Lead Optimization: Synthesis and Biological Evaluation of PBT-1 Derivatives as Novel Antitumor Agents

Abstract

Phenanthrene-based tylophorine-1 (PBT-1) was identified previously as a lead compound in an anticancer drug discovery effort based on natural Tylophora alkaloids. An expanded structural optimization using a new more efficient synthetic route provided 14 PBT-derivatives. Eleven compounds displayed obvious antiproliferative activities in cellular assays (GI₅₀ 0.55–9.32 μM). The most potent compounds 9c, 9g, and 9h (GI₅₀ < 1 μM) contained a 7-hydroxy group on the phenanthrene B-ring in addition to a pendant piperidine E-ring with different 4-substituents. Compound 9h with NH₂ as the piperidine substituent was at least 4-fold more potent against triple-negative breast cancer MDA-MB-231 than estrogen-responsible breast cancer MCF-7 cell growth. In further biological evaluations, the new active compounds induced cell cycle accumulation in the late S and G2/M phase without interfering with microtubule formation or cell morphology. These results on the optimization of the B- and E-rings of PBT-1 should benefit further development of novel antitumor agents.

With novel structural scaffolds and diverse biological activities, plant natural products are an important resource in the discovery of lead compounds for new drug development. Examples are the well-known anticancer drugs taxol and vinblastine as well as the clinical drug candidate combretastatin A-4 (CA-4). Tylophora alkaloids are a class of natural products isolated primarily from Cynanchum, Pergularia, and Tylophora species in the Asclepiadaceae family;¹ some representatives are shown in Figure 1. Tylophora alkaloids have a pentacyclic structural scaffold with a chiral center and exhibit diverse and potent biological activities, including antitumor,^2,3 anti-inflammatory,⁴ anti-arthritis,⁵ and anti-lupus effects in vivo.⁶ In a National Cancer Institute (NCI) screening program to discover potential novel antitumor agents from natural products, tylophorine and its analogues showed potent and uniform activity [mean GI₅₀ < 10^–8 M (low nM level)] against 54 human tumor cell lines, including resistant or cross-resistant sublines, and several refractory cell lines, such as melanoma and lung tumor cell lines, in a 60 human tumor cell line (HTCL) panel.⁷ On the other hand, while tylocrebrine, a positional isomer of tylophorine, advanced to clinical trials as a drug candidate, intolerable central nervous system (CNS) side effects were found.⁸ Furthermore, mechanistic studies on active Tylophora alkaloids revealed that their antitumor and anti-inflammatory activities involve several pharmacological actions, including inhibitory effects on protein synthesis, nucleic acid synthesis, RNA transcription, or nuclear factor κB (NF-κB) in the regulation of P-glycoprotein,^9,10 thus implying that Tylophora alkaloids and their analogues might be developed as novel anticancer drugs with a unique mechanism of action to overcome multidrug resistance to current anticancer chemotherapy.¹¹

Figure 1.

Structures of some Tylophora alkaloids, prior lead PBT-1, and modification strategies.

During our prior studies on the total synthesis of antofine and tylophorine analogues,^12,13 we also synthesized several series of simplified phenanthrene-based tylophorine (PBT) analogues¹⁴⁻¹⁶ related to antofine by simultaneously opening the D-ring to remove the chiral center and changing the E-ring. PBT-1 was discovered as a new promising lead compound (Figure 1) with potent antiproliferative activity (GI₅₀ values ∼ 80 nM) against a HTCL panel, including A549 (lung), DU-145 (androgen receptor-unresponsive prostate), ZR-75-1 (estrogen receptor and HER2-positive breast), KB (identical to AV-3 as a cervical carcinoma HeLa derivative), and multidrug resistant KB subline KB-VIN. Moreover, PBT-1 showed modest antitumor potency in vivo against human A549 xenograft in nude mice.¹⁵ As a potential antitumor agent for lung adenocarcinoma, mechanistic studies on PBT-1 indicated that it inhibits HSP90 and hnRNPA2/B1 to reduce the activation of AKT and Slug expression, thus suggesting that PBT-1 binds to HSP90 and/or hnRNP A2/B1 and initiates antitumor activities by affecting Slug- and AKT-mediated metastasis and tumorigenesis.¹⁷ Although PBT derivatives are structural analogues of Tylophora alkaloids, biological functional studies revealed that they have different mechanisms of action.¹⁸ Meanwhile, our prior structure–activity relationship (SAR) studies revealed the following antitumor pharmacophores of PBT derivatives. (1) A planar phenanthrene scaffold is required, but the D ring is removable; (2) a hydrophilic 6-membered N-heterocycle at the C-9 position is favorable for enhanced antiproliferative activity and fewer possible CNS side effects due to reduced penetration of the blood–brain barrier (BBB); (3) a methylene linker between the phenanthrene core and the N-hydrophilic ring is preferred over carbonyl or other groups; and (4) the 6-methoxy substituent on the phenanthrene skeleton is necessary for potency.

Because positional isomers of natural Tylophora alkaloids display different potencies and various mechanisms of action, we postulated that substituents at different positions on the phenanthrene core would affect the biological activity and targets. Thus, further PBT-1 optimization efforts should focus on the substituents on the phenanthrene core to reveal additional SAR correlations and possibly find new PBT compounds with higher antiproliferative activity. As a part of our systematic lead optimizations to complement prior structural optimizations of PBT derivatives, the current modification strategies focused successively on the introduction of additional substituents on the B-ring, alteration of the E-ring moiety, and modification of groups on the A-ring. All synthesized new PBT-1 derivatives were tested for antiproliferative activity in a HTCL panel, and new active compounds were selected for additional cell-based biological evaluations to evaluate their impact on cell cycle progression. Here, we report recent study results on 14 new PBT compounds, 9a–m and 10a–b, including chemical synthesis, evaluation of antiproliferative activity in vitro, SAR analysis, and target identification.

The current studies used a short, efficient, and slightly different synthetic route from that used previously^15,16 and led to the successful synthesis of new PBT-1 derivatives 9a–m and 10a–b as outlined in Schemes 1 and 2. To incorporate additional and different substituents on the B-ring, substituted methyl 2-bromophenylacetate (4a–c) building blocks were prepared by bromination of corresponding substituted methyl phenylacetates 3a–c, either easily synthesized (3a with 3,4,5-trimethoxy and 3c with 5-hydroxy-4-methoxy substitution) or commercially available (3b with 4,5-dimethoxy). Rather than the prior 4-step reaction sequence, a convenient one-pot synthesis using Suzuki–Miyaura coupling followed by an intramolecular aldol condensation cascade reaction¹⁹ was then applied to construct the phenanthrene scaffold. Commercially available 2-formyl-4,5-methylenedioxyphenylboronic acid (5a) was reacted with a substituted methyl 2-bromophenylacetate (4a, 4b, or 4c) in the presence of catalyst [Pd(PPh₃)₄] and K₃PO₄ in dimethoxyethane (DME) at 100 °C to afford corresponding methyl phenanthrene-9-carboxylates 6a–6c, respectively. The treatment of 6c with propargyl bromide or bromomethylcyclopropane in N,N-dimethylformamide (DMF) in the presence of anhydrous K₂CO₃ and KI (or NaI) at 70–85 °C converted its 7-hydroxy group to the corresponding 7-(prop-2-yn-1-yloxy) or 7-cyclopropylmethoxy side chain of 6d and 6e, respectively. To change the substituents on the A-ring, intermediates 6g and 6h were prepared from 4c and reacted with 4-methoxy- (5b) or 5-methoxy-2-formyl-phenylboronic (5c) acid, respectively, via the above one-pot reaction (Scheme 2). Because a free hydroxyl group might interfere in the subsequent ester reduction, compounds 6c, 6g, and 6h were first treated with acetic anhydride in CH₂Cl₂ in the presence of dimethyl aminopyridine (DMAP) and triethylamine to afford corresponding acetoxy-protected compounds 6f, 6i, and 6j, respectively, in quantitative yield. Subsequently, the 9-ester group in the 6-series compounds was reduced with LiBH₄ solution in THF at 0 °C to give a hydroxymethyl moiety, affording the corresponding 7-series compounds generally with good yields (77–99%). During this process, the protected 7-acetoxy group in 6f, 6i, and 6j was also removed simultaneously to yield the corresponding 7-hydroxy-compounds 7c, 7g, and 7h, respectively. However, reduction of 6d under the same condition generated two products in a 2:1 ratio; the predominant compound was the expected 7d (only ester reduced) and the other compound was 7c (ester reduced and unstable propargyl group in 6d lost), which was also the reduced product produced from 6f. Next, the 7-series compounds were treated individually with cyanuric chloride (TCT)/DMF^20,21 in CH₂Cl₂ at room temperature to produce corresponding chloromethyl 8-series compounds. This simple procedure allows a rapid and quantitative conversion of alcohols into the corresponding chlorides under very mild conditions; TCT is a convenient and efficient chlorinating agent in comparison with other chlorinating reagents, such as thionyl chloride, phosphorus chloride, oxalyl chloride, etc. After a simple aqueous workup, the chloromethyl intermediates were used directly in the next reaction without further purification. In the final step, the chloro group was displaced by various amine reagents in THF/isopropanol (v/v 1:3) in the presence of K₂CO₃ at 80 °C to afford corresponding target compounds 9a–g, i–m, and 10a,b respectively. The Boc group in 9g was removed by treatment with trifluoroacetic acid in CH₂Cl₂ to afford 9h. The structures of all new synthetic compounds were identified from an analysis of NMR and MS spectroscopic data.

Scheme 1.

Reaction conditions: (i) SO₂(OCH₃)₂, K₂CO₃/acetone, rt for 3a; (ii) MeOH/H₂SO₄, reflux for 3c; (iii) Br₂/CH₂Cl₂, <5 °C to rt; (iv) Pd(PPh₃)₄, K₃PO₄, CH₃OCH₂CH₂OCH₃ (DME), 100 °C, 18–24 h or 80 °C microwave 1 h; (v) RBr, K₂CO₃/KI, DMF, 70 °C; (vi) Ac₂O/CH₂Cl₂, DMAP, Et₃N, (vii) LiBH₄ THF/MeOH, 0 °C to rt; (viii) (a) cyanuric chloride (TCT)/DMF, rt, argon protection; (b) added 7 in CH₂Cl₂, rt; (ix) amine, K₂CO_3, THF/iso-PrOH (v/v 1:3), 80 °C; (x) TFA/CH₂Cl₂, rt.

Scheme 2.

Reaction conditions: (i) Pd(PPh₃)₄, K₃PO₄, DME, 100 °C; (ii) LiBH₄ in THF, MeOH, <0 °C to rt; (iii) Ac₂O, DMAP/Et₃N, CH₂Cl₂; (iv) (a) cyanuric chloride (TCT)/DMF, rt under argon protection, (b) added 7 in CH₂Cl₂, rt; (v) amine, K₂CO_3, THF/iso-PrOH (v/v 1:3), 80 °C.

The synthesized new PBT-1 derivatives 9a–m and 10a–b as well as prior lead PBT-1 were evaluated for antiproliferative activity in cellular assays against a HTCL panel, including A549, MDA-MB-231 (triple-negative breast cancer), KB, multidrug-resistant (MDR) KB subline KB-VIN, and MCF-7 (estrogen receptor-positive breast cancer), in parallel with paclitaxel and CA-4 as experimental controls. Their activity in the cellular assay was determined as GI₅₀ values by using the established sulforhodamine B (SRB) method, and related data are summarized in Table 1.

Table 1. Antiproliferative Activities of New Compounds 9 and 10 against Human Tumor Cell Lines.

		GI50 (μM)a
		A549	MDA-MB-231	KB	KB-VIN	MCF-7
R1/R2 on B-ring
9a	OCH3/OCH3	>10	>10	>10	>10	>10
9b	H/OCH3	5.74	5.76	5.90	5.78	5.97
9c	H/OH	0.56	0.58	0.55	0.80	0.66
9d	H/OCH2C≡CH	4.59	4.66	4.33	2.27	6.05
9e	H/OCH2CH(CH2)2	>10	>10	>10	>10	>10
PBT-1	H/H	0.48	0.51	0.48	0.65	0.49
X–R on E-ring
9g	CH–NHBoc	0.62	0.83	0.74	0.91	3.50
9h	CH–NH2	0.66	0.85	0.76	4.12	3.67
9i	N–SO2CH3	>10	>10	>10	>10	>10
9j	N–COCH3	2.55	3.10	2.44	4.15	4.78
9k	N–CH3	5.67	6.32	5.84	6.18	7.33
9l	N–CH2CH3	5.54	6.80	6.15	6.71	9.32
9m	N–CH2CH2OH	4.97	6.10	5.29	6.02	6.31
R′/R″ on A-ring
10a	OCH3/H	5.09	5.51	5.80	6.06	6.81
10b	H/OCH3	4.52	2.93	2.17	4.49	4.21
CA-4 (nM)b		5.5	8.2	3.6	3.8	84
paclitaxel (nM)b		0.6	7.4	4.6	1565	7.1

^aThe GI₅₀ values are the concentrations corresponding to 50% cell growth inhibition with at least three independent experiments.

^bCombretastatin A4 (CA-4) and paclitaxel served as positive controls in the same assays.

In our study of how substituents on the phenanthrene core affected the antiproliferative activity, we first introduced additional substituents (R¹ and/or R²) on the B-ring, while retaining the other structural moieties of PBT-1, to give new compounds 9a–e. Among them, the most active compound 9c (R² = OH) exhibited high antiproliferative effects in the tested HTCL panel with sub-micromolar GI₅₀ values (0.55–0.80 μM), comparable to those of prior lead PBT-1 (GI₅₀ 0.48–0.65 μM) in the same cellular assays. Compounds 9b (R² = OMe) and 9d (R² = propargyloxy) with an added 7-substituent exhibited similar low micromolar antiproliferative potency (GI₅₀ 5.74–5.97 and 2.27–6.05 μM, respectively), making them 8–10 fold less potent than 9c. However, derivatives 9a with a 5,6,7-trimethoxy B-ring and 9e (R² = cyclopropylmethoxy) with a longer side chain on the 7-position of the B-ring were inactive (GI₅₀ > 10 μM). These results indicated that a 7-OH is favorable to the antiproliferative activity and better than other tested groups (9c > 9b ≅ 9d > 9a, 9e). Subsequently, we kept the B-ring from 9c but modified the identity and N-substituent of the E-ring. With the exception of the less active 9i, new compounds 9g–m showed broad antiproliferative activity in the HTCL panel with GI₅₀ values ranging from sub-micromolar to single digit micromolar (0.62–9.32 μM). Like 9c and prior lead PBT-1, compounds 9g and 9h with a piperidine E-ring showed similar potency (GI₅₀ < 1 μM) and were more active than 9j–m with a piperazine ring against the tested cell lines, especially A549, MDA-MB-231, and KB cell lines (see Table 1). Thus, for antiproliferative activity, piperidine is preferable to piperazine as the E-ring. Although both E-rings are six-menbered heterocycles, a piperidine ring has a sp³ tetrahedral 4-C atom and likely a more stable chair conformation then a piperazine ring with a sp² trigonal planar 4-N atom. Interestingly, while 9c had similar potency against two breast cancer subtypes (GI₅₀ 0.58 μM MDA-MB-231, 0.66 μM MCF-7), 9g and 9h were 4-fold more potent against MDA-MB-231 (GI₅₀ 0.83–0.85 μM) than MCF-7 (GI₅₀ 3.50–3.67 μM) cells. We postulated that the selectivity might be related to the orientation and nature of the 4-substituent on the piperidine (E-ring) −CH₂OH in 9c, NHBoc and NH₂ in 9g and 9h, respectively. Finally, compounds 10a and 10b with a single methoxy substituent rather than a methylenedioxy group on the A-ring of the phenanthrene core were evaluated in the above HTCL panel. The GI₅₀ values of 10a (2-methoxy) were 5.09–6.81 μM, and those of 10b (3-methoxy) were 2.17–4.52 μM; thus, these two compounds were less potent than 9c (2,3-methylenedioxy). However, based on all results from the new compounds (9a–d, g,h, j–m, 10a–b), the most significant effects on the molecular antiproliferative activity resulted from changes in the 5,6,7-substituents (5-H, 6-OCH₃, 7-OH optimal) on the B-ring of the phenanthrene and in the identity (piperidine optimal) and substitution (CH₂OH, NHBoc, NH₂ optimal) of the heterocyclic E-ring.

Subsequently, we also assessed whether the new active compounds and prior lead PBT-1 share the same mechanism of action. The additional cell-based biological evaluations included impacts on cell cycle progression and cell morphology, such as microtubule formation. Since previous studies reported that prior lead PBT-1 and the natural Tylophora alkaloids have different mechanisms of action,¹⁸ selected new active compounds 9c, 9h, 9j, and 9b were further investigated for effects on cell cycle progression in MDA-MB-231 cells²² in parallel with PBT-1 and CA-4, a known tubulin polymerization inhibitor that arrests cells in the G2/M phase. Basically, all tested compounds including PBT-1 caused cell accumulation at the late S and G2/M, and the cell cycle distribution pattern was clearly distinguishable from that with CA-4 (Figure 2). These observations suggested that PBT-1 and its derivatives impact the cell cycle progression in the S and G2/M phases and likely have the same mechanism of action. Next, the effects of compounds on microtubules and cell morphology were analyzed by immunostaining (Figure 3). Like the normal cell morphology found in control cells, the microtubules were observed clearly in cells treated with PBT-1 or 9c, while the microtubules were totally depolymerized in CA-4 treated cells, suggesting that PBT-1 and 9c do not affect tubulin polymerization or cell morphology. Further investigation on the mechanism of action is required to fully explain the cell cycle accumulation between the late S to G2/M phases.

Figure 2.

Effects of compounds on cell cycle progression. MDA-MB-231 (triple-negative breast cancer) cells were treated with compounds for 24 h at equal (1× GI₅₀) or 3-fold (3× GI₅₀) concentration of their GI₅₀ as indicated. DMSO or 0.2 μM CA-4 (3× GI₅₀) was used as a vehicle control (CTRL) or a tubulin polymerization inhibitor arresting cells at G2/M, respectively. Cell cycle distributions at each phase of cell cycle were analyzed by flow cytometry (LSRII) after staining with propidium iodide (PI) in the presence of RNase.

Figure 3.

Effects of compounds on cell morphology. MDA-MB-231 cells were treated with compounds for 24 h at 3-fold (3× GI₅₀) concentration of their GI₅₀. DMSO or CA-4 was used as a vehicle CTRL or a tubulin polymerization inhibitor, respectively. Paraformaldehyde-fixed cells were stained with antibody to α-tubulin (green) and DAPI for DNA (blue) and then by observation under a confocal fluorescence microscope.

In summary, our expanded structural optimization of prior lead PBT-1 led to two series of new compounds, 9a–m and 10a–b. A new more efficient synthetic route and convenient methods were applied successfully in the current studies for all designed new target compounds. Fourteen PBT derivatives were synthesized and evaluated for antiproliferative activity in cellular assays. Eleven compounds displayed obvious antiproliferative activities with sub-micromolar to low single digit micromolar GI₅₀ values (0.55–9.32 μM), but were less potent than paclitaxel in the same cellular assays. Among them, 7-hydroxy-PBT compounds 9c, 9g, and 9h containing different 4-substituted piperidine (E-ring) moieties exhibited the highest antiproliferative activity (GI₅₀ < 1 μM), comparable to that of prior lead PBT-1. Compounds 9g and 9h were selectively effective against triple-negative breast cancer MDA-MB-231 cell growth with at least 4-fold higher potency than against estrogen-responsible breast cancer MCF-7 cell growth. Based on further biological evaluations, such as effects on cell cycle progression, microtubule formation, and cell morphology, we concluded that our new active compounds induced cell cycle accumulation in the late S and G2/M phase without interfering with microtubule formation or cell morphology. Current SAR studies revealed the following observations. (1) The B-ring of the phenanthrene scaffold is modifiable via the introduction of additional substituents, and a 7-hydroxy is a favorable group to maintain or increase antiproliferative activity, potentially improve druglike properties, and serve as a locus to make various prodrugs for further studies. (2) A six-membered piperidine ring (E-ring) is better than a piperazine moiety, perhaps due to the molecular nature and conformation. (3) The 4-substituent on the piperidine ring is likely related to the antitumor potency and selectivity against triple-negative breast cancer MDA-MB-231 cell growth. (4) The changes in groups on the A-ring affected antiproliferative potency less. The new derivatives likely have the same or similar biological mechanism of action to that of prior lead PBT-1. Therefore, these recent results will be helpful for further development of novel antitumor agents.

Glossary

Abbreviations

PBT

phenanthrene-based tylophorine

HTCL

human tumor cell line

SAR

structure–activity relationship

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsmedchemlett.1c00505.

• Synthetic experimental details, related spectroscopic data of intermediate and target compounds, and biological assay protocols (PDF)

Author Contributions

The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Conceived the project: L.X., M.G., and K.-H.L. Performed experiments: L.X., M.G., and X.C. Analyzed data: L.X. and M.G. Prepared the manuscript: L.X., M.G., S.L.M.-N., and K.-H.L. All authors reviewed the manuscript.

The authors declare no competing financial interest.

Author Status

^§ K.-H.L.: Passed away on October 24, 2021.