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Structure–antitumor activity relationship of hybrid acetogenins focusing on connecting groups between heterocycles and the linker moiety

We studied hybrid molecules of annonaceous acetogenins and mitochondrial complex I-inhibiting insecticides to develop a novel anticancer agent. A structure–antitumor activity relationship study focusing on the connecting groups between the heterocycles and the linker moiety bearing the tetrahydrofuran moiety was conducted. Eleven hybrid acetogenins with 1-methylpyrazole instead of γ-lactone were synthesized and their growth inhibitory activities against 39 human cancer cell lines were evaluated. The nitrogen atom at the 2′-position of the linker moiety was essential for inhibiting cancer growth. The 1-methylpyrazole-5-sulfonamide analog showed potent growth inhibition of NCI-H23, a human lung cancer cell line, in a xenograft mouse assay without critical toxicity. Hence, the results of this study may pave the way for the development of novel anticancer agents, with both selective and broad anticancer activities.


Introduction
Cancer is the leading cause of mortality worldwide, accounting for approximately 10 million deaths in 2020. 1 Cancer treatment typically includes chemotherapy, radiation therapy, and/or surgery. More recently, immunotherapy has also been employed for the treatment of cancer. Unfortunately, several other types of cancers remain difficult to treat with known anticancer drugs. Therefore, the development of drugs with novel mechanisms is crucial. Recently, examination of the inhibitors of mitochondrial complex I (reduced nicotinamideadenine dinucleotide-ubiquinone oxidoreductase) has garnered signicant interest. 2 For example, Kawada and coworkers reported that mitochondrial complex I inhibitors selectively suppress tumor growth through concomitant acidi-cation of the intra-and extra-cellular environment since tumor tissues comprise more stromal cells, whose energy production depends on oxidative phosphorylation in the mitochondria, rather than normal tissues. 3 One such inhibitor, darinaparsin, has been submitted to regulatory authorities designated as a treatment for relapsed or refractory peripheral T-cell lymphoma in June 2021. 4 Owing to our interest in complex I inhibitors, we have studied the structure-activity relationship (SAR) of annonaceous acetogenins and their analogs. 5 Annonaceous acetogenins are polyketides isolated from Annona plants, which show antitumor activity against human cancer cells by inhibiting mitochondrial complex I. 6,7 Most of them have one to three tetrahydrofuran (THF) rings in the center of the long hydrocarbon chain and an a,b-unsaturated-g-lactone ring at the end of the molecule. In contrast, mitochondrial complex Iinhibiting insecticides (e.g., tebufenpyrad) typically comprise N-containing heterocycles with hydrophobic side chains ( Fig. 1). 8 We hypothesized that an acetogenin analog with an Ncontaining heterocycle in place of the g-lactone ring 9 can act as a novel antitumor agent because the structural features of acetogenins and insecticides are similar; in addition, both are known to bind to the ND1 subunit in the mitochondria. 10 Solamin was selected as a mother compound because it is a mono-THF acetogenin with a simple structure; however, it comprises all characteristic components of acetogenins. 11 We have previously reported that a hybrid 12 analog 1 with 1-methylpyrazole showed 80-fold higher growth inhibitory activity against NCI-H23, a human lung cancer cell line, than solamin. 13 Subsequently, the effect of the connecting group between heterocycles and hydrophobic parts bearing the THF moiety on their biological activities was examined because 1-methylpyrazole groups of most insecticides are linked to the hydrophobic sections with an amide group. Preliminary data have shown that the connecting group signicantly inuences the biological activity; for instance, an analog linked via an amide group has a potent growth inhibitory activity against some human cancer cell lines. 14 This study reports (1) the synthesis of hybrid acetogenins with sulfonamide and ketomethylene bond as an amido bond's bioisosteres, (2) analysis of the effects of connecting groups on growth inhibitory activities against human cancer cell lines, (3) the effects of substituents on 1-methylpyrazole, and (4) in vivo mice xenogra studies.

Synthesis of hybrid acetogenins
The synthesis of all analogs 7a-7k was initiated using a common a-tetrahydrofuranyl aldehyde 2 prepared by a method previously reported by us. 15 Scheme 1 illustrates the synthesis of analogs 7a-7h. Under Carreira's conditions, 16 the asymmetric alkynylation of 2 with 10-azido-1-decyne 3 resulted in a high yield of propargyl alcohol 4, which shows a high diastereoselectivity; this threo/trans/threo stereochemical conguration on 4 is the same as that of the stereoisomer, which is reported to show the most potent anti-tumor activity. 17 The reduction of azide and the alkyne moiety of 4 was simultaneously carried out to obtain primary amine 5. Then, amine 5 was condensed to 1-methylpyrazole-5-carboxylic acid with 1-(3dimethylaminopropyl)-3-ethylcarbodiimide (EDC), resulting in a good yield of amide 6a. Substituted pyrazole-5-carboxamides 6b-6c and pyrazole-4-carboxamide 6d were also prepared using a similar procedure, resulting in moderate yields. The sulfonamides 6e-6f were synthesized from 5 using the corresponding sulfonyl chlorides in the presence of Et 3 N. Deprotection of tert-butyldimethylsilyl (TBS) ether in 6a-6f under acidic conditions resulted in moderate to good yields of the desired analogs 7a-7f. N-Methyl amide 7g was synthesized by Nmethylation of 7a using methyl iodide under basic conditions. The reduction of 7a using BH 3 $SMe 2 in THF under reux conditions produced amine 7h, albeit with a low yield. The synthesis of inverse amide 7i was initiated via the same reaction as that used for the synthesis of 4 using methyl undec-10-ynoate 8 instead of 3 to obtain propargyl alcohol 9 with high diastereoselectivity (Scheme 2). Good yields of carboxylic acid 11 were obtained via hydrogenation of propargyl alcohol 9, followed by hydrolysis under basic conditions via methyl ester 10. Condensation of 11 with 5-amino-1-methylpyrazole produced amide 12 with a 74% yield. Inverse amide 7i was synthesized via deprotection of TBS ether of 12. Scheme 3 illustrates the synthesis of the ester 7j. Ester 7j was obtained from 2 using the following steps: (1) asymmetric alkynylation with alkyne 13, (2) hydrogenation of the triple bond, (3) deprotection of TBS ethers, and (4) primary alcoholselected condensation of 1-methylpyrazole-5-carboxylic acid.

Evaluation of growth inhibitory activity against human cancer cells
The growth inhibitory activities of analogs 7a-7k against JFCR39, 18 a panel of 39 human cancer cell lines were evaluated. The results are presented in Fig. 2. The activities of analogs 1, 7l, 13   represents the logarithm of 1/GI 50 (50% growth inhibitory concentration) relative to the control values for the aforementioned 39 human cancer cell lines.
First, our comparison of the activities of 1-methylpyrazole-5yl analogs (1, 7a, 7e, and 7g-7k) revealed that the patterns could be classied into two categories: selective inhibition and broad Fig. 2 Cell growth inhibition was assessed using the sulforhodamine B colorimetric assay by measuring changes in total cellular protein levels following 48 h of treatment with a given test compound. Fingerprints were obtained by calculating 50% growth inhibitory concentration (GI 50 ) values for JFCR39 cell lines. The x-axis represents the logarithm of the 1/GI 50 values for 39 human cancer cell lines. Absence of a bar signifies that the GI 50 value of the corresponding cell line is higher than 10 À4 M. CNS ¼ central nervous system. inhibition. For instance, amide 7a showed selective and very potent activities against some cancer cells, including BSY-1, HCT-116, NCI-H23, DMS114, LOX-IMVI, MKN-B, and MKN-A, but did not inhibit the growth of 12 types of cells in this concentration range. The selective activity was also observed for alkyl 1, ester 7j, and ketomethylene 7k, although their potencies were different. Sulfonamide 7e, 1-methylamide 7g, and methylene amine 7h also showed potent inhibition of DMS114, MKN-B, and MKN-A; however, their selectivities were low because they exhibited moderate activities against all other cancer cells tested. The planarity and/or hydrogen-donating properties of the amide proton attributed to the introduction of the amide bond did not affect their growth inhibitory activity; however, these properties affected their selectivities for specic cancer cell lines. Interestingly, the activity of inverse amide 7i was decient in contrast to that of amide 7a.
In terms of structural features, analogs with a nitrogen atom at the 2 0 -position of the side chain (group A: 7a, 7e, 7g, and 7h) tended to be signicantly more active than the others (group B: 1 and 7i-7k) (Fig. 3A). These tendencies were observed against approximately half of the cell lines (for example, MKN-B in  (1) and the following correlation equations, n refers to the number of compounds, r implies correlation coefficient, s refers to standard deviation, and the gures in parentheses indicate 95% condence intervals. This equation allows for the design of novel analogs; analogs linked via boron amide might be promising since the XLOGP3 value is À0. 57. In contrast, interestingly, compounds of group B showed an inverse correlation with those of group A. For example, the activities against NCI-H23 are represented by eqn (2) Next, the effect of substituents in 1-methylpyrazole was evaluated (Fig. 2). Both 3-ethylpyrazole 7b and 4-chloro-3ethylpyrazole 7c, with substituents identical to those of tebufenpyrad, showed selective and potent growth inhibitory activity against some cells. The selective and potent growth inhibitory activity of 7b and 7c was observed against the same cell lines as those observed with 7a, except for LOX-IMVI. Therefore, the connecting groups could be more signicant than the substituents of 1-methylpyrazole observed in our analogs. It is possible that carboxylic acid 23 produced by the hydrolysis of 7c was not in its active form because of the lack of the activity, suggesting that the presence of pyrazole moiety at a certain distance from the THF moiety is vital to the activity.
Both 4-pyrazole-carboxamide 7d and 4-pyrazole-sulfonamide 7f showed signicantly more potent activities than 4-alkyl-pyrazole 7l, suggesting that the connecting groups observed in our analogs might also play an essential role when present in other analogs.
The aforementioned results revealed that the growth inhibitory activities of our analogs signicantly depended on the connecting group between the 1-methylpyrazole moiety and the hydrophobic alkyl chain bearing the THF ring. The importance of the nitrogen atom at the 2 0 -position of the side chain is unclear; however, the presence of a polar atom in this position may be essential, as 2 0 -hydroxy solamin, also known as murisolin, showed more potent activity than solamin. 22 The positive correlation between the reduction in lipophilicity and activity suggested the presence of a water-soluble pocket at the binding site of our analogs, suggesting that more potent analogs might be discovered using synthetic analogs with less lipophilicity.

In vivo studies using mice xenogras
To clarify the efficacy of our analogs as novel anticancer agents, we conducted in vivo studies using mouse xenogras. Amide 7a and sulfonamide 7e were tested since they showed distinctive ngerprints in in vitro studies: while the former demonstrated Fig. 4 Tumor growth and body weight changes in nude mice bearing a human lung cancer NCI-H23 xenograft. Nude mice were subcutaneously inoculated with a tumor fragment measuring 3 Â 3 Â 3 mm from a subcutaneous tumor developed. When the tumor reached a volume of 100-300 mm 3 , the mice were divided randomly into test groups (day 0). (A and B) Amide 7a (5 mg kg À1 ) was intraperitoneally administered at days 0, 7 and 14. (C and D) Sulfonamide 7e (100 mg kg À1 ) was intraperitoneally administered on day 0, or on both days 0 and 6. Body weight and relative tumor volume are expressed as means AE SD. *P < 0.05, two-sided Mann-Whitney U test compared with the vehicle-administered control group. Triangle: control group; open and closed circles: administered group.
selective activity, the latter showed a broad activity. The sulfonamide 7e was selected from the broad inhibitory analogs with similar potencies, 7e, 7g, and 7h, because the total yield of 7e (45% from 2) was higher than those of 7g and 7h (which were 25% and 11%, respectively). NCI-H23 was selected for inoculation into mice because the two analogs 7a and 7e showed almost the same inhibition potency (log(1/GI 50 of 7a) ¼ 7.28; log(1/GI 50 of 7e) ¼ 6.60). The results for 7a are shown in Fig. 4A and B. The maximum tolerated dose (MTD) of 7a was 5 mg kg À1 ; this nding can be attributed to its high toxicity. Three administrations of 5 mg kg À1 7a on days 0, 7, and 14 did not show signicant antitumor activity. In contrast, no death was observed aer administering 100 mg kg À1 of sulfonamide 7e, which was 20-fold higher than that of 7a (Fig. 4D). Furthermore, as shown Fig. 4C, the administration of 7e on day 0 at a dose of 100 mg kg À1 signicantly inhibited tumor growth as determined on day 16 (1.43 AE 0.71 compared to tumor size at day 0), whereas the control tumor grew more than six times during this period (5.58 AE 1.56). Hence, the average tumor size in drugtreated mice compared to those in control mice was 27%. Weight loss was observed aer the rst administration; however, this loss was recovered later, suggesting that 7e was well tolerated. When 100 mg kg À1 of 7e was administered twice on days 0 and 6, weight loss was observed aer each administration. However, the bodyweight was eventually recovered, and a more potent tumor growth inhibition effect was observed on day 16 (12% of control). The administration of 7e at a dose less than 100 mg kg À1 can also be expected to show signicant tumor growth inhibition, although this was not tested in the present study due to the lack of sufficient amounts of 7e. This result suggests that sulfonamide 7e is a promising novel anticancer lead, although further investigation of molecular properties, such as solubility, membrane permeability, and bioavailability, would be necessary before the analog can be used as an anticancer agent.
These results indicate that the connecting group of our analogs contributes not only to the potency of growth inhibitory activity against cancer cells but also to their safety. Sulfonamide groups are known as one of the bioisosteres of the amide bond. 23 It is not clear why the conversion of the amide bond into the sulfonamide bond in our analogs avoided the loss of bodyweight of mice; however, the metabolites, the primary amine and/or the carboxylic acid produced by hydrolysis of 7a, might have induced the loss of bodyweight because the sulfonamide bonds are oen used in place of amide to improve stability against amide bond hydrolysis by proteolytic enzymes. We previously reported that an acetogenin analog with thiophene in place of g-lactone in solamin showed potent antitumor activity without acute toxicity. 24 Using in vitro data to predict in vivo toxicity may not be accurate; however, we might obtain an analog via searching with amide's bioisostere, which shows a broad spectrum activity against JFCR39 since both the sulfonamide and the thiophene carboxamide analogs show wide spectrum effects. The present SAR study is valuable because it provides another approach to demonstrate that acetogenins might lead to the development of novel anticancer agents.

Conclusions
We reported the synthesis of 11 hybrid acetogenins with various connecting groups between their pyrazole and the linker bearing the THF moiety and their growth inhibitory activities against 39 human cancer cell lines. The results demonstrated that the nitrogen atom at the 2 0 -position of the linker could be crucial for the inhibition of cancer growth. However, substituents in the pyrazole ring did not affect their activities. Moreover, the connecting groups divided our analogs into two categories in terms of activity: one showed selective activity and the other showed broad activity. Amide 7a, one of the former compounds, caused substantial loss of body weight in mice and did not show signicant growth inhibition against NCI-H23 in a mouse xenogra assay, although exhibited potent activity in vitro. In contrast, sulfonamide 7e, one of the latter compounds, showed potent antitumor activity without acute toxicity. These data indicate the immense potential of our hybrid acetogenins as novel antitumor lead compounds. Investigation of their physical properties, bioavailability, mode of action, and structureactivity association are underway.

Conflicts of interest
There are no conicts to declare.