We report a new 1-6 self-immolative, traceless crosslinker derived from the natural product gallic acid. The linker acts through a pH-dependent mechanism for drug release. This 5-(hydroxymethyl)pyrogallol orthoester derivative (HMPO) was stable for 24 hours at pH values of 7.4 and 6.6 and in plasma, releasing molecules bound to the hydroxymethyl moiety under acid-dependent stimuli at pH 5.5. The linker was non-toxic and was used for the conjugation of Doxorubicin (Doxo) or Combretastatin A4 with Cetuximab. The ADCs formed showed their pH responsivity reducing cell viability of A431 and A549 cancer cells better than Cetuximab alone. © 2022 The Royal Society of Chemistry

Elena Cini

Elena Dreassi

Elena Petricci

Enrico Rango

Federica Finetti

Francesca Migliorini

Giovanni Ievoli

Giulia Macrì

Lorenza Trabalzini

Maurizio Taddei

Chemical Communications

English

Gallic acid, a key component of red wine tannins, has been transformed into a linker that releases Doxorubicine or Combretastatin A4 at pH 5.5 in cancer cells.</jats:p

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A pH-responsive crosslinker platform for antibody-drug conjugate (ADC) targeting delivery

Giulia Macrì

Archivio della Ricerca - Università degli Studi di Siena

10532 |  Chem. Commun., 2022, 58, 10532–10535 This journal is © The Royal Society of Chemistry 2022Cite this: Chem. Commun., 2022,58, 10532A pH-responsive crosslinker platform forantibody-drug conjugate (ADC) targeting delivery†Francesca Migliorini, Elena Cini, Elena Dreassi, Federica Finetti, Giovanni Ievoli,Giulia Macrı̀, Elena Petricci, Enrico Rango, Lorenza Trabalzini andMaurizio Taddei *We report a new 1–6 self-immolative, traceless crosslinker derivedfrom the natural product gallic acid. The linker acts through a pH-dependent mechanism for drug release. This 5-(hydroxymethyl)pyr-ogallol orthoester derivative (HMPO) was stable for 24 hours at pHvalues of 7.4 and 6.6 and in plasma, releasing molecules bound tothe hydroxymethyl moiety under acid-dependent stimuli at pH 5.5.The linker was non-toxic and was used for the conjugation ofDoxorubicin (Doxo) or Combretastatin A4 with Cetuximab. TheADCs formed showed their pH responsivity reducing cell viabilityof A431 and A549 cancer cells better than Cetuximab alone.Conjugation between a targeting moiety and a drug is one ofthe most efficient methods for overcoming drug selectivityproblems.1 Besides antibody-drug conjugates (ADCs),2 thereare excellent examples of conjugates containing integrin,3carbonic anhydrase ligands,4 folic acid,5 prostate specific anti-gen (PSA)6 or somatostatin.7 Conjugation allows an increase indrug concentration in the tissues where the target is locatedafter the drug cargo has been selectively activated at the site ofaction. Release is controlled by a trigger mechanism thatdepends on the linker, the key component of the conjugationsystem.8 The way the linker is removed (and the drug released)depends on the trigger mechanism. Historically, the firstexternal stimulus used to release drugs from a ADC was pH.9In cancer, mechanisms related to abnormal rapid metabolism andproliferation lead to a hypoxic microenvironment in which severalacidic molecules are produced that lower the pH to around 5.7–6.9.10 Within the tumour cell, the proton influx lowers the intracel-lular and substructural pH to 5.0–6.0, while a lower pH (4.0–5.0) isachieved in the lysosomes.11 The pH difference between tumourcells and healthy tissue has been exploited to produce pH-responsive anti-cancer nanomaterials,12 although the presence ofan acidic tumour microenvironment poses a potential problem forselectivity. At ADC, once the antibody reaches its target, the inter-nalisation of the receptor selectively transports the conjugate intothe interior of the cell to be eventually metabolised in the acidlysosome environment.2a FDA-approved acylhydrazones (found inMylotargs and Besponsas), for example, release the active ingre-dient in acidic environments, but can only be made from carbonylor hydrazine derivatives, limiting the delivery to agents that containthese functions.13 The bifunctional cross-linker N-ethoxybenzyli-midazole (NEBI) has been used as a tunable pH-sensitive linkerand used for targeted delivery of indenoisoquinoline agents ormodified Doxo to cancer tissue (Scheme 1).5d,14 In this case, abenzaldehyde or an imidazole moiety remains in the released drug.Maleimido derivatives, after hydrolytic conversion to monoamidesof maleic acid, have a proximal carboxylate group that supportsamide hydrolysis with the formation of the malic anhydride underacidic conditions.15 Although efficient, this linker is limited totransporting only primary amines (Scheme 1).1Scheme 1 Examples of acid labile linkers for drug targeting.Dipartimento di Biotecnologie, Chimica e Farmacia, Università degli Studi di Siena,Via A.Moro 2, 53100 Siena, Italy. E-mail: maurizio.taddei@unisi.it† Electronic supplementary information (ESI) available. See DOI: https://doi.org/10.1039/d2cc03052gReceived 30th May 2022,Accepted 21st June 2022DOI: 10.1039/d2cc03052grsc.li/chemcommChemCommCOMMUNICATIONOpen Access Article. Published on 31 August 2022. Downloaded on 11/4/2022 3:38:32 PM.  This article is licensed under a Creative Commons Attribution 3.0 Unported Licence.View Article OnlineView Journal  | View IssueThis journal is © The Royal Society of Chemistry 2022 Chem. Commun., 2022, 58, 10532–10535 |  10533Wagner and co-workers have determined the release kineticsof several functional groups between pH 7.4 and 5.5 andclassified their properties in terms of behaviour under neutralconditions.16 The spiro-orthoester SpiDo exhibited a hydrolysishalf-life t1/2 = 1.5 h (assuming a first-order reaction) andcomplete hydrolysis was obtained after 7 h at pH 5.5.17 How-ever, to our knowledge, the SpiDo platform has only been usedfor in vitro imaging experiments with microorganelles.In conclusion, despite the great improvements achieved inpH-sensitive linkers, a new chemistry for versatile linkers withadjustable release rate is still highly desirable for prodrugdevelopment, including ADC and gene delivery.The 1–6 self-immolative elimination process, typical of p-amino or p-hydroxybenzyl alcohol, is one of the most commonprocesses to drive the release of prodrugs and bioconjugates byexternal stimuli.18 We report here a new linker based on the 1–6self-immolative p-hydroxybenzyl alcohol platform designed forthe conjugation of a drug to a target system (Scheme 1) and thecorresponding release at acid pH. The orthoester group waschosen to provide an effective trigger at pH around 5.5, and thebenzyl alcohol moiety was used for binding drugs bearing anamine (as a carbamate) or a phenol (as an aryl benzyl ether). Inone side of the aromatic ring, we incorporated a triple bond forclick chemistry to bind the cargo to the antibody support. Thevalue of this platform was evaluated in the analysis of cytotoxicdrug delivery to cancer cells by conjugation with cetuximab(Ctx), a monoclonal antibody (mAb) specific for epidermalgrowth factor receptor (EGFR).Since orthoesters are known to be stable in plasma butsensitive to hydrolysis at pH 5.5,16,19 we developed an aromaticorthoester derived from gallic acid, a natural tannin compo-nent found in several plants and available in large quantities ata low price. Gallic acid (1, Scheme 2) already has all theproperties for our design: two OH groups to form a cyclicorthoester, another OH for the installation of a triple bondfor orthogonal couplings, and the p-carboxyl group that can beeasily reduced to benzyl alcohol. Thus, gallic acid 1 wasesterified to the corresponding ethyl ester 2, which cyclisedwith triethyl orthoformate to give the orthoester 3 in high yield(Scheme 2). The remaining free –OH reacted with propargylbromide 4 (K2CO3/KI in dry acetone for 2 h) and the propargylether was reduced with LiAlH4 in THF to give the 5-(hydroxy-methyl)pyrogallol orthoester derivative 5 (HMPO) in good over-all yield starting from 1 (Scheme 2).Compounds 8 and 11 (Scheme 2) were prepared to evaluatethe hydrolysis profile and release of the molecule linked to thebenzyloxy group. Activation of 5 with p-nitrophenyl-chloroformate and further reaction with tryptamine 7 in alka-line medium gave carbamate 8 in acceptable yields. Afterconversion of alcohol 5 to benzyl chloride 9 (see ESI†), nucleo-philic displacement with 10 gave phenyl ether 11. The beha-viour of compounds 8 and 11 at different pH values wasinvestigated by HPLC analysis. Compound 8 released the pay-load almost completely after 7 hours at pH 5.5 (95% release,t(1/2) = 3 h, Fig. S1, ESI†). A comparable kinetics was observed atthe same pH with 11 (t1/2 D 3 h and the 95% of release reachedafter 6 h, Fig. S2, ESI†). When 8 and 11 were treated at 37 1C atpH 7.4 and 6.5, no significant hydrolysis occurred within 6 h.Only after 24 h at pH 6.5 the HPLC profile of the samplesshowed the presence of 10% of tryptamine 7 and 25% of phenol10 respectively (Fig. S1 and S2, ESI†). These results agreed withthose reported for existing orthoester linkers, confirming thatHMPO is sensitive to lysosomal pH, showing an excellentstability profile at physiological and extracellular solid tumorpH values.To get a plausible picture of the disassembling mechanismfor HMPO conjugates, we studied the behavior of 11 at pH 5.5through 1H NMR analysis. The sample was incubated indeuterated acetic buffer at 37 1C and the FID recorded every30 minutes. After 5 minutes, the shifts of the aromatic signalsfrom d 7.87 to 7.92 and of the methoxy singlet from d 4.42 to4.49 suggested the release of phenol 10 in solution (Fig. S3,ESI†). Moreover, we noticed a new peak at d 8.69 assigned to thetransient assembling of a formate derivative. Finally, the char-acteristic aromatic and aliphatic signals of a 5-(hydroxymethyl)-3-(2-propynyloxy)pyrogallol (12, Scheme 3) were observed, con-firming the complete disassembling of the structure and therelease of the phenol. An ESI/MS analysis of the solutioncontained in the NMR tube confirmed the presence of com-pounds 10 and 12.Scheme 2 Synthesis and linkage of HMPO platform. Scheme 3 Proposed mechanism for disassembling of HMPO cargo.Communication ChemCommOpen Access Article. Published on 31 August 2022. Downloaded on 11/4/2022 3:38:32 PM.  This article is licensed under a Creative Commons Attribution 3.0 Unported Licence.View Article Online10534 |  Chem. Commun., 2022, 58, 10532–10535 This journal is © The Royal Society of Chemistry 2022A reasonable mechanism for cargo release from the HMPOplatform was then supposed (Scheme 3). In acid conditions, theorthoester moiety is protonated releasing EtOH. The transient(stabilized) carbocation 11b gives the pyrogallol formate 11f.The expected 1–6 elimination occurs with formation ofp-quinone methide 11g and release of the cargo 10.The alternative cleavage of the protonated 2-hydroxy-benzodioxole 11d might give formate 11h that, after esterhydrolysis, releases the cargo 10 through 1–6 elimination.We applied the HMPO platform to the conjugation of theantitumor drugs Doxo (13) and Combretastatin A4 (14) with Cetux-imab (Ctx), a monoclonal antibody specific for the epidermal growthfactor receptor (EGFR). Activation of 5 with p-nitrophenyl chloro-formate and further nucleophilic displacement with Doxo gavecarbamate 15 in good yield (73%) (Scheme 4). Compound 15 wassubjected to CuCAAC reaction with 6-azidohexanoic acid 16 in thepresence of Cu(II) acetate and sodium ascorbate in DMF/water,providing compound 17 in 64% yield (Scheme 4). Alternatively,chloride 9 reacted with Combretastatin A4 (14), deprotonated byNaH in DMF, to give compound 18 in a good yield (98%). After-wards, azide 16 gave 19 in 75% yield (Scheme 4).Molecules 15 and 18 resulted stable in buffers at pH 7.4 and6.5 (Table S1 and Fig. S4, S5, ESI†). Only a small amount of freeDoxo (4%) and Combretastaine A4 (5%) was detected after 24 hat 37 1C at pH 7.4.The stability in water was investigated to exclude falsepositive release, confirming that 15 and 18 remain untouchedafter 24 h at 37 1C in water. A test was also done in humanplasma showing a good stability of these compounds (Table 1).The payload 15 showed a t1/2 value in line with the generalbehavior of pure Doxo in plasma.20 These results confirmedthat pH-sensitive HMPO platform is stable to biological fluidsas blood. On the other hand, hydrolysis at pH 5.5 showed arapid release of drugs 13 and 14 (Fig. S4 and S5, ESI†). With 15,in the first hours a plateau was reached and almost 90% of drugwas released after 6 h. Although with a different behaviour,93% of Combretastatin A4 (14) was released from 18 after 12 h(see ESI†).For the preparation of the ADCs, carboxylic acids 17 and 19 weretransformed into the corresponding NHS-esters in PBS at pH 7.4 inpresence of Sulfo-NHS (20) and EDC, and further reacted with Ctx(Scheme 5) to obtain bioconjugates 21 and 22. After purification bydialysis, the drug antibody ratio (DAR) was determined by MALDIanalysis (Table S1, ESI†). A HPLC/MS analysis of the dialyzedsolution showed the absence of the free drugs 13 and 14 in solution.The anti-proliferative activity (MTT assay) of the conjugate 21 and 22was evaluated in A549 (human lung carcinoma, Fig. 1) and A431(epidermoid carcinoma cell line, Fig. S7, ESI†), both overexpressingthe epidermal growth factor receptor (EGFR). Treatment with thetwo conjugates showed a stronger antiproliferative effect if com-pared with unconjugated Ctx (Fig. 1). The effect was also compar-able to the activity of the free drugs confirming the release of Doxoand Combretastatin A4 from the linker in the cell culture. Theobserved differences between the release kinetics of 15 and 18 andthe cell viability assays of the corresponding conjugates 21 and 22might be due to the different mechanism of action of the two drugs(intercalation into DNA and inhibition of topoisomerase II for Doxoversus depolymerisation of tubulin for Combretastatin A4), whichprobably requires different times of action.HMPO alone wasn’t toxic, on the other hand, on cellsemployed in the tests. DAPI staining, Annexin V-FITC and PIstaining tests were also employed to investigate the pathway ofcell death. The results showed that 21 and 22 significantlyinduce apoptosis and cell death (Fig. S7 and S8, ESI†).In conclusion, we demonstrated that the new HMPO plat-form (5) can be employed for drug delivery in acid biologicScheme 4 Conjugation of Doxo and Combretastatin A4 with HMPO.Table 1 Stability tests of payloads 15 and 18Comp. H2Oa pH 7.4,a t1/2b pH 6.5,a t1/2b Plasma,a t1/2b15 99% 94%, 424 h 90%, 424 h 35%, 8.3 h18 99% 95%, 424 h 93%, 424 h 89%, 27.6 ha Value expressed as percentage of unmodified compound after 24 h ofincubation. b Half-life (t1/2) expressed as the amount of time it takesbefore half of the drug is hydrolyzed/degraded.Scheme 5 ADC preparation.ChemComm CommunicationOpen Access Article. Published on 31 August 2022. Downloaded on 11/4/2022 3:38:32 PM.  This article is licensed under a Creative Commons Attribution 3.0 Unported Licence.View Article OnlineThis journal is © The Royal Society of Chemistry 2022 Chem. Commun., 2022, 58, 10532–10535 |  10535environments resulting suitable for conjugation of the cytotoxicdrugs Doxo (a primary amine) and Combretastatin A4 (aphenol) with an antibody carrier. The resulting ADCs exhibitedimproved cytotoxic activity in cancer cells compared to theunconjugated antibody and to the cargo-linker fragment.Various primary and secondary amine-, alcohol- and phenol-containing molecules can be bound by the formation of carba-mates, carbonates or ethers. Orthogonal reactivity to thesebonds is possible by introducing azides, alkynes, tetrazines orother fragments for click chemistry to allow easy conjugationwith macromolecular supports. HMPO has the same stabilityand other typical properties as other pH-reactive linkers, but isnon-toxic and easily accessible synthetically (4 steps, 76%overall yield) from gallic acid, a cheap natural starting material.Ultimately, the low toxicity suggests a potential use of our linkerin non-oncology therapies. Application of the linker to non-canonical cytotoxic drugs for intra- and extracellular delivery isin preparation and will be reported shortly.This work was supported by the AIRC (under IG 2017 – ID.20758 project – P. I. Taddei Maurizio).Conflicts of interestThere are no conflicts to declare.Notes and references1 X. Yang, Z. Pan, M. R. Choudhury, Z. Yuan, A. Anifowose, B. Yu,W. Wang and B. Wang, Med. Res. Rev., 2020, 40, 2682–2713.2 (a) K. C. Nicolaou and S. Rigol, Angew. Chem., Int. Ed., 2019, 58,11206–11241; (b) K. Tsuchikama and Z. An, Protein Cell, 2018, 9, 33–46;(c) V. Chudasama, A. Maruani and S. Caddick, Nat. Chem., 2016, 8, 114–119.3 (a) L. Battistini, K. Bugatti, A. Sartori, C. Curti and F. Zanardi, Eur.J. Org. Chem., 2021, 2506–2528; (b) B. Alday-Parejo, R. Stupp andC. Rüegg, Cancers, 2019, 11, 978; (c) P. Zhong, X. Gu, R. Cheng,C. Deng, F. Meng and Z. Zhong, Int. J. Nanomed., 2017, 12,7913–7921; (d) D. Arosio and C. Casagrande, Adv. 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Cancer cell survival was evaluated by MTT test. Cells were exposed to increasing concentration of: (a) conjugate 21, Doxorubicin andCetuximab; (b) conjugate 22, Combretastatin A4 and Cetuximab. Data are expressed as percent of cell viability. #,*, 1 p o 0.05 vs. basal; ##,**, 11 p o 0.01vs. basal and ###,***, 111 p o 0.001 vs. basal.Communication ChemCommOpen Access Article. Published on 31 August 2022. Downloaded on 11/4/2022 3:38:32 PM.  This article is licensed under a Creative Commons Attribution 3.0 Unported Licence.View Article Online

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A pH-responsive crosslinker platform for antibody-drug conjugate (ADC) targeting delivery

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Archivio della Ricerca - Università degli Studi di Siena