Synthesis and characterization of a highly potent and selective isotopically labeled retinoic acid receptor ligand, ALRT1550

Article abstract of DOI:10.1021/jo971409i

The syntheses of two labeled homologues of (2E,4E,6E)-7-(3,5-di-tert- butylphenyl)-3-methylocta-2,4,6-trienoic acid (ALRT1550, 2), [¹³CD₃]ALRT1550 (3) and [³H]ALRT1550 (4), are described in this report. ALRT1550 is an exceptionally potent antiproliferative agent which is currently in phase I/II clinical trials for acute chemotherapy. Both homologues were prepared from commercially available 3,5-di-tert-butylbenzoic acid. Homologue [¹³CD₃]ALRT1550 was labeled at the 7-position of the trienoic acid chain via addition of [¹³CD₃]MgI to a Weinreb amide precursor. The preparation of [³H]ALRT1550 utilized novel methodology to synthesize a sterically hindered and site-specific tritium-labeled tert- butyl group. Saturation binding and Scatchard analysis of this ligand at the retinoic acid receptors are also described, along with competition binding (K(i)) values for a series of known retinoids using [³H]ALRT1550 or [³H]ATRA as the labeled probes.

Full text of DOI:10.1021/jo971409i

J . Org. Chem. 1998, 63, 543-550
543
Syn th esis a n d Ch a r a cter iza tion of a High ly P oten t a n d Selective
Isotop ica lly La beled Retin oic Acid Recep tor Liga n d , ALRT1550
Youssef L. Bennani,‡,1a Kristin S. Marron,‡ Dale E. Mais,† Karen Flatten,†
Alex M. Nadzan,‡,1b and Marcus F. Boehm*,‡
Departments of Medicinal Chemistry and Endocrine Research, Ligand Pharmaceuticals Inc., 10275
Science Center Drive, San Diego, California 92121
Received J uly 30, 1997X
The syntheses of two labeled homologues of (2E,4E,6E)-7-(3,5-di-tert-butylphenyl)-3-methylocta-
2,4,6-trienoic acid (ALRT1550, 2), [13CD3]ALRT1550 (3) and [3H]ALRT1550 (4), are described in
this report. ALRT1550 is an exceptionally potent antiproliferative agent which is currently in
phase I/II clinical trials for acute chemotherapy. Both homologues were prepared from commercially
available 3,5-di-tert-butylbenzoic acid. Homologue [13CD3]ALRT1550 was labeled at the 7-position
of the trienoic acid chain via addition of [13CD3]MgI to a Weinreb amide precursor. The preparation
of [3H]ALRT1550 utilized novel methodology to synthesize a sterically hindered and site-specific
tritium-labeled tert-butyl group. Saturation binding and Scatchard analysis of this ligand at the
retinoic acid receptors are also described, along with competition binding (Ki) values for a series of
known retinoids using [3H]ALRT1550 or [3H]ATRA as the labeled probes.
Ch a r t 1
Retinoids are small organic molecules comprising
vitamin A, its metabolites, and synthetic analogues.
These compounds act as modulators of nuclear transcrip-
tion by binding to and activating one or more of the six
characterized intracellular retinoid receptors, retinoic
acid receptors (RARR,â,γ), and retinoid X receptors
(RXRR,â,γ).2,3 This results in regulation of numerous
cellular processes such as development, reproduction,
bone formation, hematopoiesis, and immune function.4,5
These biological actions also have implications for treat-
ment of dermatological diseases and certain cancers.6,7
Endogenous retinoids such as all-trans-retinoic acid
(ATRA, 1a ) (Chart 1), a RAR-selective agonist, and 9-cis-
retinoic acid (1b, ALRT1057), a pan agonist (activates
all six retinoid receptors8-10), as well as a variety of
synthetic retinoids including 13-cis-RA, etretinate, Taz-
arotene, and Targretin, have shown clinical utility,11-13
* Author to whom correspondence should be addressed.
‡ Department of Medicinal Chemistry.
for the treatment of acne, psoriasis, acute promyelocytic
leukemia, squamous cell carcinoma, melanoma, and
Kaposi’s sarcoma.6,7 The therapeutic effects of these
compounds result from their ability to control abnormal
cellular processes by modulating cellular differentiation,
inhibiting cellular proliferation, and regulating apoptosis.
† Department of Endocrine Research.
X Abstract published in Advance ACS Abstracts, December 15, 1997.
(1) (a) Present address: Abbott Laboratories, Abbott Park, IL 60064.
(b) Present address: Chugai Biopharmaceuticals, Inc., San Diego, CA
92121.
(2) Mangelsdorf, D. J .; Umesono, K.; Evans, R. M. The Retinoids;
Academic Press: Orlando, FL, 1994; pp 319-349.
(3) Leid, M.; Kastner, P.; Chambon, P. Trends Biochem. Sci. 1992,
17, 427-433.
(4) Chemistry and Biology of Synthetic Retinoids; Dawson, M. I.,
Okamura, W. H., Eds.; CRC Press: Boca Raton, FL, 1990.
(5) The Retinoids. Biology, Chemistry and Medicine, 2nd ed.; Sporn,
M. B., Roberts, A. B., Goodman, D. S., Eds.; Raven Press: New York,
1994.
The promising clinical results obtained with retinoids
have inspired continued research to identify the next
generation of therapeutically useful retinoids. As a
consequence of our efforts to develop novel drugs, we
recently discovered (2E,4E,6E)-7-(3,5-di-tert-butylphen-
yl)-3-methylocta-2,4,6-trienoic acid (2, ALRT1550),14
a
(6) Biro, D. E.; Shalita, A. R. Skin Pharm. 1993, 6 (Suppl. 1), 24-
34.
highly potent and selective activator of the RARs in
(7) Shahidallah, M.; Tham, S. N.; Goh, C. L. Int. J . Dermatol. 1994,
33, 60-63.
binding (Table 1) and cotransfection assays.15-17 In
(8) Heyman, R. A.; Mangelsdorf, D. J .; Dyck, J . A.; Stein, R. B.;
Eichele, G.; Evans, R. M.; Thaller, C. Cell 1992, 68, 397-406.
(9) Boehm, M. F.; McClurg, M. M.; Pathirana, C.; Mangelsdorf, D.;
White, S. K.; Hebert, J .; Winn, D.; Goldman, M. E.; Heyman, R. A. J .
Med. Chem. 1994, 37, 408-414.
(12) Smith, M. A.; Parkinson, D. R.; Cheson, B. D.; Friedman, M.
A. J . Clin. Oncol. 1992, 10, 839-864.
(13) Vokes, E. E.; Weichselbaum, R. R.; Lippman, S. M.; Hong, W.
K. N. Engl. J . Med. 1993, 328, 184-194.
(10) Bennani, Y. L.; Boehm, M. F. J . Org. Chem. 1995, 60, 1195-
1200.
(14) Zhang, L.; Nadzan, A. M; Heyman, R. A; Love, D; Mais, D. E;
Croston, G; Lamph, W. L; Boehm, M. F. J . Med. Chem. 1996, 39, 2659-
2663.
(11) Orfanos, C. E.; Ehlert, R.; Gollnick, H. Drugs 1987, 34, 459-
503.
S0022-3263(97)01409-6 CCC: $15.00 © 1998 American Chemical Society
Published on Web 01/13/1998

544 J . Org. Chem., Vol. 63, No. 3, 1998
Ta ble 1. Com p etition Bin d in g Da ta for Ba cu lovir u s-Exp r essed Retin oic Acid Recep tor sa
Bennani et al.
[3H]ATRA (nM)
[3H]ALRT1550 (nM)
retinoid
RARR
RARâ
RARγ
RARR
RARâ
RARγ
ALRT1550
ATRA
TTNPB
1.07 ( 0.12 (4)
18.7 ( 1.7 (5)
20.0 ( 9.0 (4)
0.69 ( 0.26 (4)
17.4 ( 1.2 (5)
39.0 ( 8.0 (4)
1.93 ( 0.23 (5)
18.5 ( 1.9 (5)
51.0 ( 18 (4)
0.97 ( 0.19 (3)
17.7 ( 2.3 (3)
117.5 ( 72.3 (3)
0.78 ( 0.08 (3)
17.7 ( 4.3 (3)
71.2 ( 20.4 (3)
7.4 ( 2.8 (3)
32.5 ( 11.7 (3)
69.8 ( 24.1 (3)
a
Data are shown as mean ( SEM (n).
Sch em e 1a
a
(a) i. (COCl)2, CH2Cl2, cat. DMF, ii. MeO(Me)NH-HCl, Et3N, CH2Cl2 (90%); (b) Mg, 13CD3I, Et2O (90%); (c) NaH, THF, (EtO)2P(O)CH2CN
(75%); (d) DIBAL, -78 °C, CH2Cl2 (80%); (e) (EtO)2P(O)CH2C(CH3)CHCO2Et, n-BuLi, THF, DMPU (86%); (f) KOH, EtOH (85%).
addition, ALRT1550 exhibits potent antiproliferative
activity on human cervical carcinoma cells (ME180).14 As
a result of its efficacious action in vitro as well as other
favorable preclinical studies, this compound is currently
in Phase I/II clinical trials for the acute treatment of
various cancers.
Stable isotope and radiolabeled homologues of AL-
RT1550 are required for pharmacological and metabolic
characterizations such as receptor binding, identification
of metabolites,18 and stability studies. The syntheses of
two labeled homologues 7-13CD3-labeled 3 and monotri-
tiated 4 ALRT1550 homologues, are described in this
report. The preparation of the radiolabeled homologue
4 utilizes novel methodology to access sterically hindered
and site-specific tritium-labeled tert-butyl groups. Satu-
ration binding and Scatchard analysis of this ligand at
the retinoic acid receptors are also described, along with
competition binding (Ki) values for a series of retinoids,
ALRT1550, ATRA, and TTNPB (5) (see Chart 1), using
[3H]ALRT1550 or [3H]ATRA as the labeled probes.
followed by the addition of N,O-dimethylhydroxylamine
hydrochloride19 in the presence of triethylamine. Amide
7 was treated with 13CD3-MgI, generated in situ by the
treatment of magnesium turnings with 13CD3-iodometh-
ane, to give labeled methyl ketone 8 in 90% yield.
Treatment of ketone 8 with the carbanion of diethyl
(cyanomethyl)phosphonate gave the corresponding trans-
trisubstituted olefinic nitrile 9 in 75% yield. DIBAL
reduction to aldehyde 10, followed by all-trans-triene
formation using n-BuLi/diethyl [3-(ethoxycarbonyl)-2-
methylprop-2-enyl]phosphonate in the presence of DMPU,
gave ester 11 in 86% yield. Saponification of 11 in
methanol afforded the desired acid 3, which was recrys-
tallized to 99.3% purity as determined by HPLC. This
material was used as a probe for identifying ALRT1550
metabolites in vivo.
The structural features of ALRT1550 including the
conjugated trienoate, the structural density of the aro-
matic moiety, and the absence of heteroatom functional-
ity, presented significant synthetic challenges for intro-
ducing a tritium label late in the synthesis. Previously,
we observed that the natural hormone 9-cis-RA20 exhib-
ited degradation of the tetraenoic acid chain. As a result,
we decided to avoid preparing a radiolabeled homologue
of 2 with the radiolabel atom in the chain. Given the
aromatic ring meta-substitution pattern, the steric hin-
drance of its appended tert-butyl groups, and the trienoic
acid side chain, it was unlikely that direct aromatic-ring
labeling could be easily achieved, particularly with the
further criterion of introducing the radionucleus at the
penultimate stages of the synthesis. Thus, we decided
Ch em istr y
The synthesis of 7-13CD3-labeled ALRT1550 3 is shown
in Scheme 1. 3,5-Di-tert-butylbenzoic acid (6) was con-
verted to the corresponding N-methoxy-N-methylbenz-
amide 7 in 90% yield, by treatment with oxalyl chloride
(15) Mangelsdorf, D. J ; Ong, E. S; Dyck, J . A; Evans, R. M. Nature
1990, 345, 224-229.
(16) Berger, T. S; Parandoosh, Z; Perry, B. W; Stein, R. B. J . Steroid
Biochem. Mol. Biol. 1992, 41, 733-738.
(17) Umesono, K; Giguere, V; Glass, C. K; Rosenfeld, M. G; Evans,
R. M. Nature 1988, 336, 262-265.
(19) Nahm, S.; Weinreb, S. M. Tetrahedron Lett. 1981, 22, 3815-
3818.
(20) Shirley, M. A; Bennani, Y. L; Boehm, M. F; Breau, A. P;
Pathirana, C; Ulm, E. H. Drug Metab. Dispos. 1996, 24, 293-302.
(18) Blaner, W. S.; Olson, J . A. In The Retinoids: Biology, Chemistry
and Medicine, 2nd ed.; Sporn, M. B., Roberts A. B., Goodman, D. S.,
Eds.; Raven Press: New York, 1994; p 229.

Synthesis and Characterization of ALRT1550
J . Org. Chem., Vol. 63, No. 3, 1998 545
Sch em e 2
ment of ketone 22 with the lithium carbanion of diethyl-
(cyanomethyl)phosphonate (n-BuLi, 0 °C, THF) gave a
5:1 trans:cis mixture of the corresponding separable
cyano olefins 23 in 85% yield. The trans-isomer of 23
was treated with DIBAL, in dichloromethane at -78 °C,
to give aldehyde 24 (85% yield) followed by homologation
with the appropriate phosphonate, as in Scheme 2, to give
ethyl trienoate 25 in 80% yield (5:1 ratio of 2-trans- to
2-cis-olefins).
Desilylation of compound 25 followed by PCC oxidation
gave aldehyde 27 in 84% yield. Treatment of 27 with
p-toluenesulfonyl hydrazide (EtOH, catalytic HCl) af-
forded the corresponding hydrazone 28 in 80% yield with
∼15% concomitant double-bond isomerization as deter-
mined by 1H NMR. Neopentylic hydrazone 28 was
successfully reduced to the corresponding methyl group
using NaCNBH3-ZnCl2 or NaCNB2H3-ZnCl2 in reflux-
ing methanol24 to give retinoate 29, which after saponi-
fication in ethanolic KOH gave ALRT1550 (2). While the
above synthetic sequence appeared to be a viable way
for preparing the monotritiated tert-butyl group in one
radiolabeled step, the requirement of 4-5 mol equiv of
NaCNB3H3, with high specific activity, became the limit-
ing reagent.25,26
Thus, in an alternative approach (shown in Scheme
4), aldehyde 27 was reduced with readily available
NaB3H4 in methanol to the corresponding alcohol, which
was oxidized to give tritiated aldehyde 31. The radiola-
beled aldehyde was transformed to its corresponding
p-tosylhydrazone 32, which in turn was selectively
reduced using NaCNBH3-ZnCl2 in refluxing methanol
to give the corresponding radiolabeled ester 33. Potas-
sium hydroxide saponification of 33 gave the desired
monotritiated [3H]ALRT1550 (4). HPLC purification
gave 134 mCi of 99.5% pure [3H]ALRT1550, with a
specific activity of 21 Ci/mmol. Since the theoretical
specific activity that could be achieved using carrier-free
tritium gas is 29 Ci/mmol, the specific activity measured
for this synthesis most likely reflects a tritium isotope
effect during the oxidation of neopentylic alcohol 30 to
aldehyde 31 (Scheme 4).
to introduce one tritium atom, as shown for 4, at one of
the methyl functions of the arene tert-butyl substituents.
A retrosynthetic pathway to 4, Scheme 2, depicts the
introduction of a tritium atom at the neopentylic position
through tritide reduction of the fully elaborated aldehyde
12, which in turn could be derived from aldehyde 13,
starting from tert-butylbenzene-3,5-dicarboxylic acid (14).
The synthesis is shown in Scheme 3. Commercially
available tert-butylbenzene-3,5-dicarboxylic acid (14) was
reduced to diol 15 using borane-THF complex in THF
at 0 °C, in nearly quantitative yield. Diol 15 was oxidized
(PCC-Celite, CH2Cl2, 25 °C, 97% yield) to dialdehyde 16,
which was treated with methylmagnesium bromide
(THF, -78 °C) to give the corresponding bis-secondary
diol 17. Selective monsilylation21 of diol 17 (NaH, THF,
TBSCl) afforded monosilyl ether 18, in 75% yield, which
was further oxidized (PCC-Celite, CH2Cl2, 25 °C, 95%)
to give methyl ketone 19. The introduction of the
neopentylic aldehyde in 20 was achieved using Martin’s
method.22 Thus, diethyl [(N-benzylideneamino)meth-
yl]phosphonate23 was treated with n-BuLi followed by the
addition of ketone 19 (THF at -78 °C to reflux). The
resulting, crude enamine adduct was further treated with
n-BuLi followed by the addition of excess iodomethane
in THF. The crude mixture was treated with 1 N
hydrochloric acid to give, with concomitant desilylation,
hydroxyaldehyde 20, which contained the necessary
functional groups required for the tritiation experiment.
Sodium borohydride reduction of 20, followed by regio-
selective oxidation of the benzylic alcohol (MnO2, CH2Cl2),
gave neopentylic alcohol 21, which was further protected
as its TBDPS ether 22. The trienoate chain in 4 was
introduced following standard transformations. Treat-
Biologica l Ch a r a cter iza tion
Saturation binding data of [3H]ALRT1550 (4) to bacu-
lovirus-expressed RARR, RARâ, and RARγ are plotted
in Figure 1 as a function of free radioligand concentra-
tion. Radiolabeled retinoid 4 bound to the three receptors
in a saturable and protein-dependent manner. Linear
analyses of the saturation data where bound/free is
plotted versus bound are shown as a Scatchard plot
(24) Kim, S.; Oh, C.-H.; Ko, J . S.; Anh, K.-H.; Kim, Y.-J . J . Org.
Chem. 1985, 50, 1927-1932.
(25) The following reactions were unsuccessful: reduction of the
neopentylic triflate derived from alcohol 26 (see Binkley, R. W.;
Ambrose, M. G.; Hehemann, D. G. J . Org. Chem. 1980, 45, 4387-4391)
and the neopentylic iodide (prepared according to Curran, D. P.;
Rakiewicz, D. M. J . Org. Chem. 1985, 107, 1448) under n-Bu3SnH
conditions (see Parnes, H.; Pease, J . J . Org. Chem. 1979, 44, 151-
152); reduction of the tosylate derived from 26 (TsCl, Py neat, 84%)
using lithium triethylborohydride according to Binkley, R. W. J . Org.
Chem. 1985, 50, 5646-5649. For LAH reduction of neopentylic
tosylates, see: Collins, D. J .; J acobs, H. A. Aust. J . Chem. 1986, 39,
2095-2110. For Zn/AcOH reduction of neopentylic iodides, see: Ito,
M.; Kibayashi, C. Synthesis 1993, 137-140.
(21) McDougal, P. G.; Rico, J . G.; Oh, Y. I.; Condon, B. D. J . Org.
Chem. 1986, 51, 3388-3390.
(22) Martin, S. F.; Phillips, G. W.; Puckette, T. A.; Colapret, J . A.
J . Am. Chem. Soc. 1980, 102, 5866-5872.
(26) For NaCNB3H3 reduction of a steroidal neopentylic hydrazone,
see: Schenk, V. G.; Albrecht, H. P.; Lietz, H. Drugs Res. 1978, 28,
518.
(23) Davidson, S. K.; Phillips, G. W.; Martin, S. F. Org. Synth. 1987,
65, 119-134.

546 J . Org. Chem., Vol. 63, No. 3, 1998
Bennani et al.
Sch em e 3a
a
(a) BH3-THF, THF (99%); (b) PCC, Celite, CH2Cl2 (97%); (c) MeMgBr, THF, -78 °C (75%); (d) NaH, THF, TBSCl (75%); (e) PCC,
Celite, CH2Cl2 (95%); (f) i. (EtO)2 P(O)CH2NdCHPh, n-BuLi, ii. n-BuLi, iodomethane, iii. HCl, THF (68%); (g) NaBH4, MeOH (98%); (h)
MnO2, CH2Cl2 (94%); (i) TPSCl, Imid, CH2Cl2-DMF (cat.) (91%); (j) (EtO)2 P(O)CH2CN, n-BuLi, THF (85%); (k) DIBAL, CH2Cl2, - 78 °C
(85%); (l) n-BuLi, DMPU, THF, (EtO)2P(O)CH2C(CH3)CHCO2Et, -78 to 25 °C (80%); (m) TBAF, THF (85%); (n) PCC, Celite, CH2Cl2
(84%); (o) p-tosyl-SO2NHNH2, EtOH; (p) NaCNBnH3, ZnCl2, MeOH; (q) i. KOH, EtOH, ii. HCl.
Sch em e 4a
a
(a) NaB3H4, MeOH; (b) PCC, Celite, CH2Cl2; (c) p-Tol-SO2NHNH2, EtOH; (d) NaCNBH3-ZnCl2, MeOH; (e) KOH, EtOH.
(inset). The apparent Kd is determined as -1/slope. The
Scatchard analysis results indicate both high-affinity
binding of the ligand to the receptors and binding to a
single class of binding sites. The x-intercept of the
Scatchard plot is often referred to as the Bmax or maxi-
mum number of binding sites per unit of protein. The
affinities calculated from these results for RARR, RARâ,
and RARγ are 3.3 ( 0.2, 1.8 ( 0.3, and 8.2 ( 1.2 nM,
respectively. No specific binding to the three RXRs could
be observed with this ligand (data not shown).
Comparison of Ki values for a series of retinoids
(ALRT1550, ATRA, and TTNPB27) using [3H]ALRT1550
or [3H]ATRA as the labeled probes is shown in Table 1.
(27) Leoliger, P.; Bollag, W.; Mayer, H. Eur. J . Med. Chem. 1980,
15, 9-15.

Synthesis and Characterization of ALRT1550
J . Org. Chem., Vol. 63, No. 3, 1998 547
3,5-Di-ter t-bu tyl-N-m eth oxym eth ylben za m id e, 7. To a
solution of 3,5-di-tert-butylbenzoic acid (6) (5.0 g, 21.36 mmol)
in CH2Cl2 (50 mL) was added 1 drop of DMF followed by the
slow addition of oxalyl chloride (3.75 mL, 42.7 mmol) at rt.
The mixture was stirred for 2.5 h and concentrated, and the
residue was dried under high vacuum (0.05 mmHg) for 1 h
and dissolved in CH2Cl2 (25 mL) (solution A). A separate
solution of N,O-dimethylhydroxylamine hydrochloride (2.01 g,
2.16 mmol) and triethylamine (6.25 mL) in CH2Cl2 (50 mL)
was cooled to 0 °C, and solution A was added over a 60-min
period. The reaction mixture was stirred at rt for 1 h, and
water (50 mL) was added. CH2Cl2 (50 mL) was added, and
the mixture was washed with satd NH4Cl (50 mL), water (50
mL), and brine (50 mL). The organic layer was separated,
dried (MgSO4), concentrated, and purified by silica gel chro-
matography (hexanes-EtOAc, 9:1) to give 5.36 g (19.3 mmol)
of the desired amide 7 (90% yield): 1H NMR (CDCl3) δ 7.49
(s, 1H), 7.46 (s, 2H), 3.60 (s, 3H), 3.34 (s, 3H), 1.33 (s, 18H);
HRMS (EI+, 70 eV) calcd for C17H27NO2 277.2042, found
278.2128 (M + H).
2-[13CD3]-1-(3,5-d i-ter t-bu tylp h en yl)eth a n on e, 8. To a
flame-dried three-neck 50-mL flask, equipped with a condenser
under a nitrogen atmosphere, were added magnesium turnings
(540 mg, 21.0 mmol) and diethyl ether (12.0 mL) followed by
slow addition of 13CD3-iodomethane (3.0 g). Addition of
13CD3-iodomethane resulted in slow warming to reflux of the
reaction solvent. The slightly turbid solution was stirred for
30 min at rt and cooled to 0 °C, and a solution of 3,5-di-tert-
butyl-N-methoxy-N-methylbenzamide (7) (4.5 g, 17.1 mmol)
in diethyl ether (15 mL) was slowly added. The reaction
mixture was stirred at rt for 1 h and the reaction quenched
with 10% HCl (15 mL). EtOAc (50 mL) was added, and the
layers were separated. The organic layer was washed with
water (20 mL) and brine (20 mL) and dried (MgSO4). The
solution was concentrated and the residue purified over a short
pad of silica gel (hexanes-EtOAc, 9:1) to give 3.63 g (15.3
mmol) of the desired ketone 8 (90% yield): 1H NMR (CDCl3)
δ 7.80 (d, J ) 1.6 Hz, 2H), 7.65 (s, 1H), 1.37 (s, 18H); 13C NMR
(CDCl3) δ 151.4, 137.1, 127.5, 122.7, 35.1, 31.6, 26.2; HRFAB-
MS calcd for C1513CH212H3O (M + H) 237.2127, found 237.2112.
(2E)-4-[13CD3]-3-(3,5-d i-ter t-b u t ylp h en yl)b u t -2-en en i-
tr ile, 9. A suspension of NaH (854 mg, 21.3 mmol of a 60%
suspension in mineral oil) was rinsed with pentane (3 × 5 mL),
suspended in THF (10 mL), and cooled to 0 °C. A solution of
diethyl (cyanomethyl)phosphonate (4.8 g, 27.0 mmol) in THF
(30 mL) was added to the suspension. The solution was stirred
for 20 min, and a solution of ketone 8 (3.20 g, 13.55 mmol) in
THF (10 mL) was slowly added. The reaction mixture was
stirred for 12 h at rt and the reaction quenched with a satd
NH4Cl solution. EtOAc (50 mL) was added, and the organic
layer was washed with water (3 × 20 mL) and brine (3 × 20
mL) and then dried (MgSO4). The solvent was concentrated
and the residue purified by silica gel chromatography (5%
EtOAc-hexanes) to give 1.87 g (7.18 mmol) of (2E)-4-[13CD3]-
3-(3,5-di-tert-butylphenyl)but-2-enenitrile (9) as a trans:cis
mixture (4:1 by 1H NMR) (53% crude yield): 1H NMR (CDCl3)
δ 7.49 (s, 1H), 7.26 (s, 2H), 5.59 (d, J ) 8 Hz, 1H), 1.35 (s,
18H); 13C NMR (CDCl3) δ 151.5, 138.1, 124.7, 120.3, 118.0,
F igu r e 1. Binding data of [3H]ALRT1550 (4) to baculovirus-
expressed RARR, RARâ, and RARγ and Scatchard analyses
(inset) of these saturation results. The -1/slope of the Scat-
chard plot is a measure of the affinity (Kd) of the ligand for
the receptor.
These values were determined using competition binding
assays with a fixed concentration of the appropriate
labeled ligand and varying concentrations of the unla-
beled competitor. As can be seen, the Ki values deter-
mined for these retinoids are in agreement, for the most
part, and are independent of the labeled ligand utilized.
Con clu sion
We have synthesized 7-13CD3-labeled (3) and monotri-
tiated (4) homologues of the highly potent, antiprolifera-
tive, RAR-selective agent ALRT1550 (2). The radiolabel
in 4 was introduced at a neopentylic position, thus
establishing an effective methodology to prepare steri-
cally hindered and site-specific radiolabeled tert-butyl
3
groups. The positioning of the H-label on one methyl of
two tert-butyl groups reduces the potential for loss of the
radiolabel due to metabolic oxidation and/or degradation,
as compared to other sites of labeling on the targeted
molecule. We also have characterized this ligand at the
three intracellular retinoic acid receptors (RARs) and
demonstrated its ability to selectively bind to the RARs
with high affinity. Both labeled compounds 3 and 4 are
used as probes to study the distribution and metabolism
of this highly potent retinoid in vivo.
Exp er im en ta l Section
All the reactions were carried out under a nitrogen atmo-
sphere except where stated. The organic solvents were
purchased from Fisher Scientific, THF was distilled from Na
(metal) in the presence of benzophenone, and diethyl ether was
distilled from CaH2. Thin-layer chromatography was per-
formed on Merck Kieselgel 60 F-254 plates. Reactions were
monitored by TLC using both UV and an aqueous solution of
ammonium molybdate and cerium sulfate for staining. 1H,
13C, and 3H NMR spectra were recorded on Bruker 300 (3H)
and 400 MHz spectrometers. UV spectra were measured on
a Kontron Uvikon model 941 spectrometer, HPLC purification
was performed on a Waters system using a Beckman C18
ultrasphere (5 mm, 10 mm × 25 cm) column. Scintillation
counting was performed on a Beckman LS6000IC scintillation
counter using Ecoscint A scintillation solution (National
Diagnostics).
95.3, 35.18, 31.6, 19.9 (hept); HRFAB-MS calcd for C17
-
13CH222H3N (M + H) 260.2287, found 260.2267.
(2E)-4-[13CD3]-3-(3,5-d i-ter t-bu tylp h en yl)bu t-2-en a l, 10.
To a solution of nitrile 9 (1.87 g, 7.22 mmol) in anhydrous
dichloromethane (12 mL) at -78 °C was added DIBAL (8.0
mL of a 1 M solution in toluene). The reaction mixture was
stirred at -78 °C for 60 min, quenched with excess Rochelle
salt (10 mL), and then allowed to warm to rt. EtOAc (50 mL)
was added and the mixture washed with water (3 × 20 mL)
and brine (3 × 20 mL). The organic layer was dried (MgSO4)
and concentrated, and the residue was purified by silica gel
chromatography (hexanes-EtOAc, 9:1) to give 1.51 g (5.74
mmol) of (2E)-4-[13CD3]-3-(3,5-di-tert-butylphenyl)but-2-enal
(10) (80% yield): 1H NMR (CDCl3) δ 7.80 (d, J ) 1.6 Hz, 2H),
7.65 (s, 1H), 1.37 (s, 18H); 13C NMR (CDCl3) δ 191.7, 191.6,

548 J . Org. Chem., Vol. 63, No. 3, 1998
Bennani et al.
151.4, 140.2, 127.4, 124.7, 120.8, 120.7, 35.2, 31.6, 16.1;
HRFAB-MS calcd for C1713CH242H3O (M + H) 263.2283, found
263.2234.
solid thus obtained was filtered and rinsed with cold pentane
(50 mL). The solid was dried in vacuo to give 15.3 g (68.9
mmol) of the desired compound 17 (71% yield): 1H NMR
(CDCl3) δ 7.32 (s, 2H), 7.2 (s, 1H), 4.92 (m, 2H), 1.82 (d, 2 H,
J ) 2.5 Hz), 1.52 (d, 6H, J ) 6.5 Hz), 1.33 (s, 9H); 13C NMR
(CDCl3) δ 152.0, 145.9, 121.9, 119.8, 70.9, 35.0, 31.6, 25.4;
HRMS (EI+, 70 eV) calcd for C14H22O2 222.1620, found
222.1609.
(2E,4E,6E)-8-[13CD3]-7-(3,5-d i-ter t-bu tylp h en yl)-3-m eth -
ylocta-2,4,6-tr ien oic Acid, 3. A solution of diethyl [3-(ethoxy-
carbonyl)-2-methylprop-2-enyl]phosphonate (4.56 g, 17.3 mmol)
in anhydrous THF (25.0 mL) was cooled to 0 °C and treated
with anhydrous DMPU (6.5 mL) followed by n-BuLi in hexanes
(6.45 mL of 2.0 M solution). The mixture was stirred at 0 °C
for 20 min and then cooled to -78 °C. A solution of (2E)-3-
[13CD3]-4-(3,5-di-tert-butylphenyl)but-2-enal (10) (1.5 g, 5.76
mmol) in THF (20.0 mL) was slowly added, and the reaction
mixture was stirred at -78 °C for an additional 60 min. The
mixture was allowed to warm to 23 °C for 1 h with stirring. A
satd solution of ammonium chloride (5 mL) was added, and
the mixture was extracted with EtOAc (3 × 10 mL). The
organic layer was washed with water (2 × 25 mL) and brine
(50 mL), dried (MgSO4), and concentrated. The resulting
residue was purified on a short silica gel column to give 1.5 g
(4.05 mmol) of the ester 11 (86% yield).
1-{3-ter t-Bu t yl-5-[1-[(ter t-b u t yld im et h ylsila n yl)oxy]-
eth yl]p h en yl}eth a n ol, 18. Under a nitrogen atmosphere,
sodium hydride (2.75 g, 68.8 mmol of a 60% mineral oil content
mixture) was rinsed with hexanes (2 × 10 mL) and suspended
in THF (200 mL), and 5-tert-butyl-1,3-benzene-2,2′-diethanol
(17) (12.69 g, 57 mmol) was added with vigorous stirring. The
mixture was stirred for 45 min at rt to give a white slurry, to
which tert-butyldimethylsilyl chloride (8.61 g, 57 mmol) was
added. The reaction mixture was stirred for 2.5 h; water (25
mL) was added followed by extraction with EtOAc (350 mL).
The organic layer was washed with a saturated NH4Cl solution
(100 mL), water (2 × 100 mL), and brine (2 × 100 mL) and
dried (MgSO4). The solvent was concentrated and the residue
purified by silica gel chromatography to give 2.14 g of starting
material and 14.45 g (43.0 mmol) of the desired monosilylated
product 18 as an oil (75% yield): 1H NMR (CDCl3) δ 7.32 (s,
1H), 7.28 (s, 1H), 7.12 (2s, 1H), 4.92 (m, 2H), 1.85 (s, 1H), 1.5
(d, 3H, J ) 6.5 Hz), 1.43 (d, 3H, J ) 6.5 Hz), 1.35 (s, 9H), 0.9
(s, 9H), 0.05 (s, 6H); 13C NMR (CDCl3) δ 151.46, 151.41, 147.06,
147.0, 145.3, 121.8, 121.0, 120.8, 119.7, 119.6, 71.19, 71.14,
71.1, 35.0, 31.6, 27.4, 26.0, 25.8, 25.3, 18.4, 4.5; HRMS (EI+,
70 eV) calcd for C20H36O2Si (M - H) 335.2406, found 335.2389
(M - H).
1-{3-ter t-Bu t yl-5-[1-[(ter t-b u t yld im et h ylsila n yl)oxy]-
eth yl]p h en yl}eth a n on e, 19. Alcohol 18 (14.45 g, 44 mmol)
in CH2Cl2 (100 mL) was added to a vigorously stirred solution
of PCC (15.0 g, 69 mmol) and Celite (30 g) in CH2Cl2 (500 mL).
After 3 h at rt, the mixture was filtered over a short pad of
silica gel (2 in. × 4 in.) and rinsed with CH2Cl2 (500 L). The
solution was concentrated to give 14.4 g (43.1 mmol) of the
desired ketone 19 (98% yield): 1H NMR (CDCl3) δ 7.89 (s, 1H),
7.73 (s, 1H), 7.65 (s, 1H), 4.94 (q, 1H, J ) 6.3 Hz), 2.63 (s,
3H), 1.45 (d, 3 H, J ) 6.3 Hz), 1.37 (s, 9H), 0.96 (s, 9H), 0.12
(s, 3H), -0.05 (s, 3H); 13C NMR (CDCl3) δ 198.8, 151.8, 147.3,
137.1, 127.4, 123.6, 122.9, 70.8, 35.1, 31.5, 27.4, 26.9, 26.0, 18.4,
-4.5, -4.6; HRMS (EI+, 70 eV) calcd for C20H34O2Si (M + H)
335.2406, found 335.2437.
1-[3-ter t-Bu tyl-5-(1-h ydr oxyeth yl)ph en yl]-2-m eth ylpr o-
p ion a ld eh yd e, 20. A flame-dried three-neck round-bottom
flask was charged with THF (90 mL) and cooled to -78 °C
followed by slow addition of n-BuLi (12.0 mL of a 2 M solution;
24 mmol). A solution of diethyl [(N-benzylideneamino)meth-
yl]phosphonate (6.14 g, 24 mmol) in THF (15 mL) was added,
and the resulting mixture was stirred for 1 h at -78 °C. A
solution of ketone 19 (7.0 g, 20.9 mmol) in THF (15 mL) was
added, and the mixture was warmed to rt, stirred for 30 min,
and then refluxed for 2 h. The reaction mixture was cooled to
rt and concentrated. Diethyl ether (400 mL) was added, and
the solution was washed with sodium chloride (200 mL). The
aqueous layer was extracted with ether (200 mL), and the
combined organic layers were washed with brine (200 mL),
dried (MgSO4), and concentrated to give a yellow residue which
was dried under high vacuum (1 mmHg) for 1 h. THF (90
mL) was added to this residue, and the solution was cooled to
-78 °C. n-BuLi (12.0 mL of a 2 M solution; 24 mmol) was
slowly added, and the deeply colored solution was stirred for
60 min followed by addition of iodomethane (6.50 mL, 0.1 mol).
After the mixture warmed to rt and stirred for 4 h, the reaction
was quenched with HCl (3 N; 100 mL), and the biphasic
solution was stirred for 16 h. EtOAc (300 mL) was added; the
organic layer was separated, washed with water (2 × 100 mL)
and brine (2 × 100 mL), and then dried (MgSO4). The solution
was concentrated to give a residue which was purified by silica
gel chromatography to give 3.3 g (13.30 mmol) of the desired
aldehyde 20 (64% yield): 1H NMR (CDCl3) δ 9.5 (s, 1H), 7.33
(s, 1H), 7.17 (s, 1H), 7.11 (s, 1H), 4.91 (m, 1H), 1.80 (d, 1H, J
) 3.4 Hz), 1.5 (d, 3H, J ) 6.3 Hz), 1.47 (s, 3H), 1.32 (s, 9H);
Ester 11 (750 mg, 2.02 mmol) was dissolved in methanol
(20 mL), and 5 N potassium hydroxide (5 mL) was added. The
mixture was heated at reflux for 30 min, cooled to rt, and
acidified with 1 N HCl. Extraction with EtOAc (50 mL)
followed by washings with water (20 mL) and brine (20 mL)
and drying (MgSO4) gave a residue, which was recrystallized
from hot ether-hexanes. Two consecutive recrystallizations
gave 375 mg of the desired acid 3, which was 99.23% pure as
determined by HPLC: 1H NMR (CDCl3) δ 7.38 (s, 1H), 7.30
(s, 2H), 7.08 (dd, J ) 15, 11.2 Hz, 1H), 6.55 (dd, J ) 11.2, 7.6
Hz, 1H), 6.42 (d, J ) 15.1 Hz, 1H), 5.84 (s, 1H), 2.40 (s, 3H),
1.35 (s, 18H); 13C NMR (CDCl3) δ 171.6, 155.5, 150.9, 142.2,
135.6, 132.4, 132.3, 126.6, 122.4, 120.3, 117.8, 35.1, 31.7, 12.3,
14.4; HRMS (EI+, 70 eV) calcd for C2213CH292H3O2 344.2625,
found 344.2641.
[3-ter t-Bu tyl-5-(h yd r oxym eth yl)p h en yl]m eth a n ol, 15.
To a vigorously stirred solution of 5-tert-butyl-1,3-benzenedi-
carboxylic acid (14) (20.0 g, 90 mmol) in THF (200 mL) at 0
°C was added a solution of borane-THF complex in THF (190
mL) via an addition funnel over 20 min. The mixture was
warmed to rt and stirred for an additional 90 min. A solution
of 1:1 water-THF (200 mL) was slowly added followed by an
additional 200 mL of water. The mixture was extracted with
EtOAc (200 mL). The aqueous layer was extracted with EtOAc
(2 × 100 mL), and the combined organic layers were washed
with water (2 × 100 mL) and brine (2 × 100 mL) and dried
(MgSO4). The solvent was concentrated to give 17.1 g (88.2
mmol) of diol 15 (98% yield): 1H NMR (CDCl3) δ 7.32 (s, 2H),
7.19 (s, 1H), 4.69 (s, 4H), 1.75 (br s, 2H), 1.33 (s, 9H), 13C NMR
(CDCl3) δ 152.0, 141.1, 123.5, 123.1, 65.6, 34.9, 31.5; HRMS
(EI+, 70 eV) calcd for C12H18O2 194.1307, found 194.1298.
5-ter t-Bu tylben zen e-1,3-d ica r ba ld eh yd e, 16. Dimetha-
nol 15 (20.0 g, 103 mmol) was added to a vigorously stirring
mixture of PCC (66.0 g, 306 mmol) and Celite (130 g) in
dichloromethane (CH2Cl2) (500 mL). The mixture was stirred
for 3 h at rt, while monitored for completion by TLC. The
reaction mixture was filtered over a short pad of silica gel (2
in. × 4 in.) and rinsed with CH2Cl2 (1 L). The solvent was
concentrated to give 18.7 g (97.3 mmol) of the desired dialde-
hyde 16 (94% yield): 1H NMR (CDCl3) δ 10.11 (s, 2H), 8.18 (s,
3H), 1.41 (s 9H); 13C NMR (CDCl3) δ 191.6, 153.9, 137.2, 131.8,
129.2, 35.3, 31.3; HRMS (EI+, 70 eV) calcd for C12H14O2
190.0994, found 190.1022.
1-[3-ter t-Bu tyl-5-(1-h yd r oxyeth yl)p h en yl]eth a n ol, 17.
A solution of 5-tert-butylbenzene-1,3-dicarbaldehyde (16) (18.7
g, 96.4 mmol) in THF (400 mL) was cooled to -78 °C, and a
solution of methylmagnesium bromide (80.0 mL of a 3 M
solution) was slowly added. The reaction mixture was warmed
to rt and stirred for 60 min. The reaction was then quenched
with a satd NH4Cl solution (100 mL) followed by HCl (1 N, 50
mL) and the mixture extracted with EtOAc. The organic layer
was washed with water (2 × 100 mL) and brine (2 × 100 mL)
and dried (MgSO4). After concentration, the crude residue was
dissolved in hot EtOAc (30 mL) followed by pentane (200 mL).
The clear solution was cooled at -4 °C for 3 h, and the white

Synthesis and Characterization of ALRT1550
J . Org. Chem., Vol. 63, No. 3, 1998 549
13C NMR (CDCl3) δ 202.5, 152.2, 146.3, 141.3, 123.1, 121.6,
121.0, 71.0, 50.9, 35.1, 31.6, 25.5, 22.8; HRMS (EI+, 70 eV)
calcd for C16H24O2 248.1776, found 248.1769.
31.6, 26.9, 25.6, 22.8, 19.5, 16.9, 14.3; HRMS (EI+, 70 eV) calcd
for C30H35O2Si (M - t-Bu) 455.2406, found 455.2394.
E t h yl
(2E,4E,6E)-7-{3-ter t-Bu t yl-5-[2-[(ter t-b u t yld i-
1-[3-ter t-Bu tyl-5-(2-h ydr oxy-1,1-dim eth yleth yl)ph en yl]-
eth a n on e, 21. A solution of alcohol 20 (1.77 g, 7.53 mmol) in
MeOH (50 mL) was cooled to 0 °C, and NaBH4 (300 mg, 7.93
mmol) was added portionwise. The reaction mixture was
warmed to rt and stirred for 30 min. The solution was
concentrated, and the residue was taken up in EtOAc (50 mL)
and washed with HCl (10%, 3 × 10 mL), water (3 × 20 mL),
and brine (3 × 20 mL). The organic layer was dried (MgSO4)
and concentrated to give 1.76 g of the diol. The crude diol
(1.65 g, 6.78 mmol) was dissolved in CH2Cl2 (20 mL), and MnO2
(18.7 g, 0.17 mmol) was added. The reaction mixture was
vigorously stirred for 4 h and then filtered over a short pad of
Celite. The solvent was removed to give 1.63 g (6.57 mmol) of
the desired ketone 21 (87% yield): 1H NMR (CDCl3) δ 7.85 (s,
1H), 7.8 (s, 1H), 7.63 (s, 1H), 3.64 (d, 2H, J ) 4.5 Hz), 2.6 (s,
3H), 1.38 (s, 6H), 1.35 (s, 9H); 13C NMR (CDCl3) δ 151.6, 146.3,
145.6, 122.6, 120.7, 120.6, 73.3, 71.2, 40.5, 35.1, 34.8, 31.8,
31.68, 31.63, 25.6, 25.5, 22.8, 14.3; HRMS (EI+, 70 eV) calcd
for C16H24O2 248.1776, found 248.1759.
1-{3-ter t-Bu t yl-5-[3-[(ter t-b u t yld ip h en ylsila n yl)oxy]-
1,1-d im eth yleth yl]p h en yl}eth a n on e, 22. To a solution of
alcohol 21 (1.63 g, 6.96 mmol) in CH2Cl2 (30 mL) were added
imidazole (500 mg, 7.35 mmol), 1 drop of DMF, and tert-
butyldiphenylsilyl chloride (2.01 g, 7.33 mmol). The mixture
was stirred overnight at rt and the reaction quenched with
excess satd NH4Cl. CH2Cl2 (50 mL) was added, and the
organic layer was washed with water (3 × 20 mL) and then
brine (3 × 20 mL), dried (MgSO4), and concentrated to give a
residue which was purified by silica gel chromatography (5%
EtOAc-hexane) to afford 2.77 g (5.69 mmol) of the desired
ether 22 (78% yield): 1H NMR (CDCl3) δ 7.85 (s, 1H), 7.76 (s,
1H), 7.63 (1H), 7.48-7.25 (mm, 10H), 3.62 (s, 2H), 2.56 (s, 3H),
1.38 (s, 6H), 1.33 (s, 9H), 0.94 (s, 9H); 13C NMR (CDCl3) δ
199.0, 151.2, 147.9, 136.9, 135.7, 133.7, 129.7, 128.6, 127.7,
124.2, 122.8, 73.8, 40.7, 35.1, 31.6, 26.9, 25.7, 19.5; HRMS (EI+,
70 eV) calcd for C28H33O2Si (M - t-Bu) 429.2250, found
429.2280.
p h en ylsila n yl)oxy]-1,1-d im eth yleth yl]p h en yl}-3-m eth yl-
octa -2,4,6-tr ien oa te, 25. A solution of diethyl [3-(ethoxycar-
bonyl)-2-methylprop-2-enyl]phosphonate (1.20 g, 4.52 mmol)
in a 5:1 mixture of anhydrous THF-DMPU (25 mL) was cooled
to 0 °C, and 2.15 mL of a 2.0 M solution of n-BuLi in hexanes
(4.5 mmol) was added. The mixture was stirred at 0 °C for 20
min and then cooled to -78 °C. A solution of aldehyde 24 (1.25
g, 2.52 mmol) in THF (10.0 mL) was slowly added, and the
reaction mixture was stirred at -78 °C for an additional 60
min. The mixture was allowed to warm to rt for 1 h with
stirring; then a satd solution of ammonium chloride (5 mL)
was added and extracted using EtOAc (3 × 10 mL). The
combined EtOAc extracts were washed with water (2 × 25 mL)
and then brine (50 mL), dried (MgSO4), and concentrated. The
residue was purified on a short silica gel column to give 1.35
g (2.17 mmol) of the desired ester 25 (86% yield): 1H NMR
(CDCl3) δ 7.5 (d, J ) 7 Hz, 1H), 7.34 (m, 9H), 7.0 (dd, J ) 16
Hz, 1H), 6.5 (d, J ) 12 Hz, 1H), 6.34 (d, J ) 16 Hz, 1H), 5.8 (s,
1H), 4.17 (q, J ) 7.3 Hz, 2H), 3.6 (s, 2H), 2.39 (s, 3H), 2.24 (s,
3H), 1.38 (s, 6H), 1.32 (s, 9H), 1.30 (t, 3H, J ) 7.3 Hz), 0.96 (s,
9H); 13C NMR (CDCl3) δ 167.4, 150.6, 147.4, 142.2, 141.7,
135.8, 133.9, 131.4, 129.6, 127.7, 126.6, 123.3, 121.6, 120.6,
118.9, 74.0, 59.9, 40.7, 35.1, 31.7, 26.9, 25.7, 19.6, 17.0, 14.6,
14.1; HRMS (EI+, 70 eV) calcd for C41H54O3Si 622.3842, found
622.3854.
E t h yl (2E,4E,6E)-7-[3-ter t-Bu t yl-5-(2-h yd r oxy-1,1-d i-
m eth yleth yl)ph en yl]-3-m eth ylocta-2,4,6-tr ien oate, 26. The
above silyl ether 25 (1.05 g, 1.68 mmol) was dissolved in THF
(20 mL), and TBAF (17 mL of 1 M solution in THF) was added.
The reaction mixture was stirred at room temperature for 12
h; EtOAc (50 mL) was added followed by washing with water
(2 × 20 mL) and brine (20 mL). The organic layer was
separated, dried (MgSO4), and concentrated. The residue was
purified by silica gel chromatography (10% EtOAc-hexanes)
to give 479 mg (1.25 mmol) of the desired alcohol 26 (74%
yield): 1H NMR (CDCl3) δ 7.9 (d, 1H, J ) 16 Hz, 2:cis isomer,
∼15%), 7.34 (s, 2H), 7.29 (s, 1H), 7.03 (dd, 1H, J ) 16 Hz),
6.53 (d, 1H, J ) 12 Hz), 6.38 (d, 1H, J ) 16 Hz), 5.72 (s, 1H),
5.68 (s, 1H, 2:cis isomer, ∼15%), 4.15 (q, 2H, J ) 6.7 Hz), 3.62
(s 2H), 2.38 (s, 3H), 2.28 (s, 3H), 1.6 (br s, 1H), 1.37 (s, 6H),
1.33 (s, 9H), 1.26 (t, 3H, J ) 6.7 Hz); HRMS (EI+, 70 eV) calcd
for C25H36O3 384.2664, found 384.2654.
(2E)-3-{3-ter t-Bu tyl-5-[3-[(1-ter t-bu tyld ip h en ylsila n yl)-
oxy]-1,1-d im eth yleth yl]p h en yl}bu t-2-en en itr ile, 23. To a
solution of diethyl(cyanomethyl)phosphonate (1.7 g, 9.55 mmol)
in THF (30 mL) at 0 °C was added n-BuLi (4.64 mL of a 2.0 M
solution in hexanes). The solution was stirred for 10 min, and
a solution of ketone 22 (2.6 g, 5.46 mmol) in THF (10 mL) was
added. The reaction mixture was stirred for 30 min; then the
reaction was quenched with a satd NH4Cl solution. EtOAc
(50 mL) was added, and the organic layer was washed with
water (3 × 20 mL) and then brine (3 × 20 mL), dried (MgSO4),
and concentrated to give a trans:cis mixture (∼4:1 by 1H NMR)
of nitrile 23 which was purified by silica gel chromatography
affording 1.70 g (3.24 mmol) of the trans isomer in (63% yield):
1H NMR (CDCl3) δ 7.85 (s, 1H), 7.48-7.22 (mm, 13H), 5.5 (s,
1H), 3.59 (s, 2H), 2.43 (s, 3H), 1.35 (s, 6H), 1.27 (9s, 9H), 0.94
(s, 9H); 13C NMR (CDCl3) δ 161.4, 151.3, 148.1, 138.0, 135.7,
133.7, 129.8, 127.7, 125.7, 121.7, 120.5, 118.0, 95.2, 73.8, 53.6,
40.7, 35.1, 31.6, 26.9, 25.6, 20.7, 19.5; HRMS (EI+, 70 eV) calcd
for C30H34ONSi (M - t-Bu) 452.2410, found 452.2425.
(2E)-3-{3-ter t-Bu tyl-5-[2-[(ter t-bu tyldiph en ylsilan yl)oxy]-
1,1-d im eth yleth yl]p h en yl}bu t-2-en a l, 24. A solution of
nitrile 23 (1.7 g, 3.42 mmol) in anhydrous CH2Cl2 (20 mL) was
cooled to -78 °C, and DIBAL (3.5 mL of a 1 M solution in
toluene) was added dropwise. The reaction mixture was
stirred at -78 °C for 60 min, quenched with excess Rochelle
salt, and allowed to warm to rt. EtOAc (50 mL) was added,
and the mixture was washed with water (3 × 20 mL) and brine
(3 × 20 mL). The organic layer was dried (MgSO4) and
concentrated. The obtained residue was purified by silica gel
chromatography to give 1.25 g (2.44 mmol) of the desired
aldehyde 24 (75% yield): 1H NMR (CDCl3) δ 10.17 (d, 1H, J
) 8 Hz), 7.48-7.22 (mm, 13H), 6.36 (d, 1 H, J ) 8 Hz), 3.60
Eth yl (2E,4E,6E)-7-[3-ter t-Bu tyl-5-(1,1-d im eth yl-2-oxo-
eth yl)p h en yl]-3-m eth ylocta -2,4,6-tr ien oa te, 27. To a vig-
orously stirred mixture of PCC (350 mg, 1.39 mmol) and Celite
(750 mg) in CH2Cl2 (20 mL) was added a solution of alcohol
26 (330 mg, 0.889 mmol) in CH2Cl2 (10 mL). The mixture was
stirred for 3 h at rt and then filtered over a short pad of silica
gel. The solution was concentrated, and the residue was
purified by silica gel chromatography to give 290 mg (0.76
mmol) of the desired aldehyde 27 (85% yield): 1H NMR
(CDCl3) δ 9.5 (s, 1H), 7.9 (d, 1H, J ) 16 Hz, 2:cis isomer,
∼15%), 7.34 (s, 2H), 7.29 (s, 1H), 7.03 (dd, 1H, J ) 16 Hz),
6.53 (d, 1H, J ) 12 Hz), 6.38 (d, 1H, J ) 16 Hz), 5.79 (s, 1H),
5.68 (s, 1H, 2:cis isomer, ∼15%), 4.15 (q, 2H, J ) 6.7 Hz), 2.38
(s, 3H), 2.28 (s, 3H), 1.37 (s, 6H), 1.33 (s, 9H), 1.26 (t, 3H, J )
6.7 Hz).
Eth yl (2E,4E,6E)-7-[3-ter t-Bu tyl-5-(1,1-d im eth yleth yl-
2-p-tolu en esu lfon ylh ydr azon e)ph en yl]-3-m eth ylocta-2,4,6-
tr ien oa te, 28. To a solution of aldehyde 27 (250 mg, 0.67
mmol) in ethanol (5 mL) was added 130 mg (0.7 mmol) of
p-toluenesulfonyl hydrazide. The reaction mixture was heated
at 40-45 °C for 15 min followed by evaporation of the solvent
to afford a residue which was purified by silica gel chroma-
tography (10% EtOAc-hexanes) to give 330 mg (0.60 mmol)
of hydrazone 28 (89% yield): 1H NMR (CDCl3) δ 7.82 (d, 2H,
J ) 7.4 Hz), 7.5 (2s, 2H), 7.3 (d, 2H, J ) 7.4 Hz), 7.18 (s, 1H),
7.05 (s, 1H), 7.0 (dd, 1H, J ) 16 Hz), 6.45 (d, 1H, J ) 12 Hz),
6.38 (d, 1H, J ) 12 Hz), 5.82 (s, 1H), 4.2 (q, 2H, J ) 6.7 Hz),
2.42 (s, 3H), 2.38 (s, 3H), 2.28 (s, 3H), 1.37 (s, 6H), 1.33 (s,
9H), 1.26 (t, 3H, J ) 6.7 Hz): HRMS (EI+, 70 eV) calcd for
(s, 2H), 2.54 (s, 3H), 1.37 (s, 6H), 1.32 (s, 9H), 0.94 (s, 9H); 13
C
NMR (CDCl3) δ 191.6, 159.5, 151.2, 147.9, 140.2, 135.7, 133.8,
129.7, 127.7, 127.3, 125.7, 122.1, 121.0, 73.9, 40.7, 35.1, 31.8,
C32H42N2O4S 550.2865, found 550.2819.

550 J . Org. Chem., Vol. 63, No. 3, 1998
Bennani et al.
(2E,4E,6E)-7-(3,5-Di-ter t-b u t ylp h en yl)-3-m et h yloct a -
2,4,6-tr ien oic Acid (ALRT1550), 2. To a solution of sodium
cyanoborohydride (17 mg, 0.54 mmol) and zinc chloride (18
mg, 0.27 mmol) in methanol (3 mL) was added a solution of
hydrazone 28 (30 mg, 0.0545 mmol). The mixture was heated
at reflux for 8 h and then cooled to rt. The solution was
concentrated and the residue taken up in EtOAc (10 mL) and
washed with water (5 mL), satd NaHCO3 (5 mL), water (5 mL),
and brine (2 × 5 mL). The organic layer was dried (MgSO4)
and concentrated. The residue was purified by silica gel
chromatography (Rf 0.8 in 5% EtOAc-hexanes) to give 8.80
mg (0.024 mmol) of the ethyl ester as a mixture of geometric
isomers (47% yield): 1H NMR (CDCl3) δ 7.4 (d, J ) 1 Hz, 1H),
7.21 (d, J ) 1 Hz, 2H), 7.04 (m, 1H), 6.54 (d, J ) 8 Hz, 1H),
6.4 (d, J ) 8 Hz, 1H), 5.82 (s, 1H), 4.17 (m, 2H), 2.39 (s, 3H),
2.28 (s, 3H), 1.34 (s, 18H), 1.3 (t, J ) 7.7 Hz, 3H).
The above ester (8.8 mg, 0.024 mmol) in ethanol (2 mL) was
treated with 5 N KOH at 60 °C for 2 h. The mixture was
cooled to rt, acidified using 1 N HCl, and then extracted with
EtOAc. The organic layer was dried (MgSO4) and concentrated
to give ALRT1550 (2), which was purified by silica gel
chromatography (10% MeOH-CHCl3): 1H NMR (CDCl3) δ
7.39 (t, J ) 1 Hz, 1H), 7.3 (d, J ) 1 Hz, 2H), 7.08 (m, 1H), 6.54
(d, J ) 8 Hz, 1H), 6.4 (d, J ) 8 Hz, 1H), 5.84 (s, 1H), 2.4 (s,
3H), 2.29 (s, 3H), 1.35 (s, 18H); HRMS calcd for C23H32O2
340.2402, found 340.2394. Anal. (C23H32O2) C, H.
was purified by silica gel chromatography (5% EtOAc-hexane)
followed by preparative TLC (5% EtOAc-hexane). Two
compounds were isolated with the more polar compound being
the desired ester 32 (457 mCi). Ester 32 (407 mCi) was
dissolved in ethanol (5 mL), and 3 N KOH (1 mL) was added.
The mixture was heated at 70 °C for 2 h, cooled to rt, and
acidified with 3 N HCl. EtOAc extraction (3 × 10 mL) followed
by sequential washings with water (10 mL) and brine (2 × 10
mL), drying (MgSO4), and concentration gave a residue which
after purification by silica gel chromatography (20% EtOAc-
hexane) afforded 410 mCi of material. Half of this material
was further purified by HPLC using a semipreparative Beck-
man ODD column using (85% methanol-15% water containing
2% AcOH, at 6.0 mL/min, UV ) 230 nM) to give 139 mCi of
[3H]ALRT1550 (4) having a specific activity of 21 Ci/mmol.
[3H]ALRT1550 coeluted with an authentic sample of AL-
RT1550 by ODC HPLC.
Bin d in g Assa ys. Stock solutions of compounds were
prepared as 5 mM ethanol or DMSO solutions and serial
dilutions carried out in 1:1 DMSO-ethanol. Assay buffers
consisted of the following for the receptor assays: 8% glycerol,
120 mM KCl, 8 mM Tris HCl, 5 mM CHAPS, 4 mM DTT, and
0.24 mM PMSF, pH 7.4 at rt. Binding assays were performed
in a similar manner as described previously.15-17 The final
volume for binding assays was 250 µL, consisting of 10-40 µg
of extracted protein, depending upon the receptor being
assayed, plus 5 nM [3H]ATRA or 3 nM [3H]ALRT1550 and
varying concentrations of competing ligand (0-10-5 M). For
saturation analysis, varying concentrations of labeled AL-
RT1550 were used. Assays were formatted for a 96-well
minitube system. Incubations were carried out at 4 °C until
equilibrium was achieved. Nonspecific binding was defined
as that binding remaining in the presence of 1000 nM of the
appropriate unlabeled retinoid. At the end of the incubation
period, 50 µL of 6.25% hydroxylapatite was added in the
appropriate wash buffer (wash buffer consisted of 100 mM KCl,
10 mM Tris HCl, and 0.5% Triton X-100) which binds the
receptor-ligand complex. The mixture was vortexed, incu-
bated for 30 min at room temperature, and centrifuged, and
the supernatant was removed. The hydroxylapatite pellet was
washed two more times with wash buffer, and the amount of
receptor-ligand complex was determined by liquid scintillation
counting of the hydroxylapatite pellet in a Wallac Microbeta
scintillation counter.
Eth yl (2E,4E,6E)-7-[3-ter t-Bu tyl-5-(1,1-d im eth yl-2-[3H]-
2-h yd r oxyeth yl)p h en yl]-3-m eth ylocta -2,4,6-tr ien oa te, 30.
A solution of aldehyde 27 (70 mg, 0.118 mmol) in THF (1.0
mL) was added to a freshly prepared solution of sodium
borotritide (2.2 mg, 0.058 mmol, 6.4 Ci) at rt. The reaction
mixture was stirred for 1 h and then treated with 10% HCl (2
mL). The mixture was extracted with EtOAc (10 mL) and
washed with water (5 mL) and brine (5 mL). The organic layer
was dried (MgSO4) and concentrated to give 3.5 Ci of the
corresponding alcohol 30, which was used in the next step
without further purification.
Eth yl (2E,4E,6E)-7-[3-ter t-Bu tyl-5-(1,1-d im eth yl-2-[3H]-
2-oxoeth yl)p h en yl]-3-m eth ylocta -2,4,6-tr ien oa te, 31. To
a vigorously stirred mixture of PCC (74 mg, 0.29 mmol) and
Celite (150 mg) in CH2Cl2 (5.0 mL) was added a solution of
alcohol 30 (3.5 Ci) in CH2Cl2 (1.0 mL). The mixture was stirred
for 3 h and filtered over a short pad of silica gel. The solution
was concentrated, and the residue was purified by silica gel
chromatography (10% EtOAc-hexane) to give 2.3 Ci of the
aldehyde 31, which was used in the next step without further
purification.
After correcting for nonspecific binding, IC50 values were
determined. The IC50 value is defined as the concentration of
competing ligand required to decrease specific binding by 50%.
The IC50 value was determined graphically from a computer-
based log-logit plot of the data. Kd values were determined
by application of the Cheng-Prusoff equation.28
Eth yl (2E,4E,6E)-7-[3-ter t-Bu tyl-5-(1,1-d im eth yl-2-[3H]-
eth yl 2-p-tolu en esu lfon ylh yd r a zon e)p h en yl]-3-m eth yl-
octa -2,4,6-tr ien oa te, 32. To a solution of aldehyde 31 (2.3
Ci) in ethanol (2 mL) were added p-toluenesulfonyl hydrazide
(38 mg, 0.065 mmol) and concentrated HCl (5 µL). The
mixture was heated at 45 °C for 1 h and then cooled to rt. The
solution was concentrated under nitrogen flow, and the residue
was purified by silica gel chromatography (10% EtOAc-
hexanes) to afford 1.8 Ci of the desired hydrazone 32.
(2E,4E,6E)-7-[3-ter t-Bu tyl-5-(1,1-d im eth yl-2-[3H]eth yl)-
ph en yl]-3-m eth ylocta-2,4,6-tr ien oic Acid ([3H]ALRT1550),
4. A solution of hydrazone 32 (1.8 Ci) was added to a mixture
of sodium cyanoborohydride (80 mg, 0.12 mmol) and ZnCl2 (70
mg, 0.51 mmol) in methanol (2 mL). The mixture was heated
at reflux for 4 h, cooled to rt, and concentrated. The residue
was extracted with EtOAc (3 × 10 mL) and washed with water
(10 mL) and satd NaHCO3 (2 × 10 mL). The organic layer
was dried (MgSO4) and concentrated to give a residue which
Ack n ow led gm en t. The authors wish to thank Hi-
romi Morimoto of the National Tritiation Labs in
Berkeley for technical support.
1
Su p p or tin g In for m a tion Ava ila ble: Copies of H NMR
spectra for compounds 2, 3, 7-10, and 15-28 (20 pages). This
material is contained in libraries on microfiche, immediately
follows this article in the microfilm version of the journal, and
can be ordered from the ACS; see any current masthead page
for ordering information.
J O971409I
(28) Cheng, Y.-C.; Prusoff, W. F. Biochem. Pharmacol. 1973, 22,
3099-3108.