1. Introduction

ijms

International Journal of Molecular Sciences

Int. J. Mol. Sci.

1422-0067

Molecular Diversity Preservation International (MDPI)

10.3390/ijms9050821

ijms-09-00821

Article

Synthesis and Biological Evaluation of a Novel Pentagastrin-Toxin Conjugate Designed for a Targeted Prodrug Mono-therapy of Cancer

Tietze

Lutz F.

* Panknin

Olaf

Krewer

Birgit

Major

Felix

Schuberth

Ingrid

Institute of Organic and Biomolecular Chemistry, Georg-August-University Göttingen, Tammannstraße 2, D-37077 Göttingen, Germany

*Author to whom correspondence should be addressed; E-mail: ltietze@gwdg.de; Fax: +49-551-39-9476

20 5 2008

5 2008

9 5 821 837 25 4 2008 15 5 2008 16 5 2008

2008

This article is an open-access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/3.0/).

A novel carbamate prodrug 2 containing a pentagastrin moiety was synthesized. 2 was designed as a detoxified analogue of the highly cytotoxic natural antibiotic duocarmycin SA (1) for the use in a targeted prodrug monotherapy of cancers expressing cholecystokinin (CCK-B)/gastrin receptors. The synthesis of prodrug 2 was performed using a palladium-catalyzed carbonylation of bromide 6, followed by a radical cyclisation to give the pharmacophoric unit 10, coupling of 10 to the DNA-binding subunit 15 and transformation of the resulting seco-drug 3b into the carbamate 2 via addition of a pentagastrin moiety.

antibiotics antitumor agents pentagastrin prodrug prodrug monotherapy

1. Introduction

One of the major problems in the chemotherapy of cancers is the usually low differentiation between normal and malignant cells by the known antiproliferating agents, resulting in severe side effects. Several approaches have been developed to overcome this problem like the antibody-directed enzyme prodrug therapy (ADEPT) [1,2] and the prodrug monotherapy (PMT) [3]. Whereas in ADEPT artificial antibody-enzyme conjugates are needed for targeting tumor cells, in PMT specific endogenous enzymes or receptors overexpressed in cancerous tissue are addressed to allow a selective killing of tumor cells. In both approaches, a relatively untoxic prodrug is used, which is then selectively converted into the corresponding cytotoxic drug in the cancer tissue; however, for PMT the prodrug is linked to a ligand which allows a targeting of cancer cells. Among these ligands small peptides play an important role having a low immunogenicity as well as high specifities and affinities to certain receptors which are overexpressed on certain tumour cells [4]. Some of these peptides that are already successfully applied in cancer therapy, belong to the gastrin family. For example, radiolabeled gastrin derivatives have shown a high therapeutic and diagnostic potential in targeting cholecystokinin (CCK-B)/gastrin receptor expressing tumors [5]. In addition, a gastrin derivative was linked to a triazene alkylating agent [6]. However, the observed receptor-mediated cytotoxicity of this conjugate was quite low. Better results were obtained with heptagastrin linked to an ellipticine derivative [7]. A high receptor-mediated cytotoxicity could be achieved with the anthracyclines daunorubicin, doxorubicin and 2-pyrrolinodoxorubicin as well as other cytotoxic agents like melphalan, cisplatin or methotrexate coupled to peptides of the LHRH [4,8], bombesin [4,8a,9], somatostatin [4,8a,10] and neuropeptide Y [4,11] type. Recently, we have developed the pentagastrin-toxin conjugate 3a containing a seco-duocarmycin SA derivative (Scheme 1) [12].

Here, we report the synthesis of a novel pentagastrin conjugate 2 for the use in a targeted tumor therapy, which has the advantage over normal gastrin-toxin conjugates that a prodrug is used instead of a toxic drug (Scheme 1). In this concept, the pentagastrin moiety should serve not only as a targeting ligand for CCK-B/gastrin receptors, but also as a detoxifying unit. Thus, the corresponding drug 4b, which again is an analogue of the naturally occuring antibiotic (+)-duocarmycin SA (1) with an IC₅₀ value of 10 pm (L1210) [13], should be formed via 3b inside the tumor cells by cleavage of the carbamate by lysosomal enzymes after endocytosis. The antiproliferative effect of 1 and its analogues such as the CBI-drugs 4 derive most probably from a selective alkylation of N-3 of adenine in DNA by nucleophilic attack at the spirocyclopropyl-cyclohexadienone moiety as the pharmacophoric group [14]. Since we have previously shown that the formation of a drug as 4b from a seco-drug as 3b is a very fast process and that the blocking of the phenolic hydroxyl group of 3b allows a very strong reduction of its cytotoxicity, we used the seco-drug 3b as substrate for the conjugation with the pentagastrin moiety, performing the connection to the phenolic hydroxyl group via a carbamate moiety [1b,1c,15].

In our approach we did not employ the whole heptadecapeptide gastrin but the shorter β-alanine modified pentagastrin, because its β-Ala-Trp-Met-Asp-Phe-NH₂ sequence representing the C-terminal amide of the natural peptides restores the biological activity of gastrin in a comparable order of magnitude [4e,16]. As a consequence, the seco-duocarmycin moiety 3b had to be attached via the N-terminal amino functionality of pentagastrin using a carbamate. Such a carbamate substructure exists also in KW-2189 (5) (Figure 1) [17], an agent already investigated in clinical trials, and in several other anticancer agents [18].

For the formation of the carbamate moiety, we envisaged an addition of an isocyanate to the seco-drug 3b. The TMSE ester moiety was introduced to allow a better comparison with the already prepared pentagastrin-conjugate 3a. Moreover, the handle could be used for the introduction of a fluorescence dye to allow an investigation of the mode of action of such a compound employing a confocal laser scanning microscope.

2. Results and Discussion 2.1. Synthesis

As starting material for the preparation of 2 we employed the known aminonaphthalin 6 [12]. 6 was converted into TMSE ester 7 in 56 % yield by a palladium-catalyzed carbonylation reaction using a CO atmosphere (1 bar) and Mo(CO)₆ as additional CO source [19] in a mixture of 2-(trimethylsilyl)-ethanol and DMF (Scheme 2). The moderate yield of 56% of this carbonylation reaction might be due to the relatively high electron density of 6. Nevertheless, 6 had to be used in the carbonylation reaction as the Curtius rearrangement of the corresponding acid to the protected naphtholamine could not be achieved after the introduction of the TMSE ester moiety. Iodination of 7 employing NIS [20,21] with TsOH·H₂O as catalyst followed by N-alkylation of the formed 8 with 1,3-dichloropropene and subsequent radical cyclization [22] using the untoxic tris-(trimethylsilyl)-silan (TTMSS) [23] as hydride source and AIBN as radical starter provided seco-CBI derivative 10 in 65 % yield over three steps.

In order to connect 10 with the peptide unit, we used the isocyanate 13 containing an ester moiety. This was first reacted with the phenolic hydroxyl group and then bound to the peptide via an amide linkage. The required isocyanate 13 was prepared as follows: first, β-alanine (11) was converted into the corresponding benzyl ester hydrochloride 12 employing TMSCl and benzylic alcohol [24]. Then, the isocyanate moiety was introduced using solid and thus easy to handle triphosgene in refluxing toluene to give 13 in 87 % yield over two steps (Scheme 3).

Hence, the synthesis of carbamate prodrug 2 was completed in seven further steps (Scheme 4). After deprotection of the secondary amino functionality in 10 under acidic conditions in an aqueous HCl/EtOAc mixture with Et₃SiH as cation scavenger [25], the obtained hydrochloride salt 14 was directly coupled with the DNA-binding subunit TMI-CO₂H (15) to give 16 in 43 % yield over two steps. Then, the benzyl ether moiety in 16 was cleaved by transfer hydrogenolysis with an aqueous ammonium formate solution and palladium on charcoal as the catalyst [26] to yield phenol 3b which was subsequently coupled with isocyanate 13 to afford carbamate 17 in a very good yield of 83 % over two steps. The benzyl ester in 17 was cleaved again by using transfer hydrogenolytic conditions to give 18 in 90 % yield. This reaction had to be carefully monitored by TLC as the carbamate was sensitive to these conditions. Finally, carboxylic acid 18 was treated with HOSu/EDC·HCl and the resulting active ester 19 directly coupled with the fully unprotected tetrapeptide 20 [27] to yield carbamate prodrug 2 in 57 % (83 % based on recovered starting material) over two steps.

2.2. In vitro cytotoxicity tests

The in vitro cytotoxicity assays were carried out in duplicate with CCK-B/gastrin-receptor positive cells of the human pancreatic cell line MIA PaCa-2 and CCK-B/gastrin-receptor negative cells of the human bronchial carcinoma cell line A549 as control in six multiwell plates with concentrations of 10², 10³ and 10⁴ cells per cavity. Incubation with various concentrations of the seco-drug 3b and the prodrug 2 was performed in ultraculture medium (Table 1).

Prodrug 2 shows the same cytotoxicity as its corresponding seco-drug 3b in the cell culture assays using the CCK-B/gastrin-receptor positive cell line (MIA PaCa) and the CCK-B/gastrin-receptor negative cell line (A549). Thus, the obtained IC₅₀-values are almost identical in these four experiments. This indicates that prodrug 2 seems not to be stable under the used cell culture conditions. In fact, HPLC-MS-measurements revealed a decomposition of prodrug 2 under loss of the targeting pentagastrin moiety thereby forming the corresponding seco-drug 3b.

We suppose that the unstability of the carbamate moiety can be traced back to the hydrogen atom at its nitrogen which in turn is part of the β-alanine moiety of pentagastrin. We therefore plan to replace the hydrogen by a carbon moiety though it is not known whether such a modification of the pentagastrin would interfere with the binding of the conjugate to the corresponding CCK-B/gastrin-receptor.

3. Experimental Section

General: All reactions were performed in flame dried glassware under an argon atmosphere. Solvents were dried and purified according to standard procedures and redistilled prior to use. TLC chromatography was performed on precoated aluminium silica gel SIL G/UV254 plates (Macherey-Nagel & Co.) and silica gel 60 (0.040-0.063 mm) (Merck) was used for column chromatography. IR: Bruker Vector 22. UV/VIS: Perkin-Elmer Lambda 2. ¹H-NMR: Varian Mercury-200, Unity-300 (300 MHz), Unity Inova-600 (600 MHz). ¹³C-NMR: Varian Mercury-200 (50 MHz), Unity-300 (75 MHz), Unity Inova-600 (150 MHz). For ¹H and ¹³C, CDCl₃, [D₆]DMSO and [D₇]DMF were used as solvents. Chemical shifts are reported on a δ scale. Signals are quoted as s (singlet), d (doublet), t (triplet), q (quartet), m (multiplet), m_c (centered multiplet) and br (broad). MS: Finnigan MAT 95, TSQ 7000, LCQ. HRMS was performed using among others a modified peak matching technique, error ±2 ppm, with a resolution of ca. 10,000. Elemental analysis: Mikroanalytisches Labor des Institutes für Organische und Biomolekulare Chemie der Universität Göttingen.

3-Amino-1-benzyloxy-N-(tert-butoxycarbonyl)-7-[2-(trimethylsilyl)-ethoxycarbonyl]-naphthalene (7): A magnetically stirred and degassed solution of bromide 6 (3.60 g, 8.40 mmol), N(nBu)₃ (6.0 ml, 4.7 g, 25 mmol) and 2-(trimethylsilyl)-ethanol (6.0 ml, 5.0 g, 42 mmol) in DMF (35 ml) was treated with Pd(PPh₃)₂Br₂ (332 mg, 420 μmol), 1,1'-bis-(diphenylphosphino)-ferrocene (931 mg, 1.68 mmol) and Mo(CO)₆ (1.1 g, 4.2 mmol). The reaction mixture was degassed again, set under a carbon monoxide atmosphere (1 bar) and stirred for 7 h at 120 °C (preheated bath). The alcohol and tributylamine were distilled off under reduced pressure, the resulting red oil adsorbed on silica gel and subjected to column chromatography (pentane/EtOAc = 10:1 → 7:1) to afford ester 7 (2.32 g, 56 %) as orange solid. R_f = 0.43 (pentane/EtOAc = 7:1); ¹H NMR (300 MHz, CDCl₃): δ = 0.08 (s, 9 H, Si(CH₃)₃), 1.13–1.18 (m, 2 H, CH₂SiMe₃), 1.55 (s, 9 H, C(CH₃)₃), 4.43–4.49 (m, 2 H, CH₂OC=O), 5.26 (s, 2 H, CH₂Ph), 6.73 (brs, 1 H, NH), 7.08 (d, J = 1.5 Hz, 1 H, 2-H), 7.34–7.64 (m, 6 H, 5 × Ph-H, 4-H), 7.69 (d, J = 8.7 Hz, 1 H, 5-H), 8.02 (dd, J = 8.7, 2.1 Hz, 1 H, 6-H), 8.96 ppm (d, J = 2.1 Hz, 1 H, 8-H); ¹³C NMR (50 MHz, CDCl₃): δ = –1.40 (Si(CH₃)₃), 17.35 (CH₂SiMe₃), 28.32 (C(CH₃)₃), 63.09 (CH₂OC=O), 70.30 (CH₂Ph), 80.97 (C(CH₃)₃), 99.48 (C-3), 106.31 (C-1), 121.59, 125.61 (C-4a, C-6), 125.31, 126.61, 126.87, 127.45, 128.04, 128.61 (C-5, C-7, C-8, 5 × Ph-C), 136.41, 137.13, 138.65 (C-2, C-8a, Ph-C_i), 152.51, 156.23 (C-4, C=O), 167.11 ppm (C(O)OTMSE); MS (EI, 70 eV): m/z (%) = 493 (18) [M]⁺, 437 (10) [M – C₄H₉ + H]⁺, 91 (100) [C₇H₇]⁺.

2-Amino-4-benzyloxy-N-(tert-butoxycarbonyl)-1-iodo-6-[2-(trimethylsilyl)-ethoxycarbonyl]-naphthalene (8): A magnetically stirred solution of 7 (352 mg, 713 μmol) in 1:1 THF/MeOH (10 ml) was treated with a solution of TsOH·H₂O (14 mg, 71 μmol) in THF (1 mL) and N-iodosuccinimide (322 mg, 1.43 mmol). The resulting mixture was warmed to 50 °C and stirred for 1 h. The reaction mixture was quenched with NaHCO₃ (5 ml, saturated solution) and water and extracted with EtOAc (2 × 10 ml). The combined organic phases were washed with Na₂S₂O₃ (1 × 15 ml, saturated solution) and brine (15 ml), dried (MgSO₄) and concentrated under reduced pressure to give an orange oil. This material was adsorbed on silica gel and subjected to column chromatography (pentane/EtOAc = 20:1) to afford iodide 8 (320 mg, 73 %) as colorless foam. R_f = 0.65 (pentane/EtOAc = 10:1); ¹H NMR (300 MHz, CDCl₃): δ = 0.08 (s, 9 H, Si(CH₃)₃), 1.13–1.18 (m, 2 H, CH₂SiMe₃), 1.59 (s, 9 H, C(CH₃)₃), 4.44–4.49 (m, 2 H, CH₂OC=O), 5.31 (s, 2 H, CH₂Ph), 7.33–7.60 (m, 5 H, 5 × Ph-H), 8.04 (d, J = 9.3 Hz, 1 H, 8-H), 8.09 (dd, J = 9.3, 1.8 Hz, 1 H, 7-H), 8.14 (s, 1 H, 3-H), 8.95 ppm (d, J = 1.8 Hz, 1 H, 5-H); ¹³C NMR (50 MHz, CDCl₃): δ = –1.40 (Si(CH₃)₃), 17.34 (CH₂SiMe₃), 28.30 (C(CH₃)₃), 63.29 (CH₂OC=O), 70.55 (CH₂Ph), 79.16 (C(CH₃)₃), 81.53 (C-1), 100.13 (C-3), 122.77, 126.27, 136.16, 137.06 (C-4a, C-6, C-8a, Ph-C_i), 125.62, 127.90, 128.07, 128.16, 128.58, 131.39 (C-5, C-7, C-8, 5 × Ph-C), 140.53 (C-2), 152.51, 156.60 (C-4, C=O), 166.66 ppm (C(O)OTMSE); MS (EI, 70 eV): m/z (%) = 619 (14) [M]⁺, 563 (10) [M – C₄H₉ + H]⁺, 535 (18) [M – C₄H₈ – CO]⁺, 491 (5) [M – I – H]⁺, 91 (100) [C₇H₇]⁺, 57 (36) [C₄H₉]⁺.

(E/Z)-2-Amino-4-benzyloxy-N-(tert-butoxycarbonyl)-N-(3-chloro-2-propenyl)-1-iodo-6-[2-(trimethylsilyl)-ethoxycarbonyl]-naphthalene (9): A magnetically stirred solution of 8 (54 mg, 87 μmol) in DMF (1.5 ml) was treated with NaH (5.20 mg, 60 % in oil, 218 μmol). Stirring was continued for 40 min at 20 °C before (E/Z)-1,3-dichloropropene (16.0 μl, 19.0 mg, 174 μmol) was added dropwise, and it was stirred for a further 13 h at 20 °C. The ensuing mixture was then adjusted to pH 5 with NH₄Cl (saturated solution) and extracted with EtOAc (4 × 5 mL). The combined organic phases were washed with water (10 ml), brine (10 ml), dried (MgSO₄) and concentrated under reduced pressure to give an orange oil. Subjection of this material to column chromatography (pentane/EtOAc = 10:1) gave iodide 9 (59 mg, 97 %) as pale yellow foam. R_f = 0.43, 0.52 (pentane/EtOAc = 10:1); ¹H NMR (200 MHz, CDCl₃): δ = –0.14/0.10 (2 × s, 9 H, Si(CH₃)₃), 1.14–1.22 (m, 2 H, CH₂SiMe₃) 1.30/1.58 (2 × s, 9 H, C(CH₃)₃), 3.78 (dd, J = 13.8, 6.4 Hz, 1 H, 1′-H_a), 4.19–4.35 (m, 1 H, 1′-H_b), 4.46–4.54 (m, 2 H, CH₂OC=O), 5.31 (brs, 2 H, CH₂Ph), 5.92–6.15 (m, 2 H, 2′-H, 3′-H), 6.65–6.85 (m, 1 H, 3-H), 7.30–7.55 (m, 5 H, 5 × Ph-H), 8.15 (d, J = 8.0 Hz, 1 H, 8-H), 8.25 (d, J = 8.0 Hz, 1 H, 7-H), 9.01–9.10 ppm (m, 1 H, 5-H); ¹³C NMR (50 MHz, CDCl₃): δ = –1.40 (Si(CH₃)₃), 17.33 (CH₂SiMe₃), 28.19/28.46 (C(CH₃)₃), 45.76/48.97 (C-1′), 63.53/64.59 (CH₂OC=O), 70.49/70.58 (CH₂Ph), 80.88/81.39 (C(CH₃)₃), 94.52 (C-1), 107.93/108.51 (C-3), 120.87/121.96 (C-3′), 124.79, 128.26/128.29, 135.91/136.01, 137.51/137.64 (C-4a, C-6, C-8a, Ph-C_i), 125.36, 127.00/127.20, 127.90/127.94, 128.17/128.21, 128.45, 128.72/128.76, 133.06 (C-5, C-7, C-8, C-2′, 5 × Ph-C), 144.60/144.98 (C-2), 153.37/153.59 (C-4), 155.99/156.12 (C=O), 166.40/166.43 ppm (C(O)OTMSE); MS (EI, 70 eV): m/z (%) = 694 (16) [M + H]⁺, 566 (18) [M – I]⁺, 510 (88) [M – I – C₄H₉ + H]⁺, 91 (100) [C₇H₇]⁺, 57 (16) [C₄H₉]⁺.

(1R/S)-5-Benzyloxy-3-(tert-butoxycarbonyl)-1-chloromethyl-2,3-dihydro-1H-benz[e]indole-7-carboxylic acid [2-(trimethylsilyl)-ethyl] ester (10): Through a magnetically stirred solution of iodide 9 (308 mg, 444 μmol) in benzene (13 ml) was bubbled argon for 45 min. The oxygen-free solution was then treated with tris-(trimethylsilyl)-silane (124 μl, 99.0 mg, 400 μmol) and AIBN (17.0 mg, 102 μmol) and stirred for 2 h under reflux. The ensuing mixture was adsorbed on silica gel and subjected to column chromatography (pentane/EtOAc = 10:1) to afford 10 (208 mg, 92 %) as pale yellow solid. R_f = 0.44 (pentane/EtOAc = 10:1); UV/VIS (CH₃CN): λ_max (lg ε) = 217 (4.417), 271 (4.753), 354 nm (4.158); IR (KBr): ν̃ = 3388 (NH), 2955, 1700 (C=O), 1621, 1461, 1412, 1366, 1249, 1139, 928, 839 cm^–1; ¹H NMR (300 MHz, CDCl₃): δ = 0.08 (s, 9 H, Si(CH₃)₃), 1.12–1.18 (m, 2 H, CH₂SiMe₃), 1.61 (s, 9 H, C(CH₃)₃), 3.45 (t, J = 10.5 Hz, 1 H, 10-H_b), 3.88–4.02 (m, 2 H, 1-H, 10-H_a), 4.14 (dd, J = 11.4, 9.0 Hz, 1 H, 2-H_b), 4.27 (d, J = 11.4 Hz, 1 H, 2-H_a), ), 4.44–4.49 (m, 2 H, CH₂OC=O), 5.29 (s, 2 H, CH₂Ph), 7.35–7.48 (m, 3 H, 3 × Ph-H), 7.54–7.59 (m, 2 H, 2 × Ph-H), 7.64 (d, J = 8.7 Hz, 1 H, 9-H), 7.87 (brs, 1 H, 4-H), 8.08 (dd, J = 8.7, 1.5 Hz, 1 H, 8-H), 9.02 ppm (d, J = 1.5 Hz, 1 H, 6-H); ¹³C NMR (125 MHz, CDCl₃): δ = –1.41 (Si(CH₃)₃), 17.33 (CH₂SiMe₃), 28.41 (C(CH₃)₃), 41.38 (C-1), 46.36 (C-10), 53.11 (C-2), 63.14 (CH₂OC=O), 70.42 (CH₂Ph), 81.43 (C(CH₃)₃), 96.93 (C-4), 114.37, 121.50, 124.92, 132.37, 144.14 (C-3a, C-5a, C-7, C-9a, C-9b), 121.72 (C-9), 126.75 (C-6), 127.21 (C-8), 127.62, 128.07, 128.59 (5 × Ph-C), 136.34 (Ph-C_i), 152.41 (C-5), 157.25 (C=O), 166.91 ppm (C(O)OTMSE); MS (EI, 70 eV): m/z (%) = 567 (4) [M]⁺, 511 (7) [M – C₄H₉ + H]⁺, 91 (90) [C₇H₇]⁺, 57 (30) [C₄H₉]⁺; HRMS: calcd for C₃₁H₃₈ClNO₅Si: 567.2208; confirmed.

β-Alanine benzyl ester hydrochloride (12): A magnetically stirred suspension of β-alanine (11) (1.00 g, 11.2 mmol) in benzylic alcohol (56.0 ml, 58.0 g, 539 mmol) was treated dropwise over a period of 10 min with trimethylsilylchloride (3.6 ml, 3.0 g, 28 mmol) and stirring continued for a further 15 h at 20 °C. The resulting clear solution was poured into Et₂O (600 mL), the precipitate collected by filtration and washed with Et₂O (100 mL). Drying of this material under reduced pressure gave hydrochloride 12 (2.15 g, 89 %) as white solid; ¹H NMR (200 MHz, [D₆]DMSO): δ = 2.79 (t, J = 7.0 Hz, 2 H, 2-H₂), 3.03 (t, J = 7.0 Hz, 2 H, 3-H₂), 5.13 (s, 2 H, CH₂Ph), 7.32–7.41 (m, 5 H, 5 × Ph-H), 8.23 ppm (brs, 3 H, NH₃⁺); ¹³C NMR (50 MHz, [D₆]DMSO): δ = 31.34 (C-2), 34.47 (C-3), 65.87 (CH₂Ph), 127.95 (2 × Ph-C), 128.01 (Ph-C_p), 128.34 (2 × Ph-C), 135.73 (Ph-C_i), 170.05 ppm (C=O); MS (ESI): m/z (%) = 180 (100) [M – Cl]⁺, 359 (100) [2M – Cl – HCl]⁺.

3-Isocyano-propionic acid benzyl ester (13): A magnetically stirred suspension of hydrochloride 12 (2.13 g, 9.88 mmol) in toluene (15 ml) was treated with triphosgene (2.93 g, 9.88 mmol) and heated to reflux for 7.5 h (end of HCl-evolution). The resulting solution was concentrated under reduced pressure to afford isocyanate 13 (1.99 g, 98 %) as yellow liquid which was used for the next reaction without further purification. R_f = 0.21 (pentane/EtOAc = 10:1); IR (film): ν̃ = 3349, 2957, 2277 (NCO), 1736 (C=O), 1498, 1176, 823, 752, 699 cm^–1; ¹H NMR (200 MHz, CDCl₃): δ = 2.65 (t, J = 6.4 Hz, 2 H, 2-H₂), 3.61 (t, J = 6.4 Hz, 2 H, 3-H₂), 5.18 (s, 2 H, CH₂Ph), 7.35–7.40 ppm (m, 5 H, 5 × Ph-H).

(1R/S)-5-Benzyloxy-1-chloromethyl-3-(5,6,7-trimethoxyindole-2-carbonyl)-2,3-dihydro-1H-benz[e]indole-7-carboxylic acid [2-(trimethylsilyl)-ethyl] ester (16): A solution of the protected amine 10 (369 mg, 650 μmol) in CH₂Cl₂ (5 ml) was treated with HCl (18 ml of a 4 m solution in EtOAc) and Et₃SiH (105 μl, 76.0 mg, 650 μmol) and stirred for 7 h at 20 °C. The solvent was removed under reduced pressure and the ensuing residue treated with toluene (2 × 10 ml) and again concentrated under reduced pressure. The resulting crude hydrochloride 14 was dried under reduced pressure and then treated with 5,6,7-trimethoxyindole-2-carboxylic acid (15) (180 mg, 715 μmol), EDC·HCl (374 mg, 1.95 mmol) and DMF (16 ml) and stirred for 1 d at 20 °C. The reaction mixture was adjusted to pH 2 with HCl (2 n) and extracted with EtOAc (4 × 20 ml). The combined organic phases were washed with water (3 × 20 ml), brine (20 ml), dried (MgSO₄) and concentrated under reduced pressure to give a brown solid. This material was adsorbed on silica gel and subjected to column chromatography (pentane/EtOAc = 3:1) to yield 16 (196 mg, 43 % over two steps) as green solid. R_f = 0.57 (pentane/ EtOAc = 2:1); UV/VIS (CH₃CN): λ_max (lg ε) = 209 (4.758), 271 (4.470), 315 (4.465), 363 nm (4.524); IR (KBr): ν̃ = 3461 (NH), 2951, 1711 (C=O), 1624, 1527, 1459, 1408, 1309, 1107, 837, 747 cm^–1; ¹H NMR (300 MHz, CDCl₃): δ = 0.09 (s, 9 H, Si(CH₃)₃), 1.13–1.19 (m, 2 H, CH₂SiMe₃), 3.42 (dd, J = 10.8, 10.2 Hz, 1 H, 10-H_b), 3.90–4.05 (m, 11 H, 3 × OCH₃, 1-H, 10-H_a), 4.43–4.48 (m, 2 H, CH₂OC=O), 4.56 (dd, J = 11.1, 8.7 Hz, 1 H, 2-H_b), 4.71 (dd, J = 11.1, 1.8 Hz, 1 H, 2-H_a), 5.25–5.31 (m, 2 H, CH₂Ph), 6.85 (s, 1 H, 4′-H), 6.96 (d, J = 2.4 Hz, 1 H, 3′-H), 7.31–7.55 (m, 5 H, 5 × Ph-H), 7.62 (d, J = 9.0 Hz, 1 H, 9-H), 8.07 (dd, J = 9.0, 1.8 Hz, 1 H, 8-H), 8.21 (s, 1 H, 4-H), 9.03 (d, J = 1.8 Hz, 1 H, 6-H), 9.75 ppm (d, J = 2.4 Hz, 1 H, indole-NH); ¹³C NMR (75 MHz, CDCl₃): δ = –1.47 (Si(CH₃)₃), 17.26 (CH₂SiMe₃), 42.72 (C-1), 45.88 (C-10), 55.11 (C-2), 56.11, 61.00, 61.36 (3 × OCH₃), 63.18 (CH₂OC=O), 70.30 (CH₂Ph), 97.54 (C-4′), 98.87 (C-4), 106.75 (C-3′), 115.94, 123.45, 125.65, 131.71, 144.30 (C-3a, C-5a, C-7, C-9a, C-9b), 121.99 (C-9), 122.48, 125.61, 129.48, 138.72, 140.55 (C-2′, C-3a′, C-6′, C-7′, C-7a′), 126.47 (C-6), 127.12 (C-8), 127.44, 127.97, 128.49 (5 × Ph-C), 136.22 (Ph-C_i), 150.08 (C-5′), 156.65 (C-5), 160.51 (C=O), 166.62 ppm (C(O)OTMSE); MS (ESI): m/z (%) = 723 (100) [M + Na]⁺, 1423 (75) [2M + Na]⁺, 699 (100) [M – H]^–; HRMS: calcd for C₃₈H₄₁ClN₂O₇Si: 701.2444 [M + H]⁺; found: 701.2441.

(1R/S)-1-Chloromethyl-5-hydroxy-3-(5,6,7-trimethoxyindole-2-carbonyl)-2,3-dihydro-1H-benz[e]indole-7-carboxylic acid [2-(trimethylsilyl)-ethyl] ester (3b): A magnetically stirred solution of benzyl ether 16 (152 mg, 217 μmol) in 3:1 THF/MeOH (6 ml) was treated with 10 % Pd/C (62 mg) and dropwise with NH₄HCO₂ (572 μl of a 25 % solution in water, 2.26 mmol) and stirring continued for 75 min at 20 °C. The reaction mixture was filtered through a pad of Celite^® and it was thoroughly washed with MeOH and THF. The filtrate was dried (MgSO₄) and concentrated under reduced pressure to give 3b (133 mg, quant.) as yellow solid which was used for the next reaction without further purification. R_f = 0.25 (pentane/ EtOAc = 2:1); ¹H NMR (300 MHz, [D₆]DMSO): δ = 0.09 (s, 9 H, Si(CH₃)₃), 1.12–1.17 (m, 2 H, CH₂SiMe₃), 3.81–3.88 (m, 7 H, 2 × OCH₃, 10-H_b), 3.95 (s, 3 H, OCH₃), 4.02 (dd, J = 11.1, 3.1 Hz, 1 H, 10-H_a), 4.20 (m_c, 1 H, 1-H), 4.41–4.50 (m, 3 H, CH₂OC=O, 2-H_b), 4.74 (dd, J = 11.1, 9.1 Hz, 1 H, 2-H_a), 6.97 (s, 1 H, 4′-H), 7.07 (d, J = 2.0 Hz, 1 H, 3′-H), 7.91–7.98 (m, 3 H, 4-H, 8-H, 9-H), 8.82 (d, J = 1.0 Hz, 1 H, 6-H), 10.83 (s, 1 H, OH), 11.42 ppm (d, J = 2.0 Hz, 1 H, indole-NH); ¹³C NMR (75 MHz, [D₆]DMSO): δ = –1.45 (Si(CH₃)₃), 16.87 (CH₂SiMe₃), 40.72 (C-1), 47.36 (C-10), 55.09 (C-2), 55.93, 60.84, 61.00 (3 × OCH₃), 62.56 (CH₂OC=O), 98.07 (C-4′), 100.82 (C-4), 106.31 (C-3′), 115.12, 123.06, 125.45, 131.92, 144.76 (C-3a, C-5a, C-7, C-9a, C-9b), 120.95, 123.12, 130.69, 139.00, 139.94 (C-2′, C-3a′, C-6′, C-7′, C-7a′), 123.94 (C-9), 125.89 (C-8), 126.02 (C-6), 149.19 (C-5′), 155.57 (C-5), 160.39 (C=O_TMI), 165.89 ppm (C(O)OTMSE); MS (ESI): m/z (%) = 633 (100) [M + Na]⁺, 1243 (29) [2M + Na]⁺, 573 (100) [M – H – HCl]^–, 609 (22) [M – H]^–; HRMS: calcd for C₃₁H₃₅ClN₂O₇Si: 611.1975 [M + H]⁺; found: 611.1975.

(1R/S)-5-(2-Benzyloxycarbonyl-ethylcarbamoyloxy)-1-chloromethyl-3-(5,6,7-trimethoxyindo-le-2-carbonyl)-2,3-dihydro-1H-benz[e]indole-7-carboxylic acid [2-(trimethylsilyl)-ethyl] ester (17): A magnetically stirred solution of phenol 3b (198 μmol, 121 mg) in CH₂Cl₂ (15 ml) at 0 °C was treated dropwise with 13 (203 μl, 203 mg; 990 μmol) and then triethylamine (139 μl, 100 mg, 990 μmol). The reaction mixture was warmed to 20 °C and stirred for a further 16 h. The ensuing solution was cooled to 0 °C, adjusted to pH 2 with HCl (2 n) and extracted with EtOAc (4 × 10 ml). The combined organic phases were washed with brine (10 ml), dried (MgSO₄) and concentrated under reduced pressure to give a yellow solid. This material was adsorbed on silica gel and subjected to column chromatography (pentane/EtOAc = 1:1) to afford carbamate 17 (135 mg, 83 %) as yellow solid. R_f = 0.29 (pentane/EtOAc = 2:1); ¹H NMR (300 MHz, [D₆]DMSO, 100 °C): δ = 0.10 (s, 9 H, Si(CH₃)₃), 1.13–1.19 (m, 2 H, CH₂SiMe₃), 2.71 (t, J = 7.1 Hz, 2 H, 2″-H₂), 3.47–3.53 (m, 2 H, 1″-H₂), 3.85 (s, 6 H, 2 × OCH₃), 3.98 (dd, J = 11.2, 6.8 Hz, 1 H, 10-H_b), 4.00 (s, 3 H, OCH₃), 4.09 (dd, J = 11.2, 3.4 Hz, 1 H, 10-H_a), 4.39 (m_c, 1 H, 1-H), 4.45–4.50 (m, 2 H, CH₂OC=O), 4.58 (dd, J = 11.1, 2.6 Hz, 1 H, 2-H_b), 4.80 (dd, J = 11.1, 9.2 Hz, 1 H, 2-H_a), 5.17 (s, 2 H, CH₂Ph), 6.99 (s, 1 H, 4′-H), 7.09 (d, J = 2.2 Hz, 1 H, 3′-H), 7.30–7.41 (m, 5 H, 5 × Ph-H), 8.04 (dd, J = 8.7, 1.4 Hz, 1 H, 8-H), 8.09 (d, J = 8.7 Hz, 1 H, 9-H), 8.24 (s, 1 H, 4-H), 8.62 (d, J = 1.4 Hz, 1 H, 6-H), 11.02 ppm (brs, 1 H, indole-NH); ¹³C NMR (75 MHz, [D₆]DMSO, 100 °C): δ = –0.95 (Si(CH₃)₃), 17.61 (CH₂SiMe₃), 34.62/34.68 (C-2″), 37.58/37.60 (C-1″), 41.76 (C-1), 47.79/47.84 (C-10), 55.62 (C-2), 56.99 (OCH₃), 61.38 (2 × OCH₃), 63.36 (CH₂OC=O), 66.15 (CH₂Ph), 99.48 (C-4′), 107.05 (C-3′), 111.34 (C-4), 122.42, 124.17, 126.73, 132.19, 144.43 (C-3a, C-5a, C-7, C-9a, C-9b), 123.78, 126.36, 131.09, 139.56, 140.95 (C-2′, C-3a′, C-6′, C-7′, C-7a′), 124.29 (C-9), 125.41 (C-6), 126.78 (C-8), 128.23 (2 × Ph-C), 128.35 (Ph-C_p), 128.81 (2 × Ph-C), 136.72 (Ph-C_i), 149.15 (C-5′), 150.13 (OC(O)N), 156.16/1546.18 (C-5), 161.23 (C=O_TMI), 166.19 (C(O)OTMSE), 171.23 ppm (C(O)OBn); MS (ESI): m/z (%) = 611 (100) [M – C(O)NH-β-Ala-OBn + H]⁺, 838 (18) [M + Na]⁺, 1653 (14) [2M + Na]⁺, 573 (100) [M – C(O)NH-β-Ala-OBn – HCl]^–, 609 (55) [M – C(O)NH-β-Ala-OBn – H]^–.

(1R/S)-5-(2-Carboxy-ethylcarbamoyloxy)-1-chloromethyl-3-(5,6,7-trimethoxyindole-2-carbo-nyl)-2,3-dihydro-1H-benz[e]indole-7-carboxylic acid [2-(trimethylsilyl)-ethyl] ester (18): A magnetically stirred solution of benzyl ester 17 (203 mg, 249 μmol) in 3:1 THF/MeOH (10 ml) was treated with 10 % Pd/C (83 mg) and dropwise with NH₄HCO₂ (653 μl of a 25 % solution in water, 2.59 mmol). Stirring was continued for 25 min (TLC-monitoring necessary as the alanyl residue is easily cleaved off) at 20 °C. The reaction mixture was filtered through a pad of Celite^®, which was thoroughly washed with MeOH and CH₂Cl₂. The combined filtrates were concentrated under reduced pressure, the residue dissolved in CH₂Cl₂, dried (MgSO₄) and concentrated under reduced pressure again to give a yellow oil. This material was adsorbed on silica gel and subjected to column chromatography (CH₂Cl₂/ MeOH = 20:1) to afford acid 18 (163 mg, 90 %) as pale yellow solid. R_f = 0.44 (CH₂Cl₂/MeOH = 10:1); ¹H NMR (300 MHz, CDCl₃): δ = 0.06 (s, 9 H, Si(CH₃)₃), 1.12 (m_c, 2 H, CH₂SiMe₃), 2.74 (m_c, 2 H, 2″-H₂), 3.41 (m_c, 1 H, 10-H_b), 3.65 (m_c, 2 H, 1″-H₂), 3.83–4.07 (m, 11 H, 1-H, 10-H_a, 3 × OCH₃), 4.42 (m_c, 2 H, CH₂OC=O), 4.57–4.64 (m, 1 H, 2-H_b), 4.71–4.74 (m, 1 H, 2-H_a), 6.39 (brs, 1 H, NH), 6.87 (brs, 1 H, 4′-H), 6.99 (brs, 1 H, 3′-H), 7.62 (m_c, 1 H, 9-H), 8.01 (m_c, 1 H, 8-H), 8.41 (brs, 1 H, 4-H), 8.56 (brs, 1 H, 6-H), 9.99 ppm (brs, 1 H, indole-NH); ¹³C NMR (75 MHz, CDCl₃): δ = –1.46 (Si(CH₃)₃), 17.34 (CH₂SiMe₃), 34.17 (C-2″), 36.99 (C-1″), 43.01 (C-1), 45.75 (C-10), 55.17 (C-2), 56.22, 61.31, 61.49 (3 × OCH₃), 63.65 (CH₂OC=O), 97.78 (C-4′), 107.11 (C-3′), 111.78 (C-4), 121.31, 124.21, 126.85, 131.42, 143.39 (C-3a, C-5a, C-7, C-9a, C-9b), 122.50 (C-9), 123.56, 125.86, 129.29, 138.68, 140.83 (C-2′, C-3a′, C-6′, C-7′, C-7a′), 125.93 (C-6), 126.52 (C-8), 149.00 (C-5′), 150.10 (OC(O)N), 154.39 (C-5), 160.58 (C=O_TMI), 166.61 (C(O)OTMSE), 171.50 ppm (C(O)OH); MS (ESI): m/z (%) = 748 (90) [M + Na]⁺, 1473 (100) [2M + Na]⁺, 573 (96) [M – C(O)NH-β-Ala-OH – HCl]^–, 1449 (100) [2M – H]^–.

(1R/S)-1-Chloromethyl-5-[2-(N-succinimidyloxycarbonyl)-ethyl-carbamoyloxy]-3-(5,6,7-tri-methoxyindole-2-carbonyl)-2,3-dihydro-1H-benz[e]indole-7-carboxylic acid [2-(trimethylsilyl)-ethyl] ester (19): A magnetically stirred solution of acid 18 (110 mg, 152 μmol) and N-hydroxysuccinimide (26.0 mg, 228 μmol) in 1:1 THF/CH₂Cl₂ (10 ml) at 0 °C was treated with EDC·HCl (44.0 mg, 228 μmol). The reaction mixture was warmed to 20 °C and stirring continued for 15 h. The ensuing solution was then adjusted to pH 2 with HCl (2 n) and extracted with EtOAc (4 × 10 ml). The combined organic phases were washed with brine (10 ml), dried (MgSO₄) and concentrated under reduced pressure to afford 19 as yellow solid which was used for the next reaction without further purification. R_f = 0.83 (CH₂Cl₂/MeOH = 10:1); MS (ESI): m/z (%) = 845 (90) [M + Na]⁺, 1667 (100) [2M + Na]⁺, 573 (100) [M – C(O)NH-β-Ala-OSu – HCl]^–.

(1R/S)-1-Chloromethyl-5-(l-phenylalaninamidyl-l-aspartyl-l-methionyl-l-tryptophyl-β-alanyl-carbonyloxy)-3-(5,6,7-trimethoxyindole-2-carbonyl)-2,3-dihydro-1H-benz[e]indole-7-carboxylic acid [2-(trimethylsilyl)-ethyl] ester (2): A magnetically stirred solution of tetragastrin (20)^[27] (39.4 mg, 66.0 μmol) in water (1 ml) was treated with NEtiPr₂ (11.5 μl, 8.50 mg, 66.0 μmol) and dropwise with a solution of crude 19 (49 mg, 60 μmol) in DMF (3.5 ml). Stirring was continued for 7 h at 20 °C. The ensuing mixture was adjusted to pH 2 with HCl (2 n) and extracted with EtOAc (5 × 5 ml). The combined organic phases were washed with water (5 ml) and brine (5 ml), treated with toluene (5 ml) and concentrated under reduced pressure. The resulting yellow solid was adsorbed on silica gel and subjected to column chromatography (CH₂Cl₂/MeOH = 15:1 + 0.5 % HOAc → 10:1 + 0.5 % HOAc) to give 2 (45 mg, 57 % over two steps, 83 % based on recovery, 1:1 mixture of both diastereomeres) as light yellow solid. Further purification was achieved by HPLC. R_f = 0.27 (CH₂Cl₂/MeOH = 10:1 + 0.5 % HOAc); ¹H NMR (600 MHz, [D₇]DMF): δ = 0.10/0.12 (2 × s, 9 H, Si(CH₃)₃), 1.20 (m_c, 2 H, CH₂SiMe₃), 1.98–2.07 (m, 5 H, 2c-H₂, SCH₃), 2.37–2.66 (m, 6 H, 2b-H₂, 3c-H₂, 1e-H₂), 2.98–3.06 (m, 3 H, 2a-H_b, 1f-H₂), 3.17 (dd, J = 14.8, 8.4 Hz, 1 H, 2d-H_b), 3.21–3.24 (m, 1 H, 2a-H_a), 3.28–3.33 (m, 1 H, 2d-H_a), 3.88 (s, 3 H, OCH₃), 3.90 (s, 3 H, OCH₃), 3.95 (dd, J = 11.1, 7.9 Hz, 1 H, 10-H_b), 4.03 (s, 3 H, OCH₃), 4.12 (dd, J = 11.1, 3.3 Hz, 1 H, 10-H_a), 4.30 (m_c, 1 H, 1-H), 4.42–4.47 (m, 1 H, 1c-H), 4.50 (m_c, 2 H, CH₂OC=O), 4.58 (m_c, 1 H, 1a-H), 4.64 (m_c, 1 H, 1b-H), 4.67 (dd, J = 10.7, 2.2 Hz, 1 H, 2-H_b), 4.73 (m_c, 1 H, 1d-H), 4.85 (dd, J = 10.7, 9.1 Hz, 1 H, 2-H_a), 6.57 (s, 1 H, NH), 6.97 (m_c, 1 H, 5d′-H), 7.05 (s, 1 H, 4′-H), 7.06 (m_c, 1 H, 6d′-H), 7.17 (m_c, 1 H, 1 × Ph-H), 7.19 (m_c, 2 H, 3′-H, 2d′-H), 7.24–7.32 (m, 4 H, 4 × Ph-H), 7.37/7.39 (2 × d, J = 8.0 Hz, 1 H, 7d′-H), 7.57/7.63 (2 × d, J = 7.9 Hz, 1 H, 4d′-H), 7.73 (brs, 1 H, NH), 7.83 (brs, 1 H, NH), 8.02 (d, J = 8.8 Hz, 1 H, 9-H), 8.05 (dd, J = 8.8, 1.6 Hz, 1 H, 8-H), 8.11 (brs, 1 H, 4-H), 8.18–8.44 (m, 4 H, NH), 8.99 (d, J = 1.6 Hz, 1 H, 6-H), 10.82/10.87 (2 × s, 1 H, indole-NH_Trp), 11.34 ppm (s, 1 H, indole-NH_TMI); ¹³C NMR (150 MHz, [D₇]DMF): δ = –1.46/–1.42 (Si(CH₃)₃), 15.01 (SCH₃), 17.67/17.76 (CH₂SiMe₃), 28.12 (C-2d), 30.79 (C-3c), 31.88 (C-2c), 32.09 (C-1e), 35.02 (C-1f), 38.16 (C-2a), 39.40 (C-2b), 42.21 (C-1), 47.94 (C-10), 51.56 (C-1b), 53.65 (C-1c), 55.07 (C-1d), 55.27 (C-1a), 56.03 (C-2), 56.46/56.49, 61.36, 61.43 (3 × OCH₃), 63.38 (CH₂OC=O), 98.89 (C-4′), 101.95 (C-4), 107.19 (C-3′), 110.62 (C-3d′), 111.93 (C-7d′), 116.06, 124.35, 126.61, 131.78, 145.99 (C-3a, C-5a, C-7, C-9a, C-9b), 118.89 (C-5d′), 118.95 (C-4d′), 121.46/121.51 (C-6d′), 122.17, 129.86, 133.05, 139.97, 141.16 (C-2′, C-3a′, C-6′, C-7′, C-7a′), 123.75 (C-9), 124.41 (C-2d′), 126.85, 126.91 (C-6, C-8, Ph-C_p), 128.51 (C-3ad′), 128.78 (2 × Ph-C), 129.83 (2 × Ph-C), 137.33 (C-7ad′), 139.04 (Ph-C_i), 150.54 (C-5′), 152.52 (OC(O)N), 156.90 (C-5), 161.36 (C=O_TMI), 162.20 (C=O_Ala), 166.96 (C(O)OTMSE), 170.71, 171.25, 172.27, 172.33 (4 × C=O), 184.62 ppm (C(O)OH); MS (ESI): m/z (%) = 633 (100) [M – C(O)NH-pentapeptide + H + Na]⁺, 573 (100) [M – C(O)NH-pentapeptide – Cl]^–; HRMS: calcd for C₆₄H₇₄ClN₉O₁₅SSi: 1304.4556 [M + H]+; found: 1304.4558; HPLC (preparative): column: Kromasil 100 C18; eluent: 85 % MeOH, 15 % H₂O + 0.05 % TFA; flow: 12 mL/min; R_t: 31–40 min.

Cell culture: Human bronchial carcinoma cells of line A549 (ATCC CCL 185) were kindly provided by the Institut für Zellbiologie, Universität Essen, and human pancreatic carcinoma cells Mia PaCa-2 by the Universitätsklinikum Göttingen, Abteilung Hämatologie und Onkologie. Cell lines were maintained as exponentially growing cultures at 37 °C and 7.5% CO₂ in air in culture medium (DMEM (Biochrom) supplemented with 10 % fetal calf serum, 44 mm NaHCO₃ (Biochrom) and 4 mm l-Glutamine (Invitrogen)).

In vitro cytotoxicity assays: Cells of line A549 or MIA PaCa-2 were seeded in duplicates in 6 multiwell plates at concentrations of 10², 10³ and 10⁴ cells per well. After cells were allowed to adhear, cells were washed in a serum-free incubation medium (Ultraculture medium, Lonza). Incubation with compounds 2 and 3b was then performed in Ultraculture medium at various concentrations for 24 h. All substances were used as freshly prepared solutions in DMSO (Merck) diluted with incubation medium to a final concentration of DMSO of 1% in the wells. After exposure the test substance was removed and cells were washed with fresh medium. Cultivation in normal growth medium was done for 10 days. The medium was removed, the clones were dried and stained with Löffler's methylene blue (Merck) and then counted macroscopically.

The IC₅₀ values are based on the relative colony-forming rate, which was determined according to the following formula: relative colony-forming rate [%] = 100 × (number of clones counted after exposure) / (number of clones counted in the control).

Acknowledgments

This work was supported by the Deutsche Forschungsgemeinschaft and the Fonds der Chemischen Industrie. F. M. thanks the Studienstiftung des deutschen Volkes (German National Academic Foundation) for a Ph.D. scholarship. B. K. is grateful to the Deutsche Telekom Foundation for a Ph.D. scholarship.

References and Notes 1a)

Bagshawe

Antibody directed enzymes revive anti-cancer prodrugs concept

Br. J. Cancer198756531532Reviews:

10.1038/bjc.1987.237

3426915

Tietze

Feuerstein

Enzyme and Proton-Activated Prodrugs for a Selective Cancer Therapy

Curr. Pharm. Des.2003921552175

10.2174/1381612033454072

14529411

Tietze

Feuerstein

Highly Selective Compounds for the Antibody-Directed Enzyme Prodrug Therapy of Cancer

Aust. J. Chem.200356841854

10.1071/CH03036

Jung

Antibody directed enzyme prodrug therapy (ADEPT) and related approaches for anticancer therapy

Mini-Rev. Med. Chem.20011399407

10.2174/1389557013406747

12369965

Syrigos

Epenetos

Antibody directed enzyme prodrug therapy (ADEPT): a review of the experimental and clinical considerations

Anticancer Res.199919605613

10226606

Springer

Niculescu-Duvaz

Antibody-directed enzyme prodrug therapy (ADEPT): a review

Adv. Drug Deliv. Rev.199726151172

10.1016/S0169-409X(97)00032-X

10837540

2a)

Tietze

Major

Schuberth

Spiegl

Krewer

Maksimenka

Bringmann

Magull

Selective Treatment of Cancer: Synthesis, Biological Evaluation and Structural Elucidation of Novel Analogues of the Antibiotic CC-1065 and the Duocarmycins

Chem. Eur. J.20071343964409

10.1002/chem.200700113

17455190

Tietze

Major

Schuberth

Antitumor Agents: Development of Highly Potent Glycosidic Duocarmycin Analogues for Selective Cancer Therapy

Angew. Chem.200611867246727

10.1002/ange.200600936

Angew. Chem., Int. Ed.20064565746577

10.1002/anie.200600936

Tietze

Krewer

Frauendorf

Major

Schuberth

Investigation of Reactivity and Selectivity of DNA-Alkylating Duocarmycin Analogues by High-Resolution Mass Spectrometry

Angew. Chem.200611867206724

10.1002/ange.200600935

Angew. Chem., Int. Ed.20064565706574

10.1002/anie.200600935

3a)

Ganesh

Improved biochemical strategies for targeted delivery of taxoids

Bioorg. Med. Chem.20071535973623

10.1016/j.bmc.2007.03.041

17419065

Devalapally

Navath

Yenamandra

Akkinepally

Devarakonda

Directed Enzyme Prodrug Therapy/Prodrug MonoTherapy

Arch. Pharm. Res.200730723732

10.1007/BF02977634

17679550

Alaoui

Saha

Schmidt

Monneret

Florent

J-C

New Taxol®(paclitaxel) prodrugs designed for ADEPT and PMT strategies in cancer chemotherapy

Bioorg. Med. Chem.20061450125019

10.1016/j.bmc.2006.03.002

16554162

Kratz

Warnecke

Schmid

Chung

D-E

Gitzel

Prodrugs of anthracyclines in cancer chemotherapy

Curr. Med. Chem.200613477523

10.2174/092986706776055751

16515518

Schmidt

Monneret

Prodrug Monotherapy: synthesis and biological evaluation of an etoposide glucuronide-prodrug

Bioorg. Med. Chem.20031122772283

10.1016/S0968-0896(03)00108-1

12713838

Prijovich

Chen

Leu

Y-L

Chern

J-W

Roffler

Anti-tumour activity and toxicity of the new prodrug 9-aminocamptothecin glucuronide (9 ACG) in mice

Br. J. Cancer.20028616341638

10.1038/sj.bjc.6600317

12085215

Denny

Prodrug strategies in cancer therapy

Eur. J. Med. Chem.200136577595

10.1016/S0223-5234(01)01253-3

11600229

Bosslet

Straub

Blumrich

Czech

Gerken

Sperker

Kroemer

Gesson

J-P

Koch

Monneret

Elucidation of the Mechanism Enabling Tumor Selective Prodrug Monotherapy

Cancer Res.19985811951201

9515805

Bosslet

Czech

Hoffmann

Tumor Targeting199514550 4Reviews:a)

Reubi

Mäcke

Krenning

Candidates for peptide receptor radiotherapy today and in the future

J. Nucl. Med.20054667S75S

15653654

Dyba

Tarasova

Michejda

Small Molecule Toxins Targeting Tumor Receptors

Curr. Pharm. Des.20041023112334

10.2174/1381612043384024

15279611

Reubi

Peptide Receptors as Molecular Targets for Cancer Diagnosis and Therapy

Endocrine Reviews200324389427

10.1210/er.2002-0007

12920149

Reubi

Waser

Concomitant expression of several peptide receptors in neuroendocrine tumours: molecular basis for in vivo multireceptor tumour targeting

Eur. J. Nucl. Med.200330781793

10.1007/s00259-003-1184-3

Langer

Beck-Sickinger

Peptides as Carrier for Tumor Diagnosis and Treatment

Curr. Med. Chem.200117193f)

Breeman

WAP

de Jong

Kwekkeboom

Valkema

Bakker

Kooij

PPM

Visser

Krenning

Somatostatin receptor-mediated imaging and therapy: basic science, current knowledge, limitations and future perspectives

Eur. J. Nucl. Med.20012814211429

10.1007/s002590100502

11585303

Heppeler

Froidevaux

Eberle

Maecke

Receptor targeting for tumor localisation and therapy with radiopeptides

Curr. Med. Chem.20007971994

10.2174/0929867003374516

10911025

5a)

Nock

Maina

Béhé

Nikolopoulou

Gotthardt

Schmitt

Behr

Macke

CCK-2/Gastrin Receptor–Targeted Tumor Imaging with^99mTc-Labeled Minigastrin Analogs

J. Nucl. Med.20054617271736

16204724

Béhé

Behr

Cholecystokinin-B (CCK-B)/gastrin receptor targeting peptides for staging and therapy of medullary thyroid cancer and other CCK-B receptor expressing malignancies

Biopolymers (Peptide Science)200266399418

10.1002/bip.10356

Behr

Jenner

Béhé

Angerstein

Gratz

Raue

Becker

Radiolabeled Peptides for Targeting Cholecystokinin-B/Gastrin Receptor-Expressing Tumors

J. Nucl. Med.19994010291044

10452322

Behr

Jenner

Radetzky

Béhé

Gratz

Yücekent

Raue

Becker

Targeting of cholecystokinin-B/gastrin receptors in vivo: preclinical and initial clinical evaluation of the diagnostic and therapeutic potential of radiolabelled gastrin

Eur. J. Nucl. Med.199825424430

10.1007/s002590050241

9553173

Schmidt

Hernandez

Rouzer

Czerwinski

Chmurny

Michejda

Peptide-Linked 1,3-Dialkyl-3-acyltriazenes:Gastrin Receptor Directed Antineoplastic Alkylating Agents

J. Med. Chem.19943738123818

10.1021/jm00048a016

7966139

Czerwinski

Tarasova

Michejda

Cytotoxic agents directed to peptide hormone receptors: Defining the requirements for a successful drug

Proc. Natl. Acad. Sci. USA1998951152011525

10.1073/pnas.95.20.11520

9751698

8a)

Buchholz

Keller

Schally

Halmos

Hohla

Heinrich

Koester

Baker

Engel

Therapy of ovarian cancers with targeted cytotoxic analogs of bombesin, somatostatin, and luteinizing hormone-releasing hormone and their combinations

Proc. Natl. Acad. Sci. USA20061031040310407

10.1073/pnas.0602971103

16801542

Nagy

Schally

Minireview. Targeting of Cytotoxic Luteinizing Hormone-Releasing Hormone Analogs to Breast, Ovarian, Endometrial, and Prostate Cancers

Biol. Reprod.200573851859

10.1095/biolreprod.105.043489

16033997

Nagy

Schally

Armatis

Szepesházi

Halmos

Kovács

Zarandi

Groot

Miyazaki

Jungwirth

Horvath

Cytotoxic analogs of luteinizing hormone-releasing hormone containing doxorubicin or 2-pyrrolinodoxorubicin, a derivative 500-1000 times more potent

Proc. Natl. Acad. Sci. USA19969372697273

10.1073/pnas.93.14.7269

8692981

9a)

Engel

Keller

Schally

Halmos

Hammann

Nagy

Effective Inhibition of Experimental Human Ovarian Cancers with a Targeted Cytotoxic Bombesin Analogue AN-215

Clin. Cancer Res.20051124082415

10.1158/1078-0432.CCR-04-1670

15788692

Nagy

Armatis

Cai

Szepesházi

Halmos

Schally

Design, synthesis, and in vitro evaluation of cytotoxic analogs of bombesin-like peptides containing doxorubicin or its intensely potent derivative, 2-pyrrolinodoxorubicin

Proc. Natl. Acad. Sci. USA199794652656

10.1073/pnas.94.2.652

9012839

10a)

Szepesházi

Schally

Halmos

Armatis

Hebert

Sun

Feil

Kiaris

Nagy

Targeted Cytotoxic Somatostatin Analogue AN-238 Inhibits Somatostatin Receptor-positive Experimental Colon Cancers Independently of Their p53 Status

Cancer Res.200262781788

11830533

Nagy

Schally

Halmos

Armatis

Cai

Csernus

Kovács

Koppán

Szepesházi

Kahán

Synthesis and biological evaluation of cytotoxic analogs of somatostatin containing doxorubicin or its intensely potent derivative, 2-pyrrolino-doxorubicin

Proc. Natl. Acad. Sci. USA19989517941799

10.1073/pnas.95.4.1794

9465096

Langer

Kratz

Rothen-Rutishauser

Wunderli-Allenspach

Beck-Sickinger

Novel Peptide Conjugates for Tumor-Specific Chemotherapy

J. Med. Chem.20014413411348

10.1021/jm001065f

11311056

Tietze

Panknin

Major

Krewer

Synthesis of a Novel Pentagastrin-Drug Conjugate for a Targeted Tumor Therapy

Chem. Eur. J.20081428112818

10.1002/chem.200701521

18214880

13a)

Boger

Johnson

CC-1065 and the Duocarmycins: Understanding their Biological Function through Mechanistic Studies

Angew. Chem.199610815421580

10.1002/ange.19961081306

Angew. Chem. Int. Ed. Engl.19963514381474

10.1002/anie.199614381

Ichimura

Ogawa

Katsumata

Takahashi

Nakano

Duocarmycins, new antitumor antibiotics produced by streptomyces; producing organisms and improved production

J. Antibiot.19914410451053

10.7164/antibiotics.44.1045

1955385

Ichimura

Ogawa

Takahashi

Kobayashi

Kawamoto

Yasuzawa

Takahashi

Nakano

Duocarmycin, a new antitumor antibiotic from streptomyces sp

J. Antibiot.19904310371038

10.7164/antibiotics.43.1037

2211354

14a)

Boger

Bollinger

Hertzog

Johnson

Cai

Mésini

Garbaccio

Jin

Kitos

Reversed and Sandwiched Analogs of Duocarmycin SA: Establishment of the Origin of the Sequence-Selective Alkylation of DNA and New Insights into the Source of Catalysis

J. Am. Chem. Soc.199711949874998

10.1021/ja9637210

Boger

Johnson

CC-1065 and the Duocarmycins: Understanding their Biological Function through Mechanistic Studies

Angew. Chem.199610815421580

10.1002/ange.19961081306

Angew. Chem. Int. Ed. Engl.19963514381474

10.1002/anie.199614381

Hurley

Needham-VanDevanter

Covalent Binding of Antitumor Antibiotics in the Minor Groove of DNA. Mechanism of Action of CC-1065 and the Pyrrolo(1,4) benzodiazepines

Acc. Chem. Res.198619230237

10.1021/ar00128a001

15a)

Tietze

Haunert

Feuerstein

Herzig

A Concise and Efficient Synthesis of seco-Duocarmycin SA

Eur. J. Org. Chem.20033562566b)

Baird

Winstein

Neighboring Carbon and Hydrogen. Li. Dienones from Ar₁[UNK]-3 Participation. Isolation and Behavior of Spiro(2,5)octa-1,4-diene-3-one

J. Am. Chem. Soc.196385567578

10.1021/ja00888a020

16a)

Morley

Tracy

Gregory

Structure–Function Relationships in the Active C-Terminal Tetrapeptide Sequence of Gastrin

Nature196520713561359b)

Tracy

Gregory

The antral Hormone Gastrin: Physiological Properties of a Series of Synthetic Peptides structurally related to Gastrin I

Nature1964204935938

10.1038/204935a0

14248713

17a)

Alberts

Erlichman

Reid

Sloan

Ames

Richardson

Goldberg

Phase I study of the duocarmycin semisynthetic derivative KW-2189 given daily for five days every six weeks

Clin. Cancer Res.1998421112117

9748127

Nagamura

Kobayashi

Gomi

Saito

Studies on the active metabolite (DU-86) of KW-2189, a novel derivative of duocarmycin

Bioorg. Med. Chem. Lett.1996621472150

10.1016/0960-894X(96)00388-5

Nagamura

Kobayashi

Gomi

Saito

Synthesis and antitumor activity of duocarmycin derivatives: A-ring pyrrole analogues of duocarmycin B2

Bioorg. Med. Chem.1996413791391

10.1016/0968-0896(96)00132-0

8879561

Nagamura

Asai

Kanda

Kobayashi

Gomi

Saito

Synthesis and Antitumor Activity of Duocarmycin Derivatives: Modification of Segment A of Duocarmycin B2

Chem. Pharm. Bull.19964417231730

10.1248/cpb.44.1723

8855367

Yasuzawa

Muroi

Ichimura

Takahashi

Ogawa

Takahashi

Sano

Saitoh

Duocarmycins, Potent Antitumor Antibiotics Produced by Streptomyces sp. Structures and Chemistry

Chem. Pharm. Bull.199543378391

10.1248/cpb.43.378

7774022

Asai

Nagamura

Saito

A Novel Property of Duocarmycin and Its Analogs for Covalent Reaction with DNA

J. Am. Chem. Soc.199411641714177

10.1021/ja00089a004

Ray

Chaturvedi

Application of organic carbamates in drug design. Part 1: Anti-cancer agents-recent reports

Drugs Fut.200429343357

10.1358/dof.2004.029.04.787236

Kaiser

N-F

Hallberg

Larhed

In situ Generation of Carbon Monoxide from Solid Molybdenum Hexacarbonyl. A Convenient and Fast Route to Palladium-Catalyzed Carbonylation Reactions

J. Comb. Chem.20024109111

10.1021/cc010085f

11886283

Boger

Han

Tarby

Boyce

Cai

Jin

Kitos

Synthesis, Chemical Properties, and Preliminary Evaluation of Substituted CBI Analogs of CC-1065 and the Duocarmycins Incorporating the 7-Cyano-1,2,9,9a-tetrahydrocyclopropa[c]benz[e]indol-4-one Alkylation Subunit: Hammett Quantitation of the Magnitude of Electronic Effects on Functional Reactivity

J. Org. Chem.19966148944912

10.1021/jo9605298

21a)

Boger

Brunette

Garbaccio

Synthesis and Evaluation of a Series of C3-Substituted CBI Analogues of CC-1065 and the Duocarmycins

J. Org. Chem.20016651635173

10.1021/jo010309g

11463270

Boger

McKie

Cai

Cacciari

Baraldi

Synthesis and Properties of Substituted CBI Analogs of CC-1065 and the Duocarmycins Incorporating the 7-Methoxy-1,2,9,9a-tetrahydrocyclopropa[c]benz[e]indol-4-one (MCBI) Alkylation Subunit: Magnitude of Electronic Effects on the Functional Reactivity

J. Org. Chem.19966117101729

10.1021/jo952033g

11667041

22a)

Boger

Boyce

Garbaccio

Searcey

Synthesis of CC-1065 and duocarmycin analogs via intramolecular aryl radical cyclization of a tethered vinyl chloride

Tetrahedron Lett.19983922272230

10.1016/S0040-4039(98)00232-9

Patel

Andis

Enkema

Johnson

Kennedy

Mohamadi

Schultz

Soose

Spees

Total Synthesis of Seco (+)- and ent-(−)-Oxaduocarmycin SA: Construction of the (Chloromethyl)indoline Alkylating Subunit by a Novel Intramolecular Aryl Radical Cyclization onto a Vinyl Chloride

J. Org. Chem.19976288688874

10.1021/jo971880b

Boger

McKie

An Efficient Synthesis of 1,2,9,9a-Tetrahydrocyclopropa[c]benz[e]indol-4-one CBI: An Enhanced and Simplified Analog of the CC-1065 and Duocarmycin Alkylation Subunits

J. Org. Chem.19956012711275

10.1021/jo00110a034

23a)

Studer

Amrein

Silylated Cyclohexadienes: New Alternatives to Tributyltin Hydride in Free Radical Chemistry

Angew. Chem.200011231963198

10.1002/1521-3757(20000901)112:17<3196::AID-ANGE3196>3.0.CO;2-7

Angew. Chem. Int. Ed.20003930803082

10.1002/1521-3773(20000901)39:17<3080::AID-ANIE3080>3.0.CO;2-E

Chatgilialoglu

Organosilanes as radical-based reducing agents in synthesis

Acc. Chem. Res.199225188

10.1021/ar00016a003

Belshaw

Mzengeza

Lajoie

Chlorotrimethylsilane Mediated Formation of ω-Allyl Esters of Aspartic And Glutamic Acids

Synth. Commun.19902031573160

10.1080/00397919008051540

Mehta

Jaouhari

Benson

Douglas

Improved efficiency and selectivity in peptide synthesis: use of triethylsilane as a carbocation scavenger in deprotection of t-butyl esters and t-butoxycarbonyl-protected sites

Tetrahedron Lett.19923354415444

10.1016/S0040-4039(00)79116-7

26a)

Ram

Spicer

Rapid debenzylation of N-benzylamino derivatives to amino-derivatives using ammonium formate as catalytic hydrogen transfer agent

Tetrahedron Lett.198728515516

10.1016/S0040-4039(00)95769-1

Bieg

Szeja

Removal of-Benzyl Protective Groups by Catalytic Transfer Hydrogenation

Synthesis19857677 27Tetragastrin (20) was purchased from Bachem

Figures and Table Figure 1.

KW-2189 (5).

Scheme 1.

(+)-Duocarmycin SA (1), prodrug 2, seco-drugs 3 and drugs 4; a) enzymatic toxification of the novel carbamate prodrug 2 to the seco-drug 3b inside the tumor cells and b) rapid cyclisation of the seco-drugs 3 in situ to give the cytotoxic drugs 4.

Scheme 2.

Synthesis of seco-CBI compound 10. a) Mo(CO)₆, 1 bar CO, 5 mol% Pd(PPh₃)₂Br₂, 20 mol% dppf, nBu₃N, TMSEOH, DMF, 120 °C, 7 h, 56%; b) NIS, TsOH·H₂O, THF/MeOH, 50 °C, 1 h, 73%; c) NaH, 1,3-dichloropropene, DMF, 20 °C, 13.5 h, 97%; d) HSi(SiMe₃)₃, AIBN, benzene, reflux, 2 h, 92%.

Scheme 3.

Synthesis of isocyanate 13. a) TMSCl, BnOH, 20 °C, 15 h, 89%; b) triphosgene, toluene, reflux, 7.5 h, 98%.

Scheme 4.

Synthesis of carbamate prodrug 2. a) HCl/EtOAc, Et₃SiH, CH₂Cl₂, 20 °C, 7 h; b) TMI-CO₂H (15), EDC·HCl, DMF, 20 °C, 1 d, 43% (two steps); c) Pd/C, NH₄HCO₂, THF/MeOH, 20 °C, 75 min, quant.; d) isocyanate 13, NEt₃, CH₂Cl₂, 0–20 °C, 16 h, 83%; e) Pd/C, NH₄HCO₂, THF/MeOH, 20 °C, 25 min, 90%; f) HOSu, EDC·HCl, THF/CH₂Cl₂, 0–20 °C, 15 h; g) tetragastrin (20), NEtiPr₂, H₂O/DMF, 20 °C, 7 h, 57% (two steps).

Table 1.

In vitro cytotoxicity of prodrug 2 and of seco-drug 3b against CCK-B/gastrin-receptor positive cells of the human pancreatic cell line MIA PaCa-2 and CCK-B/gastrin-receptor negative human bronchial carcinoma cells (A549). Cells were exposed to various concentrations of the test substance for 24 h at 37 °C; after 10 days of incubation following the exposure to the substance, clone formation was compared to an untreated control assay and the relative colony-forming rate was determined. IC₅₀ is the drug concentration required for 50% growth inhibition of target cells.

Compound	MIA PaCa-2 IC₅₀ [nm]	A549 IC₅₀ [nm]
2	0.31	0.11
3b	0.31	0.14