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Published in final edited form as: Tetrahedron Lett. 2014 Nov 14;56(23):3101–3104. doi: 10.1016/j.tetlet.2014.11.038

A five-membered lactone prodrug of CBI-based analogs of the duocarmycins

Mika Uematsu 1, Daniel M Brody 1, Dale L Boger 1,*
PMCID: PMC4459655  NIHMSID: NIHMS642674  PMID: 26069351

Abstract

The preparation, characterization and examination of the CBI-based 5-membered lactone 5 capable of serving as a prodrug or protein (antibody) conjugation reagent are disclosed along with its incorporation into the corresponding CC-1065 and duocarmycin analog 6, and the establishment of their properties.

Keywords: CC-1065, duocarmycin, CBI, prodrug

CC-1065 (1) 1 and duocarmycin SA (2) 2 were the first isolated and are the two most widely recognized members of a class of exceptionally potent naturally occurring antitumor compounds that also include duocarmycin A (3)3 and yatakemycin (4)4 (Figure 1). They derive their antitumor properties from their sequence selective DNA alkylation properties, undergoing a stereoelectronically-controlled adenine N3 alkylation within the minor groove at specific sites within 4–5 base pair A–T rich sites.5,6 Extensive studies conducted not only with the natural products, but also their synthetic unnatural enantiomers,7 and a systematic series of key analogs have defined the fundamental features that control their DNA alkylation selectivity, efficiency, and catalysis, providing in a detailed understanding of the relationships between structure, reactivity, and biological activity.6,7,8

Figure 1.

Figure 1

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Natural products.

In early studies, synthetic phenol precursors, which have yet to undergo the Winstein Ar-3’ spirocyclization, were found to be equipotent to and indistinguishable from their cyclized cyclopropane containing counterparts in cell growth inhibition assays, DNA alkylation studies, and in vivo antitumor models. Due to this indistinguishable behavior, protection of the phenol provides an ideal site on which to prepare inactive prodrugs that can be cleaved in vivo releasing the active drug.9 Several classes of prodrugs that use this protection and release strategy have been disclosed based on a series of unique activation strategies including hydrolytic, reductive, oxidative, or enzymatic reactions.10–15 In the course of our recent studies in this area and although not the objective of the work,15 we had the opportunity to prepare, characterize and examine a CBI-based16,17 5-membered lactone capable of serving as such a prodrug or protein (antibody) conjugation reagent. Herein, we disclose the synthesis of 5, its incorporation into the corresponding CC-1065 and duocarmycin analog 6, and the establishment of their properties (Figure 2).

Figure 2.

Figure 2

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Lactone CBI prodrug or conjugation reagent.

Coupling of carboxylic acid 718 with N-methyl O-THP hydroxylamine provided 8 in excellent yield (Scheme 1, 90%). Transannular spirocyclization upon Mitsunobu activation of the secondary alcohol 8 afforded 9 (75%). Acid-catalyzed addition of chloride to the activated cyclopropane, effecting ring contraction of the fused piperidine to pyrrolidine, was carried out under conditions (1 N HCl, THF, 25 °C, 2 h) that also promoted the subsequent lactone formation in a single step without N-Boc deprotection, providing 519 directly in excellent yield (90%).

Scheme 1.

Scheme 1

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Acid-catalyzed N-Boc deprotection of 5 (4 N HCl, EtOAc, 25 °C, 1 h) followed by coupling of the crude hydrochloride salt 10 with 11 (1.2 equiv, 3 equiv of EDCI, 0.1 M DMF, 25 °C, 2 h) provided 619 (80%) in good yield (Scheme 2).

Scheme 2.

Scheme 2

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The chemical reactivity of the 5-membered lactone was probed by examining its reaction in pure MeOH (25 °C) and in pH 7 phosphate buffer (25% DMSO−phosphate buffer, 25 °C) where it displayed an interesting, somewhat divergent, behavior (Figure 3). Within 1 h in MeOH, approximately 50% of 5 was converted to the corresponding methyl ester 1220 derived from lactone methanolysis and the conversion was complete within 24 h. Extended exposure to the conditions led to slower generation of the spirocyclized methyl ester 1320 (25% at 3 d). In contrast to intuitive expectations, this reactivity was much more attenuated in pH 7 phosphate buffer.

Figure 3.

Figure 3

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Chemical reactivity of the 5-membered lactone.

Aside from characterizing the intrinsic reactivity of the 5-membered lactone toward mild nucleophiles, the studies suggest a novel protein conjugation strategy involving reaction of the lactone with an amine that can be conducted in water (buffer) where the lactone may selectively react with protein-linker amines in preference with water and that the initial seco-CBI-based product may not undergo rapid spirocyclization. Deliberate hydrolysis of 5 and 6 was accomplished in DMSO-H2O (2:1, 0.006 and 0.002 M respectively, 80 °C, 24 h) to provide the corresponding seco-CBI derivatives 14 (51%) and 16 (65%), bearing the free carboxylic acid.

The cytotoxic activity of 5 and 6 were established in a cell growth inhibition assay enlisting a mouse leukemia cell line (L1210) that has been used historically to initially assess members of this class of candidate drugs. As anticipated and analogous to the behavior of the free drugs themselves, 5 was approximately 1000-fold less active than 6. Consistent with the relatively slow release of the free drug and like the behavior of other hydrolytically released and labile prodrugs, both 5 and 6 were found to be potent inhibitors of cell growth, being only 5−10-fold less potent than the corresponding parent CBI-based free drug analogs (Figure 4). Interestingly, the corresponding carboxylic acids 14 and 16, derived from water hydrolysis of the lactone with release of the free drug, were much less active in the cell-based cell growth assay, suggesting their limited ability to penetrate either the cell or nucleus membrane.

Figure 4.

Figure 4

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Cell growth inhibition (cytotoxic) activity.

Supplementary Material

Acknowledgements

We gratefully acknowledge the financial support of the National Institutes of Health (CA042056, DLB).

Footnotes

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References and notes

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  • 19.For 5: yellow solid: [α]28D–41 (c 0.1, acetone); 1H NMR (acetone-d6, 600 MHz) δ 8.34 (d, J = 8.4 Hz, 1H), 8.19−8.05 (br, 1H), 8.03 (d, J = 6.6 Hz, 1H), 7.91 (dd, J = 8.4, 6.6 Hz, 1H), 4.34−4.27 (m, 2H), 4.23 (dd, J = 11.4, 3.6 Hz, 1H), 3.95 (dd, J = 11.4, 7.2 Hz, 1H), 1.60 (s, 9H); 13C NMR (acetone-d6, 150 MHz) δ 167.6, 152.9, 151.7, 145.9, 131.7, 129.3, 127.6, 126.7, 124.6, 122.6, 118.3, 99.0, 82.1, 54.1, 48.4, 41.4, 28.6; IR (film) νmax 1784, 1704, 1401, 1327, 1137, 670 cm−1; ESI-TOF HRMS m/z 360.0997 (M+H+, C19H18ClNO4 requires 360.0997). For 6: yellow solid; [α]25D−327 (c 0.1, acetone); 1H NMR (THF-d8, 600 MHz) δ 11.13 (s, 1H), 11.10 (s, 1H), 9.37 (s, 1H), 8.42 (s, 1H), 8.39 (d, J = 1.8 Hz, 1H), 8.25 (d, J = 8.4 Hz, 1H), 7.99 (d, J = 7.2 Hz, 1H), 7.82 (dd, J = 8.4, 7.2 Hz, 1H), 7.59 (d, J = 7.8 Hz, 1H), 7.47 (dd, J = 9.0, 1.8 Hz, 1H), 7.45 (d, J = 8.4 Hz, 1H), 7.41 (d, J = 8.4 Hz, 1H), 7.20−7.17 (m, 2H), 7.03 (t, J = 7.2 Hz, 1H), 4.90 (dd, J = 10.8, 9.0 Hz, 1H), 4.79 (dd, J = 10.8, 3.0 Hz, 1H), 4.34 (ddd, J = 12.0, 8.4, 3.6 Hz, 1H), 4.09 (dd, J = 10.8, 3.6 Hz, 1H), 3.87 (dd, J = 10.8, 8.4 Hz, 1H); 13C NMR (THF-d8, 150 MHz) 167.2, 161.7, 160.6, 151.6, 146.8, 138.3, 134.9, 133.5, 132.2, 131.4, 129.2, 129.10, 129.07, 127.8, 127.3, 124.9, 124.5, 123.1, 122.5, 120.8, 120.2, 119.9, 113.8, 112.9, 112.8, 107.3, 103.1, 101.2, 56.5, 47.8, 43.3; IR (film) νmax 2923, 2854, 1738, 1367, 1230, 669 cm−1; ESI-TOF HRMS m/z 561.1330 (M+H+, C32H21ClN4O4 requires 561.1324).
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