Syntheses and Structure-Activity Relationships in Growth Inhibition Activity against Human Cancer Cell Lines of 12 Substituted Berberine Derivatives.

In this study, quaternary berberine chloride is used as a lead compound to design and synthesize a series of berberine-12-amine derivatives to evaluate the growth inhibition activity against human cancer cell lines. Forty-two compounds of several series were obtained. The quaternary berberine-12-N,N-di-n-alkylamine chlorides showed the targeted activities with the IC50 values of most active compounds being dozens of times those of the positive control. A significant structure–activity relationship (SAR) was observed. The activities of quaternary berberine-12-N,N-di-n-alkylamine chlorides are significantly stronger than those of the reduced counterparts. In the range of about 6-8 carbon atoms, the activities increase with the elongation of n-alkyl carbon chain of 12-N,N-di-n-alkylamino, and when the carbon atom numbers are more than 6-8, the activities decrease with the elongation of n-alkyl carbon chain. The activities of the tertiary amine structure are significantly higher than that of the secondary amine structure.


Introduction
Nearly all kinds of malignant tumors (cancers) are severe diseases endangering human health. The search for effective therapies against malignancies has gone through a long historical period. Currently, surgical therapy, radiotherapy, chemotherapy (including molecular-targeted drug therapy), endocrine therapy, and biotherapy are the most commonly used therapies to treat malignancies in the clinic. However, it is very worrying that while a series of the most commonly diagnosed malignant tumors in the clinic represent one of the leading causes of diseases-related deaths worldwide, the clinical benefits of these therapies are all limited and there are no specific drugs or other therapies available for the treatment of many cancers. Poor curative effect and obvious toxicity or hypertoxicity are the outstanding defects of current anticancer drugs [1]. Thus, better treatment options against malignancies, including innovative antitumor drugs, are urgently needed.
against cancer cells was one of the major hot spots that were concerned. However, the problems of poor pharmacological effect and poor pharmacokinetic characteristics of QBC itself further limit its clinical application [17]. Research on berberine-type alkaloids is one of the hot topics in the field of medicinal chemistry at present, particularly research on structural modification and many other pharmacological activities. Our group has also been performing these investigations over the past few years, and has touched on many aspects of several berberine-type alkaloids in pharmaceutical chemistry, such as studies on the structural modifications of quaternary coptisine chloride and quaternary palmatine chloride, the exploration of the pharmacological activities of X-box-binding protein 1 (XBP1), which include transcriptional activation, anti-ulcerative colitis, and antibacterial [18][19][20][21][22]. Recently, we ran a study on the structural modification of QBC to explore the possibility of improving its activity for inhibiting human cancer cell growth, and to investigate the structure-activity relationship (SAR). In our study, the covalently connected hydrogen atom at position 12 of the QBC core was replaced by N-acylamino-, N,N-di-n-alkylamino-, and N-n-alkylamino-, respectively, and the different reduction states of the end-products were also studied in order to yield several classes of new berberine-type alkaloid derivatives as target compounds. All the synthesized compounds were screened for the in vitro inhibition of human cancer cell growth. Two classes of end-products exhibited definite activities and a structure-activity relationship, with one class of compounds showing the activity more than dozens of times that of fluorouracil (5-FU), which was used as a positive control. This article reports on the design and syntheses of the target compounds, the evaluation of the in vitro inhibition of cancer cell growth, and the SAR analysis.

Chemistry
By consulting published literature related to QBC, it was found that, in addition to the replacement of hydrogen atom at position-12 of the QBC core by halogens, there has been no research reports on the structural modification based on position-12. However, as far as organic chemistry is concerned, it is obvious that the electrophilic nitration reaction at position-12 is possibly more likely to occur due to the influence of the methoxy groups in positions-9 and -10. Following nitration, the reduction of the nitro group leads to the formation of a primary amino group, which can be capitalized on to carry out multiple other structural modifications.
Thus, in the current study, the QBC was firstly nitrated under the conditions of NaNO2 plus concentrated HNO3 to produce quaternary 12-nitroberberine chloride (2). Compound 2 was reduced under the condition of SnCl2•2H2O to yield quaternary 12-aminoberberine chloride (3). Then, compound 3 was reacted with relevant acyl chlorides to successfully synthesize the targeted quaternary berberine-12-N-acylamine chloride derivatives (4a-m) (Scheme 1). On top of the signature signals of H-1 (s, 1H), 4 (s, 1H), 8 (s, 1H), 11 (s, 1H), 13 (s, 1H), MeO-9 (s, 3H), MeO-10 (s, 3H), CH2-5 (t, 2H), CH2-6 (t, 2H), and CH2-14 (s, 2H) of the QBC core, all the synthesized target compounds showed signals that corresponded to acyls in the 1 H NMR spectra. All the positive ESIMS data was also consistent with the structures (see Materials and Methods section). Research on berberine-type alkaloids is one of the hot topics in the field of medicinal chemistry at present, particularly research on structural modification and many other pharmacological activities. Our group has also been performing these investigations over the past few years, and has touched on many aspects of several berberine-type alkaloids in pharmaceutical chemistry, such as studies on the structural modifications of quaternary coptisine chloride and quaternary palmatine chloride, the exploration of the pharmacological activities of X-box-binding protein 1 (XBP1), which include transcriptional activation, anti-ulcerative colitis, and antibacterial [18][19][20][21][22]. Recently, we ran a study on the structural modification of QBC to explore the possibility of improving its activity for inhibiting human cancer cell growth, and to investigate the structure-activity relationship (SAR). In our study, the covalently connected hydrogen atom at position 12 of the QBC core was replaced by N-acylamino-, N,N-di-n-alkylamino-, and N-n-alkylamino-, respectively, and the different reduction states of the end-products were also studied in order to yield several classes of new berberine-type alkaloid derivatives as target compounds. All the synthesized compounds were screened for the in vitro inhibition of human cancer cell growth. Two classes of end-products exhibited definite activities and a structure-activity relationship, with one class of compounds showing the activity more than dozens of times that of fluorouracil (5-FU), which was used as a positive control. This article reports on the design and syntheses of the target compounds, the evaluation of the in vitro inhibition of cancer cell growth, and the SAR analysis.

Chemistry
By consulting published literature related to QBC, it was found that, in addition to the replacement of hydrogen atom at position-12 of the QBC core by halogens, there has been no research reports on the structural modification based on position-12. However, as far as organic chemistry is concerned, it is obvious that the electrophilic nitration reaction at position-12 is possibly more likely to occur due to the influence of the methoxy groups in positions-9 and -10. Following nitration, the reduction of the nitro group leads to the formation of a primary amino group, which can be capitalized on to carry out multiple other structural modifications.
In addition, in order to explore the effect of 12 N-monosubstituted amine counterparts of the synthesized compounds 6a-l and 7a-l on the inhibition of human cancer cell growth, tertiary tetrahydroberberine-12-N-n-propylamine derivatives (8) and quaternary berberine-12-N-n-propylamine chloride (9) as representatives were also designed and synthesized by capitalizing on the steric hindrance at position-12 and, in particular, carefully controlling for the amount of aldehyde. The synthesis is indicated in Scheme 3. The tertiary 12-aminotetrahydroberberine (5) was reacted with a carefully controlled amount of propionaldehyde to obtain 8 through the same process of addition and reduction reactions as synthesizing the tetrahydroberberine-12-N,N-di-n-alkylamine derivatives. Then, the target compound, quaternary berberine-12-N-n-propylamine iodide (9), was synthesized by oxidization under the condition of iodine. All the structures of the synthesized compounds were confirmed by NMR and MS methods. To synthesize the targeted quaternary berberine-12-N,N-di-n-alkylamine chlorides, compound 2 was reduced using NaBH 4 and NiCl 2 ·6H 2 O as reagents to yield the tertiary 12-aminotetrahydroberberine (5). Compound 5 was reacted with relevant aliphatic aldehydes through a process of addition and reduction reactions to obtain tetrahydroberberine-12-N,N-di-n-alkylamine derivatives (6a-l). Compounds 6a-l were oxidized using 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ) as the reagent and then reacted with 2N hydrochloric acid (Scheme 2) to yield the targeted quaternary berberine-12-N,N-di-n-alkylamine chlorides (7a-l) (Scheme 2) [23]. All the structures of the synthesized compounds were confirmed by NMR and MS methods (see Materials and Methods section and Figures S5-S79). To synthesize the targeted quaternary berberine-12-N,N-di-n-alkylamine chlorides, compound 2 was reduced using NaBH4 and NiCl2·6H2O as reagents to yield the tertiary 12-aminotetrahydroberberine (5). Compound 5 was reacted with relevant aliphatic aldehydes through a process of addition and reduction reactions to obtain tetrahydroberberine-12-N,N-di-n-alkylamine derivatives (6a-l). Compounds 6a-l were oxidized using 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ) as the reagent and then reacted with 2N hydrochloric acid (Scheme 2) to yield the targeted quaternary berberine-12-N,N-di-n-alkylamine chlorides (7a-l) (Scheme 2) [23]. All the structures of the synthesized compounds were confirmed by NMR and MS methods. In addition, in order to explore the effect of 12 N-monosubstituted amine counterparts of the synthesized compounds 6a-l and 7a-l on the inhibition of human cancer cell growth, tertiary tetrahydroberberine-12-N-n-propylamine derivatives (8) and quaternary berberine-12-N-n-propylamine chloride (9) as representatives were also designed and synthesized by capitalizing on the steric hindrance at position-12 and, in particular, carefully controlling for the amount of aldehyde. The synthesis is indicated in Scheme 3. The tertiary 12-aminotetrahydroberberine (5) was reacted with a carefully controlled amount of propionaldehyde to obtain 8 through the same process of addition and reduction reactions as synthesizing the tetrahydroberberine-12-N,N-di-n-alkylamine derivatives. Then, the target compound, quaternary berberine-12-N-n-propylamine iodide (9), was synthesized by oxidization under the condition of iodine. All the structures of the synthesized compounds were confirmed by NMR and MS methods. (c) 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ), CH 2 Cl 2 , 2N HCl, rt, 2h. R = a-l: C n H 2n + 1 (n = 0-11).
In addition, in order to explore the effect of 12 N-monosubstituted amine counterparts of the synthesized compounds 6a-l and 7a-l on the inhibition of human cancer cell growth, tertiary tetrahydroberberine-12-N-n-propylamine derivatives (8) and quaternary berberine-12-N-n-propylamine chloride (9) as representatives were also designed and synthesized by capitalizing on the steric hindrance at position-12 and, in particular, carefully controlling for the amount of aldehyde. The synthesis is indicated in Scheme 3. The tertiary 12-aminotetrahydroberberine (5) was reacted with a carefully controlled amount of propionaldehyde to obtain 8 through the same process of addition and reduction reactions as synthesizing the tetrahydroberberine-12-N,N-di-n-alkylamine derivatives. Then, the target compound, quaternary berberine-12-N-n-propylamine iodide (9), was synthesized by oxidization under the condition of iodine. All the structures of the synthesized compounds were confirmed by NMR and MS methods (see Materials and Methods section and Figures S80 and S81).

Biological Activities
All the synthesized compounds were evaluated for the growth inhibition activity against several human cancer cell lines using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay that was modeled after our previous publication [24,25]. The cancer cells in this study included human colorectal cancer cells (HCT-8), human liver cancer cells (Bel7402), human cervical cancer cells (HeLa), human lung cancer cells (A549), and human gastric cancer cells (BGC-823). A clinically applied strong thymidylate synthetase inhibitor, 5-FU, was used as the positive control.
Experimental results showed that the intermediates quaternary 12-nitroberberine chloride (2), quaternary 12-aminoberberine chloride (3), tertiary 12-aminotetrahydroberberine (5), and tertiary tetrahydroberberine-12-N-n-propylamine (8), and the target compounds quaternary berberine-12-N-acylamine chloride derivatives (4a-m) and quaternary berberine-12-N-n-propylamine iodide (9) showed little or very little growth inhibition activity against the tested human cancer cell lines, each showing IC50 values greater than 10 µM (see Table 1, but data of 4a-m not shown). Most of the tertiary tetrahydroberberine-12-N,N-di-n-alkylamine derivatives (6a-l) also showed little or very little growth inhibition activity against the tested human cancer cell lines, but a general SAR is obvious for this series of compounds. In the range of four carbon atoms for the n-alkyl carbon chain from 12-N,N-di-n-alkylaminos of series compounds 6a-l, the title activity increases with the elongation of the n-alkyl carbon chain, and when there are more than four carbon atoms, the activity decreases with the elongation of the n-alkyl carbon chain. Tetrahydroberberine-12-N,N-di-n-butylamine (6d), as the most active compound in the 6a-l series, only showed IC50 values inhibiting the growth of HCT-8, Bel7402, HeLa, A549, and BGC-823 by 12.81 µM, 16.38 µM, 9.74µM, 11.40 µM, and 10.43 µM, respectively.

Biological Activities
All the synthesized compounds were evaluated for the growth inhibition activity against several human cancer cell lines using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay that was modeled after our previous publication [24,25]. The cancer cells in this study included human colorectal cancer cells (HCT-8), human liver cancer cells (Bel7402), human cervical cancer cells (HeLa), human lung cancer cells (A549), and human gastric cancer cells (BGC-823). A clinically applied strong thymidylate synthetase inhibitor, 5-FU, was used as the positive control.
Experimental results showed that the intermediates quaternary 12-nitroberberine chloride (2), quaternary 12-aminoberberine chloride (3), tertiary 12-aminotetrahydroberberine (5), and tertiary tetrahydroberberine-12-N-n-propylamine (8), and the target compounds quaternary berberine-12-N-acylamine chloride derivatives (4a-m) and quaternary berberine-12-N-n-propylamine iodide (9) showed little or very little growth inhibition activity against the tested human cancer cell lines, each showing IC 50 values greater than 10 µM (see Table 1, but data of 4a-m not shown). Most of the tertiary tetrahydroberberine-12-N,N-di-n-alkylamine derivatives (6a-l) also showed little or very little growth inhibition activity against the tested human cancer cell lines, but a general SAR is obvious for this series of compounds. In the range of four carbon atoms for the n-alkyl carbon chain from 12-N,N-di-n-alkylaminos of series compounds 6a-l, the title activity increases with the elongation of the n-alkyl carbon chain, and when there are more than four carbon atoms, the activity decreases with the elongation of the n-alkyl carbon chain. Tetrahydroberberine-12-N,N-di-n-butylamine (6d), as the most active compound in the 6a-l series, only showed IC 50 values inhibiting the growth of HCT-8, Bel7402, HeLa, A549, and BGC-823 by 12.81 µM, 16.38 µM, 9.74 µM, 11.40 µM, and 10.43 µM, respectively. However, the targeted quaternary berberine-12-N,N-di-n-alkylamine chloride series compounds 7a-l exhibited, to varying degrees, some or significant growth inhibition activities against the tested human cancer cell lines. By comparison, 7a-l are much more active than their reduction state counterparts 6a-l, with the IC 50 values of most compounds in the quaternary berberine-12-N,N-di-n-alkylamine chloride series being on or over the micromolar level (Table 1). This result demonstrated the impact of variations in the reduction states of the end-products on the title bioactivity. Still, the carbon chain length of the n-alkyl substituents was found to be an important factor to affect the activity of the end-products for all the tested human cancer cell lines. Quaternary berberine-12-N,N-dimethylamine chloride (7a), which was the nascent compound of the active quaternary berberine-12-N,N-di-n-alkylamine chloride series, showed growth inhibition activities against HCT-8, Bel7402, Hela, A549, and BGC-823 with IC 50 values of 53.41 µM, 50.01 µM, 8.95 µM, 13.37 µM, and 28.82 µM, respectively. Then, an obvious trend was shown that in the range of about six to eight carbon atoms; the activity increases with the elongation of the n-alkyl carbon chain of 12-N,N-di-n-alkylaminos, and when there are more than six to eight carbon atoms, the activity decreases with the elongation of the n-alkyl carbon chain. This trend is basically the same as that observed in the tetrahydroberberine-12-N,N-di-n-alkylamine derivatives series (6a-l), although the activity of the latter series is very weak (Figures 2-6). Under this demonstrated SAR, quaternary berberine-12-N,N-di-n-hexylamine chloride (7f) exhibited the most significant growth inhibition activity by IC 50 value of 0.29 µM for A549, which translated to 22

Reagents and Materials
Nuclear magnetic resonance (NMR) spectra were recorded on a Varian Mercury-400 NMR spectrometer and reported with tetramethylsilane (TMS) as an internal standard and chloroform-d (CDCl3) (D, 99.8% + 0.05% v/v TMS) or dimethyl sulfoxide-d6 (DMSO-d6) (D, 99.9% + 0.05% v/v TMS) (Cambridge Isotope Laboratories, Inc., Andover, MA, USA) as solvents. Chemical shifts (δ values) and coupling constants (J values) are given in ppm and Hz, respectively. ESIMS + were obtained using an Agilent 1100 series Liquid Chromatograph/Mass Selective Detector (LC/MSD) Trap SL mass spectrometer. QBC was purchased from the market and the purity was determined to be over 98% by high-pressure liquid chromatography (HPLC) and the structure was confirmed on the basis of chemical and spectroscopic data (data not shown). All the reagents and solvents were reagent grade or were purified by standard methods before use. Anhydrous solvents and reagents were all analytically pure and dried through routine protocols. The reaction progress was monitored by thin-layer chromatography (TLC) on a high-efficiency TLC plate with precoated silica gel (GF254) produced by Qingdao Haiyang Chemical (Qingdao, China). The spots were visualized by I2 steam or under UV light (254 nm). Column chromatography (CC) was carried out with silica gel (200-300 mesh size; Qingdao Haiyang Chemical, Qingdao, China). The concentration of solution after reactions involved the use of a rotary evaporator operated at a reduced pressure of ca. 9.0 mbar.

Reagents and Materials
Nuclear magnetic resonance (NMR) spectra ( Figures S1-S81) were recorded on a Varian Mercury-400 NMR spectrometer and reported with tetramethylsilane (TMS) as an internal standard and chloroform-d (CDCl 3 ) (D, 99.8% + 0.05% v/v TMS) or dimethyl sulfoxide-d 6 (DMSO-d 6 ) (D, 99.9% + 0.05% v/v TMS) (Cambridge Isotope Laboratories, Inc., Andover, MA, USA) as solvents. Chemical shifts (δ values) and coupling constants (J values) are given in ppm and Hz, respectively. ESIMS + were obtained using an Agilent 1100 series Liquid Chromatograph/Mass Selective Detector (LC/MSD) Trap SL mass spectrometer. QBC was purchased from the market and the purity was determined to be over 98% by high-pressure liquid chromatography (HPLC) and the structure was confirmed on the basis of chemical and spectroscopic data (data not shown). All the reagents and solvents were reagent grade or were purified by standard methods before use. Anhydrous solvents and reagents were all analytically pure and dried through routine protocols. The reaction progress was monitored by thin-layer chromatography (TLC) on a high-efficiency TLC plate with precoated silica gel (GF 254 ) produced by Qingdao Haiyang Chemical (Qingdao, China). The spots were visualized by I 2 steam or under UV light (254 nm). Column chromatography (CC) was carried out with silica gel (200-300 mesh size; Qingdao Haiyang Chemical, Qingdao, China). The concentration of solution after reactions involved the use of a rotary evaporator operated at a reduced pressure of ca. 9.0 mbar.

Growth Inhibition Activity Assay Against Human Cancer Cell Lines
The growth inhibitory activity of all the synthesized compounds against the human HCT-8, Bel7402, HeLa, A549, and BGC-823 cell lines were examined using a published method of our group [25]. The compounds were dissolved in DMSO (100 µL), and then the solutions containing the compounds were diluted to working solutions with RPMI 1640 culture medium containing 10% serum. The corresponding human tumor cells were added to 96-well microplates (100 µL/well), and cultured in an incubator with 5% CO 2 at 37 • C for 24 h. The working solutions of the compounds were added to microplates at a final concentration of 0.1 µmol/L, 1 µmol/L, 10 µmol/L, and 100 µmol/L (4 replicate wells per concentration). After 72 h, the culture solution was discarded, and RPMI 1640 culture medium (10% serum, 100 µL) containing 0.5 mg/mL MTT was added to each well. After incubating at 37 • C, 5% CO 2 for 4 h, the solution was discarded. DMSO (150 µL) was added to each well, and the plates were shaken at room temperature for 10 min to completely dissolve the blue crystals in order to detect the optical density (OD) value at 570 nm (detection wavelength) and 655 nm (reference wavelength) using a Bio-Rad 450 microplate reader (Hercules, CA, USA). The inhibition rate of the test compound was calculated according to the following formula: Growth inhibition rate % = (negative control OD − pending compound OD)/ (negative control OD − background OD) × 100% (1)

Conclusions
In this article, quaternary 12-nitroberberine chloride (2), quaternary 12-aminoberberine chloride (3), tertiary 12-aminotetrahydroberberine (5), and five series of 12-aminoberberine derivatives of different reduction states, including quaternary berberine-12-N-acylamine chlorides (4a-m), tertiary tetrahydroberberine-12-N,N-di-n-alkylamine derivatives (6a-l), quaternary berberine-12-N,N-di-n-alkylamine chlorides (7a-l), tertiary tetrahydroberberine-12-N-n-propylamine (8), and quaternary berberine-12-N-n-propylamine iodide (9) were designed and synthesized. The growth inhibition activities of these synthesized compounds against several human cancer cell lines were screened. The series of quaternary berberine-12-N,N-di-n-alkylamine chlorides (7a-l) showed some or significant growth inhibition activities against the tested human cancer cell lines, with the IC 50 values of most compounds being on or over the micromolar level. In addition, significant SAR was observed. Firstly, the activities of quaternary berberine-12-N,N-di-n-alkylamine chlorides series (7a-l) are obviously stronger than those of the reduced counterparts, the tertiary tetrahydroberberine-12-N,N-di-n-alkylamine derivatives series (6a-l). Secondly, the length of the n-alkyl carbon chain of 12-N,N-di-n-alkylaminos has a significant effect on the activities. In the range of about six to eight carbon atoms, the activity increases with the elongation of the n-alkyl carbon chain of 12-N,N-di-n-alkylaminos, and when there are more than six to eight carbon atoms, the activity decreases with the elongation of the n-alkyl carbon chain. Regarding the activity feature of 12-N,N-di-n-alkylaminos, we guess that it is relevant to their log P values and their ability to cross cell membranes; this is an area of further research for us. Thirdly, the activities are also affected by the number of n-alkyl groups on the amino nitrogen atom. The activities of the tertiary amine structure of the 12-amino are significantly higher than the secondary amine structure. These findings are very helpful for the further medicinal chemistry study of berberine-type alkaloids.