Improving the Stability of Maleimide–Thiol Conjugation for Drug Targeting

Abstract Maleimides are essential compounds for drug conjugation reactions via thiols to antibodies, peptides and other targeting units. However, one main drawback is the occurrence of thiol exchange reactions with, for example, glutathione resulting in loss of the targeting ability. A new strategy to overcome such retro‐Michael exchange processes of maleimide–thiol conjugates by stabilization of the thiosuccinimide via a transcyclization reaction is presented. This reaction enables the straightforward synthesis of stable maleimide–thiol adducts essential in drug‐conjugation applications.

In the past 50 years, the use of maleimide compounds as Michael acceptors has become ac ommon way for conjugation to thiol-bearing molecules. [1] The applications vary from peptideand antibody-drug conjugates, fluorescent-labeling of biomolecules as well as PEGylation of peptides and proteins. For example,i n2 011b rentuximab vedotin was approved by the FDA for the treatment of Hodgkin lymphoma where the highly cytotoxic antimitotic agentm onomethyl auristation Ei sc onjugated via am aleimide moiety to ac ysteine of the CD30-specific antibody. [2] Also, in the case of trastuzumab emtansine, approvedf or metastatic breast cancer, am aleimide moiety is used. In this case, the maleimide is attached to the antibody and reacts with at hiol group of the cytotoxic drug. [3] In general, the use of maleimides has many advantages, like fast kinetics, quantitative conversion and high specificity.N evertheless, the crucial factorsf or successful drug delivery,n amely stability of the conjugate and controlled release,a re not yet fully provided.T he main weakness is ap ossible thiol exchange (e.g. with glutathione;G SH) of the formed thiosuccinimide, induced by ar etro-Michael reaction. This b-elimination reactionr esults in the loss of targetingp roperties and, therefore, promotes offtargeta ctivity. [4] One of the few possibilities to diminish this problem of maleimides is to exploit the fast hydrolysis of thiosuccinimides when electron-withdrawing moieties are present, resultingi nt he formation of stable thioethers (thiosuccinimides). [5] However,t his strategy is limited to N-aryl substituted maleimides [5a, 6] or other electron-withdrawing N-substituents. [7] The much more commonly used N-alkyl-substituted derivatives show too slow hydrolysis of the formed thiol adductst ogenerate the desireds table thioethers.
In ar ecent publication, we synthesized ad rug-peptide conjugatev ia reactiono famaleimide moiety and an N-terminal cysteinef or coupling. [8] High-performance liquid chromatography/mass spectrometry( HPLC-MS)m easurements revealed that the product peak converted within severalh ours into a new peak with the same exact mass. This reaction was supposed to be aM ichael-transcyclization already known from similars ystems. [9] (Figure 1).
We assume that the formation of the six-membered ring is lockingt he thioether conjugation moiety and the transcyclization reaction is, therefore, an elegant method to prevent retro-Michael reactions. Consequently,t his strategy serves as a promising tool for the synthesis of stable maleimide-thiol drug conjugates.
Herein, we report on the detailed analysis of the postulated transcyclizationr eactionu sing am odel compound system by HPLC-MS measurements and investigated the stability in the presence of GSH. Furthermore, the feasibility of this method was confirmedf or ad rug-peptideconjugate. As af irst step, am odel reactiont os tudy and prove the suggested transcyclization reaction was developed ( Figure 2). Therefore, l-cysteine methyl ester hydrochloride (1a)w as reacted with N-ethylmaleimide (2)i np hosphate buffer (PB) at pH 7.4 ( Figure 3A). As anticipated, the instantly formed maleimide-thiol conjugate 3a (t = 0h,r t = 4.3 and 5.3min) underwent ac onversion reaction, leadingt oamore hydrophobic compound with the same mass (m/z = 261) with ar etention time of 9.1 min ( Figure 3A)a ssigned to the transcyclization product 4 (Figure 2A). The fact that the initial Michael adduct 3a appeared as two separate peaks in the extracted ion chromatogram (EIC) can be explained by the formation of diastereomers. In contrast, in case of 4 only one peak was observed ( Figure 3A). When the HPLC conditions were adjusted to af latter gradient,asecond isomer could be observed for 4 as well, however,w ithj ust % 10 %a bundancy ( Figure S1). This imbalance can probablybeexplained by steric hindrance of the substituents of the thiomorpholinone core. The ratio between the two diastereomers of 3a did not change over time revealing no preference for one diastereomer in the transcyclizationp ro-cess. The negative control reaction wasp erformed with Nacetyl l-cysteine methyl ester (1b)a nd N-ethyl maleimide (2) where the transcyclization is supposedlyp revented by the protected amino moiety ( Figure 2B). Incubation of the compounds under the same conditions resulted in the instant formation of only one peak (rt = 10.2 min, m/z = 303) attributed to the thiosuccinimide 3b which was stable for more than 24 h ( Figure 3B;e ven with al ess steep gradient, the second diastereomerc ould not be observed. However, two sets of peaks are present in the NMR spectra;s ee ESI). Consequently,a sd esired, the protection of the amino moiety prevented the transcyclization reaction.
Since the Michael adduct (3a)a nd the transcyclizationp roduct (4)p ossess the same exact mass, we also synthesized the compounds (together with 3b)a nd characterized them in detail via 1 Ha nd 13 CNMR spectroscopy to confirmt heir chemical identity.C ompound 3a was generated from 1a and 2 in MeOH to keep the free amino group protonated and avoid the ring closing reaction. In contrast, 4 was synthesized from the same educts, however in 100 mm PB solutiona tp H7.4. The NMR of 4 clearly proofed the transcyclization reaction:O nt he one hand the absence of an amine NH 2 signal, but the presence of two NH signals (8.07 and 7.89 ppm). On the other hand, ac ross peak in the heteronuclear multiple bond correlation (HMBC) spectrum of the proton located at N2 (8.07 ppm) and C6 (186.07 ppm), in line with the newly formed bond. The presence of diastereomers in case of 3a was indicated by two 13 Cs ignals for each carbon atom. Notably, the retention time of both synthesized compoundsi nt he HPLC perfectlyf itted to the co-incubation experimentsi nF igure 3, confirming the peak assignment. For the purified transcyclization product 4 a X-ray single crystal structure [18a] could be obtained (Figure 4; Ta ble S1-S3). The crystal structure showed one diastereomer [18b] namely the trans-oriented conformationsa tC 5( S)a nd C7 (R). Notably,i nt he literatureo nly the cis-conformer was reported in the case of N-phenyl maleimide and l-cysteine   [9a] However, the absence of a 1 H-1 H-NOESY-cross peak between C5 and C7 in the NMR spectrumo f4 (which is clearly visiblef or the cis-isomer 7 )s uggests the presence of the trans isomer also in solution.
To prove the superiors tabilityo fatranscyclization product under retro-Michael conditions we performed co-incubation experimentsw ith GSH. On the one hand, we compared the stabilityo ft he thiosuccinimide 3b and the transcyclization product 4.O nt he other hand we synthesized two peptidedrug conjugates as "real world" examples.W eu sed the epidermal growth factor receptor (EGFR)-binding peptide (Leu-Ala-Arg-Leu-Leu-Thr;L ARLLT) and an oxaliplatin(IV)-maleimide complex ( Figure 5). For conjugation aC ys-miniPEG linker was attached to the LARLLTp eptide sequence. We synthesized the transcyclization product 5a using 24 hi ncubation of the pep-tide and the maleimide-bearing platinum complex in PB pH 7.4, [8] resulting in~95 %conversion to 5a.For the thiosuccinimider eference complex 5b we protected the terminal Cys via acetylation. All compounds (50 mm)w erei ncubated in an aqueous phosphate buffered solution (100 mm,p H7.4, 25 8C) in the presence of 10-folde xcesso fr educed GSH for 25 ha nd the reaction was monitored via HPLC-MS.T he N-acetylated, open-chain complex 5b underwent distinctt hiol-exchange reaction with GSH ( Figure 5A)w ith formation of the oxaliplatin(IV)-thiosuccinimide-GSH speciesa tm/z = 947.I nc ontrast, the transcyclization bioconjugate 5a did not show significant GSHadduct formation even after 25 h( Figure 5B). The same picture could be observed for the model compounds: 4 was basically not affected by the presence of GSH, whereas in case of 3b the N-ethylthiosuccinimide-GSH adduct was formed (Figure S2). The conversion to the GSH adduct proceeded with 0.5 %h À1 resulting in~15 %after 25 hincubation. Ta ken together,t his data shows that in the presence of a cysteinem oiety maleimide-thiol bonds can be stabilized against ar etro-Michael thiol exchange reaction with only minimal additional effort. The respective transcyclization can be achieved simplyb ya ne xtended incubation time in buffered solution.Int he last years alot of work was put into the discovery of SH conjugation moieties other than maleimides, which also increase the stability against retro-Michael reactions. For example exo-cyclic maleimides, [10] sulfones, [11] carbonylacrylic reagents [12] or 2-formylphenylboronic acids [13] (Figure S3). Fact is, however,t hat most of the thiol-coupling reagents commercially available are still common (alkyl) maleimides:f or example, succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC) is one of the most popularl inkersf or antibody coupling, N-(g-maleimidobutyryloxy)succinimide ester (GMBS) or similard erivativesa re used for peptide coupling and dibenzoazacyclooctyne-maleimide (Mal-DBCO) for click chemistry or crosslinking reagentsw ith two or more maleimide moieties. [14]   Therefore, am ethod which still uses maleimides, but strongly enhances the stability, is of high interest. Different types of application for the new methodc an be distinguished:1 )attachment of an N-terminalc ysteinet oa ny peptide for (drug) conjugation and subsequent reactionw ith the desired maleimide. 2) Peptides which already contain ac ysteine, but where the incubation time probablyw as not sufficient to generate the stabilized transcyclization product [15] or the terminal cysteine amino group was protected. [16] 3) Targeted (drug)c onjugates where currently alkylthiols are used for maleimide coupling [17] and 4) antibody-drug conjugates where the SMCCm aleimide linker is attached to al ysine of the antibody and the drug is modified with acysteinel inker to enable transcyclization.