Synthesis and biological activity of novel retinamide and retinoate derivatives

Article abstract of DOI:10.1248/cpb.52.501

Retinoic acid and its amide derivative, N-(4-hydroxyphenyl)retinamide (4-HPR), have been proposed as chemopreventative and chemotherapeutic agents. However, their low cytotoxic activity and water solubility limit their clinical use. In this study, we synthesized novel retinoid derivatives with improved cytotoxicity against cancer cells and increased hygroscopicity. Our syntheses were preceded by selective O-acylation and N-acylation, which led to the production of retinoate and retinamide derivatives, respectively, in one pot directly from aminophenol derivatives and retinoic acid without protection. Transcription assays in COS-1 cells indicated that the N-acylated derivatives (2A - 5A) and 4-HPR (1A) were much weaker ligands for all three subtypes of retinoic acid receptor (RAR) than all-trans retinoic acid (ATRA), although they showed some selectivity for RARβ and RARγ. In contrast, the O-acylated retinoate derivatives (1B - 5B) activated all three RAR isotypes without specificity to an extent similar to ATRA. The cytotoxicity was determined using an MTT assay with HCT116 colon cancer cells, and the IC ₅₀ of N-acylated retinamide derivative 4A and O-acylated retinoate derivative 5B was 1.67 μM and 0.65 μM, respectively, which are about five and 13-fold better than that of 4-HPR (8.21 μM), a prototype N-acylated derivative. When retinoate derivative 5B was coupled to organic acid salts, the resulting salt derivatives 5C and 5D had RAR activation and cytotoxicity similar to those of 5B. These data may delineate the relationship between the structure and function of retinoate and retinamide derivatives.

Full text of DOI:10.1248/cpb.52.501

May 2004
Chem. Pharm. Bull. 52(5) 501—506 (2004)
501
Synthesis and Biological Activity of Novel Retinamide and Retinoate
Derivatives
Soo-Jong UM,a Youn-Ja KWON,b Hye-Sook HAN,b Si-Ho PARK,b Myoung-Soon PARK,b
c
,b
*
Young-Soy RHO, and Hong-Sig SIN
a Department of Bioscience and Biotechnology/Institute of Bioscience, Sejong University; b Chebigen Inc., 305-B,
c
Chungmugwan, Sejong University; Seoul 143–747, Korea: and Department of Chemistry, Chonbuk National University;
Chonju 561–756, Korea. Received August 14, 2003; accepted December 26, 2003
Retinoic acid and its amide derivative, N-(4-hydroxyphenyl)retinamide (4-HPR), have been proposed as
chemopreventative and chemotherapeutic agents. However, their low cytotoxic activity and water solubility limit
their clinical use. In this study, we synthesized novel retinoid derivatives with improved cytotoxicity against can-
cer cells and increased hygroscopicity. Our syntheses were preceded by selective O-acylation and N-acylation,
which led to the production of retinoate and retinamide derivatives, respectively, in one pot directly from
aminophenol derivatives and retinoic acid without protection. Transcription assays in COS-1 cells indicated that
the N-acylated derivatives (2A—5A) and 4-HPR (1A) were much weaker ligands for all three subtypes of retinoic
acid receptor (RAR) than all-trans retinoic acid (ATRA), although they showed some selectivity for RARb and
RARg. In contrast, the O-acylated retinoate derivatives (1B—5B) activated all three RAR isotypes without speci-
ficity to an extent similar to ATRA. The cytotoxicity was determined using an MTT assay with HCT116 colon
cancer cells, and the IC50 of N-acylated retinamide derivative 4A and O-acylated retinoate derivative 5B was
1.67 mM and 0.65 mM, respectively, which are about five and 13-fold better than that of 4-HPR (8.21 mM), a proto-
type N-acylated derivative. When retinoate derivative 5B was coupled to organic acid salts, the resulting salt de-
rivatives 5C and 5D had RAR activation and cytotoxicity similar to those of 5B. These data may delineate the re-
lationship between the structure and function of retinoate and retinamide derivatives.
Key words retinamide; retinoate; derivative; cytotoxicity; retinoic acid receptor (RAR)
The amide analogue of retinoic acid, N-(4-hydroxy- ity, a selected derivative (5B) was coupled to organic acid
phenyl)retinamide (4-HPR), was developed as a therapeutic salts. We determined the selectivity of these compounds in
agent for various skin conditions. It is currently being as- the activation of RAR subtypes and measured their cytotoxic
sessed as a drug to prevent and treat several cancers.1,2) Un- activity against HCT116 colon cancer cells. Our data may be
like all-trans retinoic acid (ATRA), 4-HPR selectively binds useful for delineating the relationship between structure and
to retinoic acid receptors (RAR) b and g and activates them function in retinoate and retinamide derivatives.
less strongly than ATRA. These properties make 4-HPR less
toxic and substantially less teratogenic than ATRA.3) There- Results and Discussion
fore 4-HPR has been used as an antitumor agent in animal
Chemistry The synthesis of retinoid derivatives 1A—
studies4,5) and as a chemopreventive agent for several cancers 5A and 1B—5B is illustrated in Charts 1 and 2. As shown in
in clinical trials.6—8) A number of in vitro biological studies Chart 1, the selective N- and O-acylation of ATRA and 4-
indicated that 4-HPR has marked cytotoxicity in various can- aminophenol (4-AP) gave either the corresponding car-
cer cells by inducing apoptosis.9,10)
boamide (method A) or carboester (method B). The coupling
However, 4-HPR has a major limitation in its clinical use. of ATRA and 4-AP in method A, which uses EDCI/DMAP
The plasma levels in patients receiving 200 mg of 4-HPR as a reagent, creates 4-HPR (1A), which contains a car-
daily are less than 1 mM, which is far less than the effective boamide bond. In contrast, the coupling of the same com-
concentration (usually 10 mM) required to induce apoptosis in pounds using method B, which uses DCC/DMAP as a
vitro. Therefore it is necessary to use higher doses of 4-HPR reagent, creates 4-APR (1B) with a carboester bond. As pre-
or to synthesize other derivatives with better clinical efficacy. viously reported,14) 4-HPR could also be prepared from
Taking the second approach, several retinamide derivatives ATRA via retinoyl chloride using method C. Both reactions
have recently been synthesized, which have hydroxyl, car- involve acylation at the carboxy moiety of ATRA. At present,
boxy, or methoxy substitutions on the terminal phenylamine it is not clear how one acylation reaction can give rise to two
ring.11,12) Of these derivatives, 3-HPR showed the most active different compounds. It is generally accepted that the cou-
growth inhibition in four bladder cancer cell lines,11) while 2- pling of an acid and amine in the presence of EDCI/DMAP
CPR, a carboxyl derivative, was the most effective in some or DCC/DMAP results in a reaction producing a carboamide
head and neck, and lung cancer cell lines.12)
bond.15—17) In preparing 4-HPR, it is plausible that
To develop other potentially potent antitumor agents, we EDCI/DMAP activates the NH2 group of 4-AP by preferen-
synthesized many retinoid derivatives. Since butyric acid has tially shifting a proton to the OH group, making the NH2
been implicated as an anticancer agent,13) we introduced group more likely to react with the carboxy moiety of ATRA.
aminophenol or its butyryl derivatives into ATRA by selec- How is 4-APR produced ? ATRA can be activated by
tive N-acylation (carboxamide) and O-acylation (carboxyl DCC/DMAP, and its activated carboxy moiety reacts with the
ester) to produce retinamide (1A—5A) and retinoate (1B— OH group of 4-AP to form 4-APR. In the presence of
5B) derivatives, respectively. To increase their water solubil- DCC/DMAP, it is likely that the NH2 group of 4-AP serves
∗ To whom correspondence should be addressed. e-mail: hssin@chebigen.com
© 2004 Pharmaceutical Society of Japan

502
Vol. 52, No. 5
Chart 1. Synthesis of Retinamide (1A, 2A) and Retinoate (1B, 2B) Derivatives
Methods A, B, C, and D are described in Experimental.
Chart 2. Synthesis of Intermediates 6—15
Conditions: (a) Cbz–Cl, TEA, DMF; (b) butyryl chloride, NMM; (c) 10% Pd/C, H2, MeOH.
Table 1. Summary of Two Different Retinoids (Retinamide and Retinoate
Derivatives) Prepared According to Methods A and B
as a base to activate the OH group of 4-AP, allowing O-acy-
lation and creating 4-APR as the sole product.18,19) Therefore
this method enables the efficient preparation of two different
types of retinoid derivative via the simple, selective coupling
of retinoic acid to the NH2 or OH group without protection.
The structures of 4-HPR and 4-APR were elucidated by IR
spectroscopy based on the carbonyl absorption frequencies
of 1634 cmꢀ1 for 4-HPR and 1716 cmꢀ1 for 4-APR. Method
D was used to introduce a butyryl group into derivatives 1A
(4-HPR) and 1B (4-APR), producing derivatives 2A and 2B
in yields of 91% and 93%, respectively.
Method Aa)
Method Bb)
Starting
materials
Products
Yield (%)
Products
Yield (%)
48
91
49
93
4-AP
1A
2A
1A
1B
2Ac)
2Bc)
To synthesize other retinamide and retinoate derivatives,
we first synthesized target compounds 13, 14, and 15, as
shown in Chart 2. To introduce selectively a butyryl group
into the ortho-OH (R2) of 4-amino resorcinol 6, a series of
reactions was performed. First, the amine of 6 was protected
with Cbz–Cl (1.2 eq)/TEA (1.2 eq) to obtain the N-Cbz 7 in
89% yield. Acylation of 7 with butyryl chloride (1.5
eq)/NMM (1.5 eq) according to method D provided the
monobutyrated compound 10 in 65% yield. Nitro com-
pounds 11 and 12 were prepared from starting materials 8
and 9 by butyrylation of the OH or NH2 group (R3) meta to
the nitro group, in 61% and 72% yield, respectively. Com-
pounds 10, 11, and 12 were catalytically hydrogenated with
10% Pd/C in MeOH, producing the corresponding com-
pounds 13, 14, and 15 in high yields. Intermediates 13—15
were coupled with ATRA by selective N- or O-acylation ac-
cording to methods A and B to give final compounds 3A—
5A and 3B—5B, respectively, as shown in Table 1. The
structures of these compounds were verified by IR spec-
troscopy.
47
52
13
14
15
3A
4A
5A
3B
4B
5B
50
51
45
43
a) Method A: EDCI (2.0 eq)/DMAP/CH2Cl2, room temperature, 4 h. b) Method
B: DCC (2.0 eq)/DMAP/CH2Cl2, room temperature, 4 h. c) 2A and 2B were prepared
from 1A and 1B, respectively, using Method D: butyryl chloride (1.2 eq)/NMM (1.2
eq)/0 °C, 1 h.
tunately, this was true for derivative 5B, which has significant
biological activity (Table 2). Derivative 5B has a terminal
NH2 group, which can form salts to increase its water solu-
bility. Although the hydrochloride and hydrobromide salts
were more hydrophilic, these compounds were very unstable
Consistent with the general notion that most synthetic
retinoids are lipophilic, our retinoid derivatives (Fig. 1) were
insoluble in aqueous solvents and precipitated in vivo. Unfor-

May 2004
503
Chart 3. Synthesis of Organic Acid Salts of Retinoate Derivative 5B
These compounds were prepared according to method E: THF, 45 °C→hexane, 10 °C.
Table 2. EC50 and IC50 Values of Retinoid Derivatives for RAR Activation
and Cytotoxicity
under strongly acidic conditions. Therefore we prepared or-
ganic salts 5C—F by reacting the free base form of 5B with
a stoichiometric amount of fumaric, maleic, tartaric, or
pamoic acid in THF according to Method E,20) as shown in
Chart 3.
EC50 (M)a) for RAR
IC50 (mM)b,c) for
HCT116 cells
Retinoids
a
b
g
Effect of Retinoid Derivatives on RARs To determine
the activity and specificity of our retinoid derivatives on
RAR subtypes, a series of conventional transcriptional assays
was performed using a retinoid responsive reporter gene in
the presence of each receptor. COS-1 cells were cotrans-
fected with a receptor expression vector (RARa, b, and g in
pSG5) and reporter gene [(RARE)3-tk-CAT], and treated
with increasing concentrations of ATRA, 4-HPR, or other
retinoids. CAT ELISA was used to measure the amount of
CAT protein induced in response to the retinoid derivatives.
For each retinoid and receptor, a dose–response curve was
plotted. The concentration required for 50% activation (EC50
value) was determined by interpolation from the curves
(Table 2). ATRA, a physiological RAR ligand, strongly acti-
vated all three RARs with an EC50 of 1.8—5.1ꢁ10ꢀ9 M. As
previously reported, 4-HPR (1A) was selectively active for
RARb and g.21,22) Retinamide derivatives 2A—5A, like 4-
HPR, were all more active for RARb and g with a lower Vmax
than ATRA, whereas retinoate derivatives 1B—5B, except
ATRA
1A (4-HPR)
5.1ꢁ10ꢀ9
5.3ꢁ10ꢀ7
8.6ꢁ10ꢀ9
4.6ꢁ10ꢀ7
4.8ꢁ10ꢀ9
6.4ꢁ10ꢀ7
3.2ꢁ10ꢀ8
8.3ꢁ10ꢀ8
7.5ꢁ10ꢀ9
ꢂꢂ10ꢀ6
3.7ꢁ10ꢀ9
7.6ꢁ10ꢀ9
3.9ꢁ10ꢀ9
1.3ꢁ10ꢀ7
5.1ꢁ10ꢀ9
8.3ꢁ10ꢀ8
5.1ꢁ10ꢀ8
3.2ꢁ10ꢀ8
8.1ꢁ10ꢀ9
ꢂ10ꢀ6
1.8ꢁ10ꢀ9
9.9ꢁ10ꢀ9
2.8ꢁ10ꢀ9
1.5ꢁ10ꢀ7
4.9ꢁ10ꢀ9
8.1ꢁ10ꢀ8
5.3ꢁ10ꢀ8
2.4ꢁ10ꢀ8
7.4ꢁ10ꢀ9
ꢂ10ꢀ6
ꢂ20
8.21
9.73
14.71
3.01
3.15
4.08
1.67
ꢂ20
ꢂ20
0.65
0.78
0.73
1B
2A
2B
3A
3B
4A
4B
5A
5B
5C
5D
4.9ꢁ10ꢀ9
4.1ꢁ10ꢀ9
5.2ꢁ10ꢀ9
4.4ꢁ10ꢀ9
5.2ꢁ10ꢀ9
6.1ꢁ10ꢀ9
8.7ꢁ10ꢀ9
4.4ꢁ10ꢀ9
6.3ꢁ10ꢀ9
a) The dose–response curves were plotted using data obtained from transcription as-
says with individual RAR subtypes in the presence of increasing concentrations of test
retinoid. EC50 values, the concentrations required for 50% activation, were determined
by interpolation of the curves. b) The dose–response curves were plotted by MTT
assay as described in Experimental. IC50 values, the concentrations required for 50%
growth inhibition, were determined by interpolation of the curves. Each experiment
was performed in triplicate and repeated a minimum of three times. c) pꢃ0.05 for all
IC50 values.
for 3B, were as active as ATRA for all three RARs with a HPR. Of the retinamide derivatives, 3A and 4A were supe-
similar Vmax. At present, it is not clear why 3B is not as active rior to 4-HPR, the parental retinamide. Retinoate derivatives
as the other derivatives. The side functional group of 3B may 2B, 3B, and 5B were more cytotoxic than 4-HPR. Retinoate
interfere with the coactivator binding required for RAR acti- derivative 4B and retinamide derivative 5A were much less
vation, but this remains to be determined using a coactivator cytotoxic than 4-HPR. Interestingly, 4B was a strong ligand
as in CBP and SRC-1.23) Organic salts 5C and 5D were as for all RARs, whereas 5A was nearly inactive for all RARs,
active as their parental compound 5B for all RARs.
suggesting that RAR activation is not correlated with cyto-
Cytotoxic Activity of Retinoid Derivatives The cyto- toxicity in HCT116 cells. Organic salts 5C and 5D were as
toxic potential of the retinoid derivatives was investigated by cytotoxic as parental compound 5B in these cells. For a bet-
determining their concentrations required for 50% growth in- ter clinical outcome, we searched for retinoid derivatives
hibition (IC50 value) for HCT116 colon cancer cells. Follow- with a submicromolar IC50 value and low side effects. Al-
ing treatment of the cells with increasing concentrations of though the IC50 value of 4-HPR is relatively high, its rela-
retinoid derivatives (0, 0.5, 1, 2.5, 5, 10 mM) for 48 h, prolif- tively few side effects made it useful in clinical trials. In this
eration was assayed using MTT, and dose–response curves regard, our derivative 5B and its organic salt forms, the most
were plotted. The IC50 value was determined by interpolation effective of the derivatives tested, are promising cancer pro-
from the curves. As summarized in Table 2, the IC50 value of drugs, although their side effects and hygroscopicities remain
derivative 5B was 0.65 mM for HCT116 cells, which was 13- to be determined in animal studies.
fold better than that of 4-HPR (8.21 mM). The IC50 values of
Using data not presented here, we found that the position
the other derivatives was slightly better than (2B, 3A, B, 4A), of the butyryl group is critical for activity, especially in the
similar to (1B, 2A), or much worse (4B, 5A) than that of 4- case of 5B. A derivative with the butyryl group in the meta

504
Vol. 52, No. 5
position of the amine group inhibited HCT116 cell growth Jꢄ8.80, 2H), 6.24 (m, 4H), 5.78 (s, 1H), 5.51 (s, 1H), 2.41 (s, 3H), 2.01
(br s, 5H), 1.72 (s, 3H), 1.60 (m, 2H), 1.46 (m, 2H), 1.02 (s, 6H). 13C-NMR
(100 MHz, DMSO-d6): d 163.93, 152.96, 147.19, 142.92, 137.72, 137.07,
136.82, 135.94, 131.04, 129.95, 129.17, 129.09, 127.16, 122.88, 120.53,
50 times more potently than did a derivative with the butyryl
in the ortho position. When the butyryl groups of 3A and 5B
were removed, the cytotoxic potential was completely abro-
114.87, 39.20, 33.85, 32.61, 28.84, 21.53, 18.79, 13.24, 12.61. IR (KBr,
cmꢀ1) 2924, 2858, 1634 (NH–CꢄO–), 1582. MS: m/z (%)ꢄ392 (Mꢅ, 100),
283 (13), 224 (7), 102 (18). HR-EI-MS m/z 391.2512 (Calcd for C26H33NO2,
391.2511).
gated. Furthermore, the number of carbons in the side func-
tional group was also important in both 3A and 5B. A deriva-
tive with four carbons at that position, i.e., a butyryl group,
was the most potent under our assay conditions.24) These
overall results suggest that increased cytotoxicity is closely
associated with side chain length. Based on the structure–
function relationship, we propose that a cellular target pro-
tein(s) responds to the four-carbon fatty acid chain by direct
association.
4-{(2E,4E,6E,8E)-[3,7-Dimethyl-9-(2,6,6-trimethyl-1-cyclohexenyl)-
nona-2,4,6,8-tetraenoylamino]}-phenyl Butanoate (2A) Using method
D, yield: 91% as a yellow solid, mp 146—148 °C. 1H-NMR (400 MHz,
CDCl3): d 7.56 (d, Jꢄ7.84, 2H), 7.19 (s, 1H), 7.04 (d, Jꢄ8.80, 2H), 7.00
(dd, Jꢄ15.00, 11.40, 1H), 6.26 (m, 4H), 5.78 (s, 1H), 2.53 (t, Jꢄ7.34, 2H),
2.41 (s, 3H), 2.01 (br s, 5H), 1.78 (m, 2H), 1.72 (s, 3H), 1.60 (m, 2H), 1.46
(m, 2H), 1.04 (t, Jꢄ7.34, 3H), 1.02 (s, 6H). 13C-NMR (100 MHz, DMSO-
d6): d 172.21, 165.01, 150.48, 146.53, 139.21, 137.60, 137.14, 135.85,
135.16, 130.30, 129.77, 129.39, 128.39, 121.77, 121.07, 120.61, 115.42,
39.69, 36.26, 34.33, 33.18, 29.03, 21.83, 19.33, 18.54, 13.85, 13.74, 12.96.
MS: m/z (%)ꢄ462 (Mꢅ, 33), 461 (100), 339 (7), 282 (18). HR-EI-MS m/z
461.2931 (Calcd for C30H39NO3, 461.2930).
Conclusion
This paper describes a new synthetic method for retinoid
derivatives that involves selectively controlling N-acylation
(carboxamide) and O-acylation (carboxyl ester). Our method
led to the facile synthesis of various retinoid derivatives in
one pot directly from retinoic acid and aminophenol deriva-
tives without protection. In terms of RAR activation, reti-
namide derivatives 2A—5A functioned like 4-HPR, i.e., they
were specific for RAR b and g, whereas, like ATRA,
retinoate derivatives 1B—5B were active for all three RARs
without specificity. In cytotoxicity assays using HCT116
cells, we found that retinamide derivatives 3A and 4A and
retinoate derivatives 2B, 3B, and 5B were superior to 4-HPR,
the parental retinamide. Data derived from derivatives 4B
and 5A indicated that RAR activation is not correlated with
cytotoxicity. Organic salts 5C and 5D were similar to parent
compound 5B in terms of RAR activation and cytotoxicity in
HCT116 cells. Although the data were not shown, the in-
creased cytotoxicity of retinoid derivatives appears to be
closely associated with the side chain length of the functional
group. From these overall results, we conclude that our deriv-
ative 5B and its organic salt forms, the most effective of the
derivatives tested, are promising cancer prodrugs.
2-{(2E,4E,6E,8E)-[3,7-Dimethyl-9-(2,6,6-trimethyl-1-cyclohexenyl)-
nona-2,4,6,8-tetraenoylamino]}-5-hydroxyphenyl Butanoate (3A) Yield:
47% as a yellow solid, mp 143—145 °C. 1H-NMR (400 MHz, CDCl3): d
9.63 (s, 1H), 7.48 (s, 1H), 7.05 (dd, Jꢄ14.90, 11.40, 1H), 6.86 (d, Jꢄ8.60,
1H), 6.73 (d, Jꢄ2.50, 1H), 6.57 (dd, Jꢄ8.60, 2.50, 1H), 6.22 (m, 4H), 5.86
(s, 1H), 2.54 (t, Jꢄ7.30, 2H), 2.41 (s, 3H), 2.01 (br s, 5H), 1.80 (m, 2H),
1.72 (s, 3H), 1.60 (m, 2H), 1.46 (m, 2H), 1.00 (m, 3H), 1.02 (s, 6H). 13C-
NMR (100 MHz, CDCl3): d 172.41, 166.90, 152.86, 150.13, 149.19, 140.07,
137.67, 137.17, 134.85, 131.47, 130.11, 129.39, 128.96, 123.96, 122.46,
119.03, 113.29, 112.94, 39.57, 36.18, 34.24, 33.10, 28.94, 21.74, 19.18,
18.43, 13.83, 13.60, 12.94. IR (KBr, cmꢀ1) 3318, 2960, 2924, 1746
(O–CꢄO–), 1635 (NH–CꢄO–), 1577. MS: m/z (%)ꢄ478 (Mꢅ, 100), 283
(40), 161 (35). HR-EI-MS m/z 477.2877 (Calcd for C30H39NO4, 477.2879).
5-{(2E,4E,6E,8E)-[3,7-Dimethyl-9-(2,6,6-trimethyl-1-cyclohexenyl)-
nona-2,4,6,8-tetraenoylamino]}-2-hydroxyphenyl Butanoate (4A) Yield:
50% as a yellow solid, mp 165—167 °C. 1H-NMR (400 MHz, CDCl3): d
7.58 (br s, 1H), 7.48 (br s, 1H), 6.92 (m, 3H), 6.60 (d, Jꢄ8.80, 1H), 6.24 (m,
4H), 5.81 (s, 1H), 2.57 (t, Jꢄ7.35, 2H), 2.41 (s, 3H), 2.01 (br s, 5H), 1.80
(m, 2H), 1.72 (s, 3H), 1.60 (m, 2H), 1.46 (m, 2H), 1.05 (m, 3H), 1.02 (s,
6H). 13C-NMR (100 MHz, CDCl3): d 171.78, 164.98, 150.68, 148.41,
140.28, 138.39, 139.34, 137.17, 136.95, 135.52, 135.46, 129.44, 129.33,
127.86, 122.29, 122.17, 110.36, 108.33, 39.52, 35.54, 33.89, 32.72, 28.61,
21.42, 18.85, 18.11, 13.34, 13.24, 12.54. MS: m/z (%)ꢄ478 (Mꢅ, 100), 391
(28), 283 (33). HR-EI-MS m/z 477.2878 (Calcd for C30H39NO4, 477.2879).
{(2E,4E,6E,8E)-[3,7-Dimethyl-9-(2,6,6-trimethyl-1-cyclohexenyl)-
nona-2,4,6,8-tetraenoylamino]}-(3-butylamino-4-hydroxy)phenylamide
1
(5A) Yield: 51% as a yellow solid, mp 186—190 °C. H-NMR (400 MHz,
Experimental
CDCl3): d 9.31 (s, 1H), 8.97 (s, 1H), 8.70 (s, 1H), 7.92 (br s, 1H), 7.25 (dd,
Jꢄ8.70, 2.31, 1H), 6.94 (dd, Jꢄ14.90, 11.40, 1H), 6.85 (d, Jꢄ8.70, 1H),
6.24 (m, 4H), 5.91 (s, 1H), 2.39 (m, 5H), 2.01 (br s, 5H), 1.72 (s, 3H), 1.67
(m, 4H), 1.46 (m, 2H), 1.02 (s, 6H), 0.98 (t, Jꢄ7.38, 3H). 13C-NMR
(100 MHz, CDCl3): d 173.19, 165.28, 149.37, 144.45, 138.82, 137.70,
137.31, 135.70, 131.27, 129.83, 129.80, 129.64, 128.26, 126.25, 122.09,
118.11, 117.75, 113.75, 39.59, 38.81, 34.24, 33.08, 28.95, 21.74, 19.23,
19.21, 13.69, 13.59, 12.87. IR (KBr, cmꢀ1) 3293, 2960, 1649 (NH–CꢄO–),
1506. MS: m/z (%)ꢄ478 (30), 477 (Mꢅ, 100), 212 (15), 175 (8). HR-EI-MS
m/z 476.3038 (Calcd for C30H40N2O3, 476.3039).
Method B. 4-Amino Phenyl (2E,4E,6E,8E)-3,7-Dimethyl-9-(2,6,6-
trimethyl-1-cyclohexenyl)-2,4,6,8-nonatetraenoate (1B, 4-APR) Yield:
49% as a yellow form. To a solution of DCC (0.14 g, 0.66 mmol) in dry
CH2Cl2 (5 ml) was added retinoic acid (0.10 g, 0.33 mmol) in dry CH2Cl2
(5 ml). The solution was stirred at room temperature for 0.5 h. To this mix-
ture were added 4-amino phenol (0.034 g, 0.33 mmol) in dry DMF (2 ml)
and DMAP (cat.), and the mixture was stirred for 4—5 h. The reaction was
quenched with NH4Cl (aq.) and extracted with EtOAc (30 ml). The extracts
were washed with H2O and brine, dried (Na2SO4), and evaporated. The
residue was purified by column chromatography (EtOAc/hexaneꢄ1/4) to
give 1B (0.064 g, 49%) as a yellow form, mp 147—150 °C. 1H-NMR
(400 MHz, CDCl3): d 7.10 (dd, Jꢄ14.90, 11.40) , 6.88 (d, Jꢄ8.74, 2H), 6.65
(d, Jꢄ8.75, 2H), 6.24 (m, 4H), 5.96 (s, 1H), 2.39 (s, 3H), 2.01 (br s, 5H),
1.72 (s, 3H), 1.61 (m, 2H), 1.46 (m, 2H), 1.02 (s, 6H). 13C-NMR (100 MHz,
CDCl3): d 166.07, 154.92, 143.88, 142.92, 140.13, 137.64, 137.19, 134.88,
131.66, 130.13, 129.41, 128.95, 122.34, 117.49, 116.14, 115.60, 39.57,
34.24, 33.09, 28.94, 21.74, 19.18, 13.99, 12.92. IR (KBr, cmꢀ1) 3462, 3375,
ATRA was purchased from Sigma Chemical Co. (Sigma-Aldrich Korea,
Seoul). The dry DMF was stored over 4-Å sieves and degassed before use by
bubbling argon through it for at least 1 h. Dry CH2Cl2 was obtained by distil-
lation from CaH2. The other commercially available reagents and solvents
were used without further purification. All reactions were conducted under
an Ar atmosphere, except for those reactions utilizing water as a solvent.
They were monitored by TLC (Merck Kieselgel 60, F254). All the products
prepared were purified by flash column chromatography on silica gel 60
(Merck, 230—400 mesh). 1H- and 13C-NMR spectra were recorded on a
Brüker AC-200F and JEOL JNM EX-400 using CDCl3 and DMSO-d6 as a
solvents. All chemical shifts (d) are quoted in ppm downfield from TMS and
coupling constants (J) are given in Hz. Mass spectra were measured on an
Agilent 1100 LC/MSD (API-ES) mass spectrometer, Micro MS-autospec/
OA Tof (HR-EI-MS). IR spectra were recorded on Perkin Elmer 16FPC FT-
IR spectrophotometer and frequencies are given in reciprocal centimeters.
Method A. (2E,4E,6E,8E)-[3,7-Dimethyl-9-(2,6,6-trimethyl-1-cyclo-
hexenyl)-nona-2,4,6,8-tetraenoylamino]-(4-hydroxy)phenylamide (1A, 4-
HPR) To a solution of EDCI (0.13 g, 0.66 mmol) in dry CH2Cl2 (5 ml) was
added retinoic acid (0.10 g, 0.33 mmol) in dry CH2Cl2 (5 ml). The solution
was stirred at room temperature for 0.5 h. To this mixture were added 4-
amino phenol (0.053 g, 0.33 mmol) in dry DMF (2 ml) and DMAP (cat.),
and the mixture was stirred for 4—5 h. The reaction was quenched with
NH4Cl (aq.) and extracted with EtOAc (30 ml). The extracts were washed
with H2O and brine, dried (Na2SO4), and evaporated. The residue was puri-
fied by column chromatography (EtOAc/hexaneꢄ1/4) to give 1A (0.062 g,
48%) as a yellow solid. mp 170—171 °C. 1H-NMR (400 MHz, CDCl3): d
7.34 (d, Jꢄ8.80, 2H), 7.13 (s, 1H), 6.98 (dd, Jꢄ14.90, 11.40, 1H), 6.77 (d,

May 2004
505
2919, 1716 (O–CꢄO–), 1582. MS: m/z (%)ꢄ393 (27), 392 (Mꢅ, 100), 283 2H). 13C-NMR (100 MHz, CDCl3ꢅDMSO-d6): d 154.32, 145.93, 136.91,
(20), 175 (8). HR-EI-MS m/z 391.2513 (Calcd for C26H33NO2, 391.2511).
4-(Butyrylamino)phenyl (2E,4E,6E,8E)-3,7-Dimethyl-9-(2,6,6-trimethyl-
1-cyclohexenyl)-2,4,6,8-nonatetraenoate (2B) Using method D, yield:
128.60, 128.40, 128.32, 128.23, 127.69, 125.89, 122.42, 117.00, 105.62,
102.73, 65.47. MS: m/z (%)ꢄ260 (Mꢅ, 100), 126 (23), 110 (21).
Method D. 2-Benzyloxycarbonylamino-5-hydroxyphenylbutanoate
93% as a yellow solid, mp 148—152 °C. 1H-NMR (400 MHz, CDCl3): d (10) Compound 7 (1.01 g, 3.85 mmol) was dissolved in dry CH2Cl2 (15 ml)
7.50 (d, Jꢄ8.75, 2H), 7.21 (s, 1H), 7.05 (m, 3H), 6.24 (m, 4H), 5.97 (s, 1H), and N-methyl morpholine (0.62 ml, 5.77 mmol) was added. To the resulting
2.40 (s, 3H), 2.32 (t, Jꢄ7.32, 2H), 2.01 (br s, 5H), 1.72 (m, 2H), 1.70 (s, mixture butyryl chloride (0.56 ml, 5.77 mmol) was added dropwise at 0 °C,
3H), 1.61 (m, 2H), 1.46 (m, 2H), 1.02 (s, 6H), 1.00 (t, Jꢄ7.32, 3H). 13C- and stirred for 30 min and the reaction was quenched with 5% NaHCO3 so-
NMR (100 MHz, CDCl3): d 171.23, 165.17, 155.63, 146.76, 140.45, 137.62,
lution and washed with 5 N HCl and H2O. The organic layer was dried over
137.14, 135.40, 134.66, 132.04, 130.20, 129.34, 129.13, 122.07, 120.73, MgSO4 and concentrated under reduced pressure. The crude product was
117.02, 39.56, 39.49, 34.23, 33.09, 28.93, 21.73, 19.17, 19.02, 14.06, 13.72,
12.94. MS: m/z (%)ꢄ462 (Mꢅ, 33), 461 (100), 339 (19), 283 (30). HR-EI-
MS m/z 461.2931 (Calcd for C30H39NO3, 461.2930).
purified by column chromatography (EtOAc/hexaneꢄ1/5) to give 0.83 g
(65%) of 10 as a white solid, mp 112—114 °C. 1H-NMR (200 MHz, CDCl3):
d 7.55 (d, Jꢄ7.24, 1H), 7.37 (m, 5H), 6.65 (d, Jꢄ2.74, 1H), 6.56 (s, 1H),
4-Amino-3-(butyryloxy)phenyl (2E,4E,6E,8E)-3,7-Dimethyl-9-(2,6,6- 6.38 (s, 1H), 5.17 (s, 2H), 2.50 (t, Jꢄ7.35, 2H), 1.72 (m, 2H). 1.00 (t,
trimethyl-1-cyclohexenyl)-2,4,6,8-nonatetraenoate (3B) Yield: 52% as a
Jꢄ7.42, 3H). 13C-NMR (100 MHz CDCl3): d 171.65, 155.01, 145.93,
yellow form, mp 119—123 °C. 1H-NMR (400 MHz, CDCl3): d 7.11 (dd, 135.87, 128.59, 128.38, 128.32, 128.25, 127.95, 122.72, 121.73, 119.57,
Jꢄ15.00, 11.40, 1H), 6.87 (s, 1H), 6.77 (m, 1H), 6.25 (m, 5H), 6.00 (s, 1H), 113.62, 109.77, 67.33, 36.00, 18.39, 13.54. MS: m/z (%)ꢄ494 (Mꢅ, 100),
3.63 (br s, 2H), 2.48 (t, Jꢄ7.34, 2H), 2.42 (s, 3H), 2.01 (br s, 5H), 1.77 (m, 391 (13), 283 (21).
2H), 1.72 (s, 3H), 1.60 (m, 2H), 1.46 (m, 2H), 1.03 (s, 6H), 1.02 (m, 3H).
2-Hydroxy-5-nitro Phenylbutanoate (11) Yield: 61% as a white solid,
13C-NMR (100 MHz, CDCl3): d 172.44, 164.55, 156.47, 152.59, 142.30, mp 108—111 °C. 1H-NMR (200 MHz, CDCl3): d 8.07 (m, 2H), 7.05 (d,
140.72, 137.79, 137.63, 137.20, 134.99, 132.40, 130.08, 129.29, 124.01, Jꢄ8.7, 1H), 2.64 (t, Jꢄ7.34, 2H), 1.82 (m, 2H), 1.02 (t, Jꢄ7.32, 3H). 13C-
119.21, 116.25, 113.23, 112.88, 39.57, 36.14, 34.25, 33.11, 29.68, 28.95, NMR (100 MHz, CDCl3): d 169.01, 156.27, 141.39, 141.12, 121.82, 117.89,
21.75, 19.18, 18.43, 14.14, 13.64, 12.94. MS: m/z (%)ꢄ478 (Mꢅ, 100), 382 117.05, 39.41, 18.83, 13.42. MS: m/z (%)ꢄ226 (Mꢅ, 100), 156 (18).
(15), 283 (65). HR-EI-MS m/z 477.2878 (Calcd for C30H39NO4, 477.2879).
N-(2-Hydroxy-5-nitrophenyl)butyramide (12) Yield: 72% as a white
4-Amino-2-(butyryloxy)phenyl (2E,4E,6E,8E)-3,7-Dimethyl-9-(2,6,6- solid, mp 122—123 °C. 1H-NMR (200 MHz, CDCl3): d 8.91 (br s, 1H), 8.80
trimethyl-1-cyclohexenyl)-2,4,6,8-nonatetraenoate (4B) Yield: 45% as a
(br s, 1H), 7.84 (m, 1H), 6.97 (dd, Jꢄ8.80, 0.88, 1H), 2.43 (t, Jꢄ7.28, 2H),
yellow form, mp 125—127 °C. 1H-NMR (400 MHz, CDCl3): d 7.11 (dd, 1.74 (m, 2H), 1.00 (t, Jꢄ7.24, 3H). 13C-NMR (100 MHz, CDCl3): d 174.56,
Jꢄ15.00, 11.40, 1H), 6.90 (d, Jꢄ8.75, 1H), 6.51 (m, 2H), 6.25 (m, 4H), 135.07, 125.72, 122.95, 118.06, 114.09, 111.16, 38.76, 19.08, 13.59. MS:
5.93 (s, 1H), 3.65 (br s, 2H), 2.48 (t, Jꢄ7.35, 2H), 2.39 (s, 3H), 2.01 (br s, m/z (%)ꢄ225 (Mꢅ, 100), 155 (31), 110 (12), 95 (11).
5H), 1.72 (s, 3H), 1.65 (m, 4H), 1.46 (m, 2H), 1.03 (s, 6H), 0.97 (t, Jꢄ7.34,
2-Amino-5-hydroxyphenylbutanoate (13) To a solution of 10 (0.23 g,
3H). 13C-NMR (100 MHz, CDCl3): d 171.80, 164.38, 156.02, 144.92, 0.70 mmol) in MeOH (5 ml) Pd/C (10%, 0.02 g) was added at room tempera-
142.68, 140.58, 137.63, 137.13, 134.57, 134.30, 132.18, 130.26, 129.31, ture. After stirring for 30 min under H2, the reaction mixture was filtered on
129.21, 123.63, 116.39, 112.68, 110.05, 39.56, 35.89, 34.24, 33.11, 29.35,
celite and evaporated in vacuo to give 13 (0.13 g, 96%) as a light brown oil.
28.95, 21.75, 19.18, 18.50, 14.06, 13.66, 12.96. MS: m/z (%)ꢄ478 (Mꢅ, 1H-NMR (200 MHz, CDCl3): d 7.18 (d, Jꢄ8.60, 1H), 6.41 (d, Jꢄ2.60, 1H),
100), 356 (16), 283 (40). HR-EI-MS m/z 477.2878 (Calcd for C30H39NO4,
477.2879).
6.32 (dd, Jꢄ8.60, 2.60, 1H), 3.70 (br s, 2H), 2.36 (t, Jꢄ7.42, 2H), 1.73 (m,
2H), 1.00 (t, Jꢄ7.42, 3H). 13C-NMR (100 MHz, CDCl3): d 168.89, 147.72,
141.13, 132.24, 116.87, 113.32, 109.40, 39.59, 18.82, 13.65. MS: m/z
(%)ꢄ196 (Mꢅ, 100), 126 (33).
4-Amino-2-(butyrylamino)phenyl (2E,4E,6E,8E)-3,7-Dimethyl-9-(2,6,6-
trimethyl-1-cyclohexenyl)-2,4,6,8-nonatetraenoate (5B) Yield: 43% as a
yellow form, mp 127—129 °C. 1H-NMR (400 MHz, CDCl3): d 7.69 (br s,
5-Amino-2-hydroxyphenylbutanoate (14) Yield: 99% as a light brown
1H), 7.27 (br s, 1H), 7.12 (dd, Jꢄ14.90, 11.40, 1H), 6.90 (d, Jꢄ8.70, 1H), oil. 1H-NMR (200 MHz, CDCl3): d 7.54 (m, 1H), 6.86 (d, Jꢄ8.80, 1H), 6.60
6.28 (m, 5H), 6.00 (s, 1H), 3.72 (br s, 2H), 2.41 (s, 3H), 2.32 (t, Jꢄ7.44,
2H), 2.01 (br s, 5H), 1.72 (s, 3H), 1.70, (m, 2H), 1.64 (m, 2H), 1.46 (m, 2H),
1.02 (s, 6H), 0.98 (t, Jꢄ7.38, 3H). 13C-NMR (100 MHz, CDCl3): d 170.96,
165.17, 156.45, 144.60, 140.86, 137.65, 137.04, 134.40, 132.51, 130.59,
(m, 1H), 2.64 (t, Jꢄ7.34, 2H), 1.82 (m, 2H), 1.02 (t, Jꢄ7.32, 3H). 13C-NMR
(100 MHz, CDCl3): d 167.84, 141.53, 140.02, 139.71, 116.23, 113.30,
109.41, 39.65, 18.95, 13.59. MS: m/z (%)ꢄ196 (Mꢅ, 100), 126 (26).
N-(5-Amino-2-hydroxyphenyl)butylamide (15) Yield: 100% as a light
130.29, 129.43, 129.19, 122.48, 116.06, 110.40, 108.32, 39.77, 39.63, 34.25, brown solid. 1H-NMR (200 MHz, CDCl3): d 7.79 (br s, 1H), 6.76 (d,
33.11, 28.93, 21.68, 19.18, 18.92, 14.16, 13.61, 12.93. IR (KBr, cmꢀ1) 3441, Jꢄ2.65, 1H), 6.58 (d, Jꢄ8.60, 1H), 6.44 (dd, Jꢄ8.60, 2.65, 1H), 2.36 (t,
3344, 2929, 1710 (O–CꢄO–), 1685 (NH–CꢄO–), 1506. MS: m/z (%)ꢄ478
(20), 477 (Mꢅ, 65), 284 (20), 283 (100), 175 (22). HR-EI-MS m/z 476.3040
(Calcd for C30H40N2O3, 476.3039).
Jꢄ7.24, 2H), 1.69 (m, 2H), 0.98 (t, Jꢄ7.24, 3H). 13C-NMR (100 MHz,
CDCl3): d 173.28, 140.76, 139.37, 126.40, 119.31, 113.89, 109.18, 39.35,
18.71, 13.43. MS: m/z (%)ꢄ195 (Mꢅ, 100), 125 (13), 110 (21).
Method C. (2E,4E,6E,8E)-[3,7-Dimethyl-9-(2,6,6-trimethyl-1-cyclo-
Method E. Fumarate Salt of 4-Amino-2-(butyrylamino)phenyl-
hexenyl)-nona-2,4,6,8-tetraenoylamino]-(4-hydroxy)phenylamide
(1A, (2E,4E,6E,8E)-3,7-dimethyl-9-(2,6,6-trimethyl-1-cyclohexenyl)-2,4,6,8-
HPR) A mixture of dry DMF (0.077 ml, 0.99 mmol) and SOCl2 (0.072 ml,
0.99 mmol) was stirred under an argon atmosphere for 1 h. ATRA was added
to the solution (0.10 g, 0.33 mmol) in dry DMF (2 ml). After stirring at 0 °C
for 45 min, in subdued light, the clear deep red retinoyl chloride solution
was added dropwise to a cooled solution of distilled triethylamine (0.14 ml,
0.99 mmol) and 4-aminophenol (0.072 g, 0.66 mmol) in dry, degassed DMF
(2 ml). The temperature was maintained at 10—15 °C during the addition.
The dark-colored reaction solution was stirred at room temperature until
TLC analysis indicated none remaining (about 2 h). The reaction was
nonatetraenoate (5C) To a solution of fumaric acid (27 mg, 0.23 mmol)
dissolved in dry THF (5 ml) at 40—50 °C was added to free base 5B
(0.10 mg, 0.21 mmol) in dry THF (2 ml). After stirring for 2 h at 40 °C, the
solution was filtered and stirred at 10 °C until crystallization starts and then
n-hexane (3 ml) was dropped slowly into the solution. The resulting mixture
was stirred for 3 h at 0 °C and the salt formed was filtered and dried to give
fumarate salt 5C (1.10 g, 89%) as a yellow powder, mp 142—146 °C. 1H-
NMR (200 MHz, CDCl3ꢅDMSO-d6): d 7.46 (br s, 1H), 7.55 (br s, 1H), 7.10
(dd, Jꢄ14.90, 11.40, 1H), 6.88 (d, Jꢄ8.70, 1H), 6.77 (s, 2H), 6.28 (m, 5H),
quenched with NH4Cl (aq.) and extracted with EtOAc. The extracts were 6.00 (s, 1H), 2.40 (s, 3H), 2.32 (t, Jꢄ7.44, 2H), 2.03 (br s, 5H), 1.72 (s, 3H),
washed with H2O and brine, dried (Na2SO4), and evaporated. The residue
was purified by column chromatography using hexane/EtOAc (3/1) as the
eluent to give 1A (0.10 g, 78%) as a yellow solid.
(2,4-Dihydroxyphenyl)-carbamic Acid Benzylester (7) TEA (trimethyl-
amine, 0.72 ml, 7.41 mmol) was added to a solution of 4-amino resorcinol
(1.0 g, 6.18 mmol) dissolved in dry DMF (5 ml). To the resulting mixture
Cbz-Cl (1.06 ml, 7.41 mmol) was added dropwise at 0 °C and stirred for 2 h.
The reaction mixture was extracted with EtOAc (50 ml) and washed with
1.65 (m, 4H), 1.46 (m, 2H), 1.02 (s, 6H), 0.98 (t, Jꢄ7.40, 3H). 13C-NMR
(100 MHz, CDCl3): d 170.94, 165.98, 164.99, 153.65, 146.26. 139.75,
137.23, 136.88, 135.02, 133.99, 131.95, 130.64, 129.85, 129.65, 128.23,
122.72, 117.99, 109.76, 109.20, 39.08, 38.87, 33.86, 33.33, 28.80, 21.53,
18.75, 18.70, 13.66, 13.47, 12.66. MS (API-ES, Neg, scan): m/z (%)ꢄ591
([Mꢀ1], 10), 476 (11), 475 (24), 406 (29), 405 (100), 283 (58), 255 (62),
115 (9).
Maleate Salt of 4-Amino-2-(butyrylamino)phenyl(2E,4E,6E,8E)-3,7-
H2O (2ꢁ30 ml). The organic layer was dried over MgSO4 and evaporated in dimethyl-9-(2,6,6-trimethyl-1-cyclohexenyl)-2,4,6,8-nonatetraenoate
vacuo. The crude product was crystallized from (EtOAc/hexaneꢄ1/3) to (5D) Yield: 91% as
a
yellow powder, mp 122—124 °C. 1H-NMR
give 7 (1.42 g, 89%), as a white solid, mp 121—123 °C. 1H-NMR (200 MHz,
CDCl3ꢅDMSO-d6): d 8.96 (br s, 1H), 8.46 (br s, 1H), 7.47 (d, 1H, NH),
(200 MHz, CDCl3ꢅDMSO-d6): d 8.78 (br s, 1H), 7.62 (br s, 1H), 7.10 (dd,
Jꢄ14.90, 11.40, 1H), 6.90 (d, Jꢄ8.70, 1H), 6.61 (dd, Jꢄ8.78, 2.63, 1H),
7.37 (m, 5H), 6.45 (d, Jꢄ2.50, 1H), 6.33 (dd, Jꢄ8.64, 2.50, 1H), 5.17 (s, 6.28 (m, 4H), 6.20 (s, 2H), 6.00 (s, 1H), 2.38 (s, 3H), 2.33 (t, Jꢄ7.44, 2H),

506
Vol. 52, No. 5
2.03 (br s, 5H), 1.72 (s, 3H), 1.63 (m, 4H), 1.46 (m, 2H), 1.02 (s, 6H), 0.94 sured spectrophotometrically at 550 nm. Each experiment was performed in
(t, Jꢄ7.40, 3H). 13C-NMR (100 MHz, CDCl3): d 170.91, 166.01, 165.03,
153.89, 146.25, 139.73, 137.21, 136.85, 134.98, 133.93, 131.92, 130.59,
129.85, 129.62, 128.23, 122.72, 118.01, 109.76, 109.21, 39.08, 38.87, 33.86,
triplicate and repeated a minimum of three times.
Acknowledgments This study was supported by a grant (02-PJ1-PG11-
33.30, 28.81, 21.53, 18.75, 18.73, 13.70, 13.47, 12.68. MS (API-ES, Neg, VN01-SV01-0019) from the Korean Ministry of Health & Welfare, Repub-
scan): m/z (%)ꢄ591 ([Mꢀ1], 12), 476 (10), 475 (21), 406 (30), 405 (100), lic of Korea. S.J.U. was supported by BK21 project from the Ministry of Ed-
255 (62), 232 (40), 115 (30).
ucation & Human Resources Development.
Tartarate Salt of 4-Amino-2-(butyrylamino)phenyl(2E,4E,6E,8E)-3,7-
dimethyl-9-(2,6,6-trimethyl-1-cyclohexenyl)-2,4,6,8-nonatetraenoate References
1
(5E) Yield: 95% as a yellow solid, mp 131—135 °C. H-NMR (200 MHz,
CDCl3ꢅDMSO-d6): d 7.89 (br s, 1H), 7.51 (br s, 1H), 7.09 (dd, Jꢄ14.90,
11.40, 1H), 6.86 (d, Jꢄ8.70, 1H), 6.24 (m, 5H), 6.02 (s, 1H), 4.52 (s, 2H),
2.39 (s, 3H), 2.32 (t, Jꢄ7.38, 2H), 2.03 (br s, 5H), 1.72 (s, 3H), 1.63 (m,
4H), 1.46 (m, 2H), 1.02 (s, 6H), 0.94 (t, Jꢄ7.40, 3H). 13C-NMR (100 MHz,
CDCl3): d 173.12, 170.98, 164.01, 153.68, 146.21, 139.77, 137.26, 136.91,
135.04, 131.98, 130.66, 129.87, 129.68, 128.25, 122.75, 118.00, 109.83,
109.27, 72.16, 39.00, 38.87, 33.88, 33.36, 28.80, 21.53, 18.73, 18.70, 13.68,
13.50, 12.69. MS (API-ES, Neg, scan): m/z (%)ꢄ625 ([Mꢀ1], 8), 476 (16),
475 (42), 406 (18), 405 (100), 283 (36), 255 (41), 149 (21).
Pamoate Salt of 4-Amino-2-(butyrylamino)phenyl(2E,4E,6E,8E)-3,7-
dimethyl-9-(2,6,6-trimethyl-1-cyclohexenyl)-2,4,6,8-nonatetraenoate (5F)
Yield: 93% as a yellow powder, mp 110—112 °C. 1H-NMR (200 MHz,
CDCl3ꢅDMSO-d6): d 8.43 (s, 2H), 8.20 (d, Jꢄ8.70, 2H), 7.70 (d, Jꢄ8.71,
2H), 7.56 (br s, 1H), 7.34 (t, Jꢄ8.71, 2H), 7.18 (t, Jꢄ8.70, 2H), 7.10 (dd,
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(100 MHz, CDCl3): d 172.55, 171.11, 164.90, 153.87, 153.66, 143.89, 11) Clifford J. L., Sabichi A. L., Zou C., Yang X., Steele V. E., Kelloff G.
139.82, 137.22, 136.89, 136.11, 134.98, 133.37, 132.04, 131.33, 130.84,
130.05, 129.84, 129.65, 128.61, 126.69, 123.32, 122.95, 120.48, 117.89,
114.72, 111.02, 110.43, 67.01, 39.08, 38.87, 33.85, 32.62, 28.78, 25.12,
21.51, 19.88, 18.77, 13.69, 13.49, 12.66. MS (API-ES, Neg, scan): m/z
(%)ꢄ864 ([Mꢀ1], 6), 476 (17), 475 (32), 406 (25), 405 (100), 387 (14), 283
(16), 255 (15).
RAR Transactivation Assay Transient transfections were carried out
using a liposome-based method with Lipofectamine, according to the manu-
facturer’s instructions (Gibco BRL). Subsequent transcription assays were
carried out using a CAT ELISA kit (Roche Molecular Biochemicals), as pre-
viously described.22) Briefly, COS-1 cells (1ꢁ106 cells) maintained in Dul-
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overnight transfection with the reporter gene [(RARE)3-tk-CAT] and recep- 18) Smith M. B., March J., “March’s Advanced Organic Chemistry,” 5th
tor expression vector (RARa, b, and g in pSG5), the cells were washed and
fed with complete medium supplemented with increasing concentrations of
ATRA, 4-HPR, or other retinoids as indicated in the text. All data presented
represent the means of at least three independent transfections.
ed., Chap. 2, 8, John Wiley & Sons, New York, 2001.
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Cytotoxicity Assay The antiproliferative effect of retinoid derivatives
was monitored using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium 21) Fanjul A. N., Delia D., Pierotti M. A., Rideout D., Yu J. Q., Pfahl M., J.
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taining various concentrations of retinoid derivatives. DMSO controls
(0.01%) did not affect cell proliferation. After 48 h, 50 ml of MTT solution
(2 mg/ml in PBS) was added to the culture medium and the reaction mixture
was incubated at 37 °C in a 5% CO2 atmosphere for 4 h. The MTT solution
was aspirated and 150 ml of DMSO was added. The optical density was mea-
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