Synthesis of ∆3-2-Hydroxybakuchiol Analogues and Their Growth Inhibitory Activity against Rat UMR106 Cells

A series of ∆3-2-hydroxybakuchiol analogues have been synthesized and tested for their growth inhibitory activity against rat UMR106 cells by using the MTT method. Some of them exhibit enhanced activities compared with the natural product, and the preliminary SAR profile shows that the chain tail on the natural product could be subtly modified to enhance the activity and the aromatic moiety or the terminal olefin on the main chain can also be modified without any evident loss of activity. The stereo-configuration of the quaternary chiral center has an important influence on the activity.


Introduction
Natural products from plants are an important source of potential therapeutic agents for human health. The medicinal plant Psoralea coryliforlia L., a member of the Leguminosae family, has been used for a long time as a Traditional Chinese Medicine for the treatment of premature ejaculation, knee pain, pollakiuria, callus, psoriasis, vitiligo and psoriasis [1]. The seed extract has been suggested as a useful remedy for bone fractures, osteomalacia and osteoporosis [2]. A number of monoterpene OPEN ACCESS phenols occurring in the plant have been isolated and demonstrated to possess interesting biological activities [3][4][5][6][7], and among them, bakuchiol (15b, Figure 1), one of the major components in the plant seed, has attracted great attention due to its diverse activities, such as antibacterial, antihelminthic, antioxidant, and especially antitumor properties [8][9][10][11][12][13][14][15][16][17][18][19][20][21][22][23]. In contrast to that, ∆ 3 -2-hydroxybakuchiol (15a), a congener of bakuchiol, has not attracted much interest among medicinal chemists, probably due to its scarcity and lability [24,25]. To the best of our knowledge, only Guo and co-workers have recently investigated its inhibitory effects against monoamine transporters, suggesting that it might be a potential psychopharmacologic agent for the treatment of psychogenic disorders [26].
Given the fact that the analog bakuchiol shows various biological activities, we have put ∆ 3 -2-hydroxybakuchiol into our natural product-based drug discovery program [27][28][29][30]. Recently, a facile asymmetric synthesis of ∆ 3 -2-hydroxybakuchiol was established and the compound was tested for antiosteoporosis effects [31,32]. Unexpectedly, this natural product did not show the desired activity, but it exhibited growth inhibitory activity against osteosarcoma cells (rat UMR106 cell) with an IC 50 value of 69 µM, suggesting that it was worthy of further investigation [33]. Herein the synthesis of ∆ 3 -2-hydroxybakuchiol analogues was described and their growth inhibitory activity against rat UMR106 cells was demonstrated. The preliminary structure-activity relationship knowledge of this natural product has been obtained by modifying the substituent (G) on the aromatic ring and the R 1 group on the chain, while keeping the main chain unchanged. In addition, we would also replace the terminal olefin with an ethyl moiety (R 2 ) in order to probe the effect of this moiety on the activity (Figure 1).

Chemistry
Our synthetic approach to the analogues of ∆ 3 -2-hydroxybakuchiol is depicted in Scheme 1. Starting from (E)-2-methylbut-2-enoyl chloride and (R)-4-isopropyloxazolidin-2-one, the α,β-unsaturated imide 3 bearing an Evans' auxiliary was prepared in excellent yield. Then, the -alkylation of 3 with tert-butyl iodoacetate afforded the fragment 4 in a moderate yield with an excellent diastereoselectivity (dr > 20: 1). After selective reduction of the imide group, the key chiral intermediate 5 was obtained in an acceptable yield. To avoid the formation of the volatile lactone from 5, the free hydroxyl group in 5 was protected with a TBS group to afford compound 6. Upon reduction of the ester group and subsequent oxidation, compound 8a was prepared in 94% yield (over two steps). Scheme 1. Synthetic approach to the analogues of ∆ 3 -2-hydroxybakuchiol.
Compound 8a successfully underwent a Wittig reaction to afford the α,β-unsaturated ester 9a. Next, the TBS group was removed and oxidized to afford the corresponding aldehyde 11a in 92% yield (over two steps). Through a Takai-Utimoto reaction with CrCl 2 and CHI 3 , the trans-iodo-olefin 12a was obtained in up to 86% yield. The iodo-olefin was coupled with different substituted aryl zinc species via the Negishi reaction to give the desired cross-coupling product 13a,c-f, respectively. After selective 1,2-addition reactions of the esters 13a, c-g with alkyllithiums, the corresponding products 14a, c-j were obtained in acceptable (up to 73%) yields.
In order to assess the effect of the terminal olefin moiety in ∆ 3 -2-hydroxybakuchiol on the biological activity, compound 14b was prepared. Hydrogenation of 7a provided saturated alcohol 7b in a quantitative yield which was successfully transformed into 14b by a method similar to that shown in Scheme 1. As positive control, bakuchiol (15b) and its methyl ether (15c), together with their corresponding enantiomers 15d and 15e, were also prepared according to our recently reported method [31].

Activity against Rat UMR106 Cell
With the various analogues of ∆ 3 -2-hydroxybakuchiol in hand, their cytotoxic activity against rat UMR106 osteosarcoma cells was tested by the MTT assay after two days of treatment [34] and the results are shown in Table 1.
When the modifications were performed only on the aromatic moiety of ∆ 3 -2-hydroxybakuchiol, the substituent effect seemed to have somewhat of an effect on the activity (compounds 14a, c, d, f). For example, the activity was still retained in the case of a para-methoxy (14a; Entry 7), but other groups such as H and para-methyl led to slightly reduced activities (14c, 14d; Entries 8 and 9). Notably, a strong electron-withdrawing group gave a slightly increased activity (14f, Entry 11). When a larger bulky group was used (compounds 14e,g; Entries 10 and 12), the activity was just slightly decreased. Surprisingly, if the phenolic hydroxyl was moved to the meta-position (14h), its IC 50 value was still up to 123 µM, with about one-fold reduced activity in contrast to ∆ 3 -2-hydroxybakuchiol (Entry 13). Modification on the R 1 group (14i,j) in the tertiary alcohol moiety suggested that this domain could accommodate a bulky hydrophobic space, and 14j gave the best result (IC 50 : 17 µM; Entries 14 and 15). In addition, the terminal double bond could be saturated without influence on the activity, implied by the almost identical activities of 14b (IC 50 : 71 µM; Entry 16) and On the other hand, bakuchiol (15b), with a slight structural difference on the chain tail of ∆ 3 -2-hydroxybakuchiol, showed almost the same potency (IC 50 : 62 µM, Entry 18). Interestingly, ent-bakuchiol (15d) had a one-fold enhanced activity (IC 50 : 33 µM, Entry 20). However, in comparison with (S-or R)bakuchiol methyl ether showed different influences on the activity, namely, (S)-bakuchiol methyl ether (15c) maintained the activity (IC 50 : 60 µM, Entry 17) but (R)-bakuchiol methyl ether (15e) gave less activity (IC 50 : 161 µM, Entry 19), indicating that the stereochemistry played an important role to the activity.

General Information
Solvents were distilled from the appropriate drying agents before use. All the reagents were purchased from Acros (

Activity Tests
The MTT assay was used to measure cell viability. All the tested compounds were dissolved in 0.5% DMSO. Before analysis, UMR 106 cells (rat osteosarcoma cell line) were cultured with or without different concentrations of compounds for 48 h, respectively. At the end of incubation 0.5% MTT (20 μL) was added to each well, followed by incubation at 37 °C in 5% CO 2 atmosphere for 4 h. The medium was then removed carefully, and DMSO (150 μL) was added to each well. The plates were shaken gently for 10 min to dissolve the blue formazan crystals. Absorbance was measured at 570 nm using an ELx-800 universal microplate reader (Bio-Tek, Winooski, VT, USA).

Conclusions
In summary, a series of the analogues of ∆ 3 -2-hydroxybakuchiol have been synthesized, and some of them have been found to possess evident cytotoxic activity against rat UMR106 cells. Furthermore, the SAR study gives a preliminary profile, including: (1) the chain tail on the natural product could be somewhat modified to enhance the activity; (2) the aromatic moiety or the terminal olefin attached on the main chain are allowed to be modified without evident loss of activity; (3) the stereoconfiguration at the quaternary chiral center has an important influence on the activity.