**2. Results**

Since the first preparation of octahydro-1,5-diazocine (**1,** 1,5-diazacyclooctane, a "bishomo-piperazine", Scheme 1) in 1939 by W. L. C. Veer [31] several routes have been suggested to this compound, among them the ring cleavage reaction of 1,5-diaminobicyclo [3.3.0]octane, the condensation of propane-1,3-diamine with 1,3-dibromopropane, and the silica-supported intramolecular cyclization of propane-1,3-diamine at 350 ◦C [32–43].

**Scheme 1.** Synthesis of octahydro-1,5-diazocine (**1**) and its dihydrobromide (**8**): Reactions and conditions: a: NH2-NH2, EtOH, reflux, 4 h; then HBr, benzaldehyde, 7.5%; b: TosCl, no solvent, 80 ◦C, 30 min, 83%; c: TosCl, pyridine, 0 ◦C, 30 min, 87%; d: NaOMe, MeOH, DMF, 80 ◦C, 12 h, 84%; then HBr (33% glacial AcOH), 80 ◦C, 3 h, 92%.

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) [44–48]. 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.

Thus, propane-1,3-diamine (**3**) was tosylated (Scheme 1) to yield **5** in an 83% yield, while the tosylation of propane-1,3-diol (**3**) gave 87% of the di-tosylate **6**. These two compounds were condensed in the presence of sodium methoxide (which proved to result in higher yields than using sodium ethoxide) to afford 84% of **7**. De-tosylation was performed with hydrobromic acid in the presence of thioanisole and the desired octahydro-1,5-diazocine was obtained as di-hydrobromide (**8**) in a 92% isolated yield.

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 (AA) had been shown to be particularly suitable with respect to cytotoxic activity and was, therefore, included in this study as a model featuring a tri-hydroxylated triterpene carboxylic acid [21].

**Figure 1.** Structure of triterpenoic acid oleanolic acid (OA), ursolic acid (UA), betulinic acid (BA), platanic acid (PA), and asiatic acid (AA); for the latter, a numbering scheme is depicted.

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 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), HER2 enriched, 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. 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 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].

**Scheme 2.** Synthesis of the rhodamine B and rhodamine 101 conjugates; reactions and conditions: a: Ac2O, DCM, NEt3, DMAP (cat.), 21 ◦C, 24 h; b: (COCl)2, DCM, DMF (cat.), in situ; c: DCM, **8**, NEt3, DMAP (cat.), 20 ◦C, 1 h; d: (COCl)2, DCM, DMF (cat.), then rhodamine B or rhodamine 101, 20 ◦C, 1 h.

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.


**Table 1.** Cytotoxicity of compounds **14**–**28** determined by SRB assay in four different breast cancer cell lines (MDA-MB-231, HS578T, MCF-7, and T47D). IC50 values were calculated after 96 h treatment. The data represent values of at least three independent experiments, which were done each in triplicate.

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 2.** Relative cell number of MDA-MB-231 and HS578T breast cancer cells. Cells were seeded in 6-well plates and treated with different concentrations of compound 28. After 72 h the number of 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 3.** FITC Annexin V (Alexa 488)-Sytox Deep Red (Alexa 700) staining of MDA-MB-231 and HS578T cells. (**A**) Dot Plots of MDA-MB-231 and HS578T cell line after treatment with 250 nM compound 28 (**B**–**E**). Quantitative analysis of cell death of MDA-MB-231 (**B**,**C**) and HS578T cells (**D**,**E**) after treatment with different concentrations of compound 28 for 48 h (**B**,**D**) and 72 h (**C**,**E**). Data represent mean values (±SD) of at least three independent experiments. Significant *p* values are highlighted with asterisks (\* *p* ≤ 0.05; \*\* *p* ≤ 0.01).

Analysis of subcellular localization of compound **28** (Figure 4A) compared to the mitochondrial targeting compound BioTracker™ 488 Green Mitochondria Dye (Figure 4B) in MDA-MB-231 cells shows an identical pattern of accumulation, indicating the mitochon-

drial 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**.

#### **3. 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**.

#### **4. Materials and Methods**

#### *4.1. 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 expressionL 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, APCI corona discharge: 4.2 μA, capillary temperature: 250 ◦C, capillary voltage: 180 V, sheath gas: N2). Thin-layer chromatography was performed on precoated silica gel plates supplied by Macherey-Nagel (Düren, Germany). IR spectra were recorded on a Spectrum 1000 FT-IR-spectrometer from Perkin Elmer (Rodgau, Germany). The UV/Vis-spectra were

recorded on a Lambda 14 spectrometer from Perkin Elmer (Rodgau, Germany); optical rotations were measured at 20 ◦C using a JASCO-P2000 instrument (JASCO Germany GmbH, Pfungstadt, Germany). The melting points (m.p.) were determined using the Leica hot-stage microscope Galen III (Leica Biosystems, Nussloch, Germany) and are uncorrected. The solvents were dried according to the usual procedures. Microanalyses were performed with an Elementar Vario EL (CHNS) instrument (Elementar Analysensysteme GmbH, Elementar-Straße 1, D-63505, Langenselbold, Germany).

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.
