = 7.1 Hz), 1.23 (s, 9H); 13C-NMR (101 MHz, CD3OD) (mixture of rotamers) δ 171.2 (C), 170.9 (C), 166.6 (CH), 166.0 (CH), 162.2 (C), 162.1 (C), 141.4 (C), 141.3 (C), 138.0 (C), 137.9 (C), 137.8 (C), 137.4 (C), 132.1 (C), 131.8 (C), 129.5 (C), 128.5 (C), 128.4 (2xCH), 127.3 (2xCH), 127.2 (2xCH), 126.9 (C), 126.7 (2xCH), 126.1 (C), 123.5 (CH), 123.3 (CH), 122.6 (CH), 122.4 (CH), 115.9 (C), 114.0 (C), 113.3 (CH), 113.2 (CH), 64.8 (CH2), 64.7 (CH2), 62.2 (CH2), 62.1 (CH2), 58.3 (CH), 54.2 (CH), 52.54 (C), 52.52 (C), 51.2 (CH2), 48.0 (CH2), 28.9 (CH3), 28.8 (CH3), 14.8 (3xCH3), 14.7 (3xCH3). 3-{2-(tert-Butylamino)-1-[formyl(4-formylbenzyl)amino]-2-oxoethyl}-6-chloro-1H-indole-2-carboxylic acid (3)
To a solution of 16 (400 mg, 0.801 mmol) in dry CH2Cl2 (16 mL), Dess-Martin
periodinane (350 mg, 0.825 mmol, 1.03 eq.) was added portion-wise. The reaction was stirred at room temperature for 3 h under a N2 atmosphere.
Then, the mixture was quenched with a solution of Na2S2O3 (10%, 6 mL),
followed by a saturated aq. solution of NaHCO3 (10 mL) and extracted with
CH2Cl2. The combined organic layer was washed with a saturated aq.
solution of NaHCO3 and a saturated aq. solution of NaCl. Then the organic
layer was dried over MgSO4, filtered and evaporated to dryness to give the
oxidized intermediate as pale yellow solid that was quickly dissolved in EtOH (8 mL). The reaction mixture was cooled down using an ice-water bath and an aq. solution of LiOH (1.0 M, 0.19 g, 8.00 mmol, 8 mL) was added dropwise. Afterwards, the ice bath was removed, and the reaction mixture was stirred at room temperature for 18 h. The reaction mixture was again cooled down using an ice-water bath, and the mixture was slowly acidified with an aq. HCl (1 M) solution to pH 6. Filtration
of the precipitate afforded compound 3 as an off-white solid (324 mg, 0.689 mmol, 86% yield). M.p. = 211–213 ° C; HRMS (ESI+) for C24H25ClN3O5 [M + H]+: 470.148 (calc.), 470.148 (found). 1H-NMR (600
MHz, DMSO-d6, major rotamer, 75%) δ 13.47 (br. s, 1H, -OH), 11.81 (br. s, 1H, indole-NH), 9.86 (s, 1H),
8.29 (s, 1H), 7.76 (d, 1H, J = 8.8 Hz), 7.73 (s, 1H, amide-NH), 7.56 (d, J = 8.2 Hz, 2H), 7.32 (d, 1H, J = 2.0 Hz), 7.09 (dd, 1H, J = 8.8, 1.9 Hz), 7.01 (d, 2H, J = 7.9 Hz), 6.14 (s, 1H), 4.97 (d, 1H, J = 16.0 Hz), 4.36 (d, 1H, J = 16.1 Hz), 1.15 (s, 9H); 13C-NMR (151 MHz, DMSO-d 6) (mixture of rotamers) δ 192.6 (CH), 192.5 (CH), 168.88 (C), 168.82 (C), 163.80 (CH), 163.55 (CH), 162.39 (C), 161.82 (C), 145.78 (C), 144.89 (C), 135.8 (C), 135.7 (C), 134.53 (C), 134.52 (C), 128.9 (C), 128.72 (2xCH), 128.66 (2xC), 128.5 (C), 128.4 (2xCH), 127.3 (2xCH), 125.9 (2xCH), 125.5 (C), 124.7 (C), 122.3 (CH), 122.1 (CH), 120.7 (CH), 120.5 (CH),
113.9 (C), 112.6 (C), 111.9 (CH), 111.7 (CH), 56.2 (CH), 51.9 (CH), 50.6 (C), 50.5 (C), 49.2 (CH2), 46.6
(CH2), 28.5 (3xCH3), 28.3 (3xCH3).
3-(2-(tert-Butylamino)-1-(N-(4-(((4-chlorophenyl)amino)methyl)benzyl)formamido)-2-oxoethyl)-6-chloro-1H-indole-2-carboxylic acid (2)
Following the general procedure, starting from 4-chloroaniline (12.7 mg, 0.0966 mmol) as amine, compound 2 was obtained as a white solid (20 mg, 0.034 mmol, 34% yield). M.p. >119 °C (degradation); HRMS (ESI+) For C30H31Cl2N3O4 [M + H]+:
581.172 (calc.), 581.171 (found). 1H-NMR (400 MHz, CD3OD,
major rotamer, 75%) δ 8.36 (s, 1H), 7.71 (d, 1H, J = 8.8 Hz), 7.46 (br. s, 1H), 7.36 (d, 1H, J = 1.8 Hz), 7.12 (d, 2H, J = 7.9 Hz), 7.04 – 6.97 (m, 5H), 6.54 (d, 2H, J = 8.7 Hz), 6.42 (s, 1H), 5.03 (d, 1H,
J = 15.1 Hz), 4.36 (d, 1H, J = 15.1 Hz), 4.18 (s, 2H), 1.15 (s, 9H), note: of the exchangeable protons only
one broad singlet was observed; 13C-NMR (101 MHz, CD3OD) δ 171.2, 170.9, 166.7, 166.0, 148.87,
148.85, 139.8, 139.6, 137.7, 137.6, 137.5, 136.8, 131.6, 130.6, 129.7, 128.9, 128.7, 127.8, 127.7, 127.0, 126.2, 123.4, 123.2, 122.3, 122.2, 122.1, 115.1, 115.0, 113.2, 113.1, 58.3, 54.3, 52.5, 52.4, 51.2, 48.3, 48.2, 28.9, 28.8.
3-{2-(tert-Butylamino)-1-[formyl(4-{[(1-naphthylmethyl)amino]methyl}benzyl)amino]-2-oxoethyl}-6-chloro-1H-indole-2-carboxylic acid (17)
Following the general procedure, starting from 1-naphthylmethylamine (21 mg, 0.13 mmol) as amine, compound 17 was obtained as a white solid (20 mg, 0.033 mmol, 26% yield). M.p. 214–217 °C; HRMS (ESI+) for C35H36ClN4O4
[M + H]+: 611.242 (calc.), 611.243 (found). 1H-NMR (400 MHz,
DMSO-d6, major rotamer, 80%) δ 11.63 (br. s, 1H), 8.28 (s, 1H),
8.14 – 8.07 (m, 1H), 7.93 (d, 1H, J = 9.4 Hz), 7.86 (d, 1H, J = 8.1 Hz), 7.68 (d, 1H, J = 8.8 Hz), 7.60 (s, 1H), 7.58 – 7.43 (m, 4H), 7.35 (d, 1H, J = 1.8 Hz), 7.17 (d, 2H, J = 8.0 Hz), 7.08 – 6.99 (m, 2H), 6.96 (d, 2H, J = 8.0 Hz), 6.30 (s, 1H), 4.86 (d, 1H, J = 15.5 Hz), 4.36 (d, 1H, J = 15.5 Hz), 4.24 (s, 2H), 3.87 (s, 2H), 1.11 (s, 9H), note: the carboxylic acid proton was not observed; 13C-NMR (101 MHz, DMSO-d6) (mixture of rotamers) δ 169.2, 169.0,
163.8, 163.5, 163.4, 137.7, 137.0, 135.6, 135.3, 133.3, 131.40, 131.35, 128.4, 128.04, 127.95, 127.8, 127.7, 127.6, 127.2, 126.83, 126.77, 126.11, 126.05, 125.8, 125.7, 125.3, 125.0, 123.9, 123.9, 122.4, 121.8, 120.0, 112.2, 111.8, 56.6, 51.5, 50.41, 50.40, 48.5, 46.7, 28.5, 28.3.
3-(1-(N-(4-(((4-Bromophenyl)amino)methyl)benzyl)formamido)-2-(tert-butylamino)-2-oxoethyl)-6-chloro-1H-indole-2-carboxylic acid (18)
Following the general procedure, starting from 4-bromoaniline (17.2 mg, 0.100 mmol) as amine, compound 18 was obtained as a white solid (12 mg, 0.019 mmol, 20% yield). M.p. >129 °C (degradation); HRMS (ESI+) for C30H31BrClN4O4 [M + H]+:
625.121 (calc.), 625.121 (found). 1H-NMR (400 MHz, CD3OD,
major rotamer, 77%) δ 8.37 (s, 1H), 7.71 (d, 1H, J = 8.8 Hz), 7.35 (d, 1H, J = 1.9 Hz), 7.16 – 7.10 (m, 4H), 7.04 – 6.98 (m, 3H), 6.50 (d, 2H, J = 8.7 Hz), 6.46 (s, 1H), 5.01 (d, 1H, J = 15.0 Hz), 4.38 (d, 1H, J = 15.0 Hz), 4.18 (s, 2H), 1.15 (s, 9H), note: the carboxylic acid and amine protons were not observed; 13C-NMR (101 MHz, CD3OD) (mixture of rotamers) δ 171.7, 171.2, 168.0, 166.7, 166.1,
149.30, 149.26, 139.8, 139.4, 137.9, 136.93, 136.86, 136.7, 132.6, 130.5, 130.4, 129.2, 128.0, 128.0, 127.4, 127.1, 126.4, 123.3, 122.9, 121.8, 121.6, 115.6, 115.5, 112.9, 112.8, 112.3, 108.9, 108.8, 58.3, 54.7, 52.3, 52.2, 51.1, 48.4, 48.3, 48.1, 28.9, 28.8.
3-(2-(tert-Butylamino)-1-(N-(4-(((2,4-dimethylphenyl)amino)methyl)benzyl)formamido)-2-oxoethyl)-6-chloro-1H-indole-2-carboxylic acid (19)
Following the general procedure, starting from 2,4-dimethylaniline (12.1 mg, 0.100 mmol) as amine, compound
19 was obtained as a white solid (19 mg, 0.033 mmol, 34% yield).
M.p. >132 °C (degradation); HRMS (ESI+) for C32H36ClN4O4,
[M + H]+ 575.242 (calc.), 575.241 (found). 1H-NMR (400 MHz,
CD3OD, major rotamer, 75%) δ 8.37 (s, 1H), 7.71 (d, 1H, J = 8.8
Hz), 7.30 (d, 1H, J = 1.8 Hz), 7.13 (d, 2H, J = 8.0 Hz), 7.05 – 6.91 (m, 3H), 6.87 – 6.72 (m, 2H), 6.43 – 6.36 (m, 2H), 5.05 (d, 1H, J = 15.0 Hz), 4.34 (d, 1H, J = 15.1 Hz), 4.25 (s, 2H), 2.16 (s, 3H), 2.14 (s, 3H), 1.15 (s, 9H), note: the carboxylic acid and amine protons were not observed; 13C-NMR (101 MHz, CD
3OD) (mixture of rotamers) δ 171.2,
166.7, 166.1, 145.1, 140.4, 140.2, 137.6, 137.0, 136.6, 131.8, 131.7, 130.7, 129.1, 128.10, 128.07, 128.0, 127.8, 126.98, 126.95, 126.4, 123.7, 123.3, 122.9, 121.9, 121.8, 113.00, 112.98, 112.9, 112.0, 111.9, 58.3, 54.5, 52.4, 52.3, 51.2, 28.9, 28.8, 20.5, 17.8.
3-(2-(tert-Butylamino)-1-(N-(4-(((4-chloro-2 methylphenyl)amino)methyl)benzyl)formamido)-2-oxoethyl)-6-chloro-1H-indole-2 carboxylic acid (20)
Following the general procedure, starting from 4-chloro-2-methylaniline (15.7 mg, 0.111 mmol) as amine, compound 20 was obtained as a white solid (13.9 mg, 0.0233 mmol, 22% yield). M.p. >123 °C (degradation); HRMS (ESI+) for C31H33Cl2N4O4 [M + H]+: 595.187 (calc.), 595.187 (found). 1H-NMR (400 MHz, CD3OD, major rotamer, 70%) δ 8.36 (s, 1H),
7.73 (d, 1H, J = 8.8 Hz), 7.34 (d, 1H, J = 1.9 Hz), 7.10 (d, 2H,
J = 7.9 Hz), 7.03 (dd, J = 8.8, 1.9 Hz, 1H), 6.99 – 6.88 (m, 4H),
6.37 (d, 1H, J = 8.6 Hz), 6.33 (s, 1H), 5.07 (d, 1H, J = 15.1 Hz), 4.33 (d, 1H, J = 15.4 Hz), 4.27 (s, 2H), 2.15 (s, 3H), 1.15 (s, 9H), note: the carboxylic acid and amine protons were not observed; 13C-NMR (101
MHz, CD3OD) (mixture of rotamers) δ 171.3 (C), 171.0 (C), 166.7 (CH), 166.1 (CH), 146.4 (C), 146.3 (C),
140.0 (C), 139.8 (C), 137.7 (C), 137.4 (C), 137.2 (C), 136.7 (C), 131.3 (C), 131.1 (C), 130.4 (CH), 129.1 (CH), 127.8 (CH), 127.6 (CH), 127.3 (CH), 127.1 (C), 127.0 (CH), 126.3 (C), 125.4 (C), 125.3 (C), 123.4 (CH), 123.0 (CH), 122.1 (CH), 122.02 (CH), 121.96 (C), 121.9 (C), 114.0 (C), 113.1 (CH), 113.0 (CH), 112.3 (CH), 112.2 (CH), 58.3 (CH), 54.4 (CH), 52.5 (C), 52.3 (C), 51.2 (CH2), 48.3 (CH2), 48.2 (CH2), 48.1 (CH2),
3-(1-(N-(4-(((4-Bromo-2-methylphenyl)amino)methyl)benzyl)formamido)-2-(tert-butylamino)-2-oxoethyl)-6-chloro-1H-indole-2-carboxylic acid (21)
Following the general procedure, starting from 4-bromo-2-methylaniline (19 mg, 0.10 mmol) as amine, compound 21 was obtained as a white solid (21 mg, 0.033 mmol, 33% yield). M.p. >141 °C (degradation); HRMS (ESI+) for C31H33BrClN4O4
[M + H]+ 639.137 (calc.), 639.137 (found). 1H-NMR (400 MHz,
CD3OD, major rotamer, 75%) δ 8.37 (s, 1H), 7.71 (d, 1H, J = 8.8
Hz), 7.34 (d, 1H, J = 1.9 Hz), 7.17 – 6.91 (m, 7H), 6.41 (s, 1H), 6.33 (d, 1H, J = 8.6 Hz), 5.05 (d, 1H, J = 15.0 Hz), 4.35 (d, 1H,
J = 15.1 Hz), 4.27 (s, 2H), 2.15 (s, 3H), 1.14 (s, 9H) , note: the carboxylic acid and amine protons were
not observed; 13C-NMR (101 MHz, CD3OD) (mixture of rotamers) δ 171.5 (C), 171.1 (C), 167.1 (C), 166.7
(CH), 166.1 (CH), 146.8 (C), 146.7 (C), 139.9 (C), 139.7 (C), 137.7 (C), 137.2 (C), 137.0 (C), 136.6 (C), 133.1 (CH), 130.9 (C), 130.7 (C), 130.31 (CH), 130.30 (CH), 129.1 (CH), 127.8 (CH), 127.6 (CH), 127.2 (C), 127.1 (CH), 126.3 (C), 125.8 (C), 125.7 (C), 123.3 (CH), 122.9 (CH), 121.9 (CH), 121.8 (CH), 113.02 (C), 112.99 (CH), 112.9 (CH), 112.82 (CH), 112.75 (CH), 112.2 (C), 108.9 (C), 108.8 (C), 58.3 (CH), 54.6 (CH), 52.4 (C), 52.2 (C), 51.1 (CH2), 48.4 (CH2), 48.1 (CH2), 48.0 (CH2), 28.9 (3xCH3), 28.8 (3xCH3), 17.6 (2xCH3). 3-(1-(N-(4-(((4-Bromonaphthalen-1-yl)amino)methyl)benzyl)formamido)-2-(tert-butylamino)-2-oxoethyl)-6-chloro-1H-indole-2-carboxylic acid (22)
Following the general procedure, starting from 1-amino-4-chloronaphthalene as amine (40.1 mg, 0.181 mmol), compound 22 was obtained as a white solid (24.4 mg, 0.0361 mmol, 21% yield) after purification by prep-HPLC (Phenomenex Kinetex EVO C18 250x10 mm, H2O/Acetonitrile (0.10% formic
acid): 0.9/0.1 --> 0.1/0.9). M.p. >168 °C (degradation); HRMS (ESI+): C34H33BrClN4O4 [M + H]+, 675.137 (calc.), 675.136
(found). 1H-NMR (400 MHz, CD3OD, major rotamer, 65%) δ
8.35 (s, 1H), 8.13 – 8.08 (m, 2H), 7.73 (d, 1H, J = 8.8 Hz), 7.58 – 7.53 (m, 1H), 7.52 – 7.43 (m, 2H), 7.31 (d, 1H, J = 1.9 Hz), 7.14 (d, 2H, J = 7.4 Hz), 7.06 (dd, 1H, J = 5.2, 1.8 Hz), 6.91 (d, 2H, J = 7.6 Hz), 6.34 – 6.27 (m, 1H), 6.23 (s, 1H), 5.09 (d, 1H, J = 14.9 Hz), 4.40 (s, 2H), 4.36 – 4.23 (m, 1H), 1.13 (s, 9H), note: the carboxylic acid and amine protons were not observed;
13C-NMR (151 MHz, CD3OD) (mixture of rotamers) δ 171.2 (C), 170.9 (C), 166.7 (CH), 166.1 (CH), 145.34
(C), 145.30 (C), 139.6 (C), 139.4 (C), 137.7 (C), 137.61 (C), 137.57 (C), 136.8 (C), 133.44 (C), 133.41 (C), 131.63 (C), 131.57 (C), 131.55 (CH), 131.53 (CH), 129.0 (2xCH), 128.2 (2xCH), 128.00 (CH), 127.97 (CH), 127.8 (2xCH), 127.7 (CH), 127.1 (CH), 126.22 (C), 126.19 (CH), 126.1 (CH), 123.5 (CH), 123.2 (CH), 122.7 (CH), 122.6 (CH), 122.4 (CH), 122.2 (CH), 113.2 (CH), 113.1 (CH), 109.6 (C), 109.4 (C), 106.13 (CH), 106.10 (CH), 58.3 (CH), 54.4 (CH), 52.5 (C), 52.4 (C), 51.2 (CH2), 48.3 (CH2), 48.2 (CH2), 48.1 (CH2), 28.9 (3xCH3), 28.7 (3xCH3).
3-[1-[{4-[(Benzylamino)methyl]benzyl}(formyl)amino]-2-(tert-butylamino)-2-oxoethyl]-6-chloro-1H-indole-2-carboxylic acid (23)
Following the general procedure, starting from benzylamine (10.7 mg, 0.0999 mmol) as amine, compound 23 was obtained as a white solid (17 mg, 31% yield). M.p.241–243 °C; HRMS (ESI+) for C31H34ClN4O4 [M + H]+: 561.226 (calc.), 561.226 (found). 1H-NMR (400 MHz, DMSO-d6, major rotamer, 82%) δ 11.51 (br.
s, 1H), 8.27 (s, 1H), 7.65 (d, 1H, J = 8.7 Hz), 7.58 (br. s, 1H), 7.41 (d, 2H, J = 7.1 Hz), 7.38 – 7.21 (m, 4H), 7.13 (d, 2H, J = 8.0 Hz), 7.02 – 6.98 (m, 1H), 6.93 (d, 2H, J = 8.0 Hz), 6.35 (s, 1H), 4.82 (d, 1H, J = 15.6 Hz), 4.35 (d, 1H, J = 15.6 Hz), 3.85 (s, 2H), 3.80 (s, 2H), 1.13 (s, 9H), note: of the exchangeable protons only two broad singlets were observed; 13C-NMR (101 MHz, DMSO-d6) (mixture
of rotamers) δ 169.3, 163.78, 163.76, 137.3, 135.5, 135.1, 129.0, 128.8, 128.3, 128.8, 128.2, 127.6, 127.2, 127.1, 125.8, 125.1, 121.7, 119.8, 111.7, 56.7, 50.7, 50.5, 50.4, 46.6, 28.5, 28.3.
4.5 Acknowledgments
Funding was granted to A.K.H.H. by the Netherlands Organization for Scientific Research (NWO-CW, VIDI grant 723.014.008) and by the Helmholtz-Association’s Initiative and Networking Fund; to A.K.H.H. and A.D. by the COFUND ALERT (grant agreement No. 665250); to T.A.H. by Grant UMO-2014/12/W/NZ1/00457 from the National Science Centre, Poland; and to A.D. by the National Institute of Health (NIH) (2R01GM097082-05), the European Lead Factory (IMI) under grant agreement number 115489, the Qatar National Research Foundation (NPRP6-065-3-012). Moreover, funding was received through ITN “Accelerated early stage drug discovery” (AEGIS, grant agreement No 675555), Hartstichting (ESCAPE-HF, 2018B012) and KWF Kankerbestrijding grant (grant agreement No. 10504).
4.6 Contributions from co-authors
M.Y. Unver designed the project. C.G. Neochoritis provided helpful suggestions for the Ugi reaction. R. van der Vlag, M.Y. Unver and T. Felicetti performed KTGS experiments. T. Felicetti synthesized 2, 17–19 and 23. Enzyme-inhibition studies were carried out by A.
Twarda-Clapa and B. Labuzek. 1H-15N HSQC analysis was performed by B. Musielak. Part of
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4.8 Supporting figures
MDM2 MDMX
Ki = 0.40 ± 0.05 µM Ki = 4.18 ± 0.27 µM
Figure S1. Inhibitory activity of compound 2 towards MDM2/MDMX.
MDM2 MDMX
Ki = 0.76 ± 0.08 µM Ki = 12.16 ± 2.15 µM
Figure S2. Inhibitory activity of compound 17 towards MDM2/MDMX.
MDM2 MDMX
Ki = 0.63 ± 0.07 µM Ki = 4.56 ± 0.49 µM
Figure S3. Inhibitory activity of compound 18 towards MDM2/MDMX. 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 1.1 1.2 1.3 0.0001 0.001 0.01 0.1 1 10 100 F ra c tio n o f b o u n d r e p o rt e r p e p tid e Inhibitor concentration (μM) 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 1.1 1.2 1.3 0.0001 0.001 0.01 0.1 1 10 100 F ra c tio n o f b o u n d r e p o rt e r p e p tid e Inhibitor concentration (μM) 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 1.1 1.2 1.3 0.0001 0.001 0.01 0.1 1 10 100 F ra c tio n o f b o u n d r e p o rt e r p e p tid e Inhibitor concentration (μM) 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 1.1 1.2 1.3 0.0001 0.001 0.01 0.1 1 10 100 F rac tio n o f b o u n d r ep o rt er p e p tid e Inhibitor concentration (μM) 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 1.1 1.2 1.3 0.0001 0.001 0.01 0.1 1 10 100 F ra c tio n o f b o u n d r e p o rt e r p e p tid e Inhibitor concentration (μM) 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 1.1 1.2 1.3 0.0001 0.001 0.01 0.1 1 10 100 F ra c tio n o f b o u n d r e p o rt e r p e p tid e Inhibitor concentration (μM)
MDM2 MDMX
Ki = 0.75 ± 0.08 µM Ki = 7.08 ± 0.52 µM
Figure S4. Inhibitory activity of compound 19 towards MDM2/MDMX.
MDM2 MDMX
Ki = 0.47 ± 0.04 µM Ki = 3.73 ± 0.30 µM
Figure S5. Inhibitory activity of compound 20 towards MDM2/MDMX.
MDM2 MDMX
Ki = 0.49 ± 0.04 µM Ki = 4.61 ± 0.39 µM
Figure S6. Inhibitory activity of compound 21 towards MDM2/MDMX. 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 1.1 1.2 1.3 0.0001 0.001 0.01 0.1 1 10 100 F ra c tio n o f b o u n d r e p o rt e r p e p tid e Inhibitor concentration (μM) 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 1.1 1.2 1.3 0.0001 0.001 0.01 0.1 1 10 100 F ra c tio n o f b o u n d r e p o rt e r p e p tid e Inhibitor concentration (μM) 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 1.1 1.2 1.3 0.0001 0.001 0.01 0.1 1 10 100 F ra c tio n o f b o u n d r e p o rt e r p e p tid e Inhibitor concentration (μM) 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 1.1 1.2 1.3 0.0001 0.001 0.01 0.1 1 10 100 F ra c tio n o f b o u n d r e p o rt e r p e p tid e Inhibitor concentration (μM) 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 1.1 1.2 1.3 0.0001 0.001 0.01 0.1 1 10 100 F ra c tio n o f b o u n d r e p o rt e r p e p tid e Inhibitor concentration (μM) 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 1.1 1.2 1.3 0.0001 0.001 0.01 0.1 1 10 100 F rac tio n o f b o u n d r ep o rt er p e p tid e Inhibitor concentration (μM)
MDM2 MDMX
Ki = 0.095 ± 0.010 µM Ki = 3.95 ± 0.46 µM
Figure S7. Inhibitory activity of compound 22 towards MDM2/MDMX.
MDM2 MDMX
Ki = 3.18 ± 0.18 µM Not active
(64% of reporter peptide bound at 50 μM)
Figure S8. Inhibitory activity of compound 23 towards MDM2/MDMX.
MDM2 MDMX
Ki = 0.55 ± 0.05 µM Ki = 11.4 ± 1.6 µM
Figure S9. Inhibitory activity of compound 3 towards MDM2/MDMX. -0.1 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 1.1 1.2 1.3 0.0001 0.001 0.01 0.1 1 10 100 F ra c tio n o f b o u n d r e p o rt e r p e p tid e Inhibitor concentration (μM) 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 1.1 1.2 1.3 0.0001 0.001 0.01 0.1 1 10 100 F ra c tio n o f b o u n d r e p o rt e r p e p tid e Inhibitor concentration (μM) 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 1.1 1.2 1.3 0.0001 0.001 0.01 0.1 1 10 100 F ra c tio n o f b o u n d re p o rt e r p e p tid e Inhibitor concentration (μM) 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 1.1 1.2 1.3 0.0001 0.001 0.01 0.1 1 10 100 F ra c tio n o f b o u n d r e p o rt e r p e p tid e Inhibitor concentration (μM) 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 1.1 1.2 1.3 0.0001 0.001 0.01 0.1 1 10 100 F rac tio n o f b o u n d r ep o rt er p e p tid e Inhibitor concentration (μM) 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 1.1 1.2 1.3 0.0001 0.001 0.01 0.1 1 10 100 F ra c tio n o f b o u n d r e p o rt e r p e p tid e Inhibitor concentration (μM)
Figure S10. UV-vis absorption spectrum of 3 (red), 22 (blue) and 24 (green) in MilliQ/ACN 50:50 (with 0.1% FA).
For comparison, data was normalized at 305 nm (λmax of 3).
Figure S11. Determining the detection limit of the UPLC-MS system of aldehyde 3 at 254 nm (close-up). From top
to bottom: 0.30, 0.10, 0.033, 0.011, 0.0037, 0.0012, 0.00041 mg/mL and blank. The peak of 3 at 0.0012 mg/mL (2.6 μM,purple) can be clearly distinguished from the blank, while the peak at 0.00041 mg/mL (cyan) cannot be reliably identified. RT = Retention Time, MA = Measured Area.
0 0.5 1 1.5 2 2.5 230 330 430 530 630 A bs orbance Wavelength (nm) 3 22 24 0 0.25 0.5 0.75 1 1.25 1.5 250 300 350 400 A bs orbance Wavelength (nm) 3 22 24
Figure S12. Determining the detection limit of the UPLC-MS system of aldehyde 3 at 305 nm (close-up). From top
to bottom: 0.30, 0.10, 0.033, 0.011, 0.0037, 0.0012, 0.00041 mg/mL and blank. The peak of 3 at 0.00041 mg/mL (0.88 μM,cyan) can be clearly distinguished from the blank. RT = Retention Time, MA = Measured Area.
Figure S13. Plot of the integrated area versus concentration of 3 in the UPLC-MS vial at wavelength 254 nm (blue)
and 305 nm (red). Linear fits are shown using dotted lines.Solving both equations for a value of three times the measured area of the blank gives an estimated detection limit of 1.56 μM and 1.07 μM for 254 and 305 nm, respectively. Based on this calculation, comparing the traces visually would result in a higher detection limit for 254 nm and a lower detection limit for 305 nm.
Table S1. Overview retention times of all compounds. Compound Ret. time
(min)
Compound Ret. time (min) A m ine s tar ti ng m at er ial s 4 5.54 R edu ct iv e a m ina ti o n pr o du ct s 2 8.72 5 6.50 18 8.79 6 5.45 19 8.60 7 6.16 17 7.44 8 7.12 20 8.92 9 7.46 21 8.99 10 8.18 22 9.21 11 1.87 23 7.28 Aldehyde 3 8.00 (±)-Nutlin-3 7.37 y = 2838x - 1088.8 R² = 1 y = 2621.1x - 1457.9 R² = 1 0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 2 0 100 200 300 400 500 600 700 M e asur e d A re a (x 10 6) Concentration of 3 in vial (µM) 254 nm 305 nm
