Diarylamines incorporating hexahydrophenalene or octahydrobenzoheptalene as Retinoid X Receptor (RXR)-specific agonists

Article abstract of DOI:10.1248/cpb.c13-00740

Selective ligands for retinoic acid receptors (RARs) and for retinoid X receptors (RXRs) are required for both biological studies and therapeutic purposes. We have synthesized a series of diarylamines incorporating hexahydrophenalene or octahydrobenzoheptalene as a hydrophobic moiety and examined their activities towards RARs and RXRs. Most of these compounds showed agonistic activity towards RXRs, but were inactive towards RARs. These RXR-specific ligands showed synergistic activity in RARα,β ligand-induced terminal differentiation of leukemia cell line HL-60.

Full text of DOI:10.1248/cpb.c13-00740

254
Regular Article
Chem. Pharm. Bull. 62(3) 254–259 (2014)
Vol. 62, No. 3
Diarylamines Incorporating Hexahydrophenalene or
Octahydrobenzoheptalene as Retinoid X Receptor (RXR)-Specific
Agonists
Yohei Amano,* Masayuki Noguchi, and Koichi Shudo
Research Foundation ITSUU Laboratory; 2–28–10 Tamagawa, Setagaya-ku, Tokyo 158–0094, Japan.
Received September 20, 2013; accepted December 21, 2013
Selective ligands for retinoic acid receptors (RARs) and for retinoid X receptors (RXRs) are required
for both biological studies and therapeutic purposes. We have synthesized a series of diarylamines incorpo-
rating hexahydrophenalene or octahydrobenzoheptalene as a hydrophobic moiety and examined their activi-
ties towards RARs and RXRs. Most of these compounds showed agonistic activity towards RXRs, but were
inactive towards RARs. These RXR-specific ligands showed synergistic activity in RARα,β ligand-induced
terminal differentiation of leukemia cell line HL-60.
Key words retinoid; retinoic acid receptor; retinoid X receptor (RXR); RXR-specific ligand
Retinoids are natural and synthetic analogues of all-trans- synthesized as follows. After coupling reaction of tricyclic
retinoic acid (ATRA), an active metabolite of vitamin A, and amine and ethyl 4-iodobenzoate catalyzed by Pd(dba)3 in the
they modulate and regulate various biological functions, in- presence of ( )-2,2′-bis(diphenylphosphino)-1,1′-binaphthyl
cluding cell differentiation, proliferation, and morphogenesis, (BINAP), N-alkylation followed by ester hydrolysis afforded
by modulating gene transcription mediated by intranuclear diphenylamine compounds 1a–d, 2a–d, and 3a–f (Fig. 2). On
retinoic acid receptors (RAR α, β, and γ) and retinoid X re- the other hand, after treatment of tricyclic amine with ethyl
ceptors (RXRα, β, and γ).1) The endogenous ligands of RARs 2-chloropyrimidine-5-carboxylate in the presence of potas-
and RXRs have been identified as ATRA and 9-cis-retinoic sium carbonate, N-alkylation followed by ester hydrolysis af-
acid (9cRA), respectively2,3) (Fig. 1), though their specificity forded (N-arylamino)pyrimidine compounds 1g–j, 2g–j, and
is not high. It is believed that the activation of RXRs alone 3g–j.
does not cause any specific transactivation; rather RXRs act
The activities of compounds 1a–3j were evaluated by
as heterodimers with RARs, peroxisome proliferator-activated means of reporter assay utilizing COS-1 cells transfected with
receptors (PPARs), liver X receptor (LXR) and other nuclear expression vectors containing GAL4-RARα, β, or γ and full-
receptors.4,5) RXR-specific agonists alone cannot activate length RXRα, β, or γ. N-Unsubstituted compounds (e.g. 1a, 2a,
RXR-RAR heterodimers, but act as retinoid synergists that 3a) and some N-methyl compounds (e.g. 1b, 2b, 3b) showed
dose-dependently and robustly enhance the potency of RAR moderate agonistic activity towards RARα and β, but most of
agonists. Thus, selective ligands specific for RARs and spe- the N-alkylated compounds had no activity towards RARs.
cific for RXRs are required for both biological studies and On the other hand, most of the compounds showed agonistic
therapeutic purposes. Recently we reported several RARα activity towards RXRs, and in particular, N-ethyl- and N-
and RARβ-specific ligands, i.e., hexahydrophenalenyl- and cyclopropylmethyl-pyrimidine-5-carboxylic acid compounds
octahydrobenzo[ef]heptalenyl-carbamoyl- or -carboxamido- (e.g. 1j, 2i, 2j, 3i, 3j) showed potent and selective activity
4-benzoic acids, that do not activate RARγ or RXRs.6) Here towards RXRα (Table 1).
we report a series of diarylamines incorporating hexahydro-
phenalene and octahydrobenzoheptalene that show RXR- tivators in the cell-based assay. Substitution on the nitrogen
specific agonist activity.
atom with a medium-sized alkyl group (more than two carbon
Several of the compounds prepared were potent transac-
An RXR ligand, LGD1069 (bexarotene), has been suggested atoms) is necessary for significant RXR activity. When the
to have a therapeutic effect in a mouse model of Alzheimer’s substituent is a hydrogen or methyl group (1a,b, 2a,b, 3a,b),
disease (AD),7) and our unpublished results also indicate that RXR activity is weak, but at the same time significant RAR
RXR ligands enhance the therapeutic effect of RAR ligands. activity was observed. These latter compounds are similar to
Indeed, RXR ligands together with RAR ligands have poten- LGD1069, which is generally accepted to be an RXR ligand
tial therapeutic value in a variety of immunological and neu- with weak RAR activity. The more potent compounds bearing
rodegenerative disorders.8–10)
a pyrimidinylcarboxylic acid moiety showed similar activity to
Many synthetic RXR-selective agonists have been re- PA024, which also bears this structure. Introduction of tricy-
ported,4,5) and diarylamine-type ligands, such as PA024, show
very potent activity.11) Therefore, building on the structures
of our previously developed potent and subtype-selective
RAR agonists, in the present work we synthesized a series
of diarylamine ligands incorporating a tricyclic hydrophobic
moiety, i.e., hexahydrophenalene or octahydrobenzoheptalene,
as candidate RXR agonists. The target compounds were
The authors declare no conflict of interest.
Fig. 1. The Structures of Endogenous RAR and RXR Ligands
© 2014 The Pharmaceutical Society of Japan
*To whom correspondence should be addressed. e-mail: yamano@itsuu.or.jp

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255
Fig. 2. The Structures of Diarylamine Derivatives
Table 1. Bioactivities of Diarylamine Derivatives
Transcriptional activation assay datab)
RXRα RXRβ
1000nM
HL-60 Differentiationc)
Compounda)
RXRγ
1000nM
RARα
1000nM
RARβ
1000nM
RARγ
1000nM
With Am80
Alone
1nM
100nM
100nM
1000nM
100nM
Blank
9cRA
1
1
1
1
10d)
2
1
1
4d)
1
1
1
5d)
6
1
1
16
71d)
63d)
73d)
20d)
13d)
10d)
N.D.e)
N.D.e)
HX630
PA024
N.D.e)
N.D.e)
N.D.e)
N.D.e)
N.D.e)
N.D.e)
35
26
31
2
5d)
73
53
54
38
38
35
35
86
1a
1b
1c
1d
1g
1h
1i
31
3
17
4
2
1
1
1
1
1
1
1
4
1
1
1
2
1
1
1
4
2
1
1
1
1
2
1
1
1
1d)
1d)
2
11
1d)
1d)
1
3
2d)
1d)
12d)
21d)
41
5
N.D.e)
N.D.e)
26
42d)
8d)
N.D.e)
1
2
60
43
2
1
1
2
25
57
2d)
7
41
15
37
2d)
17d)
30d)
36d)
6d)
3
77
2
5
1d)
1d)
9
1d)
1d)
2d)
5d)
1d)
1d)
3d)
4
1d)
1d)
29d)
31d)
2
1d)
N.D.e)
N.D.e)
N.D.e)
3
N.D.e)
N.D.e)
N.D.e)
53
1
1
42
1d)
1
1
53
6d)
1j
1
1
39
58
16d)
2d)
2a
2b
2c
2d
2g
2h
2i
31
9
22
11
4
1
4
42d)
10
N.D.e)
N.D.e)
N.D.e)
N.D.e)
N.D.e)
N.D.e)
56
1d)
33d)
32
7d)
2d)
22d)
19d)
39d)
38
N.D.e)
N.D.e)
N.D.e)
N.D.e)
N.D.e)
3
2
64
33
2
4
57
37
26
8
12
2
1d)
3d)
35
2d)
52d)
52
1d)
1d)
5d)
13d)
1d)
1d)
7
1d)
9d)
31d)
30d)
4
1d)
1d)
1
3d)
25d)
30d)
30d)
10d)
22d)
33
1
2
17d)
25d)
2d)
2j
1
2
47
52
5
76
3a
3b
3c
3d
3e
3f
22
5
15
9
N.D.e)
N.D.e)
32
7d)
13d)
50
N.D.e)
N.D.e)
2
N.D.e)
N.D.e)
85
7
3d)
1
3
38
19
2
3
26
51
6
34
18
33
2
88
2
3
21
44
5d)
1d)
1d)
3d)
14d)
6d)
27d)
2d)
8d)
22d)
32d)
27d)
15d)
1d)
26d)
9d)
N.D.e)
N.D.e)
N.D.e)
N.D.e)
2
N.D.e)
N.D.e)
N.D.e)
N.D.e)
91
2
3
1
11
3g
3h
3i
10
2
10
3
3d)
22d)
47
39d)
54d)
51
3d)
17d)
29d)
27d)
29d)
13d)
22d)
18d)
1
2
3j
1
2
42
51
4
87
a) Structures of 1a–3j are shown in Fig. 2. b) Data are mean values of fold induction (vs. DMSO) at 100 or 1000nM measured by two or more experiments. c) Data are mean
values of the percentage of differentiated cells evaluated from NBT reduction assay at 100nM. d) One experiment. e) Not determined.

256
Vol. 62, No. 3
clic hydrocarbon (hexahydrophenalene or octahydrobenzohep- submitted).
talene) instead of bicyclic hydrocarbon (tetramethyltetraline)
In conclusion, building on the structures of our previously
as a hydrophobic moiety seems to result in slight selectivity reported potent synthetic retinoids, we synthesized a series of
for RXRα (1j, 2j, 3j vs. PA024). Although it is not clear how diarylamines incorporating hexahydrophenalene or octahydro-
the RXR subtype-selectivity arises, it appears that structural benzoheptalene as a bulky hydrophobic moiety. Most of these
extension of tricyclic hydrocarbon may have the potential to compounds showed agonistic activity towards RXRs, but were
increase selectivity for RXR subtypes. This is of interest, inactive towards RARs. Thus, these RXR ligands act as reti-
because few RXR subtype-selective ligands have been devel- noid synergists. Some of them, such as 2i,j, and 3i,j (Fig. 4),
oped.4,5) These compounds do not show any activity towards are considered to be potential biological tools and candidate
RARs even at concentrations above 1000nM, and are thus therapeutic drugs, even though they are somewhat less potent
activators of RXRs. Dose–response curves of representative than the strongest known RXR ligand, PA024. They are more
compounds are shown in Fig. 3.
potent than the most widely used RXR ligand, HX630, and
These RXR ligands have a synergistic effect on RAR more RXR-selective than LGD1069 and LGD100268.
ligand-induced terminal differentiation of leukemia cell line
HL-60.11,12) The N-unsubstituted compounds showed weak
Experimental
cell-differentiating activity on their own, i.e., they act as weak
General Melting points were determined on a Yanagi-
RAR agonists. However, the N-substituted compounds them- moto micro-melting point apparatus (hot plate), and are not
1
selves showed no RAR agonist activity, but in the presence corrected. H-NMR spectra were obtained on a Varian Mer-
of a low concentration (1nM) of RARα, β ligand Am80, these cury 300 at 300MHz in CDCl3 or in DMSO-d6. The chemical
new RXR ligands showed synergistic differentiation-inducing shifts are calculated on the basis of tetramethylsilane (0ppm
1
activity in this system. This is consistent with our recent for H-NMR in CDCl3) or (CH3)2SO (2.49ppm in DMSO-d6).
finding that the RXR ligand HX630 potently enhances RAR High resolution-electrospray ionization-mass spectra (HR-ESI-
ligand activities in Alzheimer’s model animals (manuscript MS) was recorded on a Shimadzu LCMS-IT-TOF spectrom-
Fig. 3. Transcriptional Activities Measured by Means of Reporter Gene Assay in Cos-1 Cells with Full Length RXRα (■), RXRβ (●), and RXRγ
(▲)
Data are mean values of fold increase (vs. DMSO). The horizontal scale is the molar concentration of added compound.

March 2014
257
Fig. 4. The Structures of Synthetic RXR Ligands (2i, 2j, 3i, and 3j)
eter. Column chromatography was conducted on silica gel rt for 30min. MeI (0.020mL, 0.321mmol) was added to the
(Fuji Davison BW 200). Usual work-up refers to washing of whole mixture and the reaction mixture was stirred overnight
the organic phase with water or brine, drying over anhydrous at ambient temperature. The reaction mixture was poured
Na2SO4, and removal of the solvent(s) by evaporation under into saturated aqueous NH4Cl and extracted with Et2O. Usual
reduced pressure.
4-[N-(5,6,6a,7,8,9-Hexahydro-4H-2-phenalenyl)amino]- column chromatography (AcOEt–n-hexane=1:80) to afford
benzoic Acid (1a) To solution of 5,6,6a,7,8,9-hexa- ethyl 4-[N-methyl-(5,6,6a,7,8,9-hexahydro-4H-2-phenalenyl)-
work-up gave a residue, which was purified by silica gel
a
hydro-4H-2-phenalenylamine6) (0.250g, 1.33mmol) and ethyl amino]benzoate (0.033g, quant.). To a suspension of the above
4-iodobenzoate (0.360g, 1.30mmol) in benzene (5mL) was ester (0.033g, 0.0976mmol) in EtOH (10mL) was added 20%
added Pd2(dba)3 (0.024g, 0.0262mmol), ( )-BINAP (0.036g, aqueous NaOH (1mL) and the mixture was stirred overnight
0.0578mmol), and Cs2CO3 (0.455g, 1.40mmol), and the mix- at 50°C. The mixture was acidified by adding 2M aqueous
ture was stirred overnight at 80°C under an Ar atmosphere. HCl and extracted with CHCl3. Usual work-up gave a residue,
After cooling, the mixture was acidified by adding 2M aque- which was purified by recrystallization from EtOH–CHCl3
ous HCl and extracted with Et2O. Usual work-up gave a resi- to afford 1b (0.027g, 87%). 1b: Colorless needles (EtOH–
1
due, which was purified by silica gel column chromatography CHCl3); mp 288–289°C; H-NMR (CDCl3) δ: 1.29–1.42 (2H,
(AcOEt–n-hexane=1:50) to afford ethyl 4-[N-(5,6,6a,7,8,9- m), 1.72–1.86 (2H, m), 1.88–2.01 (4H, m), 2.54–2.62 (1H, m),
hexahydro-4H-2-phenalenyl)amino]benzoate (0.160g, 36%). 2.75–2.80 (4H, m), 3.31 (3H, s), 6.72 (2H, d, J=9.0Hz), 6.75
To a suspension of the above ester (0.154g, 0.459mmol) in (2H, s), 7.85 (2H, d, J=9.0Hz); HR-MS Calcd for C21H24NO2
EtOH (12mL) was added 20% aqueous NaOH (3mL) and ([M+H]+): 322.1807. Found: 322.1805.
the mixture was stirred overnight at rt. The reaction mix-
Other N-alkyl-diphenylamine compounds (1c,d, 2b–d,
ture was acidified by adding 2M aqueous HCl and extracted 3b–f) were synthesized similarly.
with CHCl3. Usual work-up gave a residue, which was puri-
4-[N-Ethyl-(5,6,6a,7,8,9-hexahydro-4H-2-phenalenyl)amino]-
fied by recrystallization from AcOEt–n-hexane to give 1a benzoic Acid (1c): Colorless needles (EtOH); mp 284–285°C;
(0.125g, 89%). 1a: Pale brown needles (AcOEt–n-hexane); mp 1H-NMR (CDCl3) δ: 1.25 (3H, t, J=7.2Hz), 1.29–1.43 (2H,
1
204–205°C; H-NMR (CDCl3) δ: 1.26–1.40 (2H, m), 1.72–1.86 m), 1.76–1.86 (2H, m), 1.88–2.00 (4H, m), 2.53–2.64 (1H,
(2H, m), 1.88–2.00 (4H, m), 2.50–2.60 (1H, m), 2.77–2.81 (4H, m), 2.77–2.81 (4H, m), 3.75 (2H, q, J=7.2Hz), 6.65 (2H, d,
m), 5.93 (1H, brs), 6.76 (2H, s), 6.94 (2H, d, J=8.7Hz), 7.95 J=8.7Hz), 6.73 (2H, s), 7.85 (2H, d, J=8.7Hz); HR-MS Calcd
(2H, d, J=8.7Hz) ; HR-MS Calcd for C20H21NO2 ([M+H]+): for C22H26NO2 ([M+H]+): 336.1964. Found: 336.1941.
308.1651. Found: 308.1651.
4-[N-Cyclopropylmethyl-(5,6,6a,7,8,9-hexahydro-4H-2-
Other diphenylamine compounds (2a, 3a) were synthesized phenalenyl)amino]benzoic Acid (1d): Colorless needles
1
similarly.
(CHCl3–n-hexane); mp 214–216°C; H-NMR (CDCl3) δ: 0.17
4-[N-(6a-Methyl-5,6,6a,7,8,9-hexahydro-4H-2-phenalenyl)- (2H, q, J=5.7Hz), 0.48–0.54 (2H, m), 1.14–1.22 (1H, m),
amino]benzoic Acid (2a): Pale brown needles (AcOEt–n- 1.29–1.43 (2H, m), 1.75–1.86 (2H, m), 1.89–2.00 (4H, m),
hexane); mp 232–234°C; 1H-NMR (CDCl3) δ: 1.18 (3H, s), 2.53–2.62 (1H, m), 2.77–2.80 (4H, m), 3.55 (2H, d, J=6.3Hz),
1.51 (2H, td, J=12.9, 4.8Hz), 1.69 (2H, dt, J=12.9, 4.1Hz), 6.68 (2H, d, J=8.7Hz), 6.75 (2H, s), 7.86 (2H, d, J=8.7Hz);
1.76–1.87 (2H, m), 1.99–2.18 (2H, m), 2.76 (2H, dt, J=17.3, HR-MS Calcd for C24H28NO2 ([M+H]+): 362.2120. Found:
8.7Hz), 2.89 (2H, ddd, J=17.3, 7.9, 3.3Hz), 5.91 (1H, brs), 362.2111.
6.74 (2H, s), 6.94 (2H, d, J=8.6Hz), 7.94 (2H, d, J=8.6Hz);
HR-MS Calcd for C21H23NO2 ([M+H]+): 322.1807. Found: phenalenyl)amino]benzoic Acid (2b): Colorless needles
322.1808.
(EtOH); mp 257–258°C; 1H-NMR (DMSO-d6) δ: 1.11 (3H,
4-[N-Methyl-(6a-methyl-5,6,6a,7,8,9-hexahydro-4H-2-
4-[N-(5,6,7,7a,8,9,10,11-Octahydro-4H-2-benzo[ef ]- s), 1.40 (2H, td, J=12.9, 4.8Hz), 1.59–1.66 (2H, m), 1.68–1.78
heptalenyl)amino]benzoic Acid (3a): Brown needles (AcOEt–n- (2H, m), 1.89–2.04 (2H, m), 2.68 (2H, dt, J=17.4, 8.7Hz),
1
hexane); mp 203–204°C; H-NMR (CDCl3) δ: 1.48–1.67 (6H, 2.82 (2H, ddd, J=17.4, 8.1, 3.3Hz), 3.21 (3H, s), 6.68 (2H, d,
m), 1.80–1.96 (6H, m), 2.74–2.87 (4H, m), 3.18–3.25 (1H, J=8.7Hz), 6.70 (2H, s), 7.69 (2H, d, J=8.7Hz); HR-MS Calcd
m), 5.92 (1H, brs), 6.78 (2H, s), 6.95 (2H, d, J=8.7Hz), 7.95 for C22H26NO2 ([M+H]+): 336.1964. Found: 336.1959.
(2H, d, J=8.7Hz); HR-MS Calcd for C22H26NO2 ([M+H]+):
336.1964. Found: 336.1965.
4-[N-Ethyl-(6a-methyl-5,6,6a,7,8,9-hexahydro-4H-2-
phenalenyl)amino]benzoic Acid (2c): Colorless needles
4-[N-Methyl-(5,6,6a,7,8,9-hexahydro-4H-2-phenalenyl)- (EtOH); mp 279–280°C; 1H-NMR (CDCl3) δ: 1.20 (3H, s),
amino]benzoic Acid (1b) To a suspension of NaH (60%, 1.24 (3H, t, J=6.9Hz), 1.48–1.58 (2H, m), 1.66–1.73 (2H, m),
0.012g, 0.300mmol) in N,N-dimethylformamide (DMF) 1.76–1.88 (2H, m), 1.99–2.16 (2H, m), 2.76 (2H, dt, J=17.1,
(3mL) was added a solution of ethyl 4-[N-(5,6,6a,7,8,9- 8.7Hz), 2.88 (2H, ddd, J=17.1, 8.1, 3.3Hz), 3.74 (2H, q,
hexahydro-4H-2-phenalenyl)amino]benzoate (0.030g, 0.0976 J=6.9Hz), 6.64 (2H, d, J=8.7Hz), 6.70 (2H, s), 7.86 (2H, d,
mmol) in DMF (2mL) and the whole mixture was stirred at J=8.7Hz); HR-MS Calcd for C23H28NO2 ([M+H]+): 350.2120.

258
Vol. 62, No. 3
Found: 350.2111.
tion was acidified by adding 2M aqueous HCl and extracted
4-[N-Cyclopropylmethyl-(6a-methyl-5,6,6a,7,8,9- with CHCl3. Usual work-up gave a residue, which was purified
hexahydro-4H-2-phenalenyl)amino]benzoic Acid (2d): Color- by recrystallization from EtOH–CHCl3 to give 1g (0.045 g,
less needles (CHCl3–n-hexane); mp 234–235°C; 1H-NMR 90%). 1g: Colorless needles (EtOH–CHCl3); mp 296–298°C;
(CDCl3) δ: 0.16 (2H, q, J=4.8Hz), 0.47–0.53 (2H, m), 1.12–1.17 1H-NMR (DMSO-d6) δ: 1.14–1.23 (2H, m), 1.63–1.71 (2H,
(1H, m), 1.19 (3H, s), 1.48–1.58 (2H, m), 1.67–1.72 (2H, m), m), 1.77–1.90 (4H, m), 2.22–2.28 (1H, m), 2.65–2.70 (4H,
1.76–1.87 (2H, m), 1.99–2.16 (2H, m), 2.75 (2H, dt, J=17.4, m), 7.17 (2H, s), 8.66 (2H, s), 9.75 (1H, s); HR-MS Calcd for
8.7Hz), 2.83–2.92 (2H, m), 3.54 (2H, d, J=6.3Hz), 6.67 (2H, C18H20N3O2 ([M+H]+): 310.1556. Found: 310.1538.
d, J=8.7Hz), 6.73 (2H, s), 7.86 (2H, d, J=8.7Hz); HR-MS
Calcd for C25H30NO2 ([M+H]+): 376.2277. Found: 376.2281.
4-[N-Methyl-(5,6,7,7a,8,9,10,11-octahydro-4H-2-benzo[ef]-
Other pyrimidinecarboxylic acid compounds (2g, 3g) were
synthesized similarly.
2-[N-(6a-Methyl-5,6,6a,7,8,9-hexahydro-4H-2-phenalenyl)-
heptalenyl)amino]benzoic Acid (3b): Colorless needles amino]pyrimidine-5-carboxylic Acid (2g): Colorless needles
(AcOEt–n-hexane); mp 244–245°C; 1H-NMR (CDCl3) δ: (EtOH–CHCl3); mp >300°C; 1H-NMR (DMSO-d6) δ: 1.06
1.53–1.71 (6H, m), 1.81–1.97 (6H, m), 2.73–2.89 (4H, m), (3H, s), 1.30–1.40 (2H, m), 1.56–1.63 (2H, m), 1.66–1.75 (2H,
3.20–3.28 (1H, m), 3.34 (3H, s), 6.73 (2H, d, J=8.7Hz), 6.78 m), 1.91–1.98 (2H, m), 2.62–2.71 (2H, m), 2.74–2.78 (2H,
(2H, s), 7.90 (2H, d, J=8.7Hz); HR-MS Calcd for C23H28NO2 m), 7.13 (2H, s), 8.76 (2H, s), 9.78 (1H, s); HR-MS Calcd for
([M+H]+): 350.2120. Found: 350.2139.
4-[N-Ethyl-(5,6,7,7a,8,9,10,11-octahydro-4H-2-benzo[ef ]-
C19H22N3O2 ([M+H]+): 324.1712. Found: 324.1698.
2-[N-(5,6,7,7a,8,9,10,11-Octahydro-4H-2-benzo[ef ]-
heptalenyl)amino]benzoic Acid (3c): Colorless needles heptalenyl)amino]pyrimidine-5-carboxylic Acid (3g): Colorless
(AcOEt–n-hexane); mp 256.5–257.5°C; 1H-NMR (CDCl3) δ: needles (EtOH–CHCl3); mp >300°C; 1H-NMR (DMSO-d6)
1.25 (3H, t, J=7.2Hz), 1.52–1.72 (6H, m), 1.81–1.97 (6H, m), δ: 1.38–1.55 (6H, m), 1.71–1.88 (6H, m), 2.66–2.81 (4H, m),
2.73–2.89 (4H, m), 3.21–3.28 (1H, m), 3.76 (2H, q, J=7.2Hz), 3.11–3.20 (1H, m), 7.26 (2H, s), 8.79 (2H, s), 9.85 (1H, s);
6.65 (2H, d, J=8.7Hz), 6.75 (2H, s), 7.87 (2H, d, J=8.7Hz); HR-MS Calcd for C20H24N3O2 ([M+H]+): 338.1869. Found:
HR-MS Calcd for C24H30NO2 ([M+H]+): 364.2277. Found: 338.1870.
364.2289.
2-[N-Methyl-(5,6,6a,7,8,9-hexahydro-4H-2-phenalenyl)-
4-[N-Cyclopropylmethyl-(5,6,7,7a,8,9,10,11-octahydro-4H-2- amino]pyrimidine-5-carboxylic Acid (1h) To a suspen-
benzo[ef]heptalenyl)amino]benzoic Acid (3d): Colorless nee- sion of NaH (60%, 0.008g, 0.200mmol) in DMF (2mL) was
1
dles (AcOEt–n-hexane); mp 222–223°C; H-NMR (CDCl3) δ: added a solution of ethyl 2-[N-(5,6,6a,7,8,9-hexahydro-4H-2-
0.15 (2H, dd, J=10.8, 5.1Hz), 0.47–0.53 (2H, m), 1.13–1.22 phenalenyl)amino]pyrimidine-5-carboxylate (0.032g, 0.0948
(1H, m), 1.52–1.71 (6H, m), 1.80–1.97 (6H, m), 2.73–2.88 (4H, mmol) in DMF (1mL) and the mixture was stirred for 10min
m), 3.21–3.28 (1H, m), 3.56 (2H, d, J=6.3Hz), 6.69 (2H, d, at rt. MeI (0.015mL, 0.241mmol) was added, and the reac-
J=9.0Hz), 6.78 (2H, s), 7.88 (2H, d, J=9.0Hz); HR-MS Calcd tion mixture was stirred overnight at ambient temperature.
for C26H32NO2 ([M+H]+): 390.2433. Found: 390.2435.
The mixture was poured into saturated aqueous NH4Cl and
4-[N-Isobutyl-(5,6,7,7a,8,9,10,11-octahydro-4H-2-benzo[ef]- extracted with Et2O. Usual work-up gave a residue, which was
heptalenyl)amino]benzoic Acid (3e): Colorless needles suspended in EtOH (5mL). To this suspension was added 20%
(EtOH); mp 250–251°C; 1H-NMR (CDCl3) δ: 0.96 (3H, s), aqueous NaOH (1mL) and the mixture was stirred at 50°C for
0.98 (3H, s), 1.53–1.69 (6H, m), 1.80–1.98 (6H, m), 2.03–2.12 3h, then acidified by adding 2M aqueous HCl and extracted
(1H, m), 2.72–2.87 (4H, m), 3.20–3.28 (1H, m), 3.51 (2H, d, with CHCl3. Usual work-up gave a residue, which was puri-
J=7.5Hz), 6.67 (2H, d, J=9.3Hz), 6.74 (2H, s), 7.85 (2H, d, fied by recrystallization from EtOH to afford 1h (0.027g,
1
J=9.3Hz); HR-MS Calcd for C26H34NO2 ([M+H]+): 392.2590. 87%). 1h: Colorless needles (EtOH); mp 255–256°C; H-NMR
Found: 392.2586.
(CDCl3) δ: 1.34–1.43 (2H, m), 1.72–1.86 (2H, m), 1.88–1.95
4-[N-Benzyl-(5,6,7,7a,8,9,10,11-octahydro-4H-2-benzo[ef]- (4H, m), 2.53–2.62 (1H, m), 2.80–2.84 (4H, m), 3.55 (3H, s),
heptalenyl)amino]benzoic Acid (3f): Colorless needles (EtOH); 6.81 (2H, s), 8.91 (2H, s); HR-MS Calcd for C19H22N3O2 ([M+
mp 228–229°C; 1H-NMR (CDCl3) δ: 1.51–1.67 (6H, m), H]+): 324.1712. Found: 324.1688.
1.79–1.93 (6H, m), 2.71–2.87 (4H, m), 3.19–3.26 (1H, m), 4.99
Other N-alkyl-pyrimidinecarboxylic acid compounds (1i,j,
(2H, s), 6.71 (2H, d, J=9.3Hz), 6.86 (2H, s), 7.23–7.33 (5H, m), 2h–j, 3h–j) were synthesized similarly.
7.82 (2H, d, J=9.3Hz); HR-MS Calcd for C29H32NO2 ([M+
2-[N-Ethyl-(5,6,6a,7,8,9-hexahydro-4H-2-phenalenyl)-
amino]pyrimidine-5-carboxylic Acid (1i): Colorless needles
H]+): 426.2433. Found: 426.2432.
2-[N-(5,6,6a,7,8,9-Hexahydro-4H-2-phenalenyl)- (CHCl3–n-hexane); mp 252–253°C; 1H-NMR (CDCl3) δ:
amino]pyrimidine-5-carboxylic Acid (1g) A mixture of 1.26 (3H, t, J=7.2Hz), 1.34–1.43 (2H, m), 1.75–1.85 (2H, m),
5,6,6a,7,8,9-hexahydro-4H-2-phenalenylamine (0.100g, 0.531 1.89–1.98 (4H, m), 2.54–2.63 (1H, m), 2.80–2.84 (4H, m), 4.02
mmol), ethyl 2-chloropyrimidine-5-carboxylate (0.101g, 0.541 (2H, q, J=7.2Hz), 6.76 (2H, s), 8.89 (2H, s); HR-MS Calcd for
mmol), and K2CO3 (0.400g) was heated overnight at 110°C. C20H24N3O2 ([M+H]+): 338.1869. Found: 338.1856.
After cooling, the reaction mixture was poured into water,
2-[N-Cyclopropylmethyl-(5,6,6a,7,8,9-hexahydro-4H-2-
and extracted with CHCl3. Usual work-up gave a residue, phenalenyl)amino]pyrimidine-5-carboxylic Acid (1j): Pale
which was purified by silica gel column chromatography yellow amorphous solid; 1H-NMR (CDCl3) δ: 0.25 (2H,
(AcOEt–n-hexane=1:20) to afford ethyl 2-[N-(5,6,6a,7,8,9- dd, J=10.8, 4.8Hz), 0.44–0.50 (2H, m), 1.15–1.23 (1H, m),
hexahydro-4H-2-phenalenyl)amino]pyrimidine-5-carboxylate 1.30–1.43 (2H, m), 1.75–1.85 (2H, m), 1.89–1.98 (4H, m),
(0.167g, 93%). To a suspension of the above ester (0.055g, 2.54–2.62 (1H, m), 2.82 (4H, brdd, J=8.1, 4.5Hz), 3.86 (2H,
0.163mmol) in EtOH (5mL) was added 20% aqueous NaOH d, J=7.2Hz), 6.80 (2H, s), 8.79 (2H, s); HR-MS Calcd for
(1mL) and the mixture was stirred at 50°C for 3h. The reac- C22H25N3O2 ([M+H]+): 364.2025. Found: 364.2009.

March 2014
259
2-[N-Methyl-(6a-methyl-5,6,6a,7,8,9-hexahydro-4H-2- benzo[ef]heptalenyl)amino]pyrimidine-5-carboxylic Acid (3j):
phenalenyl)amino]pyrimidine-5-carboxylic Acid (2h): Color- Colorless needles (n-hexane); mp 174–176°C; 1H-NMR
1
less needles (EtOH); mp >300°C; H-NMR (CDCl3) δ: 1.19 (CDCl3) δ: 0.22 (2H, dd, J=10.8, 4.5Hz), 0.43–0.50 (2H,
(3H, s), 1.52 (2H, td, J=12.9, 5.4Hz), 1.67 (2H, m), 1.77–1.86 m), 1.14–1.20 (1H, m), 1.57–1.68 (6H, m), 1.85–1.97 (6H, m),
(2H, m), 1.99–2.15 (2H, m), 2.74–2.86 (2H, m), 2.91 (2H, ddd, 2.75–2.91 (4H, m), 3.21–3.29 (1H, m), 3.87 (2H, d, J=6.9Hz),
J=17.1, 7.8, 3.3Hz), 3.54 (3H, s), 6.80 (2H, s), 8.91 (2H, s); 6.83 (2H, s), 8.88 (2H, s); HR-MS Calcd for C24H30N3O2 ([M+
HR-MS Calcd for C20H24N3O2 ([M+H]+): 338.1869. Found: H]+): 392.2338. Found: 392.2325.
338.1845.
2-[N-Ethyl-(6a-methyl-5,6,6a,7,8,9-hexahydro-4H-2-
Acknowledgments The authors thank Dr. M. Kurihara
phenalenyl)amino]pyrimidine-5-carboxylic Acid (2i): Colorless and Dr. T. Shoda, Division of Organic Chemistry, National
1
needles (CHCl3–n-hexane); mp 257–258°C; H-NMR (CDCl3) Institute of Health Sciences, for HR-ESI-MS measurements.
δ: 1.19 (3H, s), 1.24 (3H, t, J=7.2Hz), 1.53 (2H, td, J=12.9,
4.8Hz), 1.63–1.70 (2H, m), 1.77–1.86 (2H, m), 2.01–2.10
(2H, m), 2.74–2.83 (2H, m), 2.86–2.95 (2H, m), 4.01 (2H,
q, J=7.2Hz), 6.73 (2H, s), 8.89 (2H, s); HR-MS Calcd for
C21H26N3O2 ([M+H]+): 352.2025. Found: 352.2008.
2-[N-Cyclopropylmethyl-(6a-methyl-5,6,6a,7,8,9-
hexahydro-4H-2-phenalenyl)amino]pyrimidine-5-carboxylic
Acid (2j): Colorless needles (EtOH–n-hexane); mp 123–124°C;
1H-NMR (CDCl3) δ: 0.20–0.25 (2H, m), 0.43–0.49 (2H, m),
0.95–0.98 (1H, m), 1.18 (3H, s), 1.48–1.58 (2H, m), 1.63 (2H,
m), 1.74–1.85 (2H, m), 1.98–2.10 (2H, m), 2.74–2.82 (2H, m),
2.83–2.95 (2H, m), 3.85 (2H, d, J=6.9Hz), 6.77 (2H, s), 8.87
(2H, s); HRMS Calcd for C23H28N3O2 ([M+H]+): 378.2182.
Found: 378.2163.
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2-[N-Methyl-(5,6,7,7a,8,9,10,11-octahydro-4H-2-benzo[ef]-
heptalenyl)amino]pyrimidine-5-carboxylic Acid (3h): Colorless
1
needles (EtOH); mp >300°C; H-NMR (CDCl3) δ: 1.54–1.69
(6H, m), 1.82–1.98 (6H, m), 2.75–2.93 (4H, m), 3.20–3.28 (1H,
m), 3.56 (3H, s), 6.85 (2H, s), 8.91 (2H, s); HR-MS Calcd for
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1.26 (3H, t, J=7.2Hz), 1.54–1.69 (6H, m), 1.82–1.94 (6H, m),
2.75–2.92 (4H, m), 3.21–3.28 (1H, m), 4.12 (2H, q, J=7.2Hz),
6.79 (2H, s), 8.89 (2H, s); HR-MS Calcd for C22H28N3O2 ([M+
H]+): 366.2182. Found: 366.2160.
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