J. Med. Chem. 1993,36,1474-1479
1474
Antitumor Agents. 141.t Synthesis and Biological Evaluation of Novel Thiocolchicine Analogs: N-Acyl-, N-Aroyl-, and N-( Substituted benzy1)deacetylthiocolchicines as Potent Cytotoxic and Antimitotic Compounds Li Sun,$Ernest Hamel) Chii M. Lin,o Susan B. Hastie,I Amy Pyluck,l and Kuo-Hsiung Lee'J Natural Products Laboratory, Division of Medicinal Chemistry and Natural Products, School of Pharmacy, University of North Carolina, Chapel Hill, North Carolina 27599, Laboratory of Molecular Pharmacology, Developmental Therapeutics Program, Division of Cancer Treatment, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892, and Department of Chemistry, State University of New York, Vestal Parkway East, Binghamton, New York 13902-6000 Received December 28, 1992
Three series of novel thiocolchicine analogs, N-acyl-, N-aroyl-, and N-(substituted benzyl)-
deacetylthiocolchicinoids,have been synthesized and evaluated for their cytotoxicity against various tumor cell lines, especially solid tumor cell lines, and for their inhibitory effects on tubulin polymerization in vitro. Most of these compounds showed strong inhibitory effects on tubulin polymerization comparable to that obtained with thiocolchicine and greater than that obtained with colchicine. Only compounds with a long side chain a t the C(7) position, such as 22-24, did not inhibit tubulin polymerization. Several of the active N-aroyldeacetylthiocolchicineanalogs had positive optical rotations, in contrast to the negative optical rotation observed with most colchicinoids. This property might be attributed to a reversal of biaryl configuration from the normal US to aR. Therefore, the N-aroyl analogs were further evaluated by circular dichroism, which readily distinguishes between the USand aR biaryl configurations. This latter technique demonstrated that the active N-aroyl analogs do have an US configuration despite their positive optical rotations. However,comparison of lH NMR and UV spectral data of N-(substituted benzyl)deacetylthiocolchicines with those of corresponding N-aroyldeacetylthiocolchicinessuggested a different biaryl dihedral angle [even though these compounds have the same USbiarylconfigurationl. The similar tubulin binding properties of these compounds suggest that a biaryl dihedral angle of 5 3 O is not essential for colchicinoid-tubulin interaction. The increased cytotoxicity of N-(substituted benzy1)deacetylthiocolchicinescompared to the N-aroyldeacetylthiocolchicinesmay be attributed to different lipophilicity, drug uptake, or drug metabolism in the tumor cells. The side chain a t the C(7) position affects inhibition of tubulin polymerization and the cytotoxic activity of colchicinoids as a function of ita size and its contribution to lipophilicity.
Introduction Colchicine (1) (Figure 11, the major alkaloid isolated from Colchicum autumnale,2 is a well-known tubulin toxin.3 The biological effects of 1, such as ita antiinflammatory and antitumor properties, probably derive from ita tubulin binding activitya2 Binding of 1 to tubulin prevents microtubule assembly and causes cells treated with the drug to arrest in mitosise3Because of the severe toxicity of 1, many related compounds have been synthesized, with the goal of improving the therapeutic index and enhancing antitumor properties.e Studies on the binding of colchicinoids to tubulin have indicated that the configuration and conformation (including the dihedral angle of the biaryl system) composed of the trimethoxyphenylA ring and the tropolonic C ring are of major importance.' It has been proposed that compounds with an aS configured biaryl system bind readily to tubulin, while those with an aR configured biaryl system bind poorly.7 In addition, a dihedral angle of -53O may be necessary for optimal binding of colchicinoids to tubulin.* Thus, modification of the biaryl configuration or conformation of colchicinoids should influence the binding of 1 analogs to tubulin. It has been shown that For part 140, see ref 1. To whom correspondence should be addressed. Natural Products Laboratory, School of Pharmacy, University of North Carolina a t Chapel Hill. 8 Laboratory of Molecular Pharmacology, NCI. 1 Department of Chemistry, State University of New York. +
10
R2 1: R1= CH,. R2 = CCH, 2: RI=H, R2=SCH, 3: RI= CHs. R2 = SCH,
Figure 1. Structures of colchicine (l),3-demethylthiocolchicine (2), and thiocolchicine (3).
substituents at the C(1) position do affect the biaryl conformation, and these analogs have the expected reduced affinity for t ~ b u l i n .The ~ side chain at the C(7) position could also change the tubulin affinity for colchicinoids by affecting their biaryl confiiation or conformation through steric effecta.1° Changes in the biaryl configuration or conformation are usually accompanied by large changes in optical properties (e.g., optical rotation), and these properties have been attributed mainly to the asymmetry derived from the biaryl system rather than to the asymmetry derived from the C(7)position.' Thus, large changes in optical rotation could be indicative of changes in the biaryl conformation or configuration of the colchicinoids. Previous studies have focused mainly on the potent antileukemic activity of 1 and related compounds synthesized by modifications of lap6However, cytotoxicity studies against various solid tumor cell lines are limited. 3-Demethylthiocolchicine (2) (Figure 1)appears to have a broader antitumor spectrum and lower toxicity than 1
0022-262319311836-1474$04.00/0 0 1993 American Chemical Society
Novel Thiocolchicine Analogs
Journal of Medicinal Chemistry, 1993, Vol. 36, No. 10 1475
Scheme I. Syntheses of N-Acyl-, N-Aroyl-, and N-(Substituted benzy1)deacetylthiocolchicinoids
Table I. Chemical Shifta, Optical Properties, and Inhibition of Tubulin Polymerization of Corresponding N-Aroyl- and N-(Substitutedbenzy1)deacetylthiocolchicines
NaSCH3 * H20, R.T.
1
3
5 18 6 14 7 17 11 13
NH,
'81
0
RCHO glacial acetic NaBH3cN/ acid
4
SCH3 Y R g O H
Z H3CO
q
V
H
C
O
R
H3CO 0
5: R =
6: R = 7: R =
15R=
11:R= NO2
8: R =
9 NO,
16R= I7:R=
and thiocolchicine (3) (Figure lh3 Consequently, analogs of 2 may prove rewarding in a search for new chemotherapeutic agents, particularly for one directed at the colchicine site of tubulin, which has thus far not been exploited clinically in cancer treatment. In addition, the role of the substituent at the C(7) position of colchicinoids in tubulin binding is not entirely clear. In this paper we report the synthesis and biological evaluation of three series of novel thiocolchicine analogs, N-acyl-, N-aroyl-, and N-(substituted benzy1)deacetylthiocolchicinoids, which possess bulky substituents in the side chain at the C(7) position and/or different lipophilicity. These compounds were evaluated for their cytotoxicity against SR leukemiacells and seven solid human tumor cell lines, non-small cell lung cancer A549/ATCC, small cell lung cancer DMS 114, colon carcinoma HCT15, CNS carcinoma SNB-19, melanoma SK-MEL-28, ovarian carcinoma OVCAR-3, and renal carcinoma RXT631. The newly synthesized compounds were evaluated for their effects on tubulin polymerization, in comparison to 1and 3, and their optical rotation and circular dichroic properties were examined.
Chemistry As shown in Scheme I, all of the compounds were synthesized from deacetylthiocolchicine (41, which was prepared by established procedures.11J2 Acylation of 4 with various commercially available substituted acids in the presence of DCC afforded N-acyl- and N-aroyldeacetylthiocolchicinoid~.~~ Reductive alkylation of 4 by reaction with substituted benzaldehydes in the presence of sodium cyanoborohydride afforded N-(substituted benzyl)deacetylthiocolchicine~.~~ Structures of the compounds were confirmed by spectral data and elemental analysis. Conformational changes in the biaryl systems of Naroyldeacetylthiocolchicinoidsare suggested by differences
7.06 7.18 7.05 7.17 7.05 7.09 "7.05 7.13
7.24 7.42 7.23 7.42 7.24 7.30 7.23 7.37
-179 +89.5 -158 +152 -212 -41.5 -161 -23.2
-2.9(369) -3.3(367) -3.2(365) -3.2 (368) -3.0(368) -3.2(369) -3.0(365) -3.4(367)
2.8 ?2.1 2.3 1.9 2.8 3.4 2.3 2.4
[e] is the term for molar ellipticity. The nm value is the minimum of the peak and is the value at which molar ellipticitywas calculated. * ITP = inhibition of tubulin polymerization. in their lH NMR spectra and optical rotations in comparison with those of the corresponding N-(substituted benzyl)- and N-acyldeacetylthiocolchicines (as well as normal colchicinoids). The chemical shifts for protons at the C(11) and the C(12) positions of N-aroyldeacetylthiocolchicines were shifted downfield relative to both N-(substituted benzyl)- and N-acyldeacetylthiocolchicines. For the N-aroyldeacetylthiocolchicines,18,14,17, and 13, there were 0.04-0.12 (for H-11) and 0.06-0.18 (for H-12) ppm downshifts from those of the corresponding N-(substituted benzy1)deacetylthiocolchicines)5,6,7, and 11,respectively (Table I). In addition,the optical rotations of these N-aroyldeacetylthiocolchicineswere more positive compared to the corresponding N-(substituted benzyl)deacetylthiocolchicinoids, which have the large negative optical rotations common to most colchicinoids (Table I). In particular, severalN-aroyldeacetylthiocolchicinoids(14 and 18-20) were found to have positive optical rotations, even though these compounds allstronglyinhibited tubulin polymerization (seebelow, Table 11). Sincethis is opposite to the negative rotation observed with 1and 3, these potent new antitubulin agents appeared to have an aR biaryl c~nfiguration.~~~ Although unexpected, this is not unprecedented. We recently described the preparation and detailed characterization of a series of six-membered B ring analogs of 3 with aR configurations,that were potent antitubulin corn pound^.^^ The activity of the six-membered B ring agents was thought to require a rapid aR-crS equilibrium, with the minor aS species binding to tubulin. In order to confirm the aR configuration suggested by the positive optical rotations of these N-aroyl derivatives, we examined the circular dichroic properties of some of these compounds as well as those of the corresponding N-(substituted benzyl)deacetylthiocolchicines, which showed large negative optical rotations (Table I). All eight compounds show large negative bands in the near-UV region of the spectrum (Table I), indicating that the aS biaryl conformation of the phenyltropone ring system is retained in these thiocolchicinoids. The molar ellipticities of the N-benzyldeacetylthioolchicinoidsare less than or equal to the molar ellipticities of the corresponding N-aroyldeacetylthiocolchicinoids. Similarly, in the colchicine series, the molar ellipticities of several N-alkylated colchicinoids are less than the molar ellipticity of colchicine.16 Thus, the minor differences in the near-UV CD bands of the N-aroyl- and N-benzyldeacetylthiocolchicinoids may be due to different electronic properties of an amine vs an amide at this position. Alternatively, the aromatic ring at C(7) of these compounds may be positioned differently in the N-aroyl- vs the N-benzyl-
1476 Journal of Medicinal Chemistry, 1993, Vol. 36,No. 10
S u n et al.
Table 11. Biological Evaluation of N-(Substituted benzyl)-, N-Aroyl-, and N -Acyldeacetylthiocolchicine Analogs cytotoxicity (log GI50)" compd SR A549/ATCC DMS114 HCT-15 SNB-19 SK-MEL-5 OVCAR-3 RXF-631 -C
