Design, synthesis, and biological evaluation of novel water-soluble
N-mustards as potential anticancer agents
Naval Kapuriya
a,e, Rajesh Kakadiya
a, Huajin Dong
b, Amit Kumar
a, Pei-Chih Lee
a, Xiuguo Zhang
b,
Ting-Chao Chou
b, Te-Chang Lee
a, Ching-Huang Chen
a, King Lam
d, Bhavin Marvania
a,e,
Anamik Shah
e, Tsann-Long Su
a,c,⇑aInstitute of Biomedical Sciences, Academia Sinica, Taipei 11529, Taiwan
bPreclinical Pharmacology Core Laboratory, Molecular Pharmacology and Chemistry Program, Memorial Sloan-Kettering Cancer Center, New York, NY 10021, USA c
Graduate Institute of Pharmaceutical Chemistry, China Medical University, Taichung, Taiwan
d
Development Center for Biotechnology, Taipei County 221, Taiwan
e
Department of Chemistry, Saurashtra University, Rajkot, Gujarat, India
a r t i c l e
i n f o
Article history:
Received 20 September 2010 Revised 1 November 2010 Accepted 2 November 2010 Available online 5 November 2010 Keywords:
Anticancer agents
Water soluble nitrogen mustards DNA interstrand cross-linking agents Cell cycle
a b s t r a c t
A series of novel water-soluble N-mustard-benzene conjugates bearing a urea linker were synthesized. The benzene moiety contains various hydrophilic side chains are linked to the meta- or para-position of the urea linker via a carboxamide or an ether linkage. The preliminary antitumor studies revealed that these agents exhibited potent cytotoxicity in vitro and therapeutic efficacy against human tumor xeno-grafts in vivo. Remarkably, complete tumor remission in nude mice bearing human breast carcinoma MX-1 xenograft and significant suppression against prostate adenocarcinoma PC3 xenograft were achieved by treating with compound 9aa0at the maximum tolerable dose with relatively low toxicity. We also
dem-onstrate that the newly synthesized compounds are able to induce DNA cross-linking through alkaline agarose gel shift assay. A pharmacokinetic profile of the representative 9aa0in rats was also investigated.
The current studies suggest that this agent is a promising candidate for preclinical studies.
Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction
The bioavailability of drugs is one of the important factors to determine whether a drug can be successfully developed for clini-cal application. Consequently, the physicochemiclini-cal properties, including solubility, permeability, stability pKa, and lipophilicity/ hydrophilicity balance, are key factors that influence the bioavail-ability of drugs. Compounds with poor solubility usually have a higher risk of failure during the period of new drug discovery and development because these properties may affect antitumor evaluations in animal models as well as pharmacokinetic and phar-macodynamic properties of the compound. Several approved drugs possess poor water solubility, resulting in reduced bioavailability. For example, the poor water-soluble paclitaxel (mitotic spindle inhibitor) when converted into its water-soluble poly(glutamic acid) conjugates shows some evidence for increased tumor availability of the drug for targeted therapy.1–4Similar efforts in developing water-soluble derivatives of camptothecin (DNA topoisomerase I inhibitors) led to the discovery of topotecan (1,
Chart 1)5and irinotecan (CPT-11, 2),6which have been approved for clinical use.
In one of our research projects on the discovery and develop-ment of new anticancer agents, we have synthesized a series of DNA-directed alkylating agents, in which the phenyl N-mustard pharmacophore (warhead) is linked to the DNA-affinic molecule (carrier, such as 9-anilinoacridines, acridines or quinolines) via a urea, carbamate or hydrazinecarboxamide linker.7–9These linkers were previously utilized for preparing antibody-directed enzyme prodrug therapy (ADEPT),10 gene-directed enzyme prodrug ther-apy (GDEPT),11 or melanocyte-directed enzyme prodrug therapy (MDEPT). It demonstrated that these linkers are able to reduce the chemical reactivity of the reactive N-mustard moiety.12–14 We also reported that the N-mustard-DNA-affinic molecule conju-gates exhibit potent antitumor activity against various human tu-mor xenograft models. These conjugates are able to induce tu-more DNA interstrand cross-linking than other alkylating agents such as melphalan or cisplatin. However, these agents are generally lipophilic and have poor water-solubility, compromising antitumor and pharmacokinetic studies in animal models. To overcome the drawback of insufficient solubility of these agents, it is necessary to consider designing and synthesizing compounds with improved water-solubility during the period of discovery and development.
Earlier works on the developing water-soluble N-mustard deriv-atives by Denny and co-workers have introduced a variety of hydro-philic side chains to the benzene ring of 4-nitroaniline-N-mustards
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.bmc.2010.11.005
⇑ Corresponding author. Tel.: +886 2 2789 9045; fax: +886 2 2782 5573. E-mail address:[email protected](T.-L. Su).
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Bioorganic & Medicinal Chemistry
j o u r n a l h o m e p a g e : w w w . e l s e v i e r . c o m / l o c a t e / b m cderivatives (3) via a carboxamide linker to form water-soluble bior-eductive anticancer prodrugs.15Other studies also clearly demon-strated that linking a side chain containing a basic tertiary amino function can increase the aqueous solubility of drug by forming inorganic or organic acid salts.16–20These studies prompted us to design and synthesize chemical stable and water-soluble phenyl N-mustards, yet preserving the potent antitumor activity. We therefore prepared a series of water-soluble N-mustard-benzene conjugates via a urea linker. The benzene moiety contains a variety of water-soluble hydrophilic side chains including N,N-dimethyl-amino or cyclicN,N-dimethyl-amino functions, which can be converted into water-soluble derivatives by forming hydrochloride or other salts. The hydrophilic side chain in the benzene ring is located at the para-or meta-position to the urea linker via a carboxamide (compound 9aa0,bf0series,Scheme 1) or an ether linkage (compound 19aa0,be0 series,Scheme 2). These conjugates were subjected to antitumor evaluation. The results show that they exhibit potent antitumor activity in inhibiting human tumor xenografts. The chemical
synthesis, in vitro and in vivo antitumor activity as well as DNA cross-linking are described here in this paper.
2. Chemistry
The water-soluble phenyl N-mustard-benzene conjugates con-taining a hydrophilic carboxamide side-chain (9a,b series) were prepared starting from the commercially available ethyl 3- (or 4-)isocyanatobenzoate (4a,b) (Scheme 1). Thus, reactions of 4a,b with the known N,N-bis(2-chloroethyl)benzene-1,4-diamine hydrochloride (5)14afforded 6a,b. The products were hydrolyzed with concd HCl/AcOH (3:1 v/v) under reflux to yield benzoic acid derivatives 7a,b following the literature procedure.21Coupling of 7a,b with various amines (8a0–8f0) in dry DMF in the presence of DDC/HOBT/Et3N afforded the desired compounds 9aa0–9bf0in fair to good yields after purification by column chromatography. All final products obtained had poor water solubility, since they were in a free base form.
Chart 1.
Scheme 1. Reagents and conditions: (a) Et3N, THF, room temperature; (b) concd HCl/AcOH (3:1, v/v), reflux, 6 h; (c) DCC/HOBT, TEA, DMF, rt; (d) Et3N, CHCl3, rt; (e) 10% Pd/C/
Since compound 9aa0was selected for further antitumor studies based on its cytotoxicity against tumor cell lines tested (see be-low), an alternative method was developed to optimize the synthe-sis of this agent (Scheme 1). The reaction of 3-nitrobenzoyl chloride (11) with N,N-dimethylethylamine hydrochloride (8a0) yielded benzoylcarboxamides 12, which was then converted into the corresponding aniline derivatives 13 by catalytic hydrogena-tion (10% Pd/C, H2) in EtOH. Reaction of 13 with 4-[N,N-bis(2-chlo-roethyl)amino]phenylisocyanate (10)22 [freshly prepared by reacting 5 with triphosgene] in anhydrous DMF in the presence of triethylamine (TEA) afforded the desired 9aa0hydrochloride salt, which was found to be soluble in water. This synthetic method can be applied to preparing water-soluble 9aa0–9bf0hydrochloride or mesylate. For instance, we have synthesized 9ad0and 9bd0 hydro-chloride by following the same synthetic route as that for 9aa0. However, one may obtain free compounds during purification by column chromatography if a long column and a large amount of silica gel is used. We have also prepared water-soluble mesylate salt of 9aa0by treating with methane sulfonic acid in MeOH.
The synthetic route for the preparation of phenyl N-mustard-benzene conjugates containing a hydrophilic ether side-chain (19a,b series) is depicted inScheme 2. The reactions of the potas-sium salt of 3- (or 4-)nitrophenols (15a and 15b, respectively, freshly prepared from the corresponding 14a,b with KOH in EtOH) with various
x
-aminoalkyl halides (16a0–16e0) in dry toluene affor-ded nitro substituted phenyl ether derivatives 17a,b following the literature procedure.23The nitro function of 17a,b was reduced to the corresponding aniline derivatives 18a,b by catalytic hydroge-nation (10% Pd/C/H2). The products 18a,b were then reacted with freshly prepared phenylisocyanate 10 in the presence of triethyl-amine to yield the final products 19aa0–19be0. Similarly, the hydrochloride salt may be converted into its free form during purification by liquid column chromatography.It should be noted that compounds 9aa0–9bf0were synthesized as a free base form as one can identify from their1H NMR spectro-photometer spectrum. For example, the protons of NMe2 and NCH2-functions in 9aa0, 9ab0, 9ba0, and 9bb0, which exist as a free base and have poor water-solubility, are located near d 2.21 and 2.40, respectively. In contrast, the corresponding functions in 9aa0hydrochloride or mesylate salt, which are water-soluble, have a chemical shift at lower field, near d 2.82 and 3.26, respectively. Similar observations were found in the series of 19aa0–19ae0, in which the corresponding protons are located at higher fields (near d2.21 and 2.61, respectively), indicating that these agents exist in a free base form. The same protons in derivatives 19ba0–19be0have higher chemical shift values, demonstrating that they exist as
hydrochloride salt. The assignment for whether compounds form a free base or salt is further confirmed by comparison with 17aa0–17be0and 18aa0–18be0(see1H NMR spectral data in Supple-mentary data). The results demonstrated that the ether linker lo-cated at the para-position to the urea moiety (19ba0–19be0) can easily form hydrochloride salts under the reaction conditions, but that has not happened in the corresponding meta-substituted derivatives (19aa0–19ae0). Although we are able to convert the free derivatives into its hydrochloride salts by dissolving compounds in a mixture of CHCl3/MeOH followed with 20% HCl in ethyl acetate (data not shown), we surprisingly found that both free base (19aa0–19ae0) and hydrochloride salt forms (19ba0–19be0) have poor water-solubility.
3. Biological results and discussion 3.1. In vitro cytotoxicity
Human lymphoblastic leukemia (CCRF/CEM) and its drug-tant sublines (CCRF-CEM/taxol and CCRF-CEM/VBL (330-fold resis-tant to taxol and 680-fold resisresis-tant to vinblastine, respectively) were used to evaluate the cytotoxicity of tested compounds and to realize whether they had multi-drug resistance (MDR) toward taxol or vinblastine in our antitumor screening program. The antiproliferative activities of the newly synthesized N-mustard-benzene conjugates are summarized in Table 1. The structure-activity relationship study shows that the newly synthesized compounds exhibit significant cytotoxicity with IC50 values of sub-micromolar range in inhibiting CCRF/CEM cell growth in culture. There is no significant difference in term of their cytotox-icities affected by the type of side-chain (carboxamide or ether linkage) and the location of the substituent (meta- or para-posi-tion). In general, compounds having an ether linkage are slightly more potent than the corresponding compounds bearing a carbox-amide side-chain. Of this series of compounds, 9aa0is the most cyto-toxic. In the series of compounds having a carboxamide side-chain, the tertiary amino substituent (
x
-N,N-dimethylamino-, N-alkylpyr-roliny- or N-alkylpiperidinyl function) does not have a big influence to their potency. However, the N-alkylmorpholinyl derivatives (9ae0 and 9be0) are less cytotoxic against CCRF-CEM among the tested compounds. In the series of compounds having an ether linkage, derivatives having an ether side-chain located at the para-position to the urea linker are more cytotoxic than or as potent as the corre-sponding meta-substituted derivatives. Similarly, the N-alkylmorp-holinyl derivatives (19ae0 and 19be0) are slightly less cytotoxic against CCRF-CEM in comparison with other derivatives.We investigated whether the newly synthesized compounds are multidrug resistant to the distinct drugs, such as taxol or vinbla-stin. The in vitro cytotoxicity of these derivatives against CCRF-CEM/taxol and CCRF-CEM/VBL reveal that they generally have no
or little cross-resistance to these two natural products except com-pounds 9ab0, 9ac0, 9af0, 9bb0, 9bc0, and 9bf0, which have certain ex-tent of cross-resistance (Table 1). It also demonstrates that compounds having an ether side-chain (compound 19a,b series)
Table 1
The cytotoxicity of new N-mustards against human lymphoblastic leukemia (CCRF/CEM) and its drug-resistant sublines (CCRF-CEM/taxol and CCRF-CEM/VBL)a
HN N H O R O H N N(CH2CH2Cl)2 9aa'-9bf' HN OR O H N N(CH2CH2Cl)2 19aa'-19be' Compd R Cytotoxicity, IC50(lM) CCRF-CEM CCRF-CEM/taxolb CCRF-CEM/VBLb 9aa0 (CH 2)2NMe2 0.13 ± 0.002 1.22 ± 0.02 [9.4]c 0.80 ± 0.01 [6.2] 9ab0 (CH 2)3NMe2 0.29 ± 0.012 21.20 ± 0.15 [9.4] 44.03 ± 0.08 [151.8] 9ac0 (CH 2)2N 0.34 ± 0.001 25.20 ± 0.45 [73.7] 36.56 ± 0.24 [106.9] 9ad0 (CH 2)2N 0.27 ± 0.02 4.62 ± 0.01 [17.0] 10.49 ± 0.46 [43.7] 9ae0 (CH 2)2N O 0.58 ± 0.002 8.16 ± 0.18 [14.1] 16.29 ± 0.11 [28.1] 9af0 N N 0.28 ± 0.003 12.19 ± 0.66 [43.5] 20.04 ± 0.48 [71.6] 9ba0 (CH 2)2NMe2 0.23 ± 0.002 4.80 ± 0.07 [21.1] 6.74 ± 0.29 [29.6] 9bb0 (CH 2)3NMe2 0.26 ± 0.01 20.39 ± 0.80 [77.5] 32.82 ± 0.34 [124.8] 9bc0 (CH 2)2N 0.27 ± 0.010 19.67 ± 0.45 [71.8] 30.82 ± 0.34 [112.5] 9bd0 (CH 2)2N 0.36 ± 0.001 14.97 ± 1.47 [41.8] 24.28 ± 0.29 [67.8] 9be0 (CH 2)2N O 0.73 ± 0.01 19.82 ± 0.19 [27.1] 29.76 ± 0.26 [16.8] 9bf0 N N 0.22 ± 0.01 20.47 ± 0.66 [91.4] 32.18 ± 0.12 [143.7] 19aa0 (CH 2)2NMe2 1.32 ± 0.001 0.81 ± 0.001 [0.6] 1.48 ± 0.003 [1.1] 19ab0 (CH 2)3NMe2 0.32 ± 0.01 2.05 ± 0.01 [6.4] 2.37 ± 0.35 [7.4] 19ac0 (CH 2)2N 0.35 ± 0.01 1.20 ± 0.002 [3.5] 4.20 ± 0.01 [12.1] 19ad0 (CH 2)2N 0.19 ± 0.003 0.57 ± 0.02 [3.0] 0.73 ± 0.01 [3.8] 19ae0 (CH 2)2N O 0.41 ± 0.10 0.73 ± 0.07 [1.8] 1.13 ± 0.58 [2.8] 19ba0 (CH 2)2NMe2 0.10 ± 0.002 1.50 ± 0.01 [7.6] 1.19 ± 0.04 [6.02] 19bb0 (CH 2)3NMe2 0.12 ± 0.001 1.36 ± 0.001 [11.8] 1.26 ± 0.001 [10.9] 19bc0 (CH 2)2N 0.14 ± 0.001 1.57 ± 0.001 [11.5] 2.05 ± 0.10 [15.0] 19bd0 (CH 2)2N 0.12 ± 0.003 0.50 ± 0.002 [4.02] 0.72 ± 0.03 [7.47] 19be0 (CH 2)2N O 0.35 ± 0.01 0.84 ± 0.01 [2.4] 0.64 ± 0.01 [1.8] Taxol — 0.003 ± 0.0001 0.43 ± 0.04 [330] 1.27 ± 0.05 [980] Vinblastine — 0.0007 ± 0.0001 0.08 ± 0.01 [106.2] 0.50 ± 0.12 [679.5] a
Cell growth inhibition was measured by the XTT assay26
for leukemic cells and the SRB assay27
for solid tumor cells after 72-h incubation using a microplate spectro-photometer as described previously.28
Similar in vitro results were obtained by using the Cell Counting Kit-8 for the CCK-8 assays as described by technical manual of Dojindo Molecular Technologies, Inc. (Gaithersburg, MD; website:www.dojindo.com). IC50values were determined from dose-effect relationship at six or seven concentrations of
each drug by using the CompuSyn software by Chou and Martin30based on the median-effect principle and plot using the serial deletion analysis.31,32Ranges given for taxol
and vinblastine were mean ± SE (n = 4).
b
CCRF-CEM/taxol and CCRF-CEM/VBL are subcell lines of CCRF-CEM cells that are 330-fold resistant to taxol, and 680-fold resistant to vinblastine, respectively, when comparing with the IC50of the parent cell line.
c
Table 2
Cytotoxicity of new N-mustards against human solid tumors (H1299, CL1–0, CL1–5, PC3, HCT-116, MX-1 and MCF-7) cell growth in vitro Compd Cytotoxicity, IC50(lM) H1299a CL1–0a CL1–5a PC3a HCT-116b MX-1b MCF-7a 9aa0 10.37 ± 2.67 14.59 ± 2.01 6.08 ± 1.01 1.88 ± 0.73 0.69 ± 0.03 0.57 ± 0.02 0.63 ± 0.11 9ab0 ND ND ND ND 0.85 ± 0.01 2.33 ± 0.05 ND 9ac0 ND ND ND ND 0.47 ± 0.001 2.17 ± 0.01 ND 9ad0 6.22 ± 1.91 8.67 ± 1.02 4.64 ± 1.43 1.70 ± 0.67 0.31 ± 0.002 0.73 ± 0.004 1.18 ± 0.95 9ae0 ND ND ND ND 1.21 ± 0.072 1.45 ± 0.01 ND 9af0 11.47 ± 2.99 6.23 ± 1.67 8.09 ± 1.49 3.19 ± 0.99 0.81 ± 0.072 2.24 ± 0.01 3.09 ± 1.01 9ba0 ND ND ND ND 0.39 ± 0.002 1.09 ± 0.001 ND 9bb0 12.70 ± 2.99 16.2 ± 4.32 9.79 ± 2.45 5.25 ± 1.77 4.54 ± 0.02 1.11 ± 0.023 5.17 ± 1.91 9bc0 ND ND ND ND 0.36 ± 0.01 2.33 ± 0.07 ND 9bd0 ND ND ND ND 0.91 ± 0.02 4.40 ± 0.05 ND 9be0 ND ND ND ND 2.74 ± 0.001 3.39 ± 0.16 ND 9bf0 9.63 ± 2.01 15.84 ± 3.10 8.51 ± 1.56 1.91 ± 0.99 0.64 ± 0.01 3.64 ± 0.01 1.54 ± 0.85 19ad0 1.52 ± 0.79 2.73 ± 0.91 3.25 ± 1.01 1.13 ± 0.89 0.27 ± 0.01 0.19 ± 0.01 1.82 ± 0.95 19ba0 4.38 ± 1.06 4.07 ± 1.59 3.01 ± 0.53 1.67 ± 0.73 0.27 ± 0.001 0.28 ± 0.003 1.25 ± 0.99 19bb0 9.79 ± 1.56 7.09 ± 1.42 5.11 ± 1.34 0.94 ± 0.23 0.29 ± 0.001 0.26 ± 0.01 1.32 ± 0.32 19bc0 2.25 ± 0.88 3.01 ± 0.94 5.92 ± 1.56 1.78 ± 0.29 0.70 ± 0.0002 0.49 ± 0.01 1.84 ± 0.77 19bd0 1.84 ± 0.91 2.87 ± 0.68 4.75 ± 1.12 0.93 ± 0.14 0.27 ± 0.001 0.15 ± 0.01 1.24 ± 0.39 a
Cell growth inhibition was determined by the Alamar blue assay29
in a 72 h incubation using a microplate spectrophotometer as described previously.
b
Cell growth inhibition was measured by the SRB assay27
for solid tumor cells after 72-h incubation using a microplate spectrophotometer as described previously.28
Figure 1. Therapeutic effect of 9aa0in nude mice bearing human breast carcinoma MX-1 xenograft (30 mg/kg, Q2D 5, iv injection, n = 5); Treatment started on Day 8 after
tumor implantation. All treatments were carried out via iv injection. CR is referred as complete tumor remission. (A) Average tumor size changes; (B) average body weight changes.
have less MDR in comparison with the corresponding derivatives bearing a carboxamide side-chain (compound 9a,b series).
To further explore the antiproliferative activity of the new syn-thesized N-mustards, the selected compounds were studied for their cytotoxicity in inhibiting other human solid tumors such as human non-small cell lung cancer (H1299), lung adenocarcinoma (CL1–0 and CL1–5), colon cancer (HCT-116), prostate cancer (PC3) and human breast tumor (MX-1 and MCF-7) cell growth in vitro (Table 2). The results showed that these agents, in general, are more cytotoxic toward human prostate, colon and breast tu-mors among the tumor cell lines tested. It is of great interest to note that the tested compounds have almost equal potency against both MX-1 and resistant MCF-7 cell lines in vitro. Of these agents,
compounds 19ad0and 19bd0are shown to have a broad spectrum of antitumor activity against the tested tumor cell growth in culture.
3.2. In vivo therapeutic effect
In the present study, we evaluated the antitumor activity of compound 9aa0 since this agent shows to be the most cytotoxic against solid tumor cell lines tested.Figure 1 shows the thera-peutic efficacy of this agent in nude mice bearing human adeno-carcinoma MX-1 xenograft. The complete tumor remission (CR) was achieved at the maximum tolerable doses of 30 mg/kg (Q2D 5, intravenous injection), and maintained for over
Figure 2. Therapeutic effect of 9aa0in nude mice bearing human prostate cancer PC3 xenograft (30 mg/kg (Q2D 4), followed by 40 mg/kg (Q2D 3), iv injection n = 4).
70 days. The therapeutic effect of compound 9aa0 was further investigated against human prostate carcinoma PC3 xenograft in nude mice. As shown in Figure 2, more than 99% of tumor suppression was observed at the dose of 30 mg/kg (Q2D 4), followed by 40 mg/kg (Q2D 3). We have also evaluated the antitumor activity of compound 9aa0 in nude mice bearing hu-man colon cancer HCT-116 xenograft (Fig. 3). More than 95% tu-mor suppression was observed at the dose of 30 mg/kg (Q2D 5, iv injection). In comparison, the antitumor effects of 9aa0 (30 mg/kg, Q2D 6, iv injection n = 4, where n = number of mice tested per experiment) with cyclophosphamide (80 mg/kg, Q2D 6, iv injection) in nude mice bearing human glioma U87 MG xenograft (Fig. 4) revealed that the former compound was more potent, but less toxic than that of the latter drug. In all in vivo xenograft experiments, body weight is referred to total body weight minus tumor weight assuming 1 mm3= 1 mg.
3.3. DNA cross-linking study
To realize whether the newly synthesized compounds are capa-ble of cross-linking with DNA doucapa-ble strands, pEGFP-N1 DNA was reacted with compounds, 9aa0, 9ad0, 19bd0, and 19bb0 at various concentrations as indicated (1, 5, and 10
l
M) and subjected to alkaline agarose gel shifting assay after BamH1 digestion (Fig. 5).24Melphalan was used as the positive control. As shown inFig. 5, all the tested compounds were able to induce DNA inter-strand cross-linking, suggesting that DNA cross-linking may be the main mechanism of action for these agents.3.4. Cell cycle inhibition
Previous studies demonstrated that DNA-interacting agents damage DNA, inducing G2 arrest in the cell cycle through the G2
Figure 3. Therapeutic effects of 9aa0in nude mice bearing HCT-116 xenograft (30 mg/kg (Q2D 5, iv injection n = 4). (A) Average tumor size changes; (B) average body
DNA-damage checkpoint pathway.25We studied the inhibitory ef-fect of 9aa0on cell cycle distribution (Table 3). The human non-small lung carcinoma H1299 cells were treated with 9aa0 at the concentrations of 2.5, 5, and 10
l
M for 24 h. The cells were har-vested, stained with propidium iodide (PI) and analyzed with a flow cytometer. As shown inTable 3, 9aa0treatment induced sig-nificant G2/M arrest in H1299 cells. Similar G2/M arrest was previ-ously observed in SW626 cells treated with melphalan.26 Furthermore, we also found that increased sub-G1 populations were noticed in H1299 cells treated with 9aa0(Table 3).3.5. Pharmacokinetic profile of 9aa0
Prior to clinical studies, our leading compound 9aa0 were subjected to pharmacokinetic studies in healthy male Sprague
Dawley rats. A single intravenous dose was administered via an indwelling catheter in jugular vein to a group of two male rats at a dose level of 1.0 mg/kg. The formulations were prepared as a solution in 5.0% w/v DMSO with 10% w/v Cremophor in di-distilled water. Serial blood samples were collected from jugular vein catheter up to 24 h post-dose from all animals in Group. Concentration levels of 9aa0 were determined in plasma using a validated LC–MS/MS assay with a lower limit of quantification (LLOQ) of 2.5 ng/mL. The plasma concentration-time data above the LLOQ at each dose level were used in the calculation of phar-macokinetic parameters of 9aa0 using the validated program WinNonlin™, version 5.2.1. Pharmacokinetic parameters are summarized in Table 4. The result showed that the mean terminal half-life (t1/2) and mean residence time (MRT) of 9aa0 were 0.58 h and 0.11 h, respectively. The mean apparent plasma
Figure 4. Therapeutic effect of 9aa0(30 mg/kg, Q2D 6, iv injection n = 4) and cyclophosphamide (80 mg/kg, Q2D 6, iv injection n = 4) in nude mice bearing human glioma
clearance of 9aa0 was 18.0 mL/min/kg with the apparent volume of distribution at steady state of 0.15 L/kg.
4. Conclusion
In the present studies, we have designed and synthesized a ser-ies of water-soluble DNA alkylating N-mustard derivatives, in which the phenyl N-mustard pharmacophore is attached to a ben-zene ring via a urea linker. We have demonstrated that these con-jugates can be easily converted into water-soluble derivatives by forming hydrochloride or mesylate salts. The newly synthesized compounds exhibited significant antitumor activity both in vitro and in vivo against various human tumor xenografts. Detailed structure-activity relation studies revealed that the types of the hydrophilic side-chain (carboxamide or ether) linked to the ben-zene ring does not greatly affect their cytotoxicities. Moreover, we showed that these derivatives have little or no cross-resistance to either taxol or vinblastine. Among the newly synthesized deriv-atives, we selected compound 9aa0 for further in vivo antitumor evaluation since this agent shows to be the most cytotoxic against solid tumor cell lines tested. Remarkably, complete tumor remission in nude mice bearing human breast carcinoma MX-1
xenograft and significant suppression against prostate adenocarci-noma PC3 xenograft were achieved with acceptable toxicity. We also demonstrate that the newly synthesized compounds are able to induce DNA cross-linking by alkaline agarose gel shift assay. Furthermore, the pharmacokinetic study reveals that 9aa0 has a good pKaprofile in rats with a half-life of 0.58 h. The present stud-ies suggest that this agent is a promising candidate for preclinical studies.
5. Experimental section
5.1. Chemistry: general methods
All commercial chemicals and solvents were reagent grade and were used without further purification unless otherwise specified. Melting points were determined on a Fargo melting point appara-tus and are uncorrected. Column chromatography was carried out on Silica Gel G60 (70–230 mesh, ASTM; Merck and 230–400 mesh, Silicycle Inc.). Thin-layer chromatography was performed on Silica Gel G60 F254(Merck) with short-wavelength UV light for visualiza-tion. All reported yields are isolated yields after chromatography or crystallization. Elemental analyses were done on a Heraeus CHN-O Rapid instrument.1H NMR spectra were recorded on a 600 MHz, Brucker AVANCE 600 DRX and 400 MHz, Brucker Top-Spin spec-trometers in the indicated solvent. The chemical shifts were re-ported in ppm (d) relative to TMS. High performance liquid chromatography analyses for checking purity of synthesized com-pounds were recorded on a Hitachi D-2000 Elite instrument: col-umn, Mightysil RP-18 GP 250-4.6 (5
l
m); mobile phase, 90% A, 5% B, and 5% C in 25 min (mobile phase A = acetonitrile, B = THF, and C = H2O); flow rate, 1 mL/min; injected sample 10l
L, column temp, 27 °C; wavelength, 254 nm. The purity of all compounds was P95% based on analytical HPLC.5.2. Synthesis of water-soluble N-mustards having a carbox-amide linker (9aa0,bf0series)
5.2.1. Ethyl 3-[3-(4-(bis(2-chloroethyl)amino)phenyl)ureido]-benzoate (6a)
To a suspension of N,N-bis(2-chloroethyl)benzene-1,4-diamine hydrochloride (5, 6.12 g, 20 mmol) in dry chloroform (100 mL)
Figure 5. Representative DNA cross-linking gel shift assay for 9aa0, 9ad0, 19bd0, and
19bb0at various concentrations as indicated. Control lane shows single stranded
DNA (SL), while cross-linking (CL) shown in all tested lanes is DNA double-stranded cross-linking. Melphalan (1 and 10lM) was used as a positive control.
Table 4
Summary of pharmacokinetic parameters of 9aa0following intravenous injection to rats (n = 2)
Dose (mg/kg) C0(ng/mL) t1/2(h) MRT (h) AUC(0–last)(h ng/mL) AUC(0–1)(h ng/mL) CLss(mL/min/kg) Vss(L/kg)
1 11,980 0.58 0.11 925.81 934.93 18.00 0.15
Table 3
Cell cycle inhibition in human non-small cell lung adenocarcinoma H1299 by treating with compound 9aa0
Concentration (lM) 0 2.5 5 10
Sub-G1 14.1 ± 0.7 20.0 ± 4.9 19.7 ± 4.5 26.0 ± 4.7 G1 42.9 ± 0.8 2.3 ± 1.0 10.7 ± 3.9 6.5 ± 3.2
S 22.5 ± 0.3 1.8 ± 2.6 8.8 ± 1.4 5.2 ± 4.6
was added TEA (3.5 mL) at 10 °C. A solution of ethyl 3-iso-cyanatobenzoate (4a, 3.32 mL, 20 mmol) in dry chloroform was added dropwise to the above mixture at the same temperature. The reaction mixture was then allowed to stir at room temperature for 12 h. The reaction mixture was evaporated reduced pressure. The product was purified by column chromatography using CH2Cl2/MeOH (100:2, v/v) as an eluent. The fraction containing the main product were combined and evaporated to dryness. The white solid residue was triturated with hexane, collected by filtra-tion, and dried to give 6a, yield: 6.81 g (80%); mp 164–165 °C;1H NMR (DMSO-d6) d 1.32 (3H, t, J = 7.2 Hz, Me), 3.67–3.72 (8H, m, 4 CH2), 4.31 (2H, q, J = 7.2 Hz, CH2), 6.71 (2H, d, J = 8.8 Hz, 2 ArH), 7.28 (2H, d, J = 8.8 Hz, 2 ArH), 7.40 (1H, t, J = 7.2 Hz, ArH), 7.53 (1H, d, J = 8.0 Hz, ArH), 7.63–7.65 (1H, m, ArH), 8.12 (1H, s, ArH), 8.34 and 8.80 (each 1H, br s, exchangeable, 2 NH). Anal. Calcd for C20H23Cl2N3O3: C, H, N.
5.2.2. Ethyl 4-[3-(4-(bis(2-chloroethyl)amino)phenyl)ureido]-benzoate (6b)
Compound 3b was synthesized by following the same proce-dure as that for 3a and prepared from N,N-bis(2-chloroethyl)ben-zene-1,4-diamine hydrochloride (5, 7.65 g, 25 mmol) and ethyl 4-isocyanatobenzoate (4b, 4.78 g, 25 mmol). Yield 8.11 g (76%); mp 221–223 °C;1H NMR (DMSO-d
6) d 1.30 (3H, t, J = 7.2 Hz, Me), 3.70–3.71 (8H, m, 4 CH2), 4.27 (2H, q, J = 7.2 Hz, CH2), 6.72 (2H, d, J = 8.8 Hz, 2 ArH), 7.29 (2H, d, J = 8.8 Hz, 2 ArH), 7.56 (2H, d, J = 8.8 Hz, 2 ArH), 7.86 (2H, d, J = 8.8 Hz, 2 ArH), 8.46 and 8.96 (each 1H, br s, exchangeable, 2 NH). Anal. Calcd for C20H23Cl2N3O3: C, H, N.
5.2.3. 3-[3-(4-(Bis(2-chloroethyl)amino)phenyl)ureido]benzoic acid (7a)
A solution of 6a (4.253 g, 10 mmol) in mixture of concd HCl/ AcOH (100 mL, 3:2 v/v) was heated at 100 °C for 6 h. The reaction mixture was cooled to room temperature, the separated white so-lid was collected by filtration, the soso-lid cake was washed with water and dried to give 7a; yield: 3.52 g (89%); mp 191–192 °C; 1H NMR (DMSO-d
6) d 3.70 (8H, s, 4 CH2), 6.74 (2H, d, J = 8.8 Hz, 2 ArH), 7.30 (2H, d, J = 8.8 Hz, 2 ArH), 7.30 (1H, t, J = 8.0 Hz, ArH), 7.51 (1H, d, J = 7.6 Hz, ArH), 7.63–7.65 (1H, m, ArH), 8.09 (1H, s, ArH), 8.09 and 8.82 (each 1H, br s, exchangeable, 2 NH). Anal. Calcd for C18H19Cl2N3O32H2O: C, H, N.
5.2.4. 4-[3-(4-(Bis(2-chloroethyl)amino)phenyl)ureido]benzoic acid (7b)21
Compound 4b was synthesized by following the same proce-dure as that for 7a and was prepared from 6b (2.13 g, 5 mmol) in a mixture of conc. HCl/AcOH (70 mL, 3:2 v/v). Yield 1.78 g (90%); mp 238–241 °C (reported >300 °C, decomp.);21 1H NMR (DMSO-d6) d 3.69–3.73 (8H, m, 4 CH2), 6.74 (2H, d, J = 8.8 Hz, 2 ArH), 7.30 (2H, d, J = 8.8 Hz, 2 ArH), 7.54 (2H, d, J = 8.8 Hz, 2 ArH), 7.83 (2H, d, J = 8.8 Hz, 2 ArH), 8.88 and 9.39 (each 1H, br s, exchangeable, 2 NH). Anal. Calcd for C18H19Cl2N3O32H2O: C, H, N.
5.2.5. 1-[3-((2-(Dimethylamino)ethyl)carbamoyl)phenyl]-3-[4-(bis(2-chloroethyl)amino)-phenyl]urea hydrochloride (9aa0)
A mixture of 7a (0.792 g, 2 mmol), DCC (0.618 g, 3 mmol), HOBT (0.505 g, 3 mmol), TEA (0.4 mL) and N,N-dimethylethylenediamine (8a0, 0.264 g, 3 mmol) in dry DMF (25 mL) was stirred at room tem-perature for 72 h. The reaction mixture was filtered to remove the by-product urea and the filtrate was evaporated in vacuo to dry-ness. The residue was dissolved in CH2Cl2(250 mL) and succes-sively washed with water (100 mL), satd NaHCO3 aqueous solution (100 mL) and brine (100 mL), dried over Na2SO4and fil-tered. The filtrate was evaporated to dryness and the residue was
chromatographed over a silica gel column using CH2Cl2/MeOH (100:9) as an eluent. The fractions containing the main product were combined and evaporated in vacuo to dryness. The residue was crystallized from ethyl acetate to give white solid. The solid was suspended in ethyl acetate (50 mL) and treated with 2.5 M HCl in ethyl acetate (5.0 mL) at 0 °C. The excess solvent was evap-orated to dryness and solid separated was dried in vacuo to give desired product 9aa0, 580 mg (58%); mp 156–157 °C; 1H NMR (DMSO-d6) d 2.20 (6H, s, 2 NMe), 2.42 (2H, t, J = 6.8 Hz, CH2), 3.32–3.35 (2H, m, CH2), 3.68–3.70 (8H, m, 4 CH2), 6.71 (2H, d, J = 8.8 Hz, 2 ArH), 7.29 (2H, d, J = 8.8 Hz, 2 ArH), 7.34–7.39 (2H, m, 2 ArH), 7.59 (1H, d, J = 7.6 Hz, ArH), 7.83 (1H, s, ArH), 8.29 (1H, t, J = 5.2 Hz, exchangeable, CONH), 8.40 and 8.72 (each 1H, br s, exchangeable, 2 NH). Anal. Calcd for C22H29Cl2N5O2: C, H, N.
By following the same procedure as described for 9aa0, the fol-lowing compounds were synthesized.
5.2.6. 1-[3-((3-(Dimethylamino)propyl)carbamoyl)phenyl]-3-[4-(bis(2-chloroethyl)amino)-phenyl]urea (9ab0)
Compound 9ab0was prepared from 7a (0.792 g, 2 mmol), DCC (0.618 g, 3 mmol), HOBT (0.505 g, 3 mmol), TEA (0.4 mL) and N,N-dimethylpropane-1,3-diamine (8b0, 0.305 g, 3 mmol). Yield, 645 mg (65%); mp 138–140 °C;1H NMR (DMSO-d 6) d 1.61–1.66 (2H, m, CH2), 2.13 (6H, s, 2 NMe), 2.25 (2H, t, J = 6.8 Hz, CH2), 3.27 (2H, q, J = 6.8 Hz, CH2), 3.66–3.72 (8H, m, 4 CH2), 6.71 (2H, d, J = 8.8 Hz, 2 ArH), 7.29 (2H, d, J = 8.8 Hz, 2 ArH), 7.32–7.38 (2H, m, 2 ArH), 7.58 (1H, d, J = 8.0 Hz, ArH), 7.84 (1H, s, ArH), 8.40 and 8.72 (each 1H, br s, exchangeable, 2 NH), 8.46 (1H, t, J = 5.2 Hz, exchangeable, CONH). Anal. Calcd for C23H31Cl2N5O22H2O: C, H, N.
5.2.7. 1-[3-((2-(Pyrrolidin-1-yl)ethyl)carbamoyl)phenyl]-3-[4-(bis(2-chloroethyl)amino)-phenyl]urea hydrochloride (9ac0)
Compound 6ac0was prepared from 7a (0.792 g, 2 mmol), DCC (0.618 g, 3 mmol), HOBT (0.505 g, 3 mmol), TEA (0.4 mL) and 1-(2-aminoethyl)pyrolidine (8c0, 0.341 g, 3 mmol). Yield, 591 mg (56%); mp 129–131 °C;1H NMR (DMSO-d
6) d 1.87–1.91 (5H, m, CH), 3.0 (2H, t, J = 5.2 Hz, CH2), 3.32 (2H, d, J = 6.0 Hz, CH2), 3.62– 3.64 (3H, m, CH), 3.72 (8H, s, 4 CH2), 6.73 (2H, d, J = 8.8 Hz, 2 ArH), 7.30 (2H, d, J = 8.8 Hz, 2 ArH), 7.34–7.36 (2H, m, 2 ArH), 7.62 (1H, d, J = 9.2 Hz, ArH), 7.94 (1H, s, ArH), 8.78–8.79 (1H, m, exchangeable, CONH), 9.0 and 9.33 (each 1H, br s, exchangeable, 2 NH). Anal. Calcd for C24H31Cl2N5O22H2O: C, H, N.
5.2.8. 1-[3-((2-(Piperidin-1-yl)ethyl)carbamoyl)phenyl]-3-(4-(bis(2-chloroethyl)amino)-phenyl)urea hydrochloride (9ad0)
Compound 9ad0was prepared from 7a (0.792 g, 2 mmol), DCC (0.618 g, 3 mmol), HOBT (0.505 g, 3 mmol), TEA (0.4 mL) and 1-(2-aminoethyl)piperidine (8d0, 0.384 g). Yield, 683 mg (63%); mp 151–152 °C;1H NMR (DMSO-d6) d 1.38–1.51 (7H, m, CH), 2.41– 2.45 (5H, m, CH), 3.34–3.38 (2H, m, CH), 3.66–3.72 (8H, m, 4 CH2), 6.71 (2H, d, J = 8.8 Hz, 2 ArH), 7.29 (2H, d, J = 8.8 Hz, 2 ArH), 7.32–7.38 (2H, m, 2 ArH), 7.58 (1H, d, J = 7.6 Hz, ArH), 7.84 (1H, s, ArH), 8.28 (1H, t, J = 5.2 Hz, exchangeable, CONH), 8.40 and 8.70 (each 1H, br s, exchangeable, 2 NH). Anal. Calcd for C25H33Cl2N5O2: C, H, N.
5.2.9. 1-[3-((2-Morpholinoethyl)carbamoyl)phenyl]-3-[4-(bis(2-chloroethyl)amino)phenyl]- urea hydrochloride (9ae0)
Compound 9ae0was prepared from 7a (0.792 g, 2 mmol), DCC (0.618 g, 3 mmol), HOBT (0.505 g, 3 mmol), TEA (0.4 mL) and 1-(2-aminoethyl)morpholine (8e0, 0.260 g, 3 mmol). Yield, 588 mg (54%); mp 167–169 °C;1H NMR (DMSO-d6) d 2.41–2.47 (5H, m, CH), 3.36–3.38 (2H, m, CH), 3.57 (5H, br s, CH), 3.69 (8H, s,
4 CH2), 6.71 (2H, d, J = 8.8 Hz, 2 ArH), 7.29 (2H, d, J = 8.8 Hz, 2 ArH), 7.33–7.40 (2H, m, 2 ArH), 7.59 (1H, d, J = 7.2 Hz, ArH), 7.91 (1H, s, ArH), 8.30 (1H, t, J = 5.6 Hz, exchangeable, CONH), 8.34 and 8.66 (each 1H, br s, exchangeable, 2 NH). Anal. Calcd for C24H31Cl2N5O3: C, H, N.
5.2.10. 1-[4-(Bis(2-chloroethyl)amino)phenyl]-3-[3-(4-(piperi-din-1-yl)piperidine-1-carbonyl] phenyl)urea hydrochloride (9af0)
Compound 6af0 was prepared from 7a (0.792 g, 2 mmol), DCC (0.618 g, 3 mmol), HOBT (0.505 g, 3 mmol), TEA (0.4 mL) and 4-piperidino-piperidine (8f0, 0.504 g, 3 mmol). Yield, 700 mg (60%); mp 118–119 °C; 1H NMR (DMSO-d6) d 1.37–1.48 (8H, m, CH), 1.68–1.79 (2H, m, CH), 2.47–2.50 (3H, m, CH), 2.73 (1H, br s, CH), 2.98 (1H, br s, CH), 3.37 (4H, br s, CH), 3.66–3.72 (8H, m, 4 CH2), 6.71 (2H, d, J = 8.8 Hz, 2 ArH), 6.90–6.92 (1H, d, J = 7.2 Hz, ArH) 7.27 (2H, d, J = 8.8 Hz, 2 ArH), 7.29–7.32 (1H, m, ArH), 7.41 (1H, d, J = 8.0 Hz, ArH), 7.51 (1H, s, ArH), 8.42 and 8.71 (each 1H, br s, exchangeable, 2 NH). Anal. Calcd for C28H37Cl2N5O2: C, H, N.
5.2.11. 1-[4-((2-(Dimethylamino)ethyl)carbamoyl)phenyl]-3-[4-(bis(2-chloroethyl)amino)-phenyl]urea hydrochloride (9ba0)
Compound 9ba0was prepared from 7b (0.396 g, 1 mmol), DCC (0.309 g, 1.5 mmol), HOBT (0.202 g, 1.5 mmol), TEA (0.2 mL) and 8a0, (0.132 g, 1.5 mmol). Yield, 340 mg (72%); mp 189–190 °C;1 H NMR (DMSO-d6) d 2.17 (6H, s, 2 NMe), 2.38 (2H, br s, CH2), 3.28 (2H, br s, CH2), 3.69 (8H, br s, 4 CH2), 6.71 (2H, d, J = 8.8 Hz, 2 ArH), 7.29 (2H, d, J = 8.8 Hz, 2 ArH), 7.49 (2H, d, J = 8.8 Hz, 2 ArH), 7.76 (2H, d, J = 8.8 Hz, 2 ArH), 8.18, 8.47 and 8.83 (each 1H, br s, exchangeable, 3 NH). Anal. Calcd for C22H29Cl2N5O2: C, H, N.
5.2.12. 1-[4-((3-(Dimethylamino)propyl)carbamoyl)phenyl]-3-[4-(bis(2-chloroethyl)amino)-phenyl]urea (9bb0)
Compound 9bb0was prepared from 7b (0.594 g, 1.5 mmol), DCC (0.464 g, 2.25 mmol), HOBT (0.304 g, 2.25 mmol), TEA (0.3 mL) and 8b0(0.229 g, 2.25 mmol). Yield, 534 mg (69%); mp 203–205 °C;1H NMR (DMSO-d6) d 1.62–1.67 (2H, m, CH2), 2.13 (6H, s, 2 NMe), 2.25 (2H, t, J = 6.8 Hz, CH2), 3.24 (2H, q, J = 6.8 Hz, CH2), 3.69– 3.72 (8H, m, 4 CH2), 6.71 (2H, d, J = 8.8 Hz, 2 ArH), 7.29 (2H, d, J = 8.8 Hz, 2 ArH), 7.49 (2H, d, J = 8.8 Hz, 2 ArH), 7.75 (2H, d, J = 8.8 Hz, 2 ArH), 8.39 (1H, t, J = 5.2 Hz, exchangeable, CONH), 8.49 and 8.86 (each 1H, br s, exchangeable, 2 NH). Anal. Calcd for C23H31Cl2N5O22H2O: C, H, N.
5.2.13. 1-[4-((2-(Pyrrolidin-1-yl)ethyl)carbamoyl)phenyl]-3-[4-(bis(2-chloroethyl)amino) phenyl]urea hydrochloride (9bc0)
Compound 9bc0was prepared from 7b (0.594 g, 1.5 mmol), DCC (0.464 g, 2.25 mmol), HOBT (0.304 g, 2.25 mmol), TEA (0.3 mL) and 8c0(0.256 g, 2.25 mmol). Yield, 419 mg (53%); mp 183–185 °C;1H NMR (DMSO-d6) d 1.05–2.00 (4H, m, CH), 3.02–3.04 (2H, m, CH), 3.30–3.33 (2H, m, CH), 3.42–3.45 (4H, m, CH), 3.70 (8H, s, 4 CH2), 6.72 (2H, d, J = 8.8 Hz, 2 ArH), 7.29 (2H, d, J = 8.8 Hz, 2 ArH), 7.52 (2H, d, J = 8.8 Hz, 2 ArH), 7.85 (2H, d, J = 8.8 Hz, 2 ArH), 8.67–8.69 (1H, m, exchangeable, CONH), 9.01 and 9.48 (each 1H, br s, exchangeable, 2 NH). Anal. Calcd for C24H31Cl2N5O22H2O: C, H, N.
5.2.14. 1-[4-((2-(Piperidin-1-yl)ethyl)carbamoyl)phenyl]-3-(4-(bis(2-chloroethyl)amino)-phenyl)urea hydrochloride (9bd0)
Compound 9bd0was prepared from 7b (0.594 g, 1.5 mmol), DCC (0.464 g, 2.25 mmol), HOBT (0.304 g, 2.25 mmol), TEA (0.3 mL) and 8d0(0.288 g, 2.25 mmol). Yield, 400 mg (49%); mp 198–200 °C;1H NMR (DMSO-d6) d 1.37–1.39 (1H, m, CH), 1.67–1.79 (5H, m, CH), 2.98 (2H, s, CH), 3.43–3.50 (4H, m, CH), 3.60–3.63 (2H, m, CH2), 3.67–3.71 (8H, m, 4 CH2), 6.71 (2H, d, J = 8.8 Hz, 2 ArH), 7.29 (2H, d, J = 8.8 Hz, 2 ArH), 7.49 (2H, d, J = 8.8 Hz, 2 ArH), 7.75 (2H, d, J = 8.8 Hz, 2 ArH), 8.30–8.31 (1H, m, exchangeable, CONH), 8.35 and 8.66 (each 1H, br s, exchangeable, 2 NH). Anal. Calcd for C25H33Cl2N5O2: C, H, N.
5.2.15. 1-[4-((2-Morpholinoethyl)carbamoyl)phenyl]-3-[4-(bis-(2-chloroethyl)amino)phenyl]- urea hydrochloride (9be0)
Compound 9be0was prepared from 7b (0.594 g, 1.5 mmol), DCC (0.464 g, 2.25 mmol), HOBT (0.304 g, 2.25 mmol), TEA (0.3 mL) and 8e0(0.195 g, 2.25 mmol). Yield, 538 mg (61%); mp 234–235 °C;1H NMR (DMSO-d6) d 2.41–2.47 (6H, m, CH), 3.35–3.39 (2H, m, CH), 3.56–3.57 (4H, m, CH), 3.70–3.71 (8H, m, 4 CH2), 6.71 (2H, d, J = 8.8 Hz, 2 ArH), 7.28 (2H, d, J = 8.8 Hz, 2 ArH), 7.49 (2H, d, J = 8.8 Hz, 2 ArH), 7.75 (2H, d, J = 8.8 Hz, 2 ArH), 8.20–8.23 (1H, m, exchangeable, CONH), 8.42 and 8.79 (each 1H, br s, exchangeable, 2 NH). Anal. Calcd for C24H31Cl2N5O3: C, H, N. 5.2.16. 1-[4-(Bis(2-chloroethyl)amino)phenyl]-3-[4-(4-(piperi-din-1-yl)piperidine-1-carbonyl]phenyl)urea hydrochloride (9bf0)
Compound 9bf0was prepared from 7b (0.594 g, 1.5 mmol), DCC (0.464 g, 2.25 mmol), HOBT (0.304 g, 2.25 mmol), TEA (0.3 mL) and 8f0(0.378 g, 2.25 mmol). Yield, 638 mg (73%); mp 222–224 °C;1H NMR (DMSO-d6) d 1.38–1.50 (9H, m, CH), 1.75 (2H, br s, CH), 2.51 (2H, br s, CH), 2.84 (2H, br s, CH), 3.32–3.40 (4H, m, CH), 3.68–3.72 (8H, m, 4 CH2), 6.71 (2H, d, J = 8.8 Hz, 2 ArH), 7.27–7.31 (4H, m, 4 ArH), 7.48 (2H, d, J = 8.8 Hz, 2 ArH), 8.46 and 8.81 (each 1H, br s, exchangeable, 2 NH). Anal. Calcd for C28H37Cl2N5O20.8H2O: C, H, N.
5.3. Alternative method for preparing 9aa0, 9ad0, and 9bd0 hydrochloride
5.3.1. N-(2-(Dimethylamino)ethyl)-3-nitrobenzamide (12aa0) A solution of N,N-dimethylethylene diamine (8a0, 13.2 mL) in dry THF was added to a solution of 3-nitrobenzoyl chloride (11a, 18.5 g, 100 mmol) in dry THF at 0 °C. After being stirred at room temperature for 3 h, the solvent was evaporated to dryness. The oily residue was dissolved in minimum amount of water and then evaporated to dryness. The solid separated was recrystallized from water and washed with hexane to give 12aa0, 21 g, (89%); mp 89– 90 °C; MS (ESI) m/z: 238 [M+H]+.1H NMR (DMSO-d
6) d 2.19 (6H, s, 2 NMe), 2.43 (2H, t, J = 6.8 Hz, CH2), 3.40 (2H, q, J = 12.6 and 6.4 Hz, CH2), 7.78 (1H, t, J = 7.9 Hz, ArH), 8.29 (1H, d, J = 7.8 Hz, ArH), 8.37–8.39 (1H, m, ArH), 8.68 (1H, s, ArH), 8.79–8.82 (1H, m, exchangeable, NH).
By following the same procedure as described for 12aa0, the fol-lowing compounds were synthesized.
5.3.2. 3-Nitro-N-(2-(piperidin-1-yl)ethyl)benzamide (12ad0) Compound 12ad0was synthesized from 1-(2-aminoethyl)piper-idine (8d0, 3.31 g, 22.0 mmol) and 3-nitrobenzoyl chloride (11ad0, 4.0 g, 21.5 mmol). Yield, 5.5 g, (92%); mp 203–204 °C; MS (ESI) m/ z: 278 [M+H]+.1H NMR (DMSO-d6) d 1.67–1.69 (6H, m, 3 CH2), 2.91–2.92 (2H, m, CH2), 3.22–3.32 (2H, m, CH2), 3.50–3.70 (2H, m, CH2), 3.72–3.74 (2H, m, CH2), 7.76–7.80 (1H, m, ArH), 8.37– 8.43 (2H, m, 2 ArH), 8.72–8.73 (1H, m, ArH), 9.38 (1H, t, J = 5.4 Hz, exchangeable, NH). 5.3.3. 4-Nitro-N-(2-(piperidin-1-yl)ethyl)benzamide (12bd0) Compound 12bd0 was synthesized from 2-(piperidin-1-yl)eth-anamine (8d0, 4.23 g, 33 mmol) and 3-nitrobenzoyl chloride (11b, 5.56 g, 30 mmol). Yield, 7.2 g, (86%); mp 165–167 °C (lit.27 97– 99 °C). MS (ESI) m/z: 278 [M+H]+. 1H NMR (DMSO-d6) d 1.54– 1.57 (2H, m, CH), 1.81–1.82 (4H, m, CH), 3.09–3.22 (4H, m, CH),
3.24–3.27 (2H, m, CH), 3.72–3.76 (2H, m, CH), 8.21 (2H, d, J = 8.7 Hz, 2 ArH), 8.32 (2H, d, J = 8.7 Hz, 2 ArH), 9.40 (1H, t, J = 5.2 Hz, exchangeable, NH).
5.3.4. 3-Amino-N-(2-(dimethylamino)ethyl)benzamide (13aa0) 10% Palladium on charcoal (1.5 g) was suspended in a solution of 12aa0 (10.6 g, 4.5 mmol) in ethyl acetate. The mixture was hydrogenated at 35 psi for 3 h. The reaction mixture was filtered through a pad of Celite and the filtrate was evaporated in vacuo to dryness to give 13aa0, 8.62 g (93%); mp 97–100 °C. 1H NMR (DMSO-d6) d 2.16 (6H, s, 2 NMe), 2.36 (2H, t, J = 6.8 Hz, CH2), 3.40 (2H, q, J = 13 and 6.5 Hz, CH2), 5.20 (1H, br s, exchangeable, NH), 6.66 (1H, d, J = 7.8 Hz, ArH), 6.91 (1H, d, J = 7.7 Hz, ArH), 7.00 (1H, s, ArH), 7.05 (1H, t, J = 7.4 Hz, ArH), 8.05–8.08 (1H, m, exchangeable, NH). The product pure enough and was used di-rectly for the next reaction without further purification.
By following the same procedure as described for 13aa0, the compounds 13ad0and 13bd0were synthesized.
5.3.5. 3-Amino-N-(2-(piperidin-1-yl)ethyl)benzamide (13ad0) Compound 13ad0was synthesized from 12ad0(3.0 g, 10 mmol) and Pd/C (0.5 g). Yield was 2.0 g (75%). 1H NMR (DMSO-d
6) d 1.67–1.82 (6H, m, 3 CH2), 2.88–2.90 (2H, m, CH2), 3.15–3.30 (2H, m, CH2), 3.50–3.64 (2H, m, CH2), 3.66–3.69 (2H, m, CH2), 4.20 (2H, br s, exchangeable, NH2), 7.20 (1H, d, J = 7.8 Hz, ArH), 7.37 (1H, t, J = 7.8 Hz, ArH), 7.54 (1H, s, ArH), 7.62 (1H, d, J = 7.8 Hz, ArH), 8.96 (1H, t, J = 5.4 Hz, exchangeable, NH). The product pure enough and was used directly for the next reaction without further purification.
5.3.6. 4-Amino-N-(2-(piperidin-1-yl)ethyl)benzamide (13bd0) Compound 13ad0 was synthesized from 12bd0 (10.6 g, 4.5 mmol) and Pd/C (1.5 g). Yield was 8.62 g (93%); mp 109– 110 °C (lit.25118–120 °C).1H NMR (DMSO-d
6) d 1.52 (2H, m, CH), 1.76 (4H, m, CH), 3.07 (6H, m, CH), 3.58–3.59 (2H, m, CH), 5.67 (2H, s, exchangeable, NH2), 6.54 (2H, d, J = 8.4 Hz, 2 ArH), 7.64 (2H, d, J = 8.4 Hz, 2 ArH), 8.43 (1H, s, exchangeable, NH). The product pure enough and was used directly for the next reaction without further purification.
5.3.7. Compound 9aa0hydrochloride
A solution of known 4-[N,N-bis(2-chloroethyl)-amino]phenyl-isocyanate (10) [freshly prepared from N,N-bis(2-chloroethyl)-ben-zene-1,4-diamine hydrochloride (5, 2.68 g, 8.75 mmol) by treating with triphosgene (1 g, 3.4 mmol) in the presence of TEA (1.25 mL) at 10 °C] in dry DMF was added dropwise to a solution of 13aa0 (1.22 g, 5 mmol) in dry DMF containing Et3N (1.5 mL) at room tem-perature. After being stirred at room temperature for 8 h, the sol-vent was evaporated to dryness in vacuo. The residue was purified by column chromatography using CHCl3/MeOH (v/v 100:5) as the eluent. The fraction containing main products were combined and evaporated to dryness to give water-soluble 9aa0 hydrochloride, 1.5 g (78%); mp 172–173 °C;1H NMR (DMSO-d 6) d 1H NMR (DMSO-d 6) d 2.82 (6H, s, 2 NMe), 3.26 (2H, t, J = 5.8 Hz, CH2), 3.62–3.65 (2H, m, CH2), 3.66–3.71 (8H, m, 4 CH2), 6.71 (2H, d, J = 8.8 Hz, 2 ArH), 7.29 (2H, d, J = 8.8 Hz, 2 ArH), 7.32–7.37 (1H, m, ArH), 7.47 (1H, d, J = 7.6 Hz, ArH), 7.61 (1H, d, J = 8.4 Hz, ArH), 7.95 (1H, s, ArH), 8.74 (1H, t, J = 5.2 Hz, exchangeable, CONH), 8.95 and 9.26 (each 1H, br s, exchangeable, 2 NH). Anal. Calcd for C22H29Cl2N5O2HClH2O: C, H, N.
5.3.8. Compound 9aa0mesylate
A solution of 9aa0hydrochloride (900 mg) in H
2O (500 mL) was neutralized with saturated NaHCO3 aqueous solution to pH 7–8 and then extracted with CHCl3 (300 mL 3). The organic layer
was washed with water, dried (Na2SO4), and evaporated under re-duced pressure. The residue (800 mg, 1.55 mmol) was dissolved in EtOH (200 mL). Methanesulfonic acid (149 mg, 155 mmol) was also added to the solution. The mixture was stirred at room tem-perature for 3 h and the solvent was removed under reduced pres-sure. The solid residue was recrystallized from EtOH to yield 9aa0 mesylate, 645 mg 68%, mp 155–156 °C;1H NMR (DMSO) d 2.36 (3H, s, CH3SO3), 2.85 (3H, s, NMe), 2.86 (3H, s, NMe), 3.27 (2H, m, CH2), 3.60 (2H, m, CH2), 3.69 (8H, m, 4 CH2) 6.71 (2H, d, J = 9.0 Hz, 2 ArH), 7.30 (2H, d, J = 9.0 Hz, 2 ArH), 7.37 (1H, d, J = 7.7 Hz, ArH), 7.43 (1H, d, J = 7.7 Hz, ArH), 7.56 (1H, d, J = 7.7 Hz, ArH), 8.0 (1H, s, ArH), 8.49 (1H, s, NH), 8.62 (1H, br, exchangeable, NH), 8.81 (1H, s, NH), 9.27 (1H, br, NH). Anal. Calcd for C23H33N5O5Cl2S0.25H2O: C, H, N, S.
By following the same procedure as described for 9aa0 hydro-chloride, the compounds 9ad0hydrochloride and 9bd0 hydrochlo-ride were synthesized.
5.3.9. Compound 9ad0hydrochloride
Compound 9ad0 was prepared from 13ad0 (1.5 g, 6 mmol) and N-mustard isocyanate 10 [freshly prepared from 5 (4.0 g, 13 mmol) and triphosgene (2.58 g, 9.5 mmol)]. Yield, 1.4 g (46%); mp 98–99; 1H NMR (DMSO-d 6) d 1.76–1.78 (6H, m, 3 CH2), 2.90–2.91 (2H, m, CH2), 3.18–3.20 (2H, m, CH2), 3.44–3.50 (2H, m, CH2), 3.67– 3.68 (2H, m, CH2), 3.69–3.71 (8H, m, 4 CH2), 6.71 (2H, d, J = 8.1 Hz, 2 ArH), 7.29 (2H, d, J = 8.1 Hz, 2 ArH), 7.34 (1H, t, J =7.8 Hz, ArH), 7.46 (1H, d, J = 7.5 Hz, ArH), 7.60 (1H, d, J = 7.8 Hz, ArH), 7.93 (1H, s, ArH), 8.76 (1H, br s, exchangeable, NH), 8.84, 9.15 (each 1H, s, exchangeable, 2 NH). HRMS (FAB+) m/z calcd for C25H33Cl2N5O2[M+H]+: 506.4678; found: 506.2072.
5.3.10. Compound 9bd0hydrochloride
Compound 9bd0was prepared from 13bd0(0.50 g, 2 mmol) and N-mustard isocyanate 10 [freshly prepared from 5 (1.07 g, 3.5 mmol) and triphosgene (0.4 g, 1.36 mmol)]. Yield, 0.66 g (64%); mp 127–128;1H NMR (DMSO-d 6) d 1.36–1.41 (1H, m, CH), 1.68–1.79 (5H, m, CH), 2.87–2.96 (2H, m, CH), 3.20–3.23 (2H, m, CH), 3.51–3.54 (2H, m, CH), 3.63–3.66 (2H, m, CH), 3.67–3.72 (8H, m, 4 CH2), 6.73 (2H, d, J = 8.9 Hz, 2 ArH), 7.30 (2H, d, J = 8.9 Hz, 2 ArH), 7.53 (2H, d, J = 8.6 Hz, 2 ArH), 7.85 (2H, d, J = 8.6 Hz, 2 ArH), 8.75 (1H, t, J = 5.3 Hz, exchangeable, CONH), 9.01 and 9.48 (each 1H, s, exchangeable, 2 NH). HRMS (FAB+) m/z calcd for C25H33Cl2N5O2[M+H]+: 506.4678; found: 506.2076. 5.4. Synthesis of water-soluble N-mustards having an ether linker (19aa0–19be0series)
Detailed procedures for the synthesis of intermediates 15a,b, 17aa0–17be0, and 18aa0–18be0along with their spectroscopic data are provided in theSupplementary data.
5.4.1. 1-(4-(Bis(2-chloroethyl)amino)phenyl)-3-(3-(2-(dimethylamino)ethoxy)phenyl)urea (19aa0)
Triethylamine (5 mL) was added dropwise to a solution of phen-ylisocyanate 10 [freshly prepared from 5 (3.366 g, 10.8 mmol) and triphosgene (1.24 g, 4.2 mmol) in dry CHCl3 (100 mL)] at 10 °C and stirred for 30 min. A solution of 18aa0 (1.384 g, 6 mmol) in CHCl3(25 mL) containing TEA (3 mL) at 10 °C was added slowly to the above solution. The reaction mixture was then allowed to stir at room temperature for 16 h and the reaction mixture was washed successively with water (350 mL) and brine (350 mL). The organic layer was dried (Na2SO4) and filtered. The filtrate was evaporated in vacuo to dryness and the residue was chromato-graphed on a silica gel column using CH2Cl2/MeOH (100:6) as an eluent. The fractions containing the main product were combined and evaporated to dryness. The white solid residue was triturated
with hexane and then collected by filtration to give 19aa0, 0.710 g (46%); mp 143–145 °C;1H NMR (DMSO-d
6) d 2.21 (6H, s, 2 NMe), 2.61 (2H, s, CH2), 3.69 (8H, br s, 4 CH2), 4.00 (2H, s, CH2), 6.51– 6.53 (1H, m, ArH), 6.70–6.72 (2H, m, 2 ArH), 6.88–6.90 (1H, m, ArH), 7.13–7.18 (2H, m, 2 ArH), 7.26–7.28 (2H, m, 2 ArH), 8.32 and 8.52 (each 1H, br s, exchangeable, 2 NH). Anal. Calcd for C21H28Cl2N4O2: C, H, N.
By following the same procedure as described for 19aa0, the fol-lowing compounds were prepared.
5.4.2. 1-(4-(Bis(2-chloroethyl)amino)phenyl)-3-(3-(3-(dimethyl-amino)propoxy)phenyl)urea (19ab0)
Compound 19ab0 was prepared 10 [freshly prepared from 5 (3.366 g, 10.8 mmol) and triphosgene (1.246 g, 4.2 mmol)] and 18ab0 (1.384 g, 6 mmol). Yield, 1.247 g (43%); mp 127–128 °C;1H NMR (DMSO-d6) d 1.84–1.89 (2H, m, CH2), 2.21 (6H, s, 2 NMe), 2.44 (2H, t, J = 7.2 Hz, CH2), 3.69–3.70 (8H, m, 4 CH2), 3.95 (2H, t, J = 7.2 Hz, CH2), 6.48–6.51 (1H, m, ArH), 6.70 (2H, d, J = 8.8 Hz, 2 ArH), 6.86 (1H, d, J = 8.0 Hz, ArH), 7.13 (1H, t, J = 8.0 Hz, ArH), 7.20 (1H, s, ArH), 7.27 (2H, d, J = 8.8 Hz, 2 ArH), 8.40 and 8.62 (each 1H, br s, exchangeable, 2 NH). Anal. Calcd for C22H30Cl2N4O20.6H2O: C, H, N.
5.4.3. 1-(4-(Bis(2-chloroethyl)amino)phenyl)-3-(3-(2-(pyrrolidin-1-yl)ethoxy)phenyl)urea (19ac0)
Compound 19ac0 was prepared 10 [freshly prepared from 5 (3.366 g, 10.8 mmol) and triphosgene (1.246 g, 4.2 mmol)] and 18ac0 (1.456 g, 6 mmol). Yield, 1.36 g (45%); mp 124–125 °C; 1H NMR (DMSO-d6) d 1.68 (4H, s, 2 CH2), 2.50 (4H, s, 2 CH2), 2.77 (2H, s, CH2), 3.69 (8H, s, 4 CH2), 4.01 (2H, s, CH2), 6.51 (1H, d, J = 7.2 Hz, ArH), 6.70 (2H, d, J = 8.0 Hz, 2 ArH), 6.88 (1H, d, J = 7.6 Hz, ArH), 7.13 (1H, t, J = 8.0 Hz, ArH), 7.18 (1H, s, ArH), 7.27 (2H, d, J = 8.0 Hz, 2 ArH), 8.37 and 8.57 (each 1H, br s, exchangeable, 2 NH). Anal. Calcd for C23H30Cl2N4O2: C, H, N. 5.4.4. 1-(4-(Bis(2-chloroethyl)amino)phenyl)-3-(3-(2-(piperidin-1-yl)ethoxy)phenyl)urea (19ad0)
Compound 19ad0 was prepared 10 [freshly prepared from 5 (1.683 g, 5.4 mmol) and triphosgene (1.246 g, 4.2 mmol)] and 18ad0 (0.75 g, 3 mmol). Yield, 0.850 g (55%); mp 86–88 °C; 1H NMR (DMSO-d6) d 1.37 (2H, s, CH2), 1.49 (4H, s, 2 CH2), 2.42 (4H, s, 2 CH2), 2.63 (2H, s, CH2), 3.69 (8H, s, 4 CH2), 4.00 (2H, s, CH2), 6.50–6.52 (1H, m, ArH), 6.69–6.71 (2H, m, 2 ArH), 6.87–6.88 (1H, m, ArH), 7.12–7.18 (2H, m, 2 ArH), 7.26–7.28 (2H, m, 2 ArH), 8.35 and 8.54 (each 1H, br s, exchangeable, 2 NH). Anal. Calcd for C24H32Cl2N4O25H2O: C, H, N.
5.4.5. 1-(4-(Bis(2-chloroethyl)amino)phenyl)-3-(3-(2-morpholi-noethoxy)phenyl)urea (19ae0)
Compound 19ae0 was prepared 10 [freshly prepared from 5 (3.366 g, 10.8 mmol) and triphosgene (1.246 g, 4.2 mmol)] and 18ae0 (1.55 g, 6 mmol). Yield, 0.948 g (31%); mp 168–169 °C;1
H NMR (DMSO-d6) d 2.45–2.48 (4H, m, 2 CH2), 2.68 (2H, t, J = 6.0 Hz, CH2), 3.58 (4H, t, J = 4.8 Hz, 2 CH2), 3.66–3.72 (8H, m, 4 CH2), 4.04 (2H, t, J = 6.0 Hz, CH2), 6.51–6.54 (1H, m, ArH), 6.70 (2H, d, J = 9.2 Hz, 2 ArH), 6.87 (1H, d, J = 8.0 Hz, ArH), 7.13 (1H, t, J = 8.0 Hz, ArH), 7.19 (1H, s, ArH), 7.27 (2H, d, J = 9.2 Hz, 2 ArH), 8.31 and 8.50 (each 1H, br s, exchangeable, 2 NH). Anal. Calcd for C23H30Cl2N4O3HCl: C, H, N.
5.4.6. 1-(4-(Bis(2-chloroethyl)amino)phenyl)-4-(3-(2-(dimethyl-amino)ethoxy)phenyl)urea hydrochloride (19ba0)
Compound 19ba0 was prepared 10 [freshly prepared from 5 (1.683 g, 5.4 mmol) and triphosgene (0.623 g, 4.2 mmol)] and 18ba0 (0.65 g, 3 mmol). Yield, 0.864 g (60%); mp 203–205 °C;1
H NMR (DMSO-d6) d 2.83 (6H, s, 2 NMe), 3.46–3.47 (2H, m, CH2),
3.69 (8H, s, 4 CH2), 4.30 (2H, t, J = 4.9 Hz, CH2), 6.72 (2H, d, J = 8.3 Hz, 2 ArH), 6.92 (2H, d, J = 8.8 Hz, 2 ArH), 7.28 (2H, d, J = 8.5 Hz, 2 ArH), 7.38 (2H, d, J = 8.8 Hz, 2 ArH), 8.84 and 9.01 (each 1H, br s, exchangeable, 2 NH). Anal. Calcd for C21H28Cl2N4O2HClH2O: C, H, N.
5.4.7. 1-(4-(Bis(2-chloroethyl)amino)phenyl)-4-(3-(3-(dimethyl-amino)propoxy)phenyl)urea hydrochloride (19bb0)
Compound 19bb0 was prepared 10 [freshly prepared from 5 (3.366 g, 10.8 mmol) and triphosgene (1.246 g, 4.2 mmol)] and 18bb0 (1.384 g, 6 mmol). Yield, 1.70 g (58%); mp 178–180 °C;1H NMR (DMSO-d6) d 2.07–2.14 (2H, m, CH2), 2.78 (6H, s, 2 NMe), 3.17–3.22 (2H, m, CH2), 3.65–3.72 (8H, m, 4 CH2), 3.99 (2H, t, J = 6.0 Hz, CH2), 6.69 (2H, d, J = 8.9 Hz, 2 ArH), 6.86 (2H, d, J = 9.0 Hz, 2 ArH), 7.26 (2H, d, J = 8.8 Hz, 2 ArH), 7.35 (2H, d, J = 9.0 Hz, 2 ArH), 8.62 and 8.64 (each 1H, br s, exchangeable, 2 NH). Anal. Calcd for C22H30Cl2N4O2HClH2O: C, H, N.
5.4.8. 1-(4-(Bis(2-chloroethyl)amino)phenyl)-4-(3-(2-(pyrrolidin-1-yl)ethoxy)phenyl)urea (19bc0)
Compound 19bc0 was prepared 10 [freshly prepared from 5 (3.366 g, 10.8 mmol) and triphosgene (1.246 g, 4.2 mmol)] and 18bc0 (1.456 g, 6 mmol). Yield, 1.56 g (52%); mp 205–207 °C;1H NMR (DMSO-d6) d 1.87–1.90 (2H, m, CH2), 2.00 (2H, br s, CH2), 3.07–3.10 (2H, m, CH2), 3.53–3.58 (4H, m, 2 CH2), 3.65–3.72 (8H, m, 4 CH2), 4.28 (2H, t, J = 4.9 Hz, CH2), 6.69 (2H, d, J = 8.9 Hz, 2 ArH), 6.93 (2H, d, J = 8.9 Hz, 2 ArH), 7.27 (2H, d, J = 8.8 Hz, 2 ArH), 7.38 (2H, d, J = 8.8 Hz, 2 ArH), 8.70 and 8.87 (each 1H, br s, exchangeable, 2 NH). Anal. Calcd for C23H30Cl2N4O21.1HCl: C, H, N.
5.4.9. 1-(4-(Bis(2-chloroethyl)amino)phenyl)-4-(3-(2-(piperidin-1-yl)ethoxy)phenyl)urea (19bd0)
Compound 19bd0 was prepared 10 [freshly prepared from 5 (3.366 g, 10.8 mmol) and triphosgene (1.246 g, 4.2 mmol)] and 18bd0 (1.54 g, 6 mmol). Yield, 1.73 g (56%); mp 211–212 °C;1H NMR (DMSO-d6) d 1.37–1.39 (1H, m, CH), 1.67–1.70 (1H, m, CH), 1.79 (4H, br s, 2 CH2), 2.98 (2H, br s, CH2), 3.43–3.50 (2H, m, 2 CH2), 3.67–3.72 (8H, m, 4 CH2), 4.34 (2H, t, J = 4.9 Hz, CH2), 6.69 (2H, d, J = 8.9 Hz, 2 ArH), 6.92 (2H, d, J = 8.9 Hz, 2 ArH), 7.27 (2H, d, J = 8.9 Hz, 2 ArH), 7.38 (2H, d, J = 8.9 Hz, 2 ArH), 8.72 and 8.89 (each 1H, br s, exchangeable, 2 NH). Anal. Calcd for C24H32Cl2N4O2HCl: C, H, N.
5.4.10. 1-(4-(Bis(2-chloroethyl)amino)phenyl)-4-(3-(2-morpholi-noethoxy)phenyl)urea (19be0)
Compound 19be0 was prepared 10 [freshly prepared from 5 (3.366 g, 10.8 mmol) and triphosgene (1.246 g, 4.2 mmol)] and 18be0 (1.55 g, 6 mmol). Yield, 1.33 g (43%); mp 210–212 °C; 1H NMR (DMSO-d6) d 3.20 (2H, br s, CH2), 3.47–3.52 (4H, m, 2 CH2), 3.65–3.71 (8H, m, 4 CH2), 3.78–3.84 (2H, m, CH2), 3.95–3.98 (2H, m, CH2), 4.36 (2H, t, J = 4.8 Hz, CH2), 6.69 (2H, d, J = 9.0 Hz, 2 ArH), 6.93 (2H, d, J = 8.9 Hz, 2 ArH), 7.27 (2H, d, J = 8.9 Hz, 2 ArH), 7.38 (2H, d, J = 9.0 Hz, 2 ArH), 8.67 and 8.84 (each 1H, br s, exchangeable, 2 NH). Anal. Calcd for C23H30Cl2N4O3HCl: C, H, N.
5.5. Cytotoxicity assays
The in vitro cytotoxicity of the newly synthesized N-mustard derivatives were determined in T-cell acute lymphocytic leukemia (CCRF-CEM) and their resistant subcell lines (CCRF-CEM/taxol and CCRF-CEM/VBL) by the XTT assay28and human solid tumor cells (i.e., breast carcinoma MX-1 and colon carcinoma HCT-116) by the SRB assay29 in a 72 h incubation using a microplate spectro-photometer as previously described.30After the addition of
phena-zine methosulfate–XTT solution, incubated at 37 °C for 6 h and absorbance at 450 and 630 nm was detected on a microplate read-er (EL 340). The cytotoxicity of the newly synthesized compounds against non-small cell lung cancer H1299, human prostate cancer PC3, human lung adenocarcinoma (CL 1–0 and CL 1–5) and human breast adenocarcinoma MCF-7 were determined by the Alamar blue assay31in a 72 h incubation using a microplate spectropho-tometer as previously described. After the addition of Alamar blue solution, it was incubated at 37 °C for 6 h. Absorbance at 570 and 600 nm was detected on a microplate reader. IC50 values were determined from dose-effect relationship at six or seven concen-trations of each drug using the CompuSyn software by Chou and Martin32based on the median-effect principle and plot.33,34Ranges given for taxol, vinblastine and cisplatin were mean ± SE (n = 4). 5.6. In vivo studies
Athymic nude mice bearing the nu/nu gene were obtained from NCI, Frederick, MD and used for all human tumor xenografts. Male nude mice 6 weeks or older weighing 20–24 g or more were used. Compound 9aa0was administered via the tail vein for iv injection as previously described.30Tumor volume was assessed by measur-ing length width height (or width) by usmeasur-ing a caliper. Vehicle used was 50
l
L DMSO and 40l
L Tween 80 in 160l
L saline. The maximal tolerate dose of the tested compound was determined and applied for the in vivo therapeutic efficacy assay. For tumor-bearing nude mice during the course of the experiment, the body-weight referred to total body weight minus the weight of the tumor assuming 1 mm3= 1 mg. All animal studies were con-ducted in accordance with the guidelines for the National Institute of Health Guide for the Care and Use of Animals and the protocol approved by the Institutional Animal Care and Use Committee. 5.7. Alkaline agarose gel shift assayFormation of DNA cross-linking was analyzed by alkaline aga-rose gel electrophoresis. In brief, purified pEGFP-N1 plasmid DNA (1500 ng) was mixed with various concentrations (1–20
l
M) of 9aa0, 9ad0, 19bd0and 19bb0in 40l
L binding buffer (3 mM sodium chloride/1 mM sodium phosphate, pH 7.4, and 1 mM EDTA). The reaction mixture was incubated at 37 °C for 2 h. At the end of the reaction, the plasmid DNA was linearized by digestion with BamHI and followed by precipitation with ethanol. The DNA pellets were dissolved and denatured in alkaline buffer (0.5 N NaOH–10 mM EDTA). An aliquot of 20l
L of DNA solution(1l
g) was mixed with 4l
L of 6% alkaline loading dye and then electrophoretically re-solved on a 0.8% alkaline agarose gel with NaOH–EDTA buffer at 4 °C. The electrophoresis was carried out at 18 V for 22 h. After staining the gels with an ethidium bromide solution, the DNA was then visualized under UV light.5.8. Cell cycle inhibition
The effects of 9aa0on cell cycle distribution were analyzed with a flow cytometer as previously described.35 Briefly, human non-small cell lung carcinoma H1299 cells were treated with 9aa0at 2.5, 5, and 10 mM for 24 h. The attached cells were then trypsini-zed, washed with phosphate buffer saline (PBS), and fixed with ice-cold 70% ethanol for 30 min. The cells were stained with 4
l
g/ml propidium iodide (PI) in PBS containing 1% Triton X-100 and 0.1 mg/ml RNase A. The stained cells were then analyzed using the FACS SCAN flow cytometer (Becton Dickinson, San Joes, CA, USA). The percentage of the cells in each cell cycle phase was determined using the ModFit LT 2.0 software based on the DNA histograms.5.9. In vivo pharmacokinetic study/drug administration and sampling
All procedures of the present study were in accordance with the IACUC guidelines. Prior approval of the Institutional Animal Ethics Committee (IAEC) was obtained before initiation of the study. A single intravenous dose of 9aa0was administered via an indwelling catheter in jugular vein to two male Sprague Dawley rats at a dose level of 1.0 mg/kg. The formulation was prepared as a solution in 5.0% w/v DMSO with 10% w/v Cremophor in double distilled water. Blood samples were collected at 0.083, 0.166, 0.333, 0.666, 1, 2, 4, 6, and 8 h after fasted from twelve hours prior to dosing. Serial blood samples (200
l
L/each) were collected from all animals via the carotid artery cannulations. Samples were placed into tubes containing 10l
L of 350 IU/mL heparin solution. Plasma was har-vested from the blood by centrifugation at 4000 rpm for 10 min at 4 ± 2 °C. All samples were stored at approximately 80 °C until bioanalysis.5.10. Bioanalytical method
Concentration levels of 9aa0in rat plasma samples were deter-mined by partially validated LC–MS/MS method. The calibration curve was linear from 2.5 to 300 ng/mL for 9aa0. The coefficient of determination (r2) was greater than 0.999. The plasma samples were quenched by adding 90
l
L of acetonitrile (0.1% formic acid) containing the internal standard (BO-1441, 100 ng/mL) to 30l
L of sample. The plasma samples were vortex-mixed briefly at high speed, kept on ice for 10 min, and then centrifuged at 12,000 rpm for 5 min. Approximately 100l
L of the supernatant of each tube was transferred to an amber clean autosampler vial with insert for analysis. A 5l
L of the aliquot solution was subsequently in-jected into the LC–MS/MS system.Acknowledgements
We are grateful to the National Science council (Grant No. NSC-99-2320-B-001-013) and Academia Sinica (Grant No. AS-96-TP-B06) for financial support. T.-C.C. was supported by the Sloan-Ket-tering Institute General Fund. The NMR spectra of synthesized compounds were obtained at High-Field Biomolecular NMR Core Facility supported by the National Research Program for Genomic Medicine (Taiwan). We would like to thank Dr. Shu-Chuan Jao in the Institute of Biological Chemistry (Academia Sinica) for provid-ing the NMR service and the National Center for High-performance computing for computer time and facilities.
Supplementary data
Supplementary data (detailed experimental procedures for the synthesis of intermediate compounds 15a,b, 17ae0–17be0, and 18aa0–18be0 along with their spectroscopic data, and elemental analysis data of all unknown compounds) associated with this arti-cle can be found, in the online version, at doi:10.1016/j.bmc. 2010.11.005.
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