Discovery of new quinazoline derivatives as irreversible dual EGFR/HER2 inhibitors and their anticancer activities – Part 1

Overexpression of EGFR and HER2 are observed in many breast, ovarian, colon and prostate cancers. The second and third generation irreversible EGFR/HER2 dual kinase inhibitors became popular after the approval of Afatinib by FDA to overcome the mutation related problem. To find efficacious drug candidates, a series of novel quinazoline derivatives were designed, synthesized and evaluated as dual EGFR/HER2 tyrosine kinase (TK) inhibitors. Selected twenty four compounds were reported here with significant inhibitory activities against EGFR/HER2 tyrosine kinases. Several compounds showed nanomolar IC₅₀ values. In vitro studies of quinazoline derivatives were done on NCI-H1975, HCC827, A431, MDA MB-453 cell lines. The compounds 1a, 1d and 1v were found more potent compared to standard drug afatinib. In vivo efficacy study of 1d on nude mice NCI-H1975 tumour xenograft model was discussed.     

Synthesis and biological evaluation of novel tricyclic oxazine and oxazepine fused quinazolines. Part 1: Erlotinib analogs

Two series of novel tricyclic oxazine and oxazepine fused quinazolines have been designed and synthesized. The in vitro antitumor effect of the title compounds was screened on N87, A431, H1975, BT474 and Calu-3 cell lines. Compared to erlotinib and gefitinib, compounds 1a–1h were found to demonstrate more potent antitumor activities. Several derivatives could counteract EGF-induced phosphorylation of EGFR in cells, and their potency was comparable to the reference compounds. Compounds 1a–1h were chosen for further evaluation of EGFR and HER2 in vitro kinase inhibitory activity. Compounds 1b–1f, 1h effectively inhibited the in vitro kinase activity of EGFR and HER2 with similar efficacy as erlotinib and gefitinib.     

Identification of quinones as HER2 inhibitors for the treatment of trastuzumab resistant breast cancer

HER2 overexpression is associated with aggressive breast cancer with high recurrence rate and poor patient prognosis. Treatment of HER2 overexpressing patients with the HER2 targeting therapy trastuzumab results in acquired resistance within a year. The HER2/EGFR dual kinase inhibitor lapatinib was shown to inhibit some trastuzumab resistant breast cancer cell lines and is currently in clinical trials. Our group has found two new quinone compounds that show excellent inhibition of breast tumor cells expressing HER2 or the trastuzumab resistant HER2 oncogenic isoform, HER2Δ16. Compound 4 ((1R,2S,3S)-1,2,3,5,8-pentahydroxy-1,2,3,4-tetrahydroanthracene-9,10-dione) and compound 5 (5,8-dihydroxy-2,3-bis(hydroxymethyl)naphthalene-1,4-dione) showed sub-micromolar inhibition potency against these cell lines. These compounds also inhibit auto-phosphorylation of the Y1248 and Y1068 residues of HER2 and EGFR, respectively.     

Dual EGFR/HER2 inhibitors and apoptosis inducers: New benzo[g]quinazoline derivatives bearing benzenesulfonamide as anticancer and radiosensitizers

Dual targeting of EGFR and HER2 is a proven anticancer strategy for the treatment of solid tumors. An array of new N-substituted-2-(4-oxo-3-(4-sulfamoylphenyl)-3,4-dihydrobenzo[g]quinazolin-2-ylthio) acetamides 5–18 were designed and synthesized from the starting compound 4-(2-mercapto-4-oxobenzo[g]quinazolin-3(4H)-yl) benzenesulfonamide 4. The targeted compounds were screened for their cytotoxic activity against MDA-MB-231 breast cancer cell line. The IC₅₀ of all the compounds were in the range of 0.36–40.90 µM. The percentage inhibition towards EGFR was measured and found to be in the range of 63.00–16.90 %. The most potent compounds 5, 9, 15, 17 and 18 were further screened for their activity against both EGFR and HER2 receptors. The compounds showed IC₅₀ in the range of 0.64–1.81 µM for EGFR and 1.13–2.21 µM for HER2, in comparison to erlotinib, the reference drug. Compound 17, the most potent towards EGFR in this series, undergoes cell cycle analysis and was found to arrest the cycle at the G2/M phase. Measurement of the cytotoxicity of compound 17 against normal breast cell line showed mild cytotoxic activity. The most potent compounds were subjected to a single dose of 8 Gy of γ-radiation and the cytotoxicity of the tested compounds was found to increase after irradiation, thus proving the synergistic effect of γ-irradiation. Molecular docking was adopted for all the synthesized compounds to confirm their mechanism of action.     

Synthesis and investigation of novel 6-(1,2,3-triazol-4-yl)-4-aminoquinazolin derivatives possessing hydroxamic acid moiety for cancer therapy

By merging the critical pharmacophore of EGFR/HER2 and HDAC inhibitors into one compound, a novel series of EGFR, HER-2, and HDAC multitarget inhibitors were synthesized. Compounds 9a–l contained 4-anilinoquinazolines with C-6 triazole-linked long alkyl chains of hydroxamic acid and displayed excellent inhibition against these enzymes (compound 9d exhibited the best inhibitory potency on wild-type EGFR, HDAC1, and HDAC6 with IC₅₀ values 0.12 nM, 0.72 nM and 3.2 nM individually). Furthermore, compounds 9b and 9d potently inhibited proliferation of five human cancer cell lines (with IC₅₀ values between 0.49 and 8.76 μM). Further mechanistic study revealed that compound 9d also regulated the phosphorylation of EGFR and HER2 and histone H3 hyperacetylation on the cellular level and induced remarkable apoptosis in BT-474 cells. Therefore, our study suggested that a system network-based multi-target drug design strategy might provided an alternate drug design method, by taking into account the synergy effect of EGFR, HER-2 and HDAC.     

Design and synthesis of new benzoxazole/benzothiazole-phthalimide hybrids as antitumor-apoptotic agents

Herein, we synthesized a series of twelve benzoxazole and benzothiazole derivatives incorporated with phthalimide core as anticancer agents. The most active compounds were 5a and 5g against HepG2 and MCF7 cell lines with IC₅₀ = 0.011 and 0.006 μM, respectively.They evaluated against EGFR and HER2 enzymes. From cell cycle analysis, it was observed that test compounds exerted pre G1 apoptosis and cell cycle arrest at G2/M phase. The achieved results suggested that apoptosis was due to activation of caspase-7 and caspase-9. EGFR was chosen as a biological target for carrying molecular modeling study for the newly synthesized compounds.     

Discovery of 2-aryl-8-hydroxy (or methoxy)-isoquinolin-1(2H)-ones as novel EGFR inhibitor by scaffold hopping

2-Aryl-8-hydroxy (or methoxy)-isoquinolin-1(2H)-one has been proposed as a novel scaffold of EGFR inhibitor based on scaffold hoping. In the present study, a series of 2-aryl-8-hydroxy (or methoxy)-isoquinolin-1(2H)-one derivatives were synthesized. Their antiproliferative activities in vitro were evaluated via MTT assay against two human cancer cell lines, including A431 and A549. The SAR of the title compounds was preliminarily discussed. The compounds with ideal inhibition were evaluated through ELISA-based EGFR-TK assay. Compound 6c showed the best activity against A431 and EGFR tyrosine kinase. These findings suggest that title compounds are EGFR inhibitors with novel structures.     

Design of novel lipidated peptidomimetic conjugates for targeting EGFR heterodimerization in HER2 + cancer

The human epidermal growth factor receptor (EGFR) family is known to be involved in cell signaling pathways. The extracellular domain of EGFR consists of four domains, of which domain II and domain IV are known to be involved in the dimerization process. Overexpression of these receptors is known to play a significant role in heterodimerization of these receptors leading to the development of cancer. We have designed peptidomimetic molecules to inhibit the EGFR heterodimerization interaction that have shown antiproliferative activity and specificity for HER2-positive cancer cell lines. Among these, a peptidomimetic, compound 5, exhibited antiproliferative activity at low nanomolar concentrations in HER2-overexpressing cancer cell lines. To improve the stability of this peptidomimetic, we have designed and synthesized a novel conjugate of peptidomimetic compound 5 with a lipid, stearic acid. The antiproliferative activity of this conjugate was evaluated in HER2-positive cancer cell lines. Results suggested that the conjugate exhibited selective antiproliferative activity in HER2-overexpressing breast and lung cancer cell lines and was able to block HER2:HER3 heterodimerization. Also, the conjugate showed improved stability with a half-life of 5?h in human serum compared to the half-life of 2?h for parent compound 5. The binding affinity of the conjugate to HER2 protein was evaluated by SPR analysis, and the mode of binding of the lipid conjugate to domain IV of HER2 protein was demonstrated by docking analysis. Thus, this novel lipid conjugate can be used to target HER2-overexpressing cancers.     

Novel Pyrazoloquinolin-2-ones: Design,synthesis, docking studies,and biological evaluation as antiproliferative EGFR-TK inhibitors

Two new series of diethyl 2-[2-(substituted-2-oxo-1,2-dihydroquinolin-4-yl)hydrazono]-succinates 6a-g and 1-(2-oxo-1,2-dihydroquinolin-4-yl)-1H-pyrazoles 7a-f have been designed and synthesized. The structures of the synthesized compounds were proved by IR, mass, NMR (2D) spectra and elemental analyses. The target compounds were evaluated for their in vitro cytotoxic activity against 60 cancer cell lines according to NCI protocol. Consequently, seven compounds were further examined against the most sensitive cell lines, leukemia CCRF-CEM, and MOLT-4. 5-Amino-1-(6-bromo-2-oxo-1,2-dihydroquinolin-4-yl)-1H-pyrazole-3,4-dicarbonitrile (7f) was the most active product, with IC₅₀ = 1.35 uM and 2.42 uM against MOLT-4 and CCRF-CEM, respectively. Also, it showed a remarkable inhibitory activity compared to erlotinib on the EGFR TK with IC₅₀ = 247.14 nM and 208.42 nM, respectively. Cell cycle analysis of MOLT-4 cells treated with 7f showed cell cycle arrest at G2/M phase (supported by Caspases, BAX and Bcl-2 studies) with a significant pro-apoptotic activity as indicated by annexin V-FITC staining. Moreover, the docking study indicated that both the pyrazole moiety and the quinolin-2-one ring showed good fitting into EGFR (PDB code: 1M17). In order to interpret SAR of the designed compounds, and provide a basis for further optimization, molecular docking of the synthesized compounds to known EGFR inhibitors was performed. The study illustrated the effect of several factors on the compounds’ activity.     

Inhibitory effects of metachromins L–Q and its related analogs against receptor tyrosine kinases EGFR and HER2

Metachromins are a series of sesquiterpenoid quinones isolated from Okinawan marine sponges. Inhibitory effects of metachromins L–Q (1–6), sesquiterpenoid quinones with an amino acid residue, and their related analogs (7–18) prepared from metachromins A (19) and C (20) against receptor tyrosine kinases EGFR and HER2 were investigated. Two analogs 11 and 12 showed relatively stronger inhibitory activity against EGFR, while metachromins L–Q (1-6) and seven analogs (8, 10, 11, and 15–18) showed inhibitory activities against HER2.     

Hit discovery of 4-amino-N-(4-(3-(trifluoromethyl)phenoxy)pyrimidin-5-yl)benzamide: A novel EGFR inhibitor from a designed small library

Searching for hit compounds within the huge chemical space resembles the attempt to find a needle in a haystack. Cheminformatics-guided selection of few representative molecules of a rationally designed virtual combinatorial library is a powerful tool to confront this challenge, speed up hit identification and cut off costs. Herein, this approach has been applied to identify hit compounds with novel scaffolds able to inhibit EGFR kinase. From a generated virtual library, six 4-aryloxy-5-aminopyrimidine scaffold-derived compounds were selected, synthesized and evaluated as hit EGFR inhibitors. 4-Aryloxy-5-benzamidopyrimidines inhibited EGFR with IC₅₀ 1.05–5.37 μM. Cell-based assay of the most potent EGFR inhibitor hit (10ac) confirmed its cytotoxicity against different cancerous cells. In spite of no EGFR, HER2 or VEGFR1 inhibition was elicited by 4-aryloxy-5-(thio)ureidopyrimidine derivatives, cell-based evaluation suggested them as antiproliferative hits acting by other mechanism(s). Molecular docking study provided a plausible explanation of incapability of 4-aryloxy-5-(thio)ureidopyrimidines to inhibit EGFR and suggested a reasonable binding mode of 4-aryloxy-5-benzamidopyrimidines which provides a basis to develop more optimized ligands.     

Design and synthesis of pyrrolo[3,2-d]pyrimidine HER2/EGFR dual inhibitors: Improvement of the physicochemical and pharmacokinetic profiles for potent in vivo anti-tumor efficacy

During the course of our studies on a novel HER2/EGFR dual inhibitor (TAK-285), we found an alternative potent pyrrolo[3,2-d]pyrimidine compound (1a). To enhance the pharmacokinetic (PK) profile of this compound, we conducted chemical modifications into its N-5 side chain and conversion of the chemically modified compounds into their salts. Among them, 2cb, the tosylate salt of compound 2c, showed potent HER2/EGFR kinase inhibitory activity (IC₅₀: 11/11 nM) and cellular growth inhibitory activity (BT-474 cell GI₅₀: 56 nM) with a good drug metabolism and PK (DMPK) profile. Furthermore, 2cb exhibited significant in vivo antitumor efficacy in both mouse and rat xenograft models with transplanted 4-1ST gastric cancer cell lines (mouse, T/C = 0%, 2cb po bid at 100 mg/kg; rat, T/C: -1%, 2cb po bid at 25 mg/kg).     

Combination of 4-anilinoquinazoline,arylurea and tertiary amine moiety to discover novel anticancer agents

In present study, 4-anilinoquinazolines scaffold, arylurea and tertiary amine moiety were combined to design, synthesize gefitinib analogs and discover novel anticancer agents. A series of 4-anilinoquinazoline derivatives (1, 2, 3 and 4) bearing arylurea and tertiary amine moiety at its 6-position were synthesized. Their antiproliferative activities in vitro were evaluated via MTT assay against A431 cell and A549 cell. The SAR of the title compounds was discussed. The compounds 2d, 2i and 2j with potent antiproliferative activities were evaluated their inhibitory activity against EGFR-TK. Compound 2j displayed potent inhibitory activity against EGFR-TK. In addition, compound 2j, at 50 mg/kg, can completely inhibit cancer growth in established nude mouse A549 xenograft model in vivo. These results suggest that the 4-anilinoquinazoline derivatives bearing diarylurea and tertiary amino moiety at its 6-position can serve as anticancer agents and EGFR inhibitors.     

Novel inhibitors of epidermal growth factor receptor: (4-(Arylamino)-7H-pyrrolo[2,3-d]pyrimidin-6-yl)(1H-indol-2-yl)methanones and (1H-indol-2-yl)(4-(phenylamino)thieno[2,3-d]pyrimidin-6-yl)methanones

Several members of the quinazoline class of known tyrosine kinase inhibitors are approved anticancer agents, often showing selectivity for receptors of the HER/ErbB-family. Combining structural elements of this class with the bisindolylmethanone-structure led to a series of novel compounds. These compounds inhibited EGFR in the nanomolar range. Moreover, inhibition of EGFR autophosphorylation in intact A431 cells was shown, with IC₅₀ values ranging form 0.3–1 μM for compound 42, and 0.1–0.3 μM for 45. In a panel of 42 human tumor cell lines the sensitivity profile of the novel compounds was shown to be similar to that of the quinazoline class of tyrosine kinase inhibitors lapatinib and erlotinib (Tarceva®).     

Novel pyrrolo[2,1-f][1,2,4]triazin-4-amines: Dual inhibitors of EGFR and HER2 protein tyrosine kinases

A novel series of 5-((4-aminopiperidin-1-yl)methyl)-pyrrolo[2,1-f][1,2,4]triazin-4-amines with small aniline substituents at the C4 position were optimized for dual EGFR and HER2 protein tyrosine kinase inhibition. Compound 8l exhibited promising oral efficacy in both EGFR and HER2-driven human tumor xenograft models.     

Design,synthesis and biological evaluation of novel 6-alkenylamides substituted of 4-anilinothieno[2,3-d]pyrimidines as irreversible epidermal growth factor receptor inhibitors

A novel series of 6-alkenylamides of 4-anilinothieno[2,3-d]pyrimidine derivatives was designed, synthesized and evaluated as irreversible inhibitors of the epidermal growth factor receptor (EGFR). Most of the compounds exhibited good potency against EGFR wild type (EGFR wt) and EGFR T790M/L858R. Among these, the half-maximal inhibitory concentration (IC₅₀) values of 17 compounds against EGFR wt were less than 0.020 μM, and those of 12 compounds were less than 0.010 μM. The IC₅₀ values of 10 compounds against EGFR T790M/L858R were less than 0.005 μM. Compounds 8l, 9n, 9o, 9q and 9v almost completely blocked the phosphorylation of EGFR in the A431 cell line at 1 μM. Compounds 8l, 9n, 9o, 9q and 9v blocked the autophosphorylation of EGFR in NCI-H1975 cells at high concentration (1 μM), and compound 8l was confirmed to be an irreversible inhibitor through the dilution method.     

Toward discovery of mutant EGFR inhibitors; Design,synthesis and in vitro biological evaluation of potent 4-arylamino-6-ureido and thioureido-quinazoline derivatives

A new series of 4-anilinoquinazolines with C-6 ureido and thioureido side chains and various substituents at the C-4 anilino moiety was designed, synthesized and evaluated as wild type (WT) and mutant EGFR inhibitors. Most of the compounds inhibited EGFR kinase wild type (EGFR WT) with IC₅₀ values in the low nanomolar range (<0.495–9.05 nM) and displayed more potent cytotoxic effect in BaF/3 expressing EGFR WT than reference compound gefitinib. The anti-proliferative effect of all synthesized compounds against gefitinib insensitive double mutant cell lines Ba/F3 expressing Del19/T790M and Ba/F3 expressing L858R/T790M were assayed. Compounds 4d, 6f, 7e showed significant inhibition (IC₅₀ = 1.76–2.38 μM) in these mutant lines and significant Her2 enzyme inhibition (IC₅₀ = 19.2–40.6 nM) compared to lapatinib (60.1 nM). The Binding mode of compounds 6d, 6f, 7a, 7b and 8b were demonstrated. Furthermore, growth inhibition against gefitinib insensitive cell lines PC9-GR4 (Del19/T790M) were tested, compounds 6f and 7e showed about eight and three folds respectively greater potency than gefitinib. Our structure–activity relationships (SAR) studies suggested that presence of ethyl piperidino urea/thiourea at 6-position and bulky group of (3-chloro-4-(3-fluorobenzyloxy)phenyl)amino at 4-position of quinazoline may serve as promising scaffold for developing inhibitors against wild type and mutant EGFR.     

Design,synthesis and biological evaluation of 8-substituted-6-hydrazonoindolo[2,1-b]quinazolin-12(6H)-one scaffolds as potential cytotoxic agents: IDO-1 targeting molecular docking studies

Herein, we have reported the synthesis of 18 novel 8-substituted tryptanthrin analogues based on our earlier work. All these tryptanthrin analogues were well characterized by ¹H & ¹³C NMR, FT-IR, Mass Spectrometry and Elemental Analysis. All these 8-substituted analogues were screened for their anti-oxidant activity by DPPH radical scavenging assay. Out of all the tested compounds, T¹¹, T¹², T¹⁷ and T¹⁸ showed potent anti-oxidant activity. The anti-cancer activity have been performed by using MTT assay protocol and their results depicts that compounds having the 4-pyridyl or 4-carboxyphenyl substituents at the 8th position of the tryptanthrin framework are found to be the most promising cytotoxic agent against A549, MCF-7 and HeLa human cancer cell lines compared to others as well as with the standard drug cisplatin. Moreover, the comparative molecular docking studies against the three protein receptors IDO1, EGFR and HER2 strongly suggested that IDO1 is the best target protein, which exhibits lowest binding energies of ?11.73 and ?11.61 kcal mol^?1 for T¹¹ and T¹² scaffolds, respectively towards the in vitro anti-cancer activity.     

Benzo[g]quinazolin-based scaffold derivatives as dual EGFR/HER2 inhibitors

Targeting EGFR has proven to be beneficial in the treatment of several types of solid tumours. So, a series of novel 2-(4-oxo-3-(4-sulfamoylphenyl)-3,4-dihydrobenzo[g]quinazolin-2-ylthio)-N-substituted acetamide 5–19 were synthesised from the starting material 4-(2-mercapto-4-oxobenzo[g]quinazolin-3(4H)-yl) benzenesulfonamide 4, to be evaluated as dual EGFR/HER2 inhibitors. The target compounds 5–19, were screened for their cytotoxic activity against A549 lung cancer cell line. The percentage inhibition of EGFR enzyme was measured and compared with erlotinib as the reference drug. Compounds 6, 8, 10, and 16 showed excellent EGFR inhibitory activity and were further selected for screening as dual EGFR/HER2 inhibitors. The four selected compounds showed IC₅₀ ranging from 0.009 to 0.026?µM for EGFR and 0.021 to 0.069?µM for the HER2 enzyme. Compound 8 was found to be the most potent in this study with IC₅₀ 0.009 and 0.021?µM for EGFR and HER2, respectively.     

Discovery of novel thienoquinoline-2-carboxamide chalcone derivatives as antiproliferative EGFR tyrosine kinase inhibitors

Novel thienoquinoline carboxamide-chalcone derivatives were prepared via the cyclization of acylated chalcones and 2-mercaptoquinoline-3-carbaldehyde in DMF with K₂CO₃. Thienoquinolines 9a–f, h exhibited promising antiproliferative effect against all the tested cell lines and gave a significant activity as EGFR inhibitors, with IC₅₀ values ranging from 0.5 and 3.2?µM, and compounds 9e and 9f being the most active of the series. They also showed better activity than Erlotinib against melanoma cancer cell line A375. Moreover, compound 9f influenced pre G1 apoptosis and cell cycle arrest at G2/M phase. The binding mode of the best EGFR inhibitor 9e in the EGFR active site revealed that the thienoquinoline ring occupied the ATP-binding site while the chalcone moiety is located in the allosteric site and is responsible for the enhanced activity of these compounds.