Novel Crizotinib–GnRH Conjugates Revealed the Significance of Lysosomal Trapping in GnRH-Based Drug Delivery Systems

Several promising anti-cancer drug–GnRH (gonadotropin-releasing hormone) conjugates have been developed in the last two decades, although none of them have been approved for clinical use yet. Crizotinib is an effective multi-target kinase inhibitor, approved against anaplastic lymphoma kinase (ALK)- or ROS proto-oncogene 1 (ROS-1)-positive non-small cell lung carcinoma (NSCLC); however, its application is accompanied by serious side effects. In order to deliver crizotinib selectively into the tumor cells, we synthesized novel crizotinib analogues and conjugated them to a [d-Lys6]–GnRH-I targeting peptide. Our most prominent crizotinib–GnRH conjugates, the amide-bond-containing [d-Lys6(crizotinib*)]–GnRH-I and the ester-bond-containing [d-Lys6(MJ55*)]–GnRH-I, were able to bind to GnRH-receptor (GnRHR) and exert a potent c-Met kinase inhibitory effect. The efficacy of compounds was tested on the MET-amplified and GnRHR-expressing EBC-1 NSCLC cells. In vitro pharmacological profiling led to the conclusion that that crizotinib–GnRH conjugates are transported directly into lysosomes, where the membrane permeability of crizotinib is diminished. As a consequence of GnRHR-mediated endocytosis, GnRH-conjugated crizotinib bypasses its molecular targets—the ATP-binding site of RTKs— and is sequestered in the lysosomes. These results explained the lower efficacy of crizotinib–GnRH conjugates in EBC-1 cells, and led to the conclusion that drug escape from the lysosomes is a major challenge in the development of clinically relevant anti-cancer drug–GnRH conjugates.


Compound 6. 2-[4-(4-iodo-1H-pyrazol-1-yl)piperidin-1-yl]ethanol
To the solution of compound 2 (370 mg, 1.335 mmol) in dry THF (10 mL) K2CO3 (921 mg, 6.676 mmol) was added and the mixture was stirred for 30 minutes at reflux temperature under nitrogen atmosphere. Bromoethanol (469 µL, 2.670 mmol) was added, and the reaction mixture was stirred for 24 h, at reflux temperature, under nitrogen atmosphere. The reaction mixture was cooled to ambient temperature, filtered, and the precipitate was washed with THF. The filtrate was concentrated under vacuum. The crude product was dissolved in CH2Cl2 (10 mL), then cooled to 0°C and 4 M HCl-dioxane (1 mL) was added. The white suspension was filtered off, and the crude product was dissolved in H2O (10 mL) and washed with diethyl ether (5 mL).
The pH of the aqueous layer was adjusted to >10 by adding 10% (m/V) NaOH solution, and then the aqueous portion was extracted with CH2Cl2 (2*5 mL). The organic layer was dried over Na2SO4
The Alloc protecting group was removed in DCM by using tetrakis(triphenylphosphine)palladium(0) and phenylsilane under nitrogen atmosphere. After deprotection, sodium N,N-diethyldithiocarbamate in DMF was used for the removal of palladium.
Compound 5 (57 mg, 0.075 mmol) was dissolved in DMF then COMU (35 mg, 0.082 mmol) and NMM (16.5 µL, 0,150 mmol) were added. The mixture was added to the resin (150 mg) harbouring the selectively unprotected [D-Lys 6 ]-GnRH-I. The conjugation was carried out for 2 h, at 25°C. The conjugate was cleaved from the resins with TFA/phenol/water/TIPS (88:5:5:2 ratio) mixture in 2 h, at 25°C. The filtrate was precipitated in cold diethyl ether, the white precipitate was filtered and dried under vacuum to give the crude peptide conjugate. Crude conjugate was dissolved in 1:4 mixture of acetonitrile/water containing 0.1% TFA, and purified by semi-preparative HPLC using Phenomenex Gemini C18 column (

S2.1 HRMS
High resolution mass spectra were acquired on a Q Exactive TM Focus, high resolution and high mass accuracy, hybrid quadrupole-orbitrap mass spectrometer (Thermo Fisher Scientific, Bremen, Germany) in the 200-2000 m/z range. MS data were analysed by Xcalibur TM software (Thermo Fisher Scientific). Samples were dissolved in 500 µ L eluent (0.1% HCOOH in acetonitrile/water 50:50, v/v).

S2.3 Structure determination of the synthetic intermediates, crizotinib and its derivatives by 1 Hand 13 C-NMR measurements
The spectra were recorded in DMSO-d6 solution in 5 mm tubes at RT, on a Bruker DRX-500 spectrometer at 500 ( 1 H) and 125 ( 13 C) MHz, with the deuterium signal of the solvent as the lock and TMS as internal standard ( 1 H, 13 C). The 2D-HSQC and 2D-HMBC spectra were obtained by using the standard Bruker pulse programs. Figure S4. The appropriate numbering modes of atoms presented on the structures of 5, 7 and 9 are used for the assignment of 1 H-and 13 C-NMR data of all the compounds characterised in the Experimental section. For compound 2 this numbering is retained starting with atom number 14 on the pyrazole ring.
The skeletal structure and the substitution pattern of the characterised compounds were unambiguously confirmed by combined use of 1 H-and 13 C-NMR methods including 2D correlation measurements such as 1 H-13 C-HSQC, 1 H-13 C-HMBC, only the following remarks are necessary to make. The presence of the N-substituted piperidine ring in compounds 5-9 was evidenced by the HMBC cross peaks generated by three-bond correlations between signal pairs H-19/C-20 and H-19'/C-20 (for 5) and H-19/C-20 and H-20/C-19 (for 6-9). Giving further support for the N-acylation of the piperidine unit in 5, the separated H-19 and H-19' signals discernible in its 1 H NMR spectrum are significantly downfield shifted (e.g. δH-19equatorial/=4.49 ppm/ δH-19'equatorial=3.94 ppm) relative to the H-19 shifts measured for 3, 4 and 6-8 (δH-19equatorial=2.8-2.9 ppm). However, in ester 9 the downfield-shifted H-19equatorial H-19axial signals (at 3.66 ppm and 3.21 ppm, resp.) point to the fast rotation of the extended chain on the piperidine ring exerting an anisotropic deshielding effect on the aforementioned protons, which -in time average -may get in the proximity of the ester group in the backfolded oxopropanoic acid terminal. Finally, for all compounds characterised in this contribution the characteristic triplet of triplets split of the sharp H-17 signal with one large coupling constant (~11 Hz) unambiguously refers to its axial position and, consequently to equatorial position of the pyrazole fragment on the rigid piperidine ring adopting well-defined chair conformation.

S3.
Confocal laser scanning microscopy Figure S5. The GnRHR expression of human fibroblast cells is negligable, and the receptor was not detectable on the plasma membrane. permeability (cm/s) R = mass retention (percentage loss of compound due to non-specific binding to the plastic surfaces during the permeation assay) A = membrane area (cm 2 ) c0 = initial donor concentration ca = final acceptor concentration cd = final donor concentration ce = equilibrium concentration t = incubation time (sec) Va = acceptor volume (mL) Vd = donor volume (mL)