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3-ethyl and 3-methyl ethers of 19-iodocholesterol as potential adrenal scanning agents
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3-ETHYL AND 3-METHYL ETHERS OF 19-IODOCHOLEST~ROL POTENTIAL ADRENAL SCANNING AGENTS.
AS
J.A. Owoyale Faculty of Pharmaceutical Sciences, Ahmadu Bello Unlverslty,Zaria, Nigeria. and S.S. Szlnal College of Pharmacy, University of Florida, Gainesvllle, Florida, 32610. Received 5-6-80 ABSTRACT 19-Iodocholesterol- 131I, though useful as an adrenal scanning agent, was found to be unstable, However, the substitution of methoxyl and ethoxyl groups for the hydroxyl group in the 3 position rendered these derivatives much more stable in solid form than the parent compound. They showed concentration in the adrenals of dogs similar to that of 19-iodocholesterol itself and therefore may be useful as adrenal scanning agents. INTRODUCTION 19-Iodocholesterol-131I
has been used as early as
1971 as an adrenal scanning agent in man (1,2). tlons in this laboratory an unstable compound by other workers
Observa-
showed that 19-iodocholesterol
is
(3,~) and this has since been confirmed
(~,6).
In addition,
the immediate
precursor in the synthesis of 19-1odocholesterol, 19-tosyloxycholesterol
Is unstable
namely
(~).
It was therefore thought that the instability of these two compounds was probably due to the slmultane~us presence of a hydroxyl group on C-3 and a good leaving group on C-19 of the steroid molecule. An indirect evidence for this has since been found. This Is the ease with which 19-iodocholesterol its isomer,
converts to
6B-iodomethyl-19-norcholest-5(10)-en-3s-ol
(7).
Thls isomer has been shown to localize better in the adrenal
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glands (8,9) and to be more stable than 19-1odocholesterol (6).
Since a hydroxyl group is reactive, it was believed
that its conversion to an ether should lead to a more stable compound.
Thus 19-1odocholesterol 3-methyl ether
and 19-iodocholesterol 3-ethyl ether were synthesized and tested in the dog for their localization in the adrenal glands. EXP~IMENTAL General: melting points were determined on a ThomasHoover capillary melting point apparatus and are not corrected. Infrared (Jr) spectra were recorded on a Beckman infrared spectrophotometer Acculab 3. Nmr spectrum was taken in CDC13 at 90Hz in the Fourier transform mode on a Bruker HX-90 spectrometer equipped with a Nicolet 1083 computer. Mass spectrum was recorded on a DuPont Model double beam instrument by direct probe. High resolution ~91 mass spectrum was performed by Battelle's Columbus Laboratories, Columbus, Ohio. Qualitative tlc was run on Merck sillica gel using cyclohexane-ethyl acetate (9:1) as the solvent system. Elemental analyses were performed by Midwest Microlab, Indianapolis, Indiana. Structures of the various compounds were confirmed by it, nmr (IH and/or 13C) or mass spectra (~).
I
v
v17
vm Scheme A
IV
~*a-~OX~a
135
The synthesis of 19-iodocholesterol 3-methyl ether (VII) starting with commercially available cholesterol (I) or its methyl ether (II) is shown in Scheme A. $-Bromo-~-methoxy-~s-cholestan-68-ol(III) N-bromoacetamide (5'3 g, 0.039 mol) was added in portions during 15 minutes to a cold (< 20 ° ) stirred mixture of II (10 g, 0.025 mol), dioxane (125 ml), ~rater (5 ml) and aqueous perchloric acid (7.3 ml of a solution consisting of 3.15 ml of 70% HC10.~ and 5 ml H20), in an opaque flask. The reaction mzxture was allowed to warm to room temperature while stirring continued for another 3 hours. The mixture was then cooled to 5 ° and 1% Na2SgO 3 solution (ca 60 ml) and ice-water were added with stirring. The gelatin-gus ppt obtained was taken up in ether and the ether extract was washed with water, dried (Na2S04) , and evaporated to give an oil which crystallized from abs EtOH. Recrystallization in the same solvent gave 3.1 g (25%) of a solid as long white needles, mp_161-163 °, ir (KBr) 3~90cm-i (OH stretching), 1075cm -I (C-O-C antisymmetrical stretching of ether); nmr (CDCl~ 4.5 (m,3-H), 3.35 (s, 3-OCH~), 3.69 (6-H), 0.66 (s,18ZCH3) , 0.97 (s,19-CH3) , 0.91 ~d, 21-CH3) , 0.86 (d, 26-CH3), 0.86 (d,27-CH~); tlc Rf 0.53. Anal. Calcd for C28H49BrO2: C,67.58; H,9.93; Br,16.06. Found: C,67.63; H,9.96; Br,17.26. $-Bromo-~ ~-methoxy-68,19-oxido-~ -cholestane(IV) A mixture of Pb (OAc) 4 ~48.8 g, 9.11 mol, previously dried over P205 for 2~ hours), CaCO 3 (2~.2 g, 0.24 mol) and cyclohexane (1.21), was refluxed for ~0 minutes using a 500-watt lamp~ Compound III (11 g, 0.02 mol) and iodine (I~ g, 0.06 mol) were added to this mixture and refluxing continued for 1 hour. The initial purple color of the reaction mixture was usually lost by the end of the reaction period. The mixture was then cooled to room temperature, filtered over Cellte, and the filtrate was evaporated to give an oil which crystallized from abs EtOH. Recrystallization from abs EtOH yield 7.1 g (65%) Qf IV as white flat blades, mp 109-111°; ir (KBr) 1490cm -A (6 ,19-~ido group stretching); 108Ocm~l (C-O-C antisymmetrical stretching of ether), ~ (CDCl~) 3.7 (m,~-H), 3.3~ (s,3-O~H3), ~.O5 (6-H), 0.70 (s,18-C~3), 3.91 (d,19-CH2), 3.70 (d, 19-CH2), 0.90 (d,21-CH3), 0.86 (d,26-CH3) , 0.86 (d.27-CH3); tlc Rf.= 0.41. Anal. Calcd for C28Hh7Br02: C,67.86, H,9.56, Br, 16.13. Found: C,67.57; H,9.77; Br,16.40. 38-methoxy-~-cholesten-1~-ol(V) To a stirred mixture of glacial AcOH (150 ml), water ~dm~n~n~u~ I~5gf, o O ~ I m ~ } ) ~ n m ~ ~ d d ~ i 4 n g S O ~ y a m minutes. Stirring continued for an additional ~0 minutes while the temperatureof the mixture was maintained between 45 ° and 50 ° . The mixture was cooled to room temperature, filtered over Celite and the Celite-zinc cake was washed
136
~
~
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x ~
with ether-CH2Cl2 (5:1). The combined filtrate was washed with water, dried (Na2SOh) , and evaporated to give a white solid. Recrystallizatio6 from acetone gave 3.3 g (64%) of a white fluffy product, mp 171-172o; ir (KBr) 3465cm-1 (n~v OH stretching); I085cm-1 (C-O-C antlsymmetrical stretching of e t ~ ) ~ 1670cm-~ ~C=C~ nmr (CDC13) 3.0 (mp3-H), 3.35 ks,3- 3), 5.75 ( - ), .74 (s,18-CH~), 3.83 (d,19-CH2), 0.92 (d,21-CH3) , 0.86 (d,26-CHq), 0.88 (d,27-CH3) ; tlc Rf = 0.20. Anal. Calcd for C 2 ~ 4 8 0 2 : C,80.71; ~,11.61. Found: C,80.95; H,11.66. ~S-Methoxy-19-tosyloxy-~-chelestene(VI) Tosyl chloride (6.4 g, 0.034 mol) was added in one portion to a solution of V (3.2 g, 0.008 mol) in pyridine (110 ml) at 5 °. The resulting mixture was allowed to warm up to and maintained at room temperature for 5 days, after which it was poured into ice-water and extracted with ethen The ether extract was washed successively with dilute HC1, 5% NaHC03 solution, and water, then dried (Na2S04) , and evaporated to give an oil which upon triturationwith MeOH yielded white crystals. Recrystallization from CHC13-MeOH yielded 3.3 g (75%) of VI as white needles, mp 97-980; ir 1595cm-~ (phenyl); 1360cm-1 (S02 asymmetrical stretching), 1170cm-1 (SO2 split - symmetrical stretching); 1090cm-1 (C-O-C antisymmetrical stretching of ether; nmr(CDClq) 3.0 (m,3-H), 3s30 (s,3TOCH3), 5.56 ~6-H), 0.62 (s,18-~Hq), 4.12 (d,19-CH2), 7.77 (At-H), 7.33 (Ar-H), 2.45 ~,Ar-C~3), 0.89 (d,21-CH3) , 0.86 (d,26-CH3) , 0.86 (d,27-CHq), tic Rf: 0.34. Ahal. Calcd for C3~H5404S: C, 73.6~; H, 9.53; S, 5.62. Found: C, 73.43; H, 9.21; S, 5.62. 19-Iodo-3S-methoxy-5-cholestene(VIl ) A s6kution of V~ (143 mg, 0.0500 nmol) and sodium iodide (75 mg, 0.500 nmol) in isopropyl alcohol (20 ml), was refluxed for 4 hr under N2. After cooling, the solvent was reduced to about 5 ml and the resulting mixture ~as poured into ice-water and extracted with ether. The ethereal extract was washed with water (Na2S20~ solution was necessary in some other cases) dried (Na2S04) , and evaporated to give an oil which upon tritura~ion with isopropyl alcohol yielded 79.2 mg (60%) of VII as ~vhite crystals, mp 78-800; ir (KBr) 1090cm-1 (C-O-C antisymmetrical stretching due to ether); 1670cm-1 (CffiC); nmr (CDC13) 3.0 (m,3-H), 3.36 (s,3.0CHR), 5.63 (d,21-CH3), 0.86 (d,27-CH3) tlc Rf = 0.56; m/e 526 ~M+). Anal. Calc8 for C28Hh7IO: C,63.86; H, 9.00; I, 24.15. Found: C,63.47 H, 9j07; I, 24.30. 19-1odo-~-methoxy-5-cholestene-125I(VII~) A solution of VII (200 mg) and Na-lZbI (7.75 mCi) in acetone was refluxed for 4 hr under N2. The solvent was evaporated using N2 and the product was taken up in ether and water. The ether extract was washed with water, dried (Na2S04) , and evaporated (N2). The resulting product
T
]B ~ ¢ 0
137
X ]~m
(180 mg of VIII~ tlc), an oil with specific activity of 39,Ci/mg, was immediately formulated for iv in~ection. The ethyl and methyl ethers of 19-1odocholesterol were synthesized starting from the known 3B-acetoxy-19-iodo-5cholestene (IX, 1) - Scheme B. This method was later adapted by Couch et al.(10) in the synthesis of I-labeled 19-1odochnlesterol~,--
Ix
RO ~
vm Ft- CH3 X~ R'CH2C"5
vl R-c~ n
..c.ac.3
Scheme B Pure 19-iodo-~-cholesten-~-ol (X) A solution of 3-acetoxyl derivative of 19-iodocholesterol (IX, 6~0 mg, 1.154 m mol) in dioxane (25 ml) was added dropwise to a stirred alcoholic solution of NaOH (320 mg, 0.007 mmol, NaOH in 0.32 ml H20 diluted to 32 ml with MeOH). This mixture was stirred for 2 hr at room temperature and then poured into ice-water and extracted with ether. The ethereal extract was dried (Na2SOL) and the ether was removed under vacuum to give a flaky'solid which upon trituration with a very small amount of pet. ether gave a white solid. Re-precipitation at room temperature from a mixture of ether and 95% ethanol by evaoorating part of the solvent under vacuum gave 360 mg (61~J) ~f a white crystalline compound, mp 118-119o; ir (KBr) 3~000m-I (OH stretching); 1660cm-I (C=C), absence of 1708cm-1 (C=O due to IX); nmr (CDCI%) 3.35 (m,3-H), 5.62 (6-H), 0.78 (s, 18-CHq), 3.29 (d,19-C~2), 3.59 ~d,19-CH2), 0.93 (d,21TCH3), 0.86 ~d,26-CH3) , 0.86 (d,27-CH3) ; tlc = O.17 m/e 512 (M+3. The action of perchloric acid on a suspension of 19iodocholesterol in either trimethyl orthoformate or triethyl orthoformate gave in from 5 to 15 minutes the desired methyl ether or ethyl ether of 19-iodocholesterol respectively. A typical example is the preparation of 38-ethoxy-19iodo-5-cholestene (XI). ~-Ethox~-l~-iodo-~-cholestene (XI) Aq HC10~ ( 7 ~ . 325 mg, 3.499 nmol) was added dropwise
138
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to a stirred suspension of freshly prepared pure 19-1odocholesterol (X , 596 mg, 1.163 nmol) in triethyl-orthoformate (4 ml). Stirring continued for 10 minutes; then the mixture was added slowly to NaHCO 3 solution and extracted ~h ether, The ether extract was washed with waterp dried a2SO~) , and evaporated to give an oil which crystallized from abs ~tOH. Recrystalllzation from this so~vent gave 300 mg (48%) of 7d as white crystals, mp 67-68 , ir (KBr) 1110cm-1 (C-O-Cantisymmetrlcal stretching of ether); 1660cm-1 (C=C); tlc Rf = 0.6~; m/e 540 (M+). Anal. Calcd for C29H1910: C,6~.43; H,9.14; 1,23.~8. Found: C,64.39; H, 9.1~; 1,23.72. ~8-Ethoxy-19-iodo-~-cholestene- 125 I (XII) This compound was prepared in a similar manner to the methoxyl analogue (VIII). The resulting product was also immediately formulated for iv injection.
~N
Formulation. Each radioiodinated compound (VIII and XII, 50 mg) was formulated in 95% ethanol ( 4 m 1), polysorbate80 (30 drops, Sigma Chemicals) and 0.9% saline solution (ca ~5 ml to make 50 ml solution) for iv injection into dogs. Animal Studies: A total of 9 dogs weighing 18-30kg. each was employed in these studies. Each dog received about 50 mg of formulated drug (50 ml, about 60~Ci/kg) by injection into the foreleg vein. These dogs received no other drugs or treatment and they continued to feed on dog chow. Half the number of dogs receiving any one drug was sacrificed after 24 hours and the rest after 4 days by iv injection of lethal doses of sodium pentobarbital. Autopsies were carried out immediately after the death of the animals. Samples of various organs (lung, liver, spleen, heart, kidney, testis, brain and adrenal) and of fat and muscle were collected, cleaned of fat, washed with water and stored in formalin for at least 1 week to harden. The entire organs were removed from formalin, washed well with water, blotted weighed. Two to 4 block sections c_~a I x I x 2.5 cm were excised from different parts of each organ except the adrenals where the entire organ was divided into 3 or 4 sections. One section each of fat and muscle was taken for each dog. Each block section was weighed, placed in a plastic test-tube (1.5 x 15 cm) and the radioactivity in each sample was determined in a sodium iodide well crystal (using Packard auto-gamma scintillation spectrophotometer, model 3001).
dr~ and
RESULTS Unlike 19-4Lodocholesterol which started to decompose during its preparation,
the methyl and ethyl ethers of
this compound were found to be 98% pure at 9 and 5 months
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139
respectively in the solid form (3,4,11).
The decomposition
was monitored by tlc, yellow color formation (3,4) and quantitated by the use of 13C nmr (11).
Similarly the
methyl and ethyl ethers of 19-iodocholesterol when formulated were found to be stable for 6 and 15 days respectively whereas 19-iodocholesterol started decomposing during formulation (3,4). The results of the tissue distribution of 19-iodocholesterol-125I (prepared by the method of Counsell e_~ta_!l.) and the corresponding methyl and ethyl ethers in dogs are presented in Tables I and II. Table I.
Lung Liver Spleen Heart Kidney Testis Brain Muscle
Fat
Tissue Distribution Studies in Dogs One Day's Result 19-Iodocholesterol-125I a The 3-methyl The 3-ethyl ether Vill a ether (XII) b 84 + 14 73"+ 20 170 103 ~ 6 271 • 78 546 104 ~ 16 110 ~ 26 319 hO ~ 8 ~0 • 13 59 ~0 • 8 59 ~ 18 7~ 29 ; 3 27 ; 2 56 I~ • 2 16 • 4 I0 10 • 2 9 ~ 2 17
13 •
Adrenal
2
435 ~ 90
3
14
622 ~ 17
13 •
1,240
~Values represent DPM/mg + SEM for 3 dogs. Values represent DPM/mg Tor one ~og. It will be noticed from these two tables that the localization of the 3 compounds in the adrenal was higher on the 4th day than on the first day. In order to appreciate the usefulness of the methyl and ethyl ethers of 19-iodocholesterol, the adrenal-kidney concentration ratios in dogs are presented in Table III.
140
~
~
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x x,~
~able II.
Tissue Distribution Studies in Dogs Four Days' Results. 19-Iodocholesterol-1251a The 3-methyl The 3-ethyl ether (VIII) a ether (XII) b Lung 31Z ~ 24 * 5 155 + 80 Liver 34+ ~ 93 ~ 12 ~56 ~ I
Spleen Heart Kidney Testis Braim Muscle Fat Adrenal
12
13 ÷
21 + 1 - -
2+0 6 ~' I 13~ 3 1 , 0 1 b ~ 1 _ 51
26 ~' 25 "~' 3+ 5~' 16; 701 ~'
27
3 [--11 0 2 128
166 37
68 + 6.3 ~' 1'7 3~' 0 16 ~' .3 16~ 3 1,520 -- 1.35
~Values represent DPM/mg + SEM for 3 dogs. Values represent DPM/mg ~ SEM for 2 dogs. Table III.
Adrenal-Kidney Concentration Ratios
Ratios Compound One-Day Four Days ~Probabili~y) a 19-Iodocholesterol 8.66_+1.27 49.28~.52 The 3-methyl ether (VIII) 11. 0.06) The 3-ethyl ether (XII) 16.81 22.75~3.73 (P > 0.10) aprobability was obtained using the Student's t-t~st. DISCUSSION Both the methyl and ethyl ethers of 19-iodocholesterol localized in the adrenal glands.
The adrenal-kidney ratio
of the methyl and ethyl ethers of 19-iodocholesterol ~re not significantly different from that of the parent 19-iodocholesterol (p > 0.06 and p > 0.10 respectively). The methyl and ethyl ethers of 19-iodocholesterol which are more stable both in the solid form and in solution coupled with their organ distribution patterns would therefore appear to be just as useful for practical application as adrenal markers as 19-iodocholesterol.
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REFERENCES le 2. 3. 4. 5. 6. 7. 8. 9. 10.
11.
Counsell, R.E., Ranade, V.V., Blair, R.J., Beierwaltes, W.H. and Weinhold, P.A., Steroids 16, 317 (1970). Beierwaltes, W.H., Leiberman, L.M., Ansari, A.N. and Nishiyama, H., J. Am. Med. Assoc.! 216, 275 (1971). Szinai, S.S., Williams, C.M., Couch,~.W. and ~1oyale, J.A. Abstracts, 169th National Meeting of the American Chemical Society, Philadelphia Pa., April,(1975). Owoyale, J.A. Ph.D. Dissertation, University of Florida,
(1975).
Hotte, C.E. and Ice, R.D., J. Nucl. Med. f 15, 38 (1974). Scott, K.N., Mareci, T.H., Couch, M.W., an~-Williams, C.M., Steroids, 30, 2131 (1977). Kojima, M., Maeda, M., Ogawa, H., Nitta, K., and Ito, T., J.C.S. Chem. Comm., 47 (1975). Kojima, M., Maeda, M., Ogawa, H., Nitta, K., and Ito, T., J. Nucl. Med., 16, 666 (1975). Sarkar, S.D., B e i e ~ l t e s , W.H., Ice, R.D., Basmadjian, G.P., Hetzel, K.R., Kennedy, W.P. and Mason, M.M., J. Nucl. Med., 16,1038 (1975). -Couch, M.W.,-~cott, K.N., and Williams, C.M., Steroids, ~c7~t 1K areci, T.H., Couch, M.W. and Williams, C.M. Steroids, 30, 511 (1977). ADDENDUM The bromine analysis for compound III is not acceptable. The reaction conditions employed to prepare III as in the case of its 3-acetyl analog always gave more than one compound. Eecrystallization from various solvenlme.g, ethanol and dichloromethane gave different products (tlc and glc) which were difficul~ to isolate. This may be responsible for the unacceptable analysis. Accordingly, the unrecrystallized product was used to obtain IV at 65% yield. No reports on optical rotation have been made since our starting material had a 3-alkoxyl group ~nd the final products have the same group intact with only an additional R/imary iodide on C-19 which was easily detected by1:~C nmr.
133
3-ETHYL AND 3-METHYL ETHERS OF 19-IODOCHOLEST~ROL POTENTIAL ADRENAL SCANNING AGENTS.
AS
J.A. Owoyale Faculty of Pharmaceutical Sciences, Ahmadu Bello Unlverslty,Zaria, Nigeria. and S.S. Szlnal College of Pharmacy, University of Florida, Gainesvllle, Florida, 32610. Received 5-6-80 ABSTRACT 19-Iodocholesterol- 131I, though useful as an adrenal scanning agent, was found to be unstable, However, the substitution of methoxyl and ethoxyl groups for the hydroxyl group in the 3 position rendered these derivatives much more stable in solid form than the parent compound. They showed concentration in the adrenals of dogs similar to that of 19-iodocholesterol itself and therefore may be useful as adrenal scanning agents. INTRODUCTION 19-Iodocholesterol-131I
has been used as early as
1971 as an adrenal scanning agent in man (1,2). tlons in this laboratory an unstable compound by other workers
Observa-
showed that 19-iodocholesterol
is
(3,~) and this has since been confirmed
(~,6).
In addition,
the immediate
precursor in the synthesis of 19-1odocholesterol, 19-tosyloxycholesterol
Is unstable
namely
(~).
It was therefore thought that the instability of these two compounds was probably due to the slmultane~us presence of a hydroxyl group on C-3 and a good leaving group on C-19 of the steroid molecule. An indirect evidence for this has since been found. This Is the ease with which 19-iodocholesterol its isomer,
converts to
6B-iodomethyl-19-norcholest-5(10)-en-3s-ol
(7).
Thls isomer has been shown to localize better in the adrenal
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glands (8,9) and to be more stable than 19-1odocholesterol (6).
Since a hydroxyl group is reactive, it was believed
that its conversion to an ether should lead to a more stable compound.
Thus 19-1odocholesterol 3-methyl ether
and 19-iodocholesterol 3-ethyl ether were synthesized and tested in the dog for their localization in the adrenal glands. EXP~IMENTAL General: melting points were determined on a ThomasHoover capillary melting point apparatus and are not corrected. Infrared (Jr) spectra were recorded on a Beckman infrared spectrophotometer Acculab 3. Nmr spectrum was taken in CDC13 at 90Hz in the Fourier transform mode on a Bruker HX-90 spectrometer equipped with a Nicolet 1083 computer. Mass spectrum was recorded on a DuPont Model double beam instrument by direct probe. High resolution ~91 mass spectrum was performed by Battelle's Columbus Laboratories, Columbus, Ohio. Qualitative tlc was run on Merck sillica gel using cyclohexane-ethyl acetate (9:1) as the solvent system. Elemental analyses were performed by Midwest Microlab, Indianapolis, Indiana. Structures of the various compounds were confirmed by it, nmr (IH and/or 13C) or mass spectra (~).
I
v
v17
vm Scheme A
IV
~*a-~OX~a
135
The synthesis of 19-iodocholesterol 3-methyl ether (VII) starting with commercially available cholesterol (I) or its methyl ether (II) is shown in Scheme A. $-Bromo-~-methoxy-~s-cholestan-68-ol(III) N-bromoacetamide (5'3 g, 0.039 mol) was added in portions during 15 minutes to a cold (< 20 ° ) stirred mixture of II (10 g, 0.025 mol), dioxane (125 ml), ~rater (5 ml) and aqueous perchloric acid (7.3 ml of a solution consisting of 3.15 ml of 70% HC10.~ and 5 ml H20), in an opaque flask. The reaction mzxture was allowed to warm to room temperature while stirring continued for another 3 hours. The mixture was then cooled to 5 ° and 1% Na2SgO 3 solution (ca 60 ml) and ice-water were added with stirring. The gelatin-gus ppt obtained was taken up in ether and the ether extract was washed with water, dried (Na2S04) , and evaporated to give an oil which crystallized from abs EtOH. Recrystallization in the same solvent gave 3.1 g (25%) of a solid as long white needles, mp_161-163 °, ir (KBr) 3~90cm-i (OH stretching), 1075cm -I (C-O-C antisymmetrical stretching of ether); nmr (CDCl~ 4.5 (m,3-H), 3.35 (s, 3-OCH~), 3.69 (6-H), 0.66 (s,18ZCH3) , 0.97 (s,19-CH3) , 0.91 ~d, 21-CH3) , 0.86 (d, 26-CH3), 0.86 (d,27-CH~); tlc Rf 0.53. Anal. Calcd for C28H49BrO2: C,67.58; H,9.93; Br,16.06. Found: C,67.63; H,9.96; Br,17.26. $-Bromo-~ ~-methoxy-68,19-oxido-~ -cholestane(IV) A mixture of Pb (OAc) 4 ~48.8 g, 9.11 mol, previously dried over P205 for 2~ hours), CaCO 3 (2~.2 g, 0.24 mol) and cyclohexane (1.21), was refluxed for ~0 minutes using a 500-watt lamp~ Compound III (11 g, 0.02 mol) and iodine (I~ g, 0.06 mol) were added to this mixture and refluxing continued for 1 hour. The initial purple color of the reaction mixture was usually lost by the end of the reaction period. The mixture was then cooled to room temperature, filtered over Cellte, and the filtrate was evaporated to give an oil which crystallized from abs EtOH. Recrystallization from abs EtOH yield 7.1 g (65%) Qf IV as white flat blades, mp 109-111°; ir (KBr) 1490cm -A (6 ,19-~ido group stretching); 108Ocm~l (C-O-C antisymmetrical stretching of ether), ~ (CDCl~) 3.7 (m,~-H), 3.3~ (s,3-O~H3), ~.O5 (6-H), 0.70 (s,18-C~3), 3.91 (d,19-CH2), 3.70 (d, 19-CH2), 0.90 (d,21-CH3), 0.86 (d,26-CH3) , 0.86 (d.27-CH3); tlc Rf.= 0.41. Anal. Calcd for C28Hh7Br02: C,67.86, H,9.56, Br, 16.13. Found: C,67.57; H,9.77; Br,16.40. 38-methoxy-~-cholesten-1~-ol(V) To a stirred mixture of glacial AcOH (150 ml), water ~dm~n~n~u~ I~5gf, o O ~ I m ~ } ) ~ n m ~ ~ d d ~ i 4 n g S O ~ y a m minutes. Stirring continued for an additional ~0 minutes while the temperatureof the mixture was maintained between 45 ° and 50 ° . The mixture was cooled to room temperature, filtered over Celite and the Celite-zinc cake was washed
136
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~'~ ~ o
x ~
with ether-CH2Cl2 (5:1). The combined filtrate was washed with water, dried (Na2SOh) , and evaporated to give a white solid. Recrystallizatio6 from acetone gave 3.3 g (64%) of a white fluffy product, mp 171-172o; ir (KBr) 3465cm-1 (n~v OH stretching); I085cm-1 (C-O-C antlsymmetrical stretching of e t ~ ) ~ 1670cm-~ ~C=C~ nmr (CDC13) 3.0 (mp3-H), 3.35 ks,3- 3), 5.75 ( - ), .74 (s,18-CH~), 3.83 (d,19-CH2), 0.92 (d,21-CH3) , 0.86 (d,26-CHq), 0.88 (d,27-CH3) ; tlc Rf = 0.20. Anal. Calcd for C 2 ~ 4 8 0 2 : C,80.71; ~,11.61. Found: C,80.95; H,11.66. ~S-Methoxy-19-tosyloxy-~-chelestene(VI) Tosyl chloride (6.4 g, 0.034 mol) was added in one portion to a solution of V (3.2 g, 0.008 mol) in pyridine (110 ml) at 5 °. The resulting mixture was allowed to warm up to and maintained at room temperature for 5 days, after which it was poured into ice-water and extracted with ethen The ether extract was washed successively with dilute HC1, 5% NaHC03 solution, and water, then dried (Na2S04) , and evaporated to give an oil which upon triturationwith MeOH yielded white crystals. Recrystallization from CHC13-MeOH yielded 3.3 g (75%) of VI as white needles, mp 97-980; ir 1595cm-~ (phenyl); 1360cm-1 (S02 asymmetrical stretching), 1170cm-1 (SO2 split - symmetrical stretching); 1090cm-1 (C-O-C antisymmetrical stretching of ether; nmr(CDClq) 3.0 (m,3-H), 3s30 (s,3TOCH3), 5.56 ~6-H), 0.62 (s,18-~Hq), 4.12 (d,19-CH2), 7.77 (At-H), 7.33 (Ar-H), 2.45 ~,Ar-C~3), 0.89 (d,21-CH3) , 0.86 (d,26-CH3) , 0.86 (d,27-CHq), tic Rf: 0.34. Ahal. Calcd for C3~H5404S: C, 73.6~; H, 9.53; S, 5.62. Found: C, 73.43; H, 9.21; S, 5.62. 19-Iodo-3S-methoxy-5-cholestene(VIl ) A s6kution of V~ (143 mg, 0.0500 nmol) and sodium iodide (75 mg, 0.500 nmol) in isopropyl alcohol (20 ml), was refluxed for 4 hr under N2. After cooling, the solvent was reduced to about 5 ml and the resulting mixture ~as poured into ice-water and extracted with ether. The ethereal extract was washed with water (Na2S20~ solution was necessary in some other cases) dried (Na2S04) , and evaporated to give an oil which upon tritura~ion with isopropyl alcohol yielded 79.2 mg (60%) of VII as ~vhite crystals, mp 78-800; ir (KBr) 1090cm-1 (C-O-C antisymmetrical stretching due to ether); 1670cm-1 (CffiC); nmr (CDC13) 3.0 (m,3-H), 3.36 (s,3.0CHR), 5.63 (d,21-CH3), 0.86 (d,27-CH3) tlc Rf = 0.56; m/e 526 ~M+). Anal. Calc8 for C28Hh7IO: C,63.86; H, 9.00; I, 24.15. Found: C,63.47 H, 9j07; I, 24.30. 19-1odo-~-methoxy-5-cholestene-125I(VII~) A solution of VII (200 mg) and Na-lZbI (7.75 mCi) in acetone was refluxed for 4 hr under N2. The solvent was evaporated using N2 and the product was taken up in ether and water. The ether extract was washed with water, dried (Na2S04) , and evaporated (N2). The resulting product
T
]B ~ ¢ 0
137
X ]~m
(180 mg of VIII~ tlc), an oil with specific activity of 39,Ci/mg, was immediately formulated for iv in~ection. The ethyl and methyl ethers of 19-1odocholesterol were synthesized starting from the known 3B-acetoxy-19-iodo-5cholestene (IX, 1) - Scheme B. This method was later adapted by Couch et al.(10) in the synthesis of I-labeled 19-1odochnlesterol~,--
Ix
RO ~
vm Ft- CH3 X~ R'CH2C"5
vl R-c~ n
..c.ac.3
Scheme B Pure 19-iodo-~-cholesten-~-ol (X) A solution of 3-acetoxyl derivative of 19-iodocholesterol (IX, 6~0 mg, 1.154 m mol) in dioxane (25 ml) was added dropwise to a stirred alcoholic solution of NaOH (320 mg, 0.007 mmol, NaOH in 0.32 ml H20 diluted to 32 ml with MeOH). This mixture was stirred for 2 hr at room temperature and then poured into ice-water and extracted with ether. The ethereal extract was dried (Na2SOL) and the ether was removed under vacuum to give a flaky'solid which upon trituration with a very small amount of pet. ether gave a white solid. Re-precipitation at room temperature from a mixture of ether and 95% ethanol by evaoorating part of the solvent under vacuum gave 360 mg (61~J) ~f a white crystalline compound, mp 118-119o; ir (KBr) 3~000m-I (OH stretching); 1660cm-I (C=C), absence of 1708cm-1 (C=O due to IX); nmr (CDCI%) 3.35 (m,3-H), 5.62 (6-H), 0.78 (s, 18-CHq), 3.29 (d,19-C~2), 3.59 ~d,19-CH2), 0.93 (d,21TCH3), 0.86 ~d,26-CH3) , 0.86 (d,27-CH3) ; tlc = O.17 m/e 512 (M+3. The action of perchloric acid on a suspension of 19iodocholesterol in either trimethyl orthoformate or triethyl orthoformate gave in from 5 to 15 minutes the desired methyl ether or ethyl ether of 19-iodocholesterol respectively. A typical example is the preparation of 38-ethoxy-19iodo-5-cholestene (XI). ~-Ethox~-l~-iodo-~-cholestene (XI) Aq HC10~ ( 7 ~ . 325 mg, 3.499 nmol) was added dropwise
138
~
-a" J
mLo
x ~
to a stirred suspension of freshly prepared pure 19-1odocholesterol (X , 596 mg, 1.163 nmol) in triethyl-orthoformate (4 ml). Stirring continued for 10 minutes; then the mixture was added slowly to NaHCO 3 solution and extracted ~h ether, The ether extract was washed with waterp dried a2SO~) , and evaporated to give an oil which crystallized from abs ~tOH. Recrystalllzation from this so~vent gave 300 mg (48%) of 7d as white crystals, mp 67-68 , ir (KBr) 1110cm-1 (C-O-Cantisymmetrlcal stretching of ether); 1660cm-1 (C=C); tlc Rf = 0.6~; m/e 540 (M+). Anal. Calcd for C29H1910: C,6~.43; H,9.14; 1,23.~8. Found: C,64.39; H, 9.1~; 1,23.72. ~8-Ethoxy-19-iodo-~-cholestene- 125 I (XII) This compound was prepared in a similar manner to the methoxyl analogue (VIII). The resulting product was also immediately formulated for iv injection.
~N
Formulation. Each radioiodinated compound (VIII and XII, 50 mg) was formulated in 95% ethanol ( 4 m 1), polysorbate80 (30 drops, Sigma Chemicals) and 0.9% saline solution (ca ~5 ml to make 50 ml solution) for iv injection into dogs. Animal Studies: A total of 9 dogs weighing 18-30kg. each was employed in these studies. Each dog received about 50 mg of formulated drug (50 ml, about 60~Ci/kg) by injection into the foreleg vein. These dogs received no other drugs or treatment and they continued to feed on dog chow. Half the number of dogs receiving any one drug was sacrificed after 24 hours and the rest after 4 days by iv injection of lethal doses of sodium pentobarbital. Autopsies were carried out immediately after the death of the animals. Samples of various organs (lung, liver, spleen, heart, kidney, testis, brain and adrenal) and of fat and muscle were collected, cleaned of fat, washed with water and stored in formalin for at least 1 week to harden. The entire organs were removed from formalin, washed well with water, blotted weighed. Two to 4 block sections c_~a I x I x 2.5 cm were excised from different parts of each organ except the adrenals where the entire organ was divided into 3 or 4 sections. One section each of fat and muscle was taken for each dog. Each block section was weighed, placed in a plastic test-tube (1.5 x 15 cm) and the radioactivity in each sample was determined in a sodium iodide well crystal (using Packard auto-gamma scintillation spectrophotometer, model 3001).
dr~ and
RESULTS Unlike 19-4Lodocholesterol which started to decompose during its preparation,
the methyl and ethyl ethers of
this compound were found to be 98% pure at 9 and 5 months
T
~" ~ o
X
X ~ ,--
139
respectively in the solid form (3,4,11).
The decomposition
was monitored by tlc, yellow color formation (3,4) and quantitated by the use of 13C nmr (11).
Similarly the
methyl and ethyl ethers of 19-iodocholesterol when formulated were found to be stable for 6 and 15 days respectively whereas 19-iodocholesterol started decomposing during formulation (3,4). The results of the tissue distribution of 19-iodocholesterol-125I (prepared by the method of Counsell e_~ta_!l.) and the corresponding methyl and ethyl ethers in dogs are presented in Tables I and II. Table I.
Lung Liver Spleen Heart Kidney Testis Brain Muscle
Fat
Tissue Distribution Studies in Dogs One Day's Result 19-Iodocholesterol-125I a The 3-methyl The 3-ethyl ether Vill a ether (XII) b 84 + 14 73"+ 20 170 103 ~ 6 271 • 78 546 104 ~ 16 110 ~ 26 319 hO ~ 8 ~0 • 13 59 ~0 • 8 59 ~ 18 7~ 29 ; 3 27 ; 2 56 I~ • 2 16 • 4 I0 10 • 2 9 ~ 2 17
13 •
Adrenal
2
435 ~ 90
3
14
622 ~ 17
13 •
1,240
~Values represent DPM/mg + SEM for 3 dogs. Values represent DPM/mg Tor one ~og. It will be noticed from these two tables that the localization of the 3 compounds in the adrenal was higher on the 4th day than on the first day. In order to appreciate the usefulness of the methyl and ethyl ethers of 19-iodocholesterol, the adrenal-kidney concentration ratios in dogs are presented in Table III.
140
~
~
m ~ o
x x,~
~able II.
Tissue Distribution Studies in Dogs Four Days' Results. 19-Iodocholesterol-1251a The 3-methyl The 3-ethyl ether (VIII) a ether (XII) b Lung 31Z ~ 24 * 5 155 + 80 Liver 34+ ~ 93 ~ 12 ~56 ~ I
Spleen Heart Kidney Testis Braim Muscle Fat Adrenal
12
13 ÷
21 + 1 - -
2+0 6 ~' I 13~ 3 1 , 0 1 b ~ 1 _ 51
26 ~' 25 "~' 3+ 5~' 16; 701 ~'
27
3 [--11 0 2 128
166 37
68 + 6.3 ~' 1'7 3~' 0 16 ~' .3 16~ 3 1,520 -- 1.35
~Values represent DPM/mg + SEM for 3 dogs. Values represent DPM/mg ~ SEM for 2 dogs. Table III.
Adrenal-Kidney Concentration Ratios
Ratios Compound One-Day Four Days ~Probabili~y) a 19-Iodocholesterol 8.66_+1.27 49.28~.52 The 3-methyl ether (VIII) 11.
The adrenal-kidney ratio
of the methyl and ethyl ethers of 19-iodocholesterol ~re not significantly different from that of the parent 19-iodocholesterol (p > 0.06 and p > 0.10 respectively). The methyl and ethyl ethers of 19-iodocholesterol which are more stable both in the solid form and in solution coupled with their organ distribution patterns would therefore appear to be just as useful for practical application as adrenal markers as 19-iodocholesterol.
~ T ~ C O I D m
141
REFERENCES le 2. 3. 4. 5. 6. 7. 8. 9. 10.
11.
Counsell, R.E., Ranade, V.V., Blair, R.J., Beierwaltes, W.H. and Weinhold, P.A., Steroids 16, 317 (1970). Beierwaltes, W.H., Leiberman, L.M., Ansari, A.N. and Nishiyama, H., J. Am. Med. Assoc.! 216, 275 (1971). Szinai, S.S., Williams, C.M., Couch,~.W. and ~1oyale, J.A. Abstracts, 169th National Meeting of the American Chemical Society, Philadelphia Pa., April,(1975). Owoyale, J.A. Ph.D. Dissertation, University of Florida,
(1975).
Hotte, C.E. and Ice, R.D., J. Nucl. Med. f 15, 38 (1974). Scott, K.N., Mareci, T.H., Couch, M.W., an~-Williams, C.M., Steroids, 30, 2131 (1977). Kojima, M., Maeda, M., Ogawa, H., Nitta, K., and Ito, T., J.C.S. Chem. Comm., 47 (1975). Kojima, M., Maeda, M., Ogawa, H., Nitta, K., and Ito, T., J. Nucl. Med., 16, 666 (1975). Sarkar, S.D., B e i e ~ l t e s , W.H., Ice, R.D., Basmadjian, G.P., Hetzel, K.R., Kennedy, W.P. and Mason, M.M., J. Nucl. Med., 16,1038 (1975). -Couch, M.W.,-~cott, K.N., and Williams, C.M., Steroids, ~c7~t 1K areci, T.H., Couch, M.W. and Williams, C.M. Steroids, 30, 511 (1977). ADDENDUM The bromine analysis for compound III is not acceptable. The reaction conditions employed to prepare III as in the case of its 3-acetyl analog always gave more than one compound. Eecrystallization from various solvenlme.g, ethanol and dichloromethane gave different products (tlc and glc) which were difficul~ to isolate. This may be responsible for the unacceptable analysis. Accordingly, the unrecrystallized product was used to obtain IV at 65% yield. No reports on optical rotation have been made since our starting material had a 3-alkoxyl group ~nd the final products have the same group intact with only an additional R/imary iodide on C-19 which was easily detected by1:~C nmr.