J. Med. Chem. 1993,36, 2018-2025
2018
Synthesis of Phosphocholine and Quaternary Amine Ether Lipids and Evaluation of in Vitro Antineoplastic Activity Susan L. Morris-Natschke,’J Fatma Gumus) Canio J. Marasco, Jr.,s Karen L. Meyer,ll Michael Marx,l Claude Piantadosi? Matthew D. Layne,# and Edward J. Modest# School of Pharmacy, Division of Medicinal Chemistry and Natural Products, University of North Carolina, Chapel Hill, North Carolina 27599, Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Gazi University, Etiler, Ankara, Turkey, Roswell Park Memorial Institute, Grace Cancer Drug Center, Buffalo,New York 14263, College of Pharmacy, University of Oklahoma, Oklahoma City, Oklahoma 73190, ChemSyn Science Laboratories, Lenexa, Kansas 66215, and Department of Biochemistry, Boston University School of Medicine, Boston, Massachusetts 02118 Received August 13, 1992
The in vitro antineoplastic activity of many phosphorus-containing (e.g., phosphocholines) and non-phosphorus-containing(e.g., quaternary ammonium salts) ether lipids has been evaluated in the HL-60promyelocytic cell line. These compounds are analogues of ET-18-OMe (l-O-octadecyl2-O-methyl-ruc-glycero-3-phosphocholine). Structural modification of l-(alkyhnido)-, - (alkylthio)-, and (alky1oxy)propyl backbones has provided further insight into the structure-activity relationships of these lipids. In this study, a long saturated C-1 chain and a three-carbon backbone with a single short C-2 substituent were preferred. At the positively charged nitrogen of phosphocholines, fewer than three Substituents caused a significantloss of activity, and substituents larger than methyl decreased activity slightly. In the nonphosphorus compounds, many nitrogen heterocycles and also a sulfonium moiety were incorporated without changing the degree of activity; however, a thiazolium group decreased activity. The most active compound, 29 [N-[3(hexadecyloxy)-2-methoxypropyl]-3-(hydroxymethyl)pyridiniumbromide], was approximately twice as active as the reference standard, ET-M-OMe, in a trypan blue dye exclusion assay.
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Introduction Synthetic ether lipids display a wide range of biological activitiesincluding inhibition of (1)membrane-associated enzyme [protein kinase C (PKC),l*” phospholipase C,2b and sodium/potassium ATPasekY1 activity, (2) neoplastic .~~~ cell growth! and (3) infectious HIV-1 r e p l i c a t i ~ n Exact mechanisms of action for these inhibitory activities have not been established, but some interrelationships may occur. Both preferential PKC inhibition1>2 and membrane fluidization6 in the malignant cell have been proposed as mechanisms for the antineoplastic activity of ether lipids. Entry of HIV-1into cells may depend on phosphorylation of the CD4 receptor’ by PKC, suggesting a possible link between PKC inhibition and anti-HIV-1activity. Further, increased membrane fluidization has also been suggested as a mechanism by which AL721, a mixture of naturally occurring lipids, inhibits HIV infectivity.s In recent years, we have focused on the biological activity of ether lipids and have synthesized a variety of analogues, both type A phosphorus-containinfi9JO(phosphocholines and phosphoethanolamines)and type B non-phosphoruscontaining2aJl (primarily quaternary ammonium salts). In vitro biological activities of these compounds against various neoplastic cell lines,’3lB-13 membrane-linked enzymes,fLJO and/or HIV-1496 have been reported. Further structural modification of l-(alkyloxy)-,-(alkylthio)-,and -(alkylamido)propyl phospholipids and quaternary ammonium ether lipids has been made to further define the structure-activity relationships. The present paper will T o whom correspondence should be addressed. +University of North Carolina. 8 Gazi University. 1 Grace Cancer Drug Center. I University of Oklahoma. 1 ChemSyn Science Laboratories. 8 Boston University School of Medicine.
describe the antineoplastic activity of these analogues against the human HL-60 promyelocytic leukemia cell line. The standard used in all assays was l-O-octadecyl-2O-methyl-ruc-glycero3-phosphocholiie,ET-180Me. Two assays were used to determine the in vitro IDm values: a trypan blue dye exclusion assay (TB) and an assay measuring inhibition of incorporation of tritiated thymidine (TdR).
Chemistry The ether lipid structures are shown in Figures 1(type A phosphorus)and 2 (type B non-phosphorus). The type B non-phosphoruscompounds constitute three series: (1) analogues with a heterocyclic (e.g., pyridine or N-methylpyrrole) or acyclic (e.g., trimethyl) amine directly attached to the carbon backbone; (2) analogues with an inverse choline substituent (e.g., NJV-dimethyl-3’-hydroxypropylamine) on the carbon backbone; and (3) analogues with the quaternary moiety present at the end of an alkoxy spacer group. The synthesesof many of these compounds (Figure 1, compounds 1,2, 18-22; Figure 2, compounds 23 and 24) have been reported previously and for new compounds follow general procedures described in earlier p a p e r ~ . a ” ~ ~ J 3 Scheme I illustrates the preparation of thiolipids 3 and 5-7. First, 3-mercapto-l,2-propanediol was alkylated with an alkyl halide and alcoholicpotassium hydroxide.l4J5The primary hydroxyl was then protected as the trityl ether.16 For compound 3, a dicyclohexylcarbodiimide (DCC)/ dimethyl sulfoxide (DMSO) oxidationlegave a ketone at C-2, which was reacted with methylmagnesium iodide to give a tertiary alcohol at (2-2. The general reaction sequence then continued by formation of the C-2 alkyl ether with an alkyl iodide and sodium hydride. Detritylation with p-toluenesulfonic acid” reformed the free
0022-2623/93/1836-2018$04.00/00 1993 American Chemical Society
Phosphocholine and Quaternary Amine Ether Lipids Structure B
Structure A
R+XR
Structure
c
Journal of Medicinal Chemistry, 1993, Vol. 36, No.14 2019
Scheme I&* {OH
x
B
R8tarence Compounds: 0 18:o ETl8one 10 NHCO 17:O Pd s 16:o New Compounds: 3 s 1s:o 4 S CH2CHOMe(CH2)l3CH3 5 s 16:l
18:o
9
0
10
0
(CH2)gCO(CH2)5CH3 16:O
014:O
17:O
One OEt OEt OEt
H H
16:o
19e
0
16:o
20e
0
16:o
21e
NHCO NHCO
19:O
-
i7:o
structure A
x
13-15
-
c:" BL
X
ET18OMe 23d
o S
1a:o 16:O
one H one H
21d
s
18:o
one H
New Compounds:
25 26
0
l6:O
one H
0
16:O
one
27
0
8:O
one H one H One H OEt H
H
20
0
16:O
29
16:O
30
0 0
31
0
18:o
OEt
H
32
S
16:O
CH20Me
H
33
s
16:O
~n20ne H
34
s
16: 0
one H
3s 36
S
06:O
S
16: 0 16:O
3i-a
6
16: 0
30a
S
16:O
39
S
12:o
one
40
s
16:O
OEt
41
s
16:O
05:O
42
s
16:O
H
18:o
C
"coc17H3S OPO;CH2CH&
H
structure B
Z
-a E Reference Compounds:
R O '"' OF'O$H2CH2&R(")3
iii
-
Figure 1. Structures of type A phosphorus-containing ether lipids. (a) All compounds have structure A except for 11 which has structure C and 18 and 22 which have structure B. (b)PC = phosphocholine = OPOs(CH2)2NMes;all Z groups are zwitterionic. (c)Synthesisreported in ref 10. (d)Synthesisreported in 9. (e) Synthesis reported in ref Sa.
R+xR
ii or iii
Scheme 11.
OMe
H H H H H H
s
A
R'O+l
OTr
-
a (i) (1) RX, KOH, (2) TrC1; (ii) (1)DMSO, DCC, (2) MeMgI (Y = Me), (3) Et&NaH (R' = Et); or (iii) (1) R'I, NaH (Y = H,R' = Et or Pr); (iv) (1) pTSA, (2) POCS, (3) choline tosylate (R" = Me), or (2) C4P02CH2CHaBr, (3) NEt (R" = Et)." R, R'O, Y a~ given in Figure 1 for compounds 3 and S-7.
H H H
17:O 17:O 17:O 17:O
1e,e
OH
SR
{OH
3. 5-7
OEt H H H
0
zaate
H
16:o
8
14 15 16 17
PC PC PC
H
H
16:o 16:o
0
H
-
s
NHCO NHCO NHCO NHCO NMeco
OMe OEt OEt
17:O
s
12
Zb
Me H H H
6
13
X
OEt OEt OEt OPr OEt OEt OEt One
7
11L
E
-
SH
+ O A Rz
z
H
one H
Figure2. Structuresof type B non-phosphorus-containingether lipids. (a)All compounds have structure A except for 37 and 38 which have structure B. (b) All Z groups contain a positively charged quaternarynitrogen atom except for compound 26 which has a positively charged sulfur atom. (c) PC = phosphocholine = OPOs(CH2)2NMes;all Z groups are zwitterionic. (d)Synthesis reported in ref 2a. primary hydroxyl. Phosphocholines were prepared by reaction with phosphorus oxychloride followed by choline tosylate.18 For compound 7, (NJVJV4riethylamino)ethanol tosylate replaced choline tosylate, and, for phosbropholipid 8, (NJV-dimethyl-N-benzy1amino)ethanol mide was used in this phosphorylation step.
(i) CI~HSSCOC~; (ii) C1902CHzCH2Br; (iii) 2 = NMes for compound 13, pyridine for compound 14, M ~ ~ N C H ~ C H Z C Hfor ~OH compound 16.
In the synthesis of compound 4, preparation of the @-methoxy-substitutedC-1alkyl chain required additional steps that are not illustrated in Scheme I. 3-Mercapto1,2-propanediolwas reacted with 1,Zepoxyhexadecaneand KOH, the 1,2-diolwas then protected as the dimethyl ketal. After alkylation of the free side-chain hydroxyl group, the ketal was opened with acid. The remaining steps (tritylation at C-1, alkylation of C-2, detritylation, and phosphorylation) were performed as shown in Scheme I. Compound 9 was prepared by reaction of 3-O-trityl-2O-ethylglycerolwith the mesylate of 10-oxo-l-hexadecanol and sodium hydride followed by detritylation and phosphorylation as shown in Scheme I. The keto alcohol was prepared by reaction of dihe~ylcadmium~~ with the acid chloride of sebacic acid monomethyl ester.20 Formation of the dimethoxy phosphocholine, 10, began with esterification of the primary hydroxyl of 1-Ohexadecylglycerol with benzoyl chloride.ls A ketone at C-2 was generated with the DCC/DMSO oxidation shown in Scheme I and was ketalized with methanol and sulfosalicylic acid.16 Saponification of the benzyl ester was followed by formation of the phosphocholine with 2-chloro-2-oxo-1,3,2-dioxaphospholaneand trimethylaminee21This same phosphorylation procedure was used in preparation of compound 11 from 2-heptadecyl-4(hydroxymethyl)-1,3-dioxolane. The latter compoundwas formed by transacetalation of octadecanal dimethoxy acetal with glycerol.22 SchemeI1shows the synthesis of amido compounds 13, 14, and 16. 3-Octadecanamido-l-propanol was formed by reaction of 3-amino-l-propanol with stearoyl chloride in pyridine. This alcohol was reacted with 2-bromoethyl phosph~dichloridate~~ and the isolated bromide then displaced with trimethylamine, pyridine, or (NJV-dimethy1amino)ethanolto give the final products. This same phosphorylating reagent and reaction with pyridine gave the glycerophosphate 12. Amido compounds 16 and 17 were prepared from 3-amino-1,2-propanediol by the reaction sequence: amidation, tritylation at C-1, alkylation at C-2, detritylation, and phosphorylation with 2-bromo-
2020 Journal of Medicinal Chemistry, 1993, Vol. 36, No. 14
Scheme IIIab R
200 "C). lH NMR (CDCl& 0.87 (t, 3H, terminal CHd, 1.24 {m, 26H, (CHZ)l& 1.55 (m, 2H, CHZOCHzCHz), 2.7-3.05 (dd, 2H, CHC&S), 3.05 (t, 2H, SCHzCH2N),3.4-3.6 (m,5H, CHCH20CH2),3.45 (s,3H,OCHs),3.51 (a, 9H, N(CH&), 3.82 (m, 2H, CHzN). Anal. (CsHuNOzSBr) C, H, N, S. sMethyl-s(2-hydroxyethyl)-s[~( hexadecylow)-2-methoxypropyl]sulfonium Tosylate (26). 3-(Hexadecyloxy)-2(methoxypropyl)-(2'-hydroxyethyl)suMde (0.8g, 2.1 mmol) and methyl tosylate (0.8g, 4.3 mmol) were placed in 40 mL of acetone and refluxed for 8 h. An additional aliquot of methyl tosylate was added; the mixture was refluxed for 8 h and stirred for 48 h at room temperature. After concentration, the crude product was chromatographed on silica gel using CHCkMeOH (91 to 41), giving pure sulfonium salt in a 20% yield (242 mg, 0.42 mmol) as a waxy solid. lH NMR (CDCla): 0.87 (t, 3H, terminal CHs), 1.25{m,26H, (CHZ)l3],1.55 (m, 2H, CHCHZOCHZCHZ), 2.3 (s,3H,CeH4CH3),3.1(2 s,3H,SCH3),3.4 (s,3H,0CH3),3.5-4.1 (overlapping m, 11H, CHZOCH&HCH~SCH&HZ), 7.1 and 7.6 (2 d, 4H, C&). Anal. (C&IaeOeSz) C, H, S. 3- (Hexadecylthio)-2-(met hoxymethy1)-l-bromopropane. Thiscompoundwas used in the synthesisof both analogues 32 and 33. First, 3-(hexadecylthio)-2-(methoxymethyl)-l-propan01 was prepared with slight modifications of the procedure of Bosies et a1.m The bromide was then prepared with CBr4 and triphenylphosphine as described in the synthesis of 25. 1HNMR (CDCla): 0.85 (t,3H, terminal CHs), 1.25 {m,26H, (CH2)13),1.55 (m, 2H, SCHZCHZ),2.1 (m, lH, CH), 2.53 (t,2H, SCHZCHZ),2.63 (d, 2H, CHCHzS), 3.35 (8,3H, OCHs), 3.45 and 3.65 (2 dd, 4H, CH20, CHZBr).
Morris-Natschke et al. Nflfl-Trimethy 1-N-[3- (hexadecylthio)-2-(methoxymethyl)propyl]ammonium Bromide (32). Compound 32 was prepared by heating (60-65 OC) the above bromide (1.0 g, 2.4 mmol) with 40% aqueous M e a (10 mL) in THF (25 mL) for 16 h. Concentration, precipitation from acetone, and chromatography with a CHCls-MeOH gradient (965 to 1:l)gave 411 mg of pure adduct (0.85 mmol, 35% yield, mp 157-159 "C) and 248 mg of impure material. IH NMR (CDCh): 0.85 (t,3H, terminal CH& 1.25 {m,26H, (CHZ)ls),1.55 (m, 2H, SCHZCHZ), 2.4 (m, lH, CHI, 2.55 (t,2H, SCHzCHz),2.7 (d, 2H, CHCHzS), 3.35 (8,3H, OCHs), 3.48 {E, 9H, N(CH3)3),3.4-3.7 (m, 4H, CHZO,CHZN). Anal. (CaHazNOSBr) C, H, N, S. Nfl-Dimet hyl-N-(3-hydroxypropyl)-N-[3-( hexadecylthio)-2-(methoxymethyl)propyl]ammonium Bromide (33). Compound 33 was also prepared from the above bromide (1.0 g, 2.4 mmol) by reaction with (NJV-dimethy1amino)propanol(heat, 5 mL) at 60-65 "C for 16 h. Crude salt was precipitated from the reaction mixture with EhO at -20 "C. Chromatography as for 32 gave 518 mg of pure compound (1.0 mmol, 41% yield, mp 114-116 "C). 1H NMR (CDCL): 0.85 (t,3H, terminal CHa), 1.25 {m, 26H, (CH2)13), 1.55 (m, 2H, SCH&Hz), 2.1 (m, 2H, NCHzCHzCHzO), 2.45 (m, lH, CH), 2.6 (t, 2H, SCHzCHz), 2.7 (dd, 2H, CHCHzS), 3.33 {a, 6H, N(CHs)z), 3.38 (8, 3H, OCHa), 3.4-3.65 (m, 4H, CHCHzO, CHzOH), 3.9 (m, 4H, CHzNCHz). Anal. (CXHdOzSBr) C, H, N, S. The remaining ammonium salts (27-31 and 34-42) were prepared from the appropriate bromides (synthesized following the standard procedures described in refs 2a, 9, and 11)by reaction with an aliphatic or aromatic amine as described above or in refs 2a, 11,and 13. lH NMR and elemental analyses are supplied in the supplementary material.
Acknowledgment. The authors would like to thank Dr. Jefferson R. Surles and Shang-Yong Chen for their synthetic contributions. Supplementary Material Available: Chemical names, analytical data, and lH NMR data for compounds 6-7,27-31, and 34-42 (3pages). Orderinginformation is given on any current masthead page.
References (a) Helfman, D. M.; Barnes, K. C.; Kinkade, J. M.; Vogler, W. R.; Shoji, M.; Kuo, J. F. Phospholipid-Sensitive Ca*+-Dependent Protein Phosphorylation System in Various of Leukemia Cell Lines HL60 and K562, and ita Inhibition by Alkyllyeophoepholipid. Cancer Res. 1983,43,2965-2961. (b) Parker, J.; Daniel, L. W.; White, M. Evidence of Protein Kinase C Involvement in Phorbol Diester-StimulatedArachidonic Acid Release ,and Prostagandin Synthesis. J. Biol. Chem. 1987,262,5385-6393. (a)Marasco,C.J.,Jr.;Pmtadosi,C.;Meyer,K. L.;Morris-Natechlre, S.;Ishaq, K. S.;Small,G. W.;Daniel, L. W. Synthesisand Biological Activity of Quaternary Ammonium Derivatives of Alkylglycerols as Potent Inhibitore of Protein Kmase C. J. Med. Chem. 1990,33, 985-992. (b) Perella,F. W.; Piantadosi,C.;Marasco, C.J.;Modest, E. J. Inhibition of Phospholipase C and Cell Growthby Ether Lipid Analogues of Phosphotidylinositol. h o c . Am. Ausoc. Cancer Res. 1990,31,409. (c) Zheng, B.; Oishi,K.; Shoji, M.; Eibl, H.; Berdel, W. E.; Hajdu, J.; Vogler, W. R.; Kuo, J. F. Inhibition of Protein Kinase C (Sodium plus Potaaeium)-Activated Adenosine Triphosphataseand Scdium Pumpby Synthetic PhoepholipidAnalogues. Cancer Res. 1990,50, 3025-3031. (d) Diomede, L.; Bianchi, R.; Modest, E. J.; Piovani, B.; Bubba, F.; Salmona,M. Modulation of ATPase Activity by Cholesterol and Synthetic Ether Lipide in Leukemic Cella. Biochem. Pharmacal. 1992,43,803-807. (a) Berdel, W. E.; Munder, P. G. AntineoplasticActions of Ether Lipids Related to Platelet-ActivatingFactor. In Platelet-Activating Factor and Related Lipid Mediators; Snyder, F., Ed.; Plenum Press: New York, 1987; pp 449-467. (b) Berdel, W. E.; Andreesen, R.; Munder, P. G. Synthetic Alkyl-Phospholipid Analogs: A New Class of Antitumor Agents. In Phospholipids and Cellular Regulation; Kuo, J. F., Ed.;CRC Preee: Boca Raton, FL, 1985;Vol. 11, pp41-73. (c) Modest, E. J.; Daniel, L. W.; Wykle, R. L.; Berens,M. E.; Piantadosi,C.; Surles,J. R.;Morrm-Natschke, S. Novel Phospholipid Analogs as Membrane-Active Antitumor Agents. In New Avenues of Developmental Cancer Chemotherapy (Bristol-MeyersCancer Sympoeia);Harrap, K. R., Connore,T. A., Eds.; Academic Press: New York, 1987; Vol. 8, Chapter, 19, pp 387-400.
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