2.1. Anticancer Activity
Benzo[b]furan derivatives have demonstrated a fascinating array of biological and pharmaceutical activities, including antitumor properties. For instance, Flynn et al. described the discovery of 7-hydroxy-6-methoxy-2-methyl-3-(3,4,5-trimethoxybenzoyl)benzo[b]furan (BNC105,
6a, R
1 = OH, R
2 = Me), a potent and selective antiproliferative agent. They achieved the synthesis of various derivatives, many of which were obtained through a modified Larock-type coupling between o-iodophenol
1a,
b and 3-silyl-1-arylpropinone
2, yielding 2-silylbenzo[b]furans
3a,
b in 59% and 69% yields, respectively (
Scheme 1) [
32,
33]. Subsequently, silanes
3a (R
1 = H) and
3b (R
1 = Oi-Pr) underwent treatment with TBAF in methanol to remove the silyl groups. For compound
3b, an additional reaction with AlCl
3 was performed to eliminate the isopropyl group, resulting in the formation of compounds
4a (R
1 = H) and
4b (R
1 = OAc) with yields of 83% and 86%, respectively. On the other hand, compound
3a underwent bromodesilylation with a 59% yield, producing 2-bromobenzo[b]furan
5a (R
1 = H). To avoid competitive bromination of the C–4 position of benzo[b]furan during the bromodesilylation process of
3b, the isopropyl group was first exchanged for an acetyl group, yielding compound
3c. Subsequent bromodesilylation of
3c resulted in the formation of compound
5b with a 69% yield. Additionally, the brominated derivatives
5a and
5b exhibited versatile functionality allowing bromine replacement through palladium coupling or nucleophilic displacement, leading to the formation of analog series
6, which includes heterocyclic, carbocyclic, and alicyclic analogs at C–2 of benzo[b]furan (
Table 1). One striking example of the versatility of the brominated derivatives is observed in the synthesis of the biologically significant compound
6a (R
1 = OH, R
2 = Me), which was obtained via a Negishi reaction, coupling the derivative
5b with methylzinc bromide using palladium, achieving an impressive 93% yield.
A molecular docking simulation was performed to investigate the interactions of compounds
4b and
6a with the
α,
β-tubulin dimer complexed with podophyllotoxin (PDB ID code: 1SA1). During the docking study, the structures of compounds
4b and
6a were oriented, considering the structural similarities between these synthesized compounds and colchicine
7. Specifically, the study focused on the interaction of colchicine with the
β-tubulin subunit, where the 3,4,5-trimethoxyphenyl rings overlapped with similar rings in colchicine. Additionally, the C6–OMe and C7–OH substituents were examined for their interactions with the methoxy and carbonyl groups on the tropone ring of colchicine (
Figure 3). The study showed the formation of a hydrogen bond between the C7–OH group of the benzo[
b]furan (
4b and
6a) with the Asn
β258 side chain, as well as the formation of a hydrogen bond with the amide nitrogen Val 181 in the adjacent subunit of
α-tubulin. Furthermore, the orientation of the C–2 position of benzo[
b]furan toward a gap between the
α- and
β-tubulin subunits allows it to harbor large substituents.
Another conducted study aimed to evaluate the effectiveness of benzo[
b]furan
6a on various human cancer cell lines using Combretastatin-A4 (CA-4) as a standard drug (
Table 2). Interestingly, the results revealed that benzo[
b]furan
6a exhibited excellent selectivity against human aortic arterial endothelial cells (HAAECs), a characteristic not observed with CA-4. Furthermore, compound
6a demonstrated significantly higher antiproliferative activity than CA-4, with up to a 10-fold increase in potency observed across many of the tested cell lines.
On the other hand, Romagnoli et al. highlighted the significance of incorporating a 3,4,5-trimethoxybenzoyl group at the C–2 position of benzo[
b]furan in determining the antiproliferative activity of benzofuran derivatives [
34]. As depicted in
Scheme 2, they synthesized a series of amino 2-(3′,4′,5′-trimethoxybenzoyl)-benzo[
b]furan with good yields through three reaction steps: (1) a one-step cyclization reaction of nitrosalicylaldehydes or 2-hydroxyacetophenone
8 with 2-bromo-1-(3′,4′,5′-trimethoxyphenyl)ethanone and anhydrous potassium carbonate in acetone at reflux, yielding the nitro derivatives of 2-(3′,4′,5′-trimethoxybenzoyl)benzo[
b]furanone
9a–
j; (2) the subsequent reduction of the nitro group using iron in a mixture of 37% HCl in water and ethanol at reflux, leading to the formation of the amino derivatives
10a–
j; and (3) the preparation of analogs
11a–
i in good yields through a substitution reaction between
α-bromoacrylic acid and the amino benzo[
b]furanone derivatives
10a–
f and
10h–
j, using an excess of two equivalents of EDCI and BtOH in dry DMF as a solvent. The glycine prodrug
13 was obtained in a 95% yield through the reaction between the amino derivative
10h and
N-Boc-glycine using EDCI and BtOH as coupling agents, with subsequent scission of the
N-Boc protecting group with a solution of 3M HCl in ethyl acetate.
The antiproliferative activity of this series of derivatives, including amino 2-(3′,4′,5′-trimethoxybenzoyl)-benzo[
b]furans
10a–
j and
11a–
I, was evaluated against various cancer cell lines, along with CA-4 as the standard drug (
Table 3). Compound
10h (R
4,7 = H, R
3 = Me, R
5 = NH
2, R
6 = OMe) demonstrated the most promising results in the series, exhibiting significant growth inhibition against cancer cell lines L1210, FM3A/0, Molt4/C8, CEM/0, and HeLa, with IC
50 values ranging from 16 to 24 nM. Notably, compound
10h exhibited higher activity in the FM3A/0 cell line, with an IC
50 value of 24 nM, compared to the standard drug CA-4 (IC
50 = 42 nM). SAR information derived from the comparison of unsubstituted compounds
10b (R
3,4,6,7 = H, R
5 = NH
2),
10g (R
3,4,7 = H, R
5 = NH
2, R
6 = OMe), and
10i (R
3,4,6 = H, R
5 = NH
2, R
7 = OMe) vs. methyl derivatives
10c (R
4,6,7 = H, R
3 = Me, R
5 = NH
2),
10h (R
4,7 = H, R
3 = Me, R
5 = NH
2, R
6 = OMe), and
10j (R
4,6 = H, R
3 = Me, R
5 = NH
2, R
7 = OMe) showed a significant increase in antiproliferative activity against cell lines by introducing the methyl group at the C–3 position of the benzofuran ring. Also, an increase in activity can be observed when comparing the methyl derivative
10c with
10b. When comparing the activities of compounds
10g and
10h with those of
10i and
10j, higher activity can be observed in compounds with methoxy groups at position C–6 rather than at position C–7 of the benzofuran ring. According to the results, compound
10h, with a methyl group at the C–3 position and a methoxy group at the C–6 position, exhibited 2–4 times greater potency than the unsubstituted compound
10g and 3–10 times higher activity than compound
10j, which features a methoxy group at the C–7 position of the benzofuran ring. Changing the positions of the amino and methoxy groups from
10j (R
4,6 = H, R
3 = Me, R
5 = NH
2, R
7 = OMe) to
10f (R
4,6 = H, R
3 = Me, R
5 = OMe, R
7 = NH
2) resulted in a reduction in activity.
The unsubstituted
α-bromoacryloylamide derivatives
11a (R
3,5–7 = H, R
4 = X),
11b (R
3,4,6,7 = H, R
5 = X),
11d (R
3–5,7 = H, R
6 = X), and
11e (R
3–6 = H, R
7 = X) exhibited antiproliferative potency that was 10–100 times greater than their amino counterparts, demonstrating the direct relationship between the presence of
α-bromoacryloylamides and increased activity (
Table 3). Shifting the
α-bromoacryloylamide group from the C–4 at the 5 position to the C–6 at the 7 position led to decreased activity. Finally, there were no significant differences in antiproliferative activity on all cell lines observed between compound
10h and its glycine hydrochloride prodrug
13.
To explore the potential correlation between antiproliferative activity and tubulin inhibition, the most active compounds,
10g,
10h,
10i,
10j, and
11a, were evaluated in the inhibition of tubulin polymerization. Among these compounds,
10h demonstrated the highest potency, displaying an IC
50 value of 0.56 µM, which was two times higher than that of CA-4 (IC
50 = 1.0 µM). Compounds
10g and
10j exhibited IC
50 values of 1.4 and 1.6 µM, respectively, for tubulin polymerization, showing a marginal decrease in potency compared to CA-4. Compound
10i exhibited approximately half the activity of
10j. In contrast, compound
11a did not induce any alteration in tubulin assembly, even at a concentration as high as 40 µM, suggesting that the mechanism of action of this
α-bromoacryloylamide derivative does not involve interaction with tubulin. Subsequently, the effect of the selected compounds,
10g,
10h,
10j, and
11a, was assessed on the cell cycle of human myeloid leukemia cell lines HL-60 and U937 using flow cytometry. The cells were cultured for 24 h with a concentration of 100 nM for each compound, and the two most active compounds (
10g and
10h) were examined at a lower concentration of 10 nM.
Figure 4 shows the fractions of hypodiploid cells in the sub-G1 peak of each compound studied, representing apoptotic cells. Compound
10j showed a modest increase in apoptotic cells at 100 nM, while compounds
10g and
10h presented different effects on the cell cycle in the two cell lines. It was observed that compound
11a had no effect on cell cycle distribution at 100 nM, in agreement with the previous results observed in tubulin inhibition. The significant increase in the sub-G1 peak in both cell lines with increasing concentrations of compounds
10g and
10h suggests that these compounds exert their growth inhibitory effect by inducing apoptosis.
To further examine the apoptotic effects of compounds
10g,
10h,
10j, and
11a, proteolytic processing of caspases in HL-60 and U937 cells was observed by Western blot analysis (
Figure 5). Compounds
10g,
10h,
10j, and
11a allowed the cleavage of inactive procaspase-9 to the active 37 kDa fragment, while lower concentrations of compounds
10g and
10h (0.3 µM) significantly promoted procaspase-8 hydrolysis. Furthermore, compounds
10g and
10h significantly led to the cleavage of inactive procaspases-3 and -6 in both cell lines (
Figure 5). Additionally, the induction of poly(ADP-ribose)polymerase (PARP) cleavage showed that compounds
10j and
11a exhibited lower potency in inducing PARP cleavage compared to compounds
10g and
10h. The appearance of the fragment at 85 kDa coincided with caspase-3 activation, observed by a decrease in the proenzyme at 36 kDa and an increase in cleaved procaspase-3 levels at 20 and 18 kDa (
Figure 5). Dose–response studies were performed, and cytosolic preparations were analyzed by immunoblotting to investigate whether apoptosis induced by compounds
10g,
10h,
10j, and
11a in HL-60 and U937 cells involved the release of cytochrome c from mitochondria into the cytosol during the apoptotic event. The results revealed a significant increase in the amount of cytochrome c in the cytosol of both cell lines at 15 kDa (
Figure 5).
CA-4 and its analogs have been clinically termed vascular disruptors agents (VDAs) [
35], so glycine prodrug
13 was tested to see its ability as a VDA in an in vivo model in rat breast cancer tumors, using the spatial frequency optical technique (SDFI). With 10 min of administration of compound
13 (30 mg kg
−1), there is a rapid decrease in oxygen saturation in tumor tissues similar to that observed with CA-4, confirming that prodrug
13 causes vascular disruption in vivo.
Interestingly, Wellington et al. conducted the synthesis of a variety of dihydroxylated 5,6-benzo[
b]furans
16 with catechol derivative
14 using a commercial laccase, Suberase
®, under different reaction conditions [
36]. All the synthesized compounds were evaluated for their anticancer properties. The synthetic protocol consisted of reacting an equivalent of catechol
14 with an equivalent of the 1,3-dicarbonyl compound
15 at room temperature using Suberase
® in an air-open vessel at pH 7.15 (
Scheme 3). In method A, the reaction of catechol derivatives
14a–
c with 1,3-dicarbonyls compounds
15a–
e was performed at room temperature at pH 7.15 for 24 h. In method B, the reaction was conducted under similar conditions with an extended time of 44 h to investigate the potential enhancement in product yield with prolonged reaction time. In method C, a mixture of the 1,3-dicarbonyl compound and catechol, combined in a 4:1 ratio, was dissolved in DMF. Subsequently, the resulting mixture was left to react for 42 h. The results obtained from methods A–C are shown in
Table 4. In particular, method A demonstrated the most favorable outcome of the three methods, exhibiting the highest yield of 98% for compound
16j (Entry 15,
Table 4). On the other hand, method B afforded compound
16k in a 77% yield (Entry 18,
Table 4), while method C yielded 71% for compound
16g (Entry 11,
Table 4). It is worth noting that the reaction time in Method B had minimal impact on the yield, whereas the presence of DMF in method C may have potentially deactivated the laccase, Suberase
®, leading to lower yields.
Anticancer studies were performed on various types of cancer, including renal (TK10), melanoma (UACC62), breast (MCF7), and cervical (HeLa), using a sulforhodamine B (SRB) assay to determine the growth inhibitory effects of these compounds. Notably, the 5,6-dihydroxylated benzo[b]furans 16e, 16g, 16h, 16k, 16m and 16n exhibited potent cytotoxic effects against the melanoma cell line (UAC62), with GI50 values ranging from 0.77 to 9.76 µM. Among these compounds, 16h (R = OMe, R1 = R2 = CH2, R3 = H, R4 = Me) and 16n (R = OMe, R1 = R2 = CH2, R3 = H, R4 = Ph) showed better activity than the standard drug Etoposide (GI50 = 0.89 µM). Moreover, compound 16n showed potent activity (GI50 = 9.73 µM) against the renal cancer cell line (TK10), while both 16h and 16n demonstrated strong activity against the breast cancer cell line (MCF7), with GI50 values of 8.79 and 9.30 µM, respectively.
In 2013, Kamal et al. synthesized a series of benzo[
b]furans with a modification at position 5 of the benzene ring by introducing C-linked substituents to generate 2-(3′,4′,5′-trimethoxybenzoyl)benzo[
b]furan derivatives [
37]. The most biologically interesting benzo[
b]furan derivatives,
22 and
25, were synthesized through a sequence of reactions depicted in
Scheme 4, which included (a) the acylation of
17 to yield product
18; (b) methylation using methyl iodide and potassium carbonate, resulting in
19; (c) iodination with iodine and silver nitrate in a catalytic amount to produce
20; (d) cyclization with 2-bromo-1-(3,4,5-trimethoxyphenyl)ethanone and potassium carbonate in acetone to furnish the benzofuran derivative
21; (e) Sonogashira coupling reaction to obtain
22; (f) Wittig reaction with the ylide generated from methyltriphenylphosphonium bromide in the presence of LiHMDS, leading to
23; (g) Heck coupling reaction with ethyl acrylate to yield the ethyl cinnamate derivative
24; and (h) ester reduction with DIBAL resulting in the formation of (
E)-allyl alcohol
25.
A study was performed to assess the cytotoxicity of benzofuran analogs against ME-180, A549, ACHNs, HT-29, and B-16 cell lines using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and CA-4 as the standard drug (
Table 5). The results from compound
22, which contains a 4-MeO-phenylacetylene group, showed good activity, with IC
50 values in the range of 0.08–1.14 µM against all evaluated cell lines, as shown in
Table 5. However, derivative
22 was two times less active against A549 and significantly less active against all four cell lines compared to CA-4. On the other hand, analog
25 exhibited the effect of the alkenyl substituent at position 5 of benzofuran on cytotoxicity, showing higher potency compared to compound
22 against ME-180, A549, ACHN, and B-16 cancer cell lines, with IC
50 values ranging from 0.06 to 0.17 µM. These values were comparable to those obtained with CA-4 against A549 and ACHN cancer lines, with IC
50 values of 0.05 and 0.09 µM, respectively. Furthermore, compounds
22 and
25 inhibited tubulin polymerization by 37.9 and 65.4%, respectively, which is comparable to the 70.5% tubulin inhibition observed with CA-4 (
Table 5).
Apoptotic studies were performed using various assays, including Hoechst staining assay, caspase-3 activation, DNA fragmentation analysis, and Western blot analysis. Specifically, a Hoechst staining assay was utilized to study the effects of compounds
22 and
25 on nuclear condensation. Remarkably, cells treated with these compounds showed a pronounced increase in nuclear condensation compared to untreated cells, strongly suggesting their potent ability to induce cell apoptosis (
Figure 6). In addition, caspase-3 activation analysis was conducted on A549 cells, treating them with concentrations of 50 and 100 nM of compounds
22 and
25 and comparing them to CA-4 at 100 nM. The results indicated a significant increase in caspase-3 activation, ranging from 1.5- to 3-fold compared to the control experiment (50 and 100 nM), demonstrating the programmed apoptotic activity induced by compounds
22 and
25 in the A549 cells.
DNA fragmentation analysis was conducted by incubating A549 cells with a 50 nM concentration of compounds
22 and
25. The results revealed a discrete staircase pattern after 48 h of treatment, indicative of significant fragmentation associated with cell death (
Figure 7a). Additionally, Western blot analysis was realized on the same cancer cell line, treating it with the same concentration of compounds
22 and
25 as used in the DNA fragmentation analysis (
Figure 7b). After 48 h of treatment, it was observed that the anti-apoptotic protein Bcl-2 was down-regulated, while the pro-apoptotic protein Bax was up-regulated. The results provide evidence that the induction of apoptosis by compounds
22 and
25 is associated with Bcl-2 down-regulation.
In a study conducted by Frías et al., an asymmetric synthesis of diheteroarylalkanes was presented. This synthesis involved a one-pot reaction using dienamine and Friedel–Crafts reactions between aldehyde
26 and indole
27, catalyzed by the Hayashi–Jorgensen catalyst
28 (20 mol %). Various substituents at different positions on the aldehyde and indole were utilized during the reaction [
38]. When starting, materials with electron-withdrawing groups (EWGs) or electron-donating groups (EDGs) were located at the
para position to the oxygen atom, and products
29a,
g–
I showed good yields and enantioselectivity ranging from 93% to 97% toward the (
S)-enantiomer (
Table 6). However, substrates substituted at the
ortho and
meta positions also enabled the synthesis of products
29j and
29k without a decrease in the final enantioselectivity (
ee = 93% and 94%, respectively). Indoles with bromo (
29b) or methoxy (
29c) substituents exhibited satisfactory yields and enantioselectivity (>95%). Methyl groups displayed good yield and enantioselectivity in products
29d and
29e (
ee = 99% and 96%, respectively). Additionally, the introduction of the 1
H-benzo[
g]indole group resulted in the desired aldehyde
29f with excellent enantioselectivity (
ee = 98%).
The enantioselectivity of compound
29 is explained through the proposed reaction mechanism depicted in
Scheme 5. It begins with the condensation reaction between aldehyde
26 and organocatalyst
28 to form iminium ion
I. Then, the isomerization of
I gives dienamine intermediate
II, which undergoes intramolecular condensation/dehydration sequence to afford iminium ion
III. At this pivotal stage, indole
27 undergoes an attack on intermediate
III, leading to the formation of product
29 with remarkable enantioselectivity. This precise enantiocontrol is facilitated by the steric shielding offered by the bulkier group (CPhPhOTMS) present in the organocatalyst.
The synthesized products
29a–
k were evaluated for their antiproliferative activity against a panel of tumor cell lines, including HBL-100 (breast), HeLa (cervix), SW1573 (non-small-cell lung), and WiDr (colon), using the SRB assay.
Figure 8 presents the GI
50 values, comparing them with
cis-platin as the standard drug. The results highlight the significance of the substituent on the aryl moiety of the benzofuran ring in influencing the antiproliferative activity of product
29. The introduction of strong electron-withdrawing groups (EWGs) and electron-donating groups (EDGs) resulted in a decrease in activity for compounds
29h,
29i, and
29j in WiDr cell lines. Notably, compounds
29a (R
1 = R
2 = H) and
29g (R
1 = 5-Cl, R
2 = H) exhibited GI
50 values comparable to
cis-platin in the WiDr cell line, with values of 28 and 16 µM for compounds
29a and
29g, respectively. However, the product of highest biological interest was
29f (R
1 = H, R
2 = benzo[
g]), which showed the most significant activity across all cell lines, achieving similar or even better potency than
cis-platin (GI
50 = 2–18 µM).
In a separate study, Penthala et al. synthesized a series of heterocyclic analogs, including indoles, benzofurans, and benzothiophenes, based on Combretastatin, and assessed their anticancer activity against a panel of 60 human cancer cell lines [
39]. The benzo[
b]furans were synthesized by condensing benzo[
b]furanocarbaldehyde
30 (1.0 mol) with phenylacetonitrile
31 (1.1 mol) in a 5% sodium methoxide/methanol solution for 3–6 h, resulting in the successful formation of the desired product
32 (
Table 7). The evaluation of anticancer studies focused on compounds
32a,
32b, and
32d against 60 cancer cell lines. Compound
32a exhibited the most favorable results, displaying GI
50 values ranging from <0.01 to 73.4 µM across all 60 cell lines. It effectively inhibited the growth of 70% of the evaluated cancer cell lines, with a remarkable GI
50 value < 0.01 µM in almost all cases. On the other hand, substituting the 3,4,5-trimethoxyphenyl group in
32a (R = 2-CHO, R
1 = OMe, R
2 = OMe) with the 3,4-dimethoxyphenyl group in
32b (R = 2-CHO, R
1 = H, R
2 = OMe) resulted in reduced growth inhibition against 54% of the cancer cells, exhibiting GI
50 values ranging from 0.229 to 0.996 µM. Furthermore, compound
32b exhibited potent anti-proliferative activity in MDA-MB-435 melanoma cells, exhibiting a remarkable GI
50 value of 0.229 µM. Similarly, compound
32d (R = 3-CHO, R
1 = OMe, R
2 = OMe) displayed significant antiproliferative inhibition, with GI
50 values ranging from 0.237 to 19.1 µM, and effectively inhibited 52% of the evaluated cell lines with a GI
50 value < 1 µM. In addition, the evaluation of anti-leukemia activity against the MV4–11 cell line was performed for compounds
32a–
d (
Table 7), demonstrating that
32a emerged as the most active compound among the evaluated benzo[
b]furans in the leukemia cell line. Later, a molecular docking simulation was performed to investigate the interactions of compound
32a with
α/
β tubulin in complex with colchicine-DAMA (PDB ID code: 1SA0). The simulation revealed a hydrophobic interaction at the
α–
β interface, where colchicine binds, and stability was observed through van der Waals interactions with Asn101, Ser178, Thr179, and Val181 in
α-tubulin, as well as Asn258 and Lys352 in
β-tubulin (
Figure 9). The calculated free energy value for these interactions was −7.74 kcal/mol.
In the same year, Romagnoli et al. synthesized a series of compounds known as 3-(3′,4′,5′-trimethoxyanilino)benzo[
b]furan, wherein a 2-methoxy/ethoxycarbonyl group was combined with either no substituent or a methoxy group at each position of the benzene ring [
40]. The synthesis of compounds
35a–
l involved a two-step reaction process (
Scheme 6). In the first step, 2-hydroxybenzonitrile derivatives
33a–
f were condensed with methyl or ethyl bromoacetate and K
2CO
3 in DMF, leading to the formation of 3-aminobenzo[
b]furan analogs
34a–
l in high yields through a one-pot tandem cyclization method. Subsequently, compounds
35a–
l were synthesized via palladium-catalyzed C-N Buchwald–Hartwig cross-coupling between the deactivated 3-aminobenzo[
b]furans
34a–
l and 1-bromo-3,4,5-trimethoxybenzene in toluene at 100 °C, utilizing Pd(OAc)
2,
rac-BINAP, and Cs
2CO
3 as the catalyst, ligand, and base, respectively.
The in vitro antiproliferative activity was evaluated against seven cell lines, and the corresponding results are presented in
Table 8. The findings revealed a notable correlation between the presence and position of the methoxy substituent on the benzene moiety of the benzo[
b]furan system. Among the series of 2-alkoxycarbonyl derivatives, the highest activity was observed when the methoxy group was located at the C–6 position, as exemplified by compounds
35g (R = Me, R
3 = OMe, R
1,2,4 = H) and
35h (R = Et, R
3 = OMe, R
1,2,4 = H), exhibiting IC
50 values ranging from 0.3 to 27 nM for
35g and from 13 to 100 nM for
35h. On the contrary, compounds
35c (R = Me, R
1 = OMe, R
2−4 = H) and
35d (R = Et, R
1 = OMe, R
2−4 = H) displayed the lowest activity when the methoxy group was situated at the C–4 position, with IC
50 values exceeding 10 µM. Furthermore, the methoxycarbonyl group demonstrated superior efficacy compared to the ethoxycarbonyl substituent in all cell lines, except for MCF-7 cells, which exhibited equal sensitivity to both compounds. Notably, compounds
35i (R = Me, R
4 = OMe, R
1−3 = H) (average IC
50 = 370 nM) and
35j (R = Et, R
4 = OMe, R
1−3 = H) (average IC
50 = 670 nM), featuring a methoxy C–7 substituent, displayed higher activity compared to
35e (R = Me, R
2 = OMe, R
1,3,4 = H) (average IC
50 = 1.500 nM) and
35f (R = Et, R
2 = OMe, R
1,3,4 = H) (average IC
50 = 2.900 nM), which possessed a methoxy C–5 substituent. These compounds also demonstrated remarkable activity against RS 4;11 cells, with IC
50 values of 39 nM for
35e and 1 nM for
35i (R = Me, R
4 = OMe, R
1−3 = H). Additionally, in Jurkat cells, they displayed an IC
50 value of 30 nM for
35i. In contrast, compounds
35a (R = Me, R
1−4 = H) and
35b (R = Et, R
1−4 = H) exhibited IC
50 values of 3.300 and 2.600 nM, respectively. The absence of a methoxy substituent led to lower activity, highlighting the significant enhancement achieved by including a methoxy substituent at C–5. Furthermore, the substitution with ethoxycarbonyl at C–7 resulted in notably lower potency when compared to the substitution with C–7-methoxy (
35i–
l).
Compounds
35e and
35g–
j, along with CA-4, were investigated to determine their inhibitory effects on tubulin polymerization and colchicine binding to tubulin. The aim was to gain insights into their mechanisms of antiproliferative action, particularly their interaction with tubulin microtubules (
Table 9). The results revealed that compound
35g exhibited the highest potency among the tested compounds, with an IC
50 of 1.1 µM, comparable to that of CA-4. Meanwhile, compound
35h demonstrated slightly lower activity compared to CA-4. Compounds
35e,
35i, and
35j showed 6–7 times lower potency than CA-4, with IC
50 values of 7.5, 7.6, and 6.4 µM, respectively. Regarding colchicine binding studies, outcomes were observed exclusively for compounds
35g and
35h, exhibiting inhibition percentages of 83% and 74%, respectively, which are comparable to the 99% inhibition observed with CA-A. The findings underscore the intricate interplay between the inhibition of tubulin polymerization and the hindrance of colchicine binding, shedding light on their potential synergistic effects in influencing antiproliferative pathways.
A molecular docking simulation was performed to investigate the interactions of compound
35g with the colchicine site of tubulin (PDB ID code: 3HKC) (
Figure 10). This revealed that the trimethoxyphenyl ring of
35g resides near Cys241. Moreover, a potential hydrogen bond interaction was observed between the ester moiety and Ala250, consistent with other colchicine site agents. These findings underscore the potential impact of substitutions at C–4, C–5, and C–7 on the antiproliferative activity of the compounds.
Conducting Western blot studies, we aimed to explore the potential of compounds
35h and
35g in triggering apoptosis via the activation of caspase-3 and caspase-9, crucial components of the mitochondrial apoptotic pathway. Upon exposing HeLa cells to these compounds, we observed a concentration- and time-dependent activation of caspases, as depicted in
Figure 11. Moreover, both in vitro and in vivo revealed the activation of poly(ADP-ribose) polymerase (PARP), a major substrate targeted by caspase-3. In addition to these findings, we carefully examined the role of Bcl-2 and Mcl-1 proteins, well known for their capacity to counteract pro-apoptotic proteins and preserve mitochondrial membrane potential. After 48 h of treatment with concentrations of 100 and 250 nM for both compounds, a decrease in Bcl-2 protein expression was observed, while Mcl-1 showed strong down-regulation. Interestingly, at 24 h, Mcl-1 expression increased for
35g but not for
35h. These results suggest that compounds
35g and
35h effectively down-regulate anti-apoptotic proteins.
To assess angiogenesis, the vascular properties of
35g (the most potent within the series) were investigated in vitro using HUVEC endothelial cells. The endothelial cell motility and the ability of
35g to disrupt tubular structures formed by HUVECs on Matrigel were investigated. As shown in
Figure 12a,b, at a concentration of 25 nM, compound
35g exhibited significant inhibition of cell motility within just 6 h of incubation. This inhibitory effect remained highly significant at all concentrations after 24 h of incubation. Moreover, in
Figure 12c, it was observed that compound
35g disrupted the network of HUVECs compared to the control after 1 h of incubation. Remarkably, after 3 h, all tested concentrations demonstrated significant disruption of the tubular-like structures.
Expanding on the encouraging results regarding the antiproliferative and anticancer activity [
37], Kamal et al. conducted more extensive investigations on benzo[
b]furans
22 and
25 to explore their potential efficacy against breast cancer cell lines, specifically MCF-7 and MDA MB-231. These studies involved assessments of the cell cycle and the PI3K/Akt/mTOR signaling pathway, along with other complementary studies [
41].
Table 10 presents the results obtained for the antiproliferative activity of compounds
22 and
25 against the mentioned cancer cell lines. Remarkably,
22 and
25 displayed significant activity, especially in the MCF-7 cell line, exhibiting IC
50 values of 0.057 and 0.051 µM, respectively. Due to the MCF-7 cell line showing the highest anticancer activity among the tested cell lines, it was chosen for further analysis to investigate the correlation between cell growth inhibition and cell cycle arrest. In this study, MCF-7 cells were treated with the compounds
22 and
25 at concentrations of 25 nM and 50 nM for 48 h. The results revealed that these compounds induced G2/M cell cycle arrest compared to the untreated control cells. Specifically, at a concentration of 25 nM, compounds
22 and
25 caused a cell accumulation of 36.4% and 37.1%, respectively, in the G2/M phase. Moreover, at 50 nM, these percentages increased to 47.6% and 50.5%, respectively, in the same cell phase (
Figure 13).
The PI3K/Akt/mTOR signaling pathway plays a crucial role in breast tumor cell growth. Thus, the impact of compounds
22 and
25 on this signaling pathway was investigated in MCF-7 cells. The results demonstrated effective suppression of p-Akt, p-mTOR, p-p70S6K, and p-4E-BP1 expression levels after 48 h of treatment with a concentration of 50 nM (
Figure 14a). The findings strongly support the potent inhibitory activity of both compounds against the PI3K/Akt/mTOR pathway. Notably, given the involvement of this pathway in apoptosis regulation, the studies further unveiled that its inhibition resulted in the up-regulation of key apoptotic markers. These markers included the release of cytochrome c, up-regulation of p53, down-regulation of procaspase-9, cleavage of poly(ADP-ribose)polymerase (PARP), up-regulation of Bax, and down-regulation of Bcl-2 (
Figure 14b). Collectively, these results firmly establish the inhibition of the PI3K/Akt/mTOR pathway as the primary mechanism underlying the induction of apoptosis in breast cancer cells by compounds
22 and
25.
In their study, Yin et al. achieved the successful synthesis of 2,3-dihydrobenzo[
b]furan
37 in a 32% yield through the dimerization of methyl caffeate
36 using silver oxide in the presence of anhydrous benzene and acetone (
Scheme 7). The primary aim of this study was to explore the potential correlation between IL-25, an endogenous factor secreted by tumor-associated fibroblasts (TAFs), and the inhibition of metastasis in 4T1 mammary tumors in mice [
42].
The investigation into the antimetastatic effects of compound
37 involved the injection of luciferase-expressing 4T1-Luc2 transgenic mouse cells into the mammary fat pad of the experimental mice [
42]. At 15 days after tumor cell implantation, the 4T1 tumors were surgically resected in situ. Over the following 8 weeks, a comparative analysis of tumor metastatic activity and survival was conducted between the control group and the mice treated with compound
37 (
Figure 15a–c). By detection of luminescent activity of 4T1-Luc2 cells as an indicator of tumor metastasis, it was observed that treatment with
37 (≥20 µg kg
−1) significantly suppressed 4T1 cell metastasis to the lung (
Figure 15a). In addition, treatment with
37 at a relatively low dose (>20 µg kg
−1) had considerable antimetastatic activity in comparison with the treatment with Doxorubicin (2 mg kg
−1) (
Figure 15b), used as a control drug for breast cancer. Treatment with
37 also significantly increased the survival rate of mice with tumor resection (
Figure 15c). These results demonstrate that the in vivo administration of compound
37 effectively prevents breast tumor metastasis following tumor resection.
Following the previously mentioned findings, the study showed the potential regulatory effects of administering compound
37 in vivo on metastatic tissues [
42]. To assess the physiological significance of compound
37 in the modulatory activity, the researchers evaluated the expression of various cytokines secreted in vivo in the lung tissue of the test mice. Remarkably, the findings demonstrated that the administration of compound
37 (at a dosage of 100 µg kg
−1) had a stimulatory effect on IL-25 activity in pulmonary fibroblasts surrounding the pulmonary artery and vein (
Figure 16b). In contrast, little to no IL-25 expression was observed in lung fibroblasts from control and Docetaxel-treated mice, which suggests that the induction of IL-25 expression in fibroblasts of the lung tissue microenvironment specifically resulted from the administration of compound
37. This finding is intriguing as IL-25 expression is not typically considered a conventional drug target for anticancer medications.
To quantify the change in the cell population of IL-25-expressing lung fibroblasts in response to treatment with compound
37, the researchers assessed FSP-1 + ER-TR7 + IL-25 + cells from lung tissues of test mice and compared their IL-25 expression levels at 3 weeks after tumor resection. The results unveiled a remarkable increase in the IL-25 fibroblast cell population from 16.7% to 79.5% in
37-treated mice compared to those treated with PBS (
Figure 16a). Furthermore, the population of fibroblasts in FSP-1 + ER-TR7 + cells in mice treated with
37 exhibited a significant dose-dependent increase from 5.2% to 7.3% (
Figure 16a), in contrast to docetaxel treatment, which showed no augmentation in the number or level of evaluated fibroblasts. These findings solidify the evidence that compound
37 effectively stimulates lung fibroblasts in an in vivo setting.
Additionally, the researchers conducted complementary studies to assess the potential suppressive effect of IL-25 secreted by fibroblasts on the growth activity of mammary tumor cells [
42]. In this regard, they compared the levels of IL-25 in mouse (4T1) and human (MDA-MB-231) tumor cells treated with compound
37 using an immunoprecipitation method mediated by anti-IL-25 antibodies. To ensure accuracy, the samples were first immunodepleted with 3T3 (3T3-CM) and WI38 (WI38-CM) fibroblasts for IL-25, utilizing anti-rabbit IgG antibody (isotype control) as the negative control for immunodepletion (
Figure 17a). The results indicated that the levels of IL-25 secreted in media conditioned with compound
37 treated fibroblasts were significantly higher compared to untreated conditioned media (
Figure 17a). Moreover, the researchers detected only a minor fraction of unspecific binding to the protein in the IgG antibody, confirming the high specificity and efficiency of the anti-IL-25 antibody employed. On the other hand, when 4T1 and MDA-MB-231 cells were cultured with 3T3-CM, their growth was notably higher compared to cells cultured solely with fresh medium. This observation implies that in both cases, fibroblasts released critical cellular and molecular factors that contribute to the expansion of tumor cells, potentially influencing the suppression of metastatic mammary tumor cell growth (
Figure 17b,c).
In their investigation, Yin et al. conducted a comparative analysis of the in vivo treatment effects of IL-25, examining its additive vs. overlapping impact [
42]. They discovered that co-administration of compound
37 (at a dosage of 100 µg kg
−1) and IL-25 (at a dosage of 10 µg kg
−1) resulted in a similar antimetastatic activity compared to the group of mice treated solely with compound
37 (
Figure 18a). The survival rate of mice in the co-treatment group (
37 + Anti-IL-25) was also comparable to the group treated with compound
37 alone, in contrast to the untreated group (
Figure 18b). These findings suggest that the in vivo antimetastatic effect of
37 can be effectively substituted by the administration of exogenous IL-25.
Moreover, the study evaluated the combined effect of compound
37 and docetaxel in suppressing the metastatic activities of human MDA-MB-231-Luc2 cells in mice through bioluminescent studies following the resection of mammary tumor tissues in the experimental mice (
Figure 19a). The results demonstrated that the treatment with compound
37 (100 µg kg
−1) and docetaxel (5 mg kg
−1) showed significantly higher antimetastatic activity than the treatment with docetaxel alone (
Figure 19b). As a result, the test mice receiving the combination of compound
37 and docetaxel exhibited a higher survival rate compared to those receiving single treatments (
Figure 19c). These findings suggest a complementary effect on the anticancer activity of docetaxel when combined with compound
37, effectively suppressing the metastatic activities of tumor cells by modulating the tumor-associated microenvironment.
On a different note, Quan et al. conducted a molecular modeling study involving 64 Combretastatin A-4 analogs based on five-membered heterocycles. Their objective was to explore the development of novel anticancer agents by using 3D-QSAR, molecular docking, and molecular dynamic (MD) simulation [
43]. Within the 3D-QSAR approach, both CoMFA and CoMSIA models were prepared for both the training and test sets. The CoMFA model incorporated steric and electrostatic fields, while the CoMSIA model included steric, electrostatic, hydrophobic, hydrogen bond donor, and hydrogen bond acceptor fields. Three-dimensional contour maps for both models were performed using the “Stdev*Coeff” field type. By analyzing the results obtained from the study, the researchers identified essential structure–activity relationships, which highlighted substitutions that could enhance biological activity. This was summarized in A–D regions, as shown in
Figure 20. Building on this information, they designed five novel benzo[
b]furan derivatives. The structures and predicted pIC
50c values of compounds
43a–
e are provided in
Table 11. Although these data indicate the presence of inhibitory activity for the designed compounds, they were not comparable to CA-4.
Additionally, the researchers conducted 20 ns molecular dynamics (MD) simulations and binding free energy calculations using the Amber 12.0 package [
43]. The stability of the tubulin–inhibitor complex in the designed compounds
43a–
e was evaluated, employing the general Amber force field (gaff) for ligands and the ff99SB force field for proteins. In this study, binding free energy calculations were performed using both MM/GBSA and MM/PBSA methods. The results showed that the calculated binding free energies using MM/GBSA for CA-4 and the inhibitors
43a–
e were as follows: −34.32, −57.52, −54.41, −55.78, −50.77, and −55.56 kcal mol
−1, respectively. Meanwhile, the MM/PBSA results revealed the binding free energies of −21.54, −43.45, −42.03, −40.99, −39.79, and −36.23 kcal mol
−1 for CA-4 and the inhibitors
43a–
e, respectively. Among the five newly designed compounds,
43a exhibited the most negative binding free energy, suggesting it has the potential for the best inhibitory activity within the series. These computational findings provide valuable insights into the potential efficacy of the designed compounds and can guide further experimental investigations to validate their inhibitory activity against tubulin.
Using the binding free energy calculations, two of the designed compounds,
43a and
43b, were selected for synthesis. The synthetic process involved several sequential steps to obtain the desired compounds. Firstly, the synthesis began with the iodination of methoxyphenol
38 using a catalytic amount of AgOTFA in chloroform at room temperature for 24 h to afford the iodinated compound
39 (
Scheme 8). Subsequently, the acetylation of the iodine-phenol
39 was carried out in the presence of acetic anhydride in pyridine at room temperature for 4 h to yield the acetate derivative
40. Next, the Sonogashira coupling of compound
40 with either 1-ethynyl-4-ethoxybenzene or 1-ethynyl-4-methoxybenzene was conducted in the presence of catalytic PdCl
2(PPh
3)
3, leading to the formation of alkyne
41. The intermediates underwent an intramolecular cyclization reaction mediated by K
2CO
3 in methanol at 60 °C for 16 h to produce the benzo[
b]furan derivative
42. Finally, a Friedel–Crafts reaction was performed in the presence of 3,4,5-trimethoxybenzoyl chloride, followed by the addition of compound
42 and tin (IV) chloride to deliver compound
43. By employing this multi-step synthetic approach, the researchers successfully synthesized compounds
43a and
43b, paving the way for further evaluation of their potential as benzo[
b]furan-based anticancer agents.
The in vitro antiproliferative activity of compounds
43a–
b was evaluated against six human cancer cell lines, and their tubulin inhibition was assessed, using CA-4 and CA-4P as standard drugs. As shown in
Table 12, the compound
43a (R
1 = H, R
2 = Oet) exhibits the highest activity with IC
50 values of 1.37, 8.99, 1.31, and 0.91 µM against A549, HeLa, HepG2, and MCF-7 cell lines, respectively [
43]. Compound
43a exhibits comparable and even superior activity than CA-4 in the mentioned cell lines. Specifically, it is 4.4-fold more active against A549 cells and 12.2-fold more active against HepG2 cells than CA-4. In addition, compound
43a showed only comparable activity with compound CA-4P in the MCF-7 cell line. On the other hand, compound
43b (R
1 = H, R
2 = Ome) exhibited remarkable activity against A549 and HepG2 cells, with IC
50 values of 6.87 and 4.75 µM, respectively, which is comparable to CA-4 (IC
50 = 5.99 and 16.04 µM, respectively). Finally, in a tubulin polymerization assay, compound
43a demonstrated potent inhibition of tubulin polymerization, with an IC
50 value of 0.86 µM, which is comparable to CA-4 (IC
50 = 0.88 µM) and superior to CA-4P (IC
50 = 4.79 µM).
Similarly, Lauria et al. conducted the synthesis of a novel series of 3-benzoylamino-5-(1
H-imidazol-4-yl)methylaminobenzo[
b]furans
51–
53 and subsequently evaluated their potential as antitumor agents [
44]. The synthetic route involved a sequence of steps (
Scheme 9). Initially, 2-fluorobenzonitrile
44 underwent a nitration reaction in the presence of a mixture of concentrated nitric and sulfuric acids under a nitrogen atmosphere at 0 °C for 2 h to afford 2-fluoro-5-nitrobenzonitrile
45. Afterward, ethyl glycolate was utilized for nucleophilic displacement in the presence of K
2CO
3 and anhydrous DMF at 100 °C for 12 h, facilitating in situ intramolecular cyclization and giving rise to the 3-amino-benzo[
b]furan derivative
46. Following this, intermediate
46 underwent acyl substitution with benzoyl chloride
47 in pyridine, serving as both base and solvent, at room temperature for 12 h, yielding compound
48, containing amide functionality at the C–3 position. The subsequent reduction of the nitro group in compound
48 was performed through hydrogenation using a Parr hydrogenation apparatus at 500 psi in the presence of Pd/C (10%) as a catalyst in ethanol at room temperature for 2 h, yielding 5-amino-benzo[
b]furan derivative
49. Finally, the compounds
51a–
f and
52a–
f were obtained through a reductive amination with imidazole-4-carbaldehyde
50 using sodium cyanoborohydride as a selective reducing agent in a mixture of ethanol and acetic acid at room temperature for 6–24 h. In this final step, compound
53 was also isolated using carbaldehyde
50b, wherein evidence of the hydrolysis of the amide functionality was observed. These novel compounds hold promise as potential antitumor agents and warrant further investigation to assess their efficacy in cancer treatment.
The biological studies focused on the antiproliferative activity in HeLa and MCF-7 cell lines using the MTT assay, cell cycle analysis, and in silico assessment [
44].
Table 13 shows the GI
50 values of compounds
51a–
f and
52a–
f. Notably, the insertion of a methyl group in the imidazole ring increased activity for compounds
52a (R
1 = R
2 = R
3 = R
4 = H),
52e (R
1 = H, R
2 = CF
3, R
3 = R
4 = H), and
52f (R
1 = Cl, R
2 = F, R
3 = R
4 = H) without substitution on the benzoyl moiety (
52a vs.
51a), or functionalized with 4-trifluoromethyl- (
52e vs.
51e) and 3-chloro-4-fluoro- (
52f vs.
51f) substituents. Compound
52f exhibited the highest activity against HeLa and MCF-7 cell lines, with GI
50 values of 2.14 µM and 1.55 µM, respectively. In cell cycle analysis, compounds
52b,
52c,
52d, and
52f showed significant suppression of the G0/G1 phase and an accumulation of cells in G2/M at 1xGI
50 concentrations (
Figure 21). However, at 2xGI
50 concentrations, changes in the distribution profile were observed. Albeit compounds
52a and
52e did not show a significant impact on the cell cycle at the evaluated concentrations, they induced G0/G1 arrest at 2.5xGI
50 and 5xGI
50 concentrations, which correlated with their antiproliferative effects in other phases.
In the in silico studies, the researchers assessed the binding modes of each derivative at the colchicine binding site on tubulin, considering the involved amino acid residues [
44]. To facilitate this analysis, they obtained the crystal structure of tubulin bound to colchicine from the PDB database (PDB ID code: 4O2B) and extracted the tubulin dimer with colchicine (chains A and B) from the protein model.
Table 14 shows the favorable induced docking protocol (IFD) scores for all ligand–tubulin complexes, with compounds
51 and
52 showing affinity similar to colchicine and higher than CA-4. Notably, significant differences in the binding complexes were observed among the evaluated amino acids. These benzo[
b]furans displayed strong interactions with amino acids Alaα180, Serα178, Ileβ318, Alaβ316, Leuβ255, Leuβ248, and Lysβ254, as indicated by the IFD scores. Interestingly, compounds
52b–
d,
f presented an aromatic ring in contact with Cysβ241, a crucial and distinctive feature for identifying new antitubulin molecules. A more detailed illustration of these interactions is found in
Figure 22, depicting the ligand interaction maps of compounds
52b and
52e with colchicine as a reference.
Afterward, Pervaram et al. synthesized 1,2,4-oxadiazole-fused benzo[
b]furan derivatives
62a–
j and assessed their antiproliferative activity against four human cancer cell lines, including A549 (lung), MCF-7 (breast), A375 (melanoma), and HT-29 (colon), using the MTT method [
45]. The synthetic route for compounds
62a–
j is shown in
Scheme 10. The synthesis began with the reaction of 5-methoxybenzofuran-3-carbaldehyde
54 with 2-aminophenol
55 in refluxing ethanol for 4 h, leading to the 2,3-dihydrobenzo[
d]oxazole intermediate, which was oxidized to 2-(5-methoxybenzofuran-3-yl)benzo[
d]oxazole
56 by adding Pb(Oac)
4 and acetic acid at room temperature for 1 h. Next, compound
56 reacted with BBr
3 in anhydrous CH
2Cl
2 at room temperature for 5 h to give 3-(benzo[
d]oxazol-2-yl)benzofuran-5-ol
57, which is
O-alkylated with 2-bromoacetonitrile
58 and K
2CO
3 in refluxing acetone for 5 h to furnish compound
59. Subsequently, a nucleophilic addition reaction between
59 and hydroxylamine hydrochloride in the presence of K
2CO
3 in refluxing ethanol for 3 h yielded acetaminide
60. Finally, compound
60 was reacted with the different benzoyl chloride
61 using pyridine at room temperature for 4 h to obtain 1,2,4-oxadiazole-fused benzo[
b]furan derivatives
62a–
j in yields ranging from 63% to 93%.
The antiproliferative activity of all synthesized compounds
62a–
j was assessed against four cancer cell lines using the MTT method and CA-4 as the standard drug (
Table 15) [
45]. Notably, compounds
62b (R
1 = 3,4,5-
tri-(Ome)
3),
62g (R
1 = 4-NO
2),
62h (R
1 = 4-CN), and
62j (R
1 = 4-CF
3) exhibited comparable and, in some cases, even higher potency than CA-4, with IC
50 values ranging from 0.012 to 1.45 µM for these compounds, while CA-4 had IC
50 values ranging from 0.11 to 0.93 µM.
Similarly, Kwiecień et al. conducted a study on the synthesis and evaluation of functionalization at position 3 of 2-phenyl- and 2-alkylbenzo[
b]furans as potential antitumor agents [
46]. The synthesis of 2-phenylbenzo[
b]furan
67 involved a three-step reaction process (
Scheme 11). Firstly, 2-hydroxybenzaldehyde
63 was
O-alkylated with methyl 2-bromo-2-phenylacetate
64 in the presence of K
2CO
3 and DMF at 92–94 °C for 4 h to afford methyl 2-(2-formylphenoxy)-2-phenylacetate derivative
65 with yields in the range of 61–76%. Secondly, basic hydrolysis of ester
65 in methanol refluxing for 2 h, and then protonation yielded 2-(2-formylphenoxy)-2-phenylacetic acid
66 in acceptable yields (70–72%). Lastly, an intramolecular cyclization of compound
66 using a mixture of Ac
2O and AcONa at 125–130 °C for 4.5 h produced the benzo[
b]furan
67 in excellent yields (90–97%).
Later, the researchers focused on the acylation of 2-phenylbenzo[b]furan 67. Initially, the acetylation of 67 with Ac2O in the presence of Amberlyst-15 in 1,2-dichloroethane refluxing for 6 h afforded 2-phenylbenzo[b]furan-3-yl)ethan-1-ones 68a–b in acceptable yields (69–72%). Then, (4-hydroxy-3,5-dimethoxyphenyl)-(2-phenylbenzo[b]furan-3-yl)methanones 69a–b were synthesized in 68–76% yields through an AlCl3-mediated acylation with 3,4,5-trimethoxybenzoyl chloride in 1,2-dichloroethane at 45 °C for 3.5 h. Finally, the phenylbenzo[b]furan-3-yl-(3,4,5-trimethoxyphenyl)methanone 70 was obtained in a 69% yield through an Amberlyst-mediated acylation with 3,4,5-trimethoxybenzoyl chloride in 1,2-dichloroethane refluxing for 6.5 h.
A synthetic route for obtaining 3-phenyl-functionalized 2-alkylbenzo[
b]furans
72 and
73 was developed (
Scheme 12). Firstly, 1-(5-bromo-2-ethylbenzo[
b]furan-3-yl)-2-(4-hydroxyphenyl)ethanone
72 was synthesized from 5-bromo-2-ethylbenzo[
b]furan
71a through an acylation with 4-methoxyphenylacetyl chloride, followed by demethylation to convert the methoxy group into a hydroxy group. Secondly, 2-(3,5-dibromo-4-hydroxyphenyl)-1-(2-butylbenzo[
b]furan-3-yl)ethanone
73 was prepared from 2-butylbenzo[
b]furan
71b by carrying out three sequential reactions: acylation with 4-methoxyphenylacetyl chloride, demethylation of the methoxy group, and bromination.
The biological evaluation encompassed the examination of compounds
68b,
69a–
b,
70,
72, and
73, focusing primarily on antiproliferative studies, flow cytometry, confocal microscopy imaging, and the tubulin polymerization assay, among other complementary analyses [
46]. For the in vitro antiproliferative assessment, the benzo[
b]furan derivatives were tested against the A375 cancer cell line using a cell proliferation reagent WST-1 assay (
Table 16). The results revealed that compounds
69a (R
1 = H),
69b (R
1 = Ome), and
70 exhibited the most potent antiproliferative activity, displaying IC
50 values of 2.85, 0.86, and 0.09 µM, respectively. Conversely, compounds
68b (R
1 = Ome),
72, and
73 demonstrated low activity, with IC
50 values exceeding 100 µM.
Flow cytometry analysis employed an apoptosis detection kit FITC Annexin V to evaluate apoptosis and necrosis in cells after 48 h of incubation with benzo[
b]furan derivatives [
46]. The results from the flow cytometry demonstrated minimal cytotoxicity for compounds
68b,
72, and
73, with approximately 90% of live cells, which is similar to the control group. In contrast, compounds
69a,
69b, and
70 exhibited a significant increase in late apoptotic cells, with percentages reaching 57.63%, 71.21%, and 58.52%, respectively. These findings highlight the potent anticancer activity of 3-aryl-2-phenylbenzo[
b]furan derivatives.
In addition, a complementary study on cell cycle distribution was performed, revealing that the 3-aryl-2-phenylbenzo[b]furan derivatives induced the accumulation of A375 cells in a tetraploid state (4N), resulting in a decrease in the percentage of cells in the G0/G1 phase. Specifically, compounds 69a, 69b, and 70 led to 66.34%, 58.86%, and 63.62% of A375 cells in the G2/M phase, respectively. In contrast, compounds 68b, 72, and 73 did not show significant differences in the percentages of A375 cells in the G0/G1 and G2/M phases compared to untreated control cells. These findings provide valuable insights into the mode of action of the 3-aryl-2-phenylbenzo[b]furan derivatives and highlight their potential as effective anticancer agents.
After analyzing the cell cycle distribution results, Kwiencień et al. conducted a confocal microscopy analysis [
46]. Cells were incubated with the tested compounds for 7 h, fixed, and then stained for
α-tubulin and chromosomes. The control cells displayed well-defined bipolar spindle formation with chromosome alignment at the metaphase central plate or anaphase distribution (
Figure 23A,B). However, cells treated with 3-aryl-2-phenylbenzo[
b]furans exhibited diverse phenotypes, characterized by enlarged nuclei and the absence of a visible mitotic spindle (
Figure 23C–H). Notably, compound
70 showed a distinct phenotype with binuclear or enlarged nuclei. These observations suggest that the disparity between the tested compounds and control cells was specifically evident during mitosis, indicating a specific mitotic activity of the compounds. This finding sheds significant light on the potential of these compounds as specific mitosis-targeting agents.
Finally, the effects of benzo[
b]furan derivatives on tubulin polymerization were evaluated based on fluorescence [
46]. Paclitaxel (PTX), vinblastine (VBL), and DMSO (0.2%) were used as control and reference compounds. As depicted in
Figure 24, DMSO had no direct effect on tubulin polymerization. In contrast, the reference compounds (PTX and VBL) interacted with tubulin, resulting in alterations to the normal polymerization curve. Upon comparing the curves of VBL,
69a,
69b, and
70, it became evident that these compounds were the most effective in inhibiting tubulin polymerization, as indicated by a decrease in
Vmax (maximum slope values for the growth phase) and a reduction in the final mass of the protein polymer. These findings align with the observations from confocal microscopy and flow cytometry analyses, where the inhibition of tubulin polymerization led to the prevention of mitotic spindle formation, resulting in the presence of polyploid nuclei and cell cycle arrest at 4N. These collective data provide compelling evidence for the significant impact of the tested compounds on tubulin polymerization and their potential as potent agents affecting cell division and proliferation.
Anwar et al. pursued a unique approach and conducted preliminary studies, which revealed a promising anticancer activity in a benzofuran–pyrazole hybrid
77 [
47]. Encouraged by these favorable findings, the researchers explored the potential benefits of its nanorange form, aiming to investigate the influence of the nanorange and its effect on the cytotoxic potency of the hybrid
77 [
48]. The synthesis of hybrid
77 involved a three-step process: (1) the conversion of 1-(benzofuran-2-yl)ethanone
74 into the pyrazole-4-carbaldehyde
75 through the Vilsmeier–Haack reaction; (2) Claisen–Schmidt condensation of compound
75 with 2-acetylpyrrole to give chalcone
76 in an 88% yield; and, finally, (3) the cyclocondensation of compound
76 with hydrazine hydrate in acetic acid to afford benzofuran–pyrazole hybrid
77 in an 85% yield (
Scheme 13).
On the other hand, nanoparticles of the benzofuran–pyrazole hybrid 77 were synthesized using the nanoprecipitation method and exhibited sizes ranging from 3.8 to 5.7 nm. The characterization of these nanoparticles involved transmission microscopy (TEM) to confirm spherical shape and average size. Additionally, the surface charge and stability of the nanoparticles were analyzed utilizing the Malvern Zetasizer nano Zs instrument. These results indicated that the nanoparticles had an average size of 3.8–5.7 nm and a zeta potential of −27.3 mV, with a polydispersity index (PDI) of 0.77, confirming their uniformity and stability.
The anticancer activity of the benzofuran–pyrazole hybrid
77 and its nanoparticles was assessed against two breast cancer cell lines, MCF-7 and MDA-MB-231, using an MTT assay and doxorubicin as a standard drug (
Table 17) [
48]. The results showed that the nanoparticles of hybrid
77 exhibited the highest cytotoxic activity against both MCF-7 and MDA-MB-231 cell lines with IC
50 values of 1 and 0.6 nM, respectively, outperforming doxorubicin (IC
50 = 620 nM in both cases). In contrast, hybrid
77 showed lower activity than its nanoparticles against both cell lines, with IC
50 values of 7 and 10 nM. This difference in activity can be attributed to the high surface area/volume ratio of nanoparticles, which allows for selective targeting of cells and tissues, and more effective interactions compared to hybrid
77 (>100-fold). Finally, IC
50 values of hybrid
77 and its nanoparticles showed over a 1000-fold difference when targeting normal breast cells MCF-12A compared to cancer cells, indicating their safety profiles in normal cells.
The researchers performed complementary analyses to explore the cell cycle, the effect on caspase-3/p53/Bax/Bcl-2 levels, and PARP-1 cleavage [
48]. For the cell cycle analysis, the effects of hybrid
77 and its nanoparticles were evaluated against MCF-7 and MDA-MB-231 cell lines using flow cytometry, comparing the results with a DMSO control. As shown in
Figure 25a, both the hybrid
77 and its nanoparticles induced apoptotic cells, resulting in percentages of 9.18% and 21.54% for the MCF-7 line, and 11.09% and 23.17%, for the MDA-MB-231 line, respectively. These findings underscore the significantly greater potency of the nanoparticles of
77, being twice as effective as the hybrid
77 against both cell lines tested. Moreover, exposure to the hybrid
77 and its nanoparticles caused a noticeable disruption in cell cycle distribution, with percentages of 11.26% and 17.52% observed in MCF-7, and 12.11% and 19.24% in MDA-MB-231, respectively (
Figure 25b). These results suggest that the inhibitory potency was predominantly associated with the nanoparticles of
77, particularly in the G2/M phase.
In the study focusing on caspase-3/p53/Bax/Bcl-2 levels, the researchers employed an enzyme-linked immunosorbent assay (ELISA) to investigate two cancer cell lines [
48]. As shown in
Table 18, hybrid
77 exhibited a notable increase in caspase-3 levels (>5-fold) compared to untreated cells. However, the nanoparticles of
77 demonstrated an even more remarkable effect, surpassing the impact of hybrid
77, with caspase-3 levels elevated by over 17-fold vs. untreated cells. Moreover, p53 levels showed an approximately 7-fold increase for hybrid
77 and a remarkable 14-fold increase for nanoparticles of
77 against both cell lines tested. Additionally, Bax levels displayed a significant increase of 5- to 13-fold, while Bcl-2 levels decreased by 4- to 7-fold in both cell lines compared to the untreated cells.
In the final phase of the study, the researchers conducted a PARP-1 cleavage assay, using staurosporine as the standard drug [
48]. As depicted in
Table 19, hybrid
77 did not exhibit a considerable inhibitory effect on both cancer cell lines compared to staurosporine. However, the nanoparticles of
77 showed a remarkable inhibitory effect, proving to be 4 and 10 times more potent than the hybrid
77 for the MCF-7 and MDA-MB-231 cell lines, respectively. Notably, the nanoparticles of
77 exhibited an IC
50 value of 6 nM, whereas staurosporine had an IC
50 value of 8 nM for the MDA-MB-231 line.
Shikonin–benzo[
b]furan hybrids have shown potential as inhibitors of tubulin polymerization. To further explore this, Shao et al. synthesized the compound
79 using the Finkelstein reaction of salicylaldehyde
78 with ethyl bromoacetate in the presence of KI as a catalyst under mild reaction conditions (
Scheme 14) [
49]. Subsequently, compound
79 underwent condensation with K
2CO
3 in DMF at 120 °C for 3 h to afford ethyl benzofuran-2-carboxylate
80, which was then hydrolyzed with sodium hydroxide in DMF at 60 °C for 30 min, resulting in benzo[
b]furan-2-carboxylic acid
81. Finally, the esterification reaction of carboxylic acid
81 with shikonin
82 using a mixture of DCC and DMAP in dichloromethane at 0 °C for 8 h afforded shikonin–benzo[
b]furan hybrids
83a–
q in low yields (20–36%).
The antiproliferative activity of the shikonin–benzo[
b]furan hybrids
83a–
q was evaluated against five human cancer cell lines (HepG2, HT29, HCT116, MDA-MB-231, and A549) and two non-cancerous cells (293T and LO2) using the MTT assay, with colchicine, shikonin, and CA-4 as the standard drugs (
Table 20). The results demonstrated significant antiproliferative activity for the majority of the hybrids against the tested cancer cell lines. Remarkably, compounds
83c (R
1 = 3-OMe),
83o (R
1 = 3-OEt), and
83i (R
1 = 3-
tert-butyl) exhibited outstanding activity in the HT29 cell line, with IC
50 values of 0.18, 0.73, and 0.82 µM, respectively, compared to shikonin and colchicine (IC
50 = 2.80 and 1.81 µM, respectively). Surprisingly, compound
83c (IC
50 = 0.18 µM) demonstrated even higher activity than CA-4 (IC
50 = 0.31 µM) against the HT29 cell line. The impact of substitutions on antiproliferative activity was evident, as the most active shikonin–benzo[
b]furan hybrids
83c,
83i, and
83o with a substitution at position 3 of the salicylaldehyde
78 displayed greater potency than shikonin in four of the cancer cell lines tested. However, this trend was observed only in mono-substituted compounds because di-substituted compounds (
83i and
83p) exhibited lower activity than them at the same position. On the other hand, compound
83f (R
1 = 2-Br) at position 2 demonstrated significant activity comparable to that of shikonin. Furthermore, the cytotoxicity of all compounds was evaluated in two non-cancerous cells (293T and LO2) (
Table 20). The results indicated that all shikonin–benzo[
b]furan hybrids exhibited low cytotoxicity (CC
50 > 100 µM). These findings suggest their potential as promising candidates for further exploration in cancer treatment research.
Subsequently, the effect of hybrid
83c on tubulin microtubule dynamics was evaluated in the HT29 cell line, using colchicine, CA-4, and paclitaxel as the standard drugs [
49]. The hybrid
83c exhibited a similar action to colchicine, indicating its potential as a microtubulin destabilizing agent with a more potent inhibitory effect on tubulin polymerization than both colchicine and CA-4, as evidenced by their respective IC
50 values of 0.98, 2.11, and 1.12 µM (
Table 21). In addition, compound
83c displayed a competitive trend at the tubulin binding site, demonstrating a remarkable 92.42% inhibition at 4 µM, comparable to colchicine.
The results led to additional biological investigations on hybrid
83c, including cell cycle and apoptosis assays, tubulin antiangiogenesis, and antivascular assays [
49]. Flow cytometry was employed to analyze the cell cycle using the HT29 cell line at varying concentrations of hybrid
83c. The results revealed cell cycle arrest at the G2/M phase persisting over time (
Figure 26a,b). Moreover, a Western blot study was performed to assess the impact of hybrid
83c on cell-cycle-related proteins. The study revealed that hybrid
83c increased the expression of P21 and cyclin B1, alongside reducing the expression of Cdc2, p-Cdc2, and p-Cdc25c, corroborating the findings from the cell cycle analysis (
Figure 26c).
Cell apoptosis analysis was performed using the Annexin V-FITC/PI assay and confirmed by the Western blot in HT29 cells. Hybrid
83c induced cell apoptosis in a concentration- and time-dependent manner (
Figure 27a,b) [
49]. Moreover, the expression of proteins associated with cell apoptosis, including Bax, PARP, caspase-3, and caspase-9, increased, while Bcl-2 expression decreased, aligning with the observed trend in cell apoptosis (
Figure 27c).
A complementary study was conducted to explore the effect of hybrid
83c on inhibiting tubulin polymerization and determine its potential to regulate microtubule dynamics in living cells [
49]. The immunofluorescence assay was performed using the HT29 cell line, with colchicine and paclitaxel as positive control drugs (
Figure 28). The results demonstrated that cells treated with paclitaxel showed more stable microtubules, whereas those treated with colchicine disintegrated and became soluble. Similarly, cells treated with hybrid
83c showed a similar response to colchicine, inducing the collapse of microtubules in a dose-dependent manner. On the other hand, the potential antivascular activity of hybrid
83c was investigated using endothelial cells (HUVECs), which were treated with different concentrations of
83c and then seeded in Matrigel. After 6 h of treatment, cells treated with hybrid
83c exhibited a dose-dependent inhibition in the formation of HUVEC cords, suggesting that hybrid
83c can impede the formation of HUVEC tubes.
As mentioned earlier, aryl- and alkylbenzo[
b]furan groups have exhibited significant biological activity against various types of human cancer. Building upon these promising findings, Sivaraman et al. undertook the synthesis of a series of 2-aryl[
b]benzofurans, classified as lignane and neolignane, through a one-pot reaction using 2-bromobenzo[
b]furans as crucial intermediates [
50].
Lavandula agustifola served as the source for both natural (
89a,
99, and
100) and non-natural (
88a–
b,
89b,
94, and
95) compounds in the process (
Scheme 15,
Scheme 16 and
Scheme 17).
In the initial synthesis, 2,5-dibromo-6-methoxybenzo[
b]furan
85a was obtained in a 60% yield through a
gem-dibromo olefination/cyclization sequence (
Scheme 15). Subsequently, a selective C–2 acylation of the Suzuki-type reaction resulted in the formation of 4-(5-bromo-6-methoxybenzofuran-2-yl)phenol
86a. Further vinylation of
86a, followed by a hydroboration/oxidation sequence, led to the compound
88a, which was then subjected to methylation to afford compound
89a. For the tetrasubstituted benzo[
b]furans
88b and
89b, a similar reaction pathway was employed, utilizing 1-(5-bromo-2,4-dihydroxyphenyl)ethanone
84b, leading to the formation of 2,5-dibromo-3-methylbenzo[
b]furan-6-ol
85b. Subsequent methylation of
85b gave 2,5-dibromo-6-methoxy-3-methylbenzo[
b]furan
85c. By employing the identical reaction steps as previously described, compounds
88b and
89b were obtained with overall yields of 6% and 5%, respectively.
The second reaction was performed following a similar procedure as described in
Scheme 15. Initially, a one-pot cyclization of 5-bromo-2-hydroxy-3-methoxybenzaldehyde
90 yielded benzo[
b]furan
91. Subsequently, a selective C–2 arylation gave
92, and vinylation led to the formation of 4-(7-methoxy-5-vinyl-benzofuran-2-yl)phenol
93 (
Scheme 16). Afterward, a hydroboration/oxidation sequence afforded compound
94, and its methylation yielded compound
95, with overall yields of 22% and 16%, respectively.
Benzo[
b]furan derivatives
99 and
100 were synthesized by a multi-step process starting from
94. Initially, compound
94 underwent diacetylation to afford
96 (
Scheme 17). Subsequently, a regioselective iodination reaction of
96 with NIS afforded iodinated compound
97, which underwent a Suzuki reaction to give compound
98. Finally, benzofuran
98 suffered deprotection and methylation to obtain compounds
99 and
100 with overall yields of 10% and 8%, respectively.
The previously synthesized compounds were evaluated for their cytotoxic effects on five human cancer cell lines, including MCF-7, A549, PC3, HepG2, and Hep3B, utilizing the MTT assay [
50]. Notably, compounds
88a,
94, and
99 exhibited significant reductions in cell counts, indicating promising cytotoxic activity. Subsequently, comprehensive biological studies were conducted using Western blot analysis to assess the effects of compounds
88a,
94, and
99 on proteins associated in cellular processes (
Figure 29). As expected, all compounds induced the cleavage of PARP, indicative of activation of the apoptosis pathway. Additionally, MCF-7, A549, and HepG2 cells showed phosphorylation and stabilization of p53 in response to the compounds, while PC3 and Hep3B cells did not exhibit this response. Furthermore, the compounds similarly induced the p21 target gene across all cancer cell lines, regardless of the sensitivity of each cell line. This suggests that these compounds affect cancer cell survival through a combination of both p53-dependent and p53-independent mechanisms.
Similarly, Oliva et al. synthesized a novel series of 2-amino-3-(3′,4′,5′-trimethoxybenzoyl)benzo[
b]furan derivatives
103a–
o and evaluated their in vivo and in vitro anticancer activity [
51]. The synthesis involved two distinct reaction steps, as illustrated in
Scheme 18. Initially, the Knoevenagel condensation reaction of salicylaldehyde
101 with 2-azido-1-(3,4,5-trimethoxyphenyl)ethanone was conducted in methanol at room temperature for 24 h, utilizing piperidinium acetate to afford
α-azido chalcone
102. Subsequently, the chalcones were treated with PTSA (20 mol%) in refluxing acetonitrile for 12 h, resulting in the formation of 2-amino-3-(3′,4′,5′-trimethoxybenzoyl)benzo[
b]furan derivatives
103a–
n in 60–80% yields. An alternative photochemical process was also employed, where thermal heating was replaced with irradiation at room temperature using a 25 W compact fluorescent lamp (CFL) to obtain compounds
103a–
n, albeit in reduced yields. Moreover, compound
103n was obtained from
103i through hydrogenation using Pd/C (10%) as the catalyst, while compound
103o was derived from
103m through reduction using iron and ammonium chloride.
The antiproliferative activity of the compounds
103a–
l and
103n–
o was evaluated against six human cancer cell lines (HeLa, HT-29, Daoy, HL-60, SEM, and Jurkat) using the MTT assay and CA-4 as the standard drug (
Table 22). The results revealed the remarkable activity of four of the compounds (
103d,
103f,
103k, and
103l), with IC
50 values below 5 nM. Specifically, compound
103f (R
1,2,4 = H, R
3 = OEt) displayed the highest potency with an IC
50 value of 5 pM against the Daoy cell line, outperforming the other cell lines evaluated. Both compounds
103f and
103l (R
1,2,4 = H, R
3 = Me) exhibited greater potency than CA-4 in all cancer lines and demonstrated significantly higher sensitivity in Daoy, HL-60, and Jurkat cell lines, with IC
50 values ranging from 0.005 to 0.38 nM. Furthermore, a clear and consistent trend in the antiproliferative activity was observed among the methoxy-substituted compounds (
103b–
e). The compounds bearing a methoxy group at either the C–5 or C–6 position exhibited the highest activity, while those with the methoxy group at the C–4 or C–7 position displayed the lowest activity. Specifically, the order of potency was as follows: 6-OMe (
103d) > 5-OMe (
103c) > 7-OMe (
103e) > 4-OMe (
103b). In particular, compound
103b exhibited a remarkable IC
50 value ranging from 2.8 to 8.5 nM. Moreover, the substitution of the methoxy group with a methyl group at C–6 (
103d and
103l, respectively) resulted in a 3–14-fold increase in activity against HeLa, HT-9, SEM, and Jurkat cell lines for
103l compared to
103d. However, the substitution of the methoxy group with a hydroxy group at C–6 (
103n) did not enhance its activity in any significant manner. Finally, in the halide compounds, increasing the size from fluorine to bromine (
103k) led to a 71–338-fold increase in activity across all six cell lines, with particularly pronounced effects observed in the Daoy line.
The antiproliferative studies facilitated the identification of the most promising compounds from the synthesized series of 2-amino-3-(3′,4′,5′-trimethoxybenzoyl)benzo[
b]furans [
51]. Subsequently, compounds
103c–
d,
103f–
g, and
103k–
l were selected for further evaluation to assess their inhibitory effects on tubulin polymerization and [
3H]colchicine binding to tubulin (
Table 23). The results demonstrated that all compounds exhibited comparable tubulin polymerization inhibition as compared to CA-4. Specifically, compounds
103f and
103l displayed greater potency than CA-4 (IC
50 = 0.54 nM), with IC
50 values of 0.37 and 0.39 nM, respectively. Moreover, in the colchicine binding studies to tubulin, compounds
103d,
103f, and
103l showed results similar to CA-4 at concentrations of 5 and 0.5 µM. These findings suggest that the compounds in this assay are robust antiproliferative and antitubulin agents.
The potent inhibition of tubulin polymerization displayed by compounds
103f and
103l prompted the evaluation of their effects on cell cycle progression using flow cytometry with the HeLa cell line. As shown in
Figure 30a,b, both compounds caused a remarkable cell cycle arrest at the G2/M phase after 24 h of treatment at a concentration of 10 nM. Furthermore, there was a notable reduction in the number of cells in the G1 phase, while no significant effect was observed on the S phase for both compounds. These findings indicate that compounds
103f and
103l exhibited a strong influence on cell cycle dynamics, which may play a crucial role in their antiproliferative activity.
Furthermore, a study was conducted to investigate the effects of compound
103f on two proteins, Bubr1 and Mad-2, which play essential roles in the spindle assembly checkpoint (SAC), and are associated with apoptotic cell death (
Figure 31). The treatment with compound
103f resulted in a significant reduction in the expression of both Bubr1 and Mad-2 proteins, even at low concentrations as low as 10 nM, indicating a potential arrest of the mitotic checkpoint. Moreover, the study examined cyclin B, a key regulator in the G2/M phase of the cell cycle, which showed a dose-dependent increase in expression in response to compound
103f. This result aligns with the rapid accumulation of cells in the G2/M phase induced by the compound.
Considering the well-established association of tubulin-targeting agents with antivascular effects against tumor endothelium, the response of compound
103f was evaluated to assess its antivascular activity in angiogenesis in vivo using HUVEC endothelial cells [
51]. For this purpose, HUVECs were seeded on Matrigel to analyze the impact of compound
103f on the formation of “tubule-like” structures in these cells. The results indicated that after 1 h of treatment, compound
103f effectively disrupted the HUVEC network at both concentrations tested (10 and 100 nM) compared to the control cells. To quantitatively evaluate these effects, image analysis was conducted to measure parameters such as the tubule segment length, meshwork area, and number of branches. Notably, the results showed a statistically significant effect at a concentration of 10 nM on segment length and mesh area, underscoring the strong potential of compound
103f to induce vascular disruption. These findings strongly suggest that compound
103f holds promise as a potential antivascular agent for inhibiting angiogenesis.
Finally, compound
103f underwent in vivo tests to evaluate its antitumor and cytotoxic effects in syngeneic mice. The method involved injecting E0771 murine breast cancer cells into the mammary fat pads of female C57BL/6 mice. Simultaneously, compound
103f was administered intraperitoneally on alternate days at two doses (5 or 15 mg/kg). The results demonstrated a dose-dependent reduction in tumor growth upon treatment with compound
103f, achieving a decrease of 45.7% and 16.9% at 15 and 5 mg/kg, respectively (
Figure 32a). Compound
103f exhibited higher potency than the reference drug (CA-4P), which reduced tumor growth by 26.5% at 30 mg/kg. Importantly, cytotoxicity tests revealed no apparent signs of toxicity at the 15 mg/kg doses of compound
103f (
Figure 32b). These findings highlight the potential of compound
103f as a promising antitumor agent with limited toxicity in vivo.
In 2021, Xu et al. accomplished the successful synthesis of novel polycyclic heterocycles derived from Evodiamine, a quinazolinocarboline alkaloid naturally occurring in the
Evodia rutaecarpa plant native to China [
52]. This research primarily aimed to evaluate these compounds as potential inhibitors of topoisomerase I (Top 1) for treating triple-negative breast cancer (TNBC), an aggressive subtype of breast cancer. The key focus of their synthetic efforts was the preparation of the 2-(5-methoxybenzofuran-3-yl)ethanamine
110, which involved a five-step reaction sequence using 1,4-dimethoxybenzene
104 as the starting reagent (
Scheme 19). Subsequently, intermediate
110 underwent an amidation reaction with ethyl formate in reflux conditions for 12 h to afford amide
111 in a 72% yield. The following step involved an intramolecular cyclization of compound
111 using POCl
3 in dichloromethane at room temperature for 12 h to give compound
112, which was then reacted with substituted salicylic acid chlorides
114a–
f in dichloromethane at room temperature for 12 h to obtain the compounds
115a–
f in good yields. Finally, the intermediates
115a–
f underwent
O-demethylation using BBr
3 in dichloromethane at −78 °C for 6 h to furnish evodiamine derivatives
116a–
f in good yields.
The antiproliferative evaluation of evodiamine analogs
115a–
f and
116a–
f was performed on MDA-MB-435 human breast carcinoma cells using the MTT method, with evodiamine as the standard drug (
Table 24) [
52]. Notably, the results significantly favored compounds
116a–
f, exhibiting a higher percentage of inhibition at 10 µM compared to evodiamine. Building upon these promising findings, compounds
116a–
f underwent further evaluation against four human cancer cell lines: MDA-MB-435, MDA-MB-231, HCT116, and A549, using the MTT method in the presence of evodiamine and camptothecin as the standard drugs (
Table 25). The breast cancer lines showed the highest sensitivity to these compounds. For instance, compounds
116a (R
1 = H) and
116f (R
1 = 3-Cl) showed the best activity against the MDA-MB-435 cell line, with IC
50 values of 0.47 and 0.42 µM, respectively, which were comparable to the camptothecin (IC
50 = 0.31 µM). The introduction of a halogen at position 3 significantly enhanced the antiproliferative activity of the analogs, as demonstrated by
116f, which was the most active in the series with IC
50 values of 0.36, 0.42, and 0.76 µM against the MDA-MB-231, MDA-MB-435, and HCT116 cell lines, respectively.
The data presented above shed light on the main objective of this study, which involves evaluating the topoisomerase inhibitory activity of compounds
116a–
f [
52]. To achieve this, a Top-1-mediated DNA cleavage assay was performed using purified Top 1 on compounds
116a–
f. During the assay, DNA and Top 1 were incubated with or without these compounds to observe their effect on the appearance of relaxed DNA fragments. Remarkably, among the tested compounds, only
116f and camptothecin (CPT) exhibited significant inhibition of Top-1-mediated relaxation of supercoiled DNA at a concentration of 50 µM (
Figure 33a). Furthermore, in a Top 1 inhibition study, compound
116f showed activity at 20 µM, while evodiamine only displayed moderate activity up to 500 µM (
Figure 33b,c). These findings strongly suggest that
116f specifically targets Top 1 and holds great potential as a promising candidate for further investigation in cancer drug development.
Complementary biochemical studies were performed to explore the ability of the compound
116f to effectively trap Top 1–DNA cleavable complexes within cancer cells, potentially leading to cell death. To assess this, the researchers quantified the number of trapped cleavable complexes using [
3H]thymidine incorporation and SDS-K
+ precipitation methods. As shown in
Figure 34a, there was a significant increase in the formation of the cleavable complex over prolonged periods in cells treated with both camptothecin and the compound
116f. Furthermore, to confirm the formation of the cleavable complex, an immunological band depletion assay was performed to verify the presence of Top 1 in the precipitated complex, demonstrating that the complex could not migrate through the gel. Conversely, in the absence of the complex, Top 1 would have migrated through the gel. The results revealed a proportional decrease in the amount of free Top 1 with increasing time in cells treated with 15 µM of CPT and 30 µM of
116f, particularly evident after 9 h, where almost no free Top 1 was detected (
Figure 34b). These compelling findings strongly support the notion that the
116f analog effectively interacts with Top 1, leading to the formation of the cleavable complex, underscoring its potential as a promising candidate for anti-cancer drug development.
Furthermore, a comprehensive study was performed to investigate the potential of stabilizing Top 1–DNA covalent complexes through an indirect process involving ROS generation and subsequent oxidative DNA damage, as observed in other studies with staurosporine. This investigation employed a fluorogenic ROS probe in combination with flow cytometry analysis. As demonstrated in
Figure 35a, the treatment of MDA-MB-231 cells with the compound
116f resulted in a compelling, dose-dependent increase in ROS levels. Notably, even in the absence of Top 1 in MDA-MB-231 cells (siRNA-Top 1), the
116f analog induced ROS generation, indicating that the compound has the ability to generate ROS independently of Top 1 (
Figure 35b).
The cell cycle study focused on evaluating the mitochondrial dysfunction involved in the apoptosis process. To achieve this, a JC-1 fluorescent probe was employed to measure the mitochondrial membrane potential, incubated with MDA-MB-231 cells at different concentrations, and quantified by flow cytometry analysis. Mitochondrial dysfunction was found at 3.0% with 0 µM, 12.2% with 0.1 µM, 18.5% with 0.2 µM, and 25.0% with 0.4 µM of cells after treatment with the compound
116f (
Figure 36a). These data suggest a direct relationship with the mitochondrial pathway. Therefore, it was also necessary to evaluate the expression of apoptotic proteins, including Bax, Bcl-2, cytochrome C, and caspase-3, using a Western blot assay.
Figure 36b shows that following 48 h of treatment with
116f, the levels of Bax, cytochrome C, and caspase-3 proteins noticeably increased, while Bcl-2 expression significantly decreased.
Finally, in vivo studies were conducted using a mouse xenograft model with surgical residual tumor samples from a patient with TNBC to assess the anticancer potency of compound
116f in TNBC, with paclitaxel (PTX) used as the positive control. Different concentrations of
116f were administered to the mice, followed by histological analysis of tumor tissue sections using H&E staining (
Figure 37a). The results showed that the tumors from mice treated with
116f exhibited reduced cell density and increased necrosis rates compared to the untreated mice. Furthermore, immunofluorescence labeling of cell proliferation marker Ki67 showed a significant decrease in proliferating Ki67 cells in tumors treated with
116f compared to untreated tumors. This result convincingly demonstrates the potent inhibitory effect of
116f on tumor growth. Additionally, the body weight of the evaluated mice treated with compound
116f showed no significant changes, indicating the absence of apparent toxicity (
Figure 37b). These promising findings highlight the potential of compound
116f for future TNBC treatments, even at doses as low as 20 mg/kg.
Using a similar approach, Zhang et al. successfully identified potential drugs for triple-negative breast cancer (TNBC) [
53]. They focused on the derivatives of ZINCO3830212, which was selected through molecular docking studies involving pocket U of SIRT3, encompassing crucial amino acids, such as Phe157, Arg158, Ser159, Pro176, Glu177, and Glu323. This particular pocket is known for its significant role in modulating autophagy and its associations with various human cancers. To synthesize these derivatives, the researchers utilized alkanes as linkers and introduced halogen substituents to obtain compounds
117a–
r through an
O-alkylation reaction. Subsequently, all synthesized compounds
117a–
r were evaluated against four human cancer cell lines: HL60, U937, MCF-7, and MDA-MB-231, using the MTT method and ZINCO3830212 as the reference compound (
Table 26). Among these derivatives, compounds
117c–
e exhibited the most promising results in activating SIRT3. The researchers also investigated the impact of alkane length on SIRT3 activation by varying the linker length. Interestingly, they observed a decrease in activity for compound
117o (
n = 0, R
1 = I, R
2 = 1-pyrrolidinyl) with a shorter linker. Moreover, when substituents other than iodine (i.e., H, Cl, Br, and Me) were used, the activity was not favored in compounds
117n (
n = 0, R
1 = H, R
2 = 1-pyrrolidinyl) and
117p–
r (
n = 1, R
1 = Cl, Br, Me, R
2 = 1-pyrrolidinyl). However, in the case of compound
117c (
n = 1, R
1 = I, R
2 = 1-pyrrolidinyl) containing iodine, its activity surpassed that of compounds
117p–
r. Remarkably, compound
117c showed the highest antiproliferative activity against the breast carcinoma line MDA-MB-231, with an IC
50 value of 2.19 µM, compared to ZINCO3830212 (IC
50 = 33.43 µM). This significant finding highlights its potential as a promising SIRT3 activator for TNBC treatment (
Table 26).
This study compared the deacetylation activity of the compound
117c with resveratrol and honokiol, known SIRT3 activators, using molecular coupling and evaluating their antiproliferative effects [
53]. Molecular docking revealed that, unlike the compound
117c, resveratrol and honokiol tended to bind to the acetylated substrate recognition site, indicating no allosteric effect (
Figure 38a). Also, compound
117c showed significantly higher potency than the reference activators, with an IC
50 value of 2.19 µM against the MDA-MB-231 cell line. In contrast, resveratrol and honokiol showed IC
50 values of 98.89 µM and 44.89 µM, respectively, with normalized E
max values of 0.25 for resveratrol, 0.91 for honokiol, and 1.00 for compound
117c (
Table 27). Additionally, compound
117c induced the deacetylation of two tested SIRT3 substrates, MnSOD2 and p53 (
Figure 38b), further confirming its potent deacetylation and antiproliferative effects.
The conducted studies provided significant insights, demonstrating the activation capability of compound
117c on SIRT3 [
53]. To assess its selectivity toward SIRT3, a CETSA cellular thermal shift assay was performed on all SIRTs (SIRT1, SIRT2, SIRT3, and SIRT5). The collected data clearly revealed a direct interaction between compound
117c and SIRT3, while SIRT1, SIRT2, and SIRT5 remained unaffected in their thermal stability. This distinct result confirmed the specific binding affinity of compound
117c for SIRT3 in the MDA-MB-231 cell line (
Figure 39). With its selectivity established, the researchers explored the influence of SIRT3 on both short- and long-term effects of compound
117c in the MDA-MB-231 cell line, confirming a concentration- and time-dependent inhibition of tumor cell proliferation (
Figure 40a,b). Next, the antiproliferative activity was examined in the presence of SIRT3, and the findings indicated a significant impairment in the inhibitory effect of compound
117c when SIRT3 was absent. As a result, the antiproliferative effect displayed a marked reduction after SIRT3 knockdown, firmly establishing the dependence of compound
117c on SIRT3 for its activity (
Figure 40c).
On the other hand, the regulation of autophagy by SIRT3 could have a suppressive effect on tumor growth and elimination [
53]. As depicted in
Figure 41a, compound
117c inhibits autophagy, as evidenced by the increase in LC3-II expression and the decrease in p56 expression following treatment. Moreover, the inhibition of autophagy resulted in a significant increase in cell viability and a notable attenuation of tumor migration (
Figure 41b,c). This indicates that compound
117c exerts its antiproliferative effect on MDA-MB-231 cells by inducing autophagy. To delve into the role of SIRT3 in this process, the researchers employed SIRT3-specific siRNA to attenuate SIRT3 expression. Intriguingly, the attenuation of SIRT3 resulted in a significant decrease in the induction of autophagy, which is evident from the reduced levels of LC3-II and p62, along with the down-regulation of E-cadherin (
Figure 41d). These findings suggest that compound
117c regulates autophagy and tumor migration by activating SIRT3.
Finally, in vivo studies were carried out to evaluate the antitumor activity of compound
117c in TNBC mouse xenograft models at three different concentrations (25, 50, and 100 mg/kg) [
53]. After 16 days of treatment, compound
117c exhibited significant antiproliferative activity in a dose-dependent manner. Additionally, the administration of
117c resulted in a notable decrease in tumor growth, tumor volume, and tumor weight compared to the control group (
Figure 42a,b). However, when evaluating the toxicity of
117c, certain degrees of toxicity were observed in vivo at high concentrations, leading to pulmonary septum widening and other cellular abnormalities.
In addition to the previous findings, complementary in vivo studies were conducted to assess the ability of compound
117c to activate SIRT3 and induce autophagy in a mouse xenograft model using MDA-MB-231 TNBC cells [
53]. The research team measured acetyl-lysine (Ac. K) acetylation levels, specifically AcK68-MnSOD2 and AcK122-MnSOD2, and examined the expression levels of SQSTM1/p62 and LC3 (
Figure 43). The results provided compelling evidence that the acetylation levels of K68-MnSOD2 and K122-MnSOD2 precisely matched the deacetylation sites of SIRT3. Moreover, a noticeable decrease in the expression of p62 was observed, while LC3-II levels were significantly up-regulated compared to the positive control (
β-actin). These findings convincingly demonstrate the activation of autophagy by compound
117c. In conclusion, these promising in vivo results underscore the potential of compound
117c, in combination with SIRT3, as a promising alternative for the treatment of TNBC-type cancer.
In their pursuit of employing eco-friendly synthetic strategies and achieving favorable yields, Irfan et al. conducted the synthesis of benzo[
b]furan-based oxadiazole/triazole derivatives
120a–
g and
121a–
h using ultrasound and microwave irradiation, respectively (
Table 28) [
54]. In method A, benzo[
b]furan–oxadiazole derivatives
120a–
g were obtained in 60–90% yields through
S-nucleophilic substitution of 5-(benzofuran-2-yl)-1,3,4-oxadiazole-2-thiol
118a with bromoacetanilides
119a–
g utilizing pyridine in acetonitrile under ultrasound irradiation at 40 °C for 30 min. Similarly, method B was employed to synthesize benzo[
b]furan–triazole derivatives
121a–
h in 68–96% yields through a
S-nucleophilic substitution of 5-(benzofuran-2-yl)-4-phenyl-4
H-1,2,4-triazole-3-thiol
118b with bromoacetanilides
119a–
h, utilizing pyridine in DMF under microwave irradiation for 60–70 s. In summary, the microwave-assisted method B led to better yields and shorter reaction times for benzo[
b]furan derivatives than the ultrasound-assisted method A.
The hemolytic, thrombolytic, and anticancer activities of previously synthesized benzo[
b]furan-based oxadiazole/triazole derivatives were assessed (
Table 29) [
54]. Among these compounds,
121b (X = NPh, R
1 =
N-morpholinyl) exhibited the lowest cytotoxicity (0.1%), while
121g (X = NPh, R
1 = 4-chloro-
N-anilinyl) and
120b (X = O, R
1 =
N-morpholinyl) showed the highest toxicity (23.4% and 22.12%, respectively) compared to ABTS (95.9%). In the thrombolysis assay, the majority of compounds demonstrated moderate activity when compared to the positive control (ABTS). Notably, compound
121f (X = NPh, R
1 = 2,4-dimethyl-
N-anilinyl) exhibited the highest thrombolytic potential with a value of 61.4% compared to the positive control ABTS (86%).
The IC
50 value and cell viability percentage against the A549 lung cancer cell line were determined using an MTT assay, with crizotinib and cisplatin as the standard drugs (
Table 29). In summary, compound
120d (X = O, R
1 = 2-methoxy-
N-anilinyl) exhibited the highest potency with a cell viability of 27.49% and an IC
50 value of 6.3 µM, demonstrating greater activity than crizotinib (28.22% and 8.54 µM, respectively) and lower cytotoxicity than cisplatin (15.34% and 3.88 µM, respectively). In addition, compound
121h (X = NPh, R
1 = 2,4-dichloro-
N-anilinyl) showed slightly lower activity with a cell viability of 29.29% and an IC
50 value of 10.9 µM. Other oxadiazole/triazole derivatives, such as
120b–
c,
120e–
g,
121a,
121c–
e, and
121g, exhibited moderate anticancer activity, with cell viability ranging from 34.47% to 49.8%. Although compound
121f (X = NPh, R
1 = 2,4-dimethyl-
N-anilinyl) had the highest cell viability (99.1%), it did not demonstrate any activity against the A549 cell line.
A molecular docking simulation was performed to investigate the interactions of compound
120d with anaplastic lymphoma kinase (ALK) in conjunction with crizotinib (PDB ID code: 2XP2) [
54]. In summary, crizotinib displayed direct contact with ALK residues in the active site, while compound
120d exhibited even more effective binding to these ALK residues (
Figure 44a,b). Notably, the phenyl and heterocyclic rings of
120d engaged in π-sigma interactions with Leu1256 and Val1130, while the NH group formed hydrogen bonds with Gly1201 and π-anion bonds with Glu1210.
Isatin, also known as 1
H-indole-2,3-dione, is an important
N-heterocycle in medicinal chemistry and drug discovery [
55]. In their study, Mohammed et al. synthesized an isatin–benzofuran hybrid
126 and investigated its antiproliferative activity against HT29 and SW620 cancer cell lines, along with its impact on tumor and metastatic development involved in primary cellular processes [
56].
Scheme 20 shows the three-step synthesis of the isatin–benzofuran hybrid
126. Firstly, 2-hydroxyacetophenone
122 underwent cyclization with ethyl bromoacetate utilizing K
2CO
3 as a base in refluxing acetonitrile for 8 h to afford ethyl 3-methylbenzofuran-2-carboxylate
123, which then reacted with hydrazine hydrate in refluxing methanol for 4 h to give 3-methylbenzofuran-2-carbohydrazide
124. Finally, a condensation reaction between
124 and isatin
125 catalyzed by acetic acid in refluxing ethanol for 5 h afforded isatin–benzofuran hybrid
126 with an 80% yield.
Next, comprehensive biological tests and analyses were carried out, specifically targeting cell viability, real-time migration, invasion studies, cell cycle assays related to apoptosis, and cytotoxicity evaluations [
56]. In the initial phase, a cell viability, migration, and invasion assays were carried out using an xCELLigence Automated Dual Layer Real-time Cell Analyzer (RTCA-DP) with various concentrations of isatin–benzofuran hybrid
126. The results revealed a noteworthy dose-dependent reduction in cell proliferation, migration, and invasion in both cancer cell lines compared to untreated cells (
Figure 45a–c). Moreover, notable variations were observed in the inhibitory effects of hybrid
126 on the proliferation and migration of the SW620 line compared to the HT29 line, while in invasion, the HT29 line exhibited a more pronounced inhibitory effect. In addition, the impact of hybrid
126 on tumor suppression, based on the p53 protein, was evaluated. The results indicated a significant increase in p53 expression levels, 2.46-fold in HT29 cells, and 4.81-fold in SW260 cells at a concentration of 10 μM, demonstrating the potent inhibitory effect on tumor cell proliferation when utilizing hybrid
126 (
Figure 45d).
Apoptotic studies were performed to evaluate the pro-apoptotic effects of hybrid
126, involving its role in suppressing the expression levels of mitochondrial proteins Bcl-x, Bax, and cytochrome C utilizing flow cytometry [
56]. The results showed a significant suppression of the Bcl-x protein expression by 50% in the HT29 cells, and by 75% and 90% in the SW620 cells at concentrations of 5 and 10 μM, respectively, in comparison to untreated cells (
Figure 46a,b). On the other hand, a significant increase in the expression of Bax and cytochrome C was observed in both HT29 and SW620 cell lines, showing an approximately two-fold increase compared to the basal expression of untreated cells (
Figure 46a,b). Moreover, the down-regulation of Bcl-x and the up-regulation of Bax and cytochrome C in both cancer cell lines exposed to hybrid
126 were further confirmed in gene expression levels, when compared to untreated cells. These findings support the apoptotic effects of hybrid
126 on cell cycle disruption in both HT29 and SW620 cell lines (
Figure 46c,d).
In this particular study, they assessed the cytotoxic effect of hybrid
126 both alone and in combination with three anticancer drugs: irinotecan (IRI), 5-fluorouracil (5-FU), and oxaliplatin (OXA) in the HT29 and SW620 cancer cell lines at different concentrations (
Figure 47a,b) [
56]. Notably, when combining hybrid
126 with IRI in the HT29 cell line (
Figure 47a), there was a significant inhibition of cell proliferation at 10 μM (−75% vs. −50%) and 20 μM (−90% vs. −65%) compared to the single treatment with IRI alone. When combining 5-FU with hybrid
126, cell proliferation was significantly inhibited at 5 μM (−50% vs. −20%) and 10 μM (−67% vs. −55%) compared to the single drug treatment. Similarly, OXA inhibited cell proliferation at 5 μM (−55% vs. −25%) and 10 μM (−75% vs. −45%) when used in conjunction with hybrid
126. On the other hand, in the SW620 cell line (
Figure 47b), the combined treatment of IRI with
126 resulted in a substantial inhibition of cell proliferation at 5 μM (−45% vs. −15%) and at 10 μM (−75% vs. −45%). Similarly, 5-FU combined with
126 showed comparable inhibition of cell proliferation to IRI at 5 μM (−55% vs. −15%) and at 10 μM (−70% vs. −50%). Moreover, when utilizing the treatment in conjunction with OXA, a more pronounced inhibition of cell proliferation was observed at 5 μM (−55% vs. −15%), 10 μM (−65% vs. −50%), and 20 μM (−90% vs. −75%) compared to the single drug treatment. These findings demonstrate a significant enhancement in the effectiveness of the anticancer drugs employed in this study when combined with hybrid
126.
2.2. Antibacterial Activity
Antibiotic resistance presents a worldwide concern, steadily escalating in severity, and posing substantial challenges to healthcare systems globally. Novel approaches are urgently needed to address this critical problem. Recently, benzo[
b]furans and their derivatives have shown remarkable inhibitory potential against various Gram-positive bacteria, including
Staphylococcus aureus (
S. aureus),
Bacillus subtilis (
B. subtilis), and
Enterococcus spp. (
E. spp.), as well as Gram-negative bacteria, such as
Pseudomonas syringae (
P. syringae),
Klebsiella pneumoniae (
K. pneumoniae),
Salmonella typhi (
S. typhi),
Pseudomonas aeruginosa (
P. aeruginosa), and
Escherichia coli (
E. coli) [
57]. In the province of Lampang, Thailand, a noteworthy medicinal discovery was made from the root extracts of
Stemona aphylla, resulting in the isolation of alkaloids
127a–
i (
Table 30) [
58]. The extraction process involved drying 15.14 kg of ground root material, which was then extracted with 95% ethanol for 16 days at room temperature. After evaporating the extract, a portion of the residue was partitioned between 50% aqueous methanol and dichloromethane to give 8.86 g of the dichloromethane extract. The alkaloids were further isolated using column chromatography or preparative thin-layer chromatography through successive separations. The identified alkaloids and their corresponding masses are listed in
Table 30.
Antimicrobial studies were conducted on alkaloids
127a,
127c–
d,
127g, and
127i, evaluating their MIC values against two Gram-negative bacteria (
Escherichia coli and
Klebsiella pneumoniae) and three Gram-positive bacteria (
Staphylococcus aureus,
methicillin-resistant Staphylococcus aureus (
MRSA), and
Streptococcus pyogenes), with gentamicin used as the standard drug (
Table 31) [
58]. The results indicated minimal activity against Gram-negative bacteria for all tested compounds. However, alkaloids
127a (R
1 = H, R
2 = H, R
3 = Me, R
4 = H, R
5 = Me, R
6 = H),
127c (R
1 = H, R
2 = H, R
3 = Me, R
4 = Me, R
5 = Me, R
6 = H), and
127i (R
1 = H, R
2 = OMe, R
3 = Me, R
4 = Me, R
5 = Me, R
6 = H) showed significant activity against
MRSA, exhibiting MIC values of 15.6 µg/mL, outperforming the control antibiotic (MIC = 45.0 µg/mL). Compounds
127d,
127g, and
127i showed moderate activity against
S. aureus, with MIC values of 31.3 µg/mL, in comparison to gentamicin (MIC = 22.5 µg/mL).
Next, Ashok et al. synthesized a series of
E-(1)-(6-benzoyl-3,5-dimethylfuro [3′,2′: 4,5]benzo[
b]furan-2-yl)-3-(aryl)-2-propen-1-ones
130a–
g using both conventional and microwave heating protocols (
Table 32) [
59]. Firstly, they obtained 2-acetyl-3,5-dimethyl-6-benzoylbenzodifuran
129 by cyclizing 5-acetyl-2-benzoyl-6-hydroxy-3-methylbenzo[
b]furan
128 with 2-chloroacetone employing K
2CO
3 as a base in refluxing acetone for 8 h (Method A), and microwave heating at 120 °C for 4 min under solvent-free conditions (Method B). Subsequently,
bis-chalcones
130a–
g were synthesized through a Claisen–Schmidt condensation reaction of
129 with (hetero)aromatic aldehydes using NaOH as a base in refluxing ethanol for 6–8 h (Method A, 53–68%) and microwave-assisted aldol condensation at 90 °C for 4–5 min under solvent-free conditions (Method B, 87–94%). In summary, method B led to
bis-chalcones in higher yields and shorter reaction times compared to method A, which utilized conventional heating.
The antimicrobial activities of
bis-chalcones
130a–
g were evaluated using the plate count method with nutrient agar as the culture medium. The bacterial strains tested included two Gram-negative bacteria,
Escherichia coli and
Pseudomonas aeruginosa, and two Gram-positive bacteria,
Bacillus subtilis and
Staphylococcus aureus, using chloramphenicol, carbenicillin, streptomycin, and tetracycline as the standard drugs [
59]. The inhibition zones (in mm) were measured after 24 h of incubation at 37 °C (
Table 33). In summary,
bis-chalcones
130a–
g showed inhibition zones ranging from 6 to 11 mm and 7 to 17 mm against Gram-negative and Gram-positive bacterial strains, respectively. In particular, compounds
130a (Ar = Ph),
130b (Ar = 2-ClPh),
130f (Ar = 1,3-diphenyl-1
H-pyrazol-4-yl), and
130g (Ar = 1-phenyl-3-(4-bromophenyl)-1
H-pyrazol-4-yl) showed good activity against all bacterial strains, with inhibition zones in a range of 9 to 17 mm. However, none of these
bis-chalcones demonstrated higher activity than the control drugs for each bacterial strain (13–22 mm).
In a separate study, Ostrowska et al. utilized microwave irradiation to synthesize a collection of
O-alkylamino benzo[
b]furancarboxylates with good yields [
60,
61]. Firstly, the esterification reaction of 6-acetyl-5-hydroxy-2-methyl-3-benzo[
b]furancarboxylic acid
131a with methanol catalyzed by sulfuric acid afforded benzo[
b]furancarboxylate
132a (
Scheme 21). Subsequently, microwave-assisted
O-alkylation reaction of
132a with 2-chloroethyl-
N,N-diethylamine in the presence of K
2CO
3 and Aliquat 336 in acetone gave
O-alkylated benzofuran-3-carboxylate
133a. In an alternative approach, compounds
134a and
135a were synthesized under analogous conditions to the previous
O-alkylation protocol, using carboxylic acid
131a instead of ester
132a. Notably, compounds
133b–
g and
134b–
g were obtained using the corresponding benzo[
b]furancarboxylic acids
131b–
g as the initial reactants (
Table 34). Finally, the benzo[
b]furancarboxylate derivatives
133a–
d,
133f,
134a–
d,
134f, and
135a were transformed into their respective hydrochloride salts.
The antimicrobial activity of hydrochlorides of benzo[
b]furancarboxylates
133a–
d,
133f,
134a–
d,
134f–
g, and
135a was screened against six Gram-positive bacterial strains, including
Micrococcus luteus,
Bacillus cereus,
Bacillus subtilis,
Staphylococcus epidermidis,
Staphylococcus aureus, and
Enterococcus hirae, along with two Gram-negative bacterial strains, such as
Escherichia coli and
Pseudomonas aeruginosa (
Table 35). The results unveiled that the
O-alkyl-benzo[
b]furancarboxylate
133f.HCl (R
1 = COOMe, R
2 = H, R
3 = Br, R
4 = OMe, R
5 = O(CH
2)
2NEt
2) showed the highest potency, with MIC values ranging from 0.003 to 0.012 µmol/cm
3 against all Gram-positive bacterial strains. Conversely, the hydroxy-benzo[
b]furancarboxylate
134g.HCl (R
1 = COO(CH
2)
2NEt
2, R
2 = H, R
3 = H, R
4 = H, R
5 = OMe) showed the lowest potency, with an MIC value of 15.28 µmol/cm
3 in all Gram-positive bacterial strains. Additionally, it becomes evident that within the Gram-positive strains, compounds
135a.HCl and
133f.HCl exhibited the most remarkable activity against
E. coli and
P. aeruginosa with MIC values of 0.59 and 3.12 µmol/cm
3, respectively. Notably, the observed structure–activity relationships are as follows: (i) compound
133c.HCl exhibited higher activity than
133b.HCl due to the introduction of a methoxy group at the C–5 position, (ii) compound
133d.HCl displayed higher activity than
133b.HCl due to the presence of the 7-(4-methoxycinnamoyl) group, and (iii) 2-(
N,N-diethylamino)ethyl esters
134a.HCl,
134c.HCl, and
134d.HCl exhibited superior activity against Gram-positive strains compared to compounds
133a.HCl,
133c.HCl, and
133d.HCl. These variations underscore the influential role of substituents in the benzo[
b]furan moiety, as evidenced by their distinct activities against diverse bacterial strains.
Concurrently, Kenchappa et al. synthesized a series of (5-substituted-1-benzofuran-2-yl)(2,4-phenyl-substituted)methanones
139a–
i by incorporating a pharmacophore group at the 2 position of the benzo[
b]furan ring, a response to the consistent trend of enhanced antimicrobial activity observed across various studies [
62]. Initially, the synthesis commenced with the cyclization reaction of salicylaldehyde derivatives
136a–
c with
α-bromoacetophenones
137a–
c utilizing potassium carbonate as a base in refluxing acetonitrile to afford benzo[
b]furan derivatives
138a–
i (
Scheme 22). Subsequently, the Knoevenage condensation of compounds
138a–
i with Meldrum’s acid catalyzed by acetic acid at temperatures of 110–115 °C for a duration of 8–10 h resulted in the formation of (5-substituted-1-benzofuran-2-yl)(2,4-phenyl)methanones
139a–
i in 75–91% yields. It is important to emphasize that the presence of acetic acid facilitated the generation of a carbanion in Meldrum’s acid, thereby enhancing the nucleophilic addition and subsequent dehydration processes.
The antimicrobial activity of benzo[
b]furan derivatives
139a–
i was screened against one Gram-positive bacterial strain, including
Bacillus subtilis, as well as four Gram-negative bacterial strains, including
Pseudomonas syringae,
Salmonella typhi,
Klebsiella pneumoniae, and
Escherichia coli, using the agar well diffusion method [
62]. Streptomycin was employed as the standard reference. The minimum inhibitory concentration (MIC) studies were performed using a serial broth-dilution method at different concentrations, including 1, 10, 25, 50, and 100 mol/L [
62]. Based on the findings presented in
Table 36, compound
139c (R
1 = Br, R
2 = Br, R
3 = H) showed the most potent activity against the Gram-positive strain with an inhibition zone of 13 mm, closely approximating the effectiveness of streptomycin (16 mm) at a concentration of 0.5 mg/mL. Interestingly, compound
139c also emerged as the most effective against all Gram-negative strains with inhibition zones ranging from 10 to 14 mm, akin to the performance of streptomycin (13–17 mm), at a concentration of 0.5 mg/mL. As shown in
Table 37, compounds
139c (R
1 = Br, R
2 = Br, R
3 = H) and
139a (R
1 = H, R
2 = Br, R
3 = H) demonstrated remarkable activity across all bacterial strains, displaying MIC values in ranges of 14.80–16.00 µg/mL and 15.50–16.50 µg/mL, aligning closely with the efficacy of streptomycin (MIC = 14.8–16.0 µg/mL). In contrast, compounds
134b (R
1 = H, R
2 = OMe, R
3 = H) and
139f (R
1 = OH, R
2 = OMe, R
3 = H) showed reduced activity, possibly attributed to the presence of electron-donating groups at the C–5 position of the benzo[
b]furan ring. Furthermore, the introduction of a bromine group at C–4 of the benzo[
b]furan ring did not increase the activity of compound
139i (R
1 = OH, R
2 = H, R
3 = Br) (
Table 37).
Naftifine, a topical allylamine, exhibits effectiveness across an extensive spectrum of dermatophytic fungi, including
Trichophyton and
Microsporum spp., and has also shown significant efficacy against
Candida and
Aspergillus spp. [
63]. In 2016, Wang et al. undertook the synthesis of naphthalene hydrochloride (NFT) derivatives, previously recognized as potent inhibitors of the diapophytoene desaturase (CrtN) enzyme, which is a crucial molecular target against infections caused by pigmented
Staphylococcus aureus [
64]. The process of molecular design comprised several sequential stages. It commenced with an analysis of the naphthalene moiety of NFT, which served as a potential pharmacophore group. Subsequently, modifications were introduced in the
N-methyl group, involving various steric groups (region A). Concurrently, the synthesis of specific analogs was undertaken to explore the impact of different linker types within the allyl portion on inhibitory activity (region B). Finally, a meticulous design approach led to the synthesis of 21 analogs, each featuring distinct substituents (region C), as illustrated in
Scheme 23. In pursuit of this goal, several syntheses were undertaken to generate the varied analogs portrayed in
Scheme 24.
Scheme 24 sowed synthetic procedures to synthesize a series of naftifine analogs—
140,
142a–
c,
143a–
b, and
144a–
t [
64]. The synthesis began with the nucleophilic substitution of 2-iodophenol
145 utilizing 1-bromo-2,2-diethoxyethane and NaH in DMF at 90 °C to give compound
146, which was cyclized in the presence of polyphosphoric acids under refluxing toluene to yield 7-iodobenzofuran
147. Further progression involved the substitution of the iodine atom in
147 with a cyano group in DMF at 130 °C for 4 h to furnish benzofuran-7-carbonitrile
148. The subsequent reduction of the cyano group in
148 was performed with LiAlH
4 under mild reaction conditions to obtain benzofuran-7-ylmethanamine
149. Later, allylamine
142a was synthesized with an overall yield of 95% through a reductive amination reaction of compound
149 with
trans-cinnamaldehyde utilizing NaBH
4 as the reducing agent. Subsequently, the
N-alkylation of allylamine
142a was conducted using iodoethane or 2-iodopropane in the presence of NaH as a base in DMF at ambient temperature to deliver compounds
142b–
c with a purity ≥ 95%. In another synthetic strategy, amine
149 was initially protected with di-
tert-butyl dicarbonate, followed by reduction using LiAlH
4 to afford 1-(benzofuran-7-yl)-
N-methylmethanamine
151 (
Scheme 24). Simultaneously, the
α,
β-unsaturated aldehyde
152 were reduced using NaBH
4 to afford allylic alcohol
153, which was subjected to an Appel reaction utilizing PBr
3 in Et
2O at ambient temperature to afford allylic bromide
154. Finally, aliphatic nucleophilic substitution between compounds
151 and
154 gave a series of naftifine analogs, which were converted into their corresponding hydrochloride salts
140,
143a–
b, and
144a–
t.
In a similar manner, the synthesis of naftifine analog
141 involved an
O-alkylation reaction of 3-bromophenol
155 with 1-bromo-2,2-diethoxyethane to give compound
156, which was then cyclized using polyphosphoric acids, leading to the formation of two isomeric products
157, namely, 4-bromobenzo[
b]furan and 6-bromobenzo[
b]furan (
Scheme 25). Subsequently, the bromine atom within isomers
157 was substituted with a cyano group to afford isomers
158. Finally, through a series of consecutive reactions involving reduction and Boc
2O protection, reduction, and nucleophilic substitution, the naftifine analog
141 was successfully synthesized.
After obtaining the desired analogs, their inhibitory potential against
S. aureus Newman was systematically evaluated using naftifine (NFT) as the reference drug [
64]. The results indicated that compound
140 (R
3 = Me, R
5 = 7-benzofuranyl) showed the highest potency with an IC
50 value of 247.3 nM, in comparison to NFT as the standard drug (IC
50 = 296.0 nM). In contrast, the isomeric compound
141 demonstrated low activity with an IC
50 value of 758.7 nM. In the case of analogs featuring
N-methyl substitutions
142a–
c, the incorporation of an ethyl or
iso-propyl group markedly diminished activity (IC
50 > 1000 nm), as shown in
Table 38. While the incorporation of cycloalkyl substituents
144a–
b and the 2-furanyl group
144c did not improve activity (IC
50 > 1000 nM), the incorporation of 1- and 2-naphthalenyl groups
144d and
144e led to better activity (IC
50 = 887.7 and 17.1 nM, respectively). It is worth noting that the presence of electron-donating and electron-withdrawing groups attached to the aromatic ring significantly influenced the activity profile. Also, the position of the substituent on the aromatic ring affected the activity, such as compounds
144h (R
1 = 4-FPh, IC
50 = 31.2 nM) vs.
144o (R
1 = 2-FPh, IC
50 = 288.3 nM) vs.
144r (R
1 = 3-FPh, IC
50 = 513.0 nM). The same behavior was observed for compounds
144k (R
1 = 4-NO
2Ph, IC
50 = 71.1 nM) vs.
144p (R
1 = 2-NO
2Ph, IC
50 > 1000 nM) vs.
144s (R
1 = 3-NO
2Ph, IC
50 > 1000 nM). A key highlight is the exceptional activity displayed by compound
144l (R
1 = 4-CF
3Ph), showcasing an impressive IC
50 value of 4.0 nM, which is 74 times lower than NFT (IC
50 = 296.0 nM), as shown in
Table 39.
Initial investigations unveiled the most prospective analogs with the potential to inhibit diapophytoene desaturase (CrtN) enzyme in the Staphyloxanthin (STX) biosynthesis pathway. STX is a notable golden carotenoid pigment synthesized by
S. aureus, which opens up a novel avenue for treating
S. aureus or
methicillin-resistant S. aureus (MRSA) infections [
64]. As shown in
Table 40, a selection process identified five analogs (
144f,
144i,
144j,
144l, and
144t) with the highest activity against
S. aureus Newman, which were subjected to an evaluation of their capacity to inhibit the CrtN enzyme. The results revealed that five analogs displayed a remarkable 40-fold increase in inhibitory potency against CrtN compared to NFT. Interestingly, this potent inhibition of CrtN stands in contrast to their comparatively weaker impact on the enzymatic activity of pigmented
S. aureus Newman. Furthermore, an assessment was conducted on the water solubility of the five analogs, revealing NFT to possess low solubility (6.2 mg/mL). This investigation facilitated the clarification of the interplay between solubility and chemical structure. Indeed, the replacement of the naphthalene ring with a benzo[
b]furan ring generated an elevation in solubility, particularly evident in the cases of analogs
144f and
144l, showcasing solubilities 2–3 times greater than that of NFT, measuring 19.7 and 10.0 mg/mL, respectively. These findings served as the basis for advancing the assessment of the compound
144l, both in vitro and in vivo.
In their in vitro investigations, Wang et al. examined the impact of compound
144l on three MRSA strains: USA400 MW2, USA300 LAC, and Mu50 [
64]. The results revealed a reduction in color due to the inhibitory effects of
144l, evidenced by IC
50 values of 5.45, 3.39, and 0.38 nM. These findings mirrored the observations made with the
S. aureus Newman strain (
Figure 48).
In vivo studies allowed the evaluation of virulence reduction in three of four colonies. Mice were infected with mock or treated with the compound
144l with
S. aureus Newman, USA400 MW2, and Mu50 strains by retro-orbital injection [
64]. Bacterial survival within host organs was then measured. Notably, in the
S aureus Newman strain, the group treated with compound
144l displayed a reduction in bacterial survival. Kidneys and hearts showed decreases of 0.85 and 1.01 log
10CFU/organ, respectively (
Figure 49).
Regarding the MRSA strains, BPH-652 served as a reference CrtN inhibitor [
64]. For the USA400 MW2 strain, the administration of a 200 mg/kg dose of compound
144l to mice resulted in a remarkable 99.6% reduction in survival rates within hepatic organs (2.35 log
10CFU). Impressively, this outcome surpassed that of the BPH-652-treated group (1.58 log
10CFU). Furthermore, with the dosage scaled down to 50 mg/kg, the bacterial survival rate showed only a marginal increase in both scenarios while still maintaining superiority over BPH-652 by 0.71 log
10CFU in the
144l-treated group and by 0.25 log10CFU in the BPH-652-treated group (
Figure 50a). Within the renal organs, the administration of the 200 mg/kg dose resulted in a notable 96.6% decrease in staphylococcal loads of the
144l-treated group (1.47 log
10CFU), surpassing the BPH-652-treated group outcome of 1.14 log
10CFU. Upon reducing the dose to 50 mg/kg, slight increases in bacterial survival rates were noted compared to the high-dose treatment groups (
Figure 50b).
Employing the Mu50 strain, a parallel pattern emerged. Within liver organs, a dose of 200 mg/kg led to a reduction in survival rates by 3.58 log
10CFU (
144l-treated) and 2.84 log
10CFU (BPH-652-treated). Similarly, at 50 mg/kg, the survival rate diminished by 2.94 log
10CCFU (
144l-treated) and 1.87 log
10CFU (BPH-652-treated), as depicted in
Figure 51a. However, outcomes in renal organs yielded inconclusive results. With a dosage of 200 mg/kg, the decrease amounted to 1.11 log
10CFU (
144l-treated) and merely 0.30 log
10CFU (BPH-652-treated). At 50 mg/kg, survival was reduced to 0.68 log
10CFU (
144l-treated) and 0.25 log
10CFU (BPH-652-treated), as shown in
Figure 51b.
On the other hand, pyrrolobenzodiazepines (PBDs) have garnered considerable attention as promising antibacterial agents derived from natural sources. In line with this, Andriollo et al. undertook the synthesis of a series of PBDs incorporating C–8 linkers (
Scheme 26), with the primary objective of evaluating their bioactivity and elucidating the structure–activity relationship (SAR) [
65]. The synthetic methodology entails the synthesis of benzo[
b]furan-based pyrroles
163a–
b and
167, achieved through an amidation reaction between
N-methylpyrrole derivatives and benzo[
b]furans in DMF, utilizing the EDCI/DMAP coupling system. Subsequently, nitrile
163b underwent hydrolysis under reflux conditions using dioxane and H
2SO
4 to afford carboxylic acid
164 in a modest 9% yield (
Scheme 26). After acquiring these intermediate components, the synthesis of PBD derivatives
169,
171, and
172 with C–8 linkers was undertaken (
Scheme 27). In this stage, the deprotection of BOC-protected amines
163a and
170 was achieved by treating them with an acidic solution (TFA in DCM). Additionally, derivatives containing methyl esters, identified as
167, underwent hydrolysis using an aqueous NaOH solution. Subsequent to this, an amide coupling reaction was facilitated utilizing the EDCI/DMAP coupling system, effectively linking the PBD core and side chains. Lastly, employing pyrrolidine and Pd(PPh
3)
4 in DCM, the conjugates underwent deprotection, leading to the generation of PDB derivatives
169,
171, and
172, each exhibiting standard and reverse orientations of the amide bond (
Scheme 27).
The previously obtained PBD derivatives
169,
171, and
172 underwent an assessment to determine their capacity to bind to DNA and impart stability using a Förster resonance energy transfer (FRET)-based DNA fusion assay, utilizing netropsin as the positive control [
65]. To achieve this objective, two oligonucleotide sequences labeled with distinct fluorophores were utilized: sequence F1′-FAM-TAT-ATA-TAG-ATA-TTT-TTT-TAT-CTA-TAT-ATA-3′-TAMRA and sequence F2′-FAM-TAT-AGA-TAT-AGA-TAT-TAT-TTT-ATA-TCT-ATA-TCT-ATA-TCT-ATA-3′-TAMRA. Here, FAM corresponds to 6-carboxyfluorescein, and TAMRA represents 5-carboxytetramethylrhodamine. The results revealed that compound
169, featuring a conventional orientation, adeptly conferred substantial stability to both DNA sequences, akin to the notable effect seen with netropsin. In contrast, compounds
171 and
172, characterized by reversed orientations, displayed a clear inability to confer stability upon either DNA sequence, as evidenced by ΔT
m values below 1 °C. This compelling observation strongly suggests that the inversion of one or more amide bonds within these compounds markedly curtailed their DNA stabilizing efficacy, as firmly corroborated by the comprehensive data outlined in
Table 41.
Subsequently, PBD derivatives
169,
171, and
172 were subjected to antimicrobial testing against diverse Gram-positive bacterial strains, including methicillin-sensitive
S. aureus (MSSA) strain ATCC 9144, as well as two methicillin-resistant
S. aureus (MRSA) strains, namely, EMRSA-15 (strain HO 5096 0412) and EMRSA-16 (strain MRSA 252) [
65]. Additionally, the study incorporated vancomycin-sensitive
Enterococcus faecalis (VSE) strain NCTC 755, vancomycin-resistant
E. faecalis (VRE) strain NCTC 12201, and vancomycin-resistant
Enterococcus faecium (VRE) strain NCTC 12204. The results are presented in
Table 42. Compound
169, characterized by its standard orientation, exhibited remarkable antibacterial efficacy, with MIC values of 0.125 µg/mL against all assessed Gram-positive strains. In contrast, compound
171, characterized by its inverted amide bond orientation, exhibited a significant reduction in activity across all bacterial strains, particularly evident in MRSA strains, where MIC values exceeded 32 µg/mL. Notably, compound
172, characterized by its inversion of both amide bonds linked to the
N-methylpyrrole ring, exhibited enhanced antibacterial activity compared to compound
171. However, its activity was inferior to that of compound
169. It is worth considering that the assessment of the orientation’s impact on antibacterial activity is at the forefront of our analysis.
The mechanism of action of compound
172 was investigated via time–kill assays conducted on MRSA (EMRSA-15) and VRE (NCTC 12201) strains. These strains were exposed to compound
172 at a concentration of 4 × MIC for 24 h (
Figure 52). Compound
172 demonstrated a rapid and robust bactericidal effect, leading to a reduction in cell counts below the detectable limit within a 2 h period. In contrast, ciprofloxacin exhibited bacteriostatic activity (
Figure 52). Despite a modest cell population persisting in EMRSA-15 after 24 h of exposure to compound
172, it exhibited no resistance to the compound, suggesting the presence of a potential persister population. This trend was more pronounced in EMRSA-15 than in NCTC 12201, underscoring the potential role of fluoroquinolone resistance.
Moreover, an extensive in silico analysis was performed, employing ESI mutagenesis to unravel the mechanism of action inherent in the PBD derivatives. The gathered data unveiled that compound
172 exhibited a distinct interaction pattern with the ligand-binding domain of DNA gyrase, showcasing a notably robust binding affinity for both subunits of the bacterial DNA gyrase complex. The visualization of these interactions involving the bacterial gyrase from
Staphylococcus aureus (PDB ID code: 2XCT) is visually presented in
Figure 53A,B. As delineated in the 2D patterns depicted in
Figure 53C, it becomes apparent that compound
172 forms three conventional hydrogen bonds, establishing connections with serine 98, arginine 92, and glutamine 95 within DNA gyrase A subunit 1. Analogously, the interaction extends to subunit 2 of DNA gyrase A, where it interacts with serine 85, arginine 92, and serine 98 (
Figure 53D). The implications of the interaction underscore that the antibacterial activity attributed to compound
172 stems from its direct modulation of gyrase A via enzyme interaction, as opposed to its ability to stabilize DNA.
Considering the significance of Sortase A (SrtA) as a cysteine transpeptidase prevalent in most Gram-positive bacteria, its pivotal role in the infection process of these organisms is well-established. Inhibition of this enzyme has been shown to exert a discernible impact on the virulence of Gram-positive bacteria, thereby rendering them more resistant to antibiotics. Acknowledging this premise, Lei et al. embarked upon extending these insights to
Staphylococcus aureus (
S. aureus). Given its susceptibility to detection and elimination by the immune system due to its lower viscosity, inhibiting SrtA emerged as a pertinent strategy [
66]. To this end, a series of derivatives of 2-(4-(1-cyano-2-phenylvinyl)phenyl)-
N-isobutylbenzofuran-3-carboxamide
175a–
z,
175a2–
i2, and
176 were synthesized from intermediates
173 [
67], through substitution reactions involving cyanide, the compounds
174 were obtained (
Scheme 28). Ultimately, via condensation reactions with diverse aldehydes, the cyano derivatives of benzo[
b]furan
175a–
z,
175a2–
i2, and
176 were successfully generated.
Additionally, three benzofuran-3-carboxamide derivatives were synthesized to evaluate the effect of the olefin cyanide group in inhibiting SrtA activity (
Scheme 29). For this, a double reduction was carried out using Pd/C under a hydrogen atmosphere to reduce the olefinic double bond adjacent to the cyanide, followed by the removal of the benzyl group, thus obtaining the compounds
177a–
b and
178a–
b, respectively.
A comprehensive synthesis of 39 benzofuran cyanide derivatives was conducted, followed by their rigorous evaluation for in vitro inhibitory potential against SrtA in
S. aureus, with pHMB serving as a positive control. The findings, detailed in
Table 43, underscored the noteworthy activity exhibited by most of the synthesized analogs, with IC
50 values spanning the range of 3–100 μM. Among these, compounds
175a,
175e,
175g,
175i,
175m–
o,
175w, and
175h2 emerged as particularly significant performers. This performance differential might be attributed to the intricate interplay of structure–activity relationships, favoring the potency of analog
175a (R
1 = H, R
2 = H, IC
50 = 8.8 μM) over counterparts
175p (R
1 = 7-OMe, R
2 = H, IC
50 = 11.9 μM) and
175z (R
1 = 5-Cl, R
2 = H, IC
50 = 29.9 μM), primarily due to the strategic introduction of a substituent within the benzo[
b]furan ring. The influence of R
2 substitution on inhibitory activity was distinctly pronounced, as evidenced by the superiority of electron-withdrawing groups over donor groups,
175b (R
1 = H, R
2 = 4-Me) vs.
175i (R
1 = H, R
2 = 4-Cl),
175q (R
1 = 7-OMe, R
2 = 4-Me) vs.
175w (R
1 = 7-OMe, R
2 = 4-Cl), and
175b2 (R
1 = 5-Cl, R
2 = 3,4-Me) vs.
175h2 (R
1 = 5-Cl, R
2 = 4-Cl). Furthermore, the size of substituents emerged as another pivotal determinant, favoring the chlorine group in compounds
175i,
175w, and
175h2 (IC
50 = 9.8, 5.9, and 6.8 μM, respectively), in contrast to the bromide group in compounds
175j (R
1 = H, R
2 = 4-Br),
175x (R
1 = 7-OMe, R
2 = 4-Br), and
175i2 (R
1 = 5-Cl, R
2 = 4-Br) with IC
50 values of 19.1, 15.5, and 16.4 μM, respectively.
Furthermore, the selection process extended to the identification of four compounds
175a,
175o,
175h2, and
178a boasting the most noteworthy SrtA inhibition activity. Subsequently, the impact of these compounds on impeding the formation of bacterial biofilms, a significant contributor to drug resistance, was diligently evaluated. To facilitate this assessment, SPSS Software was harnessed to compute the inhibitory potential of the chosen compounds, as outlined in
Table 44. The findings underscored the inhibitory efficacy of all four compounds against the development of
S. aureus biofilms, with IC
50 values spanning a range of 2.1–54.2 μM. Remarkably, compound
175o emerged as the most potent inhibitor, displaying an impressive IC
50 of 2.1 μM. These results harmoniously align with the outcomes derived from the SrtA inhibition study. In addition, a further assay was performed to investigate the potential of the four compounds to disrupt the invasion of 293T cells (human embryonic kidney cells) by
S. aureus, utilizing a group of drug-free FITC-labeled bacteria as the control reference. Intriguingly, all four compounds demonstrated a pronounced reduction in the invasiveness of
S. aureus strains within 293T cells. Notably, compound
175o exhibited the most substantial interference, diminishing bacterial invasion by a noteworthy 24.0% at 100 μM, as contrasted with the control blank (
Table 44 and
Figure 54).