Chemistry of heterocyclic compounds is a significant contributor to the discovery and development of new drugs. Many pyrimidine scaffolds have been developed and synthesized as drug candidates with various pharmacological uses [1]. Pyrimidine derivatives find applications in many areas of chemistry, including organic, bioorganic, and medical chemistry, as well as agrochemistry. In addition, these compounds are found in both living plants and animals, including humans [2].

Thus, pyrimidines are contained in most nucleotides, including DNA and RNA, which makes them quite important. Moreover, pyrimidines are present in a broad range of other compounds, including benzenes (quinazolines), furans (furopyrimidines), pyrroles (pyrrolopyrimidines), and thiophene rings (thienopyrimidine). Among the condensed pyrimidines, thienopyrimidines are particularly important; depending on their articulation, they can have the following forms: thieno[2,3-d]pyrimidines I and thieno[3,2-d]pyrimidines II (Fig. 1A) [3], [4]. The synthesis of thieno[2,3-d]pyrimidine cognates was accomplished via several synthetic pathways with different starting chemical blocks [5]. The diversity of the synthetic pathways boosts the discovery of new candidates with significant and versatile pharmacological properties, which include antituberculosis [6], antioxidant [7], anti-inflammatory [8], antiviral [9], kinase inhibition [10], and antimicrobial [11] ones.

Heterocyclic compounds have powerful antitumor properties, quite prominent in medicinal chemistry. Their important cytotoxic effect against different types of human tumor cells also opens their application as enzymes or receptors for various kinase inhibitors. The scaffolds of thieno[2,3-d]pyrimidines I and thieno[3,2-d]pyrimidines II have exhibited effective antitumor activities [3], [12], [13], [14]. These scaffolds are prospective drug-like candidates with various additional modifications by functional groups. The bioactivity of series compounds based on these scaffolds has been widely investigated against different types of tumor cells (Fig. 1A, compounds II−V). In particular, compound III was used for human liver tumor cell line (HEPG2) [15]; compound IV has antitumor activity against non-small cell lung carcinoma (H460), human lung adenocarcinoma cell line (A549), and human malignant glioblastoma multiforme (U251) [16]; and compound V can inhibit growth of human cervix carcinoma (KB) [17].

Moreover, compounds based on thienopyrimidine derivatives are undergoing clinical trials and are further used in medical practice. For example, Relugolix (compound VI) is approved in the USA for the treatment of advanced prostate cancer [18] and in the EU for the therapy of advanced hormone-sensitive prostate cancer [19], [20]. PRX-08066 (compound VII) is another derivative of thieno[2,3-d]pyrimidine, which is a potent antagonist of serotonin 5-HT subtype 2B receptors and is used for the treatment of pulmonary diseases, i.e. for pulmonary arterial hypertension (phase II clinical trials). Its ability to inhibit fibroblast activation was also revealed, and it is actively being investigated as a potential antitumor drug [21]. Apitolisib (GDC-0980) is another example of the effective use of a derivative of thieno[2,3-d]pyrimidine for the treatment of prostate and breast tumor. It has demonstrated powerful antitumor efficacy in tumor models demonstrating PI3K hyperactivation or PTEN loss [22]. Olmutinib is an inhibitor of epidermal growth factor receptor (EGFR) tyrosine kinase, which has significant clinical activity and a controlled safety profile in patients with T790M‐positive non-small cell lung cancer [23]. The structure–activity relationships (SARs) of existing thienopyrimidine derivatives as antitumor agents were presented in Fig. 1D. The following tendency can be highlighted: (i) the formation of the pyrimidine ring is beneficial for antitumor activity (red), (ii) an aromatic or heteroaromatic ring fused with pyrimidine ring can improves the activity (green), (iii) large hydrophobic group in the pyrimidine ring increases the antitumor activity (purple), (iv) including small hydrophobic substituents in the aromatic or heteroaromatic ring is helpful to promote the activity of the compound as well (blue). The influence of SARs on the antitumor activity of thienopyrimidine derivatives was described in review papers [12], [24].

Although thienopyrimidine scaffolds are essential in the field of oncology, their synthesis for commercial aims is still challenging. In general, the synthesis of thienopyrimidine derivatives requires a multistep process with purification at each step. One-pot synthetic approach allows overcoming these disadvantages. This approach provides a reduced reaction time, low-cost procedure and increases the yield of target compounds [25], [26], [27]. These compounds exhibited a remarkable selective tumor activity. Inspired by early reported results, we modified the synthetic approach for potential manufacturing processes.

In this study, we report one-pot three-component synthesis of substituted pyrrolo[1,2-a]thieno[3,2-e]pyrimidin 12, containing additional functional groups for further modification as a new class of antitumor agents. We conducted extensive screening to reveal the candidates with the promising antitumor activity in tumor cells and tumor-bearing mice. In addition, we aimed to reveal potential molecular targets for tested compounds using molecular docking analysis.