Iodine scanning of a phenazine inhibitor of vacuolar sorting

Article abstract of DOI:10.1016/j.bmcl.2010.01.106

Affinity reagents are often used to address the target identification problem in chemical genetics. The design of such reagents so that the linker does not occlude interactions with protein targets is an ongoing challenge. This work describes a systematic approach to synthesize derivatives of a bioactive that should avoid interference with binding to targets and be readily converted to affinity reagents.

Full text of DOI:10.1016/j.bmcl.2010.01.106

Bioorganic & Medicinal Chemistry Letters 20 (2010) 1496–1499
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Iodine scanning of a phenazine inhibitor of vacuolar sorting
Marci Surpin a, Yunfan Zou b, Chiyi Xiong b, Natasha V. Raikhel a, Michael C. Pirrung b,
*
a Department of Botany and Plant Sciences and Center for Plant Cell Biology, University of California, Riverside, CA 92521, USA
b Department of Chemistry, University of California, Riverside, CA 92521, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Affinity reagents are often used to address the target identification problem in chemical genetics. The
design of such reagents so that the linker does not occlude interactions with protein targets is an ongoing
challenge. This work describes a systematic approach to synthesize derivatives of a bioactive that should
avoid interference with binding to targets and be readily converted to affinity reagents.
Ó 2010 Elsevier Ltd. All rights reserved.
Received 4 December 2009
Revised 15 January 2010
Accepted 20 January 2010
Available online 25 January 2010
Keywords:
Chemical genetics
Target identification
Gravitropism
Forward genetics based on mutagenesis and phenotypic screening
for modified function is a powerful method for the study of biological
pathways. Forward chemical genetics offers a complementary
approach in which a small molecule plays a role similar to that of a
modified (and readily identified) gene in a classical forward genetics
experiment. One aim of forward chemical genetics is to use the small
molecule to identify its protein target(s) and its role in the pathway
under study. Powerful methods for target identification based on
small molecules are therefore needed.1
Past work from one of our laboratories described the methyl
scanning approach to mapping the interactions between a bioac-
tive compound and its receptor protein.2 The activities of a family
of systematically synthesized derivatives were used to design and
prepare an affinity reagent for use in identification of unknown tar-
gets.3 While effective, this approach had the drawback that once
positions that can be substituted are identified, a de novo synthesis
of the affinity reagent is required. A streamlined approach would
use a group that could not only be scanned to identify sites that
can be modified without compromising activity, but would also
be convertible to the affinity reagent. We propose the use of iodine
for this purpose based on its van der Waals radius (2.15 Å), which
is comparable to methyl groups (1.7 Å) that proved able to perturb
small molecule–protein interactions in our initial studies. C–I
bonds are also synthetically versatile, participating in radical-
based bond formation, organometallic cross-coupling, and nucleo-
philic substitution. The method of iodine scanning developed here
was applied to a compound discovered via chemical genetics to
play a role in gravitropism in plants.
Past work from the other of our laboratories focused on the
interplay between gravitropic signal transduction and the plant
endomembrane system, which is functionally and genetically com-
plex. Plants utilize many of the same components for vesicular
trafficking through the endoplasmic reticulum as animals and fun-
gi, but also carry out unique functions, such as storage of proteins
and the biosynthesis of cell wall precursors.4 Furthermore, many of
the gene families that encode trafficking proteins in plants have
many more members than their animal counterparts. Such a mul-
tiplicity of trafficking genes has confounded our studies of the
plant endomembrane system. Many genes that encode its compo-
nents are essential for viability, limiting the utility of knockout mu-
tants.5 Conversely, many point mutants have no phenotypes.6 The
latter could be due to gene redundancy, a situation to which chem-
ical genetics may be particularly applicable.
Classical genetics screens had already demonstrated that the
plant endomembrane system is intimately involved in gravitropic
signal transduction. This pathway is not well understood, but
mutations in a number of genes that encode endomembrane sys-
tem components result in agravitropic phenotypes. Screening of
a commercial chemical library for compounds that interfere with
gravitropic responses in the model plant Arabidopsis identified four
compounds.7 They not only inhibit gravitropism via the endomem-
brane system, but also cause changes in vacuole morphology. One
such compound is 5271050 (1; N-phenylphenazin-2-amine; Fig. 1).
To discern structure–function relationships, we examined analogs
of 1 that were also available commercially. These included the
dioxide 2 and the parent ring system, phenazine itself. Neither of
these compounds was active in either phenotypic readout, gravit-
ropism or formation of aggregates of a prototypical vacuolar cargo
protein, tonoplast intrinsic protein (dTIP), which has been labeled
with green fluorescent protein (GFP) (vide infra).
* Corresponding author.
E-mail address: michael.pirrung@ucr.edu (M.C. Pirrung).
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.01.106

M. Surpin et al. / Bioorg. Med. Chem. Lett. 20 (2010) 1496–1499
1497
O
N
3'
KOH
I
N
O
I
NH2
NO2
O
N
N
4'
2'
+
I
7
8
N
N
O
NaOCl
64%
O
N
N
N
H
N
O
H
4
5
6
1
Scheme 2. Synthesis of 5/6-iodo-BFO.
ChemBridge ID 5271050
2
Figure 1. Structures of the vacuolar sorting inhibitor 5271050 (1) and its dioxide
analog 2.
these two isomers were present, it is unlikely that a substituent
distant from the site of the Beirut reaction would have a significant
effect on the cycloaddition, with the result that a mixture of regio-
chemical outcomes is expected. Therefore, both 7- and 8-iodo-
substituted derivatives of 1 should be formed. Though a mixture
of compounds is not ideal, syntheses that would give one com-
pound exclusively were much more involved.
With the 5/6 mixture in hand, the Beirut reaction with 3 was
performed as before (Scheme 3), delivering a 3:2 mixture of 7
and 8 (50% yield), which proved to be inseparable. Upon dithionite
We next became interested in applying the concept of iodine
scanning enumerated above to compound 1 so as to permit the gen-
eration of 1-based affinity reagents for identification of its target(s).
The only method reported for the preparation of 18 was unlikely to
have been used in a high-throughput synthesis for the generation
of a screening library. Both the presence of 2 in the ChemBridge li-
brary and a review of phenazine natural products9 suggested to us
a versatile synthetic route to phenazines based on the Beirut reac-
tion10 of benzofurazan-N-oxide (BFO; Scheme 1). The most common
version of the Beirut reaction is a hetero-Diels–Alder cycloaddition
between BFO and acetylenic dienophiles, after which bond reorgani-
zation and aromatization of the cycloadduct gives quinazoline-N,N’-
dioxides (Eq. 1). When the Beirut reaction of BFO is instead con-
ducted with a phenol in the presence of base, phenazine-N,N’-diox-
ides result (Eq. 2).
This approach toward 1 was readily tested, as both BFO and 4-
hydroxydiphenylamine (3) are commercial. Their reaction under
standard conditions provides 2, which could be reduced to 1 under
mild reaction conditions (Eq. 3). The wide availability of phenols
and the simplicity of this route suggest that this is very likely the
synthesis ChemBridge used to prepare 5271050.
This re-discovered route provided the convergent and modular
synthesis of 1 that is essential to efficient application of the iodine
scanning concept. The synthesis of iodinated derivatives of 1 sim-
ply required the preparation of the two building blocks of this syn-
thesis, BFO and 4-hydroxydiphenylamine, in iodine-substituted
forms. Several synthetic routes to BFOs are available; we adopted
the oxidation of o-nitroanilines, in part owing to the commercial
availability of 4-iodo-2-nitroaniline (4; Scheme 2). This compound
is oxidized with NaOCl under basic conditions to give a mixture of
the known11 5- and 6-iodinated BFOs 5 and 6.12 This mixture is a
result of the facile equilibration of the two positional isomers of
any substituted benzofurazan-N-oxide via o-dinitroso intermedi-
ates. This process is invisible when the BFO is symmetrical, but if
it is not, both isomers are routinely observed. Even if only one of
O
N
O
N
5/6
+
I
3
N
H
N
O
N
H
N
O
I
7
8
Scheme 3. Synthesis of 7/8-iodo-1.
N
N
I
N
N
I
N
H
N
H
9
10
12
N
N
I
N
I
N
H
N
H
N
11
Figure 2. Iodinated analogs of 1 that were prepared.
MeO
I
Pd°
+
BINAP
tert-BuONa
NH2
I
60%
MeO
I
HO
I
BBr3
O
N
N
N
N
N
O
R
O
CH2Cl2
65%
+
(1)
(2)
R
R
H
H
N
N
O
N
O
13
14
O
BFO
BFO / NaOH
Na2S2O4
10
O
N
20%
30%
HO
R
BFO +
BFO +
Scheme 4. Synthesis of analog 10.
R
N
O
I
50%
55%
73%
52%
28%
37%
35%
15
18
16
19
17
20
11
12
I
I
I
HO
NaOH Na2S2O4
2
1 (3)
N
H
45%
76%
44%
3
Scheme 1. Methods for phenazine synthesis based on BFO.
Scheme 5. Synthesis of analogs 11 and 12.

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M. Surpin et al. / Bioorg. Med. Chem. Lett. 20 (2010) 1496–1499
Figure 3. The effects of iodinated derivatives of 5271050 on aggregate formation in GFP:dTIP seedlings. (A) Hypocotyls were examined by laser scanning confocal microscopy
following germination and growth for seven days in the light and on the designated chemicals: DMSO control, 2 M 050, or 16 M compounds 9–12. Bars = 50 m. (B) The
l
l
l
effects of iodinated compounds on gravitropic bending. Four days old seedlings grown in the presence of 050 and its iodinated derivatives were gravistimulated for 24 h, and
the angle of curvature of the hypocotyl from a vertical position was measured. The values, in degrees standard error of the mean, for the DMSO control of each treatment
were as follows: 050, 55.4 3.2; 9, 48.7 3.7; 10, 58.2 3.3; 11, 53.9 5.2; and 12, 46.6 3.2. (C) The effects of iodinated compounds on hypocotyl growth. Values for the
DMSO control of each treatment, reported as mm standard error of the mean, were as follows: 050, 15.4 0.5; 9, 15.1 0.4; 10, 14.7 0.3; 11, 15.3 0.5; and 12, 14.6 0.4.
The results in panels (B) and (C) are reported as percent of DMSO controls, where the DMSO control was 100%.
reduction, the 7/8 mixture gave our initial target, 9, as a 4:1 mix-
ture (40% yield; Fig. 2). We cannot explain the variance of the iso-
mer ratios in these products and have not assigned the structure of
the major isomer. While in principle it should be possible to use
this route to prepare 6- and 9-iodo-substituted derivatives of 1,
substituents adjacent to the heterocycle severely compromise
reactivity of BFOs.10
The remainder of the targeted analogs should be accessible via
iodinated versions of phenol 3. This route was successfully applied
to the preparation of 10–12.
After initial investigation of some other strategies that proved
unsuccessful, a general route to iodinated 4-hydroxydiphenylam-
ines was adopted that involved coupling of p-methoxyaniline to
three diiodobenzene isomers using Buchwald–Hartwig conditions.
With p-diiodobenzene (Scheme 4), compound 13 results, from
which the methyl ether is cleaved with boron tribromide. The
known13 phenol 14 is then taken through the already proved syn-
thesis to give target 10.
By a similar sequence of reactions starting with o- and m-
diiodobenzene, the isomers 11 and 12 were accessed (Scheme 5).
Versions of 1 iodinated in the central benzene ring would in
principal be available from iodination of the phenol ring of 3, but
in practice it proved impossible to obtain such compounds cleanly,
owing largely to the strong tendency of 3 to be oxidized by electro-
philic halogenating reagents rather than being halogenated. We
were forced to be satisfied with the four analogs 9–12. While they
do not constitute a comprehensive set of iodo-substituted versions
of 1, they do at least represent functionalization at the extremities
of the molecule.
length, gravitropic bending, and aggregate formation. First, none
of the iodinated compounds reduced the hypocotyl length of trea-
ted seedlings (Fig. 3a), indicating that these four positions must
be unsubstituted for growth inhibition. The parent molecule, 1, de-
creased gravitropic bending by approximately 50% at the lowest
treatment concentration, 2
iodine atom at the 30 position, had only a slight effect on inhibition
of gravitropic bending; treatment with 2 M compound reduced
lM. Derivative 10, which features the
l
the bending angle by approximately 35%. The remaining three
derivatives had a greater impact on inhibition of gravitropism. In
their effects on inhibition of gravitropic bending, compound 9 devi-
ated only slightly from the negative DMSO control, and 10 and 11
were intermediate. Taken together, these results show that the 7/
8 positions on the molecule must be unsubstituted for inhibition
of gravitropic bending, while the 20 and 30 positions also contribute,
albeit to a lesser extent. Compound 12 (iodine in the 20 position) in-
duced aggregate formation at concentrations of 16
with 2 M for the parent 1). No effects were observed at concentra-
tions of 2, 4, or 8 M. Compound 11 showed sporadic aggregate for-
mation at 16 M, whereas no aggregates were observed for
compound 10 at this or higher concentrations. Compound 9 (iodine
in the 7/8 positions) induced aggregate formation P4 M.
lM (compared
l
l
l
l
Our conclusion from these results is twofold. First, one of the
bioactivity determinants of 5271050 for modifying the endomem-
brane system morphology surely resides in its aniline moiety. Sec-
ond, the growth/gravitropic and endomembrane morphology
phenotypes are clearly separable, implying that 5271050 has mul-
tiple targets. The observation that all of the iodinated derivatives
abrogate the growth effects and three out of the four derivatives af-
fect gravitropism inhibition of 5271050 suggests that both ends of
the molecule are important for these processes.
Treatment of Arabidopsis seedlings with compounds 9–12 re-
vealed varying degrees of efficacy (Fig. 3). Bioactivity testing of 9–
12 using the same approaches and treatment with 1 as a positive
control and DMSO as a negative control showed diverse effects of
the four compounds on three phenotypic responses: hypocotyl
We are currently cloning and characterizing two Arabidopsis
EMS mutants that do not form aggregates when treated with
5271050. Identification of these targets will further our under-

M. Surpin et al. / Bioorg. Med. Chem. Lett. 20 (2010) 1496–1499
1499
6. Avila, E. L.; Zouhar, J.; Agee, A. E.; Carter, D. G.; Chary, S. N.; Raikhel, N. V. Plant
Physiol. 2003, 133, 1673.
7. Surpin, M.; Rojas-Pierce, M.; Carter, C.; Hicks, G. R.; Vasquez, J.; Raikhel, N. V.
Proc. Natl. Acad. Sci. U.S.A. 2005, 102, 4902.
8. Paul, D. B. Aust. J. Chem. 1972, 25, 2283.
9. Laursen, J. B.; Nielsen, J. Chem. Rev. 2004, 104, 1663.
10. Haddadin, M. J.; Issidorides, C. H. Heterocycles 1993, 35, 1503.
11. Gehrz, R. C.; Britton, D. Acta Crystallogr., Sect. B 1972, 28, 1126; Uematsu, S.;
Akahori, Y. Chem. Pharm. Bull. 1978, 26, 25.
standing of early trafficking events in the plant endomembrane
system. A more complete understanding of aggregate induction
by 5271050 will provide a tool to dissect trafficking through the
endoplasmic reticulum in much the same way brefeldin A is used
to dissect trafficking through the Golgi and endosome.14
With respect to iodine scanning methodology and target identi-
fication for 5271050, the agent that is obviously of the most inter-
est for future studies is 9. This material should be readily
biotinylated using known reagents,15 and the resulting compound
should be useful to identify endoplasmic reticulum protein targets
relevant to the activity of 5271050 regarding vacuole morphology.
12. 1H NMR data: Compound 5/6: d 7.42 (br, 1H), 7.64 (br, 1H). 8.18 (br, 1H). 7/8: d
9.47 (s, 1H), 8.78–8.82 (m, 1H), 8.39–8.43 (m, 1H), 7.99–8.23 (m, 2H), 7.81 (d,
J = 2.4, 1H), 7.59–7.63 (m, 1H), 7.33–7.46 (m, 4H), 7.12–7.17 (m, 1H). 9: d 9.31
(s, 1H), 8.49–8.57 (m, 1H), 7.74–8.09 (m, 4H), 7.43–7.51 (m, 5H), 7.14–7.19 (m,
1H). 10: d 8.02–8.11 (m, 3H), 7.56–7.74 (m, 5H), 7.42 (dd, J = 9.1, 2.4, 1H), 7.05
(d, J = 8.7, 2H), 6.22 (br, 1H). 11: d 8.10–8.20 (m, 3H), 7.70–7.82 (m, 2H), 7.61–
7.68 (m, 2H), 7.45–7.54 (m, 2H), 7.32–7.38 (m, 1H), 7.08–7.14 (m, 1H), 6.30 (br,
1H). 12: d 8.12–8.20 (m, 3H), 7.70–7.82 (m, 2H), 7.61–7.68 (m, 2H), 7.45–7.54
(m, 2H), 7.32–7.38 (m, 1H), 7.08–7.14 (m, 1H), 6.30 (br, 1H). 13: d 7.45 (d,
J = 6.6, 2H), 7.05 (d, J = 6.6, 2H), 6.87 (d, J = 7.8, 2H), 6.66 (d, J = 6.3, 2H), 5.50 (br,
1H), 3.81 (s, 3H). 14: d 7.48 (d, J = 6.6, 2H), 7.01 (d, J = 6.3, 2H), 6.80 (d, J = 6.5,
2H), 6.64 (d, J = 6.6, 2H), 5.45 (br, 1H), 4.67 (br, 1H). 15: d 7.21–7.22 (m, 1H),
7.06–7.15 (m, 3H), 6.79–6.83 (m, 4H), 5.47 (br, 1H), 3.80 (s, 3H). 16: d 7.21 (s,
1H), 7.15 (d, J = 7.5, 1H), 6.80–7.02 (m, 6H), 6.06 (br, 1H), 5.50 (br, 1H). 17: d
9.49 (s, 1H), 8.46–8.55 (m, 3H), 7.81–7.92 (m, 3H), 7.60–7.69 (m, 2H), 7.47 (d,
J = 7.5, 1H), 7.40 (d, J = 7.5, 1H), 7.40 (t, J = 8.1, 1H).
Acknowledgment
This work was partially supported by NSF Grant MCB-0515963.
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