Development of a multi-targeted chemotherapeutic approach based on G-quadruplex stabilisation and carbonic anhydrase inhibition

Figures & data

Figure 1. Rationale for the development of GQ ligand–CAI derivatives as innovative chemotherapeutics to disrupt tumour cell growth at multiple levels.

Figure 2. Chemical structure of the carbonic anhydrase inhibitors discussed here.

Figure 3. Drug design and general structure of the berberine-CAI derivatives proposed here.

Figure 4. Chemical structure of the berberine derivatives as GQ ligands discussed here.

Scheme 1. Synthetic path to yield berberrubine 2 from berberine hydrochloride 1.

Scheme 2. Synthetic pathway to yield the berberine azide (3–6) and alkyne (7, 8) intermediates.

Scheme 3. Synthesis of benzenesulfonamide azide (11, 12) and alkyne (14, 16) intermediates.

Scheme 4. Synthesis of coumarin azide (18–20 and 25) and alkyne (21–23) derivatives.

Scheme 5. Synthesis of derivatives 26–39 of type 1 which include the triazole N¹ berberine oriented. CuAAC: (i) CuSO₄ and sodium ascorbate or (ii) Cu nanosized and tetramethylammonium chloride in tBuOH/H₂O at 40 °C.

Scheme 6. Synthesis of hybrid derivatives 40–45 of type 2 which include the triazole N¹ CAI oriented. CuAAC: (i) CuSO4 and sodium ascorbate or Cu nanosized and tetramethylammonium chloride in tBuOH/H₂O at 40 °C.

Scheme 7. Synthesis of berberine derivative 49 bearing an aliphatic primary sulphonamide moiety.

Table 1. Inhibition data of human CA I, II, IX, and XII isoforms with sulphonamides 26–33, 40, 41, 47 and coumarins 34–39, 42–45 derivatives and the standard sulphonamide inhibitor acetazolamide (AAZ) by a stopped-flow CO₂ hydrase assay⁴⁹.

Compound		CAI	KI (nM)^a^,b
			hCA I	hCA II	hCA IX	hCA XII
26	S-2,0		580.5 ± 32.5	59.4 ± 3.8	1.4 ± 0.1	7.3 ± 0.5
27	S-2,2		940.2 ± 52.1	6.0 ± 0.4	0.65 ± 0.05	4.4 ± 0.3
28	S-3,0		872.1 ± 55.9	66.8 ± 3.9	0.73 ± 0.04	14.0 ± 1.0
29	S-3,2		805.0 ± 60.8	62.3 ± 4.7	0.56 ± 0.03	31.2 ± 2.5
30	S-4,0		592.4 ± 36.3	55.1 ± 3.8	0.49 ± 0.03	25.5 ± 1.4
31	S-4,2		915.6 ± 57.9	71.5 ± 5.3	0.71 ± 0.04	7.2 ± 0.5
32	S-5,0		573.6 ± 35.8	15.8 ± 1.2	0.24 ± 0.02	18.3 ± 1.0
33	S-5,2		219.5 ± 16.2	41.1 ± 2.7	0.54 ± 0.03	2.9 ± 0.1
40	S-1,0m		3670 ± 22	90.1 ± 5.6	3.9 ± 0.2	24.8 ± 1.3
41	S-1,0p		829.1 ± 50.3	22.9 ± 1.4	2.7 ± 0.2	6.1 ± 0.3
47	S-3,a		75.1 ± 5.4	21.5 ± 1.6	17.8 ± 0.9	35.9 ± 2.1
34	C-3,2		>100 µM	>100 µM	66.0 ± 4.3	30.1 ± 1.5
35	C-3,3		>100 µM	>100 µM	34.5 ± 2.4	18.9 ± 0.9
36	C-3,4		>100 µM	>100 µM	58.9 ± 3.9	28.4 ± 1.7
37	C-4,2		>100 µM	>100 µM	17.3 ± 1.2	8.4 ± 0.5
38	C-4,3		>100 µM	>100 µM	30.1 ± 1.8	16.7 ± 1.2
39	C-5,2		>100 µM	>100 µM	48.6 ± 2.6	20.1 ± 1.3
42	C-1,4		>100 µM	>100 µM	35.1 ± 2.8	10.2 ± 0.8
43	C-1,5		>100 µM	>100 µM	25.5 ± 1.5	4.2 ± 0.3
44	C-1,6		>100 µM	>100 µM	9.6 ± 0.5	14.5 ± 0.9
45	C-1,0c		>100 µM	>100 µM	51.5 ± 3.6	5.4 ± 0.3
AAZ^c		–	250	12.5	25.0	5.7
SLC-0111^c		–	5080	960	45	4.5

^a Inhibition data are expressed as means ± SEM of three different assays. ^binhibitor-enzyme incubation time: 15 min for sulphonamides (S); 6 h for coumarins (C); ^cfrom ref. 16.

1. The off target hCA I was the least inhibited isoform by all sulphonamide derivatives 26–33, 40, 41, 47 with K_I values in the range 75.1–3670 nM. The p-substituted benzenesulfonamides 26–33, 41 showed a rather flat SAR, based on K_I values in the narrow range 580.5–940.2 nM, except for compound 33 (S-5,2) that reported a K_I value of 219.5 nM. The latter is endowed with the longest spacers at the triazole sides, presumably promoting the interaction within the narrow hCA I active site. As a matter of fact, the unhindered aliphatic sulphonamide 47 stood out as the most effective hCA I inhibitor, exhibiting a K_I of 75.1 nM. In contrast, the most hindered m-substituted benzenesulfonamide 40 inhibited hCA I only in the micromolar range (K_I of 3670 nM).

2. The most physiologically relevant hCA II was effectively inhibited by most sulphonamide compounds with K_Is in a low-medium nanomolar range, i.e. 6.0–90.1 nM. Again, the SAR of the p-substituted benzenesulfonamides 26–33, 41 was rather flat. Of this subset, compounds 27 (S-2,2) and 32 (S-5,0) stood out as the most effective hCA II inhibitors with K_Is of 6.0 and 15.8 nM, respectively. In the case of hCA II, the aliphatic sulphonamide derivative 47 reported a medium inhibitory trend with a K_I of 22.1 nM. The m-substituted benzenesulfonamide 40 was also the weaker inhibitor against hCA II (K_I of 90.1 nM).

3. With the only exception of derivative 47 (K_I of 17.8 nM), all sulphonamide compounds 26–33, 40, 41 were single-digit to subnanomolar inhibitors of the main target hCA IX. In detail, derivatives 26, 40, and 41 bearing the shortest spacers at the triazole sides showed the highest K_I values, which are 1.1, 3.9, and 2.7 nM, respectively. The other medium-length benzenesulfonamides unexpectedly exhibited 0.2 to 0.7 nM inhibition constants against hCA IX. Compound 32 (S-5,0) stood out as the most potent hCA IX inhibitor of the set (K_I of 0.24 nM). Such a general trend of high efficacy in the sulphonamide subset despite differences in the spacer length suggests that the berberine nucleus, as a molecular tail, positively impacts the interaction of benzenesulfonamide derivatives with the hCA IX active site. Indeed, structural studies showed that the binding cleft of the tumour-associated hCA IX is roomier and shows more accessory pockets than the ubiquitous hCAs I and II, and even the related hCA XII, making it possible to better accommodate the polycyclic berberine core. Notably, 10- to more than 100-fold improved K_I values were measured for the sulphonamide compounds here reported with respect to SLC-0111 (K_I of 45.0 nM against hCA IX).

4. The other target hCA XII was the second most inhibited isoform by most sulphonamide compounds 26–33, 40, 41 with K_Is in a low-medium nanomolar range, i.e. 2.9 – 31.2 nM. In fact, only 32 and 47 comparably or more effectively targeted hCA II vs hCA XII. In the subset of single-digit nanomolar hCA XII inhibitors, 27 (S-2,2) and 33 (S-5,2) demonstrated the most effective inhibitory properties with K_Is of 4.4 and 2.9 nM, respectively, that are comparable or better than those measured for SLC-0111 (K_I of 4.5 nM). It can be speculated that 27 and 33 have proper spacer lengths to promote the interaction of the positively charged berberine core with specific accessory pockets present in the hCA XII active site, more hydrophilic than hCAs I, II, and IX. The aliphatic derivative 47 showed the worst, though effective, hCA XII inhibition with a K_I of 35.9 nM.

5. The inhibition profiles of the coumarin derivatives 34–39, 42–45 were in line with previous evidence of this class of CAIs³⁵. In detail, inhibition of hCAs I and II was detected below 100 µM for none of the lactone compounds, whilst all of them were low-medium nanomolar inhibitors of the target hCAs IX and XII, with K_Is in the ranges 9.6–66.0 nM and 4.2–30.1 nM, respectively. The effect of spacer length at the triazole sides over the K_I values is clear for the prodrug coumarin subset, but a neat SAR cannot be ruled out. In fact, elongation of the spacer between the triazole and coumarin scaffold appears to increase the inhibitory action more than the extension of the berberine-triazole spacer, as observed with compounds 43 (C-1,5) with hCA XII (K_I of 4.2 nM) and 44 (C-1,6) with hCA IX (K_I of 9.6 nM). Nonetheless, the shortest coumarin derivative 45 (C-1,0c) produced a hCA XII inhibition comparable with 43 (C-1,5) (K_I of 5.4 nM).

6. With the only exception of derivative 47, all derivatives showed marked target/off target isoform selectivity (Table S1, Supporting Information), unlike the reference drug AAZ and prevalently above the lead SLC-0111. Whilst the selectivity indexes (SI) of the coumarin derivatives for the target hCAs over hCA I were comparable or slightly greater in the case of hCA XII (151.5–1041 vs 332.2–2381), the sulphonamide derivatives selectively targeted hCA IX over hCA I at a greater extent (307.1–2390) and hCA II (8.5–112.4), with the subset 29–31 being of great interest since displaying SI > 1000 and SI > 100, respectively. Instead, these zinc binder compounds generally showed a great hCA XII/hCA I selectivity (25.8–213.7), while only sulphonamides 26, 31 and 33 reported notable SI for hCA XII over hCA II (8.1, 9.9, 14.2). Hence, as a general tendency, coumarin derivatives stood out for isoform selectivity, whilst the sulphonamides here reported demonstrated almost unprecedented inhibition potency associated with medium to high selectivity of action.

Table 2. Changes in the G4-stabilising effect (ΔΔT_1/2) of the indicated compounds with respect to berberine, as determined by circular dichroism melting experiments.

Compound		ΔΔT1/2 (°C)^a
		Tel23	c-Kit1	c-Myc
26	S-2,0	0.5	9.0	>13.0
27	S-2,2	−2.0	5.5	6.5
28	S-3,0	−3.5	3.5	>13.0
29	S-3,2	−1.5	3.0	4.5
30	S-4,0	−0.5	7.5	>13.0
31	S-4,2	−1.5	2.5	1.0
32	S-5,0	2.5	6.0	1.0
33	S-5,2	1.0	5.5	0.5
40	S-1,0m	−4.0	3.0	>13.0
41	S-1,0p	−4.0	2.0	2.5
47	S-3,a	−3.0	2.5	0.5
34	C-3,2	1.0	7.0	6.0
35	C-3,3	−1.5	5.5	6.5
36	C-3,4	3.0	4.0	3.0
37	C-4,2	−0.5	4.5	1.0
38	C-4,3	−2.0	4.5	3.0
39	C-5,2	−1.0	7.0	>13.0
42	C-1,4	1.0	4.0	4.0
43	C-1,5	−2.5	3.5	2.0
44	C-1,6	−2.0	4.5	>13.0
45	C-1,0c	−2.5	3.5	>13.0

^a ΔΔT_1/2 values are the difference between ΔT_1/2 induced by the MTDL compounds and those induced by berberine.

Table 3. Compound-induced thermal stabilisation of the dual-labelled F-c-Kit1-T GQ measured by FRET melting competition experiments.

Compound		ΔT1/2 (°C)^a
		F-c-Kit1-T	F-c-Kit1-T + Hairpin (1:15)	F-c-Kit1-T + Hairpin (1:50)
26	S-2,0	12.4	10.2	12.4
29	S-3,2	13.2	12.8	13.2
30	S-4,0	13.6	13.8	12.6
32	S-5,0	14.4	15.0	14.6
34	C-3,2	10.6	11.4	11.2
37	C-4,2	12.0	12.8	12.6
39	C-5,2	14.6	13.2	14.0

^a ΔT_1/2 values are the difference between the T_1/2 of F-c-Kit1-T in the presence (2 molar equiv) and absence of ligands, without or with large excesses of unlabelled Hairpin (15 and 50 molar equiv with respect to GQ). The T_1/2 of F-c-Kit1-T is 57.5 (±0.2) °C. All experiments were performed at least in duplicate, and the reported values are the average of the measurements. Errors in the ΔT_1/2 values are within ± 0.5 °C.

Figure 5. (A) Monomolecular parallel GQs arranged in columns growing along the c axis. (B) OMIT electron density map contoured at 2σ level of S-3,2 (29) and skeleton of the ligand in the two disordered orientations—gray (Y orientation) and black (Z orientation); (C) and (D) interaction detail with GQ: (C) Y orientation; (D) Z orientation (T11 and T12 nucleic base atoms not shown). Final coordinates and structure factors (Table S3, Supporting Information) have been deposited with the Protein Data Bank (PDB accession number 7PNL).

Table 4. Concentrations of the investigated MTDLs required to give 50% of cell viability reduction (IC₅₀ values), by means of the MTT assay.

Compound		IC50 (µM)	95% confidence interval (µM)
26	S-2,0	>200	–
29	S-3,2	>200	–
30	S-4,0	35.0	27.3–45.5
32	S-5,0	>200	–
34	C-3,2	>50	–
37	C-4,2	40.0	38.0–46.0
39	C-5,2	37.5	28.6–53.0

Figure 6. (A) Representative fields showing G4 foci formation detected by immunofluorescence in HeLa cells treated for 24 h with 35.0 µM S-4,0, 40.0 µM C-4,2, and 37.5 µM C-5,2 or an equivalent amount of vehicle (0.1% DMSO). As a positive control, cells were treated with 2 µM RHPS4 (a well-established GQ ligand)⁶⁸ for 24 h. Scale bar: 10 µm. Upper panels: the merged channels of BG4-stained GQ structures (magenta) and Hoechst-counterstained nuclei (grey) are reported. Lower panels: enlargements from the pictures in the upper panels. (B) Quantitative analysis of the fluorescence intensity of the total number of GQ foci. An average of 50 cells were screened for each condition and the results are expressed as a fold change of fluorescence intensity (anti-GQ signal) over the negative control (DMSO). Histograms show the mean ± SD of three independent experiments. The statistical significance was calculated using a one-way ANOVA test on GraphPad Prism 8.0.2 (ns: not significant, **: p < 0.01).

Figure 7. (A) Representative fields showing G4 foci formation detected by immunofluorescence in HeLa cells pre-treated with 50 μM cobalt (II) chloride hexahydrate for 60 h to induce hypoxia and then exposed to 1 µM C-4,2 or vehicle (0.1% DMSO) for the following 12 h, still in the presence of CoCl₂. DMSO-treated normoxic HeLa cells are also shown. Scale bar: 10 µm. Upper panels: the merged channels of BG4-stained GQ structures (magenta) and Hoechst-counterstained nuclei (grey) are reported. Lower panels: enlargements from the pictures in the upper panels. (B) Quantification of the cellular BG4 foci in DMSO-treated normoxic HeLa cells, DMSO-treated hypoxic HeLa cells, and C-4,2-treated hypoxic HeLa cells. An average of 50 cells were screened for each condition. The number of GQ foci has been normalised to the mean of the corresponding control sample (DMSO). Histograms show the mean ± SD of three independent experiments. The statistical significance was calculated using a one-way ANOVA test on GraphPad Prism 8.0.2 (**: p < 0.01, ****: p < 0.0001).

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