Introduction

Inflammation and bacterial infection are closely tied to one another. Bacterial infections frequently result in pain and inflammation [1]. Essentially, inflammation is a biological response that arises in reaction to bodily stimuli that are biological, chemical, physical, and immunological [2]. As a result, inflammation is a crucial marker in a number of pathological conditions, associated with Alzheimer illness, osteoarthritis, rheumatoid arthritis, and diseases linked to obesity [3, 4].

The pharmacological actions of curcumin have been discovered to be quite diverse over time, including antibacterial [5], antifungal [6], antiviral [7], anti-Alzheimer [8], antioxidant [9], anti-inflammatory [10], anti-malarial [11], anticancer [12] and antitumor homes [13]. This absence of toxicity for molecule makes it absolutely exceptional. Because curcumin may be utilised in huge doses without becoming harmful, it is possible that this molecule will play a key role for enhancing healing. Curcumin continues to be the focus of various medical trials due to its unique properties, which make it a valuable lead molecule for therapeutic development [14]. Curcumin has been shown to protect against the negative effects of ionising radiation. As a result, it may be useful during cancer radiation [15].

Therefore, early detection and therapy of inflammatory vascular diseases depend on ability of image analysis to detect the inflammatory endothelium or the guidance of the medicine to target lesions. Diagnostic accuracy varies between imaging techniques. Accordingly, depending on the type of inflammation being studied, anatomical/structural alterations in the diseased organ are routinely seen and used to diagnose inflammatory diseases. Curcumin derivatives that have been radiolabeled show promise as imaging agents [16].

Microwave methods used in the synthesis of heterocyclic compounds are another major section of green chemistry approaches. Such approaches are gaining popularity due to their environmental friendliness, increased reaction yield and time, more convenient and simply synthesised procedures, and great energy efficiency [17, 18]. When comparing microwave irradiation technique to conventional heating methods, it is obvious that microwave technique is more environmentally tolerant, readily controllable, and ecologically benign. Many heterocyclic processes were carried out in less time, with greater yields, and under softer and cleaner conditions [19,20,21]. Because of its economy, simplicity, and gentle circumstances, green synthesis is currently regarded as an important approach in heterocyclic chemical synthesis. As previously said, further attempts are still being made to synthesise new heterocycle derivatives based on their preferred uses in biology and industry [22,23,24,25,26]. Recently, a variety of conditions have been used to conduct the Biginelli reaction, and the experimental procedures have undergone several variations. The use of Bronsted, Lewis, and protonic acids has historically been catalysed [27, 28]. In a recently released article, a solvent-free condensation of curcumin, guanidine, and aromatic benzaldehyde that was microwave-irradiated is reported.

Radiolabelled curcumin and curcumin derivatives have been investigated as possible radiotracers for the early detection of Alzheimer’s disease and cancer in the field of nuclear medicine [29]. Chemical changes were used to improve curcumin stability, as well as functionalization for tagging with various radiohalogens or metal radionuclides (fluorine-18, technetium-99m, gallium-68, and so on) [16]. Furthermore, as established by Kumar et al. [30], curcumin may be directly labelled with radioiodine (e.g., 125I) using iodogen-coated glass tubes as an oxidising agent. however, radiolabeled curcumin was produced with low radiochemical yield of 75%. When metal coordination occurs, the keto-enol group can behave as a bidentate hard chelator with two coordination oxygens producing a six membered ring. Curcumin has been shown to be a good bidentate chelating agent for hard Lewis acids such as iron [31, 32], gallium [33], and copper [34] in previous decades due to its high affinity for the metal ion. Curcumin’s most recent uses in the production of metal-based hybrid materials for medication administration [35, 36] or metal detection [37] are particularly intriguing. Radiolabeled curcumin and its analogs with radiometals (e.g., gallium − 68 and 99mTc) have been investigated in some pharmacological studies because of their potential as therapeutic agents in various human diseases. Using ″2 + 1″ ligand system, Sagnou et al. were the first to investigate [99mTc]Tc-curcumin mixed ligand complexes with a tricarbonyl core by reacting [99mTc]TcO4−with a kit including NaBH4, Na2CO3, and Na-K tartrate, as described in the literature [38]. Triantis et al. also reported the preparation the non-radiolabeled (Rhenium complexes) and radiolabeled (99mTc−complexes) of curcumin derivatives, fac-[99mTc/Re(cur)-(PPh3)2(CO)2] and fac-[99mTc/Re(cur)-(PPh3)(CO)3] [39].

Curcumin derivatives examined in vivo thus far were obviously capable of visualising the lesion but had a broad distribution in numerous organs such as the liver, kidneys, spleen, and lungs. Although significant disadvantages remain, and none of the radiolabelled curcuminoids have yet attained clinical application, the research conducted thus far give important insights and establish the groundwork for future advances. The fundamental challenge in increasing the effectiveness of these radiotracers is that the mechanism of their preferential absorption in specific malignancies has yet to be understood [16].

These findings, together with the significant accumulation in a variety of organs, prohibits direct use of these radiolabelled derivatives as diagnostic tools, however recent research sets the framework for developing further curcumin-based compounds with increased characteristics. The goal of our research was to create a synthetic approach for generating curcumin derivatives with anti-inflammatory properties utilising microwave irradiation. Furthermore, the 99mTc-radiolabeling technique was employed to investigate the biodistribution of the most effective synthesised molecule, as well as its potential as a radiotracer for in vivo imaging of inflammation.

Experimental

The open capillary tube melting points measurements of the synthesised compounds were made. All the chemical compounds utilized withinside the test had been bought from Sigma Aldrich chemical corporations and utilised as provided. Technetium-99m (99mTc) elution was generated from 99Mo/99mTc generator as ([99mTc]TcO4−), at the Radioisotope Production Facility (RPF), Egyptian Atomic Energy Authority (EAEA) (Cairo, Egypt). Microwave irradiation is now employed with the LG, MS 1927 microwave starting kit. Three hundred Watt (W) of microwave energy was produced in open air. On a Bruker Avance II, 13C NMR and 1H NMR spectra were captured using DMSO-d6 and deuterated chloroform (CDCl3) as solvents. Overall chemical shifts were expressed as a percentage of tetramethyl silane (ppm). Using a KBr pellet and a Schimadzu Prestize 21 version, IR spectra were captured at ambient temperature. A JEOL-AccuTOF JMST100LC mass spectrometer had captured mass spectra.

General synthetic process for curcumin derivatives 4a-c

As shown in scheme 1, a round bottom flask (50 mL) was filled with the following ingredients: curcumin (1, 1.0 mmol), aromatic aldehyde (2, 1.0 mmol), guanidine (3, 1.0 mmol), and chitosamine hydrochloride (0.01 mmol) [28]. The flask was then microwave-irradiated for 5 to 10 min while being kept solvent-free. The reaction was monitored.

by TLC using chloroform/methanol (9:1) as the mobile phase. After the reaction concluded, the ingredients were allowed to dissolve in ethanol, agitated for about 15 min, followed by recrystallization using the appropriate solvents:

5-(4-Hydroxy-3-methoxyphenylethylenecarbonyl)-6-(4-hydroxy,3-methoxyphenylethyl-ene)-4-(4-bromophenyl)-3,4-dihydropyrimidinimine (BHMC, 4a)

Prepared by using 4-bromobenzaldehyde, curcumin and guanidine in a prescence of chitosamine hydrochloride for 5 min. Recrystallization from a MeOH /Dimethylformamide (5:1) mixture yields a dark red powder. IR (KBr, cm−1): 3411, 3459 (N–H), 3311 (O–H), 3215 (C–H), 3102 (C–H), 2931 (C–H), 1631 (C= N), 1683 (C=O), 1598 (C=C); 1H NMR (400 MHz, CDCl3): 3.78 (s, 6 H, OCH3), 4.87 (s, 1H, HC–N), 5.01 (bs, 2 H, OH), 6.49 (d, 1H, J = 15.6 Hz), 6.69–6.83 (dd, 2 H, J = 8.4, 0.4 Hz), 7.04–7.40 (m, 9 H, HC=C), 7.45–7.59 (dd, 2 H, J = 8.4, 1.9 Hz), 7.65 (s, 2 H, NH), 7.76 (bs, 1H, NH); ESI-MS: m/z (M+) 575.

5-(4-Hydroxy-3-methoxyphenylethylenecarbonyl)-6-(4-hydroxy,3-methoxyphenylethyl-ene)-4-(4-chlorophenyl)-3,4-dihydropyrimidinimine (4b)

Prepared by using 4-chlorobenzaldehyde, curcumin and guanidine in a prescence of chitosamine hydrochloride for 5 min. Recrystallization from ethanol yields a pale yellow powder. IR (KBr, cm−1): 3400, 3449 (N–H), 3307 (O–H), 3196 (C–H), 3102 (C–H), 2910 (C–H), 1628 (C=N), 1675 (C=O), 1596 (C=C); 1HNMR (400 MHz, CDCl3): 3.79 (s, 6 H, OCH3), 4.78 (s, 1H, HC–N), 5.03 (bs, 2 H, OH), 6.49 (1H, d, J = 15.6 Hz), 6.69–6.83 (dd, 2 H, J = 8.4, 0.4 Hz), 7.04–7.36 (m, 5 H, HC = C), 7.44–7.59 (m, 6 H, Ar-H), 7.65 (s, 2 H, NH), 7.76 (bs, 1H, NH); ESI-MS: m/z (M+) 532.

5-(4-Hydroxy-3-methoxyphenylethylenecarbonyl)-6-(4-hydroxy,3-methoxyphenylethyl-ene)-4-(4-nirtrophenyl)-3,4-dihydropyrimidinimine (4c)

Prepared by using 4-nitrobenzaldehyde, curcumin and guanidine in a prescence of chitosamine hydrochloride for 10 min. Recrystallization from ethanol yields a brown powder. IR (KBr, cm−1): 3410, 3455 (N–H), 3310 (O–H), 3200 (C–H), 3111 (C–H), 2910 (C–H), 1625 (C=N), 1681 (C=O), 1591 (C=C); 1HNMR (400 MHz, CDCl3): 3.78 (s, 6 H, OCH3), 4.59 (s, 1H, HC–N), 5.01 (bs, 2 H, OH), 6.49 (d, 1H, J = 15.6 Hz), 6.69–6.83 (dd, 2 H, J = 8.4, 0.4 Hz), 7.04–7.59 (m, 9 H, HC=C), 7.65 (s, 2 H, NH), 7.76 (bs, 1H, NH), 8.07 (d, 2 H, J = 8.1 Hz); ESI-MS: m/z (M+) 542.

Evaluation of anti-inflammatory activity

The carrageenan-induced approach described in the literature [28] was used to test synthetic compounds for their anti-inflammatory properties. This method involved weighing 24 mice in total, dividing them into four groups of six mice each cage, and having them fast for two hours before the experiment. The first group acted as the negative control (normal saline solution was delivered intraperitoneally), the second group received 200 mg/kg intraperitoneally of curcumin; the third group received 100 mg/kg intraperitoneally of diclofenac; and the fourth group received 200 mg/kg body weight of 4a-c and Re-BHMC. After an hour, an injection of freshly prepared 0.1 mL of 1% suspension of carrageenan in saline solution was administered to each of the four groups of mice in order to induce acute inflammation. A plethysmometer (UGO Basile, 7140 Italy) was used to measure the paw volumes at 2 h, 3 h, and 4 h following carrageenan injection. The following formula was used to determine the percentage of inhibition:

$$\% {\text{ Inhibition }} = {\text{ }}\left( {{\text{Vc}} - {\text{Vt}}} \right)/{\text{Vc }}*{\text{ 1}}00$$
(1)

where Vc = edema volume of control; Vt = edema volume of test.

Preparation of [99mTc]Tc-BHMC complex

Radiolabeling process

The 99mTc-radiolabeling was performed on the most effective anti-inflammatory agent (BHMC, 4a) by utilizing sodium dithionite (Na2S2O4) as a suitable reducing agent to create the [99mTc]Tc-BHMC complex utilising the direct radiochemical approach. Dimethyl sulfoxide was used to dissolve precisely weighed 100–1000 µg of BHMC before being transferred to an evacuated penicillin vial containing 1–10 mg Na2S2O4. The pH of the reaction medium was adjusted to 3–9 using 0.1 M NaOH or 0.1 M HCl solutions. To the aforementioned reaction mixture, 200 µL (150 MBq) of newly eluted technetium-99m from the 99Mo/99mTc generator was included. The reaction mixture was diluted to 1 mL with distilled water, vortexed, and kept at room temperature for between 15 and 60 min.

Investigation of the [ 99m Tc]Tc-curcumin derivative complex

The thin layer chromatographic method was used to assess the radiochemical purity of 99mTc-complex utilising TLCSG strips. Acetone was used as the mobile phase in order to determine the amount of free [99mTc]TcO4− in the preparation. To evaluate decreased hydrolyzed technetium, a mobile phase consisting of a combination of 2:5:1 ethanol, water, and ammonium hydroxide was utilised. To count the fractions of 1 cm (up to 10) individually, a single-channel analyzer was connected to a well-type NaI (Tl) detector.

Prediction of the complex structure

The labeled complex rhenium analogue was synthesised (6 mg BHMC, 1 ml of 0.025 M NH4ReO4 and 60 mg of Na2S2O4 for 2 h at room temperature and pH 7 medium). Absorbance was determined at 254 nm wave length following HPLC purification into the reversed-phase column (RP18), with acetonitrile: water: triethyl amine (70:30:0.01) used as the eluent at a flow rate of 1 mL/min., the analogue was subjected to MS (ESI) spectroscopy and elemental analysis for characterization [40,41,42,43]. The MS (ESI) analysis of the Re-BHMC complex revealed peaks for its molecular ions at m/z 760 and 762 for 185Re and 187Re, respectively. 1H NMR (400 MHz, CDCl3): 3.85 (s, 6 H, OCH3), 4.86 (s, 1H, HC–N), 5.23 (bs, 2 H, OH), 6.48 (d, 1H, J = 15.6 Hz, ), 6.77 (dd, 2 H, J = 8.4, 0.5 Hz), 7.05–7.40 (m, 9 H, HC=C), 7.45–7.59 (dd, 2 H, J = 8.4, 1.9 Hz), 7.65 (s, 2 H, NH), 7.76 (bs, 1H, NH). (Figures S1, S2) Elemental analysis: Calc. for C29H26BrN3O5Re: C, 45.67; H, 3.44; O, 10.49. Found: C, 45.70; H, 3.46; O, 10.51.

Investigation of biodistribution

The study met the standards constructed by the Egyptian Atomic Energy Authority and was authorised by the animal ethics committee:

Creation of foci for infection

Using biological components, a single clinical isolate of Staphylococcus aureus was used to produce a focal infection. Turbid suspension was created by diluting specific colonies. Three mice groups each received 200 µl of the suspension intramuscularly in the left lateral thigh muscle. The infected thigh takes twenty-four hours to swell up significantly.

Induction of non-infected inflammation

A 200 ml intramuscular injection of turpentine oil that had been autoclaved at 121 °C for 20 min caused sterile inflammation in the left lateral thigh muscle of mice. Swelling began to show up two days later. The Student t test was used to examine data differences. The 2-tailed test is used to give the findings for P, and the mean SEM is displayed for each result. A significant criterion of P < 0.05 was used.

By measuring the quantitative distribution of organs, biodistribution of the 99mTc-BHMC complex was assessed. Mice were injected with 0.1 ml of a solution containing about 18 MBq of [99mTc]Tc-BHMC complex into their tail veins following a 24-hour period of bacterial induction. After the mice were killed, a heart puncture was used to remove the blood. Fresh muscle, bone, and blood samples were collected and counted into bottles that had been previously weighed. The different organs were removed, numbered, and compared to a reference solution containing the material indicated on the labels. The average percent values of administered dose/organ were computed. The assumed percentages of blood, bone, and muscles in the total body weight were 7, 10, and 40%, respectively. Adjustments were made for physical decay and background radiation during the experiment. Thighs from both the target and non-target groups were dissected and tallied. Additionally, radioactivity ratios of the target and non-target thighs were determined [44].

Results and discussion

Chemistry

The microwave technology was employed for heterocyclic synthesis to achieve ecologically acceptable techniques that adhere to green chemistry standards. Organic chemists can get closer to the ideal synthesis by using multicomponent reactions as a useful tool for sustainable organic synthesis and in concert with other green chemistry concepts [45, 46]. Curcumin served as the starting material for the synthesis of each chemical detailed in this article (Scheme 1). In an improved method, curcumin was combined with guanidine and substituted aromatic aldehydes and microwave-irradiated to produce curcumin 3,4-dihydropyrimidinimines (4a-c). The reaction was sufficiently speeding up with 0.08 g of the non-toxic acid catalyst chitosamine hydrochloride. After 5–10 min, the reactions were finished, and the desired curcumin derivatives were produced in good to outstanding yields.

Scheme 1
scheme 1

Three-component synthesis of curcumin 3,4-dihydropyrimidinimines under microwave irradiation

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Anti-inflammatory activity

The in vivo anti-inflammatory efficacy of curcumin 3,4-dihydropyrimidinimines (4a-c) has been examined with the carrageenan-induced paw edema technique at doses of 100, 200, and 300 mg/kg [47]. According to Table 1 findings, curcumin was outperformed by all synthetic substances in terms of anti-inflammatory activity. The chemicals are therefore higher achieving than curcumin at preventing the production of prostaglandin, which causes inflammation, according to the available research. BHMC (4a) and Re-BHMC showed the highest inhibition percent for inflammation. Additionally, there is no significant difference between them, which indicates that there is no change in the pharmacological properties of the original compound after the formation of the complex.

Table 1 Activity of the synthesized curcumin compounds against inflammation
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Preparation of [99mTc]Tc-BHMC complex

Thin layer chromatography (TLCSG) was used to assess the radiochemical yield of the [99mTc]Tc-BHMC complex. The proportion of free [99mTc]TcO4− that migrated with the solvent front (Rf = 1.0), leaving [99mTc]Tc-BHMC complex and colloid at the origin, was calculated using acetone and a TLCSG strip. An ethanol: water: ammonium hydroxide combination (2:5:1) was used to calculate the amount of reduced hydrolyzed technetium (colloid) that stayed at the origin (Rf = 0), whereas [99mTc]TcO4− and [99mTc]Tc-BHMC migrated with the solvent front (Rf = 1). By deducting the total percentages of colloid and free pertechnetate from 100%, the radiochemical purity was determined. The mean of the three trials is the radiochemical yield. Various parameters influencing the radiochemical yield of [99mTc]Tc-BHMC complex were investigated. These parameters included ligand content, sodium dithionite content, pH, reaction duration, and in-vitro stability.

Because of the tracer quantity, the [99mTc]Tc-BHMC complex could not be characterized. The non-radioactive rhenium compound, or its equivalent 99mTc, is thus synthesised on a macroscopic level as shown in Scheme 2. Finally, a comparison may be made between the HPLC analysis of the 99mTc-complex of chelator and its equivalent, the Re-complex. The isostructurality of [99mTc]Tc-BHMC and Re-BHMC complexes was shown by the almost same retention periods in the investigated radio and UV chromatograms (Fig. 1), which were 9.1 and 9.3 min for [99mTc]Tc-BHMC and its counterpart rhenium complex, respectively.

Fig. 1
figure 1

HPLC chromatogram for the measurement of the analogue rhenium complex and [99mTc]Tc-BHMC

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Scheme 2
scheme 2

Preparation of the analogue rhenium complex of [99mTc]Tc-BHMC

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The impact of response time

According to Fig. 2A, the radiochemical yield of the [99mTc]Tc-BHMC complex was investigated at various reaction periods (5–60 min) with pH 8 and sodium dithionite present. It is evident that by extending the reaction time from 5 to 30 min, the radiochemical yield rose from 30.5 to 96.5%. The radiochemical yield has achieved saturation and is unaffected by reaction times longer than thirty minutes which indicates its fastness and the reaction stability after 30 min.

Effect of sodium dithionite content

It is required to decrease Tc7+ to its more reactive lower oxidation states in order to label with technetium-99m which facilitates its chelation [48]. As with [99mTc]TcO4−, sodium dithionite is typically added to [99mTc]TcO4− to facilitate its reduction. The impact of a sodium dithionite concentration of 1–10 mg on the radiochemical of BHMC was investigated. Figure 2B makes it abundantly evident that employing insufficient sodium dithionite caused partial [99mTc]TcO4− reduction, which resulted in a high concentration of free [99mTc]TcO4− and a low radiochemical yield. Nonetheless, the radiochemical yield rose in tandem with the sodium dithionite concentration. It was discovered that 6 mg of sodium dithionite content was the ideal level. There is no colloidal species other than [99mTc]TcO2 obtained in the reaction which is a major advantage of using sodium dithionite as a reducing agent in preparing technetium radiopharmaceuticals [49, 50]. The quantity of reduced hydrolyzed Technetium-99m rose when too much sodium dithionite was employed, which decreased the radiochemical yield.

Effect of ligand content

The impact of ligand concentration on the [99mTc]Tc-BHMC complex radiochemical yield was examined within the 100–1000 µg range, with findings displayed in Fig. 2C. This image makes it very evident that the radiochemical yield was low at low ligand quantities. This may have been caused by not having enough ligand to bind all of the reduced technetium while a white precipitate was generated [51]. Up to 600 µg of ligand were added, and the radiochemical yield rose accordingly. Beyond this point, the radiochemical yield was constant, reaching a maximum of 96.5 ± 0.15% and high specific activity ~ 6.4 Ci/mg.

Impact of the reaction mixture pH

We looked at how the pH of reaction mixture affected the [99mTc]Tc-BHMC complex radiochemical yield in the pH range of 3–9. At pH 8, a greater percent radiochemical yield (96.5 ± 0.15%) was achieved where BHMC combined all the reduced technetium, as seen in Fig. 2D. In acidic media, a lower percent radiochemical yield because protons compete with technetium [52] and more impurities will be produced reduced hydrolyzed technetium because the reducing agent will reduce more strongly in acidic conditions [53].

Fig. 2
figure 2

Imperfections in the radiochemical yield for the [99mTc]Tc-BHMC complex as dependent variables of A Reaction time B Na2S2O4 amount C BHMC amount D pH

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Biological distribution for mice with inflammation

The biodistribution assay indicates the usefulness and effectiveness of the radiopharmaceutical. This assay allows for the evaluation and quantification of radiopharmaceutical uptake in tissues, as well as the determination of how it is excreted from the body [54]. Table 2 depicts the uptake of the [99mTc]Tc-BHMC complex in significant bodily fluids of animals given bacteria and turpentine oil. Because of the major activity was detected in the liver, the hepatobiliary route was mostly used to remove [99mTc]Tc-BHMC complex from the bloodstream. While uptake was lower in other organs. On the other side, the uptakes by the oil and bacteria models were non-specific which attributed to the increase of the blood flow in the inflammation site. After 30 min, mice with infectious lesions that had received the [99mTc]Tc-BHMC combination had the highest mean target-to-non-target (T/NT) ratio, which was 5.12 ± 0.03 between inflammatory muscle and normal muscle. When T/NT equaled 1.12, the buildup of activity at the infection site peaked 30 min after intravenous injection and then gradually decreased over the next 240 min. In contrast, 60 min after receiving the [99mTc]Tc-BHMC complex injection, animals with inflammation had a T/NT ratio of 6.85 ± 0.07. The [99mTc]Tc-BHMC complex showed a higher T/NT in sterile inflamed muscle (turpentine) than in infected muscle (bacteria) at 60 minute intervals. As therefore, the [99mTc]Tc-BHMC complex can differentiate between sterile inflammation and bacterial infections. As a result, the [99mTc]Tc-BHMC complex showed greater absorption in inflammatory tissue and could be used as an imaging tool for inflammation.

Table 2 [99mTc]Tc-BHMC biodistribution in mice with varying inflammatory types (% ID/g) at various injection time frames
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Conclusion

A series of curcumins were produced with a yield of 90–96% as a result of a one-pot multicomponent cyclocondensation reaction using green methods and solvent-free microwave irradiation. All synthetically produced curcumin derivatives were tested for anti-inflammatory efficacy. BHMC derivative, which was synthesised from 4-bromobenzaldehyde, demonstrated higher anti-inflammatory activity. This curcumin derivative was directly labelled with technetium-99m and had a high radiochemical yield. According to a comparative biodistribution study of the compounds in bacterial infection and sterile inflammation, the [99mTc]Tc-BHMC complex accumulated rapidly in the sterile inflammatory site when compared to bacterial infection sites. According to the findings, the novel [99mTc]Tc-BHMC complex is quickly eliminated from the body via the hepatobiliary pathway.