1. Introduction

2. Chemistry

2.1. Compound 1

2.1.1. The Synthesis of Salt-Containing Compound 1 (PENTA)

To find a potential complexation and/or stablization partner for retinazone [24,25] , an attempt was made to synthesize a polyammonium polycation from the adamantane [26] -derived influenza A virus inhibitor [22] and N-methyl- D -aspartate (NMDA) subtype glutamate receptor antagonist [21] amantadine × HCl. For that purpose 1-aminoadamantane hydrochloride and an 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 condensor. 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).

2.1.2. 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-m-xylene) 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 successful synthesis of compound 1 was to utilize 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 synthesis of compound 1.

Figure 1. The organic chemical structure formula of compound 1 [PT162 (NSC 796018)]. Four adamantan-1-ammonium cages are included within the four-bladed propeller-shaped polyammonium cation.

Figure 1. The organic chemical structure formula of compound 1 [PT162 (NSC 796018)]. Four adamantan-1-ammonium cages are included within the four-bladed propeller-shaped polyammonium cation.

2.1.3. Structure Elucidation of Compound 1 [PT162 (NSC 796018)]

According to the structure elucidation by nuclear magnetic resonance (NMR) spectroscopy compound 1 [PT162 (NSC 796018)] was formed from the intermediate N-[3-(chloromethyl)benzyl]-1-adamantanamine (Scheme 1). The latter intermediate quaternarized 1-aminoadamantane to give N,N,N-tris{3-[(tricyclo[3.3.1.1^3,7]decan-1-ylamino)methyl]benzyl}-1-adamantanammonium hydroxide (Scheme 1), which underwent Hofmann elimination [27] with 1-adamantanol being expelled. 1-Adamantanol is produced from adamantene, a known reaction intermediate [28–30]. The resulting tris{3-[(tricyclo[3.3.1.1^3,7]decan-1-ylamino)methyl]benzyl}amine in turn was quaternarized by another N-[3-(chloromethyl)benzyl]-1-adamantanamine to yield, after acidification with HCl, tetrakis{3-[(tricyclo[3.3.1.1^3,7]decan-1-ammonio)methyl]benzyl}ammonium pentachloride (compound 1) (Scheme 1).

The structure of salt-containing compound 1 (PENTA) was secured by analysis of its ¹H- (Figure S1) and ¹³C-NMR (Figure S2) spectra recorded in DMSO-d₆. The proton NMR of the aliphatic part of salt-containing compound 1 (PENTA) was analysed for (Figure S1): δ 1.62 (3 H, d; ²J_gem = −12.1 Hz; δ-CH_axial), 1.69 (3 H, d; ²J_gem = −12.4 Hz; δ-CH_equatorial), 2.02 (6 H, s; β-CH₂), 2.15 (3 H, s; γ-CH), 4.10 (2 H, t; ³J_vicinal = 6.3 Hz; 8-CH₂), 4.78 (2 H, s; 7-CH₂). The proton NMR of the aromatic part of salt-containing compound 1 (PENTA) was analysed for (Figure S1): δ 7.42‒7.49 (2 H, m; H-4, H-6), 7.65 (1 H, d; ³J_ortho = 7.1 Hz; H-5), 7.69 (1 H, s; H-2). The three axial δ-protons of the adamantane cage were split from the corresponding three equatorial δ-protons. A broad ammonium resonance was found at δ 9.24 (2 H, br s; 8-NH₂⁺ ammonium). The aliphatic resonances contained an adamantane part, two methylene groups and an ammonium resonance in the integrated proton ratio 15 : 2 : 2 : 2.

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). Obviously, the ammonium resonance originated from an (1-adamantyl-NH₂-R)⁺ system. Therefore, only two reasonable possibilities for the structure of compound 1 (PT162) remained. The first would be di{3-[(tricyclo[3.3.1.1^3,7]decan-1-ammonio)methyl]benzyl}ether dichloride (Figure 2), the second being expressed by the already given formula (Figure 1). The former structure could be ruled out for the following reasons: (i) the oxygen elemental analysis of salt-containing compound 1 (PENTA) showed only trace amounts of oxygen (w ≤ 0.82%) resulting from the ethanol and acetone traces contained in salt-containing compound 1 (PENTA), (ii) the absence of a commonly very strong dibenzyl ether band in the FT‒IR spectrum (Figure S3) expected at wavenumber 1090 cm^-1 for dibenzyl ether [31], (iii) the downfield methylene δ 4.78 (2 H, s; 7-CH₂) did not match the typical dibenzyl ether chemical shift found at δ 4.54 ppm (in CDCl₃) [32], and (iv) the analysis of the carbon-13 NMR spectrum of salt-containing compound 1 (PENTA) (Figure S2). The ¹³C-NMR of the aliphatic part of salt-containing compound 1 (PENTA) was analysed for (Figure S2): δ 28.50 (γ-CH), 35.25 (δ-CH₂), 37.35 (β-CH₂), 42.31 (8-CH₂), 45.84 (7-CH₂), 57.06 (α-C). The ¹³C-NMR of the aromatic part of salt-containing compound 1 (PENTA) was analysed for (Figure S2): δ 128.87 (C-4)*, 129.14 (C-6)*, 130.36 (C-2)*, 130.62 (C-5)*, 133.22 (C-3), 137.84 (C-1). The starred assignments are tentative and interchangeable (they could not be assigned unequivocally to the individual carbons). Clearly the structure proof could be gained by looking at the downfield methylene δ 45.84 (7-CH₂) which did not fit the typical dibenzyl ether α-CH₂ chemical shift found at δ 72.10 ppm (in CDCl₃) [32]. The 100.62 MHz ¹³C-Distortionless Enhancement by Polarization Transfer Including Detection of Quaternary Nuclei (DEPTQ) [33,34] (DEPTQ ¹³C-NMR) subspectrum (in DMSO-d₆) of salt-containing compound 1 (PENTA) (Figure S4) secured the given CH₂ assignments, and proved that the resonance at δ 57.06 ppm originated from a quaternary carbon. The assignments were further verified by analysis of the gradient-selected Correlation Spectroscopy (gs-COSY) two-dimensional ¹H−¹H-correlation spectrum [34] (Figure S5), the gradient-selected Heteronuclear Multiple Quantum Coherence (gs-HMQC) [34] (Figure S6), and the gradient-selected Heteronuclear Multiple Bond Correlation (gs-HMBC) [34] (Figure S7) two-dimensional ¹H‒¹³C-correlation spectra of salt-containing compound 1 (PENTA).

Scheme 1. The synthesis of the polyammonium polycation compound 1 from 1-aminoadamantane × HCl and 1,3-bis(chloromethyl)benzene. The intermediate N-[3-(chloromethyl)benzyl]-1-adamantanamine reacted with amantadine × HCl under influence of NaOH to N,N,N-tris{3-[(tricyclo[3.3.1.1^3,7]decan-1-ylamino)methyl]benzyl}-1-adamantanammonium hydroxide, which in turn underwent Hofmann elimination [27] on heating at reflux under elimination of 1-adamantanol (produced from adamantene [28−30]). Compound 1 resulted from quaternarization with another molecule N-[3-(chloromethyl)benzyl]-1-adamantanamine and acidification with hydrochloric acid (HCl).

Scheme 1. The synthesis of the polyammonium polycation compound 1 from 1-aminoadamantane × HCl and 1,3-bis(chloromethyl)benzene. The intermediate N-[3-(chloromethyl)benzyl]-1-adamantanamine reacted with amantadine × HCl under influence of NaOH to N,N,N-tris{3-[(tricyclo[3.3.1.1^3,7]decan-1-ylamino)methyl]benzyl}-1-adamantanammonium hydroxide, which in turn underwent Hofmann elimination [27] on heating at reflux under elimination of 1-adamantanol (produced from adamantene [28−30]). Compound 1 resulted from quaternarization with another molecule N-[3-(chloromethyl)benzyl]-1-adamantanamine and acidification with hydrochloric acid (HCl).

Figure 2. The structure of the dibenzyl ether derivative di{3-[(tricyclo[3.3.1.1^3,7]decan-1-ammonio)methyl]benzyl}ether dichloride. This hypothetical structure was considered as alternative reaction product in the synthesis of compound 1.

Figure 2. The structure of the dibenzyl ether derivative di{3-[(tricyclo[3.3.1.1^3,7]decan-1-ammonio)methyl]benzyl}ether dichloride. This hypothetical structure was considered as alternative reaction product in the synthesis of compound 1.

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 to these peaks could be found in both spectra. These absorptions mainly could 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 C₇₂H₁₀₀N₅Cl₅ × 1.5 NaCl. The reason for this 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 [35]. It could be speculated that the sodium content (NaCl) in salt-containing compound 1 (PENTA) led to 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 ¹H-NMR (Figure 3) and ¹³C-DEPTQ (data not shown) spectroscopy experiments, as well as elemental analysis and FT–IR spectroscopy. By ¹H-NMR, through the integration of the proton resonance peaks, and by ¹³C-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 which 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.

Figure 3. The 700.43 MHz ¹H-NMR spectrum (in DMSO-d₆) of the pure polyammonium polycation tetrakis{3-[(tricyclo[3.3.1.1^3,7] decan-1-ammonio)methyl]benzyl}ammonium pentachloride = pure (salt-free) compound 1 [PT162 (NSC 796018)] [DMSO-d₅h₁ quintet at δ 2.50 ppm, ²J_gem (²H−¹H) = +1.9 Hz; water at δ 3.35 ppm].

Figure 3. The 700.43 MHz ¹H-NMR spectrum (in DMSO-d₆) of the pure polyammonium polycation tetrakis{3-[(tricyclo[3.3.1.1^3,7] decan-1-ammonio)methyl]benzyl}ammonium pentachloride = pure (salt-free) compound 1 [PT162 (NSC 796018)] [DMSO-d₅h₁ quintet at δ 2.50 ppm, ²J_gem (²H−¹H) = +1.9 Hz; water at δ 3.35 ppm].

2.2. Compound 2

2.2.1. The Synthesis of Colchic(in)oid Compound 2 [PT166 (NSC 750423)]

Compound 2 [PT166 (NSC 750423)] was synthesized from (–)-colchicine sesquihydrate (× 1½ H₂O) and thiosemicarbazide under catalysis of sodium hydroxide (NaOH) in refluxing 90% (v/v) aqueous ethanol. The structure (Figure 4) of compound 2 was secured by X-ray crystallography (see Section 2.2.3.), ¹H-NMR (Figure 5) and ¹³C-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. Compound 2 was registered by National Cancer Institute (NCI) as NSC 750423.

Figure 4. The organic chemical structure formula of compound 2 [PT166 (NSC 750423)]. It contains a water of crystallization and ⅔ (ethyl acetate).

Figure 4. The organic chemical structure formula of compound 2 [PT166 (NSC 750423)]. It contains a water of crystallization and ⅔ (ethyl acetate).

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 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 was 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 ¹H-NMR spectroscopy. The representative ¹H-NMR spectrum of compound 2 in DMSO-d₆ is pictured (Figure 5).

Scheme 2. The synthesis of the modified colchic(in)oid 10-(2-carbamothioylhydrazinyl)-10-demethoxycolchicine monohydrate × ⅔ (ethyl acetate) (compound 2, PT166, NSC 750423) by NaOH-catalysed amidation (amino-de-alkoxylation) of the vinylogous (tropolonic) carboxylic acid methyl ester (−)-colchicine. Methanol was split off by thiosemicarbazide (TSC) from (−)-colchicine during short refluxing. Subsequently, the product compound 2 was liberated from a monosodium salt intermediate by acidification with hydrochloric acid and extraction with ethyl acetate. The term colchic(in)oid is now well accepted and documented in literature [36].

Scheme 2. The synthesis of the modified colchic(in)oid 10-(2-carbamothioylhydrazinyl)-10-demethoxycolchicine monohydrate × ⅔ (ethyl acetate) (compound 2, PT166, NSC 750423) by NaOH-catalysed amidation (amino-de-alkoxylation) of the vinylogous (tropolonic) carboxylic acid methyl ester (−)-colchicine. Methanol was split off by thiosemicarbazide (TSC) from (−)-colchicine during short refluxing. Subsequently, the product compound 2 was liberated from a monosodium salt intermediate by acidification with hydrochloric acid and extraction with ethyl acetate. The term colchic(in)oid is now well accepted and documented in literature [36].

Figure 5. The 400.13 MHz ¹H-NMR spectrum (in DMSO-d₆) [DMSO-d₅h₁ quintet at δ 2.50 ppm, ²J_gem (²H−¹H) = +1.9 Hz; water at δ 3.32 ppm] of the colchic(in)oid 10-(2-carbamothioylhydrazinyl)-10-demethoxycolchicine monohydrate × ⅔ (ethyl acetate) = compound 2 [PT166 (NSC 750423)]. Downfield of δ 11 ppm no resonances were detected.

Figure 5. The 400.13 MHz ¹H-NMR spectrum (in DMSO-d₆) [DMSO-d₅h₁ quintet at δ 2.50 ppm, ²J_gem (²H−¹H) = +1.9 Hz; water at δ 3.32 ppm] of the colchic(in)oid 10-(2-carbamothioylhydrazinyl)-10-demethoxycolchicine monohydrate × ⅔ (ethyl acetate) = compound 2 [PT166 (NSC 750423)]. Downfield of δ 11 ppm no resonances were detected.

2.2.2. The Nuclear Magnetic Resonance Spectra of Compound 2

It is known that colchic(in)oids [36] have the tendency to retain solvents, like water [37] and/or ethyl acetate [38], very firmly. Natural (–)-colchicine itself retained chloroform [39,40,41], dibromomethane/diiodomethane [42], or water as dihydrate [42,43] or sesquihydrate [41,44]. Therefore, it could be understood that compound 2 was obtained as monohydrate × ⅔ (ethyl acetate) binary solvate, as judged by ¹H-NMR and elemental analysis.

The ¹H-NMR resonances of the colchic(in)oid compound 2 were assigned with help of literature [45,46,47,48], especially [46] which gave a complete assignment of the protons in the ¹H-NMR spectrum of (–)-colchicine (in CDCl₃). Compound 2 represents a completely new compound, never synthesized before [according to Chemical Abstracts Service (CAS) SciFinder®]. Therefore, the proton NMR spectrum (Figure 5) of compound 2 was interpreted by own effort 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; ³J = 7.1 Hz; O–CH₂–CH₃ ethyl acetate), 1.85 (1 H, m; H_A-6), 1.86 (3 H, s; 17-CH₃), 1.99 (1.5 H, s; ROOC–CH₃ ethyl acetate), 2.05 (1 H, m; H_B-6), 2.19 (1 H, m; H_A-5), 2.57 (1 H, m; H_B-5), 3.51 (3 H, s; 13-OCH₃)*, 3.79 (3 H, s; 15-OCH₃)*, 3.83 (3 H, s; 14-OCH₃)*, 4.03 (1 H, q; ³J = 7.1 Hz; O–CH₂–CH₃ ethyl acetate), 4.37 (1 H, m; H-7). The three starred assignments (*) are tentative and interchangeable [they could not be assigned unequivocally to the individual methoxyl protons, because their chemical shifts did nearly coincide (Figure 5)]. The gradient-selected Correlation Spectroscopy (gs-COSY) two-dimensional ¹H−¹H-correlation spectrum [34] proton–proton couplings (data not shown) in connection with the gradient-selected Heteronuclear Multiple Quantum Coherence (gs-HMQC) [34] ¹³C–¹H couplings (data not shown) gave the required informations to assign the proton resonances of compound 2. Aromatic or troponic protons in compound 2 were identified as: δ 6.60 (1 H, d; ³J = 11.1 Hz; H-11), 6.76 (1 H, s; H-4), 7.14 (1 H, s; H-8), 7.20 (1 H, d; ³J = 10.9 Hz; H-12). The acetamide N–H, which coupled to H-7, could be recognized at δ 8.56 (1 H, d; ³J = 7.6 Hz; N–H acetamide). Exchangeable protons of the thiosemicarbazide moiety, detectable in DMSO-d₆, could be unequivocally assigned as: δ 7.56 (1 H, br s; H₂N–C=S amino, 4′-H_A), 7.96 (1 H, br s; H₂N–C=S amino, 4′-H_B), 9.06 (1 H, s; 1′-N–H), 9.59 (1 H, s; 2′-N–H). In the gradient-selected Correlation Spectroscopy (gs-COSY) two-dimensional ¹H−¹H-correlation spectrum [34] (data not shown) of compound 2 (in DMSO-d₆) no W-shaped long-range ⁴J (¹H–¹H) coupling, known as zig-zag (W) coupling, was found. This differentiates compound 2 from the thiosemicarbazones (E)-4-(dimethylamino)benzaldehyde thiosemicarbazone [24] and (E)-4-bromo-2-fluorobenzaldehyde thiosemicarbazone [24], where such a “W” coupling was observed [24]. This pointed to sterical fixation as 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, leading occasionally to the rearrangement products allocolchicine (colchicic acid methyl ester) [49,50] or colchicic acid (allocolchiceine) [51], 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 [50] and Fernholz [51].

Scheme 3. The possible side product in the synthesis of compound 2, the allocolchiceine (syn. colchinoic acid, colchicinoic acid, colchicic acid) monosodium salt, originating from hydrolytic ring C contraction by a benzilic-type rearrangement.

Scheme 3. The possible side product in the synthesis of compound 2, the allocolchiceine (syn. colchinoic acid, colchicinoic acid, colchicic acid) monosodium salt, originating from hydrolytic ring C contraction by a benzilic-type rearrangement.

The ¹³C-NMR spectrum of compound 2 in DMSO-d₆ was interpreted with help of literature data on (–)-colchicine [46] and (–)-colchiceine [52]. In addition, own experimental observations were applied in the following aliphatic carbon assignments: δ 14.05 (O–CH₂–CH₃ ethyl acetate), 20.72 (ROOC–CH₃ ethyl acetate), 22.49 (C-17, CH₃ acetamide), 29.33 (C-5), 36.34 (C-6), 51.38 (C-7), 55.84 (14-OCH₃)**, 59.72 (O–CH₂–CH₃ ethyl acetate), 60.62 (13-OCH₃, 15-OCH₃)**. The two double-starred assignments (**) are tentative and interchangeable (they could not be assigned unequivocally to the individual carbons). The aromatic and troponic carbon resonances, the carbonyl and the thiocarbonyl resonances, were detected as: δ 107.61 (C-4), 108.27 (C-11), 126.23 (C-8), 131.57 (C-1a), 134.26 (C-4a), 137.21 (C-12), 140.71 (C-3)***, 150.34 (C-1)***, 150.40 (C-10), 150.46 (C-12a), 152.61 (C-2)***, 152.73 (C-7a), 168.39 (C-16, HN–C=O acetamide), 170.30 (C=O ester carbonyl, ethyl acetate), 174.81 (C-9, C=O carbonyl), 181.86 (C-3′, C=S thiocarbonyl). The three triple-starred resonances (***) could not being assigned unequivocally to their individual carbon.

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 [53,54], 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 [55,56]. The reason for the latter irregularity could be the special tropylium oxide resonance type of tropones and tropolones [57,58,59,60,61]. Therefore, the synthesis of compound 2 clearly obeyed the common rules for chemical reactivity of tropolones, whereas the synthesis of the cyclic thiourea congener [53,54] of compound 2 did not follow the common chemical reactivity experience for tropolones.

The Fourier–transform infrared (FT–IR) absorption spectra of the colchic(in)oid 10-(2-carbamothioylhydrazinyl)-10-demethoxycolchicine monohydrate × ⅔ (ethyl acetate) = compound 2, and of the reference substance (−)-colchicine sesquihydrate [(−)-colchicine × 1½ H₂O] are given in the Supplementary Information (Figure S8) for comparison.

Interestingly, the natural colchic(in)oids (–)-colchicine, and the partialsynthetic (–)-colchicine derivative N-acetylcolchinol methyl ether (Figure 6), occur in pure atropisomeric forms (Figure 6), as was elucidated by Brossi et al. [62,63]. The natural forms have the (aS,7S)-absolute configuration (Figure 6). The correct assignment of the absolute configuration of (–)-colchicine was given as (aS,7S) by Brossi et al. [63] according to the Cahn–Ingold–Prelog (CIP) rules [64,65]. The wrong (aR,7S)-absolute configuration was firstly postulated in 1981 during studies on tubulin binding by (–)-colchicine [66], and later in 1999 by Berg & Bladh [67]. The absolute configuration at C-7 was established earlier as (7S) [68] by chemical degradation of natural (–)-colchicine to N-acetyl-L-glutamic acid.

Figure 6. The atropisomerism of natural (–)-colchicine and its degradation product N-acetylcolchinol methyl ether. Both exhibit the (aS,7S)-absolute configuration, as was proved by Brossi et al. [62,63].

Figure 6. The atropisomerism of natural (–)-colchicine and its degradation product N-acetylcolchinol methyl ether. Both exhibit the (aS,7S)-absolute configuration, as was proved by Brossi et al. [62,63].

Taken together, the structure of the modified colchic(in)oid compound 2 could be proved with considerable evidence, and biological effects, especially antineoplastic properties, are expected from biological testing of compound 2. Renewed interest in colchic(in)oid research is indicated by reports on conjugating (–)-colchicine to vitamin B₁₂ (cobalamin) [69] or paclitaxel (taxol) [70]. These colchic(in)oid conjugates were suggested for the chemotherapy of various neoplastic conditions.

2.2.3. 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 ϑ = 100 K) of the crystal and molecular structure of compound 2 (Figure 7, Figure 8, Figure 9, Figure S9, Figure S10). Compound 2 crystallized in the monoclinic space group P2₁ with ethyl acetate and water of crystallization [C₂₂H₂₆N₄O₅S × 1.5 H₂O × 0.5 (C₄H₈O₂)] (Z = 4) (Figure 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-(2-carbamothioylhydrazinyl)-1,2,3-trimethoxy-9-oxo-5,6,7,9-tetrahydrobenzo[a]heptalen-7-yl]acetamide (Figure 9).

Figure 7. The molecular view of one independent, isolated molecule of compound 2 as found in the single crystal. Compound 2 crystallized in the monoclinic space group P2₁ as sesquihydrate hemi(ethyl acetate) solvate (C₂₄H₃₃N₄O₇.₅₀S) [C₂₂H₂₆N₄O₅S × 1.5 H₂O × 0.5 (C₄H₈O₂)] (Z = 4). Compound 2 is helical stereogenic and shows the (M)-helicity. The plane of the thiourea moiety in the 10-(thiosemicarbazide) substituent shows +119.1° torsion angle towards the 2-aminotropone plane.

Figure 7. The molecular view of one independent, isolated molecule of compound 2 as found in the single crystal. Compound 2 crystallized in the monoclinic space group P2₁ as sesquihydrate hemi(ethyl acetate) solvate (C₂₄H₃₃N₄O₇.₅₀S) [C₂₂H₂₆N₄O₅S × 1.5 H₂O × 0.5 (C₄H₈O₂)] (Z = 4). Compound 2 is helical stereogenic and shows the (M)-helicity. The plane of the thiourea moiety in the 10-(thiosemicarbazide) substituent shows +119.1° torsion angle towards the 2-aminotropone plane.

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 [67]. The classification of (M)-helicity for compound 2 followed the Cahn–Ingold–Prelog (CIP) rules for assignment of molecular helicity [64,65]. The (M)-helicity of (–)-colchicine was previously assigned correctly by Brossi et al. [63]. The X-ray crystallographic structure was deposited at The Cambridge Crystallographic Data Centre (CCDC) and assigned the deposition № CCDC 1839505 (ID: RIVGOW). The crystal data of the X-ray crystallographic determination of the crystal and molecular structure of compound 2 are tabulated (Table 1).

Figure 8. Crystal packing in the monoclinic (space group P2₁) unit cell (Z = 2) of compound 2 crystallized as hydrate (ethyl acetate) solvate (2 C₂₂H₂₆N₄O₅S × 3 H₂O × C₄H₈O₂) C₄₈H₆₆N₈O₁₅S₂ (M = 1059.21 g/mol). Unit cell dimensions (a, b, c are indicated from the origin o): a = 9.1886(5) Å, b = 20.9047(10) Å, c = 13.9841(7) Å, α = 90.00°, β = 106.153(2)°, γ = 90.00°, V = 2580.1(2) ų.

Figure 8. Crystal packing in the monoclinic (space group P2₁) unit cell (Z = 2) of compound 2 crystallized as hydrate (ethyl acetate) solvate (2 C₂₂H₂₆N₄O₅S × 3 H₂O × C₄H₈O₂) C₄₈H₆₆N₈O₁₅S₂ (M = 1059.21 g/mol). Unit cell dimensions (a, b, c are indicated from the origin o): a = 9.1886(5) Å, b = 20.9047(10) Å, c = 13.9841(7) Å, α = 90.00°, β = 106.153(2)°, γ = 90.00°, V = 2580.1(2) ų.

Figure 9. The helical axis atropisomerism view of one independent, isolated molecule of compound 2 as found in the single crystal. Compound 2, the colchic(in)oid N-[(aS,7S)-10-(2-carbamothioylhydrazinyl)-1,2,3-trimethoxy-9-oxo-5,6,7,9-tetrahydrobenzo[a]heptalen-7-yl]acetamide, is helical stereogenic and shows the [M(inus)]-helicity. Priority rules [64,65] show the helical axis turn in (S).

Figure 9. The helical axis atropisomerism view of one independent, isolated molecule of compound 2 as found in the single crystal. Compound 2, the colchic(in)oid N-[(aS,7S)-10-(2-carbamothioylhydrazinyl)-1,2,3-trimethoxy-9-oxo-5,6,7,9-tetrahydrobenzo[a]heptalen-7-yl]acetamide, is helical stereogenic and shows the [M(inus)]-helicity. Priority rules [64,65] show the helical axis turn in (S).

Table 1. X-ray crystallographic data for compound 2, deposited and published as CSD Communication № CCDC 1839505 (ID: RIVGOW).

Table 1. X-ray crystallographic data for compound 2, deposited and published as CSD Communication № CCDC 1839505 (ID: RIVGOW).

Empirical formula	C48H66N8O15S2
Formula weight, Mr (g·mol–1)	1059.21
Temperature, T (K)	100(2)
Radiation, λ (Å)	MoK 0.71073
Crystal system	Monoclinic
Space group	P21
Unit cell dimensions
a (Å)	9.1886(5)
b (Å)	20.9047(10)
c (Å)	13.9841(7)
α (°)	90.00
β (°)	106.153(2)
γ (°)	90.00
Unit cell volume, V (Å3)	2580.1(2)
Formula units per unit cell, Z	2
Calculated density, ρcalc (Mg·m–3)	1.363
Absorption coefficient, μ (mm–1)	0.178
F(000)	1124
Theta (ϑ) range for collection	1.80 to 26.05°
Reflections collected	49598
Independent reflections	9686
Minimum / maximum transmission, Tmin / Tmax	0.9193 / 0.9929
Absorption correction	Multi-scan, SADABS 2008/1 (G.M. Sheldrick, 2008)
Refinement method	Full-matrix least-squares on F2
Data / parameters / restraints	9686 / 693 / 5
Goodness−of−fit on F2, S	1.021
Flack parameter, x	0.09(6)
Final R indices [I > 2σ(I)]	R1 = 0.0506, wR2 = 0.1269
R indices (all data)	R1 = 0.0594, wR2 = 0.1325
Maximum / minimum residual electron density, Δρmax / Δρmin (e·Å–3)	0.747 / –0.496

Summary of the crystal data for compound 2: C₂₄H₃₃N₄O₇.₅₀S [C₂₂H₂₆N₄O₅S × 1½ H₂O × ½ (C₄H₈O₂)], M_r = 529.61 g/mol, colorless plate, 0.48 × 0.31 × 0.04 mm³, monoclinic space group P2₁, a = 9.1886(5) Å, b = 20.9047(10) Å, c = 13.9841(7) Å, β = 106.153(2)°, V = 2580.1(2) ų, Z = 4, ρ_calcd = 1.363 g·cm^–3, μ = 0.178 mm^–1, F(000) = 1124, T = 100(2) K, x_Flack = 0.09(6), R₁ = 0.0594, wR² = 0.1325, 9686 independent reflections [2ϑ ≤ 52.1°] and 693 parameters. Computer programs utilized: APEX2 ver. 2008.3 (Bruker AXS, 2008), Saint+ ver. 7.53A (Bruker AXS, 2008), SHELXS97 (G.M. Sheldrick, 2008), SHELXL97 (G.M. Sheldrick, 2008), XP ver. 5.1 (Bruker AXS, 1998).

2.3. Compound 3

2.3.1. The Synthesis of Compound 3 [PT167 (NSC 799315)]

Compound 3 [PT167 (NSC 799315)] (Figure 10) was synthesized from compound 1 [PT162 (NSC 796018)] and compound 2 [PT166 (NSC 750423)] by trans-(thio)amidation catalysed by sodium hydroxide (NaOH) at room temperature. A similar trans-(thio)amidation originating from a thiosemicarbazide or thiosemicarbazone moiety was observed previously in the synthesis of retinazone, a retinoid thiosemicarbazone derivative [24,25]. As a result compound 2 was connected to compound 1 through a thioamide-bonding at an adamantanamine nitrogen (Scheme 4). The molecular stoichiometry of compound 3 was determined by ¹H-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 ¹H-NMR spectrum of compound 3 (Figure 11) exhibits a peculiar resonance compression [the adamantane resonances 2.00 (24 H; β-CH₂), 2.14 (12 H; γ-CH) could not being 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 by 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.

Figure 10. The organic chemical structure formula of compound 3 [PT167 (NSC 799315)] = C₁₁₆H₁₄₂N₁₁O₁₀S₂⁺ × Cl⁻ × 5 H₂O = C₁₁₆H₁₅₂ClN₁₁O₁₅S₂ (M = 2040.10 g/mol). The compound 3 contains one chloride anion (Cl⁻) and five water of crystallization (× 5 H₂O).

Figure 10. The organic chemical structure formula of compound 3 [PT167 (NSC 799315)] = C₁₁₆H₁₄₂N₁₁O₁₀S₂⁺ × Cl⁻ × 5 H₂O = C₁₁₆H₁₅₂ClN₁₁O₁₅S₂ (M = 2040.10 g/mol). The compound 3 contains one chloride anion (Cl⁻) and five water of crystallization (× 5 H₂O).

Figure 11. The 700.43 MHz ¹H-NMR spectrum of compound 3 = C₁₁₆H₁₄₂N₁₁O₁₀S₂⁺ × Cl⁻ × 5 H₂O = C₁₁₆H₁₅₂ClN₁₁O₁₅S₂ (M = 2040.10 g/mol) dissolved in DMSO-d₆. Yellow marked is the solvent DMSO-d₆ residual resonance [DMSO-d₅h₁ quintet at δ 2.50 ppm, ²J_gem (²H−¹H) = +1.9 Hz] [DMSO]. The water broad singlet resonance is found at δ 3.32 ppm. Traces of the utilized synthesis solvents ethanol (triplet, δ 1.05 ppm; quartet, δ 3.44 ppm) and acetone (singlet, δ 2.09 ppm) can be detected.

Figure 11. The 700.43 MHz ¹H-NMR spectrum of compound 3 = C₁₁₆H₁₄₂N₁₁O₁₀S₂⁺ × Cl⁻ × 5 H₂O = C₁₁₆H₁₅₂ClN₁₁O₁₅S₂ (M = 2040.10 g/mol) dissolved in DMSO-d₆. Yellow marked is the solvent DMSO-d₆ residual resonance [DMSO-d₅h₁ quintet at δ 2.50 ppm, ²J_gem (²H−¹H) = +1.9 Hz] [DMSO]. The water broad singlet resonance is found at δ 3.32 ppm. Traces of the utilized synthesis solvents ethanol (triplet, δ 1.05 ppm; quartet, δ 3.44 ppm) and acetone (singlet, δ 2.09 ppm) can be detected.

Figure 12. Molecular modeling {ACD/Chem Sketch version 12.01 with integrated ACD/3D Viewer (Advanced Chemistry Development, Inc., Toronto, Ontario, Canada) and processed with Mercury 3.1 version 3.1.1 [The Cambridge Crystallographic Data Centre (CCDC), Cambridge, UK]} of compound 3 = C₁₁₆H₁₄₂N₁₁O₁₀S₂⁺ × Cl⁻ × 5 H₂O = C₁₁₆H₁₅₂ClN₁₁O₁₅S₂ (M = 2040.10 g/mol) without the chloride counteranion (Cl⁻) and the water of crystallization (pentahydrate). 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.

Figure 12. Molecular modeling {ACD/Chem Sketch version 12.01 with integrated ACD/3D Viewer (Advanced Chemistry Development, Inc., Toronto, Ontario, Canada) and processed with Mercury 3.1 version 3.1.1 [The Cambridge Crystallographic Data Centre (CCDC), Cambridge, UK]} of compound 3 = C₁₁₆H₁₄₂N₁₁O₁₀S₂⁺ × Cl⁻ × 5 H₂O = C₁₁₆H₁₅₂ClN₁₁O₁₅S₂ (M = 2040.10 g/mol) without the chloride counteranion (Cl⁻) and the water of crystallization (pentahydrate). 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.

2.3.2. The Proton Nuclear Magnetic Resonance Spectrum (¹H-NMR) of Freshly Synthesized Compound 3

Evidence for the formulation of compound 3 as the macromolecular entity [(bis{3-[(tricyclo[3.3.1.1^3,7]decan-1-ylamino)methyl]benzyl}ammonio)bis(methanediylbenzene-3,1-diylmethanediyl)]di-2-[(aS,7S)-7-(acetylamino)-1,2,3-trimethoxy-9-oxo-5,6,7,9-tetrahydrobenzo[a]heptalen-10-yl]-N-(tricyclo[3.3.1.1^3,7]decan-1-yl)hydrazinecarbothioamide chloride pentahydrate stems from the proton NMR (¹H-NMR) spectrum of compound 3 [PT167 (NSC 799315)] dissolved in deuterated dimethyl sulfoxide (DMSO-d₆) (Figure 11).

The proton resonances were measured as chemical shifts δ (ppm): δ 1.48–1.68 (24 H, br m; δ-CH₂, adamantane), 1.83 (2 H, m; H_A-6, colch), 1.85 (6 H, s; 17-CH₃, colch), 2.01 (2 H, m; H_B-6, colch), 2.17 (2 H, m; H_A-5, colch), 2.52 (2 H, m; H_B-5, colch), 3.48 (6 H, s; 13-OCH₃, colch)*, 3.65 (2 H, br s; secondary amine N–H), 3.77 (6 H, s; 15-OCH₃, colch)*, 3.82 (6 H, s; 14-OCH₃, colch)*, 4.26 (br m; 8-CH₂, m-xylylene), 4.32‒4.39 (2 H, br m; H-7, colch), 4.76 (s; 7-CH₂, m-xylylene), 6.73 (2 H, s; H-4, colch), 7.03 (2 H, br m; H-11, colch), 7.06 (2 H, s; H-8, colch), 7.12‒7.72 (br m; H-4, H-6, H-5, H-2, m-xylylene), 7.17 (2 H, br m; H-12, colch), 8.54 (2 H, d; ³J = 7.7 Hz; N–H acetamide, colch), 9.59 (4 H, s; 1′-N–H, 2′-N–H, hydrazinecarbothioamide) [colch = the colchic(in)oid part of compound 3; * these assignments are tentative and interchangeable (they could not be assigned unequivocally to the individual methoxy groups); the adamantane resonances δ 2.00 ppm (β-CH₂) and 2.14 ppm (γ-CH) were not detected due to paramagnetic resonance compression].

Scheme 4. The synthesis of compound 3 = C₁₁₆H₁₄₂N₁₁O₁₀S₂⁺ × Cl⁻ × 5 H₂O = C₁₁₆H₁₅₂ClN₁₁O₁₅S₂ (M = 2040.10 g/mol) by trans-(thio)amidation [24,25] catalysed by sodium hydroxide (NaOH) at room temperature from compound 1 and compound 2.

Scheme 4. The synthesis of compound 3 = C₁₁₆H₁₄₂N₁₁O₁₀S₂⁺ × Cl⁻ × 5 H₂O = C₁₁₆H₁₅₂ClN₁₁O₁₅S₂ (M = 2040.10 g/mol) by trans-(thio)amidation [24,25] catalysed by sodium hydroxide (NaOH) at room temperature from compound 1 and compound 2.

2.3.3. The Proton Nuclear Magnetic Resonance Spectrum (¹H-NMR) of Compound 3 after Six Year Storage at +0−4 °C in the Refrigerator

The specimen of compound 3 which was synthesized at Saturday, May 27^th, 2017, was investigated by proton NMR (Figure 13) at Wednesday, August 23^rd, 2023, after over six years storage at +0−4 °C in the refrigerator. The specimen was newly dried over CaCl₂ in vacuo for 19 h. The proton resonances were measured as chemical shifts δ (ppm): δ 1.14 (1 H, s; hydroxytropyl radical O–H), 1.45–1.81 (24 H, br m; δ-CH₂, adamantane), 1.83 (2 H, m; H_A-6, colch), 1.85 (6 H, s; 17-CH₃, colch), 1.91 (2 H, m; H_B-6, colch), 2.02–2.14 (12 H, br m; γ-CH), 2.18 (2 H, br m; H_A-5, colch), 2.55 (2 H, br m; H_B-5, colch), 2.86–3.08 [7 H, br s; 7-CH₂, m-xylylene CH₂ including ammonium ylide R−(CH⁻)N⁺(CH₂R)₃], 3.49 (6 H, s; 13-OCH₃, colch)*, 3.67 (1 H, s; secondary amine N–H), 3.78 (6 H, s; 15-OCH₃, colch)*, 3.82 (6 H, s; 14-OCH₃, colch)*, 4.26‒4.39 (8 H, br m; H-7, colch; 3 × 8-CH₂ m-xylylene), 4.76 (2 H, s; 1 × 8-CH₂, m-xylylene at 8-NH^+• radical cation), 6.72 (2 H, s; H-4, colch), 6.99 (2 H, br s; H-11, colch), 7.06 (2 H, s; H-8, colch), 7.17 (2 H, br s; H-12, colch), 7.30‒7.37 (12 H, br m; H-4, H-6, H-2, m-xylylene), 7.51 (4 H, br s; H-5, m-xylylene), 8.14 (1 H, s; 8-NH^+• radical cation N–H), 8.48 (2 H, d; ³J = 7.9 Hz; N–H acetamide, colch), 9.56 (4 H, br s; 1′-N–H, 2′-N–H, hydrazinecarbothioamide) [colch = the colchic(in)oid part of compound 3; * these assignments are tentative and interchangeable (they could not be assigned unequivocally to the individual methoxy groups); the adamantane β-CH₂ resonance δ 2.00 ppm was not detected due to paramagnetic resonance compression].

The ¹H-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 (Figure 13) convincingly proves that (i) the substance is very pure even after six years storage, (ii) exhibits the chemical structure given by me, and (iii) points to proton transfer during storage yielding the ylide monocation (ylide monohydrochloride) with discernible resonances (7 H) for the ylide R−(CH⁻)N⁺(CH₂R)₃.

This substance bears a peculiar magnetic property. During NMR spectrometer shim it was detected that newly dried (CaCl₂, 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-d₆ solution of newly dried compound 3 had to be strongly diluted with DMSO-d₆, and that the spectrum acquisition time had to be elongated considerably. This pointed to inclusion of a paramagnetic partial structure in newly dried compound 3. Indeed, the ¹H-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) [71,72] producing in consequence the 8-NH^+• radical cation (Figure 14, in red).

Figure 13. The 499.86 MHz ¹H-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. The 499.86 MHz ¹H-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 14. The electron transfer in compound 3 yielding a partially paramagnetic molecular species. The colchic(in)oid part of the molecule picks up one electron from the ylide monocation yielding a resonance-stabilized 1-hydroxycyclohepta-2,4,6-trien-1-yl radical (hydroxytropyl radical) [71,72] producing the 8-NH^+• radical cation.

Figure 14. The electron transfer in compound 3 yielding a partially paramagnetic molecular species. The colchic(in)oid part of the molecule picks up one electron from the ylide monocation yielding a resonance-stabilized 1-hydroxycyclohepta-2,4,6-trien-1-yl radical (hydroxytropyl radical) [71,72] producing the 8-NH^+• radical cation.

2.3.4. The Liquid Chromatographic (HPLC) Investigation of Compound 3

The specimen of compound 3 which was synthesized at Saturday, May 27^th, 2017, was investigated at Friday, July 21^st, 2023, after over six years storage at +0−4 °C in the 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 t_0min = 95% eluent A/5% eluent B to t_13min = 5% eluent A/95% eluent B, t_16min = stop. A 5 µl volume of a 50 µM compound 3 solution in acetonitrile (N≡C−CH₃) was injected (0.25 nmol, 510.025 ng).

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 Table 2) which were separated due to the complicated ionization kinetics of the macromolecular substance compound 3 dissolved in acetonitrile with 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. (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 Table 2) which were separated due to the complicated ionization kinetics of the macromolecular substance compound 3 dissolved in acetonitrile with 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.

The total ion current (TIC) chromatogram is shown in Figure 15A, 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 presence of 0.1% (v/v) HCOOH. The quaternary ammonium compound 3 can be protonated once or twice, or being not protonated like the in situ substance (Table 2). Moreover, compound 3 can exist in equilibrium as a neutral nitrogen ylide [73,74,75] at the central quaternary ammonium cation. This nitrogen ylide can be protonated once or twice, or being 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).

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.

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.

HPLC peak (min)	Peak identity	ESI−MS fragment peaks (m/z) (relative peak intensity of the 100% base peak)
7.842−7.925	ammonium trication	290.1670 (100%), 678.3662 (12.5%), 135.1157 (9.6%)
8.508−8.558	ammonium dication	712.3545 (100%), 123.0807 (16.5%), 356.6799 (2.1%), 483.2713 (1.3%)
8.608−8.658	ammonium monocation	712.3550 (100%), 543.3511 (27.9%), 356.6804 (18.1%), 123.0806 (14.2%), 653.3701 (5.4%), 483.2714 (2.5%), 409.2404 (1.7%)
8.691−8.741	ammonium monocation	712.3558 (100%), 356.6814 (19.1%), 578.2448 (6.3%), 135.1165 (5.8%)
9.024−9.074	ylide dication	483.2727 (100%), 965.5391 (80.9%), 712.3534 (19.5%), 123.0798 (14.1%), 356.6793 (5.0%), 831.4260 (4.3%), 577.2110 (3.4%), 213.1784 (2.3%)
9.740−9.973	ylide monocation	227.2000 (100%), 123.0804 (47.2%), 609.8644 (31.0%), 712.3522 (6.6%), 398.7700 (5.8%), 1218.7191 (5.0%), 796.5327 (4.0%), 483.2712 (2.5%)
13.386−13.453	neutral ylide	123.0805 (100%), 637.3055 (45.6%), 227.1988 (7.1%), 439.2038 (5.8%), 712.3521 (3.2%)

2.3.5. The Electrospray Ionization (ESI) Mass Spectrometric Investigation of Compound 3 after HPLC Separation

The six structure-proofing fragment cations in the electrospray ionization (ESI) time−of−flight (ToF) mass spectrometry of compound 3 (Figure S11−S20) following HPLC separation on a RP8 column (LC/MS coupling) are shown in Figure 16 (left, dications; right, monocations). The cations (C₅₈H₇₄N₆O₅S)²⁺ m/z 483.2727 (100%) (generated from ylide dication), (C₅₈H₇₃N₆O₅S)⁺ m/z 965.5391 (80.9%) (generated from ylide dication), (C₇₄H₈₉N₇O₇S)²⁺ m/z 609.8644 (31.0%) (generated from ylide monocation), (C₇₄H₈₈N₇O₇S)⁺ m/z 1218.7191 (5.0%) (generated from ylide monocation), (C₄₀H₄₉N₄O₆S)²⁺ m/z 356.6814 (19.1%) (generated from ammonium monocation), and (C₄₀H₅₀N₅O₅S)⁺ m/z 712.3550 (100%) (generated from ammonium monocation) are the major, structure-proofing fragments of compound 3 created in the ESI−ToF mass spectrometer under the fragmentor voltage V_f = 175 V. The molecule cation (C₁₁₆H₁₄₂N₁₁O₁₀S₂)⁺ m/z 1913.0382 (or a higher protonated form) was not observed due to extensive molecule fragmentation.

The two peaks (C₇₄H₈₉N₇O₇S)²⁺ m/z 609.8644 (31.0%) (generated from ylide monocation) and (C₇₄H₈₈N₇O₇S)⁺ m/z 1218.7191 (5.0%) (generated from ylide monocation) (Figure 16) are to be formulated as ammonia (NH₃) coordination-stabilized [73,74] nitrogen ylides [75] being in equilibrium with a mass spectrometric generated species exhibiting pentavalent nitrogen [76,77,78] according to Figure 17. The fragment cations (C₁₈H₂₁NNaO)⁺ m/z 290.17 [(3-{[(adamantan-1-yl)imino-κN]methyl}benzaldehyde-κO)sodium(1+)], (C₈H₇O₂)⁺ m/z 135.12 [protonated isophthal(di)aldehyde], (C₈H₁₁O)⁺ m/z 123.08 [protonated m-xylyl alcohol (3-methylbenzyl alcohol)], and (C₁₆H₂₁N)^+• m/z 227.20 [N-phenyladamantan-1-amine radical cation], accompany the greater fragments pointing to extensive fragmentation force under the fragmentor voltage V_f = 175 V. The N-phenyladamantan-1-amine radical cation results from 1-{[(adamantan-1-yl)amino]methyliumyl}cyclopenta-2,4-dien-1-ide (C₁₆H₂₁N, a zwitterionic fulvene) created from the benzyl species N-{[3-(aminomethyl)phenyl]methyl}adamantan-1-amine (C₁₈H₂₆N₂) through loss of C₂H₅N and fulvene−to−benzene rearrangement [79,80].

In summary, the chemical structure of compound 3 could be substantiated as derivative of colchiceine hydrazide (10-hydrazinyl-10-demethoxycolchicine) [53,54] bridged over a thiocarbonyl to the compound 1 core, being quite pure. None compound 2 and compound 1, the synthesis starting substances, or colchiceine hydrazide, a possible degradation product, could be detected in the compound 3 preparation by highly sensitive detection methods.

Figure 16. The six structure-proofing fragment cations (left, dications; right, monocations) in the 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 m/z 483.2727 (100%) (generated from ylide dication), m/z 965.5391 (80.9%) (generated from ylide dication), m/z 609.8644 (31.0%) (generated from ylide monocation), m/z 1218.7191 (5.0%) (generated from ylide monocation), m/z 356.6814 (19.1%) (generated from ammonium monocation), and m/z 712.3550 (100%) (generated from ammonium monocation) are the major, structure-proofing fragments of compound 3.

Figure 16. The six structure-proofing fragment cations (left, dications; right, monocations) in the 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 m/z 483.2727 (100%) (generated from ylide dication), m/z 965.5391 (80.9%) (generated from ylide dication), m/z 609.8644 (31.0%) (generated from ylide monocation), m/z 1218.7191 (5.0%) (generated from ylide monocation), m/z 356.6814 (19.1%) (generated from ammonium monocation), and m/z 712.3550 (100%) (generated from ammonium monocation) are the major, structure-proofing fragments of compound 3.

Figure 17. The two cations (C₇₄H₈₉N₇O₇S)²⁺ m/z 609.8644 (31.0%) [generated from of compound 3 ylide monocation] and (C₇₄H₈₈N₇O₇S)⁺ m/z 1218.7191 (5.0%) [generated from of compound 3 ylide monocation] (see Figure 16) are to be formulated as ammonia (NH₃)-stabilized [73,74] nitrogen ylides [75] (left, in blue) being in equilibrium with a mass spectrometric enabled species characterized by twice pentavalent nitrogen [76−78] (right, in blue).

Figure 17. The two cations (C₇₄H₈₉N₇O₇S)²⁺ m/z 609.8644 (31.0%) [generated from of compound 3 ylide monocation] and (C₇₄H₈₈N₇O₇S)⁺ m/z 1218.7191 (5.0%) [generated from of compound 3 ylide monocation] (see Figure 16) are to be formulated as ammonia (NH₃)-stabilized [73,74] nitrogen ylides [75] (left, in blue) being in equilibrium with a mass spectrometric enabled species characterized by twice pentavalent nitrogen [76−78] (right, in blue).

3. Biological Activities

3.1. NCI 60 Cell Five-Dose Screen with the Drugs Compound 1, Compound 2 and Compound 3

3.1.1. National Cancer Institute (NCI) Developmental Therapeutics Program (DTP) 60-Cancer Cell 5-Dose Testing

First page: graphic allover presentation of second page with All Cell Lines in one graphic and molar concentrations expressed in logarithmic log₁₀ unit. The earlier the colored curves aspire to the bottom, the more potent is the drug. The more the curves strive from ±0% to –100%, the more the tumor is actually 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 log₁₀ units (−9 = 1 nM, −8 = 10 nM, −7 = 100 nM, −6 = 1 µM, −5 = 10 µM, −4 = 100 µM, −3 = 1,000 µM = 1 mM). The Percentage Growth are 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 is the tumor inhibition. Generally, it is not sufficient to reach only 0% Growth, since the tumor is still there. Ideal is –100% Growth, since the tumor cells were completely killed by the drug, but only 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 used concentrations in log₁₀ unit are given for five doses (Note: mostly these are not smooth values, but technically created values). Then the Mean Optical Densities of the used vital stain sulforhodamine B (SRB) retained in the cells are given. The higher the SRB optical density, the more is 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 > 1.00E–X (for example: > 1.00E–4 = > 100 µM) means that the corresponding defined criterion is not reached by the drug = failure to reach the defind tumor inhibition criterion (GI50, TGI, or LC50). The NSC number of the test drug is given at 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 log₁₀ units. The defined tumor inhibition criterion (GI50, TGI, or LC50) is recalculated in log₁₀ expression, and the the colored bars which indicate then the sensitivity of the individual tumor cell line to the agent are given in log₁₀ 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 Log₁₀GI50, Log₁₀TGI and Log₁₀LC50. From that values the corresponding MID is calculated. The more negative the MID, the more potent is 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 log₁₀ unit:

The Delta is defined as:

Delta = Mean — Growth 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

3.1.2. Overall NCI 60 Cell Five-Dose Screen Results with the Drugs Compound 1, Compound 2 and Compound 3

Compound 1 (PT162, 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 the Table 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 15.849 µM (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 [81,82].

3.1.3. 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 log₁₀ unit 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 log₁₀ units. The defined tumor inhibition criterion (GI50, TGI, or LC50) is recalculated in log₁₀ expression, and the the colored bars which indicate then the sensitivity of the individual tumor cell line to the agent are given in log₁₀ units.

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.

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.

3.1.4. 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 unit 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 the colored bars which indicate then the sensitivity of the individual tumor cell line to the agent are given in log₁₀ units.

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.

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.

3.1.4. 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 unit 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 the colored bars which indicate then the sensitivity of the individual tumor cell line to the agent are given in log₁₀ units.

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. 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.

3.1.5. 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. 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 compound 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 for 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. 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 compound 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 for 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.

Compound 2 failed to induce a significant difference in the cytochrome c-residing cellular compartment in PC-3 cells (Figure 21C) and, less significant, 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.

3.1.6. HIV-1_LAI Replication Reverse Transcriptase Assay with the Drug Compound 1 in Primary PBL Cells

Compound 1 (PT162, NSC 796018) is inhibitory towards human immunodeficieny virus type 1 (HIV-1) strain LAI (HIV-1_LAI) (= HIV-1_BRU = LAV-1) replication in freshly explanted, primary human peripheral blood lymphocytes (PBL cells) (Table 3). The effective inhibitory concentration 50% (EC₅₀) in PBL cells was 0.56 µM, the effective inhibitory concentration 90% (EC₉₀) in PBL cells was 4.3 µM. The cyctotoxic concentration 50% (CC₅₀) for the PBL cells was 2.2 µM, this yields a selectivity index 50% (SI₅₀) = CC₅₀/EC₅₀ of 3.9. The r² coefficient of determination (r² measure of goodness–of‒fit) on EC₅₀ and EC₉₀ was 0.93. As a positive control served AZT (zidovudine, 3′-azido-3′-deoxythymidine). The given effective inhibitory concentrations (μM ± s.d.) for the positive control AZT were averaged and treated statistically from four (n = 4) independent determinations (Table 3). Furthermore, the cytotoxicity of compound 1 (PT162, NSC 796018) towards CCRF−CEM cells (ATCC® CCL-119™, acute lymphoblastic leukemia cells, tumorigenic CD4⁺ T lymphoblasts) [83] and Vero cells [African green monkey (grivet) Chlorocebus aethiops (syn. Cercopithecus aethiops) kidney epithelial cells] was determined (Table 3). The cyctotoxic concentration 50% (CC₅₀) for the CCRF−CEM cells was < 1 µM with 60.0% growth inhibition of CCRF−CEM cells at 1 µM. The cytotoxic concentration 50% (CC₅₀) for the Vero cells was 1.8 µM.

The inhibitory action of p53 on HIV-1 replication is well established [84,85,86]. Compound 1 (PT162, NSC 796018) clearly inhibits HIV-1_LAI replication by inducing p53 and its associated cyclin-dependent kinase inhibitor p21^Waf1/p21^Cip1 [87]. The latter factor p21^Waf1/p21^Cip1 is induced by p53 activation [87] and inhibits HIV-1 replication [88,89]. In addition, tumor suppressor protein p53 interacts with HIV-1 trans-activator protein Tat (trans-activator of transcription) [90,91,92], HIV-1 Nef (negative regulatory factor) accessory protein [93], and HIV-1 Vpr (viral protein R, viral protein rapid) accessory protein [89,94]. The marked cytotoxic action of compound 1 (PT162, NSC 796018) on CCRF−CEM acute lymphoblastic leukemia cells clearly stems from its strong antileukemic/antineoplastic activity.

Table 3. Cytotoxicity and antiviral activity of tetrakis{3-[(tricyclo[3.3.1.1^3,7]decan-1-ammonio)methyl]benzyl}ammonium pentachloride (compound 1, PT162, NSC 796018) versus human immunodeficiency virus type 1 strain LAI (HIV-1_LAI) in mammalian cells.

Table 3. Cytotoxicity and antiviral activity of tetrakis{3-[(tricyclo[3.3.1.1^3,7]decan-1-ammonio)methyl]benzyl}ammonium pentachloride (compound 1, PT162, NSC 796018) versus human immunodeficiency virus type 1 strain LAI (HIV-1_LAI) in mammalian cells.

Drug	Cytotoxicity CC50 (µM) and CCRF–CEM growth inhibition at fixed 1 µM concentration (%, in parentheses)			Anti-HIV-1LAI activity EC50 (µM)/EC90 (µM) in PBM cells
	PBM cells	CCRF −CEM	Vero	EC50	EC90	SI50	r2
Compound 1 (PT162, NSC 796018)	2.2	< 1 (60.0)	1.8	0.56	4.3	3.9	0.93
AZT*	> 100	14.3	56.0	0.0044 ± 0.0018	0.031 ± 0.015	> 22,727	0.99

CC₅₀, cytotoxic concentration 50%. EC₅₀, effective inhibitory concentration 50%. EC₉₀, effective inhibitory concentration 90%. SI₅₀, selectivity index CC₅₀/EC₅₀. r², coefficient of determination (r² measure of goodness–of‒fit) on EC₅₀ and EC₉₀. AZT, zidovudine (3′-azido-3′-deoxythymidine). * The given effective inhibitory concentrations (µM ± s.d.) for the positive control AZT were averaged and treated statistically from four (n = 4) independent determinations.

4. Experimental Section

4.4. Chemical Synthesis

4.4.1. Salt-Containing Tetrakis{3-[(tricyclo[3.3.1.1^3,7]decan-1-ammonio)methyl]benzyl}ammonium Pentachloride (Compound 1 × 1.5 NaCl, PT162 × 1.5 NaCl)

2.50 g 1-Aminoadamantane hydrochloride (13.32 mmol) and 3.50 g 1,3-bis(chloromethyl)benzene (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 (40.00 mmol) in 40 ml of water was added, and the mixture was refluxed for 3 h. After 20 min and 40 min reflux, 20 ml of acetone, each, were added through the reflux condensor. After 60 min, 80 min, and 100 min reflux, 40 ml of acetone, each, were added through the reflux condensor. After 120 min reflux, additional 20 ml of acetone were added through the reflux condensor. Then, after 10 min pre-cooling at +0–2 °C, the colorless solution with few suspended impurities was warm filtrated. The filtrate (pH 9) was mixed with 100 ml of water, and was 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) were added, and the colorless solution (pH 2–3) was evaporated further in vacuo to a volume of ca. 120 ml.

Then the turbid suspension was mixed with 20 ml of water and was extracted with 100 ml of ethyl acetate (EtOAc) to remove unreacted 1,3-bis(chloromethyl)benzene (α,α′-dichloro-m-xylene). The aqueous phase was isolated and further evaporated in vacuo to a volume of ca. 50 ml. The aqueous phase was then frozen at –25 °C for 1½ h. The evolved white precipitate of crude compound 1 × 1.5 NaCl (PT162 × 1.5 NaCl) was filtered and dried over CaCl₂ in vacuo (from the filtrate additional substance could be obtained which was treated as before). 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 obtained which was treated as before.

The combined crude product (730 mg) was dissolved in 50 ml of 80% (v/v) aqueous acetone by short (5 min) refluxing, and was hot filtrated to remove few impurities. The filtrate was transferred and mixed with 40 ml of 50% (v/v) aqueous acetone. Afterwards, the filtrate was evaporated in vacuo from the acetone. The resulting solution (pH 5) was acidified to pH 0–1 by addition of 0.6 ml of 10.27 M [32% (m/v)] hydrochloric acid (6.16 mmol). Instantly, a white precipitate formed. The suspension was supplemented with 10 ml of 50% (v/v) aqueous acetone and was frozen at –25 °C for 2½ h. The evolved white precipitate of compound 1 × 1.5 NaCl (PT162 × 1.5 NaCl) was filtered and dried over CaCl₂ in vacuo.

4.4.2. Pure (Salt-Free) Tetrakis{3-[(tricyclo[3.3.1.1^3,7]decan-1-ammonio)methyl]benzyl}ammonium Pentachloride (Compound 1, PT162, NSC 796018)

1-Aminoadamantane hydrochloride (M = 187.71 g/mol, 10.075 g, 53.6732 mmol) was dissolved in water (100 ml). A solution of sodium hydroxide NaOH (2.160 g, 54.0000 mmol) in water (20 ml) was added. Residues were transferred with water (20 ml). A heavy precipitate of the free base 1-aminoadamantane formed instantly. The suspension was frozen at –25 °C for 1 h. The evolved white precipitate of the yield of 1-aminoadamantane (free base) was filtered and vacuum-sucked dry (ca. 1 h).

The still wet 1-aminoadamantane (free base) and 1,3-bis(chloromethyl)benzene (α,α′-dichloro-m-xylene) (M = 175.06 g/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. After 5 min pre-cooling at +0–2 °C, the colorless solution with few suspended impurities 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 (3,200 µl, 32.8640 mmol), and ethyl acetate (EtOAc) (200 ml), and was frozen at –25 °C for 2.5 h. Afterwards, water (200 ml) and EtOAc (1,000 ml) were added, the mixture was shaken vigorously for 1 min, and was frozen at –25 °C for 2.5 h. Then 10.27 M [32% (m/v)] hydrochloric acid (3,000 µl, 30.8100 mmol) was added, the mixture was shaken vigorously for 1 min, and it was 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 re-extracted with water (90 ml), the aqueous phase was isolated after phase separation, and was combined with the first aqueous phase. Finally, the isolated upper EtOAc phase was re-extracted with acidified {3,000 µl 10.27 M [32% (m/v)] hydrochloric acid, 30.8100 mmol} water (100 ml), 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 then evaporated in vacuo at the lowest possible temperature to a volume of 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 white crystals were filtered and dried over CaCl₂ in vacuo. The filtrate was cooled at +0–2 °C for 50 h. The evolved second yield (62 mg) of white crystals was filtered and dried over CaCl₂ in vacuo. Both yields were combined.

4.4.3. (M)-10-(2-Carbamothioylhydrazinyl)-10-demethoxycolchicine Monohydrate × ⅔ (Ethyl Acetate) = N-[(aS,7S)-10-(2-Carbamothioylhydrazinyl)-1,2,3-trimethoxy-9-oxo-5,6,7,9-tetrahydrobenzo[a]heptalen-7-yl]acetamide Monohydrate × ⅔ (Ethyl Acetate) (Compound 2, PT166, NSC 750423)

5.00 g (–)-Colchicine sesquihydrate (× 1½ H₂O) (M = 426.46 g/mol, 11.72 mmol) and 1.08 g thiosemicarbazide (M = 91.13 g/mol, 11.85 mmol) were dissolved in 25 ml of 90% (v/v) aqueous ethanol by refluxing for 5 min. After adding a solution of 0.48 g sodium hydroxide (12.00 mmol) in 2 ml of water, the deep orange-red solution was refluxed for 5 min. The cold deep orange-red solution solution, after pre-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 (11.30 mmol) which was diluted with 2 ml of water. Afterwards, the volume of the solution was reduced in vacuo approximately by one half. The reddish-brown solution was then mixed with 100 ml of water, and was titrated with 1.1 ml of 10.27 M [32% (m/v)] hydrochloric acid (11.30 mmol) which was diluted with 2 ml of water. The oily emulsion was extracted with 50 ml of ethyl acetate (EtOAc). The separated aqueous layer (pH 2) was additionally extracted with 40 ml of EtOAc. After neutralization of this aqueous phase with sodium hydrogen carbonate NaHCO₃, 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 yellow precipitate of compound 2 was filtered (1.01 g) and dried over CaCl₂ in vacuo. From the combined aqueous phases by cooling two days at +0–2 °C a second crop of compound 2 could be obtained (1.75 g). It was treated as before and combined with the main yield.

4.4.4. [(Bis{3-[(tricyclo[3.3.1.1^3,7]decan-1-ylamino)methyl]benzyl}ammonio)bis(methanediylbenzene-3,1-diylmethanediyl)]di-2-[(aS,7S)-7-(acetylamino)-1,2,3-trimethoxy-9-oxo-5,6,7,9-tetrahydrobenzo[a]heptalen-10-yl]-N-(tricyclo[3.3.1.1^3,7]decan-1-yl)hydrazinecarbothioamide Chloride Pentahydrate (Compound 3, PT167, NSC 799315) (synthesized at Saturday, May 27^th, 2017)

Materials:

• (Compound 1, PT162, NSC 796018) = tetrakis{3-[(tricyclo[3.3.1.1^3,7]decan-1-ammonio)methyl]benzyl}ammonium pentachloride (C₇₂H₁₀₀Cl₅N₅) (M = 1,212.86 g/mol) [w (n/n) ≥ 99% (¹H-NMR and elemental analysis)], synthesized at Friday, December 30^th, 2016.
• (Compound 2, PT166, NSC 750423) = N-[(7S)-10-(2-carbamothioylhydrazinyl)-1,2,3-trimethoxy-9-oxo-5,6,7,9-tetrahydrobenzo[a]heptalen-7-yl]acetamide monohydrate × ⅔ (ethyl acetate) [C₂₂H₂₆N₄O₅S × H₂O × ⅔ (C₄H₈O₂)] (M = 535.28 g/mol) [w (n/n) ≥ 98% (¹H-NMR and elemental analysis)], synthesized at Thursday, January 29^th, 2009.

Compound 1 [PT162 (NSC 796018)] (M = 1,212.86 g/mol, 300 mg, 247.3492 µmol) and compound 2 [PT166 (NSC 750423)] (M = 535.28 g/mol, 300 mg, 560.4543 µmol) were suspended in absolute ethanol (20 ml), and the yellow suspension was heated to 40–50 °C for 4 min (heatgun). Then water (1,000 µl) was added under stirring and all material dissolved to give a bright yellow solution. Afterwards, a solution of sodium hydroxide (37 mg, 925.0000 µmol) in water (2,000 µl) was added under stirring. The color of the solution changed to orange-yellow. After cooling at +0–2 °C for 12 min, the mixture was frozen at –25 °C for 10 min. After adding water (10 ml), the precipitating emulsion was frozen at –25 °C for 2 h. The evolved first yield (212 mg) of the bright yellow, amorphous substance compound 3 was filtered, carefully dried by vacuum suction for 30 min on the sintered glass filter filter, and dried over CaCl₂ in vacuo. The filtrate was transferred with water (20 ml), and was frozen at –25 °C for 30 min. After cooling at +0–2 °C for 1 h, the evolved second yield (17 mg) of the bright yellow, amorphous substance compound 3 was filtered [the initial turbid filtrate was transferred with water (10 ml), and was re-filtered on the same sintered glass filter used before], carefully dried by vacuum suction for 30 min on the sintered glass filter filter, and dried over CaCl₂ in vacuo. Both yields were combined.

5. Conclusions

Figure 22. The organic chemical structural formula of cardiolipin [1,3-bis(sn-3′-phosphatidyl)-sn-glycerol, 1,3-diphosphatidyl-sn-glycerol]. The molecule is optically active, representing the L,L-form. Pictured is the predominant dianionic species in the inner mitochondrial membrane with C_18:2 [linoleic acid, (9Z,12Z)-9,12-octadecadienoic acid] at each of the four acyl positions, as isolated from bovine heart, bovine heart cardiocyte mitochondria, and rat liver [108].

Figure 22. The organic chemical structural formula of cardiolipin [1,3-bis(sn-3′-phosphatidyl)-sn-glycerol, 1,3-diphosphatidyl-sn-glycerol]. The molecule is optically active, representing the L,L-form. Pictured is the predominant dianionic species in the inner mitochondrial membrane with C_18:2 [linoleic acid, (9Z,12Z)-9,12-octadecadienoic acid] at each of the four acyl positions, as isolated from bovine heart, bovine heart cardiocyte mitochondria, and rat liver [108].

6. Patents

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