Pyrrolizine/Indolizine-NSAID Hybrids: Design, Synthesis, Biological Evaluation, and Molecular Docking Studies

In the current study, eight new hybrids of the NSAIDs, ibuprofen and ketoprofen with five pyrrolizine/indolizine derivatives were designed and synthesized. The chemical structures of these hybrids were confirmed by spectral and elemental analyses. The antiproliferative activities of these hybrids (5 μM) was investigated against MCF-7, A549, and HT-29 cancer cell lines using the cell viability assay, MTT assay. The results revealed 4–71% inhibition of the growth of the three cancer cell lines, where 8a,e,f were the most active. In addition, an investigation of the antiproliferative activity of 8a,e,f against MCF-7 cells revealed IC50 values of 7.61, 1.07, and 3.16 μM, respectively. Cell cycle analysis of MCF-7 cells treated with the three hybrids at 5 μM revealed a pro-apoptotic increase in cells at preG1 and cell cycle arrest at the G1 and S phases. In addition, the three hybrids induced early apoptotic events in MCF-7 cells. The results of the molecular docking of the three hybrids into COX-1/2 revealed higher binding free energies than their parent compounds 5a,c and the co-crystallized ligands, ibuprofen and SC-558. The results also indicated higher binding free energies toward COX-2 over COX-1. Moreover, analysis of the binding modes of 8a,e,f into COX-2 revealed partial superposition with the co-crystallized ligand, SC-558 with the formation of essential hydrogen bonds, electrostatic, or hydrophobic interactions with the key amino acid His90 and Arg513. The new hybrids also showed drug-likeness scores in the range of 1.06–2.03 compared to ibuprofen (0.65) and ketoprofen (0.57). These results above indicated that compounds 8a,e,f deserve additional investigation as potential anticancer candidates.


Introduction
Currently, more than 150 anticancer drugs approved by the FDA for the treatment of different types of cancer are available in the market [1]. However, the rates of morbidity and mortality due to cancers are still high, even within high-income countries [2]. In addition, the low rate of survival in patients with metastatic cancers [3] also confirms the urgent need for continuous research in this field.
In addition, several successful examples of NSAIDs hybrids/conjugates in cancer research have been also reported, Figure 1. Although the parent NSAIDs have showed weak cytotoxic activities [24][25][26][27], their hybrids with the cytotoxic agents were reported to have potent anticancer activity. Among these hybrids, the riboflavin-dexibuprofen conjugates IVa-e were synthesized by Banekovich et al. [24]. A biological evaluation of these conjugates IVa-e revealed growth-inhibitory activity of MCF-7 and HT-29 at IC50 values of 8-15 μM. These results indicated that the conjugates have much higher antiproliferative activities than their parent components, dexibuprofen and tetraacetylriboflavin.
Curci et al. [25] have synthesized prodrug conjugate V of kiteplatin Pt(IV) and two molecules of ibuprofen. The new conjugate exhibited much higher cytotoxic activity compared to the parent components. It inhibited the growth of HCT15 and HCT116 cancer cell lines at IC50 of 0.45 and 0.26 μM, respectively.

Rational Design
Previously, we reported compound VIIIa-c ( Figure 2) with in vivo anti-inflammatory activities comparable with that of ibuprofen [28,29]. Only two of these hybrids (VIIIb,c) were also investigated for their cytotoxic activity against three cancer cell lines (IC 50 = 0.06-0.87 µM). These results motivated us to investigate the structure-activity relationship and the potential mechanism which mediates the cytotoxic activity of this scaffold.
Many of the NSAIDs displayed weak antiproliferative activity which could be COXdependent or independent [30]. Esterification/amidation of the carboxylic acid group in ibuprofen was associated in some cases with increases in the anti-inflammatory activity and decrease in GIT side effects [31,32]. Some of the ester and amide derivatives of ibuprofen also showed improved antiproliferative activity compared to the parent drug [30]. On a cellular level, this improvement of the antiproliferative activity could be due to the increased lipophilicity and enhancer cellular uptake [33].
Encouraged by the above findings, we have designed a new series of hybrids (scaffold B) by incorporating the pyrrolizine/indolizine derivatives (scaffold A) with NSAIDs, Figure 3. Ibuprofen was selected as the NSAID component of the new hybrids based on the promising cytotoxic activity of VIIIb,c. In addition, ketoprofen was also selected in this study to compare the effect of the two drugs on the antiproliferative activity of the new hybrids. The aim of this selection was to compare the impact of the two drugs on cytotoxic activities of the final hybrids, Figure 3. Chemical structure of compounds VIIIa-c: VIIIa, a hybrid of ibuprofen with 6amino-N-(4-bromophenyl)-7-cyano-2,3-dihydro-1H-pyrrolizine-5-carboxamide that exhibited in vivo anti-inflammatory activity [25]; VIIIb,c, ibuprofen-pyrrolizine/indolizine hybrids with cytotoxic activity [26].
Many of the NSAIDs displayed weak antiproliferative activity which could be COXdependent or independent [30]. Esterification/amidation of the carboxylic acid group in ibuprofen was associated in some cases with increases in the anti-inflammatory activity and decrease in GIT side effects [31,32]. Some of the ester and amide derivatives of ibuprofen also showed improved antiproliferative activity compared to the parent drug [30]. On a cellular level, this improvement of the antiproliferative activity could be due to the increased lipophilicity and enhancer cellular uptake [33].
Encouraged by the above findings, we have designed a new series of hybrids (scaffold B) by incorporating the pyrrolizine/indolizine derivatives (scaffold A) with NSAIDs, Figure 3. Ibuprofen was selected as the NSAID component of the new hybrids based on the promising cytotoxic activity of VIIIb,c. In addition, ketoprofen was also selected in this study to compare the effect of the two drugs on the antiproliferative activity of the new hybrids. The aim of this selection was to compare the impact of the two drugs on cytotoxic activities of the final hybrids, Figure 3.  To investigate the relationship between the chemical structure and the antiproliferative activity of the new hybrids (scaffold B), several pyrrolizine/indolizine derivatives (scaffold A) were used. In addition, the new hybrids were designed bearing electron-donating (OCH3) and electron-withdrawing (F, Cl, and Br) substituents at the para-position of ring A to compare the impact of the electronic effects of their antiproliferative activity, Figure 3. The new hybrids will be evaluated for their antiproliferative activity against a panel of cancer cell lines. An additional investigation will also be performed to evaluate the potential mechanism of their action of the most active compounds. To investigate the relationship between the chemical structure and the antiproliferative activity of the new hybrids (scaffold B), several pyrrolizine/indolizine derivatives (scaffold A) were used. In addition, the new hybrids were designed bearing electron-donating (OCH 3 ) and electron-withdrawing (F, Cl, and Br) substituents at the para-position of ring A to compare the impact of the electronic effects of their antiproliferative activity, Figure 3. The new hybrids will be evaluated for their antiproliferative activity against a panel of cancer cell lines. An additional investigation will also be performed to evaluate the potential mechanism of their action of the most active compounds.

Chemistry
The synthesis of the target hybrids 8a-i is presented in Scheme 1. Compounds 2a,b were obtained from the reaction of 1a,b with malononitrile according to the previous reports [34,35]. In addition, compounds 4a-d were prepared from the reaction of chloroacetyl chloride with the appropriate aniline derivatives 3a-d [34,35]. On the other hand, the pyrrolizine/indolizine derivatives 5a-e were prepared from the reaction of 2a,b with the appropriate acetanilide derivatives 4a-d [36]. Preparation of the target hybrids 8a-i was achieved in two steps following the previous report [29]. In the first step, the NSAIDs, ibuprofen and ketoprofen were converted into their acid chloride derivatives 7a,b using thionyl chloride. The second step takes place through an acylation of the primary amino groups in compounds 5a-e using the freshly prepared acid chloride derivatives 7a,b. Structural elucidation of the new hybrids 8a-i was performed. The spectral data of compounds 8a,b is discussed below.
The IR spectrum of pyrrolizine-ibuprofen 8a revealed absorption bands at 2221 and 1659 cm −1 indicate the cyano and carbonyl groups, respectively (Supplementary data, Figure S1). The 1 H-NMR spectrum of 8a revealed seven signals at the aliphatic region (δ 0.88-4.29 ppm) which indicate the aliphatic protons, (Supplementary data, Figure S10-S13). The two methyl groups of the isobutyl moiety (-CH2CH(CH3)2) appears as a doublet signal at δ 0.88 ppm. Another doublet signal at δ 1.61 indicates the methyl group of the propionamide moiety (-COCHCH3). In addition, four doublet signals are found at δ 6.74, 7.04, 7.21, and 7.26 ppm indicate the two para-substituted phenyl rings. Two singlet signals at δ 8.21 and 9.03 ppm indicate the two amide protons. To prepare 4-methoxy/fluoro indolizine derivatives, 2b was also refluxed with the appropriate acetanilide 4a,b for 48 h. However, the duplication of the reflux time was also associated with the formation of uncyclized derivatives rather than the target indolizines. The IR spectra of the obtained products revealed absorption bands of the geminal cyano groups, which confirms the formation of uncyclized products. These results align with the results of our previous report [35].
Preparation of the target hybrids 8a-i was achieved in two steps following the previous report [29]. In the first step, the NSAIDs, ibuprofen and ketoprofen were converted into their acid chloride derivatives 7a,b using thionyl chloride. The second step takes place through an acylation of the primary amino groups in compounds 5a-e using the freshly prepared acid chloride derivatives 7a,b. Structural elucidation of the new hybrids 8a-i was performed. The spectral data of compounds 8a,b is discussed below.
The IR spectrum of pyrrolizine-ibuprofen 8a revealed absorption bands at 2221 and 1659 cm −1 indicate the cyano and carbonyl groups, respectively (Supplementary data, Figure S1). The 1 H-NMR spectrum of 8a revealed seven signals at the aliphatic region (δ 0.88-4.29 ppm) which indicate the aliphatic protons, (Supplementary data, Figures S10-S13). The two methyl groups of the isobutyl moiety (-CH 2 CH(CH 3 ) 2 ) appears as a doublet signal at δ 0.88 ppm. Another doublet signal at δ 1.61 indicates the methyl group of the propionamide moiety (-COCHCH 3 ). In addition, four doublet signals are found at δ 6.74, 7.04, 7.21, and 7.26 ppm indicate the two para-substituted phenyl rings. Two singlet signals at δ 8.21 and 9.03 ppm indicate the two amide protons.
On the other hand, the IR spectrum of the ketoprofen hybrid 8b revealed absorption bands at 2211 and 1698 cm −1 , indicating the cyano and carbonyl groups, respectively (Supplementary data, Figure S2). A total of 6 signals at the aliphatic region (δ 1.62-4.27 ppm) in the 1 H-NMR spectrum of 8b are observed indicating the aliphatic protons, (Supplementary data, Figures S20-S23). The signal corresponding to the methyl group of the propionamide moiety (-NHCOCHCH 3 ) appears as a doublet signal at δ 1.62 ppm. The spectrum also shows one triplet signal at δ 2.83, which indicates the methylene group (CH 2 -1) of the pyrrolizine nucleus. A multiplet at δ 6.69-7.68 ppm indicates 12 of the aromatic protons of the 3 phenyl rings, while a single signal at δ 7.86 ppm indicates the aromatic proton at ortho-position of the phenyl propionamide ring (B). In addition, 2 singlet signals at δ 8.94 and 9.02 ppm indicates the two amide protons in this compound.
Supplementary data including all spectral data and copies of IR, 1 H-NMR, and 13 C-NMR spectra of the final compounds 8a-i are provided with this manuscript (Supplementary data, Figures S1-S78).

Antiproliferative Activity Screening Assay
Previously, the ibuprofen hybrids/conjugates IV-VIII exhibited antiproliferative activities against different types of cancer cell lines [24][25][26][27][28][29]. Accordingly, the new hybrids 8a-i were evaluated for their antiproliferative activity using the cell viability assay, MTT assay following the previous report [37]. In this assay, the new hybrids were evaluated against three cancer cell lines including MCF-7 breast, A549 lung, and HT-29 colon cancer cell lines. The antiproliferative activities of the starting materials 5a-e, ibuprofen and ketoprofen, were also investigated in this assay. The cancer cells were treated with the test compounds at a single dose (5 µM) for 72 h. The inhibition in the viability of the cancer cell lines was calculated relative to the untreated control. The results (growth%) are represented in Figure 4. On the other hand, the IR spectrum of the ketoprofen hybrid 8b revealed absorption bands at 2211 and 1698 cm −1 , indicating the cyano and carbonyl groups, respectively (Supplementary data, Figure S2). A total of 6 signals at the aliphatic region (δ 1.62-4.27 ppm) in the 1 H-NMR spectrum of 8b are observed indicating the aliphatic protons, (Supplementary data, Figure S20-S23). The signal corresponding to the methyl group of the propionamide moiety (-NHCOCHCH3) appears as a doublet signal at δ 1.62 ppm. The spectrum also shows one triplet signal at δ 2.83, which indicates the methylene group (CH2-1) of the pyrrolizine nucleus. A multiplet at δ 6.69-7.68 ppm indicates 12 of the aromatic protons of the 3 phenyl rings, while a single signal at δ 7.86 ppm indicates the aromatic proton at ortho-position of the phenyl propionamide ring (B). In addition, 2 singlet signals at δ 8.94 and 9.02 ppm indicates the two amide protons in this compound.
Supplementary data including all spectral data and copies of IR, 1 H-NMR, and 13 C-NMR spectra of the final compounds 8a-i are provided with this manuscript (Supplementary data, Figure S1-S78).

Screening Assay
Previously, the ibuprofen hybrids/conjugates IV-VIII exhibited antiproliferative activities against different types of cancer cell lines [24][25][26][27][28][29]. Accordingly, the new hybrids 8a-i were evaluated for their antiproliferative activity using the cell viability assay, MTT assay following the previous report [37]. In this assay, the new hybrids were evaluated against three cancer cell lines including MCF-7 breast, A549 lung, and HT-29 colon cancer cell lines. The antiproliferative activities of the starting materials 5a-e, ibuprofen and ketoprofen, were also investigated in this assay. The cancer cells were treated with the test compounds at a single dose (5 μM) for 72 h. The inhibition in the viability of the cancer cell lines was calculated relative to the untreated control. The results (growth%) are represented in Figure 4. Columns are mean of cell viability percent after treatment with compounds 5a-e, 8a-i, ibuprofen and ketoprofen at 5 μM for 72 h ±SD (n = 6). MTT cell viability assay experiment was conducted three times. p-value determined by two-tailed unpaired t-test. * indicates significant difference versus control at p < 0.05 (*), p < 0.01 (**). # indicates significant difference versus Ibuprofen at p < 0.05 (#), p < 0.01 (##). @ indicates significant difference versus Ketoprofen at p < 0.01 (@). Columns are mean of cell viability percent after treatment with compounds 5a-e, 8a-i, ibuprofen and ketoprofen at 5 µM for 72 h ±SD (n = 6). MTT cell viability assay experiment was conducted three times. p-value determined by two-tailed unpaired t-test. * indicates significant difference versus control at p < 0.05 (*), p < 0.01 (**). # indicates significant difference versus Ibuprofen at p < 0.05 (#), p < 0.01 (##). @ indicates significant difference versus Ketoprofen at p < 0.01 (@). The results of the cell viability assay of the tested hybrids against MCF-7 cells revealed 12-71% inhibition of the cancer cells' viability, Figure 4, which were much higher compared with those of ibuprofen (5%) and ketoprofen (9%). The changes in the cell viability of MCF-7 cells treated by all the new hybrids 8a-i were statistically different from the control (p < 0.01). Compounds 8a,e,f induced more than 50% inhibition of the growth of MCF-7 cells at 5 µM. In addition, the pyrrolizine derivatives 8a-f showed higher growth-inhibitory activities than their parent compounds 5a-c, however, the indolizine derivatives 8g-i exhibited lower growth-inhibitory activities than their parent compounds 5c,d. The results also indicated higher antiproliferative for the ibuprofen derivatives (8a,c,g) compared to their corresponding ketoprofen analogs (8b,d,h). Among the new hybrids, 8f exhibited the highest antiproliferative activity (71%) against MCF-7 cells, which was statistically different from those of the control and ibuprofen (p < 0.01).
The effects of the new hybrids 8a-i on the growth of A549 cancer cell line are presented in Figure 5. Except for 8h, the new hybrids exhibited 13-48% inhibition in the growth of A549 cells compared to the control (p < 0.01). The results also revealed higher antiproliferative activity for the ibuprofen hybrids than the ketoprofen analogs. However, no antiproliferative activity was observed for ibuprofen and ketoprofen against A549 cells. Among the new hybrids, 8a,e,f showed the highest antiproliferative activity. The results of the cell viability assay of the tested hybrids against MCF-7 cells revealed 12%-71% inhibition of the cancer cells' viability, Figure 4, which were much higher compared with those of ibuprofen (5%) and ketoprofen (9%). The changes in the cell viability of MCF-7 cells treated by all the new hybrids 8a-i were statistically different from the control (p < 0.01). Compounds 8a,e,f induced more than 50% inhibition of the growth of MCF-7 cells at 5 μM. In addition, the pyrrolizine derivatives 8a-f showed higher growth-inhibitory activities than their parent compounds 5a-c, however, the indolizine derivatives 8g-i exhibited lower growth-inhibitory activities than their parent compounds 5c,d. The results also indicated higher antiproliferative for the ibuprofen derivatives (8a,c,g) compared to their corresponding ketoprofen analogs (8b,d,h). Among the new hybrids, 8f exhibited the highest antiproliferative activity (71%) against MCF-7 cells, which was statistically different from those of the control and ibuprofen (p < 0.01).
The effects of the new hybrids 8a-i on the growth of A549 cancer cell line are presented in Figure 5. Except for 8h, the new hybrids exhibited 13%-48% inhibition in the growth of A549 cells compared to the control (p < 0.01). The results also revealed higher antiproliferative activity for the ibuprofen hybrids than the ketoprofen analogs. However, no antiproliferative activity was observed for ibuprofen and ketoprofen against A549 cells. Among the new hybrids, 8a,e,f showed the highest antiproliferative activity. Anti-proliferative effect of the tested compounds on the viability of A549 cells. Columns are mean of cell viability percent after treatment with compounds 5a-d, 8a-i, ibuprofen and ketoprofen at 5 μM for 72 h ± SD (n = 6). MTT cell viability assay experiment was conducted three times. p-value determined by two-tailed unpaired t-test. * indicates significant difference versus control at p < 0.05 (*), p < 0.01 (**). # indicates significant difference versus Ibuprofen at p < 0.05 (#), p < 0.01 (##). @ indicates significant difference versus Ketoprofen at p < 0.01 (@).
Finally, compounds 8a-i were also investigated for their effects on the viability of HT-29 cancer cells. The results are presented in Figure 6. Except for compound 8b, all of the tested hybrids included 4%-53% inhibition in the viability of HT-29 cells. However, both ibuprofen and ketoprofen did not show antiproliferative activity against HT-29 cells at 5 μM. Anti-proliferative effect of the tested compounds on the viability of A549 cells. Columns are mean of cell viability percent after treatment with compounds 5a-d, 8a-i, ibuprofen and ketoprofen at 5 µM for 72 h ± SD (n = 6). MTT cell viability assay experiment was conducted three times. p-value determined by two-tailed unpaired t-test. * indicates significant difference versus control at p < 0.05 (*), p < 0.01 (**). # indicates significant difference versus Ibuprofen at p < 0.05 (#), p < 0.01 (##). @ indicates significant difference versus Ketoprofen at p < 0.01 (@).
Finally, compounds 8a-i were also investigated for their effects on the viability of HT-29 cancer cells. The results are presented in Figure 6. Except for compound 8b, all of the tested hybrids included 4-53% inhibition in the viability of HT-29 cells. However, both ibuprofen and ketoprofen did not show antiproliferative activity against HT-29 cells at 5 µM. Columns are mean of cell viability percent after treatment with compounds 5a-d, 8a-i, ibuprofen and ketoprofen at 5 μM for 72 h ± SD (n = 6). MTT cell viability assay experiment was conducted three times. p-value determined by two-tailed unpaired t-test. * indicates significant difference versus control at p < 0.01 (**). # indicates significant difference versus Ibuprofen at p < 0.05 (#), p < 0.01 (##). @ indicates significant difference versus Ketoprofen at p < 0.05 (@), p < 0.01 (@@).
The results also indicated higher antiproliferative activity for the ibuprofen derivatives (8a,c,e,g) against HT-29 cells than their ketoprofen analogs (8b,d,f,h). Among the new hybrids, 8a,e,f exhibited the highest antiproliferative activity (47%-49%) against HT-29 cells compared to the control (p < 0.01).
In conclusion, the results of the cell viability assay showed the highest sensitivity for MCF-7 cells toward the new hybrids. Moreover, compounds 8a,e,f were the most active in inhibiting the proliferation of MCF-7 cells.

Cytotoxicity Assay
Based on the results of the MTT viability assay ( Figures. 4-6), three of the new hybrids (8a,e,f) were selected for further investigation of their antiproliferative activities. The MTT assay was used to determine the IC50 values of the selected hybrids following the previous report [38]. MCF-7 cell line, the most sensitive to the antiproliferative effect of the new hybrids, was selected for this study. Doxorubicin was used as a reference drug. The cancer cells were treated with the test compounds at different concentrations. The results expressed as IC50 values were determined, Table 1. The results of the MTT assay revealed that the three hybrids inhibit the growth of MCF-7 cells at IC50 values in the range of 1.07-7.61 μM compared to doxorubicin (IC50 = 2.07 μM). The IC50 value of doxorubicin was slightly higher than the reported value [39]. Columns are mean of cell viability percent after treatment with compounds 5a-d, 8a-i, ibuprofen and ketoprofen at 5 µM for 72 h ± SD (n = 6). MTT cell viability assay experiment was conducted three times. p-value determined by two-tailed unpaired t-test. * indicates significant difference versus control at p < 0.01 (**). # indicates significant difference versus Ibuprofen at p < 0.05 (#), p < 0.01 (##). @ indicates significant difference versus Ketoprofen at p < 0.05 (@), p < 0.01 (@@).
The results also indicated higher antiproliferative activity for the ibuprofen derivatives (8a,c,e,g) against HT-29 cells than their ketoprofen analogs (8b,d,f,h). Among the new hybrids, 8a,e,f exhibited the highest antiproliferative activity (47-49%) against HT-29 cells compared to the control (p < 0.01).
In conclusion, the results of the cell viability assay showed the highest sensitivity for MCF-7 cells toward the new hybrids. Moreover, compounds 8a,e,f were the most active in inhibiting the proliferation of MCF-7 cells.

Cytotoxicity Assay
Based on the results of the MTT viability assay (Figures 4-6), three of the new hybrids (8a,e,f) were selected for further investigation of their antiproliferative activities. The MTT assay was used to determine the IC 50 values of the selected hybrids following the previous report [38]. MCF-7 cell line, the most sensitive to the antiproliferative effect of the new hybrids, was selected for this study. Doxorubicin was used as a reference drug. The cancer cells were treated with the test compounds at different concentrations. The results expressed as IC 50 values were determined, Table 1. The results of the MTT assay revealed that the three hybrids inhibit the growth of MCF-7 cells at IC 50 values in the range of 1.07-7.61 µM compared to doxorubicin (IC 50 = 2.07 µM). The IC 50 value of doxorubicin was slightly higher than the reported value [39]. Among the three compounds, 8e was the most active in inhibiting the growth of MCF-7 cells, while 8a showed the lowest cytotoxicity.

Cell Cycle Analysis
Previous reports indicated that the amide derivatives of ibuprofen/ketoprofen induced cell cycle arrest at the G0/G1 or G1/S phases in cancer cell lines [30].
In the current study, the effect of compounds 8a,e,f on the cell cycle distributions of MCF-7 cells was investigated 8a,e,f were also investigated. The cancer cells were treated with the test compounds at 5 µM for 48 h. This study was performed following the previous report [36]. The results are presented in Figure 7. Among the three compounds, 8e was the most active in inhibiting the growth of MCF-7 cells, while 8a showed the lowest cytotoxicity.

Cell Cycle Analysis
Previous reports indicated that the amide derivatives of ibuprofen/ketoprofen induced cell cycle arrest at the G0/G1 or G1/S phases in cancer cell lines [30].
In the current study, the effect of compounds 8a,e,f on the cell cycle distributions of MCF-7 cells was investigated 8a,e,f were also investigated. The cancer cells were treated with the test compounds at 5 μM for 48 h. This study was performed following the previous report [36]. The results are presented in Figure 7. The result of cell cycle analysis revealed that each of the 3 compounds showed profound increase in the preG1 phase in MCF-7 cells, as an indication of their pro-apoptotic activities, especially 8e that showed 10-fold increase compared to control, Table 2. Additionally, 8a and 8e arrested cells in the S phase at the expense of G1 and G2/M phases, while 8f slightly increased cells in the G1 phase.  The result of cell cycle analysis revealed that each of the 3 compounds showed profound increase in the preG1 phase in MCF-7 cells, as an indication of their pro-apoptotic activities, especially 8e that showed 10-fold increase compared to control, Table 2. Additionally, 8a and 8e arrested cells in the S phase at the expense of G1 and G2/M phases, while 8f slightly increased cells in the G1 phase.

Annexin V-FITC/PI Apoptosis Assay
The amide derivatives of ibuprofen/ketoprofen were also reported to induce apoptosis in different types of cancer cell lines [30]. In the current study, annexin V-FITC/PI double staining protocol was used to investigate the effects of compounds 8a,e,f on apoptotic events in MCF-7 cells. The cancer cells were treated with each of the 3 hybrids at 5 µM for 48 h. The study was performed following the previous report [36]. The results were presented in Figure 8. The amide derivatives of ibuprofen/ketoprofen were also reported to induce apop sis in different types of cancer cell lines [30]. In the current study, annexin V-FITC/PI do ble staining protocol was used to investigate the effects of compounds 8a,e,f on apopto events in MCF-7 cells. The cancer cells were treated with each of the 3 hybrids at 5 μM f 48 h. The study was performed following the previous report [36]. The results were p sented in Figure 8. The 3 hybrids 8a,e,f induced increase in the early apoptotic events in MCF-7 ce ranging from 18-50 folds (11%-30%) compared to the control, Table 3. This was at t expense of late and necrotic events which were absent.

Molecular Docking Studies
The main mechanism of action that mediate the anti-inflammatory activity of ib profen/ketoprofen is the reversible inhibition of COX-1/2 isoenzymes [40,41]. In additio the mechanistic studies of the starting material 5c revealed moderate inhibition of CO The 3 hybrids 8a,e,f induced increase in the early apoptotic events in MCF-7 cells ranging from 18-50 folds (11-30%) compared to the control, Table 3. This was at the expense of late and necrotic events which were absent. The main mechanism of action that mediate the anti-inflammatory activity of ibuprofen/ketoprofen is the reversible inhibition of COX-1/2 isoenzymes [40,41]. In addition, the mechanistic studies of the starting material 5c revealed moderate inhibition of COX-1/2 with weak selectivity toward COX-2 [28]. Although several reports indicated that the antiproliferative activity of NSAIDs are COX-independent, some other reports described a correlation between COXs inhibition and this activity [30]. Moreover, many of the NSAIDs prodrugs have also displayed intrinsic anti-inflammatory activities mediated even in part by COXs inhibition [42].
Based on the above mentioned finding, we performed a molecular docking study of compounds hybrids 8a,e,f into the two COXs to compare the binding characteristics against those of their parent compounds (5a,c and ibuprofen). The docking study was performed into the crystal structure of COX-1 (pdb 1EQG) [43] and COX-2 (pdb 1CX2) [44] using AutoDock 4.2 [45]. The ligands (8a,e,f) and protein molecules were prepared into the suitable formate (pdbqt) for AutoDock following the previous report [46]. In the current study, COX-1/2 proteins were used as rigid molecules, while the ligands were docked as flexible molecules. The 2/3D binding modes of the tested compounds were generated by Discovery Studio Visualizer [47].
Firstly, ibuprofen was re-docked into the active site of COX-1 to validate the docking procedures. The 2/3D binding modes of the re-docked ibuprofen was investigated against those of the co-crystallized ligand. A RMSD of 0.87 Å was observed between the re-docked and the co-crystallized ligand. The re-docked molecule showed identical hydrogen bonds and hydrophobic interactions with those of the co-crystallized ibuprofen, Supplementary data, Figure S79.
Validation of the docking study into COX-2 (pdb: 1CX2) was also performed. the cocrystallized ligand, SC-558 was docked into the active site of COX-2. The results revealed superposition of the re-docked SC-558 with the co-crystallized ligand with RMSD of 1.30 Å. The binding mode of the re-docked SC-558 overlaid with the co-crystallized ligand is shown in Supplementary data, Figure S80.
The results of the docking study of the 3 hybrids (8a,e,f) into COX-1 binding free energies in the range of −10.36 to −10.55 kcal/mol compared to ibuprofen (energy (∆G b = −8.43 kcal/mol), Table 4. On the other hand, they exhibited higher binding free energies toward COX-2 (∆G b = −10.70 to −12.56 kcal/mol) compared to −10.78 kcal/mol for SC-558. The binding free energies of the three hybrids were also higher than their parent compounds 5a,c. Table 4. Results of the docking of 8a,e,f into ovine COX-1/2 in comparison to the parents 5a,c and the co-crystallized ligands (ibuprofen/ SC-558).  Compound 8a exhibited a binding free energy (∆G b ) of -10.36 kcal toward COX-1, which was higher than ibuprofen, Table 4. An investigation of the binding mode of 8a revealed that the best fitting conformation adopted a binding orientation superposing with the co-crystallized ibuprofen into COX-1, where the 4-methoxyaniline moiety in 8a superposed with the 4-isobutylphenyl moiety in ibuprofen, Figure 9. In addition, the carboxamide moiety at C2 of compound 8a was also superposed with the propionic acid moiety in ibuprofen. This orientation allowed 8a to interact similarly to ibuprofen with Arg120 and Tyr355 in COX-1 forming a cluster of four conventional hydrogen bonds. Compound 8a and ibuprofen exhibited similar hydrophobic interactions with Val116, Val349, Ala527, and Leu531. Compound 8a exhibited a binding free energy (ΔGb ) of -10.36 kcal toward COX-1, which was higher than ibuprofen, Table 4. An investigation of the binding mode of 8a revealed that the best fitting conformation adopted a binding orientation superposing with the co-crystallized ibuprofen into COX-1, where the 4-methoxyaniline moiety in 8a superposed with the 4-isobutylphenyl moiety in ibuprofen, Figure 9. In addition, the carboxamide moiety at C2 of compound 8a was also superposed with the propionic acid moiety in ibuprofen. This orientation allowed 8a to interact similarly to ibuprofen with Arg120 and Tyr355 in COX-1 forming a cluster of four conventional hydrogen bonds. Compound 8a and ibuprofen exhibited similar hydrophobic interactions with Val116, Val349, Ala527, and Leu531. The results of the docking study of compound 8a into COX-2 also revealed a higher binding free energy than SC-558. These results indicated that the hybrid 8a has higher affinity toward COX-2 over COX-1. An analysis of the binding mode of the best fitting conformation of compound 8a into COX-2 revealed nice superposition of the two phenyl rings with the phenyl rings of SC-558, Figure 10. The pyrrole ring in 8a was also located near the binding position of the pyrazole ring. Compound 8a exhibited one conventional hydrogen bond with Arg120 and one carbon hydrogen bond with Ser353. In addition, 8a The results of the docking study of compound 8a into COX-2 also revealed a higher binding free energy than SC-558. These results indicated that the hybrid 8a has higher affinity toward COX-2 over COX-1. An analysis of the binding mode of the best fitting conformation of compound 8a into COX-2 revealed nice superposition of the two phenyl rings with the phenyl rings of SC-558, Figure 10. The pyrrole ring in 8a was also located near the binding position of the pyrazole ring. Compound 8a exhibited one conventional hydrogen bond with Arg120 and one carbon hydrogen bond with Ser353. In addition, 8a showed one electrostatic interaction with Arg513. Based on these results, the higher binding free energy of 8a toward COX-2 could be attributed to the electrostatic interaction with Arg513 and the multiple hydrophobic interactions with the hydrophobic residues in COX-2. showed one electrostatic interaction with Arg513. Based on these results, the higher binding free energy of 8a toward COX-2 could be attributed to the electrostatic interaction with Arg513 and the multiple hydrophobic interactions with the hydrophobic residues in COX-2. Compound 8e also showed higher affinity toward COX-1 than ibuprofen. However, 8e did not show any conventional hydrogen bonds with the amino acids in COX-1, Figure  11. The results of the docking study of 8e into COX-2 also revealed higher binding free energy compared to COX-1. The 4-bromophenyl moiety in 8e superposed with the bromophenyl moiety in SC-558 into the hydrophobic pocket of COX-2, Figure 12. In addition, the pyrrolizine nucleus was also overlaid with the pyrazole ring in SC-558, while the phenyl propionamide in 8e extended with the phenyl sulfonamide moiety in SC-558 into the side pocket of COX-2. Compound 8e displayed six hydrogen bonds including four conventional hydrogen bonds with His90, Leu352, and Arg513 and two carbon hydrogen bonds with His90 and Gly526, Figure 12. Compound 8e also showed higher affinity toward COX-1 than ibuprofen. However, 8e did not show any conventional hydrogen bonds with the amino acids in COX-1, Figure 11. showed one electrostatic interaction with Arg513. Based on these results, the higher binding free energy of 8a toward COX-2 could be attributed to the electrostatic interaction with Arg513 and the multiple hydrophobic interactions with the hydrophobic residues in COX-2. Compound 8e also showed higher affinity toward COX-1 than ibuprofen. However, 8e did not show any conventional hydrogen bonds with the amino acids in COX-1, Figure  11. The results of the docking study of 8e into COX-2 also revealed higher binding free energy compared to COX-1. The 4-bromophenyl moiety in 8e superposed with the bromophenyl moiety in SC-558 into the hydrophobic pocket of COX-2, Figure 12. In addition, the pyrrolizine nucleus was also overlaid with the pyrazole ring in SC-558, while the phenyl propionamide in 8e extended with the phenyl sulfonamide moiety in SC-558 into the side pocket of COX-2. Compound 8e displayed six hydrogen bonds including four conventional hydrogen bonds with His90, Leu352, and Arg513 and two carbon hydrogen bonds with His90 and Gly526, Figure 12. The results of the docking study of 8e into COX-2 also revealed higher binding free energy compared to COX-1. The 4-bromophenyl moiety in 8e superposed with the bromophenyl moiety in SC-558 into the hydrophobic pocket of COX-2, Figure 12. In addition, the pyrrolizine nucleus was also overlaid with the pyrazole ring in SC-558, while the phenyl propionamide in 8e extended with the phenyl sulfonamide moiety in SC-558 into the side pocket of COX-2. Compound 8e displayed six hydrogen bonds including four conventional hydrogen bonds with His90, Leu352, and Arg513 and two carbon hydrogen bonds with His90 and Gly526, Figure 12. Among all the tested compounds, 8f displayed the highest binding free energy toward COX-2, Table 4. An investigation of the binding interactions of 8f revealed one conventional hydrogen bond with Arg120 and one carbon hydrogen bond with Val116, Figure 13. The orientation of the best fitting conformation of 8f into COX-2 also revealed partial overlay of the two phenyl rings with the two phenyl rings in SC-558, Figure 14. The bromophenyl moiety in 8f occupied the hydrophobic pocket of COX-2 and formed identical hydrophobic interactions with Leu384, Tyr385, and Trp387. On the other hand, the superposition of the phenyl carboxamide at C2 in 8f with the phenyl sulfonamide moiety in SC-558 into the side pocket of COX-2 allowed the two molecules to form one electrostatic interaction with His90 and two hydrophobic interactions with Ser353 and Val523. Moreover, 8f showed one carbon hydrogen bond with Arg120 compared to unfavorable donordonor interaction for SC-558. Among all the tested compounds, 8f displayed the highest binding free energy toward COX-2, Table 4. An investigation of the binding interactions of 8f revealed one conventional hydrogen bond with Arg120 and one carbon hydrogen bond with Val116, Figure 13. Among all the tested compounds, 8f displayed the highest binding free energy toward COX-2, Table 4. An investigation of the binding interactions of 8f revealed one conventional hydrogen bond with Arg120 and one carbon hydrogen bond with Val116, Fig  The orientation of the best fitting conformation of 8f into COX-2 also revealed partial overlay of the two phenyl rings with the two phenyl rings in SC-558, Figure 14. The bromophenyl moiety in 8f occupied the hydrophobic pocket of COX-2 and formed identical hydrophobic interactions with Leu384, Tyr385, and Trp387. On the other hand, the superposition of the phenyl carboxamide at C2 in 8f with the phenyl sulfonamide moiety in SC-558 into the side pocket of COX-2 allowed the two molecules to form one electrostatic interaction with His90 and two hydrophobic interactions with Ser353 and Val523. Moreover, 8f showed one carbon hydrogen bond with Arg120 compared to unfavorable donordonor interaction for SC-558. The orientation of the best fitting conformation of 8f into COX-2 also revealed partial overlay of the two phenyl rings with the two phenyl rings in SC-558, Figure 14. The bromophenyl moiety in 8f occupied the hydrophobic pocket of COX-2 and formed identical hydrophobic interactions with Leu384, Tyr385, and Trp387. On the other hand, the superposition of the phenyl carboxamide at C2 in 8f with the phenyl sulfonamide moiety in SC-558 into the side pocket of COX-2 allowed the two molecules to form one electrostatic interaction with His90 and two hydrophobic interactions with Ser353 and Val523.

Target
Moreover, 8f showed one carbon hydrogen bond with Arg120 compared to unfavorable donor-donor interaction for SC-558. The 2/3D binding modes of the parent compounds (5a,c) were illustrated in supplementary data, Figures S81 and S82.
In conclusion, the three hybrids 8a,e,f exhibited higher free energies toward COX-1/2 than the parent compounds (5a,c) and the co-crystallized ligands (ibuprofen and SC-558). In addition, their binding free energies toward COX-2 were higher than those for COX-1. An analysis of the binding modes of these hybrids into COX-2 revealed partial superposition with SC-558 and formation of hydrogen bonds, electrostatic, or hydrophobic interactions with the key amino acids His90, and Arg513.

Drug-Likeness and ADME Studies
Pharmacokinetic properties play a critical role in the discovery and development of the new drug candidates [48]. The successful transition of these new compounds to the development stage depends mainly on their pharmacokinetics and pharmacodynamics properties [49]. In this study, the molecular properties related to the pharmacokinetic and drug-likeness parameters of compounds 8a-i were evaluated and compared with those of ibuprofen, ketoprofen, and compounds 3. The study of the molecular properties was calculated using SwissADME [50] and Molsoft L.L.C. The results are presented in Table 5.  The 2/3D binding modes of the parent compounds (5a,c) were illustrated in Supplementary data, Figures S81 and S82.
In conclusion, the three hybrids 8a,e,f exhibited higher free energies toward COX-1/2 than the parent compounds (5a,c) and the co-crystallized ligands (ibuprofen and SC-558). In addition, their binding free energies toward COX-2 were higher than those for COX-1. An analysis of the binding modes of these hybrids into COX-2 revealed partial superposition with SC-558 and formation of hydrogen bonds, electrostatic, or hydrophobic interactions with the key amino acids His90, and Arg513.

Drug-Likeness and ADME Studies
Pharmacokinetic properties play a critical role in the discovery and development of the new drug candidates [48]. The successful transition of these new compounds to the development stage depends mainly on their pharmacokinetics and pharmacodynamics properties [49]. In this study, the molecular properties related to the pharmacokinetic and drug-likeness parameters of compounds 8a-i were evaluated and compared with those of ibuprofen, ketoprofen, and compounds 3. The study of the molecular properties was calculated using SwissADME [50] and Molsoft L.L.C. The results are presented in Table 5.
Based on Lipinski's rule, an orally active drug has a total number of hydrogen bond donors ≤ 5, hydrogen bond acceptors ≤ 10, logP < 5, and molecular weight < 500 da [51]. An investigation of physicochemical parameters of 8a-i revealed that their molecular weights are above 500 daltons. However, the molecular weights are less than that of compound VI. The polar surface areas of 8a-i are either comparable or lower than that of compound VI. The calculated logP values of the nine hybrids are in the range of 2.12-3.73 compared to compound VI (logP = 3.71). The results also showed that compound 8a,c have no violation from Lipinski's rule, while all of the remaining compounds showed only one violation (MW > 500).
The tested hybrids also showed drug-likeness scores (DLS) in the range of 1.06−2.03 compared to 1.01 for compound VI. The fraction of 8a-i that can undergo GIT absorption was calculated following the previous report [52]. The results revealed that 80.10-84.75% of 8a-i could be absorbed from GIT compared to compound VI (80.40%).
The detailed results of the ADME study are provided in Supplementary data, Tables S1-S18.

Conclusions
The design of anticancer-NSAID hybrids could be used to avoid some of the problems encountered in combination therapy. Many of the hybrids combining ibuprofen with anticancer agents have displayed promising in vitro cytotoxic activities. In the current study, eight hybrids bearding the NSAIDs (ibuprofen and ketoprofen) with five pyrrolizine/indolizine derivatives (5a-e) were designed and synthesized. A structural elucidation of the new hybrids was confirmed using IR, mass, 1 H-NMR, 13 C NMR and DEPT C 135 spectra. The new hybrids were evaluated for their antiproliferative activities against three (MCF-7, A549, and HT-29) cancer cell lines using a quick screening assay. The results revealed a 4-71% inhibition in the growth of the cancer cells, where MCF-7 cells were the most sensitive. Based on the results of this assay, compounds 8a,e,f, the most active against MCF-7 cells were selected for additional biological evaluations. The cytotoxic activity of 8a,e,f against MCF-7 cells revealed IC 50 values in the range of 1.07-7.61. Cell cycle analysis of MCF-7 cells treated with the three hybrids at 5 µM revealed pro-apoptotic increase in cells at preG1 and cell cycle arrest at the G1 and S phases. In addition, the three hybrids induced early apoptotic events in MCF-7 cells. A molecular docking study of the three hybrids into the active sites of COX-1/2 was also performed. The results revealed higher binding free energies for the three hybrids toward COX-1/2 compared to the parent compounds 5a,c and the co-crystallized ligands, ibuprofen and SC-558. The results also indicated the higher binding free energies of the three hybrids toward COX-2 over COX-1. An analysis of the binding modes of the best fitting conformation of the three hybrids into COX-2 revealed partial superposition with SC-558 and formation of hydrogen bonds, electrostatic, and hydrophobic interactions with the key amino acid such as His90, and Arg513. The new hybrids also showed drug-likeness score of 1.06-2.03. The above results support the future investigation of compounds 8a,e,f as potential anticancer candidates.

Chemistry
Chemical reagents and solvents were obtained from commercial sources. Solvents are dried by standard methods when necessary. The purity of the new compounds was checked with TLC. Melting points (m.p.) are uncorrected and were determined by IA 9100MK-Digital melting point apparatus (Cole-Parmer, East Bunker Ct Vernon Hills, IL, USA). Infrared spectra (IR) were recorded using BRUKER TENSOR 37 spectrophotometer (Bruker, Billerica, MA, USA). The proton magnetic spectra were recorded on BRUKER AVANCE III at 500 MHz, 13 C NMR (125 MHz), and DEPT C 135 (125 MHz). Mass spectra were recorded on Agilent UPLC/MS/MS 1260 infinity II with 6420 Triple quad LC/MS detector at Faculty of Pharmacy, Minia University, Minia, Egypt.
Compounds 2a,b [34], and 4a-d [35], and 5a-e [36] were prepared according to the previous reports. 4.1.1. General Procedure (A) for Preparation of Compounds (8a-i) The synthesis of the hybrids 8a-i was achieved using the same reaction conditions applied in the synthesis of compound 8e [28].

Preparation of Compounds 7a,b
Thionyl chloride (1 gm, 8.41 mmol) was added to 5.9 mmol of (±)-ibuprofen/ketoprofen in a dry round-bottom flask. The reaction mixture was refluxed on a steam bath. After for 2 h reflux, the excess thionyl chloride was removed under reduced pressure. The residue (ibuprofen/ketoprofen acetyl chloride) was dissolved in methylene chloride (30 mL).

Preparation of Compounds 8a-j
A solution of compound 5a-e (3 mmol) in methylene chloride (30 mL) was added dropwise to the acid chloride solution obtained from the appropriate NSAIDs. To the reaction mixture, 0.5 mL of TEA was added, and the mixture was stirred for 2 h at rt. The reaction mixture was left to stand overnight. The solvent was evaporated under reduced pressure and the solid residue obtained was washed with 10 mL of aqueous sodium carbonate 2% and left to dry. The dry residue was recrystallized from ethanol-acetone.

Screening Assay
To evaluate the antiproliferative activities of the new compounds and their parent compounds, a quick screening MTT assay was used [37]. The cancer cell lines were treated with the hybrids 8a-i, and the parent compounds, ibuprofen, ketoprofen and compounds 5a-e at 5 µM. After 72 h of treatment, the absorbance of the purple formazan was determined using multi-plate reader at 570 nm and the growth% of the treated cells was determined and compared with the control.

MTT Assay
The antiproliferative activity of compounds 8a,e,f was evaluated against MCF-7 cells using MTT assay [38]. First, 96-well plates were used for the seeding of the cancer cells for 24 h. The cancer cells were treated test compounds 8a,e,f, and doxorubicin at different concentrations (0 µM-50 µM) for 72 h. The antiproliferative activity expressed as IC 50 values for each of the four compounds were determined.

Cell Cycle Analysis
The effect of compounds 8a,e,f on cell cycle perturbation of MCF-7 cells was evaluated using flowcytometry (BD FAC SCalibur flow cytometer, BD Biosciences, Franklin Lakes, NJ, USA). The cancer cell was cultured into 6 well plates, then were treated with each of the test compounds at 5 µM for 48 h. The assay was conducted following the previous report [36]. drugs of the three hybrids 5a,c were also docked into the two COXs. The crystal structures of the two enzymes were obtained from protein data bank (http://www.rcsb.org/pdb) (accessed on 28 July 2021). AutoDock 4.2 [45] was used to dock the test compounds into the active sites of the two COXs. The study was performed following the previous report [46]. The generated 2/3D figures of the binding modes of the test compounds were created by discovery studio visualizer [47].

Drug-Likeness and ADME Studies
The molecular properties related to the pharmacokinetic/drug-likeness parameters of compounds 8a-i were evaluated by SwissADME (http://www.swissadme.ch/) (accessed on 24 September 2021) [50] and Molsoft (http://molsoft.com/mprop/) (accessed on 7 August 2021) webservers in the calculation of the physicochemical properties. The compounds were either sketched directly or imputed as SMILES. The detailed results are provided in the Supplementary data, Tables S1-S18.