Development of Indole Alkaloid-Type Dual Immune Checkpoint Inhibitors Against CTLA-4 and PD-L1 Based on Diversity-Enhanced Extracts

Cancer immunotherapy involves the use of the immune system for cancer treatment. Recently, immune checkpoint-blocking antibodies have become integral for the treatment of some cancers. However, small molecules exhibit advantages over monoclonal antibody drugs, such as cell penetration, long half-life, and low manufacturing costs, and the possibility of oral administration. Thus, it is imperative to develop small-molecule immune checkpoint inhibitors. Previously, we have screened a library of synthetic indole-alkaloid-type compounds, which are produced by diversity-enhanced extracts of Japanese cornelian cherry, and reported that an unnatural pentacyclic compound inhibits CTLA-4 gene expression. In this study, immune checkpoint inhibitors with increased potency were developed by introducing substituents and conversion of functional groups based on the unnatural pentacyclic compound. The developed compounds suppressed not only CTLA-4 and PD-L1 gene expression but also protein expression on the cell surface. Their efficacy was not as potent as that of the existing small-molecule immune checkpoint inhibitors, but, to the best of our knowledge, the developed compounds are the first reported dual small-molecule inhibitors of CTLA-4 and PD-L1.


INTRODUCTION
Cancer immunotherapy involves the use of the immune system for cancer treatment. Recently, immune checkpoint inhibitors have become integral for the treatment of some cancers (Pardoll, 2012;Sharma and Allison, 2015;Darvin et al., 2018;Robert, 2020). Immune checkpoints are negative regulators of the immune system, playing roles in autoimmunity prevention and self-tolerance maintenance. Programmed cell death-1 (PD-1) (Wherry, 2011;Schietinger and Greenberg, 2014) and cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) (Wing et al., 2008) are typical immune checkpoint proteins on T cells. Sometimes, cancer cells find ways to use these checkpoints to avoid attack by the immune system. Recently, ipilimumab (Yervoy ® ), an anti-CTLA-4 monoclonal antibody, pembrolizumab (Keytruda ® ), and nivolumab (Opdivo ® ), anti-PD-1 monoclonal antibodies, have been approved. These antibody drugs exhibit clinically significant antitumor responses; however, small molecules exhibit advantages over monoclonal antibody drugs, such as cell penetration, long half-life, and low manufacturing costs, and the possibility of oral administration. Thus, it is imperative to develop small-molecule immune checkpoint inhibitors (Adams et al., 2015;Smith et al., 2019).
Recently, we have proposed the use of "diversity-enhanced extracts" (Kikuchi et al., 2014;Kikuchi et al., 2016;Oshima and Kikuchi, 2018;Kikuchi et al., 2019), an approach for increasing the chemical diversity of natural-product-like compounds via the combination of natural product chemistry and diversity-oriented synthesis (Schreiber, 2000;Burke et al., 2003;Burke and Schreiber, 2004). Diversity-enhanced extracts are obtained from multiple chemical reactions that directly form new carbon-carbon bonds in the extracts of natural resources in order to afford diverse natural product-like library-bearing remodeled molecular scaffolds.
Several chemical transformations of natural extracts using similar methods have been reported (López et al., 2007;Ramallo et al., 2011;Ramallo et al., 2018;Solís et al., 2019;Salazar et al., 2020) (Kawamura et al., 2011;Kamauchi et al., 2015;Tomohara et al., 2016;Kamauchi et al., 2018;Guo et al., 2021;Sulistyowaty et al., 2021), but most of the methods simply provide compounds in which some functional groups have been transformed. However, we have defined diversity-oriented extracts as those natural extracts formed by reactions that produce multiple diversities similar to that observed in diversity-oriented synthesis, including the formation of new carbon-carbon bonds, modification of molecular scaffolds (Oshima and Kikuchi, 2018), and conversion of functional groups.
As reported previously (Kikuchi et al., 2016), the CTLA-4 gene expression inhibitor 1 was obtained by screening a library of unnatural indole alkaloid-type compounds produced from the diversity-enhanced extracts. In this study, we synthesized derivatives of 1, which we subsequently evaluated to identify compounds with higher potency. This led to the development of dual immune checkpoint inhibitors against PD-L1 and CTLA-4.
Next, effects of 2a-2j with substituents on the indole ring of 1 were examined on the CTLA-4 and PD-L1 expression ( Table 1). Compared to 1, a majority of the compounds exhibited a marginal change. However, 5-benzyloxy compound 2e exhibited a slightly increased inhibitory activity toward CTLA-4 and PD-L1 expression. Thus, the introduction of a bulky substituent at C-5 of the indole ring is desirable; as a result, further derivatization was conducted on the basis of the compound structure of 2e.

Synthesis of Compounds With a Bulky
Substituent at the Fifth-Position of the Indole Ring and Their Immune Checkpoint Inhibitory Activity Bulky substituents equivalent to or greater than the benzyl group and cyclohexylmethoxy (3a) and 2-naphthylmethoxy (3b) groups were introduced into the fifth-position of the indole ring ( Figure 3). In addition, compounds in which 4-methoxy (3c), 4-chloro (3d), and 4-nitro (3e) groups were introduced into the benzene ring of 2e as electron-donating, weakly electronwithdrawing, and strongly electron-withdrawing groups, respectively, were synthesized. 5-Benzyloxy derivative 2e was subjected to tosylation, followed by elimination of the tosyl group under basic conditions to afford 6′,7′-dehydro compound 5 ( Figure 4). Compound 2e was oxidized by Dess-Martin periodinane to the 7′-oxo derivative 6, which was further reduced by sodium borohydride to afford alcohol 7, corresponding to the 7′-epi-form of 2e. The C-7 stereochemistry of 7 was confirmed by the NOESY correlation between H-5′ and H-7′.
The inhibitory effects of CTLA-4 and PD-L1 on the expression of the aforementioned synthesized compounds were investigated ( Table 1). Results for 3a and 3b indicate that a bulkier substituent such as a naphthalene ring is desirable. The introduction of substituents into the benzene ring of 2e slightly improved the activity, and 3e with a 4-nitro group exhibited particularly good results. On the other hand, 5-7, in which the iridoid moiety of 2e was modified, did not give good results; particularly, when the oxygen functional group at C-7 was removed, the inhibitory effect was weakened. Therefore, it is crucial to retain the iridoid moiety in this compound.

Synthesis of Compounds Using Tryptophan Derivatives and Their Immune Checkpoint Inhibitory Activity
Finally, we modified the chemical structure of 3e, which gave the best results thus far, and we synthesized compounds using tryptophan derivatives to further improve their biological activity. We synthesized N-Boc-5-hydroxy-L-tryptophan methyl ester (8) according to a previously reported method (Zhu et al., 2015), followed by the etherification of the p-nitrobenzyl group and removal of the Boc group to afford 9 ( Figure 5). In addition, 7 was reduced to amino alcohol derivative 10, and the etherification of the p-nitrobenzyl group and removal of the Boc group were performed to obtain 11. Mixtures of iridoids, which were α-glucosidase-treated extracts of C. officinalis, were then subjected to condensation with 10 and 11 to afford diversityenhanced extracts; these extracts were separated to afford corresponding compounds 12 and 13, respectively ( Figure 1C). The inhibitory effects of compounds 12 and 13 on the expression of CTLA-4 and PD-L1 were slightly enhanced compared to compound 3e (Table 1); in particular, the inhibitory effects of 12 and 13 toward PD-L1 expression were enhanced by approximately sevenfold compared with that of 1. Thus, whether 12 and 13 suppress not only CTLA-4 and PD-L1 gene expression but also protein expression on the cell surface is investigated. The consistent change in the surface CTLA-4 expression was identified by flow cytometry analysis, where the mean fluorescence intensity (MFI) of MT-2 cells treated with 12 and 13 exhibited 73 and 29% decrease, respectively, at a concentration of 20 μM ( Figure 6). The consistent change in the surface PD-L1 expression was also identified by flow cytometry analysis, where the MFI of THP-1 cells treated with 12 and 13 exhibited 55 and 76% decrease, respectively, at a concentration of 15 μM ( Figure 7).

CONCLUSION
Based on the unnatural pentacyclic compound 1 obtained from the diversity-enhanced extracts of Japanese cornelian cherry, 12 and 13 were obtained with immune checkpoint inhibitory activities via the introduction of substituents and conversion of functional groups.
Although it is difficult to obtain these unnatural pentacyclic indole FIGURE 4 | Synthesis of iridoid moiety-modified compounds 5-7.
Frontiers in Chemistry | www.frontiersin.org November 2021 | Volume 9 | Article 766107 alkaloid-like compounds and their derivatives by other synthetic methods, they could be efficiently obtained by utilizing the diversity-enhanced extracts. Compounds 12 and 13 suppressed the CTLA-4 and PD-L1 gene expression and their protein expression on the cell surface. Although their potency is not as high as those of the existing small-molecule immune checkpoint inhibitors against CTLA-4 (Huxley et al., 2004;Zeng et al., 2013) or PD-L1 (Skalniak et al., 2017;Taylor et al., 2018;Park et al., 2021), these compounds are the first reported dual small-molecule inhibitors of CTLA-4 and PD-L1. Therefore, using these compounds can provide an option for cancer immunotherapy, either as monotherapy or in combination with monoclonal antibody-based blockers. On the other hand, since these compounds inhibit CTLA-4 and PD-L1 expression at the same time, it is unlikely that they specifically inhibit only the expression of these two proteins. Rather, they may suppress the expression of several related proteins by suppressing the expression of genes upstream of these proteins. These are issues that need to be addressed in the future.

Preparation of Mixtures of Iridoids From Cornus officinalis
Accessory fruits (500 g) of Cornus officinalis, which was purchased from Uchidawakanyaku Ltd. (Tokyo, Japan), were extracted twice with methanol (3 L) at room temperature to give the extract (105 g). This extract was partitioned with ethyl acetate and water to yield water solubles. The water solubles was subjected to activated charcoal (200 g) and then successively eluted with water (2 L), 5% ethanol-water solution (2 L) and methanol (2 L). The methanol eluent was concentrated in vacuo to give glycoside-rich fractions (16.1 g). The glycoside-rich fractions were dissolved in 0.05 M citrate buffer (pH 6.0) (600 ml), and β-glucosidase (from Sweet Almond, Toyobo Co., Ltd.) (300 mg) was added to the solution. After being stirred for 2 days at 45°C, the reaction mixture was extracted with ethyl acetate three times. The combined organic layer was washed with Frontiers in Chemistry | www.frontiersin.org November 2021 | Volume 9 | Article 766107 water, dried over sodium sulfate, and concentrated in vacuo to give a mixture of iridoids (1.03 g).

Synthesis of Compound 11
Compound 10 (200 mg, 0.653 mmol) was dissolved in acetone (3 ml), and cesium carbonate (280 mg, 0.862 mmol) and 4nitrobenzyl bromide (170 mg, 0.787 mmol) were added to this solution at 0°C. After being stirred for 3 h at 0°C, the reaction mixture was poured into saturated ammonium chloride solution and extracted with ethyl acetate three times. The combined organic layer was washed with water and brine, dried over sodium sulfate, and concentrated in vacuo. The residue was chromatographed over silica gel eluted by hexane-ethyl acetate (1:3) to afford N-Boc-5-(p-nitrobenzyl)oxy-L-tryptophanol (254 mg, 88%).