The Ugi4CR as effective tool to access promising anticancer isatin-based α-acetamide carboxamide oxindole hybrids

Considering early-stage drug discovery programs, the Ugi four-component reaction is a valuable, flexible, and pivotal tool, facilitating the creation of two new amide bonds in a one-pot fashion to effectively yield the desired α-aminoacylamides. Here, we highlight the reputation of this reaction approach to access number and scaffold diversity of a library of isatin-based α-acetamide carboxamide oxindole hybrids, promising anticancer agents, in a mild and fast sustainable reaction process. The library was tested against six human solid tumor cell lines, among them, non-small cell lung carcinoma, cervical adenocarcinoma, breast cancer and colon adenocarcinoma. The most potent compounds 8d, 8h and 8k showed GI50 values in the range of 1–10 μM.


Introduction
Meticulous attention has been given by chemists regarding process formation of new bonds and synthesis of new scaffolds.In drug discovery and development, medicinal chemists struggle everyday towards the creation of new synthetic methods, driven by the increasing complexity of the molecules and taking into consideration economic and social aspects.Multicomponent reactions (MCRs) are remarkable tools which demonstrated great potential for more sustainable production of active pharmaceutical ingredients (API's).These flexible and versatile one-pot transformations in which three or more reagents are combined to access a new complex scaffold with remarkable atom economy, cost and time-effective and mainly diminishing waste production is a conscientious boost for structural diversity and sustainability [1][2][3].The well-known Passerini, Ugi, Mannich, Biginelli, Hantzsch and Strecker reactions are some examples of the classic MCRs, representing the easygoing generation of a collection of small-molecules essential for structure-activity relationships (SAR).The isocyanidebased Ugi reaction is one of the most resourceful tools and still broadly studied MCR, generating multifunctional libraries of α-aminoacylamide derivatives, or Ugi adducts, with stereochemistry control [4,5].Unquestionable potential of application in the pharmaceutical industry is recognizable by the number of APIs obtained by this reaction approach [6,7].The oxindole framework is a privileged unit, recognized massively by its extensive biological applications [8,9].In the last few years we have been active in isatin modification using new synthetic approaches, anticipating the creation of new libraries of smallmolecule hybrids with potential as cholinesterase inhibitors [10][11][12][13], important to treat neurodegenerative diseases, and anticancer agents [14][15][16] (Figure 1).
Cancer is a complex, cureless and fatal disease, oftentimes diagnosed worldwide.Being one of the leading causes of death worldwide, it is expected an increase of 47% with 28.4 million cases diagnosed, in 2040 [17,18].Despite long years of research, there is still an urgent need to find novel, effective and safe drugs for cancer therapy.
Recently, focusing on the design of more potent anticancer drug candidates using more sustainable synthetic processes, we report a new Ugi four-component reaction approach for easy access to Ugi-derived isatin-peptoids in moderate to excellent yields (up to 99% yield).Some selected compounds were screened against five human solid tumor cell lines: lung (A549), breast (HBL-100 and T-47D), cervix (HeLa) and colon (WiDr).Preliminary SAR studies have revealed the preference of the N-benzylisatin structure over the 3,3-protected-oxindole, ali-phatic chain on the acid component and small aliphatic chain on the aldehyde component to increase the antiproliferative activity.Also, benzyl isocyanide was favored over the aliphatic one (Scheme 1A) [16].Considering the value of amide groups in drug discovery [19], the feasibility of running the isatin-based Ugi reaction [16,[20][21][22][23] and the potential of the bis-amide-oxindole type derivatives as anticancer agents, a second family was synthesized, and screened for their anticancer activities (Scheme 1B).
Resourcefulness of the Ugi4CR and preliminary SAR studies [16] lead us to synthesize a third library of oxindole derivatives, using trifluoroacetic acid (TFA), under mild reaction conditions, to afford the corresponding α-acetamide carboxamide isatin hybrids 8 from the 3-protected oxindole counterparts 5 and 7, in moderate to good yields (Scheme 4).The best yield was obtained when 3-protected oxindole derivatives 5 possess an aromatic or heterocyclic unit substituted in the 5-amide position of the oxindole ring.Compounds 8c, 8d, 8e and 8m were obtained in 83, 72, 74 and 84% yield, respectively.An exception was noticed for N-heterocycle units (pyridine and 1H-pyrrole) substituted in the same position, since compounds 8g, 8l and 8i were obtained in 56, 51 and 36% yield, respectively.The 1,2,3-triazole hybrid isatin compound 8n was obtained in 56% yield.Compounds 5ib and 5hb, with N-Boc protectedheterocycle units in the 5-amide position of the oxindole ring (Scheme 2) failed to afford the corresponding 3-deprotected isatin hybrids, since only decomposition byproducts (not identified) were obtained.

Antiproliferative activity
Considering the potential antiproliferative activity of these compounds, we screened 14 α-acetamide carboxamide isatin hybrids against six human solid tumor cell lines.The panel of cell lines comprised non-small cell lung carcinoma A549 and SW1573, cervical adenocarcinoma HeLa, breast cancer HBL-100 and T-47D, and colon adenocarcinoma WiDr.The halfmaximal growth inhibitory concentration (GI 50 ) values after 48 hours of exposure were calculated for each compound (Table Scheme 4  S1, Supporting Information File 1).The standard anticancer drug cisplatin (CDDP) was used as positive control.The results are viewed as GI 50 range plot (Figure 3).The compounds were classified in three groups according to the GI 50 range plot.The first group included the most active compounds 8d, 8h and 8k.These compounds exhibited antiproliferative effects in the range of 1-10 µM against all cell lines.The second group comprised the less potent compounds, which were 8a, 8b, 8g and 8i.In this group, the GI 50 values were higher than 10 µM in all cell lines tested.Finally, the third group enclosed the compounds that displayed a larger GI 50 range with relevant activity against some cell lines (GI 50 < 10 µM), but less potent against the others (GI 50 > 10 µM).Some structure-activity relationships derived from the GI 50 values.The presence of a nitro group at the furan moiety enhanced the activity (8d > 8m).Presumably, the nitro group made 8d the most potent analogue bearing an aromatic amide (8c, 8e, 8g, 8i, 8l-n).For the aliphatic amides, the most potent derivatives were 8h and 8k.The former is an α,β-unsaturated amide, which could react with nucleophiles inside the cell and thus explain its relative potency.The latter bears a long aliphatic side chain (thirteen carbon atoms), which could allow anchoring to cell membranes, representing a potential target.Overall, the results of the biological activity allow speculating that the compounds from the series 8a-n might exhibit diverse mode of actions.Taking all these considerations as a whole, further studies of the biological activity of compounds 8d, 8h and 8k might provide insights into the mode of action.Generally, the biological results point out the relevance of these isatin hybrids as privileged scaffolds for the development of new therapeutically relevant substances.

Conclusion
Two new families of α-acetamide carboxamide oxindole and isatin hybrids were synthesized efficiently using the sustainable and efficient Ugi4CR approach.Easy access to isatin from the 3-protected oxindole scaffold was demonstrated using mild reaction conditions.Flexibility of the carboxylic acid component and also the carbonyl one (ketone/aldehyde) was exhibited in the library of Ugi adducts obtained in moderate to good yields, in a fast and clean reaction process.Among the library of α-acetamide carboxamide isatin hybrids, 14 were tested regarding their antiproliferative activity.Compounds 8d, 8h and 8k were found to be the most potent ones, with GI 50 values in the range of 1-10 μM.Further studies on the mode of action and lead-discovery are in progress and will be reported shortly.

Figure 1 :
Figure 1: (A) Accessing libraries of oxindole hybrids using commercially available isatin as starting material and (B) most potent oxindole derivatives as BuChE inhibitors and anticancer agents, by Marques et al. [10-16].

Figure 3 :
Figure 3: GI 50 range plot against human solid tumor cell lines of investigated α-acetamide carboxamide isatin hybrids.Green most potent, yellow intermediate, red less potent.