Synthesis, In Vitro Anti-Inflammatory Activity, and HRMS Analysis of New Amphetamine Derivatives

Herein, we report the obtaining of new hybrid molecules of amphetamine with different profens (amfens). The obtained amfens are characterized by their melting points, UV, 1H–, 13C–NMR, and HRMS spectra. A complete and detailed mass spectral analysis of the newly obtained derivatives of amphetamine with ibuprofen, flurbiprofen, ketoprofen, naproxen, and carprofen was performed. In vitro inhibition of albumin denaturation of each new compound was assessed, and they showed significant activity. The IC50 values of the obtained amphetamine-profen derivatives ranged from 92.81 to 159.87 µg/mL. This indicates that the new hybrids inherit the anti-inflammatory properties of profens. Using in silico method, the toxicity was also calculated. The obtained results are given in LD50 values. Depending on the route of administration, the amfens are less toxic compared to the standard amphetamine.


Introduction
For many years, scientists have been fascinated by the synthesis of amphetamine and its derivatives [1]. To date, numerous organic synthetic transformations have been reported. The constant search for similar biofunctional molecules is the driving force behind the discovery of novel approaches to the synthesis of amphetamine and its derivatives [1].
Amphetamine is a compound that consists of a phenyl ring connected to an amino group by a two-carbon hydrocarbon chain and a methyl group at the α-carbon. It is a psychoactive substance that induces feelings of cheerfulness, strength, and lightness in people who are severely physically exhausted. The compound and its derivatives are used to treat depression, narcolepsy, and obesity [2].
Nonsteroidal anti-inflammatory drugs (NSAIDs) are used worldwide to treat pain and inflammation [6]. Profens make the most important group of nonsteroidal anti-inflammatory drugs (NSAIDs), and in many countries, some of them are freely sold. Chemically, all profens bear the characteristic 2-aryl-propionic acid moiety. They are optically active compounds due to the presence of chiral carbon (alpha-carbon of propionic acid) [7].
Some of the most well-known and widely used profens are ibuprofen, flurbiprofen, ketoprofen, naproxen, and carprofen (  This was the reason for obtaining hybrid molecules that structurally combine both the amphetamine and the profen skeleton. Those two classes of compounds can be easily combined via an amide bond.
Amides represent some of the most important and valuable organic functional groups in naturally occurring molecules, pharmaceuticals, agrochemicals, and polymers. The synthesis of amides is of great importance in organic and medicinal chemistry. Improved and innovative methods for the synthesis of amides are in high demand, both from the chemical and pharmaceutical industries. Various amides are present in about 25% of This was the reason for obtaining hybrid molecules that structurally combine both the amphetamine and the profen skeleton. Those two classes of compounds can be easily combined via an amide bond.
Amides represent some of the most important and valuable organic functional groups in naturally occurring molecules, pharmaceuticals, agrochemicals, and polymers. The synthesis of amides is of great importance in organic and medicinal chemistry. Improved and innovative methods for the synthesis of amides are in high demand, both from the chemical and pharmaceutical industries. Various amides are present in about 25% of the best-selling pharmaceutical products and in many other important medicinal compounds [8].
In this regard, we aimed to synthesize new hybrid molecules combining the powerful central nervous system stimulant actions of amphetamine with the strong analgesic properties of profens due to their anti-inflammatory effect.

Synthesis
An extensive literature review revealed that amphetamine is a structure that has piqued the interest of synthetic chemists for over a century. In this regard, we were looking for compounds that contained both an amphetamine and a profen fragment. For convenience, further, in the article, we will call them amfens (amphetamine-profens) for short.
Creating new structures that combine amphetamine and profen molecules could be useful for studying their biological activity. When the two structures are combined, a completely new molecule is formed that has the biological properties of both amphetamine and profen. This is the driving force behind our efforts to create new amfen molecules.
The classic method for the synthesis of amides was applied to obtain the final amfens, the Schotten-Baumann method, which consists of the acylation of amphetamine 1 with acid chlorides 2 (Scheme 1). Table 1 shows the yields of the obtained products. All further analyzes were performed on the diastereomeric mixtures. the best-selling pharmaceutical products and in many other important medicinal compounds [8].
In this regard, we aimed to synthesize new hybrid molecules combining the powerful central nervous system stimulant actions of amphetamine with the strong analgesic properties of profens due to their anti-inflammatory effect.

.Synthesis
An extensive literature review revealed that amphetamine is a structure that has piqued the interest of synthetic chemists for over a century. In this regard, we were looking for compounds that contained both an amphetamine and a profen fragment. For convenience, further, in the article, we will call them amfens (amphetamine-profens) for short.
Creating new structures that combine amphetamine and profen molecules could be useful for studying their biological activity. When the two structures are combined, a completely new molecule is formed that has the biological properties of both amphetamine and profen. This is the driving force behind our efforts to create new amfen molecules.
The classic method for the synthesis of amides was applied to obtain the final amfens, the Schotten-Baumann method, which consists of the acylation of amphetamine 1 with acid chlorides 2 (Scheme 1). Table 1 shows the yields of the obtained products. All further analyzes were performed on the diastereomeric mixtures.  The reaction mixture was stirred in an electromagnetic stirrer at room temperature. Тriethylamine was used as a hydrogen chloride acceptor. The progress of the reaction is monitored by thin-layer chromatography. The reaction was complete fully and no starting amphetamine was observed. The final products were isolated and additionally purified by filtration throughout neutral Al2O3.
Examining the 1 H-NMR (Supplementary Materials Figures S1-S5) data in detail, it can be seen that all the signals are fully consistent with the corresponding molecule. To describe the signals, the same were compared with the spectral data of the corresponding parent drugs. The characteristic doublet for 6 protons from the ibuprofen residue in compound 3a emerges at 0.83 ppm. The NH signal comes out for each amfen in the region between 5.09 and 5.25 ppm. The signals for the methyl groups appear as doublets. The doublet for the methyl group from amphetamine is seen in all new compounds from 0.94 to 0.98 ppm, and the -CH3 signal from profen part is seen as a doublet at 1.38-1.47 ppm.  The reaction mixture was stirred in an electromagnetic stirrer at room temperature. Тriethylamine was used as a hydrogen chloride acceptor. The progress of the reaction is monitored by thin-layer chromatography. The reaction was complete fully and no starting amphetamine was observed. The final products were isolated and additionally purified by filtration throughout neutral Al 2 O 3 .
Examining the 1 H-NMR (Supplementary Materials Figures S1-S5) data in detail, it can be seen that all the signals are fully consistent with the corresponding molecule. To describe the signals, the same were compared with the spectral data of the corresponding parent drugs. The characteristic doublet for 6 protons from the ibuprofen residue in compound 3a emerges at 0.83 ppm. The NH signal comes out for each amfen in the region between 5.09 and 5.25 ppm. The signals for the methyl groups appear as doublets. The doublet for the methyl group from amphetamine is seen in all new compounds from 0.94 to 0.98 ppm, and the -CH 3 signal from profen part is seen as a doublet at 1.38-1.47 ppm. In the region around 7 ppm, all the aromatic protons appear. 13 C-NMR spectra (Supplementary Materials Figures S6-S10) of the obtained compounds also confirmed the structure of the compounds. The UV spectra of all compounds are also available in the supplementary file (Figures S11-S15). The complete description of the structures from the NMR data was also demonstrated with the molecular ion of each compound. The performed mass spectral analysis unequivocally confirmed both the structure and the purity of the obtained amfens (Supplementary Materials Figures S16-S20). Further fragmentation allowed us to study their structures and decay pathways in detail.

Mass Analysis
We investigated the fragmentation of newly synthesized amfens with structurally different profens in this study. The profen residue distinguishes the newly synthesized compounds 3a-e. We used mass spectrometry to investigate the structure of the new amphetamine hybrid molecules. ESI was used in positive ionization mode. The fragmentation of amphetamine derivatives 3a-e follows a mechanism depicted in Scheme 2. The mass spectrum shows that the resulting ions have low intensity. The profenamide cation is stable and does not undergo further fragmentation except for ketoprofen. The fragmentation of the ketoprofen amide cation (m/z 254) is probably due to the fact that the tautomeric form (2) undergoes loss of an OH group, and a radical ion with m/z 237 is obtained (Scheme 3): The structural fragment that connects amphetamine to the various profens is -CH-NH-C(O)-C(CH 3 )-. Several fragmentation pathways originate from here. The main fragmentation pathways of compounds 3a-e are associated with the cleavage of C-N (path 1 (red)), N-C (path 2 (blue)), and C-C (path 3) bonds.
Pathway 2 is associated with cleavage of the N-C bond. It is mainly characteristic of amphetamine fragmentation. The resulting amphetamine ion of m/z 136 undergoes loss of a neutral NH 3 molecule, resulting in an ion of m/z 119. The subsequent loss of -CH 3 yields an ion of m/z 91, which is resonance stabilized to a tropylium cation. The ions with m/z 136, 119, and 91 are amphetamine-specific ions (Scheme 2). Paths 1 and 3 are characteristic only for the fragmentation of the amfens. Pathway 1 is related to the cleavage of the C-N bond, resulting in an amide cation of the corresponding profen (m/z 206 for ibuprofen, m/z 244 for flurbiprofen, m/z 254 for ketoprofen, m/z 230 for naproxen, m/z 273 for carprofen) (Supplementary Materials Figures S16-S20).
The mass spectrum shows that the resulting ions have low intensity. The profenamide cation is stable and does not undergo further fragmentation except for ketoprofen. The fragmentation of the ketoprofen amide cation (m/z 254) is probably due to the fact that the tautomeric form (2)  The mass spectrum shows that the resulting ions have low intensity. The profenamide cation is stable and does not undergo further fragmentation except for ketoprofen. The fragmentation of the ketoprofen amide cation (m/z 254) is probably due to the fact that the tautomeric form (2) undergoes loss of an OH group, and a radical ion with m/z 237 is obtained (Scheme 3):  Their stability is due to the formation of resonance-stable cations (Scheme 4a-c, 5ab). The ion intensity increases as the aromatic rings in the profen structure increase. The profens with condensed cores, naproxen and carprofen, have the highest intensity. Their stability is due to the formation of resonance-stable cations (Schemes 4a-c and 5a,b). The ion intensity increases as the aromatic rings in the profen structure increase. The profens with condensed cores, naproxen and carprofen, have the highest intensity. Their stability is due to the formation of resonance-stable cations (Scheme 4a-c, 5ab). The ion intensity increases as the aromatic rings in the profen structure increase. The profens with condensed cores, naproxen and carprofen, have the highest intensity.

Anti-Inflammatory Activity
Amfens are hybrid molecules that are made up of fragments with different properties and structures. Amphetamine is characterized by its psychotropic properties. It inhibits the enzyme monoamine oxidase (EC 1.4.3.4), a flavoenzyme that catalyzes the oxidation of biogenic amines, increasing the levels of the neurotransmitters norepinephrine, serotonin, and dopamine in the brain [9,10]. Profens are used as NSAIDs. They have the property of inhibiting COX1 and COX2, which oxidize arachidonic acid to prostaglandins [6].
Until now, no information has been found in the literature about the synthesis of hybrid molecules, which in their structures contain fragments with psychotropic and antiinflammatory properties. Applying such an approach can lead to the appearance of two effects: one is to increase or slow down the psychotropic effect and the second is that the resulting amfens exhibit good anti-inflammatory properties. What application they will find depends mostly on what properties they will inherit.
In the present work, we investigated the in vitro anti-inflammatory activity of the newly synthesized compounds 3a-e, evaluated as inhibition of albumin denaturation (IAD).
Inflammation is the response of living tissues to injury. It involves a complex array of enzyme activation, mediator release, fluid extravasation, cell migration, tissue breakdown, and repair [11]. The denaturation of proteins is a well-documented cause of inflammation in rheumatoid arthritis [12]. The obtained amfens were screened for the inhibition of albumin denaturation. This method provides information as to which albumin is protected from denaturation when heated. For this purpose, we have used human albumin.
The profens ibuprofen, flurbiprofen, ketoprofen, naproxen, and carprofen as structural fragments in amfens have proven anti-inflammatory properties, we used them as comparative standards.
The percentages of inhibition of synthesized amfens are presented in Figure 3. The results of the study are presented as IC 50 . The IC 50 values of profens estimated as IAD range from 75.43 to 126.58 µg/mL (Figure 3). The IC 50 values of the resulting amfens ranged from 92.81 to 159.87 µg/mL. Carprofen exhibits the highest anti-inflammatory activity. We assume that this is due to the fact that it contains 9H-Carbazole in its structure. Amfens showed anti-inflammatory activity, although they resulted less potent than their analogous free profens. As the number of aromatic nuclei increases, the anti-inflammatory activity decreases, i.e., the lipophilic character of the newly synthesized amfens (3a-c) is enhanced. This trend is not observed in amfens with condensed aromatic cores. Perhaps, therefore, amfens do not reproduce the same order of anti-inflammatory activity as profens. Overall, the results of the in vitro studies indicate that the amfens exhibit significant activity. We derive important information from the experiment, which confirms that the resulting amfens possess the property of protecting the albumin molecule from denaturation upon heating. The purpose of the experiment is to find out what properties the new hybrid molecules (amfens) will inherit. The experiment carried out by IAD proves that amfens inherit the properties of profens.

Prediction of Acute Rat Toxicity by Gusar Software
The toxicity of amphetamines is a widely discussed topic. Amphetamines produce both peripheral and central sympathomimetic effects. Their lethal complications include severe hyperthermia and hypertension, dysrhythmias, ischemia, dissections, and intracranial hemorrhage [13]. In order to analyze the toxicity of the newly obtained amphetamine derivatives, we used GUSAR software (Plovdiv, Bulgaria).
Gusar software was created to make QSAR/QSPR models on the basis of the appropriate training sets. The software offers in silico prediction of LD50 values for rats with four types of administration, oral, intravenous, intraperitoneal, and subcutaneous [14].
We used the software for quantitative in silico toxicity prediction of LD50 values of the obtained amfens 3a-e. Table 2 presents the obtained results as LD50 values in mg/kg. The purpose of the experiment is to find out what properties the new hybrid molecules (amfens) will inherit. The experiment carried out by IAD proves that amfens inherit the properties of profens.

Prediction of Acute Rat Toxicity by Gusar Software
The toxicity of amphetamines is a widely discussed topic. Amphetamines produce both peripheral and central sympathomimetic effects. Their lethal complications include severe hyperthermia and hypertension, dysrhythmias, ischemia, dissections, and intracranial hemorrhage [13]. In order to analyze the toxicity of the newly obtained amphetamine derivatives, we used GUSAR software (Plovdiv, Bulgaria).
Gusar software was created to make QSAR/QSPR models on the basis of the appropriate training sets. The software offers in silico prediction of LD 50 values for rats with four types of administration, oral, intravenous, intraperitoneal, and subcutaneous [14].
We used the software for quantitative in silico toxicity prediction of LD 50 values of the obtained amfens 3a-e. Table 2 presents the obtained results as LD 50 values in mg/kg. Observing the obtained data (Table 2), it can be seen that all amfens 3a-e show lower calculated toxicity compared to amphetamine 1, on the three types of administration: Intraperitoneal, subcutaneous, and oral. Amfens 3a-e seem to be with similar calculated toxicity compared to the standard amphetamine 1 when administrated intravenous (IV). This can be attributed to the fact that the intravenous drug administration provides the most complete drug availability with a minimum delay. For amfens 3a and 3b, the following order of decreasing calculated toxicity was observed depending on the route of administration: IV > SC > IP > Oral, while for amfens 3c and 3d, the order is as follows: IV > IP > SC > Oral (Figure 4). Only with compound 3e, as well as with the standard amphetamine 1, the calculated toxicity changes in the order: IV > Oral > SC > IP. The results of this study show that the oral administration of this family of drugs is more beneficial, reducing the toxic effect. Amfens 3a and 3c were seen to dramatically reduce the toxic effect when being orally administrated by 9-to 11-fold compared to amphetamine 1 (Figure 4). IP-intraperitoneal, IV-intravenous, SC-subcutaneous, Applicability domain: a-compound falls in the applicability domain of models; b-compound does not fall in the applicability domain of models. The superscript numbers show the acute rodent toxicity classification by OECD (The Organization for Economic Co-operation and Development). OECD classifies the chemicals into five categories, where 1 is highly toxic and 5 is practically non-toxic.
Observing the obtained data (Table 2), it can be seen that all amfens 3a-e show lower calculated toxicity compared to amphetamine 1, on the three types of administration: Intraperitoneal, subcutaneous, and oral. Amfens 3a-e seem to be with similar calculated toxicity compared to the standard amphetamine 1 when administrated intravenous (IV). This can be attributed to the fact that the intravenous drug administration provides the most complete drug availability with a minimum delay. For amfens 3a and 3b, the following order of decreasing calculated toxicity was observed depending on the route of administration: IV > SC > IP > Oral, while for amfens 3c and 3d, the order is as follows: IV > IP > SC > Oral (Figure 4). Only with compound 3e, as well as with the standard amphetamine 1, the calculated toxicity changes in the order: IV > Oral > SC > IP. The results of this study show that the oral administration of this family of drugs is more beneficial, reducing the toxic effect. Amfens 3a and 3c were seen to dramatically reduce the toxic effect when being orally administrated by 9-to 11-fold compared to amphetamine 1 ( Figure 4).

Synthesis
The reagents were purchased from commercial suppliers (Sigma-Aldrich S.A. and Riedel-de Haën, Sofia, Bulgaria) and used as received. A Bruker Avance Neo 400 was used for the recording of NMR spectral data (BAS-IOCCP-Sofia, Bruker, Billerica, MA, USA). All compounds were analyzed in CDCl 3 at 400 and 101 MHz for 1 H-NMR and 13 C-NMR, respectively. Chemical shifts were determined to tetramethylsilane (TMS) (δ = 0.00 ppm) as an internal standard. The coupling constants are given in Hz. Recorded NMR spectra were taken at room temperature (approx. 295 K). The MS analysis was carried out on a Q Exactive Plus high-resolution mass spectrometer (HRMS) with a heated electrospray ionization source (HESI-II) (Medical University of Sofia, Thermo Fisher Scientific, Inc., Bremen, Germany) equipped with a Dionex Ultimate 3000RSLC ultrahigh-performance liquid chromatography (UHPLC) system (Thermo Fisher Scientific, Inc., Bremen, Germany). For the TLC analysis, precoated 0.2 mm Fluka silica gel 60 plates (Merck KGaA, Darmstadt, Germany) were used.
Corresponding acyl chlorides 2a-e (1 mmol) were added to a solution of amphetamine 1 (1 mmol) in dichloromethane (15 mL) at room temperature. Triethylamine (1.2 mmol) was added after 10 min. After 30 min, the reaction mixture was washed with diluted hydrochloric acid (H 2 O:HCl = 4:1 v/v), a saturated solution of Na 2 CO 3 , and water. The organic layer was dried using anhydrous Na 2 SO 4 , concentrated, and filtered on a short column with neutral Al 2 O 3 .

Inhibition of Albumin Denaturation (IAD)
In vitro analysis of anti-inflammatory activity was assessed as inhibition of albumin denaturation (IAD). The analysis was performed according to Sakat method [16] with minor modifications [17]. The experiment was performed with human albumin. The solution of albumin (1%) was prepared in distilled water (pH 7.4). The tested compounds/standard were dissolved firstly in 1.2 mL DMF and PBS up to 25 mL, so the final concentration of the stock solution is 1000 µg/mL. Then, a series of working solutions with different concentrations (20-500 µg/mL) in PBS was prepared. The reaction mixture contained 2 mL test sample/standard of different concentrations and 1 ml albumin (1 %). The mixture was incubated at 37 o C for 15 min and then heated at 70 o C for 15 min in water bath. After cooling, the turbidity was measured at 660 nm with a spectrophotometer (Camspec M508, Leeds, England). The experiment was performed three times. Percentage inhibition of albumin denaturation (IAD) was calculated against control. The control sample is albumin with the same concentration dissolved in distilled water.

Conclusions
In conclusion, we have successfully obtained four new hybrid molecules containing an amphetamine skeleton and profen part. The newly obtained amfens are fully characterized. A detailed mass spectral analysis of the newly obtained amfens was performed. In vitro analysis showed that the amfens inherit the anti-inflammatory properties of profens. Compounds 3a-e are expected to be less toxic, especially via oral administration, based on the calculated values via GUSAR software. The obtained predicted toxicity results for oral administration of the newly synthesized amfens show much lower toxicity values in comparison with the amphetamine. This makes them promising and interesting for future biological evaluation.