Molecular symmetry and biological activities of new symmetrical tris(2-aminoethyl)amine derivatives

Article abstract of DOI:10.1248/cpb.60.408

In terms of molecular symmetry and bioactivity, new C₃- and C_S-symmetrical derivatives based on the tris(2-aminoethyl)amine scaffold were designed and synthesized. The synthesized compounds were evaluated for antiviral activity with herpes simplex virus type 1 (HSV-1) by a plaque reduction assay and for cytotoxic activity with Vero cells. Most of the compounds showed no significant anti-HSV-1 activity, but some of the symmetrical derivatives showed high levels of cytotoxic activitiy.

Full text of DOI:10.1248/cpb.60.408

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408
Note
Chem. Pharm. Bull. 60(3) 408 414 (2012)
Vol. 60, No. 3
Molecular Symmetry and Biological Activities of New Symmetrical
Tris(2-aminoethyl)amine Derivatives
Nobuko Mibu,a Kazumi Yokomizo,b Wataru Uchida,a Satoshi Takemura,a Jianrong Zhou,b
,a
Hatsumi Aki,a Takeshi Miyata,b and Kunihiro Sumoto*
a Faculty of Pharmaceutical Sciences, Fukuoka University; 8–19–1 Nanakuma, Jonan-ku, Fukuoka 814–0180,
b
Japan: and Faculty of Pharmaceutical Sciences, Sojo University; 4–22–1 Ikeda, Kumamoto 862–0082, Japan.
Received October 18, 2011; accepted December 7, 2011
In terms of molecular symmetry and bioactivity, new C3- and CS-symmetrical derivatives based on the
tris(2-aminoethyl)amine scaffold were designed and synthesized. The synthesized compounds were evaluated
for antiviral activity with herpes simplex virus type 1 (HSV-1) by a plaque reduction assay and for cytotoxic
activity with Vero cells. Most of the compounds showed no significant anti-HSV-1 activity, but some of the
symmetrical derivatives showed high levels of cytotoxic activitiy.
Key words tris(2-aminoethyl)amine; tripodal; anti-herpes simplex virus type 1; C3 symmetry; CS symmetry;
plaque reduction assay
There have been many reports on the molecular recognition
properties of symmetrical molecules,1) and it is known that the
Results and Discussion
Synthesis The synthetic strategies for two classes of
symmetrical feature is frequently observed in many biological target symmetrical compounds are outlined in Chart 1. C3-
stages. Two-fold and three-fold symmetrical macromolecu- symmetrical compounds are considered to be easily obtained
lar structures are common features in specialized biological by the reaction of TREA and electrophiles. Therefore, we
—
4)
functions.2
Regarding the molecular symmetry, small sym- planned the synthesis of two types of molecules, which con-
metrical molecules with a C2-symmetrical or C3-symmetrical tain amide or ureido functionality, by acylation of TREA with
structure have often been found in various synthetic biologi- acylating reagents or addition of TREA to an isothiocyanate,
—
7)
cally active compounds.5
respectively. The results of the synthesis of C3-symmetrical
In the course of our work on new antiviral compounds, we compounds are shown in Chart 2 and Table 1. The results
have presented a new class of antiviral candidates. For exam- for CS-symmetrical compounds are also shown in Chart 3
ple, we have already reported that most of the triarylmethane and Table 2. Thus, C3-symmetrical compounds 6a18,19) and 6b
derivatives showed a wide range of antiviral activities against were easily obtained by the reactions of TREA with the cor-
herpes simplex virus type 1 (HSV-1).8,9) In continuation with responding acyl chloride 2a or 2b using triethylamine (TEA)
our work for finding potent antiviral derivatives, synthetic (Entries 1 and 2 in Table 1). In case of the reaction with nic-
molecular modifications directed our attention to the molecu- otinoyl chloride·HCl (2c·HCl), preparation by a similar proce-
lar symmetry for the biological activity. Our recent studies on dure resulted in a low yield of 6c (10%, Entry 3). Change of
heteroaryl-substituted triarylmethane derivatives indicated that the solvent and the reagent stoichiometry resulted in improved
compounds bearing a prochiral symmetric feature (possess- yield of 6c to 70% (Entry 4). The preparation of compound 6c
ing CS symmetry) showed a higher level of antiviral activity was also achieved by condensation with carboxylic acid 2c′,
than that of the corresponding C3-symmetrical molecules9,10) though in a low yield (33%, Entry 5). Compound 7 was pre-
(see Fig. 1). In the area of molecular recognition, a tripodal pared by condensation with cinnamic acid derivative 3 (35%,
arrangement of cell-specific ligands is an ideal recoginition Entry 6). The addition reaction of TREA to isothiocyanate 5
motif for cell surfaces, and it is known that a number of re- gave the target compound 11 quantitatively (Entry 7).
ceptors share a common binding motif with three-fold sym-
For the synthesis of CS-symmetrical compounds 9 from
—
14)
metry.7,11
In order to find new promising candidates, we compound 1, we first planned a two-step method via diacyl-
have chosen tris(2-aminoethyl)amine (TREA) as a new scaf- ated TREA 8 as shown in Chart 3. In the first step, acylation
fold. TREA derivatives have been studied extensively in the of TREA by acyl chloride 2a gave tri-acylated derivative 6a
field of coordination chemistry; however, little is known about predominantly even in a 1:0.1 ratio of TREA:2a (Entry 8).
their biological activities. To our knowledge, some TREA Acylation using the free carboxylic acid 2a′ gave a better
derivatives have been reported to work as membrane anion yield (49%) of the intermediate 8 (Entry 9). The second step
transporters15,16) or as HIV-1 protease inhibitors.17) In addition
to these previous results, the flexibility of a TREA scaffold
may enable recognition of the sites of target biological mol-
ecules, and this possibility prompted us to select TREA as a
template for target tripodal-type molecules.
Here, we describe the synthesis of new symmetrical tri-sub-
stiuted TREA derivatives and the results of biological evalu-
ation of the synthesized compounds for anti-HSV-1 activity.
Fig. 1. Structures of Antiviral Compounds with C3 and CS Symmetry
*To whom correspondence should be addressed. e-mail: kunihiro@adm.fukuoka-u.ac.jp
© 2012 The Pharmaceutical Society of Japan

March 2012
409
Chart 1. Synthetic Outline for Target TREA Derivatives
the above reaction conditions of the steps through compound
8 (Entries 9 and 10) was 28%. Another method conducted in a
one-pot reaction with the inverse order of addition of reagents
(5, 2a) resulted in an increased yield of 9 (45%) together with
a byproduct 10 in 11% yield (Entry 11). In the same method,
we changed the ratio of reagents (5:2a) from 1:2 to 2:1 for
the synthesis of 10, and we obtained 10 (23%) and tri-acylated
compound 11 (28% in Entry 12).
For the synthesis of an intermediate for the target com-
pound 13, acylation of TREA by acid anhydride 4 resulted
in the formation of bis-acylated compound 12 (25%) together
with tri-acylated compound 15 (11% in Entry 13). In a manner
identical to that for the synthesis of 9, the product 13 (34%)
was obtained together with a byproduct 14 (7% in Entry 14).
As a result, we recognized that it seems to be difficult to
achieve high reaction selectivity for bis- or mono-substituted
TREA derivatives. Furthermore, the method using a one-pot
reaction with the combination of 5 and 2a or 5 and 4 gave CS-
symmetrical 9 or 13 in a moderate yield.
The C3- or CS-symmetrical structures of the target products
were easily confirmed by 13C-NMR spectroscopic analysis.
All of the structures of the synthesized compounds were also
easily confirmed by spectroscopic and elemental analyses ex-
cept for the elemental analyses data of compounds 8 and 14
(see Experimental).
Chart 2. Synthesis of C3-Symmetrical TREA Derivatives
of the reaction leading to the desired 9 by the reaction of 8
Evaluation of Antiviral Activity and Discussion The
with 5 gave a moderate yield (Entry 10). The total yield under anti-HSV-1 activities of all of the synthesized TREA deriva-
Chart 3. Synthesis of CS-Symmetrical TREA Derivatives

410
Vol. 60, No. 3
tives (except for 8, 12, 14) were evaluated by plaque reduction logarithm of the partition coefficient (logP value) is one of the
assays,20) and the derivatives were evaluated for cytotoxicity important factors for its biological activities.23,24) It is interest-
—
—
with Vero cells. The results for compounds 6a c, 7, 9 11, ing that compound 6b, which has the largest logP (shown in
13, and 15 are summarized in Table 3, and aciclovir21) is also Table 3),25) showed a high level of cytotoxicity (IC50=17.7μM).
listed as a positive control. All compounds evaluated in this In contrast, other C3-symmetrical compounds (6a, 6c, 7, 11,
study showed no distinct inhibitory concentration (EC50) val- 15), which have lower logP values (<7.41) than the value of
ues for antiviral activity less than the dose of 100μM. Among highly lipophilic 6b (logP=8.30), showed low levels of cyto-
the compounds tested, compounds 6b and 13 showed high lev- toxicity against Vero cells.
els of cytotoxicity against Vero cells (IC50=17.7, 21.5μM), and
compound 9 also showed some cytotoxicity (IC50=60.9μM).22)
It is well known that lipophilicity of the compound as the tively).26
In addition, two compounds (9 and 13) showed consider-
ably high levels of cytotoxicity (IC50=60.9, 21.5μM, respec-
—
28)
It is of great interest that the calculated logP
Table 1. Synthesis of C3-Symmetrical TREA Derivatives
Ratio of
Entry
Reagent
Conditions
TREA:Reagent:TEA or
(TPP)
Product (yield %)
°
1
2
2a
2b
0 C 1h, rt 0.5h, dry
CH2Cl2, N2
1:3.3:3.3
1:3.5:3.5
6a (68)
6b (66)
°
0 C 1h, rt 0.5h, dry
CH2Cl2, N2
°
3
4
5
2c·HCl
2c·HCl
2c′
0 C 1h, dry CH2Cl2, N2
1:3.3:3.3
1.2:1:1.4
1:3:(3)
6c (10)
6c (70)b)
6c (33)
°
0 C 1h, dry THF, N2
°
i) 1, 2c′, 70 C 1h, ii)
TPP, reflux 1d, pyridine
6
3
i) 3, NHS, rt 0.5h, N2,
1:3:3:3a)
7 (35)
ii) DCC, rt 3h, N2, iii) 1,
rt 1d, dry THF, N2
7
5
Rt 1d, dry CH2Cl2
1:4
11 (99)
a) Ratio of 1:3:NHS:DCC=1:3:3:3. b) The yield was based on the starting compound 2c·HCl.
Table 2. Synthesis of CS-Symmetrical TREA Derivatives
Ratio of
TREA:Reagent:TEA or
(CDI)
Entry
Reagent
Conditions
Product (yield %)
8
9
2a
0 C 0.5h, rt 3h, dry
CH2Cl2, N2
1:0.1:0.1
1:2:(2)
6a (80), 8 (10)a)
6a (10), 8 (49)a)
°
2a′
i) 2a′, CDI, reflux 2h, ii)
1, reflux 1d, dry CH2Cl2,
N2
10b)
11
5
Rt 3h, dry CH2Cl2
1:3.3
1:1:2:2c)
9 (58)
i) 5, ii) 2a
i) 1, 5, rt 2.5h, ii)
9 (45), 10 (11)
°
TEA, 2a, 0 C 0.5h, dry
CH2Cl2, N2
12
i) 5, ii) 2a
i) 1, 5, rt 2.5h, ii)
1:2:1:1d)
10 (23), 11 (28)
°
TEA, 2a, 0 C 0.5h, dry
CH2Cl2, N2
13
14
4
−78 C 0.5h→rt over-
5:3
12 (25), 15 (11)e)
13 (34), 14 (7)g)
°
night, dry CH2Cl2, N2
i) 5, ii) 4
i) 1, 5, rt 1h, ii) TEA, 4,
1:1:2:2f)
rt 1h, dry CH2Cl2, N2
a) The yield was based on the starting compounds 2a or 2a′. b) The starting material 8 was used in this entry. c) Ratio of 1:5:2a:TEA=1:1:2:2. d) Ratio of
1:5:2a:TEA=1:2:1:1. e) The yield was based on the starting compound 4. f) Ratio of 1:5:4:TEA=1:1:2:2. g) The yield was based on TREA (1).
Table 3. Antiviral Activity (EC50) against HSV-1, Cytotoxicity (IC50) against Vero Cells, and Calculated logP
Compd.
6a
6b
6c
7
9
10
11
13
15
Aciclovira)
1.1
EC50
(μM)
>100
N.D.
>100
>100
N.D.
>100
>100
N.D.
181.7
IC50
(μM)
>200
17.7
>200
>200
60.9
3.97
579.5
5.69
>200
21.5
3.83
227.4
2.04
>444
−0.76
logP
2.25
8.30
−0.93
2.71
7.41
a) Data were taken from ref. 21.

March 2012
411
values for these compounds were around ca. 3.9 (see Table 3) lized from 2-PrOH to give compound 6a (410mg, 0.473mmol,
—
°
and that both derivatives belong to a class of compounds with 68%) as colorless needles: mp 150 152 C (from 2-PrOH). IR
CS symmetry. In contrast, the corresponding C3-symmetrical (KBr) cm−1: 3380 (NH of amide), 2840 (ArOMe), 1610, 1505,
1
compounds (6a, 11, 15) showed no significant cytotoxicity 1255 (amide). H-NMR (DMSO-d6) δ: 2.70 (6H, t, J=6.6Hz,
(IC50=>200μM). It is also noteworthy that the cytotoxicity H2′), 3.36 (6H, brq, H1′), 3.78 (9H, s, OCH3), 6.68 (6H, d,
of CS-symmetrical compound 10, which consists of the same J=8.85Hz, H2,6), 7.75 (6H, d, J=8.85Hz, H3,5), 8.11 (3H, t,
functional groups as those of CS-symmetrical compound 9 J=5.2Hz, NH). 13C-NMR (DMSO-d6) δ: 37.43 (C1′), 53.23
with a different ratio of the groups, showed a surprisingly (C2′), 55.15 (OCH3), 113.26 (C3) 126.64 (C1), 128.76 (C2),
decreased cytotoxic property (IC50=579.5μM) (see represented 161.30 (C4), 165.75 (C=O). Positive-ion FAB-MS m/z: 549
molecular structures in Chart 3). These results apparently (M+H)+. HR-FAB-MS m/z: 549.2731 (Calcd for C30H37N4O6:
indicate that the factors for cytotoxicity of compounds in this 549.2713). Anal. Calcd for C30H36N4O6: C, 65.68; H, 6.61; N,
series may involve not only molecular symmetry but also the 10.21. Found: C, 65.64; H, 6.61; N, 10.22.
nature of introduced functionalities and logP values, which
characterizes the lipophilicity of a molecule in its natural methyl)benzamide] (6b) Entry 2: In
state. lar to that for the preparation of 6a, by the reaction
Based on the above findings in this study, we are investigat- of (293mg, 2.0mmol), TEA (708mg, 7.0mmol),
N,N′,N″-(Nitrilotri-2,1-ethanediyl)tris[3,5-bis(trifluoro-
a
manner simi-
1
ing further synthetic molecular modifications for symmetrical and 3,5-bis(trifluoromethyl)benzoyl chloride (2b, 1.936g,
tripodal types of compounds by introduction of more lipophil- 7.0mmol), the crude product was obtained, and recrys-
ic and aromatic functional groups in order to search for prom- tallization from EtOH–H2O gave compound 6b (1.144g,
—
°
ising cytotoxic lead compounds for anticancer agents. In the 1.32mmol, 66%) as colorless crystals: mp 145 147 C (from
search for antiviral compounds, we are planning to introduce EtOH–H2O). IR (KBr) cm−1: 3250 (NH of amide), 1650,
1
reversed polar (or hydrophilic) functionality in the designed 1550, 1280 (amide), 1150 (CF). H-NMR (DMSO-d6) δ: 2.78
symmetrical molecules. These results will be described in a (6H, t, J=6.3Hz, H2′), 3.45 (6H, m, H1′), 8.14 (3H, brs, H4),
separate paper.
8.32 (6H, d, J=1.2Hz, H2, 6), 8.84 (3H, t, J=5.5Hz, NH).
13C-NMR (DMSO-d6) δ: 38.03 (C1′), 53.38 (C2′), 122.90
(CF3, J=273.1Hz), 124.33 (C4), 127.72 (C2,6), 130.13 (C3,5
Experimental
Melting points were determined using a micro melting J=33.1Hz). Positive-ion FAB-MS m/z: 867 (M+H)+. HR-FAB-
point apparatus (Yanagimoto MP-S3) without correction. IR MS m/z: 867.1632 (Calcd for C33H25F18N4O3: 867.1639). Anal.
spectra were measured by a Shimadzu FTIR-8100 IR spectro- Calcd for C33H24F18N4O3: C, 45.74; H, 2.79; N, 6.47. Found: C,
photometer. Low- and high-resolution mass spectra (LR-MS 45.74; H, 2.80; N, 6.52.
and HR-MS) were obtained by a JEOL JMS HX-110 double-
N,N′,N″-(Nitrilotri-2,1-ethanediyl)tris(3-pyridinecarbox-
focusing model equipped with an FAB ion source interfaced amide) (6c) Entry 3: In a manner similar to that of the
1
with a JEOL JMA-DA 7000 data system. H- and 13C-NMR preparation of 6a, from the reaction of 1 (293mg, 2.0mmol),
spectra were obtained by JEOL JNM A-500. Chemical TEA (668mg, 6.6mmol), and nicotinoyl chloride hydrochlo-
°
shifts were expressed in δ ppm downfield from an internal ride (2c·HCl, 1.175g, 6.6mmol) at 0 C under N2 atmosphere
1
tetramethylsilane (TMS) signal for H-NMR and the carbon for 1h, the obtained crude product was recrystallized from
signal of the corresponding solvent [CDCl3 (77.00ppm) and 2-PrOH to give compound 6c (93mg, 0.20mmol, 10%) as col-
dimethyl sulfoxide (DMSO)-d6 (39.50ppm)] for 13C-NMR. orless crystals.
The signal assignments were confirmed by 2D-NMR analy-
Entry 4: The reaction of 1 (620mg, 4.2mmol), TEA (460mg,
ses, 1H–1H two-dimensional (2D) correlation spectroscopy 4.6mmol), and 2c·HCl (610mg, 3.4mmol) in dry tetrahydro-
(COSY), 1H–l3C heteronuclear multiple-quantum coherence furan (THF) was held at 0 C under N2 atmosphere for 30min
°
1
(HMQC), and H–l3C heteronuclear multiple-bond connectiv- with stirring. The reaction mixture was filtrated to remove
ity (HMBC). Microanalyses were performed with a Yanaco TEA·HCl salt. After evaporation of the solvent, centrifugal
MT-6 CHN corder. Routine monitoring of reactions was car- chromatography (CH2Cl2 :95% EtOH:28% NH3=85:18.5:0.5)
ried out using precoated Kieselgel 60F254 plates (E. Merck). gave 6c (367mg, 0.80mmol, 70% from 2c·HCl).
Centrifugal or flash column chromatography was performed
Entry 5: To a mixture of nicotinic acid (2c, 6.40g, 46mmol)
on silica gel (Able-Biott or Fuji Silysia FL40D/Kanto 60N) in pyridine (15mL) was added a solution of 1 (2.19g,
with a UV detector. Commercially available starting materials 15mmol) in pyridine (10mL) dropwise with stirring at room
were used without further purification.
temperature. After the resulting solution had been stirred for
°
N,N′,N″-(Nitrilotri-2,1-ethanediyl)tris(4-methoxybenza- 1h at 70 C, triphenylphosphite (TPP, 13.96g, 45mmol) was
mide) (6a)18,19) Entry 1: To a solution of TREA (1, 293mg, added dropwise with stirring and then the solution was re-
2.0mmol) and TEA (661mg, 6.6mmol) in dry CH2Cl2 (20mL) fluxed overnight. After cooling and evaporation of the solvent,
was added a solution of 4-methoxybenzoyl chloride (2a, the residue was dissolved in CH2Cl2 (50mL). The solution
°
1.081g, 6.3mmol) in dry CH2Cl2 (10mL) at 0 C under N2 was extracted with water (30mL×3), and to the combined
atmosphere. After stirring for 1h, the reaction mixture was aqueous layer was added NaOH (10g) and CH2Cl2 (100mL).
allowed to warm up to room temperature and then stirred The resulting mixture was separated into three layers. The
for another 30min. The resulting mixture was poured into middle layer was isolated, and 2-PrOH and ether were added.
water (60mL), and then the aqueous layer was extracted with Then the solidified crude product was separated and recrystal-
CH2Cl2 (2×30mL), the combined organic layer was washed lized from 2-PrOH to give compound 6c (2.31g, 5.00mmol,
—
°
with brine and dried over MgSO4, and the solvent was re- 33%) as colorless crystals: mp 150 152 C (from 2-PrOH).
moved in vacuo. The resulting solid material was recrystal- IR (KBr) cm−1: 3350 (NH of amide), 1610, 1510, 1255 (am-

412
Vol. 60, No. 3
1
ide). H-NMR (DMSO-d6) δ: 2.75 (6H, brt, J=6.7Hz, H2′), 6.25 (3H, brs, NHα or NHβ), 6.42 (3H, brs, NHβ or NHα).
3.40 (6H, m, H1′), 7.42 (3H, ddd, J=7.9, 4.9, 0.6Hz, H5), 8.10 13C-NMR (CDCl3) δ: 29.58 (Cc), 36.24 (Ca), 42.19 (Cb), 43.12
(3H, ddd, J=7.9, 2.1, 1.8Hz, H6), 8.54 (3H, dt, J= 5.5, 0.3Hz, (C1′), 53.59 (C2′), 54.31 (Cd), 180.62 (C=S). Positive-ion FAB-
NH), 8.66 (3H, dd, J=4.9, 1.8Hz, H4), 8.95 (3H, d, J=2.1Hz, MS m/z: 726 (M+H)+. HR-FAB-MS m/z: 726.4385 (Calcd for
H2). 13C-NMR (DMSO-d6) δ: 37.67 (C1′), 53.16 (C2′), 123.20 C39H64N7S3: 726.4389). Anal. Calcd for C39H63N7S3·H2O: C,
(C5) 129.85 (C1), 134.63 (C6), 148.2 (C2), 151.57 (C4), 164.83 62.94; H, 8.80; N, 13.18. Found: C, 62.94; H, 8.60; N, 13.09.
(C=O). Positive-ion FAB-MS m/z: 462 (M+H)+. HR-FAB-MS
N,N′-[(2-Aminoethyl)imino]di-2,1-ethanediyl]bis(4-
m/z: 462.2255 (Calcd for C24H28N4O3: 462.2254). Anal. Calcd methoxybenzamide) (8) Entry 8: This compound was ob-
for C24H27N4O3: C, 62.46; H, 5.92; N, 21.24. Found: C, 62.20; tained by a method similar to that described in Entry 1. To
H, 5.92; N, 21.05.
a stirred solution of 1 (7.45g, 51.0mmol) and TEA (506mg,
(2E,2′E,2″E)-N,N′,N″-(Nitrilotri-2,1-ethanediyl)tris[3- 5.0mmol) in dry CH2Cl2 (20mL) was added a solution of
°
2a (852mg, 5.0mmol) in dry CH2Cl2 (8mL) at 0 C under
(2,3-dimethoxyphenyl)-2-propenamide] (7) Entry 6:
A
mixture of trans-2,3-dimethoxycinnamic acid (3, 1.524g, N2 atmosphere for 30min dropwise. The reaction mixture
7.32mmol) and N-hydroxysuccinimide (NHS, 0.842g, was stirred for 3h, and then the solvent was removed in
7.32mmol) in dry THF (15mL) was stirred at room tem- vacuo. Centrifugal chromatography (CH2Cl2 :95% EtOH:28%
perature under N2 atmosphere for 30min. After addition of NH3=80:19.5:0.5) gave 6a (732mg, 1.33mmol, 80%) and
N,N′-dicyclohexylcarbodiimide (DCC, 1.51g, 7.32mmol), the compound 8 (104mg, 0.25mmol, 10%).
reaction mixture was stirred for another 3h. The precipitated
Entry 9: A solution of 2a′ and carbonyl diimidazole (CDI,
dicyclohexylurea (DCU) was removed by filtration, and to the 1.07g, 5.6mmol) in dry CH2Cl2 (50mL) was refluxed for 2h
resulting filtrate was added a solution of 1 (357mg, 2.44mmol) under N2 atmosphere. To this mixture was added dropwise a
in dry THF (2mL) dropwise over a period of 15min. This solution of 1 (395mg, 2.7mmol) and the reaction mixture was
mixture was stirred overnight at room temperature under N2 refluxed overnight. Then the reaction mixture was cooled to
atmosphere. The white precipitate was filtrated and dissolved room temperature and washed with 4M NaOH (30mL×3) after
in CHCl3 (30mL). The resulting solution was washed with addition of CH2Cl2 (50mL). The organic layer was separated,
saturated NaHCO3 (2×30mL), dried over MgSO4, and concen- dried over MgSO4, and filtered. After removal of the solvent,
trated to give an amber-colored oil. Separation by flash chro- the resulting yellow solid was purified by centrifugal chroma-
matography (CH2Cl2 :95% EtOH:28% NH3=97:2.7:0.3) gave tography (CH2Cl2 :95% EtOH:28% NH3=73:25:2) to give 6a
compound 7 (610mg, 0.851mmol, 35%). An analytical sample (140mg, 0.26mmol, 10%) and 8 (560mg, 1.35mmol, 49%) as
was obtained by recrystallization from acetonitrile as colorless a pale yellow oil.
−1
—
°
crystals: mp 128 130 C (from CH3CN). IR (KBr) cm : 3300
8: Positive-ion FAB-MS m/z: 415 (M+H)+. HR-FAB-MS
(NH of amide), 1620, 1470, 1170 (amide). H-NMR (CDCl3) δ: m/z: 415.2342 (Calcd for C22H31N4O4: 415.2345). 1H-NMR
2.63 (6H, dt, J=5.5, 0.6Hz, H2′), 3.45 (6H, dt, J=5.5, 4.9Hz, (CDCl3) δ: 2.61 (2H, brt, H2″), 2.69 (4H, t, J=5.5Hz, H2′),
H1′), 3.70 (9H, s, 2-OCH3), 3.81 (9H, s, 3-OCH3), 6.64 (3H, 2.79 (2H, brt, H1″), 3.49 (4H, dt, J=5.5, 5.2Hz, H1′), 3.54 (2H,
t, J=7.9Hz, H5), 6.69 (3H, d, J=15.9Hz, Hα), 6.75 (3H, dd, brs, NH2), 3.75 (6H, s, OCH3), 6.71 (4H, dm, J=8.9Hz, H3),
J=7.9, 1.2Hz, H4), 6.94 (3H, dd, J=7.9, 1.2Hz, H6), 7.15 7.69 (2H, brt, J=5.2Hz, NH), 7.71 (4H, dm, J=8.9Hz, H2).
(3H, t, J=4.9Hz, NH), 7.89 (3H, d, J=15.9Hz, Hβ). 13C-NMR 13C-NMR (CDCl3) δ: 38.08 (C1′), 38.77 (C1″), 53.65 (C2′),
(CDCl3) δ: 37.80 (C1′), 53.89 (C2′), 55.85 (3-OCH3), 61.05 54.21 (C2″), 55.19 (OCH3), 113.45 (C3), 126.38 (C1), 128.93
(2-OCH3), 113.27 (C4), 119.53 (C6), 122.33 (Cα) 123.94 (C5), (C2), 161.88 (C4), 167.57 (C=O).
1
129.02 (C1), 135.48 (Cβ), 148.24 (C2), 152.91 (C3), 167.04
N,N′-[[[(2-Tricyclo[3.3.1.13.7]dec-1-ylthioureido)ethyl]im-
(C=O). Positive-ion FAB-MS m/z: 717 (M+H)+. HR-FAB-MS ino]di-2,1-ethanediyl]bis(4-methoxybenzamide) (9) Entry
m/z: 717.3509 (Calcd for C39H49N4O9: 717.3500). Anal. Calcd 10: A solution of 8 (118mg, 0.28mmol) and 5 (180mg,
for C39H48N4O9: C, 64.54; H, 6.80; N, 7.72. Found: C, 64.56; 0.93mmol) in dry CH2Cl2 (3mL) was stirred at room tem-
H, 6.65; N, 7.73.
perature for 3h. After evaporation, the residue was purified
N,N′,N″-(Nitrilotri-2,1-ethanediyl)tris[N‴-tricyclo- by centrifugal chromatography (CH2Cl2 :95% EtOH:28%
[3.3.1.13,7]dec-1-ylurea] (11) Entry 7: To a stirred solution NH3=97:2.7:0.3) to give 9 (101mg, 0.17mmol, 58%) as color-
−1
―
°
of 1-adamantyl isothiocyanate (5, 1.94g, 10.0mmol) in dry less crystals: mp 78 81 C. IR (KBr) cm : 3320 (NH of am-
CH2Cl2 (40mL) was added a solution of 1 (370mg, 2.5mmol) ide), 2900 (ArOMe), 1550, 1500 (C=S–N), 1300 (C=S), 1250
1
in dry CH2Cl2 (2mL) at room temperature. After stirring for (amide). H-NMR (CDCl3) δ: 1.67 (6H, brs, a), 2.08 (3H, brs,
1d, the solvent was removed in vacuo. To the resulting pale c), 2.20 (6H, brs, b), 2.65 (4H, t, J=5.2Hz, H2′), 2.71 (2H, dt,
yellow oil, dry benzene (50mL) was added, and the solvent J=5.2, 0.3Hz, H2″), 3.47 (4H, dt, J=5.5, 5.2Hz, H1′), 3.62
was removed again by azeotropic distillation. The solidified (2H, dt, J=5.5, 5.2Hz, H1″), 3.79 (6H, s, –OCH3), 6.66 (1H,
crude product was dissolved in CH2Cl2 (ca. 5mL) and MeOH brs, NHβ or NHγ), 6.73 (4H, d, J=8.9Hz, H3), 6.75 (2H, brs,
(ca. 10mL) was added. Then the separated crystals 11 (1.50g, NHα), 7.31 (1H, br s, NHγ or NHβ), 7.53 (4H, d, J=8.9Hz, H2).
2.1mmol, 83%) were collected by filtration. The filtrate gave 13C-NMR (CDCl3) δ: 29.69 (Cc), 36.44 (Ca), 38.18 (C1′), 42.04
additional crystals 11 (0.29g, 0.4mmol, 16%). The total yield (Cb), 42.18 (C1″), 53.67 (Cd), 54.03 (C2″), 54.37 (C2′), 55.30
was nearly quantitative. Recrystallization of the product from (OCH3), 113.64 (C3), 126.15 (C1), 128.71 (C2), 162.15 (C4),
EtOH gave an analytical pure sample 11 as colorless crystals: 167.87 (C=O), 181.51 (C=S). Positive-ion FAB-MS m/z: 608
−1
mp 110 111 C (from EtOH). IR (KBr) cm : 3285 (NH of (M+H)+. HR-FAB-MS m/z: 608.3271 (Calcd for C33H46N5O4S:
—
°
amide), 2905 (ArOMe), 1545, 1455, 1360 (C=S–N), 1300 608.3271). Anal. Calcd for C33H45N5O4S: C, 64.45; H, 7.51; N,
1
(C=S). H-NMR (CDCl3) δ: 1.69 (18H, brs, a), 2.15 (27H, brs, 11.39. Found: C, 64.51; H, 7.59; N, 11.12.
c, b), 2.70 (6H, t, J=5.5Hz, H2′), 3.66 (6H, d, J=4.9Hz, H1′),
N-[2-Bis[2-[[[tricyclo[3.3.1.13,7]dec-l-ylamino]thioxometh-

March 2012
413
yl]amino]ethyl]]-4-methoxy-benzamide (10) Entry 11: To C24H35N4O4: 443.2658). Anal. Calcd for C24H34N4O4·0.7H2O:
a solution of 1 (585mg, 4.0mmol) in dry CH2Cl2 (40mL) C, 63.33; H, 7.84; N, 12.31. Found: C, 63.26; H, 7.81; N, 12.15.
−1
— °
15: mp 115 116 C. IR (KBr) cm : 3300 (NH of am-
was added a solution of 5 (773mg, 4.0mmol) in dry CH2Cl2
(10mL) at room temperature. After stirring for 2.5h, TEA ide), 3080, 2935, 2835 (ArOMe), 1640, 1245 (amide), 1175,
(810mg, 8.0mmol) was added to the solution. Then 2a 1035 (ArOMe). 1H-NMR (CDCl3) δ: 2.42 (6H, dt, J=5.5,
(1.365g, 8.0mmol) in dry CH2Cl2 (15mL) was added drop- 0.3Hz, H2′), 3.13 (6H, dt, J=5.8, 5.5Hz, H1′), 3.46 (6H, s,
°
wise with stirring for 30min at 0 C under N2 atmosphere. α-CH2), 3.75 (9H, s, OCH3), 6.52 (3H, brt, NH), 6.83 (6H,
After removal of the precipitated TEA·HCl salt, the residue dm, J=8.5Hz, H3), 7.17 (6H, dm, J=8.5Hz, H2). 13C-NMR
obtained by evaporation was purified by flash column chroma- (CDCl3) δ: 37.94 (C1′), 42.52 (Cα), 54.54 (C2′), 55.21 (OCH3),
tography (CH2Cl2 :95% EtOH:28% NH3=97:2.7:0.3) to give 114.17 (C3), 127.21 (C1), 130.29 (C2), 158.66 (C4), 172.05
9 (1.086g, 1.79mmol, 45%) and 10 (290mg, 0.43mmol, 11%) (C=O). Positive-ion FAB-MS m/z: 591 (M+H)+. HR-FAB-MS
as a colorless solid. Recrystallization of the crude 10 from i- m/z: 591.3176 (Calcd for C33H43N4O6: 591.3183). Anal. Calcd
Pr2O gave an analytical pure sample 10 as colorless crystals: for C33H42N4O6·0.3H2O: C, 66.49; H, 7.20; N, 9.40. Found: C,
−1
—
°
mp 145 147 C (from i-Pr2O). IR (KBr) cm : 3275 (NH of 66.61; H, 7.11; N, 9.37.
amide), 2910 (ArOMe), 1630 (amide), 1555, 1500 (C=S–N),
N,N′-[[[2-Tricyclo[3.3.1.13,7]-methyl]amino]ethyl]imino]
1
1295 (C=S). H-NMR (DMSO-d6) δ: 1.61 (12H, brs, a), 2.01 diethylene]bis[(4-methoxyphenyl)acetamide] (13) and N-
(6H, brs, c), 2.12 (12H, brs, b), 2.61 (4H, m, H2″), 2.65 (2H, [2-Bis[2-[[[tricyclo[3.3.1.13,7]dec-l-ylamino]thioxomethyl]-
m, H2′), 3.34 (2H, m, H1′), 3.43 (4H, m, H1″), 3.80 (3H, s, amino]ethyl]]-(4-methoxyphenyl)acetamide (14) Entry 14:
–OCH3), 6.95 (2H, brs, NHγ), 6.98 (2H, d, J=8.9Hz, H3,5), To a solution of 1 (585mg, 4.0mmol) in dry CH2Cl2 (40mL)
7.11 (2H, t, J=4.9Hz, NHβ), 7.80 (2H, d, J=8.9Hz, H2,6), was added a solution of 5 (774mg, 4.0mmol) in dry CH2Cl2
8.24 (1H, t, J=5.8Hz, NHα). 13C-NMR (DMSO-d6) δ: 28.94 (25mL) at room temperature, and the resulting mixture was
(Cc), 35.87 (Ca), 37.35 (C1′), 41.10 (C1″), 41.15 (Cb), 52.54 stirred for 1h. After addition of TEA (810mg, 8.0mmol), a
(C2″), 52.57 (Cd), 53.32 (C2′), 55.23 (OCH3), 113.37 (C3,5), solution of 4 (2.52g, 8.0mmol) in dry CH2Cl2 (20mL) was
°
126.71 (C1), 128.81 (C2,6), 161.42 (C4), 165.92 (C=O), 180.48 added dropwise at 0 C under N2 atmosphere. After stir-
(C=S). Positive-ion FAB-MS m/z: 667 (M+H)+. HR-FAB- ring for 1h at room temperature, the reaction mixture was
MS m/z: 667.3824 (Calcd for C36H55N6O2S2: 667.3828). Anal. washed with saturated NaHCO3 (50mL) and dried over
Calcd for C36H54N6O2S2·0.5i-Pr2O: C, 65.23; H, 8.56; N, 11.70. MgSO4, and the solvent was evaporated. The residue was
Found: C, 65.06; H, 8.47; N, 11.62.
purified by flash column chromatography (CH2Cl2 :95%
Entry 12: The reaction was carried out in a manner simi- EtOH:28% NH3=97:2.7:0.3 to 95:4.5:0.5) to give 13
lar to that described above except for the ratio of reagents (869mg, 1.37mmol, 34%) as colorless needles and 14 (202mg,
[1 (585mg, 4.0mmol):5 (1.55g, 8.0mmol):TEA (405mg, 0.30mmol, 7%) as colorless crystals.
−1
— °
13: mp 55 57 C. IR (KBr) cm : 3315 (NH of amide),
4.0mmol):2a (682mg, 4.0mmol)]=1:2:1:1. After evapora-
tion of the solvent, the residue was purified by flash column 2905 (ArOMe), 1650 (amide), 1540, 1510 (C=S–N), 1360
chromatography (CH2Cl2 :95% EtOH:28% NH3=92:7:1) to (C=S). H-NMR (CDCl3) δ: 1.66 (6H, brs, a), 2.06 (3H, brs,
1
give 10 (604mg, 0.91mmol, 23%) and 11 (813mg, 1.12mmol, c), 2.17 (6H, brs, b), 2.45 (4H, t, J=5.5Hz, H2′), 2.58 (2H, t,
28%) as a white solid.
J=5.5Hz, H2″), 3.19 (4H, dt, J=5.5, 5.2Hz, H1′), 3.46 (2H,
N,N′-[(2-Aminoethyl)imino]di-2,1-ethanediyl]bis[(4- m, H1″), 3.49 (4H, s, α), 3.78 (6H, s, –OCH3) 6.08 (2H, brt,
methoxyphenyl)acetamide] (12) and N,N′,N″-(Nitrilotri-2,1- NHα), 6.62 (1H, brs, NHβ or NHγ), 6.87 (4H, d, J=8.9Hz,
ethanediyl)tris[(4-methoxyphenyl)acetamide] (15) Entry H3), 6.89 (1H, brs, NHγ or NHβ), 7.18 (4H, d, J=8.9Hz, H2).
13: To a solution of 1 (731mg, 5.0mmol) in dry CH2Cl2 13C-NMR (CDCl3) δ: 29.66 (Cc), 36.39 (Ca), 38.23 (C1′),
(275mL) was added dropwise a solution of 4-methoxyphenyl- 41.98 (Cb), 42.21 (C1″), 42.84 (Cα), 53.58 (Cd or C2″), 53.69
acetic anhydride (4, 943mg, 3.0mmol) in dry CH2Cl2 (100mL) (C2″ or Cd), 54.86 (C2′), 55.30 (OCH3), 114.45 (C3), 126.75
°
at −78 C with stirring, and then stirring was continued at (C1), 130.48 (C2), 158.94 (C4), 172.40 (C=O), 181.26 (C=S).
room temperature overnight. After the resulting reaction mix- Positive-ion FAB-MS m/z: 636 (M+H)+. HR-FAB-MS m/z:
ture had been washed with saturated NaHCO3, the organic 636.3604 (Calcd for C35H50N5O4S: 636.3584). Anal. Calcd for
layer was dried over MgSO4. The filtrate was evaporated C35H49N5O4S: C, 66.11; H, 7.77; N, 11.01. Found: C, 65.92; H,
and the residue was purified by centrifugal chromatography 7.81; N, 10.86.
1
—
°
(CH2Cl2 :95% EtOH:28% NH3=93:6.5:0.5 to 73:25:2) to
14: mp 108 110 C, H-NMR (CDCl3) δ: 1.69 (12H, brs,
give 12 (163mg, 0.37mmol, 25% from 4) as a pale yellow oil a), 2.11 (6H, brs, c), 2.15 (12H, brs, b), 2.54 (2H, t, J=5.5Hz,
and 15 (62mg, 0.104mmol, 11% from 4) as a colorless solid.
H2′), 2.62 (4H, t, J=5.8Hz, H2″), 3.27 (2H, m, H1′), 3.54
12: IR (KBr) cm−1: 3285 (NH2, OH), 3070, 2935, 2835 (4H, m, H1″), 3.57 (2H, s, α), 3.80 (3H, s, –OCH3) 6.09 (1H,
1
(ArOMe), 1645, 1245 (amide), 1175, 1035 (ArOMe). H-NMR m, NHα), 6.29 (2H, br s, NHβ or NHγ), 6.52 (2H, brs, NHγ or
(CDCl3) δ: 1.90 (2H, brs, NH2), 2.39 (2H, dt, J=5.5, 0.6Hz, NHβ), 6.90 (2H, dm, J=8.7Hz, H3), 7.21 (2H, dm, J=8.7Hz,
H2″), 2.46 (4H, dt, J=5.5, 0.3Hz, H2′), 2.57 (2H, dt, J=5.5, H2). 13C-NMR (CDCl3) δ: 29.62 (Cc), 36.30 (Ca), 38.32 (C1′),
0.6Hz, H1″), 3.20 (4H, J=6.1, 5.5Hz, H1′), 3.48 (4H, s, 42.16 (Cb), 42.92 (C1″, Cα), 53.50 (C2″), 54.04 (Cd), 55.33
α-CH2), 3.76 (6H, s, OCH3), 6.61 (2H, brt, NH), 6.85 (4H, (C2′ or OCH3), 55.37 (OCH3 or C2′), 114.58 (C3), 126.44
dm, J=8.5Hz, H3), 7.19 (4H, dm, J=8.5Hz, H2). 13C-NMR (C1), 130.65 (C2), 159.03 (C4), 173.06 (C=O), 181.10 (C=S).
(CDCl3) δ: 37.80 (C1′), 39.57 (C1″), 42.56 (Cα), 54.02 (C2′), Positive-ion FAB-MS m/z: 681 (M+H)+. HR-FAB-MS m/z:
55.24 (OCH3), 56.49 (C2″), 114.18 (C3), 127.29 (C1), 130.37 681.3979 (Calcd for C37H57N6O2S2: 681.3984).
(C2), 158.67 (C4), 171.85 (C=O). Positive-ion FAB-MS
Antiviral Activity Assay and Cytotoxicity The antiviral
m/z: 443 (M+H)+. HR-FAB-MS m/z: 443.2653 (Calcd for activities of synthesized compounds (6a c, 7, 9 11, 13, 15)
—
—

414
Vol. 60, No. 3
were measured by using a plaque reduction assay20) as de-
scribed in our previous paper.29) Results of antiviral activity
(EC50) and cytotoxicity (IC50) with Vero cells are summarized
in Table 3.
14) Kim J., Kim S.-G., Seong H. R., Ahn K. H., J. Org. Chem., 70,
—
7227 7231 (2005).
15) Boon J. M., Lambert T. N., Smith B. D., Beatty A. M., Ugrinova
—
V., Brown S. N., J. Org. Chem., 67, 2168 2174 (2002) and related
references cited therein.
—
16) Berezin S. K., Davis J. T., J. Am. Chem. Soc., 131, 2458 2459
Acknowledgements We thank Ms. Ai Saito and Ms.
Nami Ueki for their valuable technical assistance.
(2009).
17) Ma C. M., Nakamura N., Hattori M., Chem. Pharm. Bull., 49,
—
915 917 (2001).
References and Notes
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Supramol. Chem., 18, 55 58 (2006).
783 (2011).
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(London), 468, 705 708 (2010).
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4) Balzarini J., Antiviral Res., 71, 237 247 (2006).
—
T., Uyeda M., Biol. Pharm. Bull., 28, 1779 1781 (2005).
“
”
5) Contreras J.-M., Sippl W., The Practice of Medicinal Chemistry,
22) In calorimetric pre-experiments for sugar recognition of C3-
symmetrical cytotoxic compound 6b with some sugars derivatives,
3rd ed., ed. by Wermuth C. G., Elsevier Academic Press, London,
—
2008, pp. 380 414.
binding constants (Ka) between 6b and guest sugars were in the
−1
—
6) Gibson S. E., Castaldi M. P., Angew. Chem. Int. Ed., 45, 4718
—
range of ca. 50 400M in aqueous 25% 2-propanol solution at
4720 (2006).
°
25 C. The results of sugar recognition of the products will be de-
7) Humblet V., Misra P., Bhushan K. R., Nasr K., Ko Y.-S., Tsukamoto
scribed elsewhere in detail.
T., Pannier N., Frangioni J. V., Maison W., J. Med. Chem., 52,
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Drug Deliv. Rev., 23, 3 25 (1997).
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53, 1171 1174 (2005).
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A., Bond J. A., Henderson R. F., Crit. Rev. Toxicol., 19, 185 226
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—
Chem., 47, 1434 1438 (2010) and references are cited therein.
25) LogP were calculated by using CAChe (v.6.1.12).
26) We have already observed that some of the compounds with high
levels of cytotoxicity against Vero cells also show an interesting an-
titumor activity. Therefore, this assay for cytotoxicity of compounds
may provide a conventional pre-screening procedure for such com-
pounds.27,28)
10) Through our previous studies on triarylmethane derivatives, we
arrived at a working hypothesis that a C3-symmetrical molecule
having a tripodal scaffold may be an efficient structural feature
for molecular recognition such as recognition of sugar chains in
macromolecules or receptor sites. The rigidity of the molecules and
nature of functional groups (such as H-bond donors or acceptors for
suprafacial interactions) in symmetrical target molecules may play
important roles for its biological properties.
27) Thongthoom T., Promsuwan P., Yenjai C., Eur. J. Med. Chem., 46,
—
3755 3761 (2011).
28) Sen S., Sarker K., Ghosh A., Mishra S., Saha A., Goswami D., Jha
—
11) Abe H., Aoyagi Y., Inouye M., Org. Lett., 7, 59 61 (2005).
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T., Gupta J. K., De A. U., Int. J. Pharm. Sci. Rev. Res., 10, 118 124
—
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(2011).
—
13) Kim H.-J., Kim Y.-H., Hong J.-I., Tetrahedron Lett., 42, 5049 5052
29) Mibu N., Yokomizo K., Kashige N., Miake F., Miyata T., Uyeda M.,
(2001).
—
Sumoto K., Chem. Pharm. Bull., 55, 111 114 (2007).