Synthesis and biological evaluation of 3-amino-propan-1-ol based poly(ether imine) dendrimers

Article abstract of DOI:10.1016/j.tet.2005.02.045

A general synthetic strategy for the rapid construction of poly(ether imine) dendrons and dendrimers with a nitrogen core, originating from 3-amino-propan-1-ol, is described. A new trifunctional monomer, namely, 3-[bis-(3-hydroxypropyl)amino]propan-1-ol, was used in a divergent synthesis of dendrimers up to the third generation. This method permitted installation of, either alcohol, amine, nitrile, ester or carboxylic acid groups at the peripheries the dendrimers. Cytotoxicity studies on water-soluble carboxylic acid terminated dendrimers were conducted and these studies revealed that poly(ether imine) dendrimers were non-toxic. These results illustrate that poly(ether imine) dendrimers are useful for biological studies.

Full text of DOI:10.1016/j.tet.2005.02.045

Tetrahedron 61 (2005) 4281–4288
Synthesis and biological evaluation of 3-amino-propan-1-ol based
poly(ether imine) dendrimers
Thatavarathy Rama Krishna,a Samta Jain,b Utpal S. Tatub,
*
and Narayanaswamy Jayaramana,
*
aDepartment of Organic Chemistry, Indian Institute of Science, Bangalore 560012, India
bDepartment of Biochemistry, Indian Institute of Science, Bangalore 560012, India
Received 20 October 2004; revised 29 January 2005; accepted 17 February 2005
Available online 18 March 2005
Abstract—A general synthetic strategy for the rapid construction of poly(ether imine) dendrons and dendrimers with a nitrogen core,
originating from 3-amino-propan-1-ol, is described. A new trifunctional monomer, namely, 3-[bis-(3-hydroxypropyl)amino]propan-1-ol,
was used in a divergent synthesis of dendrimers up to the third generation. This method permitted installation of, either alcohol, amine,
nitrile, ester or carboxylic acid groups at the peripheries the dendrimers. Cytotoxicity studies on water-soluble carboxylic acid terminated
dendrimers were conducted and these studies revealed that poly(ether imine) dendrimers were non-toxic. These results illustrate that
poly(ether imine) dendrimers are useful for biological studies.
q 2005 Elsevier Ltd. All rights reserved.
1. Introduction
involving four reactions, namely, (i) Michael addition to
form a nitrile; (ii) reduction of the nitrile to an amine;
(iii) double Michael addition of the amine with an acrylate
and (iv) reduction of the resulting ester to an alcohol.5 We
herein describe the synthesis, characterization and cyto-
toxicity of new poly(ether imine) dendrimers with a
nitrogen core. The poly(ether imine) (PETIM) dendrimers
herein are denoted as PETIM–m(CO2R)n, PETIM–m(OH)n
PETIM–m(CN)n and PETIM–m(CO2H)n, wherein ‘m’ and
‘n’ refer to the generation number and the number of
terminal groups, respectively.
Hyperbranched dendritic macromolecules have consider-
able interest in a number of chemical, biological and
materials related studies.1,2 Dendrimers with biocompatible
building blocks has led to the emergence of a new class of
dendritic polymers called ‘biodendrimers’.3 Linear
polymers such as poly(ethylene glycol) and poly(ethylene
imine) are important materials in biological studies4 and we
were interested to combine the molecular features of these
two polymers in order to derive new types of biocompatible
dendrimers. Herein, we report a facile synthesis of
poly(ether imine) dendrons and dendrimers up to the third
generation, containing 24 peripheral functionalities, with a
nitrogen core, and the evaluation of their cytotoxicity
profiles.
The iterative reaction sequence was initiated with the
Michael addition of acrylonitrile to an alcohol in the
presence of aq NaOH. The addition of acrylonitrile to
the alcohol has to be carried out slowly, so as not to increase
the temperature of the reaction mixture above 30 8C and the
reaction was continued for about 6 h. After completion of
the reaction, excess acrylonitrile was removed in vacuo,
neutralized with aq HCl (1 M) and extracted with CHCl3.
Upon a work-up procedure, the required Michael addition
product was obtained in excellent yields. Hydrogenation of
the nitrile groups with Ra-Co and H2 (w40 bar) in
water/MeOH solvent system afforded the desired primary
amine-terminated dendrimers. Subsequent Michael addition
of tert-butyl acrylate to the amine proceeded readily and the
reaction led to almost quantitative yield. Reduction of
the tert-butyl ester at the peripheries of dendrons and
dendrimers to the corresponding alcohols was performed
2. Results and discussion
2.1. General synthetic methods
For a successful synthesis of dendrimers, high efficiencies
and yields of the reactions are essential. In a synthetic
strategy, we have identified iterative synthetic sequences
Keywords: Cytotoxicity; Dendrimers; Michael addition; Raney cobalt.
*
Corresponding authors. Tel.: C91 80 2293 2406/2403; fax: C91 80 2360
0529; e-mail: jayaraman@orgchem.iisc.ernet.in
0040–4020/$ - see front matter q 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2005.02.045

4282
T. R. Krishna et al. / Tetrahedron 61 (2005) 4281–4288
Scheme 1. (i) tert-butyl acrylate, MeOH, 6 h; (ii) LAH, THF, 0 8C; (iii) H2C]CH–CN (2 M equiv), cat. NaOH, 6 h, rt; (iv) Ra-Co, H2, H2O/MeOH (99:1),
70 8C, 40 bar.
with LAH in THF and the Michael addition reaction
followed, so as to obtain the corresponding nitrile. Although
our procedure previously5 offered a convenient entry to
synthesize PETIM dendrons, the present procedure is
significantly better, primarily because the synthesis herein
is devoid of any protection/deprotection synthetic sequence.
2.2. Synthesis of dendrons
Monomer 1 was synthesized by treatment of 3-amino-
propan-1-ol with tert-butyl acrylate in quantitative yields
(Scheme 1). Reduction of 1 with LAH led to the isolation of
3-[bis-(3-hydroxypropyl)amino]propan-1-ol (2), after
Scheme 2. (i) tert-butyl acrylate, MeOH, 6 h; (ii) LAH, THF, 0 8C; (iii) H2C]CH–CN, cat. NaOH, 6 h, rt; (iv) Ra-Co, H2, H2O/MeOH (99:1), 70 8C, 40 bar;
(v) AcCl, H2O, CH2Cl2, 6 h, rt.

T. R. Krishna et al. / Tetrahedron 61 (2005) 4281–4288
4283
quenching the reaction mixture with ice. Alcohol 2 was
subjected to Michael addition to afford bis-nitrile 3. Apart
from 3, mono-nitrile 4 and tris-nitrile 5 were also obtained.
1H NMR spectrum of mono-, bis- and tris-nitrile exhibited
resonances at w3.63 and w3.54 ppm corresponding to
CH2CN and OCH2 protons, respectively. Mono-, bis- and
tris-nitriles were identified by comparison of the
intensities of the chemical shifts of CH2CN and OCH2
protons. The ratio was 1:4 for mono-nitrile 4; 2:5 for bis-
nitrile 3 and 1:2 for tris-nitrile 5. Bis-nitrile 3 was
separated from the mixture of tris- and mono-nitrile product
(alumina, CHCl3/MeOH). Reduction of the nitrile groups in
3 with Ra-Co, followed by double Michael addition with
tert-butyl acrylate, afforded the second-generation dendron
6 with a free hydroxyl group at the focal point in excellent
yields.
Scheme 3. (i) LAH, THF, 0 8C; (ii) H2C]CH–CN, cat. NaOH, 6 h, rt; (iii) Ra-Co, H2, H2O/MeOH (99:1), 72 8C, 40 bar; (iv) tert-butyl acrylate, MeOH, 6 h;
(v) AcCl, H2O, CH2Cl2, 6 h, rt.

4284
T. R. Krishna et al. / Tetrahedron 61 (2005) 4281–4288
2.3. Synthesis of dendrimers
acrylonitrile to afford PETIM–2(CN)12 (16). The nitrile
groups in 16 were reduced (Ra-Co, H2) and the resulting
crude amine was subjected to Michael addition with tert-
butyl acrylate to afford PETIM–3(CO2R)24 (17), with 24
ester groups at the periphery of the dendrimer (Scheme 4).
In order to generate CO2H groups at the periphery of
dendrimers, the tert-butyl ester protecting groups in
dendrimers 7, 9, 13 and 17 were deprotected (acetyl
chloride, H2O) to afford the free carboxylic acid-containing
dendrimers 8, 10, 14 and 18, respectively, in excellent
yields.
An alternative strategy was undertaken in order to
synthesize PETIM dendrimers in which the nitrogen atom
was the core. The required starting material, ester 7, was
obtained upon treatment of NH3 with tert-butyl acrylate in
MeOH (Scheme 2).6 Reduction of 7 to 3-[bis-(3-hydroxy-
propyl)amino]-propan-1-ol (2) was carried out with LAH in
quantitative yields. Treatment of 2 with acrylonitrile
afforded tris-nitrile 5 in excellent yields. Hydrogenation of
nitrile 5 in a high-pressure autoclave resulted in amine,
which was treated with excess tert-butyl acrylate, so as to
afford PETIM–1(CO2R)6 (9). Ester 9 was subjected to
sequential reactions: (i) reduction with LAH to afford
PETIM–1(OH)6 (11) and (ii) treatment of 11 with
acrylonitrile to afford PETIM–1(CN)6 (12). Reduction of
12 to an intermediate amine, followed by treatment with
tert-butyl acrylate afforded PETIM–2(CO2R)12 (13) in
overall good yields (Scheme 3). An iterative synthetic
sequence of Michael addition and reduction reactions was
continued with PETIM–2(CO2R)12 (13), so as to obtain
PETIM–2(OH)12 (15), which was then treated with
2.4. Characterization
Characterization of the divergently grown dendrons and
dendrimers and their intermediates was performed by
spectroscopic and spectrometric techniques. Functional
group conversion to nitrile (n 2251), amine (n 3460), tert-
butyl ester (n 1727) and alcohol (n 3390) were followed by
IR spectroscopy. In 1H NMR spectroscopy, separate
resonances were observed for different types of protons,
which helped in assigning the structure and constitution of
Scheme 4. Reagents and conditions: (i) Ra-Co, H2, H2O/MeOH (99:1), 72 8C, 40 bar; (ii) tert-butyl acrylate, MeOH, 6 h; (iii) AcCl, H2O, CH2Cl2, 6 h, rt.

T. R. Krishna et al. / Tetrahedron 61 (2005) 4281–4288
4285
dendrons and dendrimers. Characteristic triplet of protons at
w3.60 ppm for CH2CN, w2.30 for CH2–CO2 tBu, w1.70
and 3.70 ppm for CH2CH2OH were used to check the
completion of the reaction and growth of symmetrical
dendrimers. 13C NMR chemical shifts could also be used to
monitor the functional group transformation at the periphery
of dendrons and dendrimers. Specifically, the appearance of
chemical shifts for nitrile group at w117 ppm, carbonyl at
w172 ppm and w62 ppm for CH2OH group were used to
follow the progress of the reaction and growth of the
dendrimers. The mass spectrometric analysis for dendrimers
and dendrons exhibited the calculated molecular ion peak as
an intense peak in most of the compounds. For dendrons and
dendrimers with molecular weight less than 1000 Da, high-
resolution ES-MS analysis could be performed successfully
with the error less than 5 ppm between the observed
and calculated mass position. Higher generation dendrimers
16–18 could not be analyzed by mass spectrometry. Also,
reliable elemental analysis could not be obtained for the
dendrons and dendrimers, due to the hygroscopic nature of
these compounds.
to 100 mg/mL of the dendrimer solutions in both the cell
lines T47D as well as CV-1. The MTT assay reveals that no
measurable cytotoxicity is observed for these water-soluble
dendrimers on both the cell lines.
3. Conclusion
We have developed a new method for the rapid synthesis of
dendrons and dendrimers with a nitrogen as the core. The
propyl ether–imine and carboxylic acid surface group
functionalities of the dendrimers reported herein impart
water solubility. In vitro cytotoxicity studies reveal that
these new PETIM dendrimers are non-toxic. In the context
of their facile synthesis and non-toxic properties, PETIM
dendrimers have potential for a range of applications such as
drug delivery, scaffolds for biomimetic chemistry and other
biological studies, apart from their utility in chemical and
materials related studies.
4. Experimental
2.5. Evaluation of the cytotoxic properties of dendrimers
4.1. General remarks
PETIM dendrimers synthesized herein are similar in
chemical constitution to polyether and polyamine polymers,
which are studied extensively in biological studies.4
Dendrimers with carboxylic acid groups at the peripheries
were studied for hemolytic cleavage of red blood cells.7 In
vitro cytotoxic studies were carried out by MTT assay8 on
water soluble dendrimers incubated on two different cell
lines human breast cancer T47D and African green monkey
kidney CV-1. Cells were seeded at the density of 10,000
cells per well in a 96 well microtitre plate using bovine
serum-containing medium. Cells were left to recover for
24 h, before the addition of fresh medium containing
dendrimers and then incubated for 24 h. MTT was added
and the cells were left for 2 h. The formazan formed was
dissolved in optical grade DMF (20% w/v SDS in 50%
DMF) and plates were read using a scanning multi-well
spectrophotometer set at 570 nm. The viability of the cells
exposed to dendrimers was expressed as percentage of the
viability of cells with respect to control cell culture grown in
the absence of dendrimers. Figure 1 shows the relative CV-1
cell viability of dendrimers 8, 10, 14 and 18 at different
concentrations. The cell survival rate was more than 98% up
Chemicals tert-butyl acrylate, LAH and Ra-Co (Raneyw
2700 cobalt) were purchased and were used as received.
Acrylonitrile was purified by passing through neutral
alumina before use. Solvents were dried and distilled
according to the literature procedures. Analytical TLC was
performed on commercial plates coated with aluminium
oxide 60 F254 neutral (type E, 0.2 mm) or silica gel GF254
(0.25 mm). Silica gel (100–200 mesh) and basic alumina
were used for column chromatography. Infrared spectra
were recorded either neat or as KBr samples. Matrix-
assisted laser desorption ionization-time-of-flight mass
spectra (MALDI-TOF-MS) was performed using gentisic
acid matrix. Electrospray and high-resolution mass spectra
were performed on a Q-TOF mass spectrometer. 1H and 13
C
NMR spectral analysis were performed either on a
spectrometer operating at 300 and 75 MHz, respectively,
or on a 400 spectrometer operating at 400 and 100 MHz,
respectively, with residual solvent signal acting as the
internal standard. The following abbreviations are used to
explain the multiplicities: s, singlet; d, doublet; t, triplet; m,
multiplet; band, several overlapping signals; b, broad.
Figure 1. In vitro cytotoxicity studies of compounds 8, 10, 14 and 18 on African green monkey kidney CV-1 cell line, in the presence of dendrimers, at various
concentrations.

4286
T. R. Krishna et al. / Tetrahedron 61 (2005) 4281–4288
4.2. General procedure for Michael addition of
acrylonitrile (I)
4.5.2. Compound 2. A solution of 1 (21.0 g, 63.4 mmol) in
THF (25 mL) was added drop-wise to a suspension of LAH
(7.22 g, 0.19 mol) in THF (150 mL), left stirring for 1 h and
worked up as described in the general procedure III to obtain
2 a colorless liquid (12.0 g, 99%). Alternatively, a solution
of 7 (21.0 g, 52.2 mmol) in THF (25 mL) was added drop-
wise to a suspension of LAH (7.22 g, 0.19 mol) in THF
(150 mL), left stirring for 1 h and worked up as described in
the general procedure III to obtain 2 as a colorless liquid
(10.0 g, 99%). FT-IR (neat) n: 3349, 1466, 1375, 1058. CI-
MS m/z: 192 [M]C (100%); 146 [MK1KCH2CH2OH]C
(40%). 1H NMR (CDCl3) d: 1.74 (q, 6H, JZ6.3 Hz), 2.60 (t,
6H, JZ6.3 Hz), 3.72 (t, 6H, JZ6.3 Hz). 13C NMR (CDCl3)
d: 28.7, 52.1, 61.8. HRMS m/z: calcd for C9H21O3N:
192.1599. Found: 192.1602.
Acrylonitrile was added to a mixture of alcohol and aqueous
NaOH (40%) while maintaining the temperature below
30 8C. The reaction mixture was stirred for 6 h at room
temperature, neutralized with aq HCl (1 M) and diluted with
CHCl3. The organic layer was washed with aqueous NaOH
(5%) followed by brine, evaporated in vacuo and dried
thoroughly to afford the desired Michael addition product.
4.3. General procedure for the reduction of nitrile using
Ra-Co catalyst and subsequent Michael addition of
resulting amine (II)
The nitrile derivative in MeOH was transferred to a
hydrogenation reactor vessel and was admixed with
Ra-Co catalyst in H2O (40 mL). The mixture was
hydrogenated (H2, 40 atm) at 70 8C for 1 h, cooled,
decanted and the solvents were evaporated to afford the
amine as a colorless oil. A solution of the crude amine in
MeOH (5 mL) was treated with tert-butyl acrylate and
stirred for 6 h. Excess tert-butyl acrylate, solvents were
removed in vacuo and the resulting product was purified
(alumina).
4.5.3. Compound 3. Acrylonitrile (3.36 g, 63.4 mmol) was
added to a mixture of 2 (6.0 g, 31.3 mmol) and aq NaOH
(40%) (0.31 mL), stirred for 6 h and worked up as given in
the general procedure I to afford 3 as a colorless liquid
(3.58 g, 38%). FT-IR (neat) n: 3452, 2251, 1466, 1222,
1112, 1060. CI-MS m/z: 298 [M]C (100%). 1H NMR
(CDCl3) d: 1.69–1.79 (m, 6H), 2.52–2.64 (m, 10H), 3.52 (t,
4H, JZ6.3 Hz), 3.63 (t, 4H, JZ6.3 Hz), 3.76 (t, 2H, JZ
6.3 Hz). 13C NMR (CDCl3) d: 18.9, 26.9, 27.8, 50.7, 55.1,
64.5, 65.3, 69.2, 117.9. HRMS m/z: calcd for C15H27O3N3:
298.2130. Found: 298.2131.
4.4. General procedure for the reduction of tert-butyl
esters (III)
4.5.4. Compound 4. Colorless liquid (1.53 g, 20%). FT-IR
(neat) n: 3394, 2251, 1466, 1222, 1116, 1060. EI-MS m/z:
243 [MK1]C (2%); 199 [MKCH2CH2OH]C (24%); 155
[MK1K2!CH2CH2OH]C (39%). 1H NMR (300 MHz,
CDCl3) d: 1.69–1.79 (m, 6H), 2.52–2.64 (m, 8H), 3.52 (t,
2H, JZ6.3 Hz), 3.63 (t, 2H, JZ6.3 Hz), 3.73 (t, 4H, JZ
6.3 Hz). 13C NMR (75.5 MHz, CDCl3) d: 18.9, 26.9, 28.5,
50.7, 52.8, 62.6, 65.3, 69.2, 117.9.
To a suspension of LAH in THF at 0 8C, the ester in THF
was added drop-wise for w30 min. The suspension was
stirred at 0 8C for 30 min., brought to room temperature and
continued the stirring for additional 1 h. The reaction
mixture was cooled to 0 8C, quenched with ice (w1 g),
Na2SO4 (solid) was added and left aside till the grey residue
became a free white suspension and settled. The suspension
was passed through a plug of celite, the filtrate was dried and
concentrated to obtain the alcohol as colorless liquid.
4.5.5. Compound 5. Colorless liquid (2.19 g, 20%).
Alternatively, 5 was synthesized upon reaction of 2
(12.0 g, 62.6 mmol) with acrylonitrile (9.95 g, 0.18 mol)
and aq NaOH (40%) (0.62 mL) for 6 h. After work up as
given in the general procedure I, 5 was isolated, as a
colorless liquid (21.2 g, 96%). FT-IR (neat) n: 2251, 1470,
1368, 1116. ES-MS m/z: 351 [M]C (100%). 1H NMR
(300 MHz, CDCl3) d: 1.78 (q, 6H, JZ6.3 Hz), 2.58–2.64
(m, 12H), 3.54 (t, 6H, JZ6.3 Hz), 3.64 (t, 6H, JZ6.3 Hz).
13C NMR (75.5 MHz. CDCl3) d: 18.9, 26.7, 50.6, 65.3, 69.1,
118.0. HRMS: calcd for C18H30O3N4: m/z: 351.2396 [M]C.
Found: 351.2395 [M]C.
4.5. General procedure for the hydrolysis of tert-butyl
esters (IV)
To a solution of the ester in CH2Cl2, AcCl and H2O were
added slowly. The solution was stirred at room temperature
for 8 h. Solvents were removed in vacuo, the resulting
residue triturated several times with hexane and CH2Cl2 to
afford the desired acid as a white foamy solid.
4.5.1. Compound 1. A mixture of 3-aminopropan-1-ol
(4.91 g, 65.4 mmol) and tert-butyl acrylate (21.87 g,
171.0 mmol) in MeOH (25 mL) were stirred at room
temperature for 6 h. Excess of tert-butyl acrylate and
solvent were removed in vacuo. The crude product was
diluted with CHCl3 and washed with brine. The organic
layer was dried and concentrated to obtain 1 as colorless
viscous liquid (21.3 g, 98%). FT-IR (neat) n: 3429, 1729,
1459, 1368, 1158. ES-MS m/z: 332 [M]C (100%); 276 [MC
1KCMe3]C (53%). 1H NMR (CDCl3) d: 1.42 (s, 18H), 1.66
(q, 2H, JZ6.0 Hz), 2.37 (t, 4H, JZ7.2 Hz), 2.60 (t, 2H, JZ
6.0 Hz), 2.72 (t, 4H, JZ7.2 Hz), 3.71 (t, 2H, JZ6.0 Hz).
13C NMR (CDCl3) d: 28.1, 28.4, 33.3, 49.4, 50.4, 63.2, 80.7,
171.7. HRMS m/z: calcd for C17H33O5N: 332.2437. Found:
332.2440.
4.5.6. Compound 6. The bis-nitrile 3 (0.50 g, 1.67 mmol) in
MeOH (0.5 mL) was added with Ra-Co catalyst in H2O
(40 mL). The reaction was stirred for 1 h and the reaction
mixture was worked up as given in the general procedure II
to afford the amine intermediate. A solution of crude amine
(0.50 g, 1.65 mmol) in MeOH (5 mL) was treated with tert-
butyl acrylate (4.37 g, 34.1 mmol), and the reaction was
followed as given in general procedure II, to afford, after
purification (alumina) (hexane/EtOAc, 85:15) 6, as a
colorless liquid (1.28 g, 94% combined yield for nitrile
reduction and Michael addition). FT-IR (neat) n: 3440,
1729, 1461, 1367, 1157. ES-MS m/z: 819 [M]C (100%). 1H
NMR (CDCl3) d: 1.43 (s, 36H), 1.68 (b, 10H), 2.34 (t, 8H,

T. R. Krishna et al. / Tetrahedron 61 (2005) 4281–4288
4287
JZ7.2 Hz), 2.47 (m, 8H), 2.64 (t, 2H, JZ5.7 Hz), 2.68 (t,
8H, JZ7.2 Hz), 3.38 (b, 8H), 3.77 (t, 2H, JZ5.4 Hz). 13C
NMR (CDCl3) d: 27.1, 27.6, 28.1, 29.7, 33.8, 49.4, 50.5,
51.0, 54.9, 64.4, 69.0, 80.2, 172.1. HRMS m/z: calcd for
C43H83O11N3: 818.6106. Found: 818.6103.
and worked up as described in the general procedure III to
obtain 11, as a colorless liquid (2.27 g, 99%). FT-IR (neat)
n: 3378, 1465, 1375, 1113, 1061. ES-MS m/z: 712 [MC1]C
1
(56%); 356 [MC1/2]C (100%). H NMR (CDCl3) d: 1.74
(m, 24H), 2.46 (m, 18H), 2.57 (t, 6H, JZ6.3 Hz), 3.37–3.42
(m, 12H), 3.68 (t, 12H, JZ5.7 Hz). 13C NMR (CDCl3) d:
26.8, 27.1, 28.6, 50.7, 50.9, 52.8, 62.7, 68.7, 69.1. HRMS
m/z: calcd for C36H78O9N4: 711.5847. Found: 711.5855.
4.5.7. Compound 7. Ammonia (0.90 g, 53 mmol) and tert-
butyl acrylate (21.87 g, 171.0 mmol) in MeOH (25 mL)
were stirred at room temperature for 6 h. Excess of tert-
butyl acrylate and solvent were removed in vacuo, dried and
concentrated to obtain 7, as colorless viscous liquid (21.0 g,
98%). FT-IR (neat) n: 1730, 1456, 1368, 1154. EI-MS m/z:
402 [M]C (100%); 424 [MCNa]C (55%); 440 [MCK]C
(45%). 1H NMR (CDCl3): d: 1.42 (s, 27H), 2.34 (t, 6H, JZ
7.2 Hz), 2.71 (t, 6H, JZ7.2 Hz). 13C NMR (CDCl3) d: 28.1,
33.9, 49.2, 80.2, 171.9. HRMS m/z: calcd for C21H39O6N:
402.2855. Found: 402.2842.
4.5.12. Compound 12. Acrylonitrile (0.89 g, 16.8 mmol)
was added to a mixture of 11 (1.66 g, 2.34 mmol) and aq
NaOH (40%, 0.15 mL), stirred for 6 h and worked up as
given in the general procedure I to obtain crude product
which was purified on alumina (CHCl3/MeOH, 98:2) to
afford compound 12, as a colorless liquid (2.35 g, 97%).
FT-IR (neat) n: 2251, 1652, 1368, 1118. ES-MS m/z: 1030
[MC1]C (100%); 750 [MKC15H26N3O2]C (55%); 515
[MC1/2]C (62%). 1H NMR (CDCl3) d: 1.65–1.75 (m,
24H), 2.43–2.50 (m, 24H), 2.60 (t, 12H, JZ6.3 Hz), 3.40
(t, 12H, JZ6.3 Hz), 3.52 (t, 12H, JZ6.3 Hz), 3.63 (t, 12H,
JZ6.3 Hz). 13C NMR (CDCl3) d: 18.8, 27.3, 27.4, 50.4,
50.7, 65.2, 69.0, 69.4, 117.9. HRMS m/z: calcd for
C54H96O9N10: 1029.7440. Found: 1029.7428.
4.5.8. Compound 8. To a solution of 7 (0.85 g, 2.1 mmol) in
CH2Cl2 (25 mL), AcCl (4.6 mL) and H2O (1.08 mL) were
added. The deprotection reaction was continued and worked
up as given in the general procedure IV for the hydrolysis of
tert-butyl esters to afford 8 as a white foamy solid (0.49 g,
99%). FT-IR (KBr) n: 3096, 1754, 1716, 1402, 1180. ES-
MS m/z: 234 [MC1]C (100%); 256 [MCNa]C (20%); 174
4.5.13. Compound 13. Hexa-nitrile 12 (0.35 g, 0.34 mmol)
in MeOH (0.5 mL) was added with Ra-Co catalyst in H2O
(40 mL). The reaction was stirred for 1 h and the reaction
mixture was worked up as given in the general procedure II
to afford the amine intermediate. A solution of crude amine
(0.36 g, 0.34 mmol) in MeOH (5 mL) was treated with tert-
butyl acrylate (4.37 g, 34.1 mmol), and the reaction was
followed as given in general procedure II, to afford, after
purification (alumina) (hexane/EtOAc, 60:40) 13, as a
colorless liquid 0.88 g (70% combined yield for nitrile
reduction and Michael addition). FT-IR (neat) n: 1729,
1461, 1367, 1255, 1157. ES-MS m/z: 2591 [MC1]C (14%).
1H NMR (CDCl3) d: 1.44 (s, 108H), 1.68 (q, 36H, JZ
6.6 Hz), 2.34 (t, 24H, JZ6.9 Hz), 2.46 (b-t, 36H, JZ
6.6 Hz), 2.71 (t, 24H, JZ6.9 Hz), 3.39 (app t, 36H). 13C
NMR (CDCl3) d: 27.4, 27.7, 28.1, 33.8, 49.4, 50.6, 50.9,
68.9, 69.3, 80.2, 172.0.
1
[MKCH2CO2H]C (36%). H NMR (D2O) d: 2.85 (t, 6H,
JZ6.0 Hz), 3.37 (t, 6H, JZ6.0 Hz). 13C NMR (D2O) d:
28.1, 50.0, 174.5.
4.5.9. Compound 9. Tris-nitrile 5 (0.80 g, 2.2 mmol) in
MeOH (0.5 mL) was added with Ra-Co catalyst in H2O
(40 mL). The reaction was stirred for 1 h and the reaction
mixture was worked up as given in the general procedure II
to afford the amine intermediate. A solution of crude amine
(0.82 g, 2.2 mmol) in MeOH (5 mL) was treated with tert-
butyl acrylate (4.37 g, 34.1 mmol), and the reaction was
followed as given in general procedure II, to afford, after
purification (alumina) (hexane/EtOAc, 95:5) 9, as a color-
less liquid (2.33 g, 90% combined yield for nitrile reduction
and Michael addition). FT-IR (neat) n: 1729, 1457, 1367,
1158. ES-MS m/z: 1132 [M]C (75%); 872 [MK2(CH2-
CH2CO2 Bu)]C (46%). 1H NMR (CDCl3): d: 1.41 (s, 54H),
t
1.66 (m, 12H), 2.32 (t, 12H, JZ7.2 Hz), 2.44 (t, 12H, JZ
7.2 Hz), 2.68 (t, 12H, JZ7.2 Hz), 3.37 (m, 12H). 13C NMR
(CDCl3) d: 28.1, 33.7, 49.4, 50.5, 68.9, 80.2, 172.1.
4.5.14. Compound 14. To a solution of 13 (0.70 g,
0.27 mmol) in CH2Cl2 (25 mL), AcCl (1.4 mL) and H2O
(0.32 mL) were added. The deprotection reaction was
continued and worked up as given in the general procedure
IV for the hydrolysis of tert-butyl esters to afford 14, as a
foamy precipitate (0.51 g, 99%). FT-IR (neat) n: 2568,
1729, 1416, 1186. MALDI-TOF-MS m/z: 1946 [MC2C
Na]C (65%). 1H NMR (D2O) d: 1.83 (b, 36H), 2.78 (t, 24H,
JZ6.3 Hz), 3.14 (b, 36H), 3.33 (t, 24H, JZ6.3 Hz), 3.37
(b-t, 36H). 13C NMR (D2O) d: 24.0, 24.2, 29.1, 50.3, 51.9,
53.1, 68.3, 68.8, 174.8.
4.5.10. Compound 10. To a solution of 9 (0.85 g, 2.1 mmol)
in CH2Cl2 (25 mL), AcCl (4.6 mL) and H2O (1.08 mL)
were added. The deprotection reaction was continued and
worked up as given in the general procedure IV for the
hydrolysis of tert-butyl esters to afford 10, as a white foamy
precipitate (0.49 g, 99%). FT-IR (neat) n: 2568, 1729, 1416,
1186. ES-MS m/z: 796 [MC1]C (100%); 723 [MK
CH2CH2CO2H]C (51%); 651 [MC1K2!CH2CH2CO2-
H]C (34%). 1H NMR (D2O) d: 1.85 (b, 12H), 2.80 (t, 12H,
JZ6.4 Hz), 3.14 (b-t, 6H), 3.21 (t, 6H, JZ6.4 Hz), 3.37 (t,
12H, JZ6.4 Hz), 3.46 (t, 6H, JZ6.0 Hz), 3.50 (t, 6H, JZ
6.0 Hz). 13C NMR (100 MHz, D2O) d: 24.0, 24.2, 29.2,
50.3, 51.4, 53.0, 68.4, 68.8, 175.1.
4.5.15. Compound 15. A solution of 13 (0.27 g, 0.1 mmol)
in THF (5 mL) was added drop wise to a suspension of LAH
(0.06 g, 1.4 mmol) in THF (100 mL) left stirring for 1 h and
worked up as described in the general procedure III to obtain
15 a colorless liquid (0.12 g, 66%). FT-IR (neat) n: 3372,
1464, 1365, 1113, 1062. ES-MS m/z: 1750 [M]C. 1H NMR
(CDCl3) d: 1.67–1.75 (m, 60H), 2.50–2.63 (m, 36H), 2.61 (t,
24H, JZ6.3 Hz), 3.46–3.48 (m, 36H), 3.69–3.75 (m, 24H).
4.5.11. Compound 11. A solution of 9 (3.78 g, 3.3 mmol) in
THF (15 mL) was added dropwise to a suspension of LAH
(0.90 g, 23.7 mmol) in THF (100 mL), left stirring for 1 h

4288
T. R. Krishna et al. / Tetrahedron 61 (2005) 4281–4288
13C NMR (CDCl3) d: 26.7, 28.5, 28.7, 29.8, 50.8, 52.0, 52.7,
61.0, 61.6, 62.3, 68.7.
DMF, pH 4.7) was added. Absorbance was measured at
570 nm after an overnight incubation at 37 8C.
4.5.16. Compound 16. Acrylonitrile (0.07 g, 1.4 mmol)
was added to a mixture of 15 (0.18 g, 0.1 mmol) and aq
NaOH (40%, 14 mL), stirred for 6 h and worked up as given
in the general procedure I to obtain crude product which was
purified (alumina) (CHCl3/MeOH, 98:2) to afford com-
pound 16, as a colorless liquid (0.19 g, 77%). FT-IR (neat)
Acknowledgements
We are grateful to the Department of Science and
Technology, New Delhi, for financial support. TRK thanks
the University Grants Commission, New Delhi, for a
research fellowship.
1
n: 2251, 1652, 1368, 1118. H NMR (CDCl3) d: 1.67–1.76
(m, 60H), 2.45–2.52 (m, 60H), 2.60 (t, 24H, JZ6.3 Hz),
3.41 (m, 12H, JZ6.3 Hz), 3.52 (t, 24H, JZ6.3 Hz), 3.64 (t,
24H, JZ6.3 Hz), 3.72 (t, 24H, JZ6.3 Hz). 13C NMR
(CDCl3) d: 18.8, 27.3, 27.4, 50.5, 50.8, 65.9, 69.0, 69.4,
117.4.
References and notes
´
1. For recent reviews: (a) Tomalia, D. A.; Frechet, J. M. J. J.
Polym. Sci., Part A: Polym. Chem. 2002, 40, 2719–2728. (b)
4.5.17. Compound 17. The nitrile 16 (0.09 g, 0.037 mmol)
in MeOH (0.5 mL) was added with Ra-Co catalyst in H2O
(40 mL). The reaction was stirred for 1 h and the reaction
mixture was worked up as given in the general procedure II
to afford the amine intermediate. A solution of crude amine
(0.09 g, 0.034 mmol) in MeOH (5 mL) was treated with
tert-butyl acrylate (4.37 g, 34.1 mmol) and the reaction was
followed as given in general procedure II, to afford, after
purification (alumina) (hexane/EtOAc, 40:60) 17, as a
colorless liquid 0.04 g (18% combined yield for nitrile
reduction and Michael addition). FT-IR (neat) n: 1729,
1458, 1367, 1254, 1158. 1H NMR (CDCl3) d: 1.44 (s,
216H), 1.68 (m, 84H), 2.34 (t, 48H, JZ7.2 Hz), 2.46 (app t,
84H), 2.71 (t, 48H, JZ7.2 Hz), 3.39 (m, 84H). 13C NMR
(CDCl3) 27.6, 28.1, 29.7, 33.8, 49.4, 50.5, 50.8, 68.9, 80.2,
172.0.
¨
Newkome, G. R.; Vogtle, F.; Moorefield, C. N. Dendrimers and
Dendrons; Wiley: West Sussex, 2001. (c) Astruc, D.; Chardac,
F. Chem. Rev. 2001, 101, 2991–3023. (d) Majoral, J. P.;
Caminade, A. M.; Maraval, V. Chem. Commun. 2002, 2929–
2942. (e) Smith, D. K.; Diederich, F. Chem. Rev. 1998, 4, 1353–
1361. (f) Bosman, A. W.; Janssen, H. M.; Meijer, E. W. Chem.
Rev. 1999, 99, 1665–1688. (g) Hearshaw, M. A.; Moss, J. R.
Chem. Commun. 1999, 1–8.
2. Few representative reviews and articles on chemical, biological
and materials studies on dendrimers, see: (a) D’Emanuele, A.;
Attwood, D.; Abu-Rmaileh, R. In Dendrimers; Swarbrickle, J.,
Boylan, J. C., Eds.; Encyclopedia of Pharmaceutical Tech-
nology, issue 3, 2nd ed.; Marcel Dekker: New York, 2003;
pp 1–21. (b) Fuchs, S.; Kapp, T.; Otto, H.; Schoeneberg, T.;
Franke, P.; Gust, R.; Schlueter, A. D. Chem. Eur. J. 2004, 10,
1167–1192. (c) Wiener, E. C.; Brechbeil, M. W.; Brothers, H.;
Magin, R. L.; Gansow, O. A.; Tomalia, D. A.; Lauterbur, P. C.
Magn. Reson. Med. 1994, 31, 1–11. (d) Nithyanandhan, J.;
Davis, R.; Das, S.; Jayaraman, N. Chem. Eur. J. 2004, 10,
689–698. (e) Kaanumalle, L. S.; Nithyanandhan, J.; Pattabira-
man, M.; Jayaraman, N.; Ramamurthy, V. J. Am. Chem. Soc.
2004, 126, 8999–9006.
4.5.18. Compound 18. To a solution of 17 (0.028 g,
0.5 mmol) in CH2Cl2 (25 mL), AcCl (52 mL) and H2O
(12 mL) were added. The deprotection reaction was
continued and worked up as given in the general procedure
IV to afford 18, as a foamy precipitate (0.021 g, 99%). FT-
IR (neat) n: 2568, 1729, 1416, 1186. 1H NMR (D2O) d: 1.83
(b, 84H), 2.78 (t, 48H, JZ6.3 Hz), 3.14 (b, 84H), 3.33 (t,
48H, JZ6.3 Hz), 3.37 (b-t, 84H). 13C NMR (D2O) d: 24.0,
24.2, 29.1, 29.8, 49.8, 51.3, 51.4, 52.2, 53.6, 68.4, 68.5,
174.8.
3. Grinstaff, M. W. Chem. Eur. J. 2002, 8, 2839–2846.
4. (a) Boussif, O.; Lezoualch, F.; Zanta, M. A.; Mergny, M. D.;
Scherman, D.; Demeneix, B.; Behr, J. P. Proc. Natl. Acad. Sci.
U. S. A. 1995, 92, 7297–7301. (b) Choi, J. S.; Joo, D. K.; Kim,
C. H.; Kim, K.; Park, J. S. J. Am. Chem. Soc. 2000, 122,
474–480.
4.6. Cytotoxicity studies (MTT assay)
African green monkey kidney CV-1 or human breast cancer
T47D cells were seeded at a density of 10,000 cells/well in
96-well plate and grown in 95 mL of culture medium
supplemented with 10% fetal bovine serum for 24 h.
Following exposure of the cells with 5 mL of aqueous
solutions of dendrons or dendrimers for 24 h, 25 mL of
5 mg/mL stock solution of 3-[4,5-dimethylthiazol-2-yl]-2,5-
diphenyltetrazolium bromide (MTT) were added to each
well. After 2 h of incubation at 37 8C, 100 mL of extraction
buffer (containing 20% w/v SDS in a solution of 50% of
5. Krishna, T. R.; Jayaraman, N. J. Org. Chem. 2003, 68,
9694–9703.
6. Tomalia, D. A.; Baker, H.; Dewald, J.; Hall, M.; Kallos, G.;
Martin, S.; Roeck, J.; Ryder, J.; Smith, P. Polym. J. 1985, 17,
117–132.
7. For toxicity studies of PAMAM and PPI dendrimers, see:
Malik, R.; Wiwattanapatapee, R.; Klopsch, R.; Lorentz, K.;
Frey, H.; Weener, J. W.; Meijer, E. W.; Paulus, W.; Duncan, R.
J. Controlled Release 2000, 65, 133–148.
8. Mosmann, T. J. Immun. Methods 1983, 65, 55–63.