Solid phase-assisted synthesis and screening of a small library of N-(4-hydroxyphenyl)retinamide (4-HPR) analogs

Article abstract of DOI:10.1016/j.bmcl.2006.10.050

Using solid phase-assisted synthesis and purification, a 49 member library of analogs of the mammary tumor chemopreventive retinoid N-(4-hydroxyphenyl)retinamide (4-HPR) has been prepared. After prescreening for growth inhibitory activity in human mammary tumor cells (MCF-7) in culture, most of those analogs which showed activity (12 of them) were assayed for apoptosis-inducing activity in the MCF-7 cells. At least 3 of the analogs (13, 24, and 28) showed activity approaching that of 4-HPR.

Full text of DOI:10.1016/j.bmcl.2006.10.050

Bioorganic & Medicinal Chemistry Letters 17 (2007) 836–840
Solid phase-assisted synthesis and screening of a small library
of N-(4-hydroxyphenyl)retinamide (4-HPR) analogs
Serena M. Mershon,a Allyson L. Anding,b Jason S. Chapman,b
Margaret Clagett-Dame,b,c Laura A. Stonerocka and Robert W. Curley, Jr.a,*
aDivision of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, The Ohio State University, Columbus, OH 43210, USA
bDepartment of Biochemistry, College of Agricultural and Life Sciences, University of Wisconsin-Madison, Madison, WI 53706, USA
cPharmaceutical Sciences Division, School of Pharmacy, University of Wisconsin-Madison, Madison, WI 53706, USA
Received 7 September 2006; revised 19 October 2006; accepted 23 October 2006
Available online 25 October 2006
Abstract—Using solid phase-assisted synthesis and purification, a 49 member library of analogs of the mammary tumor chemopre-
ventive retinoid N-(4-hydroxyphenyl)retinamide (4-HPR) has been prepared. After prescreening for growth inhibitory activity in
human mammary tumor cells (MCF-7) in culture, most of those analogs which showed activity (12 of them) were assayed for apop-
tosis-inducing activity in the MCF-7 cells. At least 3 of the analogs (13, 24, and 28) showed activity approaching that of 4-HPR.
ꢀ 2006 Elsevier Ltd. All rights reserved.
O
The synthetic retinoid N-(4-hydroxyphenyl)retinamide
(4-HPR; 1, Fig. 1) was developed a number of years
ago and has shown promise as a breast cancer chemo-
preventive agent in animals.1 This analog is a simple
amide derivative of the naturally occurring parent reti-
noid, all-trans retinoic acid (atRA; 2), but (1) is less toxic
and substantially less teratogenic than 2.2 In addition to
breast tumor cells, 4-HPR inhibits the growth of and
induces apoptosis in a number of other tumor cell lines
including prostate,3 bladder,4 head and neck squamous
carcinoma,5 and neuroblastoma cell lines.6 This breadth
of activity has led to its exploration in human clinical
trials as a breast cancer chemopreventive agent7 and in
animal studies as an antitumor agent.8
R
1 R = HN
OH
2 R = OH
3 R = HN
HOOC
The mechanism through which 4-HPR acts remains un-
clear despite its structural resemblance to 2. Most reti-
noids are thought to act by binding to the nuclear
retinoic acid receptors (RARs), which bind atRA. The
receptors function as ligand-dependent transcription
factors9 and some researchers have reported that 4-
HPR can activate RARs in transactivation assays.10
We and others find, however, that 4-HPR has low affin-
ity for the retinoid receptors.11–13 4-HPR has also been
4 R = HN
OH
Cl
Cl
O
CHCl2
6 R =
[(Ph)3PCl]+2 Cl2CCOCCl2
2-
Keywords: Library; Solid phase synthesis; Retinoid; 4-HPR; Mammary
tumor cell; Chemoprevention.
5
*
Corresponding author. Tel.: +1 614 292 7628; fax: +1 614 292
2435; e-mail: curley.1@osu.edu
Figure 1. Structures of retinoids and chlorinating agent.
0960-894X/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2006.10.050

S. M. Mershon et al. / Bioorg. Med. Chem. Lett. 17 (2007) 836–840
837
shown to induce apoptosis in both atRA-sensitive and -
resistant cells, pointing to a mode of action that is inde-
pendent of the RAR.14
ArNH2/6.2 pyridine; 0 ꢁC ! rt, 1–2 h; (3) 1.25 TFPA,
then H2O, then 600 mg (2.8 meq) of Amberliteꢂ A-21
resin. A goal of this method was to avoid any other puri-
fication of product retinamides prior to activity screen-
ing. Unfortunately, in all instances, whether solution
phase16 or solid phase methods were employed, a small
quantity (0.5–1%) of a non-polar retinoid impurity was
formed. After numerous NMR spectroscopic and mass
spectrometric experiments, the structure of this impurity
was determined to be the retinoate ester 6. Extensive
experiments established that formation of this undesired
ester requires the generation of retinoyl chloride and the
presence of activated hexachloroacetone and presumably
resulted from reaction of the enolate of tetrachloroace-
tone dianion (see 5) with retinoyl chloride. Since the pres-
ence of 6 could confound activity screens and no method
was found to suppress its formation, all retinamides were
Despite continued interest in 4-HPR as a chemopreven-
tive agent, relatively few 4-HPR analogs have been
reported. The majority of these showed good activity
in early studies of their ability to reverse squamous
metaplasia in vitamin A-deficient hamster tracheae.15
A number of these analogs are being reinvestigated in
an effort to find more potent retinamides given the rela-
tively modest activity 1 has shown in a breast cancer pre-
vention trial.7 In particular, it has been suggested that
N-(2-carboxyphenyl)retinamide (2-CPR; 3) has more
pronounced activity than 1 in some head and neck squa-
mous carcinoma cell and lung cancer cell lines,5 and N-
(3-hydroxyphenyl)retinamide (3-HPR; 4) is more potent
in bladder cancer cell lines.4 This suggests the possibility
that in breast cancer cells, a similar improvement in
activity might be obtainable.
subjected to a rapid silica gel chromatography
(EtOAc/hexanes) to give the final products.
Using these synthetic and purification methods summa-
rized in the previous paragraph, and commercially avail-
able anilines or their simple derivatives, a total of 49
retinamides (43 previously unreported) were synthe-
sized. Table 1 shows the majority of the prepared retina-
Because of the limited range of 4-HPR related structures
that have been prepared and uncertainties about the
molecular target(s) for this molecule, preparation of a li-
brary of 1-like compounds appeared to be warranted.
Due to the sensitivity of retinoids, synthesis of a series
of arylamides of RA would be facilitated by a method
of activating retinoic acid that generates little acid and
could be adapted to solid phase methods to simplify
isolation. To this end, it was reasoned that the mild,
acid-free procedure of Villeneuve and Chan would suf-
fice.16 In this method, acid chlorides are generated by
treatment of a dry THF solution of the carboxylic acid
with one equivalent of triphenylphosphine, 0.5 equiv
of hexachloroacetone, and 6 equivalents of pyridine at
ꢀ78 ꢁC. It is thought that triphenylphosphine and hexa-
chloroacetone react to form the chlorinating reagent 5
which converts the carboxylic acid (RA here) to the acid
chloride in the absence of any HCl generation, which
would otherwise isomerize RA. Polymer supported
phosphines and CCl4 can also be used to form acid
chlorides at an elevated temperature in high yield.16
Adaptation of this latter strategy for use with hexa-
chloroacetone appeared feasible as did removal of unre-
acted starting materials using solid phase trapping
reagents. Parlow and coworkers have used the reactive
tetrafluorophthalic anhydride (TFPA) to trap unreacted
aniline as a polyamine resin removable phthalic acid
monoamide.17 Others have used similar anion exchange
resins to remove chloride ion and unreacted carboxylic
acid from library mixtures.18–20 Thus, it was hoped that
library purification would be accomplished by filtering
off the phosphine resin, treating with TFPA, stirring
with amine anion exchange resin, and evaporation of
volatiles.
mides. Table
2
shows the small number of
naphthylamine amides that were synthesized and the
N-methyl analog of 4-HPR (47) was also prepared.
All structures were assigned based on the analysis of
mass spectra, 1H and 13C NMR spectra, and COSY
1
and/or H–13C correlation spectra. Product purity was
assessed by reverse-phase HPLC (82% or 86% metha-
nol/H2O) and found to be 91–99% (mean = 95 2%)
for all analogs except 12, 25, and 45 (85–89%) and 38,
which decomposed prior to analysis. Yields varied great-
ly (20–94%) and were generally the poorest for those
analogs with electron withdrawing ring substituents,
especially if the substituents sterically crowded the
amine moiety. In particular, synthesis of 24 using 2-ami-
no-5-nitrophenol also resulted in formation of substan-
tial amounts of the retinoate ester which was removed
chromatographically, while use of 2-amino-3-nitrophe-
nol provided no amide and led exclusively to formation
of the ester, which was not biologically evaluated.
All of the analogs except 38 and 41 were initially evalu-
ated for their ability to inhibit growth of the human
MCF-7 breast cancer cells in culture using a fluorescein
diacetate cell viability assay.21 The number of live cells
was assessed 48 h after the administration of a single
dose of the analogs shown in Tables 1 and 2, analog
47 or 4-HPR (McNeil Pharmaceuticals), all at a final
concentration of 10ꢀ5 M, or vehicle (0.2% ethanol).
The resulting effect on MCF-7 cell growth is summa-
rized in Tables 1 and 2. Analog 47, which was prepared
in 68% yield, showed modest activity (+ at best; see
Table 1 legend) in this assay.
Initial efforts to optimize reaction and purification condi-
tions were conducted on a 0.1 mmol scale for the synthe-
sis of 4-HPR. The conditions investigated included
reagent stoichiometry, order of reagent addition, temper-
ature, time, and choice of anion exchange resin with the
following conditions found to be optimal: (1) 1.0 RA/3.0
Ph3P-resin/THF, then 0.5 HCA; 0 ꢁC, 1 h; (2) 1.5
Since 4-HPR is known to induce apoptosis in these
MCF-7 breast cancer cells, compounds showing cell
growth inhibitory activity (Tables 1 and 2: + to +++,
or those analogs with P25% of 4-HPR’s activity) were
evaluated for apoptotic activity after 48 h of treatment

838
S. M. Mershon et al. / Bioorg. Med. Chem. Lett. 17 (2007) 836–840
Table 1. Anilinamide analogs prepared
R5
R6
R4
R3
O
N
H
R2
Compound
R2
R3
R4
R5
R6
% Yield
Growth inhibitiona
1b
7
H
H
OH
OH
OH
CH3
H
H
H
94
71
+++
ꢀ
CH3
CH3
OH
OH
OH
OH
H
H
H
H
8
H
CH3
H
H
85
66
ꢀ
9
H
H
++
ꢀ
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28b
29
30c
31d
32e
33
34
35
36
37
38
39
40
41b
3b
H
CH3
C(CH3)3
H
80
75
H
H
H
ꢀ
H
H
H
CH3
H
83
80
ꢀ
OH
OH
OH
H
CH3
H
H
+++
ꢀ
CH3
H
H
H
71
66
OCH3
OH
OH
H
H
H
ꢀ
Cl
H
H
H
75
70
ꢀ
Cl
H
H
++
++
ꢀ
OH
H
H
Cl
Cl
Cl
Br
Cl
H
H
59
75
OH
OH
CH3
OH
OH
NO2
H
H
H
Br
Cl
H
75
31
ꢀ
OH
NO2
H
H
ꢀ
H
H
70
74
ꢀ
NO2
H
H
H
ꢀ
OH
OH
OH
OH
OH
OH
OH
OH
H
H
H
28
20
+++
+
H
NO2
H
H
CO2CH3
CO2H
H
H
H
64
77
ꢀ
H
H
H
ꢀ
CO2CH3
CO2H
H
H
H
86
+
H
H
H
Quant.
59
36
ꢀ
H
H
CO2CH3
CO2H
H
ꢀ
H
H
H
ꢀ
OH
OH
CO2CH3
CO2H
H
CO2CH3
CO2H
OH
OH
H
H
61
61
ꢀ
H
H
H
ꢀ
H
H
H
81
76
+
H
H
H
ꢀ
OH
OH
CH2OH
H
CO2CH3
H
H
Quant.
75
57
ꢀ
H
SO2CH2CH3
H
H
ꢀ
H
H
H
NDf
+
CH2OH
H
H
H
H
80
76
H
NH2
H
H
H
++
ND
+
CO2CH3
CO2H
H
H
H
H
64
74
H
H
OCH2CH3
H
H
42b,g
H
H
H
63
ꢀ
a Activity versus 4-HPR standard, all at 10ꢀ5M: +++, P100% the activity of standard; ++, P50% the activity of standard; +, P25% the activity of
standard; ꢀ, activity equivalent to vehicle.
b Previously reported analog.
c At 5.9 · 10ꢀ6M.
d At 3.9 · 10ꢀ6M.
e At 6.6 · 10ꢀ6 M.
f Not determined.
g At 7.4 · 10ꢀ6 M.
using a terminal dUTP nick-end labeling (TUNEL)
assay.21 Cells were treated with vehicle or a single dose
of standard 4-HPR, or one of 11 of the library analogs
and synthetic 4-methoxyphenyl retinamide (4-MPR; 50)
which is a known active 4-HPR metabolite, all at
10ꢀ5 M. The % of TUNEL-labeled cells was assessed
48 hrs later. As shown in Figure 2, analog 24 showed
activity comparable to that of 4-HPR while 13 and the
previously known analog 28 showed lesser but signifi-
cant apoptotic activity. To further evaluate the activity
of 24 relative to 4-HPR, an 8 day cell viability assay
was conducted at several doses of compound in MCF-
7 cells, Figure 3 shows that the activity of 24 is similar
to that of 1, with no live cells remaining after 8 days
of exposure of cells to either compound at 10ꢀ5 M.
While no obvious pattern has yet emerged to build a
hydroxyphenylretinamide structure–activity relation-
ship, it is encouraging that other analogs of 4-HPR
(notably 24, 13, and perhaps 28) can be prepared which
retain significant mammary tumor cell growth inhibitory

S. M. Mershon et al. / Bioorg. Med. Chem. Lett. 17 (2007) 836–840
839
Table 2. Naphthylamine amide analogs
5
H
Ret
N
OH
2
1
Compound
Position
% Yield
Growth inhibitiona
Ret NH
OH
43
44
45
46
1
2
4
5
3
37
26
43
88
ꢀ
ꢀ
ꢀ
ꢀ
1
1
2
aAs in Table 1.
OH
O
N
CH3
47 (+)
and apoptotic activity. It is notable that only several of
the analogs prepared have any significant activity in
inhibiting cell growth, demonstrating that the effect of
these analogs must be specific, and not simply due to a
non-specific cytotoxicity of this general class of agents.
180
160
140
120
100
80
In an effort to enhance activity and explore greater
structural diversity, the solid phase-assisted amide syn-
thesis was used to prepare another series of retinamides
(Table 3). The principal strategy used to select these tar-
gets was the batchwise Topliss tree approach,22 which is
a manual Hansch approach designed to rapidly improve
the activity of lead compounds that contain monosubsti-
tuted aromatic rings (compound 48). In this method,
different tree ‘branches’ are followed based on the ana-
logs they are being compared to. The branch choice de-
pends on whether the analog has lower, equivalent, or
more activity than the preceding compound in the
branching scheme. Recommended substitutions are
based on a systematic variation of the physico-chemical
properties of the substituent and thus the molecule.
After establishment of structure by the methods
60
40
20
0
3
9
13 17 18 24 25 28 34 39 40
4MPR
Retinamide (1x10-5 M)
Figure 2. TUNEL staining in MCF-7 cells 48 h after exposure to
10ꢀ5 M of compound. The results shown represent mean values
(+SEM) from three independent cell culture experiments (intra-assay
variability); samples with the highest proportion of dead cells always
showed the greatest assay variability (see supplementary materials).
5
4
3
2
1
0
Table 3. Topliss tree developed anilinamides
R4
O
N
H
R3
Compound
R3
R4
% yield
Growth inhibitiona
1b
48
H
H
H
H
H
Cl
H
OH
94
27
30
37
45
39
10
++
+
H
Cl
-7
-6 -5
-7
-6 -5
#24
49
+
50b
51
OCH3
CH3
Cl
+
V
4-HPR
Log Concentration (M)
ꢀ
+
52
53
Figure 3. The number of live MCF-7 cells remaining after 8 days of
treatment with either compound 24 or 1 is similar over a range of
concentrations (10ꢀ7 to 10ꢀ5 M).
N(CH3)3
ꢀ
a,bAs in Table 1.

840
S. M. Mershon et al. / Bioorg. Med. Chem. Lett. 17 (2007) 836–840
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H2O) showed product purity of 91–99% (mean =
97 3%) except for analog 53 (86%). Growth inhibition
assessment in MCF-7 cells as above gave the results
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Acknowledgment
Financial support in the form of a Grant (CA 49837)
from the National Cancer Institute is gratefully
acknowledged.
Supplementary data
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Supplementary data associated with this article can be
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