New pemetrexed-peptide conjugates: synthesis, characterization and in vitro cytostatic effect on non-small cell lung carcinoma (NCI-H358) and human leukemia (HL-60) cells Zsanett Miklán, a,{ Erika Orbán, a,{ Zoltán Bánóczi a and Ferenc Hudecz a,b * Pemetrexed (Pem) is a novel antimetabolite type of anticancer drug that demonstrated promising clinical activity in a wide variety of solid tumors, including non-small cell lung carcinoma and malignant pleural mesothelioma. It inhibits enzymes involved in the folate pathway, for which the presence of its free carboxylic groups is necessary. The heteroaromatic ring system of Pem has a modifiable amino group, which opens a possibility to apply a new strategy to conjugate Pem to carrier molecules. Considering this as well as the necessity of untouched carboxylic groups of Pem in the new conjugates, we developed a new synthesis strategy. Here, we describe the synthesis and the characterization of new Pem-peptide conjugates in which cell-penetrating octaarginine or/and lung-targeting H-Ile-Glu-Leu-Leu-Gln-Ala-Arg-NH 2 peptide is attached to the drug by thioether bond. The conjugates characterized by RP-HPLC and MS exhibited cytostatic effect in vitro on non-small cell lung carcinoma as well as on human leukemia cell lines. The IC 50 values of the conjugates were similar, but the conjugates with H-Ile-Glu-Leu-Leu-Gln-Ala-Arg-NH 2 sequence were slightly more effective. Our data show that the in vitro cytostatic effect of the free Pem was essentially maintained after conjugation with cell-penetrating or cell-targeting peptides. Thus, the conjugation strategy reported could lead to the development of a new generation of active Pem conjugates. Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd. Keywords: synthesis of pemetrexed-oligoarginine conjugates; pemetrexed; IELLQAR lung-targeting peptide; in vitro cytostatic effect of conjugates; non-small cell lung carcinoma; human leukemia Introduction Perturbation of nucleotide metabolism for cancer therapy has primarily focused on the utilization of nucleoside and nucleic acid base analogues, which compete with their physiological counter- parts for incorporation into DNA and RNA, as well as antifolate agents. One of the first antifolate agents was methotrexate, and still it is in clinical use [1]. Much is known about its mode of action and the mechanisms by which tumors exhibit inherent or acquired resistance to this drug [2]. During the preparation of new analogues, a promising agent, pemetrexed (Pem, LY231514 or Alimta, Figure 1.), has been developed by Eli Lilly and Company (Indianapolis, IN, USA). Pem is an antimetabolite inhibiting at least three enzymes (thymidylate synthase, dihydrofolate reductase and glycinamide ribonucleotide formyltransferase) involved in the folate pathway [3,4]. After being transported into the cell via the reduced folate car- rier, Pem binds to folate receptor-a with a very high affinity [5]. In the cells, Pem is polyglutamated to the active pentaglutamide by a reaction catalyzed by folylpolyglutamate synthase. Pem proved to be a better substrate for folylpolyglutamate synthase as compared with methotrexate [6]. The polyglutamate deriva- tives (the pentaglutamate is the predominant intracellular form) are potent inhibitors of thymidylate synthase and are also weaker inhibitors of glycinamide ribonucleotide formyltransferase. Pem and its polyglutamate derivatives also inhibit dihydrofolate reductase but with less potency than methotrexate. Compared with methotrexate, Pem polyglutamates have an increased intracellular half-life, resulting in prolonged drug action in malignant cells [7]. Pem has demonstrated promising clinical activity in a wide variety of solid tumors, including non-small cell lung (NSCLC), breast, colorectal, pancreatic, gastric, bladder and malignant pleural mesothelioma [8]. NSCLC is the most common form of lung cancer, which is the leading cause of cancer mortality in men worldwide. First-line therapy of NSCLC is based on platinum agents, and docetaxel is the standard second-line treatment option. Pem was compared * Correspondence to: Ferenc Hudecz, Research Group of Peptide Chemistry, Hungarian Academy of Sciences, Eötvös Loránd University, Pázmány P. sétány 1A, H-1117 Budapest, Hungary. E-mail: fhudecz@elte.hu a Research Group of Peptide Chemistry, Hungarian Academy of Sciences, Eötvös Loránd University, Budapest, Hungary b Department of Organic Chemistry, Eötvös Loránd University, Budapest, Hungary { These authors contributed equally to this work. J. Pept. Sci. 2011; 17: 805–811 Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd. Research Article Received: 6 June 2011 Revised: 28 June 2011 Accepted: 28 June 2011 Published online in Wiley Online Library: 6 October 2011 (wileyonlinelibrary.com) DOI 10.1002/psc.1407 805