Triazine dendrimers with orthogonally protected amines on the periphery. Masking amines with Dde and BOC groups provides an alternative to carrying protected alcohols and disulfides through an iterative synthesis

Article abstract of DOI:10.1021/jo701320h

(Chemical Equation Presented) An orthogonally protected dendrimer based on melamine displaying 24 Boc-protected amines (Boc is t-butoxycarbonyl) and 12 Dde-protected (Dde is N-2-(4,4-dimethyl-2,6-dioxocyclohexylidene)ethyl) amines was synthesized using a

Full text of DOI:10.1021/jo701320h

Triazine Dendrimers with Orthogonally Protected Amines on the
Periphery. Masking Amines with Dde and BOC Groups Provides an
Alternative to Carrying Protected Alcohols and Disulfides through
an Iterative Synthesis
Alona P. Umali, Hannah L. Crampton, and Eric E. Simanek*
Department of Chemistry, Texas A&M UniVersity, College Station, Texas 77843-3255
simanek@tamu.edu
ReceiVed June 25, 2007
An orthogonally protected dendrimer based on melamine displaying 24 Boc-protected amines (Boc is
t-butoxycarbonyl) and 12 Dde-protected (Dde is N-2-(4,4-dimethyl-2,6-dioxocyclohexylidene)ethyl) amines
was synthesized using a convergent route in four linear steps in 43% overall yield to provide 5 g of
product. Postsynthetic manipulation of this dendrimer produced a 57 kDa macromolecule bearing poly-
(ethylene glycol) groups and pyridyldisulfide groups linked via carbamate and amide bonds, respectively.
These orthogonally protected amines provide more reactive handles for chemical modification when
compared to other groups, notably hydroxyls, that have been explored. In addition, the Dde group proves
to be more stable to the synthetic methods employed than do the disulfides used previously.
Monochlorotriazine and dichlorotriazine intermediates are invaluable as a route for eliminating unwanted
byproducts arising from over-substitution of the triazine ring. Routes requiring three reactions on a simple,
generation zero core versus six reactions on a generation one core are compared.
Introduction
the development of materials for organic electrooptical purposes.
The creation of electronic gradients within a macromolecule is
possible when the electron source and the electron sink are on
the same macromolecule.4
The ability of dendrimers to present multiple functional
groups upon a well-defined structure with very low polydis-
persity offers enormous advantages to these macromolecules
and opportunities for multiple uses.1 Examples of asymmetric
or heterogeneously functionalized dendrimers or macromol-
ecules are replete in the literature.2 For instance, in the
development of systems for drug delivery, the desire to
covalently incorporate different species such as drug molecules,
targeting moieties, radiolabels, and solubilizing groups neces-
sitates the development of macromolecular scaffolds having
surface groups amenable to covalent modifications.3 A similar
utility is seen on the other end of the application spectrum in
While these targets can be obtained through convergent or
divergent approaches, historically, heterogenously functionalized
dendrimers were derived from a divergent strategy. Although
the exact structure of the resulting heterogeneously function-
alized dendrimers cannot be assigned due to the statistical nature
of the reactions employed, utility and alteration of the physical
properties of the dendrimers are observed. Early work from
Newkome and co-workers is representative: amine-terminated
poly(propylene)imine dendrimers were functionalized with
known ratios of possible monomers (isocyanates derived from
Behera’s amine) leading to combinatorially generated libraries
of macromolecules.5 Convergent approaches successfully reduce
* Corresponding author. Fax: (979) 845-9452. Phone: (979) 845-4242.
(1) (a) Gillies, E. R.; Freche´t, J. M. J. J. Am. Chem. Soc. 2002, 124,
14137-14146. (b) Grayson, S. M.; Freche´t, J. M. J. Chem. ReV. 2001, 101,
3819-3867. (c) Fre´chet, J. M. J. Macromol. Symp. 2003, 201, 11-22. (d)
Stiriba, S.-E.; Frey, H.; Haag, R. Angew. Chem., Int. Ed. 2002, 41, 1329-
1334. (e) Aulenta, F.; Hayes, W.; Rannard, S. Eur. Polym. J. 2003, 39,
1741-1771. (f) Cloninger, M. J. Curr. Opin. Chem. Biol. 2002, 6, 742-
748.
(3) Ihre, H. R.; Padilla De Jesus, O. L.; Szoka, F. C., Jr.; Freche´t, J. M.
J. Bioconjugate Chem. 2002, 13, 443-452.
(4) Kimoto, A.; Cho, J.; Higuchi, M.; Yamamoto, K. Macromolecules
2004, 37, 5531-5537.
(5) (a) Newkome, G. R.; Childs, B. J.; Rourk, M. J.; Baker, G. R.;
Moorefield, C. N. Biotechnol. Bioeng. 1999, 61, 243-253. (b) Newkome,
G. R.; Soo Yoo, K.; Hwang, S.-H.; Moorefield, C. N. Tetrahedron 2003,
59, 3955-3964.
(2) (a) Morgan, J. R.; Cloninger, M. J. Curr. Opin. Drug DiscoVery DeV.
2002, 5, 966-973. (b) Dykes, G. M. J. Chem. Technol. Biotechnol. 2001,
76, 903-918.
10.1021/jo701320h CCC: $37.00 © 2007 American Chemical Society
Published on Web 12/01/2007
9866
J. Org. Chem. 2007, 72, 9866-9874

Triazine Dendrimers with Orthogonally Protected Amines
SCHEME 1a
a (a) Cyanuric chloride, DIPEA, THF, 0 °C, then 1, then 2; (b) 5 equiv of piperazine, THF, rt overnight; (c) 0.5 equiv of cyanuric chloride, THF, 4 equiv
1
of DIPEA, 0 °C to rt overnight. Labels refer to assignments in the H (red) and 13C (blue) NMR spectra shown in Figure 2.
the heterogeneity. Whether the divergently derived, narrowly
disperse mixtures of macromolecules are as effective as single-
entity molecules will likely be dependent on their use. These
comparisons, however, rest on the availability of practical routes
for the latter single-entity macromolecules derived conver-
gently.6
at a 20 g scale. Substitution of this monochlorotriazine with
piperazine provides 4 in good yield, and iterative reaction with
cyanuric chloride provides 5. Of note: the synthesis of 4 from
sequential addition of 1, 2, and piperazine can be achieved in
one pot by manipulating the temperatures at which the addition
is performed, but a side product with identical physical
properties to 4 was obtained. This material, corresponding to a
triazine substituted with piperazine and two Dde-bearing amines,
could not be separated by chromatography. Indeed, the only
evidence for this trace impurity came from mass spectrometry:
the mass difference is 25 Da. This impurity persists through
the synthesis and, interestingly, was most readily identified in
the mass spectra of the intended product 11. The intermediacy
of 3 proves to be critical in obtaining products of high purity.
The core of the dendrimer, 10, was prepared from triazines
6 and 8 (Scheme 2). Both building blocks can be obtained from
the same reaction pot from cyanuric chloride, but each can be
favorably obtained by controlling the temperature. Deprotection
of surface amines of 6 and reaction with 8 at elevated
temperatures in the presence of excess K2CO3 provided 9.
Hunig’s base was found to be less efficient than K2CO3 for
this reaction.10 Deprotection of 9 with trifluoroacetic acid gave
the hexaamine in poor yields, a situation that is remedied using
methanolic HCl.
Synthesis of the Target Dendrimer. To prepare the target
dendrimer 11, a slight excess of 5 and 10 were reacted in the
presence of the polymer-supported base, 1,5,7-triazabicyclo-
[4.4.0]dec-5-ene (p-TBD). With 6.6 equiv of 5 and 12 equiv of
the base, a complete substitution of all the amines of the triazine
core was observed. This reaction was monitored by thin layer
chromatography (TLC) as well as MALDI-TOF MS of the crude
reaction mixture (Figure 1). The top trace shows a predominant
line for the starting material, 5, at the left side of the trace and
intermediates along the route to complete the desired product
(right-most line, m/z 10476) that include substitution of three,
four, and five amines of the core with dendron 5.
The incorporation of orthogonal protecting groups into a
dendrimer provides opportunities for the systematic modification
of these molecules after their convergent construction. The
molecule described bears two reactive amine groups that are
orthogonally protected. Our motivation for examining these
groups rested in the belief that the greater nucleophilicity of
the pendant amines would lead to higher conversions during
postsynthetic manipulations of these materials. Previous efforts
with masked alcohols suffered from low conversions attributed,
in part, to the low nucleophilicity of the hydroxyl group.7 Here,
the moderate-scale synthesis of a third generation dendrimer
based on melamine having two orthogonally protected amine
groups is described. The Dde8 and BOC groups are orthogonal
protecting groups that can be used to differentiate amines. To
establish this role in dendrimers, the Dde groups of 11 were
unmasked with hydrazine and acylated with a disulfide-bearing
group. Following deprotection of the BOC groups with acid,
poly(ethylene glycol) (PEG) chains were installed. We pursued
PEGylation over more trivial manipulations as it improves the
water solubility, biocompatibility, and biodistribution of these
molecules for our intended pursuit, drug delivery.9
Results and Discussion
Synthesis of the Core and Dendrons. An accelerated
approach relying on the synthesis of an orthogonally protected
dendron and a hexavalent, first generation core was adopted.1b
The synthesis is shown in Scheme 1. Selective protection of
the triamine and 4-(aminomethyl)piperidine proceeded smoothly
to provide 1 and 2, respectively. Stepwise reaction of these
amines with cyanuric chloride proceeded in 75% yield after
chromatography to provide dendron 3. This step can be executed
Although the difference in retention factors of 11 and the
excess reactant 5 as measured in TLC by preferred solvent
systems (i.e., 9:1 CH2Cl2/CH3OH) is not substantial to effect
an efficient separation by flash column chromatography,
purification of the product was possible using lower concentra-
tions of methanol in dichloromethane (99:1). However, for large-
scale reactions, the observed behavior of 11 in flash column
(6) Grayson, S. M.; Freche´t, J. M. J. J. Am. Chem. Soc. 2000, 122,
10335-10344.
(7) (a) Steffensen, M. B.; Simanek, E. E. Org. Lett. 2003, 5, 2359-
2361. (b) Lim, J.; Simanek, E. E. Mol. Pharmacol. 2005, 2, 273-277.
(8) Chhabra, S. R.; Hothi, B.; Evans, D. J.; White, P. D.; Bycroft, B.
W.; Chan, W. C. Tetrahedron Lett. 1998, 39, 1603-1606 and references
cited therein.
(9) (a) Greenwald, R. B. J. Controlled Release 2001, 74, 159-171. (b)
Hamilton, J. M.; Chess, R. B. Nat. ReV. 2003, 2, 214-221.
(10) Chen, H.-T.; Neerman, M. F.; Parrish, A. R.; Simanek, E. E. J.
Am. Chem. Soc. 2004, 126, 10044-10048.
J. Org. Chem, Vol. 72, No. 26, 2007 9867

Umali et al.
FIGURE 1. MALDI-TOF MS of the reaction mixture at 24 h (top) and 48 h (bottom). In addition to starting material (major species), the sequential
addition of dendrons can be inferred (light arrows) and observed (dark arrows) in the top spectrum. The bottom spectra reveal complete conversion
to the desired product. Loss of BOC groups (arrows) occurs during ionization in the MALDI-TOF experiment.
SCHEME 2a
a (a) 1:1 TFA/CH2Cl2, rt overnight and (b) 6 equiv of 8, K2CO3, CHCl3, THF, rt overnight.
chromatography was different. The majority of the dendrimer
eluted before excess 5, although some of it was retained and
eluted in the manner predicted by TLC. Calculated yields refer
to the early eluting fraction.
Attempts to synthesize 11 using a tris(piperazine) triazine core
and a third generation dendron bearing four Dde-protected and
eight Boc-protected amines using Hunig’s base were unsuc-
cessful. We attribute this failure to steric crowding and the lower
basicity of Hunig’s base relative to the piperazine amines in
the small triazine core (Scheme 3).10 Using K2CO3 as well as
the unsupported form of TBD were equally unsuccessful.
Instead, a product or products with very high polarity formed,
as shown by the thin layer chromatogram of the reaction
mixture, which we attribute to the loss of the Dde group. Unlike
most of the chemistries we have pursued, the incorporation of
Dde groups into this material led us to be more rigorous in the
exclusion of water: ordinarily, these reactions are carried out
in wet solvents in air. These considerations and the expense of
9868 J. Org. Chem., Vol. 72, No. 26, 2007

Triazine Dendrimers with Orthogonally Protected Amines
SCHEME 3. Preliminary Route toward Synthesis of 11a
a Generation three dendron is available in 87% overall yield from 4 in the two-step sequence commencing with piperazine, followed by treatment with
cyanuric chloride. Reaction to form 11 was plagued with impurities.
SCHEME 4a
a (a) 30% NH2NH2 in MeOH; (b) SPDP, CH2Cl2; (c) 1:1 TFA/CH2Cl2, then dialysis, then 4-nitrophenylPEGOMe.
polymer-supported TBD represent practical limitations of scale
with regard to this approach.
methanol. Surprisingly, purification of the resulting polyamine,
12, can be accomplished by flash column chromatography using
silica gel.
Postsynthetic Modification. The orthogonally protected
amines of 11 were unmasked and reacted sequentially to afford
target 14, a macromolecule containing 12 thiopyridyl groups
and 24 methoxy-terminated PEG groups (Scheme 4). To avoid
the known N f N intramolecular migration of Dde groups,8
Dde-protected amines were first unmasked using hydrazine in
The disulfide groups were installed using 24 equiv of the
NHS ester of thiopyridyl-protected 3-thiolpropanoic acid (SPDP)
in dichloromethane at room temperature. While the reaction can
be monitored using TLC, MALDI-TOF MS was more useful
in ensuring complete substitution. Mass spectra of the crude
J. Org. Chem, Vol. 72, No. 26, 2007 9869

Umali et al.
SCHEME 5a
a (a) 1.0 equiv of cyanuric chloride, 1.5 equiv of DIPEA, THF, 0 °C, 1 h; (b) 1.0 equiv of Dde-AMP, 3.0 equiv of DIPEA, THF, rt overnight; (c) 5.0 equiv
of piperazine, 2.0 equiv of DIPEA, THF, rt overnight. Overall yield for steps b and c was 92%.
SCHEME 6a
a (a) 0.5 equiv of cyanuric chloride, 4 equiv of DIPEA, THF, rt; (b) 5.0 equiv of piperazine, 2.0 equiv of DIPEA, THF, rt overnight; (c) 1.0 equiv of
cyanuric chloride, 2.0 equiv of DIPEA, THF, 0 °C, 4 h; (d) 1.0 equiv of dendron 4, THF, rt overnight.
reaction mixture on the third day of the reaction indicated a
complete functionalization. Dendrimer 13 was purified by flash
chromatography. Deprotection of the Boc groups and PEGy-
lation using a known procedure11 afforded 14 after 10 days.
Complete PEGylation required 52 mol of activated methoxy-
PEG per mole of the dendrimer. MALDI-TOF of this product
showed a molecular mass centered at 57 kDa.
Alternate Route to 4. Success at reducing levels of impurities
through the intermediacy of monochlorotriazine 3 led us to
examine alternative routes that furthered these aims. Specifically,
we pursued isolating dichlorotriazine 15 (Scheme 5) before
installing the Dde-protected amine 2. Compound 15 formed
rapidly and precipitated cleanly, with no need for column
chromatography, thus eliminating the possibility of multiple
Dde-amine substitutions on the triazine ring. Dendron 3 was
still isolated before reaction with piperazine, but no column
chromatography was necessary. The overall yield for this
scheme is comparable to that shown in Scheme 1, but
fortuitously, one chromatographic step is eliminated.
This facile isolation strategy can be readily extended. Dendron
16 with four Boc-protected amines was made from bis-Boc-
protected diethylenetriamine and cyanuric chloride in 99% yield
without chromatography (Scheme 6). Piperazine can be added
to form compound 17, which was used to synthesize the
dichlorotriazine 18 in 58% yield after precipitation. We attribute
the low yield to the precipitation protocol and byproduct that
result when piperazine substitutes two chlorine atoms. Dendron
19 was formed by reaction of dichlorotriazine 18 with dendron
4 in a process that employed only two chromatographic
purifications.
Characterization. All intermediates and products gave
1
satisfactory H and 13C NMR spectra and traces by ESI and
MALDI-TOF MS. Throughout the discussion of the NMR
traces, uppercase letters refer to proton NMR assignments.
Lowercase letters refer to carbon NMR assignments. To
underscore this difference, these assignments are shown in red
and blue, respectively, throughout the schemes and figures.
1
The three singlets at ∼δ 1.0, 2.3, and 2.5 in the H NMR
spectra of 3-5 and 11 (A, B, and C in Figure 2) correspond to
the three sets of unique and independent methyl groups of the
Dde group (Scheme 1). Derivatization of 3 with piperazine led
to new triplet peaks, O and P, corresponding to the methylene
protons of piperazine, at δ 2.9 and 3.8 for 4, which overlapped
at δ 3.8 in the monochloride 5. Protons of 4-(aminomethyl)-
piperidine were assigned using 2-D NMR (not shown). The axial
and equatorial protons of F and G (Scheme 1) had different
chemical shifts that did not change throughout the synthesis.
In the 13C NMR spectra of these intermediates, characteristic
peaks due to carbonyl, vinyl, allyl, and quaternary carbons of
the Dde group (d and e and f and g, and b; Scheme 1) and Boc
group (l shown) were diagnostic for assignment. Appearance
of the piperazine carbons in the spectra of 4 was observed (r1
and r2, Figure 2). Installation of the triazine ring to yield 5 led
to peak overlap. The expected doubling of lines for the
methylene carbons c and the carbonyl carbons d of the cyclic
Dde group was established with an HMQC spectra of dendron
1
4 (Supporting Information). The H and 13C NMR spectra of
11 were very similar to those of 5. Most noteworthy, the line
at δ 169.6 corresponding to the triazine carbon bearing chlorine
was replaced with one at δ 165 as a result of substitution with
the amine of the core in forming 11 (Supporting Information).
The intermediates in the postsynthetic modification of 11 were
also characterized by NMR spectroscopy. Loss of the charac-
(11) Kojima, C.; Kono, K.; Maruyama, K.; Takagishi, T. Bioconjugate
Chem. 2000, 11, 910-917.
9870 J. Org. Chem., Vol. 72, No. 26, 2007

Triazine Dendrimers with Orthogonally Protected Amines
FIGURE 2. NMR spectra of 12-14 and intermediates 3-11 monitor the iterative reaction. Column I, 1H NMR spectra (δ 0-6 ppm) and column
II, 13C NMR spectra (δ 10-60 ppm).
TABLE 1. Mass of 11-14 as Obtained by MALDI-TOF MS
teristic peaks due to Dde was evident in the spectra of 12 (Figure
1
M+ calculated
M+ found
2). Furthermore, the H NMR spectra of this product showed
an upfield shift of proton D, the exocyclic R proton of
(4-aminomethyl)piperidine, in the TOCSY spectra of 12 (Sup-
porting Information). The line for the corresponding carbon, h,
also shifted upfield. Substitution of the free amines of 12 to
give the target 13 derivatized with pyridyl disulfide groups led
to a downfield shift of the same protons. The corresponding
carbon, however, shifted upfield. Peaks due to aromatic protons
compound
11
12
13
14
10435
8466
10830
56909
10454
8468
10847
56709
compounds during the postsynthetic modification. The discrep-
ancies observed reflect the analytical limits of the instrument
when used in linear mode and are not necessarily a failure of
the chemistries described. Gas phase molecular simulations of
target 14 were performed to evaluate the extent of steric
hindrance that the PEG chains might communicate (Figure 3).
The low energy structure shown illustrates that PEGylation does
not necessarily shield all of the dendrimer core nor the disulfide
groups from solvent.
1
and carbons appeared in all the spectra. The H and 13C NMR
spectra of 14 showed the loss of the peaks due to the loss of
Boc, but peaks due to PEG overwhelmed a majority of the
signals associated with the dendrimer. The aromatic peaks of
pyridyl groups were, however, discernible. The mass spectra
of these compounds can be found in the Supporting Information.
Table 1 summarizes the calculated and found masses for
J. Org. Chem, Vol. 72, No. 26, 2007 9871

Umali et al.
FIGURE 3. Molecular model of 14. Yellow: pyridyl disulfide groups; blue: dendrimer scaffold; and orange: PEG groups.
4.5 kV. The nozzle skimmer potential was set to +10 V to minimize
fragmentation in that region. TOF voltages were tuned to optimize
the resolving power over the mass range observed, but usually the
following parameters were used: grid -338 V, plate +360 V,
mirror +960 V, and liner +4000 V. Acquisition and data analysis
were performed with the Analyst QS software.
Conclusion
A dendrimer based on melamine having orthogonally pro-
tected terminal amine groups was synthesized in moderate scale.
Postsynthetic manipulation can lead to dendrimers with structur-
ally diversified surface groups in a well-defined manner.
PEGylation and loading with thiopyridyl groups were illustrated
in this study. The resulting dendrimer had a mass of about
57 kDa while having 12 thiopyridyl groups reactive toward thiol-
containing molecules. We have shown previously that small
molecules and peptides can be appended to these architectures
through thiol-disulfide exchange.12
Intermediate 1.16 Diethylenetriamine (1.00 g, 9.70 mmol, 1.0
equiv) and triethylamine (2.94 g, 29.1 mmol, 3.0 equiv) were
dissolved in 50 mL of THF and cooled in an ice bath. 2-(Boc-
oxyimino)-2-phenylacetonitrile (BOC--ON) (4.78 g, 19.40 mmol,
2 equiv), dissolved in 20 mL of THF, was added dropwise to this
mixture, which was stirred for an hour in an ice bath and then at
room temperature for another hour. The solvent was evaporated,
and the residue was dissolved in 100 mL of dichloromethane. The
mixture was washed with 5% NaOH. The product was purified by
flash column chromatography (silica, 9:1 DCM/MeOH, 1% NH4-
OH), to give 2.47 g (85%). Rf ) 0.63 (4:1 DCM/MeOH). 1H NMR
(300 MHz, CDCl3) δ 3.19 (q, J ) 5.7 Hz, 4H), 2.70 (t, J ) 5.7
Hz, 4H), 1.85 (br, 2H) 1.30 (s, 18H). 13C NMR (75 MHz, CDCl3)
δ 156.4, 79.4, 49.0, 40.4, 28.6. MS: Calcd, 304.0 (M+); Found,
304.1 (M+).
Experimental Section
General. Triazine trichloride, piperazine, 4-(aminomethyl)-
piperidine (TCI), diisopropylethylamine or DIPEA, and BOC-ON
were used as-is. 1,5,7-Triazabicyclo[4.4.0]dec-5-ene bound to
polystyrene cross-linked with 2% DVB (2.6 mmol/g resin) was
purchased from a chemical supplier. MALDI-TOF mass spectra
(in the positive mode) were acquired on a Voyager-DE STR mass
spectrometer equipped with a pulsed nitrogen laser emitting at 337
nm. Samples were analyzed in linear mode using an extraction delay
time set at 350 ns and an accelerating voltage operating at 25 kV
utilizing trihydroxyacetophenone as the matrix. To improve the
signal-to-noise ratio, 100 single shots were averaged for each mass
spectrum, and typically, four individual spectra were accumulated
to generate a summed spectrum. An external mass spectrum
calibration was performed using the calibration mixture 3 of the
Sequazyme Protein Mass Standards Kit, including known protein
standards in a mass range from 5 to 25 kDa. Electrospray mass
spectra were acquired in the positive ion mode using a MDS Sciex
API Qstar Pulsar using an Ionwerks time-to-digital converter,
TDCx4, for data recording at 625 ps time resolution. Samples were
electrosprayed from acetonitrile at 50 mM under the conditions
listed next. The ion spray (needle) voltage was held constant at
Intermediate 2. 4-Aminomethylpiperidine (1.66 g, 14.5 mmol)
and 2-acetyldimedone (2.64 g, 14.5 mmol) were dissolved in 100
mL of absolute ethanol and were refluxed for 3 h. The solvent was
evaporated, and the product was purified by flash column chro-
matography (silica, 4:1 DCM/MeOH, 1% NH4OH), to give a yield
1
of 3.50 g (87.5%). Rf ) 0.075 (9:1 DCM/MeOH). H NMR (500
MHz, CDCl3) δ 3.27 (t, J ) 6.0 Hz, 2H), 2.62 (m, 2H), 2.55 (s,
3H), 2.36 (br, 4H), 1.77 (m, 3H), 1.24 (m, 2H), 1.02 (s, 6H). 13C
NMR (125 MHz, CD3OD) 199.9 (br), 175.8, 108.9, 53.6, 50.5, 46.7,
37.5, 32.0, 31.2, 28.5, 18.0. MS: Calcd, 279.2 (M+); Found, 279.2
(M+).
Intermediate 3. Cyanuric chloride (1.73 g, 9.38 mmol) and 3
mL of DIPEA was dissolved in 100 mL of THF and cooled to
0 °C. Intermediate 1 (2.85 g, 9.38 mmol) was dissolved in 50 mL
of THF and added dropwise to the cold solution of cyanuric chloride
and DIPEA. After all of the amine had reacted, as monitored by
TLC, another 3 mL of DIPEA and intermediate 2 (2.61 g, 9.38
(12) Umali, A. P.; Simanek, E. E. Org. Lett. 2003, 5, 1245-1247.
9872 J. Org. Chem., Vol. 72, No. 26, 2007

Triazine Dendrimers with Orthogonally Protected Amines
mmol) were added, and the reaction mixture was stirred at room
temperature overnight. The solvent was evaporated, and the residue
was dissolved in about 50 mL of CH2Cl2. The resulting solution
was washed with water in three 100 mL portions. The CH2Cl2 layer
was dried with MgSO4, and the solvent was evaporated. The product
was isolated by flash column chromatography (silica, 3:1 DCM/
Intermediate 8. Cyanuric chloride (0.44 g, 2.41 mmol) and
mono(t-butoxycarbonyl)piperazine (0.90 g, 4.82 mmol) were dis-
solved in about 75 mL of THF. The mixture was stirred overnight
at room temperature. The solvent was removed in vacuo. The
residue was then dissolved in DCM and washed with three 100
mL portions of water. DCM was removed after drying the mixture
with MgSO4. Flash column chromatography (50:1 DCM/MeOH)
1
EtOAc) (4.87 g, 75%). Rf ) 0.30 (3:1 DCM/EtOAc). H NMR
1
(500 MHz, CD3OD) δ 4.77 (br, 2H), 3.65 (m, 4H), 3.46 (m, 2H),
3.31 (m, 4H), 2.93 (t, J ) 12.5 Hz, 2H), 2.59 (s, 3H), 2.39 (s, 4H),
2.01 (br, 1H), 1.89 (d, J ) 10.5 Hz, 2H), 1.42 (s, 18H), 1.30 (m,
2H), 1.05 (s, 6H). 13C NMR (75 MHz, CDCl3) δ 199.2, 196.8,
173.7, 169.4, 165.8, 163.9, 156.3, 156.0, 108.0, 79.3, 79.2, 53.5,
52.3, 48.9, 47.9, 47.5, 43.3, 39.5, 39.1, 36.4, 30.2, 29.9, 29.7, 28.6,
28.4, 28.4, 28.3,18.0. MS: Calcd, 693.39 (M+); Found, 693.39
(M+), 699.40 (M + Li+), 715.37 (M + Na+).
gave 2.17 g of product (88%). Rf ) 0.34 (50:1 DCM/MeOH). H
NMR (300 MHz, CDCl3) δ 3.77 (m, 8H), 3.46 (m, 8H), 1.48 (s,
18H). 13C NMR (75 MHz, CDCl3) δ 169.9, 164.7, 154.9, 80.5,
43.5, 28.6. MS: Calcd, 484.24 (M+); Found (ESI), 484.24 (M+).
Intermediate 9. Intermediate 7 (1.00 g, 3.00 mmol) and
intermediate 8 (4.50 g, 9.30 mmol) were dissolved in about 50 mL
of CHCl3. K2CO3 (3.73 g, 27.0 mmol) was added to the mixture,
which was then stirred at 80 °C for 2 days or until the triazine
tris(piperazine) was exhausted as indicated by TLC. The reaction
mixture was washed with three 50 mL portions of water, and the
chloroform layer was dried with MgSO4. The solvent was evapo-
rated, and the product was purified by flash column chromatography
Intermediate 4. Intermediate 3 (3.49 g, 5.04 mmol) and
piperazine (2.17 g, 25.2 mmol) were dissolved in about 150 mL of
THF. The mixture was stirred at room temperature overnight. The
solvent was evaporated, and the product was obtained by flash
column chromatography (19:1 DCM/MeOH) (3.20 g, 90%). Rf )
1
(silica, 24:1 DCM/MeOH, Rf ) 0.16) to give 4.12 g (82%). H
1
0.33 (9:1 DCM/MeOH). H NMR (500 MHz, CDCl3) δ 5.79 (br,
NMR (300 MHz, CDCl3) δ 3.81 (s, 24H), 3.76 (t, J ) 5.0 Hz,
24H), 3.45 (t, J ) 5.1 Hz, 24H), 1.49 (s, 56H). 13C NMR (75 MHz,
CDCl3) δ 165.7, 165.6, 155.1, 80.1, 43.3, 43.3, 28.7. MS: Calcd,
1676.02 (M+), 838.52 (M + 2H)2+; Found (ESI), 838.53 (M +
1H), 5.69 (br, 1H), 4.78 (m, 2H), 3.74 (m, 4H), 3.64 (br, 4H), 3.30
(m, 5H), 2.89 (m, 4H), 2.75 (m, 2H), 2.55 (s, 3H), 2.36 (br, 5H),
1.88 (m, 3H), 1.39 (s, 18H), 1.22 (m, 2H), 1.02 (s, 6H). 13C NMR
(75 MHz, CDCl3) δ 199.0, 196.8, 173.5, 166.5, 165.4, 165.1, 164.8,
164.5, 156.1, 107.9, 78.9, 68.1, 61.5, 53.3, 52.2, 49.0, 46.3, 45.5,
43.6, 40.0, 36.5, 29.8, 28.2, 17.9. MS: Calcd, 743.49 (M+); Found
(ESI), 743.46 (M+).
2H)2+
.
Intermediate 10. To a 15 mL solution of protected core 9
(1.02 g, 0.609 mmol) in DCM was added 15 mL of TFA. This
was stirred at room temperature for 3 h. The solvent was evaporated,
and the residue was made basic with 5 M NaOH (about pH 14).
The product was extracted with five 50 mL portions of chloroform.
The organic layer was dried with MgSO4, chloroform was
evaporated off, and the residue was dried in vacuo to give 0.365 g
Intermediate 5. Intermediate 4 (2.82 g, 3.79 mmol) and 3 mL
of DIPEA were dissolved in 150 mL of THF. The resulting solution
was placed in an ice bath. Cyanuric chloride (0.35 g, 1.90 mmol)
was added, and the reaction mixture was stirred at room temperature
overnight. The solvent was removed, and the residue was dissolved
in DCM. The resulting mixture was washed with three 50 mL
portions of distilled water. The DCM layer was dried with MgSO4,
and DCM was removed in vacuo. Flash column chromatography
(silica, 9:1 DCM/MeOH) gave 2.18 g of the product (79%). Rf )
1
of product (56%). H NMR (300 MHz, CDCl3) δ 3.76 (br, 24H),
3.72 (t, J ) 5.0 Hz, 24H), 2.84 (t, J ) 5.0 Hz, 24H). 13C NMR (75
MHz, CDCl3) 165.6, 46.3, 44.6, 43.3. MS: Calcd, 334.25 (M+);
Found (ESI), 335.25 (M+).
Dendrimer 11. Intermediate 10 (35.3 mg, 0.033 mmol) and
intermediate 5 (0.35 g, 0.22 mmol) were completely dissolved in
4:1 CHCl3/MeOH. Polystyrene-supported 1,5,7-triazabicyclo[4.4.0]-
dec-5-ene (12 equiv, 0.20 mmol) was added to the mixture which
was refluxed at 80 °C. Loss of the amine core was detected by
TLC (9:1 DCM:MeOH) after 48 h. The polymer-supported base
was filtered off, and the product was obtained by flash column
chromatography (silica, 40:1 and then 19:1 DCM/MeOH) to give
1
0.63 (9:1 DCM/MeOH). H NMR (500 MHz, CDCl3) δ 5.65 (br,
2H), 5.58 (br, 2H), 4.76 (d, J ) 12.9 Hz, 4H), 3.80 (m, 16H), 3.63
(br, 8H), 3.29 (m, 12H), 2.76 (t, J ) 12.3 Hz, 4H), 2.54 (s, 6H),
2.34 (m, 8H), 1.84 (m, 6H), 1.34 (s, 36H), 1.23 (m, 4H), 1.01 (s,
12H). 13C NMR (125 MHz, CDCl3) δ 199.4, 196.9, 173.6, 169.8,
166.6, 165.0, 164.7, 164.6, 156.2, 108.1, 79.1, 68.0, 53.6, 52.4,
49.1, 46.7, 43.5, 43.0, 40.4, 36.7, 30.2, 29.9, 28.5, 25.7, 18.0. MS:
Calcd, 1596.94 (M+); Found (ESI), 798.96 (M + 2H+).
1
0.23 g, 66% yield. H NMR (300 MHz, CDCl3) δ 5.89, 5.69 (br,
36H), 4.78 (br, 24H), 3.82 (br, 168H), 3.62 (br, 48H), 3.28 (br,
72H), 2.76 (m, 24H), 2.51 (s, 36H), 2.31 (m, 48H), 1.83 (m, 36H),
1.35 (s, 216H), 1.21 (m, 24H), 0.98 (s, 72H). 13C NMR (125 MHz,
CDCl3) δ 173.8, 166.8, 165.7, 165.6, 165.2, 164.8, 156.4, 108.2,
79.2, 68.2, 53.7, 52.5, 49.3, 46.7, 43.4, 43.2, 40.7, 40.5, 36.8, 30.3,
30.1, 28.7, 28.7, 28.5, 25.8, 18.2. MS: Calcd, 10,435.45 (M+);
Intermediate 6. Cyanuric chloride (0.85 g, 4.59 mmol) and
mono(t-butoxycarbonyl)piperazine (2.65 g, 14.2 mmol) were dis-
solved in 100 mL of THF in a round-bottomed flask and stirred at
80 °C in the presence of K2CO3 (5.7 g, 41.3 mmol) overnight. THF
was evaporated off, and the resulting residue was dissolved in
dichloromethane. The mixture was washed with three 50 mL
portions of water. The dichloromethane layer was dried with MgSO4
and concentrated in vacuo. Flash column chromatography (silica,
4:1 DCM/EtOAc) of the residue gave 2.59 g of product (86%). Rf
) 0.64 (9:1 DCM/MeOH). 1H NMR (500 MHz, CDCl3) δ 3.72 (t,
J ) 5 Hz, 12H), 3.42 (t, J ) 5 Hz, 12H), 1.46 (s, 27H). 13C NMR
(125 MHz, CDCl3) 165.5, 155.0, 80.0, 76.2, 43.2, 28.6. MS: Calcd,
634.40 (M+); Found (ESI), 634.40 (M+).
Found (MALDI-TOF), 10,454.15 (M+), 5228.26 (M + 2H)2+
.
Dendrimer 12. Dendrimer 11 (2.54 g, 0.243 mmol) was
dissolved in 50 mL of 1:3 DCM/MeOH. Anhydrous hydrazine (25
mL) was added and stirred at room temperature for 7 h or until all
of 11 had reacted and was monitored by TLC. The solvent was
evaporated, and the residue was dissolved in 100 mL of chloroform.
This mixture was then washed with 50 mL portions of distilled
water 3 times. The chloroform layer was dried with MgSO4 and
evaporated. Flash chromatography using DCM from silica gave
1.74 g of product (84%). 1H NMR (300 MHz, CDCl3) δ 5.96, 5.86
(br, 24H) 4.74 (m, 24H), 3.82, 3.65 (br, 216H), 3.32 (br, 48H),
2.77 (m, 24H), 2.58 (br, 24H), 1.76 (br, 24H), 1.60 (m, 36H), 1.39
(s, 216H), 1.14 (m, 24H). 13C NMR (75 MHz, CDCl3) δ 166.6,
165.4, 165.3, 164.8, 164.5, 156.0, 78.8, 47.8, 46.2, 43.1, 40.4, 39.6,
29.7, 28.4. MS: Calcd, 8466.44 (M+); Found (MALDI-TOF),
8468.68 (M+), 8491.08 (M + Na+).
Intermediate 7. Intermediate 6 (2.01 g, 3.18 mmol) was
dissolved in 25 mL of 1:1 TFA/DCM solution and was stirred at
room temperature for 3 h. The solvent was evaporated, and the
residue was treated with 5 M NaOH until highly basic (pH 14 using
pH paper). The product was extracted with three 50 mL portions
of chloroform. The organic layer was dried with MgSO4, and
chloroform was evaporated off. The residue was dried in vacuo,
1
giving 1.01 g of the amine (95%). H NMR (300 MHz, CD3OD)
δ 4.04 (t, J ) 5.3 Hz, 12H), 3.23 (t, J ) 5.1 Hz, 12H). 13C NMR
(75 MHz, CD3OD) 166.6, 44.4, 41.2. MS: Calcd, 334.25 (M+);
Found (ESI), 335.25 (M+).
Dendrimer 13. Dendrimer 12 (0.397 g, 0.0468 mmol) and
N-succinimidyl 3-(2-pyridyldithio)propionate (0.427 g, 1.37 mmol)
J. Org. Chem, Vol. 72, No. 26, 2007 9873

Umali et al.
were dissolved in 1.0 mL of 1:1 dichloromethane/chloroform and
stirred at room temperature for 3 days. The reaction was monitored
by TLC and MALDI-TOF MS. The product was isolated by flash
column chromatography using dichloromethane as solvent. Rf )
0.28 (9:1 DCM/MeOH), 0.304 g (60%). 1H NMR (500 MHz,
CDCl3) δ 8.42 (m, 12H), 7.62 (m, 24H), 7.11 (m, 12H), 6.85 (br,
12H), 5.81 (br, 24H), 4.71 (br, 24H), 3.82, 3.64 (br, 216H), 3.31
(br, 48H), 3.19 (br, 24H), 3.06 (m, 24H), 2.76 (m, 24H), 2.61 (m,
24H), 1.78 (br, 24H), 1.38 (br, 216H), 1.18 (br, 24H). 13C NMR
(75 MHz, CDCl3) δ 170.9, 166.5, 165.3, 165.2, 164.8, 164.4, 159.4,
156.1, 149.4, 121.0, 120.2, 78.9, 46.3, 45.2, 43.1, 40.3, 36.7, 35.7,
34.9, 29.6, 28.4. MS: Calcd, 10830.42 (M+); Found (MALDI-
TOF), 10847.93 (M+).
THF, and the solution was stirred at room temperature for 2 days.
The solvent was removed in vacuo, and the residue was purified
by silica column chromatography with 4:1 DCM/MeOH. Rf ) 0.65,
4.450 g, 92%. 1H NMR (300 MHz, CDCl3) δ 5.715 (br, 3H), 5.258
(s, 1H), 3.686 (br, 4H), 3.611 (br, 4H), 3.536 (br, 4H), 3.271 (br,
8H), 2.840 (br, 4H). NMR (75 MHz, CDCl3) δ 166.1, 164.7, 156.4,
79.4, 47.5, 46.8, 46.0, 43.4, 40.8, 38.6, 28.6. MS: Calcd, 767.5018
(M+); Found (ESI), 768.5382 (M + H)+.
Intermediate 18. Cyanuric chloride (1.07 g, 5.782 mmol) was
dissolved in 50 mL of THF and cooled to 0 °C in an ice bath.
Intermediate 17 (3.7 g, 4.82 mmol) was dissolved in 50 mL of
THF and added dropwise to the cold cyanuric chloride solution.
The mixture was stirred at 0 °C for 4 h, and the solvent was
removed in vacuo. The residue was dissolved in dichloromethane
and washed with three 50 mL portions of water. The organic layer
was dried with Na2SO4 and filtered, and the solvent was removed
in vacuo. The solids were dissolved in a minimal amount of DCM,
and hexanes were added to precipitate the product (2.566 g, 58%
Dendrimer 14. Dendrimer 13 (0.234 g, 0.0216 mmol) was
dissolved in 10 mL of a 2:1 TFA/CH2Cl2 mixture and stirred
overnight. The solvent was removed, and the residue was dissolved
in a minimum amount of methanol. The salt product was precipi-
tated out and washed with diethylether. After drying, the depro-
tection product was dissolved in DMSO and stirred with MeO-
PEG 4-nitrophenylcarbonate13 (3.07 g, 72 equiv) and 7 mL of
triethylamine for 10 days. The reaction mixture was dissolved in
10 mL of water and dialyzed using a dialysis bag with a molecular
weight cutoff of 12-14 kDa in 1:1 water/methanol for 24 h, in 2:1
water/methanol for 48 h, and then in water alone for another 48 h,
changing the solution as often as possible. Water was removed,
1
yield). H NMR (300 MHz, CDCl3) δ 5.580 (br, 3H), 5.287 (s,
1H), 3.907 (m, 4H), 3.844 (m, 4H), 3.652 (br, 4H), 3.579 (br, 4H),
3.313 (m, 8H), 1.400 (s, 36H). 13C NMR (75 MHz, CDCl3) δ 170.6,
166.0, 164.4, 156.5, 156.3, 79.5, 47.7, 47.1, 44.3, 40.7, 38.5, 28.7,
28.6. MS: Calcd, 914.44 (M+); Found (MALDI-TOF), 915.25 (M
+ H)+, 937.24 (M + Na)+.
1
and the product was vacuum-dried (1.14 g, 94%). H NMR (500
Intermediate 19. Intermediate 18 (0.122 g, 0.1331 mmol) and
intermediate 4 (0.099 g, 0.1331 mmol) were dissolved in 20 mL
of THF. DIPEA was added (0.07 mL, 0.3993 mmol), and the
mixture was stirred for 16 h at room temperature. The solvent was
removed in vacuo. The resulting residue was dissolved in 15 mL
of DCM and was washed with 10 mL of water (2 times) and with
10 mL of brine (1 time). The organic layer was dried with Na2SO4
and filtered, and the solvent was removed in vacuo. The resulting
residue was dissolved in a minimal amount of dichloromethane,
and hexanes were added to form a precipitate (0.1575 g, 73%). 1H
MHz, CDCl3) δ 8.35 (br), 7.56 (br), 7.03 (br), 4.08 (br), 3.70 (m),
3.57 (m), 3.48 (m), 3.42 (m), 3.30 (m), 3.16 (m), 2.98 (br), 2.66
(br), 2.53 (br), 1.68 (br), 1.17 (m), 1.05 (m). 13C NMR (75 MHz,
CDCl3) δ 170.8, 165.0, 159.4, 156.3, 149.3, 137.0, 120.8, 119.8,
80.5, 73.3, 72.3, 71.6, 71.3, 70.3, 70.0, 69.3, 67.2, 64.1, 63.3, 61.4,
58.8, 45.0, 42.8, 40.8, 35.3, 34.4, 29.5. MS: Calcd, 56909 (Mav+);
Found (MALDI-TOF), 56798 (Mav+).
Intermediate 15. Cyanuric chloride (2.91 g, 15.8 mmol) was
dissolved in 100 mL of THF and cooled to 0 °C in an ice bath.
DIPEA (4.25 mL, 23.7 mmol) was added to the cold solution.
Intermediate 1 (4.8 g, 15.8 mmol) was dissolved in 100 mL of
THF and added dropwise to the cold cyanuric chloride solution
over 1 h. The mixture was stirred at 0 °C for an additional 45 min.
The solvent was removed in vacuo, and the resulting solids were
dissolved in 50 mL of dichloromethane. The solution was washed
with 50 mL of water (2 times) and with 50 mL of brine (1 time).
The organic layer was dried with Na2SO4 and filtered, and the
solvent was removed in vacuo. The resulting residue was dissolved
in a minimal amount of dichloromethane, and 100 mL of hexanes
was added slowly. The resulting precipitate was filtered and dried
under vacuum overnight (6.814 g, 96%). Rf ) 0.51 (3:2 DCM/
EtOAc). 1H NMR (300 MHz, CDCl3) δ 5.273 (s, 0.5H), 5.015 (br,
1.5H), 3.711 (t, 3JH-H ) 6 Hz, 4H) 3.365 (m, 4H), 1.361 (s, 18H).
13C NMR (75 MHz, CDCl3) δ 170.2, 165.9, 156.3, 79.9, 48.9,
38.8, 28.5. MS: Calcd, 450.15 (M+); Found (ESI), 457.16 (M + Li)+.
Intermediate 16. Cyanuric chloride (0.260 g, 1.41 mmol) and
intermediate 1 (0.86 g, 2.83 mmol) were dissolved in 20 mL of
THF. DIPEA (1.01 mL, 5.65 mmol) was added, and the mixture
was stirred at room temperature for 16 h. The solvent was removed
in vacuo, and the residue was dissolved in DCM. The organic layer
was washed with 20 mL of water (2 times) and with 20 mL of
brine (1 time). The organic layer was dried with Na2SO4 and filtered,
and the solvent was removed in vacuo to yield a white solid (0.9996
g, 99%). Rf ) 0.44 (3:2 DCM/EtOAc). 1H NMR (300 MHz, CDCl3)
δ 5.607 (br, 1.5 H), 5.195 (br, 1.5 H), 3.602 (m, 8H), 3.291 (m,
8H), 1.384 (s, 36H). 13C NMR (75 MHz, CDCl3) δ 169.1, 165.5,
156.4, 79.5, 48.1, 47.4, 39.7, 37.9, 28.6. MS: Calcd, 717.40 (M+);
Found (ESI), 718.40 (M + H)+.
3
NMR (300 MHz, CDCl3) δ 5.632 (br, 5H), 4.766 (d, JH-H ) 12
Hz, 2H), 3.806 (br, 16H), 3.592 (br, 12H), 3.305 (br, 14H), 2.782
3
(t, JH-H ) 12 Hz, 2H), 2.539 (s, 3H), 2.349 (s, 4H), 2.018 (br,
3
1H), 1.849 (d, JH-H ) 12 Hz, 2 H), 1.398 (br, 54H), 1.237 (br,
2H), 1.015 (s, 6H). 13C NMR (75 MHz, CDCl3) 199.2, 196.9, 173.8,
169.9, 166.0, 164.7, 156.4, 156.3, 108.2, 79.3, 53.7, 49.3, 47.7,
46.9, 46.7, 43.6, 43.2, 40.8, 40.5, 38.5, 36.8, 31.8, 30.3, 30.1, 28.6,
22.9, 18.2. MS: Calcd, 1620.95 (M+); Found (MALDI-TOF),
1621.68 (M + H)+, 1643.67 (M + Na)+, 1659.63 (M + K)+.
Molecular Modeling. The energy-minimized structure of 18 (gas
phase) was obtained using the software package Cerius2 4.6. The
pcff second-generation force field in the open force field (OFF)
program was used in minimizations and dynamics. The structure
was initially minimized in the fully extended conformation. Constant
volume and temperature (NVT) molecular dynamics (MD) calcula-
tions were then performed on the minimized extended structure
via simulated annealing. The simulated annealing was carried out
for 840.0 ps, over a temperature range of 300-1000 K, with ∆T
) 50 K, using the Nose´ temperature thermostat, a relaxation time
of 0.1 ps, and a time step of 0.001 ps. The dendrimer was minimized
after each annealing cycle, resulting in 300 minimized structures
of 14.
Acknowledgment. This work was supported by the N.I.H.
(GM 65460) and the Welch Foundation (A 1439). H.L.C. thanks
the N.S.F. for a predoctoral fellowship.
Intermediate 17. Intermediate 16 (4.5 g, 6.26 mmol) and
piperazine (2.698 g, 31.32 mmol) were dissolved in 180 mL of
Supporting Information Available: 1H and 13C NMR and mass
spectra. This material is available free of charge via the Internet at
http://pubs.acs.org.
(13) Westerberg, D. A.; Carney, P. L.; Rogers, P. E.; Kline, S. J.; Johnson,
D. K. J. Med. Chem. 1996, 32, 236-243.
JO701320H
9874 J. Org. Chem., Vol. 72, No. 26, 2007