1812
J . Med. Chem. 1990,33, 1812-1818
Synthesis of Quaternary Amine Ether Lipids and Evaluation of Neoplastic Cell Growth Inhibitory Properties Susan L. Morris-Natschke, Karen L. Meyer, Canio J. Marasco, Jr., Claude Piantadosi,* Fiona Rossi,' Patrick L. Godwin,' and Edward J. Modestt,* University of N o r t h Carolina, School of Pharmacy, Division of Medicinal Chemistry and Natural Products, Chapel Hill, N o r t h Carolina 27599, and Bowman Gray School of Medicine, W a k e Forest University, Department of Biochemistry, Winston-Salem, N o r t h Carolina 27103. Received August 11, 1989
Novel quaternary amine ether lipids have been synthesized and tested for inhibition of neoplastic cell proliferation with the HL-60 promyelocytic leukemia cell line. These compounds contain a positively charged quaternary amine functional group attached either directly to the glycerol backbone or at the end of an alkoxy chain. The biological testing has identified several analogues with activity equivalent to or greater than that exhibited by the reference compound in this assay, ET-18-OMe (l-0-octadecyl-2-0-methyl-rac-glycero-3-phosphocholine). Among the most bromide] and active analogues are compounds 11, [N,NJV-triethyl-3-(hexadecyloxy)-2-ethoxy-l-propylammonium 22 [N-[4-[3-(hexadecyloxy)-2-ethoxypropoxy]-l-butyl]pyridinium bromide], which are approximately 3 times as active as the reference standard.
Ether lipids derived from glycerol display a wide variety of biological a c t i v i t i e ~ . ' ~One ~ of these is the growth inhibition of various neoplastic cell lines exhibited by synthetic alkyl ether phospholipid^.^-^ The mechanism for this antineoplastic activity is not fully understood and multiple hypotheses have been proposed."13 Membrane i n t e r a ~ t i o n ' ~ Jand ~ J ~protein kinase C inhibition8J6J7are considered to be two of the many possible modes of action which may be involved in inhibition of malignant cell growth. The alkyl ether phospholipid ET-18-OMe (1-0octadecyl-2-0-methyl-rac-glycero-3-phosphocholine) has been found to both accumulate in the plasma membrane of neoplastic cells"J5 and also to selectively inhibit protein kinase C over other kinases present in HL-60 cells.17 Further, quite recently, ether lipids have also been shown to possess antiviral activity against human immunodeficiency virus type 1 (HIV-l).'* This laboratory has previously reported a series of quaternary amine ether lipids (inverse cholines) which contain an ammonium functionality but lack the phosphate group present in ether phospholipids. These analogues were found to be inhibitors of protein kinase C.19 Since inhibition of this kinase has been linked to antineoplastic activity, this series of lipids was tested for inhibition of malignant cell proliferation. The present paper will report the results of this study on the growth inhibition of HL-60 human leukemic cells in vitro. Two additional series of quaternary amine compounds which are structurally related to the inverse cholines were also prepared and evaluated to further investigate the structural features needed for PKC inhibition and antineoplastic activity. Figure 1 shows an overview of the basic compound structures and of ET-lg-OMe, the reference compound used in this assay. Each of the compound classes shares in common a long chain alkyl ether at the carbon-1 position and a short chain alkyl ether at the carbon-2 position of a three-carbon backbone. The carbon-3 position is different in each class of compounds. (1) Alkyl ether phospholipids such as the reference Et-18-OMe contain a phosphocholinegroup. (2) Series I quaternary amine ether lipids (inverse cholines) contain a N,N-dimethyl+ hydroxyethyl or N,N-dimethyl-3-hydroxypropylammonium group. (3) Series I1 quaternary amine ether lipids contain an ammonium group directly attached to carbon-3
* To whom correspondence should be addressed. Wake Forest University. Present address: Department of Biochemistry, Boston University School of Medicine, Boston, MA 02118. t
Scheme Insb XRi
R 2 c :
R2CxR1 - R2C Z'
Br
Br
"(i) (1)MsCl, Et3N, (2) LiBr, acetone or CBr,, Ph,P; (ii) (CH3)2N(CH2)2-30H or N(CH& or N(CHZCH,), or pyridine or 4hydroxy-N-methylpiperidine. bX,R1, Rz, and Z are given in Tables 1-111.
Scheme I P b
OH
..CXR' O(CH2),Br
..c XR,
O(CH,).Z'B
t
"(i) for n = 3-10, Br(CH2),Br, NaH, NaI; (ii) N(CH3), or pyridine. *X, R1, R2 and Z are given in Tables 1-111. Scheme 111"
-
200 "C dec. NMR (CDCI,): 6 0.86 (t, 3 H, H, SCHz),2.63 (overlapping d of d, 2 H, CHCHzS), 3.45 ( s , 9 H, terminal CHJ, 1.2-1.3 (m, 28 H, (CH2)14), 2.37 (q, 2 H, N(CH3)& 3.4-3.7 (overlapping m, 9 H, CHCH20CH2CH2CHzCOCH2CH2CH2N),3.31 and 3.50 (2 t, 4 H, CH20CHz),5.11 (t, 2 HzN, OCHzCH3). Anal. (Cz8HmNSOZBr.H20) C, H, N. H, CHzN),8.08 (m, 2 H, pyridinium H), 8.48 (m, 1 H, pyridinium (*) -N,N,N-Trimethy l-N- [ lo-[ 3-(hexadecyloxy)-2-methH), 9.44 (m, 2 H, pyridinium H). Anal. (C24HuNOBr.0.67Hz0) oxypropoxyl-1-decyllammonium Bromide (20). NMR C, H, N. (CDCl,): 6 0.85 (t, 3 H, terminal CH,), 1.2-1.4 (m, 34 H, (CH2)13, (*)-N-[4-[3-( Hexadecyloxy)-2-ethoxypropoxy]-1-butyl]O(CH2)3(CHzCHz)2(CH2)3N), 1.55 and 1.75 and 1.90 (3 m, 10 H, pyridinium Bromide (22). Yield 8590. NMR (CDC13): 6 0.85 OCHZCHZ, OCHzCH2CH2(CHzCH2)zCHzCHzCHzN),3.45 (8,12 (t, 3 H, terminal CHJ, 1.18 (t, 3 H, OCHzCH3),1.2-1.3 (m, 26 H, O W 3 , N(CH3),), 3.4-3.7 (overlapping m, 11 H, H, (CH2)13), 1.54 (m, 2 H, OCHzCHz), 1.61 (m, 2 H, CHz0CHzCHCH20CH2(CH2CHz)4CHzN). Anal. (C33H70N03OCH2CHzCHzCH2N),2.13 (m, 2 H, OCH2CHzCHzCHzN),3.2-3.8 Br.0.50Hz0) C, H, N. (overlapping m, 11H, CH20CH2,OCHzCH3,CHCHzOCH2),5.10 (&)-N,N,N-Trimethyl-N-[4434 hexadecyloxy)-2-ethoxy(t,2 H, CHzN),8.1 (t, 2 H, pyridinium H), 8.45 (t,1H, pyridinium propoxyl-1-butyllammoniumBromide (21). NMR (CDC13): H), 9.60 (d, 2 H, pyridinium H). Anal. (C3,,HSeNO3Br'HZ0)C, 6 0.85 (t, 3 H, terminal CH,), 1.2 (t, 3 H, OCHzCH,), 1.2-1.4 (m, H, N. 26 H, (CH2)13), 1.55 (m, 2 H, OCH2CH2),1.70 and 1.90 (m, 4 H, The syntheses of (*)-N,N,N-trimethyl-N-[3-( hexadecylOCHzCHzCHzCH2N),3.45 (s,9 H, N(CH,),), 3.4-3.8 (overlapping thio)-2-methoxy-1-propyllammoniumbromide (9) and m, 13 H, CH20CHzCHCH20CHzCH2CHzCHzN, OCHzCH3). Anal. (*)-N,N,N-trimet hyl-N-[ 2 4 3 4 hexadecyloxy)-2-methoxy(C~~H~NO~BPO.BHZO) C, H, N. propoxy]ethyl]ammonium bromide (16) are reported in ref NJVJVN-Trimethyl-N-[ 3 4 hexadecy1oxy)- 1-propyl]ammo19. nium Bromide (13). l-(Hexadecyloxy)-3-bromopropane(0.5 g, (f)-N-Methyl-N-[3-(hexadecyloxy)-2-ethoxy- 1-propyl]0.001 mol) was dissolved in 13 mL of CHC13/2-propanol/DMF j3-hydroxyethylamine (26). This compound was prepared by ( 3 5 5 ) , and 1mL of 40% aqueoue trimethylamine was added. The reaction of l-(hexadecyloxy)-2-ethoxy-3-bromopropane (0.60 g, reaction mixture was heated a t 60 "C and then stirred a t room 0.0015 mol) with 2-(methy1amino)ethanol (0.225 g, 0.003 mol) in temperature overnight. Thin-layer chromatography indicated an acetonitrile a t 50 "C for 24 h. After cooling, the solvent was incomplete reaction and an additional 0.5 mL of trimethylamine removed and the residue was partitioned between diethyl ether was added and the reaction continued a t 60 "C for 2 h. After and water. The ether was dried over sodium sulfate and then cooling, the solvent was removed and the product was purified evaporated and the product (0.1 g, 0.0002 mol, 14% yield) was by repeated recrystallization with ether. The salt was obtained purified by column chromatography on silica gel with CHC13/ as a white solid in 75% yield, decomposing a t 200-203 "C. NMR MeOH (9:l) as eluant. NMR (CDClJ: 6 0.87 (t, 3 H, terminal (CDCI,): 6 0.87 (t,3 H, terminal CH,), 1.2-1.3 (m, 26 H, (CH2)13), CH,), 1.2 (t,3 H, OCH2CH3)1.2-1.3 (m, 26 H, (CH2)13),1.55 (m, 1.53 (m, 2 H, OCH2CHz),2.08 (m, 2 H, OCH2CHzCHzN),3.2-3.48 2 H, OCH2CHz),2.33 (5, 3 H, NCH,), 2.5-2.6 (overlapping m, 4 (overlapping m, 13 H, CHzOCHz,N(CH3),), 3.66 (m, 2 H, CH2N). H, CH2NCHz),3.4-3.7 (overlapping m, 9 H, CHzOCHzCHOCHz, Anal. (CzzH48NOBr)C, H, N. CH20H). Anal. (C24H51N03)C, H, N. (k)-N,N,N-Trimethyl-[ 443 4 hexadecyloxy)-2-methoxyAcknowledgment. T h e protein kinase C inhibition propoxyl-1-butyllammoniumBromide (18). NMR (CDC13): studies were performed b y George W. Small u n d e r t h e 6 0.85 (t, 3 H, terminal CH,), 1.2-1.4 (m, 26 H, (CHz)13),1.55 (m, direction of Dr. Larry W. Daniel at Bowman Gray School 2 H, OCH2CHd, 1.70 and 1.90 (m, 4 H, OCH2CHzCH2CHzN), 3.45 of Medicine, Winston-Salem, NC. T h i s research was (8, 12 H, OCH,, N(CH3),), 3.4-3.8 (overlapping m, 11 H, supported in part by National Institute of Health Research CHz0CHzCHCHzOCH2CH2CHzCH2N). Anal. (C2,HSNO3Br. O.5HzO) C, H, N. G r a n t CA 42216 a n d American Cancer Society Research
J. Med. Chem. 1990,33, 1818-1823
1818
Grant RD 262 (to University of North Carolina) and National Cancer Institute Research Grants CA 41314 and BRSG-RR 05404 (to Bowman Gray School of Medicine, Wake Forest University). Registry No. 1, 124581-78-8;2, 124581-79-9;3, 124581-80-2; 4, 124581-94-8;5, 126614-04-8;5 (starting iodide), 126614-27-5; 6,124581-82-4;7,126614-05-9; 8,124581-83-5;9, 124581-81-3; 10, 126614-06-0; 10 (starting bromide), 126614-21-9; 11,126614-07-1; 11 (startingbromide), 126614-36-6;12,126614-08-2;13, 12664453-9; 13 (starting bromide), 126614-26-4; 14, 126614-09-3; 15, 126614-10-6;15 (starting bromide), 126614-32-2; 16,124581-92-6; 17, 126614-11-7;17 (startingbromide), 126614-28-6;18, 126614-
12-8; 18 (starting bromide), 126614-23-1; 19, 126614-13-9; 19 (starting bromide), 126614-29-7; 20, 126614-14-0; 20 (starting bromide), 126614-33-3;21, 126614-15-1;21 (starting bromide), 126614-34-4;22,126614-16-2;23,126614-17-3;23 (starting bromide), 126614-30-0; 24, 126614-18-4; 24 (starting bromide), 126614-35-5;25,126614-19-5;25 (starting bromide), 126614-31-1; 12661426,126614-20-8; (I)-MeS03CH2CH(OMe)CH20C16H39, 22-0; ( ~ ) - H O C H 2 C H ( O M e ) C H 2 0 C 1 6 H 111188-59-1; ~, (&)H2C=CHCH2CH20CH2CH(OMe)CH20C16H33, 126614-24-2;
H~Cl~OCH2CH(OMe)CH20(CH2)rOCH2CH(OMe)CH20C16H39, 126614-25-3;Br(CH2)Br,110-52-1;HO(CH2),0C1&s3,23377-40-4; CBr,, 558-13-4; MeNHCH2CH20H, 109-83-1; N-methyl-4hydroxypiperidine, 106-52-5.
Syntheses and Platelet Aggregation Inhibitory and Antithrombotic Properties of [2-[ (a-Aminoalkoxy)phenyl]ethyl]benzenes Ryoji Kikumoto, Hiroto Hara,* Kunihiro Ninomiya, Masanori Osakabe, Mamoru Sugano, Harukazu Fukami, and Yoshikuni Tamao Pharmaceuticals Laboratory, Research Center, Mitsubishi Kasei Corporation, 1000, Kamoshida-cho, Midori-ku, Yokohama, Japan. Received June 2, 1989
A series of [2- [ (u-aminoalkoxy)phenyl]ethyl] benzene derivatives were synthesized and evaluated for their ability to inhibit collagen-inducedplatelet aggregation in vitro and to protect experimentalthrombosis in mice. The resulk showed that the compounds were in vitro inhibitors of collagen-inducedplatelet aggregation. Most of them were also effectivein the mouse antithrombotic assay. The compounds were found to be potent antagonists to S2serotonergic receptor, and good correlation (r = 0.85) between their S2serotonergic receptor antagonism and their potency as [2-[2platelet antiaggregatory drugs was observed. Among the compounds studied, mono[2-(dimethylamino)-l-[ (3-methoxyphenyl)ethyl]phenoxy]methyl]ethyl] succinate hydrochloride (12b, MCI-9042) was selected for further
pharmacological and toxicological evaluation. The intravascular formation of platelet aggregates is an important pathogenic factor in the development of cardiovascular disease state such as atherosclerosis, myocardial infarction, transient ischemic attacks, and stroke.l-* Hence, one rational approach in the research for antithrombotic drugs is to search for inhibitors of platelet aggregation. Platelets are activated by exposed collagen on an injured vessel wall and subsequently aggregate to form a platelet plug and secrete intracellular granules containing serotonin, ADP, and some active substances. Since the first trigger of platelet aggregation in vivo is the activation by collagen of subendothelial tissues, we focused on an agent which would inhibit collagen-induced platelet aggregation. In our efforts to search for inhibitors of collagen-induced platelet aggregation, we have discovered a novel class of benzenes (I). compounds, [2-(w-aminoalkoxy)phenyl]ethyl]
Scheme I
38: Rz- H. 2-OH
OH 4a: R, = R2 = H. 2-OH
OH
5a: R1 = R2 = H, 2-OH I / O(CHz)J,
I ( A ; Ring A , B; Ring B)
In this paper are described the relationship between the structure of [2 4 (w-aminoalkoxy)phenyl]ethyl]benzenesand the inhibition of in vitro collagen-induced platelet aggregation and in vivo thrombus formation in mice. During the course of study with the compounds, we found that this series of compounds exhibited a potent S2serotonergic (1) Woolf, N. Br. Med. Bull. 1978, 34, 137. (2) Mitchell, J. R. A. Br. Med. Bull. 1978, 34, 103. (3) Bouchier-Hayes,D.Ir. Med. J . 1983, 76, 101. (4) Koch-Weser, J. N . Engl. J . Med. 1978, 298, 1403.
receptor antagonism, which highly correlated with an inhibitory effect on in vitro collagen-induced platelet aggregation.
0022-2623/90/1833-l818$02.50/00 1990 American Chemical Society
