Antibacterial single-bead screening

Article abstract of DOI:10.1016/j.tet.2003.10.070

Triazine based antibiotics were prepared by the attachment of cyanuric chloride onto a Marshall-type safety catch linker, followed by successive aromatic nucleophilic substitutions, linker activation and nucleophilic cleavage. High-loading dendrimer beads allowed the release of sufficient amount of compound from a single bead to give clear inhibition.

Full text of DOI:10.1016/j.tet.2003.10.070

Tetrahedron 59 (2003) 10213–10222
Antibacterial single-bead screening
Sylvain Lebreton,a Nicholas Newcombeb and Mark Bradleya
,
*
aDepartment of Chemistry, University of Southampton, Southampton, Hampshire SO17 1BJ, UK
bAstraZeneca, Mereside, Alderley Park, Macclesfield, Cheshire SK10 4TG, UK
Received 7 February 2003; revised 2 October 2003; accepted 23 October 2003
Abstract—Triazine based antibiotics were prepared by the attachment of cyanuric chloride onto a Marshall-type safety catch linker,
followed by successive aromatic nucleophilic substitutions, linker activation and nucleophilic cleavage. High-loading dendrimer beads
allowed the release of sufficient amount of compound from a single bead to give clear inhibition.
q 2003 Elsevier Ltd. All rights reserved.
Bead-based screening, exploiting the one-bead one-
compound concept of split and mix combinatorial library
synthesis, is a powerful method for the synthesis and
screening of large libraries either on or off beads.1 Screening
peptide-based libraries has been carried out with the peptide
tethered to the solid support, whereas libraries of small
organic compounds have usually been screened in solution
after cleavage, where the molecule is fully exposed in the
assay medium.2 Among the numerous solution-phase assays
available, gel-permeation assays for antimicrobial screening
can be easily adapted to the screening of bead-based
combinatorial libraries.3 Jayawickreme et al. presented the
first evidence that single-bead activity from a large peptide-
based library could be detected following cleavage from
single beads and gel based assay4 and this was followed by
the screening of a large non-peptidic type library in a similar
lawn assay.5 These assays using porous gel matrices
(primarily agar or agarose) consisted of spreading the
library beads across the gel matrix and cleaving the
compounds from the solid-phase support into the gel
containing the appropriate cells. Zones of inhibition
corresponding to the lack of cell growth indicate the
presence of active compounds.6 However, a critical issue is
the limited amount of compound available on single beads.
For instance, a standard single polystyrene bead bearing
approximately 500 pmol of compound will only yield a
5 mM solution in a 100 ml volume. This limitation may be
critical to the viability of the assay and its sensitivity. Here,
we report on the use of high-loading dendrimerised resin
beads for single-bead antibacterial screening.
1. Results and discussion
Our studies targeted compounds containing a decorated
1,3,5-triazine. Such structures have recently been found to
inhibit DNA Gyrase,7 an enzyme responsible for DNA
super-coiling. This represents an attractive antibacterial
target because it is vital to bacteria yet has no direct
counterpart in mammals. Moreover, 1,3,5-triazine rep-
resents a useful template for the construction of heterocycles
and has been widely reported for library generation.5,8 We
recently reported a novel 1!3 C-branched isocyanate-type
monomer which was used to prepare high-loading beads via
a divergent dendrimerisation process.9 Using 250–300 mm
1% DVB polystyrene resin beads generation 1.0 and 2.0
resin-bound dendrimers were prepared with a loading of 46
and 119 nmol/bead, respectively (Fig. 1).
The safety-catch linker of Marshall and Liener10 was chosen
for our synthesis as it is easily converted into the activated
nucleophile-sensitive arylsulfone using mild oxidative
conditions. The utility of this strategy has been reported
for the synthesis of peptides,10,11 ureas,12 and heterocyclic
compounds.13 Prior to embarking on the solid-phase library
synthesis, solution-phase models were investigated.
Benzylamine, used as a starting point for the synthesis,
was reacted with the Marshall-type linker 1 using DCC and
HOBt (Scheme 1). Reaction of 2a with cyanuric chloride in
THF afforded the 3,5-dichloro-triazine 3a in 98% yield (no
product was formed when the reaction was performed in
DMSO or DMF, which are usually the first choice solvents
for dendrimerised resins due to their efficient swelling of the
resins which thus maximises site accessibility and mini-
mises cross-coupling). The first aromatic nucleophilic
substitution with p-fluoroaniline worked successfully in
both THF and DMF although further substitution of the
remaining chloride appeared to be a problem. A 0.2 M
solution of p-fluoroaniline in THF reacted with
Keywords: beads; dendrimers; triazines; antibiotic; linker.
*
Corresponding author. Tel.: þ44-2-380-593-598; fax: þ44-2-380-596-
766; e-mail: mb14@soton.ac.uk
0040–4020/$ - see front matter q 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2003.10.070

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S. Lebreton et al. / Tetrahedron 59 (2003) 10213–10222
Figure 1. Generation 2.0 resin-bound dendrimer.
Scheme 1. Solution model and solid-phase triazine synthesis strategy (PS¼Polystyrene resin). (i) HOBt, DIC/DMF; (ii) cyanuric chloride/THF; (iii) aniline
derivative (R1), DIPEA/DMF or DMF/THF (1:1); (iv) aniline derivative (R2), DIPEA/DMF; (v) CH3CO3H/DCM; (vi) morpholine or piperidine or
N-methylpiperazine or pyrrolidine/DMSO, 1008C. See Figure 3 for the monomers used in library synthesis.

S. Lebreton et al. / Tetrahedron 59 (2003) 10213–10222
10215
Figure 2. (a) Rate of reaction of p-fluoroaniline onto 3a. Mono-substitution (complete after 30 min) versus di-substitution. (b) Rate of addition of p-
methoxyaniline onto 3-chloro-5-(p-fluororaniline)-triazine.
reduced thus for library synthesis purposes, it was reasoned
Table 1. Rate of triazine cleavages at 60, 100, and 1308C
that the first set of anilines to be reacted onto 3,5-dichloro-
triazine scaffold should be the least electron-donating, while
the second set much more electron rich.
T (8C)
Arylsulfide 4aa
Arylsulfone 5aa
60
100
130
Not cleaved
,120 h
,40 h
10 h
1
,40 min
Oxidation of the arylsulfide 4a was performed with
peracetic acid in DCM. The electron-donating character of
the sulfide increases the electron density within the aromatic
triazine system, which makes it more inert towards
nucleophilic attack. Once oxidised, the electron-withdraw-
ing character of the sulfoxide renders the triazine ring much
more electrophilic and thus more labile to aromatic
nucleophilic substitution at the ipso position. Initial
oxidation of the sulfide to the sulfonyl was found to be
fast (,10 min) followed by further oxidation to the sulfone.
After 3–4 h, the ratio of sulfone/sulfonyl as determined by
LC/MS was 90:10. This did not increase despite adding
more oxidising agent. However, the presence of the
sulfoxide product was not a problem as it is also electron
withdrawing and was displaced efficiently.
a
Time required for complete cleavage as determined by LC/MS.
Table 2. Secondary amine mediated triazine cleavage at 1008C
Amine
Timea (min)
Piperidine
N-Methylpiperazine
Pyrrolidine
40
45
5
a
Time required for complete cleavage as determined by LC/MS.
3,5-dichlororo-triazine 3a (3 equiv. of p-fluoroaniline with
respect to 3a; i.e. library conditions) and the relative rates of
mono and di-analine substitution were monitored by LC/MS
(Fig. 2(a)). The optimal reaction time was observed to be
30 min, at which time 100% of the mono-substituted
triazine was formed. Longer exposure resulted in additional
substitution.
The non-oxidised and oxidised forms of 3-(p-fluoroaniline)-
5-(p-methoxy aniline)-triazine derivatives (4a and 5a,
respectively) were reacted with morpholine in DMSO at
60, 100, and 1308C and monitored by LC/MS (Table 1) and
showed a significant increase in reactivity when the linker
was activated.
Similarly, the addition of a 0.2 M solution of p-methoxyani-
line in DMF onto the 3-chloro-5-(p-fluororaniline)-triazine
scaffold was monitored (Fig. 2(b)). The reaction reached
completion after 10 h. As expected p-methoxyaniline
reacted much faster than p-fluororaniline due to the
enhanced electron-rich character of the aniline. On going
from the first to the second aromatic substitution, the
electrophilic character of the aromatic triazine ring is
Cleavage with a range of cyclic secondary amines
proceeded with high efficiency at 1008C (Table 2), all
triazines being completely cleaved within 1 h.
Following optimisation in solution, the chemistry was
transferred to polystyrene resin and gave purities of cleaved
Figure 3. Building blocks used for triazine library synthesis.

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S. Lebreton et al. / Tetrahedron 59 (2003) 10213–10222
Initial synthesis and cleavage from generation 1.0 dendri-
merised resin afforded the expected triazines with purities
between 55 and 75%. Close monitoring of the reaction
cleavage with all four cyclic amines (R3, Fig. 3) showed that
in most cases a compound of mass Mþ16 was being
generated at a much faster rate than the expected triazine
(which eluted before the desired triazine on LC/MS). This
suggested that the impurity was an oxidised form of the
triazine due to the long exposure to peracetic acid. The
N-oxide triazine would also withdraw electron density from
the triazine system, which would result in faster nucleo-
philic attack and cleavage compared to the non-oxidised
triazine. To verify this, the oxidation step was carried out for
7 rather than 18 h in order to reduce N-oxidation. Cleavage
in this case gave all triazines in purities between 81 and
94%. The beads were found to be robust throughout the
synthesis, even to the high temperature cleavages.
Figure 4. Structure of the triazine chosen for single-bead loading
quantification.
Table 3. Single-bead loading determinations (results shown in nmol/bead)
Gen. 0.0
Gen. 0.0 recycled
Gen. 1.0
Gen. 2.0
Bead 1
Bead 2
Bead 3
Bead 4
15.9
10.4
14.2
11.5
11.8
24.0
23.3
21.2
48.6
47.4
51.3
16.9
16.3
17.2
Average 16.6 (92%) 12.0 (67%)
22.8 (50%) 49.1 (41%)
Construction of the single-bead based triazines on gener-
ation 2.0 resin began by reacting a 0.5 M solution of
cyanuric chloride (50 equiv.) with resin-bound linker 2d
followed by amine displacement as shown in Scheme 1.
Oxidation to the sulfone with peracetic acid was followed
by nucleophilic aromatic substitution with morpholine in
DMSO at 1008C to afford the expected triazine 7 in 77%
purity (HPLC, l¼254 nm) (Fig. 4).
Based on an HPLC calibration analysis, loading measure-
ments were carried out on the triazines 5b,c,d (Table 3).
Cleavage from a generation 2.0 single bead released
49.1 nmol of product, corresponding to a 41% yield based
on the full loading of generation 2.0 resin-bound dendrimer.
Figure 5. Zone-based diffusion assay.
A 96-compound library (6R1£4R2£4R3, Fig. 4) was
synthesised starting from the 3,5-dichloro-triazine resin 3.
The resin was distributed into a 96-well filter-plate where
reaction steps were carried out. Once the linker was
activated beads were transferred into a Bohdan Mini-
blocke to allow cleavage at 1008C. Removal of the solvent
and excess of amines under vacuum using a Genevace
afforded the triazine derivatives. The library was then split
into two daughter plates for quality assessment and
compounds between 83 and 98%. Triazine cleavage
resulted in the formation of the phenolic resin 6b, which
could be reused by repeating all synthetic steps (i.e. addition
of cyanuric chloride followed by subsequent aniline
additions) to afford cleaved triazines with purities
between 58 and 63%, but the pre-oxidised linker made
all displacements much faster and
discriminating.
a little less
Figure 6. Most active library members showing antibacterial activity.

S. Lebreton et al. / Tetrahedron 59 (2003) 10213–10222
10217
extensively studied and optimised through solution and
solid-phase studies. A library of triazines prepared on
aminomethyl polystyrene resin allowed the identification of
active compounds, which inhibited bacteria growth in a
zone-based assay. High-loading dendrimer beads allowed
the release of sufficient amount of compound from a single
bead to give clear inhibition, which was not the case for
non-dendrimerised single beads. In conclusion, the high-
loading dendrimerised beads developed in our group
continue to act as valuable synthetic supports for single-
bead screening and analysis.
Figure 7. Single and multi-bead screening for compound D9 on generation
0.0, 1.0, and 2.0 resins.
screening. Quality control of all library members was
achieved by LC/MS, and all expected compounds were
found with purities between 80 and 100% (l¼254 nm).
Only the 3,5-dimethoxyaniline building block did not give
the expected compound. Instead, the major compound was
found to correspond to the triazine core bearing one R1
substituent, no R2 (3,5-dimethoxyaniline) and two R3
substituents.
2. Experimental
2.1. General
NMR spectra were recorded using a Bruker DPX 400
spectrometer operating at 400 MHz for 1H and 100 MHz for
13C. Chemical shifts were reported on the d scale in ppm and
were referenced to residual non-deuterated solvent reson-
ances. Mass spectra were obtained on a VG Platform single
quadrupole mass spectrometer in electrospray mode. IR
spectra were obtained on a BioRad FTS 135 spectrometer
with a Golden gate accessory, with neat compounds as oils
or solids with stretches reported in cm21. Melting points are
uncorrected. Commercially available reagents were used
without further purification. THF was freshly distilled under
nitrogen from a solution of sodium and benzophenone.
Purifications by column chromatography were achieved on
silica gel 60 (230–400 mesh) purchased from Merck.
Analytical HPLC data were obtained using a Hewlett–
Packard HP1100 Chemstation, using a Phenomenex C18
prodigy 5 mm (150 mm£3 mm) column with a flow rate of
1 ml/min monitoring at the wavelengths of 254 nm and ELS
(PL-ELS 1000 from Polymer Laboratories) and eluting with
(A) 0.1% TFA in water and (B) 0.042% TFA in acetonitrile,
gradient 0% (B) to 100% (B) over 20 min.
The second library daughter plate was prepared for screen-
ing by dissolving the library member in DMF and spotting
onto discrete 6 mm diameter filter papers, which were then
placed on top of 1% agar seeded with Macrococcus luteus
and incubated at 378C for 48 h (Fig. 5). Assay revealed the
presence of several inhibitors exhibiting zones of
inhibition (DMF alone did not affect bacteria growth).
Similar activities were obtained with Bacillus subtilis
bacteria. The top five members of the library chosen for in
depth screening and chemical analysis (Fig. 6) were
re-synthesised in solution en masse from cyanuric chloride
and three, sequential, amine displacements.
Compound D9, which gave the best inhibition, was
re-synthesised on Generation 1.0 and Generation 2.0 resins.
Cleavages from 1, 2, 3, 5, and 10 beads were carried out.14
Each sample was then screened using the same procedure as
described above. The results are shown in Figure 7.
Thus cleavage from a generation 2.0 single bead released
enough product to give a clear zone of inhibition. It was
found that at least three beads for generation 1.0 and ten
beads for generation 0.0 were necessary to give similar
zones of inhibition. Single-bead analysis by LC/MS showed
that the product was of good purity (Fig. 8).
LC/MS spectra were obtained on one of the following
spectrometers. Micromass ZMD spectrometer in electro-
spray positive mode using a Phenomenex Synergi 4m MAX-
RP 80A (50 mm£4.60 mm i.d.) column (Method A: from 5
to 95% MeCN in 1% HCOOH in water over 5 min). Waters
ZMD spectrometer in electrospray positive mode using a
Waters XTerra MS C18 5m (50 mm£3.0 mm i.d) column
(Method B: from 5 to 100% MeOH in 0.1% HCOOH in
water over 10 min; Method C: from 5 to 100% MeOH in
0.1% HCOOH in water over 15 min). The preparation of
resin-bound dendrimers has been reported elsewhere.15
Zone-based antibacterial assays were successfully
developed to screen triazine based antibiotics released
from single-beads. Synthesis, based on the attachment of
cyanuric chloride onto a Marshall-type safety catch linker,
followed by successive aromatic nucleophilic substitutions
and linker activation prior to nucleophilic cleavage, was
Figure 8. LC/MS analysis from a single-bead cleavage (l¼254 nm).

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S. Lebreton et al. / Tetrahedron 59 (2003) 10213–10222
Inhibition assays. Solutions of each compound in DMF at a
concentration between 50 and 1000 mg/ml were spotted
onto discrete filter papers (6 mm in diameter) and placed
onto seeded agar plates
J¼9 Hz, 2H), 7.15 (d, J¼9 Hz, 2H), 7.24–7.31 (m, 5H),
8.45 (t, J¼6 Hz, 1H), 9.58 (s, 1H); 13C NMR (d6-DMSO) d
39.1, 42.3, 116.0, 123.3, 126.6, 127.1, 128.1, 132.9, 139.1,
157.0, 168.1; IR (neat) nmax 1626, 749, 698; MS (ESþ) m/z
274.4 (100%) [MþH]þ, 296.3 [MþNa]þ; elemental
analysis found C 65.99%, H 5.77%, N 5.78%, S 11.15%;
calcd C 65.91%, H 5.53%, N 5.12%, S 11.73%.
Antibacterial dilutions (mg/ml)
800 700 600 500 400 300
1000 900
F7 10þþ 7.5þþ 8þþ 8þþ 8þþ 7þ 7þ
2.1.3. 2-[[4-[(4,6-Dichloro-1,3,5-triazin-2-yl)oxy]-
phenyl]thio]-N-(phenylmethyl)-acetamide (3a). To
B7 8þ
8þ
7þ
a
D9 8þþ 8þþ
D5 7.5þ 7.5þ
A9 9þþ 8þþ
7.5þ 7þ
7þ
solution of cyanuric chloride (177 mg, 0.96 mmol) in THF
(2.5 ml) stirred at 08C was added dropwise a solution of 2a
(250 mg, 0.91 mmol) in THF (0.5 ml) followed by the
dropwise addition of a solution of DIPEA (124 mg,
0.96 mmol) in THF (0.5 ml). The resulting mixture was
stirred at ice-bath temperature for another hour before
warming to room temperature, where it was stirred for 18 h.
The reaction mixture was concentrated in vacuo to give an
oil which was extracted with DCM (50 ml) and water
(50 ml). The organic phase was washed with water
(2£50 ml) and the combined aqueous phases were extracted
with DCM (2£100 ml). The combined organic phases were
dried over MgSO4 and concentrated in vacuo to give 3a as a
white solid (379 mg, 0.90 mmol, 98% yield): mp 142–
7.5þ 7.5þ 7.5þ 7þ 7þ
8þ
7.5þ 7.5þ
Zones of inhibition (mm); þþ, clear crisp zone of inhibition; þ, non-clear
zone of inhibition.
Agar medium was cooled to 45–508C before the addition of
the bacteria (between 10 and 100 ml per 40 ml of LB Agar
medium of log phase M. luteus or B. subtilis). This was
shaken gently for a few seconds before pouring into a
130 mm plate. When the medium had hardened completely,
the plates were inverted and stored at 48C.17
2.1.1. 2-(4-Hydroxyphenylthio)acetic acid (1). To a stirred
solution of 4-mercaptophenol (10 g, 79.25 mmol) in THF
(200 ml) was added DIPEA (12 g, 118.8 mmol). The
reaction mixture was stirred for 10 min. As the mixture
became viscous with the formation of a white precipitate,
more THF (100 ml) was added. Bromoacetic acid (16.5 g,
118.8 mmol) was added, and the solution was stirred for
20 h. The solvent was removed in vacuo, and the resulting
solid was dissolved in AcOEt (300 ml) and washed with
acidified water (2£300 ml, pH 1) and water (2£300 ml).
The combined water phases were extracted with AcOEt
(1£500 ml). The combined organic phases were dried over
MgSO4 and concentrated in vacuo to give a yellowish solid.
Recrystallisation from AcOEt afforded 1 as a white solid
(12.75 g, 69.21 mmol, 87% yield): mp 1388C (lit. 141–
1428C);16 TLC Rf¼0.36 (DCM/MeOH/AcOH, 9:1:0.05);
1H NMR (d6-DMSO) d 3.67 (s, 2H), 6.85 (d, J¼8.5 Hz, 2H),
7.36 (d, J¼8.5 Hz, 2H), 9.69 (s, 1H), 12.65 (br s, 1H); 13C
NMR (d6-DMSO) d 38.2, 116.5, 123.5, 133.3, 157.5, 171.3;
IR (neat) nmax 3119, 1686; MS (ES2) m/z 183.9 (75%)
[M2H]2.
1
1448C; LC/MS 3.78 min (98%); method A; H NMR (d6-
DMSO) d 3.70 (s, 2H), 4.44 (d, J¼6 Hz, 2H), 7.01 (t,
J¼6 Hz, 1H), 7.10 (d, J¼8.5 Hz, 2H), 7.09–7.14 (m, 2H),
7.25–7.27 (m, 3H), 7.33 (d, J¼8.5 Hz, 2H); 13C NMR (d6-
DMSO) d 37.4, 43.8, 122.0, 127.6, 128.7, 129.7, 133.4,
137.6, 149.7, 167.3, 170.9, 173.2; IR (neat) nmax 1640; MS
(ESþ) m/z 421.1 (100%) [MþH]þ.
2.1.4. 2-[[4-[[4-Chloro-6-[(4-fluorophenyl)amino]-1,3,5-
triazin-2-yl]oxy] phenyl] thio]-N-(phenylmethyl)-aceta-
mide (4a). To a solution containing 3a (1.3 g, 3.086 mmol)
in THF (30 ml) stirred at 08C was added a solution of
p-fluoroaniline (343 mg, 3.086 mmol) in THF (1.5 ml)
followed by the addition of a solution of DIPEA (399 mg,
3.086 mmol) in THF (1.5 ml) at such a rate that the
temperature always remained below 58C. After stirring for
2 h at ice-bath temperature, the reaction mixture was
concentrated in vacuo to give an oil which was extracted
with DCM (100 ml) and water (100 ml). The organic phase
was washed with water (2£100 ml) and the combined
aqueous phases were extracted with DCM (1£250 ml). The
combined organic phases were dried over MgSO4 and
concentrated in vacuo. The resulting foam was purified
by column chromatography (AcOEt/isohexane, 2:3) to give
2-[[4-[[4-chloro-6-[(4-fluorophenyl)amino]-1,3,5-triazin-2-
yl]oxy] phenyl] thio]-N-(phenylmethyl)-acetamide as a
white foam (1.36 g, 2.75 mmol, 89% yield): mp 86–878C;
LC/MS 3.95 min (98%); method A; 1H NMR (d6-DMSO) d
3.78 (s, 2H), 4.31 (d, J¼6 Hz, 2H), 7.06 (t, J¼9 Hz, 2H),
7.19–7.33 (m, 9H), 7.44–7.51 (m, 2H); 13C NMR (d6-
DMSO) d 37.0, 42.9, 115.7, 122.8, 123.9, 127.2, 127.6,
128.7, 129.7, 134.0, 134.4, 139.5, 150.4, 158.0, 160.5,
164.9, 170.2, 171.2, 168.3; IR (neat) nmax 1631; MS (ESþ)
m/z 496.4 (100%) [MþH]þ, 518.5 [MþNa]þ.
2.1.2. 2-[(4-Hydroxyphenyl)thio]-N-(phenylmethyl)-
acetamide (2a). To a solution of 2-(4-hydroxyphenylthio)-
acetic acid 1 (2 g, 10.86 mmol) and HOBt (1.61 g,
11.95 mmol) in DMF (50 ml) was added benzylamine
(1.28 g, 11.95 mmol). The reaction mixture was stirred for
5 min before adding DCC (2.7 g, 13.03 mmol). A white
precipitate formed within hours. The reaction mixture was
stirred for another 17 h. The mixture was filtered and
washed with DMF. The filtrate was concentrated in vacuo
and then extracted with DCM (250 ml) and water (250 ml).
The aqueous phase was extracted with DCM (2£200 ml)
and the combined organic phases washed with water
(3£200 ml). The organic phase was dried over MgSO4
and concentrated in vacuo. After purification by column
chromatography (AcOEt/isohexane, 1:1), 2a was isolated as
a white solid (2.73 g, 9.99 mmol, 92% yield): mp 107–
To a stirred solution of 2-[[4-[[4-chloro-6-[(4-fluorophenyl)-
amino]-1,3,5-triazin-2-yl]oxy] phenyl] thio]-N-(phenyl-
methyl)-acetamide (500 mg, 1.01 mmol) in DMF (10 ml)
was added a solution of p-anisidine (149 mg, 1.21 mmol) in
1
1088C; LC/MS 2.72 min (98%); method A; H NMR (d6-
DMSO) d 3.53 (s, 2H), 4.27 (d, J¼6 Hz, 2H), 6.73 (d,

S. Lebreton et al. / Tetrahedron 59 (2003) 10213–10222
10219
DMF (1.0 ml) followed by the addition of a solution of
DIPEA (156 mg, 1.21 mmol) in DMF (1.0 ml). The reaction
mixture was stirred for 22 h before being concentrated in
vacuo to give an oil which was extracted with DCM (80 ml)
and water (80 ml). The organic phase was washed with
water (3£80 ml) and the combined aqueous phases were
extracted with DCM (2£150 ml). The combined organic
phases were dried over MgSO4 and concentrated in vacuo.
Purification by column chromatography (DCM to
DCM/MeOH, 95:5) afforded the title compound as a
yellowish oil which crystallised within a few hours
(584 mg, 1.00 mmol, 99% yield): mp 1008C; LC/MS
cooled to 258C. Cyanuric chloride (902 mg, 4.89 mmol) was
then slowly added followed by dropwise addition of a
solution of DIPEA (178 mg, 1.38 mmol) in THF (1 ml) at
such a rate that temperature never exceeded 258C. The
reaction mixture was shaken for another 20 h and the resin
filtered and washed with THF, THF/DCM, and DCM
(5£10 ml each). The resin was finally dried in a vacuum
oven at 608C for 24 h to afford 1.12 g of resin; IR nmax 1627;
elemental analysis: S 3.18%, which corresponds to
0.99 mmol/g.
2.1.8. [(4-Fluorophenyl)amino]-6-[(4-methoxyphenyl)-
amino]-1,3,5-triazine resin (4b). A suspension of resin
3b (500 mg, 0.515 mmol) in THF (3 ml) was cooled in an
ice-bath. A solution of p-fluoroaniline (60 mg, 0.541 mmol)
in THF (1 ml) was added dropwise. A white precipitate
formed which dissolved upon dropwise addition of a
solution of DIPEA (70 mg, 0.541 mmol) in THF (0.5 ml).
The syringe was left at ice-bath temperature for 1 h with
occasional shaking and then shaken at rt for 8 h. The
1
4.10 min (100%); method A; H NMR (d6-DMSO) d 3.69
(s, 2H), 3.79 (s, 3H), 4.45 (d, J¼6 Hz, 2H), 6.82 (br d, 2H),
6.94 (br d, 2H), 7.06 (t, J¼6 Hz, 1H), 7.10–7.13 (m, 2H),
7.23–7.29 (m, 3H), 7.32 (d, J¼7.0 Hz, 2H), 7.34–7.38 (m,
2H), 7.39–7.44 (m, 2H); 13C NMR (d1-chloroform) d 38.2,
43.8, 55.5, 114.1, 114.9, 115.3, 122.6, 123.2, 127.6, 128.7,
130.1, 130.8, 134.0, 137.7, 151.4, 156.5, 158.0, 160.4,
165.7, 170.9, 167.7; IR (neat) nmax 1654; MS (ESþ) m/z
583.2 (100%) [MþH]þ.
(4-chloro-6-[(4-fluorophenyl)amino]-1,3,5-triazine
resin
was subsequently washed with THF, THF/DCM, and
DCM (5£3 ml each) before being dried in a vacuum
oven at 608C overnight. 553 mg of resin was obtained;
IR nmax 1645; elemental analysis: S 3.07%, which
corresponds to 0.96 mmol/g; F 1.69%, which corresponds
to 0.89 mmol/g.
2.1.5. 2-[[4-[[4-[(4-Fluorophenyl)amino]-6-[(4-methoxy-
phenyl)amino]-1,3,5-triazin-2-yl]oxy]phenyl]sulfonyl]-
N-(phenylmethyl)-acetamide (5a). To a stirred solution of
4a (50 mg, 0.086 mmol) in DCM (0.5 ml) was added a
solution of peracetic acid (39% in AcOH, 43 mg,
0.218 mmol). The reaction mixture was stirred for 7 h
before being extracted with DCM (20 ml) and water
(20 ml). The organic phase was washed with water
(2£20 ml) and the combined aqueous phases were extracted
with DCM (1£50 ml). The combined organic phases were
dried over MgSO4 and concentrated in vacuo. Purification
by column chromatography (AcOEt/isohexane, 3:2)
afforded the title compound as a yellowish oil (48.8 mg,
0.079 mmol, 92% yield): LC/MS 4.01 min (100%); method
A; 1H NMR (d6-DMSO) d 3.72 (s, 3H), 4.15 (s, 2H), 4.32 (d,
J¼6 Hz, 2H), 6.75 (br d, J¼8 Hz, 2H), 6.86–6.90 (m, 2H),
7.20–7.29 (m, 5H), 7.29–7.33 (d, J¼9 Hz, 2H), 7.44 (br d,
2H), 7.54 (br d, 2H), 7.87 (d, J¼9 Hz, 2H), 8.42 (br t, 1H);
13C NMR (d1-chloroform) d 43.6, 55.4, 61.9, 113.7, 114.8,
115.1, 122.7, 123.1, 127.3, 127.8, 128.5, 130.1, 131.7,
135.0, 137.9, 155.9, 157.0, 157.4, 159.8, 161.1, 165.6,
170.1; IR (neat) nmax 1659, 1292, 1151; MS (ESþ) m/z 615.2
(100%) [MþH]þ.
To a suspension of 4-chloro-6-[(4-fluorophenyl)amino]-
1,3,5-triazine resin (200 mg, 0.192 mmol) in DMF (1 ml)
was added a solution of p-anisidine (33 mg, 0.269 mmol) in
DMF (0.5 ml) followed a solution of DIPEA (35 mg,
0.269 mmol) in DMF (0.5 ml). The resin was shaken for
18 h, and then subsequently washed with THF, THF/DCM,
and DCM (5£2 ml each) before being dried in a vacuum
oven at 608C overnight. 216 mg of resin was obtained; IR
n
max 1645; elemental analysis: S 2.73%, which corresponds
F 1.53%, which corresponds to
to 0.85 mmol/g;
0.81 mmol/g.
2.1.9. [(4-Fluorophenyl)amino]-6-[(4-methoxyphenyl)-
amino]-1,3,5-triazine resin (5b). To a suspension of resin
4b (110 mg, 0.093 mmol) in DCM (1 ml) was added 39%
peracetic acid in acetic acid (73 mg, 0.372 mmol). The resin
turned from yellow to orange. After shaking for 18 h, the
resin was subsequently washed with DCM, DCM/AcOH
(1:1), and DCM (5£2 ml each), and dried in vacuum oven at
608C overnight. 118 mg of resin was obtained; IR nmax
1650, 1284, 1147; elemental analysis: S 2.78%, which
corresponds to 0.87 mmol/g; F 1.43%, which corresponds to
0.75 mmol/g.
2.1.6. 4-Hydroxythiophenol resin (2b). To 1% DVB
polystyrene aminomethyl resin (250–300 mm, 1 g,
1.38 mmol) was added a solution of 2-(4-hydroxy-
phenylthio)acetic acid linker (508 mg, 2.76 mmol) and
HOBt (372 mg, 2.76 mmol) in DMF (10 ml). After shaking
the reaction vessel for 5 min DIC (436 mg, 3.45 mmol) was
added. The resin was shaken for 18 h and subsequently
washed with DMF, DMF/THF, THF, THF/DCM, and
DCM (5£10 ml each) before being dried in a vacuum
oven at 608C for 24 h. A Ninhydrin test gave a negative
result; IR nmax 1625; MAS-NMR (CDCl3) d 3.48 (br s, 2H);
elemental analysis: S 3.64%, which corresponds to
1.14 mmol/g.
2.2. Generation 1.0 4-hydroxythiophenol resin (2c)
To generation 1.0 dendrimerised polystyrene resin (250–
300 mm, 664 mg, 1.06 mmol) was added a solution of the
linker 1 (584 mg, 3.17 mmol) and HOBt (429 mg,
3.17 mmol) in DMF (7 ml). After shaking the reaction
vessel for 5 min DIC (534 mg, 4.24 mmol) was added. The
resin was shaken for 36 h and subsequently washed with
DMF, DMF/THF, THF, THF/DCM, and DCM (5£10 ml
each) before being dried in a vacuum oven at 408C
overnight. A ninhydrin test was negative.
2.1.7. 4,6-Dichloro-1,3,5-triazine resin (3b). A suspension
of 4-hydroxythiophenol resin 2b (950 mg, 1.06 mmol) in
THF (10 ml) was heated at 508C under N2 for 20 min and

10220
S. Lebreton et al. / Tetrahedron 59 (2003) 10213–10222
2.3. Generation 1.0 4,6-dichloro-1,3,5-triazine resin (3c)
solution of cyanuric chloride (415 mg, 2.25 mmol) in THF
(2.0 ml) followed by dropwise addition of a solution of
DIPEA (79 mg, 0.45 mmol) in THF (1 ml). The resin was
left unstirred at ice-bath temperature for 1 h and then shaken
at rt for 18 h. The resin was subsequently washed with
DMF, DMF/THF, THF, THF/DCM, and DCM (5£5 ml
each). The resin was finally dried in a vacuum oven at 608C
for 24 h to afford 64 mg of resin.
To a suspension of resin 2c (255 mg, 0.321 mmol) in THF
(1 ml) cooled at 0–58C under N2 was slowly added a
solution of cyanuric chloride (296 mg, 1.61 mmol) in THF
(1.0 ml) followed by the dropwise addition of a solution of
DIPEA (166 mg, 1.28 mmol) in THF (1 ml). The resin was
left unstirred on an ice-bath temperature for 1 h and then
shaken at rt for 22 h. The resin was subsequently washed
with DMF, DMF/THF, THF, THF/DCM, and DCM
(5£5 ml each). The resin was finally dried in a vacuum
oven at 608C for 24 h to afford 307 mg of resin; elemental
analysis: S 3.46%, which corresponds to 1.08 mmol/g.
2.8. Generation 2.0 [(4-fluorophenyl)amino]-6-[(4-
methoxyphenyl)amino]-1,3,5-triazine resin (4d)
To a suspension of resin 3d (30 mg, 0.216 mmol) in DMF/
THF (1:1, 0.2 ml) at 0–58C was added a solution of
p-fluoroaniline (6.2 mg, 0.056 mmol) in DMF/THF (1:1,
0.1 ml) followed by DIPEA (7.3 mg, 0.056 mmol). The
resin was shaken at rt for 8 h before being subsequently
washed with DMF. DMF/THF, THF, THF/DCM, and DCM
(5£2 ml each). After drying the resin in a vacuum oven at
608C overnight, 200 mg of Generation 2.0 4-chloro-6-[(4-
fluorophenyl)amino]-1,3,5-triazine resin was obtained. To a
suspension of this resin (0.023 mmol) in DMF (0.2 ml) was
added a solution of p-anisidine (11 mg, 0.09 mmol) in DMF
(0.1 ml) followed by DIPEA (12 mg, 0.09 mmol). The resin
was shaken for 36 h plus 2 h at 508C, then subsequently
washed with DMF, DMF/THF, THF, THF/DCM, and DCM
(5£2 ml each), and finally dried in a vacuum oven at 608C
overnight.
2.4. Generation 1.0 [(4-fluorophenyl)amino]-6-[(4-
methoxyphenyl)amino]-1,3,5-triazine resin (4c)
To a suspension of resin 3c (200 mg, 0.216 mmol) in
DMF/THF (1:1, 2 ml) at 0–58C was added a solution of
p-fluoroaniline (25 mg, 0.224 mmol) in DMF/THF (1:1,
0.3 ml) followed by a solution of DIPEA (29 mg,
0.224 mmol) in DMF/THF (1:1, 0.3 ml). The Generation
1.0 4-chloro-6-[(4-fluorophenyl)amino]-1,3,5-triazine resin
was shaken at rt for 8 h before being subsequently washed
with DMF, DMF/THF, THF, THF/DCM, and DCM
(5£5 ml each). After drying the resin in a vacuum oven at
608C overnight, 200 mg of resin was obtained. To the dry
Generation 1.0 4-chloro-6-[(4-fluorophenyl)amino]-1,3,5-
triazine resin (30 mg, 0.03 mmol) was added a solution of
p-anisidine (15 mg, 0.12 mmol) in DMF (0.3 ml) followed
by DIPEA (8 mg, 0.06 mmol). The resin was shaken for
15 h, then heated for 2 h at 508C, and subsequently washed
with DMF, DMF/THF, THF, THF/DCM, and DCM
(5£2 ml each), and finally dried in a vacuum oven at 608C
overnight. 33 mg of resin was obtained.
2.9. Generation 2.0 [(4-fluorophenyl)amino]-6-[(4-
methoxyphenyl)amino]-1,3,5-triazine resin (5d)
To a suspension of resin 4d (0.023 mmol) in DCM (0.2 ml)
was added 39% peracetic acid in acetic acid (23 mg,
0.12 mmol). After shaking for 7 h, the resin was sub-
sequently washed with DCM, DCM/AcOH (1:1), and DCM
(5£2 ml each), and dried in vacuum oven at 608C overnight.
2.5. Generation 1.0 [(4-fluorophenyl)amino]-6-[(4-
methoxyphenyl)amino]-1,3,5-triazine resin (5c)
2.10. Library synthesis on aminomethyl polystyrene
resin
To a suspension of resin 4c (33 mg, 0.03 mmol) in DCM
(0.3 ml) was added 39% peracetic acid in acetic acid
(29 mg, 0.15 mmol). After shaking for 7 h, the resin was
subsequently washed with DCM, DCM/AcOH (1:1), and
DCM (5£2 ml EACH), and dried in vacuum oven at 608C
overnight. 36 mg of resin was obtained.
4,6-dichloro-1,3,5-triazine resin 3b was distributed in a 96-
well filter plate (,0.025 mmol/well). After swelling the
resin in 0.10 ml of dry THF, solutions of the first set of
aniline derivatives in dry THF (0.06 mmol/well) was added
followed by DIPEA (0.06 mmol/well). The plate was sealed
and shaken on an horizontal shaker for 5 h. The resins were
washed with THF and DMF, before adding solutions of the
second set of aniline derivatives (0.06 mmol/well) and
DIPEA (0.06 mmol/well). The plate was sealed and shaken
for 13 h, plus 2 h in a water bath at 558C. The resins were
washed with DMF, THF and DCM before being dried in a
vacuum oven at 408C overnight. The resin was swollen in
0.3 ml DCM. A 39% solution of peracetic acid in acetic acid
(0.114 mmol/well) was added and the plate was sealed and
shaken for 5 h. The resins were washed with DCM, DCM/
AcOH (1:1), and DCM before being dried in a vacuum oven
at 408C overnight. Aliquots of beads (50) were transferred
into a 48-well MiniBlocke where they were swollen in
0.1 ml of DMSO before adding the corresponding solution
of cyclic secondary amine (0.06 mmol/well). The plate was
sealed and shaken at 1008C for 5 h. The filtrates were
collected in a 96-well plate. The resins were washed
2.6. Generation 2.0 4-hydroxythiophenol resin (2d)
To generation 2.0 dendrimerised polystyrene resin (250–
300 mm, 300 mg, 0.357 mmol) was added a solution of acid
linker 1 (329 mg, 1.79 mmol) and HOBt (241 mg,
1.79 mmol) in DMF (3 ml). After shaking the reaction
vessel for 5 min DIC (270 mg, 2.14 mmol) was added. The
resin was shaken for 36 h and subsequently washed with
DMF, DMF/THF, THF, THF/DCM, and DCM (5£5 ml
each) before being dried in a vacuum oven at 408C
overnight. The reaction was repeated until a satisfactory
ninhydrin test was obtained. 397 mg of resin was obtained.
2.7. Generation 2.0 4,6-dichloro-1,3,5-triazine resin (3d)
To a suspension of resin 2d (50 mg, 0.045 mmol) in THF
(1 ml) cooled at 2108C under N2 was slowly added a

S. Lebreton et al. / Tetrahedron 59 (2003) 10213–10222
10221
thoroughly with DMSO. DMSO and excess of low-boiling
point secondary amine removal was carried out on a
Genevace under vacuum.14
powder (1.09 g, 2.30 mmol, 85%): mp 171–1728C; LC/MS
1
7.96 min (100%); method B; H NMR (DMSO) d 2.33 (s,
3H), 2.48 (t, J¼4.5 Hz, 4H), 3.87 (t, J¼4.5 Hz, 4H), 6.09 (s,
2H), 6.95 (d, J¼8.5 Hz, 1H), 7.17 (d, J¼8.5 Hz, 1H), 7.57
(s, 1H), 7.70 (d, J¼8.5 Hz, 2H), 8.07 (d, J¼8.5 Hz, 2H),
9.26 (s, 1H), 9.66 (s, 1H); 13C NMR (CDCl3) d 42.7, 45.6,
54.2, 100.6, 102.5, 107.6, 112.8, 119.1, 121.1, 121.4, 125.4,
134.1, 142.0, 143.9, 146.8, 163.9, 164.3; MS (ESþ) m/z
474.2 (100%) [MþH]þ; elemental analysis found C
55.83%, H 4.65%, N 20.56%, F 11.79%; calcd C 55.81%,
H 4.68%, N 20.70%, F 12.04%.
2.10.1. Generation 2.0 single-bead cleavage and analysis
for D9. LC–MS (ESþ) 8.98 min (100%); method C; HR-
MS (ESþ) for C21H25N7F found 394.2154; calcd 394.2150.
2.10.2. Generation 2.0 single-bead cleavage and analysis
for F7. LC–MS (ESþ) 7.35 min (87%); method B; HR-MS
(ESþ) for C22H23N7O2F3 found 474.1858; calcd
474.1861.
2.11.3. N-(4-Methoxyphenyl)-6-(4-methylpiperazin-1-
yl)-N0-(4-trifluoromethylphenyl)-[1,3,5]triazine-2,4-dia-
mine (B7). Trituration with diethylether afforded the title
compound as white crystals (1.12 g, 2.44 mmol, 90%): mp
153–1568C; LC/MS 7.34 min (100%); method B; 1H NMR
(DMSO) d 2.33 (s, 3H), 2.47 (t, J¼4.5 Hz, 4H), 3.85 (s, 3H),
3.87 (t, J¼4.5 Hz, 4H), 7.00 (d, J¼4.5 Hz, 2H), 7.70 (d,
J¼4.5 Hz, 4H), 8.08 (d, J¼4.5 Hz, 2H), 9.20 (s, 1H), 9.61
(s, 1H); 13C NMR (CDCl3) d 43.3, 46.2, 54.9, 55.6, 114.1,
119.6, 121.6, 121.9, 122.5, 126.0, 133.3, 144.6, 155.2,
164.5, 165.0; MS (ESþ) m/z 460.4 (100%) [MþH]þ;
2.10.3. Generation 2.0 single-bead cleavage and analysis
for B7. LC–MS (ESþ) 7.32 min (94%); method B; HR-MS
(ESþ) for C22H25N7OF3 found 460.2066; calcd
460.2067.
2.10.4. Generation 2.0 single-bead cleavage and analysis
for A9. LC–MS (ESþ) 8.84 min (92%); method B; HR-MS
(ESþ) for C20H22N6O2F found 397.1780; calcd 397.1783.
2.10.5. Generation 2.0 single-bead cleavage and analysis
for D5. LC–MS (ESþ) 7.71 min (94%); method B; HR-MS
(ESþ) for C21H23N6O2 found 391.1874; calcd 391.1877.
elemental analysis found
C 57.17%, H 4.99%; N
21.03%, F 11.93%; calcd C 57.51%, H 5.26%, N 21.33%,
F 12.40%.
2.11. Synthesis of active triazines—general procedure
2.11.4. N-(3-Fluorophenyl)-N0-(4-methoxyphenyl)-6-
morpholin-4-yl-[1,3,5]triazine-2,4-diamine (A9). Purifi-
cation by column chromatography (neat DCM) afforded the
title compound as an off white powder (657 mg, 1.66 mmol,
61%): mp 161–1638C; LC/MS 8.06 min (100%); method B;
1H NMR (DMSO) d 3.53 (t, J¼4.5 Hz, 4H), 3.61–3.63 (m,
7H), 6.61 (dt, J¼2.8, 8.5 Hz, 1H), 6.75 (d, J¼9.0 Hz, 2H),
7.13 (q, J¼8.0 Hz, 1H), 7.33 (d, J¼7.0 Hz, 1H), 7.46 (d,
J¼9.0 Hz, 2H), 7.68 (d, J¼10.5 Hz, 1H), 8.98 (s, 1H), 9.21
(s, 1H); 13C NMR (CDCl3) d 43.9, 55.6, 66.4, 106.6, 106.9,
108.1, 108.3, 114.1, 115.7, 122.5, 130.1, 130.2, 133.3,
142.6, 142.7, 155.2, 161.5, 163.9, 164.4, 165.1; MS (ESþ)
m/z 397.4 (100%) [MþH]þ.
To a stirred slurry solution of cyanuric chloride (500 mg,
2.71 mmol) in DCM (10 ml) at 2108C was added the first
aniline derivative (2.71 mmol) followed by DIPEA
(350 mg, 2.71 mmol). The reaction mixture was stirred at
2108C for 1 h. The second aniline (2.71 mmol) was added
followed by DIPEA (350 mg, 2.71 mmol). The reaction
mixture was stirred for 18 h. The corresponding cyclic
amine (2.98 mmol) was added followed by DIPEA (385 mg,
2.98 mmol), the reaction was stirred for 18 h, extracted with
DCM (200 ml) and water (200 ml). The aqueous phase was
extracted with DCM (2£100 ml) and the combined organic
phases were washed with water (2£200 ml) and concen-
trated in vacuo.
2.11.5. N-Benzo[1,3]dioxol-5-yl-N0-phenyl-6-piperidin-1-
yl-[1,3,5]triazine-2,4-diamine (D5). Purification by
column chromatography (neat DCM) afforded the title
compound as a light yellow powder (853 mg, 2.18 mmol,
2.11.1. N-(3-Fluorophenyl)-6-(4-methyl-piperazin-1-yl)-
N0-p-tolyl-[1,3,5]triazine-2,4-diamine (D9). Purification
by column chromatography (DCM/MeOH, 96:4) afforded
the title compound as white crystals (851 mg, 2.16 mmol,
80%): mp 186–1878C; LC/MS 6.94 min (100%); method B;
1H NMR (DMSO) d 2.09 (s, 3H), 2.14 (s, 3H), 2.23 (t,
J¼4.5 Hz, 4H), 3.64 (t, J¼4.5 Hz, 4H), 6.62 (dt, J¼2.0,
8.0 Hz, 1H), 6.96 (d, J¼8.0 Hz, 2H), 7.14 (q, J¼8.0 Hz,
1H), 7.35 (d, J¼8.0 Hz, 1H), 7.46 (d, J¼8.0 Hz, 2H), 7.69
(d, J¼12.0 Hz, 1H), 9.02 (s, 1H), 9.22 (s, 1H); 13C NMR
(CDCl3) d 20.8, 43.2, 46.2, 54.8, 106.8, 106.9, 108.1, 108.3,
115.7, 120.8, 129.3, 130.1, 130.2, 131.3, 137.8, 142.6,
142.7, 161.5, 163.9, 164.5, 164.9; MS (ESþ) m/z 394.3
(100%) [MþH]þ; elemental analysis found C 63.38%, H
6.10%, N 24.61%, F 4.82%; calcd C 64.10%, H 6.15%, N
24.91%, F 4.83%.
1
81%): mp 1358C; LC/MS 7.49 min (100%); method B; H
NMR (DMSO) d 1.63–1.64 (m, 4H), 1.74–1.75 (m, 2H),
3.86 (t, J¼5.0 Hz, 4H), 6.08 (s, 2H), 6.92 (d, J¼8.5 Hz, 1H),
7.05 (t, J¼7.5 Hz, 1H), 7.18 (d, J¼8.5 Hz, 1H), 7.37 (t,
J¼8.0 Hz, 2H), 7.63 (s, 1H), 7.84 (d, J¼7.5 Hz, 2H), 9.11
(s, 1H), 9.19 (s, 1H); 13C NMR (DMSO) d 24.8, 25.9, 44.2,
101.2, 102.9, 108.2, 113.0, 120.3, 122.0, 128.8, 135.2,
140.8, 142.3, 147.4, 146.5, 146.7; MS (ESþ) m/z 391.3
(100%) [MþH]þ; elemental analysis found C 64.43%, H
5.76%, N 21.41%; calcd C 64.60%, H 5.68%, N 21.51%.
Acknowledgements
2.11.2. N-Benzo[1,3]dioxol-5-yl-6-(4-methylpiperazin-1-
yl)-N0-(4-trifluoromethylphenyl)-[1,3,5]triazine-2,4-dia-
mine (F7). Purification by column chromatography (DCM/
MeOH, 96:4) afforded the title compound as a brownish
We would like to thank John Ellard for helpful advice on
bacterial screening. We also thank the EPSRC (JIF),
BBRSC and AstraZeneca for funding.

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S. Lebreton et al. / Tetrahedron 59 (2003) 10213–10222
L. E. J. Am. Chem. Soc. 2001, 123, 2889–2890. (i) Arya, P.;
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14. Single beads or multiple beads were placed in a 50 ml glass
vial insert. 20 ml of DMSO was added followed by one drop of
the corresponding secondary amine. The insert was introduced
into a HPLC vial with a teflon cap. Cleavage was carried out in
an oil bath at 1108C for 5 h, after which time DMSO and
excess amine were removed on a Genevace 15 ml of DMF
was added to each insert, which were then sonicated for
20 min at 408C. These solutions were used for screening and
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