Controlled Self-Immolative Release of β-Lapachone via an Opti-mized para-Hydroxybenzyl Linker for Targeted Pancreatic Cancer Therapy

1 Controlled Self-Immolative Release of β-Lapachone via an Optimized para-Hydroxybenzyl Linker for Targeted Pancreatic Cancer Therapy Julie B. Becher1‡, Nisita Dutta1,2,3‡, Claudio D. Navo4,5, Lavinia Dunsmore1, Roman Misteli1, Enrique Gil de Montes1, Grant G. Simpson1, Christine C. Alewine2†, Gonzalo Jiménez-Osés4,5*, Gonçalo J. L. Bernardes1,6* 1Yusuf Hamied Department of Chemistry, University of Cambridge, Cambridge, CB2 1EW, UK 2Laboratory of Molecular Biology, National Cancer Institute, National Institutes of Health, Bethesda, Maryland, 20892, USA 3Medical Scientist Training Program, University of Maryland School of Medicine, Baltimore, Maryland, 21201, USA 4Center for Cooperative Research in Biosciences (CIC bioGUNE), Basque Research and Technology Alliance (BRTA), Derio, 48160, Spain 5Ikerbasque, Basque Foundation for Science, Bilbao, 48013, Spain 6Translational Chemical Biology Group, Spanish National Cancer Research Centre 26 (CNIO), Madrid, 28029, Spain KEYWORDS: ortho-quinone, prodrug, pancreatic cancer, NQO1,  -glucuronide,  -glucuronidase, para-hydroxybenzyl linker,  -lapachone ABSTRACT: Methodologies to target cancer cells without affecting healthy surrounding cells have the potential to vastly improve both patient outcomes and quality of life. ortho-Quinone natural products such as -lapachone have exhibited great therapeutic potential, but their clinical use has been limited thus far due to systemic toxicity, making them ideal candidates for targeted prodrug development. Previously developed -lapachone prodrugs suffered from suboptimal release rates, low solubility, and poor tumor specificity that have hindered their advancement into clinic. This work explores the development of an optimized β-lapachone small molecule prodrug platform that utilizes a β-glucuronide-protected para-hydroxybenzyl (PHB) moiety alkylated to one of β-lapachone’s carbonyls via an indium-mediated Barbier-type reaction. The β-glucuronide moiety detoxifies and solubilizes the ortho-quinone systemically and triggers specific release in the tumor microenvironment (TME). Additionally, the drug release rate is largely controlled by pH and can be fine-tuned via addition of electron donating/withdrawing groups to the PHB self-immolative linker. The prodrug is rationally designed to have numerous layers of pancreatic cancer targeting via the tumor-selective overexpression of the key β-lapachone activating enzymes NQO1 and 5-LO, the TME expression pattern of the release-triggering enzyme -glucuronidase, and the opportunity for modular attachment to carrier systems targeting pancreatic cancer membrane proteins. Various derivatives of this prodrug have shown promising stability, release, and efficacy against pancreatic cancer cell lines. This unique prodrug platform allows us to mask the toxicity of an ortho-quinone payload and utilize several layers of tumor specificity in the hope of effectively treating pancreatic cancer patients while minimizing toxic side effects. INTRODUCTION The ortho-quinone functional group is a natural product scaffold that has under-utilized cancer treatment potential. Ortho-quinones have repeatedly exhibited cytotoxicity against numerous cancer cell lines,1–6 but have been excluded from clinical development due to both pan-assay interference (PAINS) character and dose-limiting systemic toxicities caused by indiscriminate mechanisms of action. Many metabolic processes are known to reduce quinones to either their semiquinone or hydroquinone forms.7,8 In contrast to clinically relevant para-quinones, for many ortho-quinones, these reduced forms are highly unstable and spontaneously undergo re-oxidation. This futile redox cycling generates reactive oxygen species (ROS) that damage DNA, lipids, and proteins, consume reducing substrates (glutathione, NAD(P)H), and induce cellular oxidative stress.8,9 In order to combat the toxic systemic effects of ortho-quinones, specialized prodrug formats can be synthesized that facilitate highly-specific release of these payloads in the tumor microenvironment (TME), allowing for effective dosing with limited clinical side effects for patients suffering from various cancers. While ortho-quinones show promising anti-cancer activity against multiple diseases, one with a large unmet therapeutic need is pancreatic ductal adenocarcinoma (PDAC). PDAC is a major oncologic disease that is currently the 2 third-leading cause of cancer-related deaths in the U.S., with a five-year survival rate of just 8% in 2025.10 The lack of improvement in PDAC patient survival rates over the decades is in part due to the limited effective therapies for this fatal disease. Current first-line chemotherapy regimens are substantially toxic with neutropenia occurring in about 40-50% of patients.11–15 Figure 1. Previous -lapachone prodrugs. (a) The first-generation PAB -lapachone prodrug developed by Dunsmore et al.30 and (b) the ROS-triggered prodrug developed by Gong et al.31 are shown. (c) The -lapachone prodrug platform developed in this work involves a -glucuronidase-triggered release that is kinetically modulated by the PHB linker substituents. Pro-moiety (blue), linker (green), benzyl ketol core (yellow), and drug release triggering enzyme/conditions (red) for each prodrug platform are indicated. The key C–C bond (red) connecting the linker to the ketol stops the redox cycling of the quinone and is cleaved during a 1,6elimination mechanism (see arrows) to release the active drug. Additionally, due to the dense stroma of the PDAC TME, large molecule therapeutics have had little efficacy in penetrating these tumors and eliminating cancer cells.16 Thus, new, effective small molecule therapeutics are desperately needed to improve survival rates and preserve quality of life for PDAC patients. The ortho-quinone natural product β-lapachone (1) originates from the bark of the Central and South American Lapacho tree.17 This small molecule (MW = 242) has been shown to have microto sub-micromolar cytotoxicity against a range of cancer cell lines including colon,18 breast,18–22 NSCLC,17,23 prostate,4,20,24 and of particular interest, PDAC.19,25–29 β-lapachone’s mechanism of action involves hijacking NAD(P)H:quinone oxidoreductase 1’s (NQO1) natural cytoprotective function to kill the malignant cells that overexpress this enzyme (Figure S1).21 When β-lapachone is reduced by NQO1 into its hydroquinone form (2), its instability causes it to swiftly and spontaneously re-oxidize back to the ortho-quinone.17,21 Each step of the re-oxidation process generates ROS, and further interaction with NQO1 perpetuates a futile redox cycle. This allows for a small amount of the ortho-quinone molecule to cause drastic oxidative stress to cells.23 Eventually, this process leads to a unique pattern of cell death termed “programmed necrosis” or “necroptosis” (Figure S1).17 Importantly, this method of cell death does not rely on 3 caspase or oncogenic driver mutation (e.g., KRAS, p53) pathways that are often defunct in cancer cells.17,27 In addition, β-lapachone kills cells regardless of their cell cycle status, making it effective against both actively dividing and growth-arrested stem-like malignant cells.27,30 This manner of cytotoxicity could be beneficial to overcoming cancer drug resistance by breaking the 1:1 drug to target ratio.27,31 For PDAC, >90% of cases exhibit NQO1 overexpression due to mutant KRAS-driven transcription.26,27,32,33 Importantly, NQO1 expression is correlated to both disease progression and poor patient prognosis in pancreatic cancer as well as various other cancers.27,34–38 Beyond causing oxidative stress, -lapachone has also been identified as an inhibitor for the 5-lipoxygenase (5-LO) enzyme,39 which is overexpressed in >90% of PDAC cases.40–46 5-LO is part of the arachidonic acid pathway that is implicated in inflammation-associated carcinogenesis for numerous cancers including PDAC.43,45,47–49 Additionally, there is a correlation between increased 5-LO expression and PDAC disease progression.42,46 β-lapachone has two main shortcomings—insolubility in water50 and systemic dose-limiting toxicity.51 It advanced to clinical trials for PDAC in two solubilizing formulations, ARQ 501 (phase II)52 and ARQ 761 (phase I/Ib),51,53 which both showed moderate efficacy by stabilizing disease in patients. However, major dose-limiting side effects of anemia and methemoglobinemia were observed due to off-target redox cycling of the ortho-quinone pharmacophore by interaction with the b5 reductase 1 (CYB5R1) enzyme expressed ubiquitously in mammalian erythrocyte cells.51 Thus, additional layers of target specificity must be added to -lapachone to clinically utilize the ortho-quinone scaffold. Previous -lapachone prodrugs developed by Boothman, Gao, and co-workers involved esterasecleavable hydroquinone alkyl esters,54,55 and pH-sensitive aryl imine,56 acyl hydrazone, ketal,57 aminoalkyl alcohol and amino aromatic phenol derivatives.58 However, these prodrug strategies failed to advance to clinic due to their drawbacks—esters and hydrazones are too labile under physiological conditions in circulation, while ketals are not sufficiently labile in tumors.57 A more sophisticated ortho-quinone prodrug strategy was developed in our group using a targeted carrier-linked prodrug scaffold with a site-specific trigger-spacer-drug design.59 The trigger and spacer can then be designed to improve the low solubility of the drug through the addition of polar or ionizable groups. Dunsmore’s first generation prodrug attached a para-aminobenzyl (PAB) selfimmolative linker (SIL) via C-alkylation to one of the orthoquinone carbonyls to form a para-aminobenzyl ketol species (Figure 1a). While the PAB linker was attached, the prodrug exhibited no redox activity, decreased hemolysis, and decreased methemoglobinemia formation in in vitro assays, indicating this C-alkylation strategy is successful at stopping the off-target toxicity of the pharmacophore in circulation. Upon enzymatic or chemical cleavage of a protecting group from the PAB linker, the ortho-quinone was released via a C–C bond cleaving 1,6-elimination mechanism.59 While this first-generation prodrug represented a breakthrough in ortho-quinone prodrug technology, it exhibited suboptimal solubility and slow-release rates at physiologically relevant pHs. A similar boronate ester - lapachone prodrug (Figure 1b) was developed by the Zhang group, but relied on ROS as the trigger for prodrug activation rather than a tumor-specific enzyme.60 We hypothesized that switching the peptide-protected PAB linker to a -glucuronide-protected para-hydroxybenzyl (PHB) self-immolative linker would facilitate both faster and more controlled drug release at physiologically relevant pHs via the much lower pKa of the phenol in comparison to the PAB aniline and increased solubility via the charged, hydrophilic -glucuronide trigger moiety (Figure 1c). Our novel prodrug approach addresses several limitations of our predecessors, improving solubility, minimizing offtarget toxicity, and increasing the specificity of prodrug activation to target tissues. The -glucuronidase/-glucuronide enzyme/prodrug pair is thought to be optimal for tumor-specific enzyme prodrug therapy (EPT) due to the highly advantageous features imparted by both the glucuronide pro-moiety and the triggering enzyme.61,62 The glucuronide moiety is both strongly solubilizing63–67 and detoxifying66,67 by stopping passive cellular uptake. The -glucuronidase enzyme is widely expressed in endosomes and lysosomes in nearly all tissues, but is specifically found extracellularly in the necrotic areas of the TME where it is excreted by monocytes, granulocytes, and necrotic/apoptotic cells.68,69 Pancreatic cancer exhibits significant β-glucuronidase overexpression in tumor and stromal tumor bed cells, and in the necrotic tumor extracellular space.70 A β-lapachone β-glucuronide prodrug would not only take advantage of the target specificity imparted by NQO1 and 5-LO overexpression in PDAC, but also the tumorspecific activation given by the expression of βglucuronidase. In addition, the β-glucuronide moiety used in conjunction with a redox-masking C-alkylated PHB SIL should drastically improve the off-target toxicity and solubility of the β-lapachone prodrugs, widening their therapeutic window (TW). By changing the substituents attached to the PHB linker, the release rate of the orthoquinone can be optimized for fast release in the TME while maintaining stability in circulation. When compared to both the first-generation C-alkylated prodrug developed by Dunsmore et al.59 and the Zhang group’s boronate ester prodrug60, the prodrug derivatives in this study demonstrated vastly improved pharmacological properties, advancing ortho-quinone drugs closer to clinical relevancy. RESULTS AND DISCUSSION Preliminary modeling of pH-dependent cleavage of PHB vs PAB linkers To explore the hypothesis that a PHB -lapachone prodrug could be faster releasing than a PAB -lapachone prodrug, quantum mechanical calculations were performed for PHB--lapachone (3a) in comparison to PAB-- lapachone (4) (Figure 2). Similar to what was found for the PAB linker,59 the active releasing species for the PHB linker were the deprotonated PHB phenol (3PhO-, ΔG‡ = 19.1 kcal mol-1) and the zwitterion species (3Zw1, ΔG‡ = 5.6 kcal mol1) (Figure 2a,b and Supporting Information). In this case, 4 no C–C breaking transition state (TS) was found for the protonated ketol carbonyl species (3OH+) due to the lower nucleophilicity of the neutral phenol group in comparison to the aniline group (Figure 2b). The pH-dependent release rate profile could be calculated (Figure 2c) using the equations shown in Figure 2d. Due to the fact that the pKa of the PHB phenol (~10) is much lower than that of the PAB aniline (~25), the release rate of -lapachone from 3a is predicted to be ~50 times faster than the release rate of - lapachone from 4 at pH 6. Most importantly, the release rate of 3a shows the opposite pH dependence than the release rate for 4 (Figure 2c). In the case of 4, release is slowest at pH 7, increasing slightly as pH decreases due to the larger concentration of the protonated ketol carbonyl species (4OH+) in solution. In the case of 3a, in contrast, the release rate is predicted to increase rapidly as the pH (and therefore the concentration of the deprotonated phenol species 3PhO-) increases, exceeding that of 4 at pH 5.3 and above (Figure 2c). Figure 2. Prediction of the reactivity of PHB vs PAB -lapachone prodrugs. (a) General chemical scheme showing nine different species denoted as Q (Q = 3a or 4, where subscript indicates neutral, cationic, anionic and zwitterionic) that are in equilibrium for the PHB (X=O) and PAB (X=NH) ketol derivatives of -lapachone in an aqueous solution. Structures in black denote the major species in solution at pH 1–14 (i.e., they have a pKa in that range). Structures in grey denote the minor (negligible) species in solution at pH 1–14 (i.e., they have a pKa outside that range). Species inside a dashed box (QX– and QZw1) are those for which a 1,6elimination (i.e., C–C breaking) TS structure was found. (b) Theoretical potential energy surface (PES) calculated with PCM(H2O)/M06-2X/6-31+G(d,p) for the elimination of species 3, 3PhO-, 3OH+, 3Zw1, and 3Oalong the breaking C–C bond (marked in 5 dotted green in the chemical structure). White squares denote stationary points (i.e., TS or local minima). (c) Comparison of the experimental elimination rate constants (kobs) in logarithmic form previously reported for compound 4 (orange) vs. those theoretically predicted for compound 3a at different pHs using the reduced (light blue) or simplest equation (dark blue). Theoretical intrinsic reaction rate constants for the phenolate (k– = 6.2·10-2 s-1) and zwitterionic (kzw = 4.9·108 s-1) species were calculated quantum mechanically. Theoretical pKa values for the phenolphenolate (3PhO-, 9.6) and ketolketolate (3O-, 10.9) species were predicted using MolGpKa.71 (d) The complete equation 1 (orange) describes the effect of pH on the observed kinetic rate (kobs) considering all species represented in panel a. This equation can be approximated (equation 2, light blue) by considering only those species that are predominant at pH 1-14 and/or enable elimination (i.e., the zwitterion 3Zw1), and even further simplified (equation 3, dark blue) by considering only the dominant phenol/phenolate equilibrium, as the contribution of zwitterion 3Zw1 to kobs proved to be negligible due to its very low abundance despite being highly reactive (light vs. dark blue plots in panel c). Design and synthesis of prodrug derivatives Based on these predictions, a library of PHB prodrug derivatives was designed. Two opposing strategies were explored to modulate the release rate of -lapachone from the PHB SIL. The first approach aimed at lowering the pKa of the PHB phenol by attaching electron withdrawing groups (EWGs). This would increase the concentration of the actively releasing negatively charged phenolate species in solution, at the expense, however, of deactivating the aromatic ring towards 1,6-elimination. According to the simplified equation shown in Figure 2d, lowering the pKa of the phenol (predicted using MolGpKa71) from 9.5 to 6.5 while maintaining the intrinsic activation barrier should accelerate the reaction ~1,000 times at pH 5, but only 32fold at pH 8. Of note, this effect would quickly vanish at sufficiently basic pH values around 10 (Figure S2). In principle, such pH-controlled acceleration would be ideal to quickly release the payload only when reaching the acidic TME, potentially allowing for tumor selective chemotherapy delivery depending on the circulating dose of the prodrug. Four derivatives were designed towards this goal and compared to the unsubstituted PHB linker derivative (16a; predicted pKa 9.8)—one with two electronwithdrawing fluorine atoms attached ortho to the phenol (16b; predicted pKa 7.8), one with four fluorine atoms at the ortho and meta positions (16c; predicted pKa 6.3), one with two fluorine atoms meta to the phenol (16d; predicted pKa 8.3), and one with a sulfonamide ortho to the phenol to act as a potential carrier attachment point (16e; predicted pKa 7.6). The second strategy explored reducing the intrinsic activation barrier by stabilizing the 1,6-elimination TS. A transient positive charge is hypothesized to form mainly at the benzylic position and meta to the phenol in the PHB linker during the elimination reaction. Adding electron donating groups (EDGs) at these positions would stabilize the transient positive charge, thereby reducing the intrinsic activation barrier of the rate-limiting step, while only slightly altering the phenol’s pKa .72,73 As a drawback, no pHcontrolled acceleration can be achieved through this approach (Figure S2). Five additional derivatives were designed to test this hypothesis—one with a methoxy group meta to the phenol (16f; predicted pKa 9.5), one with two methoxy groups meta to the phenol (16g; predicted pKa 9.3), one with two methoxy groups ortho to the phenol (16h; predicted pKa 9.8), one with a methyl group at the benzylic position (16i; predicted pKa 9.8), and one with a 2-methoxyethoxy group attached meta to the phenol (16j; predicted pKa 9.5). Derivative 16j also enables the evaluation of a PEG spacer attached to the meta oxygen atom. Based on the findings by Rose et al. with self-immolative hydroxybenzyl linkers releasing amine payloads via fast C– N bond breaking,74 the distal alkoxy group oxygen could promote drug release by forming a transient seven membered ring with the benzylic position of the quinone methide formed after 1,6-elimination, in the unlikely case that the C–C bond breaking step is not rate-limiting (Figure S3). The synthesis of these prodrug derivatives started with a commercially available para-hydroxybenzaldehyde or para-hydroxybenzyl ketone (11). For derivatives c and e, the corresponding PHB alcohols (6 and 10 respectively) had to be synthesized first as they were not commercially available. 6 was synthesized by reducing 5 with borane (Scheme 1a). 10 was synthesized in 27% yield over 5 steps. First, 7 was chlorosulfonated and coupled with Nmethylpropylamine to give 8, which was then deprotected (BBr3) with a basic workup giving 9 and reduced (borane) to give 10 (Scheme 1b). Prodrug synthesis then followed six general steps (Scheme 1c). First 11, 6 or 10 were glycosylated with the acetyl-protected glucuronide moiety using modified Koenigs-Knorr conditions to give 12 for derivatives a, b, d, f, g, h, and i, or 13 for derivatives c and e. Glycosylation of 6 and 10 exhibited complete regioselectivity at the phenol oxygen rather than the benzylic alcohol, (confirmed by cross peaks in the HMBC spectra between the anomeric proton (H1’) and the phenol carbon (C1) (Figures S4, S5)). For derivative j, 11j was first glycosylated to give 12j with good regioselectivity. The one-unit PEG tether was then attached via a strictly anhydrous Ag2O-activated SN2 reaction to give 17. For derivatives a, b, d, f, g, h, i, and j, the benzyl aldehyde or ketone 12 was then reduced into 13 using sodium borohydride over silica gel at 0 C to preserve the glucuronide acetyl protecting groups. Next, the benzyl alcohols (13) were activated via chlorination (SOCl2) or bromination (PBr3) to form the benzyl halide species (14). The development of a new ortho-quinone alkylation procedure was necessary because the strongly basic aqueous conditions used by Dunsmore et al. (20 equiv KOH),59 Gong et al. (8 equiv K2CO3),60 and Kaye et al. (3 equiv NaOH)75 to C-alkylate ortho-quinones would remove the glucuronide protecting groups. A neutral, anhydrous reaction was necessary. Nair et al. reported a facile indiummediated Barbier alkylation that enabled C-benzylation of 9,10-phenanthrenequinone using benzyl bromide, sodium iodide, and powdered indium (0) metal in anhydrous DMF with ultra sonication.76 A modified version of this Barbier reaction using the benzyl halide linkers (14) and - 6 lapachone enabled the synthesis of the C-alkylated - lapachone prodrugs (15) in serviceable yields. The yield was lowest for the secondary benzyl bromide linker 14i (4%), likely due to steric hindrance from the benzylic methyl group. The C-alkylated -lapachone prodrugs (15) were isolated as a mixture of two diastereomers, with only the two major product isomers isolated for 15i. Alkylation occurred exclusively at the carbonyl adjacent to the benzene ring in -lapachone (Figure S6), which is rationalized to be the more electrophilic carbonyl (Figure S7), leading to the calculated thermodynamically more stable regioisomer (Figure S8). Interestingly, this Barbier procedure reverses the polarity of the reaction components in comparison to the previously reported base-activated reactions, making the benzyl halide indium reagent the nucleophile and the ortho-quinone the electrophile (Figure S9). Finally, the glucuronide acetyl protecting groups were removed using LiOH, and the final prodrugs (16) were purified using semi-preparative HPLC. All prodrugs except 16e were isolated as a mixture of two diastereomers. The two diastereomers of 16e had adequate retention time resolution under the semi-preparative HPLC conditions to be isolated separately, with the major product isomer being the one that was mainly studied. Derivative 16g proved to be unstable during purification and isolation, with trace lapachone present after lyophilization. For all prodrug diastereomers, the -anomer was preserved throughout the entire synthesis based on the JH1”-H2” coupling constant for the anomeric proton being > 6 Hz (: 2-5 Hz, : 6-10 Hz77) (Figure S6, Table S3.1). An optimized version of Dunsmore’s PAB prodrug was also synthesized for comparison (Scheme 1d). As lowering the pKa of the PAB aniline to physiologically relevant values is impractical, only a TS stabilization derivative was explored. 7 Scheme 1. Synthesis of prodrug derivatives. (a) First 6 was synthesized by reducing 5. i: BH3·THF, anhyd. THF. (b) Then 10 was synthesized from 7 over five steps. i: chlorosulfonic acid; ii: N-methylpropyl amine, TEA, anhyd. DCM; iii: BBr3, anhyd. DCM, -78 C; iv: 10% aq. NaOH; v: BH3·THF, anhyd. THF; (c) Finally, 11, 6, or 10 were glycosylated, reduced, chlorinated or brominated, and reacted with -lapachone in an indium-mediated Barbier reaction. Removal of the sugar protecting groups furnished the prodrugs 16 after HPLC purification. i: Acetobromo-α-D-glucuronic acid methyl ester, 11, Ag2O, anhyd. ACN; ii: NaBH4, silica, CHCl3:IPA 5:1; 8 iii: BrCH2CH2OMe, Ag2O, K2CO3, DCM:DMF 1:1; iv: SOCl2, anhyd. DCM; v: NaBH4, MeOH:THF 1:1; vi: PBr3, anhyd. DCM or ether; vii: 14, indium (0) powder, NaI, 1, anhyd. DMF, sonicated at 40 C for 12-24 h; viii: LiOH monohydrate, MeOH:THF 1:1, purified by HPLC. (d) Penicillin G amidase-triggered model compound 22 was synthesized from 18 in four steps. i: Phenylacetic acid, EEDQ, anhyd. DCM; ii: LiBH4, anhyd. 1:9 MeOH:THF; iii: PBr3, anhyd. DCM; iv: 21, indium (0) powder, NaI, 1, anhyd. DMF, sonicated at 40 C for 24 h, purified by HPLC; (e) The prodrug developed by Gong et al., 24, was synthesized from 23 and 1 using an indium-mediated Barbier reaction. i: 23, indium (0) powder, NaI, 1, anhyd. DMF, sonicated for 1 h. The PAB linker was designed with a methoxy group meta to the aniline. The aniline in 18 was first protected with a penicillin G amidase (PenG)-labile phenyl acetyl moiety to give 19, which was reduced (20), brominated (21), and attached to -lapachone using the indium-mediated Barbier reaction to furnish 22. 22 serves as an enzymetriggered model compound to measure the release rate of 1 from the modified PAB linker. For comparison purposes, the pinacol boronate ester prodrug (24) developed by Gong et al.60 was also synthesized (Scheme 1e). Using the indium Barbier reaction procedure, 24 was synthesized in high yield (88%) from 23 and 1. These reaction conditions have several advantages over those used by Gong et al. First, no base or water is necessary. Both of those conditions together accelerate hydrolysis of the boronate ester and benzyl bromide. Secondly, only a short 1 h sonication was necessary rather than a 6 h heating step. Thirdly, the -lapachone was completely consumed during the indium reaction, making purification of 24 by silica flash column chromatography facile since 1 and 24 tend to coelute. Finally, the isolated yield of 24 obtained using the indium Barbier reaction (88%) was higher than that reported by Gong et al. (71%)60. The indium Barbier reaction is therefore superior to the dithionite/base reaction conditions used by Gong et al. on a practical level to furnish 24. Release rate comparison of self-immolative linkers The mechanism of lapachone release from the PHB or PAB prodrugs is shown in Figure 3a,b. First, the purified mixture of two prodrug diastereomers (16a-j) for each PHB derivative was incubated with -glucuronidase. This enabled full removal of the glucuronide moiety from both diastereomers of the prodrug within 2-15 min, leaving a mixture of two PHB--lapachone enantiomers (3a-j, Figure 3c). For the PAB compound 22, penicillin G amidase was used in place of -glucuronidase to give a mixture of two PAB--lapachone enantiomers (25). The 3 (or 25) solution was then spiked into citrate-phosphate buffers from pH 310, and release of 1 from 3 (or 25) was measured at various timepoints by analytical HPLC. A representative example of the HPLC traces from the release experiment of 3g is shown in Figure 3d. A single peak for the two enantiomers of 3 was observed after glucuronide removal, and thus the measured kobs was for both enantiomers in the mixture. Over time, the 3/25 peak decreased, while the 1 peak increased (Figure 3d). A standard curve for -lapachone (Figure S22) was prepared, enabling the quantification of the released -lapachone. Release of 1 from the PHB linker was significantly slower than the initial removal of the glucuronide moiety by the -glucuronidase enzyme and oxidation of the released hydroquinone, so the ratelimiting step of drug release for the prodrugs is the 1,6elimination of PHB/PAB from 3/25 via the mechanisms shown in Figure 3a,b. As expected, a clear pH dependence for the release rate was observed (Figure 3e,f, S46), and thus, the computationally predicted opposite pH dependence behavior of PHB linkers in comparison to PAB linkers proved to be experimentally supported (Figure 3f). PHB linkers generally release slowest at pH 4-5, with a marginal increase in release rate as pH is decreased further. As pH increases above 5, release rate drastically speeds up (Figure 3e). These trends confirm the prediction that the main species contributing to the observed release rate for PHB linkers is the deprotonated phenolate (Figure 2c). This trend held true for all PHB derivatives except tetra-fluorinated 3c, whose acidic phenol group is largely deprotonated near neutral pH, rapidly reaching its upper rate limit set by the intrinsic activation barrier. In contrast, both PAB linkers examined (4 and 25) showed the opposite pH dependence trends. Release was slowest at pH 7 and above, increasing rapidly as pH decreased (Figure 3f). Despite the observed general beneficial effect of running each individual reaction at higher pH and thus increasing the concentration of the active phenolate species, lowering the pKa of the PHB phenol with EWGs was not successful at increasing the observed lapachone release rate from the PHB linker. While 3b and 3c showed a moderate release rate increase over 3a at pH < 6-7, all other derivatives with phenol pKa’s lower than that of 3a released -lapachone significantly slower than 3a. This effect was especially pronounced for 3c and 3d which had EWGs meta to the phenol. Unfortunately, attaching EWGs to the phenol ring proved to be detrimental for the release reaction, with ring deactivation outweighing the effect of increasing phenolate concentration and slowing drug release drastically. These results highlight the importance of accounting for multiple, potentially counteracting parameters when optimizing a chemical reaction. Conversely, as anticipated, the incorporation of EDG groups into the phenol ring significantly promoted lapachone release; unlike with EWG substituents, this effect was uniform across the full pH range, reflected by parallel upward shifts of the kobs vs. pH curves (Figure 3e). Hence, the release rate from 3f was around two orders of magnitude faster than from 3a. The half-life of drug release was decreased from 87.8 ± 3.8 h for 3a to 1.9 ± 0.1 h for 3f at pH 7.5. Di-meta-substituted 3g was even faster releasing than 3f, achieving ~2,200 and ~800-fold acceleration at pH 5 and 7.5 respectively, while di-ortho-substituted 3h showed the weakest acceleration of the TS stabilizing derivatives. The addition of an intramolecular “quenching tether” (3j, Figure S3) demonstrated none of the release rate increase in comparison to its non-tethered analogue 3f that was observed by Rose et al.74 for an analogous but mechanistically distinct reaction. The kinetics of 3j instead demonstrated that the attachment of a PEG spacer to the meta oxygen atom has very little effect on quinone release rate, further proving that in our case the reaction is kinet- 9 ically controlled, and that 1,6-elimination is the ratelimiting step rather than quinone methide quenching. To examine whether the structure-activity relationship (SAR) findings made with the PHB linker could translate to PAB--lapachone prodrugs as well, the PAB meta methoxy derivative 25 was compared to the first-generation plain PAB prodrug (4) developed by Dunsmore.59 Figure 3. Release rate comparison for PHB and PAB prodrugs. The mechanisms of release for the -glucuronide PHB prodrug derivatives (a) and the PAB derivative (b) are shown. -glucuronidase or penicillin G amidase deprotects the PHB or PAB linkers, yielding 3 or 25 respectively. A 1,6-elimination then occurs, releasing the hydroquinone form of -lapachone, which then oxidizes into the ortho-quinone. (c) The substituents on the PHB/PAB linker for each derivative are shown and classified into which strategy they employed, lowering phenolate pKa or stabilizing the elimination TS. BLap = -lapachone in its α-hydroxy ketol form (refer 16 AsPC-1 respectively. Therefore, we expected the derivatives that showed fast release of β-lapachone to have a similar IC50s to these values. The original PHB derivative, 16a, had IC50 values of 11 µM (PANC-1, Figure 7a) and 16 µM (AsPC-1, Figure 7b) upon treatment with - glucuronidase, demonstrating poor payload release throughout the long incubation time (72 h). Those derivatives with slower release rates than 16a, like 16e, had less efficacy in cells comparatively (Table 2). However, for the best performing derivatives with the fastest release rates, we observed IC50 values equal to or even lower than those of pure β-lapachone, demonstrating complete drug release during the incubation period. Prodrugs 16f and 16i showed IC50 values of 700 nM and 800 nM in PANC-1 respectively (Figure 7a), which is two to three-fold more toxic than β-lapachone in this cell line. We also see that this increased toxicity is more pronounced in the AsPC-1 cell line, reducing the IC50 from 5.4 µM of 1 to 900 nM of 16i. When comparing the measured half-lives of the derivatives to their IC50 values in cell studies, we see a strong positive correlation between the two variables, signaling that β-lapachone release rate is the main predictor in cell killing efficacy (Figure S92). Table 2. PDAC Cell Viability with Prodrug Treatment for All Derivatives. Prodrug Derivative PANC-1 IC50 (µM)* AsPC-1 IC50 (µM)* 1 1.8  0.2 5.4  0.7 27 70 - 16c Inactive Inactive 16e major product 25 ± 3 59 ± 12 16d 21 ± 3 - 16b 11 ± 2 24 16a 9.6 ± 0.9 19 ± 3 16h 1.2 ± 0.4 3.2 ± 1.7 16j 1.1 ± 0.4 2.8 ± 1 16g 1.2 ± 0.3 1.6 ± 0.6 16f 0.7 ± 0.2 2.9 ± 0.06 16i 0.8 ± 0.5 0.9 ± 0.5 *Average IC50 values ± standard deviation of at least two biological replicates for each prodrug on the PANC-1 and AsPC1 cell lines are shown. All derivatives were tested on the PANC-1 cell line, and only those that performed better than 16a were tested in replicates on both the PANC-1 and AsPC-1 cell lines. Values with no error represent only one biological replicate and empty values indicate no viability assays were run for that condition. To assess the TW of the prodrug derivatives, we tested each of them in both the PANC-1 and AsPC-1 cell lines up to 100 µM without the β-glucuronidase enzyme present. In agreement with the HPLC stability assay data, we saw no toxicity in all stable prodrug derivatives up to this concentration (Figure 7c,d). Compound 16g, which showed instability in previous assays (Figure 5b,c,e-g), also emulated the same behavior in cell culture, with an IC50 of 26 ± 6 µM in PANC-1 and 75 ± 7 µM AsPC-1 when no releasetriggering enzyme was present. We also saw in PANC-1 that a non-releasing benzyl lapachone control compound (27), which does not contain a glucuronide moiety, had an IC50 of 70 µM (Figure 7c). In comparison, all the stable glucuronide prodrug derivatives retained 100% viability at 100 µM. This confirms that the β-glucuronide sugar employed in our prodrug format increases the TW of the prodrug by preventing passive cellular uptake.67,85–87 This data indicates that the best performing, stable derivatives have a therapeutic index (TI) of greater than 110, since we were unable to reach any killing effects up to 100 µM. It is generally considered that a drug has a good therapeutic safety profile if the TI is greater than 10, which our best derivatives far exceed, signaling that our prodrug is safe to deliver systemically in future in vivo experiments and should not cause any adverse effects in areas where βglucuronidase is not overexpressed.88,89 Cellular mechanism of prodrug β-lapachone’s established mechanism of action by generating ROS species through the NQO1 enzyme as well as the additional inhibition of 5-LO shown through our group’s work, drove our interest in determining if the NQO1 and 5-LO pathways are responsible for our prodrugs’ killing effect in PDAC cells. We measured NQO1 and 5-LO protein expression levels via Western blot analysis in both the PANC-1 and AsPC-1 cell lines (Figure 8a, S94S96). The analysis shows that both AsPC-1 and PANC-1 express the NQO1 enzyme, while only AsPC-1 expresses the 5-LO enzyme (Figure 8a). We also see that PANC-1 seems to exhibit lower levels of NQO1 expression compared to AsPC-1. This is an interesting observation since the cell viability assays in Figure 7 show that the prodrugs and β-lapachone itself are consistently more toxic in the PANC-1 cell line compared to the AsPC-1 cell line. This indicates a potential alternative mechanism of action occurring in the PANC-1 cell line that is responsible for βlapachone’s toxic effects. Literature shows that there are other enzymes that also reduce β-lapachone in a similar manner to NQO1, including ferroptosis suppressor protein 1 (FSP1)90 and cytochrome P450 reductases.91 Additionally, new research has demonstrated that β-lapachone inhibits the activity of thioredoxin reductase 1 (TrxR1),92 which allows cells to maintain redox balance and recover from oxidative stress.93 Both FSP1 and TrxR1 have been found to be expressed in the PANC-1 and AsPC-1 cell lines and could contribute to the difference in potency of - lapachone seen between the cell lines.94,95 β-glucuronidase is found within lysosomes of most cells,69 so we expected to also find this enzyme expressed within the two PDAC cell lines tested. Intracellular βglucuronidase expression can prove useful in determining the potential β-glucuronidase levels released by necrotic cells in vivo when they excrete their cellular contents into the TME. Additionally, if an internalizing carrier is attached to our prodrugs via bioconjugation handles incorporated into the design, then they would make use of this intracellular lysosomal -glucur-onidase expression to trigger drug release. Both PDAC cell lines tested are shown to have β-glucuronidase expressed intracellularly (Figure 8a), with PANC-1 having slightly higher levels. Thus, the 17 prodrugs can take advantage of a self-amplifying release mechanism. Once β-lapachone is released from the prodrugs by extracellular β-glucuronidase, it will exert a killing effect on a portion of the tumor. The necrotic cells will then release their intracellular β-glucuronidase into the TME, which can then act as the enzymatic trigger to release more β-lapachone from the prodrug. The confirmation of β-glucuronidase levels within these cell lines helps inform us of ideal targeting carriers and in vivo PDAC models to test the prodrugs with for future experiments. Since increased β-glucuronidase levels have already been established in PDAC tumors, this finding indicates that we expect additional β-glucuronidase release from initial cell killing, allowing for selective prodrug activation and a selfamplified cell killing effect at the site of the tumor. Additionally, we were able to show that ROS species are generated in both the PANC-1 and AsPC-1 cell lines after treatment with one of the lead compounds 16f for 24 h. Without extracellular β-glucuronidase enzyme added, 16f generates significantly less ROS than β-lapachone alone, supporting the conclusion that C-alkylation of the orthoquinone carbonyl blocks the redox cycling of the pharmacophore59 (Figure 8b, S93). The restored generation of ROS after -glucuronidase addition corroborates that the prodrug mechanism of cell killing is through ROS generation by the released, active, -lapachone drug, most likely via interaction of a combination of the various reductive enzymes mentioned including NQO1, FSP1, and cytochrome P450 reductases. Lastly, as seen in Figure 7, we hypothesize that the increased cytotoxic effects of the prodrugs in cell lines compared to β-lapachone alone results from an additional toxic mechanism of the linker itself. The highly electrophilic quinone methide intermediate produced from the PHB linker upon 1,6-elimination during β-lapachone release is quenched by a nucleophile and aromatized into an alkylated phenol/phenolate product (Figure 8c).72,87,96 In a cellular environment, these nucleophiles likely include essential DNA, RNA, proteins, and lipids which form alkylated products that are toxic to cells.87 Through this mechanism, the PHB SIL can act as an additional toxic agent in our prodrug format, allowing for even more potency than βlapachone alone (Figure 8c). Figure 8. Target Expression, ROS Production, and Quinone Methide Toxicity in PDAC Cell Lines. Panel (a) shows representative western blot bands of varying expression levels of the prodrug target enzymes NQO1, 5-LO, and β-glucuronidase from whole cell lysates of PANC-1 and AsPC-1 with β-actin as a loading control. The A549 cell line was used as a positive control for NQO1 enzyme expression, and the HL-60 cell line was used as a positive control for 5-LO enzyme expression. For β-glucuronidase expression, two consecutive technical replicates are shown for each biological replicate, with all 3 biological replicates represented for both cell lines. Panel (b) displays the reactive oxygen species generation after treatment with 16f with and without β-glucuronidase enzyme, compared to β-lapachone for 24 h. Results were normalized to baseline cellular ROS activity as 0 and positive control tertbutyl hydrogen peroxide (TBHP) as 100. Adjusted p-values of 0.0012 (**) and <0.0001 (****) are shown for statistically significant comparisons. Panel (c) shows the hypothesized mechanism of increased cellular toxicity from the quinone methide intermediate. This mechanism must be investigated further to better understand its cytotoxic role and how specific cell lines may be more susceptible towards this mechanism of action. CONCLUSION In summary, we designed an improved -lapachone prodrug platform using -glucuronide-triggered selfimmolative para-hydroxy benzyl linkers attached via Calkylation of the ortho-quinone carbonyl. Development and application of an indium-mediated Barbier reaction to attach the glucuronide linker to the ortho-quinone carbon- 18 yl proved essential to facilitate synthesis of these prodrug derivatives. The -glucuronide moiety not only helped to solubilize the prodrugs, but also widened their TW by minimizing off-target cellular uptake. Upon glucuronide removal by extracellular - glucuronidase which is found exclusively in the TME,66– 69,77,97,98 a pH-dependent 1,6-elimination occurs, breaking the C–C bond between the quinone and the linker and liberating the active -lapachone drug. By attaching EDGs to the SIL, the release rate of -lapachone was increased by several orders of magnitude, reaching clinically relevant rates and fully restored cellular efficacy for the lead compounds 16f and 16i. This computer-aided design also proved successful at accelerating the release rate of the PAB linker in model compound 22 without altering its pH dependence profile, suggesting a prodrug using this optimized linker design would be superior relative to the first generation prodrugs developed by Dunsmore et al.59 The glucuronide prodrugs were then compared to the previously reported boronate prodrug 24.60 16a, analogous in intermediate structure to 24, released at approximately the same rate as 24, contradicting the release rate findings reported by Gong et al.60 24 also rapidly hydrolyzed into 26, which was less stable at physiological pH relative to all the glucuronide derivatives studied. All prodrug derivatives performed as expected based on kinetics and stability data in two highly characterized pancreatic cancer cell line models. Additionally, we were able to further investigate potential mechanisms of action of the prodrug in these cell lines through -lapachone target enzyme expression and ROS analysis. These findings highlight the potential of this prodrug-linker platform for translation into targeted therapies for pancreatic cancer. The optimized -lapachone prodrugs developed in this study represent the bestperforming ortho-quinone prodrugs developed to date, with bioconjugation to PDAC targeted carriers and in vivo testing of the identified lead compounds underway. ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website. Additional experimental details, compound synthesis and characterization (HPLC, mass spectrometry and NMR) and supplemental figures (PDF) AUTHOR INFORMATION Corresponding Author *[email protected] (Gonçalo J. L. Bernardes) *[email protected] (Gonzalo Jiménez-Osés) Correspondence should be addressed to Gonçalo J. L. Bernardes. Present Addresses †Dartmouth Health/ Dartmouth Cancer Center/ Geisel School of Medicine, One Medical Center Drive, Lebanon, NH, 03753, USA Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. ‡These authors contributed equally. Notes Gonçalo J. L. Bernardes is a scientific advisor to Basinnov Life Sciences (FHC Group). G.J.L.B., J.B.B., L.D., and E.G. dM. are inventors in a patent (PCT/EP2023/064139 (WO/2023/227757)) that covers the discoveries reported in this work. C.A. discloses drug-only support for clinical trials from Minnemarita, ProDa, AstraZeneca and a cooperative research and development agreement with the institution from HCW Biologics. N.D. and C.A. were supported in part by the Intramural Research Program of the National Institutes of Health (NIH). The contributions of the NIH authors were made as part of their official duties as NIH federal employees, are in compliance with agency policy requirements, and are considered Works of the United States Government. However, the findings and conclusions presented in this paper are those of the authors and do not necessarily reflect the views of the NIH or the U.S. Department of Health and Human Services. ACKNOWLEDGMENT We thank Basinnov Life Sciences (FHC Group), the Herchel Smith Fund, the NIH Oxford/Cambridge Scholars Program, the Intramural Research Program of the National Institutes of Health, the National Cancer Institute Center for Cancer Research (Project No. ZIA BC 011652), the Gates Cambridge Trust (Bill & Melinda Gates Foundation, grant No. OPP1144), the National Cancer Institute, the NIDUS studentship, the UKRI (BBSRC DTP studentship BB/M011194/1), a Marie Sklodowska-Curie Fellowship (grant No. 101023887), and MCIN/AEI/10.13039/501100011033 (grants PID2024160774OB-I00, PID2021-125946OB-I00, CEX2021-001136-S) for funding for this project. We also thank the NMR and mass spectrometry technicians at the University of Cambridge Yusuf Hamied Department of Chemistry for their advice and expertise. REFERENCES (1) Dias, R. B.; de Araújo, T. B. S.; de Freitas, R. D.; Rodrigues, A. C. B. da C.; Sousa, L. P.; Sales, C. B. S.; Valverde, L. de F.; Soares, M. B. P.; dos Reis, M. 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