Development and application of novel scaffolds in drug discovery
Boltjes, André
DOI:
10.33612/diss.98161351
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Publication date: 2019
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Boltjes, A. (2019). Development and application of novel scaffolds in drug discovery: the MCR approach. Rijksuniversiteit Groningen. https://doi.org/10.33612/diss.98161351
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Chapter 2
Fragment based Library
Generation for the Discovery of
a Peptidomimetic p53-Mdm4
Inhibitor
André Boltjes, Yijun Huang, Rob van de Velde, Laurie Rijkee, Siglinde Wolf, James Gaugler,Katarzyna Lesniak, Katarzyna Guzik, Tad A. Holak,d Alexander
Dömling
Abstract
Based on our recently resolved first co-crystal structure of Mdm4 in complex with a small molecule inhibitor (PDB ID: 3LBJ), we devised an approach for the generation of potential Mdm4 selective ligands. We performed the Ugi four-com-ponent reaction (Ugi-4CR) in 96-well plates with an indole fragment, which is specially designed to mimic Trp23, a key amino acid for the interaction between p53 and Mdm4. Generally the reaction yielded mostly precipitates collected by 96-well filter plates. The best hit compound was found to be active and selective for Mdm4 (Ki = 5 µM, 10 fold stronger than Mdm2). This initial hit may serve as the starting point for designing selective p53-Mdm4 inhibitors with higher affinity.
2
Introduction
The protein-protein interaction between the transcription factor p53 and the neg-ative regulator Mdm2 is an important recent oncology target.1 The interaction is crystallographically characterized and druggable and several compounds are in late preclinical and early clinical evaluation.2 The Mdm4 protein is a closely re-lated protein to Mdm2 and it also binds to the same epitope of p53. The sequence homology, the shape, dimension and size is similar. Nevertheless all current com-pound scaffolds characterized by co-crystal structure analysis are highly specific for Mdm2 and show no or very little Mdm4 binding which, is surprising and not well understood regarding the great similarity between the two proteins (Figure 1).3-4 Nutlin-3a 1, for example, binds to Mdm4 too but with a roughly 1000-fold lower affinity of about 25 µM.5 Several Novartis compounds 2-4 show weak low µM Mdm4 affinity while being very potent binders to Mdm2 (again ~1000-fold difference, Figure 2).6-7 Other described Mdm4 selective compounds are either covalent binders or not validated (5, 6).8-9 Surprisingly, pyrazolone compound6
5 loses activity to Mdm4 in the presence of a reducing reagent, dithiothreitol
(DTT). Incubation of these compounds with Mdm4 under non-reducing condi-tions lead to a time dependent change of Mdm4 structure determined by NMR; concomitantly, the MS analysis showed the presence of covalent adducts of the compound with Mdm4. Additionally, we have found out, by 1H NMR, that the pyrazolone reacts with β-mercaptoethanol in chloroform.
the M>V exchange. The key amino acids of Mdm2, Mdm4 and p53 are shown as sticks. Selective Mdm4 antagonists are highly sought after since Mdm4 and Mdm2 pro-teins are differentially over-expressed in several cancers and both play a promi-nent but presumably different role in apoptosis induction.10 Herein, we describe the discovery of B1, a selective p53-Mdm4 inhibitor (with ~5 µM affinity to Mdm4 but 54 µM affinity to Mdm2) with reversed selectivity compared with most p53-Mdm2 inhibitors, which we believe is a good starting point to elaborate Mdm4 selective compounds.
2
Results and Discussion
Based on our previously generated insight into the binding of small molecules into Mdm2 and Mdm4 and our recently developed Mdm2 and Mdm4 fluo-rescence polarization assay, we planned to synthesized libraries of potential Mdm2/4 binding compounds.5, 11-21 Thus, we generated a 96-member library of peptidomimetic small molecules via Ugi four-component reaction (Ugi-4CR) (Scheme 1). Each compound contains the indole or p-halobenzyl fragment to mimic the Trp23 “anchor”, which is the key anchor residue in the p53 Mdm2 and Mdm4 protein-protein interaction interface, respectively. Figure 3 illustrates the structure of amine and isocyanide inputs, as well as the experimental setting in a 96-well microliter plate. Since the reaction products regularly precipitated, the compounds were collected by a 96-well filter plate, and washed with ether to remove unreacted starting materials. The yields of the isolated products were between low (6%) and good (56%) with an average of 28% over all 96 wells. In addition, the purities of the isolated materials were considered sufficient for an initial screening. The collected samples were dissolved as a 10 mM stock solution in DMSO for the screening.
HCHO R2NH 2 R1NC N O R2 O N H R1 NH OH O N H
Scheme 1. Ugi-4CR for high throughput synthesis
Compound B1 was identified as a p53/Mdm4 inhibitor (Ki = 19 µM) via our re-cently reported fluorescence polarization assay. The hit compound was re-syn-thesized and purified by flash chromatography, which was further confirmed by the binding with Mdm4 (Ki = 5.5 µM), as shown in Figure 4.5 Although the
p53-binding sites within the Mdm4 and Mdm2 proteins are closely related, known Mdm2 small-molecule inhibitors have been shown experimentally not or very poorly to bind to its homolog Mdm4. This hit compound may provide a new avenue for the design of potential selective inhibitors of the p53-Mdm4 interaction. Further studies are ongoing in our lab to uncover the puzzle of the Mdm2 and Mdm4 selectivity.
A NH2 Cl NH2 Cl NH2 F NH2 NH2 NH2 NH2 1 2 3 4 5 6 7 8 9 10 11 12 NH2 NH2 NH2 Cl Cl NH2 Br NH2 O B NC NC NC NC NC A B C D E F G H NC CO2Me Ph Ph NC NC O N
Figure 3. Parallel synthesis of “anchor” biased compound library via
Ugi-4CR. Structures of the amine A and isocyanide B starting materials used.
For further optimizing purposes a second library was synthesized, that follows the structure of hit compound B1, yielding a total of 38 new compounds. Mi-nor changes were made in the indole moiety (from the carboxylic acid com-ponent) and different halogen substituted benzylamines were employed, keeping the cyclohexyl fragment intact, as shown in figure 5. This time a se-quential approach was used, which made it possible to run 1 mmol scale reac-tions as opposed to 0.2 mmol scale in the 96-well plate. Increased yields up to 79% were observed, in average 46%, which confirms that larger scale Ugi re-actions in general give better yields. Unfortunately, all the other compounds synthesized in Figure 5 showed worse (>50 µM) or no activity in the FP assay.
A B C N O OHN Cl N H
Figure 4. Hit compound as p53/Mdm4 inhibitor. A: Structure of B1; B: Ki = 54 µM (Mdm2)
2
A H N O O S COOH HO COOH MeO COOHCOOH COOH COOH
H N COOH N COOH H N COOH N H N COOH Cl HO I J K L M N O P Q R N N N COOH COOH S T S COOH COOH Cl H N COOH U V W B H2N CF3 H2N F H2N Cl Cl 13 14 15
Figure 4. Starting materials for the second library of compounds with the structures of A
the carboxylic acids and B the amines.
Conclusions
In summary, this work demonstrates that the Ugi four-component reaction (Ugi-4CR) can be used to address the requirements for efficient high-throughput syn-thesis of diverse compounds in a cost- and time-effective manner. Integrated with biochemical screening assays, a peptidomimetic p53-Mdm4 inhibitor B1 was identified from a 96-membered library generated via Ugi-4CR of an indole fragment. This approach provides an efficient strategy for the discovery of small molecule probes selectively targeting protein-protein interactions. Further opti-mization studies on B1 are ongoing and will be reported in due course.
Experimental procedures and Spectral Data
All reagents were purchased from commercial sources and used without fur-ther purification. Proton and carbon NMR spectra were determined on Bruker Avance™ 600 MHz NMR spectrometer. Chemical shifts are reported as δ values in parts per million (ppm) as referenced to residual solvent. 1H NMR spectra are tabulated as follows: chemical shift, number of protons, multiplicity (s = singlet, br.s = broad singlet, d = doublet, t = triplet, q = quartet, m = multiplet), and cou-pling constant. High Resolution Mass spectra were obtained at the University of Pittsburgh Mass Spectrometry facility. LC-MS analysis was performed on an SHIMADZU instrument (reverse-phase HPLC coupled to electrospray ioniza-tion-mass spectrometry), using an analytical C18 column (Dionex Acclaim 120 Å, 2.1 × 50 mm, 3.0 µm) coupled to an Applied Biosystems API2000 mass spectrom-eter (ESI-MS). Acetonitrile/water mixtures were used as the mobile phase for reverse-phase HPLC, the flow rate was maintained at 0.2 mL/min. The column temperature was kept at 40 °C. The UV detection was at 254 nm.
High throughput synthesis of a 96-member library.
Add indole-2-carboxylic acid (200 µL, 1M methanol stock solution; 0.2 mmol), amine (200 µL, 1M methanol stock solution; 0.2 mmol), isocyanide (200 µL, 1M methanol stock solution; 0.2 mmol), formaldehyde (120 µL, 2M methanol stock solution; 0.24 mmol) into each well of 96 deep well plate with printed labeling (VWR D108839, 1.2 mL).The plated was sealed by aluminum foil sealing film and was sonicated for 1h, then stand overnight under RT. After the partial evapora-tion of the solvent, the product was collected by filtraevapora-tion and washed by ether (AcroPrepTM 96 filter plate from PALL (0.2 um GHP, 1 mL well)) was used, 96 deep well plate was used as the receiver). For the second library equal conditions were used, but performed in 4 mL vials.
N-(4-chlorobenzyl)-N-(2-(cyclohexylamino)-2-oxoethyl)-1H-indole-2-carbox-amide (B1) NH O N O H N
Cl White solid, 20.6 mg; yield: 24%. HPLC/MS: tR = 11.61 min; m/z = 424.2 [M+H]+. HRMS: C 24H26N3O2ClNa, 446.1611 (calcd.), 446.1634 (found). 1H NMR (600 MHz, d6-DMSO): 1.18-1.27 (m, 5H), 1.53-1.74 (m, 4H), 3.58 (m, 1H), 3.94 (m, 1H), 4.18 (m, 1H), 4.66 (m, 1H), 5.00 (m, 1H), 6.53 (m, 1H), 6.75 (m, 1H), 7.03 (m, 1H), 7.18 (m, 1H), 7.37-7.56 (m, 4H), 7.84 (m, 1H), 8.05 (m, 1H), 11.70 (m, 1H). 13C NMR (150 MHz, d6-DMSO): 24.4, 25.1, 32.2, 47.5, 49.7, 51.2, 54.9, 103.9, 111.99, 112.04, 119.8, 121.4, 123.5, 126.8, 128.3, 129.8, 131.8, 135.9, 136.3, 163.8, 166.9. N-(tert-butyl)-N-(2-(cyclohexylamino)-2-oxoethyl)-1H-indole-2-carboxamide (B7):
2
H N O N O H N 1H NMR (600 MHz, DMSO): 1.13-1.21 (m, 3H), 1.27-1.33 (m, 2H), 1.46 (s, 9H), 1.55-1.57 (m, 1H), 1.68-1.70 (m, 2H), 1.77-1.79 (m, 2H), 3.63-3.66 (m, 1H), 4.21 (s, 2H), 6.67 (s, 1H), 7.00-7.02 (t, 1H, J = 7.2Hz), 7.14-7.16 (t, 1H, J = 7.5 Hz), 7.37-7.38 (d, 2H, J = 8.4Hz), 7.53-7.54 (d, 1H, J = 8.4Hz), 7.93-7.94 (d, 1H, J = 7.8Hz), 11.52 (s, 1H). 13C NMR (150 MHz, DMSO): 24.9, 25.7, 28.0, 32.6, 48.0, 50.6, 58.0, 100.0, 103.3, 112.4, 120.0, 121.7, 123.4, 127.2, 133.2, 136.2, 166.0, 169.9. HRMS: C21H29N3O2Na, 378.2157 (calcd.), 378.2140 (found). N-(2-(benzylamino)-2-oxoethyl)-N-(tert-butyl)-1H-indole-2-carboxamide (C7): N H O N O N H 1H NMR (600 MHz, DMSO): 1.47 (s, 9H), 4.32 (s, 2H), 4.37-4.38 (d, 2H), 6.60 (s, 1H), 7.02 (m, 1H), 7.15 (m, 1H), 7.26-7.39 (m, 5H), 7.37-7.39 (d, 1H, J = 8.4Hz), 7.47-7.48 (d, 1H, J = 8.4Hz), 8.58-8.60 (t, 1H, J = 6.0Hz), 11.51 (s, 1H). 13C NMR (150 MHz, DMSO): 28.0, 42.8, 50.7, 58.0, 103.1, 112.4, 120.1, 121.7, 123.4, 127.2, 127.4, 127.9, 128.8, 133.0, 136.2, 139.8, 165.9, 170.9. HRMS: C22H25N3O2Na, 386.1844 (calcd.), 386.1812 (found).N-(tert-butyl)-N-(2-(mesitylamino)-2-oxoethyl)-1H-indole-2-carboxamide (E7): H N O N O H N 1H NMR (600 MHz, DMSO): 1.29 (s, 9H), 1.90 (s, 6H), 2.00 (s, 3H), 4.27 (s, 2H), 6.52 (s, 1H), 6.67 (s, 2H), 6.78-6.81 (t, 1H, J = 7.2Hz), 6.92-6.95 (t, 1H, J = 7.2Hz), 7.16-7.17 (d, 1H, J = 8.4Hz), 7.26-7.28 (d, 1H, J = 8.4Hz), 9.16 (s, 1H), 11.29 (s, 1H). 13C NMR (150 MHz, DMSO): 17.9, 20.5, 27.5, 57.5, 62.8, 102.7, 111.9, 119.7, 121.0, 123.0, 126.7, 128.6, 131.9, 132.6, 134.8, 135.6, 135.8, 165.5, 169.3. HRMS: C24H30N3O2, 392.2338 (calcd.), 392.2341 (found). N-(2-(cyclohexylamino)-2-oxoethyl)-N-phenethyl-1H-indole-2-carboxamide (B12): NH O N O N H 1H NMR (600 MHz, DMSO): 1.13-1.29 (m, 6H), 1.55-1.57 (d, 1H, J = 12.6Hz), 1.68-1.70 (m, 2H), 1.75-1.77 (d, 2H, J = 12.0Hz), 2.92 (s, 2H), 3.03 (s, 1H), 3.64 (s, 1H), 3.89 (m, 1H), 4.09 (s, 1H) 4.24 (s, 1H), 6.68 (s, 1H) 7.03-7.05 (t, 1H, J = 7.32Hz), 7.18-7.26 (m, 2H), 7.26-7.36 (m, 4H), 7.42-7.44 (d, 1H, J = 8.4Hz), 7.57 (m, 1H), 8.11 (m, 1H), 11.64 (s, 1H). 13C NMR (150 MHz, DMSO): 24.5, 25.2, 32.3, 41.3, 47.6, 49.6, 52.0, 103.5, 112.0, 119.7, 121.3, 123.3, 126.2, 126.9, 128.5, 128.7, 135.8, 163.3, 167.4. HRMS: C H NO, 404.2338 (calcd.), 404.2328 (found).
Table 1. The obtained U-4CR products.
Compound Structure Mass (mg) Yield (%) LC/MS
A1 N O OHN Cl N H 22.3 28 tR = 11.61 min; m/z = 398.3 [M+H]+ B1 N O O NH Cl H N 20.6 24 tR = 11.61 min; m/z = 424.2 [M+H]+ C1 N O OHN Cl N H 32.4 38 tR = 11.48 min; m/z = 432.3 [M+H]+ D1 N O OHN Cl N H 15.6 18 tR = 11.65 min; m/z = 446.2 [M+H]+ E1 N O OHN Cl N H 28.6 31 tR = 12.01 min; m/z = 460.2 [M+H]+ F1 N O OHN Cl N H CO2Me 34.7 42 tR = 10.75 min; m/z = 414.3 [M+H]+ G1 N O OHN Cl N H N O 30.8 34 tR = 10.63 min; m/z = 453.2 [M+H]+
2
H1 N O OHN Cl N H Ph Ph 19.7 19 tR = 12.18 min; m/z = 508.2 [M+H]+ A2 N O OHN N H 25.4 35 tR = 11.18 min; m/z = 364.4 [M+H]+ B2 N O OHN N H 30.1 39 tR = 11.44 min; m/z = 390.4 [M+H]+ C2 N O OHN N H 10.5 13 tR = 11.10 min; m/z = 398.4 [M+H]+ D2 N O OHN N H 12.3 15 tR = 11.32 min; m/z = 412.3 [M+H]+ E2 NOHN O N H 12.8 15 tR = 11.66 min; m/z = 426.4 [M+H]+ F2 N O OHN N H CO2Me 27.6 36 tR = 10.31 min; m/z = 380.2 [M+H]+ G2 N O OHN N N O 34.2 41 tR = 10.44 min; m/z = 419.4A3 N O OHN N H Cl 41.6 51 tR = 11.78 min; m/z = 412.2 [M+H]+ B3 N O OHN N H Cl 23 26 tR = 11.98 min; m/z = 438.2 [M+H]+ C3 N O OHN N H Cl 5.5 6 tR = 11.65 min; m/z = 446.3 [M+H]+ D3 N O OHN N H Cl 7.4 8 tR = 11.82 min; m/z = 460.3 [M+H]+ E3 N O OHN N H Cl 17.2 18 tR = 12.15 min; m/z = 474.3 [M+H]+ F3 N O OHN N H CO2 Me Cl 30.2 35 tR = 10.90 min; m/z = 428.3 [M+H]+ G3 N O OHN N H N O Cl 37.7 40 tR = 10.40 min; m/z = 467.4 [M+H]+ H3 N O OHN N H Ph Ph Cl 10.4 10 tR = 12.33 min; m/z = 522.3 [M+H]+ A4 N O OHN N H F 23.9 31 tR = 11.24 min; m/z = 382.2 [M+H]+
2
B4 N O OHN N H F 12.4 15 tR = 11.48 min; m/z = 408.4 [M+H]+ C4 N O OHN N H F 21.2 26 tR = 11.16 min; m/z = 416.3 [M+H]+ D4 N O OHN N H F 10.7 12 tR = 11.38 min; m/z = 430.1 [M+H]+ E4 N O OHN N H F 49.2 56 tR = 11.69 min; m/z = 444.4 [M+H]+ F4 N O OHN N H F CO2Me 30.5 38 tR = 10.39 min; m/z = 398.3 [M+H]+ G4 N O OHN N H F N O 19 22 tR = 10.31 min; m/z = 437.4 [M+H]+ H4 N O OHN N H F Ph Ph 19.5 20 tR = 11.88 min; m/z = 492.3 [M+H]+B5 N O OHN N H 10.6 13 tR = 11.97 min; m/z = 396.5 [M+H]+ C5 N O OHN N H 18.3 23 tR = 11.62 min; m/z = 404.3 [M+H]+ D5 N O OHN N H 27.9 33 tR = 11.84 min; m/z = 418.2 [M+H]+ E5 NOHN O N H 27.4 32 tR = 12.18 min; m/z = 432.4 [M+H]+ F5 N O OHN N H CO2Me 17.6 23 tR = 10.82 min; m/z = 386.3 [M+H]+ G5 N O OHN N H N O 14.5 17 tR = 10.68 min; m/z = 425.3 [M+H]+ H5 N O OHN N H Ph Ph 26.3 27 tR = 12.34 min; m/z = 480.4 [M+H]+ A6 N O O NH N H 26.9 38 tR = 11.42 min; m/z = 356.4 [M+H]+ B6 N O OHN N H 14.7 19 tR = 11.63 min; m/z = 382.2 [M+H]+ C6 N O OHN N H 28.8 37 tR = 11.28 min; m/z = 390.3 [M+H]+
2
D6 NOHN O N H 32.8 41 tR = 11.51 min; m/z = 404.2 [M+H]+ E6 NO O NH H N 33 40 tR = 11.88 min; m/z = 418.5 [M+H]+ F6 N O OHN N H CO2Me 23.7 32 tR = 10.43 min; m/z = 372.3 [M+H]+ G6 OHNN O N H N O 21 26 tR = 10.34 min; m/z = 411.4 [M+H]+ H6 N O OHN N H Ph Ph 25.1 27 tR = 12.07 min; m/z = 466.5 [M+H]+ A7 N O OHN N H 7.2 11 tR = 11.23 min; m/z = 330.5 [M+H]+ B7 N O OHN N H 11.8 17 tR = 11.44 min; m/z = 356.2 [M+H]+ C7 NOHN O N H 15.7 22 tR = 10.98 min; m/z = 364.3 [M+H]+ D7 N O OHN N H 14.8 20 tR = 11.22 min; m/z = 378.4 [M+H]+ E7 OHN 28.8 37 tR = 11.58 min; m/z = 392.3G7 N O OHN N H N O 22.1 29 tR = 9.90 min; m/z = 385.3 [M+H]+ H7 OHNN O N H Ph Ph 16.6 19 tR = 11.81 min; m/z = 440.4 [M+H]+ A8 N O OHN N H MeO 8.3 13 tR = 10.30 min; m/z = 332.2 [M+H]+ B8 N O OHN N H MeO 13.5 19 tR = 10.60 min; m/z = 358.5 [M+H]+ C8 N O OHN N H MeO 9.6 13 tR = 10.32 min; m/z = 366.2 [M+H]+ D8 NOHN O N H MeO 14.9 20 tR = 10.56 min; m/z = 380.3 [M+H]+ E8 NOHN O N H MeO 36.5 46 tR = 10.92 min; m/z = 394.3 [M+H]+ F8 N O OHN N H MeO CO2Me 30.1 43 tR = 9.35 min; m/z = 448.3 [M+H]+ G8 N O OHN N H MeO N O 32.6 42 tR = 9.28 min; m/z = 387.2 [M+H]+ H8 N O OHN N H MeO Ph Ph 19.1 22 tR = 11.48 min; m/z = 442.3 [M+H]+
2
A9 N O OHN N H 21.7 33 tR = 10.80 min; m/z = 328.4 [M+H]+ B9 N O OHN N H 17.8 25 tR = 11.03 min; m/z = 354.3 [M+H]+ C9 N O OHN N H 13.8 19 tR = 10.73 min; m/z = 362.3 [M+H]+ D9 N O OHN N H 9.4 13 tR = 10.93 min; m/z = 376.4 [M+H]+ E9 N O OHN N H 29.4 38 tR = 11.32 min; m/z = 390.4 [M+H]+ F9 N O OHN N H CO2Me 35.2 51 tR = 9.84 min; m/z = 344.5 [M+H]+ G9 OHNN O N H N O 25.8 34 tR = 9.75 min; m/z = 383.2 [M+H]+ H9 N O OHN N H Ph Ph 15.1 17 tR = 11.58 min; m/z = 438.4 [M+H]+ A10 N O OHN N H 30.2 34 tR = 11.68 min; m/z = 442.1C10 N O OHN N H Br 44.9 47 tR = 11.58 min; m/z = 476.2 [M+H]+ D10 N O OHN N H Br 24.3 25 tR = 11.76 min; m/z = 490.2 [M+H]+ E10 N O OHN N H Br 28.7 29 tR = 12.11 min; m/z = 504.3 [M+H]+ F10 N O OHN N H Br CO2Me 28.3 31 tR = 10.88 min; m/z = 458.2 [M+H]+ G10 N O OHN N H Br N O 25.8 26 tR = 10.73 min; m/z = 497.1 [M+H]+ H10 N O OHN N H Br Ph Ph 17.9 16 tR = 12.27 min; m/z = 552.3 [M+H]+ A11 N O OHN N H Cl Cl 12.1 14 tR = 11.93 min; m/z = 432.0 [M+H]+ B11 N O OHN N H Cl Cl 51.4 56 tR = 12.13 min; m/z = 458.4 [M+H]+ C11 N O OHN N H Cl Cl 17.8 19 tR = 11.80 min; m/z = 466.3 [M+H]+
2
D11 N O OHN N H Cl Cl 22 23 tR = 11.98 min; m/z = 480.3 [M+H]+ E11 N O OHN N H Cl Cl 33.7 34 tR = 12.28 min; m/z = 494.2 [M+H]+ F11 N O OHN N H Cl Cl CO2Me 38 42 tR = 11.06 min; m/z = 448.1 [M+H]+ G11 N O OHN N H Cl Cl N O 30.2 31 tR = 10.91 min; m/z = 487.2 [M+H]+ H11 N O OHN N H Cl Cl Ph Ph 22.4 21 tR = 12.47 min; m/z = 542.3 [M+H]+ A12 N O OHN N H 28.9 38 tR = 11.32 min; m/z = 378.3 [M+H]+ B12 N O OHN N H 28.5 35 tR = 11.55 min; m/z = 404.3 [M+H]+ C12 N O OHN N H 14.2 17 tR = 11.23 min; m/z = 412.2E12 N O OHN N H 48.2 55 tR = 11.76 min; m/z = 440.3 [M+H]+ F12 N O OHN N H CO2Me 19.9 25 tR = 10.48 min; m/z = 394.2 [M+H]+ G12 N O OHN N H N O 21 24 tR = 10.34 min; m/z = 433.3 [M+H]+ H12 N O OHN N H Ph Ph 31.3 32 tR = 11.96 min; m/z = 488.3 [M+H]+
2
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