Cytotoxicity and Antimicrobial Activity of GaMF1 Analogs

TL;DR

Scientists modified a compound originally targeting tuberculosis to reduce its toxicity and found it also works well against certain parasites and cancer cells.

Contribution

Structural modifications of GaMF1 reduced cytotoxicity and revealed new antiparasitic and antiproliferative properties.

Findings

Structural modifications of GaMF1 reduced its cytotoxicity against human cells.

Some GaMF1 analogs showed strong antiparasitic activity against Trypanosoma species.

Selected derivatives exhibited antiproliferative effects against tumor cell lines.

Abstract

Recent studies have identified the mycobacterial adenosine triphosphate synthase inhibitor GaMF1 and its structural analogs as compounds with noteworthy antituberculosis activity. Despite these promising results, a significant limitation remains their cytotoxicity against human cells, which, in its current state, overshadows the therapeutic potential. Therefore, addressing this off‐target toxicity is essential for the further development of these compounds as viable drug candidates. In this study, we systematically explored structural modifications of the original GaMF1 scaffold with the primary aim of reducing its inherent cytotoxicity. Individual regions of the parent structure were progressively replaced, enabling the identification of substituents that effectively attenuate cytotoxic effects. Importantly, these structural refinements also led to the emergence of pronounced antiparasitic activity, particularly against trypanosomal species such as Trypanosoma cruzi, Trypanosoma brucei brucei, and Trypanosoma brucei rhodesiense. These findings suggest that the biological potential of this compound class extends beyond what has previously been described. Furthermore, we evaluated the cytotoxicity of selected derivatives against a panel of tumor cell lines, where some compounds showed encouraging antiproliferative effects.

GaMF1 is a reported, highly potent inhibitor of mycobacterial ATP synthase. Structural modification of this scaffold significantly changes biological activity. We present GaMF1 analogues with strong antiparasitic effects, potential antiproliferative properties, and discuss the selectivity limitations of the parent compound and its analogues. Notably, our most active compounds demonstrate higher activity against Trypanosoma cruzi than drugs used to treat Chagas disease.© 2026 WILEY‐VCH GmbH

Figures

Recently reported compound GaMF1 (a) [13, 14, 15] and the herein reported structural analogs (b).

Synthesis of GaMF1 analogs 4. Reagents and conditions: (i) amine (R1), DIPEA, EtOH, 50°C, 20 h; (ii) p‐phenylenediamine, EtOH, MW, 200 W, 90°C, 30 min; (iii) 3‐bromobenzolchloride, K2CO3, THF, rt, 90 min.

Synthesis of derivative 4h. Reagents and conditions: (i) HCl/MeOH, quant.

Synthesis of derivative 4s. Reagents and conditions: (i) benzylazide, CuSO4 · 5H2O, (+)‐sodium l‐ascorbate, H2O/THF (1:1; v/v), 87%.

A perspective view of the crystal structure of 4d drawn with 50% probability of thermal ellipsoids. Color code: bromine ‐ brown, carbon ‐ light brown, hydrogen ‐ white, nitrogen ‐ light blue, oxygen ‐ red, sulfur ‐ yellow.

Synthesis of GaMF1 analogs 6. Reagents and conditions: (i) ethylamine, DIPEA, EtOH, 50°C, 20 h; (ii) diamine, (EtOH), MW, 200 W, 90°C, 30–120 min; (iii) 3‐bromobenzoylchloride, K2CO3 or Cs2CO3 THF, rt, 90 min.

Tables

TABLE 1. Overview of synthesized derivatives 4.

Entry	R 1	Yield a , %
4a		62
4b		45
4c		56
4d		68
4e		71
4f		62
4g		48
4h		quant.b
4i		39
4j		37
4k		37
4l		61
4m		48
4n		74
4o		69
4p		57
4q		40
4r		34
4s		87c

TABLE 2. Overview of synthesized derivatives 6.

Entry	Central part	Yield a , %
6a		44
6b		44
6c		51
6d		68
6e		64
6f		66
6g		67
6h		59
6i		65
6j		61
6k		32

TABLE 3. Cytotoxic activity of derivatives 4 and 6 (mean IC50 in µM).

Entry	MRC‐5	MCF7	MV4‐11
4a	1.28	6.69	14.21
4b	1.83	3.34	13.16
4c	1.28	1.76	3.56
4d	1.46	5.33	17.55
4e	1.31	3.52	7.99
4f	2.42	7.08	1.63
4g	0.86	16.51	10.64
4h	1.21	>25	24.23
4i	1.19	1.37	6.11
4j	1.08	1.31	8.16
4k	0.41	1.71	3.20
4l	0.45	4.19	2.49
4m	0.56	4.69	4.29
4n	1.24	1.55	3.45
4o	1.09	3.90	9.90
4p	0.25	4.55	3.47
4q	0.25	2.23	6.12
4r	20.41	16.64	10.47
4s	0.41	0.92	1.43
6a	12.97	>25.0	22.48
6b	1.18	6.85	6.53
6c	1.67	8.11	7.36
6d	>64	4.00	2.61
6e	1.49	1.39	4.30
6f	>64	21.10	16.69
6g	1.66	11.59	15.56
6h	1.83	17.72	16.95
6i	39.76	>25	>25
6j	7.89	>25	>25
6k	1.32	>25	>25

TABLE 4. Antimicrobial activity of derivatives 4 and 6 (IC50 in µM).a

Entry	MRC‐5	Mtb H37Ra	M. abscessus	L. inf	T. cruz	T. b. bruc	T. b. rhod	PMM cytotoxicity	S. aureus	E. coli
4r	20.41	>64	>64	2.03	2.11	1.64	0.84	2.00	>64	>64
6a	12.97	>64	25.40	27.27	8.27	25.08	9.04	32.00	64	>64
6d	>64	>64	>64	>64	1.82	1.83	1.42	>64	>64	>64
6f	>64	>64	>64	7.10	1.53	0.15	0.13	8.00	>64	>64
6i	39.76	>64	>64	>64	>64	>64	46.01	>64	>64	>64

Full text

Introduction

1

Mycobacterial adenosine triphosphate (ATP) synthase is a validated and essential target for tuberculosis therapy, as it plays a crucial role in maintaining cellular energy balance in both replicating and dormant Mycobacterium tuberculosis (Mtb) [1, 2]. Inhibiting this enzyme disrupts ATP production through oxidative phosphorylation, resulting in rapid bacterial death, even in low‐oxygen conditions. The discovery of bedaquiline, the first‐in‐class diarylquinoline approved for treating multidrug‐resistant tuberculosis, confirmed the therapeutic potential of targeting the ATP synthase complex—specifically, its membrane‐embedded subunit c [3]. Since then, several structural analogs and novel compounds have been developed to enhance potency, selectivity, and pharmacokinetic properties while minimizing toxicity [1].

Bedaquiline, the first clinically approved compound that targets the c subunit of the F_1_F_0_‐ATP synthase, effectively inhibits ATP synthesis in multidrug‐resistant Mtb. However, its use is limited by long half‐life, high lipophilicity, and cardiotoxicity. Structural analogs, particularly C‐pyridyl derivatives, have been developed to improve pharmacokinetic properties and reduce cardiotoxicity while maintaining strong antimycobacterial activity (MIC_90_ = 0.01–0.02 µg/ml) [4, 5]. Other scaffolds like thiazolidinones [6] or squaramides [7, 8, 9, 10, 11, 12] also demonstrate nanomolar potency against ATP synthase. These inhibitors not only validate ATP synthase as a crucial vulnerability of Mtb, but they also establish a foundation for designing next‐generation antituberculosis drugs that are effective against drug‐resistant and latent infections.

In 2020, Gerhard Grüber and his colleagues introduced a novel inhibitor of mycobacterial ATP synthase, referred to as GaMF1 (see Figure 1a) [13]. GaMF1 exhibited remarkable efficacy against multidrug‐resistant strains, including those resistant to bedaquiline. Furthermore, the authors demonstrated that GaMF1 targets a specific loop in the γ‐subunit of mycobacterial ATP synthase, representing a new mechanism to combat mycobacteria that is distinct from previously known derivatives. This discovery holds particular significance, as there is currently only one clinically approved drug, bedaquiline, that targets mycobacterial ATP synthase, and some strains have already developed resistance. The identification of the γ‐subunit loop as a mycobacterium‐specific structural motif absent in human ATP synthase introduced a unique and selective therapeutic target. GaMF1 was shown to bind directly to this regulatory loop, inhibiting ATP synthesis without disrupting proton translocation or oxygen consumption, thereby avoiding the off‐target mitochondrial effects.

Recently reported compound GaMF1 (a) [13, 14, 15] and the herein reported structural analogs (b).

In 2022, Grüber and his team made a noteworthy advancement in the field by demonstrating that GaMF1 is also effective against Mycobacterium abscessus [14]. This finding highlights the potential of GaMF1 as a treatment option for nontuberculous mycobacterial (NTM) infections, enhancing our understanding of its therapeutic applications and underscoring its importance in addressing NTM infections. Given the intrinsic resistance of NTMs to many standard antimycobacterial agents, the demonstration of GaMF1's efficacy against M. abscessus was particularly important. This rapidly growing mycobacterium is a clinically challenging pathogen associated with pulmonary and skin infections, for which current therapies are largely unsatisfactory.

Continued development highlighted the enhanced efficacy of structural analogs of GaMF1 when used in combination with various drugs, including clofazimine, telacebec, and TBAJ‐8776 [15]. This research provides valuable insights into potential therapeutic strategies and highlights the importance of combining treatments to achieve more effective treatment outcomes. These findings support a multi‐target approach to suppress mycobacterial energy metabolism, suggesting that GaMF1 analogs could serve as valuable components in future combinatorial regimens against tuberculosis and related infections.

Over the last few years, our group has become involved in developing mycobacterial ATP synthase inhibitors [8, 10, 11, 12]. Building on the insights gained from this research and the growing interest in novel inhibitors of this enzyme, we have decided to explore the GaMF1 analogs further. In this study, we present a comprehensive structure–activity relationship (SAR) study aimed at identifying the intrinsic limitations of these structural analogs and uncovering additional biological potential beyond antimycobacterial activity (see Figure 1b).

Our findings reveal that the original GaMF1 derivative, although highly potent, suffers from pronounced cytotoxicity against MRC‐5 cells, which precludes its therapeutic use. Consequently, the design of new analogs with improved safety profiles has become a central focus of our research. Through targeted structural modifications, we achieved attenuation of cytotoxic effects while simultaneously expanding the biological spectrum of this type of scaffold. Remarkably, several of the newly synthesized analogs displayed previously unreported antiparasitic activity, particularly against trypanosomes, as well as notable antitumor properties.

African trypanosomes are vector‐borne kinetoplastid parasites transmitted by tsetse flies that have evolved sophisticated immune evasion strategies, enabling persistent infections in both human and animal hosts. T. brucei rhodesiense is a causative agent of human African trypanosomiasis, commonly known as sleeping sickness, and is responsible for the acute form of the disease in East and Southern Africa [16, 17]. Although it accounts for only approximately 8% of human infections, with the majority caused by T. brucei gambiense, this parasite also represents a major veterinary burden, as livestock infections are widespread and often severe [18]. T. brucei brucei causes animal African trypanosomiasis, also known as nagana, a disease that severely affects livestock, including cattle, camels, and equids [19]. Alongside T. brucei brucei, other causative agents such as T. congolense and T. vivax further contribute to the economic and agricultural impact of this disease across sub‐Saharan Africa. The third representative, T. cruzi, is the etiological agent of American trypanosomiasis (Chagas disease), which affects an estimated 6–7 million people worldwide [20]. This chronic infection frequently leads to progressive cardiac damage and can ultimately be fatal [21]. Treatment of trypanosomal diseases remains highly challenging and is often limited by severe drug‐related toxicity, insufficient therapeutic efficacy, and the emergence of pathogen resistance, underscoring the urgent need for safer and more effective treatment options [22].

These results highlight, for the first time, that rational reengineering of the GaMF1 framework can transform its pharmacological profile, yielding compounds with reduced toxicity and promising activity in entirely new therapeutic domains.

Results and Discussion

2

Synthesis of Suggested Analogs

2.1

Our initial studies focused on synthesizing GaMF1 analogs 4 with modified side chains R^1^ (Scheme 1). The synthetic sequence commenced with the reaction of commercially available 2,4‐dichloro‐6‐methylpyrimidine 1 with the corresponding amine in the presence of N,N‐diisopropylethylamine (DIPEA). The reaction was conducted in ethanol at 50°C for 20 h, after which complete consumption of the starting material was confirmed by thin‐layer chromatography (TLC) and liquid chromatography‐mass spectroscopy (LC‐MS) analysis. Notably, the transformation yielded a mixture of two regioisomers in an approximate 2:1 ratio. Both regioisomers were successfully isolated and characterized by nuclear magnetic resonance (NMR) spectroscopy and high‐resolution mass spectrometry (HRMS), see Supporting Information, Table S1. These data for the minor regioisomers, which were not pursued further in the synthetic pathway, are provided in the Supporting Information.

Synthesis of GaMF1 analogs 4. Reagents and conditions: (i) amine (R1), DIPEA, EtOH, 50°C, 20 h; (ii) p‐phenylenediamine, EtOH, MW, 200 W, 90°C, 30 min; (iii) 3‐bromobenzolchloride, K2CO3, THF, rt, 90 min.

The reaction of intermediates 2 with p‐phenylenediamine to afford intermediates 3 was conducted under microwave irradiation (200 W) in EtOH, using an equimolar amount of p‐phenylenediamine. The transformation proceeded smoothly, with complete conversion of the starting material to the desired products 3 achieved always within 30 min, as confirmed by LC‐MS analysis. Owing to the high purity of the resulting intermediates 3, the crude reaction mixtures were only filtered through a layer of silica gel using a DCM/MeOH/TEA (9:1:0.1) eluent. The filtrates were concentrated, and the obtained products 3 were used directly in the final acylation step without further purification. The convenient conversion of intermediates 2 with p‐phenylenediamine can also be successfully achieved in EtOH under conventional reaction conditions by refluxing the reaction mixture for 16 h. However, the microwave‐assisted approach was preferred for the synthesis of the target derivatives 3 due to the substantially reduced reaction times.

The final acylation of intermediates 3 was performed using 3‐bromobenzoyl chloride in anhydrous THF with K_2_CO_3_ as the base. The reaction proceeded efficiently for all synthesized intermediates 3, without the notable formation of any side products. As a result, the target derivatives 4 were typically obtained in high isolated yields ranging from 34% to 74% (over the two reaction steps). All final compounds 4 are summarized in Table 1.

Upon completing the entire synthetic sequence, derivative 4g was subjected to a final deprotection step. This step was conducted using a saturated solution of hydrochloric acid (HCl) in methanol, as illustrated in Scheme 2. This method effectively removed the protecting group, allowing for the isolation of the final product 4h in its desired form.

Synthesis of derivative 4h. Reagents and conditions: (i) HCl/MeOH, quant.

We set out to explore additional synthetic strategies for expanding the side chain on the pyrimidine ring, specifically utilizing cycloaddition reaction using precursor 4m (refer to Scheme 3). This method presents valuable opportunities for the preparation of various analogs, thereby enhancing our synthetic repertoire and contributing to future research endeavors.

Synthesis of derivative 4s. Reagents and conditions: (i) benzylazide, CuSO4 · 5H2O, (+)‐sodium l‐ascorbate, H2O/THF (1:1; v/v), 87%.

To definitively confirm the formation of regioisomers 2 as a major product of the first step of our synthetic sequence (see Scheme 1), we prepared single crystals suitable for single‐crystal X‐ray diffraction analysis, specifically for derivative 4d (Figure 2). This compound crystallized as a DMSO solvate, with the solvent molecules forming relatively strong N—H···O hydrogen bonds with one of the amino groups (d(N···O) = 2.831(4) Å). Another amino group forms an N—H···O hydrogen bond with the keto oxygen atom of an adjacent molecule of 4d (d(N···O) = 2.935(4) Å), resulting in the formation of a one‐dimensional supramolecular substructure (Figure S129).

A perspective view of the crystal structure of 4d drawn with 50% probability of thermal ellipsoids. Color code: bromine ‐ brown, carbon ‐ light brown, hydrogen ‐ white, nitrogen ‐ light blue, oxygen ‐ red, sulfur ‐ yellow.

Following the initial series 4, which introduced structural variation through the use of an amine substituent, we shifted our focus to synthesizing analogs with different diamine linkers (see Scheme 4). To achieve this, we used intermediate 2a, which has an ethylamine substituent, as a precursor in our synthesis. This approach allowed us to evaluate the individual contributions of various molecular segments to the overall biological activity of the compounds.

Synthesis of GaMF1 analogs 6. Reagents and conditions: (i) ethylamine, DIPEA, EtOH, 50°C, 20 h; (ii) diamine, (EtOH), MW, 200 W, 90°C, 30–120 min; (iii) 3‐bromobenzoylchloride, K2CO3 or Cs2CO3 THF, rt, 90 min.

The reaction conditions for a reaction of 2a with various diamines differed based on the nucleophilicity of the amine functional group. Derivatives 5b, 5d–5g, and 5i–5k were synthesized under conditions analogous to those used for reactions with p‐phenylenediamine in the previous series. Specifically, the reactions were conducted in EtOH at 90°C under microwave irradiation for 30–120 min, depending on the reactivity of the diamine. These transformations proceeded with complete conversion of the starting material to the desired products 5 and minimal formation of by‐products, as confirmed by TLC and LC‐MS analysis.

In contrast, these conditions proved to be ineffective for the aliphatic diamines used to prepare intermediates 5a and 5c, as no product formation was observed. Given that both diamines are liquids, the reactions were subsequently performed solvent‐free, using the diamines themselves as the reaction medium in large excess. Under these modified conditions and identical microwave parameters (200W, 90°C, 120 min), complete conversion of 2a to intermediates 5a and 5c was achieved, again without detectable formation of significant by‐products.

An exception to the developed general reaction conditions was encountered with intermediate 5h, which could not be obtained under microwave‐assisted synthesis. When the reaction was attempted in EtOH at 90°C using microwave irradiation, the desired product 6h was formed in the presence of unidentified impurities, rendering this approach unsuitable. Consequently, a conventional synthesis was used, and the reaction was carried out by refluxing 2a with the corresponding diamine in EtOH. The reaction was monitored over time, and after 90 h, a satisfactory conversion to 5h was detected without significant contamination by side‐products. The reduced reactivity of 2,3,5,6‐tetramethylbenzene‐1,4‐diamine relative to other diamines is likely attributable to its increased steric hindrance. As with the other intermediates in this series, 5h was obtained in high purity, allowing its direct use in the subsequent acylation step without chromatographic purification. Instead, the crude reaction mixture was filtered through a silica gel pad using a DCM/MeOH/TEA (9:1:0.1) eluent. Following solvent evaporation, the resulting intermediates 5 were carried forward without further purification to the final acylation step, affording the target derivatives 6.

The acylation of intermediates 5 was performed under conditions analogous to those used for intermediates 3, employing 3‐bromobenzoyl chloride in anhydrous THF with K_2_CO_3_ as the base. A notable exception was observed for the synthesis of the indole derivative 6k, where these standard conditions proved insufficient. In this case, the reaction required the use of an excess of the acyl chloride and Cs_2_CO_3_ as the base, along with an extended reaction time of 30 h to achieve an acceptable conversion.

All final products 6 were isolated in relatively high yields, summarized in Table 2 (yields reported over the two synthetic steps). A significant reduction in yield was observed for 6k, which can be attributed to the formation of side products during the acylation process. Furthermore, due to the undesired by‐product formation, the reaction had to be quenched before full conversion of the starting material 5k, further contributing to the reduced yield.

Cytotoxicity

2.2

The prepared compounds were evaluated for their in vitro cytotoxicity against human lung fibroblast cells (MRC‐5) as well as two cancer cell lines, MCF‐7 (human breast adenocarcinoma) and MV4−11 (biphenotypic B‐myelomonocytic leukemia). We observed a high degree of cytotoxicity against MRC‐5 cells (including the original GaMF1 analog 4a), which was unexpected given the relatively advanced development of these molecules as inhibitors of M. tuberculosis ATP synthase and the absence of detectable genotoxicity in assays using the human embryonic stem cell (hESC) line E3, a highly sensitive pluripotency reporter (Table 3) [13]. This observation may pose a significant challenge for further therapeutic development, as the observed toxicity may outweigh the potential biological benefits.

Within the series tested, only five derivatives, specifically 4r, 6a, 6d, 6f, and 6i, exhibited acceptable cytotoxicity profiles against MRC‐5 cells (determined as > 10 µM). In particular, compounds 4, which contain a p‐phenylenediamine linker, consistently demonstrated high toxicity, suggesting a potential link between this structural motif (or its oxidative metabolism in cells) and cytotoxic effects. However, as several derivatives of 6 (with replaced p‐phenylenediamine moiety) also showed significant cytotoxicity, it can be inferred that additional features of the scaffold contribute to the observed effects, and that the linker is not the sole driver of MRC‐5 toxicity.

On the other hand, all tested compounds displayed some degree of antiproliferative activity against both MCF‐7 and MV4−11 cancer cell lines. These findings suggest that this compound class may also hold potential in the field of cancer treatment. Nevertheless, the primary limitation lies in their lack of selectivity. Among the tested compounds, 6f and particularly 6d emerged as the most promising candidates. Both exhibited antiproliferative effects against MCF‐7 and MV4−11 cell lines, while showing no detectable cytotoxicity toward MRC‐5 fibroblasts.

In particular, compound 6d demonstrated micromolar activity (4.00 μM against MCF‐7 and 2.61 μM against MV4−11) alongside favorable selectivity, making it an attractive candidate for further development. The compound's selective cytotoxicity profile is intriguing, suggesting that rational structural modifications may enhance its pharmacological properties. However, this scaffold also appears to be highly sensitive to even minor structural changes, as evidenced by the loss of selectivity observed for its isomeric analog 6e.

Although the nontoxic nature of compound 6f is also noteworthy, its antiproliferative activity was substantially lower. Moreover, from a medicinal chemistry perspective, 6f may be considered structurally problematic due to the presence of a Michael acceptor moiety, which may raise concerns regarding potential off‐target reactivity or long‐term safety.

Antimicrobial Activity

2.3

The five compounds (4r, 6a, 6d, 6f, 6i) exhibiting cytotoxicity below the established threshold against MRC‐5 cells (>10 µM) were subsequently evaluated for their antimicrobial and antiparasitic potential (Table 4). Specifically, these compounds were tested for their in vitro antibacterial (against Gram‐negative E. coli and Gram‐positive S. aureus), antimycobacterial (M. tuberculosis, M. abscessus), and antiparasitic (Trypanosoma cruzi, Trypanosoma brucei rhodesiense, T. brucei brucei, and Leishmania infantum) activities. None of the tested derivatives exhibited inhibitory activity against bacterial strains or mycobacteria. The observed loss of antimycobacterial activity can be attributed to the replacement of key structural motifs responsible for critical interactions with the target enzyme, specifically the F subunit of mycobacterial ATP synthase.

By contrast, several compounds demonstrated promising antiparasitic activity, particularly against Trypanosoma spp. and L. infantum. Derivative 4r displayed notable activity across all three Trypanosoma species tested, as well as against L. infantum. However, concurrent cytotoxicity toward primary peritoneal mouse macrophages (PMM) was observed, indicating nonselective biological effects that would need to be addressed in the context of further optimization. Particularly noteworthy was compound 6d, which not only retained significant trypanocidal activity but also demonstrated a favorable selectivity profile, with minimal PMM cytotoxicity and no observable effect against L. infantum. Among the compounds evaluated, 6d emerges as a strong lead for further development targeting trypanosomal infections.

Compound 6f exhibited the highest antiparasitic effect overall, with submicromolar IC_50_ values, rivaling those of approved therapeutic agents. Unfortunately, its development potential is compromised by marked PMM cytotoxicity (IC_50_ = 8.00 µM). Furthermore, as previously noted, the presence of a Michael acceptor motif in its structure renders it less attractive from a medicinal chemistry perspective due to associated undesirable reactivity concerns.

Although the intended antimycobacterial activity of compounds 4 and 6 could not be confirmed, these findings reveal an unreported and compelling biological potential of GaMF1‐inspired scaffolds against parasitic pathogens. The widespread prevalence of parasitic diseases caused by Trypanosoma cruzi (Chagas disease), Trypanosoma brucei rhodesiense (Human African trypanosomiasis), and Trypanosoma brucei brucei (animal African trypanosomosis) represents a major obstacle to socioeconomic development in many low‐ and middle‐income countries, particularly in sub‐Saharan Africa.

Animal trypanosomiasis severely compromises livestock health, leading to reduced agricultural productivity, lower crop yields, and significant financial losses [23]. Chagas disease and human African trypanosomiasis particularly impair human health and productivity, limiting participation in essential activities such as agriculture, livestock farming, and land management [24, 25]. In addition, environmental changes associated with deforestation and agricultural expansion can further exacerbate disease transmission. Current treatments for these infections are often prolonged, costly, and associated with significant adverse effects, further underscoring the urgent need for improved therapeutic options.

This shift in biological profile, described above, uncovered through our systematic SAR efforts, highlights a novel application space for this compound class. Nonetheless, as with their antiproliferative effects, the key challenge to be addressed remains their selectivity, which will be a primary focus in future optimization.

Conclusions

3

In this study, we synthesized and systematically evaluated a series of GaMF1 analogs to expand our understanding of their biological potential beyond antimycobacterial applications. Despite the lack of activity against M. tuberculosis and M. abscessus, several derivatives demonstrated notable antiparasitic effects, unveiling a previously unreported activity profile for this scaffold.

Among the compounds tested, analog 6d emerged as the most promising lead, combining potent and selective antiproliferative and trypanocidal activities with minimal cytotoxicity toward nonmalignant MRC‐5 fibroblasts. In contrast, compound 6f exhibited exceptional antiparasitic potency but was limited by pronounced cytotoxicity, probably due to the presence of a Michael acceptor motif, which raises potential safety concerns.

Despite the successful identification of hit compounds 6d and 6f displaying potent activity against trypanosomal pathogens and reduced or negligible cytotoxicity within the tested concentration range, the majority of the synthesized and evaluated derivatives 6 exhibited pronounced cytotoxic effects. This was observed even in cases where significant antiparasitic activity was detected in parallel. Consequently, achieving an improved balance between potency and selectivity emerges as a critical requirement for the further development of this compound class. Addressing this challenge through targeted structural optimization will therefore constitute a central focus of future research efforts.

Overall, our findings indicate that GaMF1‐inspired scaffolds represent a versatile chemotype with broader biological relevance than previously recognized. The observed shift in activity from antimycobacterial to antiparasitic targets highlights the adaptability of this structural framework and its potential as a foundation for the development of selective antiparasitic or anticancer agents. Future work will focus on structural optimization aimed at enhancing selectivity, reducing off‐target toxicity, and elucidating the molecular mechanisms underlying their biological effects.

Experimental Section

4

Materials and Methods

4.1

Solvents and chemicals were purchased from Sigma–Aldrich (St. Louis, Missouri, USA) or Fluorochem (UK). Analytical TLC was performed using aluminum plates precoated with silica gel 60 F254. Synthesis was carried out on Domino Blocks in disposable polypropylene reaction vessels (Torviq, Tucson, AZ). All reactions were carried out at room temperature (21°C) unless stated otherwise.

The LC‐MS analyses were carried out on the ultra‐high performance liquid chromatography‐mass spectroscopy (UHPLC‐MS) system (Waters). This system consists of a UHPLC chromatograph Acquity with a photodiode array detector and a single quadrupole mass spectrometer and uses an XSelect C18 column (2.1 × 50 mm) at 30°C and a flow rate of 600 μl/min. The mobile phase was (A) 10 mM ammonium acetate in HPLC‐grade water and (B) HPLC‐grade acetonitrile. A gradient was formed from 10% A to 80% B in 2.5 min and kept for 1.5 min. The column was re‐equilibrated with a 10% solution of B for 1 min. The electrospray ionization (ESI) source operated at a discharge current of 5 μA, vaporizer temperature of 350°C, and capillary temperature of 200°C.

NMR ^1^H/^13^C spectra were recorded on JEOL ECX‐500SS (500 MHz) or JEOL ECA400II (400 MHz) spectrometers at magnetic field strengths of 11.75 T and 9.39 T at ambient temperature (∼21°C). Chemical shifts (δ) are reported in parts per million (ppm), and coupling constants (J) are reported in Hertz (Hz). NMR spectra were recorded in DMSO‐d 6 or CDCl_3_.

HRMS analysis was performed on an LC chromatograph (Dionex UltiMate 3000, Thermo Fischer Scientific, MA, USA) with a mass spectrometer Exactive Plus Orbitrap high‐resolution (Thermo Fischer Scientific, MA, USA) operating in positive scan mode in the range of 1000–1500 m/z. Electrospray was used as a source of ionization. Samples were diluted to a final concentration of 0.1 mg/ml in a solution of water and acetonitrile (50:50, v/v). The samples were injected into the mass spectrometer following HPLC separation on a Phenomenex Gemini column (C18, 50 × 2 mm, 3 µm particle) using an isocratic mobile phase of 0.01 M MeCN/ammonium acetate (80/20) at a flow rate of 0.3 ml/min.

Chemistry

4.2

General Procedure for the Synthesis of Intermediates 2

4.2.1

2,4‐dichlor‐6‐methylpyrimidine (500 mg, 3.07 mmol) was dissolved in EtOH (5 ml), followed by the addition of amine (3.07 mmol; 1 eq.) and finally DIPEA (1335.7 μl, 7.67 mmol; 2.5 eq.). The reaction mixture was stirred at 50°C for 20 h. Subsequently, approximately 1 g of silica gel was added, and the solvent was removed under reduced pressure to adsorb the crude material onto the silica gel. The residue was purified by column chromatography (Hex/EtOAc, grad.), affording the desired product 2 as the major regioisomer, along with a minor regioisomer 2′. Both regioisomers were successfully isolated and purified. Detailed experimental data for the major regioisomer 2 are provided below, whereas the corresponding data for the minor regioisomer 2′ are included in the Supporting Information.

2‐Chloro‐N‐ethyl‐6‐methylpyrimidin‐4‐amine 2a

4.2.2

Product 2a was obtained as a white solid. Yield: 191.0 mg (36%). ^1^H NMR (400 MHz, Chloroform‐d) δ 6.05 (s, 1H), 5.50 (br s, 1H), 3.31 (s, 2H), 2.30 (s, 3H), 1.22 (t, J = 7.2 Hz, 3H). ^13^C NMR (101 MHz, Chloroform‐d) δ 167.9, 164.1, 160.2, 99.4, 36.5, 23.9, 14.5. HRMS: m/z: calcd for C_7_H_11_ClN_3_ ^+^: 172.0636 [M + H]^+^; found: 172.0636.

2‐Chloro‐6‐methyl‐N‐propylpyrimidin‐4‐amine 2b

4.2.3

Product 2b was obtained as a yellow gel. Yield: 238.4 mg (42%). ^1^H NMR (400 MHz, Chloroform‐d) δ 6.04 (s, 1H), 5.73 (br s, 1H), 3.21 (s, 2H), 2.28 (s, 3H), 1.59 (h, J = 7.4 Hz, 2H), 0.92 (t, J = 7.4 Hz, 3H). ^13^C NMR (101 MHz, Chloroform‐d) δ 167.8, 164.3, 160.1, 99.5, 43.4, 23.8, 22.4, 11.3. HRMS: m/z: calcd for C_8_H_13_ClN_3_ ^+^: 186.1793 [M + H]^+^; found: 186.1795.

2‐Chloro‐N‐hexyl‐6‐methylpyrimidin‐4‐amine 2c

4.2.4

Product 2c was obtained as a pale yellow gel. Yield: 296.6 mg (40%). ^1^H NMR (400 MHz, Chloroform‐d) δ 6.05 (s, 1H), 5.34 (br s, 1H), 3.25 (s, 2H), 2.31 (s, 3H), 1.58 (p, J = 7.2 Hz, 2H), 1.37 – 1.25 (m, 6H), 0.88 (t, J = 7.5 Hz, 3H). ^13^C NMR (101 MHz, Chloroform‐d) δ 168.0, 164.2, 160.3, 99.0, 41.8, 31.5, 29.2, 26.6, 23.9, 22.6, 14.1. HRMS: m/z: calcd for C_11_H_19_ClN_3_ ^+^: 228.1262 [M + H]^+^; found: 228.1264.

2‐Chloro‐N,N‐diethyl‐6‐methylpyrimidin‐4‐amine 2d

4.2.5

Product 2d was obtained as a yellow gel. Yield: 254.1 mg (41%). ^1^H NMR (400 MHz, Chloroform‐d) δ 6.09 (s, 1H), 3.46 (s, 4H), 2.29 (s, 3H), 1.15 (t, J = 7.1 Hz, 6H). ^13^C NMR (101 MHz, Chloroform‐d) δ 166.9, 162.4, 160.4, 99.3, 42.5, 24.0, 12.7. HRMS: m/z: calcd for C_9_H_15_ClN_3_ ^+^: 200.0949 [M + H]^+^; found: 200.0948.

2‐Chloro‐4‐methyl‐6‐(piperidin‐1‐yl)pyrimidine 2e

4.2.6

Product 2e was obtained as a white solid. Yield: 313.9 mg (48%). ^1^H NMR (400 MHz, Chloroform‐d) δ 6.21 (s, 1H), 3.58 (t, J = 5.5 Hz, 4H), 2.29 (s, 3H), 1.71 – 1.56 (m, 6H). ^13^C NMR (101 MHz, Chloroform‐d) δ 167.5, 163.1, 160.5, 99.6, 45.4, 25.6, 24.6, 24.1. HRMS: m/z: calcd for C_10_H_15_ClN_3_ ^+^: 212.0949 [M + H]^+^; found: 212.0950.

4‐(2‐Chloro‐6‐methylpyrimidin‐4‐yl)morpholine 2f

4.2.7

Product 2f was obtained as a white solid. Yield: 287.4 mg (44%). ^1^H NMR (400 MHz, Chloroform‐d) δ 6.22 (s, 1H), 3.76 – 3.72 (m, 4H), 3.60 (t, J = 5.2 Hz, 4H), 2.33 (s, 3H). ^13^C NMR (101 MHz, Chloroform‐d) δ 168.2, 163.6, 160.5, 99.7, 66.5, 44.4, 24.1. HRMS: m/z: calcd for C_9_H_13_ClN_3_O^+^: 214.0742 [M + H]^+^; found: 214.0743.

Tert‐butyl 4‐(2‐chloro‐6‐methylpyrimidin‐4‐yl)piperazine‐1‐carboxylate 2g

4.2.8

Product 2g was obtained as a white solid. Yield: 415.8 mg (43%). ^1^H NMR (400 MHz, Chloroform‐d) δ 6.16 (s, 1H), 3.52 (dd, J = 6.7, 4.0 Hz, 4H), 3.40 (dd, J = 6.5, 4.0 Hz, 4H), 2.21 (s, 3H), 1.36 (s, 9H). ^13^C NMR (101 MHz, Chloroform‐d) δ 167.9, 163.0, 160.1, 154.4, 99.7, 80.2, 43.6, 43.0, 28.2, 23.9. HRMS: m/z: calcd for C_14_H_22_ClN_4_O_2_ ^+^: 313.1426 [M + H]^+^; found: 313.1424.

2‐Chloro‐N‐cyclopentyl‐6‐methylpyrimidin‐4‐amine 2i

4.2.9

Product 2i was obtained as a yellow gel. Yield: 280.2 mg (43%). ^1^H NMR (400 MHz, Chloroform‐d) δ 6.07 (s, 1H), 5.14 (br s, 1H), 3.95 (br s, 1H), 2.31 (s, 3H), 2.06 – 1.98 (m, 2H), 1.75 – 1.60 (m, 4H), 1.52 – 1.40 (m, 2H). ^13^C NMR (101 MHz, Chloroform‐d) δ 167.9, 163.7, 160.3, 99.5, 53.1, 33.3, 24.0, 23.8. HRMS: m/z: calcd for C_10_H_15_ClN_3_ ^+^: 212.0949 [M + H]^+^; found: 212.0951.

2‐Chloro‐N‐cyclohexyl‐6‐methylpyrimidin‐4‐amine 2j

4.2.10

Product 2j was obtained as a yellow gel. Yield: 275.9 mg (40%). ^1^H NMR (400 MHz, Chloroform‐d) δ 6.03 (s, 1H), 5.12 (s, 1H), 3.50 (s, 1H), 2.29 (s, 3H), 1.98 – 1.92 (m, 2H), 1.73 (dt, J = 13.1, 3.8 Hz, 2H), 1.65 – 1.58 (m, 1H), 1.43 – 1.31 (m, 2H), 1.26 – 1.15 (m, 3H). ^13^C NMR (101 MHz, Chloroform‐d) δ 167.7, 163.3, 160.4, 99.8, 50.1, 32.8, 25.5, 24.6, 23.9. HRMS: m/z: calcd for C_11_H_17_ClN_3_ ^+^: 226.1106 [M + H]^+^; found: 226.1104.

2‐Chloro‐N‐cyclooctyl‐6‐methylpyrimidin‐4‐amine 2k

4.2.11

Product 2k was obtained as a yellow gel. Yield: 307.1 mg (39%). ^1^H NMR (400 MHz, Chloroform‐d) δ 6.00 (s, 1H), 5.11 (br s, 1H), 3.69 (br s, 1H), 2.30 (s, 3H), 1.89 – 1.82 (m, 2H), 1.71 – 1.50 (m, 12H). ^13^C NMR (101 MHz, Chloroform‐d) δ 167.8, 163.1, 160.4, 99.9, 51.4, 31.9, 27.3, 25.6, 24.0, 23.6. HRMS: m/z: calcd for C_13_H_21_ClN_3_ ^+^: 254.1419 [M + H]^+^; found: 254.1417.

2‐Chloro‐N‐cyclododecyl‐6‐methylpyrimidin‐4‐amine 2l

4.2.12

Product 2l was obtained as a yellow gel. Yield: 528.2 mg (56%). ^1^H NMR (400 MHz, Chloroform‐d) δ 5.98 (s, 1H), 5.08 (br s, 1H), 2.25 (s, 3H), 1.65 – 1.54 (m, 2H), 1.40 – 1.22 (m, 20H). ^13^C NMR (101 MHz, Chloroform‐d) δ 168.2, 163.5, 160.3, 98.6, 48.5, 29.6, 24.1, 23.9, 23.3, 23.2, 21.1. HRMS: m/z: calcd for C_17_H_29_ClN_3_ ^+^: 310.2045 [M + H]^+^; found: 310.2044.

2‐Chloro‐6‐methyl‐N‐(prop‐2‐yn‐1‐yl)pyrimidin‐4‐amine 2m

4.2.13

Product 2m was obtained as a white solid. Yield: 218.0 mg (39%). ^1^H NMR (400 MHz, Chloroform‐d) δ 6.21 (s, 1H), 6.00 (br s, 1H), 4.13 (d, J = 3.8 Hz, 2H), 2.35 (s, 3H), 2.26 (t, J = 2.5 Hz, 1H). ^13^C NMR (101 MHz, Chloroform‐d) δ 168.4, 163.8, 160.2, 101.0, 78.9, 72.3, 31.3, 24.0. HRMS: m/z: calcd for C_8_H_9_ClN_3_ ^+^: 182.0480 [M + H]^+^; found: 182.0479.

N‐Benzyl‐2‐chloro‐6‐methylpyrimidin‐4‐amine 2n

4.2.14

Product 2n was obtained as a white solid. Yield: 301.0 mg (42%). ^1^H NMR (400 MHz, Chloroform‐d) δ 7.39 – 7.26 (m, 5H), 6.07 (s, 1H), 4.53 (d, J = 4.7 Hz, 2H), 2.29 (s, 3H). ^13^C NMR (101 MHz, Chloroform‐d) δ 168.2, 164.3, 160.3, 137.3, 129.0, 127.9, 127.4, 100.2, 45.7, 23.9. HRMS: m/z: calcd for C_12_H_13_ClN_3_ ^+^: 234.0793 [M + H]^+^; found: 234.0792.

2‐Chloro‐6‐methyl‐N‐(pyridin‐2‐ylmethyl)pyrimidin‐4‐amine 2o

4.2.15

Product 2o was obtained as a white solid. Yield: 240.2 mg (33%). ^1^H NMR (400 MHz, Chloroform‐d) δ 8.45 (d, J = 4.6 Hz, 1H), 7.60 (td, J = 7.6, 1.8 Hz, 1H), 7.23 (d, J = 7.8 Hz, 1H), 7.13 (dd, J = 7.2, 5.1 Hz, 1H), 7.01 (br s, 1H), 6.11 (s, 1H), 4.59 (s, 2H), 2.21 (s, 3H). ^13^C NMR (101 MHz, Chloroform‐d) δ 167.1, 163.9, 160.1, 156.0, 148.9, 136.9, 122.5, 121.9, 102.7, 45.9, 23.6. HRMS: m/z: calcd for C_11_H_12_ClN_4_ ^+^: 235.0745 [M + H]^+^; found: 235.0745.

2‐Chloro‐N‐(furan‐2‐ylmethyl)‐6‐methylpyrimidin‐4‐amine 2p

4.2.16

Product 2p was obtained as a white solid. Yield: 306.4 mg (45%). ^1^H NMR (400 MHz, Chloroform‐d) δ 7.29 (dd, J = 1.7, 0.6 Hz, 1H), 6.27 (dd, J = 3.3, 1.8 Hz, 1H), 6.20 (dd, J = 3.3, 0.6 Hz, 1H), 6.14 (s, 1H), 4.49 (d, J = 5.9 Hz, 2H), 2.26 (s, 3H). ^13^C NMR (101 MHz, Chloroform‐d) δ 167.8, 164.0, 160.0, 150.7, 142.3, 110.5, 107.7, 100.5, 38.5, 23.7. HRMS: m/z: calcd for C_10_H_11_ClN_3_O^+^: 224.0585 [M + H]^+^; found: 224.0587.

N‐(Benzo[d][1,3]dioxol‐5‐ylmethyl)‐2‐chloro‐6‐methylpyrimidin‐4‐amine 2q

4.2.17

Product 2q was obtained as a white solid. Yield: 360.9 mg (42%). ^1^H NMR (400 MHz, Chloroform‐d) δ 6.76 (s, 1H), 6.75 (s, 2H), 6.05 (s, 1H), 5.93 (s, 2H), 4.41 (d, J = 5.8 Hz, 2H), 2.28 (s, 3H). ^13^C NMR (101 MHz, Chloroform‐d) δ 168.5, 164.2, 160.2, 148.2, 147.3, 131.1, 120.7, 108.6, 107.9, 101.3, 100.3, 45.5, 23.9. HRMS: m/z: calcd for C_13_H_13_ClN_3_O_2_ ^+^: 278.0691 [M + H]^+^; found: 278.0690.

2‐Chloro‐6‐methyl‐N‐(4‐morpholinophenyl)pyrimidin‐4‐amine 2r

4.2.18

Product 2r was obtained as a yellow solid. Yield: 297.7 mg (32%). ^1^H NMR (400 MHz, Chloroform‐d) δ 8.02 (s, 1H), 7.14 (d, J = 8.6 Hz, 2H), 6.86 (d, J = 9.0 Hz, 2H), 6.18 (s, 1H), 3.80 (t, J = 5.0 Hz, 4H), 3.10 (t, J = 5.0 Hz, 4H), 2.20 (s, 3H). ^13^C NMR (101 MHz, Chloroform‐d) δ 168.1, 163.9, 159.9, 149.5, 129.1, 125.8, 116.2, 100.4, 66.7, 49.1, 23.7. HRMS: m/z: calcd for C_15_H_18_ClN_4_O^+^: 305.1164 [M + H]^+^; found: 305.1165.

General Procedure for the Synthesis of Intermediates 3

4.2.19

A solution of intermediate 2 (0.50 mmol) in EtOH (5 ml) was treated with the corresponding diamine (0.50 mmol, 1.0 eq.). The reaction mixture was stirred at 90°C for 30 min under microwave irradiation (200 W). Upon completion of the reaction, as indicated by TLC/LC‐MS, the mixture was filtered through a short pad of silica gel using DCM/MeOH/TEA (9:1:0.1, v/v/v) as the eluent. The filtrate was concentrated under reduced pressure, and the crude product was used directly in the subsequent acylation step to afford the final compounds 4 without further purification.

General Procedure for the Synthesis of Final Products 4a‐g and 4i‐r (Derivatives 4h and 4s were Prepared by Modifications of 4h and 4m Described Below this Procedure)

4.2.20

In a pre‐dried Schlenk flask under an argon atmosphere, intermediate 3 (0.50 mmol, obtained from the preceding step) was dissolved in anhydrous THF (8 ml), followed by the addition of K_2_CO_3_ (172.8 mg, 1.25 mmol, 2.5 eq.). The reaction mixture was cooled to 0°C, and 3‐bromobenzoyl chloride (66.0 μl, 0.50 mmol, 1.0 eq.) was added dropwise. The mixture was stirred at room temperature for 1 h. Upon completion of the reaction (as confirmed by TLC/LC‐MS), water (20 ml) was added, and the resulting mixture was extracted with DCM (3 × 30 ml). The combined organic layers were washed with brine (20 ml), dried over anhydrous MgSO_4_, and concentrated under reduced pressure. The crude product was purified by column chromatography using DCM/MeOH (grad.) to afford the desired compound.

The final derivatives 4h and 4s were subsequently obtained by cycloaddition or deprotection reactions, as described below.

Deprotection Providing the Final Product 4h

4.2.21

Compound 4g (28.4 mg, 0.05 mmol) was dissolved in a saturated solution of HCl in MeOH (2 ml) and stirred at room temperature for 56 h, with the reaction progress monitored by LC–MS. The resulting precipitate was collected by filtration and washed with MeOH (≈10 ml), affording the desired product 4h.

Cycloaddition Leading to Final Product 4s

4.2.22

Benzyl azide (66.6 mg, 0.50 mmol) was dissolved in a THF/H_2_O mixture (1:1, v/v), followed by the sequential addition of compound 4m (218.2 mg, 0.50 mmol, 1.0 eq.), CuSO_4_ · 5H_2_O (16.0 mg, 0.10 mmol, 20 mol%), and (+)‐sodium l‐ascorbate (15.8 mg, 0.08 mmol, 16 mol%). The reaction mixture was stirred at room temperature for 22 h. Water (15 ml) was then added, resulting in the formation of a precipitate, which was collected by filtration and washed with water (≈10 ml) to afford the product 4s.

3‐Bromo‐N‐(4‐((4‐(ethylamino)‐6‐methylpyrimidin‐2‐yl)amino)phenyl)benzamide 4a

4.2.23

Product 4a was obtained as a yellow solid. Yield: 132.9 mg (62%) over two reaction steps. ^1^H NMR (400 MHz, DMSO‐d 6) δ 10.17 (s, 1H), 8.89 (s, 1H), 8.13 (t, J = 1.9 Hz, 1H), 7.95 (dt, J = 7.8, 1.3 Hz, 1H), 7.80 – 7.75 (m, 3H), 7.61 – 7.57 (m, 2H), 7.49 (t, J = 7.9 Hz, 1H), 6.95 (s, 1H), 5.77 (s, 1H), 3.31 (t, J = 8.0 Hz, 2H), 2.12 (s, 3H), 1.16 (t, J = 7.2 Hz, 3H). ^13^C NMR (101 MHz, DMSO‐d 6) δ 163.6, 163.3, 163.1, 159.6, 138.0, 137.3, 134.0, 131.6, 130.6, 130.1, 126.7, 121.6, 120.8, 118.2, 95.2, 34.9, 23.4, 14.7. HRMS: m/z: calcd for C_20_H_21_BrN_5_O^+^: 428.0904 [M + H]^+^; found: 428.0906.

3‐Bromo‐N‐(4‐((4‐methyl‐6‐(propylamino)pyrimidin‐2‐yl)amino)phenyl)benzamide 4b

4.2.24

Product 4b was obtained as a yellow gel. Yield: 98.2 mg (45%) over two reaction steps. ^1^H NMR (400 MHz, Chloroform‐d) δ 7.99 (t, J = 1.8 Hz, 1H), 7.90 (s, 1H), 7.77 (d, J = 7.8 Hz, 1H), 7.65 – 7.59 (m, 3H), 7.52 (d, J = 8.8 Hz, 2H), 7.32 (t, J = 7.9 Hz, 1H), 7.12 (s, 1H), 5.72 (s, 1H), 4.89 (s, 1H), 3.27 (q, J = 6.7 Hz, 2H), 2.24 (s, 3H), 1.63 (p, J = 7.3 Hz, 2H), 0.98 (t, J = 7.4 Hz, 3H). ^13^C NMR (101 MHz, DMSO‐d 6) δ 163.4, 163.2, 162.7, 159.1, 137.7, 137.3, 134.0, 131.8, 130.6, 130.1, 126.7, 121.6, 120.8, 118.4, 95.4, 42.1, 23.1, 22.3, 11.5. HRMS: m/z: calcd for C_20_H_21_BrN_5_O^+^: 442.1060 [M + H]^+^; found: 442.1056.

3‐Bromo‐N‐(4‐((4‐(hexylamino)‐6‐methylpyrimidin‐2‐yl)amino)phenyl)benzamide 4c

4.2.25

Product 4c was obtained as a yellow solid. Yield: 135.6 mg (56%) over two reaction steps. ^1^H NMR (400 MHz, DMSO‐d 6) δ 10.16 (s, 1H), 8.89 (s, 1H), 8.12 (t, J = 1.9 Hz, 1H), 7.94 (d, J = 7.9 Hz, 1H), 7.79 – 7.74 (m, 3H), 7.61 – 7.56 (m, 2H), 7.49 (t, J = 7.9 Hz, 1H), 6.99 (s, 1H), 5.77 (s, 1H), 3.31 – 3.21 (m, 2H; overlapped with H_2_O signal), 2.11 (s, 3H), 1.54 (p, J = 7.1 Hz, 2H), 1.37 – 1.25 (m, 6H), 0.87 (t, J = 6.4 Hz, 3H). ^13^C NMR (101 MHz, DMSO‐d 6) δ 163.3, 163.2, 159.6, 137.9, 137.3, 134.0, 131.6, 130.6, 130.1, 126.7, 121.6, 120.8, 118.2, 95.0, 31.1, 29.1, 26.3, 23.4, 22.1, 13.9. HRMS: m/z: calcd for C_24_H_29_BrN_5_O^+^: 484.1530 [M + H]^+^; found: 484.1525.

3‐Bromo‐N‐(4‐((4‐(diethylamino)‐6‐methylpyrimidin‐2‐yl)amino)phenyl)benzamide 4d

4.2.26

Product 4d was obtained as a yellow solid. Yield: 154.8 mg (68%) over two reaction steps. ^1^H NMR (400 MHz, DMSO‐d 6) δ 10.17 (s, 1H), 8.94 (s, 1H), 8.14 (t, J = 1.8 Hz, 1H), 7.95 (dt, J = 7.8, 1.3 Hz, 1H), 7.79 – 7.72 (m, 3H), 7.62 – 7.57 (m, 2H), 7.49 (t, J = 7.9 Hz, 1H), 5.94 (s, 1H), 3.49 (q, J = 7.2 Hz, 4H), 2.18 (s, 3H), 1.14 (t, J = 7.0 Hz, 6H). ^13^C NMR (101 MHz, DMSO‐d 6) δ 164.8, 163.3, 161.3, 159.3, 137.9, 137.3, 134.0, 131.6, 130.6, 130.1, 126.7, 121.6, 120.8, 118.1, 92.5, 41.6, 23.7, 12.9. HRMS: m/z: calcd for C_22_H_25_BrN_5_O^+^: 456.1217 [M + H]^+^; found: 456.1211.

3‐Bromo‐N‐(4‐((4‐methyl‐6‐(piperidin‐1‐yl)pyrimidin‐2‐yl)amino)phenyl)benzamide 4e

4.2.27

Product 4e was obtained as a yellow gel. Yield: 164.8 mg (71%) over two reaction steps. ^1^H NMR (400 MHz, DMSO‐d 6) δ 10.14 (s, 1H), 8.92 (s, 1H), 8.09 (t, J = 1.8 Hz, 1H), 7.91 (dt, J = 7.9, 1.2 Hz, 1H), 7.73 (ddd, J = 7.9, 2.0, 1.0 Hz, 1H), 7.68 – 7.61 (m, 2H), 7.62 – 7.52 (m, 2H), 7.45 (t, J = 7.8 Hz, 1H), 6.08 (s, 1H), 3.55 (t, J = 5.3 Hz, 4H), 2.14 (s, 3H), 1.64 – 1.55 (m, 2H), 1.53 – 1.38 (m, 4H). ^13^C NMR (101 MHz, DMSO‐d 6) δ 165.1, 163.4, 162.4, 159.2, 137.6, 137.3, 134.0, 131.8, 130.6, 130.1, 126.7, 121.6, 120.8, 118.5, 93.2, 44.7, 25.1, 24.3, 23.7. HRMS: m/z: calcd for C_23_H_25_BrN_5_O^+^: 468.1217 [M + H]^+^; found: 468.1209.

3‐Bromo‐N‐(4‐((4‐methyl‐6‐morpholinopyrimidin‐2‐yl)amino)phenyl)benzamide 4f

4.2.28

Product 4f was obtained as a white solid. Yield: 146.0 mg (62%) over two reaction steps. ^1^H NMR (400 MHz, DMSO‐d 6) δ 10.19 (s, 1H), 9.03 (s, 1H), 8.13 (t, J = 1.8 Hz, 1H), 7.95 (dt, J = 7.9, 1.1 Hz, 1H), 7.77 (ddd, J = 8.0, 2.0, 1.0 Hz, 1H), 7.70 – 7.66 (m, 2H), 7.63 – 7.59 (m, 2H), 7.49 (t, J = 7.9 Hz, 1H), 6.14 (s, 1H), 3.68 (t, J = 5.2 Hz, 4H), 3.55 (t, J = 5.4 Hz, 4H), 2.20 (s, 3H). ^13^C NMR (101 MHz, DMSO‐d 6) δ 165.8, 163.4, 163.0, 159.2, 137.5, 137.3, 134.0, 131.9, 130.6, 130.1, 126.7, 121.6, 120.9, 118.6, 93.3, 65.9, 44.1, 23.8. HRMS: m/z: calcd for C_22_H_23_BrN_5_O_2_ ^+^: 470.1009 [M + H]^+^; found: 470.1006.

Tert‐butyl 4‐(2‐((4‐(3‐bromobenzamido)phenyl)amino)‐6‐methylpyrimidin‐4‐yl)piperazine‐1‐carboxylate 4g

4.2.29

Product 4g was obtained as a pale yellow solid. Yield: 136.4 mg (48%) over two reaction steps. ^1^H NMR (500 MHz, DMSO‐d 6) δ 10.19 (s, 1H), 9.02 (s, 1H), 8.13 (t, J = 1.8 Hz, 1H), 7.95 (dt, J = 7.8, 1.3 Hz, 1H), 7.77 (ddd, J = 8.0, 1.9, 0.9 Hz, 1H), 7.70 – 7.66 (m, 2H), 7.64 – 7.59 (m, 2H), 7.49 (t, J = 7.9 Hz, 1H), 6.14 (s, 1H), 3.59 (t, J = 4.5 Hz, 4H), 3.42 (t, J = 4.5 Hz, 4H), 2.20 (s, 3H), 1.43 (s, 9H). ^13^C NMR (101 MHz, DMSO‐d 6) δ 165.8, 163.4, 162.6, 159.2, 153.9, 137.5, 137.3, 134.0, 131.9, 130.6, 130.1, 126.7, 121.6, 120.9, 118.6, 93.4, 79.1, 43.3, 42.7, 28.0, 23.8. HRMS: m/z: calcd for C_27_H_32_BrN_6_O_3_ ^+^: 569.1693 [M + H]^+^; found: 569.1699.

3‐Bromo‐N‐(4‐((4‐methyl‐6‐(piperazin‐1‐yl)pyrimidin‐2‐yl)amino)phenyl)benzamide hydrochloride 4h

4.2.30

Product 4h was obtained as a white solid in quantitative yield by deprotection of 4g. ^1^H NMR (400 MHz, Deuterium Oxide) δ 7.67 – 7.63 (m, 1H), 7.56 (t, J = 6.1 Hz, 1H), 7.41 (t, J = 6.2 Hz, 1H), 7.19 (q, J = 7.1 Hz, 1H), 7.10 – 6.99 (m, 4H), 6.23 (s, 1H), 3.91 (s, 4H), 3.46 (s, 4H), 2.26 (s, 3H). ^13^C NMR (101 MHz, Deuterium Oxide with DMSO‐d 6 addition for reference) δ 165.4, 163.0, 156.5, 151.2, 136.3, 135.9, 135.2, 133.8, 131.6, 131.5, 127.3, 123.4, 122.4, 122.0, 96.3, 44.4, 42.8, 20.1. HRMS: m/z: calcd for C_22_H_24_BrN_6_O^+^: 469.1164 [M + H]^+^; found: 469.1164 (calculated and detected as a free amine).

3‐Bromo‐N‐(4‐((4‐(cyclopentylamino)‐6‐methylpyrimidin‐2‐yl)amino)phenyl)benzamide 4i

4.2.31

Product 4i was obtained as a yellow gel. Yield: 91.3 mg (39%) over two reaction steps. ^1^H NMR (400 MHz, DMSO‐d 6) δ 10.17 (s, 1H), 8.94 (s, 1H), 8.14 (t, J = 1.8 Hz, 1H), 7.95 (dt, J = 7.8, 1.1 Hz, 1H), 7.80 – 7.75 (m, 3H), 7.61 – 7.57 (m, 2H), 7.49 (t, J = 7.8 Hz, 1H), 7.00 (s, 1H), 5.78 (s, 1H), 4.20 (br s, 1H), 2.12 (s, 3H), 2.00 – 1.91 (m, 2H), 1.73 – 1.62 (m, 2H), 1.61 – 1.43 (m, 4H). ^13^C NMR (101 MHz, DMSO‐d 6) δ 163.3, 163.2, 162.8, 159.4, 137.9, 137.3, 134.0, 131.6, 130.6, 130.1, 126.7, 121.6, 120.8, 118.3, 95.5, 51.8, 32.4, 23.5, 23.3. HRMS: m/z: calcd for C_23_H_25_BrN_5_O^+^: 468.1217 [M + H]^+^; found: 468.1216.

3‐Bromo‐N‐(4‐((4‐(cyclohexylamino)‐6‐methylpyrimidin‐2‐yl)amino)phenyl)benzamide 4j

4.2.32

Product 4j was obtained as a yellow gel. Yield: 89.7 mg (37%) over two reaction steps. ^1^H NMR (400 MHz, DMSO‐d 6) δ 10.16 (s, 1H), 8.92 (s, 1H), 8.14 (t, J = 1.8 Hz, 1H), 7.95 (dt, J = 7.9, 1.1 Hz, 1H), 7.80 – 7.75 (m, 3H), 7.62 – 7.57 (m, 2H), 7.49 (t, J = 7.9 Hz, 1H), 6.90 (s, 1H), 5.76 (s, 1H), 3.78 (br s, 1H), 2.10 (s, 3H), 1.96 (dd, J = 12.0, 3.3 Hz, 2H), 1.77 (dt, J = 12.4, 3.7 Hz, 2H), 1.63 (dt, J = 12.9, 3.6 Hz, 1H), 1.41 – 1.30 (m, 2H), 1.26 – 1.14 (m, 3H). ^13^C NMR (101 MHz, DMSO‐d 6) δ 163.33, 163.19, 162.81, 159.36, 137.88, 137.27, 134.01, 131.62, 130.57, 130.10, 126.70, 121.64, 120.77, 118.28, 95.49, 51.81, 32.44, 23.49, 23.29. HRMS: m/z: calcd for C_24_H_27_BrN_5_O^+^: 482.1373 [M + H]^+^; found: 482.1371.

3‐Bromo‐N‐(4‐((4‐(cyclooctylamino)‐6‐methylpyrimidin‐2‐yl)amino)phenyl)benzamide 4k

4.2.33

Product 4k was obtained as a yellow gel. Yield: 94.2 mg (37%) over two reaction steps. ^1^H NMR (400 MHz, DMSO‐d 6) δ 10.17 (s, 1H), 8.95 (s, 1H), 8.13 (t, J = 1.7 Hz, 1H), 7.95 (d, J = 7.9 Hz, 1H), 7.81 – 7.75 (m, 3H), 7.59 (d, J = 9.0 Hz, 2H), 7.48 (t, J = 7.9 Hz, 1H), 6.93 (s, 1H), 5.75 (s, 1H), 4.13 (br s, 1H), 2.10 (s, 3H), 1.87 – 1.77 (m, 2H), 1.73 – 1.51 (m, 12H). ^13^C NMR (101 MHz, DMSO‐d 6) δ 163.3, 163.2, 162.1, 159.5, 137.9, 137.3, 134.0, 131.6, 130.6, 130.1, 126.7, 121.6, 120.7, 118.1, 95.4, 49.4, 32.1, 26.7, 25.4, 23.6, 23.3. HRMS: m/z: calcd for C_26_H_31_BrN_5_O^+^: 510.1686 [M + H]^+^; found: 510.1687.

3‐Bromo‐N‐(4‐((4‐(cyclododecylamino)‐6‐methylpyrimidin‐2‐yl)amino)phenyl)benzamide 4l

4.2.34

Product 4l was obtained as a yellow solid. Yield: 172.9 mg (61%) over two reaction steps. ^1^H NMR (400 MHz, DMSO‐d 6) δ 10.17 (s, 1H), 8.95 (s, 1H), 8.13 (t, J = 1.7 Hz, 1H), 7.95 (d, J = 7.9 Hz, 1H), 7.81 – 7.75 (m, 3H), 7.59 (d, J = 9.0 Hz, 2H), 7.48 (t, J = 7.9 Hz, 1H), 6.93 (s, 1H), 5.75 (s, 1H), 4.13 (br s, 1H), 2.10 (s, 3H), 1.87 – 1.77 (m, 2H), 1.73 – 1.51 (m, 20H). ^13^C NMR (101 MHz, DMSO‐d 6) δ 163.3, 163.0, 159.7, 137.9, 137.4, 133.9, 131.7, 130.6, 130.1, 126.7, 121.6, 120.7, 118.1, 95.3, 45.7, 29.9, 23.4, 23.3, 23.2, 22.9, 21.7. HRMS: m/z: calcd for C_30_H_39_BrN_5_O^+^: 510.1686 [M + H]^+^; found: 510.1687.

3‐Bromo‐N‐(4‐((4‐methyl‐6‐(prop‐2‐yn‐1‐ylamino)pyrimidin‐2‐yl)amino)phenyl)benzamide 4m

4.2.35

Product 4m was obtained as a yellow gel. Yield: 104.6 mg (48%) over two reaction steps. ^1^H NMR (400 MHz, DMSO‐d 6) δ 10.18 (s, 1H), 9.01 (s, 1H), 8.14 (t, J = 1.7 Hz, 1H), 7.95 (dt, J = 7.8, 1.3 Hz, 1H), 7.81 – 7.76 (m, 3H), 7.62 – 7.58 (m, 2H), 7.49 (t, J = 7.9 Hz, 1H), 7.36 (t, J = 5.9 Hz, 1H), 5.85 (s, 1H), 4.10 (d, J = 3.4 Hz, 2H), 3.10 (t, J = 2.4 Hz, 1H), 2.15 (s, 3H). ^13^C NMR (101 MHz, DMSO‐d 6) δ 164.3, 163.3, 162.6, 159.4, 137.7, 137.3, 134.0, 131.8, 130.6, 130.1, 126.7, 121.6, 120.8, 118.5, 95.2, 81.9, 72.6, 29.5, 23.4. HRMS: m/z: calcd for C_21_H_19_BrN_5_O^+^: 438.0747 [M + H]^+^; found: 438.0747.

N‐(4‐((4‐(Benzylamino)‐6‐methylpyrimidin‐2‐yl)amino)phenyl)‐3‐bromobenzamide 4n

4.2.36

Product 4n was obtained as a yellow solid. Yield: 179.6 mg (74%) over two reaction steps. ^1^H NMR (400 MHz, DMSO‐d 6) δ 10.15 (s, 1H), 8.93 (s, 1H), 8.13 (t, J = 1.8 Hz, 1H), 7.95 (dt, J = 7.8, 1.3 Hz, 1H), 7.77 (ddd, J = 8.0, 2.1, 1.0 Hz, 1H), 7.66 (d, J = 8.6 Hz, 2H), 7.60 – 7.51 (m, 3H), 7.49 (t, J = 7.9 Hz, 1H), 7.36 – 7.31 (m, 4H), 7.27 – 7.20 (m, 1H), 5.86 (s, 1H), 4.55 (s, 2H), 2.13 (s, 3H). ^13^C NMR (101 MHz, DMSO‐d 6) δ 163.9, 163.3, 163.2, 159.5, 140.1, 137.7, 137.3, 134.0, 131.6, 130.5, 130.1, 128.2, 126.9, 126.7, 126.6, 121.6, 120.8, 118.3, 95.2, 43.5, 23.4. HRMS: m/z: calcd for C_25_H_23_BrN_5_O^+^: 490.1060 [M + H]^+^; found: 490.1057.

3‐Bromo‐N‐(4‐((4‐methyl‐6‐((pyridin‐2‐ylmethyl)amino)pyrimidin‐2‐yl)amino)phenyl)benzamide 4o

4.2.37

Product 4o was obtained as a pale yellow solid. Yield: 169.2 mg (69%) over two reaction steps. ^1^H NMR (400 MHz, DMSO‐d 6) δ 10.14 (s, 1H), 8.93 (s, 1H), 8.55 (ddd, J = 4.8, 1.7, 0.9 Hz, 1H), 8.13 (t, J = 1.8 Hz, 1H), 7.94 (dt, J = 7.9, 1.4 Hz, 1H), 7.79 – 7.72 (m, 2H), 7.69 – 7.46 (m, 6H), 7.32 (dt, J = 7.9, 1.1 Hz, 1H), 7.25 (ddd, J = 7.3, 4.8, 1.1 Hz, 1H), 5.91 (s, 1H), 4.62 (d, J = 4.4 Hz, 2H), 2.14 (s, 3H). ^13^C NMR (101 MHz, DMSO‐d 6) δ 163.9, 163.3, 163.2, 159.5, 148.8, 137.6, 137.3, 136.6, 134.0, 131.6, 130.6, 130.1, 126.7, 121.9, 121.6, 120.7, 120.7, 118.2, 95.3, 45.6, 23.4. HRMS: m/z: calcd for C_24_H_22_BrN_6_O^+^: 491.1013 [M + H]^+^; found: 491.1018.

3‐Bromo‐N‐(4‐((4‐((furan‐2‐ylmethyl)amino)‐6‐methylpyrimidin‐2‐yl)amino)phenyl)benzamide 4p

4.2.38

Product 4p was obtained as a yellow gel. Yield: 135.5 mg (57%) over two reaction steps. ^1^H NMR (400 MHz, DMSO‐d 6) δ 10.17 (s, 1H), 8.97 (s, 1H), 8.13 (t, J = 1.9 Hz, 1H), 7.95 (dt, J = 7.9, 1.3 Hz, 1H), 7.77 (ddd, J = 8.0, 2.0, 1.0 Hz, 1H), 7.78 – 7.68 (m, 2H), 7.64 – 7.53 (m, 3H), 7.54 – 7.42 (m, 2H), 6.39 (dd, J = 3.1, 1.8 Hz, 1H), 6.27 (dd, J = 3.2, 0.7 Hz, 1H), 5.86 (s, 1H), 4.52 (d, J = 4.4 Hz, 2H), 2.13 (s, 3H). ^13^C NMR (101 MHz, DMSO‐d 6) δ 164.0, 163.3, 162.9, 159.5, 153.0, 141.9, 137.7, 137.3, 134.0, 131.7, 130.6, 130.1, 126.7, 121.6, 120.8, 118.4, 110.4, 106.6, 95.1, 37.0, 23.4. HRMS: m/z: calcd for C_23_H_21_BrN_5_O_2_ ^+^: 480.0857 [M + H]^+^; found: 480.0853.

N‐(4‐((4‐((Benzo[d][1,3]dioxol‐5‐ylmethyl)amino)‐6‐methylpyrimidin‐2‐yl)amino)phenyl)‐3‐bromobenzamide 4q

4.2.39

Product 4q was obtained as a yellow gel. Yield: 106.3 mg (40%) over two reaction steps. ^1^H NMR (400 MHz, DMSO‐d 6) δ 10.16 (s, 1H), 8.93 (s, 1H), 8.13 (t, J = 1.8 Hz, 1H), 7.94 (dt, J = 7.8, 1.4 Hz, 1H), 7.77 (ddd, J = 8.0, 2.0, 0.9 Hz, 1H), 7.69 (d, J = 8.7 Hz, 2H), 7.58 – 7.53 (m, 2H), 7.49 (t, J = 7.9 Hz, 2H), 6.91 – 6.79 (m, 3H), 5.97 (s, 2H), 5.84 (s, 1H), 4.44 (s, 2H), 2.12 (s, 3H). ^13^C NMR (101 MHz, DMSO‐d 6) δ 163.4, 163.3, 163.1, 159.5, 147.2, 145.9, 137.7, 137.3, 134.0, 131.6, 130.6, 130.1, 126.7, 121.6, 120.8, 120.1, 118.3, 108.0, 107.6, 100.7, 95.3, 43.3, 23.4. HRMS: m/z: calcd for C_26_H_23_BrN_5_O_3_ ^+^: 534.0958 [M + H]^+^; found: 534.0964.

3‐Bromo‐N‐(4‐((4‐methyl‐6‐((4‐morpholinophenyl)amino)pyrimidin‐2‐yl)amino)phenyl)benzamide 4r

4.2.40

Product 4r was obtained as a white solid. Yield: 94.4 mg (34%) over two reaction steps. ^1^H NMR (400 MHz, DMSO‐d 6) δ 10.20 (s, 1H), 9.04 (s, 1H), 8.97 (s, 1H), 8.13 (t, J = 1.9 Hz, 1H), 7.95 (dd, J = 7.9, 1.1 Hz, 1H), 7.78 (ddd, J = 8.0, 1.9, 0.9 Hz, 1H), 7.71 (d, J = 9.1 Hz, 2H), 7.64 – 7.54 (m, 2H), 7.50 (t, J = 7.9 Hz, 1H), 7.45 (d, J = 8.6 Hz, 2H), 6.97 – 6.86 (m, 2H), 5.98 (s, 1H), 3.74 (t, J = 4.7 Hz, 4H), 3.07 (t, J = 4.7 Hz, 4H), 2.18 (s, 3H). ^13^C NMR (101 MHz, DMSO‐d 6) δ 163.4, 161.3, 159.5, 146.8, 137.4, 137.3, 134.0, 132.2, 132.0, 130.6, 130.1, 126.7, 122.0, 121.6, 120.8, 119.0, 115.6, 66.1, 49.1, 23.5. HRMS: m/z: calcd for C_28_H_28_BrN_6_O_2_ ^+^: 561.1431 [M + H]^+^; found: 561.1444.

N‐(4‐((4‐(((1‐Benzyl‐1H‐1,2,3‐triazol‐4‐yl)methyl)amino)‐6‐methylpyrimidin‐2‐yl)amino)phenyl)‐3‐bromobenzamide 4s

4.2.41

Product 4s was obtained as a yellow gel (by cycloaddition from 4m). Yield: 248.9 mg (87%) over two reaction steps. ^1^H NMR (400 MHz, DMSO‐d 6) δ 10.17 (s, 1H), 8.95 (s, 1H), 8.13 (t, J = 1.8 Hz, 1H), 7.98 – 7.91 (m, 2H), 7.77 (ddd, J = 8.0, 1.9, 0.9 Hz, 1H), 7.71 (d, J = 9.0 Hz, 2H), 7.59 – 7.54 (m, 2H), 7.52 – 7.42 (m, 2H), 7.34 – 7.24 (m, 5H), 5.84 (s, 1H), 5.55 (s, 2H), 4.56 (d, J = 5.7 Hz, 2H), 2.12 (s, 3H). ^13^C NMR (101 MHz, DMSO‐d 6) δ 163.9, 163.3, 162.9, 159.5, 145.8, 137.7, 137.3, 136.1, 134.0, 131.7, 130.6, 130.1, 128.6, 128.0, 127.8, 126.7, 122.9, 121.6, 120.8, 118.5, 95.2, 52.7, 35.8, 23.4. HRMS: m/z: calcd for C_28_H_26_BrN_8_O^+^: 571.1387 [M + H]^+^; found: 571.1381.

General Procedure for the Synthesis of Intermediates 5

4.2.42

Intermediate 2a (0.50 mmol) was dissolved in EtOH (5 ml), followed by the addition of the corresponding diamine (0.50 mmol, 1.0 eq.). The reaction mixture was stirred at 90°C for 30–120 min (depending on the diamine used) under microwave irradiation (200 W). Upon completion of the reaction, as confirmed by TLC/LC‐MS, the mixture was concentrated under reduced pressure, and the obtained residue was filtered through a short pad of silica gel using DCM/MeOH/TEA (9:1:0.1, v/v/v) as the eluent. The filtrate was concentrated under reduced pressure, and the crude product was used directly in the subsequent acylation step to afford the final compounds 6 without further purification.

For reactions with propane‐1,3‐diamine and 1,3‐phenylenedimethanamine, the procedure was performed in neat diamine (3 ml, used in excess) without EtOH under otherwise identical conditions (90°C, 120 min, 200 W).

In the case of 2,3,5,6‐tetramethylbenzene‐1,4‐diamine, the reaction was carried out under conventional heating at 100°C for 90 h instead of microwave conditions with the use of EtOH as solvent (5 ml).

General Procedure for the Synthesis of Final Products 6a‐j

4.2.43

In a pre‐dried Schlenk flask under an argon atmosphere, intermediate 5 (0.50 mmol, obtained from the preceding step) was dissolved in anhydrous THF (8 ml), followed by the addition of K_2_CO_3_ (172.8 mg, 1.25 mmol, 2.5 eq.). The reaction mixture was cooled to 0°C, and 3‐bromobenzoyl chloride (66.0 μl, 0.50 mmol, 1.0 eq.) was added dropwise. The mixture was stirred at room temperature for 1 h. Upon completion of the reaction (as confirmed by TLC/LC‐MS), water (20 ml) was added, and the resulting mixture was extracted with DCM (3 × 30 ml). The combined organic layers were washed with brine (20 ml), dried over anhydrous MgSO_4_, and concentrated under reduced pressure. The crude product was purified by column chromatography using DCM/MeOH (grad.) to afford the desired compound 6a‐j.

3‐Bromo‐N‐(3‐((4‐(ethylamino)‐6‐methylpyrimidin‐2‐yl)amino)propyl)benzamide 6a

4.2.44

Product 6a was obtained as a colorless gel. Yield: 87.2 mg (44%) over two reaction steps. ^1^H NMR (500 MHz, DMSO‐d 6) δ 8.72 (t, J = 5.4 Hz, 1H), 8.04 (t, J = 1.8 Hz, 1H), 7.87 (dt, J = 7.8, 1.2 Hz, 1H), 7.72 (ddd, J = 8.0, 1.9, 0.9 Hz, 1H), 7.43 (t, J = 7.9 Hz, 1H), 5.77 (s, 1H), 3.42 – 3.19 (m, 6H; overlapped with H_2_O), 2.12 (s, 3H), 1.77 (p, J = 6.7 Hz, 2H), 1.05 (t, J = 7.2 Hz, 3H). ^13^C NMR (101 MHz, DMSO‐d 6) δ 164.6, 162.5, 136.7, 133.8, 130.5, 129.8, 126.3, 121.6, 94.7, 38.1, 36.9, 35.0, 28.9, 19.3, 14.1. HRMS: m/z: calcd for C_17_H_23_BrN_5_O^+^: 394.1058 [M + H]^+^; found: 394.1060.

3‐Bromo‐N‐(3‐((4‐(ethylamino)‐6‐methylpyrimidin‐2‐yl)amino)phenyl)benzamide 6b

4.2.45

Product 6b was obtained as a brown gel. Yield: 67.9 mg (32%) over two reaction steps. ^1^H NMR (400 MHz, DMSO‐d 6) δ 10.22 (s, 1H), 8.95 (s, 1H), 8.21 (s, 1H), 8.13 (t, J = 1.8 Hz, 1H), 7.95 (dt, J = 7.9, 1.3 Hz, 1H), 7.78 (ddd, J = 7.9, 1.9, 1.0 Hz, 1H), 7.58 – 7.52 (m, 1H), 7.49 (t, J = 7.9 Hz, 1H), 7.18 (d, J = 5.3 Hz, 2H), 6.96 (s, 1H), 5.78 (s, 1H), 3.36 – 3.25 (m, 2H; overlapped with water), 2.13 (s, 3H), 1.12 (t, J = 7.2 Hz, 3H). ^13^C NMR (101 MHz, DMSO‐d 6) δ 163.7, 163.3, 163.1, 159.5, 141.8, 138.6, 137.3, 134.1, 130.6, 130.2, 128.1, 126.8, 121.6, 114.6, 113.4, 111.6, 95.4, 34.9, 23.3, 14.7. HRMS: m/z: calcd for C_20_H_21_BrN_5_O^+^: 428.0904 [M + H]^+^; found: 428.0905.

3‐Bromo‐N‐(3‐(((4‐(ethylamino)‐6‐methylpyrimidin‐2‐yl)amino)methyl)benzyl)benzamide 6c

4.2.46

Product 6c was obtained as a yellow gel. Yield: 116.2 mg (51%) over two reaction steps. ^1^H NMR (500 MHz, DMSO‐d 6) δ 9.2 (t, J = 6.0 Hz, 1H), 8.1 (t, J = 1.8 Hz, 1H), 7.9 (ddd, J = 7.8, 1.6, 1.1 Hz, 1H), 7.7 (ddd, J = 8.0, 2.0, 0.9 Hz, 1H), 7.4 (t, J = 7.9 Hz, 1H), 7.3 (s, 1H), 7.2 (d, J = 7.5 Hz, 1H), 7.2 – 7.2 (m, 2H), 5.7 (s, 1H), 4.5 (t, J = 5.7 Hz, 4H), 3.2 (p, J = 6.7 Hz, 2H; overlapped with water), 2.1 (s, 3H), 1.0 (t, J = 7.2 Hz, 3H).^13^C NMR (101 MHz, DMSO‐d 6) δ 164.6, 162.8, 140.5, 139.2, 136.4, 133.9, 130.6, 129.9, 128.1, 126.4, 126.2, 125.8, 125.6, 121.7, 94.2, 43.9, 42.7, 34.9, 21.3, 14.4. HRMS: m/z: calcd for C_22_H_25_BrN_5_O^+^: 456.1210 [M + H]^+^; found: 456.1217.

3‐Bromo‐N‐(4‐(4‐((4‐(ethylamino)‐6‐methylpyrimidin‐2‐yl)amino)benzyl)phenyl)benzamide 6d

4.2.47

Product 6d was obtained as a yellow solid. Yield: 176.3 mg (68%) over two reaction steps. ^1^H NMR (400 MHz, DMSO‐d 6) δ 10.27 (s, 1H), 8.81 (s, 1H), 8.13 (t, J = 1.8 Hz, 1H), 7.94 (dt, J = 7.8, 1.3 Hz, 1H), 7.78 (ddd, J = 8.0, 1.9, 0.9 Hz, 1H), 7.73 – 7.69 (m, 2H), 7.69 – 7.65 (m, 2H), 7.49 (t, J = 7.9 Hz, 1H), 7.19 (d, J = 8.5 Hz, 2H), 7.05 (d, J = 8.5 Hz, 2H), 6.92 (br s, 1H), 5.75 (s, 1H), 3.83 (s, 2H), 3.31 – 3.21 (m, 2H; overlapped with H_2_O), 2.10 (s, 3H), 1.14 (t, J = 7.2 Hz, 3H). ^13^C NMR (101 MHz, DMSO‐d 6) δ 163.7, 163.4, 163.0, 159.6, 139.6, 137.5, 137.1, 136.7, 134.2, 132.9, 130.6, 130.2, 128.7, 128.4, 126.8, 121.6, 120.5, 118.4, 94.9, 34.9, 23.4, 14.7. HRMS: m/z: calcd for C_27_H_26_BrN_5_O^+^: 518.1373 [M + H]^+^; found: 518.1365.

3‐Bromo‐N‐(3‐(3‐((4‐(ethylamino)‐6‐methylpyrimidin‐2‐yl)amino)benzyl)phenyl)benzamide 6e

4.2.48

Product 6e was obtained as a yellow gel. Yield: 164.4 mg (64%) over two reaction steps. ^1^H NMR (400 MHz, DMSO‐d 6) δ 10.28 (s, 1H), 8.85 (s, 1H), 8.12 (t, J = 1.8 Hz, 1H), 7.93 (dt, J = 7.9, 1.1 Hz, 1H), 7.83 (s, 1H), 7.77 (ddd, J = 8.0, 1.9, 0.9 Hz, 1H), 7.65 – 7.61 (m, 2H), 7.49 (q, J = 8.8, 7.9 Hz, 2H), 7.28 (d, J = 7.7 Hz, 1H), 7.12 (t, J = 7.8 Hz, 1H), 6.99 (dt, J = 7.7, 1.4 Hz, 1H), 6.93 (s, 1H), 6.74 (dt, J = 7.4, 1.3 Hz, 1H), 5.75 (s, 1H), 3.87 (s, 2H), 3.26 (p, J = 6.7 Hz, 2H), 2.09 (s, 3H), 1.12 (t, J = 7.2 Hz, 3H). ^13^C NMR (101 MHz, DMSO‐d 6) δ 163.8, 163.3, 163.0, 159.5, 141.9, 141.7, 140.9, 138.9, 137.1, 134.2, 130.6, 130.2, 128.5, 128.2, 126.8, 124.4, 121.6, 120.7, 120.7, 118.7, 118.1, 116.2, 95.3, 41.8, 34.9, 23.3, 14.7. HRMS: m/z: calcd for C_27_H_26_BrN_5_O^+^: 518.1373 [M + H]^+^; found: 518.1370.

(E)‐3‐Bromo‐N‐(4‐(4‐((4‐(ethylamino)‐6‐methylpyrimidin‐2‐yl)amino)styryl)phenyl)benzamide 6f

4.2.49

Product 6f was obtained as a yellow solid. Yield: 174.4 mg (66%) over two reaction steps. ^1^H NMR (400 MHz, DMSO‐d 6) δ 10.38 (s, 1H), 9.04 (s, 1H), 8.16 (t, J = 1.7 Hz, 1H), 7.97 (d, J = 8.0 Hz, 1H), 7.84 (d, J = 8.7 Hz, 2H), 7.82 – 7.75 (m, 3H), 7.55 (d, J = 8.7 Hz, 2H), 7.50 (t, J = 7.9 Hz, 1H), 7.45 (d, J = 8.8 Hz, 2H), 7.16 – 6.94 (m, 3H), 5.80 (s, 1H), 3.35 – 3.27 (m, 2H; overlapped with H_2_O), 2.14 (s, 3H), 1.17 (t, J = 7.2 Hz, 3H). ^13^C NMR (101 MHz, DMSO‐d 6) δ 164.3, 164.2, 163.6, 160.0, 141.8, 138.4, 137.6, 134.8, 133.9, 131.2, 130.8, 129.7, 128.1, 127.1, 127.9, 125.4, 122.2, 121.0, 118.8, 96.0, 35.5, 23.9, 15.2,. HRMS: m/z: calcd for C_28_H_27_BrN_5_O^+^: 530.1367 [M + H]^+^; found: 530.1373.

3‐Bromo‐N‐(4‐((4‐(ethylamino)‐6‐methylpyrimidin‐2‐yl)amino)‐2,5‐dimethylphenyl)benzamide 6g

4.2.50

Product 6g was obtained as a white solid. Yield: 151.4 mg (67%) over two reaction steps. ^1^H NMR (400 MHz, DMSO‐d 6) δ 9.88 (s, 1H), 8.15 (t, J = 1.8 Hz, 1H), 7.97 (d, J = 7.8 Hz, 1H), 7.82 (s, 1H), 7.80 – 7.76 (m, 1H), 7.66 (s, 1H), 7.49 (t, J = 7.9 Hz, 1H), 7.07 (s, 1H), 6.91 (s, 1H), 5.73 (s, 1H), 3.23 (p, J = 6.9 Hz, 2H), 2.20 (s, 3H), 2.15 (s, 3H), 2.09 (s, 3H), 1.11 (t, J = 7.2 Hz, 3H). ^13^C NMR (101 MHz, DMSO‐d 6) δ 163.9, 163.7, 163.1, 160.1, 136.9, 136.9, 134.1, 130.9, 130.6, 130.2, 128.2, 127.7, 126.7, 125.0, 121.7, 94.5, 34.7, 23.3, 17.7, 17.6, 14.8. HRMS: m/z: calcd for C_22_H_25_BrN_5_O^+^: 456.1217 [M + H]^+^; found: 456.1210.

3‐Bromo‐N‐(4‐((4‐(ethylamino)‐6‐methylpyrimidin‐2‐yl)amino)‐2,3,5,6‐tetramethylphenyl)benzamide 6h

4.2.51

Product 6h was obtained as a white solid. Yield: 143.5 mg (59%) over two reaction steps. ^1^H NMR (400 MHz, DMSO‐d 6) δ 9.90 (s, 1H), 8.18 (t, J = 1.4 Hz, 1H), 8.01 (dt, J = 8.1, 0.9 Hz, 1H), 7.93 (br s, 1H), 7.80 (ddd, J = 7.9, 2.1, 1.1 Hz, 1H), 7.51 (t, J = 7.9 Hz, 1H), 7.15 – 6.33 (m, 1H), 5.62 (s, 1H), 3.05 (br s, 2H), 2.15 – 1.95 (m, 15H), 1.05 (s, 3H). ^13^C NMR (101 MHz, DMSO‐d 6) δ 163.8, 163.6, 163.4, 160.9, 136.7, 135.9, 134.1, 132.3, 132.3, 131.1, 130.7, 130.1, 126.6, 121.8, 93.3, 34.9, 23.2, 15.5, 15.2, 14.6. HRMS: m/z: calcd for C_24_H_29_BrN_5_O^+^: 484.1530 [M + H]^+^; found: 484.1524.

3‐Bromo‐N‐(2,5‐dibromo‐4‐((4‐(ethylamino)‐6‐methylpyrimidin‐2‐yl)amino)phenyl)benzamide 6i

4.2.52

Product 6i was obtained as a yellow solid. Yield: 191.0 mg (65%) over two reaction steps. ^1^H NMR (400 MHz, DMSO‐d 6) δ 10.20 (s, 1H), 8.88 (s, 1H), 8.16 (t, J = 1.5 Hz, 1H), 7.97 (d, J = 7.9 Hz, 1H), 7.82 (dd, J = 7.8, 1.0 Hz, 1H), 7.77 (s, 1H), 7.60 (s, 1H), 7.51 (t, J = 7.9 Hz, 1H), 7.28 (s, 1H), 5.90 (s, 1H), 3.32 – 3.22 (m, 2H), 2.15 (s, 3H), 1.16 (t, J = 7.2 Hz, 3H). ^13^C NMR (101 MHz, DMSO‐d 6) δ 164.2, 163.7, 163.0, 158.6, 137.5, 136.0, 134.5, 131.8, 130.7, 130.3, 130.2, 126.8, 123.9, 121.7, 120.0, 111.3, 97.0, 34.9, 23.3, 14.7. HRMS: m/z: calcd for C_20_H_19_Br_3_N_5_O^+^: 595.9088 [M + H]^+^; found: 595.9093.

3‐Bromo‐N‐(5‐((4‐(ethylamino)‐6‐methylpyrimidin‐2‐yl)amino)naphthalen‐1‐yl)benzamide 6j

4.2.53

Product 6j was obtained as a brown solid. Yield: 144.9 mg (61%) over two reaction steps. ^1^H NMR (400 MHz, DMSO‐d 6) δ 10.52 (s, 1H), 8.73 (s, 1H), 8.28 (t, J = 1.9 Hz, 1H), 8.09 (d, J = 8.0 Hz, 2H), 7.90 (d, J = 7.5 Hz, 1H), 7.84 (ddd, J = 8.0, 1.8, 0.8 Hz, 1H), 7.70 (d, J = 8.4 Hz, 1H), 7.56 – 7.46 (m, 4H), 6.87 (s, 1H), 5.77 (s, 1H), 3.18 (p, J = 6.7 Hz, 2H), 2.11 (s, 3H), 1.06 (t, J = 7.2 Hz, 3H). ^13^C NMR (101 MHz, DMSO‐d 6) δ 164.7, 163.9, 163.2, 160.8, 136.7, 136.6, 134.3, 133.5, 130.7, 130.4, 130.1, 128.9, 126.9, 125.6, 124.4, 123.9, 122.0, 121.7, 120.3, 118.3, 94.6, 34.7, 23.4, 14.7. HRMS: m/z: calcd for C_24_H_23_BrN_5_O^+^: 478.1060 [M + H]^+^; found: 478.1061.

Synthesis of (3‐Bromophenyl)(5‐((4‐(ethylamino)‐6‐methylpyrimidin‐2‐yl)amino)‐1H‐indol‐1‐yl)methanone 6k

4.2.54

In a pre‐dried Schlenk flask under an argon atmosphere, intermediate 5k (0.50 mmol) was dissolved in anhydrous THF (8 ml), followed by the addition of Cs_2_CO_3_ (407.3 mg, 1.25 mmol, 2.5 eq.). The reaction mixture was cooled to 0°C, and 3‐bromobenzoyl chloride (198.2 μL, 1.50 mmol, 3.0 eq.) was added dropwise. The mixture was stirred at room temperature for 30 h. Upon completion of the reaction (as confirmed by TLC/LC‐MS), water (20 ml) was added, and the mixture was extracted with DCM (3 × 30 ml). The combined organic layers were washed with brine (20 ml), dried over anhydrous MgSO_4_, and concentrated under reduced pressure. The crude product was purified by column chromatography using DCM/MeOH (grad.) to afford the desired compound 6k. Product 6k was obtained as a white solid. Yield: 72.7 mg (32%) over two reaction steps. ^1^H NMR (400 MHz, DMSO‐d 6) δ 11.16 (s, 1H), 7.63 (t, J = 1.8 Hz, 1H), 7.57 (ddd, J = 8.0, 1.8, 0.9 Hz, 1H), 7.43 (dt, J = 7.8, 1.3 Hz, 1H), 7.40 – 7.36 (m, 3H), 7.27 (t, J = 7.8 Hz, 1H), 7.23 (t, J = 5.6 Hz, 1H), 6.94 (dd, J = 8.6, 2.0 Hz, 1H), 6.43 (t, J = 2.5 Hz, 1H), 5.99 (s, 1H), 2.93 (s, 2H), 2.00 (s, 3H), 0.84 (s, 3H). ^13^C NMR (101 MHz, DMSO‐d 6) δ 170.3, 164.5, 163.6, 162.0, 140.9, 135.0, 133.8, 133.1, 130.8, 130.7, 128.3, 126.8, 121.7, 119.5, 112.0, 101.9, 101.2, 35.0, 23.7, 14.7. HRMS: m/z: calcd for C_22_H_21_BrN_5_O^+^: 452.0897 [M + H]^+^; found: 452.0904.

Cell Cultures and Cytotoxicity Assay

4.3

Human cancer cell lines were obtained from the European Collection of Authenticated Cell Cultures (MCF7) or Cell Lines Service (MV4−11) and cultured under standard conditions recommended by the suppliers. Specifically, MCF7 cells were maintained in Dulbecco's Modified Eagle Medium (DMEM), while MV4−11 cells were grown in RPMI‐1640 medium. All media were supplemented with 10–20% fetal bovine serum, 100 U/ml penicillin, and 0.1 mg/ml streptomycin. Cells were cultured at 37°C in a humidified incubator with 5% CO_2_.

For viability testing, cells were exposed to the tested compounds for 72 h. Following treatment, resazurin solution (Merck) was added to each well and incubated for 4 h. Cytotoxic effects were evaluated indirectly via measurement of cell viability using the resazurin assay. The fluorescence of resorufin, corresponding to the number of metabolically active (viable) cells, was measured using a Fluoroskan Ascent microplate reader (Labsystems) at an excitation/emission wavelength of 544/590 nm. IC_50_ values, defined as the compound concentration causing 50% cytotoxicity, were determined from the resulting dose–response curves.

Supporting Information

The Supporting Information contains additional schemes and experimental procedures for the preparation of pyrimidine intermediates 2 and 2′, as well as the synthetic procedure for benzyl azide. It also includes experimental data for the minor regioisomers 2′ and copies of the NMR spectra for all compounds described in this article.

Author Contributions

Jan Chasák performed the majority of the synthetic experiments, analyzed the experimental data, and wrote the chemistry part of the manuscript. Petr Vyvlečka synthesized a few derivatives 4. Ivan Nemec performed SC‐XRD measurements and refinement of the crystal structure. An Matheeussen performed the antimycobacterial and cytotoxicity assays and analyzed the experimental data. Natascha Van Pelt performed the antiparasitic and cytotoxicity assays. Paul Cos supervised the antimycobacterial and cytotoxicity assays, participated in the interpretation of the data, and reviewed the manuscript draft. Guy Caljon supervised the antiparasitic assays, participated in the interpretation of the data, and reviewed the manuscript draft. Vladimír Kryštof designed and supervised cytotoxicity assays. Lucie Brulíková initiated the project, led the project team, was responsible for funding acquisition regarding the synthetic part, designed the synthetic experiments, and analyzed the results. Lucie Brulíková wrote the paper with input from all authors. All authors have approved the final version of the manuscript.

Conflicts of Interest

The authors declare no conflicts of interest.

Supporting information

Supplementary Material