Novel Antineoplastic Inducers of Mitochondrial Apoptosis in Human Cancer Cells

I propose a new strategy to suppress human cancer completely with two entirely new drug compounds exploiting cancer’s Warburg effect characterized by a defective mitochondrial aerobic respiration, substituted by cytosolic aerobic fermentation/glycolysis of D-(+)-glucose into L-(+)-lactic acid. The two essentially new drugs, compound 1 [P(op)T(est)162] and compound 3 (PT167), represent new highly symmetric, four-bladed propeller-shaped polyammonium cations. The in vitro antineoplastic highly efficacious drug compound 3 represents a covalent combination of compound 1 and compound 2 (PT166). The intermediate drug compound 2 is an entirely new colchic(in)oid derivative synthesized from colchicine. Compound 2’s structure was determined using X-ray crystallography. Compound 1 and compound 3 were active in vitro versus 60 human cancer cell lines of the National Cancer Institute (NCI) Developmental Therapeutics Program (DTP) 60-cancer cell testing. Compound 1 and compound 3 not only stop the growth of cancer cells to ±0% (cancerostatic effect) but completely kill nearly all 60 cancer cells to a level of almost −100% (tumoricidal effect). Compound 1 and compound 3 induce mitochondrial apoptosis (under cytochrome c release) in all cancer cells tested by (re)activating (in most cancers impaired) p53 function, which results in a decrease in cancer’s dysregulated cyclin D1 and an induction of the cell cycle-halting cyclin-dependent kinase inhibitor p21Waf1/p21Cip1.


Introduction
Adenosine 5 ′ -triphosphate (ATP) is required for normal cell proliferation and survival and comes primarily from two sources.The first is glycolysis, which comprises a series of reactions that metabolizes D-(+)-glucose to pyruvate in the cytoplasm to produce a net of 2 molecules ATP from each D-(+)-glucose.The other is the citric acid cycle (CAC), also known as the Krebs cycle, Szent Györgyi−Krebs cycle or the tricarboxylic acid cycle (TCA cycle), which uses pyruvate formed from glycolysis in a series of reactions that donate electrons via nicotinamide adenine dinucleotide (NADH/H + ) and flavin adenine dinucleotide (FADH 2 ) to the respiratory chain complexes in the mitochondria.With oxygen (O 2 ) serving as the final electron acceptor, electron transfer across the mitochondrial inner membrane creates a proton gradient, which forms in summary about 30-32 molecules ATP per one D-(+)-glucose molecule by catalysis of F 1 F O -ATP synthase.In conditions of oxygen limitation, such as within muscles under prolonged exercise, pyruvate is not utilized in the citric acid cycle (CAC) but is converted into L-(+)-lactic acid ('Fleischmilchsäure') by lactate dehydrogenase (LDH) in a process termed anaerobic glycolysis.
In 1930, Otto Heinrich Warburg (1883Warburg ( -1970) ) published [1] his theory on the origin of cancer cells based on a series of preceding investigations [2][3][4].He summarized his theory on the origin of cancer cells in 1956 [5].This theory was coined the Warburg hypothesis [6].The essence of this hypothesis on the origin of cancer is that, under aerobic conditions, malignant tissues metabolize approximately tenfold more glucose to lactate in a constant time window than primary tissues, a phenomenon known as the Warburg 'malignant' effect.Warburg claimed that cancer cells heavily rely on aerobic glycolysis as an energy source for malignant growth rather than aerobic respiration, thereby claiming a defect in cancer cell respiration.The Warburg hypothesis has been heavily debated for many years since its introduction [6], especially regarding the respiratory defect misinterpreted by Warburg himself [6], and only in recent years many details of the Warburg hypothesis were confirmed as correct [7][8][9][10][11][12].Importantly, the Warburg effect itself, which was proved to constitute a fact in vivo [13], has to be differentiated from the complete Warburg hypothesis, which was the cause of ongoing scientific debate [14][15][16].
Many cancer cells consume D-(+)-glucose heavily and produce L-(+)-lactic acid rather than catabolizing D-(+)-glucose via the citric acid cycle (CAC), which is normal for generating ATP in non-hypoxic healthy cells.The avid uptake of D-(+)-glucose by tumors is the prerequisite for the detection and monitoring of human tumors by 2-deoxy-2-[ 18 F]fluoroglucose positron emission tomography (PET).More than 90 years ago, Otto Heinrich Warburg observed that thin slices of human and animal tumors ex vivo displayed high levels of D-(+)-glucose uptake and L-(+)-lactate production.The shift toward L-(+)lactate production in cancers, even in the presence of adequate oxygen, is termed the Warburg effect or aerobic glycolysis [1][2][3][4][5][6][7][8].These observations have been confirmed, although the nuances of aerobic glycolysis and its molecular characteristics are still under investigation.Tumors display aerobic glycolysis partly through activation of proto-oncogenes or loss of tumor suppressors, which is then further intensified by stabilization of the evolutionary conserved hypoxia-inducible factor (HIF) via adaptive response to hypoxic microenvironment or via pathways that stabilize HIF under non-hypoxic conditions.
In 2002, it was shown that human cancer cells indeed suffer from defects in cellular respiration, either a marked depletion in cellular mitochondrial content or a selective repression in expression of the catalytic β-subunit of mitochondrial Complex V F-type ATPase (β-F 1 -ATPase) concurrent with an increase in the expression of the glycolytic enzyme glyceraldehyde 3-phosphate dehydrogenase [7].Both mechanisms impair mitochondrial respiration and give support to the Warburg hypothesis [7].In rat rhabdomyosarcoma, R1H cells' mitochondrial function was found to be deficient by a dysregulation of the mitochondrial protein-to-cardiolipin ratio [10].Mitochondrial respiration of R1H cells was significantly impaired, joined by the incapacity of the rhabdomyosarcoma cells to differentiate into mature striated skeletal muscle cells [10].The intriguing abnormalities in cardiolipin content were confirmed in vivo utilizing brain tumors grown in mice [9].The compositional cardiolipin abnormalities involved an abundance of immature molecular species and deficiencies of mature molecular species, suggesting major defects in cardiolipin synthesis and remodeling in rodent brain tumor tissue [9].The tumor cardiolipin abnormalities were also associated with significant reductions in both individual and linked electron transport chain activities [9].The acidic phospholipid cardiolipin [1,3-bis(sn-3 ′ -phosphatidyl)-sn-glycerol, 1,3-diphosphatidyl-sn-glycerol] is known to anchor cytochrome c together with cytochrome c oxidase at the inner mitochondrial membrane [17][18][19][20].Abnormalities in cardiolipin content and composition thus strongly impair cellular mitochondrial respiration by impairment of cytochrome c/cytochrome c oxidase function(s) of Complexes III (coenzyme Q-cytochrome c reductase) and IV (cytochrome c oxidase) of the electron transport chain [9].It was reported in 2015 that the acyl fatty acid composition of cardiolipin correlates with prostate cancer PC-3 cell proliferation [21].Cardiolipin was reviewed in 2020 to play a generally important role in cancer [22].Integrated lipidomics and proteomics revealed in 2021 acyl fatty acid alterations in pancreatic cancer stem cell cardiolipin [23], an upregulation of hydroxyacyl-coenzyme A (CoA) dehydrogenase/3-ketoacyl-CoA thiolase/enoyl-CoA hydratase (trifunctional protein HADHA, the α-subunit of mitochondrial long chain fatty acid β-oxidation trifunctional enzyme) in pancreatic cancer stem cells [23], and upregulation of long chain fatty acid elongation enzymes in pancreatic cancer stem cells [23].The data indicated a crucial role of fatty acid elongation and alteration in cardiolipin acyl chain composition in pancre-atic cancer stem cells, suggesting these characteristics as attractive therapeutic targets in pancreatic ductal adenocarcinomas [23].In 2023, it was reported that mass spectrometry imaging revealed abnormalities in cardiolipin composition and distribution in banked human astrocytoma/glioblastoma brain tumor tissues in comparison to healthy control brain cortex tissue [24].Hence, there is accumulating growing evidence that cardiolipin abnormalities represent a characteristic trait of cancer as its signature phospholipid both in vitro and in vivo.
The intrinsic pathway of programmed cell death (apoptosis [25][26][27][28][29][30][31]) triggers cytochrome c release from the inner mitochondrial membrane into the cytosol [30], where cytochrome c associates with apoptotic protease activating factor 1 (Apaf-1, apoptotic peptidase activating factor 1) and procaspase 9 to form the apoptosome [30].The apoptosome, in turn, activates the effector caspases 3, 6, and 7 to execute programmed cell death [30].In cancer cells this mechanism is defective or inefficient [31].Therefore, in cancer therapy, one possibility to selectively and completely kill cancer cells is to induce their apoptosis over the intrinsic pathway by pharmacological intervention [31].Targeting mitochondria with antineoplastic drugs was suggested as a novel strategy for cancer therapy [31].Recently, I isolated a lipophilic polyammonium cation, compound 1, as a by-product of the reaction of 1-aminoadamantane (amantadine) with 1,3-bis(chloromethyl)benzene (α,α ′ -dichlorom-xylene) in refluxing absolute ethanol.Amantadine is known as an antiparkinsonian (N-methyl-D-aspartate ionotropic glutamate receptor antagonist) drug [32] and chemotherapeutic antiviral drug inhibiting influenza A virus M2 protein transmembrane proton channel [33].This synthesis was inspired by the potential binding of a polyammonium cationic drug to DNA and/or p53 tumor suppressor protein tetramerization domain [34].It was found that compound 1 (PT162, NSC 796018), a new compound never synthesized before [according to Chemical Abstracts Service (CAS ® ) SciFinder ® search], induced apoptosis in all cell lines of the National Cancer Institute (NCI) Developmental Therapeutics Program (DTP) 60-cancer cell 5-dose testing, excluding leukemia cell lines, in the micromolar range of growth inhibition 50% (GI50).I decided to merge compound 1 with the colchic(in)oid compound 2 (PT166, NSC 750423), which showed submicromolar GI50 in the NCI DTP 60-cancer cell 5-dose testing but did not induce cancer cell apoptosis.Compound 2 was synthesized from colchicine and thiosemicarbazide in a one-step procedure and represented a new compound never synthesized before [according to Chemical Abstracts Service (CAS ® ) SciFinder ® search], just as compound 1.Compound 1 reacted with compound 2 under the impact of sodium hydroxide (NaOH) to give compound 3. Compound 3 (PT167, NSC 799315), a new compound never synthesized before [according to Chemical Abstracts Service (CAS ® ) SciFinder ® search], showed submicromolar GI50 in the NCI DTP 60-cancer cell 5-dose testing constantly in most cell lines including leukemia cells.Importantly, compound 3 was able to induce apoptosis in all investigated cancer cells, including leukemia cell lines, with a Mean of Inhibition Data (MID) for total growth inhibition (TGI, growth inhibition 100%) of 4.57 µM and a MID for lethal concentration 50% (LC50) of 15.85 µM.I report here the chemistry and NCI DTP 60-cancer cell 5-dose testing data for compound 1, compound 2, and compound 3, and demonstrate the apoptotic release of cytochrome c into the cytosol and activation of effector caspases induced by compounds 1 and 3. I propose that compound 1 and compound 3 induce apoptosis according to the Warburg hypothesis of pre-damaged respiration as a hallmark of cancer by exploiting the defect in mitochondrial cardiolipin-cytochrome c association in cancer cells.

Compound 1 2.1.1. The Synthesis of Salt-Containing Compound 1 (PENTA)
To find a potential complexation and/or stabilization partner for retinazone [35,36], an attempt was made to synthesize a polyammonium polycation from the adamantane [37]derived influenza A virus inhibitor [33] and N-methyl-D-aspartate (NMDA) subtype glutamate receptor antagonist [32] amantadine × HCl.For that purpose, 1-aminoadamantane hydrochloride and a 1.5-fold molar excess of 1,3-bis(chloromethyl)benzene were dissolved in aqueous ethanol.A solution of sodium hydroxide in water (3-fold molar excess) was added, and the mixture was refluxed for 3 h.Successively, acetone was added through the reflux condenser.After filtration, dilution with water, acidification with HCl, and volume reduction, the reaction mixture was extracted with ethyl acetate to remove unreacted 1,3bis(chloromethyl)benzene.Following additional volume reduction of the aqueous phase, a crude product could be isolated by freezing.The crude product was dissolved in refluxing aqueous acetone and was hot filtrated.The filtrate was evaporated from the acetone and was acidified with HCl.Instantly, a white precipitate formed, which represented the salt-containing compound 1 (PENTA).

The Synthesis of Pure Compound 1 [PT162 (NSC 796018)]
Pure compound 1 [PT162 (NSC 796018)] (Figure 1) was synthesized from 1-aminoadamantane (amantadine) with 1,3-bis(chloromethyl)benzene (α,α ′ -dichloro-mxylene) in refluxing absolute ethanol.Compound 1 was a by-product of the synthesis, obtained in 12.4% yield.The lipophilic main product was not isolated and removed by extraction with ethyl acetate.Crucial for the successful synthesis of compound 1 was the utilization of the free base 1-aminoadamantane instead of its commercial hydrochloride.Preceding synthesis, the free base 1-aminoadamantane was prepared from the hydrochloride by neutralization with NaOH, and this free base was used in situ for the synthesis of compound 1.

The Synthesis of Salt-Containing Compound 1 (PENTA)
To find a potential complexation and/or stabilization partner for retinazone [35,36], an attempt was made to synthesize a polyammonium polycation from the adamantane [37]-derived influenza A virus inhibitor [33] and N-methyl-D-aspartate (NMDA) subtype glutamate receptor antagonist [32] amantadine × HCl.For that purpose, 1-aminoadamantane hydrochloride and a 1.5-fold molar excess of 1,3-bis(chloromethyl)benzene were dissolved in aqueous ethanol.A solution of sodium hydroxide in water (3-fold molar excess) was added, and the mixture was refluxed for 3 h.Successively, acetone was added through the reflux condenser.After filtration, dilution with water, acidification with HCl, and volume reduction, the reaction mixture was extracted with ethyl acetate to remove unreacted 1,3-bis(chloromethyl)benzene.Following additional volume reduction of the aqueous phase, a crude product could be isolated by freezing.The crude product was dissolved in refluxing aqueous acetone and was hot filtrated.The filtrate was evaporated from the acetone and was acidified with HCl.Instantly, a white precipitate formed, which represented the salt-containing compound 1 (PENTA).
This pointed to a symmetric ammonium molecule with two different m-xylylene methylene groups, which were derived from 1,3-bis(chloromethyl)benzene (m-xylylene dichloride).
ules 2024, 29, x FOR PEER REVIEW chemical shift found at δ 4.54 ppm (in CDCl3) [43], and (iv) the analysi NMR spectrum of salt-containing compound 1 (PENTA) (Figure S2).Th aliphatic part of salt-containing compound 1 (PENTA) was analyzed 28.50 (γ-CH), 35 [44,45] (DEPTQ 13 C-NMR) subspectrum salt-containing compound 1 (PENTA) (Figure S4) secured the given CH proved that the resonance at δ 57.06 ppm originated from a quaternar signments were further verified using analysis of the gradient-sel Spectroscopy (gs-COSY) two-dimensional 1 H-1 H-correlation spectrum the gradient-selected Heteronuclear Multiple Quantum Coherence (Figure S6), and the gradient-selected Heteronuclear Multiple B (gs-HMBC) [45] (Figure S7) two-dimensional 1 H-13 C-correlation spectra compound 1 (PENTA).Conclusively, the second structure possibility, the true formula, mu compound 1 (Figure 1) because all other possibilities would not agree ocal NMR data.Finally, the FT-IR spectrum of salt-containing compoun compared to the corresponding spectrum of amantadine hydrochlo Certain characteristics shared by both spectra could be recognized.The band in salt-containing compound 1 (PENTA) at wavenumber 2925/2 ated by aliphatic ν(C-H) stretching vibrations.In amantadine hydr sorption could be found at wavenumber 2914/2853 cm −1 .Aliphatic p associated with these peaks could be found in both spectra.These Conclusively, the second structure possibility, the true formula, must be depicted by compound 1 (Figure 1) because all other possibilities would not agree with the unequivocal NMR data.Finally, the FT-IR spectrum of salt-containing compound 1 (PENTA) was compared to the corresponding spectrum of amantadine hydrochloride (Figure S3).Certain characteristics shared by both spectra could be recognized.The strong absorption band in salt-containing compound 1 (PENTA) at wavenumber 2925/2850 cm −1 was created by aliphatic ν(C-H) stretching vibrations.In amantadine hydrochloride this absorption could be found at wavenumber 2914/2853 cm −1 .Aliphatic progression bands associated with these peaks could be found in both spectra.These absorptions could mainly be ascribed to the adamantane cages present in both compounds.Typical bands were found at wavenumbers 1610/1585/1459/1074 cm −1 [salt-containing compound 1 (PENTA)], in amantadine hydrochloride at wavenumbers 1626/1594/1500/1086 cm −1 .They probably originate from the 1-adamantanammonium structure shared by both compounds.In addition, salt-containing compound 1 (PENTA) clearly showed the m-xylylene aromatic linker resonances at wavenumbers 794/762/731/693 cm −1 , which are not seen with amantadine hydrochloride (Figure S3).
The elemental analysis of salt-containing compound 1 (PENTA) revealed its sodium chloride content by calculating within ±0.3% for carbon and hydrogen.The NaCl obviously was co-precipitated in the final isolation step, which is not surprising when considering the polyammonium chloride character of compound 1.The nitrogen value was found to be 1.13% lower than calculated for C 72 H 100 N 5 Cl 5 × 1.5 NaCl.The reason for this deficit could be the [N(CH 2 R) 4 ] + tetrasubstituted ammonium structure of the central ammonium nitrogen.Compounds of this type are known to cause combustion problems [46].It could be speculated that the sodium content (NaCl) in salt-containing compound 1 (PENTA) led to the formation of only partially combustible sodium nitrate (NaNO 3 ).Mutually, both effects could be responsible for the wrong nitrogen analysis of salt-containing compound 1 (PENTA).Since the other analytical data of salt-containing compound 1 (PENTA) are all very conclusive, the unsatisfactory nitrogen analysis should not be overrated.Taken together, the structure of salt-containing compound 1 (PENTA) could be demonstrated without doubt, especially when evaluating the unequivocal NMR data.
The structure of pure (salt-free) compound 1 [PT162 (NSC 796018)] was secured by 1 H-NMR (Figure 3) and 13 C-DEPTQ (data not shown) spectroscopy experiments, as well as elemental analysis and FT-IR spectroscopy.By 1 H-NMR, through the integration of the proton resonance peaks, and by 13 C-NMR, the highly symmetric structure of compound 1 was proved.Compound 1 represents a pentaammonium cation with a quaternary center and four secondary amine functions.The four functional bridges are m-xylylene linkers that connect the central quaternary ammonium cation chloride with four N-substituted 1-adamantanammonium chloride moieties.The resulting chemical structure of compound 1 is given (Figure 1).Compound 1 was registered by the National Cancer Institute (NCI) as NSC 796018.
Molecules 2024, 29, x FOR PEER REVIEW 8 of 47 deficit could be the [N(CH2R)4] + tetrasubstituted ammonium structure of the central ammonium nitrogen.Compounds of this type are known to cause combustion problems [46].It could be speculated that the sodium content (NaCl) in salt-containing compound 1 (PENTA) led to the formation of only partially combustible sodium nitrate (NaNO3).Mutually, both effects could be responsible for the wrong nitrogen analysis of salt-containing compound 1 (PENTA).Since the other analytical data of salt-containing compound 1 (PENTA) are all very conclusive, the unsatisfactory nitrogen analysis should not be overrated.Taken together, the structure of salt-containing compound 1 (PENTA) could be demonstrated without doubt, especially when evaluating the unequivocal NMR data.
The structure of pure (salt-free) compound 1 [PT162 (NSC 796018)] was secured by 1 H-NMR (Figure 3) and 13 C-DEPTQ (data not shown) spectroscopy experiments, as well as elemental analysis and FT-IR spectroscopy.By 1 H-NMR, through the integration of the proton resonance peaks, and by 13 C-NMR, the highly symmetric structure of compound 1 was proved.Compound 1 represents a pentaammonium cation with a quaternary center and four secondary amine functions.The four functional bridges are m-xylylene linkers that connect the central quaternary ammonium cation chloride with four N-substituted 1-adamantanammonium chloride moieties.The resulting chemical structure of compound 1 is given (Figure 1).Compound 1 was registered by the National Cancer Institute (NCI) as NSC 796018.Compound 2 [PT166 (NSC 750423)] was synthesized from (-)-colchicine sesquihydrate (×1½ H 2 O) and thiosemicarbazide under catalysis of sodium hydroxide (NaOH) in refluxing 90% (v/v) aqueous ethanol.The structure (Figure 4) of compound 2 was secured using X-ray crystallography (see Section 2.2.3.), 1 H-NMR (Figure 5) and 13 C-NMR spectroscopy (data not shown) experiments, as well as elemental analysis and FT-IR spectroscopy (Figure S8).The thiosemicarbazide moiety is attached at the former position of the 10-methoxy group in colchicine.The point of connection is the terminal nitrogen of the hydrazinyl moiety of thiosemicarbazide.Equimolar quantities of (-)-colchicine and thiosemicarbazide were dissolved in 90% (v/v) aqueous ethanol by refluxing for 5 min.After adding a slight excess of sodium hydroxide dissolved in water, the deep orange-red solution was refluxed for 5 min.The cold, deep orange-red solution, after pre-cooling, was nearly neutralized by the dropwise addition of hydrochloric acid.Afterwards, the volume of the solution was reduced in vacuo.The reddish-brown solution was then mixed with water and acidified with aqueous hydrochloric acid.The oily emulsion was extracted with ethyl acetate (EtOAc).The separated aqueous layer (pH 2) was additionally extracted with a second volume of EtOAc.After neutralization of this aqueous phase with sodium hydrogen carbonate, the aqueous phase (pH 7-8) was extracted twice with EtOAc each.The EtOAc phases were combined and washed twice with water.The washed EtOAc phase, which already precipitated, was mixed with acetone and frozen at -25 °C.If precipitation did not start spontaneously, the volume of the solution was reduced in vacuo until coagulation started.The evolved yellow crystalline precipitate of compound 2 was filtered and dried.From the combined aqueous phases by cooling, a second crop of compound 2 could be obtained.The underlying molecular reactions for this synthesis are pictured (Scheme 2).Compound 2 was quite pure, as judged by using 1 H-NMR spectroscopy.The representative 1 H-NMR spectrum of compound 2 in DMSO-d6 is pictured (Figure 5).

The Nuclear Magnetic Resonance Spectra of Compound 2
It is known that colchic(in)oids [47] have the tendency to retain solvents, like water [48] and/or ethyl acetate [49], very firmly.Natural (-)-colchicine itself retained chloroform [50][51][52], dibromomethane/diiodomethane [53], or water as dihydrate [53,54] or sesquihydrate [52,55].Therefore, it could be understood that compound 2 was obtained as monohydrate × ⅔ (ethyl acetate) binary solvate, as judged by using 1 H-NMR and elemental analysis.Equimolar quantities of (-)-colchicine and thiosemicarbazide were dissolved in 90% (v/v) aqueous ethanol by refluxing for 5 min.After adding a slight excess of sodium hydroxide dissolved in water, the deep orange-red solution was refluxed for 5 min.The cold, deep orange-red solution, after pre-cooling, was nearly neutralized by the dropwise addition of hydrochloric acid.Afterwards, the volume of the solution was reduced in vacuo.The reddish-brown solution was then mixed with water and acidified with aqueous hydrochloric acid.The oily emulsion was extracted with ethyl acetate (EtOAc).The separated aqueous layer (pH 2) was additionally extracted with a second volume of EtOAc.After neutralization of this aqueous phase with sodium hydrogen carbonate, the aqueous phase (pH 7-8) was extracted twice with EtOAc each.The EtOAc phases were combined and washed twice with water.The washed EtOAc phase, which already precipitated, was mixed with acetone and frozen at -25 • C. If precipitation did not start spontaneously, the volume of the solution was reduced in vacuo until coagulation started.The evolved yellow crystalline precipitate of compound 2 was filtered and dried.From the combined aqueous phases by cooling, a second crop of compound 2 could be obtained.The underlying molecular reactions for this synthesis are pictured (Scheme 2).Compound 2 was quite pure, as judged by using 1 H-NMR spectroscopy.The representative 1 H-NMR spectrum of compound 2 in DMSO-d 6 is pictured (Figure 5).
The 1 H-NMR resonances of the colchic(in)oid compound 2 were assigned with the help of literature [56][57][58][59], especially [57], which gave a complete assignment of the protons in the 1 H-NMR spectrum of (-)-colchicine (in CDCl3).Compound 2 represents a completely new compound, never synthesized before [according to Chemical Abstracts Service (CAS) SciFinder ® (Columbus, OH, USA)].Therefore, the proton NMR spectrum (Figure 5) of compound 2 was interpreted to the point it was possible without doubt.Aliphatic proton resonances of compound 2 dissolved in DMSO-d6 could be differentiated as: δ 1.18 (1.5 H, t;  N-H).In the gradient-selected Correlation Spectroscopy (gs-COSY) two-dimensional 1 H-1 H-correlation spectrum [45] (data not shown) of compound 2 (in DMSO-d6), no W-shaped long-range 4 J ( 1 H-1 H) coupling, known as zig-zag (W) coupling, was found.This differentiates compound 2 from the thiosemicarbazones (E)-4-(dimethylamino)benzaldehyde thiosemicarbazone [35] and (E)-4-bromo-2-fluorobenzaldehyde thiosemicarbazone [35], where such a "W" coupling was observed [35].This pointed to sterical fixation as a prerequisite for observable W couplings in (E)-4-(dimethylamino)benzaldehyde thiosemicarbazone and (E)-4-bromo-2-fluorobenzaldehyde thiosemicarbazone, which obviously is not realized in compound 2.This proved that compound 2 is not a thiosemicarbazone.Furthermore, the protons of the tropone (ring C) could be unequivocally assigned, and their coupling constants secured that no benzilic-type rearrangement happened to the tropolone, a reaction seen with colchic(in)oids under certain (alkaline) conditions, occasionally leading to the rearrangement products allocolchicine (colchicic acid methyl ester) [60,61] or colchicic acid (allocolchiceine) [62], both being aromatic in ring C.Under the reaction conditions employed for the synthesis of compound 2, the allocolchiceine sodium salt (Scheme 3) could be expected as a side product, but was not observed.This benzilic-type rearrangement (Scheme 3) was elucidated by Šantavý [61] and Fernholz [62].By these analyses, it was found that the amidation product of (-)-colchicine, the substituted thiosemicarbazide compound 2, was not cyclic with regard to the thiosemicarbazide unit at ring C of compound 2.This was quite surprising since the reaction product of (-)-colchicine with thiourea was cyclic with respect to the thiourea substitution in ring C [64,65], which seemed surprising, in turn, because the tropolonic C-9 carbonyl group in (-)-colchicine did not react with common carbonyl reagents like hydroxylamine or semicarbazide [66,67].The reason for the latter irregularity could be the special tropylium oxide resonance type of tropones and tropolones [68][69][70][71][72]. Therefore, the synthesis of compound 2 clearly obeyed the common rules for the chemical reactivity of tropolones, whereas the synthesis of the cyclic thiourea congener [64,65] of compound 2 did not follow the common chemical reactivity experience for tropolones.

The X-ray Crystallographic Crystal and Molecular Structure Determination of Compound 2
Compound 2 was crystallized from ethyl acetate, and a single crystal was selected for X-ray crystallographic determination (at T = 100 K) of the crystal and molecular structure of compound 2 (Figures 7-9, Figures S9 and S10).Compound 2 crystallized in the monoclinic space group P2 1 with ethyl acetate and water of crystallization S9).The crystal packing (Figure 8) with indicated hydrogen bonds (Figure S10) in the unit cell (Z = 2) of compound 2 is depicted.It should be noted that the molecule is helical stereogenic and shows the [M(inus)]-helicity as N-[(aS,7S)-10-(2carbamothioylhydrazinyl)-1,2,3-trimethoxy-9-oxo-5,6,7,9-tetrahydrobenzo[a]heptalen-7-yl] acetamide (Figure 9).The helical axis atropisomerism view of one independent, isolated molecule of compound 2 as found in the single crystal is depicted (Figure 9).This stands in contradiction to a report that claimed the [P(lus)]-helicity (aR) for (-)-colchicine [78].The classification of (M)-helicity for compound 2 followed the Cahn-Ingold-Prelog (CIP) rules for the assignment of molecular helicity [75,76].The (M)-helicity of (-)-colchicine was previously assigned correctly by Brossi et al. [74].The X-ray crystallographic structure was deposited at The Cambridge Crystallographic Data Centre (CCDC) and assigned the deposition № CCDC 1,839,505 (ID: RIVGOW).The crystal data of the X-ray crystallographic determination of the crystal and molecular structure of compound 2 are tabulated (Table 1).The helical axis atropisomerism view of one independent, isolated molecule of compound 2 as found in the single crystal is depicted (Figure 9).This stands in contradiction to a report that claimed the [P(lus)]-helicity (aR) for (-)-colchicine [78].The classification of (M)-helicity for compound 2 followed the Cahn-Ingold-Prelog (CIP) rules for the assignment of molecular helicity [75,76].The (M)-helicity of (-)-colchicine was previously assigned correctly by Brossi et al. [74].The X-ray crystallographic structure was deposited at The Cambridge Crystallographic Data Centre (CCDC) and assigned the deposition № CCDC 1,839,505 (ID: RIVGOW).The crystal data of the X-ray crystallographic determination of the crystal and molecular structure of compound 2 are tabulated (Table 1).Empirical Formula C48H66N8O15S2 Formula weight, Mr (g•mol x Flack = 0.09(6), R 1 = 0.0594, wR 2 = 0.1325, 9686 independent reflections [2 ϑ ≤ 52.originating from a thiosemicarbazide or thiosemicarbazone moiety was observed previously in the synthesis of retinazone, a retinoid thiosemicarbazone derivative [35,36].As a result, compound 2 was connected to compound 1 via a thioamide bonding at an adamantanamine nitrogen (Scheme 4).The molecular stoichiometry of compound 3 was determined using 1 H-NMR (Figure 11) spectroscopy experiments, as well as elemental analysis.Compound 1 recieved two molecules of compound 2 by trans-amidation to yield compound 3.The 1 H-NMR spectrum of compound 3 (Figure 11) exhibits a peculiar resonance compression [the adamantane resonances 2.00 (24 H; β-CH 2 ), 2.14 (12 H; γ-CH) could not be detected] induced by the large (macro)molecular structure of compound 3.This points to intramolecular (hydrophobic) interaction between the β-methylene and γ-methine structural elements of the compound 1-derived adamantanamine cages with the tropone ring (especially H-11) of the colchic(in)oid part of compound 3.An intramolecular interaction between the β-methylene and γ-methine structural elements of the adamantanamine cages with H-11 of the 10-(thiosemicarbazide)-substituted tropone ring in compound 3 could be demonstrated using molecular modeling (Figure 12).The resulting overall chemical structure of compound 3 is given (Figure 10).Compound 3 was registered by the National Cancer Institute (NCI) as NSC 799315.
Molecules 2024, 29, x FOR PEER REVIEW 17 of 47 using molecular modeling (Figure 12).The resulting overall chemical structure of compound 3 is given (Figure 10).Compound 3 was registered by the National Cancer Institute (NCI) as NSC 799315.The proton resonances were measured as chemical shifts δ (ppm): δ 1.48    This substance bears a peculiar magnetic property.During NMR spectrometer shim, it was detected that newly dried (CaCl 2 , in vacuo, 19 h) compound 3 is, in part, paramagnetic because there were considerable difficulties in shimming the NMR spectrometer probe magnetic field (personal communication Robbin Schnieders).The operator told me that the DMSO-d 6 solution of newly dried compound 3 had to be strongly diluted with DMSO-d 6 and that the spectrum acquisition time had to be elongated considerably.This pointed to the inclusion of a paramagnetic partial structure in newly dried compound 3. Indeed, the 1 H-NMR spectrum (Figure 13) gives evidence for that interpretation, which is depicted (Figure 14).The colchic(in)oid part of the molecule picks up one electron from the ylide monocation (ylide monohydrochloride) (Figure 14, in blue), yielding a resonance-stabilized 1-hydroxycyclohepta-2,4,6-trien-1-yl radical (hydroxytropyl radical) [82,83] producing, in consequence, the 8-NH +• radical cation (Figure 14, in red).
refrigerator.The high-performance liquid chromatography (HPLC) (Figure 15A,B) was performed with a reversed-phase C8 (RP8, n-octyl) column and gradient elution with eluent A = water/0.1% (v/v) formic acid (HCOOH) and eluent B = acetonitrile/0.1%(v/v) formic acid (HCOOH).The flowrate was 0.5 mL/min, and the linear eluent gradient was t0min = 95% eluent A/5% eluent B to t13min = 5% eluent A/95% eluent B, t16min = stop.A 5 µL volume of 50 µM compound 3 solutions in acetonitrile (N≡C-CH3) was injected (0.25 nmol, 510.025 ng).The total ion current (TIC) chromatogram is shown in Figure 15A, and the chromatogram with UV detection at λ = 335 nm is depicted in Figure 15B.Multiple ionic species of compound 3 were separated (Table 2) due to the complicated ionization kinetics of compound 3 dissolved in acetonitrile (N≡C-CH3) with the presence of 0.1% (v/v) HCOOH.The quaternary ammonium compound 3 can be protonated once or twice or not protonated like the in situ substance (Table 2).Moreover, compound 3 can exist in equilibrium as a neutral nitrogen ylide [84-86] at the central quaternary ammonium cation.This nitrogen ylide can be protonated once or twice or not protonated (neutral nitrogen ylide) (Table 2).The substance compound 3 was quite pure, as judged from the chromatogram with UV detection at λ = 335 nm (Figure 15B).2), which were separated due to the complicated ionization kinetics of the macromolecular substance compound 3 dissolved in acetonitrile with the presence of 0.1% (v/v) HCOOH in the HPLC eluents.(B) The analogous liquid chromatographic (HPLC) investigation of compound 3 with UV detection at λ = 335 nm.
Table 2.The liquid chromatographic (HPLC)/electrospray ionization (ESI) time-of-flight (ToF) mass spectrometric investigation of compound 3 after storing the substance over six years at +0-4 • C in the refrigerator.Given are the HPLC peak regions in the total ion current (TIC) chromatogram (Figure 15A) which were scanned in the ESI mass spectrometer with the observed fragmentation peaks (in parentheses: relative peak intensity of the 100% base peak), and the presumed ionization identities of the ionized species of compound 3 dissolved in acetonitrile with presence of 0.1% (v/v) HCOOH in the HPLC eluents.The total ion current (TIC) chromatogram is shown in Figure 15A, and the chromatogram with UV detection at λ = 335 nm is depicted in Figure 15B.Multiple ionic species of compound 3 were separated (Table 2) due to the complicated ionization kinetics of compound 3 dissolved in acetonitrile (N≡C-CH 3 ) with the presence of 0.1% (v/v) HCOOH.The quaternary ammonium compound 3 can be protonated once or twice or not protonated like the in situ substance (Table 2).Moreover, compound 3 can exist in equilibrium as a neutral nitrogen ylide [84-86] at the central quaternary ammonium cation.This nitrogen ylide can be protonated once or twice or not protonated (neutral nitrogen ylide) (Table 2).The substance compound 3 was quite pure, as judged from the chromatogram with UV detection at λ = 335 nm (Figure 15B).bridged over a thiocarbonyl to the compound 1 core, being quite pure.None of compound 2 and compound 1, the synthesis starting substances, or colchiceine hydrazide, a possible degradation product, could be detected in the compound 3 preparation using highly sensitive detection methods.The two peaks (C 74 H 89 N 7 O 7 S) 2+ m/z 609.8644 (31.0%) (generated from ylide monocation) and (C 74 H 88 N 7 O 7 S) + m/z 1218.7191(5.0%) (generated from ylide monocation) (Figure 16) are to be formulated as ammonia (NH 3 ) coordination-stabilized [84,85] nitrogen ylides [86] being in equilibrium with a mass spectrometric generated species exhibiting pentavalent nitrogen [87][88][89] according to Figure 17.The fragment cations (C 18 H 21 NNaO) + m/z 290.17    16) are to be formulated as ammonia (NH 3 )-stabilized [84,85] nitrogen ylides [86] (left, in blue) being in equilibrium with a mass spectrometric enabled species characterized by twice pentavalent nitrogen [87][88][89] (right, in blue).
In summary, the chemical structure of compound 3 could be substantiated as a derivative of colchiceine hydrazide (10-hydrazinyl-10-demethoxycolchicine) [64,65] bridged over a thiocarbonyl to the compound 1 core, being quite pure.None of compound 2 and compound 1, the synthesis starting substances, or colchiceine hydrazide, a possible degradation product, could be detected in the compound 3 preparation using highly sensitive detection methods.First page: graphic allover presentation of the second page with All Cell Lines in one graphic and molar concentrations expressed in logarithmic log 10 unit.The earlier the colored curves aspire to the bottom, the more potent the drug.The more the curves strive from ±0% to −100%, the more the tumor is killed by the drug.
Second page: inhibition curves for tumor cell lines summarized in terms of tumor type.The Sample Concentrations are given at the x-axis in log 10 units (−9 = 1 nM, −8 = 10 nM, −7 = 100 nM, −6 = 1 µM, −5 = 10 µM, −4 = 100 µM, −3 = 1000 µM = 1 mM).The Percentage Growth is given at the y-axis in % and spans from +100% to -100%.0% Growth means that the tumor is still there but not growing anymore.−100% Growth means that the tumor cells are all dead and had died by apoptosis or necrosis; this is the ideal outcome.The smoother the curve, the more reliable the tumor inhibition.Generally, it is not sufficient to reach only 0% Growth since the tumor is still there.The ideal is −100% Growth since the tumor cells were completely killed by the drug, but only a few antineoplastic drugs in clinical use reach this.Cytostatics in clinical use generally reach only 0% Growth; this is called cytostasis (therefore the name cytostatics).
Third page: the actual concentrations used in log 10 unit are given for five doses (Note: most of these are not smooth values, but technically created values).Then, the mean optical densities of the vital stain sulforhodamine B (SRB) retained in the cells are given.The higher the SRB optical density, the higher the number of living tumor cells.Then, the Percent Growth is given again.The GI50 (Growth Inhibition 50%), TGI (Total Growth Inhibition = 0% Growth), and LC50 (Lethal Concentration 50% = −50% Growth) are given in linear concentration (E-6 = 10 −6 M = µM, E-5 = 10 −5 M. ..) units.A > 10 −X (for example: >10 −4 = >100 µM) means that the corresponding defined criterion is not reached by the drug = failure to reach the defined tumor inhibition criterion (GI50, TGI, or LC50).The NSC number of the test drug is given in the heading.The NSC Number is a standardized system of all anticancer compounds tested by NCI.The NSC number can be used for unequivocal identification and definition of all anticancer drugs if registered and tested by NCI.
Fourth page: this is the summary of the results expressed in log 10 units.The defined tumor inhibition criterion (GI50, TGI, or LC50) is recalculated in log 10 expression, and the colored bars, which indicate then the sensitivity of the individual tumor cell line to the agent, are given in log 10 units.Bar to the left: less sensitive than mean; Bar to the right: more sensitive than mean.The most important feature is at the bottom of the page: the MID (Mean of Inhibition Data) indicates the mean concentration for all tested cell lines required for the drug to reach the defined tumor inhibition criterion (GI50, TGI, or LC50).It is given in Log 10 GI50, Log 10 TGI, and Log 10 LC50.From those values, the corresponding MID is calculated.The more negative the MID, the more potent the drug.The MID can be transformed from logarithmic into linear concentrations by the formula: c = 10 MID .The Delta and Range of the MID correspond to these definitions, expressed in logarithmic log 10 unit: The Delta is defined as:  S1.Compound 1 and compound 3 generally started to inhibit cancer cell growth in the submicromolar range, whereby compound 3 was slightly more potent than compound 1.The responses of compound 1 and compound 3 regarding inhibition of cancer cell growth were remarkably smooth, regular, and consistent.Nearly all cancer cell lines were inhibited by compound 1 and compound 3 in a very consistent fashion, including leukemia cell lines for compound 3.In contrast, the inhibiting effect of compound 2 on cancer cell growth was widely variable.Importantly, compound 1 and compound 3, including leukemia cell lines for compound 3, induced consistent cancer cell death in almost all cancer cell lines.In contrast, compound 2 failed to induce cancer cell death in nearly all cancer cell lines.The GI50 for compound 1 was 1.288 µM, for compound 2 0.933 µM, and for compound 3 1.349 µM (Table S1).The TGI for compound 1 was 4.677 µM, for compound 2 32.359 µM, and for compound 3 4.571 µM (Table S1).The LC50 for compound 1 was 16.596 µM, for compound 2 95.499 µM, and for compound 3 15.849µM (Table S1).As can be clearly seen from these data, compound 2 failed to induce cancer cell death and acts only cytostatic, whereas compound 1 and compound 3 successfully induced cancer cell death to nearly −100% cancer cell growth and can be classified as tumoricidal.
Compound 1 and compound 3 were consistently active versus wild-type p53-containing cancer cell lines and cancer cell lines with mutant or lost p53 protein (Table S1).The p53 status of the individual cancer cell lines in the NCI DTP 60-cancer cell 5-dose testing cell line panel was taken as published [92,93].

National Cancer Institute (NCI) Developmental Therapeutics Program (DTP) 60-Cancer Cell 5-Dose Testing Results with Compound 1
The four pages of the National Cancer Institute (NCI) Developmental Therapeutics Program (DTP) 60-cancer cell 5-dose testing results for compound 1 (PT162, NSC 796018) are given in succession.First page (Figure S21): graphic allover presentation of second page with All Cell Lines in one graphic.Second page (Figure 18): inhibition curves for tumor cell lines arranged/ordered for general tumor type.Third page (Figure S22): the mean optical densities of the utilized vital stain sulforhodamine B (SRB) retained in the cells, the actual used concentrations in log 10 units for five doses, and the Percent Growth are given.The GI50 (Growth Inhibition 50%), TGI (Total Growth Inhibition = 0% Growth), and LC50 (Lethal Concentration 50% = −50% Growth) are given in linear concentration units.Fourth page (Figure S23): this is the summary of the results expressed in log 10 units.The defined tumor inhibition criterion (GI50, TGI, or LC50) is recalculated in log 10 expression, and the colored bars, which indicate then the sensitivity of the individual tumor cell line to the agent, are given in log 10 units.

Cytochrome c Assay (Mitochondrial and Cytosolic) with the Drugs Compound 1, Compound 2, and Compound 3 in the Human Prostate Cancer Cell Lines PC-3 and DU-145
To assess the nature of the cancer cell death-inducing effect of the drugs compound 1 (PT162, NSC 796018), compound 2 (PT166, NSC 750423), and compound 3 (PT167, NSC 799315), and to verify the failure to induce cancer cell death of compound 2, the cellular compartment of cytochrome c in the prostate cancer cell lines PC-3 and DU-145 under the action of compound 1, compound 2, and compound 3 was investigated (Figure 21).Compound 1 readily induced cytochrome c translocation from mitochondria into the cytosol at 25.0 µM concentration in PC-3 cells (Figure 21A) and 5.0 µM concentration in DU-145 cells (Figure 21B).Compound 2 failed to induce a significant difference in the cytochrome c-residing cellular compartment in PC-3 cells (Figure 21C) and, less significantly, in DU-145 cells (Figure 21D).Compound 3 readily induced cytochrome c translocation from mitochondria into the cytosol at 5.0 µM concentration in PC-3 cells (Figure 21E) and 25.0 µM concentration in DU-145 cells (Figure 21F).At 25.0 µM concentration, compound 1 in DU-145 cells (Figure 21B) and compound 3 in PC-3 cells (Figure 21E) both induced complete cell death, which resulted in massive depletion of cytochrome c in the cytosol, very probably Compound 2 failed to induce a significant difference in the cytochrome c-residing cellular compartment in PC-3 cells (Figure 21C) and, less significantly, in DU-145 cells (Figure 21D).Compound 3 readily induced cytochrome c translocation from mitochondria into the cytosol at 5.0 µM concentration in PC-3 cells (Figure 21E) and 25.0 µM concentration in DU-145 cells (Figure 21F).At 25.0 µM concentration, compound 1 in DU-145 cells (Figure 21B) and compound 3 in PC-3 cells (Figure 21E) both induced complete cell death, which resulted in massive depletion of cytochrome c in the cytosol, very probably by apoptotic degradation of cytochrome c protein by effector caspases.That means that cytochrome c apoprotein is digested by the cytosolic caspases executing apoptosis.

Discussion
p53 was identified in 1979 by Sir David P. Lane and Lionel V. Crawford [106], Arnold J. Levine and Daniel I.H.Linzer [107], and Lloyd J. Old, Albert B. DeLeo and Colleagues [108].p53 had been assumed before to exist as the target of the SV40 virus (Polyomaviridae, Betapolyomavirus; simian vacuolating virus 40, simian virus 40), which is tumorigenic.SV40 virus' oncogenic potential involves suppression of the transcriptional properties of p53 [106,107] (and retinoblastoma) tumor suppressor protein(s) by the SV40 large T antigen [106,107].The name p53 was coined in 1979 [108], describing the apparent molecular mass of the protein on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS−PAGE).p53 plays an important role in the regulation of, or progression through, the cell cycle, in apoptosis and maintenance of genomic stability, by several mechanisms: p53 can activate DNA repair proteins when DNA has suffered sustained damage.Thus, it may be an important factor in aging.
p53 can arrest growth by holding the cell cycle at the G 1 /S boundary on DNA damage recognition-if it stops the cell cycle here for a long enough time, the DNA repair machinery will have time to fix the damage, and the cells will be allowed to continue the cell cycle.p53 can initiate apoptosis (genetically programmed cell death) if DNA damage proves to be irreparable.
p53 is essential for the senescence response to shortened telomeres.p53 acts as an inhibitor of angiogenesis generally mediated by vascular endothelial growth factor A (VEGF-A).
p53 plays an important role in the differentiation and maintenance of human stem cells throughout embryonal development and adult human life.

Experimental Section
Following drug addition, the plates are incubated for an additional 48 h at 37 • C, 5% CO 2 , 95% air, and 100% relative humidity.For adherent cells, the assay is terminated by the addition of cold TCA.Cells are fixed in situ by the gentle addition of 50 µL of cold 50% (m/v) TCA (final concentration, 10% TCA) and incubated for 60 min at 4 • C. The supernatant is discarded, and the plates are washed five times with tap water and air dried.Sulforhodamine B (SRB) solution [100 µL at 0.4% (m/v) in 1% acetic acid] is added to each well, and plates are incubated for 10 min at room temperature.After staining, unbound dye is removed by washing five times with 1% acetic acid, and the plates are air dried.Bound stain is subsequently solubilized with 10 mM trizma ® base [2-amino-2-(hydroxymethyl)-1,3-propandiol, tris base, trometamol], and the absorbance is read on an automated plate reader at a wavelength of λ = 515 nm.For suspension cells, the methodology is the same, except that the assay is terminated by fixing settled cells at the bottom of the wells by gently adding 50 µL of 80% TCA (final concentration, 16% TCA).Using the seven absorbance measurements [time zero, (T z ), control growth, (C), and test growth in the presence of the drug at the five concentration levels (T i )], the percentage growth is calculated at each of the drug concentration levels.Percentage growth inhibition is calculated as: [(T i − T z )/(C − T z )] × 100 for concentrations for which T i ≥ T z [(T i − T z )/T z ] × 100 for concentrations for which T i < T z Three dose-response parameters are calculated for each experimental agent.Growth inhibition of 50% (GI50) is calculated from [(T i − T z )/(C − T z )] × 100 = 50, which is the drug concentration resulting in a 50% reduction in the net protein increase (as measured using SRB staining) in control cells during the drug incubation.The drug concentration resulting in total growth inhibition (TGI) is calculated from T i = T z .The LC50 (concentration of drug resulting in a 50% reduction in the measured protein at the end of the drug treatment as compared to that at the beginning) indicating a net loss of cells following treatment is calculated from [(T i − T z )/T z ] × 100 = −50.Values are calculated for each of these three parameters if the level of activity is reached; however, if the effect is not reached or is exceeded, the value for that parameter is expressed as greater or less than the maximum or minimum concentration tested.
An outline for reading the NCI 60 Cell Five-Dose Screen data was given in Section 2.4.1.The numerical screening data are given in Supplementary Information (Table S1).

Cytochrome c Assay (Mitochondrial and Cytosolic)
Cell numbers of 2 × 10 6 PC-3 and DU-145 cells were seeded into a 100-well plate.The next day, cells were treated with either dimethyl sulfoxide (DMSO) or six concentrations of compounds 1, 2, and 3 (500 nM, 1 µM, 5 µM, 25 µM, 50 µM, 75 µM) dissolved in DMSO for a time of 48 h.The concentrations of 25 µM, 50 µM, and 75 µM of compounds 1, 2, and 3 were found to be highly toxic for both cell lines 48 h post-treatment.The enzymelinked immunosorbent assay (ELISA) for cytochrome c was performed according to the manufacturer's instructions [Cytochrome c (human), ELISA kit (Enzo Life Sciences, Inc., Farmingdale, NY, USA)] with two fractions (mitochondrial and cytosolic) of each cell line at the concentrations of compound 1, 2, and 3 of 500 nM, 1 µM, 5 µM, and 25 µM.The protein content for each fraction was calculated to determine the mass content of cytochrome c in each fraction (pg cytochrome c/mg total protein).The mass content of cytochrome c (pg cytochrome c/mg total protein) was normalized towards the corresponding blank DMSO control and is given in percent (%) of blank DMSO control.

Cytotoxicity and HIV-1 LAI Replication Reverse Transcriptase Assays with Compound 1
The cytotoxicity and human immunodeficiency virus type 1 (HIV-1) strain LAI replication assays were performed in freshly explanted primary human peripheral blood mononuclear cells (PBM cells) according to published procedures [36].The assays were conducted at least in triplicate and treated statistically (if possible).HIV-1 LAI (= HIV-1 BRU = LAV-1) was assayed in primary human peripheral blood lymphocyte (PBL) cells in the presence of a drug being evaluated.The parameter for antiviral activity was a reduction in reverse transcriptase (RT) activity in the cell supernatant after Triton X-100-mediated lysis of released virions, as measured using [5α-3 H]dTTP (5α-tritiated thymidine 5 ′ -triphosphate) incorporation into poly(rA)•poly(dT) directed by the primed RNA template poly(rA)•oligo(dT).It should be noted that the assay did not detect RT inhibition by potential RT inhibitors per se but indirectly quantified the amount of released HIV-1 in the supernatant.The detailed assay methodology was reported by Schinazi et al. [120] as based on an older assay system of Spira et al. [121].The experiments were conducted in triplicate and treated statistically using regression curve analysis (r 2 coefficient of determination).The RT inhibitor AZT (zidovudine, 3 ′ -azido-3 ′ -deoxythymidine; RETROVIR™) served as a positive control.Cytotoxicity on PBL and the other cells (CCRF−CEM, Vero) exerted by the test compounds was determined as described by Stuyver et al.The crystal data of compound 2 were collected on a Bruker X8 APEX-II diffractometer with a CCD area detector and multi-layer mirror monochromated Mo Kα radiation.The structure was solved using direct methods, refined with the SHELX software package (SHELXS97, SHELXL97) [123], and expanded using Fourier techniques.All non-hydrogen atoms were refined anisotropically.Hydrogen atoms were assigned to idealized positions and were included in structure factor calculations.The Flack parameter x Flack [124] was near zero, indicating that the right absolute configuration was solved.Masses of 2.50 g 1-adamantanammonium chloride (1-aminoadamantane hydrochloride) (M = 187.71g/mol, 13.32 mmol) and 3.50 g 1,3-bis(chloromethyl)benzene (M = 175.06g/mol, m-xylylene dichloride, α,α ′ -dichloro-m-xylene) (19.99 mmol) were dissolved in 30 mL of 90% (v/v) aqueous ethanol.A solution of 1.60 g sodium hydroxide (NaOH) (40.00 mmol) in 40 mL of water was added, and the suspension was refluxed for 3 h.After 20 min and 40 min of reflux, 20 mL of acetone was added at each time using the reflux condenser.After 60 min, 80 min, and 100 min reflux, 40 mL of acetone was added at each time in the same way.After 120 min reflux, an additional 20 mL of acetone was added in the same way.Then, after 10 min cooling at +0-2 • C, the colorless suspension was warm filtrated through filter paper.The filtrate (pH 9) was mixed with 100 mL of water and evaporated in vacuo from the acetone to a volume of ca.170 mL.Afterwards, 2 mL of 10.27 M [32% (m/v)] hydrochloric acid (20.54 mmol) was added, and the colorless solution (pH 2-3) was evaporated in vacuo to a volume of ca.120 mL.
The now turbid suspension was mixed with 20 mL of water and was extracted with 100 mL of ethyl acetate (EtOAc) to remove residual 1,3-bis(chloromethyl)benzene.The aqueous phase was isolated and evaporated in vacuo to a volume of ca.50 mL.This aqueous phase was then frozen at −25 • C for 1½ h.The crystalline white precipitate of crude compound 1 × 1.5 NaCl (PT162 × 1.5 NaCl) was filtered and dried over CaCl 2 in vacuo (from the filtrate additional substance could be obtained, which was combined with the main yield).From the separated EtOAc phase by cooling at +0-2 • C for 3 h, additional crude compound 1 × 1.5 NaCl (PT162 × 1.5 NaCl) could be isolated, which was combined with the main fraction.
The combined crude product (730 mg) was dissolved in 50 mL of 80% (v/v) aqueous acetone by short (5 min) reflux and was hot filtrated through filter paper to remove a few cloudy impurities.The filtrate was transferred and mixed with 40 mL of 50% (v/v) aqueous acetone.Afterward, the filtrate was evaporated in vacuo from the acetone.The resulting solution (pH 5) was acidified to pH 0-1 by the addition of 0.6 mL of 10.27 M [32% (m/v)] hydrochloric acid (6.16 mmol).Immediately, a fine white crystalline precipitate formed.The suspension was mixed with 10 mL of 50% (v/v) aqueous acetone and was frozen at −25 • C for 2½ h.The fine white crystals (small needles) of compound 1 × 1.5 NaCl (PT162 × 1.5 NaCl) were filtered and dried over CaCl 2 in vacuo.
The still wet 1-aminoadamantane (free base) and 1,3-bis(chloromethyl)benzene (mxylylene dichloride, α,α ′ -dichloro-m-xylene) (M = 175.06g/mol, 7.498 g, 42.8310 mmol) were suspended in absolute ethanol (200 mL).The suspension was refluxed for 3 h.After 40 min reflux, a clear colorless solution formed in the heat.After short (5 min) cooling at +0-2 • C, the colorless suspension was hot filtrated through one layer of filter paper.Residues were transferred and rinsed with absolute ethanol (10 mL) and acetone (30 mL).The filtrate was mixed with acetone (300 mL), 10.27 M [32% (m/v)] hydrochloric acid (3200 µL 32.8640 mmol), and ethyl acetate (EtOAc) (200 mL).The mixture was frozen at -25 • C for 2.5 h.Then water (200 mL) and EtOAc (1000 mL) were added, and the mixture was shaken vigorously for 1 min and was frozen at -25 • C for 2.5 h.Afterward, 10.27 M [32% (m/v)] hydrochloric acid (3000 µL 30.8100 mmol) was added, and the mixture was shaken vigorously for 1 min and was additionally frozen at -25 • C for 30 min.The upper phase was then decanted, and the lower aqueous phase was isolated.The isolated upper EtOAc phase was extracted with water (90 mL), and the aqueous phase was isolated after phase separation and combined with the first aqueous phase.Finally, the isolated upper EtOAc phase was re-extracted with water (100 mL), which was acidified with 3000 µL 10.27 M [32% (m/v)] hydrochloric acid (30.8100 mmol).The aqueous phase was isolated after phase separation and was combined with the two prior aqueous phases.The combined aqueous phases (V = 500 mL) were evaporated in vacuo at the lowest possible temperature to a volume of ca.200 mL until heavy crystallization started.The crystallizing suspension was then cooled at +0-2 • C for 6 h and frozen at −25 • C for 20 min to complete crystallization.The evolved first yield (1.543 g) of the white, fine needles was filtered and dried over CaCl 2 in vacuo.The filtrate was additionally cooled at +0-2 • C for 50 h.The evolved second yield (62 mg) of the white, fine needles was filtered and dried over CaCl 2 in vacuo.Masses of 5.00 g (-)-colchicine sesquihydrate (× 1½ H 2 O) (M = 426.46g/mol, 11.72 mmol) and 1.08 g thiosemicarbazide (M = 91.13g/mol, 11.85 mmol) were dissolved in 25 mL of 90% (v/v) aqueous ethanol by refluxing (5 min).Then, a solution of 0.48 g sodium hydroxide (12.00 mmol) in 2 mL of water was added, and the deep orange-red solution was refluxed for 5 min.The cold, deep orange-red solution, after cooling at −25 • C for 20 min, was titrated by dropwise addition of 1.1 mL of 10.27 M [32% (m/v)] hydrochloric acid (11.30mmol), which was diluted with 2 mL of water.Successively, the volume of the solution was reduced in vacuo by one-half to a volume of 15 mL.The reddish-brown solution was mixed with 100 mL of water and was titrated with 1.1 mL of 10.27 M [32% (m/v)] hydrochloric acid (11.30mmol), which was diluted with 2 mL of water.The oily emulsion was extracted with 50 mL of ethyl acetate (EtOAc) in a separation funnel.The separated aqueous phase (pH 2) was additionally extracted with 40 mL of EtOAc.After neutralization of this aqueous phase with sodium hydrogen carbonate NaHCO 3 , the aqueous phase (pH 7-8) was extracted twice with 40 mL of EtOAc each.The EtOAc phases were combined and washed twice with 100 mL of water each.The washed EtOAc phase, which already precipitated, was mixed with 50 mL of acetone and was frozen at −25 • C for 10 h.If precipitation did not start spontaneously, the volume of the solution was reduced in vacuo until coagulation started.The evolved bright yellow, fine crystalline precipitate of compound 2 was filtered (1.01 g) and dried over CaCl 2 in vacuo.From the combined aqueous phases by cooling in the refrigerator (+0-2 • C, 2 days), a second yield of compound 2 could be obtained (1.75 g).It was combined with the main yield.

2. 2 .
Compound 2 2.2.1.The Synthesis of Colchic(in)oid Compound 2 [PT166 (NSC 750423)] Compound 2 was registered by the National Cancer Institute (NCI) as NSC 750423.Molecules 2024, 29, x FOR PEER REVIEW 9 of 47 the 10-methoxy group in colchicine.The point of connection is the terminal nitrogen of the hydrazinyl moiety of thiosemicarbazide.Compound 2 was registered by the National Cancer Institute (NCI) as NSC 750423.

47 Figure 13 .
Figure 13.The 499.86 MHz 1 H-NMR spectrum of newly dried (CaCl2, in vacuo, 19 h) compound 3 in DMSO-d6 after storing the substance over six years at +0-4 °C in the refrigerator.The spectrum was measured at ϑ = 35 °C on a Varian Unity INOVA 500 instrument.Yellow marked are the solvent DMSO-d6 residual resonance [DMSO] and the water broad singlet resonance at δ 3.25 ppm [Water].Only traces of the utilized synthesis solvent acetone (singlet, δ 2.08 ppm) are present.

Figure 13 .
Figure 13.The 499.86 MHz 1 H-NMR spectrum of newly dried (CaCl 2 , in vacuo, 19 h) compound 3 in DMSO-d 6 after storing the substance over six years at +0-4 • C in the refrigerator.The spectrum was measured at ϑ = 35 • C on a Varian Unity INOVA 500 instrument.Yellow marked are the solvent DMSO-d 6 residual resonance [DMSO] and the water broad singlet resonance at δ 3.25 ppm [Water].Only traces of the utilized synthesis solvent acetone (singlet, δ 2.08 ppm) are present.

Figure 13 .
Figure 13.The 499.86 MHz 1 H-NMR spectrum of newly dried (CaCl2, in vacuo, 19 h) com in DMSO-d6 after storing the substance over six years at +0-4 °C in the refrigerator.The s was measured at ϑ = 35 °C on a Varian Unity INOVA 500 instrument.Yellow marked are th DMSO-d6 residual resonance [DMSO] and the water broad singlet resonance at δ 3.25 ppm Only traces of the utilized synthesis solvent acetone (singlet, δ 2.08 ppm) are present.

Figure 14 .
Figure 14.The electron transfer in compound 3 yields a partially paramagnetic molecular The colchic(in)oid part of the molecule picks up one electron from the ylide monocation, y

Figure 15 .
Figure 15.(A) The total ion current (TIC) chromatogram of the liquid chromatographic (HPLC)/electrospray ionization (ESI) time-of-flight (ToF) mass spectrometric investigation of compound 3 after storing the substance over six years at +0-4 °C in the refrigerator.The high-performance liquid chromatography (HPLC) of compound 3 was performed on a reversed-phase C8 (RP8, n-octyl) column with gradient elution.Indicated are the presumed multiple ionic species of compound 3 (see Table2), which were separated due to the complicated ionization kinetics of the macromolecular substance compound 3 dissolved in acetonitrile with the presence of 0.1% (v/v) HCOOH in the HPLC eluents.(B) The analogous liquid chromatographic (HPLC) investigation of compound 3 with UV detection at λ = 335 nm.

Figure 15 .
Figure 15.(A) The total ion current (TIC) chromatogram of the liquid chromatographic (HPLC)/ electrospray ionization (ESI) time-of-flight (ToF) mass spectrometric investigation of compound 3 after storing the substance over six years at +0-4 • C in the refrigerator.The high-performance liquid chromatography (HPLC) of compound 3 was performed on a reversed-phase C 8 (RP8, n-octyl) column with gradient elution.Indicated are the presumed multiple ionic species of compound 3 (see Table2), which were separated due to the complicated ionization kinetics of the macromolecular substance compound 3 dissolved in acetonitrile with the presence of 0.1% (v/v) HCOOH in the HPLC eluents.(B) The analogous liquid chromatographic (HPLC) investigation of compound 3 with UV detection at λ = 335 nm.

2. 4 .
NCI 60 Cell Five-Dose Screen with the Drugs Compound 1, Compound 2, and Compound 3 2.4.1.National Cancer Institute (NCI) Developmental Therapeutics Program (DTP) 60-Cancer Cell 5-Dose Testing The NCI DTP 60-cancer cell 5-dose testing is a free-of-charge, standardized oncologic test platform offered publicly by NCI as part of the National Institutes of Health (NIH) and financed by the United States of America government (U.S.Department of

2. 4 .
NCI 60 Cell Five-Dose Screen with the Drugs Compound 1, Compound 2, and 3 2.4.1.National Cancer Institute (NCI) Developmental Therapeutics Program (DTP) 60-Cancer Cell 5-Dose Testing The NCI DTP 60-cancer cell 5-dose testing is a free-of-charge, standardized oncologic test platform offered publicly by NCI as part of the National Institutes of Health (NIH) and financed by the United States of America government (U.S.Department of Health and Human Services).The results of the antineoplastic in vitro screening on 60 standard tumor cell lines of test substances delivered to NCI DTP are obtained as a summary consisting of four pages, which are described here:

1 , 2 , and Compound 3 Compound 1
Percent of the drug's most inhibited cell line The Range is defined as: Range = (Growth Percent of the drug's least inhibited cell line − Mean) + Delta 2.4.2.Overall NCI 60 Cell Five-Dose Screen Results with the Drugs Compound Compound NSC 796018), compound 2 (PT166, NSC 750423), and compound 3 (PT167, NSC 799315) were screened in the NCI DTP 60-cancer cell 5-dose testing program.The results are summarized in Table

Figure 18 .
Figure 18.Graphic presentation of the antineoplastic activity exhibited by the pure drug compound 1 (PT162, NSC 796018) in the National Cancer Institute (NCI) Developmental Therapeutics Program (DTP) 60-cancer cell 5-dose testing with the inhibition curves for sixty human tumor cell lines arranged/ordered in terms of general tumor type.2.4.4.National Cancer Institute (NCI) Developmental Therapeutics Program (DTP) 60-Cancer Cell 5-Dose Testing Results with Compound 2 The four pages of the National Cancer Institute (NCI) Developmental Therapeutics Program (DTP) 60-cancer cell 5-dose testing results for compound 2 (PT166, NSC 750423) are given in succession.First page (Figure S24): graphic allover presentation of second page with All Cell Lines in one graphic.Second page (Figure 19): inhibition curves for tumor cell lines arranged/ordered for general tumor type.Third page (Figure S25): the mean optical densities of the utilized vital stain sulforhodamine B (SRB) retained in the cells, the actual used concentrations in log10 units for five doses, and the Percent Growth are given.The GI50 (Growth Inhibition 50%), TGI (Total Growth Inhibition = 0% Growth), and LC50 (Lethal Concentration 50% = −50% Growth) are given in linear concentration units.Fourth page (Figure S26): this is the summary of the results expressed in log10 units.The defined tumor inhibition criterion (GI50, TGI, or LC50) is recalculated in log10 expression, and the colored bars, which indicate then the sensitivity of the individual tumor cell line to the agent, are given in log10 units.

Figure 18 .
Figure 18.Graphic presentation of the antineoplastic activity exhibited by the pure drug compound 1 (PT162, NSC 796018) in the National Cancer Institute (NCI) Developmental Therapeutics Program (DTP) 60-cancer cell 5-dose testing with the inhibition curves for sixty human tumor cell lines arranged/ordered in terms of general tumor type.2.4.4.National Cancer Institute (NCI) Developmental Therapeutics Program (DTP) 60-Cancer Cell 5-Dose Testing Results with Compound 2 The four pages of the National Cancer Institute (NCI) Developmental Therapeutics Program (DTP) 60-cancer cell 5-dose testing results for compound 2 (PT166, NSC 750423) are given in succession.First page (Figure S24): graphic allover presentation of second page with All Cell Lines in one graphic.Second page (Figure 19): inhibition curves for tumor cell lines arranged/ordered for general tumor type.Third page (Figure S25): the mean optical densities of the utilized vital stain sulforhodamine B (SRB) retained in the cells, the actual used concentrations in log 10 units for five doses, and the Percent Growth are given.The GI50 (Growth Inhibition 50%), TGI (Total Growth Inhibition = 0% Growth), and LC50 (Lethal Concentration 50% = −50% Growth) are given in linear concentration units.Fourth page (Figure S26): this is the summary of the results expressed in log 10 units.The defined tumor inhibition criterion (GI50, TGI, or LC50) is recalculated in log 10 expression, and the colored bars, which indicate then the sensitivity of the individual tumor cell line to the agent, are given in log 10 units.

Figure 19 .
Figure 19.Graphic presentation of the antineoplastic activity exhibited by the drug compound 2 (PT166, NSC 750423) in the National Cancer Institute (NCI) Developmental Therapeutics Program (DTP) 60-cancer cell 5-dose testing with the inhibition curves for sixty human tumor cell lines arranged/ordered in terms of general tumor type.2.4.5.National Cancer Institute (NCI) Developmental Therapeutics Program (DTP) 60-Cancer Cell 5-Dose Testing Results with Compound 3 The four pages of the National Cancer Institute (NCI) Developmental Therapeutics Program (DTP) 60-cancer cell 5-dose testing results for compound 3 (PT167, NSC 799315) are given in succession.First page (Figure S27): graphic allover presentation of second page with All Cell Lines in one graphic.Second page (Figure 20): inhibition curves for tumor cell lines arranged/ordered for general tumor type.Third page (Figure S28): the mean optical densities of the utilized vital stain sulforhodamine B (SRB) retained in the cells, the actual used concentrations in log10 units for five doses, and the Percent Growth are given.The GI50 (Growth Inhibition 50%), TGI (Total Growth Inhibition = 0% Growth), and LC50 (Lethal Concentration 50% = −50% Growth) are given in linear concentration units.Fourth page (Figure S29): this is the summary of the results expressed in log10 units.The defined tumor inhibition criterion (GI50, TGI, or LC50) is recalculated in log10 expression, and the colored bars, which indicate then the sensitivity of the individual tumor cell line to the agent, are given in log10 units.

Figure 19 .
Figure 19.Graphic presentation of the antineoplastic activity exhibited by the drug compound 2 (PT166, NSC 750423) in the National Cancer Institute (NCI) Developmental Therapeutics Program (DTP) 60-cancer cell 5-dose testing with the inhibition curves for sixty human tumor cell lines arranged/ordered in terms of general tumor type.2.4.5.National Cancer Institute (NCI) Developmental Therapeutics Program (DTP) 60-Cancer Cell 5-Dose Testing Results with Compound 3 The four pages of the National Cancer Institute (NCI) Developmental Therapeutics Program (DTP) 60-cancer cell 5-dose testing results for compound 3 (PT167, NSC 799315) are given in succession.First page (Figure S27): graphic allover presentation of second page with All Cell Lines in one graphic.Second page (Figure 20): inhibition curves for tumor cell lines arranged/ordered for general tumor type.Third page (Figure S28): the mean optical densities of the utilized vital stain sulforhodamine B (SRB) retained in the cells, the actual used concentrations in log 10 units for five doses, and the Percent Growth are given.The GI50 (Growth Inhibition 50%), TGI (Total Growth Inhibition = 0% Growth), and LC50 (Lethal Concentration 50% = −50% Growth) are given in linear concentration units.Fourth page (Figure S29): this is the summary of the results expressed in log 10 units.The defined tumor inhibition criterion (GI50, TGI, or LC50) is recalculated in log 10 expression, and the colored bars, which indicate then the sensitivity of the individual tumor cell line to the agent, are given in log 10 units.

Figure 20 .
Figure 20.Graphic presentation of the antineoplastic activity exhibited by the drug compound 3 (PT167, NSC 799315) in the National Cancer Institute (NCI) Developmental Therapeutics Program (DTP) 60-cancer cell 5-dose testing with the inhibition curves for sixty human tumor cell lines arranged/ordered in terms of general tumor type.

Figure 20 .
Figure 20.Graphic presentation of the antineoplastic activity exhibited by the drug compound 3 (PT167, NSC 799315) in the National Cancer Institute (NCI) Developmental Therapeutics Program (DTP) 60-cancer cell 5-dose testing with the inhibition curves for sixty human tumor cell lines arranged/ordered in terms of general tumor type.

2. 5 .
Cytochrome c Assay (Mitochondrial and Cytosolic) with the Drugs Compound 1, Compound 2, and Compound 3 in the Human Prostate Cancer Cell Lines PC-3 and DU-145 To assess the nature of the cancer cell death-inducing effect of the drugs compound 1 (PT162, NSC 796018), compound 2 (PT166, NSC 750423), and compound 3 (PT167, NSC 799315), and to verify the failure to induce cancer cell death of compound 2, the cellular compartment of cytochrome c in the prostate cancer cell lines PC-3 and DU-145 under the action of compound 1, compound 2, and compound 3 was investigated (Figure 21).Compound 1 readily induced cytochrome c translocation from mitochondria into the cytosol at 25.0 µM concentration in PC-3 cells (Figure 21A) and 5.0 µM concentration in DU-145 cells (Figure 21B).

Figure 21 .
Figure 21.Compound 1 (PT162, NSC 796018) and compound 3 (PT167, NSC 799315) induce cytochrome c translocation from mitochondria into the cytosol, a hallmark of the intrinsic pathway of apoptosis, whereas compound 2 (PT166, NSC 750423) fails to induce apoptotic cytochrome c translocation.The cytochrome c assays (mitochondrial and cytosolic) were performed with compounds 1, 2, and 3 in the human prostate cancer cell lines PC-3 and DU-145.The protein content for each cellular fraction was calculated to determine the mass content of cytochrome c in each fraction (pg cytochrome c/mg total protein).The mass content of cytochrome c (pg cytochrome c/mg total protein) was normalized towards the corresponding blank DMSO control and is given in percent (%) of blank DMSO control.Each experiment was representative of the effects of compound 1, compound 2, and compound 3 in the human prostate cancer cell lines PC-3 and DU-145.(A) Experiment compound 1 in PC-3 cells.(B) Experiment compound 1 in DU-145 cells.(C) Experiment compound 2 in PC-3 cells.(D) Experiment compound 2 in DU-145 cells.(E) Experiment compound 3 in PC-3 cells.(F) Experiment compound 3 in DU-145 cells.

Figure 21 .
Figure 21.Compound 1 (PT162, NSC 796018) and compound 3 (PT167, NSC 799315) induce cytochrome c translocation from mitochondria into the cytosol, a hallmark of the intrinsic pathway of apoptosis, whereas compound 2 (PT166, NSC 750423) fails to induce apoptotic cytochrome c translocation.The cytochrome c assays (mitochondrial and cytosolic) were performed with compounds 1, 2, and 3 in the human prostate cancer cell lines PC-3 and DU-145.The protein content for each cellular fraction was calculated to determine the mass content of cytochrome c in each fraction (pg cytochrome c/mg total protein).The mass content of cytochrome c (pg cytochrome c/mg total protein) was normalized towards the corresponding blank DMSO control and is given in percent (%) of blank DMSO control.Each experiment was representative of the effects of compound 1, compound 2, and compound 3 in the human prostate cancer cell lines PC-3 and DU-145.(A) Experiment compound 1 in PC-3 cells.(B) Experiment compound 1 in DU-145 cells.(C) Experiment compound 2 in PC-3 cells.(D) Experiment compound 2 in DU-145 cells.(E) Experiment compound 3 in PC-3 cells.(F) Experiment compound 3 in DU-145 cells.