Spectral Characterization of Prospidium Chloride Using Complementary Analytical Techniques

Abstract

The clinical efficacy of chemotherapy against rapidly proliferating cells stimulates both the development of new agents and the reassessment of established drugs. Spectroscopic methods (UV, FT-IR, and ¹H NMR) were applied to characterize prospidium chloride and related substances. The FT-IR spectrum of prospidium chloride, arising from vibrational transitions within the alkyl fragments of the dispirotripiperazinium cation, is reported with band assignments. Electronic transitions between molecular orbitals are analyzed using quantum–mechanical selection rules (Laporte and spin selection rules). The n→σ* transition (ΔS = 0) corresponds to the absorption maximum at λmax = 282 ± 0.40 nm (ε = 3.89 ± 0.08 L·mol⁻¹·cm⁻¹). A ¹H NMR spectrum (700 MHz) was used to assign chemical shifts δ (ppm), J-coupling constants (Hz), and gauche conformational features of prospidium chloride and its dihydroxy and epoxy impurities. Quantitative ¹H NMR (qNMR) was applied to determine the content of the active pharmaceutical ingredient and related substances. The methods provide complementary structural information for the characterization of prospidium chloride.

1. Introduction

Oncological diseases (from the Greek “ὄγκος”—tumor, volume, or mass, Galen, 130–200 AD) have accompanied humankind throughout its entire history [1,2,3]. The origin of the word “cancer” is attributed to the “father of medicine”, the Greek physician Hippocrates (460–370 BC), who used the terms “carcinos” and “carcinoma” (from the Greek “karkinos”—crab) to describe non-ulcerating and ulcerating tumors [4]. This unusual terminology was later explained by a physician from Alexandria (Stephanus, VII AD): “…it is called karkinos (crab) either because of the aggressivity of the animal, or because the veins located in the affected part resemble tentacles, as the legs of a crab do” [5,6]. Until 1900, surgery remained the only effective treatment modality for malignant diseases, and its success critically depended on early tumor detection [7]. At the beginning of the 20th century, radiotherapy emerged as an alternative to surgery for certain types of cancer, following the discovery of X-rays by Wilhelm Conrad Röntgen in 1895 and radioactivity by Marie and Pierre Curie [8,9]. However, the major breakthrough in cancer therapy in the mid-20th century was the advent of chemotherapy, understood as the use of specific cytostatic chemotherapeutic agents (CChtIs). The first CChtI, nitrogen mustard (NtrM), was developed in the 1940s by Yale pharmacologists Alfred Gilman and Louis Goodman based on the vesicant warfare agent Yperite (sulfur mustards (SlfM)—mustard gas) [10]. Earlier, in 1917, pathologists had reported profound bone marrow depletion in victims of sulfur mustard poisoning. Sulfur mustard was synthesized in 1916 by W. Lommel and W. Steinkopf from ethylene and sulfur chloride and was initially named “LoSt”, after the first letters of the surnames of these German chemists [11].

1.1. Mechanism of Chemotherapeutic Action

The molecule of sulfur mustard (bis(2-chloroethyl) sulfide, SlfM), a halogenated derivative of ethylsulfanylethane, is a yellow-brown, oily liquid with a mustard-like odor (boiling point 217 °C with decomposition; aqueous solubility approximately 8 × 10⁻⁴ g·mL⁻¹ at 20 °C; hydrolyzed in water within about 5 min at 37 °C) (Figure 1). Sulfur mustard a blister-causing agent, belongs to the category of chemical warfare agents (CWA) and is classified as a Group 1 carcinogen. According to the United Nations Office for Disaster Risk Reduction (UNDRR), it acts initially as an irritant and subsequently as a cellular poison upon inhalation and contact, affecting the eyes, respiratory tract, and skin [12].

Figure 1 illustrates the mechanism of the alkylating action of SlfM and the drugs synthesized on its basis as follows: depolarization of the C–Cl bond leads to heterolytic cleavage with elimination of the nucleofuge (chloride ion, Cl⁻) and the formation of one of the intermediates, either a carbocation or a sulfonium ion [SR₃]⁺. This extremely high reactivity of SlfM is associated with a nucleophilic substitution (SN) pathway and with in vivo interaction with nucleophilic (Nu) NH groups of nucleotide residues in DNA (Scheme S1) [13,14]:

S(CH₂CH₂Cl)₂ + 2NuH → S(CH₂CH₂Nu)₂ + 2HCl,

Then, an exceptionally important conclusion was reached, namely that this substance markedly suppresses rapidly dividing cells, which marked the beginning of the era of chemotherapy for malignant diseases [15]. After intravenous administration of nitrogen mustard (NtrM) to several patients with advanced-stage lymphoma (previously treated exclusively with irradiation and glucocorticoids), a rapid clinical improvement was observed which, although temporary, nevertheless prompted researchers to search for new chemotherapeutic cytotoxic/cytostatic agents (CChtIs). Since the chloroalkyl terminal fragment of the mustard molecule is considered to be the pharmacophoric group responsible for the cytostatic effect, this finding triggered the synthesis of next-generation CChtIs based on the principles of rational drug design [16,17,18] (

Table 1. Evolution of active pharmaceutical ingredients (APIs) within the group of alkylating cytostatics (AlkCyts) (the full dataset, including ethylenamine and methylenamine derivatives, alkyl sulfonates, nitrosourea derivatives, triazenes, and platinum compounds, is presented in Table S1 [19,20,21,22,23,24,25,26,27]).

Structure, IUPAC Name, Molecular Weight (g.mol−1)	Molecular Complexity (MC) *	LogP	Toxicity Data and Details [19]	Carcinogen Classification [20]
Mechlorethamine 2-chloro-N-(2-chloroethyl)-N-methylethanamine 156.05	43.7	0.91	LD50 = 10 mg/kg (oral, for a rat).	Group 2A: probably carcinogenic to humans
Cyclophosphamide N,N-bis(2-chloroethyl)-2-oxo-1,3,2λ5-oxazaphosphinan-2-amine 261.08	212	0.8	LD50 = 420 mg/kg (intraperitoneal, for a mouse); Hemorrhagic cystitis, NDI/syndrome of inappropriate antidiuretic hormone secretion (SIADH).	Group 1: carcinogenic to humans
Ifosfamide N,3-bis(2-chloroethyl)-2-oxo-1,3,2λ5-oxazaphosphinan-2-amine 261.08	218	0.86	LD50 = 415 mg/kg (intraperitoneal, for a mouse); LD50 = 2600 mg/kg (oral, for a mouse); Salt wasting, hyponatremia-SIADH, hemorrhagic cystitis, AKI (ATN), CKD, renal tubular acidosis, glomerular disease, tubulointerstitial disease.	Group 3: not classifiable regarding its carcinogenicity to humans
Melphalan (2S)-2-amino-3-[4-[bis(2-chloroethyl)amino]phenyl]propanoic acid 305.20	265	0.4	LD50 = 30 mg/kg (intraperitoneal, for a rat); Hyponatremia-SIADH, glomerular dysfunction.	Group 1: carcinogenic to humans
Chlorambucil 4-[4-[bis(2-chloroethyl)amino]phenyl]butanoic acid 304.2	250	1.7	LD50 = 14 mg/kg (intraperitoneal, for a rat); LD50 = 76 mg/kg (oral, for a rat); Acute encephalopathy, seizures.	Group 1: carcinogenic to humans

* “complexity” is a numerical descriptor of molecular structural complexity that incorporates such features as the number of sp³-hybridized carbon atoms, the number of actual stereocenters, the presence of non-aromatic rings and heavy atoms, as well as ring-system complexity and side-chain branching [28,29]. Analysis of FDA-approved drugs demonstrates an overall increase in molecular complexity over the course of drug evolution, with higher complexity values correlating with improved protein-binding properties [30].

).

According to The International Union of Basic and Clinical Pharmacology (IUPHAR)/British Pharmacological Society (BPS) Guide to Pharmacology [31,32,33], in vivo exposure to alkylating antitumor agents leads to the formation of covalent bonds via a donor–acceptor mechanism between an electron-deficient chloroalkyl carbocation and the N7 atom of guanine or the N3 atom of adenine in tumor cell DNA. As a result, intra- and intermolecular cross-links are formed, which disrupt the structure and function of DNA and ultimately trigger programmed cell death (Figure 2) [34,35].

Despite the well-established efficacy of chemotherapy against rapidly proliferating cells, the use of cytotoxic (alkylating) agents is associated with a number of serious adverse effects (see Table 1). Therefore, there is a continued need for both the development of new cytostatics and for the re-evaluation of existing agents in order to gain a more detailed understanding of their properties [36].

1.2. Prospidium Chloride as a Chemotherapeutic Alkylating Agent

The search for effective alkylating agents with low systemic toxicity and high specificity toward tumor cells led to the development of prospidin [37]. Its synthesis involves the reaction of N-benzoylpiperazine hydrochloride with 2-chloroethanol under basic conditions, followed by the addition of triethylamine and heating with hydrochloric acid to afford the intermediate N, N’-dispirotripiperazinium dichloride (DSpTPpCl₂) (Figure 3a). The subsequent alkylation of DSpTPpCl₂ with epichlorohydrin yields the final product, prospidium dichloride (PrsCl₂), a saturated, polycyclic, spiro-fused, organic compound containing two shared (spiro) nitrogen atoms (Figure 3b).

Each of the spiro atoms (⁺N6 и ⁺N9) is bound by four covalent bonds to the adjacent cyclic carbon atoms, thus forming an onium ion. The piperazine nitrogen atoms, ⁚N3 and ⁚N12, which bear a lone electron pair, confer the properties of a dibasic amine. The mutually perpendicular arrangement of the ring planes forming the dispirotripiperazinium system of PrsCl₂ could, in principle, give rise to chirality; however, due to the overall mirror-symmetric molecular structure, optical activity is canceled [38].

Prospidin, an alkylating cytostatic agent (AlkCyt), is used for the treatment of various malignant neoplasms, including laryngeal cancer, skin cancer, melanoma, Kaposi sarcoma, Zaidel’s ascitic hepatoma, and others [39]. Owing to its immunosuppressive effect, prospidium chloride (PrsCl₂) is also employed as a low-toxicity therapeutic agent in refractory rheumatoid arthritis [40]. According to data on dispirotripiperazine–core compounds [41,42], prospidin is characterized by minimal myelotoxicity and a broad spectrum of antitumor activity at relatively low reported toxicity: LD₅₀ (intraperitoneal, in rat) =1200 mg/kg [43]. These features clearly distinguish prospidin from many currently available anticancer agents and provide it with important therapeutic advantages (see Table 1 and see Table S1).

However, the structural features of dispirotripiperazine salts in general, and prospidin in particular (see Figure 3), predispose them to hydrolytic degradation and the formation of transformation products, which decrease the content of the active pharmaceutical ingredient (API) and increase the levels of related impurities [44]. This necessitates the development and application of new physicochemical, high-precision, analytical methods to confirm the identity of the API and to characterize the resulting degradation products.

The aim of this study was to develop new analytical approaches, based on spectroscopic methods, for investigating the structural and physicochemical properties of prospidium chloride, a representative dispirotripiperazine-type alkylating chemotherapeutic agent, for quality control purposes.

2. Materials and Methods

2.1. Object of Research

The object of the study was the active pharmaceutical ingredient (API) prospidium chloride monohydrate PrsCl₂·H₂O (3,12-Bis(3-chloro-2-hydroxypropyl)-3,12-diaza-6,9-diazoniadispiro[5.2.5.2]hexadecane dichloride monohydrate, Prospidine, C₁₈H₃₆C_l4N₄O₂·H₂O, Mr = 500,3); manufacturer: Scientific and Technological Park «Unitechprom BSU», Minsk, Republic of Belarus, batch No. 271222, expiry date December 2027; API content ω = 100.5% on a dried basis (see Figure 3b).

The PrsCl₂·H₂O substance is a white crystalline, hygroscopic powder that is readily soluble in water and practically insoluble in 96% ethanol and chloroform; logP = −0.3; molecular complexity is 389 [45,46].

2.2. Method of Research

2.2.1. Fourier Transform Infrared (FT-IR) Spectroscopy

The vibrational spectrum of prospidium chloride in the range 4000 to 400 cm⁻¹ was obtained using an Agilent Cary 630 FT-IR spectrophotometer (Agilent, Santa Clara, CA, USA) equipped with a diamond crystal attachment in Attenuated Total Reflectance (ATR) mode and basic software; the resolution is 2 cm⁻¹, the accuracy of the wavenumber is ±0.05 cm⁻¹, the reproducibility of the wavenumber is ±0.005 cm⁻¹, and the thickness of the absorbing layer is 1.5 nm.

Procedure: approximately 1 mg of the prospidium chloride substance was placed on the surface of the internal reflection element of the FT-IR spectrophotometer to ensure the tightest and most complete contact with the entire crystal surface, until a mechanical “click” was reached.

2.2.2. Electronic Absorbtion Spectroscopy

The electronic absorption spectra (200–350 nm) of aqueous PrsCl₂ solutions in the concentration range 0.05–2.00 mol·L⁻¹ were recorded using an Agilent Cary 60 UV–Vis spectrophotometer (Agilent, Santa Clara, CA, USA) and an analytical system based on an SF-200 spectrophotometer (OKB “Spektr”, Saint Petersburg, Russia) at the Shared Research Facilities Center of Physicochemical and Biological Studies, ARRIP at T = 22.0 ± 0.5 °C).

The molar absorptivity, as an intrinsic function of the substance, was calculated from the obtained data using the Bouguer–Lambert–Beer (BLB) law (Eqution (1)):

$$\epsilon ={\displaystyle \frac{A}{C·l}} ,$$

(1)

where A is absorbance at λ_max, nm, ε (L·mol⁻¹ cm⁻¹) is the apparent specific absorbance, c (mol·L⁻¹) is the molar concentration, and l (cm) is the path length.

2.2.3. ¹H NMR Spectroscopy*

¹H NMR spectra were recorded on an NMR Bruker Avance NEO 700 spectrometer equipped with a Prodigy Cryoprobe (Bruker, Switzerland) operating at 700 MHz for protons at 298 K under the following conditions: relaxation delay—10 s; number of data points—65,536; spectral width—22 ppm; acquisition time—2.1 s; number of scans—16; pulse angle—30°. Subsequent spectral processing and identification of the target analytes were performed using TopSpin, version 4.1.3 (Bruker, Zürich, Switzerland).

Procedure: For sample preparation, accurately weighed portions of the investigated substance samples, including related impurities (Scheme S1), of approximately 50 mg were dissolved in D₂O (deuterium oxide, deuterium content ≥ 99.8%; Solvex, Moscow, Russia; CAS 7789-20-0), followed by the addition of an accurately weighed portion of about 2.5 mg of the internal standard, sodium 3-(trimethylsilyl)propionate-d₄ TSP-d₄) (CAS № 24493-21-8, Merk, Darmstadt, Germany). The sample was agitated for 5 min in a laboratory shaker (IKA VXR S000, Staufen, Germany) and centrifuged (Eppendorf Mini Spin, Hamburg, Germany) at 14.000 rpm for 5 min. The resulting mixture was transferred to a 5 mm NMR tube for analysis.

The choice of this instrumentation is justified by the fact that the ¹H nucleus is present in almost all organic compounds, including PrsCl₂, and is the most sensitive among the NMR-active nuclei.

2.3. Loss on Drying (LOD)

Determination of loss on drying (LOD) of the substance, associated with the content of crystallization/adsorbed water and other volatile components, was performed according to [47].

Procedure: an accurately weighed portion of the PrsCl₂·H₂O substance was placed into a porcelain evaporating dish that was previously dried to constant weight. Drying was carried out at 105 ± 5 °C for 3 h in a forced-convection drying oven DION SIBLAB 500/350 (DION LLC, Novosibirsk, Russia) of the Center for Collective Use of Physico-Chemical and Biological Research of ARRIP until constant mass was achieved (Δm ≤ 0.5 mg). The mass of the dish with the sample was determined and recorded every hour by removing the dish from the oven and allowing it to cool to room temperature in a desiccator for 30 min. The LOD value (%) was calculated according to Equation (2) (

Table 2. Loss on drying of the PrsCl₂·H₂O tested substance.

t, min	m1, g	m2, g	m3, g	Loss on Drying (LOD), %
180	39.9535	40.1948	40.18425	4.37

):

$$\mathsf{\omega } = {\displaystyle \frac{m_2-m_3}{m_2-m_1}}·100\%,$$

(2)

where m₁ is the weight of the measuring cup brought to a constant weight (g); m₂ is the weight (g) of the measuring cup containing the tested substance of PrsCl₂·H₂O before drying; m₃ is the weight (g) of the measuring cup containing the tested PrsCl₂ sample after drying.

Statistical Analysis

All results obtained were analyzed using statistical methods as means ± standard deviation (SD) of different and independent experiments (n ≥ 3) with the assistance of software packages provided by OriginPro 2021 9.80.200 (OriginLab Corporation, Northampton, MA, USA); p values < 0.05 were considered significant.

3. Results and Discussion

This section presents the results of specific spectral and spectroscopic analytical methods, as well as a biological assay, designed to complement and compensate for the inherent limitations of each individual technique in determining the properties, composition, and structure of the chemotherapeutic alkylating agent prospidium chloride (PrsCl₂).

3.1. Spectral Methods

3.1.1. FT-IR Spectroscopy Analysis

Despite the long-standing clinical use of prospidin and the existence of quality standards, the lack of spectral methods in its quality control remains a significant limitation [48]. In this context, data collection and the analysis of spectra arising from vibrational (FT-IR) and electronic (UV) transitions within the dispirotripiperazinium alkyl framework provide a reliable basis for rapid identification (first and second identification in the sense of pharmacopoeial requirements). The result of the interaction of incident electromagnetic radiation with the quantized vibrational levels of the prospidium chloride monohydrate molecule is shown in Figure 4 [49].

The FT-IR spectrum of prospidium chloride exhibits a broad transmittance band characteristic of O–H groups (alcohols), together with a medium-intensity band of N–H groups (amines) in the DSpTPpCl₂ spectrum in the 3550–3200 cm⁻¹ region. The observed changes in the width and shape of the band in this frequency range are associated with factors such as the relaxation rate and dephasing of the vibrational excited state, as well as inhomogeneity in the energy gap between the ground and first excited states due to differences in the molecular environment [50]. These states are described by the second derivative of the vibrational potential energy, which is proportional to the force constant k and, consequently, to the vibrational frequency in the IR spectrum (Equation (3)):

$${\left.{\displaystyle \frac{d^{2}U}{{dx}^2 }} \right|}_{x=0}=k,$$

(3)

where U(x) is the vibrational potential energy, expanded as a function of nuclear displacement x from equilibrium in the harmonic oscillator model, and k is the bond force constant.

Both spectra show similar features around 3000 cm⁻¹, which is typical for ammonium salts. A particularly intense transmittance band is observed near 900 cm⁻¹, arising from vibrational transitions of the C–Cl group of halo compounds (

Table 3. Transmittance bands in the FT-IR spectra of prospidium chloride* [51].

Wavenumber, cm−1	Group	Compound Class	Appearance
3550–3200	O-H stretching	alcohol intermolecular bonded	strong, broad
3350–3310 *	N-H stretching	secondary amine	medium
near 3000 *	N-R stretching	amine salt	strong, broad
near 2850	C-H stretching	alkane	medium
1650 *	N-H bending	amine	medium
near 1500 *	C-H bending	alkane methylene group	medium
1385–1465 *	C-H bending	methylene group (other, acyclic)	medium
1250–1020 *	C-N stretching	amine	medium
near 900 *	C-Cl stretching	halo compound	strong
1124–1087	C-O	secondary alcohol	strong
900–700	C-H bending	1,2-disubstituted 1,2,3-trisubstituted	strong

* including the DSpTPpCl₂ structure.

).

The results of the spectroscopic analysis showed that the energy of the absorbed radiation in the mid-IR region predominantly affects the vibrations of covalent bonds in polar amine N–R and C–N groups, alcoholic O–H and C–O groups of prospidine, as well as C–Cl bonds and ionogenic N⁺–Cl⁻ groups. Comparison of the FT-IR spectra of the parent dispirotripiperazinium structure (DSpTPpCl₂) and the derived prospidium chloride structure (PrsCl₂) clearly demonstrates differences in the intensity and position of transmission bands in the high-frequency region (3000–3500 cm⁻¹), which are associated with changes in the nature of the –NH and –OH groups and with rovibrational transitions between closely spaced molecular energy levels, manifesting as the fine P-, Q-, and R-branch structure of the rotational components of the fundamental vibrational absorption band (see Figure 4) [52].

Thus, FT-IR spectroscopic analysis provides important structural information on the functional groups, types of chemical bonds, and specific features of their interactions in the prospidium chloride molecule.

3.1.2. Electronic Absorption Spectrophotometry

In contrast to infrared spectrometry, UV absorption spectrophotometry provides valuable information on specific structural features and the presence of chromophores by monitoring electronic transitions between molecular orbitals (MOs). For a long time, the apparent absence of chromophores with conjugated multiple bonds in the PrsCl₂ molecule (see Figure 3) was considered a reason for the inapplicability of absorption spectrophotometry to the characterization of its physicochemical properties [53,54]. However, analysis of the prospidium structure as a heteroatomic N,N′-bis(2-hydroxy-3-chloropropyl) dispirotripiperazinium cation, containing nonbonding n orbitals (on N3, N12 and the OH group at C2) and σ* antibonding orbitals (C–Cl, N⁺–C, O–H), allows rationalization of formally Laporte-forbidden n→σ* electronic transitions (Figure 5) [55,56,57].

The energy of the absorbed light corresponds to the energy (ΔE) difference between the ground state and the excited state of the molecule (Equation (4))

$$\mathsf{\Delta }\mathrm{E}={\displaystyle \frac{\mathrm{h}\mathrm{c}}{\lambda }},$$

(4)

where h is Planck’s constant (6.625 × 10⁻³⁴ J·s), c is the speed of light (2.998 × 10⁸ m/s), and λ is the wavelength, in nm.

Electronic transitions in a molecular system from one quantum state to another are restricted, among other factors, by the spin selection rule [58]. However, the conservation of spin multiplicity for the n → σ* transition from the ground to the excited state (total spin condition ΔS = 0, spin-allowed transition) enables photon absorption (Figure 6) and gives rise to an absorption band in the spectrum of prospidium chloride (Figure 7) [59].

The absorption spectrum of prospidium chloride for a series of aqueous solutions with concentrations of 0.20–0.05 mol·L⁻¹, also obtained within the framework of intermediate precision assessment, is characterized by a maximum at λ = 282 ± 0.40 нм and a minimum at λ = 255 ± 0.40 нм (

Table 4. Linearity parameters of absorption spectrophotometry results for PrsCl₂ solutions and the analytical range.

Linear Dependence Parameters    y = ax + b
Constant (Free) Term, b $\pm$ SD	Slope, a $\pm$ SD	Coefficient of Determination, (R-Square)	Adjusted R 2	Pearson’s Coefficient, r
2.77 ± 0.02	0.092 ± 0.007	0.975	0.000039	0.9998
y = 2.77x + 0.092

) [60].

The results obtained by absorption spectrophotometry formed the basis of a new procedure for the identification of PrsCl₂ using the molar absorption coefficient (MAC), which depends on the nature of the substance (See Equation (1)) (

Table 5. Metrological characteristics of the mean molar absorption coefficient values.

Number of Levels, N	Total Sample Size, n	$\overline{\mathit{\epsilon }}$, L·mol−1 cm−1	SD	RSD	$\overline{\mathit{\epsilon }}\pm ∆\mathit{\epsilon }$ L·mol−1 cm−1	$\overline{\mathsf{\epsilon }}$, %
1–3	n = 35 t =2.03 * пpи f = 34, p = 0.975)	3.89	0.24	0.06	3.89 ± 0.08	2.1

* Critical values of Student’s t-distribution [62].

, Table S3) [61].

Theoretical and experimental investigation of the excited states of the PrsCl₂ molecule is important for elucidating many physical and chemical processes associated with changes in its electronic structure. This knowledge enables not only the development of new optical spectroscopic methods for the identification and assay of the active pharmaceutical ingredient, but also the study of the reactivity of PrsCl₂ in different media.

3.2. ¹H NMR Spectroscopy

To overcome the limitations associated with the lack of classical spectroscopic methods that would confirm not only the identity of prospidium chloride (PrsCl₂) but also the structures of closely related substances (RSs), nuclear magnetic resonance (NMR) spectroscopy was employed as a primary method for elucidating the structure of organic compounds without prior chromatographic separation of the analyte mixture into individual components. NMR spectroscopy is highly sensitive to subtle structural and conformational changes, which enables the identification of impurity species in pharmaceutical preparations and provides insight into the main pathways of their formation [63]. Moreover, NMR spectroscopy allows for a quantitative evaluation of both the active pharmaceutical ingredient and its impurities without the need for certified reference standards [64]. In this section, the key results of applying a pharmacopoeial ¹H NMR method for the identification of the chemotherapeutic alkylating agent prospidium chloride are presented.

Figure 8 shows a high-resolution ¹H NMR spectrum obtained for a solution of prospidium chloride in D₂O, a chemically inert and spectroscopically suitable solvent that does not react with the sample and produces no significant interfering signals in the NMR spectrum. For this purpose, a 700 MHz NMR spectrometer was used on protons for the first time, in contrast to earlier work [65]. The use of a 700 MHz instrument was dictated by the high molecular complexity of the prospidium chloride molecule (see Section 2.1) and the need for the accurate resolution and assignment of signals with closely spaced chemical shifts.

The signals of protons adjacent to electronegative and/or electron-withdrawing atoms (e.g., Cl) in prospidium chloride (see inset in Figure 8) are deshielded and appear at higher chemical shifts δ as w:

• δ 4.28–3.92 (br. s, 10H, -CH₂- 8,7,15,16, 2 x -CH-β)—the most deshielded protons, located next to the quaternary ammonium nitrogen N⁺ of the central ring and the CH(OH) groups (inductive effect of OH). The broad singlet is due to free rotation and conformational lability.
• δ 3.91–3.72 (br. s, 8H, -CH₂- 1,5,10,14)—a broad singlet arising from methylene protons adjacent to the ammonium nitrogen N⁺, deshielded by the +I effect of N⁺ (δ~3.8 ppm, which is typical for [R₄N]⁺CH₂-).
• δ 3.68 (dd, J = 11.7, 3.8 Hz, 2H, -CH₂-γ_a)—γ-methylene protons of the CH₂Cl groups showing geminal coupling ²J = 11.7 Hz (protons on the same carbon atom) and vicinal coupling ³J = 3.8 Hz (protons on adjacent carbon atoms). The small spin−spin coupling constants ³J (J-coupling) is consistent with a gauche conformation (groups separated by a torsion angle of approximately 60°).
• δ 3.58 (dd, J = 11.7, 5.6 Hz, 2H, -CH₂-γ_b)—signal of the diastereotopic γ-methylene protons of the CH₂Cl group with ³J = 5.6 Hz, also indicative of a gauche conformation.
• δ 3.11–2.85 (m, 8H, -CH₂- 2,4,11,13)—methylene protons of the spiro fragments (–CH₂–N–CH₂–), multiplet.
• δ 2.70–2.61 (m, 4H, 2 x -CH₂-α)—the least deshielded methylene protons in α-position to N⁺ (CH₂–N⁺), shielded by the methylene groups of the spiro ring.

All proton signals exhibit multiplet structures due to J-couplings with neighboring nuclei.

Overall, the ¹H NMR spectrum of prospidium chloride displays the characteristic pattern of a diamine with quaternary onium centers and a pronounced gradient of chemical shifts (Δδ = 1.6 ppm) from CH₂Cl to CH₂–N⁺, thereby confirming the electronic effects of Cl/N⁺/OH in the molecule. The structure of prospidium chloride was also confirmed by ¹H-¹H COSY NMR experiment (Figure S1).

¹H NMR for the Detection of PrsCl₂ Impurities

Many impurities are present in pharmaceutical substances at concentration levels on the order of parts per million (ppm) or parts per billion (ppb), which makes their detection by standard analytical methods highly challenging [66]. According to the literature, prospidium chloride (PrsCl₂) is prone to the formation of related substances upon interaction of the API with aqueous media (Figure 9) [67].

API-related impurities are considered to have potential toxic or even genotoxic properties, which necessitates particular caution in the development of methods for their detection and control (Quantitative Structure–Activity Relationship, QSAR) [68]. High-resolution ¹H NMR spectra of prospidium chloride and structurally related impurities were therefore recorded (Figure 10).

For the dihydroxy derivative (diol) (¹H NMR, 700 MHz, D₂O), the proton signals are observed at: δ 4.06 (br. s, 8H, -CH₂-8,7,15,16), 3.88–3.83 (m, 2H, 2 x -CH-β), 3.83–3.71 (br. s, 8H, -CH₂-1,5,10,14), 3.56 (dd, J = 11.7, 4.2 Hz, 2H, -CH₂-γ_a), 3.47 (dd, J = 11.8, 6.0 Hz, 2H, -CH₂- γ_b), 3.05–2.84 (m, 8H, -CH₂- 2,4,11,13), 2.60–2.52 (m, 4H, 2 x -CH₂-α).

A downfield shift change in Δδ = 0.22 ppm relative to the parent PrsCl₂ spectrum (-CH₂Cl → –CH₂OH) confirms the replacement of chlorine atoms by hydroxyl groups in the molecular structure of the dihydroxy derivative.

For the epoxy derivative (¹H NMR, 700 MHz, D₂O): δ 4.09 (br. s, 8H, -CH₂- 8,7,15,16), 3.82 (br. s, 8H, -CH₂-1,5,10,14), 3.24 (m, 2H, 2-CH-β), 3.07 (dd, J = 13.9, 2.6 Hz, 2H, -CH- αa), 3.01 (br. s, 8H, -CH₂- 2,4,11,13), 2.91–2.93 (m, 2H, -CH₂-γa), 2.68–2.64 (m, 2H, -CH₂-γb), 2.37 (dd, J = 13.9, 7.8 Hz, 2H, -CH-αb).

In the obtained ¹H NMR spectra, an additional signal of an unidentified impurity (u.i.) is also observed at δ 2.18 (br. s, u.i.) (see Figure 9 and Figure 10). The literature data [69] indicate that this signal is consistent with acetone, which is used in the precipitation and isolation stages of PrsCl₂ synthesis [70] и and may therefore be present as a residual solvent in amounts not exceeding the ICH Q3C Class 3 (<5000 ppm) [71].

Because the -CH₂-α protons of the diol at 2.60–2.52 ppm (m, 4H) and the -CH-α_b at 2.37 ppm (dd, J = 13.9, 7.8 Hz, 2H) and -CH-β at 3.24 ppm (m, 2H) in the epoxy derivative do not overlap with other signals in the spectrum, these resonances can be used for the selective identification and quantitative determination of the above impurities (See Figure 10).

The results of quantitative ¹H NMR spectroscopy (qNMR) for the determination of the content of related impurities (diol and epoxy) and API in the PrsCl₂·H₂O substance, according to Equations (5) and (6), are summarized in

Table 6. qNMR results for the analysis of prospidium chloride substance (n ≥ 3).

The Research Subject	δ (ppm)	Assignment	Content
			$\overline{\mathit{m}}\pm \mathit{S}\mathit{D}$, mg	$\overline{\mathit{\omega }}\pm \mathit{S}\mathit{D},\mathbf{\%}$
PrsCl2	3.58	-CH2Cl	46.27 ± 0.89	88.43
diol	2.60–2.52	-CH2-α	1.54 ± 0,03	2.94
epoxy	2.37	-CH-αb	1.38 ± 0.03	2.64

.

$$m_X=n_{IS}{\displaystyle \frac{I_X}{I_{IS}}}{\displaystyle \frac{N_{IS}}{N_X}}{·M}_X,$$

(5)

$${\omega }_X={\displaystyle \frac{m_X}{m_{sample}}}·100\%,$$

(6)

where n_IS is the amount of the internal standard TSP-d₄, ммоль; I_X represents the integral intensities of the proton signals PrsCl₂·H₂O at 3.58 ppm, with the diol at 2.60–2.52 ppm and the epoxy derivative at 2.37 ppm; I_IS represents the integral intensity of the methyl protons signal of TSP-d₄ at 0.0 ppm; N_IS represents the nuclei number of equivalent nuclei of the internal standard TSP-d₄ (9H); N_X represents the nuclei number of PrsCl₂·H₂O (2H), the diol (4H) and the epoxy derivative (2H); M_X represents the molecular mass of PrsCl₂·H₂O and its diol and epoxy derivatives.

Overall, ¹H NMR spectroscopy at 700 MHz in D₂O demonstrated its effectiveness as a primary method for the structural identification of PrsCl₂ and its related substances without chromatographic separation, overcoming the limitations of classical spectroscopic methods for structurally similar impurities.

The characteristic gradient of chemical shifts (Δδ = 1.6 ppm) from δ 4.28–3.92 (-CH₂-N⁺) to δ 2.61 (-CH₂-α), detailed J-couplings (²J = 11.7 Hz, ³J = 3.8–5.6 Hz, gauche-conformations), and diastereotopic dd patterns unambiguously confirm the identity of the API and the main degradation pathways (Cl → OH/epoxide) under alkaline conditions. The original qNMR approach provides a quantitative assessment of the API and its related impurities without the use of dedicated reference standards, which makes this methodology attractive for the pharmacopoeial control of cytotoxic/cytostatic drugs with high molecular complexity and potentially genotoxic impurities.

4. Conclusions

This work characterizes prospidium chloride, a chemotherapeutic alkylating agent, using UV, FT-IR, and ¹H NMR spectroscopy. The applied methods provide structural and quantitative information without requiring certified reference standards. UV spectroscopy shows absorption at 282 nm (ε = 3.89 L·mol⁻¹·cm⁻¹). FT-IR spectroscopy reveals vibrational transitions of alkyl and spirocyclic fragments. ¹H NMR spectroscopy (700 MHz) enables the detailed assignment of chemical shifts, coupling constants, and conformational analysis, as well as the quantification of related substances (dihydroxy- and epoxy-derivatives) [72]. The methods complement each other by probing different aspects of molecular structure and enable routine quality control of medicinal products containing prospidium chloride.

5. Patents

Kladiev Ant.A, Kladiev A.A, Uspenskaya E.V.—method for the determination of authenticity and content of the active substance in prospidium chloride substance by ultraviolet spectrophotometry. Russian Federation patent RU 2837428 C1, 2025 https://i.moscow/patents/ru2837428c1_20250331 (accessed on 6 March 2025).