Green synthesis of highly functionalized heterocyclic bearing pyrazole moiety for cancer-targeted chemo/radioisotope therapy

New derivatives of heterocyclic bearing pyrazole moiety were synthesized (eight new compounds from 2 to 9) via green synthesis methods (microwave-assisted and grinding techniques). 4,6-Diamino-1,3-diphenyl-1H-pyrazolo[3,4-b]pyridine-5-carbonitrile (2) shows high anti-cancer activity against both HepG2 and HCT-116 with IC50 of 9.2 ± 2.8 and 7.7 ± 1.8 µM, respectively, which referenced to 5-Fu which is showing activity of 7.86 ± 0.5 and 5.35 ± 0.3 against both HepG2 and HCT-116, respectively. The cytotoxic activity against HCT-116 and HepG2 was slightly decreased and slightly increased, respectively, by a different pyrazole moiety (compound 5). Pharmacokinetics of compound 2 was carried out using the radioiodination technique in tumour-bearing Albino mice which shows good uptake at the tumour site. The biodistribution showed high accumulation in tumour tissues with a ratio of 13.7% ID/g organ after one hour in comparison with 2.97% ID/g organ at normal muscle at the same time point. As I-131 has maximum beta and gamma energies of 606.3 and 364.5 keV, respectively, therefore the newly synthesized compound 2 may be used for chemotherapy and TRT. Supplementary Information The online version contains supplementary material available at 10.1186/s13065-023-01053-7.


Introduction
Heterocyclic chemistry is a significant field of organic chemistry and has received a great deal of interest for its potential applications in industry, biology, and the advancement of human society [1][2][3].Heterocyclic compounds typically have at least one heteroatom in their cyclic structures, such as nitrogen, sulfur, or oxygen can be made both naturally and artificially [3][4][5].Molecules which provided by a heterocyclic nucleus mainly have improvements in salt formation properties and solubility which make an improvement in pharmacokinetic parameters [6,7].Additionally, it makes it easier to settle the pharmacophores and create several biologically active compounds such as anti-inflammatory, anti-tumor, antiviral and anti-bacterial compounds [8][9][10][11][12][13].Compounds containing pyrazole moiety have received widely attention among heterocyclic compounds as a result of its extensive use in drug development research.
Pyrazole compounds are one of the most important heterocyclic derivatives widely used in petrochemical industry [14], catalytic and polymer manufacturing [15,16].Pyrazole derivatives have partially harmful effects on environment and humans and there are requirements for converting them into safe and useful products [17,18].During the last decades, derivatives of pyrazole showed wide-range of biological and pharmacological activities such as: anti-inflammatory, antagonist, analgesic, anthelmintic, anticancer, herbicidal, antiviral, antimitotic, antioxidant, insecticidal and antimicrobial activities [19][20][21][22][23][24][25][26].Furthermore, synthetic heterocyclic compounds containing nitrogen atoms have proven to have significant and diverse therapeutic potential for cancer which is one of the major causes of death worldwide [27].
Green chemistry is the chemical processes design science that eliminate or reduce the hazardous compounds generation.It prevents the pollution at a molecular level.Microwave and grinding techniques use in heterocyclic compounds synthesis are also important branch from green chemistry techniques.One-pot multicomponent reactions [28,29] are one of the most important tools for synthesis with facile execution, the effectiveness for its productivity and highly diverse products generation in a single running and from easily starting materials.So, such techniques have much more attention because of its safe on the environment, improvement of the reactions yield and time, more convenient, and easily synthetic procedures which are highly energy efficient.Comparing both microwave irradiation and grinding techniques with the conventional heating method; it's clear that microwave irradiation and grinding techniques are more environmentally tolerant, easily controlled and friendly environmental.As advantage, many heterocyclic reactions were carried out in shorter reaction time, higher yield, and milder and cleaner conditions [30][31][32].So now, this green synthesis type is considered as significant technique in heterocyclic chemistry synthesis because of its economy, simplicity and mild conditions.as aforementioned about heterocycles derivatives, and according to their favorite applications in biology and industry; more efforts in synthesizing novel heterocycles derivatives still continued [33][34][35][36][37][38][39][40].
Nuclear medicine technology is essential for developing both the search for disease treatments and the development of novel drugs.Iodine-131 is one of the most used radioisotopes in nuclear medicine.I-131 decays to the extremely stable xenon-131 with a half-life of 8.03 days.A negative beta particle as well as gamma photons are released during the decay process.The major beta emission has average and maximum energies of 191.5 keV and 606.3 keV, respectively, while the primary photon (gamma emission) has an energy of 364.5 keV [41], therefore it used in targeted radionuclide therapies (TRTs) [42].
In this study, designing and synthesizing enamino pyrazole derivative was reported as starting material via one-pot multicomponent reaction using microwave and grinding techniques.On the other hand, the reactions were pressed using thermal method according to literature method [43,44].Comparison between the percentage yields and consumed times, which resulting from the two techniques were performed.The yield economy "YE", atom economy "AE", reaction mass efficiency "RME" and optimum efficiency "OE" were used for comparison between the consumed times and the percent of the yields resulting from these techniques.The various synthesized compounds were illustrated by using different spectroscopic and analytical tools.The newly compounds were screened in-vitro anticancer against two cancer cell lines, namely, hepatocellular carcinoma (HepG2), and colorectal carcinoma (HCT-116).Radioiodination technique was used to study the pharmacokinetic of the most effective synthesized compound and also to be used as a potential agent as TRT for cancer tissues (Scheme 1).

Synthesis
All solvents, reagents and chemicals were bought from Sigma Aldrich.TLC (Merck Kiesel gel 60F254, BDH) to monitor the progress of all synthesized compounds.Microwave reactions were carried out with microwave reactor Anton Paar (monowave 300).All melting points were measured on a digital Stuart electric melting point apparatus "SMP3".Infrared spectra measurements were determined using KBr disks on PerkinElmer 293 spectrophotometer.The 1 H-NMR and 13 C-NMR spectra were measured on a Varian Mercury 75 MHz spectrometer.A GC-2010 Shimadzu Gas chromatography mass spectrometer (EI, 70 eV) was used for Mass spectrometry measurements.A Perkin-Elmer CHN-2400 analyzer was used for elemental microanalyses (CHN).No-carrieradded [ 131 I] NaI was received as a gift from RPF (Radioisotopes-Production-Facility), Egyptian Atomic Energy Authority (EAEA).A NaI (Tl) scintillation counter (Scaler Ratemeter SR7 model, the United Kingdom) was used for γ-ray radioactivity measurement.

Comparison between conventional, grinding and microwave methods
The conventional, grinding and microwave reaction times were showed in Table 1.The yield economy (YE) was used as a term to determine the conventional, grinding, and microwave synthetic different efficiencies of the same reaction.Calculation of YE was occurred through: E = yield% Reaction time"min" .YE was used to provide the yields obtained conclusively enhanced under the conventional, grinding and microwave conditions.RME = Wt of isolated product Wt of reactants .OE was used for the direct comparisons between the two reaction types and can be calculated throughOE = RME AE x100 .So, the yield economy (YE) can be considered as a metric to enhancing the conversion efficiencies of these two different synthetic methods of the same reaction.The reaction theoretical maximum efficiency was represented by using AE, while, RME gives the observed mass efficiency.The grinding and microwave reactions atom economy (AE) have the same values due to using two different reaction conditions to obtain the same desired compounds, as shown in Table 1.

In vitro anticancer evaluation Cell line
Hepatocellular carcinoma (HepG2), and colorectal carcinoma (HCT-116).The cell lines were obtained from ATCC via Holding company for biological products and vaccines (VACSERA), Cairo, Egypt.

Chemical reagents
The reagents RPMI-1640 medium, MTT and DMSO (sigma co., St. Louis, USA), Fetal Bovine serum (GIBCO, UK).5-Fluorouracil was used as a standard anticancer drug for comparison.

MTT assay (1)
HepG2 and (HCT-116 were used to determine the inhibitory effects of compounds on cell growth using the MTT assay.The relative cell viability in percentage was calculated as (A570 of treated samples/A570 of untreated sample) X 100.

Preparation of [ 131 I]iodo-pyrazole derivative
Radioiodination of compound 2 was performed using different values of the synthesized pyrazole derivative (25-400 µg) solutions, then 200 µL of chloramine-T (CAT) solution -freshly prepared-containing (100-600 µg).Then, 10 µL of (4 MBq) [ 131 I]NaI was dropped to the reaction mixture, after that pH values were studied in a range from 4 to 9. The mixture was kept at ambient temperature for 24 h.

Assessment of radiochemical purity
Formation of [ 131 I]iodo-pyrazole derivative was assessed using TLC in a mobile phase chloroform: methanol (3: 1). 5 µL from reaction was added at the spotted point at the lower edge of the paper strip, then in ascending manner TLC was developed prepared using chloroform: methanol (3: 1 v/v).[ 131 I] iodo-pyrazole derivative was migrate to R f = 0.9; while free iodide remained at the spotted area.Then RCP was calculated.

In-vivo evaluation of [ 131 I]iodo-pyrazole derivative
Biodistribution studies was performed under the Animal Ethics Committee criteria, EAEA [45][46][47].Swiss Albino mice aged three months weight 20-25 g were taken as a gift from the animal house of Egyptian Atomic Energy Authority.Ehrlich-Ascites-Carcinoma cell line (EAC) was taken from a female mouse mammary carcinoma, then used for induction of the tumour [34,48].50 µL from diluted EAC cell line was injected intramuscularly in the right Albino swiss mouse thigh 5 days.Six groups of three mice each were injected intravenously at the mice tail.The substrate concentration (compound 2) in the intravenous injection was 6.14 × 10 -4 mM, while the injected activity in each mice equal 0.4 MBq ([ 131 I]NaI).Then the mice were anaesthetized with isoflurane, weighted and sacrificed using cervical dislocation method at different time intervals (0.25, 0.5, 1, 2, 3, and 24 h) and the organs were dissected, weighed and counted.Then results were calculated as % ID/g.

Synthesis
Microwave and grinding techniques were used for heterocyclic synthesis to obtain an environmentally friendly methods are in accordance with the green chemistry principles [49][50][51].Our objective was to develop a synthetic method for the reaction of 5-amino-1,3-diphenyl-1H-pyrazole-4-carbonitrile (1) with different nucleophilic and electrophilic reagents using microwave and grinding techniques.The reactions were repeated using conventional technique with the similar outcome.
The bifunctional starting material 1 can be used as a key intermediate in interesting bicyclic compounds synthesis, via its irradiation with different nucleophilic reagents namely malononitrile, cyanoacetamide and/or chloroacetic acid in presence of sodium ethoxide producing pyrazolopyridine and pyrazolopyrrole derivatives 2-4, respectively.Formation of di-enaminonitrile derivative 2 occurred through the condensation reaction between the amino group and malononitrile to form the intermediate (A), which undergo ring closure formed the expected compound.The structure of compound 2 was confirmed from its IR spectrum, which displayed bands at 3442, 3315, 3290 cm −1 characteristics for the NH 2 group.Its 1 H-NMR spectrum showed a peak corresponding to 2NH 2 at 10.32 ppm.Its mass spectrum revealed a molecular ion peak at 326 (M + ) corresponding to C 19 H 14 N 6 .However, formation of compound 3 may takes place through the nucleophilic attack of the amino group on the cyano group of cyanoacetamide to form the intermediate (B), which underwent ring closure to produce the expected derivative.While the structure of compound 3 was supported from its IR spectrum, which was devoid of CN band and displayed band at 1658 cm While, pyrazolopyrimidin-6-one 6 was obtained through the condensation reaction between the amino group and urea to form the intermediate (D) through the elimination of one molecule of ammonia which undergo ring closure formed the expected compound.However, the structure of compound 6 was illustrated from its IR spectrum, which was devoid of CN band and revealed band at 1688 specifics for C = O group.Its 1 H-NMR spectrum display peaks at 9.55 and 10.28 ppm corresponding to NH 2 and NH, respectively.Its 13 C-NMR spectrum showed a peak corresponding to C = O at 159.8 ppm.Its mass spectrum revealed a molecular ion peak at 303 (M + ) corresponding to C 17 H 13 N 5 O. Pyrazolopyrimidin-6-thione 7 was furnished in the same manner through the intermediate (E).While the structure of compound 7 was supported from its IR spectrum, which was devoid of CN band and displayed band at 3200 and 1287 corresponding to NH and C = S groups, respectively.Its 1 H-NMR spectrum display peaks at 10.33 and 13.14 ppm corresponding to NH 2 and NH, respectively.Its 13 C-NMR spectrum showed a peak corresponding to C = S at 189.1 ppm.Its mass spectrum revealed a molecular ion peak at 319 (M + ) corresponding to C 17 H 13 N 5 S. Noteworthy, pyrazolopyrimidine derivative 8 was formed through the compound 1 irradiation with benzaldehyde in the presence of sodium ethoxide.Furthermore, the structure of compound 8 was illustrated from its 1 H-NMR spectrum display peaks at 1.31 and 4.20 corresponding to CH 3 and CH 2 groups, respectively.Its 13 C-NMR spectrum showed a peak corresponding to CH 3 and CH 2 at 15.9 and 56.8 ppm, respectively.Its mass spectrum revealed a molecular ion peak at 392 (M + ) corresponding to C 25 H 20 N 4 O.However, compound 9 formation takes place through the two molecules of compound 1 condensation, where the first molecule amino group attacks the second molecule cyano group, producing the intermediate (F) followed by ring closure (Scheme 2).However, the structure of compound 9 was illustrated from its 1 H-NMR spectrum display peaks at 9.41 and 10.57ppm corresponding to 2NH 2 groups.Its mass spectrum revealed a molecular ion peak at 520 (M + ) corresponding to C 32 H 24 N 8 .

In-vitro anticancer evaluation
The synthesized pyrazole derivatives were in-vitro examined for its cytotoxicity against two types of human cancer cells (HepG2 and HCT-116) as shown in Table 2. their cytotoxic ability was compared with 5-Fu as a reference.Results showed that the tested compound 2 has the highest cytotoxic effect against the two assessed cell lines showing an obvious cytotoxic activity.The obtained IC 50 values of compound 2 were 9.2 ± 2.8 and 7.7 ± 1.8 µM against HepG2 and HCT-116, respectively.

Structure-Activity Relationship (SAR)
In this study, a series of heterocyclic bearing Pyrazole moiety derivatives were synthesized and their cytotoxicity were evaluated against two cancer cell lines (HepG2 and HCT-116).Various derivatives were synthesized  from 5-amino-1,3-diphenyl-1H-pyrazole-4-carbonitrile (compound 1).The tested compounds results showed that exhibited very strong, strong, moderate, or weak anticancer activities against the tested cell lines.First, malononitrile was added forming pyridine moiety having diamino and cyano group (compound 2) appeared to have the highest biological activity against both cell lines.Further modifications were implemented which resulted in drastic decrease in activity.The activity of compounds 2 may be related to the core of compound which is pyrazolopyridine ring in addition to the cyano group presence at position 5 of the pyridine ring which is an electronwithdrawing group, in addition to the two amino groups presence at positions 4 and 6 of compound 2.However, the addition of hydrazine hydrate having another pyrazole moiety (compound 5) caused slight decrease in the cytotoxic activity against HCT-116 and slight increase against HepG.While the activity of compounds 5 may be related to the pyrazolopyrazole ring in addition to the two amino groups presence at positions 3 and the NH of the ring of compound 5.However, the activity of compounds 7 may be related to pyrazolopyrimidine ring in addition to the C = S group presence at position 7 of the pyrimidine ring, which is a rich by electron centres, in addition to the two amino groups presence at position 4 of compound 7. Further modifications using cyanoacetamide, chloroacetic acid, thiourea, benzaldehyde, and TEA caused some decrease in the cytotoxic activity.

Radioiodination of pyrazole derivative
Compound 2 can be used as a chemotherapeutic agent because it is the highest cytotoxic synthesized compound against both HepG2 and HCT-116 cell lines.Compound 2 was radiolabeled with iodine-131 to evaluate its biodistribution in animal models and also to be studied to carry the therapeutic radioisotope (iodine-131) to the target site (cancer tissues) for targeted radio therapy (TRT).
The highest radiochemical purity of radioiodinated compound 2 was obtained by optimization of all parameters affecting the radioiodination process.The electrophilic substitution of iodine-131 was carried out in the presence of an oxidizing agent (chloramine-T).Chloramine-T, which converts iodide ion to iodonium ion [52], has a clear impact on the process of radioiodination, also an electrophilic substitution was happen by the permission from the oxidizing agent.400 µg from CAT was the optimum amount required to create the highest RCP of 91.74% (Fig. 1).Different CAT amounts may cause undesirable oxidative byproduct and Insufficient oxidation of I-131 by increasing or decreasing of its value, respectively [53].pH value is a main factor which affect the RCP.The RCP was decreased in acidic media and increased around neutral values then decreases again towards basic media (Fig. 2).The formation of hypoiodite (IO − ) ion and iodate (IO 3

−
) ions may be the cause of this decrease in RCP value [54].Figure 3 illustrate the effect of compound 2 amount on the RCP.The maximum RCP was obtained at 100 µg from compound 2, this amount can react with all ions of iodonium in the reaction.By increasing the amount of substrate, the RCP still stable around the highest yield.Figure 4 showed the time effect on the radioiodination process which indicates its fastness.The reaction stability study was done for 24 h.

Conclusion
Pyrazole-based analogs play a significant role in the creation of numerous medications and have a wide spectrum of pharmacological actions.Pyrazole derivatives were synthesized via green methods to be tested for in-vitro chemo-and TRT.These substances were synthesized as a result of their noteworthy biological activities, which have made them prospective candidates in the discovery and development of new drugs.The most effective synthesized pyrazole derivative against the two tested cancer cell lines is 4,6-Diamino-1,3-diphenyl-1H-pyrazolo[3,4b]pyridine-5-carbonitrile which shows high effective action against both HepG2 and HCT-116 cell lines and also it has high accumulation at cancer site which guide us to conclude that this derivative (compound 2) could be used as cancer chemotherapeutic agent.Also, radioiodination of this compound using I-131 -which is used as therapeutic radioisotope-making this compound to be used for TRT.In conclusion the synthesized new pyrazole derivative can be used for chemo/radioisotope therapy after further preclinical studies.

Table 1
Show the comparison in terms of physical data between the synthesized compounds under grinding and microwave techniques *Lit.literature value, YE yield economy, RME reaction mass efficiency, OE optimum efficiency, AE atom economy, Con.conventional, G. grinding, M.W. microwave −1 characteristics for the C = O group.Its 1 H-NMR spectrum display peaks corresponding to 2NH 2 and CONH 2 at 8.89 and 11.24 ppm, respectively.Its 13 C-NMR spectrum showed a peak corresponding to C = O at 162.3 ppm.Its mass spectrum revealed a molecular ion peak at 344 (M + ) corresponding to C 19 H 16 N 6 O. Also, in case of compound 4 formation, chloroacetic acid reacted with the amino group forming the intermediate (C) through the elimination of one HCl molecule and then ring closure takes place.Also, IR spectrum of compound 4 showed disappearance of CN band in its IR spectrum and displayed bands at 3223 and 1709 cm −1 characteristics for the NH and C = O groups, respectively.Its 1 H-NMR spectrum display peaks at 5.08, 10.00 and 10.40 ppm corresponding to NH 2 , NH and OH, respectively.Its mass spectrum revealed a molecular ion peak at 318 (M + ) corresponding to C 18 H 14 N 4 O 2 .Furthermore, compound 1 was subjected to the reaction with hydrazine hydrate in microwave to produce pyrazolopyrazole derivative 5 (Scheme 2).Furthermore, the structure of compound 5 was illustrated from its IR spectrum, which was devoid of CN band and revealed bands at 3385, 3313 and 3201 specifics for NH 2 and NH groups, respectively.Its 1 H-NMR spectrum display peaks at 8.68 and 10.31 ppm corresponding to NH and NH 2 , respectively.Its mass spectrum revealed a molecular ion peak at 275 (M + ) corresponding to C 16 H 13 N 5 .