Selective inhibition of EGFR and VEGFR2 tyrosine kinases controlled by a boronic acid substituent on 4-anilinoquinazolines†
Hiroyuki
Nakamura
*,
Ryoji
Horikoshi
,
Taikou
Usui
and
Hyun Seung
Ban
Department of Chemistry, Faculty of Science, Gakushuin University, Mejiro, Toshima-ku, Tokyo 171-8588, Japan. E-mail: hiroyuyki.nakamura@gakushuin.ac.jp; Fax: +81 3 3986 0221; Tel: +81 3 5992 1029
First published on 8th September 2010
Abstract
Boronic acid-containing 4-anilinoquinazolines were synthesized as selective inhibitors of EGFR and VEGFR2 tyrosine kinases. The substituted position of the boronic acid is essential for control of both kinase inhibitions, and the boronic acid substituted at the para position of the aniline moiety exhibited significant inhibition of VEGFR2 tyrosine kinase.
Growth factor receptor tyrosine kinases play an important role for the signal transduction pathway in cell proliferation. Deregulation of the signaling pathway has been observed in many human tumors, thus these kinases have been investigated as potential targets for cancer therapy.1 The kinase inhibitors competitively bind to the ATP binding site of the protein kinases, which is relatively conserved, therefore enzymatic selectivity has been required for development of the kinase inhibitors. Among the various protein tyrosine kinase inhibitors reported,2 the 4-anilinoquinazolines have proved to have great potential for selectivity and potency.3 In 1994, Fry and coworkers first found the 4-anilinoquinazoline (PD153035) to be a specific inhibitor of epidermal growth factor receptor (EGFR) tyrosine kinase.4 Based on their findings, ZD-1839 (Iressa™)5,6 and OSI-774 (Tarceva™)7,8 have been developed as EGFR tyrosine kinase inhibitors and approved for non-small cell lung cancer (NSCLC) therapy. In the meantime, various 4-anilinoquinazoline framework-based protein kinase inhibitors have been reported including cyclin-dependent kinase 2 (CDK2),9 Src and Abl kinases,10 vascular endothelial growth factor receptor (VEGFR) tyrosine kinases,11 and platelet-derived growth factor receptor (PDGFR) kinase.12
Although organoborons have been developed as nucleophilic reagents for C–C bond formation in organic synthesis,13 the use of boron as a candidate functional group to interact with a target protein is an attractive strategy for drug design in medicine. A boron atom has a vacant orbital and interconverts with ease between the neutral sp2 and the anionic sp3 hybridization states, which generates a new stable interaction between a boron atom and a donor molecule through a covalent bond. In particular, several boronic acid compounds have been studied as enzyme inhibitors including thrombin,14 lactamases,15 dipeptidyl peptidases,16 and others.17–20 However, the most promising achievement in the area of boron pharmaceutics is the development of bortezomib (PS341), a proteasome inhibitor,21 recently approved for clinical treatment of relapsed multiple myeloma and mantle cell lymphoma. The X-ray crystal structure of the 20S proteasome in complex with bortezomib was reported. In this structure, the boronic acid of bortezomib covalently interacts with the Thr-1 hydroxyl in the active site of the 20S proteasome, forming the hybridized borate.22 Recently, we reported the prolonged inhibitory activity of a boron-conjugated 4-anilinoquinazoline toward the EGFR tyrosine kinase. The quantum mechanical docking simulation revealed that the boronic acid moiety substituted at the 6 position of the quinazoline with a benzyl linker formed a covalent B–O bond with Asp800 in addition to hydrogen bonds with Asp800 and Cys797, which may cause the prolonged inhibition of the compound toward EGFR tyrosine kinase.23 In this paper, we introduce a boronic acid, as an alternative candidate for a functional group, into the 4-anilinoquinazoline framework in pharmaceutical drug design and found that the selective inhibition of EGFR and VEGFR tyrosine kinases was controlled by a boronic acid substituent on 4-anilinoquinazolines (Fig. 1).
 | ||
| Fig. 1 Design of boronic acid-containing 4-anilinoquinazolines. | ||
The synthesis of the diboron coupling precursors 3 and 5 is shown in Scheme 1. Substitution of 4-chloroquinazoline 1 with 3-chloroaniline and 3-chloro-4-fluoroaniline gave the corresponding 4-anilinoquinazlines 3a and 3b in 69 and 87% yields, respectively.24 In a similar manner, 4-chloroquinazoline 2 was reacted with 3-chloroaniline and 3-chloro-4-fluoroaniline, and the resulting 4-anilinoquinazolines 4a and 4b were treated with aqueous ammonia followed by anhydrous trifluoromethane sulfonic acid to afford the corresponding triflates 5a and 5b in 15 and 66% yields, respectively, from 2 in three steps.
 | ||
| Scheme 1 Reagents and conditions: (a) anilines, isopropanol, reflux, 3a (69%) or 3b (87%); (b) i) 25% NH3 aq; ii) Tf2O, Py. | ||
The Suzuki–Miyaura diboron coupling reaction was employed for 3 or 5 in the presence of palladium(II) catalysts in dimethylformamide (DMF) at 80 °C to give 6a–d in 21–58% yields. Finally, deprotection of the pinacol boronate moiety was carried out under transesterification conditions using KHF2 and phenylboronic acid to afford the corresponding boronic acids 7a–d in 32–48% yields (Scheme 2).
 | ||
| Scheme 2 Reagents and conditions: (a) pinacolatodiboron, PdCl2, dppf, KOAc, DMF, 80 °C; (b) i) KHF2, MeOH–H2O; ii) PhB(OH)2. | ||
Meanwhile, the boron-conjugated 4-anilinoquinazolines, in which a boronic acid moiety was substituted on the aniline ring, were synthesized by the substitution reaction of chloroquinazolines 8a–c and boronated anilines as shown in Scheme 3. The m-substituted boronic acids 9a–c were obtained from 8a–c with (3-aminophenyl)boronic acid under acidic conditions. On the contrary, the p-substituted boronic acids 12a–c were obtained from 8a–c with commercially availrable pinacolatoboronic ester 10 under acidic condition. The resulting boronic esters 11a–c were treated with KHF2 to afford the corresponding boronic acids 12a–c.
 | ||
| Scheme 3 Reagents and conditions: (a) i) (3-aminophenyl)boronic acid, conc. HCl, isopropanol; ii) NaHCO3, MeOH–H2O; (b) 10, conc. HCl, isopropanol; (c) KHF2, MeOH–H2O. | ||
Inhibitory activity of the boron-conjugated 4-anilinoquinazolines 6, 7, 9 and 12 against EGFR, HER2, Flt-1 and KDR tyrosine kinases was determined by measuring the levels of phosphorylation of the tyrosine kinase-specific peptides (poly(Glu:Tyr) substrate) in vitro.25 As shown in Table 1, the boron-conjugated 4-anilinoquinazolines 6a–d and 7a–d, which have a boronate ester or boronic acid group substituted at the C-6 position of the quinazoline framework, selectively suppressed EGFR tyrosine kinase activity without inhibiting HER2, Flt-1 (VEGFR1 tyrosine kinase catalytic domain) or KDR (VEGFR2 tyrosine kinase catalytic domain) kinases, and their IC50 values against EGFR tyrosine kinase were at the range of 0.46–0.80 μM. On the contrary, the compounds 9a–c, and 12a–c, which have a boronic acid group substituted at the aniline ring of the quinazolines, displayed selective inhibition toward KDR tyrosine kinase. In particular, a boronic acid group substituted at the para position of the aniline, such as the compounds 12a–c, is potent for significant inhibitory activity of KDR. The IC50 values of 12a and 12b are 0.036 and 0.037 μM, respectively, which is similar to that of a known KDR inhibitor, AAL993 (IC50 = 0.014 μM).26
| Compds | IC50/μM a | |||
|---|---|---|---|---|
| EGFR | HER2b | Flt-1b | KDRb | |
| a The drug concentrations required to inhibit the phosphorylation of the poly(Glu:Tyr) substrate by 50% (IC50) were determined from semilogarithmic dose–response plots, and results represent mean ± s.d. of triplicate samples. Percent of inhibition at 1 μM concentration was indicated in parentheses. b -, no inhibitory effect at 1 μM. | ||||
| 6a | 0.59 ± 0.03 | >1 (31) | >1 (12) | >1 (-) |
| 6b | 0.57 ± 0.04 | >1 (-) | >1 (8) | >1 (-) |
| 6c | 0.49 ± 0.08 | >1 (-) | >1 (-) | >1 (-) |
| 6d | 0.52 ± 0.07 | >1 (-) | >1 (-) | >1 (-) |
| 7a | 0.65 ± 0.02 | >1 (15) | >1 (7) | >1 (-) |
| 7b | 0.80 ± 0.07 | >1 (-) | >1 (-) | >1 (-) |
| 7c | 0.46 ± 0.05 | >1 (-) | >1 (-) | >1 (-) |
| 7d | 0.51 ± 0.01 | >1 (-) | >1 (-) | >1 (-) |
| 9a | >1 (46) | >1 (-) | >1 (-) | 0.39 ± 0.03 |
| 9b | >1 (24) | >1 (-) | >1 (-) | 0.19 ± 0.02 |
| 9c | >1 (45) | >1 (-) | >1 (19) | 0.18 ± 0.02 |
| 12a | >1 (40) | >1 (-) | >1 (49) | 0.036 ± 0.006 |
| 12b | >1 (36) | >1 (-) | >1 (41) | 0.037 ± 0.012 |
| 12c | >1 (28) | >1 (-) | >1 (-) | 0.86 ± 0.12 |
| Tarceva | 0.047 ± 0.003 | >1 (-) | >1 (-) | >1 (38) |
| AAL993 | >1 (30) | >1 (-) | >1 (-) | 0.014 ± 0.002 |
We next examined the effects of the boron-conjugated 4-anilinoquinazolines on the EGF-induced tyrosine phosphorylation of EGFR and the signaling cascades in A431 cells by immunoblot analysis. Treatment of A431 with EGF (10 ng ml−1) rapidly induced autophosphorylation of EGFR, and the level of the phosphorylation reached a maximum at 10 min after EGF stimulation. Under these conditions, the boron-conjugated 4-anilinoquinazolines, 6a–d and 7a–d, potently suppressed the EGF-induced phosphorylation of EGFR at 1 μM concentration of compounds, whereas compounds 9a–c and 12a–c did not affect the EGFR phosphorylation at this concentration (Fig. 2A). It has been reported that the autophosphorylation of EGFR tyrosine kinase activated various downstream kinases including ERK and Akt, which play an important role in the regulation of cell proliferation and apoptosis, respectively.27 Therefore, we examined the effects on the EGFR-dependent activation of downstream signaling pathways. The boron-conjugated 4-anilinoquinazolines, 6a–d and 7a–d, also significantly suppressed the EGF-induced phosphorylation of ERK and Akt in parallel with the inhibition of EGFR autophosphorylation. However, these inhibitions were not observed in compounds 9a–c and 12a–c. These results indicate that the boron-conjugated 4-anilinoquinazolines, 6a–d and 7a–d, also induce the inhibitory effect on autophosphorylation of EGFR tyrosine kinase in cells as well as EGFR kinase in vitro, and arrest the downstream signaling pathway. Furthermore, we examined the effects of the boron-conjugated 4-anilinoquinazolines on the VEGF-induced phosphorylation of KDR in HUVECs. As shown in Fig. 2B, boron-conjugated 4-anilinoquinazolines 9b, 9c, 12b and 12c significantly suppressed VEGF-induced KDR phosphorylation. Although compounds 9a and 12a potently inhibited KDR kinase activity (Table 1), their inhibitory effect on KDR phosphorylation in HUVECs was not observed. The discrepancy of results between kinase assay and immunoblotting analysis might be induced by the weak membrane permeability property of dimethoxy groups in compounds 9a and 12a.
 | ||
| Fig. 2 Inhibition of the EGF-induced phosphorylation of EGFR and VEGF-induced phosphorylation of KDR. (A) A431 cells were incubated with the boron-conjugated 4-anilinoquinazolines (1 μM) or Tarceva (0.3 μM) and then stimulated with EGF (10 ng mL−1). (B) HUVECs were incubated with the boron-conjugated 4-anilinoquinazolines (1 μM) or AAL993 (1 μM) and then stimulated with VEGF (20 ng ml−1). The levels of each kinase were detected by immunoblot analysis with the specific antibody. | ||
Since the substitution of a boronic acid group on the aniline ring has been observed to be essential for selective inhibition of KDR tyrosine kinase, we next synthesized various functional groups-substituted 4-anilinoquinazolines from 4-chloroquinazoline 8 and anilines, and examined their effects on tyrosine kinase activity of EGFR, HER2, Flt-1, and KDR. As shown in Scheme 4, the substitution reaction proceeded and the corresponding 4-anilinoquinazolines 13 and 15–20 were obtained in high yields, except 4-(4′-hydroxymethylanilino)quinazoline 14 and 4-(3′-aminoanilino)quinazoline 21. The compound 14 generated from 8 and 4-hydroxymethylaniline was gradually decomposed in air and the reaction of 8 with 1,4-diaminobenzene gave the corresponding 4-anilinoquinazoline 21 in only 17% yield. In the synthesis of 4-(4′-aminoanilino)quinazoline 23, the first substitution reaction of 8 with Boc-protected 1,4-diaminobenzene was carried out and the resulting compound 22 was treated with trifluoromethanesulfonic acid (TFA) to give 23 in quantitative yield.
 | ||
| Scheme 4 Reagents and conditions: (a) anilines, isopropanol, reflux; (b) 4-(BocNH)C6H4NH2, isopropanol, reflux, 87%; (c) TFA, CH2Cl2, >99%; (d) conc. HCl, CH3OH. | ||
Table 2 shows the inhibition of a variety of functional groups substituted on the aniline moiety of 4-anilinoquinazolines, such as hydroxymethyl,28 carboxylic acid, methyl ester, cyano, and amine groups, toward various tyrosine kinase activity. Interestingly, compound 19, which has a cyano group substituted on a meta position of the aniline moiety, displayed high inhibition of EGFR tyrosine kinase (80%) similar to Tarceva (82%) at 1 μM concentration, indicating that both cyano and acetylene groups can be similarly bound to the kinase pocket. In all cases except AAL993, significant inhibition of KDR tyrosine kinase was not observed at 1 μM.
| Compds | % Inhibition at 1 μM a | |||
|---|---|---|---|---|
| EGFRb | HER2b | Flt-1b | KDRb | |
| a Percent of inhibition of the phosphorylation of the poly(Glu:Tyr) substrate at 1 μM concentration was indicated. b —, no inhibitory effect at 1 μM. | ||||
| 12a | 40 | — | 49 | 99 |
| 12b | 36 | — | 41 | 99 |
| 13 | 35 | — | — | 11 |
| 15 | — | — | 32 | 7 |
| 16 | 16 | — | — | 23 |
| 17 | 7 | — | — | 6 |
| 18 | 10 | — | — | 21 |
| 19 | 80 | — | — | 9 |
| 20 | 5 | — | — | — |
| 21 | 52 | — | — | 8 |
| 23 | 27 | 5 | — | 4 |
| 24 | 72 | 57 | 32 | 88 |
| 25 | 59 | — | — | 31 |
| Tarceva | 82 | — | — | 38 |
| AAL993 | 30 | — | 34 | 95 |
Since a boronic acid has been observed as an essential function for selective KDR kinase inhibition, we next examined whether the boronic acids 9a–c and 12a–c are active species in the kinase assay. It has been reported that an aryl boronic acid is able to undergo oxidation with reactive oxygen species (ROS) in cells to be converted to phenols.29 Furthermore, 4-(3′-hydroxyanilino)quinazoline 24, which is considered as an oxidation product of the corresponding boronic acid 9a, was reported as a selective KDR inhibitor (IC50 = 0.05 μM),30 and 4-(4′-hydroxyanilino)quinazoline 25, which may be generated from the corresponding boronic acid 12a in a similar manner, was reported as a selective JAK3 inhibitor (IC50 = 9.1 μM).31 Therefore, we synthesized the meta-hydroxyl derivative 24 and the para-hydroxyl derivative 25 according to the literature procedures30,32 and examined the inhibition toward various tyrosine kinase activity. As shown in Table 2, the meta-hydroxyl derivative 24 exhibited inhibitory potency of broad-spectrum tyrosine kinases, whereas the para-hydroxyl derivative 25 did not display significant inhibitory activity toward these kinases at 1 μM concentration. These results indicate that a boronic acid substituted on the aniline ring of the 4-anilinoquinazoline framework is essential for the selective inhibition of KDR tyrosine kinase.
Conclusions
We developed boronic acid-containing 4-anilinoquinazolines as selective inhibitors of EGFR and VEGFR2 tyrosine kinases. The substituted position of a boronic acid is essential for control of both kinase inhibitions. Since a boron atom is not observed in the living body, we believe that the current findings are promising for the utility of the boron atom as an alternative element in pharmaceutical drug design.Notes and references
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Footnote |
| † Electronic supplementary information (ESI) available: Experimental procedures and full characterisation for all new compounds. See DOI: 10.1039/c0md00115e |
| This journal is © The Royal Society of Chemistry 2010 |