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Synthesis of Oxindole-Based Bioorganometallic Kinase Inhibitors Incorporating One or More Ferrocene Groups Jahangir Amin,† Irina S. Chuckowree,† Minghua Wang,‡ Graham J. Tizzard,§ Simon J. Coles,§ and John Spencer*,† †

Department of Chemistry, School of Life Sciences, University of Sussex, Falmer, Brighton, East Sussex BN1 9QJ, U.K. Terrence Donnelly Center for Cellular and Biomolecular Research, University of Toronto, Toronto, Ontario M5S 3E1, Canada § UK National Crystallography Service, School of Chemistry, University of Southampton, Highfield, Southampton SO171BJ, U.K. ‡

S Supporting Information *

ABSTRACT: A series of oxindole-containing ferrocenes has been synthesized, studied in the solid state by X-ray crystallography, and tested for in vitro kinase inhibition. Many compounds show low or submicromolar activities against DYRK isoforms and VEGFR2, which in certain cases have been rationalized by molecular docking studies.

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INTRODUCTION As exemplified by the clinically approved sunitinib and the latestage clinical candidate SU5416,1 oxindole-based kinase inhibitors are a successful class of anticancer agents that target aberrant kinase activity (protein phosphorylation) in cancer cells, with many acting as ATP antagonists (Figure 1).2

submicromolar inhibition of VEGFR2 and low micromolar cytotoxicity in cancer cell lines. Furthermore, studies in Xenopus laevis on a number of analogues showed that angiogenesis can be inhibited in vivo.5 We have investigated structural variation and SAR in metallocene-substituted oxindoles (1−4; Figure 2). For example, replacement of the ferrocenyl group by more sterically demanding metallocene units such as 1,2,3,4,5-pentaphenylferrocene and (η4-tetraphenylcyclobutadiene)(η5-cyclopentadienyl)cobalt as in 4 led to a total loss of kinase inhibition, attributed to the steric bulk of the complex, preventing binding to the ATP pocket of the kinase.6 Initial investigations centered

Figure 1. Binding mode of oxindole-based VEGFR2 kinase inhibitors.

Bioorganometallic kinase inhibitors offer the potential for the selective inhibition of kinases, since the unique coordination properties of transition metals enable a broader scope in binding to the kinase pocket.3 In recent publications we have described thermal- and microwave-mediated Knoevenagel condensations of oxindoles with metallocene-containing carboxaldehydes, affording methylidene-substituted oxindoles as often separable mixtures of E and Z isomers.4 In vitro studies of these compounds have shown, in many cases, low or © 2013 American Chemical Society

Figure 2. Examples of metallocene-containing methylidene-substituted oxindoles (only E isomer shown for brevity). Special Issue: Ferrocene - Beauty and Function Received: April 25, 2013 Published: June 26, 2013 5818

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Scheme 1. Synthesis of Disubstituted Ferrocene Oxindoles

Figure 3. Oxindoles used in this study.

Figure 4. ADP plot for complex (E)-9a with ellipsoids drawn at the 50% probability level.

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Figure 5. ADP plot for complex (Z)-9b with ellipsoids drawn at the 50% probability level.

solid state. Examination by single-crystal X-ray diffraction showed that the compound crystallized in the triclinic space group P 1,̅ with two independent molecules in the asymmetric unit (Figure 4). (E)-9a has two H-bond acceptors and two Hbond donors, and in the crystal structure this results in four unique hydrogen bonds (N1···O102 = 2.763(8) Å, ∠N1− H1···O102 = 149.1°; N2···O101 = 2.838(8) Å, ∠N2− H2···O102 = 159.9°; N101···O2 = 2.761(8) Å, ∠N101− H101···O2 = 153.7°; N102···O1 = 2.890(8) Å, ∠N102− H102···O1 = 159.8°), leading to a complex structure with alternate layers of each of the independent molecules parrallel to the ab plane (see also Figure S1 in the Supporting Information). Likewise, crystals of the complex (Z)-9b were grown and analyzed by single-crystal X-ray diffraction. The structure crystallized in the orthorhombic space group Pca21, and two independent molecules were observed in the asymmetric unit (Figure 5). The hydrogen-bonding arrangement is simpler than in (E)-9a, with the oxindole moieties of the two independent units forming a dimer (N2···O102 = 2.893(13) Å, ∠N2− H2A···O102 = 174.8°; N102···O2 = 2.888(13) Å, ∠N102− H10A···O2 = 178.8°). Additionally, the amino group of the pyrrole moiety forms an intramolecular H bond with the carbonyl of the oxindole moiety (N3···O2 = 2.746(11) Å, ∠N3−H3A···O2 = 144.0°; N103···O102 = 2.751(12) Å, ∠N103−H10C···O102 = 144.9°) so that both moieties are coplanar in each individual molecule (see also Figure S2 in the Supporting Information). Finally, the structure of (E)-9g crystallized in the monoclinic space group C2/c with one water of crystallization and showed the expected stereochemistry (Figure 6). As for (Z)-9b, a dimer

on varying the substituents on the oxindole motif have also been carried out and have yielded interesting SAR, notably with a number of complexes displaying activity against isoforms of DYRK,5 a kinase implicated in many forms of cancer.7 Here, we describe further studies in this direction.

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RESULTS AND DISCUSSION

Using the aniline-containing (Z)-3 as a lead (IC50 = 1.1 and 4.5 μM against DYRK3,4 isoforms, respectively; Table 1, vide infra) we wished to explore amide coupling chemistry as a means of introducing substituents to potentially improve inhibition against DYRK isoforms. Hence, the reaction of the acid chloride 5a with the aniline 6a was performed in a microwave reactor in the presence of triethylamine (TEA) (Scheme 1). A standard Knoevenagel condensation with ferrocenecarboxaldehyde afforded the separable isomers (E)and (Z)-9a, which contain two distinct ferrocenyl units. Next, a library of oxindoles was synthesized (Figure 3). For one series, we opted for a Knoevenagel condensation of heterocycle-containing aldehydes with the ferrocene-containing 7a,b, since there is ample precedence for high levels of stereocontrol in this step.8 Hence, we obtained the pyrroles 9b,c as Z isomers, whereas the furan 9d was formed as exclusively the E isomer. Another series of oxindoles were formed where ferrocenecarboxaldehyde was used for the condensation reaction. Hence the fluoro-substituted 9e,f were formed as geometrical isomer mixtures, as was the phenolic analogue 9g. To further analyze these organometallic complexes, the solidstate structures of a number of derivatives were determined. Hence, the structure of (E)-9a was further investigated in the 5820

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in kinases. From Figure 7, VEGFR2 has a well-defined pocket to accommodate the oxindole core, as illustrated in panel (a).9 However, there is no space to fit a large ferrocene group at the R1 position in this orientation. Therefore, (Z)-9b can only be docked with a flipped oxindole core placing the pyrrole group inside the pocket (panel (b)). There was no productive pose produced when (Z)-9a was docked. This confirms the lack of activity observed for (Z)-9a against VEGFR2 (Table 1, vide infra). DYRK2 has a relatively open pocket. Both (Z)-9a and (Z)-9b can be docked with the oxindole core placed similarly to that observed for VEGFR2, as depicted in panels (c) and (d), respectively. The ferrocene groups in (Z)-9a and (Z)-9b can be accommodated well in the pocket and so were predicted to be active toward DYRKs. (Z)-9c and (Z)-9g were also docked, with the former adopting a pose similar to that of (Z)-9b in both VEGFR2 and DYRK2, consistent with the similarity of structures between (Z)-9c and (Z)-9b ((Z)-9c has a sulfonamide linkage of the ferrocene group at the R1 position). (Z)-9g cannot be docked with the key H-bond interaction kept in place in VEGFR2, and this could account for its loss of activity in comparison to sunitinib. The binding pose adopted uses the hydroxyl group to make a H bond to the hinge region and, for DRYK2, no productive pose can be obtained, consistent with the inactivity of (Z)-9g toward DYRK2. None of the above complexes gave any appreciable activity (>20 μM) in K562 (leukemia) cell lines (a chronic myelogenous leukemia (CML) cell line11), possibly due to poor cellular penetration.

Figure 6. ADP plot for complex (E)-9g with ellipsoids drawn at the 50% probability level.

is formed between neighboring oxindole moieties (N1···O1 = 2.896(5) Å, ∠N1−H1···O1 = 137.0°), which in turn stack in alternate orientations to form channels parallel to the crystallographic c axis (see also Figure S3 in the Supporting Information). These contain the waters of crystallization, which exhibit intermolecular hydrogen bonding to the carbonyl of the oxindole moiety and hydroxyl substituent (O1W···O1 = 2.692(5) Å, ∠O1W−H12W···O1 = 174°; O1W···O2 = 2.878(5) Å, ∠O1W−H11W···O2 = 175°; O2···O1W = 2.643(5) Å, ∠O2−H2···O1W = 175.1°). The crystal structures of 9a,b have rather high Rint values (11% and 23%). Despite several crystals being tried and several recrystallization attempts, the relatively poor data obtained are due to data quality rather than refinement. For 9a, this was due to the small size of the crystals available, and for 9b, the problem of small size was even worse and the data were collected at station I19 of the Diamond Light Source synchrotron. An initial biological screen revealed the pyrrole (Z)-9b to be a moderate inhibitor of VEGFR2 with an IC50 value of 120 nM (in comparison with 220 nM for (Z)-1 (Table 1) and 1.5 nM for sunitinib1c). The importance of the pyrrole group for biological activity is emphasized, since formally replacing it by a ferrocene as in (E)- or (Z)-9a led to no appreciable kinase inhibition. (Z)-9c, somewhat related to (Z)-9b, was also evaluated and showed single-digit VEGFR2 inhibition. (Z)-9b and (Z)-9c displayed encouraging inhibition of some DYRK isoforms, although other analogues tested showed little activity. For comparison, the furan 9d, the fluoro-substituted (E)- and (Z)9e,f, and the phenol 9g (synthesized from the corresponding oxindoles 7d−g, respectively) gave weaker VEFGR2 inhibition and were not subjected to further evaluation. Molecular docking studies were next employed to garner information of possible binding modes of the above complexes

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CONCLUSION A series of oxindoles based around the original ferrocenecontaining oxindole leads 1 and 3 have been synthesized and evaluated as kinase inhibitors. Complexes (E)- and (Z)-9a, containing two ferrocenes, show poor VEGFR-2 inhibition yet inhibit some DYRK isoforms, notably DYRK3,4. (Z)-9b displays nM inhibition of VEGFR2 and single-digit micromolar inhibition of DYRK2−4 and adds to the growing list of useful metal-based anticancer agents.12

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EXPERIMENTAL SECTION

Experimental and spectroscopic methods as well as biological experiments have been outlined in previous papers.4a,10 All experiments were conducted in air using reagent grade solvents, and all chemicals were from commercial sources and were used without further purification. NMR spectra were measured on a JEOL EX270 spectrometer at 270 MHz (1H), and CHN elemental analysis was performed on a CE Instruments Eager 300 apparatus. Microwave reactions were carried out with a CEM Explorer microwave unit. Biological assays were performed at Cerep (www.cerep.com), and docking studies were performed as previously outlined.13

Table 1. Kinase Inhibition Data for Ferrocene-Containing Complexes kinase inhibition IC50 (μM)a VEGFR2 DYRK1a DYRK2 DYRK3 DYRK4

sunitinib

(E)-1b

(Z)-1b

(Z)-3b

(E)-9a

(Z)-9a

(Z)-9b

(Z)-9c

(E)-9g

(Z)-9g

0.0015 nr nr nr nr

0.21 ia ia ia 0.47

0.22 ia ia 0.39 1.1

ia ia ia 1.1 4.5

ia ia ia ia 1.5

ia ia ia 0.96 0.65

0.12 ia 1.1 0.9 2.7

1.3 6.8 12 2 1.9

1.3 ia ia ia ia

1.9 ia ia ia ia

Abbreviations: ia, inactive (ca. 30% at 1 μM concentration were re-evaluated in a dose response assay). a

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Figure 7. Comparison of ligand poses in VEGFR2 (pdb accession code: 4agd) (a) cocrystallized ligand (sunitinib); (b) (Z)-9b; (e) (Z)-9c; (g) (Z)9g. Comparison of ligand poses in DYRK2 (pdb accession code: 4azf): (c) (Z)-9a; (d) (Z)-9b; (f) (Z)-9c. Color scheme for the atom types: carbon, yellow; nitrogen, blue; oxygen, red; iron, cyan. Docking methods have been previously reported.10 Synthesis of 5a. At 0 °C, under a nitrogen atmosphere, to a stirred solution of triphosgene (3.12 g, 11.52 mmol) in methylene dichloride (100 mL) was added dropwise a dichloromethane solution (35 mL) of ferrocenecarboxylic acid (2.52 g, 10.96 mmol), reagent-grade triethylamine (1.60 mL, 11.52 mmol), and DMAP (0.70 g, 5.78 mmol).14 The resulting mixture was stirred at 0 °C for 1.5 h and then warmed to room temperature and stirred overnight. The solution was filtered over Celite, and the filtrate was evaporated in vacuo to dryness. Trituration with hot hexane (100 mL) afforded a precipitate, which was filtered. The filtrate was concentrated under reduced pressure, and the resulting residue was crystallized from hexane to give the red

crystalline solid 5a (1.67 g, 61%), which was used as such for the next reaction. Mp: 50−52 °C. 1H NMR (δ, 270.0 MHz, CDCl3): 4.32 (5H, s, C5H5), 4.63 (2H, br s, Fc), 4.91 (2H, br s, Fc). Synthesis of 5-Ferrocenylamido-1,3-dihydroindol-2-one (7a). In an oven-dried microwave tube equipped with a stirrer, ferrocenoyl chloride (5a; 1.20 g, 4.83 mmol) and 5-amino-1,3-dihydroindol-2-one (0.84 g, 5.64 mmol) were combined in anhydrous THF (5 mL). Reagent grade triethylamine (1.65 mL, 12.45 mmol) was then added before the reaction mixture was placed in a CEM Explorer microwave unit and heated (ramped) to 90 °C, where it was held at 150 W for 30 min by moderation of the microwave power (Caution! 5822

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4.23 (5H, s, C5H5), 4.43 (2H, br s, Fc), 4.99 (2H, br s, Fc), 6.36 (1H, m, ArCH), 6.35 (1H, d, J = 8.1 Hz, ArCH), 6.91 (1H, s, ArCH), 7.31− 7.36 (2H, m, ArCH), 7.67 (1H, s, ArCH), 7.97 (1H, s, ArCH), 9.40 (1H, s, NH), 10.85 (1H, s, NH); 13C NMR (δ, 67.0 MHz, DMSO-d6) 68.5 (2C), 69.4 (5C), 70.3 (2C), 76.6, 109.3, 111.4, 112.2, 117.0, 120.3, 120.6, 125.0, 125.7, 126.2, 129.5, 133.2, 135.1, 167.8, 169.3; HRMS (m/z, HNESP) for C24H19N3O2Fe [M + H]+ calcd 438.0899, obsd 438.0896. Anal. Calcd for C24H18N3O2Fe: C, 66.1; H, 4.2; N, 9.6. Found: C, 66.0; H, 4.4; N, 9.4. Synthesis of (Z)-9c. Intermediate 7b was first synthesized in situ by dissolving aminoferrocene (0.10 g, 0.50 mmol), 2-oxoindoline-5sulfonyl chloride (0.14 g, 0.60 mmol), and triethylamine (0.28 mL, 2.00 mmol) in anhydrous pyridine (1 mL). The reaction mixture was then heated to 150 °C at 150 W for 10 min in a CEM Explorer microwave. Once the mixture was cooled to room temperature, the solvent was removed under reduced pressure. 3,5-Dimethyl-1Hpyrrolecarboxaldehyde (0.061 g, 0.50 mmol) followed by ethanol (1 mL) and piperidine (0.025 mL) were then added, and the reaction mixture was heated to 150 °C at 150 W for 20 min in the microwave. After the mixture was cooled to room temperature, ethanol was removed under reduced pressure. The crude reaction mixture was given an aqueous workup using ethyl acetate (10 mL) and washed with brine (10 mL) and then dried using magnesium sulfate. The solvent was reduced to 1/10th of its original volume, and hexane (50 mL) was carefully added. The product was collected by filtration to give (Z)-9c (0.02 g, 10%). (Z)-9c: mp 242−248 °C (solid crystallized from 1/20 ethyl acetate/ hexane); IR (thin film) νmax 1717 cm−1 (CO stretch); 1H NMR (δ, 270.0 MHz, DMSO-d6) 2.35 (6H, s, 2xCH3), 3.90 (2H, br s, Fc), 3.99 (5H, s, C5H5), 4.00 (1H, br s, NH), 4.09 (2H, br s, Fc), 6.08 (1H, s, ArCH), 7.00 (1H, d, J = 8.1 Hz, ArCH), 7.48 (1H, d, J = 8.1 Hz, ArCH), 7.72 (1H, s, ArCH), 8.14 (1H, s, ArCH), 9.10 (1H, br s, NH), 11.23 (1H, br s, NH); 13C NMR (δ, 67.0 MHz, DMSO-d6) 11.5, 13.6, 62.3 (2C), 64.5 (2C), 68.9 (5C), 95.0, 109.0, 110.6, 113.3, 116.9, 124.7, 125.1, 126.1, 127.0, 132.4, 133.8, 137.6, 140.7, 168.0; HRMS (m/z, HNESP) for C25H23N3O3FeS [M]+ calcd 501.0804, obsd 501.0797. (E)-9d was isolated using purification method ii to give a gray solid (0.38 g, 86%). (E)-9d: mp 175−177 °C (solid crystallized from 1/20 ethyl acetate/hexane); IR (Nujol mull): νmax 1695 cm−1 (CO stretch); 1H NMR (δ, 270.0 MHz, DMSO-d6) 4.09 (5H, s, C5H5), 4.24 (2H, br s, Fc), 5.00 (2H, br s, Fc), 6.85 (2H, m, CH), 7.27 (1H, d, J = 2.7 Hz, CH), 7.34 (1H, s, CH), 7.48 (1H, d, J = 8.1 Hz, CH), 8.03 (1H, s, CH), 8.66 (1H, s, CH), 9.44 (1H, s, NH), 10.51 (1H, s, NH); 13C NMR (δ, 67.0 MHz, DMSO-d6) 68.5 (2C), 69.4 (5C), 70.3 (2C), 76.6, 109.3, 113.7, 118.7, 119.4, 120.5, 121.2, 122.7, 123.3, 133.0, 138.7, 146.8, 150.7, 168.0, 169.4; HRMS (m/z, HNESP) for C24H19N2O3Fe [M + H]+ calcd 439.0740, obsd 439.0749. Anal. Calcd for C24H18N2O3Fe·0.21(hexane): C, 66.3; H, 4.9; N, 6.1. Found: C, 66.3; H, 5.2; N, 6.2. (E)-9e (0.50 g, 72%) was obtained as a red solid and (Z)-9g (0.11 g, 16%) as a purple solid, after silica gel column chromatography (9/1 dichloromethane/ethyl acetate). (E)-9e: mp 198−200 °C (solid crystallized from 1/20 ethyl acetate/ hexane); IR (thin film) νmax 1700 cm−1 (CO stretch); 1H NMR (δ, 500.0 MHz, CDCl3) 4.40 (5H, s, C5H5), 4.79 (2H, t, J = 2.2 Hz, Fc), 4.91 (2H, t, J = 2.2 Hz, Fc), 6.94 (1H, dd, J = 8.6, 4.3 Hz, ArCH), 7.08 (1H, td, J = 8.6, 2.7 Hz, ArCH), 7.86 (1H, dd, J = 8.6, 2.7, ArCH), 7.87 (1H, s, CH), 7.92 (1H, s, NH); δC 13C NMR (δ, 75.0 MHz, DMSOd6) 70.4 (5C), 72.0 (2C), 72.5 (2C), 78.3, 109.7 (d, 2JFC 23.3 Hz, ArC), 110.9 (d, 3JFC 8.3 Hz, CC), 115.3 (d, 2JFC 23.3 Hz, ArC). 123.4, 138.7, 139.2, 157.9 (d, 1JFC 202.5 Hz, ArC-F), 169.5; HRMS (m/z, HNESP) for C19H15FFeNO [M + H]+ calcd 346.0528, obsd 346.0534. Anal. Calcd for C19H14FFeNO·0.1H2O: C, 65.4; H, 4.1; N, 4.0. Found: C, 65.3; H, 4.0; N, 4.1. (Z)-9e: mp 189−191 °C (solid crystallized from 1/20 ethyl acetate/ hexane); IR (thin film) νmax 1700 cm−1 (CO stretch); 1H NMR (δ, 500.0 MHz, CDCl3); 4.21 (5H, s, C5H5), 4.63 (2H, t, J = 2.2 Hz, Fc), 5.34 (2H, t, J = 2.2 Hz, Fc), 6.73 (1H, dd, J = 8.6, 4.3 Hz, ArCH), 6.90

Hot sealed tube, high pressures; use an appropriately ventilated fumehood with the sash down). After it was cooled, the reaction mixture was extracted with ethyl acetate (25 mL) and washed with deionized water (25 mL) followed by saturated brine (25 mL). The organic layer was dried over magnesium sulfate and then filtered over fluted filter paper. The organic layer was concentrated to 1/10th of its initial volume before hexane (40 mL) was added. The precipitate was collected by filtration and washed with dichloromethane (5 mL) and hexane (5 mL) to afford the light brown solid 7a (1.88 g, 84%). Mp: 228−230 °C (solid crystallized from 1/20 ethyl acetate/hexane). IR (Nujol mull): νmax 1694 cm−1 (CO stretch). 1H NMR (δ, 270.0 MHz, DMSO-d6): 3.32 (2H, s, CH2), 4.21 (5H, s, C5H5), 4.42 (2H, t, J = 2.7 Hz, Fc), 4.97 (2H, t, J = = 2.7 Hz, Fc), 6.76 (1H, d, J = 8.1 Hz, ArCH), 7.42 (1H, d, J = 8.1 Hz, ArCH), 7.60 (1H, s, ArCH), 9.32 (1H, s, NH), 10.31 (1H, s, NH). 13C NMR (δ, 67.0 MHz, DMSO-d6): 36.0, 68.5 (2C), 69.4 (5C), 70.3 (2C), 76.6, 108.7, 118.0, 119.9, 125.9, 133.2, 139.4, 167.7, 176.3. HRMS (m/z, HNESP): for C19H17N2O2Fe [M + H]+ calcd 361.0634, obsd 361.0632. Anal. Calcd for C19H16N2O2Fe·0.7H2O: C, 61.2; H, 4.7; N, 7.5. Found: C, 61.2; H, 5.1; N, 7.7. General Procedure for the MW-Mediated Knoevenagel Condensation of Oxindoles. A mixture of the appropriate oxindole (one of 5-ferrocenylamido-1,3-dihydroindol-2-one, 5-fluorooxindole, 5,7-difluoro-2-oxindole, 5-hydroxy-1,3-dihydroindol-2-one), an appropriate aldehyde (one of ferrocenecarboxaldehyde, 1H-pyrrole-2carbaldehyde, 3,5-dimethyl-1H-pyrrole-2-carbaldehyde, furan-2-carbaldehyde (1.2 mmol)), ethanol (5 mL), reagent grade piperidine (0.05 mL), and a stirrer bar were placed in a microwave tube (35 mL volume). The vessel was then sealed with a septum and placed in the microwave cavity of a CEM Explorer. The reaction mixture was heated (ramped) to 150 °C and held at that temperature for 30 min at 150 W with moderation of microwave power. The crude reaction mixture, once cooled, was worked up as above. The E and Z isomers were separated using either (i) silica gel column chromatography (hexane/ ethyl acetate as the eluant) or (ii) precipitation using a 1/15 ratio of ethyl acetate to hexane and separating the supernatant from the precipitate. Compound 9a was isolated using silica gel column chromatography using 1/9 ethyl acetate/hexane as the eluant, giving (E)-9a as a dark red solid (0.28 g, 50%) and (Z)-9a as a dark purple solid (0.053 g, 10%), respectively. (E)-9a: mp 285−287 °C (solid crystallized from 1/20 ethyl acetate/ hexane); IR (Nujol mull) νmax 1695 cm−1 (CO stretch); 1H NMR (δ, 270.0 MHz, DMSO-d6) 4.23 (5H, s, C5H5), 4.27 (5H, s, C5H5), 4.45 (2H, t, J = 2.7 Hz, Fc), 4.68 (2H, t, J = 2.7 Hz, Fc), 4.93 (2H, t, J = 2.7 Hz, Fc), 4.99 (2H, t, J = 2.7 Hz, Fc), 6.82 (1H, d, J = 8.1 Hz, ArCH), 7.37 (1H, dd, J = 8.1, 2.7 Hz, ArCH), 7.47 (1H, s, CH), 8.33 (1H, d, J = 2.7 Hz, ArCH), 9.43 (1H, s, NH), 10.41 (1H, s, NH); 13C NMR (δ, 67.0 MHz, DMSO-d6) 69.2 (2C), 70.0 (5C), 70.3 (5C), 70.9 (2C), 72.1 (2C), 72.2 (2C), 77.1, 78.4, 109.9, 117.1, 122.3, 122.4, 123.6, 133.2, 137.5, 138.4, 166.5, 169.7; HRMS (m/z, HNESP) for C30H25N2O2Fe2 [M + H]+ calcd 557.0609, obsd 557.0600. Anal. Calcd for C30H24N2O2Fe2: C, 64.8; H, 4.4; N, 5.0. Found: C, 64.9; H, 4.7; N, 4.8. (Z)-9a: mp 270−272 °C (solid crystallized from 1/20 ethyl acetate/ hexane); IR (Nujol mull) νmax 1696 cm−1 (CO stretch); 1H NMR (δ, 270.0 MHz, DMSO-d6) 4.21 (5H, s, C5H5), 4.25 (5H, s, C5H5), 4.45 (2H, t, J = 2.7 Hz, Fc), 4.62 (2H, t, J = 2.7 Hz, Fc), 5.01 (2H, t, J = 2.7 Hz, Fc), 5.41 (2H, t, J = 2.7 Hz, Fc), 6.76 (1H, d, J = 8.1 Hz, ArCH), 7.41 (1H, dd, J = 8.1, 2.7 Hz, ArCH), 7.50 (1H, s, CH), 7.94 (1H, d, J = 2.7 Hz, ArCH), 9.40 (1H, s, NH), 10.37 (1H, s, NH); 13C NMR (δ, 67.0 MHz, DMSO-d6) 68.3 (2C), 69.3 (5C), 69.4 (5C), 70.2 (2C), 71.7 (2C), 73.2 (2C), 76.6, 77.2, 108.8, 112.0, 120.6, 122.0, 125.0, 132.9, 135.5, 137.7, 167.5, 167.7; HRMS (m/z, HNESP) for C30H25N2O2Fe2 [M + H]+ calcd 577.0609, obsd 557.0604. Anal. Calcd for C30H24N2O2Fe2: C, 64.8; H, 4.4; N, 5.0. Found: C, 64.5; H, 4.6; N, 4.7. (Z)-9b was obtained as a bright yellow solid after recrystallization from hexane (0.29 g, 66%). (Z)-9b: mp 160−162 °C; IR (Nujol mull) νmax 1634 cm−1 (CO stretch); 1H NMR (δ, 270.0 MHz, DMSO-d6) 5823

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Organometallics

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(1H, td, J = 8.6, 2.7 Hz, ArCH), 7.19 (1H, dd, J = 8.6, 2.7 Hz, ArCH), 7.37 (1H, s, CH), 7.49 (1H, br s, NH); 13C NMR (δ, 100.0 MHz, DMSO-d6) 70.2 (5C), 72.7 (2C), 73.9 (2C), 77.6, 106.6 (d, 2JFC 25.0 Hz, ArC), 110.2, 113.9 (d, 2JFC = 25.0 Hz, ArC), 122.0, 127.3, 136.2, 140.6, 158.6 (d, 1JFC = 233.0 Hz, ArC-F), 168.0; HRMS (m/z, HNESP) for C19H15FFeNO [M + H]+ calcd 346.0528, obsd 346.0536. Anal. Calcd for C19H14FFeNO: C, 65.7; H, 4.1; N, 4.0. Found: C, 65.5; H, 4.2; N, 4.0. (E)-9f (0.26 g, 70%) was obtained as a red solid and (Z)-9h (0.10 g, 28%) as a purple solid after silica gel column chromatography (9/1 dichloromethane/ethyl acetate). (E)-9f: mp 226−228 °C; IR (thin film) νmax 1598 cm−1 (CO stretch); 1H NMR (δ, 270.0 MHz, CDCl3) 4.26 (5H, s, C5H5), 4.67 (2H, t, J = 2.7 Hz, Fc), 4.76 (2H, t, J = 2.7 Hz, Fc), 6.79 (1H, td, J = 8.1, 2.7 Hz, ArCH), 7.54 (1H, dd, J = 8.1, 2.7 Hz, ArCH), 7.79 (1H, s, CH), 8.03 (1H, br s, NH); 13C NMR (δ, 67.0 MHz, DMSO-d6) 69.8 (5C), 71.5 (2C), 72.2 (2C), 77.3, 103.4 (dd, 2JFC = 25.5 Hz, ArC), 105.2 (dd, 2JFC = 25.5, 4JFC = 4.0 Hz, ArC), 121.7 (dd, 4JCC = 3.4 Hz, CC), 124.7 (dd, 3J 10.7 Hz, ArC), 125.1 (dd, 3JFC = 13.4, 4JFC = 3.4 Hz, ArC), 140.4, 145.8 (dd, 1JFC = 239.2, 3JFC = 10.1 Hz, ArCF), 156.2 (dd, 1JFC = 239.2, 3JFC = 10.1 Hz, ArCF), 168.5; HRMS (m/z, HNESP) for C19H14OFeNOF2 [M + H]+ calcd 364.0434, obsd 364.0437; Anal. Calcd for C19H13OFeNOF2: C, 62.5; H, 3.6; N, 3.8. Found: C, 62.7; H, 3.6; N, 3.7. (Z)-9f: mp 220−222 °C; IR (thin film) νmax 1539 cm−1 (CO stretch); 1H NMR (δ, 270.0 MHz, DMSO-d6); 4.21 (5H, s, C5H5), 4.68 (2H, br s, Fc), 5.33 (2H, br s, Fc), 7.08 (1H, t, J = 8.1 Hz, ArCH), 7.47 (1H, d, J = 8.1 Hz, ArCH), 7.78 (1H, s, ArCH), 10.9 (1H, s, NH); 13C NMR (δ, 75.0 MHz, DMSO-d6); 69.8 (5C), 72.6 (2C), 73.6 (2C), 76.6, 101.9 (dd, 2JFC = 23.3 Hz, ArC), 102.3 (dd, 2JFC = 23.3, 2 JFC = 3.8 Hz, ArC), 120.4 (dd, 4JCC = 3.0 Hz, CC), 122.4 (dd, 3J = 12.0, 4JFC = 3.0 Hz, ArC), 128.7 (dd, 3JFC = 10.5 Hz, ArC), 142.1, 143.7 (dd, 1JFC = 236.3, 3JFC = 9.8 Hz, ArCF), 157.2 (dd, 1JFC = 236.3, 3 J FC = 9.8 Hz, ArCF), 167.0; HRMS (m/z, HNESP) for C19H14OFeNOF2 [M + H]+ calcd 364.0434, obsd 364.0436. Anal. Calcd for C19H13OFeF2NO: C, 62.5; H, 3.6; N, 3.8. Found: C, 62.5; H, 3.5; N, 3.6. (E)-9g (0.07 g, 24%) and (Z)-9i (0.03 g, 10%) were obtained as red-brown solids after using silica gel column chromatography (3/7 ethyl acetate/dichloromethane). (E)-9g: mp 210−212 °C; IR (thin film) νmax 1667 cm−1 (CO stretch); 1H NMR (δ, 270.0 MHz, DMSO-d6) 4.25 (5H, s, C5H5), 4.66 (2H, br s, Fc), 4.81 (2H, br s, Fc), 6.65−6.61 (2H, m, ArCH), 7.39 (2H, s, CH, ArCH), 8.99 (1H, s, NH), 10.11 (1H, s, OH); 13C NMR (δ, 67.0 MHz, DMSO-d6) 69.4 (5C), 71.0 (4C), 77.9, 99.2, 109.8, 115.0, 123.9, 133.0, 134.1, 135.8, 151.7, 168.8; HRMS (m/z, HNESP) for C19H15FeNO2 [M + H]+ calcd 346.0525, obsd 346.0533. Anal. Calcd for C19H15FeNO2·0.2CH2Cl2: C, 63.7; H, 4.3; N, 3.9. Found: C, 63.7; H, 4.6; N, 3.8. (Z)-9g: mp 195−197 °C; IR (Nujol mull) νmax 1669 cm−1 (CO stretch); 1H NMR (δ, 270.0 MHz, DMSO-d6) 4.18 (5H, s, C5H5), 4.58 (2H, br s, Fc), 5.33 (2H, br s, Fc), 6.61−6.55 (2H, m, ArCH), 7.00 (1H, s, ArCH), 7.43 (1H, s, CH), 8.90 (1H, s, NH), 10.07 (1H, br s, OH); 13C NMR (δ, 67.0 MHz, DMSO-d6) 69.4 (7C), 71.5, 73.1, 77.3, 106.0, 109.5, 114.1, 132.1, 133.3, 137.7, 152.1, 157.6, 168.6; HRMS (m/z, HNESP) for C19H16FeNO2 [M + H]+ calcd 346.0525, obsd 346.0529. Anal. Calcd for C19H15FeNO2·0.2CH2Cl2: C, 63.9; H, 4.3; N, 3.9. Found: C, 63.9, H 4.5, N 3.6. X-ray Crystallography. Single-crystal X-ray diffraction analyses of (E)-9a and (E)-9g were performed using a Nonius-Kappa CCD area detector mounted at the window of an FR591 rotating anode generator with an Mo anode (λ = 0.71073 Å) and equipped with an Oxford Cryosystems cryostream device. The crystals were mounted on glass fibers, and the data were collected at 120 K. Data were processed using Collect,15 and the unit cell parameters were refined against all data. An empirical absorption correction was carried out using SADABS.16 The single-crystal X-ray diffraction analysis of (Z)-9b was performed on data collected on Station I19 of the Diamond Light Source, using a Crystal Logics κ-geometry diffractometer and a Rigaku Saturn 724+ CCD detector with a Cryostream cooler (at 100 K);

Rigaku CrystalClear was used for recording images and for data integration.17 The synchrotron X-ray wavelength was 0.6889 Å. The structures were solved either by direct methods using SHELXS-9718 ((E)-9a and (E)-9g) or by charge flipping using SUPERFLIP19 ((Z)9b) and refined on Fo2 by full-matrix least-squares refinement using SHELXL-97. All non-hydrogen atoms were refined with anisotropic displacement parameters. Hydrogen atoms were added at calculated positions or, in the case of the hydrogen atoms of the water molecule in (Z)-9b, located from the difference map and refined using a riding model with isotropic displacement parameters based on the equivalent isotropic displacement parameter (Ueq) of the parent atom. Figures were produced using OLEX2.20 The CIFs for all crystal structures have been deposited with the CCDC and have been given the deposition numbers 897102 ((E)-9g), 897103 ((E)-9a), and 908419 ((Z)-9b).

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ASSOCIATED CONTENT

S Supporting Information *

Tables, figures, and CIF files giving experimental details, selected bond angles and lengths, packing diagrams, and detailed crystallographic parameters for the crystal structures obtained. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*J.S.: e-mail, [email protected]; fax, +44 (0)1273 876687. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The EPSRC Mass Spectrometry Unit (Swansea) is thanked for HRMS data. S.J.C. and G.J.T. acknowledge funding from the EPSRC for the X-ray facilities at Southampton.21 We thank the reviewers for their encouraging comments.

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ABBREVIATIONS: DYRK,dual specificity tyrosine-phosphorylation regulated; VEGFR2,vascular endothelial growth factor receptor-2; SAR,structure/activity relationship

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REFERENCES

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