Skip to main content
NCBI home page
NIHPA Author Manuscripts logoLink to NIHPA Author Manuscripts
. Author manuscript; available in PMC: 2009 Sep 15.
Published in final edited form as: J Med Chem. 2008 Aug 5;51(16):4948–4956. doi: 10.1021/jm8002526

Inhibitors of Src Homology-2 Domain containing Protein Tyrosine Phosphatase-2 (Shp2) Based on Oxindole Scaffolds

Harshani R Lawrence 1,3,*,†, Roberta Pireddu 1,†, Liwei Chen 2,†, Yunting Luo 3,†, Shen-Shu Sung 3, Ann Marie Szymanski 2, M L Richard Yip 3,‡, Wayne C Guida 3,4,5, Saïd M Sebti 1,4, Jie Wu 1,2,4,6, Nicholas J Lawrence 1,4,*
PMCID: PMC2744494  NIHMSID: NIHMS86088  PMID: 18680359

Abstract

Screening of the NCI diversity set of compounds has led to the identification of 5 (NSC-117199) which inhibits the protein tyrosine phosphatase (PTPa) Shp2 with an IC50 of 47 μM. A focused library incorporating an isatin scaffold was designed and evaluated for inhibition of Shp2 and Shp1 PTP activities. Several compounds were identified that selectively inhibit Shp2 over Shp1 and PTP1B with low to sub-micromolar activity. A model for the binding of the active compounds is proposed.

Introduction

The function of many proteins, particularly those involved in signal transduction pathways, is dependent upon their tyrosine phosphorylation status, which is finely regulated by the action of protein tyrosine kinases and protein tyrosine phosphatases. Many protein tyrosine kinases such as Bcr-Abl, c-kit, ErbB, and VEGFR are validated drug targets for cancer therapy.1 Less attention has been paid to the development of protein tyrosine phosphatase inhibitors as an alternative strategy to modulate key target protein phosphorylation states.2, 3 The protein tyrosine phosphatase (PTP) Shp2 is a non-receptor PTP that plays a positive role in cell signaling by growth factors and cytokines and is involved in regulation of cell proliferation, differentiation, and migration.4, 5 In particular, Shp2 mediates activation of Erk1 and Erk2 MAP kinases by receptor tyrosine kinases such as ErbB1, ErbB2, and c-Met that are involved in the pathogenesis of human carcinoma.6–11 Furthermore, gain-of-function Shp2 mutants are found in childhood hematological malignancies such as juvenile myelomonocytic leukemia, some cases of solid tumors, and are associated with ~50% cases of Noonan syndrome.12 It has also been reported that Shp2 is a key mediator of the oncogenic CagA protein of Helicobactor pylori, which causes gastric cancer.13, 14 These studies suggest Shp2 PTP is a molecular target for cancer therapy and prompted us to develop Shp2 inhibitors. Furthermore, the development of small molecule Shp2 inhibitors would allow us to investigate the role of Shp2 in normal and malignant processes.

The structure of the Shp2 phosphatase has been determined by X-ray crystallography at 2.0 Å resolution.15 Shp2 contains two Src homology-2 (SH2) domains (N-SH2, C-SH2) at its N-terminal region.4 In the absence of a tyrosine-phosphorylated binding partner, the N-SH2 domain blocks the catalytic domain until the upstream signaling effectors bind to Shp2.15 Consequently, Shp2 is basally inactive due to autoinhibition and inhibition of Shp2 by a small molecule is not predicted to affect resting cells, making it an attractive target for drug design.

Several compounds have been reported to non-selectively inhibit Shp2 (Figure 1). These include the potent PTP1B inhibitor 1,16 the bis(trifluorosulfonamide) 2 which also inhibits PTP1B17 and the tetrazole 3 (NAT6-297775)18 discovered from a screen of a natural product-like library. Recently, we found that 4 (NSC-87877)26, identified as a hit from the NCI Diversity set, is a potent Shp2 and Shp1 inhibitor.19 The development of a Shp2-specific inhibitor that does not cross-inhibit Shp1 is an important goal of the present study. Shp1 is mostly expressed in hematopoietic and epithelial cells and functions as a negative regulator of signaling pathways in lymphocytes.20, 21 The crystal structure of ligand-free Shp1 shows a similar arrangement of tandem SH2 domains that adopt a conformation blocking the PTP catalytic site.22 Shp1 and Shp2 share 60% overall sequence identity and approximately 75% similarity in their PTP domains.15,22 The Shp1 and Shp2 PTP domains are known to have different substrate specificity,23,24 indicating that the domains are not identical. Furthermore, the surface electrostatic potential of the catalytic cleft is much more positive in human Shp2 than in human Shp1.25 Although amino acid residues presented at the base of Shp1 and Shp2 PTP catalytic clefts are identical, all four sides of the catalytic cleft contain one or more residues that are different between Shp1 and Shp2. These differences suggest that the development of a Shp2-specific PTP inhibitor, whilst challenging, is a realistic endeavor.

Figure 1.

Figure 1

Open in a new tab

Known non-selective inhibitors of Shp2.

Inhibitor design and synthesis

We now report the development of another class of Shp2 inhibitors based on a hit from our initial screen of the NCI Diversity set. The oxindole 5 (NSC-117199)26 was found to be a hit with only moderate potency (IC50 47 μM). Nevertheless 5 was considered a reasonable hit since it can be easily modified for library synthesis. Compounds with an oxindole core have been studied by other groups as potential therapeutic agents.27, 28 Based on 5, we have developed PTP inhibitors that display selectivity for Shp2 over Shp1 inhibition. Our primary aim, in preparing the first generation library based on the oxindole 5, was to improve the Shp2 activity and Shp2/Shp1 selectivity. Inspection of 5 docked to Shp2 furnished a number of guidelines for analog design. The model was obtained by docking ligands to the Shp2 PTP domain (from pdb 2SHP15) using GLIDE,29 and methods we have described previously.19 The model reveals the hydrazone aromatic ring system is pointing into the active site PTP signature motif30 (VHCSAGIGRTG) with the polar nitro group mimicking the substrate phosphate group (Figures 2 and 3). The sulfonic acid group of the 5 is hydrogen bonded with the basic residues Arg362 and Lys366 (Figure 2A).

Figure 2.

Figure 2

Open in a new tab

Figure 2A. Model of 5 representing possible important structural features for activity. 2B. Structure 6 represents the oxindole phamacophore for new inhibitor design.

Figure 3.

Figure 3

Open in a new tab

Overlay of 5 (blue) and 14a (green) docked in the Shp2 PTP active site.

The ortho-nitro group exhibited hydrogen bonding interactions with backbone atoms of the PTP loop residues Ser460, Cys459 and Arg465. The oxindole 5 appeared to fit well in the catalytic site suggesting that the hydrazone unit should not be replaced by longer spacer groups (Figures 2 and 3). The model also suggested the sulfonic acid could be replaced with small polar groups e.g. sulfonamide and carboxylamide. Thus, our library was therefore biased to include small polar replacements of the nitro and sulfonic acid groups to interact with the two polar binding sites (Figure 2B). Other larger replacements (e.g. benzyl sulfonamides and benzylamides at the 5-position) were included to test to the model, and to see whether only a single polar group was required. These modifications were mostly at the 2 and 3 positions of the A-ring and the C5 of the indole ring, respectively. Inclusion of sulfonamide or carboxylamide groups at the 5-position, accessible from readily available precursors, adds a useful point of diversity into the oxindole pharmacophore. The hydrogen bonding interactions of the oxindole N-H group with Asp425 was thought to be optimal, and variation of N-H was limited to N-Me. The hydrazone linker was varied by inclusion of enamine analogs (X = CH).

Several libraries of hydrazones (10, 14 and 16) were prepared by combining a 5-substituted isatin with commercially available hydrazines; the sulfonyl and carboxyl groups were elaborated with a further set of amines to provide sulfonamides and amides. The novel oxindole hydrazone sulfonamide library 10 was developed using commercially available building blocks as shown in Scheme 1. The oxindolesulfonyl chloride 8 was obtained from commercially available isatin-5-sulfonic acid according to a literature reported procedure.31 Isatin-5-sulfonyl chloride was coupled to a series of requisite amines to obtain the sulfonamide library 9. We were able to isolate and analyze (NMR, Mass) some members of the isatin library 9. Attempts to purify and isolate other members of the library 9 was not successful. However use of the crude isatinsulfonamides 9 was successful. The hydrazone library 10 was obtained by microwave assisted coupling of the crude library 9 with an appropriate set of hydrazines in moderate yields.

Scheme 1.

Scheme 1

Open in a new tab

Reagents and conditions: a) POCl3, tetramethylenesulfone, Ar, 60 °C, 3 h; b) THF, DIPEA, RNH2, Ar, rt, overnight; c) EtOH, ArNHNH2, HCl (aq. 1 M, 2 drops) 80 °C, 4 h; d) EtOH, ArNHNH2, HCl (aq. 1M, 2 drops), μw, 120 °C, 15 min.

The carboxylic acid library 14 was prepared from methyl indole-5-carboxylate 11a (Scheme 2). Treatment of 11a with NBS provided the dibromooxindole intermediate32 12 in moderate yield. This was then converted into the desired library 14 in good yields by microwave assisted coupling with the requisite hydrazines. This one pot process occurs by hydrolysis of 12 to give the intermediate isatin 13. A series of carboxylamides 15 was prepared from the pentafluorophenyl ester33 of indole-5-carboxylic acid 11b (Scheme 2). These amides were then reacted with NBS to form the dibromooxindole32 intermediates which were subsequently reacted with the requisite hydrazines to obtain the final library 16 as shown in Scheme 2. The syntheses of the most potent compounds are highlighted in Schemes 1 and 2. The 5-unsubstituted oxindoles 18 were synthesized in high yield by microwave assisted coupling of isatin and the required hydrazines as shown in Scheme 2. The libraries 10, 14, and 16 were analyzed by NMR (1H and 13C), low and high resolution mass spectroscopy. The 1H NMR spectra of these compounds indicated formation a single stereoisomer with > 95% purity. We did not observe hydrolysis of the hydrazones in routine handling and analysis of the samples. The compounds are chemically and configurationally stable in DMSO for extended periods of time at room temperature as determined by NMR (e.g. 10c and 10h). Isatin hydrazones have been reported to exist in the Z configuration in solution, presumably due to the intramolecular hydrogen bonding between NH of the hydrazone linkage and the carbonyl group of the indolinone.33 The analysis of the 13C NMR spectra of the final compounds of the library 10 (10a–e, 10h, 10i, 10m, 10o, 10q) revealed oxindole carbonyl chemical shifts around 163 ppm, indicative of the Z-hydrazone stereochemical configuration. Similarly, 13C NMR analysis of the library 14 also revealed carbonyl shifts around 163 ppm indicating that the members are also configured Z. However, the reaction of isatin 17 with 2-hydrazinobenzoic acid afforded a mixture of isomers. The 1H NMR of the compound 18b indicated formation of a mixture of stereoisomers approximately 1:3 ratio. Upon modifying the reaction conditions (microwave heating, 120 °C, 2 min.), we see the exclusive formation (by 1H and 13C NMR) of the E-configured hydrazone that was previously the minor isomer (18c) in 48% yield. The assignment of stereochemistry was based on observation of an nOe between the hydrazone NH and H-4 of the isatin (see supporting information). For comparison, no such nOe was observed for isatin 5. So far we have not been able to produce the major isomer of the 18b as a single compound. The enamine analogs 6 (X = CH) were prepared using the methods described by Kuyper and co-workers.33 None of the enamine compounds were active (synthetic details will be presented elsewhere).

Scheme 2.

Scheme 2

Open in a new tab

Reagents and conditions: a) NBS, isopropanol:H2O (95:5), 0 °C, 45 min; b) i: HCl (aq. 4M, 1–3 mL), μw, 150 °C, 5 min, ii: HCl (aq. 4M, 1–3 mL), ArNHNH2, μw, 150 °C, 5 min; c) NaOH, EtOH:H2O (1:1), 7 96%; d) pentafluorophenyl trifluoroacetate, pyridine, DMF, inert conditions, room temp, 70%; e) RNH2, pyridine, acetonitrile, room temp; f) NBS, isopropanol:H2O (95:5), 0 °C, 30 min; g) dry MeOH, ArNHNH2, μw, 150 °C, 5 min (yield from 20–60%); h) HCl (aq. 1M, 2 drops) EtOH, μw, ArNHNH2, 120 °C, 15 min (for Z-18b irradiation time was only 2 min).

Results and Discussion

The libraries were evaluated for Shp2 PTP inhibition by in vitro PTP assay using DiFMUP as the substrate.34 The compounds that displayed low micromolar inhibitory activity against Shp2 are shown in Table 1. These compounds were further screened against Shp1 and PTP1B to determine their selectivity. It was found that the carboxylic acid, sulfonamides, and carboxylamides at the 5-position of the oxindole moiety and nitro or carboxylic acid functional groups at the ortho-, meta- or para-positions of the phenylhydrazone moiety gave rise to the best Shp2 PTP inhibitory activity. Compounds 10a–r from the sulfonamide library with either carboxylic acid or nitro (ortho, meta or para) groups on the aromatic hydrazine moiety showed good Shp2 inhibitory activities; IC50 = 1–10 μM with > 5 fold Shp2 over Shp1 selectivity. Compounds from the sulfonamide library 10 displayed better solubility under the assay conditions. The bis-carboxylic acid derivatives 14a and 14b displayed IC50 0.8 and 15 μM inhibitory activity, respectively, with 20 and 5 fold Shp2 selectivity. The compounds that lack the 5-position carboxylic acid, carboxyamides or sulfonamide groups (18a–e, Scheme 2) showed poor activity (IC50 > 60 μM, not shown in Table 1) indicating that the 5-substitution with polar groups (carboxylic acid, sulfonamide or carboxyamide) is important for activity and suggesting that the interactions in this region with Lys366 and Arg362 (Figure 3) are pivotal. Members of both libraries 10 and 14 that possessed hydrogen, halogens (chloro or fluoro), primary amide or alkyl groups on the aromatic hydrazine moiety showed poor Shp2 inhibitory activity (not reported here) further demonstrating the importance of a carboxylate moiety (phosphotyrosine mimic) at this position, and its critical role in interacting with Cys459 and Arg465. The carboxyl group when positioned at the 3 and 4 positions for the sulfonamide library generally leads to better activity. It is likely that a number of binding modes are possible since the shallow binding pocket contains multiple positively charged residues available for interaction with a carboxyl group (e.g. Lys364, Lys366, Arg 465, Arg362, Arg 465, His426, Lys280). Varying degrees of selectivity for Shp2 versus PTP1B phosphatase inhibition were observed. For example the potent compounds 14a and 10f show a 2-fold Shp2:PTP1B selectivity, whereas halosulfonamides 10g, 10h and 10m show a better 4 to15-fold difference.

Table 1.

Shp2 active compounds from the isatin library

graphic file with name nihms86088f6.jpg
Compound R1 R2 IC50 (μM)a
Shp2 Shp1 PTP1B
5 SO3H 2-NO2 46.8+10.2 68±17.1 96.7±29.9
14a CO2H 2-CO2H 0.8±0.2 15.4±2.1 1.5±0.6
14b CO2H 3-CO2H 15.8±6.6 72.5±19.3 38.2±13.9
16 CONHCH2(4-ClC6H4) 3-CO2H 22.3±5.2 19.4±1.8 >300
10a SO2NHiPr 3-CO2H 4.5±1.7 15.7±1.7 37.1±4.9
10b SO2NHiPr 4-CO2H 4.5±1.0 27.7±9.0 9.3±1.6
10c SO2NH2 2-NO2 11.9±1.5 103.7±17.4 156.6±14.1
10d SO2NHCH2(4-ClC6H4) 2-NO2 4.4±2.4 40.9±15.3 9.8±2.6
10e SO2NHCH2(4-MeC6H4) 3-CO2H 5.0±1.9 32.6±8.5 11.3±4.1
10f SO2NHCH2C6H5 4-CO2H 1.3±0.9 7.1±1.1 2.5±1.5
10g SO2NHCH2(3-ClC6H4) 3-CO2H 3.8±0.2 42.5±7.2 15.4±6.2
10h SO2NHCH2(4-ClC6H4) 3-CO2H 1.4±0.6 18.0±7.2 18.8±5.1
10i SO2NHCH2(4-ClC6H4) 4-CO2H 5.9±1.4 18.1±3.7 7.5±1.1
10j SO2NHCH2(2-ClC6H4) 4-CO2H 7.4±2.4 60.8±26.1 11.7±2.4
10k SO2NHCH2(2-ClC6H4) 3-CO2H 5.5±0.4 15.9±1.7 19.4±10.2
10l SO2NHCH2(3-CF3-4-Cl-C6H4) 2-CO2H 22.9±7.0 32.4±17.9 25.7±6.6
10m SO2NHCH2(4-FC6H4) 3-CO2H 1.0±0.2 18.3±5.2 14.5±1.5
10n SO2NHCH2(4-FC6H4) 4-CO2H 6.3±2.3 10.8±4.1 11.4±0.4
10o SO2NHCH2(3-Cl-4-F-C6H4) 3-CO2H 4.8±0.6 122.8±74.5 54.9±13.3
10p SO2NH(CH2)2(2-Cl-4-ClC6H3) 3-CO2H 5.2±1.7 73.4±36.9 19.3±13.8
10q SO2NHCH2(3-CF3-4-Cl-C6H4) 4-CO2H 8.3±1.7 43.1±7.2 32.8±8.0
10r SO2NHCH2(3-CF3-4-Cl-C6H4) 3-CO2H 10.6±1.5 74.2±8.0 63.1±24.7
Open in a new tab
a

Values are means and standard deviations of at least 4 experiments, each performed in duplicate.

The comparison of the docking modes of 5 and 14a is shown in Figure 3A. The 5-substituents of the oxindole ring are superimposed and in the model display favorable interactions with both Lys366 and Arg362 residues and most likely contribute towards the Shp2 affinity of the ligands. The lack of activity found for all five derivatives of 18 (IC50 > 60 μM, Scheme 2) which lack any 5-substituents is consistent with this observation.

The orientation of the hydrazine-aromatic ring positions the carboxylic acid moiety of 14a (Figure 3) so that it is capable of undergoing additional hydrogen bond interactions with Cys459, Gly464 and Ilu463 in the catalytic site. This site binds substrate phosphotyrosine residues, therefore it is not surprising that the carboxylate ion binds better than the nitro group. These interactions may explain the 40 fold increased potency of 14a compared to 5 (IC50 values of 47 μM vs 0.8 μM). The indole nitrogen atoms do not overlay well, but both show a hydrogen bonding interaction with Shp2 amino acid residues (Asp425 in the case of 5 and Glu361 with 14a). These observations are in agreement with our design strategy to include both a polar group and phosphate mimic as a model for Shp2 inhibition.

In summary, we have identified a series of isatins capable of selectively and potently inhibiting (one compound 14a with sub micromolar activity) Shp2 over Shp1 in vitro. Furthermore the p-halosulfonamides 10h and 10m also display significant Shp2:Shp1 and Shp2:PTP1B selectivity. The SAR developed herein provides the basis for further optimization of the isatin library, which will be reported in due course.

Experimental section

General

All reagents were purchased from commercial suppliers and used without further purification. Melting points were determined using a Barnstead international melting point apparatus and remain uncorrected. 1H NMR spectra were recorded on a Varian Mercury 400 MHz spectrometer with CDCl3 or DMSO-d6 as the solvent. 13C NMR spectra are recorded at 100 MHz. All coupling constants are measured in Hertz (Hz) and the chemical shifts (δH and δC) are quoted in parts per million (ppm) relative to TMS (δ 0), which was used as the internal standard. High resolution mass spectroscopy was carried out on an Agilent 6210 LC/MS (ESI-TOF). Microwave reactions were performed in CEM 908005 model and Biotage initiator 8 machines. HPLC analysis was performed using a JASCO HPLC system equipped with a PU-2089 Plus quaternary gradient pump and a UV-2075 Plus UV-VIS detector, using an Alltech Kromasil C-18 column (150 × 4.6 mm, 5 μm). Thin layer chromatography was performed using silica gel 60 F254 plates (Fisher), with observation under UV when necessary. Anhydrous solvents (acetonitrile, dimethyl formamide, ethanol, isopropanol, methanol and tetrahydrofuran) were used as purchased from Aldrich. HPLC grade solvents (methanol, acetontrile and water) were purchased from Burdick and Jackson for HPLC and mass analysis.

2,3-Dioxoindoline-5-sulfonyl chloride 8

This compound was prepared according to a procedure reported by Lee et al.31 Mp = 200–202 °C (lit. Mp = 188–190 °C)35; 1H NMR (400 MHz, CD3CN:CDCl3 1:1) δ 9.49 (br s, NH, 1H ), 8.24 (dd, J = 8.4, 2.0 Hz, 1H), 8.16 (d, J = 2.0 Hz, 1H), 7.22 (d, J = 8.4 Hz, 1H).

Synthesis of isatin library 9

The DIPEA (2.0 mmol) and appropriate amine (1.3 mmol) were added to a solution of 8 (1.0 mmol) in anhydrous THF (8 ml) at 0 °C under inert atmosphere. The reaction mixture was warmed to r.t. and stirred overnight (some reactions required 3 days for completion). The crude reaction mixture was poured into water (10 ml) and extracted with EtOAc (3 × 15 ml). The organic phase was dried (MgSO4) and evaporated to obtain a beige/yellow solid. This crude product was taken to next stage without purification in the majority of the cases (see general procedure for library 10), except compounds 9a, 9b, 9c, which were purified by trituration with cold ethyl acetate.

2,3-Dioxo-2,3-dihydro-1H-indole-5-sulfonamide (9a)

Yellow solid,36 47%. Mp = 198–200 °C, decomposed; 1H NMR (400 MHz, DMSO-d6) δ 7.02 (d, J = 8.2 Hz, 1H), 7.38 (s, 2H), 7.82 (d, J = 1.8 Hz, 1H), 7.95 (dd, J = 8.2, 1.8 Hz, 1H), 11.35 (s, 1H); 13C NMR (DMSO-d6) δ 112.90, 118.50, 122.44, 135.74, 139.13, 153.34, 160.26, 184.00; HRMS (ESI+ve) m/z calculated for C8H7N2O4S (M+H)+ 227.0127, found 227.0133.

2,3-Dioxo-2,3-dihydro-1H-indole-5-sulfonic acid isopropylamide (9b)

Yellow solid, 70%. Mp = 184–186 °C; 1H NMR (400 MHz, DMSO-d6) δ 0.94 (d, J = 6.8 Hz, 6H), 3.11 (sept, J = 6.8 Hz, 1H), 7.04 (d, J = 8.2 Hz, 1H), 7.58 (d, J = 6.8 Hz, 1H), 7.77 (d, J = 1.8 Hz, 1H), 7.94 (dd, J = 8.2, 1.8 Hz, 1H), 11.38 (s, 1H); 13C NMR (DMSO-d6) δ 23.90, 45.99, 113.15, 118.73, 123.00, 136.53, 136.65, 153.65, 160.20, 183.90; HRMS (ESI+ve) m/z calculated for C11H13N2O4S (M+H)+ 269.0596, found 269.0594.

2,3-Dioxo-2,3-dihydro-1H-indole-5-sulfonic acid 4-chlorobenzylamide (9c)

Yellow solid, 57%. Mp = 250 °C, decomposed; 1H NMR (400 MHz, DMSO-d6) δ 3.97 (d, J = 6.2 Hz, 2H), 7.00 (d, J = 8.1 Hz, 1H), 7.20 (d, J = 8.4 Hz, 2H), 7.29 (d, J = 8.4 Hz, 2H), 7.68 (d, J = 1.9 Hz, 1H), 7.89 (dd, J = 8.1, 1.9 Hz, 1H), 8.22 (t, J = 6.2 Hz, 1H), 11.41 (s, 1H); 13C NMR (DMSO-d6) δ 46.08, 113.10, 118.57, 123.24, 128.85, 130.20, 132.40, 135.52, 136.76, 137.12, 153.75, 160.15, 183.72; HRMS (ESI+ve) m/z calculated for C15H12ClN2O4S (M+H)+ 351.0206, found 351.0203.

The general procedure for synthesis of isatin library 10

Method A

A mixture of the crude intermediate from library 9 (100 mg) and hydrazinobenzoic acid (1 eq.) in ethanol (1 ml) with hydrochloric acid (2 drops 1M aq.) was heated in the Biotage microwave reactor at 120 °C for 15 min. A yellow solid precipitated on cooling the reaction vial in an ice bath. The solid obtained was filtered and washed with methanol to give the pure product 10. The yields for these 2 steps were in the range of 15–80%.

Method B

A mixture of the pure isatin 9a–c (0.419 mmol) and the appropriate hydrazine (0.461 mmol) in ethanol (3 ml) with hydrochloric acid (2 drops 1M aq.) was heated in the Biotage microwave reactor at 120 °C for 15 min. A yellow solid was precipitated on cooling in an ice bath. The solid obtained was filtered and washed with methanol to provide the pure final product.

(Z)-3-(2-(2-Nitrophenyl)hydrazono)-2-oxoindoline-5-sulfonic acid 5

Isatin-5-sulfonic acid (7) (400 mg, 1.72 mmol) and 2-nitrophenylhydrazine (296 mg, 1.65 mmol) were suspended in ethanol (10 ml), to which was added hydrochloric acid (aq. 4M, 4 drops). The mixture was heated under reflux for 12 h. The bright orange precipitate obtained was filtered and washed with ethanol to provide the required product 5 (0.597 g, 93%). Mp = 272 °C decomposed; 1H NMR (400 MHz, DMSO-d6) δ 14.21 (s, 1H, disappeared on D2O shake), 11.23 (s, 1H, disappeared on D2O shake), 8.24 (dd, J = 8.4, 0.8 Hz, 2H), 8.21 (dd, J = 8.4, 1.2 Hz, 1H), 7.84 (appd, J = 1.6 Hz, 1H), 7.79 (t, J = 7.6 Hz, 1H), 7.57 (dd, J = 8.0, 1.6 Hz, 1H), 7.19-7.14 (m, 1H), 6.87 (d, J = 8.0 Hz, 1H); 13C NMR (DMSO-d6) δ 110.61, 116.59, 118.11, 120.25, 122.31, 126.46, 128.58, 133.59, 133.86, 137.33, 140.02, 142.14, 143.31, 163.38; HPLC 100% (Rt = 1.38, 80% methanol in water); HRMS (ESI-ve) m/z calculated for C14H9N4O6S (M-H)− 361.0248, found 361.0250.

3-[N’-(5-Isopropylsulfamoyl-2-oxo-1,2-dihydro-indol-3-ylidene)hydrazino]-benzoic acid (10a)

By method B. Yellow solid, 57%. Mp = 275 °C decomposed; 1H NMR (400 MHz, DMSO-d6) δ 12.76 (s, 1H, disappeared on D2O shake), 11.43 (s, 1H, disappeared on D2O shake), 8.05–8.06 (m, 1H), 7.92 (d, J = 2.0 Hz, 1H), 7.67–7.70 (m, 3H), 7.47–7.51 (d, J = 7.6 Hz, 2H), 7.45 (d, J = 8.0 Hz, 1H), 7.05 (d, J = 8.4 Hz, 1H), 3.21 (sept, J = 6.8 Hz, 1H), 0.93 (d, J = 6.8 Hz, 6H)); 13C NMR (DMSO-d6) δ 23.88, 45.88, 111.18, 115.36, 117.44, 119.75, 122.20, 124.74, 127.90, 127.97, 130.43, 132.79, 135.89, 143.24, 143.30, 163.76, 167.72; HPLC 99% (Rt = 5.20, 75% methanol in water); HRMS (ESI-ve) m/z calculated for C18H17N4O5S (M-H)− 401.0925, found 401.0929.

4-[N’-(5-Isopropylsulfamoyl-2-oxo-1,2-dihydro-indol-3-ylidene)hydrazino]benzoic acid (10b)

By method B. Yellow solid, 75%. Mp = 290 °C decomposed; 1H NMR (400 MHz, DMSO-d6) δ 12.77 (s, 1H, disappeared on D2O shake), 12.74 (bs, 1H, disappeared on D2O shake), 11.47 (s, 1H, disappeared on D2O shake), 7.95 (m, 1H), 7.94 (d, J = 8.0 Hz, 2H), 7.70 (d, J = 8.0 Hz, 1H), 7.56 (1H, d, J = 8.0 Hz, 2H), 7.48 (d, J = 7.2 Hz, 1H), 7.07 (d, J = 8.0 Hz, 1H), 0.93 (d, J = 6.0 Hz, 6H); 13C NMR (DMSO-d6) δ 23.91, 45.90, 111.35, 114.77, 117.83, 121.95, 125.72, 128.35, 129.08, 131.78, 136.02, 143.59, 146.57, 163.70, 167.56; HPLC 99.9 % (Rt = 1.20, 90% methanol in acetonitrile); HRMS (ESI-ve) m/z calculated for C18H17N4O5S 401.0925 (M-H)−, found 401.0932.

3-[(2-Nitrophenyl)hydrazono]-2-oxo-2,3-dihydro-1H-indole-5-sulfonamide (10c)

By method B. Yellow solid, 58%. Mp > 300 °C; 1H NMR (400 MHz, DMSO-d6) δ 14.24 (s, 1H, disappeared on D2O shake), 11.53 (s, 1H, disappeared on D2O shake), 8.21–8.24 (m, 1H), 8.07 (d, J = 1.6 Hz, 1H), 7.81–785 (m, 1H), 7.77 (dd, J = 8.0, 2.0 Hz, 1H), 7.30 (s, 2H), 7.19–7.24 (m, 1H), 7.03 (d, J = 8.4 Hz, 1H); 13C NMR (DMSO-d6) δ 111.27, 116.44, 118.04, 121.18, 122.78, 126.56, 128.56, 132.74, 133.87, 137.31, 138.72, 139.63, 144.25, 163.26; HPLC 99% (Rt = 2.28, 90% acetonitrile in water); HRMS (ESI-ve) m/z calculated for C14H10N5O5S (M-H)− 360.0408, found 360.0412.

3-[(2-Nitrophenyl)hydrazono]-2-oxo-2,3-dihydro-1H-indole-5-sulfonic acid 4-chlorobenzylamide (10d)

By method B. Yellow solid, 43%. Mp > 300 °C; 1H NMR (400 MHz, DMSO-d6) δ 14.23 (s, 1H), 11.56 (s, 1H), 8.23–8.28 (m, 2H), 8.13 (t, J = 6.4 Hz, 1H), 7.90 (d, J 1.6 Hz, 1H), 7.82 (t, J = 8.4 Hz, 1H), 7.70 (dd, J = 8.2, 1.8 Hz, 1H), 7.23–7.27 (m, 5H), 7.06 (d, J = 8.0 Hz, 1H), 3.98 (d, J = 6.4 Hz, 2H); 13C NMR (DMSO-d6) δ 46.14, 111.62, 116.69, 118.94, 121.39, 122.89, 126.58, 128.78, 129.59, 130.18, 132.38, 132.58, 133.95, 135.11, 137.32, 137.34, 139.69, 144.73, 163.26; HPLC 99% (Rt = 2.55, 90% acetonitrile in water); HRMS (ESI+ve) m/z calculated for C21H20ClN6O5S (M+NH4)+ 503.0904, found 503.0909; calculated for C21H17ClN5O5S (M+H)+ 486.0639, found 486.0643.

(Z)-3-(2-(5-(N-(4-Methylbenzyl)sulfamoyl)-2-oxoindolin-3-ylidene)hydrazinyl)benzoic acid (10e)

By method A. Yellow solid, 27%. Mp = 297 °C decomposed; 1H NMR (400 MHz, DMSO-d6) δ 12.77 (s, 1H disappeared on D2O shake), 11.43 (s, 1H disappeared on D2O shake), 8.07 (s, 1H), 8.02 (t, J = 6.4 Hz, NH, 1H disappeared on D2O shake), 7.84 (s, 1H), 7.72-7.62 (m, 3H), 7.49 (t, J = 8.0 Hz, 1H), 7.09 (d, J = 8.4 Hz, 2H), 7.05-7.02 (m, 3H), 5.75 (s, 1H disappeared on D2O shake), 3.90 (d, J = 6.0 Hz, 2H, CH2, singlet on D2O shake), 2.19 (s, CH3, 3H); HPLC 92% (Rt = 1.57, 90% methanol in acetonitrile); HRMS (ESI-ve) m/z calculated for C23H19N4O5S (M-H)− 463.1082, found 463.1087.

(Z)-4-(2-(5-(N-Benzylsulfamoyl)-2-oxoindolin-3-ylidene)hydrazinyl)benzoic acid (10f)

By method A. Yellow solid, 79%. Mp > 300 °C; 1H NMR (400 MHz, DMSO-d6) δ 12.78 (s, 1H disappeared on D2O shake), 11.48 (s, 1H disappeared on D2O shake), 8.06 (t, J = 8.0 Hz, NH, 1H disappeared on D2O shake), 7.95 (d, J = 8.0 Hz, 2H), 7.93 (s, 1H), 7.70 (dd, J = 8.0, 2.0 Hz, 1H), 7.57 (d, J = 8.0 Hz, 2H), 7.29-7.19 (m, 5 H), 7.06 (d, J = 8.0 Hz, 1H), 5.70 (s, 1H disappeared on D2O shake), 3.96 (d, J = 8.0 Hz, 2H, CH2, singlet on D2O shake); 13C NMR (DMSO-d6) δ 46.84, 111.33, 114.78, 118.01, 121.99, 125.73, 127.77, 128.28, 128.33, 128.54, 128.88, 131.77, 134.81, 138.28, 143.71, 146.57, 163.70, 167.56; HPLC 99% (Rt = 1.15, 90% methanol in acetonitrile); HRMS (ESI-ve) m/z calculated for C22H17N4O5S (M-H)− 449.0925, found 449.0940.

(Z)-3-(2-(5-(N-(3-Chlorobenzyl)sulfamoyl)-2-oxoindolin-3-ylidene)hydrazinyl)benzoic acid (10g)

By method A. Yellow solid, 40%. Mp = 295 °C, decomposed; 1H NMR (400 MHz, DMSO-d6) δ 12.77 (s, 1H disappeared on D2O shake), 11.44 (s, 1 H disappeared on D2O shake), 8.17 (t, J = 6.4 Hz, NH, 1H disappeared on D2O shake), 8.06 (s, 1H), 7.85 (d, J = 1.6 Hz, 1H), 7.70 (d, J = 6.4 Hz, 1H), 7.63 (d, J = 8.0 Hz, 2H), 7.49 (t, J = 8.0 Hz, 1H), 7.28-7.17 (m, 4 H), 7.01 (d, J = 8.4 Hz, 1H), 5.75 (s, 1H), 4.00 (d, J = 6.4 Hz, 2H, CH2, singlet on D2O shake); HPLC 99% (Rt = 1.63, 90% methanol in acetonitrile); HRMS (ESI-ve) m/z calculated for C22H16ClN4O5S (M-H)− 483.0535, found 483.0550.

(Z)-3-(2-(5-(N-(4-Chlorobenzyl)sulfamoyl)-2-oxoindolin-3-ylidene)hydrazinyl)benzoic acid (10h)

By method A. Yellow solid, 57%. Mp > 300 °C; 1H NMR (400 MHz, DMSO-d6) δ 12.77 (s, 1H disappeared on D2O shake), 11.43 (s, 1 H disappeared on D2O shake), 8.14 (t, J = 6.4 Hz, NH, 1H disappeared on D2O shake), 8.06 (s, 1H), 7.82 (s, 1H), 7.71 (d, J = 8.4 Hz, 1H), 7.64 (t, J = 7.2 Hz, 2H), 7.49 (t, J = 8.0 Hz, 1H), 7.25 (dd, J = 19.2, 8.4 Hz, 4 H), 7.03 (d, J = 8.4 Hz, 1H), 5.75 (s, 1H disappeared on D2O shake), 3.96 (d, J = 6.0 Hz, 2H CH2, singlet on D2O shake); 13C NMR (DMSO-d6) δ 46.12, 111.12, 115.46, 117.59, 119.77, 122.19, 124.76, 127.77, 128.14, 128.75, 130.45, 130.46, 132.65, 132.82, 134.68, 137.36, 143.36, 143.77, 163.74, 167.72; HPLC 98.9 % (Rt = 1.18, 90% methanol in acetonitrile); HRMS (ESI-ve) m/z calculated for C22H16N4O5SCl (M-H)− 483.0535, found 483.0536.

(Z)-4-(2-(5-(N-(4-Chlorobenzyl)sulfamoyl)-2-oxoindolin-3-ylidene)hydrazinyl)benzoic acid (10i)

By method A. Yellow solid, 55%. Mp > 300 °C; 1H NMR (400 MHz, DMSO-d6) δ 12.78 (s, 1H disappeared on D2O shake), 11.47 (s, 1H disappeared on D2O shake), 8.12 (t, J = 6.4 Hz, NH, 1H disappeared on D2O shake), 7.94 (d, J = 8.0 Hz, 2H,), 7.84 (s, 1H), 7.66 (d, J = 8.0 Hz, 1H), 7.57 (d, J = 8.0 Hz, 2H,), 7.25 (dd, J = 17.6, 8.4 Hz, 4H), 7.04 (d, J = 7.6 Hz, 1H), 5.75 (s, 1H disappeared on D2O shake), 3.97 (d, J = 6.4 Hz, 2H, CH2, singlet on D2O shake); 13C NMR (DMSO-d6) δ 46.12, 11.29, 114.79, 117.96, 121.94, 125.73, 128.76, 128.92, 130.15, 131.79, 131.82, 132.34, 134.82, 137.34, 143.71, 146.61, 163.68, 167.57; HRMS (ESI-ve) m/z calculated for C22H16ClN4O5S (M-H)− 483.0535, found 483.0540.

(Z)-4-(2-(5-(N-(2-Chlorobenzyl)sulfamoyl)-2-oxoindolin-3-ylidene)hydrazinyl)benzoic acid (10j)

By method A. Yellow solid, 28%. Mp > 300 °C; 1H NMR (400 MHz, DMSO-d6) δ 12.77 (s, 1H, disappeared on D2O shake), 11.47 (s, 1H, disappeared on D2O shake), 8.14 (t, J = 5.6 Hz, NH, 1H disappeared on D2O shake), 7.94 (d, J = 8.8 Hz, 2H), 7.92 (s, 1H,), 7.69 (dd, J = 8.4, 1.6 Hz, 1H), 7.56 (d, J = 8.8 Hz, 2H), 7.42 (d, J = 7.6 Hz, 1H), 7.34 (dd, J = 7.6, 1.6 Hz, 1H), 7.29-7.21 (m, 2H), 7.05 (d, J = 8.0 Hz, 1H), 4.04 (d, J = 5.6 Hz, 2H, CH2, singlet on D2O shake); HRMS (ESI-ve) m/z calculated for C22H16ClN4O5S (M-H)− 483.0535, found 483.0538.

(Z)-3-(2-(5-(N-(2-Chlorobenzyl)sulfamoyl)-2-oxoindolin-3-ylidene)hydrazinyl) benzoic acid (10k)

By method A. Yellow solid, 35%. Mp > 300 °C; 1H NMR (400 MHz, DMSO-d6) δ 12.77 (s, 1H, disappeared on D2O shake), 11.44 (s, 1H, disappeared on D2O shake), 8.16 (t, J = 5.6 Hz, NH, 1H disappeared on D2O shake), 8.07 (s, 1H,), 7.90 (d, J = 1.2 Hz, 1H,), 7.69 (dt, J = 8.4, 2.0 Hz, 2H), 7.63 (d, J = 8.0 Hz, 1H), 7.49 (t, J = 8.0 Hz, 1H), 7.38 (dd, J = 7.6, 2.0 Hz, 1H), 7.33 (dd, J = 7.2, 1.2 Hz, 1H), 7.29-7.21 (m, 2H), 7.05 (d, J = 8.0 Hz, 1H), 5.75 (s, 1H, disappeared on D2O shake), 3.96 (d, J = 5.6 Hz, 2H, CH2, singlet on D2O shake); HRMS (ESI-ve) m/z calculated for C22H16ClN4O5S (M-H)− 483.0535, found 483.0541.

(Z)-2-(2-(5-(N-(4-Chloro-3-(trifluoromethyl)benzyl)sulfamoyl)-2-oxoindolin-3-ylidene)hydrazinyl)benzoic acid (10l)

By method A. Yellow solid, 35%. Mp > 300 °C; 1H NMR (400 MHz, DMSO-d6) δ 14.25 (s, 1H disappeared on D2O shake), 11.30 (s, 1H, disappeared on D2O shake), 8.24 (t, J = 6.4 Hz, NH, 1H disappeared on D2O shake), 8.04 (d, J = 8.4 Hz, 1H), 7.95 (dd, J = 8.0, 1.6 Hz, 1H), 7.74 (d, J = 1.6 Hz, 1H), 7.66, (t, J = 7.6 Hz, 1H,), 7.60-7.52 (m, 4H), 7.12 (t, J = 7.6 Hz, 1H), 6.95 (d, J = 8.0 H, 1H), 4.14 (d, J = 6.4 Hz, 2H, CH2, singlet on D2O shake); HRMS (ESI-ve) m/z calculated for C23H15ClF3N4O5S (M-H)− 551.0409, found 551.0414.

(Z)-3-(2-(5-(N-(4-Fluorobenzyl)sulfamoyl)-2-oxoindolin-3-ylidene)hydrazinyl)benzoic acid (10m)

By method A. Yellow solid, 40%. Mp > 300 °C; 1H NMR (400 MHz, DMSO-d6) δ 12.76 (s, 1H disappeared on D2O shake), 11.43 (s, 1H disappeared on D2O shake), 8.10 (t, J = 6.4 Hz, NH, 1H disappeared on D2O shake), 8.06 (s, 1H), 7.86 (s, 1H), 7.70 (d, J = 7.6 Hz, 1H), 7.64 (t, J = 8.8 Hz, 2H), 7.49 (t, J = 7.6 Hz, 1H), 7.25 (dd, J = 8.0, 5.6 Hz, 2H), 7.08-7.02 (m, 3H), 3.94 (d, J = 6.4 Hz, 2H, CH2, singlet on D2O shake); 13C NMR (DMSO-d6) δ 46.07, 111.12, 115.42, 115.55 (d, J = 21 Hz), 117.56, 119.78, 122.20, 124.76, 127.75, 128.16, 130.29 (d, J = 8 Hz), 130.45, 132.79, 134.49 (d, J = 3 Hz), 134.64, 143.31, 143.35, 161.97 (d, J = 241 Hz), 163.72, 167.72; HPLC 99% (Rt = 1.45, 90% methanol in acetonitrile); HRMS (ESI-ve) m/z calculated for C22H16FN4O5S (M-H)− 467.0831, found 467.0844.

(Z)-4-(2-(5-(N-(4-Fluorobenzyl)sulfamoyl)-2-oxoindolin-3-ylidene)hydrazinyl)benzoic acid (10n)

By method A. Yellow solid, 41%. Mp > 300 °C; 1H NMR (400 MHz, DMSO-d6) δ 12.77 (s, 1H disappeared on D2O shake), 11.46 (s, 1 H disappeared on D2O shake), 8.07 (t, J = 6.4 Hz, NH, 1H disappeared on D2O shake), 7.93 (d, J = 8.8 Hz, 2H), 7.88 (s, 1H), 7.67 (dd, J = 8.4, 1.2 Hz, 1H), 7.56 (d, J = 8.4 Hz, 2H), 7.27-7.23 (m, 2H), 7.08-7.03 (m, 3H), 3.95 (d, J = 6.4 Hz, 2H, CH2, singlet on D2O shake); HPLC 96% (Rt = 1.43, 90% methanol in acetonitrile); HRMS (ESI-ve) m/z calculated for C22H16FN4O5S (M-H)− 467.0831, found 467.0846.

(Z)-3-(2-(5-(N-(3-Chloro-4-fluorobenzyl)sulfamoyl)-2-oxoindolin-3-ylidene)hydrazinyl)benzoic acid (10o)

By method B. Yellow solid, 87%. Mp = 293–295 °C; 1H NMR (400 MHz, DMSO-d6) δ 12.76 (s, 1H disappeared on D2O shake), 11.43 (s, 1 H, disappeared on D2O shake), 8.17 (t, J = 5.6 Hz, NH, 1H disappeared on D2O shake), 8.01 (s, 1H), 7.79 (s, 1H), 7.70 (d, J = 6.8 Hz, 1H), 7.64-7.58 (m, 2H), 7.49 (t, J = 7.6 Hz, 1H), 7.30 (d, J = 8.0 Hz, 1H), 7.24-7.22 (m, 2H), 6.99 (d, J = 8.8 Hz, 1H), 3.99 (d, J = 5.6 Hz, 2H, CH2, singlet on D2O shake); 13C NMR (DMSO-d6) δ 45.59, 111.06, 115.44, 117.03, 117.24, 117.51, 119.61, 119.79, 122.17, 124.77, 127.68, 128.11, 128.98 (d, J = 7 Hz), 130.35 (d, J = 15 Hz), 132.79, 134.72, 136.15 (d, J = 3 Hz), 143.34 (d, J = 6 Hz), 156.88 (d, J =244 Hz), 163.70, 167.72; HRMS (ESI-ve) m/z calculated for C22H15ClFN4O5S (M-H)− 501.0441, found 501.0437.

(Z)-3-(2-(5-(N-(2,4-Dichlorophenethyl)sulfamoyl)-2-oxoindolin-3-ylidene)hydrazinyl)benzoic acid (10p)

By method A. Yellow solid, 14%. Mp = 290–292 °C; 1H NMR (400 MHz, DMSO-d6) δ 12.77 (s, 1H disappeared on D2O shake), 11.43 (s, 1H disappeared on D2O shake), 8.07 (s, 1H), 7.82 (s, 1H), 7.71 (d, J = 6.4 Hz, 2H), 7.64-7.61 (m, 2H, integrated to1H on D2O shake), 7.50 (t, J = 8.4 Hz, 1H), 7.44 (d, J = 1.6 Hz 1H), 7.31-7.25 (m, 2H), 7.03 (d, J = 8.4 Hz, 1H), 5.75 (s, 1H disappeared on D2O shake), 2.99 (q, J = 6.4 Hz, 2H, CH2, triplet on D2O shake), 2.76 (t, J = 6.4 Hz, 2H, CH2); HRMS (ESI-ve) m/z calculated for C23H17Cl2N4O5S (M-H)− 531.0302, found 531.0304.

(Z)-4-(2-(5-(N-(4-Chloro-3-(trifluoromethyl)benzyl)sulfamoyl)-2-oxoindolin-3-ylidene)hydrazinyl)benzoic acid (10q)

By method A. Yellow solid, 34%. Mp > 300 °C; 1H NMR (400 MHz, DMSO-d6) δ 12.76 (s, 1H disappeared on D2O shake), 11.44 (s, 1 H disappeared on D2O shake), 8.25 (t, J = 6.4 Hz, NH, 1H disappeared on D2O shake), 7.94 (d, J = 8.8 Hz, 2H), 7.70 (s, 1H), 7.58-7.52 (m, 6H), 6.95 (d, J = 8.4 Hz, 1H), 5.75 (s, 1H disappeared on D2O shake), 4.13 (d, J = 6.4 Hz, 2H, CH2, singlet on D2O shake); 13C NMR (DMSO-d6) δ 45.72, 111.15, 114.76, 117.90, 121.79, 125.73, 126.04 (q, J = 272 Hz), 126.64, 126.94, 127.46 (d, J = 5 Hz), 128.37, 128.72, 131.76, 131.93, 134.10, 134.93, 138.27, 143.68, 146.59, 163.64, 167.58; HRMS (ESI-ve) m/z calculated for C23H15ClF3N4O5S (M-H)− 551.0409, found 551.0404.

(Z)-3-(2-(5-(N-(4-Chloro-3-(trifluoromethyl)benzyl)sulfamoyl)-2-oxoindolin-3-ylidene)hydrazinyl)benzoic acid (10r)

By method A. Yellow solid, 40%. Mp = 292–294 °C; 1H NMR (400 MHz, DMSO-d6) δ 12.75 (s, 1H disappeared on D2O shake), 11.41 (s, 1 H disappeared on D2O shake), 8.28 (t, J = 6.4 Hz, NH, 1H disappeared on D2O shake), 8.05 (s, 1H), 7.70-7.68 (m, 2H), 7.63 (d, J = 7.6 Hz, 1H), 7.57-7.47 (m, 5H), 6.95 (d, J = 8.0 Hz, 1H), 4.12 (d, J = 6.4 Hz, 2H, CH2, singlet on D2O shake); HRMS (ESI-ve) m/z calculated for C23H15ClF3N4O5S (M-H)− 551.0409, found 551.0420.

3,3-Dibromo-2-oxo-2,3-dihydro-1H-indole-5-carboxylic acid methyl ester (12)

N-Bromosuccinimide (13.41 g, 74.91 mmol) was added portion wise to a solution of 5-methyl indole-2-carboxylate (11a) (4.50 g, 25.71 mmol) in isopropanol:H2O (95:5, 350 ml) over 45 minutes under argon at room temperature. After the addition, the solvent was removed under reduced pressure and the solid residue was triturated with cold acetone (150 ml) to give the pure product as a yellow solid (4.90 g, 55%). Mp > 300 °C; 1H NMR (400 MHz, DMSO-d6) δ 11.71 (s, 1H), 8.05 (d, J = 1.5 Hz, 1H), 7.98 (dd, J = 8.2, 1.5 Hz, 1H), 7.06 (d, J = 8.2 Hz, 1H), 3.86 (s, 3H); 13C NMR (DMSO-d6) δ 52.92, 111.96, 125.37, 126.83, 132.08, 134.15, 143.28 (2C), 165.88, 171.27.

(Z)-3-(2-(2-Carboxyphenyl)hydrazono)-2-oxoindoline-5-carboxylic acid (14a)

Methyl dibromo-oxindole carboxylate 9 (40 mg, 0.114 mmol) was suspended in HCl (aq. 4 M, 2.00 ml) in a microwave vial and heated at 150 °C for 5 min. The intermediate 13 was not isolated (Scheme 2). 2-Hydrazinylbenzoic acid (23 mg, 0.126 mmol) was added to the reaction mixture and heated using the Biotage microwave reactor at 150 °C for 15 min. The solid obtained upon cooling the reaction vial was filtered and washed with DCM to obtain the pure 14a (15 mg, 40%) as an off white solid. Mp > 300 °C; 1H NMR (400 MHz, DMSO-d6) δ 14.22 (s, 1H, disappeared on D2O shake), 11.27 (s, 1H, disappeared on D2O shake), 8.11 (appd, J = 1.2 Hz, 1H), 8.07 (d, J = 8.4 Hz, 1H), 7.93 (dd, J = 8.0, 1.2 Hz), 7.87 (dd, J = 8.0, 1.2 Hz, 1H); 7.63 (t, J = 8.4 Hz, 1H), 7.09 (t, J = 7.2 Hz, 1H), 6.99 (d, J = 8.4 Hz, 1H); 13C NMR (DMSO-d6) δ 110.94, 114.55, 114.88, 120.71, 121.93, 122.48, 124.98, 129.82, 131.53, 131.97, 135.21, 144.74, 145.26, 163.04, 167.82, 168.91; HPLC 100% (Rt = 1.55, 80% methanol in water); HRMS (ESI-ve) m/z calculated for C16H10N3O5 (M-H)− 324.0625, found 324.0622.

(Z)-3-(2-(3-Carboxyphenyl)hydrazono)-2-oxoindoline-5-carboxylic acid (14b)

The procedure was same as for the compound 14a. 3-Hydrazinylbenzoic acid (23 mg, 0.126 mmol) was used to obtain the final compound 14b (20 mg, 54%) as an off white solid. Mp > 300 °C; 1H NMR (400 MHz, DMSO-d6) δ 12.97 (broad s, 1H, disappeared on D2O shake), 12.70 (s, 1H, disappeared on D2O shake), 11.37 (s, 1H, disappeared on D2O shake), 8.06 (s, 1H), 8.00 (s, 1H) 7.86 (dd, J = 8.0, 1.2 Hz, 1H), 7.72 (d, J = 8.0 Hz, 1H), 7.60 (d, J = 7.6 Hz, 1H), 7.49-7.45 (m, 1H), 7.00 (d, J = 8.4 Hz, 1H); HPLC 100% (Rt = 1.60, 80% methanol in water); HRMS (ESI-ve) m/z calculated for C16H10N3O5 (M-H)− 325.0625, found 324.0635.

Pentafluorophenyl 1H-indole-5-carboxylate; intermediate for 15

To a solution of 5-indolecarboxylic acid (0.5 g, 3.10 mmol) in DMF (3.00 ml) was added pentafluorophenyl trifluoroacetate (1.068 ml, 6.20 mmol) followed by pyridine (0.281 ml). The reaction mixture (a suspension was obtained at this stage) was stirred at room temperature under inert atmosphere for approximately 30 minutes. The reaction mixture was poured into ether (40 ml) and diluted with ethyl acetate (2 × 50 ml). The organic phase was washed with water, dried (Na2SO4) and concentrated to obtain an off white solid (720 mg, 70%), t.l.c. Rf = 0.71 (EtOAc: hexane, 1:1). No purification was necessary: 1H NMR (400 MHz, DMSO-d6) δ 11.71 (s, 1H, NH), 8.49 (s, 1H), 7.85 (dd, J = 8.4, 2.0 Hz, 1H), 7.59-7.55 (m, 2H), 6.67 (broad t, 1H).

N-(4-Chlorobenzyl)-1H-indole-5-carboxamide (15)

The pentafluorophenyl ester (200 mg, 0.61 mmol) from the above experiment was suspended in dry acetonitrile under argon, pyridine (0.075 ml, 0.85 mmol) was added followed by 4-chlorobenzylamine (121 mg, 0.85 mmol) and stirred overnight (approximately 12 h). The resulting cloudy solution was diluted with EtOAc and washed with HCl (4M aq., 6 ml). The organic phase was separated, washed with water, dried (Na2SO4), and concentrated to give the intermediate 15 (252 mg) as an orange solid. This compound was used in the next step without further purification. 1H NMR (400 MHz, DMSO-d6) δ 11.40 (s, 1H, disappeared on D2O shake), 11.32 (s, 1H), 8.91 (t, J = 6.4 Hz, 1H, changed on D2O shake), 8.15 (s, 1H), 7.64 (dd, J = 8.4, 1.2 Hz, 1H) 7.40-7.31 (m, 5H) 6.51 (m, 1H), 4.45 (d, J = 6.0 Hz, 2H, singlet on D2O shake).

3,3-Dibromo-N-(4-chlorobenzyl)-2-oxoindoline-5-carboxamide, Intermediate for 16

The indole 15 (252 mg, 0.89 mmol) was dissolved in aqueous isopropanol:H2O (95:5, 5 ml), and NBS (0.471 g, 2.65 mmol) was added portion wise over 30 min. with stirring under argon atmosphere. The reaction was monitored by t.l.c (EtOAc: hexane, 1:1). T.L.C. indicated the disappearance of the starting material. The reaction mixture was concentrated at room temperature and diluted with ether (approximately 10 ml). The succinimide precipitate was filtered and washed with ether. The ether phase was concentrated to obtain a pale yellow solid (225 mg, 55%). Attempts to purify this intermediate were not successful. The crude compound was used in the next step.

(Z)-3-(2-(5-(4-Chlorobenzylcarbamoyl)-2-oxoindolin-3-ylidene)hydrazinyl)benzoic acid (16)

The crude dibromoisatinamide intermediate from the above experiment (70 mg, 0.152 mmol) was suspended in dry MeOH (2.0 ml) in a microwave vial (CEM, 10.0 ml), and 3-hydrazinylbenzoic acid (31 mg, 0.166 mmol) was added and irradiated for 5 minutes at 150 °C in the CEM microwave reactor. The reaction vial was left in an ice bath until a precipitate formed. The compound 16 (15 mg, 22%) was isolated as a pale yellow solid. Mp > 300 °C; 1H NMR (400 MHz, DMSO-d6) δ 13.10 (broad s, 1H, disappeared on D2O shake), 12.79 (s, 1H, disappeared on D2O shake), 11.31 (s, 1H, disappeared on D2O shake), 9.13 (broad t, 1H), 8.12 (s, 1H), 8.04 (s, 1H), 7.84 (d, J = 8.4 Hz, 1H), 7.67 (d, J = 6.8 Hz, 1H), 7.61 (d, J = 7.6 Hz, 1H), 7.48 (t, J = 6.8 Hz, 1H), 7.37-7.33 (m, 4H), 6.99 (d, J = 8.0 Hz, 1H), 4.45 (d, J = 5.2 Hz, 2H,); HPLC 95% (Rt = 1.70, 80% methanol in water); LRMS (ESI+ve) m/z 449 [100, (M+H+)]; HRMS (ESI-ve) m/z calculated for C23H17ClN4O4 (M-H)− 447.0860, found 447.0861.

(Z)-3-(2-(2-Oxoindolin-3-ylidene)hydrazinyl)benzoic acid (18a)

A mixture of isatin (0.111 g, 0.775 mmol) and 3-carboxylphenylhydrazine (0.085 g, 0.160 mmol) and HCl (aq. 1 M, 2 drops) in ethanol (3 mL) was heated in the Biotage microwave at 120 °C for 15 min. After cooling to room temperature, pure product 18a was collected as a yellow precipitate by filtration and dried in vacuo (0.184 g, 0.65 mmol, 84%). Yellow solid, 84%. Mp > 300 °C; 1H NMR (400 MHz, DMSO-d6) δ 13.05 (broad s, 1H), 12.77 (s, 1H), 11.04 (s, 1H), 9.91 (d, J = 8.0 Hz, 1H), 7.96 (s, 1H), 7.54–7.64 (m, 3H), 7.44 (t, J = 8.0 Hz, 1H), 7.24 (t, J = 7.2 Hz, 1H), 7.04 (t, J = 8.0 Hz, 1H); 13C NMR (100 MHz, DMSO-d6) δ 111.23, 115.06, 119.16, 119.54, 121.66, 122.62, 124.12, 129.24, 129.52, 130.39, 132.74, 140.77, 143.57, 163.75, 167.75; HRMS (ESI-ve) m/z calculated for C15H10N3O3 (M-H)− 280.0727, found 280.0736.

(E) and (Z)-2-(2-(2-Oxoindolin-3-ylidene)hydrazinyl)benzoic acid (18b)

A mixture of isatin (17)(0.074 g, 0.517 mmol) and 2-carboxylphenylhydrazine (0.107 g, 0.568 mmol) and HCl (aq. 1 M, 2 drops) in ethanol (3 mL) was heated in the Biotage microwave reactor at 120 °C for 15 min. After cooling to room temperature hydrazone 18b (0.057 g, 0.20 mmol, 72%) was collected as a mixture of isomers (Z:E 3:1) as a yellow precipitate by filtration and dried in vacuo. 1H NMR (400 MHz, DMSO-d6) δ 14.19 (s, 1H), 10.92 (s, 1H), 8.01 (d, J = 8.0 Hz, 1H), 7.92 (d, J = 7.6 Hz, 1H), 7.58–7.69 (m, 2H, overlap with the minor isomer), 7.25 (t, J = 7.6 Hz, 1H), 7.21-7.11 (m, 2H, overlap with the minor isomer), 6.90 (d, J = 6.8 Hz, 1H); Minor isomer (E) (25%): 12.39 (s, 1H), 10.72 (s, 1H), 8.04 (d, J = 8.0 Hz, 1H), 7.97 (dd, J = 8.0 Hz, 1H), 7.85 (d, J = 8.4 Hz, 1H), 7.58–7.69 (m, 1H, overlap with the major isomer), 7.37 (t, J = 7.2 Hz, 1H), 7.21-7.11 (m, 2H, overlap with the major isomer), 6.94 (d, J = 7.2 Hz, 1H); HRMS (ESI-ve) m/z calculated for C15H10N3O3 (M-H)− 280.0727, found 280.0732.

(E)-2-(2-(2-Oxoindolin-3-ylidene)hydrazinyl)benzoic acid (18b)

A mixture of isatin (17)(0.060 g, 0.419 mmol) and 2-carboxylphenylhydrazine (0.085 g, 0.461 mmol) and HCl (aq. 1 M, 2 drops) in ethanol (3 mL) was heated in the Biotage microwave reactor at 120 °C for 2 min. After cooling to room temperature, pure product 18b was collected as a yellow precipitate by filtration and dried in vacuo (0.057 g, 0.20 mmol, 48%). Mp > 300 °C; 1H NMR (400 MHz, DMSO-d6) δ 12.41 (s, 1H), 10.71 (s, 1H), 8.04 (d, J = 6.8 Hz, 1H), 7.97 (dd, J = 1.2, 8.0 Hz, 1H), 7.85 (d, J = 8.4 Hz, 1H), 7.64–7.68 (m, 1H), 7.37 (t, J = 7.6 Hz, 1H), 7.06 (m, 2H), 6.94 (d, J = 7.6 Hz, 1H); 13C NMR (DMSO-d6) δ 111.33, 133.33, 114.38, 116.43, 121.97, 122.21, 122.82, 131.72, 131.97, 132.72, 135.56, 142.87, 146.20, 165.60, 170.63; HRMS (ESI-ve) m/z calculated for C15H10N3O3 (M-H)− 280.0727, found 280.0736.

(Z)-3-(2-(2-Nitrophenyl)hydrazine)indolin-2-one (18c)

A mixture of isatin (17) (200 mg, 1.35 mmol) and 2-nitrophenylhydrazine (226 mg 1.48 mmol) and HCl (aq. 4 M, 4 drops) in ethanol (26.0 mL) was heated under reflux for 2 h. The reaction mixture was cooled to room temperature and the yellow solid was filtered and washed with ethanol to obtain the pure product 18c as an orange solid (145 mg, 38%). Mp = 279 °C, decomposed; 1H NMR (400 MHz, DMSO-d6) δ 11.56 (s, 1H, disappeared on D2O shake), 10.84 (s, 1H, disappeared on D2O shake), 8.22 (d, J = 7.2 Hz, 1H), 8.01 (d, J = 8.4 Hz, 1H), 7.87-7.80 (m, 2H), 7.44-7.40 (m, 1H), 7.18 (q, J = 14.4, 7.2 Hz, 2H), 6.96 (d, J = 7.6 Hz, 1H); 13C NMR (DMSO-d6) δ 111.76, 116.27, 116.66, 122.44, 122.68, 123.69, 126.60, 132.92, 134.18, 135.96, 137.43, 140.57, 143.85, 165.17; HRMS (ESI+ve) m/z calculated for C14H11N4O3 (M+H)+ 283.0826, found 283.0834.

(Z)-3-(2-(3-Nitrophenyl)hydrazine)indolin-2-one (18d)

A mixture of isatin (17) (200 mg, 1.35 mmol) and 2-nitrophenylhydrazine (226 mg, 1.48 mmol) and HCl (aq. 4 M, 4 drops) in ethanol (26.0 ml) was heated under reflux for 2 h. The reaction mixture was cooled to room temperature and the yellow solid was filtered and washed with ethanol to obtain the pure product 18d (279 mg, 73%) as a yellow solid. Mp = 267–269 °C; 1H NMR (400 MHz, DMSO-d6) δ12.75 (s, 1H), 11.07 (s, 1H), 8.25 (t, J = 4.0 Hz, 1H), 7.85 (ddd, J = 8.0, 2.8, 0.8 Hz, 1H), 7.81 (ddd, J = 8.0, 2.0, 0.8 Hz, 1H), 7.62-7.57 (m, 2H), 7.26 (td, J =7.6, 1.2 Hz, 1H), 7.04 (td, J = 7.2, 1.2 Hz, 1H), 6.90 (d, J = 7.6 Hz, 1H); 13C NMR (DMSO-d6) δ 108.91, 111.30, 117.34, 119.95, 121.14, 122.71, 130.07, 130.53, 131.53, 141.24, 144.24, 144.80, 149.46, 163.49; HRMS (ESI+ve) m/z calculated for C14H11N4O3 (M+H)+ 283.0826, found 283.0835.

Phosphatase activity

PTP activity was measured using the fluorogenic 6, 8-difluoro-4-methylumbelliferyl phosphate (DiFMUP, from Molecular Probes) as the substrate. Each reaction contained 25 mM MOPS (pH 7.0), 50 mM NaCl, 0.05% Tween-20, 1 mM DTT, 20 μM DiFMUP, 10 nM Microcystin LR, 20 nM PTP (Shp2, Shp1 or PTP1B)19, and 5 μl test compound or dimethyl sulfoxide (DMSO, solvent) in a total reaction volume of 100 μl in black 96-well plate. Reaction was initiated by addition of DiFMUP and the incubation time was 30 min at room temperature. DiFMUP fluorescence signal was measured at an excitation of 355 nm and an emission of 460 nm with a Wallac Victor2 1420 plate reader. IC50 was defined as the concentration of an inhibitor that caused a 50% decrease in the PTP activity. For IC50 determination, 8 concentrations of compounds at 1/3 dilution (~ 0.5 log) were tested. Each experiment was performed in duplicate and IC50 data were derived from at least four independent experiments. The curve-fitting program Prism 4 (GraphPad Software) was used to calculate the IC50 value.

Acknowledgments

This work was supported by NIH Grants P01CA118210 and R01CA077467 and P30CA076292-10 (Cancer Center Support Grant).

Footnotes

Supporting Information Available: Characterization data, mass and NMR spectra and HPLC traces of compounds 5, 10a–r, 14a–b, 17 and 18a–e, nOe and COSY data for 5 and 18c and a description of the molecular modeling studies. This information is available free of charge via the Internet at http://pubs.acs.org.

a

Abbreviations: Bcr-Abl, breakpoint cluster region-Abelson; CagA, cytotoxin-associated gene A; c-MET, hepatocyte growth factor receptor; DiFMUP, 6,8-difluoro-4-methylumbelliferyl phosphate; DIPEA, Diisopropylethylamine; ErbB, avian erythroblastosis oncogene B; ERK, Externally Regulated Kinase; GLIDE, Grid-based Ligand Docking with Energetics; NBS, N-Bromosuccinimide; PTP, Protein Tyrosine Phosphatase; SH2, Src homology-2; VEGFR, Vascular endothelial growth factor receptor.

Note added in proof: Birchmeier and co-workers have recently described the virtual screening based discovery of several phenylhydrazonpyrazolone derivatives as Shp2 inhibitors. See Hellmuth, K.; Grosskopf, S.; Lum, C. T.; Wuertele, M.; Roeder, N.; von Kries, J. P.; Rosario, M.; Rademann, J.; Birchmeier, W. Specific inhibitors of the protein tyrosine phosphatase Shp2 identified by high-throughput docking. Proc. Natl. Acad. Sci. U. S. A. 2008, 105, 7275–7280.

References and Notes

  • 1.Bridges AJ. Chemical inhibitors of protein kinases. Chem Rev. 2001;101:2541–2571. doi: 10.1021/cr000250y. [DOI] [PubMed] [Google Scholar]
  • 2.(a) Bialy L, Waldmann H. Inhibitors of protein tyrosine phosphatases: Next-generation drugs? Angew Chem-Int Ed. 2005;44:3814–3839. doi: 10.1002/anie.200461517. [DOI] [PubMed] [Google Scholar]; (b) Jiang ZX, Zhang ZY. Targeting PTPs with small molecule inhibitors in cancer treatment. Cancer Metast Rev. 2008;27:263–272. doi: 10.1007/s10555-008-9113-3. [DOI] [PMC free article] [PubMed] [Google Scholar]; (c) Tautz L, Pellecchia M, Mustelin T. Targeting the PTPome in human disease. Expert Opin Ther Targets. 2006;10:157–177. doi: 10.1517/14728222.10.1.157. [DOI] [PubMed] [Google Scholar]; (d) Dewang PM, Hsu NM, Peng SZ, Li WR. Protein tyrosine phosphatases and their inhibitors. Curr Med Chem. 2005;12:1–22. doi: 10.2174/0929867053363504. [DOI] [PubMed] [Google Scholar]; Tautz L, Mustelin T. Strategies for developing protein tyrosine phosphatase inhibitors. Methods. 2007;42:250–260. doi: 10.1016/j.ymeth.2007.02.014. [DOI] [PubMed] [Google Scholar]
  • 3.Alonso A, Sasin J, Bottini N, Friedberg I, Friedberg I, Osterman A, Godzik A, Hunter T, Dixon J, Mustelin T. Protein tyrosine phosphatases in the human genome. Cell. 2004;117:699–711. doi: 10.1016/j.cell.2004.05.018. [DOI] [PubMed] [Google Scholar]
  • 4.(a) Chan G, Kalaitzidis D, Neel BG. The tyrosine phosphatase Shp2 (PTPN11) in cancer. Cancer Metast Rev. 2008;27:179–192. doi: 10.1007/s10555-008-9126-y. [DOI] [PubMed] [Google Scholar]; (b) Mohi MG, Neel BG. The role of Shp2 (PTPN11) in cancer. Curr Opin Genet Dev. 2007;17:23–30. doi: 10.1016/j.gde.2006.12.011. [DOI] [PubMed] [Google Scholar]; (c) Mannell H, Kroetz F. The role of SHP-2 in cell signaling and human disease. Curr Enzyme Inhib. 2007;3:264–272. [Google Scholar]; (d) Neel BG, Gu H, Pao L. The ‘Shp’ing news: SH2 domain-containing tyrosine phosphatases in cell signaling. Trends Biochem Sci. 2003;28:284–93. doi: 10.1016/S0968-0004(03)00091-4. [DOI] [PubMed] [Google Scholar]
  • 5.(a) Dance M, Montagner A, Salles JP, Yart A, Raynal P. The molecular functions of Shp2 in the Ras/Mitogen-activated protein kinase (ERK1/2) pathway. Cell Signalling. 2008;20:453–459. doi: 10.1016/j.cellsig.2007.10.002. [DOI] [PubMed] [Google Scholar]; (b) Chong ZZ, Maiese K. The Src homology 2 domain tyrosine phosphatases SHP-1 and SHP-2: diversified control of cell growth, inflammation, and injury. Histol Histopathol. 2007;22:1251–1267. doi: 10.14670/hh-22.1251. [DOI] [PMC free article] [PubMed] [Google Scholar]; (c) Xu R. Shp2, a novel oncogenic tyrosine phosphatase and potential therapeutic target for human leukemia. Cell Res. 2007;17:295–297. doi: 10.1038/cr.2007.18. [DOI] [PubMed] [Google Scholar]; (d) Feng GS. Shp-2 tyrosine phosphatase: signaling one cell or many. Exp Cell Res. 1999;253:47–54. doi: 10.1006/excr.1999.4668. [DOI] [PubMed] [Google Scholar]
  • 6.Nishida K, Hirano T. The role of Gab family scaffolding adapter proteins in the signal transduction of cytokine and growth factor receptors. Cancer Sci. 2003;94:1029–33. doi: 10.1111/j.1349-7006.2003.tb01396.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 7.Gu H, Neel BG. The “Gab” in signal transduction. Trends Cell Biol. 2003;13:122–30. doi: 10.1016/s0962-8924(03)00002-3. [DOI] [PubMed] [Google Scholar]
  • 8.Deb TB, Wong L, Salomon DS, Zhou G, Dixon JE, Gutkind JS, Thompson SA, Johnson GR. A common requirement for the catalytic activity and both SH2 domains of SHP-2 in mitogen-activated protein (MAP) kinase activation by the ErbB family of receptors. J Biol Chem. 1998;273:16643–6. doi: 10.1074/jbc.273.27.16643. [DOI] [PubMed] [Google Scholar]
  • 9.Cunnick JM, Dorsey JF, Munoz-Antonia T, Mei L, Wu J. Requirement of SHP2 binding to Grb2-associated binder-1 for mitogen-activated protein kinase activation in response to lysophosphatidic acid and epidermal growth factor. J Biol Chem. 2000;275:13842–8. doi: 10.1074/jbc.275.18.13842. [DOI] [PubMed] [Google Scholar]
  • 10.Maroun CR, Naujokas MA, Holgado-Madruga M, Wong AJ, Park M. The tyrosine phosphatase SHP-2 is required for sustained activation of extracellular signal-regulated kinase and epithelial morphogenesis downstream from the met receptor tyrosine kinase. Mol Cell Biol. 2000;20:8513–25. doi: 10.1128/mcb.20.22.8513-8525.2000. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 11.Furge KA, Zhang YW, Vande Woude GF. Met receptor tyrosine kinase: enhanced signaling through adapter proteins. Oncogene. 2000;19:5582–9. doi: 10.1038/sj.onc.1203859. [DOI] [PubMed] [Google Scholar]
  • 12.Bentires-Alj M, Kontaridis MI, Neel BG. Stops along the RAS pathway in human genetic disease. Nat Med. 2006;12:283–285. doi: 10.1038/nm0306-283. [DOI] [PubMed] [Google Scholar]
  • 13.Higashi H, Tsutsumi R, Muto S, Sugiyama T, Azuma T, Asaka M, Hatakeyama M. SHP-2 tyrosine phosphatase as an intracellular target of Helicobacter pylori CagA protein. Science. 2002;295:683–6. doi: 10.1126/science.1067147. [DOI] [PubMed] [Google Scholar]
  • 14.Meyer-ter-Vehn T, Covacci A, Kist M, Pahl HL. Helicobacter pylori activates mitogen-activated protein kinase cascades and induces expression of the proto-oncogenes c-fos and c-jun. J Biol Chem. 2000;275:16064–72. doi: 10.1074/jbc.M000959200. [DOI] [PubMed] [Google Scholar]
  • 15.Hof P, Pluskey S, Dhe-Paganon S, Eck MJ, Shoelson SE. Crystal structure of the tyrosine phosphatase SHP-2. Cell. 1998;92:441–50. doi: 10.1016/s0092-8674(00)80938-1. [DOI] [PubMed] [Google Scholar]
  • 16.Shen K, Keng YF, Wu L, Guo XL, Lawrence DS, Zhang ZY. Acquisition of a specific and potent PTP1B inhibitor from a novel combinatorial library and screening procedure. J Biol Chem. 2001;276:47311–9. doi: 10.1074/jbc.M106568200. [DOI] [PubMed] [Google Scholar]
  • 17.Huang P, Ramphal J, Wei J, Liang C, Jallal B, McMahon G, Tang C. Structure-based design and discovery of novel inhibitors of protein tyrosine phosphatases. Bioorg Med Chem. 2003;11:1835–49. doi: 10.1016/s0968-0896(03)00039-7. [DOI] [PubMed] [Google Scholar]
  • 18.Nören-Müller A, Reis-Corrêa I, Prinz H, Rosenbaum C, Saxena K, Schwalbe HJ, Vestweber D, Cagna G, Schunk S, Schwarz O, Schiewe H, Waldmann H. Discovery of protein phosphatase inhibitor classes by biology-oriented synthesis. Proc Nat Acad Sci USA. 2006;103:10606–10611. doi: 10.1073/pnas.0601490103. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 19.Chen L, Sung SS, Yip MLR, Lawrence HR, Ren Y, Guida WC, Sebti SM, Lawrence NJ, Wu J. Discovery of a novel Shp2 protein tyrosine phosphatase inhibitor. Mol Pharmacol. 2006;70:562–570. doi: 10.1124/mol.106.025536. [DOI] [PubMed] [Google Scholar]
  • 20.Neel BG, Tonks NK. Protein tyrosine phosphatases in signal transduction. Curr Opin Cell Biol. 1997;9:193–204. doi: 10.1016/s0955-0674(97)80063-4. [DOI] [PubMed] [Google Scholar]
  • 21.Poole AW, Jones ML. A SHPing tale: perspectives on the regulation of SHP-1 and SHP-2 tyrosine phosphatases by the C-terminal tail. Cell Signalling. 2005;17:1323–1332. doi: 10.1016/j.cellsig.2005.05.016. [DOI] [PubMed] [Google Scholar]
  • 22.Yang J, Liu L, He D, Song X, Liang X, Zhao ZJ, Zhou GW. Crystal structure of human protein-tyrosine phosphatase SHP-1. J Biol Chem. 2003;278:6516–20. doi: 10.1074/jbc.M210430200. [DOI] [PubMed] [Google Scholar]
  • 23.Tenev T, Keilhack H, Tomic S, Stoyanov B, Stein-Gerlach M, Lammers R, Krivtsov AV, Ullrich A, Böhmer FD. Both SH2 domains are involved in interaction of SHP-1 with the epidermal growth factor receptor but cannot confer receptor-directed activity to SHP-1/SHP-2 chimera. J Biol Chem. 1997;272:5966–73. doi: 10.1074/jbc.272.9.5966. [DOI] [PubMed] [Google Scholar]
  • 24.O’Reilly AM, Neel BG. Structural determinants of SHP-2 function and specificity in Xenopus mesoderm induction. Mol Cell Biol. 1998;18:161–77. doi: 10.1128/mcb.18.1.161. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 25.Yang J, Liang X, Niu T, Meng W, Zhao Z, Zhou GW. Crystal structure of the catalytic domain of protein-tyrosine phosphatase SHP-1. J Biol Chem. 1998;273:28199–207. doi: 10.1074/jbc.273.43.28199. [DOI] [PubMed] [Google Scholar]
  • 26.The NCI-Diversity set was obtained from the Developmental Therapeutics Program of the NCI/NIH. NSC refers to National Service Center. See Milne GWA, Feldman A, Miller JA, Daly GP, Hammel MJ. The NCI Drug Information System. 2. DIS pre-registry. J Chem Inf Comput Sci. 1986;26:159–68. doi: 10.1021/ci00052a003.
  • 27.Fong TAT, Shawver LK, Sun L, Tang C, App H, Powell TJ, Kim YH, Schreck R, Wang X, Risau W, Ullrich A, Hirth KP, McMahon G. SU5416 is a potent and selective inhibitor of the vascular endothelial growth factor receptor (Flk-1/KDR) that inhibits tyrosine kinase catalysis, tumor vascularization, and growth of multiple tumor types. Cancer Res. 1999;59:99–106. [PubMed] [Google Scholar]
  • 28.Sun L, Tran N, Liang C, Tang F, Rice A, Schreck R, Waltz K, Shawver LK, McMahon G, Tang C. Design, synthesis, and evaluations of substituted 3-[(3- or 4-carboxyethylpyrrol-2-yl)methylidenyl]indolin-2-ones as inhibitors of VEGF, FGF, and PDGF receptor tyrosine kinases. J Med Chem. 1999;42:5120–5130. doi: 10.1021/jm9904295. [DOI] [PubMed] [Google Scholar]
  • 29.Friesner RA, Banks JL, Murphy RB, Halgren TA, Klicic JJ, Mainz DT, Repasky MP, Knoll EH, Shelley M, Perry JK, Shaw DE, Francis P, Shenkin PS. Glide: A new approach for rapid, accurate docking and scoring. 1. Method and assessment of docking accuracy. J Med Chem. 2004;47:1739–1749. doi: 10.1021/jm0306430. [DOI] [PubMed] [Google Scholar]
  • 30.Andersen JN, Mortensen OH, Peters GH, Drake PG, Iversen LF, Olsen OH, Jansen PG, Andersen HS, Tonks NK, Møller NP. Structural and evolutionary relationships among protein tyrosine phosphatase domains. Mol Cell Biol. 2001;21:7117–36. doi: 10.1128/MCB.21.21.7117-7136.2001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 31.Lee D, Long SA, Murray JH, Adams JL, Nuttall ME, Nadeau DP, Kikly K, Winkler JD, Sung CM, Ryan MD, Levy MA, Keller PM, DeWolf WE., Jr Potent and selective nonpeptide inhibitors of Caspases 3 and 7. J Med Chem. 2001;44:2015–2026. doi: 10.1021/jm0100537. [DOI] [PubMed] [Google Scholar]
  • 32.Parrick J, Yahya A, Ijaz AS, Yizun J. Convenient preparation of 3,3-dibromo-1,3-dihydroindol-2-ones and indole-2,3-diones (isatins) from indoles. J Chem Soc, Perkin Trans. 1;1989:2009–2015. [Google Scholar]
  • 33.Bramson HN, Corona J, Davis ST, Dickerson SH, Edelstein M, Frye SV, Gampe RT, Jr, Harris PA, Hassell A, Holmes WD, Hunter RN, Lackey KE, Lovejoy B, Luzzio MJ, Montana V, Rocque WJ, Rusnak D, Shewchuk L, Veal JM, Walker DH, Kuyper LF. Oxindole-based inhibitors of cyclin-dependent kinase 2 (CDK2): Design, synthesis, enzymatic activities, and X-ray crystallographic analysis. J Med Chem. 2001;44:4339–4358. doi: 10.1021/jm010117d. [DOI] [PubMed] [Google Scholar]
  • 34.(a) Gee KR, Sun WC, Bhalgat MK, Upson RH, Klaubert DH, Latham KA, Haugland RP. Fluorogenic Substrates Based on Fluorinated Umbelliferones for Continuous Assays of Phosphatases and β-Galactosidases. Anal Biochem. 1999;273:41–48. doi: 10.1006/abio.1999.4202. [DOI] [PubMed] [Google Scholar]; (b) Montalibet J, Skorey KI, Kennedy BP. Protein tyrosine phosphatase: enzymatic assays. Methods. 2005;35:2–8. doi: 10.1016/j.ymeth.2004.07.002. [DOI] [PubMed] [Google Scholar]
  • 35.Chu W, Zhang J, Zeng C, Rothfuss J, Tu Z, Chu Y, Reichert ED, Welch JM, Mach HR. N-Benzylisatin sulfonamide analogues as potent caspase –3 inhibitors: synthesis, in vitro activity and molecular modeling studies. J Med Chem. 2005;48:7637–7647. doi: 10.1021/jm0506625. [DOI] [PubMed] [Google Scholar]
  • 36.Somasekhara S, Dighe VS, Suthar GK, Mukherjee SL. Chlorosulphonation of isatins. Curr Sci. 1965;34:508. [Google Scholar]

RESOURCES