Synthesis and biological activity of N4-phenylsubstituted-6-(2,4-dichloro phenylmethyl)-7H-pyrrolo[2,3-d]pyrimidine-2,4-diamines as vascular endothelial growth factor receptor-2 inhibitors and antiangiogenic and antitumor agents

A series of eight N⁴-phenylsubstituted-6-(2,4-dichlorophenylmethyl)-7H-pyrrolo[2,3-d]pyrimidine-2,4-diamines 8–15 were synthesized as vascular endothelial growth factor receptor-2 (VEGFR-2) inhibitors with varied substitutions in the phenyl ring of the 4-anilino moiety. In addition, five N⁴-phenylsubstituted-6-phenylmethylsubstituted-7H-pyrrolo[2,3-d]pyrimidin-4-amines 16–20 were synthesized to evaluate the importance of the 2-NH₂ moiety for multiple receptor tyrosine kinase (RTK) inhibition. Cyclocondensation of α-halomethylbenzylketones with 2,6-diamino-4-hydroxypyrimidine afforded 2-amino-6-(2,4-dichlorophenylmethyl)-3,7-dihydro-4H-pyrrolo[2,3-d]pyrimidin-4-one, 23 and reaction of α-bromomethylbenzylketones with ethylamidinoacetate followed by cyclocondensation with formamide afforded the 6-phenylmethylsubstituted-3,7-dihydro-4H-pyrrolo[2,3-d]pyrimidin-4-ones, 40–42, respectively. Chlorination of the 4-position and displacement with appropriate anilines afforded the target compounds 8–20. Compounds 8, 10 and 14 were potent VEGFR-2 inhibitors and were 100-fold, 40-fold and 8-fold more potent than the standard semaxanib, respectively. Previously synthesized multiple RTK inhibitor, 5 and the VEGFR-2 inhibitor 8 from this study, were chosen for further evaluation in a mouse orthotopic model of melanoma and showed significant inhibition of tumor growth, angiogenesis and metastasis.     

Design,synthesis and evaluation of 2-amino-4-m-bromoanilino-6-arylmethyl-7H-pyrrolo[2,3-d]pyrimidines as tyrosine kinase inhibitors and antiangiogenic agents

A series of 2-amino-4-m-bromoanilino-6-benzyl pyrrolo[2,3-d]pyrimidines analogues 4–12 were synthesized and evaluated as inhibitors of receptor tyrosine kinases (RTKs). These analogues were synthesized from the appropriate α-bromomethylbenzylketones via cyclocondensation with 2,6-diamino-4-pyrimidone to afford the 2-amino-4-oxo-6-substituted benzyl pyrrolo[2,3-d]pyrimidines. Chlorination at the 4-position followed by displacement with 3-bromoaniline or 3-bromo-N-methylaniline and methylation of the 7-NH afforded the target compounds. Remarkably, dimethylation of both the 4-N and N7 afford whole cell EGFR inhibitors that are more cytotoxic than clinically used erlotinib and mono-methylation at the 4-N or N7 affords more cytotoxic whole cell PDGFR-β inhibitors than clinically used sunitinib. Methylation at either the 4-N or N7 position was detrimental to whole cell VEGFR-2 inhibition. The inhibitory data against the RTKs in this study demonstrates that methylation of the 4-NH and/or the 7-NH influences both the specificity and potency of RTK inhibition.     

Novel 5-substituted, 2,4-diaminofuro[2,3-d]pyrimidines as multireceptor tyrosine kinase and dihydrofolate reductase inhibitors with antiangiogenic and antitumor activity

Recent evidence suggests that combination therapy of cancer with receptor tyrosine kinase (RTK) inhibitors, which are usually cytostatic, with conventional chemotherapeutic agents, which are usually cytotoxic, provide an improved treatment option. We have designed, synthesized, and evaluated a series of novel 2,4-diamino-5-substituted furo[2,3-d]pyrimidines with RTK and dihydrofolate reductase (DHFR) inhibitory activity in single molecules, as potential cytostatic and cytotoxic agents with antitumor activity. These compounds were synthesized from 2,4-diamino-5-chloromethyl furo[2,3-d]pyrimidine and aryl methyl ketones using the Wittig reaction to afford the C-8-C-9 unsaturated analogs followed by catalytic reduction to the corresponding saturated compounds. The saturated and unsaturated C-8-C-9 bridged compounds were evaluated as inhibitors of vascular endothelial growth factor receptor (VEGFR-2, Flk, KDR), epidermal growth factor receptor, and platelet-derived growth factor receptor-beta (PDGFR-beta). Selected analogs were also evaluated as antiangiogenic agents in the chicken embryo chorioallantoic membrane (CAM) assay. The compounds were also evaluated as inhibitors of human (h) DHFR and Toxoplasma gondii (tg) DHFR. In each evaluation, a known standard compound was used as a comparison. Of the compounds evaluated, compound 32 was as potent as the standard compounds against VEGFR-2 and PDGFR-beta, showing dual inhibitory activity against RTK. This analog was also highly effective in the CAM assay. A second analog 18 also demonstrated dual VEGFR-2 and PDGFR-beta inhibitory activity as well as potent antiangiogenic activity in the CAM assay. Four additional analogs were also effective against PDGFR-beta and in the CAM assay. An unsaturated C-8-C-9 moiety was necessary for RTK inhibitory activity. Compound 32 also showed inhibitory activity against hDHFR and tgDHFR, illustrating the multitarget inhibitory potential of these analogs. The biological activity of these analogs also suggests the necessity of an unsaturated C-8-C-9 bridge for dual RTK and DHFR inhibitory activity. Compounds 18 and 32 were also evaluated in a B16 melanoma mouse model and were found to be more active as antitumor agents than methotrexate. In addition, both 18 and 32 were also active in decreasing lung metastases in a mouse model of B16 melanomas.     

7-Phenyl-pyrido[2,3-d]pyrimidine-2,4-diamines: Novel and highly selective protein tyrosine phosphatase 1B inhibitors

High throughput screening of the Roche compound collection led to the identification of diaminopyrroloquinazoline series as a novel class of PTP1B inhibitors. Structural modification of diaminopyrroloquinazoline series resulted in pyrido[2,3-d]pyrimidine-2,4-diamine series which was further optimized to give compounds 5 and 24 as potent, selective (except T-cell phosphatase) PTP1B inhibitors with good mouse PK properties.     

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.     

The discovery and optimisation of pyrido[2,3-d]pyrimidine-2,4-diamines as potent and selective inhibitors of mTOR kinase

We describe a novel series of potent inhibitors of the kinase activity of mTOR. The compounds display good selectivity relative to other PI3K-related kinase family members and, in cellular assays, inhibit both mTORC1 and mTORC2 complexes and exhibit good antiproliferative activity.     

Design,synthesis and biological activity of N4-phenylsubstituted-7H-pyrrolo[2,3-d]pyrimidin-4-amines as dual inhibitors of aurora kinase A and epidermal growth factor receptor kinase

Simultaneous inhibition of multiple kinases has been suggested to provide synergistic effects on inhibition of tumour growth and resistance. This study describes the design, synthesis and evaluation of 18 compounds incorporating a pyrrolo[2,3-d]pyrimidine scaffold for dual inhibition of epidermal growth factor receptor kinase (EGFR) and aurora kinase A (AURKA). Compounds 1–18 of this study demonstrate nanomolar inhibition of EGFR and micromolar inhibition of AURKA. Compounds 1–18 allow for a structure–activity relationships (SAR) analysis of the 4-anilino moiety for dual EGFR and AURKA inhibition. Compound 6, a 4-methoxyphenylpyrrolo[2,3-d]pyrimidin-4-amine, demonstrates single-digit micromolar inhibition of both AURKA and EGFR and provides evidence of a single molecule with dual activity against EGFR and AURKA. Compound 2, the most potent inhibitor of EGFR and AURKA from this series, has been further evaluated in four different squamous cell head and neck cancer cell lines for downstream effects resulting from AURKA and EGFR inhibition.     

Synthesis and biological evaluation of pyrido[2,3-d]pyrimidine-2,4-dione derivatives as eEF-2K inhibitors

A small molecule library of pyrido[2,3-d]pyrimidine-2,4-dione derivatives 6–16 was synthesized from 6-amino-1,3-disubstituted uracils 18, characterized, and screened for inhibitory activity against eukaryotic elongation factor-2 kinase (eEF-2K). To understand the binding pocket of eEF-2K, structural modifications of the pyrido[2,3-d]pyrimidine were made at three regions (R¹, R², and R³). A homology model of eEF-2K was created, and compound 6 (A-484954, Abbott laboratories) was docked in the catalytic domain of eEF-2K. Compounds 6 (IC₅₀ = 420 nM) and 9 (IC₅₀ = 930 nM) are found to be better molecules in this preliminary series of pyrido[2,3-d]pyrimidine analogs. eEF-2K activity in MDA-MB-231 breast cancer cells is significantly reduced by compound 6, to a lesser extent by compound 9, and is unaffected by compound 12. Similar inhibitory results are observed when eEF-2K activity is stimulated by 2-deoxy-d-glucose (2-DOG) treatment, suggesting that compounds 6 and 9 are able to inhibit AMPK-mediated activation of eEF-2K to a notable extent. The results of this work will shed light on the further design and optimization of novel pyrido[2,3-d]pyrimidine analogs as eEF-2K inhibitors.     

Design,synthesis, and X-ray crystal structures of 2,4-diaminofuro[2,3-d]pyrimidines as multireceptor tyrosine kinase and dihydrofolate reductase inhibitors

To optimize dual receptor tyrosine kinase (RTK) and dihydrofolate reductase (DHFR) inhibition, the E- and Z-isomers of 5-[2-(2-methoxyphenyl)prop-1-en-1-yl]furo[2,3-d]pyrimidine-2,4-diamines (1a and 1b) were separated by HPLC and the X-ray crystal structures (2.0 and 1.4 Å, respectively) with mouse DHFR and NADPH as well as 1b with human DHFR (1.5 Å) were determined. The E- and Z-isomers adopt different binding modes when bound to mouse DHFR. A series of 2,4-diaminofuro[2,3-d]pyrimidines 2–13 were designed and synthesized using the X-ray crystal structures of 1a and 1b with DHFR to increase their DHFR inhibitory activity. Wittig reactions of appropriate 2-methoxyphenyl ketones with 2,4-diamino-6-chloromethyl furo[2,3-d]pyrimidine afforded the C8–C9 unsaturated compounds 2–7 and catalytic reduction gave the saturated 8–13. Homologation of the C9-methyl analog maintains DHFR inhibitory activity. In addition, inhibition of EGFR and PDGFR-β were discovered for saturated C9-homologated analogs 9 and 10 that were absent in the saturated C9-methyl analogs.     

The design and discovery of water soluble 4-substituted-2,6-dimethylfuro[2,3-d]pyrimidines as multitargeted receptor tyrosine kinase inhibitors and microtubule targeting antitumor agents

The design, synthesis and biological evaluations of fourteen 4-substituted 2,6-dimethylfuro[2,3-d]pyrimidines are reported. Four compounds (11–13, 15) inhibit vascular endothelial growth factor receptor-2 (VEGFR-2), platelet-derived growth factor receptor β (PDGFR-β), and target tubulin leading to cytotoxicity. Compound 11 has nanomolar potency, comparable to sunitinib and semaxinib, against tumor cell lines overexpressing VEGFR-2 and PDGFR-β. Further, 11 binds at the colchicine site on tubulin, depolymerizes cellular microtubules and inhibits purified tubulin assembly and overcomes both βIII-tubulin and P-glycoprotein-mediated drug resistance, and initiates mitotic arrest leading to apoptosis. In vivo, its HCl salt, 21, reduced tumor size and vascularity in xenograft and allograft murine models and was superior to docetaxel and sunitinib, without overt toxicity. Thus 21 affords potential combination chemotherapy in a single agent.     

Straightforward entry to pyrido[2,3-d]pyrimidine-2,4(1H,3H)-diones and their ADME properties

A straightforward synthesis of pyrido[2,3-d]pyrimidine-2,4(1H,3H)-diones was developed starting from 2-chloropyridine-3-carboxylic acid by esterification, nucleophilic aromatic substitution and amide formation in one step, and ring closure allowing their synthesis with two identical or two different group attached to nitrogen. The structural diversity of these [2,3-d]pyrimidine-2,4(1H,3H)-diones resulted in significant variation in the biopharmaceutical properties. This was reflected by the broad range in fasted state simulated intestinal fluid solubility values (12.6 μM to 13.8 mM), Caco-2 permeability coefficients (1.2 × 10⁻⁶ cm/s to 90.7 × 10⁻⁶ cm/s) and in vitro-predicted human in vivo intrinsic clearance values (0 to 159 ml/min/kg).     

3-Arylpiperazinylethyl-1H-pyrrolo[2,3-d]pyrimidine-2,4(3H,7H)-dione derivatives as novel, high-affinity and selective alpha(1)-adrenoceptor ligands

The discovery of a new series of selective and high-affinity alpha(1)-adrenoceptor (alpha(1)-AR) ligands, characterized by a 1H-pyrrolo[2,3-d]-pyrimidine-2,4(3H,7H)-dione system, is described in this paper. Some synthesized compounds, including 20, 22, and 30, displayed affinity in the nanomolar range for alpha(1)-ARs and substantial selectivity with respect to 5-HT(1A) and dopaminergic D(1) and D(2) receptors. Functional assays, performed on selected derivatives, showed antagonistic properties.     

Synthesis,Pim kinase inhibitory potencies and in vitro antiproliferative activities of diversely substituted pyrrolo[2,3-a]carbazoles

The synthesis of new pyrrolo[2,3-a]carbazole derivatives diversely substituted at the C-6 to C-9 positions is described. These compounds were tested for their kinase inhibitory potencies toward three kinases (Pim-1, Pim-2, Pim-3) as well as for their in vitro antiproliferative activities toward a human fibroblast primary culture and three human solid cancer cell lines (PC3, DU145, and PA 1). Moreover, molecular docking studies were performed to explain the enhanced inhibitory activity of the most active compound 3d.     

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.     

Identification of new pyrrolo[2,3-d]pyrimidines as potent VEGFR-2 tyrosine kinase inhibitors: Design,synthesis, biological evaluation and molecular modeling

Vascular endothelial growth factor receptor-2 (VEGFR-2) plays a crucial role in cancer angiogenesis. In the current study, a series of novel pyrrolo[2,3-d]pyrimidine based-compounds was designed and synthesized as VEGFR-2 inhibitors, in accordance to the structure activity relationship (SAR) studies of known type II VEGFR-2 inhibitors. The newly synthesized compounds were evaluated for their ability to inhibit VEGFR-2 kinase enzyme in vitro. All the tested compounds demonstrated highly potent dose-related VEGFR-2 inhibition with IC₅₀ values in nanomolar range. Among these compounds, pyrrolo[2,3-d]pyrimidine derivatives carrying biaryl urea moieties (12d and 15c) exhibited IC₅₀ values of 11.9 and 13.6 nM respectively. Additionally, most of the newly synthesized final compounds were tested on 60 human cancer cell lines. Docking of these compounds into the inactive conformation of VEGFR-2 was performed which showed comparable binding modes to that of the FDA approved VEGFR-2 kinase inhibitors. These newly discovered potent kinase inhibitors could be considered as potential candidates for the development of new targeted anticancer agent.     

Synthesis and biological activity of 5-chloro-N4-substituted phenyl-9H-pyrimido[4,5-b]indole-2,4-diamines as vascular endothelial growth factor receptor-2 inhibitors and antiangiogenic agents

Inhibition of receptor tyrosine kinase (RTK) signaling pathways is an important area for the development of novel anticancer agents. Numerous multikinase inhibitors (MKIs) have been recently approved for the treatment of cancer. Vascular endothelial growth factor receptor-2 (VEGFR-2) is the principal mediator of tumor angiogenesis. In an effort to develop ATP-competitive VEGFR-2 selective inhibitors the 5-chloro-N⁴-substituted phenyl-9H-pyrimido[4,5-b]indole-2,4-diamine scaffold was designed. The synthesis of the target compounds involved N-(4,5-dichloro-9H-pyrimido[4,5-b]indol-2-yl)-2,2-dimethylpropanamide) as a common intermediate. A nucleophilic displacement of the 4-chloro group of the common intermediate by appropriately substituted anilines afforded the target compounds. Biological evaluation indicated that compound 5 is a potent and selective VEGFR-2 inhibitor comparable to sunitinib and semaxinib.     

In vitro cytotoxic,antibacterial, antifungal and urease inhibitory activities of some N4- substituted isatin-3-thiosemicarbazones

A series of 15 previously reported N⁴-substituted isatin-3-thiosemicarbazones 3a-o has been screened for cytotoxic, antibacterial, antifungal and urease inhibitory activities. Compounds 3b, 3e and 3n proved to be active in cytotoxicity assay; 3e exhibited a high degree of cytotoxic activity (LD₅₀ = 1.10 × 10^{? 5} M). Compound 3h exhibited significant antibacterial activity against B. subtilis, whereas compounds 3a, 3k and 3l displayed significant antifungal activity against one or more fungal strains i.e. T. longifusus, A. flavus and M. canis. In human urease enzyme inhibition assay, compounds 3g, 3k and 3m proved to be the most potent inhibitors, exhibiting relatively pronounced inhibition of the enzyme. These compounds, being non-toxic, could be potential candidates for orally effective therapeutic agents to treat certain clinical conditions induced by bacterial ureases like H. pylori urease. This study presents the first example of inhibition of urease by isatin-thiosemicarbazones and as such provides a solid basis for further research on such compounds to develop more potent inhibitors.     

Synthesis, antimicrobial and antimycobacterial evaluation of [2-(substituted phenyl)-imidazol-1-yl]-pyridin-3-yl-methanones

A series of [2-(substituted phenyl)-imidazol-1-yl]-pyridin-3-yl-methanones (1-11) were synthesized and screened for their antimicrobial and antimycobacterial activities. Further, a series of [2-(substituted phenyl)-benzimidazol-1-yl]-pyridin-3-yl-methanones (12-20) reported in our earlier study was also screened for their antimycobacterial activity. The antimycobacterial activity results indicated that [2-(4-Nitro-phenyl)-imidazol-1-yl]-pyridin-3-yl-methanone (8, minimum inhibitory concentration [MIC]?=?3.13 μg) was equipotent as standard drug ciprofloxacin and [2-(4-Nitro-phenyl)-benzimidazol-1-yl]-pyridin-3-yl-methanone (16, MIC?=?1.56 μg) was equipotent as standard drug ethambutol. The results of antimicrobial screening demonstrated that 2-[1-(Pyridine-3-carbonyl)-1H-imidazol-2-yl]-benzoic acid (compound 11, MIC?=?0.002 μg) was two times more effective than standard drug ciprofloxacin (MIC?=?0.004 μg) against tested bacterial strains and [2-(2,5-Dimethyl-phenyl)-imidazol-1-yl]-pyridin-3-yl-methanone (compound 3, MIC?=?0.005 μg) was equipotent to the reference compound, fluconazole against tested fungal strains.     

Discovery of substituted 3H-pyrido[2,3-d]pyrimidin-4-ones as potent,biased, and orally bioavailable sst2 agonist

The discovery of a novel 3H-pyrido[2,3-d]pyrimidin-4-one series as potent and biased sst2 agonists is described. This class of molecules exhibits excellent sst2 potency and selectivity against sst1, sst3, and sst5 receptors, and they are significantly more potent at inhibiting cAMP production than inducing internalization. The orally bioavailable 6-(3-chloro-5-methylphenyl)-3-(3-fluoro-5-hydroxyphenyl)-5-({methyl[(2S)-pyrrolidin-2-ylmethyl]amino}methyl)-3H,4H-pyrido[2,3-d]pyrimidin-4-one (36) also suppresses GH secretion in GHRH-challenged rats in a dose-dependent manner.