Mitochondria-Targeting 1,5-Diazacyclooctane-Spacered Triterpene Rhodamine Conjugates Exhibit Cytotoxicity at Sub-Nanomolar Concentration against Breast Cancer Cells

1,5-Diazacyclooctane was prepared by a simple synthetic sequence and coupled to pentacyclic triterpenoic acids oleanolic acid, ursolic acid, betulinic acid, platanic acid, and asiatic acid; these amides were activated with oxalyl chloride and reacted with rhodamine B or rhodamine 101 to yield conjugates. The conjugates were screened in SRB assays with various human breast cancer cell lines (MDA-MB-231, HS578T, MCF-7, and T47D) and found to exert cytotoxic activity even at a low concentration. Therefore, for an asiatic acid rhodamine 101 conjugate (28), an IC50 = 0.60 nM was determined and found to induce apoptosis in MDA-MB-231 and HS578T cells. Extra experiments showed the compound to act as a mitocan and to induce inhibition of proliferation or growth arrest in MDA-MB-231 cells at lower doses followed by an induction of apoptosis at higher doses. Furthermore, differential responses to proliferation inhibition and apoptosis induction may explain differential sensitivity of mammary cell lines to compound 28.


Introduction
Breast cancer is the most common type of tumor disease and, despite recent advances in cancer therapy, it remains the leading cause of tumor-related death in women [1][2][3][4][5][6][7][8]. While traditional treatments like surgery, chemotherapy, radiation, and hormone therapy are effective [9], they often cause severe side effects and may not be suitable for all patients. Therefore, there is a need to develop new and effective treatment options. One highly promising approach is the use of natural products derived compounds as anticancer agents, especially pentacyclic triterpenoids, which have emerged as a class of phytochemicals with potential anticancer activity. Several studies have demonstrated their ability to cause apoptosis, reduce clonogenic survival and migration, and enhance the radiosensitivity of human breast cancer cells [10][11][12][13]. These effects have been attributed to their ability to modulate various signaling pathways involved in cancer progression.
Pentacyclic triterpenoic acids linked with lipophilic cations, such as rhodamines [13][14][15][16][17][18][19][20][21][22][23][24][25][26][27], are known to act as mitocans even at low nanomolar concentrations by inhibiting their synthesis of ATP [21]. In this context, the mitochondrial targeting function of rhodamine seems particularly worth mentioning [28][29][30]. Therefore, the use of an amine spacer is crucial for enhancing their cytotoxicity, whereby secondary amines are favored over primary amines to prevent lactamization and maintain their cationic structures. Furthermore, incorporating a homopiperazinyl spacer leads to more cytotoxic compounds than those analogs with a piperazinyl spacer. Therefore, we have been interested in the use of a starting from propane-1,3-diamine and propane-1,3-diol, two starting materials that are available in larger quantities and commercially cheap. In this process, both starting materials are first tosylated and then condensed by a double nucleophilic substitution. An alternative is the reaction of 1,3-dibromopropane (2) with hydrazine. This route would have the advantage of yielding the desired product in a one-pot procedure. However, it very quickly became apparent that many byproducts were formed in this reaction so that the maximum yield of pure 1 was 7.5% only. Working with larger quantities of hydrazine poses an additional risk.
As an alternative, one could also imagine the reduction of the bis-lactam 1,5-diazocane-2,6-dione; the latter compound is accessible either via Staudinger ring closure reactions and Beckmann and Schmidt rearrangements, however, usually under very drastic conditions (e.g., fuming sulfuric acid) [44][45][46][47][48]. All these routes are not very suitable, since their mostly drastic conditions make the preparation of larger amounts on a laboratory scale quite difficult.
Special attention, therefore, is deserved for the only recently proposed [49] route starting from propane-1,3-diamine and propane-1,3-diol, two starting materials that are available in larger quantities and commercially cheap. In this process, both starting materials are first tosylated and then condensed by a double nucleophilic substitution. An alternative is the reaction of 1,3-dibromopropane (2) with hydrazine. This route would have the advantage of yielding the desired product in a one-pot procedure. However, it very quickly became apparent that many byproducts were formed in this reaction so that the maximum yield of pure 1 was 7.5% only. Working with larger quantities of hydrazine poses an additional risk.
However, the published synthesis using propane-1,3-diamine (3) and propane-1,3-diol (4) could not be reproduced in terms of the yields obtained either, so we decided to optimize this synthetic route on our own.
The starting materials for the preparation of the spacered rhodamine conjugates were the triterpene carboxylic acids oleanolic acid (OA, Figure 1), ursolic acid (UA), and the lupanes betulinic acid (BA) and platanic acid (PA); in previous works, asiatic acid The triterpenoic acids were acetylated to yield the acetates 9-13 (Scheme 2). Rhodamine B and rhodamine 101 were chosen as representative examples of rhodamines. The former compound has been shown in previous studies to be an essential component of mitocan-acting triterpene carboxylic acid amide conjugates; the latter differs from the former in having a somewhat higher lipophilicity (consensus log Po/w 2.21 and 3.96, respectively; from www.swiss.adme.ch, accessed on 2 May 2023), which we consider advantageous for possible interactions with biological membranes. Thus, the reaction of acetates 9-13 with oxalyl chloride followed by the addition of 8 furnished amides 14-18. Rhodamine B and rhodamine 101 were transformed with oxalyl chloride in situ into their corresponding acid chlorides that were reacted with amides 14-18 to yield rhodamine Bderived conjugates 19-23 and rhodamine 101-derived hybrids 24-28.
Compounds 14-28 were screened in sulforhodamine B assays employing the breast cancer cell lines MDA-MB-231, HS578T, MCF-7, and T47D (Table 1). Breast cancer could be distinguished into different molecular subtypes: luminal-like (luminal A or B), HER2enriched, and basal-like, which differ in biology, treatment response, patients' survival, and clinical outcome. These subtypes are also found in cell lines and our investigated breast cancer cell lines have been characterized before. Breast cancer cell lines MDA-MB-231 and HS578T are basal and so-called triple negative, which means neither estrogen receptor (ER) and progesterone receptor (PR) nor human epidermal growth factor receptor 2 (HER2) are expressed. Basal breast cancers are mostly high-grade tumors and no therapeutic targeted therapy can be applied, thus resulting in a poor prognosis for patients although they are relatively sensitive for chemotherapy. MCF-7 and T47D breast cancer cells are luminal A and positive for ER and PR. Breast cancers of this type are often low-grade tumors, which are characterized by chemotherapy resistance, but hold good responses to hormone therapy, resulting in better clinical outcomes compared to basal breast cancers. The triterpenoic acids were acetylated to yield the acetates 9-13 (Scheme 2). Rhodamine B and rhodamine 101 were chosen as representative examples of rhodamines. The former compound has been shown in previous studies to be an essential component of mitocan-acting triterpene carboxylic acid amide conjugates; the latter differs from the former in having a somewhat higher lipophilicity (consensus log P o/w 2.21 and 3.96, respectively; from www.swiss.adme.ch, accessed on 2 May 2023), which we consider advantageous for possible interactions with biological membranes. Thus, the reaction of acetates 9-13 with oxalyl chloride followed by the addition of 8 furnished amides 14-18. Rhodamine B and rhodamine 101 were transformed with oxalyl chloride in situ into their corresponding acid chlorides that were reacted with amides 14-18 to yield rhodamine B-derived conjugates 19-23 and rhodamine 101-derived hybrids 24-28.
Compounds 14-28 were screened in sulforhodamine B assays employing the breast cancer cell lines MDA-MB-231, HS578T, MCF-7, and T47D (Table 1). Breast cancer could be distinguished into different molecular subtypes: luminal-like (luminal A or B), HER2enriched, and basal-like, which differ in biology, treatment response, patients' survival, and clinical outcome. These subtypes are also found in cell lines and our investigated breast cancer cell lines have been characterized before. Breast cancer cell lines MDA-MB-231 and HS578T are basal and so-called triple negative, which means neither estrogen receptor (ER) and progesterone receptor (PR) nor human epidermal growth factor receptor 2 (HER2) are expressed. Basal breast cancers are mostly high-grade tumors and no therapeutic targeted therapy can be applied, thus resulting in a poor prognosis for patients although they are relatively sensitive for chemotherapy. MCF-7 and T47D breast cancer cells are luminal A and positive for ER and PR. Breast cancers of this type are often low-grade tumors, which are characterized by chemotherapy resistance, but hold good responses to hormone therapy, resulting in better clinical outcomes compared to basal breast cancers.
As a result, amides of triterpenoic acids 14-18 (Table 1) show cytotoxicity at a low micromolar range for all investigated breast cancer cell lines. IC 50 values of about 0.5-50 µM were determined. As expected, conjugation of rhodamine B (compounds 19-23) or rhodamine 101 (compounds 24-28) led to increased cytotoxicity (in the nanomolar range) in all breast cancer cell lines (Table 1). In the investigated breast cancer cell lines, the IC 50 values of all homopiperazinyl-spacered rhodamine B derivatives are in a low nano-molar range with rhodamine 101 conjugates being even more cytotoxic. An asiatic acid derivatized rhodamine 101 amide (compound 28) is the most cytotoxic conjugate in all screened breast cancer cells. The IC 50 values are in a low nanomolar range (0.6-126 nM). Comparing breast cancer cell lines, the HS578T cell line is the most resistant cell line for rhodamine B or rhodamine 101 conjugates (IC 50 between 216 nM and 356 nM and between 126 nM and 1.3 µM). Our previous work showed that compounds of this class are also highly able to discriminate between malignant and nonmalignant cells [13,23] and affect mitochondrial ATP synthesis [23]. Future studies will also investigate whether changes in the expression of programmed death ligand-1 (PD-L1) can be observed [50]. As a result, amides of triterpenoic acids 14-18 (Table 1) show cytotoxicity at a low micromolar range for all investigated breast cancer cell lines. IC50 values of about 0.5-50 µM were determined. As expected, conjugation of rhodamine B (compounds 19-23) or rhodamine 101 (compounds 24-28) led to increased cytotoxicity (in the nanomolar range) in all breast cancer cell lines (Table 1). In the investigated breast cancer cell lines, the IC50 values of all homopiperazinyl-spacered rhodamine B derivatives are in a low nano-molar range with rhodamine 101 conjugates being even more cytotoxic. An asiatic acid derivatized rhodamine 101 amide (compound 28) is the most cytotoxic conjugate in all screened breast cancer cells. The IC50 values are in a low nanomolar range (0.6-126 nM). Comparing breast cancer cell lines, the HS578T cell line is the most resistant cell line for rhodamine B or rhodamine 101 conjugates (IC50 between 216 nM and 356 nM and between 126 nM and 1.3 µM). Our previous work showed that compounds of this class are also highly able to discriminate between malignant and nonmalignant cells [13,23] and affect In addition to studying the cytotoxicity of 28 in the above-mentioned cell lines, we investigated its ability to overcome resistance. While the IC 50 of 28 in A2780 cells was 0.72 nM, the resistant A2780cis cells exhibited an IC 50 of 1.82 nM. Although complete resistance reversal was not achieved, the results highlight the promising potential to partially overcome resistance. We also assessed its selectivity by comparing the cytotoxicity in nonmalignant fibroblasts CCD18Co. The IC 50 value of 28 in CCD18Co cells was 503.2 nM, which was approximately 800-fold higher than the IC 50 value observed in the MDA-MB-231 cells. The most cytotoxic compound, 28, was used for further investigations of proliferation and cell death in sensitive MDA-MB-231 and resistant HS578T breast cancer cells. In MDA-MB-231 cells, compound 28 caused a strong inhibition of proliferation (under 20% compared to the control cells) after treatment with at least 250 nM ( Figure 2). However, in HS578T cells, treatment with 250 nM of compound 28 resulted in a less decrease of proliferation by about 50%, but with 500 nM, compound 28 cell number was reduced by up to 20% compared to control cells ( Figure 2). In addition to studying the cytotoxicity of 28 in the above-mentioned cell lines, we investigated its ability to overcome resistance. While the IC50 of 28 in A2780 cells was 0.72 nM, the resistant A2780cis cells exhibited an IC50 of 1.82 nM. Although complete resistance reversal was not achieved, the results highlight the promising potential to partially overcome resistance. We also assessed its selectivity by comparing the cytotoxicity in nonmalignant fibroblasts CCD18Co. The IC50 value of 28 in CCD18Co cells was 503.2 nM, which was approximately 800-fold higher than the IC50 value observed in the MDA-MB-231 cells.
The most cytotoxic compound, 28, was used for further investigations of proliferation and cell death in sensitive MDA-MB-231 and resistant HS578T breast cancer cells. In MDA-MB-231 cells, compound 28 caused a strong inhibition of proliferation (under 20% compared to the control cells) after treatment with at least 250 nM ( Figure 2). However, in HS578T cells, treatment with 250 nM of compound 28 resulted in a less decrease of proliferation by about 50%, but with 500 nM, compound 28 cell number was reduced by up to 20% compared to control cells ( Figure 2).  Figure 3A. Cells stained negative for both annexin V and Sytox Deep Red were viable (Q3). Early apoptotic cells stained positive for annexin V but negative for Sytox Deep Red (Q4), whereas late apoptotic or dead cells stained positive for both annexin V and Sytox Deep Red (Q2). Necrotic cells are indicated as negative for annexin V but positive for Sytox Deep Red (Q1).
viable cells was counted. Data represent mean values (±SD) of at least three independent experiments. All data were referred to DMSO-treated cells (=100%). Significant p values are highlighted with asterisks (** p ≤ 0.01).
Cell death analyses were done by use of FITC annexin V-Sytox Deep Red staining in MDA-MB-231 (IC50 = 0.6 nM) and HS578T (IC50 = 126 nM) breast cancer cell lines to discriminate apoptotic and necrotic cells. An example of the evaluation of cell death via annexin V-Sytox Deep Red staining in the sensitive breast cancer cell line MDA-MB-231 and the resistant breast cancer cell line HS578T is shown in Figure 3A. Cells stained negative for both annexin V and Sytox Deep Red were viable (Q3). Early apoptotic cells stained positive for annexin V but negative for Sytox Deep Red (Q4), whereas late apoptotic or dead cells stained positive for both annexin V and Sytox Deep Red (Q2). Necrotic cells are indicated as negative for annexin V but positive for Sytox Deep Red (Q1).   Figure  4B) in MDA-MB-231 cells shows an identical pattern of accumulation, indicating the mitochondrial targeting of 28. Using a quantitative analysis of the respective integrated fluorescence intensity, a mitochondrial uptake of about 56% could be determined.
In summary, the determination of proliferation and cell death indicates that compound 28 induces inhibition of proliferation or growth arrest at a lower dose, and with increasing dose treatment with compound 28 causes an induction of apoptosis. Furthermore, differential responses to proliferation inhibition and apoptosis induction may explain the differential sensitivity of mammary cell lines to compound 28.

Discussion
1,5-Diazacyclooctane was synthesized through a straightforward synthetic pathway and subsequently linked with pentacyclic triterpenoic acids, namely oleanolic acid, ursolic acid, betulinic acid, platanic acid, and asiatic acid. These resulting amides were activated with oxalyl chloride and reacted with either rhodamine B or rhodamine 101 to form conjugates. These conjugates were then subjected to screening using SRB assays on various breast cancer cell lines, namely MDA-MB-231, HS578T, MCF-7, and T47D. The findings revealed that the conjugates exhibited cytotoxic activity even at low concentrations. Notably, the asiatic acid rhodamine 101 conjugate 28 displayed an IC50 = 0.60 nM and demonstrated the ability to induce apoptosis in MDA-MB-231 and HS578T cells. Further investigations demonstrated that the compound acted as a mitocan, resulting in the inhibition of proliferation or growth arrest in MDA-MB-231 cells at lower doses, followed by the induction of apoptosis at higher doses. Moreover, the differential responses observed in terms of proliferation inhibition and apoptosis induction could potentially explain the varying sensitivity of mammary cell lines to compound 28.

General
NMR spectra were recorded using the Varian spectrometers (Darmstadt, Germany) DD2 and VNMRS (400 and 500 MHz, respectively). MS spectra were taken on an Advion expression L CMS mass spectrometer (Ithaca, NY, USA; positive ion polarity mode, solvent: methanol, solvent flow: 0.2 mL/min, spray voltage: 5.17 kV, source voltage: 77 V, In summary, the determination of proliferation and cell death indicates that compound 28 induces inhibition of proliferation or growth arrest at a lower dose, and with increasing dose treatment with compound 28 causes an induction of apoptosis. Furthermore, differential responses to proliferation inhibition and apoptosis induction may explain the differential sensitivity of mammary cell lines to compound 28.

Discussion
1,5-Diazacyclooctane was synthesized through a straightforward synthetic pathway and subsequently linked with pentacyclic triterpenoic acids, namely oleanolic acid, ursolic acid, betulinic acid, platanic acid, and asiatic acid. These resulting amides were activated with oxalyl chloride and reacted with either rhodamine B or rhodamine 101 to form conjugates. These conjugates were then subjected to screening using SRB assays on various breast cancer cell lines, namely MDA-MB-231, HS578T, MCF-7, and T47D. The findings revealed that the conjugates exhibited cytotoxic activity even at low concentrations. Notably, the asiatic acid rhodamine 101 conjugate 28 displayed an IC 50 = 0.60 nM and demonstrated the ability to induce apoptosis in MDA-MB-231 and HS578T cells. Further investigations demonstrated that the compound acted as a mitocan, resulting in the inhibition of proliferation or growth arrest in MDA-MB-231 cells at lower doses, followed by the induction of apoptosis at higher doses. Moreover, the differential responses observed in terms of proliferation inhibition and apoptosis induction could potentially explain the varying sensitivity of mammary cell lines to compound 28.
All dry solvents were distilled over respective drying agents except for DMF which was distilled and stored under argon and a molecular sieve. Reactions using air-or moisture-sensitive reagents were carried out under an argon atmosphere in dried glassware. Triethylamine was stored over potassium hydroxide. Biological assays were performed as previously reported. The parent triterpenoic acids were obtained from local vendors.

General Procedure for Acetylation (GP 1)
To a solution of the parent triterpenoic acid (1 equiv.) in dry DCM, acetic anhydride (3 equiv.), dry triethylamine (3 equiv.), and DMAP (catal. amounts) were added, and the mixture was stirred at 20 • C for one day. The usual aqueous work-up followed by re-crystallization from ethanol furnished the corresponding acetates 9-13. Their respective m.p., [α] 20 D values, 1 H, and 13 C NMR spectra, as well as ESI MS data, correspond to the literature values.

General Procedure for the Synthesis of Amides 14-18 (GP 2)
To a solution of acetates 9-13 (1 equiv.) in dry DCM (100 mL), oxalyl chloride (5 equiv.) and DMF (2 drops) were added and the mixture was stirred at 20 • C for 2 h. The volatiles were removed under diminished pressure and the residue was dissolved in dry DCM (100 mL). This solution was slowly added to a solution of the corresponding amine (3 equiv.) in dry acetonitrile (100 mL) in the presence of DMAP (catal. amounts). The mixture was stirred at 20 • C for 1 day, the volatiles were removed under diminished pressure, and the residue was subjected to column chromatography (silica gel) to afford products 14-18.

General Procedure for the Synthesis of the Rhodamine Conjugates 19-28 (GP 3)
To a solution of the rhodamine (rhodamine B or rhodamine 101, 1 equiv.) in dry DCM (100 mL), oxalyl chloride (7 equiv.) and dry DMF (2 drops) were added, and the mixture was stirred at 20 • C for 1 h. The volatiles were removed under diminished pressure and the residue was dissolved in dry DCM (100 mL). A solution of the corresponding amine (1 equiv.) in dry DCM (100 mL) was added, followed by the addition of catal. amounts of triethylamine and DMAP. The mixture was stirred at 20 • C for 1 h (TLC showed completion of the reaction), the solvents were removed in vacuo, and the residue was subjected to column chromatography (silica gel, CHCl 3 /MeOH) to afford products 19-28.