Pyrimidine nucleotides play crucial roles in the biosynthesis of DNA, RNA, glycoproteins and membrane lipids [1]. Human dihydroorotate dehydrogenase (hDHODH), located in the inner membrane of mitochondria is a flavin-dependent enzyme and plays a key role in the de novo pyrimidine biosynthesis [2]. This redox enzyme is rate-limiting enzyme that catalyzes the oxidation of dihydroorotate to orotate in the fourth of the six conserved enzymatic reactions involved in the de novo pyrimidine biosynthetic pathway. Inhibition of pyrimidine biosynthesis by targeting hDHODH could be a promising strategy for the treatment of cancer [3], anti-infective diseases [4] and autoimmune diseases, such as rheumatoid arthritis, psoriatic arthritis and multiple sclerosis [5].

Pyrrolo[3,4-c]quinoline-1,3-diones incorporate N-substituted cyclic imide moiety, an important structural motif and well known building block for synthesis of drugs and pharmacologically active molecules. The drugs with cyclic imide structure are thalidomide and its analogues, lenalidomide, pomalidomide and iberdomide, known as immunomodulatory drugs (IMiDs) with ability to bind cereblon (CRBN) promoting degradation of IKZF1 and IKZF3 through ubiquitination-dependent proteasome pathway. They can modulate function of T cells and NK cells by production of cytokines including interleukin-2 and interferon γ (Fig. 1) [6].

The effective inhibitors of dihydroorotate dehydrogenase are also immunomodulatory drugs because they modulate pyrimidine biosynthesis. One of the most prominent hDHODH inhibitors, vidofludimus calcium, is selective oral immunomodulator that inhibit the intracellular metabolism of activated immune T-cells by blocking enzyme hDHODH [7].

A variety of inhibitors targeting human DHODH have been reported over the years, among which brequinar, leflunomide and teriflunomide have been deeply analyzed (Fig. 2). Leflunomide and its active metabolite teriflunomide have been FDA-approved as human DHODH-targeting drugs for the treatment of rheumatoid arthritis and multiple sclerosis [8]. However, the long-term use of leflunomide can cause serious side effects, such as hypertension, interstitial lung disease, gastrointestinal distress, liver damage, nausea and hair loss [5]. Brequinar, fluorinated quinoline-4-carboxylic acid derivative, is one of the most potent hDHODH inhibitors with significant antitumor and immunosuppressive activities. Unfortunately, brequinar failed in all clinical trials for solid tumors [9]. C44 is a very potent quinoline-4-carboxylic acid derivative that inhibits human DHODH with an IC50 of 1 nM [10]. Many innovative hDHODH inhibitors have new scaffolds in their structure, such as Vidofludimus, BAY 2402234 and S416 (Fig. 2). Vidofludimus is a selective and potent hDHODH inhibitor with no structural similarity to other known inhibitors and was considered for the treatment of rheumatoid arthritis, multiple sclerosis, inflammatory bowel disease and systemic lupus erythematosus [11]. BAY 2402234 is a novel and selective hDHODH inhibitor (IC50 = 1.2 nM) for treating myeloid malignancies and is currently being evaluated in phase I trial [12]. The hDHODH inhibitor S416 with a favorable pharmacokinetic profile showed antiviral effects against a variety of RNA viruses, including the coronavirus SARS-CoV-2 [13].

Many hDHODH inhibitors contain fluorine in their structure in order to improve activity towards the selected biological targets. However, the novel investigations show “dark side of fluorine” because drug-metabolizing enzymes can catalyze oxidative defluorination and release fluoride generating highly toxic effects [14], [15].

In our previous works, numerous quinoline derivatives with various structural modifications, targeting hDHODH were synthesized and tested for their enzyme inhibitory activity [16], [17], [18]. The current interest of our research group is directed to synthetic ways and biological potential of fused quinoline heterocycles, especially pyrrolo[3,4-c]quinoline-1,3-diones. These compounds exhibit a wide range of biological activities, for example, inhibitory activities against caspase-3 [19], HCV polymerase for treatment of hepatitis C virus [20] and as highly selective agonists, antagonists or inverse agonists of g-aminobutyric acid (GABA) brain receptors (Fig. 3) [21].

Considering all the above facts, we envisioned that small molecules, N-substituted pyrrolo[3,4-c]quinoline-1,3-diones, containing cyclic imide scaffold could also be good hDHODH inhibitors with favorable pharmacological profile. Consequently, we have prepared twenty N-substituted pyrrolo[3,4-c]quinoline-1,3-diones using simple synthetic protocol recently developed in our lab [22]. To our delight, hDHODH is validated as a very promising target for our compounds and here we also report cytotoxicity toward non-cancerous human keratinocyte cells HaCaT as well as human melanoma cancer cells A375, a toxicological evaluation of the three most active compounds and their comparison with the reference drug leflunomide.