Synthesis of New β-Lactam Analogs and Evaluation of Their Histone Deacetylase (HDAC) Activity Seikwan Oha, Jae-Chul Jungb, and Mitchell A. Averyc a Department of Neuroscience and Medical Research Institute, School of Medicine, Ewha Womans University, Seoul 158-710, South Korea b Ewha Global Challenge, BK21 and Medical Research Institute, School of Medicine, Ewha Womans University, Seoul 158-710, South Korea c Department of Medicinal Chemistry, School of Pharmacy, National Center for Natural Products Research & Department of Chemistry, University of Mississippi, University, MS 38677-1848, USA Reprint requests to Dr. Jae-Chul Jung. Fax: +82-02-2650-5791. E-mail: [email protected] Z. Naturforsch. 2007, 62b, 1459 – 1464; received April 16, 2007 A simple synthesis of the β-lactams 11 – 13 and 16 – 17 as novel histone deacetylase (HDAC) inhibitors is described. The key synthetic strategies involved the O-alkylation of 6-APA and the cou- pling reactions of freshly prepared N-carbobenzyloxy-L-prolines 5 and 6 and 6-aminopenicillanates 8 – 10 and 15 in high yields. It was found that all compounds show potent growth inhibitory activity on human tumor cell lines, the most potent compound 16 exhibiting an IC50 =2.1µ M in vitro. Key words: β-Lactams, Histone Deacetylase, Coupling Reaction, Anticancer, Synthesis Introduction (HDAC) as well as its acetylase inhibitors [11]. The small lactam (β and γ) derivatives are privileged struc- Since the introduction of β-lactam antibiotics, β- tures for enzyme inhibition, mainly due to their ability lactam substrates have stimulated significant interest to trap serine or cysteine residues in the DNA binding due to their wide range of intriguing biological activ- domain. ities [1] such as antibiotic [2], antioxidant [3], antivi- In a continuation of our medicinal chemistry pro- ral [4], and anticancer [5] properties. The variety of gram connected with the synthesis of new β-lactam the pharmacological activities of these β-lactams and moieties and evaluation of their biological proper- their unique structural features, including azetidin-2- ties, we required Cbz-protected L-prolines 5 – 6 and one rings, generated a great deal of interest among 6-aminopenicillanates 8 – 10, 15 as important frag- synthetic chemists and biologists. Recently, β-lactam ments in order to generate novel histone acetylase in- substrates with significant biological activities such hibitors. We wish to report herein a simple synthesis as serine-dependent enzyme inhibitors [6], matrix- and the evaluation of the anticancer activity of the 6- metalloprotease inhibitors [7], cysteine protease in- acylaminopenicillanates 11 – 13 and 16 – 17, starting hibitors [8], and apoptosis inductors [9] were reported from 6-aminopenicillanic acid (6-APA, 1) via O-alk- in the literature. Furthermore, they have served as syn- ylation and coupling reactions. thons in the preparation of various heterocyclic com- Results and Discussion pounds and potent anticancer agents such as paclitaxel, epothilones, and their analogs [10]. Current research To generate the Cbz-protected L-prolines 5 and 6, priorities for the β-lactam moieties are focused on pro- which are well known as a pharmacophore for HDAc viding better antibacterial efficacy and on biochemi- inhibitors, the commercially available L-proline (1) cal features as enzyme inhibitors including apoptos- and trans-4-hydroxy-L-proline (2) were treated with is-inducing properties. In addition, efforts to develop chlorotrimethylsilane (Me3SiCl) in the presence of di- optimal β-lactams as anticancer agents are underway. isopropylethylamine (DIPEA) in dichloromethane to More recently, histone acetylation was reasonably ac- yield the silyl-protected L-prolines 3 and 4,whichwere cepted as a mechanism of chromatin remodeling. It used in the next step without purification. Intermedi- is highly governed by the antagonistic activity of his- ates 3 and 4 were subsequently treated with benzyloxy- tone acetyltransferases (HAT) and histone deacetylase carbonyl chloride (Cbz-Cl) to give the Cbz-protected 0932–0776 / 07 / 1100–1459 $ 06.00 © 2007 Verlag der Zeitschrift f¨ur Naturforschung, T¨ubingen · http://znaturforsch.com 1460 S. Oh – J.-C. Jung · Synthesis of New β-Lactam Analogs and Evaluation of Their HDAC Activity Scheme 1. Synthesis of Cbz-protected L-prolines 5 and 6. Scheme 2. (a) For 8: bromoacetonitrile, TEA, acetone, r. t. 24 h, 42 %; for 9: allyl bromide, TEA, acetone, r. t. 36 h, 55 %; for 10: benzyl bromide, TEA, 4-DMAP, acetone, r. t. 48 h, 65 %; (b) 6, HATU, DIPEA, CH Cl ,r.t.16h,(68%for11,75% ◦ 2 2 for 12,76%for13); (c) dimethyldioxirane, CH2Cl2,0 Ctor.t.1h;then,CH2N2,CH2Cl2,r.t.1h;(d)CH2N2,CH2Cl2,r.t. 1h,95%;(e)5 – 6, HOBt, EDCI, TEA, CH2Cl2, r. t. 3 h, (80 % for 16,82%for17). L-prolines 5 and 6 in 97 and 95 % two-step yields, re- mide, and benzyl chloromethyl ether under SN2- spectively (Scheme 1). type reaction conditions including K2CO3/DMF [13], The β-lactam moieties 11 – 13 were prepared from DBU/CH3CN [14], and NaH/THF [15], but these readily generated 6-aminopenicillanates 8 – 10 [12] reactions were all unsatisfactory, and for the most with N-carbobenzyloxy-L-proline (5)andN-carbo- part the starting material was recovered. Com- benzyloxy-L-4-hydroxyproline (6) via common con- pounds 8 – 10 were readily coupled with 6 in the densation reactions. 6-Aminopenicillanic acid (6- presence of 2-(7-aza-1H-benzotriazol-1-yl)-1,1,3,3- APA, 7) was treated with several alkylating reagents tetramethyluronium hexafluorophosphate (HATU) and such as bromoacetonitrile, allyl bromide, and ben- DIPEA in dichloromethane to afford 11 – 13 in good zyl bromide to give esters 8 – 10. At this stage, yields (Scheme 2). we tried to generate the various esters using sev- In addition, 6-APA (7) was treated with freshly pre- eral alkylating reagents such as bromochlorometh- pared diazomethane [16] in dichloromethane to af- ane, chloromethyl methyl ether, 4-methoxybenzyl bro- ford ester 15, in high yield. On the other hand, in S.Oh–J.-C.Jung· Synthesis of New β-Lactam Analogs and Evaluation of Their HDAC Activity 1461 an effort to prepare the sulfoxide 14,6-APA(7)was Table 1. HDAC and growth inhibiting potency of novel β- smoothly treated with 3,3-dimethyldioxirane (DMDO) lactam moities 6 – 10. a b [17] in dichloromethane to generate a sulfoxide, which Compound IC50 enzyme (µ M) IC50 cells (µ M) was then readily treated with diazomethane [18]. Un- 11 44.0 68.4 12 40.0 32.7 fortunately, these reactions failed to afford sulfox- 13 12.8 4.0 ide 14, leaving only starting material and/or decom- 16 6.3 2.1 posed products. Compound 15 was condensed with 17 11.8 5.5 acids 5 (Z-Hyp) or 6 (Z-Hyp-OH) using ethyl(di- Sodium butyratec – 140 c methylaminopropyl)carbodiimide (EDCI) and 1-hydr- Trichostatin A – 0.0046 a HDAC enzyme assay; b the values are means of three experiments oxybenzotriazole (HOBt) to generate 16, 17 in high [20]; c materials for comparison. yields [13]. At this stage, coupling of 15 with 5, 6 was also accomplished by dicyclohexylcarbodiimide with ethyl acetate, and radioactivity was measured by (DCC)/CH2Cl2,HATU/CH2Cl2, and bis(2-oxo-3-oxa- a liquid scintillation counter. zolidinyl)phosphinic chloride (BOP-Cl) in CH2Cl2. The in vitro anticancer activity of β-lactam moieties Although the latter conditions were more convenient, 11 – 13 and 16, 17 were evaluated in human tumor cell the HOBt/EDCI method afforded a superior yield. lines, and the results are summarized in Table 1. It was found that all compounds showed potent growth in- In vitro inhibition of histone deacetylase hibitory activity on human tumor cell lines with the most potent compound 16 exhibiting IC50 =2.1µ M. A histone deacetylase fraction was prepared as de- In addition, the methoxy esters 16, 17 (Table 1, entries scribed by Yoshida et al. [19]. Human leukemia K562 4, 5) or benzyl ester 13 (Table 1, entry 3) exhibited (2.5 × 108) cells were disrupted in buffer-A [15 mM higher in vitro growth inhibitory activity when com- of potassium phosphate buffer (pH 7.5) containing pared to cyanomethyl or allyl esters 11, 12. In addition, 5 % glycerol and 0.2 mM EDTA (15 mL)]. The nu- the novel β-lactam moieties 11 – 13 and 16, 17 showed clei were collected by centrifugation (35000 g, 10 min) better HDAC activity than sodium butyrate. However, and resuspended with buffer-A (15 mL) containing 1 M all prepared sulfonamides exhibited less HDAC activ- (NH4)2SO4. After sonication, the supernatant was col- ity than trichostatin A. lected by centrifugation, and ammonium sulfate was In conclusion, a simple preparation of new histone added to make the final concentration 3.5 M. After stir- deacetylase (HDAC) inhibitors has been described. ◦ ring for 1 h at 0 C, the precipitate was collected by Compound 16 exhibited the most potent anticancer ac- centrifugation, dissolved with buffer-A (4 mL), and di- tivity among these analogs. We expect that simple syn- alyzed against buffer-A (2000 mL). The dialysate was theses of new β-lactam moieties and key fragments loaded onto a mono Q HR 5/5 column (Pharmacia) are useful for the modification of histone acetylase in- equilibrated with buffer-A, and eluted with a linear gra- hibitors. dientof0–1 M NaCl in buffer-A (30 mL). A single peak of histone deacetylase activity was eluted around Experimental Section ◦ 0.4 M NaCl, and the fraction was stored at −80 C until Reactions requiring anhydrous conditions were performed use. Inhibition of histone deacetylase was estimated as with the usual precautions for rigorous exclusion of air and described by Yoshida et al. with slight modifications. moisture. Tetrahydrofuran was distilled from sodium ben- 3H-labeled histone was prepared as reported, K562 zophenone ketyl prior to use.