Effects of immunomodulatory derivatives of thalidomide (IMiDs) and their analogs on cell-differentiation, cyclooxygenase activity and angiogenesis

Article abstract of DOI:10.1248/cpb.54.855

Various analogs of known immunomodulatory derivatives of thalidomide (1) (IMiDs: 3, 5) were synthesized, focusing on cell-differentiation-inducing, cyclooxygenase-inhibitory and anti-angiogenesis activities. Among the prepared compounds, NIDO-33 (14) showed cell differentiation-inducing activity on HL-60 cells and anti-angiogenic activity on human umbilical vein endothelial cells (HUVEC). AIDO-00 (7) also showed anti-angiogenic activity. NIDO-11 (8) showed an enhancing effect on all-trans retinoic acid (ATRA)-induced HL-60 cell differentiation, and AIDO-30 (13) exhibited cyclooxygenase (COX)-inhibitory activity.

Full text of DOI:10.1248/cpb.54.855

June 2006
Chem. Pharm. Bull. 54(6) 855—860 (2006)
855
Effects of Immunomodulatory Derivatives of Thalidomide (IMiDs) and
Their Analogs on Cell-Differentiation, Cyclooxygenase Activity and
Angiogenesis
Haruka FUJIMOTO, Tomomi NOGUCHI, Hisayoshi KOBAYASHI, Hiroyuki MIYACHI, and
Yuichi HASHIMOTO*
Institute of Molecular & Cellular Biosciences, The University of Tokyo; 1–1–1 Yayoi, Bunkyo-ku, Tokyo 113–0032, Japan.
Received January 30, 2006; accepted March 6, 2006
Various analogs of known immunomodulatory derivatives of thalidomide (1) (IMiDs: 3, 5) were synthesized,
focusing on cell-differentiation-inducing, cyclooxygenase-inhibitory and anti-angiogenesis activities. Among the
prepared compounds, NIDO-33 (14) showed cell differentiation-inducing activity on HL-60 cells and anti-angio-
genic activity on human umbilical vein endothelial cells (HUVEC). AIDO-00 (7) also showed anti-angiogenic ac-
tivity. NIDO-11 (8) showed an enhancing effect on all-trans retinoic acid (ATRA)-induced HL-60 cell differentia-
tion, and AIDO-30 (13) exhibited cyclooxygenase (COX)-inhibitory activity.
Key words thalidomide; immunomodulatory derivative of thalidomide (IMiD); differentiation; angiogenesis; cyclooxygenase
Thalidomide [a-(N-phthalimido)glutarimide, 1] was origi- dipeptidylpeptidase type IV (DPP-IV), puromycin-sensitive
nally used as a sedative/hypnotic drug, but was banned in the aminopeptidase (PSA), histone deacetylase (HDAC), and m-
1960s because of its serious teratogenicity.1—4) Remarkably, calpain],3—6) anti-androgens,3,4,7,8) cell differentiation induc-
thalidomide (1) was subsequently discovered to have various ers,9) and other agents based on the thalidomide structure.
biological activities, including anti-inflammatory and anti- During these studies, we found several previously unknown
angiogenic properties, and was identified as an effective biological activities of thalidomide (1), including enhancing
agent for the treatment of multiple myeloma (MM).1,2) In activity on all-trans retinoic acid (ATRA)-induced cell differ-
1998, the drug was approved in the U.S.A. for the treatment entiation of human leukemia cell line HL-6010) and COX-in-
of Hansen’s disease, under critical control known as hibitory activity.11) Concerning the former activity, enhance-
S.T.E.P.S. (system for thalidomide education and prescribing ment of HL-60 cell differentiation-induction is observed in
safety), and has been used world-wide for the treatment of the presence of the physiological concentration of ATRA,
various cancers. Although the precise molecular basis of the implying that thalidomide (1) may act as a cell differentiation
drug’s activities is not yet fully established, inhibition of inducer in the human body to elicit its anti-tumor activity.10)
tumor necrosis factor (TNF-a) production elicited by Concerning the latter activity, the relationship(s) between
thalidomide (1) was initially considered to be one of the key COX inhibition and antiangiogenic activity has been well
action mechanisms.1ꢀ4) Recently, a series of compounds has documented, i.e., COX inhibition results in inhibition of the
been created by chemical modification of thalidomide, focus- production of prostaglandin E2, an inducer of vascular en-
ing on the TNF-a production-inhibitory activity, to overcome dothelial growth factor (VEGF), which plays a major role in
the original devastating side effects.1—4) Among them, 4- angiogenesis.12—14) The antiangiogenic activity of thalido-
amino analogs of thalidomide (1), i.e., CC-4047 (3) and CC- mide (1) has been suggested to play a major role in the anti-
5013 (5), have been shown to possess potent anti-cancer and tumor action of the drug,1,2) and we also established that it
anti-inflammatory activities with little or none of the toxicity (and its derivatives) inhibit tube formation of human umbili-
of thalidomide (1), and these compounds are known as cal vein endothelial cells (HUVEC).15) These facts led us
IMiDs (immunomodulatory derivatives of thalidomide).1,2) prepare various derivatives of IMiDs (3, 5)/thalidomide (1),
CC-5013 (5) appeared to be non-teratogenic when tested in i.e., compounds 2, 4, and 6—17, and to investigate the ef-
the thalidomide-sensitive New Zealand rabbit preclinical fects of these compounds (1—17) on HL-60 cell differentia-
model, in which thalidomide-associated teratogenicity can be tion, COX activity, and HUVEC tube formation.
detected.1) Consequently, IMiDs (3, 5) are under clinical de-
velopment for the treatment of the myelodysplastic syn- Results and Discussion
dromes (MDS) and various cancers, including multiple
Chemistry Compounds 1—17 (Fig. 1) were prepared by
usual organic synthetic methods (Chart 1) and gave analyti-
myeloma (MM) and prostate cancer.1,2)
In spite of the established activity of thalidomide (1) and cal values close to those expected. Thalidomide (1) was pre-
IMiDs (3, 5) as immunomodulatory agents, their mecha- pared as previously reported.16) The 4-amino derivative of
nism(s) of action remain unclear.1—4) Concerning thalido- thalidomide (1), i.e., CC-4047 (3), and its decarbonylated
mide (1), studies of its TNF-a production-inhibitory activity analog, CC-5013 (5), were prepared as described by Muller
and antiangiogenic activity have been reported.1—4) We have et al.17) The 4-nitro derivative of thalidomide (1), i.e., 4NT
engaged in extensive structural development studies of (2), and its decarbonylated analog, H4NT (4), are intermedi-
thalidomide (1), and have synthesized TNF-a production ates in the synthesis of CC-4047 (3) and CC-5013 (5), re-
regulators,3,4) inhibitors of various enzymes [cyclooxygenase spectively. Briefly, condensation of 3-aminopiperidine-2,6-
(COX), nitrogen monoxide synthase (NOS), a-glucosidase, dione with 3-nitrophthalic anhydride gave 4NT (2), and simi-
∗ To whom correspondence should be addressed. e-mail: hashimot@iam.u-tokyo.ac.jp
© 2006 Pharmaceutical Society of Japan

856
Vol. 54, No. 6
Fig. 2. HL-60 Cell Differentiation-Inducing Activity of Compounds 1—
17 (10 mM)
Fig. 1. Structures of the Compounds Studied in This Paper
Fig. 3. HL-60 Cell Differentiation-Inducing Activity of NIDO-33 (14)
atives [AIDO-00 (7), AIDO-11 (9), AIDO-22 (11), AIDO-30
(13) and AIDO-33 (15)]. Similar reactions using methyl (2-
bromoethyl-6-nitro) benzoate instead of methyl (2-bro-
moethyl-3-nitro)benzoate gave INIDO-33 (16) and IAIDO
(17), which are regioisomers of NIDO-33 (14) and AIDO-33
(15), respectively.
Effects on HL-60 Cell Differentiation First we exam-
ined the HL-60 cell differentiation-inducing activity of the
prepared compounds (1—17); this was estimated in terms of
Chart 1
lar condensation with methyl (2-bromoethyl-3-nitro)benzoate the nitroblue tetrazolium (NBT)-reducing activity of the cells
gave H4NT (4).17) as previously reported.18) Of course, the measured NBT-posi-
Our previous structural development studies of thalido- tive percentage values differed from experiment to experi-
mide (1) have indicated that replacement of the glutarimide ment, but the results were basically reproducible. A typical
moiety of thalidomide (1) with a non-substituted, a 2-alkyl- set of data is presented in Fig. 2. As shown in the figure, al-
substituted or a 2,6-dialkyl-substituted phenyl ring affords most all the compounds, except NIDO-33 (14), were inactive
compounds which partially retain various biological activi- or had very weak HL-60 cell differentiation-inducing activity
ties of thalidomide (1).3,4) Therefore, amino- or nitrophthal- (2—6% NBT-positive cells) at 10 mM under the experimental
imides with a N-phenyl, N-2-alkylphenyl or N-2,6-di- conditions used (generally, non-treated HL-60 cells contain
alkylphenyl substituent (compounds 6—17) were designed. 2—5% NBT-positive cells). Only NIDO-33 (14) showed ap-
Reactions similar to the condensation reaction used for the parent HL-60 cell differentiation-inducing activity (ca. 25%
preparation of H4NT (4) with methyl (2-bromoethyl-3- NBT-positive cells at 10 mM). This activity was confirmed to
nitro)benzoate using aniline, 2,6-dimethylaniline, 2,6-di- be dose-dependent, as shown in Fig. 3. The HL-60 cell dif-
ethylaniline, 2-isopropylaniline and 2,6-diisopropylaniline, ferentiation-inducing activity of NIDO-33 (14) could be ob-
instead of 3-aminopiperidine-2,6-dione, gave NIDO-00 (6), served at a concentration as low as 1 mM, and almost all the
NIDO-11 (8), NIDO-22 (10), NIDO-30 (12) and NIDO-33 cells were differentiated at the concentration of 23 mM (Fig.
(14), respectively. Reduction of these nitro compounds with 3). To examine the features of the cells differentiated with
hydrogen gas over Pd/C gave the corresponding amino deriv- NIDO-33, Wright-Giemsa staining and fluorescence-acti-

June 2006
857
Fig. 4. Effects of Compounds 1—13 and 15—17 on ATRA-Induced HL-60 Cell Differentiation
vated cell sorter (FACS) analysis using granulocyte/mono- of 4ꢁ2, and 5ꢁ3. In this series of compounds (2—5), the 4-
cyte-specific CD11b FITC (fluorescein isothiocyanate) con- amino analogs (3, 5) are more potent than the corresponding
jugate and monocyte-specific CD14 phycoerythrin conjugate 4-nitro analogs (2, 4, respectively). On the other hand, in the
were performed as described previously.9,19) The results indi- N-arylphthalimide analog series (6—13, 16, 17), there is no
cated that NIDO-33 (14) induces HL-60 cell differentiation clear tendency for 4-amino analogs to be more potent than
to mature monocytes, as does 1a,25-dihydroxyvitamin D3 the corresponding 4-nitro analogs. Remarkably, the most po-
[1,25-(OH)2-VD3]. Typical HL-60 monocytic differentiation tent compound among those prepared was the 4-nitro analog,
inducers are rather complex molecules, including 1,25- NIDO-11 (8), which enhanced the 0.6 nM ATRA-induced
(OH)2-VD3 and 12-O-tetradecanoylphorbol 13-acetate HL-60 cell differentiation ca. 5-fold (almost 100% NBT-pos-
(TPA), while HL-60 granulocytic differentiation inducers in- itive cells) at 10 mM (Fig. 4). The cell differentiation induc-
clude quite simple compounds, such as dimethylsulfoxide tion-enhancing activity of NIDO-11 (8) was shown to be
(DMSO).20) NIDO-33 (14) can be classified as a rather sim- dose-dependent. Wright-Giemsa staining and FACS data in-
ple/small molecule, so it can be considered as unusual for a dicated that NIDO-11 (8) enhances ATRA-inducing HL-60
HL-60 monocytic differentiation inducer. The fact that only cell differentiation to mature granulocytes, though the mech-
NIDO-33 (14) was found to be active among the various anism of the enhancing activity is not known. Similar en-
structurally related derivatives suggests that the structure of hancement of ATRA-induced HL-60 cell differentiation has
the compound is critically recognized in the cell differentia- been found with TPA and tubulin disruptors at low concen-
tion-inducing assay system, though the mechanism involved trations.10,21) However, none of the compounds (1—13, 15—
is not known.
17) possessed TPA-like activity (i.e., monocytic differentia-
Next we investigated the enhancing effect of the prepared tion-inducing activity toward HL-60 cells), nor did they af-
compounds (1—17) on ATRA-induced HL-60 cell differenti- fect tubulin polymerization.
ation (Fig. 4). Although compounds 1—13 and 15—17
Although we found a HL-60 cell differentiation inducer,
themselves do not possess apparent HL-60 cell differentia- NIDO-33 (14), and a potent cell differentiation-induction en-
tion-inducing activity at 10 mM (Fig. 2), they showed potent hancer, NIDO-11 (8), the structure–activity relationship(s)
HL-60 cell differentiation-inducing activity at the same con- remain to be established. The HL-60 cell differentiation-in-
centration in the presence of a physiological concentration ducing activity and the enhancement of this activity de-
(0.6 nM) of ATRA (Fig. 4). In the figure, the level of NBT- scribed here appear to be independent phenomena, because
positive cells (%) that appeared in the presence of 0.6 nM the most active compound in the two assay systems was dif-
ATRA alone (ca. 20%) was defined as unity (1.0), and the ferent, i.e., NIDO-33 (14) and NIDO-11 (8), though their
relative values are presented on the vertical scale. Thalido- structures are very similar. NIDO-33 (14) and NIDO-11 (8)
mide (1) enhanced 0.6 nM ATRA-induced HL-60 cell differ- appear to be unique lead compounds for the development of
entiation ca. 3.2-fold, which is in accordance of our previous novel types of agents which might be useful for the differen-
report.10) As for the IMiDs, CC-5013 (5) showed more potent tiation-inducing therapy of cancers.
activity than thalidomide (1), but CC-4047 (3) was less po-
COX-Inhibitory Activity COX is an enzyme which cat-
tent than thalidomide (1), suggesting that this cell differentia- alyzes the synthesis of prostaglandins from arachidonic acid,
tion-enhancing activity is not the basis for the superiority of and is well-known as a target molecule of non-steroidal anti-
IMiDs as anti-tumor agents compared with thalidomide (1), inflammatory drugs (NSAIDs), including aspirin.22—24) There
though this activity is likely contribute at least in part to the are two isoforms of COX. COX-1 is constitutively expressed
effectiveness of IMiDs.
in most tissues, whereas COX-2 is inducible. Overexpression
Exchange of one carbonyl group of the phthalimide moi- of COX has been detected in various tumors and its role
ety of 4NT (2) and CC-4047 (3) to a methylene group in carcinogenesis and angiogenesis has been well-docu-
[H4NT (4) and CC-5013 (5), respectively] resulted in an in- mented.12—14,24—26) In the past, we suspected that COX is an-
crease of the activity, i.e., the activity decreased in the order other target molecule of thalidomide (1), because the drug is

858
Vol. 54, No. 6
Fig. 5. COX-Inhibitory Activities of Compounds 1—17 (100 mM)
effective against colon and prostate cancers and possesses Table 1. HUVEC Tube Formation-Inhibiting Activity of Compounds 1—
anti-angiogenic activity,27,28) in which COX plays an impor-
17 (100 mM)
tant role. Indeed, we found that thalidomide (1) itself pos-
Compounds
Inhibition of tube formation (%)
sesses COX-inhibiting activity.11) We therefore examined the
COX-inhibitory activity of IMiDs (3, 5) and their derivatives
(Fig. 5). Thalidomide (1) showed weak inhibitory activity to-
ward both COX-1 and COX-2, as already reported.11) IMiDs
(3, 5), however, showed only very weak or no COX-in-
hibitory activity, suggesting that COX inhibition might not
be related to the superior immunomodulatory activity of
IMiDs (3, 5).
Thalidomide (1)
4NT (2)
CC-4047 (3)
H4NT (4)
26
32
21
25
32
8
33
28
19
0
8
0
0
33
22
21
16
CC-5013 (5)
NIDO-00 (6)
AIDO-00 (7)
NIDO-11 (8)
AIDO-11 (9)
NIDO-22 (10)
AIDO-22 (11)
NIDO-30 (12)
AIDO-30 (13)
NIDO-33 (14)
AIDO-33 (15)
INIDO-33 (16)
IAIDO-33 (17)
Among the compounds prepared here, compounds 8—17
showed rather potent COX-1-inhibiting activity, and com-
pounds 6, 12, and 13 showed rather potent COX-2-inhibiting
activity (Fig. 5). As regards COX-1-inhibitory activity,
AIDO-30 (13) was the most potent, having a higher potency
than that of aspirin (Fig. 5). The amino analogs (9, 11, 13,
15, 17) were more potent than the corresponding nitro
analogs (8, 10, 12, 14, 16, respectively). Among the 4-amino
analogs, a mono-isopropyl substituent at the ortho position
of the N-phenyl ring seemed to be the best, i.e., the activity
decreased in the order of mono-isopropyl [AIDO-30 test compounds according to the reported method,15) and a
(13)]ꢁ2,6-diethyl [AIDO-22 (11)]ꢁ2,6-dimethyl [AIDO-11 typical set of data is shown in Table 1. Thalidomide (1)
(9)]ꢁ2,6-diisopropyl [AIDO-33 (15)]. On the other hand, showed moderate activity, which is in consistent with our
among the 4-nitro analogs, the mono-isopropyl-substituted previous report.15) One of the IMiDs, CC-5013 (5), showed
analog [NIDO-30 (12)] is almost the least potent, and 2,6-di- more potent activity than thalidomide (1), while the activity
ethyl substitution seemed to be the best, i.e., the activity de- of the other IMiD, CC-4047 (3), was comparable to that of
creased in the order of 2,6-diethyl [NIDO-22 (10)]ꢁ2,6-di- thalidomide (1). The nitro analog, 4NT (2), showed potent
isopropyl [NIDO-33 (16)]ꢁ2,6-dimethyl [NIDO-11 (8)]ꢂ activity, comparable with that of CC-5013 (5).
mono-isopropyl [NIDO-30 (12)].
Among the N-aryl derivatives prepared (6—17), NIDO-22
As for COX-2-inhibitory activity, AIDO-30 (13) was also (10), (12) and AIDO-30 (13) were completely inactive, but
the most potent among the prepared compounds. Its COX-2- the others showed moderate to potent tube formation-in-
inhibitory activity was higher than that of aspirin (Fig. 5), hibitory activity on HUVEC (Table 1). AIDO-00 (7) and
though it would still be classified as a COX-1-selective in- NIDO-33 (14) showed potent activity, comparable with that
hibitor. AIDO-30 (13) should be a useful lead compound for of CC-5013 (5). No clear structure–activity relationship
the development of a unique class of COX inhibitors, be- could be extracted from our data, but, at least, the activity
cause the structure of the compound is rather simple, and it (Table 1) does not seem to correlate with the COX-inhibitory
possesses basic nature, which is rare among reported COX activity of the compounds (Fig. 5). The molecular basis of
inhibitors.
the HUVEC tube formation inhibition observed in our exper-
Anti-angiogenic Activity As mentioned above, the rela- iments should therefore be different from that of the activity
tionship between COX-inhibitory activity and antiangiogenic exhibited by NSAIDs. Further investigation of the mecha-
activity has been well-documented.12—14) So, we examined nism of HUVEC tube formation inhibition and structural de-
the antiangiogenic activity of the prepared compounds by velopment studies to elucidate the structure–activity relation-
means of assay of tube formation-inhibitory activity toward ships are in progress.
HUVEC cells. The HUVEC cells were treated with 100 mM

June 2006
859
Conclusion
1.23 (dd, Jꢂ6.7, 4.9 Hz, 12H), 2.74 (sept, Jꢂ6.7 Hz, 2H), 4.62 (s, 2H), 7.27
(d, Jꢂ8.4 Hz, 2H), 7.42 (t, Jꢂ8.4 Hz, 2H), 7.84—7.74 (m, 3H). Anal. Calcd
for C20H22N2O3: C, 70.99; H, 6.55; N, 8.28. Found: C, 70.97; H, 6.56; N,
8.21. mp: 238—240.
We examined the effects of thalidomide (1), IMiDs (3, 5)
and their analogs (2, 4, 6—17) on HL-60 cell differentiation,
COX activity and angiogesis. Although we could not eluci-
date the structural basis for the superiority of IMiDs (3, 5)
over thalidomide, we identified analogs with HL-60 cell dif-
ferentiation-inducing activity [NIDO-33 (14)], enhancing ac-
tivity on ATRA-induced HL-60 cell differentiation [NIDO-
11 (8)], COX-inhibitory activity [AIDO-30 (13)], and
HUVEC tube formation-inhibitory activity [AIDO-00 (7)
and NIDO-33 (14)]. Thalidomide (1) possesses HL-60 cell
differentiation-enhancing, COX-inhibitory, and anti-angio-
genic effects, and IMiDs (3, 5) possess HL-60 cell differenti-
ation-enhancing and anti-angiogenic effects. In this sense, it
can be said that separation of these activities by structural de-
velopment based on IMiDs (3, 5) has been partially success-
ful. In addition, NIDO-33 (14) showed HL-60 cell differenti-
ation-inducing activity, which is not elicited by thalidomide
(1) or IMiDs (3, 5). Our results imply that further structural
1
IAIDO-33 (17): MS (FAB): Mꢃ1ꢂ309. H-NMR (500 MHz/CDCl3/d):
1.21 (d, Jꢂ6.6 Hz, 12H), 2.82 (sept, Jꢂ6.6 Hz, 2H), 4.48 (s, 2H), 6.65 (d,
Jꢂ8.1 Hz, 1H), 6.75 (d, Jꢂ7.3 Hz, 1H), 7.39—7.23 (m, 4H). Anal. Calcd for
C20H24N2O: C, 77.89; H, 7.84; N, 9.08. Found: C, 77.96; H, 7.86; N, 9.00.
mp: 190—191.5.
Cell Culture HL-60 cells were cultured in RPMI1640 medium supple-
mented with 10% heat-inactivated fetal bovine serum (FBS; Gibco BRL) at
37 °C under a 5% CO2 atmosphere. HUVECs were cultured in EBM-2
medium supplemented with growth factors (hEGF, VEGF, hFGF-B, and R3-
IGF-1, as well as FBS) at 37 °C under a 5% CO2 atmosphere.
Assay of Cell Differentiation-Inducing Activity Measurement of HL-
60 cell differentiation was performed as described previously.9,18) Briefly,
HL-60 cells were incubated in RPMI 1640 medium in the presence or ab-
sence of a test compound (10 mM) with or without 0.6 nM ATRA for 3 d.
Treated HL-60 cells were mixed with phosphate-buffered saline (PBS) con-
taining 0.2% nitroblue tetrazolium (NBT) and 20 mM TPA in a 1 : 1 (v/v)
ratio and incubated at 37 °C for 20 min. NBT positivity was measured by
counting 200—300 cells and the results were expressed as the percentage of
NBT-positive cells. The cell differentiation was also confirmed morphologi-
development studies of thalidomide (1) and IMiDs (3, 5) cally by microscopy after Wright-Giemsa staining, using all-trans retinoic
acid (ATRA) and 1a,25-dihydroxyvitamin D3 [1,25-(OH)2-VD3] as positive
control compounds, which have been established to induce differentiation of
HL-60 cells to mature granulocytes and monocytes, respectively. The treated
HL-60 cells were analyzed by means of FACS to characterize the differenti-
ated cell type.19) Briefly, HL-60 cells (1ꢄ106 cells), treated or not treated
with a test compound (10 mM), were washed with phosphate-buffered saline
(PBS) and incubated with fluorescent agent-conjugated antibody [mono-
clonal anti-human CD11b FITC (fluorescein isothiocyanate) conjugate,
mouse IgG1 isotype, Sigma (FITC-CD11b) or monoclonal anti-human
CD14 clone UCHM-1 PE (R-phycoerythrin) conjugate, mouse immunoglob-
ulin, Sigma (PE-CD14)] in PBS containing 0.5% bovine serum albumin
(BSA) and 0.1% NaN3 (staining buffer) at 4 °C for 30 min. After the incuba-
tion, the cells were washed with staining buffer, treated with paraformalde-
hyde (1% in PBS), and then analyzed with a flow cytometer (Cytomics
FC500, Beckman Coulter).9,19)
Assay of Anti-angiogenesis Activity HUVECs were plated on Matrigel
and treated with test compounds (100 mM) for 6 h, and tube formation was
measured as previously reported.15,29) Briefly, six-well plates were coated
with 1.5 ml of the Matrigel basement membrane matrix (Becton Dickinson)
and allowed to gel at 37 °C under under 5% CO2 in air for 30 min. Then,
HUVECs were plated at 5.0ꢄ105 cells/well in DMEM containing the vehi-
cle (0.5% DMSO) with 10% FBS in the presence or absence of a test com-
pound and incubated at 37 °C under 5% CO2 in air for 6 h. After incubation,
each well was photographed using a ꢄ5 objective to analyze tube formation.
The corresponding area was measured (as the number of pixels) using Meta-
Morph software (Universal Imaging, Downingtown, PA, U.S.A.).
might allow us to fully separate these biological activities,
leading to a range of unique biologically active compounds.
Further structural development studies and investigation of
the molecular mechanisms of action are in progress.
Experimental
1
Chemicals NIDO-00 (6): MS (FAB): Mꢃ1ꢂ255. H-NMR (500 MHz/
CDCl3/d): 5.35 (s, 2H), 7.26 (t, Jꢂ7.7 Hz, 1H), 7.48 (t, Jꢂ7.7 Hz, 2H),
7.76 (t, Jꢂ7.7 Hz, 1H), 7.91 (d, Jꢂ7.7 Hz, 2H), 8.28 (d, Jꢂ7.7 Hz, 1H), 8.47
(d, Jꢂ7.7 Hz, 1H). Anal. Calcd for C14H10N2O3: C, 66.14; H, 3.96; N, 11.02.
Found: C, 65.91; H, 4.18; N, 10.91. mp: 131—133.5.
AIDO-00 (7): MS (FAB): Mꢃ1ꢂ225. 1H-NMR (500 MHz/CDCl3/d):
4.47 (s, 2H), 5.51 (s, 2H), 6.82 (d, Jꢂ7.3 Hz, 1H), 6.95 (d, Jꢂ7.3 Hz, 1H),
7.16 (t, Jꢂ7.3 Hz, 1H), 7.20 (t, Jꢂ7.9 Hz, 1H), 7.43 (t, Jꢂ7.9 Hz, 2H), 7.86
(d, Jꢂ7.9 Hz, 2H). Anal. Calcd for C14H12N2O: C, 74.98; H, 5.39; N, 12.49.
Found: C, 74.93; H, 5.57; N, 12.45. mp: 181—182.
NIDO-11 (8): MS (FAB): Mꢃ1ꢂ283. 1H-NMR (500 MHz/CDCl3/d):
2.21 (s, 6H), 5.07 (s, 2H), 7.19 (d, Jꢂ7.2 Hz, 2H), 7.25 (t, Jꢂ7.2 Hz, 1H),
7.78 (t, Jꢂ7.7 Hz, 1H), 8.32 (d, Jꢂ7.7 Hz, 1H), 8.48 (d, Jꢂ7.7 Hz, 1H).
Anal. Calcd for C16H14N2O3: C, 68.07; H, 5.00; N, 9.92. Found: C, 67.81; H,
5.14; N, 9.82. mp: 138.5—139.5.
AIDO-11 (9): MS (FAB): Mꢃ1ꢂ253. 1H-NMR (500 MHz/CDCl3/d):
2.20 (s, 6H), 3.75 (s, 2H), 4.44 (s, 2H), 6.90 (d, Jꢂ7.5 Hz, 1H), 7.15 (d,
Jꢂ7.5 Hz, 2H), 7.21 (t, Jꢂ7.5 Hz, 1H), 7.35 (t, Jꢂ7.5 Hz, 1H), 7.42 (d,
Jꢂ7.9 Hz, 1H). Anal. Calcd for C16H16N2O: C, 76.16; H, 6.39; N, 11.10.
Found: C, 76.12; H, 6.50; N, 11.16. mp: 211—212.5.
Assay of Cyclooxygenase-Inhibitory Activity Inhibitory activity of
test compounds (100 mM) on COX-1 and COX-2 was determined with the
use of a Colorimetric COX (ovine) Inhibitor Screening Assay Kit (Cayman,
No. 760111), according to the protocol recommended by the supplier.11)
NIDO-22 (10): MS (FAB): Mꢃ1ꢂ311. 1H-NMR (500 MHz/CDCl3/d):
1.22 (t, Jꢂ7.7 Hz, 6H), 2.57—2.43 (m, 4H), 7.25 (d, Jꢂ7.7 Hz, 2H), 7.38 (t,
Jꢂ7.7 Hz, 1H), 7.79 (t, Jꢂ7.7 Hz, 1H), 8.32 (d, Jꢂ7.7 Hz, 1H), 8.49 (d,
Jꢂ7.7 Hz, 1H). Anal. Calcd for C18H18N2O3: C, 69.66; H, 5.85; N, 9.03.
Found: C, 69.69; H, 5.99; N, 8.86. mp: 160.5—162.
Acknowledgements The first two authors of this paper (H.F. and T.N.)
contributed equally to this work. We thank Mr. Ryo Kikuchi (Univ. Tokyo)
for helpful discussions and technical assistance. The work described in this
paper was partially supported by Grants-in-Aid for Scientific Research from
The Ministry of Education, Culture, Sports, Science and Technology, Japan,
and the Japan Society for the Promotion of Science.
AIDO-22 (11): MS (FAB): Mꢃ1ꢂ281. 1H-NMR (500 MHz/CDCl3/d):
1.18 (t, Jꢂ7.7 Hz, 6H), 2.56—2.40 (m, 4H), 3.72 (s, 2H), 4.43 (s, 2H), 6.89
(d, Jꢂ7.7 Hz, 1H), 7.20 (d, Jꢂ7.7 Hz, 2H), 7.31 (t, Jꢂ7.7 Hz, 1H), 7.34 (t,
Jꢂ7.7 Hz, 1H), 7.40 (d, Jꢂ7.7 Hz, 1H). Anal. Calcd for C18H18N2O·
1/8H2O: C, 76.50; H, 7.22; N, 9.91. Found: C, 76.75; H, 7.25; N, 9.72. mp:
226—227.
NIDO-30 (12): MS (FAB): Mꢃ1ꢂ297. 1H-NMR (500 MHz/CDCl3/d):
1.25 (d, Jꢂ6.9 Hz, 6H), 2.94 (sept, Jꢂ6.8 Hz, 1H), 5.18 (s, 2H), 7.22 (d,
Jꢂ7.5 Hz, 1H), 7.30 (t, Jꢂ7.5 Hz, 1H), 7.42 (d, Jꢂ7.5 Hz, 1H), 7.46 (t,
Jꢂ7.5 Hz, 1H), 7.78 (t, Jꢂ7.7 Hz, 1H), 8.30 (d, Jꢂ7.7 Hz, 1H), 8.48 (d,
Jꢂ7.7 Hz, 1H),. Anal. Calcd for C17H16N2O3: C, 68.91; H, 5.44; N, 9.45.
Found: C, 68.75; H, 5.54; N, 9.42. mp: 157—158.
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