Convenient synthesis of an N-(1-alkoxyl-9-fluorenyl)serine acridine conjugate

Article abstract of DOI:10.1080/00397910600978531

We report here the synthesis of an N-(1-alkoxyl-9-fluorenyl)serine acridine conjugate, which was achieved by a three-component assembly approach via an intramolecular reductive amination process. Copyright Taylor & Francis Group, LLC.

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Convenient Synthesis of an
N‐(1‐Alkoxyl‐9‐fluorenyl)serine Acridine
Conjugate
Jifeng Dai a & Qibing Zhou a
a Department of Chemistry , Virginia Commonwealth University , Richmond,
Virginia, USA
Published online: 25 Jul 2007.
To cite this article: Jifeng Dai & Qibing Zhou (2007) Convenient Synthesis of an
N‐(1‐Alkoxyl‐9‐fluorenyl)serine Acridine Conjugate, Synthetic Communications: An International Journal for
Rapid Communication of Synthetic Organic Chemistry, 37:1, 129-135, DOI: 10.1080/00397910600978531
To link to this article: http://dx.doi.org/10.1080/00397910600978531
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Synthetic Communicationsw, 37: 129–135, 2007
Copyright # Taylor & Francis Group, LLC
ISSN 0039-7911 print/1532-2432 online
DOI: 10.1080/00397910600978531
Convenient Synthesis of an N-(1-Alkoxyl-9-
fluorenyl)serine Acridine Conjugate
Jifeng Dai and Qibing Zhou
Department of Chemistry, Virginia Commonwealth University,
Richmond, Virginia, USA
Abstract: We report here the synthesis of an N-(1-alkoxyl-9-fluorenyl)serine acridine
conjugate, which was achieved by a three-component assembly approach via an intra-
molecular reductive amination process.
Keywords: acridine conjugate, 1-alkoxyl-9-fluorenylamine, serine
9-Fluorenylamines have recently been demonstrated as important chemo-
phores for biological activity in a variety of organic compounds.[1–4] For
example, hydroxylated or methoxylated 9-fluorenylamino-pentanenitriles
have potent anti-multidrug-resistant activity for cancer therapy,[3] and
1-hydroxyfluorenyl dinitrophenyl hydrazone is highly cytotoxic in breast,
ovary, colon, and melanoma cancer cells.[4] The synthesis of 9-fluorenylamino
derivatives has been accomplished either through reductive amination of
9-fluorenones,[3] or nucleophilic substitution of 9-bromofluorene by
amines.[5] However, in the synthesis of a-substituted amino acid derivatives
of 1-alkoxy(or hydroxyl)-fluorenylamine for potential drug candidates, we
found that neither method produced desired products. Unsuccessful attempts
of reductive amination were probably due to steric interaction between
a-amino acids and 1-substituted fluorenones, and basic conditions for SN2-
type reactions were not compatible with the benzyl ester of amino acid deriva-
tives. Thus, an alternative synthetic approach was needed. We report here a
Received in USA April 12, 2006
Address correspondence to Qibing Zhou, Department of Chemistry, 1001 West
Main Street, Virginia Commonwealth University, Richmond, Virginia 23284-2006,
USA. E-mail: qzhou@vcu.edu
129

130
J. Dai and Q. Zhou
convenient synthetic approach via an intramolecular reductive amination
process to obtain 1-alkoxyl-9-fluorenylaminoserine acridine, conjugate 1
(Scheme 1). In addition, this method could be easily modified to conjugate
with other bioactive chemophores besides acridine which is a DNA intercala-
tor and is used in this study for potential additional biological activity.[6]
The synthetic design involved the coupling of serine derivative 4 and
fluorenone 2 through an ether linkage to facilitate the reductive amination
process (Scheme 1). Acridine 3 was conjugated at later synthetic steps.
Initially, the benzyl ester of serine without glycine was used but was found
not to be able to couple with fluorenone 2 in the subsequent Mitsunobo
step. Thus, t-Boc-serine-glycine benzyl ester 4 was used instead, which was
conveniently synthesized by coupling t-Boc-protected serine 5 and glycine
benzyl ester using 1-(3-dimethylaminopropyl)-3-ethylcarbodimide hydro-
chloride (EDCI) and 1-hydroxybenzotriazole (HOBt) in 69% yield.
9-Fluorenone 2 was synthesized via a Suzuki coupling, followed by an
acid-catalyzed acylation (Scheme 2). Boronic acid-7 for the Suzuki
coupling was obtained from bromide 6 and was used immediately in the
Suzuki reaction because of its high instability.[7,8] Triflate 8 was synthesized
using reported procedures.[8] For the Suzuki coupling of 7 and 8, we found that
a combination of Cs2CO3 and THF afforded a higher yield (88%) than other
conditions including Na2CO3, K3PO4, dioxane, or acetonitrile (,45%).
Hydrolysis of the resulting biphenyl 9 to a free acid was carried out using
Scheme 1. Synthetic design of fluorenylaminoserine acridine conjugate 1.

Scheme 2. Conditions and reagents: (i) nBuLi, THF, 2788C then B(O-i-Pr)3; (ii) triflate 8, Pd(PPh3)4, KBr, Cs2CO3, THF-H2O, reflux for 16 h; (iii)
(a) NaOH, THF-H2O; (b) TFA, TFAA, 08C; (iv) PPh3, 4, DIAD, THF, sonication; (v) (a) TFA; (b) silica gel, reflux in CH3CN, 16 h; (c) NaBH3CN;
(vi) (a) 5% HCOOH in MeOH, Pd/C; (b) acridine 3, HBTU, DIPEA, DMF, 3 h.

132
J. Dai and Q. Zhou
NaOH in a mixed THF–water solution. Acid-catalyzed acylation was
achieved with trifluoroacetic acid and anhydride (2:1 ratio) at 08C,
affording fluorenone 2 as the major isomer in 72% yield. The structure of
fluorenone 2 was confirmed by the observed coupling pattern of a singlet
1
and four doublets in the aromatic region of the H NMR spectrum.
Initial attempts to couple serine 4 and fluorenone 2 under normal
Mitsunobu reaction conditions[9] with diethyl azodicarboxylate (DEAD) or
diisopropyl azodicarboxylate (DIAD) produced no desired product 10.
Recently, Lepore and He reported that under sonication, Mitsunobu
reactions of steric hindered alcohols proceeded smoothly at high concen-
trations.[10] Indeed, we found that the coupling was successful only under
this specific condition with serine derivative 4 in 40% yield. The subsequent
intramolecular reductive amination of 9-fluorenone 10 was accomplished first
by the removal of the t-Boc protecting group at 08C, then the formation of
imine through refluxing with silica gel, and finally the reduction with
NaBH3CN.[11] A comparable yield was obtained when 3-A molecular sieves
˚
were used as the dehydrating agent.
For incorporation of the acridine moiety, the benzyl protecting groups of
9-aminofluorene 11 were removed by Pd/C in a 5% HCOOH solution.
Acridine 3 was coupled through an amide linkage using O-benzotriazol-1-
yl-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU) and N,N-diiso-
propylethylamine (DIPEA) in DMF (Scheme 2). The progress of the
reaction was monitored by high performance liquid chromatography
(HPLC) analysis. Conjugate 1 was finally isolated in 24% yield by HPLC sep-
aration using a gradient condition of 10–70% CH3CN in a triethylamine
1
acetate buffer (pH 6) over 30 min. Conjugate 1 was confirmed by both H
and 13C NMR analyses and showed a molecular ion of 574.00 m/z for
M þ Hþ (calculated for C34H32N5O4, 574.25) in the ESI-MS analysis.
In conclusion, the synthesis of 1-alkoxyl-9-fluorenylaminoserine acridine
was accomplished by a three-component assembly approach through an intra-
molecular reductive amination process. We are currently investigating the
potential biological activity of this novel fluorenylamine compound.
EXPERIMENTAL PROCEDURES
Compound 4: DIPEA (314 mg, 2.43 mmol), t-butoxycarbonylserine (500 mg,
2.39 mmol), HOBt (395 mg, 2.92 mmol), and EDCI (489 mg, 2.55 mmol)
were added to 5 (863 mg, 2.43 mmol) in 20 mL of CH2Cl2. The resulting
reaction was stirred under N2 for 16 h and worked up with CH2Cl2. Upon con-
centration, an analytically pure product (587 mg) was obtained in 69% yield as
a colorless oil. 1H NMR (CDCl3, 300 MHz): d 7.42–7.34 (m, 5H), 7.20 (br s,
1H), 5.63 (d, J ¼ 7.2 Hz, 1H), 5.16 (s, 2H), 4.22 (br s, 1H), 4.15–08 (m, 3H),
3.79–3.67 (m, 1H), 1.44 (s, 9H). ESI-MS calcd. for C17H25N2O6 (M þ Hþ)
353.17; found 353.03.

N-(1-Alkoxyl-9-fluorenyl)serine Acridine Conjugate
133
Compound 9: Compound 7 (387 mg, 1.69 mmol), Pd(PPh3)4 (90 mg,
0.08 mmol), KBr (146 mg, 1.22 mmol), and Cs2CO3 (590 mg, 1.81 mmol)
were added to 8 (395 mg, 1.21 mmol) in dry THF (18 mL). The mixture
was stirred for 15 min, and then 0.5 mL of water was added. The resulting
mixture was refluxed for 16 h. The reaction was worked up with brine and
ether. The desired product 9 (383 mg) was purified by a flash column in
88% yield as a colorless oil. 1H NMR (CDCl3, 300 MHz): d 7.63
(t, J ¼ 7.7 Hz, 1H), 7.58 (d, J ¼ 6.8 Hz, 2H), 7.52 (t, J ¼ 7.2 Hz, 2H), 7.45
(t, J ¼ 7.7 Hz, 2H), 7.14–7.05 (m, 5H), 5.21 (s, 2H), 1.91 (s, 6H). ESI-MS
calcd. for C23H21O4 (M þ Hþ) 361.14; found 361.07.
Compound 2: NaOH (70 mg) and 0.5 mL water were added to 9 (94 mg,
0.26 mmol) in 10 mL of THF. The reaction mixture was refluxed for 16 h and
then acidified to pH 4–5 with diluted HCl. The reaction was worked up with
ether. Upon concentration, the resulting residue was dissolved in 5 mL of
CH2Cl2. Trifluoroacetic acid (2 mL) and trifluoroacetic anhydride (1 mL)
were then added at 08C. After 2 h, the reaction was worked up with ether.
The desired product 2 (56.4 mg) was purified by a flash column in 72%
1
yield as a yellow solid (mp 103–1048C). H NMR (CDCl3, 300 MHz): d
8.48 (s, 1H), 7.58 (d, J ¼ 8.3 Hz, 1H), 7.46–7.31 (m, 6H), 7.10 (s, 1H),
6.98 (d, J ¼ 7.2 Hz, 1H), 6.81 (d, J ¼ 7.8 Hz, 1H), 6.76 (d, J ¼ 8.6 Hz, 1H),
5.16 (s, 2H). ESI-MS calcd. for C20H15O3 (M þ Hþ) 303.10; found 302.99.
Compound 10: Serine 4 (300 mg, 0.85 mmol) and PPh3 (260 mg,
0.99 mmol) were added to 2 (200 mg, 0.66 mmol) in dry THF (0.8 mL).
The mixture was sonicated for 15 min, and then DIAD (143 mL,
0.73 mmol) was added under sonication. The reaction mixture was
sonicated for another 40 min and worked up with CH2Cl2. The desired
product (168 mg) was purified by a flash column in 40% yield as a yellow
1
oil. H NMR (CDCl3, 300 MHz): d 8.40 (br s, 1H), 7.52 (d, J ¼ 8.2 Hz, 1H),
7.47–7.34 (m, 6H), 7.26–7.22 (m, 5H), 7.12–7.09 (m, 2H), 6.81
(d, J ¼ 2.2 Hz, 1H), 6.78 (d, J ¼ 8.4 Hz, 1H), 5.90 (br s, 1H), 5.16 (s, 2H),
5.06 (d, J ¼ 4.1 Hz, 2H), 4.62 (br s, 2H), 4.25 (dd, J1 ¼ 6.7 Hz, J2 ¼ 17.6 Hz,
1H), 4.10 (dd, J1 ¼ 5.30 Hz, J2 ¼ 17.6 Hz, 1H), 3.82 (br t, J ¼ 8.6 Hz, 1H),
1.46 (s, 9H). ESI-MS calcd. for C37H37N2O8 (M þ Hþ) 637.25; found 637.00.
Compound 11: Trifluoroacetic acid (1 mL) was added to 10 (90 mg,
0.14 mmol) in dry CH2Cl2 (2 mL) at 08C. After 3 h, the solvent was
removed by vacuum. The resulting brown residue was dissolved in a
mixture of 3 mL of CH2Cl2 and 9 mL of CH3CN, followed by the addition
of 460 mg of silica gel. The resulting mixture was refluxed for 16 h under
N2. After cooling to room temperature, NaBH3CN (96 mg, 1.5 mmol) was
added. The reaction was stirred for 2 h, and acetic acid (170 mg) was
added. After 3 h, the reaction was worked up with CH2Cl2. The desired
product (34 mg) was purified by a flash column in 47% yield as a yellow
1
oil. H NMR (CDCl3, 300 MHz): d 7.94 (br s, 1H), 7.58 (d, J ¼ 8.2 Hz,
1H), 7.48–7.26 (m, 13 H), 6.95 (d, J ¼ 7.4 Hz, 2H), 5.27 (d, J ¼ 13.2 Hz,
1H), 5.17 (s, 2H), 5.15 (s, 2H), 4.85 (s, 1H), 4.17–4.10 (m, 3H), 3.64 (br t,

134
J. Dai and Q. Zhou
J ¼ 12.0 Hz, 1H). ESI-MS calcd. for C32H29N2O5 (M þ Hþ), 521.21; found
520.97.
Compound 1: To 11 (20 mg, 0.038 mmol) in 5% HCOOH in MeOH
(2 mL), 10% Pd/C (80 mg) was slowly added under N2. After 20 min, the
solid was removed with a 50 mm filter, and then 0.5 mL of trifluoroacetic
acid was added to the filtrate. Upon concentration, the free acid (11 mg)
was obtained. For the coupling, acridine 3 (14 mg, 0.04 mmol), HBTU
(41 mM in DMF, 1.1 mL), and DIPEA (41 mM in DMF, 2.6 mL) were
added to the resulting free acid (5 mg, 0.015 mmol) in DMF (1 mL). After
3 h, the desired product 1 (5.3 mg) was purified by a HPLC separation
using a Microsorb MV C18 column (10 ꢀ 250 mm, 8 mm) from Varian
1
(Walnut Creek, CA) in 24% yield as a yellow oil. H NMR (MeOH-d4,
300 MHz): d 8.37 (d, J ¼ 6.6 Hz, 2H), 7.83 (d, J ¼ 6.3 Hz, 2H), 7.74
(t, J ¼ 6.6 Hz, 2H), 7.50 (d, J ¼ 6.0 Hz, 1H), 7.41 (t, J ¼ 5.7 Hz, 2H), 7.35
(d, J ¼ 5.4 Hz, 1H), 7.24 (t, J ¼ 5.7 Hz, 1H), 7.12 (s, 1H), 6.84
(d, J ¼ 5.7 Hz, 1H), 6.74 (d, J ¼ 6.0 Hz, 1H), 4.97 (d, J ¼ 10.5 Hz, 1H),
4.81 (s, 1H), 4.07 (d, J ¼ 5.4 Hz, 1H), 3.98 (t, J ¼ 4.8 Hz, 2H), 3.87
(d, J ¼ 4.2 Hz, 2H), 3.74–3.65 (m, 1H), 3.43–3.37 (m, 2H), 2.29–1.96
(m, 2H). ESI-MS calcd. for C34H32N5O4 (M þ Hþ) 574.25; found 574.00.
ACKNOWLEDGMENT
We thank the Thomas F. and Kate Miller Jeffress Memorial Trust (J-723) for
the financial support.
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