Toward the development of chemoprevention agents. Part II: Chemo-enzymatic synthesis and anti-inflammatory activities of a new class of 5-amino-2-substitutedphenyl-1,3-dioxacycloalkanes

Article abstract of DOI:10.1016/j.bmc.2007.06.018

A new series of optically pure 5-amino-2-substitutedphenyl-1,3-dioxacycloalkanes were designed and synthesized via a chemo-enzymatic combined method to develop new chemoprevention agents. Twenty-four of newly synthesized compounds significantly inhibited xylene-induced rat ear edema and exhibited comparable or better anti-inflammatory activities than the reference drug aspirin. Treatment of these anti-inflammatory agents did not prolong the tail bleeding time in rat. In addition, 5-amino-2-substitutedphenyl-1,3-dioxacycloalkanes exhibited good membrane permeability based on in vitro Caco-2 cell monolayer permeability assay. Furthermore, some preliminary structure-activity relationships were further analyzed among these compounds. Taken together, 5-amino-2-substitutedphenyl-1,3-dioxacycloalkanes may represent a new class of anti-inflammatory drugs with safer pharmacological profile.

Full text of DOI:10.1016/j.bmc.2007.06.018

Bioorganic & Medicinal Chemistry 15 (2007) 6273–6290
Toward the development of chemoprevention agents. Part II:
Chemo-enzymatic synthesis and anti-inflammatory activities of
a new class of 5-amino-2-substitutedphenyl-1,3-dioxacycloalkanes
b
c,
a,
*
Keli Gu,a Lanrong Bi,a Ming Zhao,a,* Chao Wang, Jingfang Ju and Shiqi Peng
*
aCollege of Pharmaceutical Sciences, Capital Medical University, Beijing 100054, PR China
bCollege of Pharmaceutical Sciences, Peking University, Beijing 100083, PR China
cMitchell Cancer Institute-USA, 307 N. University Blvd, Mobile AL 36688-0002, USA
Received 19 March 2007; revised 7 June 2007; accepted 8 June 2007
Available online 13 June 2007
Abstract—A new series of optically pure 5-amino-2-substitutedphenyl-1,3-dioxacycloalkanes were designed and synthesized via a
chemo-enzymatic combined method to develop new chemoprevention agents. Twenty-four of newly synthesized compounds signif-
icantly inhibited xylene-induced rat ear edema and exhibited comparable or better anti-inflammatory activities than the reference
drug aspirin. Treatment of these anti-inflammatory agents did not prolong the tail bleeding time in rat. In addition, 5-amino-2-sub-
stitutedphenyl-1,3-dioxacycloalkanes exhibited good membrane permeability based on in vitro Caco-2 cell monolayer permeability
assay. Furthermore, some preliminary structure–activity relationships were further analyzed among these compounds. Taken
together, 5-amino-2-substitutedphenyl-1,3-dioxacycloalkanes may represent a new class of anti-inflammatory drugs with safer phar-
macological profile.
ꢀ 2007 Elsevier Ltd. All rights reserved.
1. Introduction
Chemoprevention is an exciting research field in cancer
research. In recent years, aspirin and other NSAIDs
show promise as chemoprevention agents.1 Possible tar-
gets include cancers of the colon, stomach, breast, and
lung. The most convincing evidence is from colorectal
cancer. Compelling data from epidemiological studies
suggest that NSAIDs reduce the risk of colorectal cancer
by around a half.7 For this reason, the US food and
Drug Administration has approved the use of celecoxib
in the prevention of colorectal polyps in patients with
familial adenomatous polyposis.6
Inflammation can originate from both infectious and
non-infectious processes of chronic injury or irritation.1
The inflammatory response leads to the recruitment of
mast cells and leukocytes to the site of damage with sub-
sequent release of free radicals, including reactive oxy-
gen species.1 These free radicals are known to damage
macromolecules, including lipids and DNA.2,3 In addi-
tion to the cellular and genomic damage that occurs as
part of free-radical activity and other by-products, the
release of eicosanoids triggers cell proliferation.4 The
combination of these processes facilitates carcinogene-
sis.4 Inflammation is now considered a well-established
cancer risk factor. So, it is not surprising that the regular
use of aspirin and other non-steroidal anti-inflammatory
drugs (NSAIDs) is related to a decreased risk of several
types of cancer.5–7
An ideal chemoprevention agent should be safe and
non-toxic over a long term use. Current NSAIDS have
serious toxicity rate that quickly exceeds any benefit
from cancer chemoprevention. In contrast, low-dose
aspirin is worth evaluating, especially because of the po-
tential for simultaneous cardiovascular risk reduction.
In addition, development of new NSAIDS, with less tox-
icity, is another approach toward making the toxicity-
benefit ratio more favorable for the use of these agents
for cancer chemoprevention.
Keywords: Chemoprevention; Chemo-enzymatic synthesis; 2,5-Disub-
stituted-dioxacycloalkanes; Anti-inflammatory activity; Structure–ac-
tivity relationship study.
*
Corresponding authors. Tel.: +1 251 460 7393; fax: +1 251 460 6994
(J.J.); tel.: +86 10 8391 1528; fax: +86 10 8391 1528 (S.P.).; e-mail
addresses: sqpeng@bjmu.edu.cn; jju@usouthal.edu
1,3-Dioxane derivatives have been reported recently
having anti-inflammatory, anti-cancer, and reperfusion
0968-0896/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2007.06.018

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K. Gu et al. / Bioorg. Med. Chem. 15 (2007) 6273–6290
injury protection effects through their anti-proliferative
and anti-inflammatory activities in human neutrophils
and tumor cells.8 We have been investigating 2,5-
disubstituted-1,3-dioxanes as a new class of anti-inflam-
matory agents.9–12 In contrast to classical, acidic
non-steroidal anti-inflammatory agents (NSAIDs), these
new analogues of 1,3-dioxanes are basic. To further im-
prove the efficiency and safety of anti-inflammatory
agents, in this study a new series of optically pure 5-ami-
no-2-substitutedphenyl-1,3-dioxacycloalkanes were syn-
thesized via a chemo-enzymatic combined method.
The in vivo anti-inflammatory activities of these com-
pounds were evaluated using the xylene-induced mouse
ear edema model. In addition, we have reported here
the preliminary structure–activity relationship studies.
these compounds (5a–j, 6 and 60a,b, 7 and 70c–e, 8 and
80c–e). We hypothesized that this may be due to the ra-
pid exchange of hydrogens between both the 5-NH2 and
2-substitution with CHCl3-d solvent, resulting in the
hydrogens unable to participate in subsequent NOE
experiment.
To make the stereochemistry of 5a–j assignable they
were then converted to 9a–j, of which the benzoyllation
on the 5-NH2 group and consequently stabilize the
hydrogen atom of 5-NH2 for participation in the
NOE difference spectra. For example, a positive NOE
effect was observed in 9a–e between the NH at the
5-position and the proton of the phenyl at the 2-posi-
tion, indicating that these two substituents on the ring
were arranged in a syn-configuration. Thus, based on
the configuration of 9a–e, the substituents at both 2- and
5-positions of 5a–e were accordingly assigned in a
syn-arrangement. Similarly, for 9f–j, the positive NOE
signals were observed between the substituents at the
2-, 4-, and 5-positions, which indicated that both substit-
uents on the rings of 9f–j are in syn-arrangement. Fur-
thermore, based on the configuration of 9f–j, the
substituents at the 2-, 4-, and 5-positions on the ring
of 5f–j were then assigned to be in syn-arrangement. In
a similar manner, to assign the stereochemistry of 6
and 60a,b, 7 and 70c–e, 8 and 80c–e, both compounds
again underwent benzoylacetylation reactions to afford
10 and 100a,b, 11 and 110c–e, 12 and 120c–e, respectively.
As a result, the stereochemistry of 6 and 60a,b, 7 and
70c–e, 8 and 80c–e was assigned based on the configura-
tion of 10 and 100a,b, 11 and 110c–e, 12 and 120c–e,
respectively.
2. Results and discussion
2.1. Synthesis of aminodiols12
Starting from the optically active amino acids 1a–d, in
the presence of thionyl chloride and methanol, the cor-
responding methyl esters 2a–d were readily prepared
with high yields, which then were subjected to KBH4
reduction. Accordingly, aminodiols 3a–d were obtained
in moderate yields (39–62%). Similarly, after treatment
of 2a–d with phenylacetyl chloride, followed by KBH4
reduction, a series of phenylacetamidodiols 30a–d were
obtained in excellent yields (91–93%). To avoid any pos-
sible racemization all the reactions involved in the prep-
aration of the aminodiols should be carried out at 0 ꢁC
to room temperature (Scheme 1).
2.2. Synthesis of 5-amino-2-substitutedphenyl-1,3-diox-
acycloalkanes via the acetalization
2.3. Synthesis of 5-amino-2-substitutedphenyl-1,3-dioxane
via chemo-enzymatic method
Using p-toluenesulfonic acid as catalyst, aminodiols
3a–d were subjected to acetalization with substituted
benzaldehydes 4a–e. As a result, a series of 5-amino-2-
substitutedphenyl-1,3-dioxacycloalkanes, 5a–j, 6 and
60a,b, 7 and 70c–e, 8 and 80c–e, were obtained in low
to moderate yields (Scheme 2).
It was worth to point out, a mixture of cis- and trans-
isomers was obtained when 3c,d were treated with
substituted benzaldehydes 4a–e. In contrast, when 3a,b
were treated with aldehyde, 4a–e exclusively gave cis-
products 5a–j. No more sterically demanding trans-
products were observed even changing the mole ratio
of the starting materials and the reaction conditions
(e.g., catalysts and reaction temperature). To enable a
more precise elucidation of the structure–activity rela-
tionship, we attempted different approaches to prepare
the trans-products of 5-amino-2-substitutedphenyl-1,3-
dioxane.
However, through nuclear Overhauser effect (NOE)
experiments, no positive NOE signals were observed in
R
H
R
H
R
H
ii
i
C6H5CH2CONH
H2N
CO2CH3
CO2CH3
CH2OH
H2N
CO2H
Our previous study indicated the stereoselectivity of 2,5-
disubstituted-1,3-dioxanes could be tuned by the substit-
uents of their precursors. On the basis of this finding,
aminodiols 3a,b were replaced by 2-phenylacetamido-
1,3-diols 30a,b, which were then subjected to the acetaliza-
tion with 4a–e. As we expected, a pair of (cis)- and
(trans)-products, 13 and 130a–j, was obtained in good
yield (Scheme 3).12
2'a-d
2a-d
1a-d
iii
iii
R
R
C6H5CH2CONH
H2N
CH2OH
H
H
3'a-d
3a-d
Scheme 1. Synthetic route to 3 and 30a–d. Reagents and conditions: (i)
SOCl2 and MeOH; (ii) C6H5CH2COCl/THF; (iii) KBH4, THF. In 1a,
2a, 20a, 3a, and 30a: R = CH2OH; 1b, 2b, 20b, 3b, and 30b:
R = CH(CH3)OH; 1c R = CH2CO2H; 1d R = CH2CH2CO2H; 2c and
20c: R = CH2CO2CH3; 2d and 20d: R = CH2CH2CO2CH3; 3c and 30c:
R = CH2CH2OH; 3d and 30d: R = CH2CH2CH2OH.
Next, difficulties were encountered during the deprotec-
tion of N-phenylacetyl group of 13 and 130a–j. It was
observed that racemization occurred to some extent
under the strong basic deprotection condition. In

K. Gu et al. / Bioorg. Med. Chem. 15 (2007) 6273–6290
6275
Scheme 2. Synthetic route to 5a–j, 6 and 60a,b, 7 and 70c–e, 8 and 80c–e. Reagents and condition: (i) CHCl3/THF, rt, p-toluenesulfonic acid (catalyst),
anhydrous Na2SO4; (ii) PhCOCl/TEA, CHCl3, 0 ꢁC to rt. In 4a: R = H; 4b: R = 4-CH3; 4c: R = 4-Cl; 4d: R = 4-NO2; 4e: R = 3-NO2; 6a, 60a, 10a and
100a R0 = H; 6b, 60b, 10b and 100b R0 = CH3.
the removal of N-phenylacetyl protection group of
the dioxacycloalkanes.
NH2
R''
NHCOCH2C6H5
ii
ii
R''
O
O
H
H
H
H
O
R'
O
R'
H
5a-j
H
13a-j
3'a,b
i
Often, the substrate specificity of enzymes limits their
application in synthetic organic chemistry. However,
penicillin acylase from Escherichia coli can recognize a
broad range of substrates. Besides benzylpenicillin, it ac-
cepts a range of substituted phenylacetylamides with
high specificity for the phenylacetyl group but low spec-
ificity for the amine moiety. In fact, the mild and neutral
enzymatic method provided reliable access to our de-
sired compounds. It was noticed, even in the presence
of organic co-solvents like methanol, penicillin acylase
could recognize a broad range of N-phenylacetyl
protected dioxacycloalkanes. Seemingly, it tolerated
variations in the substituents and ring size of diox-
acycloalkanes. Nevertheless, the hydrolysis rates varied
with the steric and electronic factors of the 2-substitu-
tion of dioxacycloalkanes, as well the solubility of the
substrates under the reaction conditions.
CHO
R
NHCOCH2C6H5
NH2
R''
4a-e
R''
O
O
O
H
O
H
13'a-j
R'
R'
5'a-j
Scheme 3. Synthesis of 5 and 50a–j via a chemo-enzymatic combined
method. Reagents and conditions: (i) CHCl3/THF, rt, p-toluenesulf-
onic acid (catalyst), anhydrous Na2SO4; (ii) penicillin acylase, rt, pH
6.0, 4 h.
search of a mild deprotection condition, the use of
enzymatic method attracted our attention.13–19 We at-
tempted to remove the N-phenylacetyl protecting group
of these compounds with enzymatic hydrolysis. We dis-
covered that penicillin acylase is a valuable agent for

6276
K. Gu et al. / Bioorg. Med. Chem. 15 (2007) 6273–6290
Upon treatment of an aqueous solution of 13 and 130a–j
with the penicillin acylase at pH 6.0 and 37 ꢁC, the
N-phenylacetyl protecting group was removed smoothly
to give the expected 5 and 50a–j without any undesired
side reaction. HPLC analysis indicated no racemization
occurred during the enzymatic hydrolysis.
respectively. Accordingly, 7 and 70c–e and 8 and 80c–e
were obtained in good to excellent yields.
In summary, we have developed a straightforward and
efficient chemo-enzymatic method to produce a series
of new 5-amino-2-substitutedphenyl-1,3-dioxacycloalk-
anes with high optical purity and good yields. This
method was easily scaled up to yield gram scale quantity
of this new class of compounds. These results suggested
that the enzymatic removal of the N-phenylacetyl pro-
tecting group of dioxacycloalkanes could advanta-
geously be applied in heterocyclic chemistry.
Under the similar condition, the acetalization of
(E)-phenylvinyl aldehyde 4f with 2-phenylacetamido-
1,3-diols 30a,b stereoselectively provided two pairs of
(cis)- and (trans)-products, namely 14 and 140a,b which
were then subjected to enzymatic hydrolysis.12 Accord-
ingly, the de-protected products 6 and 60a,b were
obtained in good yields (Scheme 4).
2.4. In vivo anti-inflammatory activities evaluated in the
ear edema model and SAR studies
To test the generality of the enzymatic hydrolysis, we
then expanded this method for the large scale prepara-
tion of optically pure 7- and 8-membered 5-amino-2-
substitutedphenyl-1,3-dioxacycloalkanes (Scheme 5).
Specifically, the acetalization of benzaldehyde 4c–e and
2-phenylacetamido-1,4-diol 30c provided a series of iso-
mers containing 7-membered dioxacycloalkanes, 15
and 150c–e. Under similar condition, a series of 8-mem-
bered dioxacycloalkanes, 16 and 160c–e, were prepared
stereoselectively. Subsequently, 15 and 150c–e and 16
and 160c–e were subjected to enzymatic hydrolysis,
All the newly synthesized 5-amino-2-substitutedphenyl-
1,3-dioxacycloalkanes were then evaluated for their
anti-inflammatory activity by using a xylene-induced
ear edema model assay. Briefly, the in vivo assay in-
volved the test compounds being administerted orally
in 0.5% carboxymethyl cellulose (CMC) suspension.
Each compound was initially tested at a concentration
of 20 mg/kg. It was observed that 24 of the tested com-
pounds showed significant inhibition against xylene-in-
duced inflammation in mice as compared with the
NH2
R
NHCOCH2C6H5
R
H
C
H
C
O
O
H
H
ii
O
C
H
C6H5
O
C
H
C6H5
H
H
6a,b
H
C
14a,b
3'a,b
i
C
H
CHO
NHCOCH2C6H5
NH2
R
ii
4f
R
O
O
H
H
O
H
H
O
H
H
C
H
C
C6H5
C
H
C
C6H5
14'a,b
6'a,b
Scheme 4. Synthesis of 6 and 60a,b via chemo-enzymatic combined method. Reagents and conditions: (i) CHCl3/THF, rt, p-toluenesulfonic acid
(catalyst), anhydrous Na2SO4; (ii) penicillin acylase, rt, pH 7.5, 4–5 h.
NH2
NHCOCH2C6H5
O
ii
O
H
H
H
H
R
H
R
O
O
H
R
H
3'c
i
15c-e
7c-e
NH2
NHCOCH2C6H5
O
ii
O
H
O
O
R
15'c-e
7'c-e
CHO
R
NHCOCH2C6H5
O
NH2
4c-e
ii
O
H
H
H
O
R
O
R
3'd
H
H
16c-e
8c-e
i
NH2
NHCOCH2C6H5
O
ii
O
H
O
H
O
H
R
R
8'c-e
16'c-e
Scheme 5. Synthesis of 6a,b, 7c–e, 70c–e, 8c–e, and 80c–e via chemo-enzymatic combined method. Reagents and conditions: (i) CHCl3/THF, rt,
p-toluenesulfonic acid (catalyst), anhydrous Na2SO4; (ii) penicillin acylase, rt, pH 7.5.

K. Gu et al. / Bioorg. Med. Chem. 15 (2007) 6273–6290
6277
control (Table 1), indicating that these compounds pos-
sess potent anti-inflammatory activity. It was especially
noted that these compounds exhibited even higher
anti-inflammatory activities than that of the standard
reference drug aspirin. Subsequently, those compounds
with significant anti-inflammatory activity (5 and
50h–j) were administerted in a series of lower concentra-
tion doses to enable a detailed pharmacological activity
profile (Table 2).
the anti-inflammatory activities of the compounds con-
taining 7-membered dioxacycloalkanes (7c: 32.1%; 7d:
21.8%; 7e: 24.7%; 70c: 31.1%; 70d: 23.1%; 70e: 23.7%)
were apparently decreased over their corresponding 6-
membered counterparts (5c: 64.1%; 5d: 41.0%; 5e:
56.7%; 50c: 64.7%; 50d: 42.0%; 50e: 56.1%). Similar
Table 2. Anti-inflammatory activities of 5 and 50h–j at different doses
against xylene-induced ear edema in mice
Structure–activity relationship studies were also per-
formed on a series of phenylacetaminodiol compounds
to assess whether the presence of 1,3-dioxacycle rings
is required for anti-inflammatory activity. Observations
that phenylacetaminodiols 30a–d were ineffective as anti-
inflammatory compounds suggested that the rigid 1,3-
dioxacycle ring should be retained. This observation
was consistent with our previously reported results.
Dose
(mg/kg)
Edema weight
(X SD mg)
Inhibition
(%)
CMC
Aspirin
5h
3.12 0.55
1.85 0.72
1.89 0.48
1.80 0.51
2.20 0.56
2.19 0.49
1.87 0.50
2.21 0.53
1.42 0.49b
1.31 0.50b
1.46 0.48b
1.68 0.53b
1.35 0.49b
1.72 0.46b
1.04 0.29a
0.67 0.52c
1.20 0.42a
1.07 0.35c
0.69 0.51c
1.22 0.44a
30
5
40.7
39.4
42.3
29.4
29.8
40.1
29.2
54.5
58.0
53.2
46.2
56.7
44.9
66.7
78.5
61.5
65.7
77.9
60.9
5i
5
5j
5
50h
50i
50j
5h
5
5
To examine the importance of the spatial disposition, all
of the isomers with different stereochemistries were com-
pared. For example, compounds 5a–j and 50a–j, their
comparable anti-inflammatory activities indicated the
stereochemical constraints of these 5-amino-2-substitut-
edphenyl-1,3-dioxacycloalkanes having little or no effect
on their anti-inflammatory activities (5a: 51.6% vs 50a:
50.6%; 5b: 50.0% vs 50b: 49.4%; 5c: 64.1% vs 50c:
64.7%; 5d: 41.0% vs 50d: 42.0%; 5e: 56.7% vs 50e:
56.1%; 5f: 52.9% vs 50f: 51.9%; 5g: 54.5% vs 50g: 53.8;
5h: 66.7% vs 50h: 67.9%; 5i: 78.5% vs 50i: 77.9; 5j:
61.5% vs 50j: 60.9%).
5
10
10
10
10
10
10
20
20
20
20
20
20
5i
5j
50h
50i
50j
5h
5i
5j
50h
50i
50j
N = 11.
a Compare to 10 mg/kg group P < 0.05.
b Compare to 5 mg/kg group P < 0.05.
c Compare to 10 mg/kg group P < 0.01.
To investigate the effect of heterocyclic ring sizes, the
anti-inflammatory activities of 6-, 7-, and 8-membered
dioxacycloalkanes were compared. It was noticed that
Table 1. Anti-inflammatory activities of 5-amino-2-substitutedphenyl-1,3-dioxacycloalkanes against xylene-induced ear edema in mice
Agents
Edema weight (X SD mg)
Inhibition (%)
Agents
Edema weight (X SD mg)
Inhibition (%)
CMC
5a
5b
5c
5d
5e
5f
3.12 0.55
Aspirin
50a
50b
50c
50d
50e
50f
1.85 0.72b
1.54 0.55b
1.58 0.54b
1.10 0.53b,c
1.81 0.70b
1.37 0.52b
1.50 0.52b
1.44 0.53b
1.00 0.35b,d
0.69 0.51b,d
1.22 0.44b,c
1.54 0.50b,
1.51 0.52b
2.15 0.53b
2.40 0.55b
2.38 0.55b
2.22 0.52b
2.41 0.53b
2.35 0.54b
40.7
50.6
49.4
64.7
42.0
56.1
51.9
53.8
67.9
77.9
60.9
50.6
51.6
31.1
23.1
23.7
28.8
22.8
24.7
1.51 0.53b
1.56 0.21b
1.12 0.57b,c
1.84 0.73b,
1.35 0.51b
1.47 0.50b
1.42 0.35b
1.04 0.29b,d
0.67 0.52b,d
1.20 0.42b,c
1.56 0.52b
1.47 0.48b
2.12 0.56b
2.48 0.53a
2.35 0.52b
2.24 0.54b
2.38 0.55b
2.37 0.56b
51.6
50.0
64.1
41.0
56.7
52.9
54.5
66.7
78.5
61.5
50.0
52.9
32.1
21.8
24.7
28.2
23.7
24.0
5g
5h
5i
50g
50h
50i
5j
50j
6a
6b
7c
7d
7e
8c
8d
8e
60a
60b
70c
70d
70e
80c
80d
80e
For 1,3-dioxacycloalkanes dose = 20 mg/kg, for aspirin dose = 30 mg/kg; n = 11.
a Compare to control, P < 0.05.
b Compare to control, P < 0.01.
c Compare to aspirin, P < 0.05.
d compare to aspirin, P < 0.01.

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K. Gu et al. / Bioorg. Med. Chem. 15 (2007) 6273–6290
tendency was also observed in the case of 8-membered
dioxacycloalkanes (8c: 28.2%; 8d: 23.7%; 8e: 24.0%;
80c: 28.8%; 80d: 22.8%; 80e: 24.7%). These observations
indicated that, the anti-inflammatory activity of
1,3-dioxane was more potent than either 1,3-dioxacyclo-
heptane or 1,3-dioxacyclooctane, suggesting that 6-mem-
bered dioxacycloalkanes cannot be further maximized.
inflammatory activity with aspirin at a dose of 5.0 mg/kg.
Therefore, compounds 5 and 50h–j exhibited dose-
dependent anti-inflammatory action (Table 2).
2.6. Tail bleeding time measurement
The use of conventional NSAIDs is associated with a
number of significant adverse events, including gastroin-
testinal (GI) bleeding, impaired platelet function, and
prolonged bleeding time. The clinical effects of NSAID-
induced platelet dysfunction consist of an increased
bleeding, prolonged surgical bleeding, and additive risk
of significant or life-threatening bleeding in patients tak-
ing anticoagulants. Both surgical andnon-surgical studies
have shown that conventional NSAIDs increase bleeding
time and blood loss. To develop safer anti-inflammatory
agents, it is critical to evaluate if these newly synthetic
agents have deleterious effect on normal haemostasis
leading to bleeding complications. The cutaneous bleed-
ing time model is the most common method used in ani-
mal experiments to investigate bleeding potential in
humans.20–28
It was of interest to observe that the substitution on the
phenyl ring was important for anti-inflammatory activ-
ity. In the case of 5a–j and 50a–j, it seemed that the elec-
tron-withdrawing groups on the phenyl ring endowed
these compounds with good anti-inflammatory profiles
(i.e., 5i: 78.5%; 5j: 61.5%; 50i: 77.9%; 50j: 60.9%). Inter-
estingly, in the same series, the chlorine substitution
on the phenyl ring induced a superior anti-inflammatory
effect (5c: 64.1; 50c: 64.7%; 5h: 66.7%; 50h: 67.9%).
In addition, it was noticed that, compared with N-phenyl-
acetyl and N-benzyl substituted counterparts (9a–j, 10
and 100a,b, 11 and 110c–e, 13 and 130a–j, 14 and
140a,b, 15 and 150c–e, 16 and 160c–e), the free state of
5-amino of 1,3-dioxacycloalkanes (5 and 50a–j, 6 and
60a,b) led to a significant improvement of the anti-
inflammatory activity. This observation suggested that
the amino group was optimal for the anti-inflammatory
potency. Nevertheless, the formation of the hydrogen
bond with the amino moiety might be critical during
the receptor binding process; we strongly suspect the
better anti-inflammatory activities of compounds result
from facile protonation of the amine moieties, which,
in turn, enable the molecules to easily access cellular
membrane. A minor structural alteration can lead to a
marked change in anti-inflammatory activity, which im-
plied that the improved biological profile might result
from a synergic effect.
Male mice (18–22 g) were orally administered with the
new anti-inflammatory agents. After 30, 45, 60, and
90 min of administration, a mouse was placed in a tube
holder with its tail protruding, and a 2-mm cut was
made on the tail. Flowing blood until it stopped was
gently wiped away with a tissue every 30 s until bleeding
ceased and the time recorded. Baseline bleeding times
were similar between treatment groups with an average
of 113–121 s. The treatment with the new anti-inflam-
matory agents at the high doses (200 mg/kg) did not sig-
nificantly prolong bleeding time at any time point (30,
45, 60, and 90 min) compared with NS (Table 3).
2.7. In vitro membrane permeation study
Furthermore, it was noted that the most potent com-
pounds (5 and 50h–j) all exhibited anti-inflammatory
activities much higher than that of the standard refer-
ence aspirin. The ear edema inhibition for 5 and 50h–j
at the dose of 20 mg/kg is calculated to be 66.7%,
78.5%, 61.5%, 65.7%, 77.9%, and 60.9%, respectively,
compared with that of aspirin 40.7% (30 mg/kg). Conse-
quently, they were administered in a series of lower con-
centration doses to enable a detailed pharmacological
activity profile (data summarized in Table 2).
Oral administration is the most commonly used drug-
dosing route. Therefore, the ability to predict the extent
of absorption of drug candidates after oral administra-
tion is crucial during the preclinical evaluation. The oral
bioavailability of drug candidates is influenced by many
factors, including dissolution, absorption, pre-systemic
and systemic metabolism, and elimination. However,
the intestinal mucosa is a significant barrier to oral
delivery of drugs into the systemic circulation. Caco-2
cells possess many structural and functional similarities
to human enterocytes. Recently, Caco-2 cell permeabil-
ity test has been generally used as a screening tool for
assessing drug oral bioavailability during the early stage
of drug development.29–33
2.5. Dose-dependent anti-inflammatory activities
Oral administration of compounds 5 and 50h–j was ob-
served and all produced a dose-dependent anti-inflamma-
tory response in the xylene-induced mouse ear edema test.
For example, compound 5i at doses of 5, 10.0, and
20.0 mg/kg, the anti-inflammatory effect was observed
to be 42.3%, 58.0%, and 78.5%, respectively. Apparently,
5i demonstrated a significant enhancement in its anti-
inflammatory activity with doses above 5.0 mg/kg. Aspi-
rin, by comparison, had anti-inflammatory effect of
40.7% at a dose of 30 mg/kg. Similarly, the anti-inflamma-
tory activity for compound 50i was 40.1%, 56.7%, and
77.9%, at doses of 5, 10.0, and 20.0 mg/kg, respectively.
Additionally, compound 5h exhibited comparable anti-
Caco-2 cell monolayer was utilized as an in vitro
model of the intestinal mucosa to assess the mem-
brane permeability for these newly synthesized com-
pounds. Drug permeability is difficult to measure,
thus this assay provides a convenient way to measure
permeability based on the apparent permeability coef-
ficient (Papp). Papp (A ! B) is the permeability from
the apical to the basolateral side (intestine to blood),
and Papp (B ! A) is the permeability from the baso-
lateral to the apical side (blood to intestine). It was

K. Gu et al. / Bioorg. Med. Chem. 15 (2007) 6273–6290
Table 3. Effect of 1,3-dioxacycloalkanes on the tail bleeding time ðX ꢁ SDsÞ of mice
6279
Compound
Before drug administration
Post-drug administration
30 min
45 min
60 min
90 min
NS
5a
117.9 8.2
118.4 8.4
117.2 7.9
119.3 8.5
120.3 8.6
118.6 7.9
119.4 8.2
119.9 8.7
117.3 7.6
118.2 7.8
118.8 7.9
119.1 8.2
118.2 8.0
120.3 8.4
119.5 8.3
117.5 7.4
119.1 8.7
118.8 8.0
116.9 7.6
118.0 8.0
117.2 7.8
119.5 7.9
116.6 7.7
119.5 8.3
117.9 8.0
119.2 8.5
117.5 7.7
118.7 7.5
116.7 7.5
120.5 8.8
117.3 9.2
117.7 7.5
116.9 7.6
118.2 8.1
116.7 7.9
117.7 8.0
118.8 8.0
119.2 8.5
118.3 8.1
118.5 8.4
119.4 8.7
119.3 8.7
120.9 9.0
118.3 7.9
118.4 8.6
118.9 7.6
119.3 8.1
118.5 8.0
119.0 8.3
119.5 8.7
118.8 8.4
118.7 8.2
120.4 8.5
119.6 8.7
117.8 8.0
118.9 8.3
118.5 8.0
117.2 7.8
117.9 8.0
120.7 8.9
117.7 7.9
118.5 8.1
117.9 7.8
118.4 7.6
117.8 8.0
120.1 8.5
117.9 8.5
118.6 7.9
117.8 8.2
119.4 8.5
118.6 8.3
119.3 9.4
118.7 7.9
118.8 7.6
119.0 8.7
118.9 7.9
118.7 8.6
118.2 8.4
121.0 9.2
118.9 8.0
119.2 8.3
119.2 8.0
119.2 8.5
119.2 8.7
118.9 8.5
117.7 8.2
118.0 7.7
118.9 7.8
118.5 8.2
118.6 8.3
118.1 8.1
118.2 8.1
119.3 8.5
118.6 8.2
119.2 8.4
120.2 9.2
118.5 8.1
119.0 8.4
118.6 8.1
119.6 8.4
118.5 7.9
118.7 8.1
118.7 9.0
120.3 8.4
117.9 8.1
117.5 9.1
119.0 9.1
118.2 9.0
119.2 8.1
117.6 8.0
118.7 7.9
120.9 8.6
120.6 8.7
119.1 8.6
119.8 8.7
118.5 8.1
117.2 8.0
117.4 8.5
119.7 8.8
119.5 8.9
118.7 8.2
118.6 8.0
118.4 8.2
118.6 8.2
118.9 8.6
118.7 8.2
118.8 8.3
119.5 8.6
117.8 8.0
118.4 8.1
119.5 8.3
117.6 8.3
119.2 8.8
118.6 8.1
119.0 8.4
118.6 8.4
117.9 7.6
118.5 8.3
119.1 8.5
118.5 9.1
121.5 9.2
118.4 8.2
118.4 8.2
118.5 8.5
118.9 8.6
118.7 8.8
118.4 8.0
119.4 8.3
119.4 8.1
120.2 8.8
118.7 8.5
118.7 8.3
118.9 8.5
118.3 8.6
119.6 8.0
117.8 8.2
119.6 8.7
118.9 8.5
118.7 8.6
118.9 8.4
117.7 7.6
118.6 8.5
119.0 8.7
118.2 8.1
118.9 7.9
119.2 8.2
118.4 8.1
117.7 7.9
118.6 8.6
117.9 7.9
118.5 8.7
119.0 8.7
117.2 7.8
118.6 7.9
118.3 7.9
117.7 8.2
118.0 8.3
117.8 7.9
117.6 7.4
119.3 8.7
5b
5c
5d
5e
5g
5h
5i
5j
50a
50b
50c
50d
50e
50f
50g
50h
50i
50j
6a
6b
60a
60b
7c
7d
7e
70c
70d
70e
8c
8d
8e
80c
80d
80e
N = 10, dose = 200 mg/kg.
initiated by adding the test solution to the apical or
basolateral side of the monolayer. The various newly
synthesized compounds across Caco-2 cell monolayers
were valuated (n = 3) in the apical to basolateral
(A ! B) and basolateral to apical directions
(B ! A). The influence of efflux carriers on the perme-
ability of the different compounds was also examined
by comparing the permeability ratio Pratio of absorp-
tive transport Papp (A ! B) to the secretory one Papp
(B ! A). Permeability coefficients for the synthetic
compounds are summarized in Table 4.
Table 4. Apparent permeability coefficients of 5 and 50h,i, 6 and 60a,b,
13 and 130h,i, 14 and 140a,b
Compound
Papp · 10ꢀ6(cm/s)
A ! B
B ! A
A ! B/B ! A
5h
11.89
12.99
11.72
12.86
10.22
10.25
10.18
10.31
10.22
10.40
10.15
10.32
8.97
7.01
6.96
6.88
6.90
7.05
7.17
7.07
7.06
7.00
6.98
6.86
6.93
7.87
7.51
7.32
7.15
1.70
1.87
1.70
1.86
1.45
1.43
1.44
1.46
1.46
1.49
1.48
1.49
1.14
1.20
1.21
1.23
5i
50h
50i
6a
6b
60a
60b
13h
13i
130h
130i
14a
14b
140a
140b
According to the previous study, the compound with
permeability coefficients Papp < 1 · 10ꢀ6, 1–10 · 10ꢀ6
,
and >10 · 10ꢀ6 cm/s was defined as poorly, moderately,
and well absorbed, respectively.31 The compounds 14
and 140a,b exhibited moderate permeation through the
Caco-2 cell monolayer. In contrast, after removing the
NH2 protecting group, that is, when 14 and 140a,b was
converted into 6 and 60a,b the permeation was increased,
that is, 6 and 60a,b belong to the well-absorbed com-
pounds. A similar tendency was observed between the
compounds 5 and 50h,i and 13 and 130h,i.
9.01
8.86
8.80
The standard deviations were generally less than 10% (n = 4).
A ! B, from apical side to basolateral side.
B ! A, from basolateral side to apical side.

6280
K. Gu et al. / Bioorg. Med. Chem. 15 (2007) 6273–6290
Table 5. Composition of standard solution used for protracting standard curve
Tube number
1
2
3
4
5
6
7
8
9
10
a
Reactive solution 1( ll)
Reactive solution 2( ll)
Reactive solution 3( ll)
Buffer C (ll)
200
200
40
200
200
36
200
200
32
200
200
28
200
200
24
200
200
20
200
200
16
200
200
12
200
200
8
200
200
4
b
80
20
84
18
88
16
92
14
96
12
100
10
104
8
108
6
112
4
116
2
ATP (nmol)
a For reactive tube.
b For control tube.
the mechanism underlying the enzyme inhibition pro-
cesses should prove beneficial for future development
of chemoprevention agents.
Table 6. Inhibition effect of 1,3-dioxacycloalkanes on PKC
Agents IC50 Agents IC50 Agents IC50 Agents IC50
(mM)
(mM)
(mM)
(mM)
5a
5b
5c
1.62
1.72
1.22
1.87
1.44
1.48
1.54
1.10
0.89
1.23
1.67
1.62
2.49
2.68
2.66
2.71
2.76
2.81
2.59
2.42
2.43
2.58
1.83
2.78
2.48
2.59
2.48
50a
50b
50c
50d
50e
50f
1.64 10h
1.76 10i
1.30 10j
1.88
1.65
1.78
2.64
2.57
2.62
2.80
1.99
2.68
2.61
2.71
2.64
1.94
1.71
1.82
2.69
2.77
2.81
2.73
2.87
2.94
2.92
2.86
2.93
3.01
2.89
3.00
100h
100i
1.94
1.67
1.92
2.66
2.77
2.48
2.70
1.96
2.92
2.70
2.76
2.71
2.01
1.72
1.98
2.71
2.85
2.90
2.86
2.91
2.98
2.95
2.98
3.01
3.10
3.13
3.20
3. Conclusion
100j
5d
5e
190
11a
1.48 11b
1.70 12a
1.66 12b
1.14 12c
0.93 12d
1.35 12e
1.68 12f
1.70 12g
2.55 12h
2.73 12i
2.72 12j
2.74 13c
2.79 13d
2.85 13e
2.63 14c
2.67 14d
2.52 14e
2.62 15c
1.91 15d
2.80 15e
2.56 16c
2.64 16d
2.55 16e
110a
110b
120a
120b
120c
120d
120e
120f
120g
120h
120i
We have identified 5-amino-2-substitutedphenyl-1,3-
dioxacycloalkanes as a new series of potent anti-inflam-
matory agents. Twenty-four of the newly synthesized
compounds provided the most interesting compounds
in terms of in vivo anti-inflammatory activity. In addi-
tion, these compounds did not prolong tail bleeding time
even at high doses (200 mg/kg). In conclusion, these
newly synthesized compounds display a favorable phar-
macological profile relative to the reference drug. Con-
sidering their efficacy and safety profiles, these newly
synthesized compounds are worth to work further to de-
tail the possible link of the inflammatory inhibition and
cancer prevention.
5f
5g
5h
5i
50g
50h
50i
5j
50j
6a
6b
7c
60a
60b
70c
7d
7e
70d
70e
80c
80d
80e
90a
90b
100a
100b
100c
100d
100e
100f
100g
120j
8c
130c
130d
130e
140c
140d
140e
150c
150d
150e
160c
160d
160e
8d
8e
9a
9b
10a
10b
10c
10d
10e
10f
10g
4. Experimental
All reactions were carried out under nitrogen (1 bar).
Melting points are uncorrected. Unless otherwise stated,
all reactions were carried out under a nitrogen atmo-
sphere (1 bar). The agents used in this work were pur-
chased
from
Sigma
Chemical
Co.
(USA).
1-(5-Isoquinolylsulfonyl)-2-methylpiperazine was used as the positive
control and its IC50 = 23 lM.
Chromatography was performed on Qingdao silica gel
H (Qingdao of China). The purities of the intermediates
and the products were confirmed by TLC (Merck silica
gel plates of type 60 F254, 0.25 mm layer thickness, Ger-
many) and HPLC (Waters, C18 column 4.6 · 150 mm,
USA). NMR spectra were recorded at 300 MHz on a
VXR-300 instrument or at 500 MHz on a Bruker Am-
500 instrument in CDCl3 with tetramethylsilane as inter-
nal standard. EI-MS was determined by Trace MS System
(Thermo Finnigan, USA). Optical rotations were deter-
mined with a Schmidt + Haensch Polartromic D instru-
ment (Germany). The statistical analysis of all the
biological data was carried out by use of ANOVA test,
p < 0.05 is considered significant.
The in vivo anti-inflammatory effect of 5-amino-2-sub-
stitutedphenyl-1,3-dioxacycloalkanes most likely occurs
via the inhibition of certain key enzymes involved in
inflammation and/or cell signaling pathways. 1,3-Diox-
ane derivatives have been reported recently to possess
PKC inhibitory activity and exert anti-inflammatory,
anti-cancer, and reperfusion injury protection effects
through their anti-proliferative and anti-inflammatory
activities in human neutrophils and tumor cells. There-
fore, we attempted to investigate the acting mechanism
of 5-amino-2-substitutedphenyl-1,3-dioxacycloalkanes
in PKC inhibition assay.34,35 However, the preliminary
results indicated that, most of these newly synthesized
compounds displayed weak PKC inhibitor activities
(Table 6), suggesting PKC might not be the target pro-
tein. Experiments are currently underway to ascertain
the protein target(s) of the dioxacycloalkane com-
pounds, in which other possible protein targets may also
include cyclooxygenase and lipoxygenase, and phospho-
inositide 3-kinase. Identification and understanding of
4.1. HClÆL-Ser-OCH3 (2a)
At 0 ꢁC to 50 ml of anhydrous methanol, 3.75 ml
(47.5 mmol) of thinly chloride was added dropwise. The
solution was stirred at 0 ꢁC for 30 min and then 5.0 g
(47.6 mmol) of L-Ser was added. The reaction mixture
was stirred at room temperature for 24 h and
TLC(CH3Cl/CH3OH, 9:1) indicated complete disappear-

K. Gu et al. / Bioorg. Med. Chem. 15 (2007) 6273–6290
6281
ance of L-Ser. The reaction mixture was evaporated under
4.8. N-Phenyl-L-Glu-(OCH3) (20d)
reduced pressure and the residue was triturated with
petroleum ether repeatedly to provide 7304 mg (99%) of
HClÆL-Ser-OCH3 as a colorless powder (mp 161–162 ꢁC)
which was directly used for the next reaction.
With the same procedure as that used for the prepara-
tion of 20a from 1274 mg (6.0 mmol) of HClÆL-Glu-
(OCH3)2 1433 mg (81%) of N-phenyl-L-Glu-(OCH3)2
was obtained as a colorless powder (mp 111–113 ꢁC).
4.2. HClÆL-Thr-OCH3 (2b)
4.9. General procedure for the preparation of aminodiols
3a–e and 30a–e
With the same procedure as that used for the prepara-
tion of 2a from 4.998 g (47.5 mmol) of L-Thr 7867 mg
(98%) of HClÆL-Thr-OCH3 was obtained as a colorless
powder (mp 140–142 ꢁC).
To the suspension of 200 mg (5.6 mmol) of KBH4 in
10 ml of THF the solution of 1.0 mmol of HClÆL-AA-
OCH3 in 20 ml of THF was added. The reaction mix-
ture was stirred at room temperature for 24 h and TLC
(C2H5OH/H2O, 7:1) indicated complete disappearance
of HClÆL-AA-OCH3. At 0 ꢁC the reaction mixture
was adjusted to pH 10 with hydrochloric acid (2 mol/
L) and evaporated under reduced pressure. The residue
was purified by chromatography (CH3Cl/CH3OH,
19:1).
4.3. HClÆL-Asp- (OCH3)2 (2c)
With the same procedure as that used for the prepara-
tion of 2a from 6.332 g (47.5 mmol) of L-Asp 7.495 mg
(98%) of HClÆL-Asp-(OCH3)2 was obtained as a color-
less powder (mp 149–151 ꢁC).
4.4. L-Glu-(OCH3)2 (2d)
4.9.1. 2-Aminopropane-1,3-diol hydrochloride (3a). Yield:
62%.
With the same procedure as that used for the prepara-
tion of 2a from 6.983 g (47.5 mmol) of L-Glu 8146 mg
(98%) of HClÆL-Glu-(OCH3)2 was obtained as a color-
less powder (mp 136–138 ꢁC).
4.9.2. (2R,3R)-2-Aminobutane-1,3-diol hydrochloride
(3b). Yield: 58%.
4.5. N-Phenyl-L-Ser-OCH3 (20a)
4.9.3. (2S)-2-Aminobutane-1,4-diol hydrochloride (3c).
Yield: 39%.
At 0 ꢁC, to a suspension of 1.0 g (6.4 mmol) of HClÆ
L-Ser-OCH3 in 30 ml of THF, 25 ml of saturated
aqueous sodium carbonate was added. Under stirring
to the solution 1.106 ml (8.3 mmol) of phenylacetyl
chloride was added dropwise and the pH of the reac-
tion mixture was adjusted to 8–9 by adding saturated
aqueous sodium carbonate. The reaction mixture was
stirred at room temperature for 3 h and TLC
(CH3Cl/CH3OH, 19:1) indicated complete disappear-
ance of L-Ser-OCH3. The reaction mixture was evapo-
rated under reduced pressure and the residue was
dissolved in 50 ml of ethyl acetate and then washed
with saturated aqueous sodium bicarbonate (3·
10 ml), saturated aqueous of KHSO4 (3· 10 ml), and
saturated aqueous of NaCl (3· 10 ml) successively.
The solution was evaporated under reduced pressure
and the residue was purified by chromatography
(petroleum ether/ethyl acetate, 5:1) to provide
1140 mg (75%) of C6H5CH2CO-L-Ser-OCH3 as a col-
orless powder (mp 77–79 ꢁC).
4.9.4. (2S)-2-Aminoheptane-1,5-diol hydrochloride (3d).
Yield: 42%. IR(film): m/cmꢀ1 = 3335, 3339, 3348, 1634,
1275, 1050. 1H NMR (DMSO-d6): d/ppm = 7.623 (s,
2H), 4.228 (d, J = 6.21 Hz, 2H), 3.651 (m, J = 5.31 Hz,
1H), 3.526 (t, J = 5.44 Hz, 2H), 2.004 (s, 2H), 1.992 (m,
J = 5.42 Hz, 2H), 1.562 (m, J = 5.42 Hz, 2H). 13C
NMR (DMSO-d6): d/ppm = 68.404, 63.027, 51.898,
D
28.587, 27.292. FAB-MS (m/e) 121 [M+H]+. ½aꢂ ꢀ11.2
20
(c 4.0, H2O). Anal. Calcd for C5H15ClNO2: C, 49.97;
H, 11.74; N 11.66. Found: C, 50.11; H, 11.89; N, 11.76.
4.9.5. N-[2-Hydroxy-1-(hydroxymethyl)ethyl]phenylace-
tamide (300b). Yield: 91%.
4.9.6. (1S)-N-[3-Hydroxy-1-(hydroxymethyl)propyl]phenyl-
acetamide (30c). Yield: 93%.
4.9.7. (1S)-N-[4-Hydroxy-1-(hydroxymethyl)butyl]phenyl-
acetamide (30d). Yield: 93%.
4.6. N-Phenyl-L-Thr-OCH3 (20b)
4.10. General procedure for the preparation of 5-amino-2-
substitutedphenyl-1,3-dioxacycloalkanes
With the same procedure as that used for the prepara-
tion of 20a from 1010 mg (6.0 mmol) of HClÆL-
Thr-OCH3 1171 mg (77%) of N-phenyl-L-Thr-OCH3
was obtained as a colorless powder (mp 90–92 ꢁC).
A mixture of 2.0 mmol of aminodiol, 2.6 mmol of substi-
tutedbenzaldehyde, 30 mg of tolylsulfonylic acid, 100 mg
of anhydrous NaSO4, 50 ml of chloroform, and 4 ml of
THF was stirred at rt overnight until TLC(CHCl3/
CH3OH, 20:1) indicated complete disappearance of
aminodiol. The reaction mixture was then adjusted to
pH 7 with sodium carbonate and then filtered. The
filtrate was concentrated under reduced pressure,
and the residue was purified by chromatography on silica
gel.
4.7. N-Phenyl-L-Asp-(OCH3) (20c)
With the same procedure as that used for the prepara-
tion of 20a from 1182 mg (6.0 mmol) of HClÆL-Asp-
(OCH3)2 1345 mg (80%) of N-phenyl-L-Asp-(OCH3)2
was obtained as a colorless powder (mp 90–92 ꢁC).

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K. Gu et al. / Bioorg. Med. Chem. 15 (2007) 6273–6290
4.11. General procedure for the preparation of 5-amino-2-
substitutedphenyl-1,3-dioxacycloalkanes via enzymatic
hydrolysis
for C10H12N2O2: C, 53.57; H, 5.39; N, 12.49. Found: C,
53.44; H, 5.34; N, 12.60.
4.11.5. (cis)-2-[30-Nitrophenyl]-1,3-dioxan-5-yl]amine (5e).
Yield: 44%. Mp 50–52 ꢁC. IR(KBr): m/cmꢀ1 = 3247, 3186,
1645, 1637, 1600, 1581, 1522, 1502, 1458, 1351, 1048, 861,
773, 701. 1H NMR (CDCl3): d/ppm = 8.367 (s, 1H), 8.194
(d, J = 8.22 Hz, 1H), 7.816 (d, J = 8.00 Hz, 1H), 7.534 (t,
J = 8.00 Hz, 1H), 5.574 (s, 1H), 4.181 (d, J = 12.61 Hz,
2H), 4.082 (d, J = 11.40 Hz, 2H), 2.882 (m, J = 11.00 Hz,
1H), 2.126 (s, 2H). 13C NMR (DMSO-d6): d/ppm =
146.117, 140.048, 132.169, 129.260, 123.780, 121.406,
99.969, 73.209, 45.649. FAB-MS (m/e) 225 [M+H]+. Anal.
Calcd for C10H12N2O2: C, 53.57; H, 5.39; N, 12.49. Found:
C, 53.46; H, 5.44; N, 12.38.
A suspension of 1.0 mmol of 5-phenylacetylamino-2-sub-
stitutedphenyl-1,3-dioxacycloalkanes, 500 mg of the
powder of penicillin acylase, and 20 ml of methanol/water
was adjusted to pH 6.0 with hydrochloric acid and then
shaken (37 ꢁC, 50 times/1 min) for 4 h. TLC (CHCl3/
CH3OH, 10:1) indicated the complete disappearance of
5-phenylacetylamino-2-substitutedphenyl-1,3-dioxacyclo-
alkanes. The reaction mixture was filterted and the filtrate
was adjustedtopH 8–9 withthe aqueoussodium bicarbon-
ate solution (5%). The solution was purified on the column
of Sephadex G10 to remove the NaCl and then evaporated
under reduced pressure to give 5-amino-2-substitutedphe-
nyl-1,3-dioxacycloalkanes.
4.11.6.
(S,4S,5R)-(2-Phenyl-4-methyl-1,3-dioxan-5-yl)-
amine (5f). Yield: 40%. Mp 48–50 ꢁC. IR(KBr):
4.11.1. (cis)-(2-Phenyl-1,3-dioxan-5-yl)amine (5a). Yield:
40%. Mp 52–54 ꢁC. IR(KBr): m/cmꢀ1 = 3245, 3188, 1647,
1639, 1601, 1579, 1502, 1459, 1051, 743, 692. H NMR
m/cmꢀ1 = 3247, 3185, 1645, 1637, 1601, 1582, 1500, 1455,
1
1382, 1048, 742, 694. H NMR (CDCl3): d/ppm = 7.472
1
(d, J = 6.72 Hz, 2H), 7.330 (t, J = 6.68 Hz, 1H), 7.330 (t,
J = 6.67 Hz, 2H), 5.490 (s, 1H), 4.081 (m, J = 6.61 Hz,
1H), 4.020 (d, J = 4.52 Hz, 2H), 2.499 (t, J = 4.52 Hz,
1H), 1.671 (s, 2H), 1.237 (d, J = 4.50 Hz, 3H). 13C NMR
(DMSO-d6): d/ppm = 136.153, 128.757, 128.123, 125.865,
(CDCl3): d/ppm = 7.478 (d, J = 7.04 Hz, 2H), 7.363 (t,
J = 7.56 Hz, 1H), 7.332 (t, J = 7.56 Hz, 2H), 5.510 (s,
1H), 4.141 (d, J = 11.71 Hz, 2H), 4.017 (d, J = 10.51 Hz,
2H), 2.787 (m, J = 10.32 Hz, 1H), 1.934 (s, 2H). 13C
NMR (DMSO-d6): d/ppm = 138.136, 128.964, 128.271,
125.865, 101.791, 73.300, 45.772. FAB-MS (m/e) 180
[M+H]+. Anal. Calcd for C10H13NO2: C, 67.02; H, 7.31;
N, 7.82. Found: C, 67.12; H, 7.23; N, 7.94.
101.560, 75.698, 73.868, 48.764, 17.611. FAB-MS (m/e)
D
194 [M+H]+. ½aꢂ ꢀ8.1 (c 1.00, CHCl3). Anal. Calcd for
20
C11H15NO2: C, 68.37; H, 7.82; N, 7.25. Found: C, 68.50;
H, 7.90; N, 7.14.
4.11.2. (cis)-[2-(40-Methylphenyl)-1,3-dioxan-5-yl]amine (5b).
Yield: 35%. Mp 55–56 ꢁC. IR(KBr): m/cmꢀ1 = 3247, 3189,
1645, 1635, 1602, 1580, 1503, 1461, 1382, 1048, 822. H
NMR (CDCl3): d/ppm = 7.363 (d, J = 7.82 Hz, 2H), 7.163
(d, J = 7.82 Hz, 2H), 5.470 (s, 1H), 4.134 (d, J = 4.41 Hz,
2H), 4.008 (d, J = 4.30 Hz, 2H), 2.786 (m, J = 4.41 Hz, 1H),
2.326 (s, 3H), 1.894 (s, 2H). 13C NMR (DMSO-d6):
d/ppm = 138.755, 135.367, 128.939, 125.766, 101.890,
73.316, 45.838, 21.237. FAB-MS (m/e) 194 [M+H]+. Anal.
Calcd for C11H15NO2: C, 68.37; H, 7.82; N, 7.25. Found: C,
68.44; H, 7.71; N, 7.36.
4.11.7.(2S,4S,5R)-2-[(40-Methylphenyl)-4-methyl-1,3-diox-
an-5-yl]amine (5g). Yield: 45%. Mp 63–64 ꢁC. IR(KBr):
m/cmꢀ1 = 3257, 3190, 1649, 1635, 1602, 1583, 1500, 1457,
1
1
1382, 1049, 822. H NMR (CDCl3): d/ppm = 7.362 (d,
J = 8.41 Hz, 2H), 7.154 (d, J = 8.12 Hz, 2H), 5.487 (s,
1H), 4.119 (d, J = 4.55 Hz, 1H), 4.084 (d, J = 4.22 Hz,
1H), 4.040 (m, J = 6.10 Hz, 1H), 2.538 (m, J = 4.20 Hz,
1H), 2.318 (s, 3H), 1.649 (s, 2H), 1.257 (d, J = 6.60 Hz,
3H). 13C NMR (DMSO-d6): d/ppm = 138.656, 135.466,
128.914, 125.865, 101.783, 75.788, 73.967, 48.978, 21.212,
D
17.759. FAB-MS (m/e) 208 [M+H]+. ½aꢂ ꢀ8.0 (c 1.00,
20
CHCl3). Anal. Calcd for C12H17NO2: C, 69.54; H, 8.27;
N, 6.76. Found: C, 69.47; H, 8.22; N, 6.62.
4.11.3. (cis)-[2-(40-Chlorophenyl)-1,3-dioxan-5-yl]amine (5c).
Yield: 36%. Mp 76–78 ꢁC. IR(KBr): m/cmꢀ1 = 3252, 3189,
1645, 1637, 1600, 1582, 1503, 1456, 1051, 821. H NMR
1
4.11.8. (2S,4S,5R)-2-[(40-Chlorophenyl)-4-methyl-1,3- diox-
an-5-yl]amine (5h). Yield: 45%. Mp 45–46 ꢁC. IR(KBr):
m/cmꢀ1 = 3259, 3189, 1649, 1636, 1601, 1582, 1501, 1381,
(CDCl3): d/ppm = 7.405 (d, J = 7.12 Hz, 2H), 7.288 (d,
J = 7.12 Hz, 2H), 5.483 (s, 1H), 4.122 (d, J = 3.72 Hz,
2H), 4.029 (d, J = 6.41 Hz, 2H), 2.795 (s, 1H), 1.765
(s, 2H). 13C NMR (DMSO-d6): d/ppm = 136.719, 134.749,
128.453, 127.389, 100.991, 73.407, 45.756. FAB-MS
(m/e) 214 [M+H]+. Anal. Calcd for C10H12ClNO2: C,
56.21; H, 5.66; N, 6.56. Found: C, 56.14; H, 5.74; N,
6.64.
1
1457, 1049, 822. H NMR (CDCl3): d/ppm = 7.407 (t,
J = 8.01 Hz, 2H), 7.326 (t, J = 8.01 Hz, 2H), 5.493 (s,
1H), 4.119 (m, J = 6.91 Hz, 1H), 4.077 (d, J = 5.70 Hz,
2H), 2.575 (m, J = 4.52 Hz, 1H), 1.668 (s, 2H), 1.267 (d,
J = 6.60 Hz, 3H). 13C NMR (DMSO-d6): d/ppm =
136.768, 134.683, 128.436, 127.488, 100.909, 75.920,
D
20
74.000, 48.880, 17.734. FAB-MS (m/e) 228 [M+H]+. ½aꢂ
4.11.4. (cis)-2-[40-Nitrophenyl]-1,3-dioxan-5-yl]amine (5d).
Yield: 43%. Mp 112.5–113.5 ꢁC. IR(KBr): m/cmꢀ1 = 3249,
3186, 1645, 1637, 1602, 1581, 1522, 1503, 1457, 1352, 1048,
ꢀ8.0 (c 1.00, CHCl3). Anal. Calcd for C11H14ClNO2: C,
58.03; H, 6.20; N, 6.15. Found: C, 58.12; H, 6.29; N, 6.27.
1
823. H NMR (CDCl3): d/ppm = 8.216 (d, J = 8.74 Hz,
4.11.9. (2S,4S,5R)-2-[(40-Nitrophenyl)-4-methyl-1,3-diox-
an-5-yl]amine (5i). Yield; 48%. Mp 80–82 ꢁC. IR(KBr):
m/cmꢀ1 = 3248, 3187, 1648, 1638, 1601, 1582, 1525,
2H), 7.661 (d, J = 8.74 Hz, 2H), 5.573 (s, 1H), 4.179 (d,
J = 4.51 Hz, 2H), 4.052 (d, J = 4.32 Hz, 2H), 2.839 (m,
J = 4.28 Hz, 1H), 1.733 (s, 2H). 13C NMR (DMSO-d6):
d/ppm = 148.191, 144.614, 127.084, 123.450, 100.077,
73.522, 45.707. FAB-MS (m/e) 225 [M+H]+. Anal. Calcd
1
1504, 1457, 1381, 1351, 1049, 822. H NMR (CDCl3):
d/ppm = 8.198 (d, J = 9.01 Hz, 2H), 7.659 (d, J =
9.01 Hz, 2H), 5.589 (s, 1H), 4.145 (m, J = 6.22 Hz, 1H),

K. Gu et al. / Bioorg. Med. Chem. 15 (2007) 6273–6290
6283
1
4.109 (d, J = 5.71 Hz, 2H), 2.594 (m, J = 6.22 Hz, 1H),
1.593 (s, 2H), 1.289 (d, J = 6.32 Hz, 3H). 13C NMR
(DMSO-d6): d/ppm = 148.101, 144.697, 127.142,
1350, 1049, 821. H NMR (CDCl3): d/ppm = 8.330 (d,
J = 8.44 Hz, 2H), 7.855 (d, J = 8.44 Hz, 2H), 5.768 (s,
1H), 4.394 (d, J = 4.54 Hz, 2H), 4.354 (d, J = 4.36 Hz,
2H), 2.985 (m, J = 4.42 Hz, 1H), 1.788 (s, 2H). 13C
NMR (DMSO-d6): d/ppm = 148.642, 145.041, 127.493,
123.745, 100.987, 74.152, 46.187. FAB-MS (m/e) 225
[M+H]+. Anal. Calcd for C10H12N2O2: C, 53.57; H,
5.39; N, 12.49. Found: C, 53.66; H, 5.50; N, 12.30.
123.425, 99.945, 76.184, 74.165, 48.830, 17.668. FAB-
D
MS (m/e) 239 [M+H]+. ½aꢂ ꢀ8.3 (c 1.00, CHCl3). Anal.
20
Calcd for C11H14N2O2: C, 55.46; H, 5.92; N, 11.76.
Found: C, 55.37; H, 5.84; N, 11.58.
4.11.10.
(2S,4S,5R)-2-[(30-Nitrophenyl)-4-methyl-1,3-
dioxan-5-yl]amine (5j). Yield: 41%. Mp 50–52 ꢁC.
4.11.15. (trans)-2-[30-Nitrophenyl]-1,3-dioxan-5-yl]amine
IR(KBr): m/cmꢀ1 = 3249, 3186, 1645, 1637, 1601, 1582,
1522, 1502, 1458, 1381, 1351, 1048, 743, 699. H NMR
(50e). Yield: 95%. Mp 56–58 ꢁC. IR(KBr): m/cmꢀ1
=
1
3249, 3187, 1646, 1635, 1602, 1580, 1525, 1501, 1456,
1350, 1049, 862, 770, 700. 1H NMR (CDCl3):
d/ppm = 8.416 (s, 1H), 8.285 (d, J = 8.20 Hz, 1H),
7.955 (d, J = 8.01 Hz, 1H), 7.841 (t, J = 8.01 Hz, 1H),
5.740 (s, 1H), 4.399 (d, J = 12.01 Hz, 2H), 4.275 (d,
J = 11.00 Hz, 2H), 2.996 (m, J = 10.81 Hz, 1H), 2.234
(s, 2H). 13C NMR (DMSO-d6): d/ppm = 146.548,
140.582, 132.687, 130.126, 124.178, 121.690, 100.314,
73.557, 46.164. FAB-MS (m/e) 225 [M+H]+. Anal.
Calcd for C10H12N2O2: C, 53.57; H, 5.39; N, 12.49.
Found: C, 53.66; H, 5.51; N, 12.60.
(CDCl3): d/ppm = 8.361 (s, 1H), 8.191 (d, J = 8.42 Hz,
1H), 7.820 (d, J = 7.83 Hz, 1H), 7.527 (t, J = 8.02 Hz,
1H), 5.598 (s, 1H), 4.181 (m, J = 4.30 Hz, 1H), 4.111(d,
J = 2.74 Hz, 2H), 2.612 (m, J = 3.05 Hz, 1H), 1.695 (s,
2H), 1.298 (d, J = 4.65 Hz, 3H). 13C NMR (DMSO-d6):
d/ppm = 148.117, 140.180, 132.277, 129.252, 123.722,
121.480, 99.887, 76.135, 74.082, 48.830, 17.685. FAB-
D
MS (m/e) 239 [M+H]+. ½aꢂ ꢀ8.1 (c 1.00, CHCl3). Anal.
20
Calcd for C11H14N2O2: C, 55.46; H, 5.92; N, 11.76.
Found: C, 55.60; H, 5.86; N, 11.86.
4.11.11. (trans)-(2-Phenyl-1,3-dioxan-5-yl)amine (50a).
Mp 58–60 ꢁC. IR(KBr): m/cmꢀ1 = 3283, 3196, 2925,
2860, 1651, 1620, 1601, 1588, 1521, 1452, 1352, 1190,
4.11.16. (2R,4S,5R)-(2-Phenyl-4-methyl-1,3-dioxan-5-yl)-
amine (50f). Yield: 90%. Mp 58–60 ꢁC. IR(KBr):
m/cmꢀ1 = 3255, 3189, 1646, 1635, 1602, 1581, 1502, 1456,
1
1
1052, 741, 699. H NMR (CDCl3): d/ppm = 7.620 (d,
1380, 1049, 741, 695. H NMR (CDCl3): d/ppm = 7.730
J = 7.00 Hz, 2H), 7.502 (t, J = 7.50 Hz, 1H), 7.460 (t,
J = 7.50 Hz, 2H), 5.614 (s, 1H), 4.273 (d, J = 11.40 Hz,
2H), 4.135 (d, J = 10.00 Hz, 2H), 2.941 (m,
J = 10.01 Hz, 1H), 1.969 (s, 2H). 13C NMR (DMSO-
d6): d/ppm = 138.635, 129.443, 128.714, 126.694,
102.179, 73.871, 46.262. FAB-MS (m/e) 180 [M+H]+.
Anal. Calcd for C10H13NO2: C, 67.02; H, 7.31; N,
7.82. Found: C, 67.08; H, 7.26; N, 7.92.
(d, J = 6.74 Hz, 2H), 7.516 (t, J = 6.62 Hz, 1H), 7.512 (t,
J = 6.65 Hz, 2H), 5.687 (s, 1H), 4.318 (m, J = 6.63 Hz,
1H), 4.290 (d, J = 4.54 Hz, 2H), 2.701 (t, J = 4.54 Hz,
1H), 1.851 (s, 2H), 1.315 (d, J = 4.52 Hz, 3H). 13C NMR
(DMSO-d6): d/ppm = 136.655, 129.175, 128.530, 126.157,
102.165, 76.202, 74.223, 49.320, 18.261. FAB-MS (m/e)
D
194 [M+H]+. ½aꢂ 9.6 (c 1.00, CHCl3). Anal. Calcd for
20
C11H15NO2: C, 68.37; H, 7.82; N, 7.25. Found: C, 68.28;
H, 7.70; N, 7.36.
4.11.12.
(trans)-[2-(40-Methylphenyl)-1,3-dioxan-5-yl]-
amine (50b). Yield: 94%. Mp 70–72 ꢁC. IR(KBr):
4.11.17. (2R,4S,5R)-[2-(40-Methylphenyl)-4-methyl-1,3-
dioxan-5-yl]amine (50g). Yield: 92%. Mp 68–70 ꢁC.
IR(KBr): m/cmꢀ1 = 3259, 3191, 1647, 1633, 1601, 1581,
1502, 1455, 1381, 1051, 821. 1H NMR (CDCl3):
d/ppm = 7.515 (d, J = 8.21 Hz, 2H), 7.336 (d,
J = 8.10 Hz, 2H), 5.713 (s, 1H), 4.356 (d, J = 4.58 Hz,
1H), 4.298 (d, J = 4.26 Hz, 1H), 4.240 (m, J = 6.00 Hz,
1H), 2.834 (m, J = 4.25 Hz, 1H), 2.420 (s, 3H), 1.946
(s, 2H), 1.302 (d, J = 6.55 Hz, 3H). 13C NMR
(DMSO-d6): d/ppm = 138.968, 135.970, 129.294,
m/cmꢀ1 = 3249, 3186, 1643, 1633, 1601, 1582, 1501, 1451,
1381, 1049, 820. H NMR (CDCl3): d/ppm = 7.536 (d,
1
J = 7.80 Hz, 2H), 7.365 (d, J = 7.80 Hz, 2H), 5.740 (s,
1H), 4.432 (d, J = 4.40 Hz, 2H), 4.265 (d, J = 4.32 Hz,
2H), 2.968 (m, J = 4.40 Hz, 1H), 2.634 (s, 3H), 1.997 (s,
2H). 13C NMR (DMSO-d6): d/ppm = 139.145, 135.738,
129.193, 126.142, 102.189, 73.625, 46.383, 21.727. FAB-
MS (m/e) 194 [M+H]+. Anal. Calcd for C11H15NO2: C,
68.37; H, 7.82; N, 7.25. Found: C, 68.48; H, 7.91; N, 7.13.
126.275, 102.301, 76.278, 74.175, 49.285, 21.920,
D
4.11.13.
(trans)-[2-(40-Chlorophenyl)-1,3-dioxan-5-yl]-
18.275. FAB-MS (m/e) 208 [M+H]+. ½aꢂ 9.7 (c 1.00,
20
CHCl3). Anal. Calcd for C12H17NO2: C, 69.54; H,
8.27; N, 6.76. Found: C, 69.68; H, 8.40; N, 6.85.
amine (50c). Yield: 92%. Mp 70–72 ꢁC. IR(KBr):
m/cmꢀ1 = 3254, 3187, 1642, 1635, 1602, 1580, 1501,
1454, 1050, 823. H NMR (CDCl3): d/ppm = 7.764 (d,
1
J = 7.10 Hz, 2H), 7.513 (d, J = 7.10 Hz, 2H), 5.725 (s,
1H), 4.356 (d, J = 3.74 Hz, 2H), 4.219 (d, J = 6.32 Hz,
2H), 2.975 (s, 1H), 1.868 (s, 2H). 13C NMR (DMSO-d6):
d/ppm = 137.179, 135.194, 128.741, 127.610, 101.296,
73.744, 46.165. FAB-MS (m/e) 214 [M+H]+. Anal. Calcd
for C10H12ClNO2: C, 56.21; H, 5.66; N, 6.56. Found: C,
56.31; H, 5.77; N, 6.44.
4.11.18. (2R,4S,5R)-2-[(40-Chlorophenyl)-4-methyl-1,3-
dioxan-5-yl]amine (50h). Yield: 94%. Mp 51–53 ꢁC.
IR(KBr): m/cmꢀ1 = 3258, 3186, 1645, 1633, 1600, 1580,
1502, 1382, 1455, 1051, 821. 1H NMR (CDCl3):
d/ppm = 7.672 (t, J = 8.00 Hz, 2H), 7.624 (t, J =
8.00 Hz, 2H), 5.700 (s, 1H), 4.350 (m, J = 6.94 Hz, 1H),
4.310 (d, J = 5.72 Hz, 2H), 2.835 (m, J = 4.56 Hz, 1H),
1.845 (s, 2H), 1.380 (d, J = 6.62 Hz, 3H). 13C NMR
(DMSO-d6): d/ppm = 137.186, 135.090, 128.763,
4.11.14. (trans)-2-[40-Nitrophenyl]-1,3-dioxan-5-yl]amine
(50d). Yield: 93%. Mp 104–106 ꢁC. IR(KBr): m/cmꢀ1
3251, 3188, 1644, 1636, 1601, 1580, 1525, 1501, 1456,
=
127.983, 101.292, 76.284, 74.623, 49.307, 18.341. FAB-
D
MS (m/e) 228 [M+H]+. ½aꢂ 9.6 (c 1.00, CHCl3). Anal.
20

6284
K. Gu et al. / Bioorg. Med. Chem. 15 (2007) 6273–6290
Calcd for C11H14ClNO2: C, 58.03; H, 6.20; N, 6.15.
Found: C, 57.92; H, 6.09; N, 6.08.
3187, 3032, 1675, 1646, 1636, 1602, 1580, 1501, 1455,
1049, 741, 698. 1H NMR (500 MHz, CDCl3):
d/ppm = 8.003 (s, 2H), 7.548 (q, J = 7.10 Hz, 2H), 7.550
(t, J = 7.14 Hz, 2H), 7.302 (t, J = 7.14 Hz, 1H), 6.802 (d,
J = 13.00 Hz, 1H), 6.526 (d, J = 13.00 Hz, 1H), 5.634 (d,
J = 7.34 Hz, 1H), 4.638 (d, J = 11.51 Hz, 4H). 13C
NMR (CDCl3): d/ppm = 135.344, 128.612, 128.230,
127.174, 126.503, 124.326, 106.232, 73.603, 52.876.
FAB-MS (m/e) 206 [MH]+. Anal. Calcd for
C12H16ClNO2: C, 59.63; H, 6.67; N, 5.79. Found: C,
59.49; H, 6.58; N, 5.67.
4.11.19.
(2R,4S,5R)-2-[(40-Nitrophenyl)-4-methyl-1,3-
dioxan-5-yl]amine (50i). Yield: 95%. Mp 85–87 ꢁC.
IR(KBr): m/cmꢀ1 = 3255, 3189, 1647, 1636, 1602, 1580,
1523, 1502, 1455, 1382, 1350, 1048, 823. 1H NMR
(CDCl3): d/ppm = 8.316 (d, J = 9.00 Hz, 2H), 7.915 (d,
J = 9.00 Hz, 2H), 5.810 (s, 1H), 4.328 (m, J = 6.24 Hz,
1H), 4.387 (d, J = 5.74 Hz, 2H), 2.816 (m, J = 6.24 Hz,
1H), 1.738 (s, 2H), 1.334 (d, J = 6.34 Hz, 3H). 13C
NMR (DMSO-d6): d/ppm = 148.665, 144.984, 127.723,
123.752, 100.155, 76.726, 74.650, 49.216, 18.166. FAB-
4.11.24. (2R,4S,5R)-[2-(E-Phenylvinyl)-4-methyl-1,3-diox-
an-5-yl]amine (60b). Yield: 90%. Mp 82–84 ꢁC. IR(KBr):
m/cmꢀ1 = 3252, 3188, 3030, 1672, 1645, 1635, 1601,
1580, 1501, 1454, 1381, 1051, 741, 698. 1H NMR
(500 MHz, CDCl3): d/ppm = 7.993 (s, 2H), 7.596 (d,
J = 7.20 Hz, 2H), 7.560 (t, J = 7.20 Hz, 2H), 7.435 (t,
J = 7.20 Hz, 1H), 6.763 (d, J = 11.25 Hz, 1H), 6.538 (d ,
J = 11.22 Hz 1H), 5.559 (d, J = 6.00 Hz, 1H), 4.799 (m,
J = 4.74 Hz, 1H), 4.579 (d, J = 4.76 Hz, 2H), 3.993 (m,
J = 4.86 Hz, 1H), 1.387 (d, J = 4.86 Hz, 3H). 13C NMR
(CDCl3): d/ppm = 135.713, 128.714, 128.112, 127.394,
D
MS (m/e) 239 [M+H]+. ½aꢂ 10.8 (c 1.00, CHCl3). Anal.
20
Calcd for C11H14N2O2: C, 55.46; H, 5.92; N, 11.76.
Found: C, 55.58; H, 6.04; N, 11.82.
4.11.20. (2R,4S,5R)-2-[(30-Nitrophenyl)-4-methyl-1,3-diox-
an-5-yl]amine (50j). Yield: 95%. Mp 57–59 ꢁC. IR(KBr):
m/cmꢀ1 = 3252, 3189, 1647, 1636, 1602, 1580, 1524, 1500,
1
1456, 1382, 1350, 1049, 741, 698. H NMR (CDCl3):
d/ppm = 8.398 (s, 1H), 8.224 (d, J = 8.40 Hz, 1H), 8.012
(d, J = 7.80 Hz, 1H), 7.711 (t, J = 8.00 Hz, 1H), 5.725 (s,
1H), 4.317 (m, J = 4.32 Hz, 1H), 4.297 (d, J = 2.76 Hz,
2H), 2.656 (m, J = 3.24 Hz, 1H), 1.733 (s, 2H), 1.328 (d,
J = 4.66 Hz, 3H). 13C NMR (DMSO-d6): d/ppm =
148.627, 140.597, 132.588, 129.563, 124.222, 122.009,
126.587, 126.692, 105.835, 73.217, 72.946, 19.665. FAB-
D
MS (m/e) 220 [M+H]+. ½aꢂ 14.5 (c 1.0, H2O). Anal.
20
Calcd for C13H8ClNO2: C, 61.05; H, 7.09; N, 5.48.
Found: C, 61.16; H, 7.01; N, 5.36.
100.387, 76.552, 74.385, 49.083, 18.159. FAB-MS (m/e)
D
239 [M+H]+. ½aꢂ 13.2 (c 1.00, CHCl3). Anal. Calcd for
4.11.25. (2S,5S)-2-(40-Chlorophenyl-1,3-dioxaheptan-5-yl)-
amine (7c). Colorless powder, mp 48–49 ꢁC. IR(KBr):
m/cmꢀ1 = 3345, 3044, 2960, 2915, 2843, 1644, 1605, 1552,
1464, 1370, 1176, 1052, 821, 742, 691. 1H NMR (CDCl3):
d/ppm = 7.415 (d, J = 8.51 Hz, 2H), 7.326 (d, J = 8.51 Hz,
2H), 5.713 (s, 1H), 3.821 (d, J = 4.38 Hz, 2H), 3.543 (t,
J = 6.01 Hz, 2H), 3.089 (s, 1H), 1.889 (m, J = 3.72 Hz,
2H), 1.457 (s, 2H). 13C NMR (CDCl3): d/ppm = 147.886,
20
C11H14N2O2: C, 55.46; H, 5.92; N, 11.76. Found: C,
55.36; H, 6.03; N, 11.65.
4.11.21. (cis)-[2-(E-Phenylvinyl)-1,3-dioxan-5-yl]amine (6a).
Mp 65–67 ꢁC. IR(KBr): m/cmꢀ1 = 3249, 3188, 3030, 1673,
1645, 1635, 1601, 1581, 1500, 1457, 1048, 740, 696. H
1
NMR (500 MHz, CDCl3): d/ppm = 7.881 (s, 2H), 7.278
(q, J = 7.12 Hz, 2H), 7.263 (t, J = 7.16 Hz, 2H), 7.151 (t,
J = 7.16 Hz, 1H), 6.653 (d, J = 13.10 Hz, 1H), 6.257(d,
J = 13.10 Hz, 1H), 5.404 (d, J = 7.32 Hz, 1H), 4.443 (d,
J = 11.71 Hz, 4H). 13C NMR (CDCl3): d/ppm =
135.030, 128.323, 127.656, 126.867, 126.112, 123.967,
105.882, 73.287, 52.537. FAB-MS (m/e) 206 [M+H]+.
Anal. Calcd for C12H16ClNO2: C, 59.63; H, 6.67; N,
5.79. Found: C, 59.74; H, 6.81; N, 5.91.
146.407, 127.465, 123.383, 99.276, 69.684, 61.269, 49.879,
D
38.688. FAB-MS (m/e) 229 [M+H]+. ½aꢂ 8.0 (c 1.00,
20
CHCl3). Anal. Calcd for C11H12ClNO2: C, 58.03; H, 6.20;
N, 6.15. Found: C, 58.17; H, 6.09; N, 6.22.
4.11.26. (2R,5S)-2-(40-Chlorophenyl-1,3-dioxaheptan-5-
yl)amine (70c). Colorless powder, mp 56–57 ꢁC. IR(KBr):
m/cmꢀ1 = 3342, 3041, 2966, 2917, 2840, 1641, 1602, 1542,
1450, 1376, 1169, 1053, 822, 742, 694. 1H NMR (CDCl3):
d/ppm = 7.431 (d, J = 8.01 Hz, 2H), 7.334 (d,
J = 8.07 Hz, 2H), 5.695 (s, 1H), 3.894 (t, J = 3.22 Hz,
2H), 3.724 (d, J = 4.01 Hz, 2H), 3.134 (s, 1H), 2.023 (m,
J = 2.43 Hz, 2H), 1.602 (m, J = 3.52 Hz, 2H), 1.469 (s,
2H). 13C NMR (CDCl3): d/ppm = 147.886, 146.695,
4.11.22. (2S,4S,5R)-[2-(E-Phenylvinyl)-4-methyl-1,3-diox-
=
an-5-yl]amine (6b). Mp 77–79 ꢁC. IR(KBr): m/cmꢀ1
3250, 3190, 3031, 1675, 1644, 1636, 1602, 1581, 1503,
1455, 1380, 1049, 740, 696. 1H NMR (500 MHz, CDCl3):
d/ppm = 7.815 (s, 2H), 7.279 (d, J = 7.21 Hz, 2H), 7.246
(t, J = 7.22 Hz, 2H), 7.169 (t, J = 7.21 Hz, 1H), 6.565
(d, J = 11.87 Hz, 1H), 6.266 (d, J = 11.87 Hz 1H), 5.364
(d, J = 6.04 Hz, 1H), 4.597 (m, J = 4.71 Hz, 1H), 4.386
(d, J = 4.74 Hz, 2H), 3.816 (m, J = 4.84 Hz, 1H), 1.211
(d, J = 4.84 Hz, 3H). 13C NMR (CDCl3): d/ppm =
135.320, 128.401, 127.720, 127.072, 126.240, 126.187,
127.705, 123.651, 99.652, 70.574, 62.040, 50.462, 38.987.
D
FAB-MS (m/e) 229 [M+H]+. ½aꢂ ꢀ6.0 (c 1.00, CHCl3).
20
Anal. Calcd for C11H12ClNO2: C, 58.03; H, 6.20; N,
6.15. Found: C, 58.12; H, 6.19; N, 6.27.
4.11.27. (2S,5S)-2-(40-Nitrophenyl-1,3-dioxaheptan-5-yl)-
amine (7d). Colorless powder, mp 76–78 ꢁC. IR(KBr):
m/cmꢀ1 = 3345, 3042, 2970, 2919, 2851, 1642, 1600,
1561, 1521, 1462, 1372, 1352, 1171, 1050, 821, 742, 693.
1H NMR (CDCl3): d/ppm = 8.216 (d, J = 9.04 Hz, 2H),
7.665 (d, J = 8.52 Hz, 2H), 5.793 (s, 1H), 3.862 (d,
J = 4.31 Hz, 2H), 3.610 (t, J = 6.04 Hz, 2H), 3.153 (s,
1H), 1.821 (s, 2H), 1.789 (t, J = 3.95 Hz, 2H). 13C NMR
105.543, 72.805, 72.684, 19.285. FAB-MS (m/e) 220
D
[M+H]+. ½aꢂ ꢀ12.0 (c 1.0, H2O). Anal. Calcd for
20
C13H8ClNO2: C, 61.05; H, 7.09; N, 5.48. Found: C,
61.12; H, 7.17; N, 5.53.
4.11.23. (trans)-[2-(E-Phenylvinyl)-1,3-dioxan-5-yl]amine
(60a). Yield: 90%. Mp 70–72 ꢁC. IR(KBr): m/cmꢀ1 = 3252,

K. Gu et al. / Bioorg. Med. Chem. 15 (2007) 6273–6290
6285
(CDCl3): d/ppm = 147.886, 146.407, 127.465, 123.383,
C, 59.63; H, 6.67; N, 5.79. Found: C, 59.49; H, 6.59; N,
5.92.
D
99.276, 69.684, 61.269, 49.879, 38.688. ½aꢂ 8.0 (c 1.00,
20
CHCl3). FAB-MS (m/e) 239 [M+H]+. Anal. Calcd for
C11H14N2O4: C, 55.46; H, 5.92; N, 11.76. Found: C,
55.36; H, 5.88; N, 11.82.
4.11.32. (2R,5S)-2-(40-Chlorophenyl-1,3-dioxaoctan-5-yl)-
amine (80c). Colorless powder, mp 62–64 ꢁC. IR(KBr):
m/cmꢀ1 = 3284, 3197, 2962, 2913, 2845, 1604, 1547,
1455, 1371, 1167, 1048, 822. 1H NMR (CDCl3):
d/ppm = 7.401 (d, J = 7.50 Hz, 2H), 7.243 (d, J =
7.50 Hz, 2H), 5.773 (s, 1H), 3.726 (d, J = 4.40 Hz, 2H),
3.546 (t, J = 5.12 Hz, 2H), 3.004 (m, J = 4.40 Hz, 1H),
2.301 (s, 2H), 1.576 (t, J = 5.10 Hz, 2H), 1.751 (t,
J = 5.10 Hz, 2H). 13C NMR (CDCl3): d/ppm = 135.973,
133.168, 129.725, 129.132, 105.009, 72.761, 64.421,
4.11.28. (2R,5S)-2-(40-Nitrophenyl-1,3-dioxaheptan-5-yl)-
amine (70d). Colorless powder, mp 61–63 ꢁC. IR(KBr):
m/cmꢀ1 = 3330, 3031, 2964, 2916, 2848, 1644, 1604,
1
1521, 1453, 1374, 1355, 1168, 1049, 821, 741, 693. H
NMR (CDCl3): d/ppm = 8.221 (d, J = 9.04 Hz, 2H),
7.679 (d, J = 8.04 Hz, 2H), 5.764 (s, 1H), 3.912 (m,
J = 3.45 Hz, 2H), 3.702 (d, J = 5.85 Hz, 2H), 3.160 (s,
1H), 1.832 (m, J = 3.52 Hz, 2H), 1.401 (s, 2H). 13C
52.357, 31.684, 26.059. FAB-MS (m/e) 242 [M+H]+.
D
NMR (CDCl3): d/ppm = 147.886, 146.695, 127.705,
½aꢂ ꢀ7.4 (c 1.00, CHCl3). Anal. Calcd for C12H16ClNO2:
20
C, 59.63; H, 6.67; N, 5.79.Found: C, 59.80; H, 6.79; N,
5.66.
D
20
123.651, 99.652, 70.574, 62.040, 50.462, 38.987. ½aꢂ
ꢀ16.0 (c 1.00, CHCl3). FAB-MS (m/e) 239 [M+H]+. Anal.
Calcd for C11H14N2O4: C, 55.46; H, 5.92; N, 11.76.
Found: C, 55.60; H, 6.02; N, 11.57.
4.11.33. (2S,5S)-2-(40-Nitrophenyl-1,3-dioxaoctan-5-yl)-
amine (8d). Colorless powder, mp 81–83 ꢁC. IR(KBr):
m/cmꢀ1 = 3284, 3197, 2965, 2913, 2846, 1605, 1564,
1520, 1464, 1368, 1351, 1176, 1052, 821. 1H NMR
(CDCl3): d/ppm = 8.154 (d, J = 8.92 Hz, 2H), 7.473 (d,
J = 8.92 Hz, 2H), 5.468 (s, 1H), 3.581 (d, J = 4.34 Hz,
2H), 3.368 (t, J = 5.92 Hz, 2H), 2.931 (m, J = 4.36 Hz,
1H), 2.022 (s, 2H), 1.503 (m, J = 3.38 Hz, 2H), 1.457
(m, J = 3.38 Hz, 2H). 13C NMR (CDCl3): d/ppm =
4.11.29. (2S,5S)-2-(30-Nitrophenyl-1,3-dioxaheptan-5-yl)-
amine (7e). Colorless powder, mp 68–70 ꢁC. IR(KBr):
m/cmꢀ1 = 3322, 3041, 2962, 2920, 2850, 1642, 1614,
1556, 1521, 1462, 1364, 1352, 1160, 1052, 861, 773, 742,
1
698. H NMR (CDCl3): d/ppm = 8.374 (s, 1H). 8.194
(d, J = 7.82 Hz, 1H), 7.822 (d, J = 7.52 Hz, 1H), 7.546
(t, J = 8.01Hz, 1H), 5.796 (s, 1H), 3.855 (d,
J = 4.34 Hz, 2H), 3.604 (t, J = 6.22 Hz, 2H), 3.142 (s,
1H), 1.976 (m, J = 4.84 Hz, 2H), 1.674 (s, 2H). 13C
NMR (CDCl3): d/ppm = 148.191, 141.730, 132.639,
147.482, 143.341, 128.470, 123.503, 104.561, 72.412,
D
63.493, 51.897, 31.369, 25.874. ½aꢂ 9.2 (c 1.00, CHCl3).
20
FAB-MS (m/e) 253 [M+H]+. Anal. Calcd for
C12H16N2O4: C, 57.13; H, 6.39; N, 11.10. Found: C,
57.24; H, 6.44; N, 11.21.
129.194, 123.318, 121.719, 99.071, 69.525, 61.160,
D
49.844, 38.627. ½aꢂ 12.0 (c 1.00, CHCl3). FAB-MS (m/e)
20
239 [M+H]+. Anal. Calcd for C11H14N2O4: C, 55.46;
H, 5.92; N, 11.76. Found: C, 55.33; H, 5.78; N, 11.92.
4.11.34. (2R,5S)-2-(40-Nitrophenyl-1,3-dioxaoctan-5-yl)-
amine (80d). Colorless powder, mp 66–68 ꢁC. IR(KBr):
m/cmꢀ1 = 3286, 3195, 2962, 2911, 2844, 1601, 1556,
4.11.30. (2R,5S)-2-(30-Nitrophenyl-1,3-dioxaheptan-5-yl)-
amine (70e). Colorless powder, mp 65–67 ꢁC. IR(KBr):
m/cmꢀ1 = 3324, 3036, 2963, 2918, 2846, 1645, 1609,
1558, 1522, 1459, 1371, 1351, 1162, 1051, 862, 774, 739,
699. 1H NMR (CDCl3): d/ppm = 8.330 (s, 1H), 8.145 (d,
J = 8.42 Hz, 1H), 7.789 (t, J = 7.31 Hz, 1H), 7.505 (d,
J = 7.34 Hz, 1H), 5.739 (s, 1H), 3.757 (d, J = 5.25 Hz,
2H), 3.475 (t, J = 5.24 Hz, 2H), 3.096 (m,
J = 5.28 Hz,1H), 1.775 (m, J = 5.16 Hz, 2H), 1.456 (s,
2H). 13C NMR (CDCl3): d/ppm = 147.638, 141.985,
1
1458, 1370, 1349, 1165, 1051, 820. H NMR (CDCl3):
d/ppm = 8.243 (d, J = 8.90 Hz, 2H), 7.768 (d, J =
8.90 Hz, 2H), 5.845 (s, 1H), 3.764 (d, J = 4.36 Hz, 2H),
3.683 (t, J = 5.90 Hz, 2H), 2.989 (m, J = 4.35 Hz, 1H),
2.041 (s, 2H), 1.576 (m, J = 3.39 Hz, 2H), 1.497 (m,
J = 3.39 Hz, 2H). 13C NMR (CDCl3): d/ppm = 147.876,
143.901, 129.047, 124.012, 105.165, 73.011, 63.937,
D
52.314, 31.693, 26.176. ½aꢂ ꢀ11.7 (c 1.00, CHCl3).
20
FAB-MS (m/e) 253 [M+H]+. Anal. Calcd for
C12H16N2O4: C, 57.13; H, 6.39; N, 11.10. Found: C,
57.24; H, 6.34; N, 11.22.
132.953, 129.863, 123.142, 120.800, 98.593, 69.870,
D
62.631, 50.287, 38.096. ½aꢂ ꢀ6.0 (c 1.00, CHCl3). FAB-
20
MS (m/e) 239 [M+H]+. Anal. Calcd for C11H14N2O4: C,
55.46; H, 5.92; N, 11.76. Found: C, 55.54; H, 5.81; N,
11.90.
4.11.35. (2S,5S)-2-(30-Nitrophenyl-1,3-dioxaoctan-5-yl)-
amine (8e). Colorless powder, mp 72–74 ꢁC. IR(KBr):
m/cmꢀ1 = 3287, 3193, 2958, 2925, 2856, 1640, 1619,
1559, 1526, 1464, 1369, 1349, 1166, 1048, 861, 771, 702.
1H NMR (CDCl3): d/ppm = 8.126 (s, 1H), 8.121 (d,
J = 8.51 Hz, 1H), 7.576 (d, J = 8.52 Hz, 1H), 7.449 (t,
J = 8.51Hz, 1H), 5.469 (s, 1H), 3.586 (d, J = 4.35 Hz,
2H), 3.376 (t, J = 4.28 Hz, 2H), 2.936 (d, J = 4.38 Hz,
2H), 2.142 (s, 2H), 1.509 (d, J = 4.33 Hz, 2H), 1.486 (m,
J = 4.30 Hz, 2H). 13C NMR (CDCl3): d/ppm = 148.289,
4.11.31. (2S,5S)-2-(40-Chlorophenyl-1,3-dioxaoctan-5-yl)-
amine (8c). Colorless powder, mp 56–58 ꢁC. IR(KBr):
m/cmꢀ1 = 3282, 3194, 2964, 2918, 2848, 1600, 1557,
1
1524, 1461, 1373, 1171, 1049, 820. H NMR (CDCl3):
d/ppm = 7.213 (d, J = 7.51 Hz, 2H), 7.150 (d, J =
7.51 Hz, 2H), 5.467 (s, 1H), 3.562 (d, J = 4.41 Hz, 2H),
3.334 (t, J = 5.14 Hz, 2H), 2.895 (m, J = 4.41 Hz, 1H),
2.231 (s, 2H), 1.506 (t, J = 5.14 Hz, 2H), 1.457 (t,
J = 5.14 Hz, 2H). 13C NMR (CDCl3): d/ppm = 135.286,
132.878, 129.013, 128.891, 104.505, 72.343, 63.524,
138.037, 133.690, 129.319, 122.781, 122.171, 103.483,
D
63.586, 51.874, 31.337, 25.768. ½aꢂ 8.7 (c 1.00, CHCl3).
20
FAB-MS (m/e) 253 [M+H]+. Anal. Calcd for
C12H16N2O4: C, 57.13; H, 6.39; N, 11.10. Found: C,
57.01; H, 6.25; N, 11.22.
51.727, 31.305, 25.878. FAB-MS (m/e) 242 [M+H]+.
D
½aꢂ 9.3 (c 1.00, CHCl3). Anal. Calcd for C12H16ClNO2:
20

6286
K. Gu et al. / Bioorg. Med. Chem. 15 (2007) 6273–6290
4.11.36. (2R,5S)-2-(30-Nitrophenyl-1,3-dioxaoctan-5-yl)-
amine (80e). Colorless powder, mp 74–76 ꢁC. IR(KBr):
m/cmꢀ1 = 3287, 3194, 2955, 2922, 2853, 1614, 1554,
4.12.7. (cis)-2-[40-Nitrophenyl]-1,3-dioxan-5-yl]benzamide
(9d). Colorless powder, mp 220–224 ꢁC. In a NOE
experiment, a positive NOE between the NH at the 5-
position and the proton of the phenyl at the 2-position
was observed.
1
1522, 1462, 1366, 1351, 1164, 1050, 861, 773, 701. H
NMR (CDCl3): d/ppm = 8.217 (s, 1H), 8.153 (d,
J = 8.50 Hz, 1H), 7.775 (d, J = 8.50 Hz, 1H), 7.598 (t,
J = 8.50Hz, 1H), 5.865 (s, 1H), 3.687 (d, J = 4.36 Hz,
2H), 3.546 (t, J = 4.26 Hz, 2H), 2.978 (d, J = 4.35 Hz,
2H), 2.147 (s, 2H), 1.584 (d, J = 4.35 Hz, 2H), 1.502
(m, J = 4.32 Hz, 2H). 13C NMR (CDCl3): d/ppm =
4.12.8. (trans)-2-[40-Nitrophenyl]-1,3-dioxan-5-yl]benz-
amide (90d). Colorless powder, mp 120–122 ꢁC. In a
NOESY experiment, no NOE was observed.
148.930, 138.742, 134.025, 129.913, 123.017, 122.706,
4.12.9. (cis)-2-[30-Nitrophenyl]-1,3-dioxan-5-yl]benzamide
(9e). Mp 144–146 ꢁC. In a NOE experiment, a positive
NOE between the NH at the 5-position and the proton
of the phenyl at the 2-position was observed.
D
103.845, 64.007, 52.162, 31.741, 26.140. ½aꢂ ꢀ7.4 (c
20
1.00, CHCl3). FAB-MS (m/e) 253 [M+H]+. Anal. Calcd
for C12H16N2O4: C, 57.13; H, 6.39; N, 11.10. Found: C,
57.24; H, 6.40; N, 11.22.
4.12.10. (trans)-2-[30-Nitrophenyl]-1,3-dioxan-5-yl]benz-
amide (90e). Mp 130–132 ꢁC. In a NOESY experiment,
no NOE was observed. In a NOESY experiment, no
NOE was observed.
4.12. General procedure for the synthesis of 5-benzoyl-
amino-2-substitutedphenyl-1,3-dioxacycloalkanes12
At 0 ꢁC, to the solution of 0.48 mmol of 5-amino-2-sub-
stitutedphenyl-1,3-dioxacycloalkanes, 49 mg (0.48 mmol)
of triethylamine, and 3 ml chloroform, 67 mg
(0.48 mmol) of benzoyl chloride was added drop-wise.
The reaction mixture was stirred at room temperature
for 3 h and TLC (CHCl3/MeOH, 20:1) indicated com-
plete disappearance of starting material. The reaction
mixture was evaporated and the residue was dissolved
in 3 ml ether. The solution was stirred at room temper-
ature for 1 h and then filtered. The filtrate was evapo-
rated and the residue was purified by silica gel
chromatography (CHCl3/MeOH, 15:1) to provide the
title compound as a colorless powder.
4.12.11. (2S,4S,5R)-(2-Phenyl-4-methyl-1,3-dioxan-5-yl)-
benzamide (9f). Colorless powder, mp 125–127 ꢁC. In
NOESY experiment, NOEs between the CH3 at the
4-position and the proton of phenyl at the 2-position,
and between the CH3 at the 4-position and the NH at
the 5-position were observed.
4.12.12. (2R,4S,5R)-(2-Phenyl-4-methyl-1,3-dioxan-5-yl)-
benzamide (90f). Colorless powder, mp 133–135 ꢁC. In a
NOESY experiment, no NOE was observed.
4.12.13. (2S,4S,5R)-[2-(40-Methylphenyl)-4-methyl-1,3-
dioxan-5-yl]benzamide (9g). Colorless powder, mp 152–
154 ꢁC. In NOESY experiment, NOEs between the
CH3 at the 4-position and the proton of phenyl at the
2-position, and between the CH3 at the 4-position and
the NH at the 5-position were observed.
4.12.1. (cis)-(2-Phenyl-1,3-dioxan-5-yl)benzamide (9a).
Colorless powder, mp 133–135 ꢁC. In a NOE experiment,
a positive NOE between the NH at the 5-position and the
proton of the phenyl at the 2-position was observed.
4.12.2. (trans)-(2-Phenyl-1,3-dioxan-5-yl)benzamide (90a).
Colorless powder, mp 146–148 ꢁC. In a NOESY experi-
ment, no NOE was observed.
4.12.14. (2R,4S,5R)-[2-(40-Methylphenyl)-4-methyl-1,3-
dioxan-5-yl]benzamide (90g). Colorless powder, mp
144–146 ꢁC. In a NOESY experiment, no NOE was
observed.
4.12.3. (cis)-2-[40-Methylphenyl]-1,3-dioxan-5-yl]benzam-
ide (9b). Colorless powder, mp 134–136 ꢁC. In a NOE
experiment, a positive NOE between the NH at the 5-
position and the proton of the phenyl at the 2-position
was observed.
4.12.15. (2S,4S,5R)-(2-(40-Chlorophenyl)-4-methyl-1,3-
dioxan-5-yl)benzamide (9h). Colorless powder, mp 125–
127 ꢁC. In NOESY experiment, NOEs between the CH3
at the 4-position and the proton of phenyl at the 2-posi-
tion, and between the CH3 at the 4-position and the NH
at the 5-position were observed.
4.12.4. (trans)-2-[40-Methylphenyl]-1,3-dioxan-5-yl]benz-
amide (90b). Colorless powder, mp 146–148 ꢁC. In a
NOESY experiment, no NOE was observed.
4.12.16. (2R,4S,5R)-(2-(40-Chlorophenyl)-4-methyl-1,3-
dioxan-5-yl)benzamide (90h). Colorless powder, mp
120–122 ꢁC. In a NOESY experiment, no NOE was
observed.
4.12.5. (cis)-2-[40-Chlorophenyl]-1,3-dioxan-5-yl]benzam-
ide (9c). Colorless powder, mp 168–170 ꢁC. In a NOE
experiment, a positive NOE between the NH at the 5-po-
sition and the proton of the phenyl at the 2-position was
observed.
4.12.17. (2S,4S,5R)-(2-(40-Nitrophenyl)-4-methyl-1,3-diox-
an-5-yl)benzamide (9i). Colorless powder, mp 90–92 ꢁC. In
NOESY experiment, NOEs between the CH3 at the 4-posi-
tion and the proton of phenyl at the 2-position, and between
the CH3 at the 4-position and the NH at the 5-position were
observed.
4.12.6. (trans)-2-[40-Chlorophenyl]-1,3-dioxan-5-yl]benz-
amide (90c). Colorless powder, mp 133–135 ꢁC. In a
NOESY experiment, no NOE was observed.

K. Gu et al. / Bioorg. Med. Chem. 15 (2007) 6273–6290
6287
4.12.18. (2R,4S,5R)-(2-(40-Nitrophenyl)-4-methyl-1,3-diox-
an-5-yl)benzamide (90i). Colorless powder, mp 112–114 ꢁC.
In a NOESY experiment, no NOE was observed.
4.12.31. (2S,5S)-2-(40-Chlorophenyl-1,3-dioxaoctan-5-yl)-
benzamide (12c). Colorless powder, mp 113–115 ꢁC. In
NOESY experiment, NOE between the NH at the 5-position
and the proton of phenyl at the 2-position was observed.
4.12.19. (2S,4S,5R)-[2-(30-Nitrophenyl)-4-methyl-1,3-diox-
an-5-yl]benzamide (9j). Colorless powder, mp 140–142 ꢁC.
In NOESY experiment, NOEs between the CH3 at the 4-po-
sition and the proton of phenyl at the 2-position, and
between the CH3 at the 4-position and the NH at the 5-posi-
tion were observed.
4.12.32. (2R,5S)-2-(40-Chlorophenyl-1,3-dioxaoctan-5-yl)-
benzamide (120c). Colorless powder, mp 118–120 ꢁC. In a
NOESY experiment, no NOE was observed.
4.12.33. (2S,5S)-2-(40-Nitrophenyl-1,3-dioxaoctan-5-yl)-
benzamide (12d). Colorless powder, mp 133–135 ꢁC. In
NOESY experiment, NOE between the NH at the 5-posi-
tion and the proton of phenyl at the 2-position was
observed.
4.12.20. (2R,4S,5R)-[2-(30-Nitrophenyl)-4-methyl-1,3-diox-
an-5-yl]benzamide (90j). Colorless powder, mp 130–132 ꢁC.
In a NOESY experiment, no NOE was observed.
4.12.21. (cis)-[2-(E-Phenylvinyl)-1,3-dioxan-5-yl]benzam-
ide (10a). Colorless powder, mp 119–121 ꢁC. In a NOE
experiment, a positive NOE between the NH at the 5-
position and the proton of the phenyl at the 2-position
was observed.
4.12.34. (2R,5S)-2-(40-Nitrophenyl-1,3-dioxaoctan-5-yl)-
benzamide (120d). Colorless powder, mp 116–118 ꢁC. In
a NOESY experiment, no NOE was observed.
4.12.35. (2S,5S)-2-(30-Nitrophenyl-1,3-dioxaoctan-5-yl)-
benzamide (12e). Colorless powder, mp 124–126 ꢁC. In
NOESY experiment, NOE between the NH at the 5-posi-
tion and the proton of phenyl at the 2-position was
observed.
4.12.22. (trans)-[2-(E-Phenylvinyl)-1,3-dioxan-5-yl]benz-
amide (100a). Colorless powder, mp 115–117 ꢁC. In a
NOESY experiment, no NOE was observed.
4.12.23. (2S,4S,5R)-[2-(E-Phenylvinyl)-4-methyl-1,3-diox-
an-5-yl]benzamide (10b). Colorless powder, mp 123–
125 ꢁC. In NOESY experiment, NOEs between the CH3
at the 4-position and the proton of phenyl at the 2-posi-
tion, and between the CH3 at the 4-position and the NH
at the 5-position were observed.
4.12.36. (2R,5S)-2-(30-Nitrophenyl-1,3-dioxaoctan-5-yl)-
benzamide (120e). Colorless powder, mp 127–129 ꢁC. In
a NOESY experiment, no NOE was observed.
4.12.37. (cis)-(2-Phenyl-1,3-dioxan-5-yl)phenylacetamide
(13a). Colorless powder, mp 150–152 ꢁC. In NOESY
experiment, NOE between the NH at the 5-position
and the proton of phenyl at the 2-position was observed.
4.12.24. (2R,4S,5R)-[2-(E-Phenylvinyl)-4-methyl-1,3-diox-
an-5-yl]benzamide (100b). Colorless powder, mp 116–
118 ꢁC. In a NOESY experiment, no NOE was observed.
4.12.38. (trans)-(2-Phenyl-1,3-dioxan-5-yl)phenylaceta-
mide (130a). Colorless powder, mp 194–195 ꢁC. In a
NOESY experiment, no NOE was observed.
4.12.25. (2S,5S)-2-(4-Chlorophenyl-1,3-dioxaheptan-5-yl)-
benzamide (11c). Colorless powder, mp 129–131 ꢁC. In
NOESY experiment, NOE between the NH at the 5-posi-
tionand the protonof phenylat the2-positionwasobserved.
4.12.39. (cis)-[2-(40-Methylphenyl)-1,3-dioxan-5-yl]phenyl-
acetamide (13b). Colorless powder, mp 185–187 ꢁC. In
NOESY experiment, NOE between the NH at the 5-posi-
tion and the proton of phenyl at the 2-position was
observed.
4.12.26. (2R,5S)-2-(4-Chlorophenyl-1,3-dioxaheptan-5-yl)-
benzamide (110c). Colorless powder, mp 134–136 ꢁC. In a
NOESY experiment, no NOE was observed.
4.12.40. (trans)-[2-(40-Methylphenyl)-1,3-dioxan-5-yl]phenyl-
acetamide (130b). Colorless powder, mp 181–183 ꢁC. In a
NOESY experiment, no NOE was observed.
4.12.27.
(2S,5S)-2-(4-Nitrophenyl-1,3-dioxaheptan-5-
yl)benzamide (11d). Colorless powder, mp 121–123 ꢁC.
In NOESY experiment, NOE between the NH at the
5-position and the proton of phenyl at the 2-position
was observed.
4.12.41. (cis)-[2-(40-Chlorophenyl)-1,3-dioxan-5-yl]phenyl-
acetamide (13c). Colorless powder, mp 170–172 ꢁC. In
NOESY experiment, NOE between the NH at the 5-posi-
tion and the proton of phenyl at the 2-position was
observed.
4.12.28.
(2R,5S)-2-(4-Nitrophenyl-1,3-dioxaheptan-5-yl)-
benzamide (110d). Colorless powder, mp 124–126 ꢁC. In a
NOESY experiment, no NOE was observed.
4.12.42. (trans)-[2-(40-Chlorophenyl)-1,3-dioxan-5-yl]phenyl-
acetamide (130c). Colorless powder, mp 256–258 ꢁC. In a
NOESY experiment, no NOE was observed.
4.12.29. (2S,5S)-2-(30-Nitrophenyl-1,3-dioxaheptan-5-yl)-
benzamide (11e). Colorless powder, mp 114–116 ꢁC. In
NOESY experiment, NOE between the NH at the 5-posi-
tionand the protonof phenylat the2-positionwasobserved.
4.12.43. (cis)-2-[40-Nitrophenyl]-1,3-dioxan-5-yl]phenyl-
acetamide (13d). Colorless powder, mp 201–203 ꢁC. In
NOESY experiment, NOE between the NH at the 5-po-
sition and the proton of phenyl at the 2-position was
observed.
4.12.30. (2R,5S)-2-(30-Nitrophenyl-1,3-dioxaheptan-5-yl)-
benzamide (110e). Colorless powder, mp 117–119 ꢁC. In a
NOESY experiment, no NOE was observed.

6288
K. Gu et al. / Bioorg. Med. Chem. 15 (2007) 6273–6290
4.12.44. (trans)-2-[40-Nitrophenyl]-1,3-dioxan-5-yl]pheny-
lacetamide (130d). Colorless powder, mp 107–109 ꢁC. In
a NOESY experiment, no NOE was observed.
147–149 ꢁC. In NOESY experiment, NOEs between the
CH3 at the 4-position and the proton of phenyl at the
2-position, and between the CH3 at the 4-position and
the NH at the 5-position were observed.
4.12.45. (cis)-2-[30-Nitrophenyl]-1,3-dioxan-5-yl]pheny-
lacetamide (13e). Mp 128–132 ꢁC. In NOESY experi-
ment, NOE between the NH at the 5-position and the
proton of phenyl at the 2-position was observed.
4.12.56. (2R,4S,5R)-[2-(30-Nitrophenyl)-4-methyl-1,3-diox-
an-5-yl]phenylacetamide 130j. Colorless powder, mp 140–
142 ꢁC. In a NOESY experiment, no NOE was observed.
4.12.46. (trans)-2-[30-Nitrophenyl]-1,3-dioxan-5-yl]pheny-
lacetamide (130e). Mp 141–143 ꢁC. In a NOESY experi-
ment, no NOE was observed.
4.12.57. (cis)-[2-(E-Phenylvinyl)-1,3-dioxan-5-yl]phenyl-
acetamide (14a). Colorless powder, mp 135–136 ꢁC. In
NOESY experiment, NOE between the NH at the 5-posi-
tion and the proton of phenyl at the 2-position was
observed.
4.12.47. (2S,4S,5R)-(2-Phenyl-4-methyl-1,3-dioxan-5-yl)-
phenylacetamide (13f). Colorless powder, mp 144–
146 ꢁC. In NOESY experiment, NOEs between the
CH3 at the 4-position and the proton of phenyl at the
2-position, and between the CH3 at the 4-position and
the NH at the 5-position were observed.
4.12.58. (trans)-[2-(E-Phenylvinyl)-1,3-dioxan-5-yl]phenyl-
acetamide (140a). Colorless powder, mp 150–152 ꢁC. In a
NOESY experiment, no NOE was observed.
4.12.59. (2S,4S,5R)-[2-(E-phenylvinyl)-4-methyl-1,3-diox-
an-5-yl]phenylacetyl amide (14b). Colorless powder, mp
125–127 ꢁC. In NOESY experiment, NOEs between the
CH3 at the 4-position and the proton of phenyl at the 2-po-
sition, and between the CH3 at the 4-position and the NH
at the 5-position were observed.
4.12.48. (2R,4S,5R)-(2-Phenyl-4-methyl-1,3-dioxan-5-yl)-
phenylacetamide (130f). Colorless powder, mp 156–
158 ꢁC. In a NOESY experiment, no NOE was observed.
4.12.49. (2S,4S,5R)-2-(40-Methylphenyl)-4-methyl-1,3-
dioxan-5-yl]phenylacetamide (13g). Colorless powder,
mp 159–161 ꢁC. In NOESY experiment, NOEs between
the CH3 at the 4-position and the proton of phenyl at
the 2-position, and between the CH3 at the 4-position
and the NH at the 5-position were observed.
4.12.60. (2R,4S,5R)-[2-(E-phenylvinyl)-4-methyl-1,3-diox-
an-5-yl] phenylacetyl amide (140b). Colorless powder, mp
141–143 ꢁC. In a NOESY experiment, no NOE was
observed.
4.12.61. (2S,5S)-2-(40-Chlorophenyl-1,3-dioxaheptan-5-
yl)phenylacetamide (15c). Colorless powder, mp 132–
134 ꢁC. In NOESY experiment, NOE between the NH
at the 5-position and the proton of phenyl at the 2-posi-
tion was observed.
4.12.50. (2R,4S,5R)-[2-(40-Methylphenyl)-4-methyl-1,3-
dioxan-5-yl]phenylacetamide (130g). Colorless powder,
mp 125–127 ꢁC. In a NOESY experiment, no NOE
was observed.
4.12.51. (2S,4S,5R)-(2-(40-Chlorophenyl)-4-methyl-1,3-di-
oxan-5-yl)phenylacetamide (13h). Colorless powder,
mp 137–139 ꢁC. In NOESY experiment, NOEs be-
tween the CH3 at the 4-position and the proton of
phenyl at the 2-position, and between the CH3 at
the 4-position and the NH at the 5-position were
observed.
4.12.62. (2R,5S)-2-(40-Chlorophenyl-1,3-dioxaheptan-5-yl)-
phenylacetamide (150c). Colorless powder, mp 139–141 ꢁC.
In a NOESY experiment, no NOE was observed. 13C
NMR (CDCl3): d/ppm = 169.957, 135.998, 135.634,
133.017, 129.986, 129.367, 129.048, 128.967, 127.891,
105.034, 69.591, 58.720, 45.565, 33.472. EI-MS (m/e)
D
345 [M]+. ½aꢂ ꢀ15.0 (c 0.02, CHCl3). Anal. Calcd for
20
C19H20ClNO3: C, 65.99; H, 5.83; N, 4.05. Found: C,
65.89; H, 5.79; N, 4.02.
4.12.52. (2R,4S,5R)-(2-(40-Chlorophenyl)-4-methyl-1,3-
dioxan-5-yl)phenylacetamide (130h). Colorless powder,
mp 129–131 ꢁC. In a NOESY experiment, no NOE was
observed.
4.12.63. (2S,5S)-2-(40-Nitrophenyl-1,3-dioxaheptan-5-yl)-
phenylacetamide (15d). Colorless powder, mp 125–
127 ꢁC. In NOESY experiment, NOE between the NH
at the 5-position and the proton of phenyl at the 2-posi-
tion was observed.
4.12.53. (2S,4S,5R)-[2-(40-Nitrophenyl)-4-methyl-1,3-diox-
an-5-yl]phenylacetamide (13i). Colorless powder, mp 127–
129 ꢁC. In NOESY experiment, NOEs between the CH3
at the 4-position and the proton ofphenyl atthe 2-position,
and between the CH3 at the 4-position and the NH at the 5-
position were observed.
4.12.64. (2R,5S)-2-(40-Nitrophenyl-1,3-dioxaheptan-5-yl)-
phenylacetamide (150d). Colorless powder, mp 129–
131 ꢁC. In a NOESY experiment, no NOE was observed.
4.12.54. (2R,4S,5R)-[2-(40-Nitrophenyl)-4-methyl-1,3-diox-
an- 5-yl]phenylacetamide (130i). Colorless powder, mp 120–
122 ꢁC. In a NOESY experiment, no NOE was observed.
4.12.65. (2S,5S)-2-(30-Nitrophenyl-1,3-dioxaheptan-5-yl)-
phenylacetamide (15e). Colorless powder, mp 127–
129 ꢁC. In NOESY experiment, NOE between the NH
at the 5-position and the proton of phenyl at the 2-posi-
tion was observed.
4.12.55. (2R,4S,5R)-2-(30-Nitrophenyl)-4-methyl-1,3-diox-
an-5-yl]phenylacetamide (13j). Colorless powder, mp

K. Gu et al. / Bioorg. Med. Chem. 15 (2007) 6273–6290
6289
4.12.66. (2R,5S)-2-(30-Nitrophenyl-1,3-dioxaheptan-5-yl)-
phenylacetamide (150e). Colorless powder, mp 135–
137 ꢁC. In a NOESY experiment, no NOE was observed.
was carried out by use of ANOVA test, p < 0.05 is con-
sidered significant.
4.14. Tail bleeding time measurement
4.12.67. (2S,5S)-2-(40-Chlorophenyl-1,3-dioxaoctan-5-yl)-
phenylacetamide (16c). Colorless powder, mp 120–
122 ꢁC. In NOESY experiment, NOE between the NH at
the 5-position and the proton of phenyl at the 2-position
was observed.
The new anti-inflammatory agents were orally adminis-
tered to male mice (body weight 18–22 g). After 30, 45,
60, and 90 min of administration, a mouse was placed
in a tube holder with its tail protruding, and a 2-mm
cut was made on the tail. Flowing blood until it stopped
was gently wiped away with a tissue every 30 s until
bleeding ceased and the time recorded.
4.12.68. (2R,5S)-2-(40-Chlorophenyl-1,3-dioxaoctan-5-yl)-
phenylacetamide (160c). Colorless powder, mp 130–
132 ꢁC. In a NOESY experiment, no NOE was observed.
4.15. PKC inhibition assay
4.12.69. (2S,5S)-2-(40-Nitrophenyl-1,3-dioxaoctan-5-yl)-
phenylacetamide (16d). Colorless powder, mp 136–
138 ꢁC. In NOESY experiment, NOE between the NH
at the 5-position and the proton of phenyl at the 2-posi-
tion was observed.
4.15.1. Preparation of PKC. At 4 ꢁC, 500 g of fresh bo-
vine brains was excised, immersed and rinsed in 1 L of
ice-cold buffer A (20 mM Tris–HCl, pH 7.5, 3 mM
EDTA, 50 mM 2-mercaptoethanol, 1 mM PhMeSO2F,
and 10 mg/L aprotinin). The tissue was homogenized
in 2.5 L of buffer A for 2 min and centrifuged at 9000g
for 20 min. To the supernatant ammonium sulfate
(21%, w/v) was slowly added. The mixture was centri-
fuged at 9000g for 20 min to remove the formed precip-
itates. To the supernatant additional ammonium sulfate
was added to a final concentration of 45% (w/v). After
centrifugation at 9000g for 20 min, the recovered precip-
itates were dissolved in 150 ml buffer B (20 mM Tris–
HCl, pH 7.5, 2 mM EDTA, 50 mM 2-mercaptoethanol).
The solution was dialyzed in 20-fold volume of buffer B
for 3 days and then centrifuged at 30,000g for 30 min.
The supernatant was applied to a 1-L DEAE–cellulose
column equilibrated with buffer B. The column was
washed with 2.5 L of buffer B at a flow rate of 125 ml/h.
The enzyme was eluted from the column by a linear gra-
dient of 0–0.3 M NaCl in buffer B at a flow rate of 60 ml/h.
The fractions of 50 ml each were collected and were
assayed for PKC activity. The fractions with high
PKC activity were pooled and concentrated to 112 ml
by ultrafiltration, adjusted to 30% glycerol, and stored
at ꢀ85 ꢁC.
4.12.70. (2R,5S)-2-(40-Nitrophenyl-1,3-dioxaoctan-5-yl)-
phenylacetamide (160d). Colorless powder, mp 150–
152 ꢁC. In a NOESY experiment, no NOE was observed.
4.12.71. (2S,5S)-2-(30-Nitrophenyl-1,3-dioxaoctan-5-yl)-
phenylacetamide (16e). Colorless powder, mp 131–
133 ꢁC. In NOESY experiment, NOE between the NH
at the 5-position and the proton of phenyl at the 2-posi-
tion was observed.
4.12.72. (2R,5S)-2-(30-Nitrophenyl-1,3-dioxaoctan-5-yl)-
phenylacetamide (160e). Colorless powder, mp 138–
140 ꢁC. In a NOESY experiment, no NOE was observed.
4.13. In vivo anti-inflammatory assay
4.13.1. Animals. Male Kunming mice (about 25 g) were
inbred and grown in the animal room at the college of
Pharmacy, Peking University. The animal room was
maintained at 23 2 ꢁC with a 12 h light/dark cycle. Food
and tap water were supplied ad libitum. The ethical guide-
lines described in the NIH guide for care and use of Lab-
oratory Animals were followed throughout the
experiments.
4.15.2. Determination of PKC activity.34,35
4.15.2.1. Basic theory of method. After activation by
phosphatidylserine (PS), diacylglycerol (DG), and Ca+
PKC catalyzes the phosphorylation of Histone (III-S)
by consuming ATP and produces ADP or AMP. Thus
PKC specific activity may be defined as ATP’s consump-
tion rate as the result of per milligram enzymes catalyz-
ing the phosphorylation of Histone (III-S).
Consequently PKC activity may be determined via
ATP’s consumption by ion-pair reversed phase HPLC.
The unit of PKC activity may be represented as nmol/
min/mg.
4.13.2. Xylene-induced ear edema.11,12 Male Kunming
mice were randomly divided into three groups of 12
mice, namely the test group, vehicle control group,
and positive control group. The mice in vehicle control
group were administerted orally with a suspension of
Aspirin in CMC at a dosage of 20 mg/kg, while the mice
in the test group were administerted orally a suspension
of the tested compounds in CMC at dosages of 20, 10,
and 5 mg/kg. Thirty minutes later, 0.03 ml of xylene
was applied to both the anterior and posterior surfaces
of the right ear. The left ear was considered as control.
Two hours after xylene application, mice were sacrificed
and both ears were removed. Using a cork borer with a
diameter of 7 mm, several circular sections were taken
and weighed. The increase in weight caused by the irri-
tant was measured through subtracting the weight of
the untreated left ear section from that of the treated
right ear section. The statistical analysis of the data
4.15.2.2. Buffer and reactive solution. Buffer C: Tris–
HCl 20 mM, pH 7.4; reactive solution 1: Tris–HCl
20 mM, PS 100 lg/ml, DG 10 lg/ml, MgCl2 10 mM,
CaCl2 1 mM, Histone(III-S) 1 mg/ml, DTT 1 mM, pH
7.4; reactive solution 2: Tris–HCl 20 mM, MgCl2
10 mM, Histone(III-S) 1 mg/ml, DTT 1 mM, EGTA
0.1 mg/ml, pH 7.4; reactive solution 3: ATP 0.5 mM;
enzymes: contained protein 5 mg/ml.

6290
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4. Coussens, L. M.; Werb, Z. Nature 2002, 420, 860–867.
4.15.2.3. HPLC conditions. Reversed-phase Rainbow
5. Giovannucci, E. Biomed. Pharmacother. 1999, 53, 303–
308.
6. Arber, N.; Eagle, C. J.; Spicak, J., et al. N. Engl. J. Med.
2006, 355, 885–895.
7. Raju, R.; Correa, M. C. Dis. Colon. Rectum. 2006, 49,
113–124.
C18 column (Kromasil 4.6 · 150 mm), mobile phase in-
cluded tetrabutyl ammonium hydroxide 5 mmol/L,
methanol, and KH2PO4 (20:80, v/v, pH 7.0), flow rate:
1.0 ml/min, detected at 259 nm.
4.15.2.4. Protracting standard curve. According to
Table 5 various solutions were added to control or reac-
tive tube (1.5 ml plastic centrifugal tube). The tubes were
kept at 30 ꢁC for 10 min, and then at 100 ꢁC for 1 min.
After cooling, 300 ll of chloroform was added. The
solution was surged for 1 min and centrifuged at 2000g
for 5 min. To the supernatant 300 ll of chloroform
was added. The solution was surged for 1 min and then
centrifuged at 2000g for 5 min. Onto the HPLC column
20 ll of supernatant was injected, detected at 259 nm.
The standard curve was protracted with ATP content
as X-coordinate, peak area as Y-coordinate.
8. Jiang, J. B.; Johnson, M. G.; Nichols, J. U.S. Patent
5,360,818, 1994.
9. Bi, L.; Zhao, M.; Wang, C.; Peng, S. Eur. J. Org. Chem.
2000, 2669–2676.
10. Bi, L.; Zhao, M.; Wang, C.; Peng, S.; Winterfeldt, E.
J. Org. Chem. 2002, 67, 22–26.
11. Bi, L.; Zhang, Y.; Zhao, M.; Wang, C.; Tok, J.; Peng, S.
Bioorg. Med. Chem. 2005, 13, 5640.
12. Gu, K.; Bi, L.; Zhao, M.; Wang, C.; Kao, M.; Tok, J.;
Peng, S. Bioorg. Med. Chem. 2006, 14, 1339–1347; Gu, K.;
Bi, L.; Zhao, M.; Wang, C.; Ju, J.; Peng, S. Bioorg. Med.
Chem. 2007. doi:10.1016/j.bmc.2007.05.013.
´
13. Arroyo, M.; De la Mata, I.; Acebal, C.; Castillon, M. P.
Appl. Microbiol. Biotechnol. 2003, 60, 507.
4.15.2.5. Solution of 1,3-dioxacycloalkanes. Each of
the synthesized 1,3-dioxacycloalkanes was dissolved in
buffer C to make 1,3-dioxacycloalkane containing solu-
tion for determination of PKC activity.
´
14. Fernandez-Lafuente, R.; Rosell, C. M.; Piatkowska, B.;
´
Guisan, J. M. Enzyme Microb. Technol. 1996, 19, 9.
15. Danzig, J.; Tischer, W.; Wandrey, C. Ind. J. Chem. 1993,
32, 40.
16. Illanes, A.; Altamirano, C.; Zun˜iga, M. E. Biotechnol.
Bioeng. 1996, 50, 609.
4.15.2.6. Determination of PKC activity. With the same
procedure as used for protracting standard curve in the
reactive tube the solution of tested compound was added.
To the reactive tube 200 ll of reactive solution 1, 40 ll of
reactive solution 3, 20 ll of PKC, and 60 ll of 1,3-diox-
acycloalkane containing buffer C were added. The final
concentration of 1,3-dioxacycloalkane was 0.01, 0.10,
1.00, 5.00, and 10.00 lmol/ml. To the controlled tube
200 ll of reactive solution 2, 40 ll of reactive solution 3,
20 ll of PKC, and 60 ll of buffer C were added. The tubes
were kept at 30 ꢁC for 10 min, and then at 100 ꢁC for
1 min. After cooling 300 ll of chloroform was added.
The solution was surged for 1 min and centrifuged at
2000g for 5 min. To the supernatant 300 ll of chloroform
was added. The solution was surged for 1 min and then
centrifuged at 2000g for 5 min. 20 ll of supernatant was
injected to the HPLC column, detected at 259 nm. PKC
specific activity was calculated according to standard
curve. Their IC50 values were calculated by software of
statistics.
17. Azevedo, A. M.; Fonseca, L. P.; Prazeres, D. M. F.
J. Chem. Technol. Biotechnol. 1999, 74, 1110.
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¨
19. Diender, M. B.; Straathof, A. J. J.; Heijnen, J. J. Biocatal.
Biotransform. 1998, 16, 275.
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