An alternative synthesis of 3?,4?-diaminoflavones to evaluate their antioxidant ability and cell apoptosis of zebrafish larvae

Article abstract of DOI:10.3390/molecules17078206

We described herein a concise synthesis of 3?,4?-diaminoflavone 10. This new, three-step synthetic approach is more efficient than the conventional seven-step synthetic method. The route is shortened significantly by introducing the amino moieties early and eliminating the need for nitro group reduction. The other two analogues, 5,7-dihydroxy-3?,4?-diaminoflavone 11 and 5,7-dimethoxy-3?,4?-diaminoflavone 12, were also synthesized similarly. The above three compounds, along with flavone, were evaluated for their antioxidant and UVB-protection abilities on zebrafish larvae. The data showed that compound 10 exhibited the best result, with ?102.3% of ROS-scavenging rate.

Full text of DOI:10.3390/molecules17078206

Molecules 2012, 17, 8206-8216; doi:10.3390/molecules17078206
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
An Alternative Synthesis of 3′,4′-Diaminoflavones to
Evaluate Their Antioxidant Ability and Cell Apoptosis of
Zebrafish Larvae
Tzenge-Lien Shih *, Chih-Ang Hsiao †, Zi-Yu Lin † and Yau-Hung Chen *
Department of Chemistry, Tamkang University, Tamsui Dist., 25137 New Taipei City, Taiwan;
E-Mails: z90107@yahoo.com.tw (C.-A.H.); shes060215@yahoo.com.tw (Z.-Y.L.)
†
These authors contributed equally to this work.
* Authors to whom correspondence should be addressed; E-Mails: tlshih@mail.tku.edu.tw (T.-L.S.);
yauhung@mail.tku.edu.tw (Y.-H.C.); Tel./Fax: +886-2-8631-5024 (T.-L.S.).
Received: 1 June 2012; in revised form: 2 July 2012 / Accepted: 6 July 2012 /
Published: 9 July 2012
Abstract: We described herein a concise synthesis of 3′,4′-diaminoflavone 10. This new,
three-step synthetic approach is more efficient than the conventional seven-step synthetic
method. The route is shortened significantly by introducing the amino moieties early and
eliminating the need for nitro group reduction. The other two analogues, 5,7-dihydroxy-
3′,4′-diaminoflavone 11 and 5,7-dimethoxy-3′,4′-diaminoflavone 12, were also synthesized
similarly. The above three compounds, along with flavone, were evaluated for their
antioxidant and UVB-protection abilities on zebrafish larvae. The data showed that
compound 10 exhibited the best result, with −102.3% of ROS-scavenging rate.
Keywords: antioxidant; Baker-Venkataraman rearrangement; diaminoflavones;
ROS-scavenging; zebrafish larvae
1. Introduction
Over 6,500 flavonoids are known [1], and many naturally occurring flavonoids show bioactivity [2,3].
Flavonoids are widely distributed in vegetables and plants [4–6], and their diverse biological roles
have been extensively investigated. For example, many flavonoids show antioxidant activity [2,3,7],
are metal chelators [8,9], cell-membrane protectors [10–12], or oxidase inhibitors [13]. Certain

Molecules 2012, 17
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synthetic flavonoid analogues have shown to prevent inflammation [14,15], cancer [1619], or
cardiovascular diseases [20].
The hydroxyl groups presented in flavonoids play an important role in their activities [21]. It has
been claimed that the amino groups in flavonoids have the same behaviors as hydroxyl groups as
hydrogen bond donors and acceptors [22]. Therefore, replacement of the hydroxyl groups with amino
groups in flavonoids may help to develop more soluble salt form of flavonoids while sustaining or
even improving their biological activities [22].
Few natural or synthetic aminoflavonoids have been reported however [2132]. The most common
method includes the reduction of the nitro [23,24,26,28,29] or azido [27,32] group(s) on the aromatic
rings, followed by protection and deprotection of the resulting amino moieties [25,28,30,31]. As a part
of our ongoing interest in the various aminoflavonoids, we are interested in the roles of their amino
groups. We have selected for study compounds 10, 11 and 12, which bear amino groups at the 3′ and 4′
positions in the B ring and various substituents (H, OH, OMe) at the A rings. We wished to understand
more the roles of amino groups on the A ring of flavones to compare with the existing potent
antioxidants, such as luteolin [2]. This should allow us to evaluate their antioxidant ability. Among
these molecules, we have prepared in three steps (~24.7% total yield) compound 10, which was
previously synthesized by Göker et al. in seven steps (~11.5% total yield) [23]. Based on the same
strategy, compounds 11 and 12 could be easily prepared in an efficient manner.
In order to rapidly screen the above diaminoflavones, we selected the zebrafish as an excellent
model organism for chemical and toxicological studies because of its physiological similarity to
mammals. In particular, the rapid developed and optical transparency of zebrafish embryos allow
non-invasive cellular ROS detection in vivo. In this regard, the antioxidant abilities of flavone and
newly synthesized diaminoflavones 10, 11, and 12 were evaluated on zebrafish larvae.
2. Results and Discussion
2.1. Chemistry
An altenative synthesis of 3,4-diaminoflavone 10 is depicted in Scheme 1. Unlike the early reported
method for the synthesis of compound 10 [23], we chose commercially available free diamino
compound 1 as the starting material. Compound 1 was coupled with 2 by EDCI in DMF to afford 4 in
35% yield. Although the DMF increased the solubility of starting materials, the lower yield was due to
the formation of 5 (8%) and recovered unreacted 2 (53% conversion). Compound 5 was derived from
the reaction of one of the amino groups of 4 with DMF. The structure elucidation of 5 was based on its
HMQC and HMBC spectra. When DMF was replaced with DMSO, the coupling yield of 4 was
enhanced significantly, up to 73%, and only small amounts of 2 were recovered (92% conversion).
Compound 4 underwent smoothly the Baker-Venkataraman rearrangement [28,3335] under
KOH/pyridine conditions [28] at 50 °C to afford 8 in a keto/enol form (1/3.1) mixture. Compound 8
was treated with InBr3 (0.5 equiv.) [36] under reflux conditions to afford compound 9 in 36% yield.
The yield was enhanced to 49% when 8 was heated under reflux condition in 48% HBr solution.

Molecules 2012, 17
Scheme 1. Syntheses of 3′,4′-diaminoflavone 10 and analogues 11 and 12.
8208
NH2
4'
3'
HO2C
NH2
NH2
O
R2
OH
NH2
NH2
NH2
KOH
EDCI, DMAP
R2
O
O
7
1
pyridine
50 oC
+
DMF or DMSO
see text
R2
OH
R1
O
O
5
R1
keto/enol
R1
O
4. R1=R2=H (35%, DMF; 73%, DMSO)
6. R1=R2=OMe (14%, DMF; 25%, DMSO)
8. R1=R2=H (69%, keto/enol=1:3.1)
9. R1=R2=OMe (72%, keto/enol=2.8:1)
2. R1=R2=H
3. R1=R2=OMe
NH2
4'
InBr3
B
NH2
R2
O
7
NH
3'
2
A
or HBr
or 20% H2SO4
C
O
N CH NMe2
3
5
R1
R2
O
O
O
10. R1=R2=H (36%, InBr3; 49%, HBr)
11. R1=R2=OH (47%, HBr)
12. R1=R2=OMe (50%, 20% H2SO4)
5. R1=R2=H (8%)
7. R1=R2=OMe (1%)
R1
The same strategy was also employed for the synthesis of 11 which possesses two hydroxyl groups
at C5 and C7 of the A ring. The two hydroxyl groups of 2′,4′,6′-trihydroxyacetophenone can be
selectively protected [37] to furnish 3, which was subjected to coupling with 1 to give 6 in 25% yield
in DMSO (38% conversion of 3). When DMF was used as solvent, compound 6 was isolated in
14% yield (22% conversion of 3) and a trace amount of 7 (1%) was also formed. We found out the
electron-donating groups in A ring affects the coupling yields. The Baker-Venkataraman rearrangement
of 6 to 9 gave comparable yields as the preparation of 8, but the keto/enol ratio was 2.8:1, which is
opposite the result seen for 8 (1:3.1). Sequential cyclization and demethylation of compound 9 by
heating in HBr afforded 11 in 47% yield. The best yield of compound 12 was isolated in 50% when
compound 9 was heated under reflux in 20% H2SO4 solution.
2.2. Comparison of the ROS-Scavenging Ability of Flavone and Aminoflavones 10, 11, and 12
We have previously developed a protocol to detect the level of ROS-scavenaging in zebrafish
embryos [38]. The same protocol was employed to evaluate the newly synthesized diaminoflavones
10, 11, and 12 with flavone for comparison. As shown in Figure 1, the detected ROS-scavenging rates
in flavone-treated zebrafish embryos were decreased in a concentration dependent manner by −24.4%
(1 ppm of flavone) and −94.5% (10 ppm of flavone) in comparison with that of the UV group (without
addition of flavone). As previously described [38,39], negative ROS-scavenging rates indicated that
the testing flavones possesses of ROS-scavenging activities. Similar dose-dependent results were
obtained when zebrafish embryos were treated by different concentrations (1 and 10 ppm,
individually) of diaminoflavones 10, 11, and 12 (ROS-scavenging rates: −39% to −102.3%). These
data clearly demonstrated that diaminoflavone 10 exhibited the highest ROS-scavenging ability
compared with those of flavone, 11 and 12 in low (1 ppm) as well as high concentrations (10 ppm).

Molecules 2012, 17
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Figure 1. Comparison of ROS-scavenging ability of flavone and 3′,4′-diaminoflavones 10, 11, and 12.
2.3. Flavone and Diaminoflavones Protecting Zebrafish Cells from UV-Induced Apoptosis
Zebrafish is an efficient model for evaluating sun-protective compounds because embryonic
zebrafish fins are very sensitive to UVB radiation [38,39]. Using the zebrafish model, we have shown
that flavone could protect zebrafish fins from UVB-induced apoptosis [40]. Therefore, we used the
same protocol on flavone, diaminoflavones 10, 11, and 12 to compare with their UVB-protection
ability. Results showed that no apoptotic signals were observed in the mock control embryos (no UVB,
Figure 2A). After exposing fish to UVB, many apoptotic signals accompanying with malformed fin
phenotypes were observed in the embryos (arrow in Figure 2B). However, few signals at 1 ppm or no
signals at 10 ppm were found when those embryos were co-exposed to UVB with flavone or
diaminoflavones 11 and 12 (Figure 2C,D,G–J). The concentrations of aminoflavone 10 at either
1 ppm or 10 ppm were enough protecting fin cells from apoptosis. We concluded that compound 10
might have the highest UVB-protection ability among these flavones (Figure 2E,F). On the basis of
these observations, we suggest that flavone, diaminoflavones 10, 11, and 12 are able to protect
UVB-damaged fin cells from apoptosis.
Figure 2. UVB exposure results for cell apoptosis in the fin region. Lateral views of mock
control embryos without (A) and with UVB exposure (B) after TUNEL assay staining.
(C, E, G, I) Lateral views of embryos derived from UVB + 1 ppm flavones group, or
(D, F, H, J) UV+10 ppm flavones after TUNEL assay staining. Arrows indicate the
apoptotic cells.

Molecules 2012, 17
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3. Experimental
3.1. General
Melting points were determined on a MP-2D apparatus and were not corrected. All chemicals were
commercially available and used without further purification except otherwise mentioned. 1H (600 MHz)
13
and C (150 MHz) NMR spectra were recorded on a Bruker 600 MHz instrument and the units were
reported in δ (ppm). Mass spectra (LRMS and HRMS) were recorded on a Finnigan MAT 95S
spectrometer.
3.2. Synthetic Procedures for the Key Intermediates and Aminoflavones 10, 11, and 12
2′-(3,4-Diaminobenzoyloxy)acetophenone (4). A solution of 3,4-diaminobenzoic acid (1, 1.264 g,
8.307 mmol) in DMSO (25 mL) was sequentially added 2′-hydroxyacetophenone (2, 0.50 mL,
4.154 mmol), EDCI (1.991 g, 10.385 mmol), and DMAP (0.254 g, 2.077 mmol) at 0 °C. This mixture
was stirred for 17.5 h at ambient temperature. The reaction was diluted by Na2S2O3 (sat’d). The brown
solid was filtered and the filtrate was extracted by EtOAc (×3). The organic layer was dried (MgSO4)
and concentrated. Purification by flash column chromatography (200–350 mesh amino SiO2,
hex/EtOAc = 2/1–1/2) afforded a dark red syrup and recovered 2 (0.105 g). This syrup was crystallized
from a mixture of hex/EtOAc (v/v = 7/10) solution to give a pale yellow solid (0.820 g). Yield = 73%.
1
MP = 156.5–158.5 °C. H-NMR (C6D6) δ 7.82 (d, J = 8.1 Hz, 1H), 7.65 (d, J = 7.9 Hz, 1H), 7.49
(s, 1H), 7.02 (d, J = 4.3 Hz, 2H), 6.84 (dt, J = 7.9, 4.3 Hz, 1H), 6.19 (d, J = 8.1 Hz, 1H), 2.99 (s, 2H),
2.44 (s, 2H), 2.27 (s, 3H). 13C-NMR (C6D6) δ 197.2, 165.6, 150.8, 142.2, 134.1, 133.2, 133.1, 126.0, 124.8,
124.7, 120.5, 119.5, 115.1, 39.2. HRMS (ESI) ([M+]) calcd for C15H14N2O3 270.1004. Found: 270.1002.
2′-Acetylphenyl-2-(dimethylamino)-1H-benzo[d]imidazole-6-carboxylate (5). Purification by flash
column chromatography (230400 mesh SiO2, hex/EtOAc = 2/1–0/1) provided a white solid.
Yield = 8%. Mp = 148.0–151.0 °C. 1H-NMR (CDCl3) δ7.82 (dd, J = 7.7, 1.2 Hz, 1H), 7.75 (dd, J = 8.2,
1.8 Hz, 1H), 7.69 (s, 1H), 7.56 (d, J = 1.7 Hz, 1H), 7.54 (dd, J = 8.6, 1.5 Hz, 1H), 7.31 (t, J = 7.4 Hz,
1H), 7.21 (d, J = 8.1 Hz, 1H), 6.70 (d, J = 8.2 Hz, 1H), 4.65 (br s, 2H), 3.05 (s, 6H), 2.54 (s, 3H). 13C-NMR
(CDCl3) δ 198.2, 165.4, 153.1, 150.0, 146.7, 137.6, 133.1, 132.0, 129.9, 126.9, 126.1, 124.0, 119.1,
117.8, 112.9, 40.3, 34.5, 30.4. HRMS (EI) ([M+]) calcd for C18H17N3O3 325.1426. Found: 325.1429.
1-(2-Hydroxyphenyl)-3-[3′,4′-diamino]propane-1,3-dione (8). To a solution of 4 (0.539 g, 1.993 mmol)
in pyridine (10 mL) was added KOH (0.168 g, 2.989 mmol) and the mixture was stirred at 50 °C for
3 h. The reaction was adjusted to pH 3 by 2 N HCl and extracted with EtOAc (×3). The organic
layer was dried (MgSO4), filtered through celite, and concentrated. Purification by flash column
chromatography (230–400 mesh SiO2, hex/EtOAc = 2/1–0/1) afforded a red solid (0.369 g).
Yield = 69%. MP = 168.0–170.0 °C. (major, enol form): 1H-NMR (CDCl3) δ 7.95 (d, J = 7.9 Hz, 1H),
7.41 (t, J = 7.7 Hz, 1H), 7.29 (d, J = 8.3 Hz, 1H), 7.25 (s, 1H), 7.08 (s, 1H), 6.976.90 (m, 2H), 6.58
(d, J = 8.3 Hz, 1H), 5.61 (NH2), 4.74 (br s, NH2). (minor, keto form). 1H-NMR (CDCl3) δ 7.80 (d, J = 7.9 Hz,
1H), 7.50 (t, J = 7.7 Hz, 1H), 7.19 (d, J = 8.2 Hz, 1H), 7.14 (s, 1H), 6.97–6.90 (m, 2H), 6.53 (d, J = 8.1 Hz,
1H), 5.52 (NH2), 4.59 (br s, NH2). (major, enol form). 13C-NMR (CDCl3) δ 201.1, 185.7, 183.4, 159.2,

Molecules 2012, 17
8211
141.7, 134.1, 134.0, 129.0, 121.4, 120.0, 119.5, 119.2, 117.5, 112.9, 112.5, 92.7. (minor, keto form).
13C-NMR (CDCl3) δ 192.2, 160.5, 136.1, 133.9, 131.3, 125.3, 120.9, 117.6, 113.5, 112.2, 50.1. HRMS
(ESI) ([M+]) calcd for C15H14N2O3 270.1004. Found: 270.1002.
2-(3,4-Diaminophenyl)-4H-1-benzopyran-4-one (10). A solution of compound 8 (0.110 g, 0.407 mmol)
was dissolved in 48% HBr (5 mL) and heated under reflux for 15 h. The mixture was slowly poured
into Na2S2O3 saturated solution and extracted with EtOAc (×3). The organic layer was dried (MgSO4) and
concentrated. Purification by flash column chromatography (230–400 mesh SiO2, hex/EtOAc = 1/1–0/1)
furnished a red solid which was washed several times with methanol to give a yellow-red solid (0.050 g).
1
Yield = 49%. MP = 256.0–258.0 °C. lit.23 237 °C (dec.). H-NMR (DMSO-d6) δ 8.00 (d, J = 7.7 Hz,
1H), 7.77 (t, J = 7.4 Hz, 1H), 7.65 (d, J = 8.3 Hz, 1H), 7.45 (t, J = 7.4 Hz, 1H), 7.23 (d, J = 8.4 Hz,
1H), 7.22 (s, 1H), 6.62 (d, J = 7.9 Hz, 1H), 6.59 (s, 1H), 5.39 (s, 2H, NH2), 4.75 (s, 2H, NH2).
13C-NMR (DMSO-d6) δ 176.5, 164.5, 155.5, 140.0, 134.6, 133.8, 125.1, 124.7, 123.5, 118.3, 118.0,
117.3, 113.5, 111.4, 103.0. HRMS (ESI) ([M+]) calcd for C15H12N2O2 252.0899. Found: 252.0899.
4′,6′-Dimethoxy-2′-(3,4-diaminobenzoyloxy)acetophenone (6). Compound 1 (3.726 g, 24.486 mmol)
and compound 3 (2.826 g, 14.402 mmol) were dissolved in DMSO (85 mL) at 0 °C. To this cold
solution was added EDCI (5.522 g, 28.804 mmol) and DMAP (1.231 g, 10.083 mmol) and stirred for
48 h. The reaction was added Na2S2O3 saturated solution. The resulting brown solid was filtered and
the filtrant was extracted by EtOAc (×3). The organic layer was dried (MgSO4), concentrated, and
purified by flash column chromatography (230–400 mesh SiO2, hex/EtOAc = 2/1–1/2) to provide an
1
orange solid (1.192 g) and recovered 3 (1.760 g). Yield = 25%. MP = 174.0–176.0 °C. H-NMR
(CDCl3) δ dd, J = 8.2, 1.9 Hz, 1H), 7.47 (d, J = 1.9 Hz, 1H), 6.68 (d, J = 8.2 Hz, 1H), 6.37 (d,
J = 2.2 Hz, 1H), 6.34 (d, J = 2.2 Hz, 1H), 3.84 (s, 3H), 3.80 (s, 3H), 2.46 (s, 3H). 13C-NMR (CDCl3) δ
199.8, 165.0, 162.0, 158.8, 149.9, 141.5, 133.1, 124.3, 119.5, 118.9, 117.7, 114.8, 100.1, 96.4, 55.9,
55.6, 31.9. HRMS (ESI) ([M+]) calcd for C17H18N2O5 330.1216. Found: 330.1214.
2-Acetyl-3,5-dimethoxyphenyl-2-(dimethylamino)-1H-benzo[d]imidazole-6-carboxylate (7). The same
procedure as in preparation of 5 was used to give a white solid. MP = 58.0–62.0 °C. 1H-NMR (CDCl3)
δ 7.72–7.68 (br d, 2H), 7.54 (s, 1H), 6.67 (d, J = 8.3 Hz, 1H), 6.37 (d, J = 2.1 Hz, 1H), 6.36 (d, J = 2.1
Hz, 1H), 3.84 (s, 3H), 3.81 (s, 3H), 3.10 (s, 6H), 2.46 (s, 3H). 13C-NMR (CDCl3) δ 199.9, 165.3, 161.9,
158.6, 153.1, 150.0, 146.5, 137.4, 127.0, 119.1, 118.0, 117.9, 112.9, 100.1, 96.4, 55.9, 55.6, 40.3, 34.5,
31.9. HRMS (EI) ([M+]) calcd for C20H21N3O5 385.1638. Found: 385.1631
1-(3,4-Diaminophenyl)-3-(2-hydroxy-4,6-dimethoxyphenyl)propane-1,3-dione (9). The same procedure
as in preparation of 8 was used. Purification by flash column chromatography (230–400 mesh SiO2,
hex/EtOAc = 2/1–0/1) provided a pale orange solid. Yield = 72%. MP = 207.0209.0 °C. (major, keto
1
form): H-NMR (DMSO-d6) δ 13.76 (s, 1H), 7.16–7.12 (m, 2H), 6.54 (d, J = 8.1 Hz, 1H), 6.10 (d,
J = 2.3 Hz, 1H), 6.00 (d, J = 2.3 Hz, 1H), 5.45 (s, 2H), 4.68 (br s, 2H), 4.38 (s, 2H), 3.80 (s, 3H), 3.44
1
(s, 3H). (minor, enol form): H NMR (DMSO-d6) δ 12.75 (s, 1H), 7.12–7.10 (m, 1H), 6.98 (s, 1H),
6.57 (d, J = 8.8 Hz, 1H),(d, J = 2.2 Hz, 1H), 6.08 (d, J = 2.2 Hz, 1H), 5.55 (s, 2H), 4.77 (br s,
2H), 3.90 (s, 3H), 3.79 (s, 3H) (major, keto form). 13C-NMR (DMSO-d6) δ 200.8, 192.5, 166.5, 166.2,
162.2, 141.3, 133.8, 125.4, 120.3, 113.3, 112.3, 105.3, 93.8, 90.7, 55.8, 55.7, 53.9 (minor, enol form).

Molecules 2012, 17
8212
13C-NMR (DMSO-d6) δ 188.6, 179.5, 164.1, 163.8, 160.9, 134.1, 120.8, 118.6, 113.1, 112.1, 104.3,
95.3, 94.2, 91.0, 56.1, 55.5. HRMS (ESI) ([M+]) calcd for C17H18N2O5 330.1216. Found: 330.1213.
2-(3,4-Diaminophenyl)-5,7-dihydroxy-4H-chromen-4-one (11). The same procedure as in preparation of
10 was used. Purification by flash column chromatography (230–400 mesh SiO2, hex/EtOAc =
2/1–0/1) provided an orange-yellow solid. The resulting solid was repeatedly washed with MeOH to
1
afford a pale orange-yellow solid. Yield = 47%. MP = 323 °C (dec.). H-NMR (DMSO-d6) δ 13.09
(-OH), 7.17–7.14 (m, 2H), 6.59 (d, J = 8.1 Hz, 1H), 6.44 (s, 1H), 6.33 (d, J = 1.6 Hz, 1H), 6.09 (d,
13
J = 1.6 Hz, 1H), 5.40 (s, 2H, -NH2), 4.73 (br s, 2H, -NH2). C-NMR (DMSO-d6) δ 181.2, 165.1,
164.9, 161.4, 157.3, 140.2, 134.5, 117.9, 117.4, 113.5, 111.2, 103.1, 100.7, 98.9, 93.8. HRMS (ESI)
([M+]) calcd for C15H12N2O4 284.0797. Found: 284.0792.
2-(3,4-Diaminophenyl)-5,7-dimethoxy-4H-chromen-4-one (12). Compound 9 was heated under reflux
in 20% H2SO4 for 12 h. The reaction mixture was slowly poured into a cold saturated NaHCO3
solution. The mixture was extracted with EtOAc, dried (MgSO4) and purified by flash column
chromatography (230–400 mesh SiO2, hex/EtOAc = 1/20/1) to provide an orange-red solid.
1
Yield = 50%. MP = 152.0–154.0 °C. H-NMR (DMSO-d6) δ 7.13–7.11 (m, 2H), 6.69 (d, J = 2.3 Hz,
1H), 6.58 (d, J = 8.7 Hz, 1H), 6.45 (d, J = 2.3 Hz, 1H), 6.29 (s, 1H), 5.25 (s, 2H), 4.68 (s, 2H), 3.87 (s,
13
3H), 3.80 (s, 3H). C-NMR (DMSO-d6) δ 175.5, 163.3, 161.5, 160.2, 159.1, 139.4, 134.5, 118.1,
116.6, 113.5, 111.1, 108.3, 104.5, 96.0, 93.1, 56.0, 55.9. HRMS (EI) ([M+]) calcd for C17H16N2O4
312.1110. Found: 312.1110.
3.3. Evaluation
3.3.1. Methods for Fish Embryos Maintenance, Chemicals Treatment and Survival Rates Analysis
The procedures for zebrafish culture and embryo collection used in this study have been described
previously [41,42]. For chemicals treatment, flavone and diaminoflavones 10, 11 and 12 were
individually dissolved in DMSO to the designated concentrations (1, and 10 ppm). Thirty embryos
were collected and treated with different concentrations of aminoflavones for 3 h (7275 hpf) then
counted for their survival rates.
3.3.2. UVB Exposure, ROS Detection and Data Analysis
Modified procedures were used in this study for UVB exposure and embryo collection [38,39].
After UVB exposure, all embryos were cultivated in 6-well cell culture plates until the analysis of their
ROS levels. To detect the accumulation of ROS in zebrafish embryos, embryos from the UVB-only
group (no flavone added), UVB + flavone and embryos from the UVB + diaminoflavone groups (10,
11, and 12) were incubated with 500 ng/mL dihydrodichlorofluorescein diacetate (H2DCFDA,
Molecular Probes, Eugene, OR, USA). After a 150 min incubation period at 28 °C, the fluorescence
intensity of the embryo was measured at excitation/emission = 485/530 nm. All data were presented
as “ROS-scavenging rates” calculated by the equation described previously [43]. A positive
ROS-scavenging rate indicates that the treatment with the flavone leads to the generation of ROS. A
negative ROS-scavenging rate means that the tested flavone compound exhibits ROS-scavenging
activities.

Molecules 2012, 17
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4. Conclusions
In conclusion, we have developed an improved three-step synthesis of 10. The same procedure was
used to prepare two new compounds 11 and 12. It is worth noting that the solvent plays an important
role in the coupling yields, whereby DMSO was shown to be superior to DMF as solvent. In particular,
compounds 10, 11 and 12 have shown increased ROS scavenging ability along with UV-protecting
abilities to compare with flavone. In this article, we established a rapid screening model of
diaminoflavones by zebrafish larvae. We are also synthesizing a series of derivatives with diamino
groups on the A ring of flavones along with compounds 10–12 to compare their bioactivities. The
details will be published in due course. We conclude that the diaminoflavones have potential to
develop as antioxidants and drugs.
Acknowledgments
The authors gratefully acknowledge the National Science Council (NSC98-2119-M-032-004-MY3)
and Tamkang University for supporting this work. We thank the National Center for High-Performing
Computing for assistance.
Conflict of Interest
The authors declare no conflict of interest.
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Sample Availability: Samples of the compounds 412 are available from the authors.
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