Design, synthesis and biological evaluation of substituted pyrrolo[2,3-d]pyrimidines as multiple receptor tyrosine kinase inhibitors and antiangiogenic agents

Direct and indirect involvement of receptor tyrosine kinases (RTKs) in tumor growth and metastasis makes them ideal targets for anticancer therapy. A paradigm shift from inhibition of single RTK to inhibition of multiple RTKs has been recently demonstrated. We designed and synthesized eight N(4)-phenylsubstituted-6-(2-phenylethylsubstituted)-7H-pyrrolo[2,3-d]pyrimidine-2,4-diamines as homologated series of our previously published RTK inhibitors. We reasoned that increased flexibility of the side chain, which determines potency and selectivity, would improve the spectrum of RTK inhibition. These compounds were synthesized using a bis-electrophilic cyclization to afford substituted pyrrolo[2,3-d]pyrimidines followed by chlorination and substitution at the 4-position with various anilines. Five additional compounds of this series were previously reported by Gangjee et al.(1) with activities against IGFR only. Their synthesis, characterization and biological activities against a variety of other RTKs are reported in this study for the first time. The biological evaluation, in whole cell assays, showed several analogs had remarkable inhibitory activity against epithelial growth factor receptor (EGFR), vascular endothelial growth factor receptor-1 (VEGFR-1), platelet-derived growth factor receptor-beta (PDGFR-beta), the growth of A431 cells in culture, and in the chicken embryo chorioallantoic membrane (CAM) angiogenesis assay. The inhibitory data against the RTKs in this study demonstrate that variation of the 6-ethylaryl substituents as well as the N(4)-phenyl substituents of these analogs does indeed control both the potency and specificity of inhibitory activity against RTKs. In addition, homologation of the chain length of the 6-substituent from a methylene to an ethyl increases the spectrum of RTK inhibition. New multi-RTK inhibitors (8, 12) and potent inhibitors of angiogenesis (15, 19) were identified with the best compound, N(4)-(3-trifluromethylphenyl)-6-(2-phenylethyl)-7H-pyrrolo[2,3-d]pyrimidine-2,4-diamine (15), with an IC(50) value of 30nM in the CAM angiogenesis inhibition assay.     

N⁴-(3-Bromophenyl)-7-(substituted benzyl) pyrrolo[2,3-d]pyrimidines as potent multiple receptor tyrosine kinase inhibitors: design, synthesis, and in vivo evaluation

With the goal of developing multitargeted receptor tyrosine kinase inhibitors that display potent inhibition against PDGFRβ and VEGFR-2 we designed and synthesized eleven N(4)-(3-bromophenyl)-7-(substitutedbenzyl) pyrrolo[2,3-d]pyrimidines 9a-19a. These compounds were obtained from the key intermediate N(4)-(3-bromophenyl)-7H-pyrrolo[2,3-d]pyrimidine-2,4-diamine 29. Various arylmethyl groups were regiospecifically attached at the N7 of 29 via sodium hydride induced alkylation with substituted arylmethyl halides. Compounds 11a and 19a were potent dual inhibitors of PDGFRβ and VEGFR-2. In a COLO-205, in vivo tumor mouse model 11a demonstrated inhibition of tumor growth, metastasis, and tumor angiogenesis that was better than or comparable to the standard compound TSU-68 (SU6668, 8).     

Antiangiogenic and antitumor agents. Design, synthesis, and evaluation of novel 2-amino-4-(3-bromoanilino)-6-benzylsubstituted pyrrolo[2,3-d]pyrimidines as inhibitors of receptor tyrosine kinases

Several different classes of growth factor receptors containing tyrosine kinases (RTK) are directly or indirectly involved in angiogenesis. Inhibition of these RTKs has provided a new paradigm in the treatment of tumors by restricting their growth and metastasis. We have designed, synthesized and evaluated eleven novel 2-amino-4-(3-bromoanilino)-6-substituted benzyl pyrrolo[2,3-d]pyrimidines as the first in a series of RTK inhibitors. These analogues were synthesized from appropriate alpha-bromomethylbenzyl ketones by cyclocondensation with 2,6-diamino-4-pyrimidone to afford the 2-amino-4-oxo-6-substituted benzyl pyrrolo[2,3-d]pyrimidines. Chlorination of the 4-position followed by displacement with 3-bromoaniline afforded the target compounds. In some instances, the 2-amino moiety of the pyrrolo[2,3-d]pyrimidines was protected prior to the chlorination and displacement followed by deprotection. The compounds were evaluated as inhibitors of vascular endothelial growth factor receptors VEGFR-2 (Flk-1, KDR) and VEGFR-1 (Flt-1); epidermal growth factor receptor (EGFR); and platelet-derived growth factor receptor-beta (PDGFR-beta). Selected compounds were also evaluated against the growth of A431 cells (which overexpress EGFR) in culture and as inhibitors of angiogenesis in the chicken embryo chorioallantonic membrane (CAM) assay. In each evaluation, a known standard compound was used as a comparison. Of the 11 analogues, five were more potent or equipotent as compared to standard compounds against the growth factor receptors. Two analogues showed superior inhibition of A431 cells in culture compared to the standard compounds. Three analogues were equipotent with the standard compound in the CAM assay and four of the analogues were dual inhibitors of RTKs. The structure-activity relationship for inhibition of different RTKs was quite distinct and different, and for VEGFR-2 and EGFR diametrically opposite. The inhibitory data against the RTKs in this study demonstrates that variation of the substituent(s) in the benzyl ring of these 2-amino-4-anilino 6-benzyl pyrrolo[2,3-d]pyrimidines does indeed control both the potency and specificity of inhibitory activity against RTKs.     

Synthesis, antiproliferative, and antiplatelet activities of oxime- and methyloxime-containing flavone and isoflavone derivatives

Certain oxime- and methyloxime-containing flavone and isoflavone derivatives were synthesized and evaluated for their antiproliferative activity against three solid cancer cells, human cervical epithelioid carcinoma (HeLa), hepatocellular carcinoma (SKHep1), and oral squamous cell carcinoma (SAS), which are commonly seen in Asian countries, including Taiwan. Selective compounds were also evaluated in the full panel of 60 human tumor cell lines and their mean GI50 values were obtained. The preliminary assays indicated flavone-6-yl derivatives are the most cytotoxic while isoflavone-7-yl derivatives are the best antiplatelet agents. Among them, (E)-6-(2-methoxyiminopropoxy)-2-phenyl-4H-1-benzopyran-4-one (14), (Z)-6-(2-hydroxyimino-2-phenylethoxy)-2-phenyl-4H-1-benzopyran-4-one (18a), and (Z)-6-[2-hydroxyimino-2-(4-methoxyphenyl)ethoxy]-2-phenyl-4H-1-benzopyran-4-one (18c) are three of the best antiproliferative agents with GI50 values of 0.8, 0.7, and 0.8 microM, respectively, against the growth of SKHep1; 0.9, 0.8, and 1.0 microM, respectively, against the growth of HeLa cells. Compound 18c is not only the most cytotoxic with a mean GI50 value of 0.08 microM against the full panel of 60 human tumor cell lines but also the only flavone derivative that exhibited a GI50 value of less than 1 microM against the growth of SAS. Flow cytometric analyses revealed that growth inhibition by 18c was due to accumulation in G2/M phase arrest and followed by apoptosis.     

Pyrrolo[2,1-d][1,2,3,5]tetrazine-4(3H)-ones,a new class of azolotetrazines with potent antitumor activity

Pyrrolo[2,1-d][1,2,3,5]tetrazinones 10a-o, compounds that hold the deaza skeleton of the antitumor drug temozolomide, were prepared by reaction of 2-diazopyrroles 9 and isocyanates. Such a synthetic route represents, among those leading to azolotetrazinones reported so far, the only possible one since attempts to cyclize to the title ring system 2-amino-1-carbamoylpyrroles 11 or the mono substituted 2-triazenopyrrole 12 failed. Compounds 10 were screened at the National Cancer Institute (NCI) for their activity against a panel of about 60 human tumor cell lines. Most of them possess remarkable antineoplastic activity having GI(50) values in the low micromolar or sub-micromolar range and reaching, in the case of compound 10d, nanomolar concentrations. The most sensitive cell lines were MDA-N and MDA-MB-435 of the breast sub-panel, and SR, K-562, HL60 (TB) and CCRF-CEM of the leukaemia sub-panel. SAR evaluation and COMPARE computations indicate, for compounds 10, a mechanism of action different from that of temozolomide.     

Synthesis and antileukemic activity of 16E-[4-(2-carboxy)ethoxybenzylidene]-androstene amides

In order to determine the structural requirements for cytotoxicity against various tumor cell lines, a new series of 16E-arylidene androstene amides with varying degrees of unsaturation in ring A has been synthesized. Characterization and invitro cytotoxic studies of the newly synthesized compounds are discussed. The compounds on evaluation against various tumor cell lines exhibited significant growth inhibition on leukemia cell lines. 3-Chloro-16E-{[4-(4-methylpiperazin-1-yl)-2-oxoethoxy]benzylidene}androst-5-en-17-one (10) emerged as the most potent compound of the series with GI(50) values of 3.94, 2.61, 6.90 and 1.79μM against CCRF-CEM, K-562, RPMI-8226 and SR leukemia cell lines, respectively.     

Antitumor agents 187: synthesis and cytotoxicity of substituted 8,8-dimethyl-2H,8H-pyrano[6,5-h]quinoline-2-one and related compounds

Several substituted 8,8-dimethyl-2H,8H-pyrano[6,5-h]quinoline-2-ones and related compounds were synthesized and evaluated for their in vitro cytotoxicity against a panel of human tumor cell lines. The most active compound (3) showed significant cytotoxic activity with GI50 values in the micromolar range.     

Synthesis, structural characterization, and in vitro antitumor activity of novel N-(6-chloro-1,1-dioxo-1,4,2-benzodithiazin-3-yl)arylsulfonamides

A new series of N-(6-chloro-1,1-dioxo-1,4,2-benzodithiazin-3-yl)arylsulfonamides 23-48 have been synthesized as potential anticancer agents. All compounds were screened for their cytotoxic activity against six human tumor cell lines. The selected compounds 23-27, 30, 31, 33, 35, 38, 42, 45, and 46 were further tested at the US National Cancer Institute for their in vitro activities against a panel of 53-59 human tumor cell lines. The compounds 23-26, 30, 31, 33, 38, 42, 45, and 46 showed 50% growth inhibitory activity in low micromolar concentration (GI(50)=0.03-4.9 microM) against one or more human tumor cell lines (Table 3). The prominent compound with remarkable activity (GI(50)=0.03 microM, TGI=1.3 microM) and selectivity toward melanoma UACC-257 cell line was N-(6-chloro-7-cyano-1,1-dioxo-1,4,2-benzodithiazin-3-yl)-N-(phenyl)-5-bromothiophene-2-sulfonamide 46.     

Synthesis of a new class of 2-anilino substituted nicotinyl arylsulfonylhydrazides as potential anticancer and antibacterial agents

A series of N'-1-[2-anilino-3-pyridyl]carbonyl-1-benzenesulfonohydrazide derivatives (7a-i) was synthesized and five of them were selected by the National Cancer Institute (NCI) and evaluated for their in vitro anticancer activity. Three of the investigated compounds 7d, 7f and 7g exhibited significant anticancer activity in the primary assay and further tested against a panel of 60 human tumour cell lines. Compound 7g showed 50% growth inhibitory activity in leukaemia, melanoma, lung cancer, colon cancer, renal cancer and breast cancer cells with GI(50) value of 3.2-9.6 microM. The synthesized compounds (7a-i) were also evaluated for their antibacterial activity against various Gram-positive and Gram-negative strains of bacteria. Most of these compounds showed better inhibitory activity in comparison to the standard drugs.     

Synthesis and evaluation of the antiproliferative activity of novel isoindolo[2,1-a]quinoxaline and indolo[1,2-a]quinoxaline derivatives

A novel series of isoindolo[2,1-a]quinoxaline and indolo[1,2-a]quinoxaline derivatives was synthesized and evaluated in vitro against various human cancer cell lines for antiproliferative activity. These new compounds displayed activity against leukemia and breast cancer cell lines in the 3- to 18-μM concentration range.     

Benzimidazole bearing oxadiazole and triazolo-thiadiazoles nucleus: Design and synthesis as anticancer agents

Two new series of benzimidazole bearing oxadiazole[1-(1H-benzo[d]imidazol-2-yl)-3-(5-substituted-1,3,4-oxadiazol-2-yl)propan-1-ones (4a-l)] and triazolo-thiadiazoles[1-(1H-benzo[d]imidazol-2-yl)-3-(6-(substituted)-[1,2,4]triazolo[3,4-b][1,3,4]thiadiazol-3-yl)propan-1-one (7a-e)] have been synthesized successfully from4-(1H-benzo[d]imidazol-2-yl)-4-oxobutanehydrazide (3) with an aim to produce promising anticancer agents. In vitro anticancer activities of synthesized compounds were screened at the National Cancer Institute (NCI), USA, according to their applied protocol against full NCI 60 human cell lines panel; results showed good to remarkable anticancer activity. Among them, compound (4j, NCS: 761980) exhibited significant growth inhibition and further screened at 10-fold dilutions of five different concentrations (0.01, 0.1, 1, 10 and 100μM) with GI(50) values ranging from 0.49 to 48.0μM and found superior for the non-small cell lung cancer cell lines like HOP-92 (GI(50) 0.49, TGI 19.9,LC(50) >100 and Log(10)GI(50) -6.30, Log(10)TGI -4.70, Log(10)LC(50) >-4.00).     

The synthesis and SAR of 2-amino-pyrrolo[2,3-d]pyrimidines: a new class of Aurora-A kinase inhibitors

A new class of Aurora-A inhibitors have been identified based on the 2-amino-pyrrolo[2,3-d]pyrimidine scaffold. Here, we describe the synthesis and SAR of this novel series. We report compounds which exhibit nanomolar activity in the Aurora-A biochemical assay and are able to inhibit tumor cell proliferation. This study culminates in compound 30, an inhibitor with potent activity against Aurora A (IC50=0.008 microM), anti-proliferative activity against several tumor cell lines and induces polyploidy in H460 cells.     

Synthesis and cytotoxic activity evaluation of indolo-, pyrrolo-, and benzofuro-quinolin-2(1H)-ones and 6-anilinoindoloquinoline derivatives

Certain indolo-, pyrrolo-, and benzofuro-quinolin-2(1H)-ones 4a,b, 6, 8, 16a-c and 6-anilinoindoloquinoline derivatives 10a,b, 11a,b, 12a,b have been synthesized and evaluated in vitro against a 3-cell lines panel consisting of MCF7 (Breast), NCI-H460 (Lung), and SF-268 (CNS). Those active compounds 4a,b, 6, 8, 10a,b, 11a,b, 12a,b were then evaluated in the full panel of 60 human tumor cell lines derived from nine cancer cell types. The results have shown that cytotoxicity decreases in the order of 6-anilinoindoloquinolines>indoloquinolin-2(1H)-ones>pyrroloquinolin-2(1H)-ones>benzofuroquinolin-2(1H)-ones. Among them, 1-[3-(11H-indolo[3,2-c]quinolin-6ylamino)phenyl]ethanone oxime hydrochloride (11a) and its 2-chloro derivative (11b) were most active, with mean GI(50) values of 1.70 and 1.35 microM, respectively. Both compounds 11a,b were also found to inhibit the growth of SNB-75 (CNS cancer cell) with a GI(50) value of less than 0.01 microM, and, therefore, were selected for further evaluation for in vivo antitumor activity.     

Syntheses and cytotoxicity evaluation of bis(indolyl)thiazole, bis(indolyl)pyrazinone and bis(indolyl)pyrazine: analogues of cytotoxic marine bis(indole) alkaloid

2,4-Bis(3'-indolyl)thiazoles, 3,5-bis(3'-indolyl)-2(1H)pyrazinone and 3,6-bis(3'-indolyl)pyrazine were synthesized and evaluated for cytotoxic activity against diverse human cancer cell lines by the National Cancer Institute. These compounds demonstrated significant inhibitory effects in the growth of a range of cancer cell lines. 2,4-Bis(3'-indolyl)thiazole displayed selective cytotoxicity against certain leukemia cell lines with GI50 values in the low micromolar range while the substituted derivatives showed a broad spectrum of cytotoxic activity. 3,5-Bis(3'-indolyl)-2(1H)pyrazinone and 3,6-bis[3'-(N-methyl-indolyl)]pyrazine possessed strong inhibitory activity against a wide range of human tumor cell lines. The mechanism of action remained unknown. The results suggested that 2,4-bis(3'-indolyl)thiazoles, 3,5-bis(3'-indolyl)-2(1H)pyrazinone and 3,6-bis[3'-(N-methyl-indolyl)] pyrazine offer potential as lead compounds for the discovery of anticancer agents.     

Synthesis and biological evaluation of novel steroidal[17,16-d][1,2,4]triazolo[1,5-a]pyrimidines

The preparation of steroidal[17,16-d][1,2,4]triazolo[1,5-a]pyrimidines and their biological evaluation as potential anticancer agents are herein reported. These novel heterosteroids (2, 4) were prepared through the condensation reaction of 3-amino-1,2,4-triazole with 16-arylidene-17-ketosteroids (1, 3). All the synthesized compounds were evaluated for their anticancer activity in vitro against PC-3 (human prostatic carcinoma), MCF-7 (human breast carcinoma) and EC9706 (human esophageal carcinoma) cell lines. Among the screened compounds, 2i, 2n and 4f showed significant inhibitory activity against all the three human cell lines.     

Antitumor agents. Part 3: synthesis and cytotoxicity of new trans-stilbene benzenesulfonamide derivatives

A new series of trans-stilbene benzenesulfonamide derivatives were designed and synthesized as potential antitumor agents. These new compounds were evaluated in the National Cancer Institute's 60 human tumor cell line in vitro screen. Compounds 9-13 were cytotoxic against several cell lines. Notably, two compounds, 9 and 12, demonstrated selective cytotoxic activity against BT-549 breast cancer (GI(50)=0.205 microM) and HT-29 colon cancer (GI(50)=0.554 microM), respectively.     

Design and Synthesis of Novel Thiazolo[5,4‐d]pyrimidine Derivatives as Potential Angiogenesis Inhibitors

Vascular endothelial growth factor receptor‐2 (VEGFR‐2) plays an important role in both vasculogenesis and angiogenesis. Inhibition of VEGFR‐2 has been demonstrated as a key method against tumor‐associated angiogenesis. Thiazolopyrimidine is an important analog of the purine ring, and we choose the thiazolopyrimidine scaffold as the mother nucleus. Two series of thiazolo[5,4‐d]pyrimidine derivatives were synthesized and evaluated for their antiproliferative activity. In HUVEC inhibition assay, compounds 3l (=1‐(5‐{[2‐(4‐chlorophenyl)‐5‐methyl[1,3]thiazolo[5,4‐d]pyrimidin‐7‐yl]amino}pyridin‐2‐yl)‐3‐(3,4‐dimethylphenyl)urea) and 3m (=1‐(5‐{[2‐(4‐chlorophenyl)‐5‐methyl[1,3]thiazolo[5,4‐d]pyrimidin‐7‐yl]amino}pyridin‐2‐yl)‐3‐(4‐methoxyphenyl)urea) exhibited the most potent inhibitory effect (IC₅₀=1.65 and 3.52 μm , respectively). Compound 3l also showed the best potency against VEGFR‐2 at 50 μm (98.5 %). These results suggest that further investigation of compound 3l might provide potential angiogenesis inhibitors.     

Synthesis and antitumoral activity of novel 3-(2-substituted-1,3,4-oxadiazol-5-yl) and 3-(5-substituted-1,2,4-triazol-3-yl) beta-carboline derivatives

Several novel 1-substituted-phenyl beta-carbolines bearing the 2-substituted-1,3,4-oxadiazol-5-yl and 5-substituted-1,2,4-triazol-3-yl groups at C-3 were synthesized and evaluated for their in vitro anticancer activity. The assay results pointed thirteen compounds with growth inhibition effect (GI(50)<100 microM) for all eight different types of human cancer cell lines tested. The beta-carbolines 7a and 7h, bearing the 3-(2-metylthio-1,3,4-oxadiazol-5-yl) group, displayed high selectivity and potent anticancer activity against ovarian cell line with GI(50) values lying in the nanomolar concentration range (GI(50)=10 nM for both compounds). The 1-(N,N-dimethylaminophenyl)-3-(5-thioxo-1,2,4-triazol-3-yl) beta-carboline (8g) was the most active compound, showing particular effectiveness on lung (GI(50)=0.06 microM), ovarian and renal cell lines. The potent anticancer activity presented for synthesized compounds 7a, 7h, and 8g, together with their easiness of synthesis, makes these compounds promising anticancer agents.     

Synthesis and in vitro antitumor evaluation of some indeno[1,2-c]pyrazol(in)es substituted with sulfonamide, sulfonylurea(-thiourea) pharmacophores, and some derived thiazole ring systems

The synthesis of a series of 3-(4-chlorophenyl)-[1,2-c]pyrazol(in)es substituted with benzenesulfonamide, N1,N3-disubstituted sulfonylurea, sulfonylthiourea pharmacophores, and some derived thiazolidinone and thiazoline ring systems is described. All the newly synthesized target compounds were subjected to the NCI-in vitro disease-oriented antitumor screening to be evaluated for their antitumor activity. Eight compounds namely; 2-4, 7, 8, 10, 13, and 16; showed promising broad spectrum antitumor activity against most of the tested subpanel tumor cell lines (GI50 < 100 microM). Compound 3, 4-(3-(4-chlorophenyl)-4H-indeno[1,2-c]pyrazol- 2-yl)-benzenesulfonamide; although it did not show the highest growth inhibitory value (GI50 (MG-MID) 13.2 microM), it proved to be the most active analog in this study with the highest cytostatic and cytotoxic potentials (TGI and LC50 (MG-MID) concentrations of 33.1 and 66.1 microM, respectively). In general, the oxidized pyrazoles displayed better antitumor activity than their parent pyrazoline analogs, whereas the benzenesulfonamides and the N1, N3-disubstituted sulfonylureas showed significant better antitumor spectrum than the sulfonylthioureido and the derived thiazole analogs.     

Synthesis of new indeno[1,2-e]pyrimido[4,5-b][1,4]diazepine-5,11-diones as potential antitumor agents

Novel racemic indeno[1,2-e]pyrimido[4,5-b][1,4]diazepine-5,11-diones 3-29 were obtained regioselectivily from the reaction of 5,6-diamino-3,4-dihydropyrimidin-4-ones 1 and 2-arylideneindandiones 2 as reagents. These compounds have been evaluated at the US National Cancer Institute (NCI) for their ability to inhibit approximately 60 different human tumor cell lines, where 5 and 6 presented remarkable activity against 57 and 48 cancer cell lines, respectively, with the most important GI(50) values ranging from 0.49 to 1.46 microM, in vitro assay.