Pergamon Bioorganic & Medicinal Chemistry Letters, Vol. 5, No. 5, pp. 507-512, t995 Copyright © 1995 Elsevier Science Ltd Printed in Great Britain. All rights reserved 0960-894X/95 $9.50+0.00 0960-894X(95)00062-3 NOVEL TRITYL LINKED DRUG IMMUNOCONJUGATES FOR CANCER THERAPY Vinod F. Patel*, Julie N. Hardin, James J. Starling and John M. Mastro Lilly Research Laboratories, Eli Lilly & Company, Indianapolis, Indiana 46285 Abstract: Trityl linkers were utilized in the preparation of acid labile nucleoside monoclonal antibody conjugates, COL 1-N6-Trityl-207702 (1 a-f). Conjugation led to high levels of drug incorporation into the MoAb with retention of good immunoreactivity. A strong correlation was found between the cytotoxicity of the constructs and substituents R and R' on the aromatic rings of the trityl linker. A major objective of cancer chemotherapy is to destroy malignant cells, while minimizing damage to normal cells. Although various antitumor agents have been found effective against certain tumors, there is still a great need for oncolytics which kill cancer cells more efficiently and selectively. For instance, doxorubicin is widely administered for the treatment of haematological malignancies and solid tumors, 1 however, its use is dose limited due to its cardiac toxicity and myelosuppression. 2 With the discovery of lymphocyte hybridoma technology by Kohler and Milstein 3 in the mid 1970's, which allowed the production of unlimited quantities of monoclonal antibodies, researchers gained a new tool to devise novel methods of delivering cytotoxic agents to target sites. For example, the administration of a drug linked to a monoclonal antibody (MoAb), that reacts with cell surface, tumor-associated antigens, offers an attractive approach to "selective" chemotherapy. 4 Drug immunoconjugates are composed of three distinct entities: (i) the monoclonal antibody for targeting, (ii) the cytotoxic drug and (iii) the linker which attaches the drug to the antibody. To maximize chemotherapeutic value, the drug conjugates must retain good immunoreactivity, possess potent antitumor activity and display minimal systemic toxicity. Early efforts in the field focused on the optimization of the monoclonal antibody and drug entities in search of an effective construct. 5 More recent studies have demonstrated that the linker, by facilitating the timely release of drug at the target site, 6 plays an equally critical role in the overall biological function of drug conjugates. R I N N 1-12N "N" -I~1 NO F (1) Herein, we report the use of acid-labile trityl groups as versatile linkers in monoclonal antibody drug conjugates. It is known that the pH of human tumors averages 0.8 units lower than that of the surrounding normal tissues, mainly due to the anaerobic glycolysis of carbohydrates by malignant tumor cells. 7 Thus, the trityl moiety seemed a particularly attractive group for linking drugs to monoclonal antibodies since the acidic 507
`
`IMMUNOGEN 2326, pg. 1
`Phigenic v. Immunogen
`IPR2014-00676
`
`

`

`508 V.F. PATEL et al. conditions in the tumor would facilitate site specific release of the drug. The ability to introduce substituents on the aromatic rings of the trityl group offered an ideal opportunity to electronically tune the dissociation of drug to a rate which would complement the targeting properties of the antibody. Furthermore, the incorporation of an activated ester on the linker would allow standard attachment to the protein, via formation of a stable amide bond to the epsilon amino group of lysine residues on the antibody. 8 To demonstrate these features of trityl linkers, MoAb (N6-Yrityl-207702)z conjugates (1) were synthesised in which the drug is represented by LY207702 (2), 9 a potent nucleoside antitumor antimetabolite and the MoAb (13) by a non-internalizing, murine monoclonal antibody, COL 1.10 R NH2 ( ~,mor_R,__ 0 Nil HO F HO-~ R' HO F (2) (3) (4) (i) n_Bu4NC104, 2,4,6-collidine, DMF:CH2CI 2, r.t. Table 1: Preparation of Tritylated LY207702 derivatives (a) R-DMT (b) ~MMeT (c) p_-MMT (d) I~-MeT (e) p_-T (f) m-DMT R R' Yield (%) (3) (4) OMe OMe OMe Me OMe H Me H H H 49 93 30 84 36 74 74 56 39 54 27 77 OMe OMe Trityl chlorides (3a-f) were synthesized according to the procedure previously described by Glidea. 11 The methodology was extended to prepare a range of trityl derivatives (DiMethoxyTrityl, MethoxyMethylTrityl, MonoMethoxyTrityl, MethylTrityl, Trityl) in which the substituents on the aromatic ring were systematically varied in a manner to allow study of the electronic effects of acid-mediated dissociation of the drug. In the case of trityl chlorides (3b,c,d), where RCR', the reagent was used as a racemic mixture. Accordingly, LY207702 (2) was alkylated 12 with 1.1 equivalents of trityl perchlorate, generated in situ from the chloride (3) and _nBuC10 4, to provide a 54-93% yield of the desired mono-tritylated product (4) 13 as stable solids (Table 1). As expected, alkylated products (4b,c,d) were obtained as mixtures of unseparable diastereoisomers. The presence of the N-hydroxysuccinimide ester group in the product was evident from inspection of IH NMR spectrum which showed a singlet resonance at d 2.87 ppm. Furthermore, treament of active ester (4) with isopropylamine led to the corresponding isopropylamide with the concomitant formation of N-hydroxysuccinimide. 14 The determination of N6 regioselective alkylation was based on the knowledge that
`
`IMMUNOGEN 2326, pg. 2
`Phigenic v. Immunogen
`IPR2014-00676
`
`

`

`Novel trityl linked drug immunoconjugates 509 Scheme 1 NHR' R,HN .'J~N,.~'~ N / ~o~ RO F R=Bz, R'=Piv ~ R=H, R'=Piv (5) (v, I -~ R=R'=H ~ (ii) OMe (2) (6) ~.o_~~o NH H2N .J'~N,,,u~. N ~ HO (iii)~ X=ONHS (4a) F X=NHiPr (9) OMe NHR' MeO O: X 0 (ii) x= 0-/~ (iii) (7) NHiPr (8) (iv) OMe NH2 "eO-Q---O~to d O' NHiPr (1o) (i) KOtBu, THF, r.t. (ii) TrCI(3), see Table 1 (iii) iPrNH2, CH2CI 2, r.t. I (iv) NaOMe (3eq), MeOH, reflux (v) NaOMe (6eq), MeOH, reflux I
`
`IMMUNOGEN 2326, pg. 3
`Phigenic v. Immunogen
`IPR2014-00676
`
`

`

`510 V.F. PATEL et al. the N2 amino group in purine base was far less nucleophilic than the N6 amino group and that tritylation of the secondary 2'OH was significantly slower than the primary 5'OH of the ribose sugar.15 However, in order to distinguish between N6 and 5'OH regioisomers, amides (9) and (10) were prepared. Thus, protected nucleoside (5) 16 was debenzoylated with KOtBu to give diol (6) which was then selectively tritylated at the 5'OH with trityl chloride (3a) to provide derivative (7) as the sole product. Subsequent conversion of (7) to the corresponding isopropylamide, (8), followed by deprotection of N2 and N6 pivalolyl groups using NaOMe led to amide (10) (Scheme 1). Comparison of amides (9), obtained by reacting ester (4a) with iPrNH2, and (10) by tic and 1H NMR 17 clearly indicated that the nucleosides were different, leading to the conclusion that amide (9) and therefore active ester (4a) resulted from N6 tritylation of purine nucleoside (2). Thus, regioselective N6 mono-tritylation under these alkylating conditions proved to be a particularly useful reaction which circumvents the need for prior protection of nucleoside (2). (13) R R o O o~R']y ~l_ys- NH R' ! H2N N N HO F HO F (4) (1) where y = Molar Input Ratio (MIR) where z = Conjugation Ratio (CR) MoAb (N6-Trityl-207702)z (i) 0.1M Borate buffer pH -8.6, MIR = 8, 7.5% DMF, r.t., lh The final step in the synthesis of drug conjugates (la-f) was accomplished by reacting active ester (4a-t), at a molar input ratio (MIR) of 8, with anti-CEA antibody COL1 (13), in a pH 8.60 buffered solution for lh at room temperature, followed by isolation of the product using a G-25 Sephadex desalting column.18 The conjugation ratio (CR), antibody and drug concentrations and the protein yield of the sterile- filtered drug conjugates (la-f) were determined by UV spectroscopy. The conjugation led to constructs (la-f) with high percentage of drug (2) incorporation (56-94%) and yielded good protein recovery (Table 2). Furthermore, characterization on a size exclusion Superose 12 column indicated the conjugates (1 a-f) consisted of 92-96% of the desired monomeric form, with the remainder being 3-6% low molecular weight (M.W.-3X105) and 1-2% high M.W.(>IX106) aggregates. 19 Furthermore, no free drug was detected in the conjugate preparations. Evaluation of the drug immunoconjugates (la-f) in direct and competitive binding assays showed 80-90% immunoreactivity with the target CEA antigen compared to unconjugated COL1 (13) indicating that the antigen binding region of the antibody was relatively unaffected by the conjugation procedure. Antitumor activity of drug conjugates (la-f) was assessed in an in vitro cytotoxicity assay and compared to free drug (2) and unconjugated antibody COL1 (13) (Table 2).
`
`IMMUNOGEN 2326, pg. 4
`Phigenic v. Immunogen
`IPR2014-00676
`
`

`

`Novel trityl linked drug immunoconjugates 511 Table 2: Analytical and biological data for COL l-(N6-Trityl-207702)z Conjugates (2) LY207702 CR i ( 1)COL1 -N6-Trityl-207702 (a) m-DMT* 6.44 (b) 12 -DMT 7.44 (c) 12 -MMeT 7.49 (d) 12 -MMT 5.73 (e) 12 -MeT 5.20 (f) 12 -T 4.44 (13) COL1 Protein 2 IC5o 3 Yield (%) (ug/ml) 0.260 52 0.352 65 0.270 44 2.71 61 4.94 47 6.04 5O 10 >330 (1) CR - Conjugation Ratio (= moles of drug/mole of antibody for MIR- Molar Input Ratio=8) was determined by U.V. spectroscopy at drug ~,max=254nm (*Xmax=263nm) (2) Determined by U.V. spectroscopy at Lmax=279nm and where A280=1.40 at 1.0mg/ml of protein (3) Cytotoxicity assay was performed by incubating LS174T (CEA +ve) Human Colon Carcinoma cells with drug for 48h and measuring 3H-Leucine uptake. IC50 is defined as the concentration of drug required to inhibit the incorporation of 3H-Leucine to 50% of control uptake. The above preliminary results show that the relative acid lability of the linkers, 14 which is dictated via the electronic stablisation of the intermediate trityl cation by substituents R and R' on the aromatic rings of the trityl group, correlates well with the potency of the conjugates (i.e. laD MT= rnDMT>12MMeT>pMMT>12MeT>>I2T). The controllable and predictable releasing features of trityl linkers should, therefore, allow one to couple the selective tumor targeting characteristics of a monoclonal antibody with the cytotoxic activity of an oncolytic in a synergistic manner to provide a more selective antitumor agent with an improved therapeutic index. Extensive in vivo studies are underway to identify triyl linked drug conjugates which exhibit both tumor selectivity and antitumor activity, the results of which will be reported in due course. Acknowledgment: We wish to thank C.D. Jones and T.S. Chou for use of unpublished procedures. References and Notes: 1. (a) Weinstein, H.J.; Mayer, R.J.; Rosenthal, D.S.N. Engl. J. Med.,1980, 303, 473-478 (b) McCredie, K.B.; Bodey, G.P.; Freireich, E.J.; Hester, J.P.; Rodriguez, V.; Keating, M.J.Cancer, 1981,47, 1256-1261 (c) Smalley, R.V.; Bartalucci, A.A. Eur. J. Cancer (suppl 1), 1980, 145-146 (d) Bruckner, H.W.; Cohen, C.J.; Goldberg, J.D.; Kabakon, B.; Wallach, R.C.; Deppe, G.; Greenspan, E.M.; Grusberg, S.B.; Holland, J.F. Cancer, 1981, 47, 2288-2294 2. Carter, S.K. Adriamycin - a review J.Natl. Cancer Inst., 1975, 55, 1265-1274 3. (a) Kohler, G.; Howe, S.C.; Milstein C. Eur. J. Immunol, 1976, 6, 292-5 (b) Kohler, G. Nobel lecture, Biosci. Rep.,1985,5(7), 533 (c) Kohler, G. Science,1986, 233 (4770), 1281 4. (a) Koppel G.Bioconjugate Chemistry,1990, 1, 13-23 (b) Barton R.L.; Briggs S.L.; Koppel G.,DN&P ,1991, March, 73-88(c) Baldwin R.W. Monoclonal Antibody Targeting of Anticancer Agents: Muhlbock Memorial Lecture Eur. J. Cancer Clin. 0ncol.,1985, 21(11), 1281-1285 (d)Frankel A.F.; Houston L.L.; Issell B.; Fathman G. Annu. Rev. Med. ,1986,37, 125-142 (e) Reisfeld R.A.; Cherish D.A. Cancer Surveys, 1985, 4(1), 271-290 (f)GhoseT.I.; Blair A.H.; Vaughan K.;Kulkarni P. Targeted Drugs; Goldberg E.P.,Ed. ; John Wiley and Sons: New York,1983, ppl-22 5. For example: (a) Rowland, G.F.; Axton, C.A.; Baldwin, R.W.; Brown, J.P.; Corvalan, J.R.F.;
`
`IMMUNOGEN 2326, pg. 5
`Phigenic v. Immunogen
`IPR2014-00676
`
`

`

`512 V.F. PATEL et al. Embleton, M.J.; Gore, V.A.; Hellstrom, I.; Hellstrom, K.E.; Jacobs, E.; Marsden, C.H.; Pimm, M.V.; Simmonds. R.G.; Smith, W. Cancer Immunol. lmmunother., 1985,19,1 6. (a) Kaneko, T.; Willner, D.; Monkovic, I.; Knipe; J.O., Braslawsky; G.R.; Greenfield, R.S.; Vyas, D.M. Bioconjugate Chemistry, 1991, 2(3), 133-141 (b)Mueller B.M., Wrasidlo W.A., Reisfeld Bioconjugate Chemistry, 1990, 1,325-330 7. Lavie, E.; Hirschberg, D.L.; Screiber, G.; Thor, G.; Hill, L.; Hellstrom, I.; Hellstrom, K-E. Cancer lmmunol. Immunother., 1991, 33, 223-230 8. Blair A.H.; Ghose T.I.J.lmmunological Methods ,1983,59, 129-143 9. LY207702 - 2,6-Diamino-9-(2'-deoxy-2'2'-difluoro-b-D-ribofuranosyl)purine: (a) Andis S.L., Bewley J.R., Boder G.B., et al. Medicinal Chemistry of Difluoropurines, Seminars in Oncology, in press. (b) Grindey G.B.; Hertel L.W.; Grossman C.S. Eur. Patent Application 0576227A2 10. COL- 1 represents a member of a panel of monoclonal antibodies acquired from an NCI-Lilly CRADA; COLI: Muraro R.; Wunderlich D.; Thor A.; Lundy J.; Noguchi P.;Cunningham R.;Schlom J. Cancer Res., 1985, 45, 5769-5780 11. Glidea B.D.; Coull J.M.; Koster H., Tetrahedron Lett., 1990, 31 (49), 7095-7098 12. General experimental procedure for preparation of (4) LY207702 (0.331mM) was dissolved in a solvent mixture of dry DMF: CH2C12 (l:l; 6ml) at room temperature and then tetra-n-butylammonium perchlorate (TBAPC) (0.397mM) and dry collidine (0.497mM) were added.The solution was stirred for 5mins and then trityl chloride derivative (0.331 raM) was added portionwise over 3rains.The resulting solution was stirred at room temperature, under dry nitrogen,until all the starting material was consumed (judged by tlc).The crude reaction solution was diluted with ethyl acetate (1 vol ) and directly applied onto a silica column and purified by chromatography (gradient elution EtOAc-iPrOH/EtOAc) to give the active ester. Representative physical data: (4a) 1H (d6-DMSO) 8 7.95(d,J10,2ArH), 7.87, (s,H8), 7.62 (d,J8,2ArH), 7.27 (d,J8,4ArH), 6.91 (s,NH), 6.80 (dd,J3 and 10,4ArH), 6.72 (brs,NH2), 6.25 (d,J6,3'OH), 5.65-5.44 (m,Hl'), 5.19 (t,J6,5'OH), 4.44-4.26 (m,H3'), 3.83-3.63 (m,3H,H4'5'5'), 3.70 (s,6H,2OMe), 2.85 (s,4H,2CH2) ppm; Calcd. for C36H33N709F2 requires MM_746; Found FDMS m/z 746 13. All compounds were fully characterized by spectroscopic methods. 14. Patel V.F., Hardin J. N., Grindey G.B. and Schultz R.M., Bioorg. Med. Chem Lett., submitted. 15. Greene T.W. Protective Groups in Organic Synthesis; J.Wiley and Sons, 1981, p34 16. Protected nucleoside (5) was synthesised according to the procedure developed by C.D. Jones and T.S. Chou at Lilly Research Laboratories-unpublished work. 17. Data: (10) 1H (CDCI3/d4-MeOD) 8 7.83 (s,H8), 7.62 (d,J8,2ArH), 7.40 (d,J8,2ArH), 7.19 (d,J8,4ArH), 6.74 (d,J8,4ArH), 5.53-5.45 (m,Hl'), 4.65-4.55 (m,2H,CH,H3'), 4.35-4.15 (m,3H,H4'5'5'), 3.74 (s, 6H, 2OMe), 1.19 (d,J7,6H,2Me) ppm; Calcd. for C35H37N706F2 requires M689;Found FDMS m/z 689 (11) 1H (d6-Acetone) ~57.80 (s,H8), 7.78 (d,J8,2ArH), 7.55 (d,J6,2ArH), 7.40 (brs,NH), 7.36 (dd,J2and8,4ArH), 6.90 (dd,J4and 12,4ArH), 6.26-6.14 (m,3H,NH,H 1'), 5.47-5.32 (m,3H,NH2,OH), 4.88-4.74 (m,H3'), 4.30-4.12 (m,2H,CH,H4'), 3.80 (s,6H,2OMe), 3.60-3.44 (m,H5'5'), 1.22 (d,J7,6H,2Me) ppm; Calcd. for C35H37N706F2 requires M689; Found FDMS m/z 689 18. Typical experimental procedure for conjugation: Into a round-bottom flask equipped with a magnetic stirrer was added a solution of monoclonal antibody ( 1 mole equivalent) in 0.1M borate buffer pH=8.60.To this was added, with stirring, a solution of N6-Trityl-207702 active ester (8 mole equivalents i.e. MIR=8) in distilled dimethyl- formamide (7.5% of final reaction volume).The milky solution was gently stirred at room temperature for lh, transferred to a centrifuge tube and centrifuged at 2000xG for 10 mins. The supernatant was fractionated using a Sephadex G-25 column by eluting with pH7.4 phosphate buffered saline (PBS). The fractions containing protein were pooled, concentrated (Amicon stirred- cell apparatus) and, sterile-filtered (0.2mm filter). The final conjugate solution was analyzed by Superose-12 column (eluted with 15% acetonitrile/PBS) for aggregrate content and by U.V. spectroscopy for determination of protein and drug concentrations. 19. U.V. analysis of pure monomeric, low M.W. and high M.W. forms of the conjugates showed drug had been incorporated in each of these species. (Received in USA 9 November 1994; accepted 25 January 1995)
`
`IMMUNOGEN 2326, pg. 6
`Phigenic v. Immunogen
`IPR2014-00676
`
`